JPS6363556B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates generally to novel hybrid cell lines, and more particularly to hybrid cell lines for the production of monoclonal antibodies directed against certain antigens found in normal human cytotoxic and suppressor T cells. , the antibodies so produced, and therapeutic, diagnostic methods and compositions using the antibodies. Fusion of mouse myeloma into splenocytes from immunized mice by Kohler and Milstein in 1975 [Nature 256, 495-497
(1975)] were the first to demonstrate that it was possible to obtain continuous cell lines that produced homogeneous (so-called ``monoclonal'') antibodies. Since this basic research, much effort has been devoted to the generation of various hybrid cells (so-called "hybridomas") and the use of antibodies produced by these hybridomas in various scientific studies. . For example, see Current
Topics in Microbiology and Immunology,
Volume 81−“Lymphocyte Hybridomas”, F.
Melchers, M. Patter, and N. Warner.
Editors, Springer-Verlag, 1978, and references contained therein; CJ Barnstable, et al.,
Cell, 14, 9-20 (May, 1978); P. Parham and WFBodmer, Nature 276 , 397-399
(November, 1978); Handbook of
Experimental Immunology, Third Edition
Volume 2, DMWier, Editor, Blackwell,
1978, Chapter 25; and Chemical and
Engineering News, January 1, 1979, 15−
17. These documents simultaneously demonstrate the benefits and complexities that can be gained by attempting to generate monoclonal antibodies from hybridomas. Although the general technique is conceptually well understood, many difficulties are encountered and modifications are required in each particular case.
In fact, before attempting to generate a given hybridoma, it is necessary to determine whether the desired hybridoma will be obtained, whether it will produce antibodies if obtained, or whether the antibodies so produced will have the desired specificity. I can't be sure. The degree of success is primarily influenced by the type of antigen used and the selection technique used to isolate the desired hybridomas. Only a few attempts to generate monoclonal antibodies against antigens on the surface of human lymphocytes have been reported. For example, Current Topics in
See Microbiology and Immunology, ibid, 66-69 and 164-169. The antigens used in these reported experiments were cultured lymphoblastoid leukemia and human chronic lymphocytic leukemia cell lines. The many hybridomas obtained appeared to produce antibodies against various antigens of all human cells. None of the hybridomas produced antibodies against predefined classes of human lymphocytes. It should be understood that there are two main classes of lymphocytes included in the human and animal immune systems. The first class of these (thymic derived cells, or T cells) are differentiated in the thymus from hemopoietin stem cells. Cells that differentiate while in the thymus are named "thymocytes." Mature T cells emerge from the thymus and enter tissues,
Circulate between lymph vessels and bloodstream. These T cells form a large proportion of the pool of small lymphocytes that recirculate. They have immunological specificity and participate directly as effector cells in cell-mediated immune responses (such as tissue transplant injections). T cells do not secrete humoral antibodies, but are sometimes required for the secretion of these antibodies by the second class of lymphocytes discussed below. Some types of T cells serve regulatory functions in other aspects of the immune system. The mechanism of this cellular synergistic process is not yet completely understood. The second class of lymphocytes (myeloid-derived cells, or B cells) are those that secrete antibodies. They also arise from hemopoietin stem cells, but their differentiation is not determined by the thymus.
In birds, they are differentiated in a thymus-like organ called the Bursa of Fabricius. However, no equivalent organ has been discovered in mammals.
These B cells are thought to differentiate within the bone marrow. Here, T cells are divided into at least several subtypes called "helper,""suppressor," and "killer" T cells, which (respectively) promote responses. It was recognized that the compound has the ability to suppress reactions or kill (separate) foreign cells. Although these subclasses are well understood for the murine lineage, they have only recently been described for the human lineage. See, for example: RLEvans, et al.
Journal of Experimental Medicine, Volume
145, 221−232, 1977; and L. Chess and S.F.
Schlossman−”Functional Analysis of
Ditinct Human T-Cell Subsets Bearing
Unique Differentiation Antigens”
“Contemporary Topics in Immunobiology”
O. Stutman, Editor, Plenum Press, 1977,
Volume 7, 363-379. The ability to identify or suppress classes or subclasses of T cells is important for the diagnosis or treatment of various immune dysregulation or conditions. For example, certain leukemias and lymphomas
They have different prognosis depending on whether they originate from B cells or T cells. Thus, assessment of disease prognosis relies on distinguishing between these two classes of lymphocytes. See, for example: ACAisenbery and JCLong, THe.
American Journal of Medicine, 58:300
(March, 1975); D. Belpomme, et al.
