FI75229C - DIAGNOSTIC FOERFARANDE, VID VILKET MAN ANVAENDER EN NY MONOCLONAL ANTIKROPP SPECIFIK MOT MAENNISKANS T-CELLER. - Google Patents

Description

1 75229

Diagnostic method using a novel monoclonal antibody specific for human T cells

By division separated from the application 800 846 5

The present invention relates to a diagnostic method for distinguishing patients with acute T-cell lymphoblastic leukemia (T-ALL) from patients with chronic T-cell lymphoblastic leukemia (T-CLL) and using a monoclonal cell found on the surface of all normal human T cells. an antibody specific for the antigen. The antibody is made using a new hybrid cell line.

In 1975, Kohler and Milstein combined mouse mye-15 animal cells with spleen cells from immunized mice (Nature 256 (1975) 495-497), when 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 hydride 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 Hybridomas", edited by F. Melchers, M. Potter and N. Warner, Springer-Verlag 1978 and references cited in 25; 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, paragraph 25; and Chemical and Engineering News, January 30, 1 (1979) 15-17.

These references show the results obtained and the difficulties encountered in trying to prepare a monoclonal antibody from hybridomas. Although the manufacturing method is in principle well known, there are many difficulties and each case is different. It cannot be guaranteed in advance that an attempt to produce the desired hybridoma will succeed in 75229 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 to produce a monoclonal antibody specific for human lymphocyte surface antigens have been made in only a few cases. See, e.g., Current 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 lymphocytic leukemia cell lines. Many of the Hydromats thus prepared produced an antibody that acted on various surface antigens of all human cells. None of these 15 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 cell derived from the thymus, or T cell) differs in the thymus from hematopoietic stem cells. The differentiating cells in the cat's ear 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 responses (e.g., transplant rejection). Although T cells do not secrete the antibody into body fluids, another group of lymphocytes that will be handled later will, in some cases, require a T cell and be able to secrete the antibodies themselves. Some T-cell species act in a regulatory manner in other functions of the immune system. The mechanism of this form of cell interaction is not yet fully understood.

35 The second group of lymphocytes (cells derived from the bone marrow, or B cells) includes those that secrete 3,752,29 antibodies. They also develop from hematopoietic stem cells, but their differentiation is not regulated by the thymus. In birds, they differentiate in an organ analogous to the thymus, called the "Bursa of Fabricius." However, no similar organ has been found in suckling-5 cells, 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 either accelerate or slow the reaction or kill (dissolve) foreign cells. These subclasses are well known in the body of mice, 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. Schloss-15 man - "Functional Analysis of Distinct Human T-cell Subsets Bearing Unique Dirrefentiation Antigens", in "Contemporary Topice 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 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 that the two groups of lymphocytes must be able to be distinguished from each other. 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 Leukemia and Lymphomas," edited by S.

30 Thiefelder et al., Springer, Heidelberg 1977, 33-45; and D. Belpomme et al., British Journal of Hematology 38 (1978) 85. In certain disease states (e.g., juvenile rheumatoid arthritis and certain forms of leukemia), T cell subsets have become unbalanced. It has been suggested that in autoimmune diseases, "helper" T cells are generally present in excess and certain "attenuator" T cells are deficient, 4 75229, while in malignant disease states, "weaker" T cells are generally present in excess. In certain forms of reading chemistry, an excess of stagnant T cells is formed. The success of the diagnosis can thus depend on whether this imbalance or excess can be detected. See, e.g., 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 in si-10. .

There are indications that the administration of antibodies specific for an excess of a subset of T cells to patients with autoimmune or malignant diseases has a beneficial effect on disease progression. 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 (transplant rejection mechanism), obtaining an antibody specific for T cells prevents or slows this rejection process. See, e.g., Cosimi et al., "Randomized Clinical Trial of ATG in Cadaver Renal Allgraft Recipients:" Importance of 25 T Cell Monitoring "Surgery 40 (1976) 155-163, and references therein.

To date, spontaneous autoantibodies or human T cell-selective anti-30 sera prepared by immunizing animals with human T cells, removing blood from animals to obtain serum, and adsorbing antiserum, for example, to autologous cells, have been used to identify and attenuate human T cell species and subspecies. 1a with non-allogeneic B cells to remove antibodies with undesired reactivity. The preparation of these antisera 35 is very difficult, especially in the adsorption and purification steps. In addition to the desired antibody, the adsorbed and purified antisera 75229 also contain many impurities for several reasons. One reason is that the serum contains millions of antibody molecules even before immunization with T cells. Second, immunization results in the formation of antibodies that affect a variety of 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 10 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 1/106.

