FI75231B - DIAGNOSTIC FOERFARANDE VID VILKET ANVAENDS EN MONOCLONAL ANTIKROPP SPECIFIK MOT MAENNISKANS CYTOTOXISKA OCH SUPRESSOR-T-CELLER. - Google Patents

Description

1 75231

Diagnostic method using a monoclonal antibody specific for human cytotoxic and attenuating T cells 5 Divided by application 802917

The invention relates to a diagnostic method for detecting a deficiency or excess of residual T cells in a human. The method utilizes a monoclonal antibody specific for an antigen found on the surface of 10 normal human cytotoxic and attenuating T cells. The antibody is made using a new hybrid cell line. This diagnostic method can be applied, e.g., to the detection of an acute mononucleosis infection.

In 1975, Kohler and Milstein combined mouse myeloma cells into spleen cells from immunized mice (Nature 256 (1975) 495-497), where it was first discovered that it was possible to produce a continuous cell line to produce a homogeneous (so-called "monoclonal") antibody.

Since then, many attempts have been made to produce various hybrid cells (so-called hybridomas) and to try to apply the antibody produced by these hybridomas to scientific use. See, e.g., Current Topics in Microbiology and Immunology, Volume 81 - "Lymphosyte Hybrido-25 mas", edited by F. Melchers, M. Potter and N. Warner,

Springer-Verlag 1978 and references in the publication; C.J. Barnstable et al., Cell 14 (May 1978) 9-20? P. Parham and W.F. Bodmer, Nature 276 (1978) 397-399; Handbook of Experiment Immunology, 3rd Edition, 30 Volume 2, edited by D.M. Wier, Blackwell 1978, paragraph 25; and Chemical and Engineering News, January 1 (1979) 15-17.

These references show the results obtained and the difficulties encountered in trying to prepare a hybrid-35 monoclonal antibody. Although the manufacturing method is in principle well known, there are many difficulties with 2,75231 and each case is different. It cannot be guaranteed in advance / that an attempt will be made to try to produce the desired hybridoma and that, if successful, the hybridoma will produce an antibody or that the antibody thus formed will have the desired specificity. Success depends mainly on the type of antigen used and the selection technique used to isolate the desired hybridoma.

Attempts have been made to produce a monoclonal antibody specific for human lymphocyte surface antigens in only a few cases. See, e.g., Current Topic 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 of the hybridomas thus prepared produced an antibody that acted on different surface antigens of all human cells. None of these hybridomas 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. The second of these (a 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 25 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, participate directly in cell-mediated immunological responses (e.g., transplant rejection). Although T cells do not secrete antibodies into body fluids, another group of lymphocytes that will be treated later will in some cases require T cells to be able to secrete antibodies themselves. Some T cell species act as regulators of 35 immune system functions. The mechanism of this form of cell interaction is not yet fully understood.

75231

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 the thymus does not regulate their different 5 propagation. In birds, they differentiate in an organ analogous to the thymus, called the "Bursa of Fabri-cius." However, no similar organ has been found in mammals, and thus its B cell count is differentiated 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 mouse-15 environment, 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 Bearing Unique Dirrefentia-20 tion Antigens", in "Contemporary Topics in Immuno Biology", edited by O. 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 immunoregulatory disorders.

For example, the prognosis of certain forms of leukemia and lymphoma differs depending on whether they originate from B or T cells. Thus, in assessing the prognosis of the disease, it is important to be able to distinguish between the 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 Lyhmphomas," Towers S. Thier-Felder et al., Springer, Heidelberg 1977, 33-45; and D. Bel-35 pomme et al., British Journal of Hematology 38 (1978) 85.

In certain disease states (e.g., juvenile rheumatoid arthritis, 4,75231 malignancies, and agammaglobulinemia), T cell subgroups have become unbalanced. It has been suggested that in autoimmune diseases, "helper" T cells are generally present in excess and certain "hi-5 field" T cells are deficient, whereas in agammaglobulinemia, certain "attenuator" T cells are present in excess or "helper" T cells. cells are deficient. In malignant disease states, "attenuator" T cells are usually present in excess.

10 In certain forms of leukemia, an excess of T cells in the stagnant stage of development is formed. The success of the diagnosis may thus depend on whether this imbalance or excess can be detected. See, e.g., J. Kersey et al., "Surface Markers Define Human 15 Lymphoid Malignancies with Differing Prognoses," in Hematology and Blood Transfusion, Volume 20, Springer-Verlag 1977, 17-23, and references therein.

