FI75230C - DIAGNOSTIC FOERFARANDE, VID VILKET ANVAENDS EN NY MONOCLONAL ANTIKROPP SPECIFIK MOT MAENNISKANS HJAELPARE-T-CELLER OCH I FOERFARANDET ANVAENDBAR COMPOSITION. - Google Patents

Description

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A diagnostic method using a novel monoclonal antibody specific for human helper T cells and a composition of matter useful in the method 5 Divided by application 801 343.

The present invention relates to a diagnostic method for detecting an excess or deficiency of a patient's helper T cells using a monoclonal antibody specific for an antigen found on the surface of all human nor-10 target helper T cells. The invention also relates to a diagnostic composition comprising this antibody. The antibody is made using a new hybrid cell line.

In 1975, Kohler and Milstein combined 15 myeloma cells into the spleen cells of immunized mice (Nature 256 (1975) 495-497), where it was first discovered that it was possible to prepare a continuous cell line to produce a homogeneous (so-called "monoclonal") antibody. Since then, many attempts have been made to prepare various hybrid cells (so-called hybridomas) and to try to apply the antibody produced by these hybridomas to scientific use. See for example:

Current Topics in Microbiology and Immunology, Volume 81 - "Lymphosyte Hybridomas", edited by F. Melchers, 25 M. Potter and N. Warner, Springer-Verlag 1978 and references in C.J. Barnstable et al., Cell 14 (May 1978) 9-20; P. Parham and W.F. Bodmer, Nature 276 (1978) 397-399; Handbook of Experimental Immunology, 3rd Edition, Volume 2, edited by D.M. Wier, Blackwell 1978, Chapter 25; and Chemical and 30 Engineering News, January 1 (1979) 15-17.

These references show the results obtained and the difficulties encountered in trying to prepare a hybrid monoclonal antibody. Although the manufacturing method is in principle well known, a lot of difficulties arise and each case is different. It cannot be guaranteed in advance that an attempt to produce the desired hybridoma will succeed and that, if successful, the hybridoma will produce an antibody or that the antibody thus formed will have the desired specificity. Success depends on the type of antigen used and the selection technique used to isolate the desired hybridoma.

Attempts to produce a monoclonal antibody specific for human lymphocyte surface antigens have been made in only a few cases. See, e.g., Current 10 Topics in Microbiology and Immunology, Volume, 81, 66-69, and 164-169. The antigens used in these experiments were cultured human lymphoblastoid leukemia cell lines and chronic lymphocyte leukemia cell lines. Many hybridomas thus prepared produced an antibody that acted upon various surface antigens of all human cells. None of these hybrids produced an antibody specific for a particular human lymphocyte species.

Two main groups of lymphocytes are involved in the immunological functions of the human and animal body. Of these, the second (thymus-derived cell, or T-cell) differs in the thymus from hematopoietic stem cells. The differentiating cells in the thymus are called "thymocytes." Mature T cells detach from the thymus and then enter the bloodstream, tissues, and lymphoid tissue. These T cells make up a large part of the small lymphocytes circulating in the body. They are immunologically specific and, as effector cells, are directly involved in cell-mediated immunological reactions (e.g., rejection at the time of transplantation). Although T cells do not secrete antibodies into body fluids, another group of lymphocytes to be treated later will, in some cases, require T cells to be able to secrete antibodies themselves. Some T cell species act in a regulatory manner in other immunological functions. The mechanism of this form of cell interaction is not yet fully understood.

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The second group of lymphocytes (bone marrow-derived cells, or B cells) includes those that secrete antibodies. They also develop from hematopoietic stem cells, but their thyroidism is not regulated by the thymus. In birds, they differentiate in an organ analogous to the thymus, which is called the "Bursa of Fabri-cius." However, no similar organ has been found in mammals, and thus B cells are thought to differentiate in the bone marrow.

T cells are divided into at least three subspecies, termed "helper", "attenuator", and "killer" T cells, which relatively either accelerate or slow the reaction or kill (dissolve) foreign cells. These subclasses are well known in the body of hii-15, but only recently have their functions been described in the human body. See, e.g., R.L.