See, for example, the above-mentioned article by Chess and Schlossman (from page 365 onwards) and the above-mentioned 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 capable of producing a novel monoclonal antibody specific for virtually all surface antigen found in normal human peripheral T cells has now been discovered. 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 antisera known hitherto (which naturally contain as impurities antibodies which react). -30 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 to date for the preparation of even impure antisera.

A sample of the present hybridoma has been deposited with the ATCC Depository, 12301 Parklawn Drive, Rockville, 5 MD, 20852, on March 13, 1979, under ATCC Deposit No. CRL 8000.

The preparation of a hybridoma and the antibody produced by it is described in FI patent application 800 846.

The diagnostic method according to the invention, wherein the above-mentioned hybridoma and the antibody are used, is characterized by mixing the leukemia cells of a patient with either disease with a monoclonal antibody produced by a hybridoma having an accession number of ATCC CRL 8000 and monitoring whether the reaction occurs. the monoclonal antibody used is characterized in that it a) reacts with virtually all normal human peripheral T cells but does not react with normal human peripheral B cells, null cells 20 and macrophages, b) reacts 5-10% normal human thymocytes, c) reacts with human leukemia cells with chronic T-cell lymphoblastic leukemia, but does not react with human leukemia cells with acute T-cell lymphoblastic leukemia, d) reacts with human T-cell as shown in Figure 4. , CEM and HSB-2, (e) does not react with Epstein-Barr - with human B cell lines transformed with the virus.

Example 1 Characterization of OKT1 antibody reactivity A. Isolation of lymphocyte populations

Peripheral mononuclear blood cells from normal volunteer blood donors (ages 15-35 to 40 years) were separated by Ficoll-Hypaque density gradient centrifugation 75229 (Pharmacia Fine Chemicals, Piscataway, NH).

As proposed by Boyum, Scand. J. Glin. Lab. Invest.

21 (suppl. 97) (1968) 77. Unfractionated mononuclear cells were separated into surface Ig + (B) and surface Ig ”(T- and zero-5 cell) populations using Sepahdex G-200 anti-F (ab ') 2 column chromatography by the method previously described by Chess et al., J. Immunol. 113 (1974) 1113. T cells were harvested by E-rosetting the Ig ”population with 5% sheep erythrocytes (Microbiological Associates, 10 Bethesda, MD). The rosetted mixture was centrifuged in a Ficoll

In a Hypaque centrifuge, 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 Ig ”(nol laser cell) population was separated from the Ficoll interface. This latter population was> 5% E-positive and 12% slg-positive. The surface Ig + (B) population was separated from the Sephadex-G-200 column by elution with human gamma-20 globulin as previously described. This population was> 95% surface Ig-positive and <5% E-positive.

Normal human macrophages were separated from the mononuclear population by attachment to polystyrene. At this time, 25 mononuclear cells were resuspended in final culture medium (RPMI 1640, 2.5 mM HEPES / 4- (2-hydroxyethyl) -1-piperazine-propanesulfonic acid7 buffer, 200 mM L-glutamine and penicillin-streptomycin, and 20% by heating inactivated AB serum) at 2 x 10® cells and incubated in plastic petri dishes (100 x 200 mm) (Falcon Tissue Culture Dish, Falcon, Exnard, CA) at 37 ° C overnight. The Petrimal plates were washed thoroughly to remove non-adherent cells, and the plastic-bound population was removed by washing vigorously with a cold solution containing 2.5 mM EDTA without serum, and occasionally 8,752,229 by scraping the Petri dish with the rubber tip of a disposable syringe. More than 85% of the cell population was able to feed latex particles, and Wrigt-Giemsa staining showed morphological characteristics of monocytes.

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 Ka-10 thymus was dissected, and the pieces were immediately placed in 199 (Gibco) medium with 5% fetal calf serum, finely chopped with forceps and scissors, and then a suspension of 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 i90% E-rosette positive.

C. Cell Lines 20 Epstein-Barr virus (EBV) transformed B cell lines from four healthy individuals were prepared as previously described. T cell lines CEM, HSB-2, and HJD-1 were provided by Dr. H. Lazarus, Sideny Farber Cancer Institute, Boston, MA.

25 D. T-cell acute lymphoblastic leukemia (T-ALL) cells and T-cell chronic lymphocytic leukemia (T-CLL) cells

Leukemia cells were obtained from twelve patients with acute lymphoblastic leukemia. These individual cells had been identified as belonging to T cells in previous experiments because the cells spontaneously formed rosettes with sheep erythrocytes (> 20% E +) and because they responded to T cell-specific heteroantisera, anti-HTL (anti-BK) and With A99 antiserum, such as 35 Schlossman et al. have previously described, Proc. Nat.