Agammaglobulinemia, a disease state in which no immunoglobulin is formed at all, includes at least two different forms of the disease. An excess of "weakening" T cells in Form I prevents the formation of immunoglobulin, whereas in Form II, 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 deficient; However, B cell function is impaired or "not helped," resulting in a significant reduction or complete 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.

There are indications that the administration of antibodies specific for an excess subset of T cells to patients with autoimmune or malignant diseases has a beneficial effect on the development of the disease. For example, helper T cell cancer (certain skin tissue tumors and certain forms of acute T cell lymphoblastic leukemia) can be treated with an antibody specific for the helper T cell antigen.

5 Autoimmune diseases caused by excess T cells can also be treated in the same way. Diseases caused by an excess of attenuating T cells (e.g., malignancies or agammaglobulinemia type I) can be treated by administering to the patient 10 antibodies to the attenuating T cell antigen.

Antisera that act on 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 20 Clinical Trial of ATG in Cadaver Renal Allgraft Recipients: Importance of T Cell Monitoring," Surgery 40 (1976) 155-163, and references therein.

To date, spontaneous auto-25 antibodies or antisera selective for human T cells, prepared by immunizing animals with human T cells, removing blood from animals to obtain serum, and adsorbing antiserum, have been used to identify and attenuate human T cell species and subspecies. autologous non-allogeneic B cells to remove antibodies with undesired reactivity. The preparation of these anti-sera 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 serum contains millions of antibody molecules even before immunization with T cells. Second, immunization causes the formation of antibodies that affect several species of antigens found in all human T cells used for immunization. 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 article by Chess and Schlossman, cited above (from page 365 onwards) and the article cited in The Chemical and Engineering News, which describe the shortcomings of the antisera used hitherto and the advantages of the monoclonal antibody.

A subset of T cells identified by known antisera is termed the TH2 + subset and has been found to contain both cytotoxic effector cells required for cell-mediated lymphysis and immunoregulatory attenuator T cells that attenuate both T cell and B cell function. This subset contains about 20-30% of human peripheral T cells. See, e.g., E.L. Reinherz, et al., V. Immunol. 123 (1979) 83 and New Engl. V. Med. 300 (1979) 1061.

A new hybridoma (called 0KT5) has now been discovered, which is already capable of producing a novel monoclonal antibody specific for normal human peripheral cytotoxic and attenuating TH2 + T cells (approximately 20% of normal human peripheral T cells). le surface antigen. The antibody thus formed is monospecific for a single surface antigenic determinant of normal human cytotoxic and attenuating TH2 + cells, and contains virtually no other anti-human immunoglobulins, in contrast to hitherto known antisera (containing inherently as impurities are antibodies that 75231 react with a large number of human antigens) and hitherto known monoclonal antibodies (which are not monospecific for a single human cytotoxic or attenuating T cell antigen). In addition, these 5 hybridomas 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 to date for the preparation of even impure antisera.

The present hybridoma and the antibody formed are hereinafter referred to as "OKT5"; it is clear from the context which is meant in each case.

The present hybridoma OKT5 is deposited at 15 "The American Type Culture Collection", 1201 Parklawn

Drive, Rockville, Maryland 20852, September 18, 1979 and September 28, 1979, and has been assigned ATCC Accession Nos. CRL 8013 and CRK 8016.

The preparation of the hybridoma and the antibody produced by it is described in FI patent application 802917.

The diagnostic method of the invention using said hybridoma and antibody is characterized in that a T cell sample taken from a patient is reacted with a diagnostically effective amount of IgG class monoclonal antibody produced by a hybrid cell line having an ATCC accession number. is CRL 8013 or 8016 prepared by combining cells of a mouse myeloma cell line with spleen cells of a mouse immunized with human thymocytes, and which antibody 30 a) reacts with more than 90% of cytotoxic and attenuating TH2 + cells (20-30% of all normal human peripheral T cells) but does not react with normal human peripheral B cells, null cells or macrophages, 35 b) reacts with about 80% of normal human thymocytes, 8 75231 c) does not react with T cells and does not react with 0KT4 + T cells but reacts with 68% of 0KT4 T cells, d) defines a T cell population (0KT5 +) that is highly cytotoxic and lower than normal levels in primary cirrhosis, multiple sclerosis and hyper-IgE, normal levels in all stages of Hodgkins disease and higher than normal The T cell population reacts with the antibody.