Evans et al., Journal of Experimental Medicine 145 (1977) 221-232, and L. Chess and S.F. Schlossman - "Functional Analysis of Distinct Human T-cell Subsets 20 Bearing Unique Diffefentiation Antigens", in "Contemporary Topics in Immunobiology", edited by 0. Stutman, Plenum Press 1977, Volume 7, 363-379.

The identification or attenuation of different T cell species or subtypes is important in the diagnosis and treatment of various immuno-regulatory disorders.

For example, the prognosis of certain forms of leukemia and lymphoid tissue differs depending on whether they originate from B or T cells. Thus, when assessing disease prognosis, it is important to be able to distinguish between these two groups of lymphocytes. See, e.g., A.C. Aisenberg and J.C. Long, The American Journal of Medicine 58 (1975) 300; D. Belpomme et al., In "Immunological Diagnosis of Leukemias and Lymhomas", edited by S. Thierfelder et al., Springer, Heidel-35 Berg 1977, 33-45; and D. Belpomme et al., British Journal of Hematology 38 (1978) 85.

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In certain disease states (e.g., juvenile rheumatoid arthritis, certain forms of leukemia, and agammaglobulinaemia), T cell subsets have become unbalanced. It is possible that in autoimmune diseases, there is generally an excess of 5 "helper" T cells and a deficiency of certain "attenuator" T cells, whereas in agammaglobulemia certain "attenuator" T cells are present in excess or "helper". -T cells are deficient. In malignant disease states, "attenuator" T cells are usually present in excess.

In certain forms of leukemia, an excess of stagnant T cells are formed. The success of the diagnosis may thus depend on whether this imbalance or excess can be detected. See, for example, Example 15: 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 cited therein.

Agammaglobulinemia, a disease state in which no immunoglobulin is formed, includes at least two different forms of the disease. An excess of "attenuator" T cells in Form I prevents the formation of immunoglobulin, whereas in Form I, the same disorder is caused by a deficiency of "helper" T cells. In both forms of the disease, the patient's B cells, or lymphocytes, which are responsible for the actual secretion of the antibody, are completely normal and lacking; However, B cell function is impaired or "not helped," resulting in a significant reduction or cessation of immunoglobulin production. The type of agammaglobulinemia in a patient can thus be determined by experiments to determine whether there is an excess of attenuating T cells or whether there is a deficiency of helper T cells.

35 On the therapeutic side, there are indications that the administration of antibodies specific for the excess 75230 subset of T cells to patients with autoimmune or malignant diseases has a beneficial effect on disease progression. For example, helper T cell cancer (certain skin tissue tumors and certain acute forms of T cell lymphoblastic leukemia) can be treated with an antibody specific for helper T cell antigens. Autoimmune diseases caused by excess T cells can also be treated in the same manner.

10 Antisera that affect all human T cells (so-called anti-human thymocyte globulin, or ATG) have been shown to be therapeutically useful in the treatment of transplant patients. Because T cells affect cell-mediated immunological responses (the mechanism of rejection of transplanted organs), obtaining an antibody specific for T cells prevents or slows down this rejection process. See, e.g., Cosimi et al., "Randomized Clinical Trial of ATG in Cadaver Renal Allgraft 20 Recipients: Importance of T Cell Monitoring," Surgeery 40 (1976) 155-163, and references therein.

To date, spontaneous autoantibodies or antisera selective for human T cells, prepared by immunizing animals with human T cells, removing blood from animals to obtain serum, and adsorbing the antiserum with, for example, autologous non-autologous cells, have been used to identify and attenuate human T cell species and subspecies. -allogeneic B cells to remove antibodies with undesired reactivity. The preparation of these antisera is very difficult, especially in the adsorption and purification steps. Adsorbed and purified antisera also contain many impurities in addition to the desired antibody for a number of reasons. One reason is that the serum already contains mil-35 g of antibody molecules before immunization with T cells. Second, immunization causes the formation of antibodies to 6,75230 against several antigen species found in all injected human T cells.

It is not possible to selectively produce an antibody specific for a particular antigen. Third, the titer of a specific antibody prepared by these methods is usually quite low (e.g., at dilutions greater than 1: 100, the antibody is inactive) and the ratio of specific to non-specific antibody is less than 1/10®.