Acad. Sci. 73 (1976) 1288. Tumor cells from three individuals reacted actively with (TH2 +) rabbit and / or equine anti-TH2, but cells from nine-month individuals did not. Leukemia cells obtained from two 5 patients with TH2-T-CLL leukemia were also used. 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% human AB serum until they were characterized on their surface. The tumor populations analyzed were 10> 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-bound goat anti-mouse IgG / G / M FITC (Meloy Laboratories), using cell fluorography FC200 / 4800A Instruments. In this case, 1-2 x 10 6 cells 20 were treated with 0.15 ml of antibody at a 1: 1000 dilution of 1: 1000, 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 (1:40 dilution) at 4 ° C for 30 minutes, the reaction product was centrifuged and washed three to 25 times. These cells were then analyzed on a cell fluorograph, in which the fluorescence intensity induced by each cell was recorded on a pulse height analyzer. The reactivity was similar for a 1: 30,000 dilution, but no reaction occurred at higher dilutions. The intensity of background 30 staining was determined with 0.15 ml of a 1: 1000 diluted aqueous humor obtained intraperitoneally from a BAlb / cJ mouse immunized with a non-producing hybrid clone.

In experiments to differentiate between 0KT1 + and 35 OKT1 cells, 100 x 10 6 unfractionated mononuclear cells or thymocytes were labeled with 4 ml of 10 75229 1: 1000 diluted antibody OKT1 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 activation 5-based cell sorter (FACS-1) (Becton-Dickinson, Mountain View, CA), lymphocytes were divided into 0KT1 + and OKT1 ”populations and / or T cells were fractionated into weakly responsive OKT1 + T cells (fluorescence less than 20%). highly reactive OKT1 + cells (fluorescence greater than 10 20%). Species viability was> 95% as determined by Trypan blue in all cases. The purity of all isolated populations was 2.95%.

Example 3

Study of Cell Function The mitogenic response of unfractionated and FACS-fractionated lymphocytes was examined in microculture at optimal doses of Concanavalin A (Con A) (Cal-Biochem, La Jolla, CA) and phytemaqglutinin (PHA) Chessin et al. by the method described. The alloantigenic prololi-20 ferritic response was determined simultaneously for these same populations using mitomycin-treated Laz 156, an EBV-transformed human B-lymphoblastodis cell line supplied by Dr. H. Lazarus, as a stimulant. Proliferation in the presence of tetanus toxoid (Massachusetts Department of Public Healt Biological Laboratories, Boston, MA) was tested according to Evans et al. by the method described (J. Immunol. 129 (1978) 1423) using a final concentration of 10 μm / ml. 5% of the macrophages obtained by the method described above were added to all polylations when starting to grow in vitro cultures. After four days, mitogen-stimulated cultures were treated with 0.2 μCi of tritiated thymidine (specific activity 1.9 Ci / mM, Schwartz-Mann Division of Becton-Dickinson, Orangeburg, NY), and the products were separated for 18 hours. 35 lutes with MASH II equipment (Microbiological Associates, Bethesda, MD). The activity of the tritiated thymidine compound U 75229 was measured with a Packard scintillation counter (Packard Instrument Company, Downer'a Grove, IL). The activity of the background tritiated thymidine compound was measured by replacing the mitogen with medium. Cultures stimulated with tetanus toxoid and alloantigen after 5 days were treated with tritiated thymidine for 18 hours, the products were separated, and activity was measured as described above.

Figure 1 shows the fluorescence spectrum obtained from a cell fluorograph for cell populations reacted with OKT1 (dilution 1: 1000) and G / M FITC.

Figure 2 shows the fluorescence spectrum obtained from a cell fluorograph for human thymocytes reacted with OKT1 and G / M FITC.

Figure 3 shows the fluorescence resistance spectrum obtained from a cell fluorograph for leukemia cells obtained from patients with both acute lymphoblastic leukemia and chronic lymphoblastic leukemia who have been reacted with OKT1 and G / M FITC.

Figure 4 shows the fluorescence resistance spectrum obtained from a cell fluorograph for human T cell lines reacted with OKT1 and G / M FITC.

Figure 5 shows the fluorescence spectrum obtained from a cell fluorograph for the B lymphoblastoid cell line Laz 007 reacted with OKT1 and G / M FITC.