Example 1 Characterization of OKT5 Antibody Reactivity A. Isolation of Lymphocyte Populations Peripheral mononuclear blood cells were isolated from the blood of normal volunteer donors (ages 15-40 years) by Ficoll-Hypaque density gradient centrifugation (as described by Scandia Fineway, NH), Pharmacia Fine Chemicals, Pisv . J. Clin. Lab. Invest. 20 21 (suppl. 97) (1968) 77. Unfractionated mononuclear cells were separated into surface Ig + (B)) and surface IG (T- and nol glass cell) populations using Sephadex G-200 anti-F (ab ') 2 ~ column chromatography by the method previously described by Chess et al., J. Immunol. 113 (1974) 25 1113. T-beer was recovered by E-rosetting the Ig strain with 5% sheep erythrocytes (Microbiological Associates, Bethesda, MD). The rosetted mixture was centrifuged in a Ficoll-Hypa-que centrifuge and the recovered E + pellet was treated with 0.155 M NH 4 Cl (10 mL per 10® cell). The T cell population thus obtained was <2% EAC rosette positive and> 95% E rosette positive as determined by standard methods. In addition, the non-rosette-forming Ig (nol glass cell) population was separated from the Ficoll interface. This latter population was <5% E-positive 35 and ¢ 2% slg-positive. The surface Ig + (B) population was separated from the Sephadex-G-200 column by elution with normal -9,75231 human gamma globulin as previously described. This population was> 95% surface Ig-positive and <5% E-positive.

Normal human macrophages were isolated 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 / 4- (2-hydroxyethyl) -1-piperazine-propanesulfonic acid? Buffer, 200 mM L-glutamine and penicillin streptomycin 10, and 20% heat-inactivated AB cells were counted at 2 x 10 6 cells and incubated in plastic petri dishes (100 x 20 mm) (Falcon Tissue Culture Dish, Falcon, Exnard, CA) at 37 ° C overnight. The petrimal dishes were washed thoroughly to remove non-adherent cells, and the plastic-attached 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.

20 More than 85% of the cell line was able to use latex particles for food, and Wright-Giemsa staining showed that they were morphologically characterized by monocytes.

B. Isolation of thymocytes

A normal human thyroid gland was obtained from a poti-25 who had undergone corrective heart surgery and ranged in age from two months to 14 years. The thyroid gland was dissected, and the pieces were immediately placed in 199 (Gibco) medium containing 5% fetal calf serum, finely chopped with forceps and sak-30, 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 Ficoll-Hypaque centrifuge and washed as described in A. The thymocytes thus obtained were> 95% viable and 9090% 35 E rosette positive.

75231 10 C. Cell line

A B cell line (Laz 156) transformed with Epstein-Barr virus (EBV) from a healthy individual was obtained from Dr. H. Lazarus, Sidney Farber Institute, Boston, MA.

5 Example 2

Cell fluorographic analysis and cell separation

Cell fluorographic analysis of all cell populations and reaction products of monoclonal antibodies was performed by an indirect immunofluorescence method in which cells were stained with fluorescein-conjugated goat anti-mouse IgG (G / M FITC). Instruments). At this time, 1-2 x 10 6 cells were treated with 0.15 ml of OKT5 antibody at a 1: 500 dilution, incubated at 4 ° C for 30 minutes, and washed twice. The cells were then reacted with 0.15 ml of G / M FITC (1:40 dilution) at 4 ° C for 30 minutes, the reaction product was centrifuged and washed three times. These cells were then analyzed with 20 cell fluorographs, in which the fluorescence intensity induced by each cell was recorded on a pulse height analyzer. The reactivity was similar at a dilution of 1: 10,000, but at higher dilutions no reaction occurred. The intensity of background staining was determined with 0.15 ml of a 1: 500 dilution of aqueous humor obtained intraperitoneally from a CAF1 mouse immunized with a non-productive hybrid clone. The reactivity of lymphoid populations with equine anti-TI and equine normal IgG was determined as described above (Reinherz et al.).

In experiments to distinguish subgroups of T cells, 100 x 10 unfractionated T cells were labeled with 4 ml of 1: 500 diluted antibody OKT4 or OKT5 and developed with G / M FITC. OKT4 35 had previously been found to react specifically with 55-60% of peripheral blood T lymphocytes, a group representing 11,75231 human helper T cells. Using a fluorescence-activated cell sorter (FACS-1) (Becton-Dickinson, Mountain View, CA), T cells obtained from the same individual were divided into 0KT4 + and 0KT4 populations, and 0KT5 + -5 and OKT5 populations. Species viability was> 95% as determined by Trypan blue in all cases. The purity of all isolated populations was ^ 95%.