10 See, for example, the above article by Chess and Schlossman (from page 365 onwards) and the above article in the Chemical and Engineering News, which describe the shortcomings of the antisera used so far and the advantages of the monoclonal antibody.

A new hybridoma (called OKT4) has now been discovered that is capable of producing a novel monoclonal antibody specific for the surface antigen found in virtually all normal human peripheral helper T cells (approximately 55% of healthy human peripheral T cells). The antibody thus formed is monospecific for a single surface antigenic determinant of normal human T cells and contains virtually no other anti-human immunoglobulins, in contrast to hitherto known antisera (which naturally contain antibodies which react well with many human antigens) and hitherto known monoclonal antibodies (which are not monospecific for a single human T cell antigen). In addition, this hybridoma can be cultured to produce an antibody, so that the use of animals to produce the antibody is not necessary, nor are the complex adsorption and purification steps required in previous methods for preparing crude antisera.

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Both the present hybridoma and the antibody produced by it are referred to herein as "OKT4", it being apparent from the context that which is meant in each case. The present hybridoma was deposited on April 26, 1979 at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, and has been assigned ATCC Accession No. CRL 8002.

The preparation of the hybridoma and the antibody produced by it is described in FI patent application 801343.

The diagnostic method of the invention using said hybridoma and antibody is characterized in that a patient T cell sample is reacted with a diagnostically effective amount of a monoclonal antibody produced by a hybridoma having accession number ATCC CRL 8002 and the substance a) reacts with virtually all normal human peripheral helper T cells, which is about 55% of all healthy human peripheral T cells, but does not react with normal human peripheral B cells, null cells or macrophages, b) reacts with about With 80% of normal human thymocytes, c) does not react with chronic T-cell lymphoblastic leukemia, B-cell chronic lymphoblastic leukemia, T-cell acute lymphoblastic leukemia or zero-cell acute lymphoblastic leukemia) but mut-30 does not react with human T cell lines HJD-1 , Laz 191 and HM1, e) does not react with Epstein-Barr virus transformed human B cell lines Laz 007, Laz 156, Laz 256 or SB, 35 f) reacts with about 55% of Rhesus monkey peripheral T cells.

8 75230 g) binds complement, and h) determines a population of T cells (OKT4 +) that does not react with anti-TH2 serum and has very low cytotoxicity, 5 and determines how much of the total peripheral T cell population responds to said antibody. with the substance.

Example 1 Characterization of OKT4 Antibody Reactivity A. Isolation of Lymphocyte Populations Peripheral mononuclear blood cells were isolated from the blood of normal volunteer donors (15-40 years of age) by the Ficoll-Hypaque density gradient centrifugation method (Scandia, Boy), Pharmacia Fine Chemicals, P . J.

15 Clin. Lab. Invest. 21 (suppl. 97) (1968) 77. Unfractionated mononuclear cells were separated into surface Ig + (B) and surface Ig ”(T and zero cell) populations using Sephadex G-200 anti-F (ab1) 2- column chromatography by the method previously described by Ches et al., 20 J. Immunol. 113 (1974) 1113. T cells were harvested by E-rosetting the Ig ”population with 5% sheep erythrocytes (Microbiological Associates, Bethesda, MD). The rosetted mixture was centrifuged in a Ficoll-Hypaguw centrifuge and the recovered E + pellet was treated with 0.115 M NH 2 Cl (10 mL per 10® cell). The T cell population thus obtained was 2% EAC-positive and> 95% E-positive as determined by standard methods. In addition, a non-rosette-formed Ig (null cell) population was separated from the Ficoll interface. This latter population was <5-% E-positive and £ 2-% SIg-positive. The surface Ig + (B) population was separated from the Sephadex-G-200 column by elution with normal human gammoglobulin as previously described. This population was 95% rises-35 ti surface Ig-positive and <5-% r-E-positive.