Immunological Diagnosis of Leukemias and
Lymphomas, S. Thierfelder, et al., Editors,
Springer, Heidelberg, 1977, 33−45; and
D. Belpomme, et al., British Journal of
Haematology, 1978, 38 , 85. Certain disease states (eg, juvenile rheumatoid arthritis and certain leukemias) involve an imbalance of T cell subclasses. It has been suggested that autoimmune diseases are generally associated with an excess of "helper" T cells or a deficiency of certain "suppressor" T cells, whereas malignant diseases are generally associated with an excess of "suppressor" T cells. In some types of leukemia, excess T cells are produced in a developmentally arrested state. Diagnosis could thus depend on being able to detect this imbalance, ie, excess. See, for example: J. Kersey, et al., “Surface
Markers Define Human Lymphoid
Malignancies with Differing Prognoses”,
Haematology and Blood Transfusion
Volume 20, Springer-Verlag, 1977, 17-24,
and references contained therein. Acquired agammaglobulinosis, a disease state that does not produce immunoglobulins, has at least two
It consists of two distinct types. In this type, the inability to produce immunoglobulins is due to a lack of suppressor T cells, whereas in the other type, the inability to produce immunoglobulins is due to a lack of helper T cells. In both types, there appears to be no shortage or deficiency of the patient's B cells, the lymphocytes involved in the actual secretion of antibodies. However, these B cells are suppressed or "unhelped" so that immunoglobulin production is greatly reduced or absent. This type of acquired agammaglobulinopathy is therefore associated with an excess of suppressor T cells or T helper
This can be determined by testing for the absence of cells. On the therapeutic side, there is some suggestion, although not yet definitively proven, that administering antibodies directed against subclasses of T cells in excess amounts may be therapeutically beneficial in autoimmune or malignant diseases. For example, T helper cell cancers (certain cutaneous T cell lymphomas and certain T cell acute lymphoblastic leukemias) can be treated with antibodies directed against helper T cell antigens. Treatment of autoimmunity caused by an excess of helper cells can also be achieved in the same way. Diseases due to an excess of suppressor T cells (eg, malignancies or acquired agammaglobulinopathy) can be treated by administering antibodies to suppressor T cell antigens. Antisera against all classes of human T cells (so-called anti-human thymocyte globulin, or ATG) have been reported to be therapeutically useful in patients receiving transplants. Since the cellular immune response (the mechanism for rejecting transplanted tissue) is dependent on T cells, administration of antibodies against T cells prevents or slows this rejection process. See, for example, Cogimi, et al., “Randomized
Clinical Trial of ATG in Cadaver Renal
Allgraft Recipients：Importance of T Cell
"Monitoring", Surgery 40 :155-163 (1976) and references contained therein.Identification and suppression of classes and subclasses of human T cells has traditionally been accomplished by immunizing animals with human T cells and
The animal can be bled to obtain serum, and this antiserum can be adsorbed with (for example) autologous, non-xenogeneic B cells to remove antibodies with unwanted reactivity. This was also achieved by using spontaneous autoantibodies or selective antisera for human T cells. The production of these antisera is extremely difficult, especially in the adsorption and purification steps. Even adsorbed and purified antisera contain many impurities in addition to the desired antibodies for several reasons. Firstly,
Serum contains millions of antibody molecules before immunization with T cells. Second, this immunization method generates antibodies against the various antigens found on all human cells injected. Antibodies against a single antigen are not produced selectively. Third, the titers of specific antibodies obtained with such methods are usually very low (e.g., inactive at dilutions greater than 1:100) and the ratio to nonspecific antibodies is less than ^1/10 . It is. See, for example, the aforementioned Chess and Schlossmam literature (see pages 365 onwards) and the aforementioned
See Chemical and Engineering News. Disadvantages of prior art antisera and advantages of monoclonal antibodies are described here. One of the T cell subpopulations identified by such prior art antisera has been labeled the TH ₂ ⁺ subset, which is responsible for both cytotoxic effector cells for cellular lympholysis. It has been shown to contain both T cells and immunoregulatory suppressor T cells that suppress the function of B cells. This subset contains approximately 20-30% of human peripheral T cells. References, e.g., Ref. ELReinherz, et al., J.
Immunol.123:83 (1979) and New Engl.J.
Med. 300 :1061 (1979). This time, normal human peripheral cytotoxicity and inhibitory fluid
TH ₂ ⁺ T cells (approximately 20% of normal human peripheral T cells)
A new hybridoma (designated OKT5) has been discovered that is capable of producing a novel monoclonal antibody against an antigen found in . The antibodies so generated are monospecific against a single determinant for normal human cytotoxic and suppressor _TH2 ⁺ T cells;
and are essentially free of other anti-human immunoglobulins, in contrast to prior art antisera that are inherently contaminated with antibodies reactive against a large number of human antigens, and prior art monoclonal antibodies. is not monospecific for human cytotoxic and suppressor T cell antigens. Additionally, this hybridoma can be cultured to produce antibodies. In this case it is not necessary to carry out the lengthy adsorption and purification steps necessary even when immunizing the animal, killing it and then obtaining the impure antisera of the prior art. Accordingly, one object of the present invention is to provide hybridomas that produce antibodies against antigens found on normal human cytotoxic and suppressor TH ₂ ⁺ T cells. In yet another aspect of the invention, methods of producing these hybridomas are provided. Another object of the invention is to provide essentially homogeneous antibodies directed against antigens found on normal human cytotoxic and suppressor TH ₂ ⁺ T cells. Yet another objective is to provide methods for treating or diagnosing diseases using these antibodies. Other objects and benefits of the present invention will become apparent upon consideration of the present disclosure. To achieve the aforementioned objects and benefits, the present invention provides hybridomas that produce novel antibodies against antigens found on normal human cytotoxic and suppressor TH ₂ ⁺ T cells, the antibodies themselves, and the antibodies. Diagnostic and therapeutic methods using the method are provided. The hybridoma is
Manufactured generally according to the method of Milstein and Kohler. After immunizing mice with normal human thymocytes, splenocytes from the immunized mice are fused with cells from a mouse myeloma line, and the resulting hybridomas are combined with normal E rosette-positive human T cells. The supernatants containing antibodies that selectively bind to were used to screen for them. The desired hybridomas were subsequently cloned and characterized. As a result, a hybridoma was obtained that produced an antibody (designated OKT5) against an antigen for normal human cytotoxic and suppressor TH ₂ ⁺ T cells. This antibody reacts with normal human peripheral toxic and suppressor TH ₂ ⁺ T cells, but not with other normal peripheral blood lymphoid cells, including helper T cells.
Furthermore, the cell surface antigen recognized by this antibody is detected on approximately 80% of normal human thymocytes. In view of the difficulties exhibited in the prior art and the lack of success reported using malignant cell lines as antigens, it was surprising that the method of the present invention provided the desired hybridomas. It should be noted that this unpredictable nature of hybrid cells does not allow extrapolation from one antigen or line to another. In fact, the inventors have found that when a malignant cell line of T cells is used as an antigen, hybridomas are formed that do not produce the desired antibodies. Attempts to use purified antigen isolated from the cell surface have also been unsuccessful. Both the subject hybridoma and the antibodies produced thereby are designated herein as "OKT5".