Example 3 Analysis of T cell subsets with equine anti-10 TH2

According to Example 2, equine anti-TH9 was used to separate TH2 and TH2 cells by FACS, with 60 x 10 6 unfractionated T cells being labeled with 0.12 ml of equine anti-TH2 and 0.1 ml of R / H FITC (Cappel 15 Laboratories, Downington, PA) Reinherzin et al. as described. The purity and viability of the sorted cells were similar to those of the sorted populations described above. FACS-sorted T cell fractions isolated with equine anti-TH2, OKT4, or 20 0KT5 were cultured for 48 hours at 37 ° C in a humidified atmosphere containing 5% CO 2 in RPMI 1640 medium containing 20% CO 2. human AB-s erum, 1% penicillin-streptomycin, 200 mM L-glutamine, 25 mM HEPES buffer (Microbiological Associates) and 0.5% narium bicarbonate. The cells were then analyzed by cell fluorography as described above. Background staining was determined by replacing the specific antibody with normal equine IgG and performing staining as described above.

30 Example 4

Investigation of cell function A. Proliferative studies

The mitogenic response of unfractionated and FACS-fractionated T-lymphocytes (10) was studied in 35 microcultures with optimal doses of Concanavalin A (Con A) (Calbiochem, La Jolla, CA) and phytohemagglutinin (PHA) (Burroughs-Wellcome Company, Greenville, NC).

75231 12

The alloantigenic proliferative response was determined simultaneously for these same populations using mitomycin-treated Laz 156, an EBV-transformed human N-lymphoblastoid cell-5, as a stimulant. Profiling in the presence of tetanus toxoid (Massachusetts Department of Public Health Biological Laboratories, Boston, MA) and mumps antigen (Microbiological Associates) was tested using a final concentration of 10 μg / ml for tetanus toxoid and a 1: 20.5% dilution for mumps antigen obtained as described above. macrophages were added to all populations at the start of growing in vitro cultures. After four days, mitogen-stimulated cultures were treated with 3 0.2 μCi of H-thymidine (3H-TdR) (specific activity 1.9 Ci / mM) (Schwarz-Mann Division of Becton-Dickinson,

Orangeburg, NY) and the products were separated after 18 hours on MASH II equipment (Microbiological Associates, Bethes- 3 ...

da, MD). The activity of the H-thymidine compound was measured with a Pacard scintillation counter (Packard Instrument Company, Dow-20's Grove, IL). The activity of the background 1 H-thymidine compound was measured by replacing the mitogen with a medium. After 5 days, cultures stimulated with soluble antigen and cell surface alloantigen were treated with H-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 and mitomycin-treated stimulator cells to the wells of a microtiter plate at a concentration of 2 x 10b cells / ml for different cell types. After 5 days, unfractionated T cells were fractionated into OKT5 + and OKT5 T cell fractions by 51 FACS. These T cell fractions were then added to Na 2 CrO 2 -labeled target cells, and the cells were incubated for six to 35 hours, after which specific cytotoxicity was determined. Percent cytotoxicity was calculated using the following formula: 13,75231

Experimentally released ^ Cr-spontaneously released 51Cr - x 100

Freezing / thawing-released xCr-spontaneously released 51cr ^ Assays were performed on three replicates and the results were reported as an average of these.

C. Activation of attenuator cells by Con A and attenuation of MLC

Unfractionated T cells were activated by the addition of 10 μg of Concanaval A (Con A) (Calbiochem) per 10 cells. These Con A-treated cells were cultured in 2 25 cm tissue culture flasks (Falcon, Oxnard, CA) in RPMI 1640 medium (Grand Island Biological Company) containing 20% human AB-15 serum, 1% penicillin- streptomycin, 200 mM L-glutamine, 25 mM HEPES buffer (Microbiological Associates) and 0.5% sodium bicarbonate for 48 hours at 37 ° C in a humidified atmosphere containing 5% CO 2. Untreated control T cells were cultured in the same manner, but without Con A. The cells were then centrifuged, washed five times, and added to fresh autologous responder cells in unidirectionally mixed lymphocyte culture (MLC) as described above.

In the MLC attenuation assay, culture was performed in 25 round-bottom microtiter plates (Linbro Chemical Company, New Haven, CT) in three wells, each containing 0.05 x 10 responder lymphocytes (all mononuclear), 0.05 x 10 6 with either inactivated or Con A activated autologous unfractionated or 30 fractionated T cells and 0.1 x 10 6 mitomycin-treated stimulated cells (Laz 156). After 5 days, the cultures were treated with 0.2 H-TdR, and after 18 hours the cells were harvested. The proliferation inhibition capacity (%) of MLC was then calculated using the following formula: 75231 14

Inhibitory capacity (%) = (1 - cPm Con_en_ +} χ 100 cpm ^ 2 where cpm Con A denotes the results obtained when treating MLC with H-TdR when adding Con A-activated autologous T cells or T cell fractions, and cpm denotes the results obtained with the addition of Akti-10 helpless autologous T cells.