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Normal human macrophages were separated from the mononuclear population by attachment to polystyrene. At this time, mononuclear cells were resuspended in final culture medium (RPMI 1640, 2.5 mM HEPES 5 ZJ4- (2-hydroxyethyl) -1-piperazine-propanesulfonic acid / buffer, 200 mM L-glutamine and penicillin streptomycin, and 20% by heating inactivated AB). ) at 2 x 10® cells and incubated in plastic petri dishes (100 x 20 mm), 10 (Falcon Tissue Culture Dish, Falcon, Exnard, CA) at 37 ° C overnight. Petri dishes were washed thoroughly to remove non-adherent cells, and the plastic-adhered population was removed by washing vigorously with a cold solution containing 2.5 mM EDTA without serum, and occasionally scraping the petri dish with the rubber tip of a disposable syringe. More than 85% of the cell line was able to feed latex particles, and Wright-Giemsa staining showed morphological characteristics of monocytes.

20 B. Normal thymus

Normal human thyroid gland was obtained from patients who had undergone corrective heart surgery and ranged in age from two months to 14 years. The thyroid gland was cut, and the pieces were immediately placed in 199 (Gibco) medium containing 5% fetal calf serum, finely chopped with forceps and scissors, and then a suspension containing one cell species was formed by squeezing the mixture through a steel sieve. Next, the cells were centrifuged in a Gicoll-Hypaque-centri-30 fug and washed as described in A. The thymocytes thus obtained were> 95% viable and 90% E-rosent positive.

C. Cell lines

Epstein-Barr-35 virus (EBV) transformed B cell lines obtained from four healthy units (Laz 007, 75230 10

Laz 156, Laz 256, and SB) were prepared as previously described, and T cell lines CEM, HJD-1 Laz 191, and HM1 obtained from leukemia patients were provided by Dr. H. Lazarus, Sidney Farber Cancer Institute, Boston, MA.

5 D. T-cell acute lymphobolast leukemia (T-ALL) cells and T-cell chronic lymphocytic leukemia (T-CLL) cells

Leukemia cells were obtained from twelve patients with acute lymphoblastic leukemia. The cells of these 10 individuals had been found to be T cells in previous experiments because they spontaneously formed rosettes with sheep erythrocytes (20% E +) and because they respond to T cell-specific hetero-antisera, anti-HTL (anti-BK) and A99 with antiserum for 15 min, as described by Schlossman et al. have previously described, Proc. Nat. Acad. Sci. 73 (1976) 1288. Tumor cells from three individuals reacted actively with THp rabbit and / or equine anti-Ti, but cells from nine other individuals did not react with these. Also used from two patients with TH 2 -T-CLL leukemia Both acute and chronic T-cell leukemia cells were cryopreserved at -196 ° C in the liquid nitrogen vapor phase in 10% dimethyl sulfoxide and 20% 25 human AB serum until surface-characterized. The tumor populations analyzed were 90% blasts with Wright-Giemsa morphology in all cases.

Example 2 Cell fluorographic analysis and cell separation

Cell fluorographic analysis of all cell populations was performed by an indirect immunofluorescence method in which cells were stained with fluorescein-conjugated goat anti-mouse IgG 35 (G / M FITC) (Meloy Laboratories) using FC200 / 4800A cell fluorography (Orth In addition to 11,75230 cells, 1-2 x 10 6 cells were treated with 0.15 ml of antibody at a 1: 1,000 dilution of OKT4, the cells were incubated at 4 ° C for 30 minutes and washed twice. The cells were then reacted with 0.15 ml of G / M FITC 5 (1:40 dilution) at 4 ° C for 30 minutes, the reaction product was centrifuged and washed three times. These cells were then analyzed with a cell fluorograph, in which the fluorescence intensity induced by each cell was recorded on a pulse height analyzer. The reactivity was the same at 10 at a dilution of 1:50,000, but at higher dilutions no reaction occurred. The intensity of background staining was determined with 0.15 ml of a 1: 1,000 diluted aqueous humor obtained intraperitoneally from 15 Balb / cJ mice immunized with a non-producing hybrid clone.

In experiments to differentiate between 0KT4 + and 0KT4 “cells, 100 x 10 4 unfractionated mononuclear cells or thymocytes were labeled with 4 ml of 1: 1,000 diluted antibody 20 OKT4 and developed with G / M FITC. . A similar staining method was used to prepare human T cells isolated as described in Example IA. Using a fluorescence-activated cell sorter (FACS-1) (Becton-Dickinson, Mountain 25 view, CA), lymphocytes were separated into OKT4 + and OKT4 ”populations and / or T cells were fractionated into weakly responsive OKT1 + T cells 20%) and highly reactive OKT1 "T cells (fluorescence greater than 20%). Species viability was> 95%. As determined by Trypan-si-30 in all cases. Purity of all isolated populations was> 95%.