The specific substances called and referred to will be clear from the context. The preparation and properties of this hybridoma and the resulting antibodies will be better understood from the following description and examples. The method for producing hybridomas generally consists of the following steps: A. Immunize a mouse with normal human thymocytes. Although female CAF ₁ mice were found to be preferred, other mouse strains could be used. The immunization schedule and concentration of thymocytes should be such as to produce an effective amount of suitably primed splenocytes. Three immunizations, 14 days apart, using 2×10 ⁷ cells/mouse/injection in 0.2 ml of phosphate-buffered saline solution were found to be effective. B. Remove the spleen from the immunized mouse and prepare a spleen suspension in an appropriate medium. Approximately 1 ml/spleen of medium is sufficient. The techniques for these experiments are well known. C. The suspended splenocytes are fused with mouse myeloma cells from the appropriate cell line by use of an appropriate fusion promoter. The preferred ratio of splenocytes to myeloma cells is about 5:1. A total of about 0.5-1.0 ml of fusion medium for about 10 ⁸ splenocytes is appropriate. Many murine myeloma cells are known and generally available to members of the academic community or to various depositories, such as The Salk.
the Salk Institute Cell
Distribution Center, La Jolla, Ca). The cell lines used should preferably be of the so-called "drug-resistant" type, such that unfused myeloma cells do not survive in the chosen medium, while hybridized cells do. The most common class is 8-azaguanine-resistant cell lines, which lack the enzyme hypoxanthine guanine phophoribosyl transferase and therefore HAT (hypoxanthine, aminobuterin,
and thymidine) are not supported by the medium. It is also generally preferred that the myeloma cell line used is of the so-called "non-secreting" type, ie it does not itself produce resistance, although secreting types can be used. However, in some cases a secreting myeloma line may be preferred. Preferred fusion promoters have an average molecular weight of about
Polyethylene glycol having a molecular weight of 1000 to about 4000 (commercially available as PEC1000 and the like) is preferred, although other fusion promoters known in the art can be used. D. In a separate container, the mixture of unfused splenocytes, unfused myeloma cells, and fused cells is diluted in a selective medium that does not support unfused myeloma cells; Culture for sufficient time to kill (approximately 1 week). This dilution can be of a limited type, in which the volume of diluent is statistically calculated in each separate container (e.g., each well of a microtiter plate). number of cells (e.g. 1
~4) is isolated. The vehicle is drug resistant (e.g. 8-azaguanine resistant) and does not support unfused myeloma cell lines (e.g.
HAT medium). Therefore, these myeloma cells die. Unfused splenocytes are non-malignant and therefore have only a finite number of generations. Thus, after a period of time (approximately one week) these unfused splenocytes do not regenerate. The fused cells, in contrast, have the malignant potential of the myeloma parent and can survive in the selective medium of the splenocyte parent.
Continue playing. The supernatant in each container (well) containing the E hybridoma was collected from purified human T that was E rosette positive.
Measure for antibodies against the cells. F Hybridomas that produce the desired antibodies are selected (eg, by limited dilution) and divided into branch systems. Once the desired hybridomas have been selected and cloned, the final antibody can be produced in one of two ways.
It can be generated with. The purest monoclonal antibodies can be obtained by culturing the desired hybridoma in a suitable medium for an appropriate length of time;
The desired antibody is then produced by recovering it from the supernatant. Suitable media and appropriate lengths of incubation times are known or easy to determine. This in vitro technique is essentially free of anti-human immunoglobulins of other specificities;
Monospecific monoclonal antibodies are essentially generated. Because the medium contains exogenous serum (eg, fetal calf serum), small amounts of other immunoglobulins will be present. However, this in vitro method does not produce sufficient quantities or concentrations, as the concentration of monoclonal antibody is only about 50 μg/ml. To generate very large concentrations of slightly less pure monoantennary antibodies, the desired hybridoma can be injected into mice, preferably congenital or semicongenital mice. The hybridomas form a tumor of antibody production after an appropriate incubation period, resulting in high concentrations of the desired antibody (approximately 5 to 20
mg/ml). Although these host mice also have normal antibodies in their bloodstream and ascites, the concentration of these normal antibodies is only about 5% of the monoclonal antibody concentration. Moreover, since these normal antibodies are not anti-human in specificity, monoclonal antibodies obtained from harvested ascites or serum are essentially free of contaminating anti-human immunoglobulins. This monoclonal antibody has a high titer (active at dilutions of 1:50,000 or higher) and a high specific to nonspecific immunoglobulin ratio (approximately 1/20). K
The resulting immunoglobulins that incorporate lightweight myeloma chains are non-specific, "nonsensical" peptides that merely dilute the monoclonal antibody without reducing its specificity. EXAMPLE Production of Monoclonal Antibodies A Immunization and Somatic Crossings Female CAF ₁ mice (Jackson Laboratories; 6-8 weeks old) were incubated with 2 x 10 ⁷ human cells in 0.2 ml of phosphate-buffered saline. Immunizations were made intraperitoneally with thymocytes at 14 day intervals. After the third immunization, the spleen was removed from the mouse and a single cell suspension was created by passing the tissue through a stainless steel tube. Cell fusion was performed according to the method of Kohler and Milstein. 1 x 10 ⁸ splenocytes,
RPMI1640 media (Gibco, Grand Island, NY)
2 x 10 ⁷ in 0.5 ml of fusion medium consisting of 35% polyethylene glycol (PEG1000) and 5% dimethyl sulfoxide in
P3×63Ag8U1 myeloma cells (Br, M.Scharff,
Albert Einstein College of Medicine;
Bronx, NY). These myeloma cells secrete IgG1k light chains. B Hybridoma Selection and Growth After cell fusion, cells were grown in HAT medium (hypoxanthine, aminoptyline, and thymidine).