Figure 1 in the top row shows the fluorescence spectrum from a cell fluorograph for normal human peripheral T cells, B cells, null cells, and macrophages reacted with OKT5 (1: 1000 dilution) and G / M FITC. For comparison, the results obtained using equine anti-TI® are shown in the bottom row.

Figure 2 shows the fluorescence spectrum obtained from a cell fluorograph for human thymocytes reacted with OKT5 and G / M FITC (A) and equine anti-Tβ (B).

Figure 3 shows the fluorescence spectrum obtained from a cell fluorograph for equine anti-T1 fractionated T cell fractions reacted with OKT5. Figure 4 shows the fluorescence spectrum obtained from a cell fluorograph for OKT4-fractionated T cell fractions reacted with 0KT5.

Figure 5 shows the potency of unfractionated T cells and OKT5-fractionated MLC-sensitized T cell fractions as a cytotoxic agent.

As shown in the top row of Figure 1, approximately 20% of peripheral T blood cells from a normal individual react with OKT5, but B cells, null cells, and macrophages isolated from the same individual do not react at all with OKT5. The 35-horse anti-Ti 2 also reacted with 24% of peripheral T cells, but it also did not react with B cells, null cells, or macrophages. The monoclonal antibody is thus characterized by reacting with a surface antigen found in 20% of healthy human peripheral T cells, but not by any of the surface antigens of the other three cell types mentioned. As mentioned later, the OKT5-positive portion of human peripheral T cells belong to cytotoxic and attenuator T cells. This difference in reactivity can be used to detect the OKT5 of that antibody and distinguish it from other antibodies.

Figure 2 shows that approximately 80% of normal thymocytes in a six-month-old child react with OKT5. Similar results (80% activity) were obtained using six other thymus samples 15 obtained from normal individuals ranging in age from two months to 19 years. The same value was obtained for equine anti-TH2: He. The reactivity formula of Figure 2 also makes it possible to detect this antibody OKT5 and distinguish it from other antibodies.

Figure 3 shows that the OKT5 antibody reacts with TH2 + T cells but not with TH2 T cells. About 5-10% of the TH2 + fraction did not react with OKT5. The reactivity formula of Figure 3 also makes it possible to detect the OKT5 antibody and distinguish it from other antibodies.

Figure 4 shows that the OKT4 T cell fraction does not react at all with 0KT5. In contrast, the 0KT4 T cell fraction is mostly OKT5-positive (6800 of the 10,000 cells tested are OKT5-positive). These results indicate that the 0KT5 + fraction of T cells as well as the previously defined TH2 + fraction are opposite to the OKT4 + fraction. The OKT4 + fraction contains helper T cells, while the OKT5 fraction (as well as the TH2 + fraction) contains cytotoxic and attenuator T cells. With this reactivity formula according to Figure 4, it is also possible to detect OKT5 and distinguish it from other antibodies.

75231 16

Figure 5 shows that the OKT5 + T cell fraction affects CML. The degree of degradation caused by this population is higher than that of the unfractionated T cell population. In contrast, the degradation caused by the 0KT5 T cell population is very low. Unfractionated T cells were activated by MLC against Laz 156 and then fractionated into 0KT5 and 0KT5 fractions. Both unfractionated and fractionated T cells were analyzed for CML-labeled Laz 156 targets in CML using different effector / -10 target ratios (E: T). OKT5 + T cell fractions contained an effector population in cell-mediated lymphysis. At E: T ratios of 5: 1, 10: 1, and 20: 1, the specific degrading effect of the OKT5 + T cell population was 40, 58, and 77%. The lysis efficiency of the isolated OKT5 + fraction was significantly higher than that of the unfractionated T cell population. In addition, the disruptive effect of the 0KT5 T cell population was significantly lower at all E: T ratios studied. In view of the previous finding that the TH2 + T cell fraction in humans affected their CML, the present finding would support the hypothesis that the T1 ^ * and 0KT5 T cell populations defined the same functionally active T lymphocyte populations. With this different cytotoxic effect, it is also possible to identify the OKT5 antibody and distinguish it from mucus antibodies.

Functional studies were performed on lymphoid populations fractionated with a fluorescence-activated cell sorter (FACS). The results are shown in Tables I ... III and further support the methods described for the characterization of the monoclonal antibody OKT5.