As shown in Figure 1, all peripheral T blood cells of a normal human react with OKT1, but B cells, null cells, and macrophages isolated from the same individual do not react at all with OKT1. Similar results were obtained for lymphocyte populations isolated from fifteen other normal individuals. The monoclonal antibody is thus characterized in that it reacts with the antigen found on the surface of virtually all normal human peripheral T cells, but does not react with any of the surface anti-35 genes of the other three cell types mentioned. This difference in reactivity can be used to detect the OKT1 of that antibody and distinguish it from other antibodies.

75229 12

Figure 2 shows that most normal thymocytes in a six-month-old child do not react at all with OKT1, with only 5-10% of thymocytes being reactive. From this, it can be concluded that in the 5 blue differentiation process in which stem cells develop into mature T cells, at some point the thymocytes receive the same surface antigen found in T cells and react with OKT1. It is likely that tymo-cytes appear in the subsequent stages of differentiation just 10 before they are released from the thymus into the bloodstream. Similar results (5-10% activity) were obtained using six other thymus samples obtained from normal individuals ranging in age from two months to 19 years. Using the reactivity formula in Figure 2, it is also possible to detect this antibody 0KT1 and distinguish it from other antibodies.

The use of the present antibody for diagnostic purposes is illustrated in Figure 3, which shows that leukemia cells in patients with acute lymphoblastic leukemia did not react with OKT1, but leukemia cells in patients with chronic lymphoblastic leukemia did. The antibody in question thus makes it possible to distinguish between the two forms of leukemia. Because these forms of leukemia are difficult to distinguish at certain stages, and because their prognosis and methods of treatment differ substantially, it is clear that this simple method of distinguishing between these two forms of leukemia using the present antibody is a significant advance.

The present antibody 0KT1 can still be

characterize based on how it reacts with different human T cell lines, this is illustrated in Figure 4. The figure shows that the reactivity of this antigen with human T cell lines varied, the reaction with cell line 35 HJD-1 was strong, cell line CEM

with a mild and no reaction with the HSB-2 cell line. With this different reactivity, it is also possible to characterize 0KT1.

It can be seen from Figure 5 that 0KT1 does not react with the human B cell line Laz 007. 5 other EBV-transformed B cell lines studied also behaved in a similar manner. This further supports the notion that OKT1 does not react with peripheral blood B cells of a normal unit, and also in this way it is possible to characterize OKT1 and distinguish it from other antibodies.

10 The function of this antibody was examined in lymphod populations fractionated with a fluorescence-based cell sorter (FACS). The results are shown in Tables I-III, which further support the Karak-15 terization results of the present monoclonal antibody presented above.

It can be seen from Table 1 that virtually all mixed lymphocyte culture (MLC) cells that react with PHA, Con A, soluble antigens, and alloantigens belong to a cell population that reacts with OKT1. The population that did not react with 0KT1 did not induce any of these T cell functions, the minor positive reaction possibly caused the population to contain OKT1 + cells as impurities. These functional studies further illustrate that the antigen that reacts with 0KT1 is present only in T cells because the positively responsive population functions like T cells, but no T cell functions have been found in the negatively responsive population. It can be seen from Table II that in the presence of mitogen or alloantigen 30, there is no functional difference between FACS-fractionated OKT1 strongly reactive and OKT1-poorly reactive with OKT1. Both populations increased in the same manner as the unfractionated T cell population. It can be concluded from Table III that the surface antigen that reacts with 35 OKT1 is present only in fully developed thymocytes, because the activity of the entire thymocyte 75229 14 series in MLC analysis is almost entirely due to the part of the thymocyte population that reacts with OKT1. Table III also shows the functional differences between OKT1 + lymphocytes and peripheral OKT1 + T cells, since the former does not react in the presence of mitogen.

The antibody 0KT1 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 in the structure of an antibody specific for a particular antigen) there is 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 the antibody subsequently reacts with other agents, e.g., complement or anti-mouse antibodies.

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Claims (1)

18 Claim 7 5 2 2 9 Diagnostic method for distinguishing patients with acute T-cell lymphoblastic leukemia (T-ALL) 5 from patients with chronic T-cell lymphoblastic leukemia (T-CLL), characterized by mixing a patient with either disease leukemia cells to a monoclonal antibody produced by a hybridoma with accession number ATCC CRL 10 8000, and monitoring for a reaction in which the monoclonal antibody used is characterized in that it a) reacts with virtually all normal human peripheral T cells, but does not react with normal human peripheral B cells, null cells or macrophages, b) reacts with 5-10% of normal human thymocytes, c) reacts with human leukemia cells with chronic T lymphoblastic leukemia, but does not react T-cells in human leukemia cells with acute lymphoblastic leukemia d) reacts with human T cell lines HJD-1, CEM and HSB-2 as shown in Figure 4, e) does not react with Epstein-Barr virus transformed human B cell lines.