The cells were cultured in a humid atmosphere at 37° C. with 5% CO ₂ . After several weeks, 40-100μ of the supernatant from the culture medium containing the hybridomas was collected according to Mendes (J. Immunol. 111 :860, 1973).
into pellets of ¹⁰⁶ peripheral lymphocytes, divided into E rosette positive (E ⁺ ) and E rosette negative (E ⁻ ) populations, prepared from the blood of healthy human donors as described by added. Detection of mouse hybridoma antibodies binding to these cells was determined by radioimmunoassay and/or indirect immunofluorescence. Goat anti-mouse fluoresced cells cultured in culture supernatant
IgG (G/M FITC) (Meloy Institute,
stained with Springfield, VA; F/P = 2.5);
The fluorescent antibody-coated cells were subsequently coated with a Cytofluorograf FC200/4800A.
(Ortho Instruments, Westwood, MA) was analyzed as described in the Examples. E ⁺
Hybridoma cultures containing antibodies that specifically react with lymphocytes (T cells) were selected and cloned twice by limited dilution in the presence of feeder cells. The clones were subsequently treated with 2,6,10,14-tetramethylpentadecane (trade name from Aldrich Chemical Company).
CAF ₁ prepared with Pristine (commercially available)
1 × 10 ⁷ cells (0.2 ml) of a constant clone in mice
volume) was implanted intraperitoneally. Malignant ascites from these mice was then used to characterize lymphocytes as described below in the Examples. Thematic hybrid antibodies
OKT5 was proven to be of the IgG1 subclass by standard techniques. EXAMPLES Characterization of OKT5 Reactivity A Isolation of Lymphocyte Populations Human peripheral blood mononuclear cells were collected from healthy volunteer donors (15-40 years old) according to Boyum, Scand.
J.Clin.Lab.Lnvest.21(Suppl.97):77, 1968
−Hypaque) density gradient centrifugation (Pharmacia
Fine Chemicals, Piscataway, NJ). Unsorted mononuclear cells, Chess, et al.
Sephadax G-200 anti-F as previously described in al. J. Immunol. 113:1113 (1974).
(ab′) By _two- column chromatography, the surface
Separated into Ig ⁺ (B) and Ig ^- (T plus Null) populations. T cells were isolated from Ig ^- population by 5% sheep red blood cells (Microbiological Associates, Bethesda,
MD) to form an E rosette. The rosette mixture was layered on a Ficoll-Hypaque and the recovered E ⁺ pellets were mixed with 0.155 mol of NH ₄ Cl (10
ml/ ¹⁰⁸ cells). The T cell population thus obtained was <2% EAC rosette positive and >95% E rosette positive, as determined by standard methods. In addition, non-rosette Ig ^-
(Null cell) population was harvested from the Ficoll surface. This latter population was <5% E ⁺ and 2% sIg ⁺ . The surface Ig ⁺ (B) population was obtained from Sephadex G-200 column chromatography and subsequent elution with normal human gamma globulin as described above. This population was >95% surface Ig ⁺ and <5% E ⁺ . Normal human macrophages were obtained from a mononuclear population by attachment to polystyrene. Thus, mononuclear cells were cultured in the final medium (RPMI1640, 2.5 mmol HEPES [4-(2-hydroxyethyl)-1
- piperazine propane sulfonic acid] buffer,
0.5% sodium bicarbonate, 200 mmol L-
glutamine, and 1% penicillin-streptomycin, 20% heat-inactivated human
Resuspend cells at a concentration of 2 x ¹⁰ cells in a plastic Petri dish (100 x 20 mm) (supplemented with AB serum).
(Falcon Tissue Culture Dish; Falcon,
Oxnard, CA) overnight at 37°C. After washing thoroughly to remove non-adherent cells,
Adherent populations were detached by vigorous washing with cold serum-free medium containing 2.5 mmol EDTA and occasional tugging with the plunger rubber tip of a disposable syringe. 85% more cell population
Latex particles could be ingested and had the morphological characteristics of monocytes by Wrignt-Giemsa staining. B. Isolation of Thymocytes Normal human thymus glands were obtained from patients aged between February and 14 years under corrective surgery of the heart. Freshly obtained sections of the thymus were immediately placed in 5% fetal calf serum in medium 199 (Gibco), cut into small pieces with forceps and scissors, and subsequently made into a single cell suspension by pressing through a wire mesh. I made it. The cells were then layered with Ficoll-Hypac, spun, and washed as described in the previous section A. The thymocytes thus obtained were >95% viable and 90 rosette positive. C Cell lines B cell lines transformed from normal individuals by Epstein-Barr Virus (EBV) are derived from Dr. H. Lazarus (Sidney
Farber Cancer Institute, Boston, MA). EXAMPLE Cytofluorographic analysis and cell separation Cytofluorographic analysis of monoclonal antibodies using all cell populations was performed on a Cytofluorgraf FC200/4800A (Orthofluorographic).
Fluorescence-conjugated goat anti-mouse IgG (G/M FITC) (Meloy
The test was performed by indirect immunofluorescence using a laboratory (Laboratories). In summary, 1-2 x ¹⁰⁶ cells in 0.15ml
of OKT5 at 1:500 dilution and at 4 °C.
Incubate for 30 minutes and wash twice. Then cells 0.15
ml of 1:40 dilution of G/M FITC at 4°C.