In these studies, the unfractionated T cell population was treated with OKT5 (1: 500 dilution) and G / M FITC and fractionated by FACS into OKT5 + and OKT5 fractions. To these fractionated populations, 5% macrophages were added (purity of populations equal to or greater than 95% was taken into account) prior to in vitro culture. Unfractionated T cell populations and isolated OKT5 + and OKT5 T cell fractions were stimulated with PHA,

Con A: 11a, soluble antigens and alloantigens to determine the proliferative response of populations in vitro.

The proliferative response of unfractionated T cell populations in the presence of PHA and Con A is shown in Table I. The proliferative response of an unfractionated T cell population is maximal at 1 μg PHA per 10 μg cell, using 0.5 μg or 0.1 μg PHA per 10 μg cell, the response was impaired. When treatment of fraction-free T cells with OKT5 and goat FITC, the proliferative response remained the same. In contrast, the isolated 0KT5 + and OKT5 T cell fractions behaved differently in the presence of PHA. The OKT5 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 OKT5 + cells was significantly lower at all doses of PHA studied. In addition, it was found that at a dose of 20 PHA per 0.1 μg per 10 μg cells, OKT5 + T cells did not increase at all, while the OKT5 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 response in the presence of alloantigen in MLC as well as in the presence of soluble antigens was examined. Table II shows that in the presence of Laz 156 in MLC, the response of the unfractionated T cell population, the unfractionated T cell population treated with OKT5 and G / M FITC, and both OKT5 and OKT5 T cell fractions was similar. However, the two cell fractions differed most markedly in studying their proliferative response in the presence of soluble antigens. In all cases studied, the 0KT5 + T cell fraction increased very little in the presence of soluble antigens, tetanus toxoid and mumps antigen, while the 0KT5 ~ T cell fraction increased well.

Previous studies have shown that a TH2 + population of T-lym-5 phosphites in combination with Con A would cause a decrease in the proliferation of autologous lymphocytes in MLC. To elucidate a possible similar attenuating effect of the 0KT5 + T cell fraction, T cells were activated for 48 hours with Con A and then sorted with monoclonal antibodies OKT5 and OKT4 into different T cell fractions. These fractionated T cell fractions were then added to autologous responder lymphocytes to initiate MLC. Table III shows that unfractionated Con A-activated T cells impaired autologous cell proliferation in MLC. When T cells were treated with monoclonal antibody and G / M FITC, the result was the same. Although both OKT4 + and OKT5 + T cells proliferated when activated with Con A, only OKT5 + T cells when activated with Con A had an attenuating effect.

The 20 OKT4 + fractions had no detrimental effect. In addition, the debilitating effect of the OKT5 population containing OKT4 + and OKT4 -Ti ^ fractions was very small compared to the strongly debilitating OKT5 + population.

Table IV shows the relationship between the concentrations of peripheral T cells and 25 T cell fractions in different disease states. These ratios can be used for diagnostic purposes (e.g., for the detection of acute infectious mononucleosis) by taking a blood sample from a person suspected of having one of these diseases and determining the concentration of T cells and T cell fractions in the blood sample. Using the ratios shown in the table, the 0KT5 antibody can also be detected and distinguished from other antibodies.

Diagnostic methods using other monoclonal antibody-producing hybridomas (OKT1, OKT3 and OKT4) produced by the same applicants are described in the following FI patent applications 844703, 844704 and 844705.

75231 19

The antibody 0KT5 was found to belong to the IgG1 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 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, IhG2b or IgG1, or to the classes IgM or IgA or other Ig classes. Differences in these classes or subclasses do not affect the selectivity of antibody reactivity, but may affect how the antibody subsequently reacts with other agents, for example, complement or anti-mouse antibodies.

The present antibody can be used to detect an excess of cytotoxic or attenuating T cells by reacting a T cell sample taken from a patient with an OKT5 antibody. An excess of cytotoxic or attenuating T cells is present if more than 20-30% of the total peripheral T cell population reacts with 0KT5. In this diagnostic method, the OKT5 antibody may be used as such or a combination of OKT5 and other antibodies (e.g., 0KT3 and OKT4) may be used, as shown in Table IV. With such reactivity formulas between different antibodies and T cells or T cell fractions, certain disease states can be detected more accurately than using previous diagnostic methods.