Example 3 By analyzing FACS fractionated OKT4 + and OKT4 fractions with equine anti-T1I, 35 OKT4 + and OKT4 ~ cells were separated by FACS and cultured in RPMI 1640 medium (Grand Island 75230

Biological Company) containing 20% human AB serum, 1% penicillin streptomycin, 200 mM L-glutamine, 25 mM HEPES buffer (Microbiolgoical Associates) and 0.5% sodium tricarbonate, the cell concentration in the culture mass was 2 x 10 6 cells / ml. After 24 hours, 1-2 x 10 10 cells from each population were reacted with equine anti-TI at 37 ° C in a humidified atmosphere with a carbon dioxide content of 5%, and the cells were stained with 10 μl of anti-horse IgG prepared in a fluorescein-coupled rabbit. (Cappel Laboratories, Downington, PA) by the method described by Reinherz and Schlossman, J. Immunol. 122 (1979) 1335-1341. Background staining was performed by replacing the specific antibody with normal equine IgG, which was treated as described above. 15 Example 4

Investigation of cell function A. Proliferative studies

The ability of unfractionated and FACS-fractionated lymphocytes to be distributed in microculture was studied at 20 optimal and suboptimal doses of Concanavalin A (Con A) (Calbiochem, La Jolla, CA) and phytohemagglutinin (PHA) (Burroughs-Wellscome, . The alloantigenic proliferative response was determined simultaneously for these same populations using mitomycin c-treated Laz 156, an EBV-transformed human B-lymphablastoid cell line, as a stimulus. Proliferation in the presence of tetanus toxoid (Massachusetts Department of Public Health Biological Laboratories,

Boston, MA) was tested using a final concentration of 10 μg / ml. The Herpes-Zoster antigen was kindly supplied by Dr. John Zaia (Harvard Medical School, Boston, MA) and was used at a 1: 6 dilution. 5% of the macrophages obtained by the method described above were added to all populations at the start of growing in vitro 35 cultures. After four days, mitogen-stimulated cultures were treated with 0.2 μCi of 1 H-thymine-13,75230 din (3H-TdR) (specific activity 1.9 Ci / mM) (Schwarts-Mann Division of Becton-Dickinson, Orangeburg, NY). and the products were separated after 18 hours on a MASH II instrument (Microbiological Associates, Bethesda, MD). The activity of the β-H-5 thymidine compound was measured on a Packard instrument counter (Packard Instrument Company, Downer's Grove, IL). The activity of the background 1 H-thymidine compound was measured by replacing the mitogen with a medium. Cultures stimulated with soluble antigen and allon antigen

O

After 5 days, the cells were treated with JH-thymidine for 18 hours, the products were separated, and the activity was measured as described above.

B. Cytotoxicity Studies Cultures sensitized to cell-mediated lymphysis were performed by adding unfractionated T cells, FACS-fractionated OKT4 + and 0KT4 ~ -T cell fractions, or recombined OKT4 + and OKT4 ratios to different wells of the microtiter plate. treated stimulator cells at a concentration of 2 x 10 6 cells / ml in 20 different cell types. After 5 days, non-viable cells were removed by Ficoll-Hypaque centrifugation. Unfractionated and fractionated T cell populations were then added to Na25 '*' CrO4-labeled target cells, and the cells were incubated for six hours, after which the released chromium was determined. In other experiments, unfractionated T cells were sensitized with mitomycin-treated stimulator cells as described above, and the cells were incubated for five days in MLC, after which they were fractionated by FACS into OKT4 + and OKT4 fractions and the chromium released was determined. Percent cytotoxicity was calculated using the following formula: 5 Cr Cr released in the experiment - spontaneously released ^ Cr released on cold treatment ^ Cr - spontaneously released "^ Cr x 100 75230 14 Assays were performed on three replicates and the results were reported as the average of these. The amount of Cr in all cases was less than 20% of the maximum amount of chromium released in the experiments.