The reaction mixture was allowed to react for 30 minutes, centrifuged, and washed three times. The cells were then analyzed cytofluorographically and the fluorescence intensity/cell was recorded on a pulse height analyzer. A similar reactivity pattern was observed at a dilution of 1:10000, but reactivity was lost at further dilutions. Background staining was obtained by substituting a 0.15 ml aliquot of 1:500 ascites from a Balb/cJ mouse immunized retrogradely with a nonproductive crossbred clone. The reactivity of lymphoid cell populations with equine anti-TH ₂ and normal equine IgG was determined as described in Reinherz, et al., supra. In experiments designed to isolate subpopulations of T cells, 100 × 10 ⁶ unsorted T cells were labeled with 4 ml of a 1:500 dilution of OKT4 or OKT5;
Developed with G/M FITC. OKT4 is a peripheral blood T lymphocyte that collects the helper subpopulation in humans.
It was shown to be specifically reactive with 60%. Fluorescence-activated cell sorter (FACS-1)
(Becton-Dickinson, Mountain View, CA)
Using OKT4 ⁺ and ^OKT4- subsets of T cells
from the same population.
Separated into OKT5 ⁺ subset and OKT5 ^- subset. Vitality of the latter species was >95% with Trypan blue exclusion in all cases. Purity of all isolated populations is ≥95%
It was hot. EXAMPLE Analysis of T Cell Subpopulations Using Equine Anti- _TH2 In a manner similar to that described in the Example, using equine anti- _TH2 , 60 x 10 cells were isolated as described in Reinherz et al., ^supra . Differentiate T cells into 0.12 ml of horse anti-
TH ₂ and 0.1ml R/H FITC (Coppel
Laboratories, Downingtown, PA) and separated into _TH2 ⁺ and _TH2- T cells by ^FACS . The purity and viability of the sorted cells were similar to the previously sorted populations. Horse medium TH ₂ ,
FACS sorted subsets of T cells isolated with OKT4 or OKT5 were incubated with 20% human AB serum and 1%
penicillin-streptomycin, 200 mmol L-glutamine, 25 mmol HEPES buffer (Microbiological Associates), and 0.5%
The cells were incubated for 48 hours at 37° C. and in a humidified atmosphere of 5% CO ₂ using RPMI 1640 containing 5% sodium bicarbonate. These cultured cells were then analyzed by cytofluorography as described above. Background staining was determined for the specific antibodies and staining described above by substituting normal horse IgG. EXAMPLES Functional Study A Proliferation Study The mitotic response of 10 ⁵ unisolated and FACS-sorted T lymphocytes was measured in microculture with Concanavalin A (Con A).
(Calbiochem, La, Jolla, Ca) and phytohemagglutinin
(PHA) (Burroughs-Wellcome Company,
Greenville, NC) was tested for optimal dosing. Heteroantigen proliferative responses were simultaneously measured on these same populations using mitomycin-treated Laz 156 and an EBV-transformed human B lymphoblastoid cell line as the stimulus. Tetanus toxoid (Massachusetts Department of
Public Health Biological Laboratories,
Boston, MA) and mumps antigen (Micobiological Associates) were tested using a final concentration of 10 μg/ml and a dilution of 1:20, respectively. 5% macrophages obtained as described above were added to all populations at the beginning of in vitro culture. After 4 days of mitogen stimulated culture
0.2 μCi of ³ H-thymidine ( ³ H-TdR;
specific activity of 1.9Ci/mmol) (Schwartz Mann
Division of Becton-Dickinson,
Orangeburg, NY) and after 18 hours.
MASH equipment (Microbiological Associates)
Bethesda, MD). ³ H-TdR was incorporated into a Packard scintillation counter (Packard Instrument Company,
Downer's Grove, IL). Background ³ H-TdR incorporation was obtained using medium instead of mitomeres. Soluble and cell surface xenoantigen cultures were harvested after 5 days as previously described.
Pulsed with ^3H -TdR for 18 hours, harvested, and counted. B Cytotoxicity Studies Sensitized cultures for cellular lympholysis (CML) were grown in large numbers of microtiter plates, all at 2 x 10 ⁶ cells/ml, together with mitomycin-treated stimulator cells of unsorted T cells. This was done by placing it in a well.
At the end of 5 days, unsorted T cells were analyzed by FACS.
OKT5 ⁺ and ^OKT5- T cell subsets were sorted. A subset of these T cells are then
⁵¹ Cr sodium chromate labeling was added to target cells and specific cytotoxicity was determined after culturing the cells for 6 hours. The percentage cytotoxicity was determined by the following formula: ⁵¹ Cr experimentally released - 51 Cr spontaneously released ^{/ 51} Cr released by freeze-thaw - ⁵¹ Cr spontaneously released x 100 all. Samples were obtained in three replicate experiments and results were expressed as average mean standard deviation. C ConA activation and MLC in suppressor cells
Suppression of unsorted T cells were activated with concanaparin A (ConA) (Colbiochem) at 2 μg/10 ⁶ cells.
These ConA-treated cells were placed in 25 cm ² surface area tissue culture flasks (Falcon, Oxnard, CA).
RPMI1640 (Grand Island Riological
The cells were cultured upright for 48 hours at 37° C. in a humidified atmosphere containing 5% CO ₂ in a company.
RPMI 164 contained 20% human AB serum, 1% penicillin-streptomycin, 200 mmol L-glutamine, 25 mmol HEPES buffer (Microbiological Associates) and 0.5% sodium bicarbonate. Untreated control T
Cells were cultured in the same manner but without ConA. Cells were subsequently spun, washed five times, and added to new native responder cells in one-way mixed lymphocyte cultures (MLC) as described above. In assays of inhibition, unidirectional MLC was tested using round-bottom microtiter plates (Linbro Chemical Company, New Haven;
CT) in triplicate wells, each well containing 0.05 x 10 ⁶ responder lymphocytes (total mononuclear); 0.05 x 10 ⁶ unactivated or ConA-activated autologous unsorted cells. Also differentiated T cells and 0.1×
It contained 10 ⁶ mitomycin-treated stimulated cells (Laz156). After 5 days, cultures were pulsed with 0.2 μCi of ³ H-TdR as described above.