The OKT5 antibody can be included in diagnostic compositions comprising effective amounts of the OKT5 antibody and dianostatically acceptable carriers.

| 75231 20 * H * 01 oo rH cn m oo vo • X <N r — I 00 i — i vo vo

<0 CN CO rH 04 VO

"D £ *" CN

^ Smvomr ^^ rrHoj 0 0 + l +1 +1 +1 +1 + l +1

1 I m m vo ctv fH

E-ι I σι vo σν vo o in ovi £> νοσ »νο (ΝΐηοιΗ δ S co r — I m oo σ o • ηθιηιηο400Γ0 * 3 ·

•B

0

B

• P

Tt) r- ~ o r-ι o <n

Ih m r- m oo oo y-ι σ> o vo> h σν

4J oo CN

in ^ σι rH ι-H CO Γ0 I — I

s? Q +1 +1 +1 +1 +1 +1 +1

O W

I <T \ »H CT \ CO <T \ CO \ £) TO -f rH CM O ') VO O CO Γ0 rHLn ^ roocoor ^ oo. — 1

"φ ^ Ή O 'O' VO

¢) Ο Ή σ \ ro ro

• S

cd ro 4J: <d

tfl rH

at pH

> S g m h. ro in σ oo vo ro

Qjtjovovooomo 'tn_orococNor' h -h S en _! \ r-H ro σ r — i t} · 04 O (0 vo

-¾ ro -H +1 +1 -H +1 +1 -H

"do σι rH rH <n -vr tn pH

i — li — ΐιηΓ'Οοοσ cno m 3 Λί i vo oo σ> vo r-t rro Q 56

Enco ^ R ^ p-ir ^ r ^ f N

g '-' un'vrcNoo · '»^' ro ro c dl ro 3 m σν r» ro ro en i — l ro σν t "σν H vo

0 CN VO I-H VO Γ "I — I

tn ^ votncNCNCNinp '

„Φ -M +1 +1 -H +1 -H -H

3 4-> 4-> d) tn 3 σνιη «a * pH o r-« h -h ro ή 'a · ro m Γ' vo r- ~ oo g> O i — i ov r- ro · * τ m 4-ie J i — t oo σ> ph oo 4-l®fH VO'S · rH oh <ro 1 .3 -1 £

O S \ \ -H

• r | φ I vo \ Co tji \ 3 4-Ιζτ> 0 · Ηθ CP \ 3 3 m p

X Q H 4 rH 3 tr, \ \ 3 H

l5 4) + JO \ \ 3 \ Φ U -H m 4J Dl - \ - o -—- in —- -> tistd-P 3 mtd (dmtdcNtdord MS \ ^ 3 rH 3 in 3 rH 3in 3 Φ _ CD td O <I 'rH' - '| - |' - rH '-' rH 3 ro

44 H rH - - Q O O O O 0 H C

h 3 '—ro in'— tn <tn <tni <tnro5i pH h ^ Ή Q) QH <rH * £ VO <VO e VO C VO 3 VO * —I 3 fi 4-> ΚΟΚΟ ΚΟΟΟΟΟΟΟ'Λ-Η ( S 0) OitOCUrHlirHOrHOrHOrH ^ ro 75231 21 n «sr τ I ITI O Γ" o vo M oi — im oo o

C 2 ro IT>! —I H

JD rH n (N ΓΟ I — 1

'O o m l "ro M

• h tn +1 +1 +1 +1 Φ I +1 +1 +1 +1 .V En VO T 00 'S ·

f0 I ro O I — I CO VO Γ ~ ^ ΐ · rH

• n l vo m tn mainio 3 in i — l o vo r * H E-ι m σ \ h O o m m co m r- 'ω O ή eri id m § ί -m 0)

2 m Γ "oo n o (H

-P r — i o oo r ~ o m • HO -P m m oo cm 0: ra 2 'a *

H C rH in O Γ '· νΓ T

-P W Q rH

M: td tn -H +1 +1 +1 +1 +1 -H

p rH I „+1 H eh com m m m oo ip c l m τ τ τ m r- · m in Φ + oo τ m γ» co t rn nJ -h m m

i — I C E "* ID O 'i — I

i — I Φ rH i — I rH

Φ Φ ö M rH

1 s' 3 t! 1 at 0: <ΰ

-P rH rH

O) rH rH

(0 (0 ··> - rtJ U r-l nJ -n H m i — i oo h rH MO o tn vo _ m r "vo

M rH C tl · · T rH 00 OmiOrH

Φ Φ „C. n- vo in" vd O tn -h 2 rH C om Λί -hc \ Φ mmmm ri> · η m 3 s | i. «-H +1 +1 +1 8 + 1 + 1 + i + i 2 C-h1 h · mo r- «H vd m τ to o +> in rH ao <tmm n- mr ~ M rHc Eh τ η- m rH r ~ nm -¾ to * OO ιΗίηττ mt ~~ c/o