Figure 1 shows the fluorescence spectrum obtained from a cell fluorograph for normal human peripheral T cells reacted with OKT4 (1: 1,000 dilution) and G / M FITC.

For comparison, Figures 1-5 show the results obtained for monoclonal antibodies 0KT1 and OKT3 under similar conditions.

Figure 2 shows the fluorescence spectrum obtained from a cell fluorograph for human thymocytes reacted with 0KT4 and G / M FITC.

Figure 3 shows the fluorescence spectrum obtained from a cell fluorograph for leukemia cells obtained from patients with chronic B-cell lymphoblastic leukemia who have been reacted with OKT4 and G / M FITC.

Figure 4 shows the fluorescence spectrum obtained from a cell fluorograph. to the human T cell line HJD-1 reacted with OKT4 and G / M FITC.

Figure 5 shows the fluorescence spectrum from a cell fluorograph for the human T cell line CEM reacted with OKT4 and G / M FITC.

Figure 6 shows the results obtained by reacting T cell populations with equine anti-T1 serum.

Figure 7 shows the potency of unfractionated T cells and fractionated T cell fractions as cytotoxic. The coordinate represents the percentage specific lysis of the cells and the effector / target (EIT) ratio in the abscissa.

As shown in Figure 1, approximately 45% of its normal human 35 peripheral T blood cells react with OKT4, but B cells, null cells, and 75,230 macrophages isolated from the same individual do not react at all with OKT4. Similar results were obtained for lymphocyte populations isolated from fifteen other normal humans. The monoclonal antibody is thus characterized in that it reacts with an antigen found in 55% of healthy human peripheral T cells, but does not react with any of the surface antigens of the other three cell types mentioned. As mentioned later, the OKT4-positive portion of human peripheral T cells 10 belong to helper T cells. This difference in reactivity can be used to detect the OKT4 of that antibody and distinguish it from other antibodies.

Figure 2 shows that approximately 80% of normal thymocytes in a six-month-old child reacted with 15 0KT4. Similar results (80% activity) were obtained using six other thymus samples obtained from normal people ranging in age from two months to 19 years. The reactivity formula of Figure 2 also makes it possible to detect said antibody OKT4 and distinguish it from other antibodies.

Figure 3 shows that leukemia cells in patients with B-cell chronic lymphoblastic leukemia do not react with the present antibody.

The present antibody 0KT4 can be further characterized by its reaction with different human T cell lines, as illustrated in Figures 4 and 5. The figure shows that the reactivity of this antigen with human T cell lines varied, the reaction with the CEM cell line was may-30 lying, but with the cell line HJD-1 no reaction occurred at all. OKT4 also did not react with cell lines Laz 191 and HM1. With this different reactivity, it is also possible to characterize 0KT4.

Figure 6 shows how the fractions fractionated with OKT4 reacted with anti-TH2. About 25% of the unfractionated T cell population reacts with anti-TH2.

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In contrast, the OKT4 + population does not contain any anti-TH2-reactive cells, whereas the majority of the OKT4 + population is TH2-positive and all TH2 + cells in the unfractionated T cell population belong to the OKT-5 population. This showed that the OKT4 + fraction does not include TH2 + cells and the TH2 + fraction does not contain OKT4 + cells and thus these fractions are completely separate.

Figure 7 shows the potency of unfractionated T cells, OKT4 + T cells and OKT4 ~ T cells as cytotoxic.

10 The OKT4 + population acts as a cytotoxic to a very small extent, whereas the cell-destroying effect of the OKT4 ”population is greater than that of the unfractionated T cell population. The different reactivities of the OKT4 + and OKT4 'cell populations shown in Figures 6 and 7 also make it possible to characterize the present antibody.

The results shown in Figures 1-5 are summarized in Table I, which also contains some additional information. The table compares the monoclonal antibodies produced by the OKT1, OKT3, and 0KT4 hybridomas (the latter being described in the present application). In addition to the results shown in Figures 1-5, it is also seen from Table I that OKT4 does not react with normal human peripheral T cells, null cells, or macrophages, nor with T cells and nucleus cells from patients with acute lymphoblastic leukemia or B cell lines. In contrast to OKT1, 0KT4 does not react with leukemia cells in patients with chronic T-cell lymphoblastic leukemia.