Harvested after 18 hours. The percentage inhibition of MLC proliferation was then calculated using the following formula: % inhibition = (1−cpm Con A ⁺ /cpm × 100) where cpmConA ⁺ is ConA-activated autologous T cells or T cells. represents the results of ^3H -TdR incorporation in MLC when adding a subset of
Shows the results when adding cells. Generation of hybridomas and generation and characterization of the resulting monoclonal antibodies were performed as in the examples above. Large amounts of the subject antibodies were prepared by intraperitoneal injection of the subject hybridomas into mice and harvesting of malignant ascites; the hybridomas were grown in vitro by techniques well known in the art, and the antibodies were extracted from the supernatant. It is clearly conceivable that it can be removed. As shown in Figure 1, approximately 20% of the human peripheral blood T cell population of a given normal individual is reactive with OKT5, whereas all B cells isolated from the same individual are reactive with OKT5. Characteristic cell and macrophage populations
Non-reactive with OKT5. Similarly, equine anti- _TH2 reacts with 24% of peripheral T cells and is also non-reactive with B cells, uncharacterized cells and macrophages. Monoclonal antibodies are thus reactive with antigen contained on the surface of approximately 20% of normal human peripheral T cells, but non-reactive with antigen on the surface of the other three cell types mentioned above. It can be characterized as. As discussed below, the OKT5 ⁺ portion of the human peripheral T cell population is included in the cytotoxic and suppressor T cell subsets. This specific reactivity is one test by which the subject antibody OKT1 can be detected and distinguished from other antibodies. As shown in Figure 2, approximately 80% of normal human thymocytes from 6 month old infants are reactive with OKT5. Similar results (approximately 80% reactivity) were obtained using additional thymus samples from normal individuals aged 2 months to 19 years. This value is the same as for horse anti- _TH2 . The pattern of reactivity in Figure 2 provides a second method of detecting the subject antibody OKT5 and distinguishing it from other antibodies. As shown in FIG. 3, the subject antibodies react with TH ₂ ⁺ but not with TH ₂ ⁻ T cells. Approximately 5-10% of the _TH2 ⁺ subpopulation is non-reactive with OKT5.
The reactivity pattern in Figure 3 shows the subject antibody OKT5.
provides a third method for detecting and distinguishing it from other antibodies. As shown in Figure 4, the OKT4 ⁺ T cell subpopulation is completely non-reactive with OKT5. In contrast, the OKT4 ⁻ T cell subset is largely OKT5 ⁺
(6800 out of 10000 cells tested). These results indicate that the OKT5 ⁺ subset of T cells is similar to the previously demonstrated TH ₂ ⁺ subset, is reciprocal, and is distinct from the OKT4 ⁺ subpopulation.
The OKT4 ⁺ subset contains helper T cells, whereas the OKT5 ⁺ subset (similar to the TH ₂ ⁺ subpopulation) contains cytotoxic and suppressor T cells. This reactivity pattern in Figure 4 is
Provides additional methods for identifying OKT5 antibodies and distinguishing them from other antibodies. Figure 5 shows that OKT5 ⁺ T cells influence CML. The degree of lysis mediated by this population is greater than that mediated by the unsorted T cell population. In contrast,
OKT5 ^- T cell population is minimally lytic.
After activation against Laz156 in MLC, unsorted T cells were separated into OKT5 ⁺ and OKT5 ^- subsets. Both undifferentiated and differentiated T cells are
^Various effector:targets (E:
T) ratio was analyzed. The OKT5 ⁺ T cell subset contained the effector population in cellular lympholysis. 5:1, 10:1 and
At an E:T ratio of 20:1, the OKT5 ⁺ T cell population underwent specific lysis of 40%, 50% and 77%, respectively. The lytic capacity of the isolated OKT5 ⁺ subset was significantly greater than that of the unsorted T cell population. Furthermore, the OKT5 ⁻ T cell population performed significantly lower lysis at any E:T ratio tested. In light of earlier observations that human TH ₂ ⁺ T cell subsets affected CML, our findings suggest that TH ₂ ⁺ and OKT5 ⁺ T cell subpopulations are similar individuals of functionally active lymphocytes. This would support the view that a group was defined. This unique cytotoxic ability provides yet another way to identify OKT5 and distinguish it from other antibodies. Functional studies were performed on lymphoid populations separated by fluorescence-activated cell separators (FACS). The results of these studies are shown in the Tables below and further support the foregoing characterization of the subject monoclonal antibodies. In these studies, unsorted T cell populations were treated with 1:500 dilution of OKT5 and G/M FITC and separated by FACS in OKT5 ⁺ and OKT5 ^- subsets. Given the purity of the obtained population (>95%), 5% macrophages were added to the isolated population before in vitro culture. Then,
Unsorted T cell populations and isolated OKT5 ⁺ and
OKT5 ^- a subset of T cells stimulated with PHA, ConA, soluble antigen and alloantigen,
Their in vitro growth responses were assayed. The proliferative response of unsorted T cells to PHA and ConA is shown in the table. The maximal proliferative response by the undifferentiated T cell population was obtained with 1 μg/10 ⁶ cells of PHA, and reduced responses were obtained with 0.5 μg/10 ⁶ cells and 0.1 μg/10 ⁶ cells of PHA. Treatment of unsorted T cells with OKT5 and goat-mouse FITC without subsequent fractionation did not change the proliferative response. In contrast, PHA
Differences in responses to were obtained using isolated subsets of OKT5 ⁺ and OKT5 ^- cells.