^ g ^ Tm m t vd rH

ta -h m O

S -H 00 VD rH

• r ~ irH m m vo oo t τ 2 vd vo M t ^ in • h ai r ~ m t

0 -P mm rH TT rH

tn tn ι-n m mt ^ -rHrH

^> + l + l +1 + i +1 + | + | + i CC 2 r'- vo o vo moorH · ^ · Φ Φ · η h1 oo oo mm τ vd • H c 0 inmmrH vd m in c * H tn Q> j vo rH τ mm vo -P-HH omm τ vo m

r -H rH

1 *

O φ I -H II

S a s u s -s a jj 5B -g. ϋ g £ a jh jo i 4J '3 φ V "-P φ« 9 3 # 1 9 3 5

H i S O -S '-H S o S -H

o-h u + »ji + iM y + j λφμ s iä ^ tn § ^ g tn tn ^ 22 s 75231 Ό rH (Τι 00 <N ιΗ

-MIC— t— Γ- (VI

swim

B

(OF

O

"ro> # vo m m c m m r- r" m o o in t "oo io tr gi -h O e o oo cn o oo

«-Rl rH rH

[d +1 +1 +1 + i +1 +1 SE CT \ 00 ro rH (n in

> 1 (Tl 00 CTi rH (M CO

4-> r-l r- ~ CO Γ 'VO ro ® <Ti m oo σι o CO 00 (N I — I I — I 00 r ~ * r-l ϋ H c

B

o y £ i jj -¾ 20 M roro i — I (N m

(O Ui Q I f «lO 00 | rH

H .8 $ .S "β <00 VO 00 VO (N (Tl

CN rH 00 CO 00 CO

e -h σν r ~ r ~ (ΝΓ'-οο O -H r — I (N rl <f in: g O ^ + l + l + l + l + l + t: m "EO 'm ro i tj > oo • P C & r ^ voo oo mm

-P -P CO O O Γ ~~ (Tl rH

• h a> i .H OK oo rH σι -vr <ti r-

• (0 M ro Γ '(N CM rl ID

i> s

• n I

3 -rl

rH O

0 -H I

Ui -P -P P Q I

^ S ä <IS !, + 1: (0 in - * - »e -H '-' <<<

m tn -P 5 -P c O

§ * £ „1 ° II! * Ί § S S

Sv S 8 S3 i3l | - ° 0n +, 'm öä äS * 8 «S8? £ E S s S8 £ g bb fiiSSB * ä 23

Table IV 75231

Peripheral T cell concentrations in different disease states T cell concentrations__ 5 Disease status 0KT3 + 0KT4 + 0KT5 +. .... ............. ^^

Primary hepatic N + cirrhosis (2)

Multiple sclerosis - N - (advanced) (8) 10 Myasthemia Gravis (not treated early) (3)

Acute transplantation (3) 0 or - - 0

agammaglobulinemia

Type I + 15 Type ii o

Hyper-IgE (4) - N O or -

Acute infectious mono- + 0 or - ++ nucleosis (4) x)

Hodgkins disease

20 Steps I & II N N N

Steps III & IV - N N

N = normal concentration 0 = completely absent 25 + = significantly higher than normal ++ = significantly higher than normal - = lower than normal - = significantly lower than normal 30 x) these concentrations return to normal approximately one week before the clinical symptoms disappear "Values in brackets indicate the number ".

Claims (2)

24 Claims 7 5 2 31 A diagnostic method for detecting a deficiency or excess of attenuating T cells in a human, comprising reacting a T cell sample from a patient with a diagnostically effective amount of an IgG class monoclonal antibody produced by a hybrid cell line produced by the ATCC. accession number is CRL 8013 or 8016, prepared by combining cells of 10 mouse myeloma cell lines with spleen cells of a mouse immunized with human thymocytes, and which antibody a) reacts with more than 90% of cytotoxic and helper-TH2 + T cells (consisting of 20 -30% of all normal human peripheral T cells) but does not react with normal human peripheral B cells, null cells or macrophages, b) reacts with about 80% of normal human thymocytes, c) does not react with -T- cells and not 0KT4 + T cells, but reacts with 68% of OKT4 T cells, d) defines a T cell population (0KT5 +) that is highly cytotoxic and yy lower than normal concentrations in primary cirrhosis, multiple sclerosis and hyper-IgE, normal concentrations in all stages of Hodgkins disease and higher than normal concentrations in type I agam-maglobulinaemia and acute mononucleosis infection, from the cell population reacts with the antibody. 2. A diagnostic method for detecting an acute mononucleosis infection in a human, comprising reacting a T cell sample from a patient with a diagnostically effective amount of at least one monoclonal antibody, OKT3, OKT4 or OKT5, and determining the proportion of the total peripheral The T cell population reacts with the antibody.