Cell function studies were performed on lymphocyte populations fractionated with a fluorescence-activated cell sorter (FACS). The results of these studies are shown in Tables II, III, and IV, and the results further support the 35 characterization results of the present monoclonal antibody presented above.

17 75230 In these studies, an unfractionated T cell population was treated with OKT4 (1: 1000 dilution) and G / M FITC and fractionated by FACS into OKT4 + and OKT4 “fractions. To these fractionated populations, 5 5% macrophages were added (purity of populations equal to or greater than 95% was taken into account) prior to in vitro culture. PHA, Con A, soluble an-10 antigens, and allon antigens were added as a stimulator to the unfractionated T cell population and isolated OKT4 + and QKT4 subfractions to determine the proliferative responses of the populations in vitro.

The proliferative response of unfractionated T cell populations in the presence of PHA and Con A is shown in Table II. The proliferative response of the unfractionated T cell population is maximal with 1 pg PHA per 10 cell, with 0.5 pg or 0.1 pg PHA per 10 cell, the response decreased. When unfractionated T cells were treated with OKT4 and goat-mouse FITC, the proliferative response remained the same. The isolated OKT4 + -20 and OKT4 “-T subfractions, on the other hand, behaved differently in the presence of PHA. The OKT4 + cell population behaved in the same manner as the unfractionated T cell population at all doses of PHA used. In contrast, the proliferative response of OKT4 “cells was significantly lower at all doses of PHA studied. In addition, it was found that at a dose of PHA of 0.1 pg per 10 cells, OKT4 + T cells did not increase at all, while the OKT4 + T cell fraction and unfractionated cells further increased. On the other hand, the proliferative response of these fractions in the presence of Con A was similar, so that the two cell fractions could not be distinguished from each other or from the unfractionated T cell population.

Next, the proliferative response in the presence of alloantigen in MLC as well as in the presence of soluble antigens was examined. Table III shows that in the presence of Laz 156 in MLC, the response of the unfractionated 18,75230 T cell population, the unfractionated T cell population treated with 0KT4 and G / M FITC, and both OKT4 + and OKT4 “T cell subsets was similar. It should be noted, however, that out of 5 of the six individuals studied, two OKT4 cells bound less 1 H-TdR than the corresponding OKT4 + fraction (data not shown), although they proliferated abundantly in MLC. However, the two cell fractions differed more clearly when studying their proliferative response in the presence of soluble antigens. In all cases studied, the OKT + T cell fraction was abundantly increased in the presence of soluble antigens, te-Tanus toxoid, and Herpes-Zoster, whereas the OKT “T cell fraction was virtually non-existent.

Table IV shows that the OKT4 ”T cell fraction causes almost no cell lysis when sensitized as such in MLC. Although OKT4 ~ T cells then became cytotoxic / effector in an unfractionated allo-sensitized T cell population, as such, they were unable to act as mediators in CML. When the OKT4 + population was sensitized in MLC without OKT4® T cells, they caused moderate but significant cell lysis in MLC. The recombinant mixture of 0KT4 + and OKT4 “T cells, on the other hand, caused the most 25 cells to lyse, as did the unfractionated T cell population. These findings indicate that the OKT4 ”subframe as such does not function as the cytotoxic in the most efficient way possible, but an OKT4 + population is also required for this. This T-T interaction is analogous to the T-T interaction that occurs between the TH2-T cell fraction and the TH2 + T cell fraction when they act as the most effective cytotoxic agent.

The 0KT4 antibody in question was found to belong to the IgG2 subclass, which is one of the four subclasses of mouse IgG. These subclasses of immunoglobulin G differ from the so-called from its "fixed" regions, although the structure of an antibody specific for a particular 19,75230 antigen has a so-called a "variable" region that is functionally similar regardless of which immunoglobulin G subclass it belongs to. That is, a monoclonal antibody having the characteristics described above may belong to the subclasses IgG1, IgG2a, IgG2b or IgG3 or to the classes IgM or IgA or other classes of Ig. Differences in these classes or subclasses do not affect the selectivity of antibody reactivity, but may affect how antibody 10 subsequently reacts with other agents, e.g., complement or anti-mouse antibodies.