The OKT5 ⁻ population of cells responded to all amounts of PHA in a manner similar to the unseparated T cell population. However, the proliferative response of OKT5 ⁺ cells is
It was significantly lower in all amounts of PHA tested. Furthermore, in an amount of 0.1 μg/10 ⁶ cells,
OKT5 ⁺ T cells did not proliferate rapidly, whereas OKT5 ^- T cell subsets and unsorted cells were still responsive. A subset of these
The proliferative response to ConA, on the other hand, was similar and the two subsets of cells were indistinguishable from each other or from the undifferentiated T cell population. Next, responses to foreign and soluble antigens in MLC were examined. As shown in the table, unsorted T cell populations, unsorted T cell populations treated with OKT5 and G/M FITC, and both
OKT5 ⁺ and ^OKT5- cell subsets
Responded to Laz156 in a similar manner at MLC. In contrast, the proliferative response to soluble antigen provided a clear distinction between these subsets. In all cases tested,
The OKT5 ⁺ T cell subpopulation proliferated minimally to soluble antigen, tetanus toxoid and mumps, whereas the OKT5 ^- T cell subset responded well. Previous studies have revealed that the TH ₂ ⁺ population of T lymphocytes can be induced by ConA to suppress native lymphocytes in MLCs.
To determine whether the OKT5 ⁺ T cell subset could similarly express suppressor function, T cells were activated with ConA for 48 hours and then
OKT5 and OKT4 monoclonal antibodies were used to classify distinct T cell subsets. Subsequently, these isolated cell subpopulations were added to known responding lymphocytes at the beginning of MLC. As shown in the table, unsorted T cells activated with ConA suppressed the proliferation of autologous cells in MLC. Treatment with monoclonal antibody and G/M FITC did not change this result. both OKT4 ⁺ and
OKT5 ⁺ T cells proliferated against ConA;
Only OKT5 ⁺ T cells, when activated with ConA,
He became restrained. OKT4 ⁺ subset was not suppressive. In addition, OKT4 ⁺ and OKT4 ^- TH ₂ ^-
The OKT5 subpopulation, containing both subpopulations, was minimally suppressive compared to the highly suppressive OKT5 ⁺ population. The table shows the relationship between levels of peripheral T cells and T cell subsets and various disease states. These relationships can be examined for diagnostic purposes (e.g.
can be used to detect infectious mononucleosis) by analyzing blood samples from individual patients with one of these diseases to determine the levels of T cells and T cell subsets. These relationships can also be used for therapeutic purposes when the disease state is due to high levels of a T cell subset (eg, type I acquired agamma albuminosis). For therapeutic use, administration of appropriate monoantennary antibodies to patients with elevated levels of T cell subsets can reduce or eliminate this excess. The relationships shown in the table are yet another method by which OKT5 antibodies can be detected and distinguished from other antibodies. Other monoclonal antibodies producing hybridomas (OKT1,
OKT3 and OKT4) are described and claimed in the following U.S. patent applications: 1979
Filed on March 20, 1979 SN22, 132; Filed on April 26, 1979
SN33639; and SN33669 filed April 26, 1979.
These applications are incorporated herein by reference. According to the invention, a hybridoma capable of producing antibodies against antigens found on normal human cytotoxic and suppressor T cells, a method for producing this hybridoma, a monoclonal antibody against antigens found on human cytotoxic and suppressor T cells, Antibodies, methods of producing the antibodies, and methods of treating or diagnosing diseases using the antibodies are provided. As used herein, "Hyper IgE" means that the amount of this antibody is higher than the normal value. This is generally manifested in allergic predisposition. In addition, the "levels of peripheral T cells in diseased states" in the "Table" below were measured in the same manner as the levels of peripheral T cells in normal blood described in the Examples and section above. That is, instead of analyzing blood from normal healthy individuals, blood from patients diagnosed with a certain disease was analyzed.

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[Table] Type +

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Table: Although only a single hybridoma has been described that produces a single monoclonal antibody directed against human cytotoxic and suppressor T cell antigens, the present invention is considered to encompass all monoclonal antibodies exhibiting the properties described herein. It will be done. thematic antibody
OKT5 was determined to belong to subclass IgG ₁ , one of the four subclasses of murine IgG. These subclasses of immunoglobulin G differ from each other in so-called "fixed"regions; antibodies directed against a specific antigen have so-called "variable" regions, which are independent of which subclass of immunoglobulin G belongs to it. are functionally identical. That is, monoclonal antibodies exhibiting the properties described herein are subclass IgG ₁ ,
IgG ₂ a, IgG ₂ b, or IgG ₃ or class
It can be IgM, IgA, or other known Ig classes. Differences between these classes or subclasses do not affect the selectivity of the antibody's reaction pattern, but may affect other reactions of the antibody with other substances, such as complementary antibodies or anti-mouse antibodies. There will be. The subject antibody is
Although specific to IgG ₁ , antibodies having the pattern of reactivity exemplified herein are considered to be encompassed within the scope of the invention regardless of the immunoglobulin class or subclass to which they belong. Additionally, included within the scope of the invention are methods of producing the monoclonal antibodies described above using the hybridoma technology exemplified herein. Although only one example of a hybridoma has been described herein, those skilled in the art will recognize that other hybridomas can be produced, following immunization, fusion and selection methods provided herein, to produce antibodies with the reactive properties described herein. It is thought that it is possible to obtain similar results. Since individual hybridomas generated from known myeloma cell lines from known species of mice cannot be further identified except by reference to the antibodies produced by this hybridoma, they produce antibodies with the aforementioned reactive properties. All hybridomas are considered to be encompassed within the scope of this invention, as are methods of making the antibodies using these hybridomas. Other aspects of the invention include monoclonal antibodies.
A method of treating or diagnosing disease using OKT5 or other monoantennary antibodies that exhibit the pattern of reactivity revealed herein. The subject antibodies can be used to detect excessive cytotoxic or suppressor T cell activity by reacting T cell compositions from individual antibodies with the OKT5 antibody.
Excessive cytotoxic or suppressor T cell activity is indicated by the presence of 20-30% or more of the total peripheral T cell population reacting with OKT5. This diagnostic technique uses the OKT5 antibody alone or with other antibodies (e.g., OKT3 and
It can be used in combination with OKT4). Patterns of reactivity with panels of antibodies directed against T cells or T cell subsets will allow for more precise detection of certain disease states than is possible using prior art diagnostic methods. Treatment of disease states characterized by excess suppressive activity (e.g., malignancies) by administering therapeutically effective amounts of OKT5 antibodies to individual patients at the time of treatment. I can do it. By selectively reacting with the antigen of suppressor T cells, an effective amount of OKT5 antibody reduces the excess amount of suppressor T cells, thus alleviating the effects of suppressor T cell excess. Diagnostic and therapeutic compositions comprising an effective amount of an OKT5 antibody and a diagnostically or therapeutically acceptable antibody are also included within the scope of the invention. The subject hybridoma OKT5 was purchased from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD;
20852) on September 18, 1979 and September 28, 1979, with ATCC numbers CRL8013 and CRL8016, respectively.

[Brief explanation of drawings]

FIG. 1 shows the fluorescence pattern obtained on a cytofluorograph after reacting normal human peripheral T cells, B cells, featureless cells and macrophages with OKT5. For comparison, results obtained using equine anti- _TH2 are shown in the bottom row. Figure 2 shows the fluorescence pattern obtained on a cytofluorograph after reactivity of human lymphocytes with (A) OKT5 and G/M FITC and (B) equine anti- _TH2 . . Third
The figure shows a subset of equine anti- _TH2 isolated T cells.
The fluorescence pattern obtained on the cytofluorograph after reaction with OKT5 is shown. Figure 4 is OKT4
The fluorescence pattern obtained on the cytofluorograph after reacting a subset of isolated T cells with OKT5 is shown. FIG. 5 shows the cytotoxic potential of unsorted T cells and T cell subsets separated with OKT5 after allosensitization in MLC.

Claims (1)

Claims: 1. Produced by a hybridoma formed by the fusion of cells from a murine myeloma line and splenocytes from a mouse previously immunized with human thymocytes, the cytotoxic and suppressor TH ₂ ⁺
A mouse monoclonal antibody that reacts with more than 90% of human T cells, but does not react with normal human peripheral B cells, uncharacterized cells or macrophages. 2 Generated by a hybridoma formed by the fusion of cells from a murine myeloma line and splenocytes from a mouse previously immunized with human thymocytes, producing (a) cytotoxic and suppressor TH ₂ ⁺ T cells; (approximately 20-30% of all normal human peripheral B cells)
(b) reacts with approximately 80% of normal human thymocytes; (c) reacts with approximately 80% of normal human thymocytes; ( _c ) ^- does not react with T cells or OKT ₄ ⁺ T cells, but reacts with about 68% of OKT ₄ ^- T cells, (d) is strongly cytotoxic and is associated with early cirrhosis, multiple sclerosis and T-cell population (OKT ₅ ⁺ ) lower than normal levels in hyper-IgE, normal levels in all stages of Hodgkins disease, and higher than normal levels in acquired agammaglobulinosis and acute infectious mononucleosis A monoclonal antibody of class IgG according to claim 1, which defines: 3. A monoclonal antibody according to claim 1 belonging to subclass IgG ₁ . 4. A monoclonal antibody according to claim 1, produced from a hybridoma formed by the fusion of splenocytes from a CAF ₁ mouse previously immunized with P3X63Ag8U1 myeloma cells and human thymocytes. 5. (i) immunizing a mouse with human thymocytes, (ii) removing the spleen from the mouse and forming a suspension of splenocytes, and (iii) immunizing the splenocytes with mouse myeloma cells and a fusion promoter. (iv) the fused cells are diluted and cultured in separate wells in a medium that does not support unfused myeloma cells; (v) the supernatant in each well containing the hybridomas is fused in the presence of (vi) selecting and separating hybridomas that produce the desired antibodies into clones; and (vii) recovering the antibodies from the supernatant of the clones. and cytotoxic and suppressor _TH2 ⁺ T cells (approximately 20-30% of all normal human peripheral B cells)
A method for producing a mouse monoclonal antibody that reacts with more than 90% of normal human peripheral B cells, noncharacteristic cells, or macrophages. 6. The method according to claim 5, wherein the mouse belongs to strain CAF ₁ and the myeloma cells are P3X63Ag8U1. 7. A method for producing a monoclonal antibody according to claim 5, which comprises culturing the hybridoma ATCC CRL8016 in a suitable medium and collecting a liquid from the supernatant on the hybridoma. 8 (i) immunizing a mouse with human thymocytes, (ii) removing the spleen from the mouse and forming a suspension of splenocytes, and (iii) immunizing the splenocytes with mouse myeloma cells and a fusion promoter. (iv) the fused cells are diluted and cultured in separate wells in a medium that does not support unfused myeloma cells; (v) the supernatant in each well containing the hybridomas is fused in the presence of (vi) selecting and dividing the hybridomas that produce the desired antibody into clones; (vii) recovering the antibody from the supernatant above the clone; (viii) collecting the antibody from the supernatant above the clone; intraperitoneally into a mouse ^, and (ix) harvesting the malignant ascites fluid or serum containing the desired antibodies from _the mouse. Approximately 20-30% of normal human peripheral B cells)
Reacts with more than 90% of normal human peripheral B
A method for producing mouse monoclonal antibodies that do not react with cells, non-characteristic cells or macrophages. 9. The production method according to claim 8, wherein the mouse belongs to strain CAF ₁ and the myeloma cells are P3X63Ag8U1. 10. A method for producing a monoclonal antibody according to claim 8, which comprises injecting the hybridoma ATCC CRL8016 into a mouse and collecting the antibody from malignant ascites or serum of the mouse.