The present antibody can be used to detect type II agammaglobulinemia by reacting the antibody with a patient's T cell sample.

15 If less than 55% of the total peripheral T cell population reacts with OKT4, there are too few helper T cells or so-called are completely absent. This assay can also be used in general to detect a deficiency or excess of helper T cells. Diagnostic compositions comprising diagnostically effective amounts of OKT4 antibody in admixture with diagnostically acceptable carriers are also within the scope of the present invention.

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Table I

Reactivity and properties of monoclonal antibodies 5% reactivity with the following cell types: CKT1 OKT3 CKT4

Peripheral T cells (10 samples) 95% 95% 55%

Peripheral B cells (10 samples) 2% 2% 2%

Peripheral null cells (10 samples) 2% 2% 2%

Thymocytes x (8 samples) 5-1Q% 5-10% 80% 10

Reactivity with the following cells and cell lines: T-cell chronic lymphocytic leukemia (3 cases) + + (1) ;-( 2) T-cell acute lymphocytic leukemia (8 cases) - - - 1 5

Zero-cell acute lymphocytic leukemia (15 cases) - B-cell chronic lymphocytic leukemia (6 cases) + (4) ;-( 2) - - B-cell lines + (4) - - - 20 T-cell lines + HJI> -1 + ( +) CEM Ψ · +

Laz 191 + HM1 +

IgG subclass IgG, IgG, IgG_ 25 Complement binding capacity + + 1

Obtained from patients ranging in age from 2 months to 18 years.

+ Obtained from Dr. H. Lazarus, Sidney Farber Cancer Center. 30 B cell lines Laz 256, 156, 007 and SB were obtained by transformation of human peripheral B cells with Epstein-Barr virus, and T cell lines HJD-1, CEM, Laz 191 and HM1 were obtained from leukemia patients.

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Claims (2)

75230 24 A diagnostic method for detecting an excess or deficiency of a patient's helper T cells, for example a deficiency of a helper T cell due to type II agammaglobulinemia, characterized in that the patient's T cell sample is reacted with a diagnostically effective amount of a monoclonal antibody. produced by a hybridoma with accession number ATCC CRL 10 8002, in which the antibody a) reacts with virtually all normal human peripheral helper T cells, which is about 55% of all normal human peripheral T cells, but does not react normal human peripheral 15 with B cells, null cells and macrophages, b) reacts with about 80% of normal human thymocytes, c) does not react with T-cell chronic lymphoblastic leukemia, B-cell chronic lymphoblastic leukemia, T-cell acute lymphoblastic leukemia d) reacts with the human T cell line CEM but does not react with the human T cell lines HJD-1, Laz 191 and HM1, e) does not react with the human B cell lines transformed with Epstein-Barr virus cell lines Laz 007, Laz 156, Laz 256 and not SB, f) reacts with about 55% of Rhesus monkey peripheral T cells, g) binds complement, and h) determines the T cell population (OKT4 +), which does not react with anti-TI serum and has very low cytotoxicity, and determines how much of the total peripheral T cell population reacts with said antibody. A diagnostic composition for detecting a helper T cell deficiency 752 30 25, characterized in that it comprises the amount of monoclonal antibody required to detect a helper T cell deficiency mixed with a diagnostically acceptable carrier produced by the antibody. 5 hybridoma with accession number ATCC CRL 8002, which antibody a) reacts with virtually all normal human peripheral helper T cells, which is about 55% of all normal human peripheral T cells, but does not with normal human peripheral B cells, null cells and macrophages, b) reacts with about 80% of normal human thymocytes, c) does not react with chronic T-cell lymphoblastic leukemia, chronic B-cell lymphoblastic leukemia, acute T-cell with human leukemia cells with lymphoblastic leukemia or zero-cell acute lymphoblastic leukemia, d) reacts with 20 reacts with human T cell lines HJD-1, Laz 191 and HM1, e) does not react with Epstein-Barr virus transformed human B cell lines Laz 007, Laz 156, Laz 256 and SB, 25 f) reacts about 55%: with rhesus monkey peripheral T cells, g) binds complement, and h) defines a T cell population (OKT4 +) that does not react with anti-Tfβ serum and has very low cytotoxicity. 75230 26