FI75435C - DIAGNOSTIC FOERFARANDE, VID VILKET MAN UTNYTTJAR EN NY MONOCLONAL ANTIKROPP SPECIFIK MOT MAENNISKANS T-CELLERS ANTIGEN OCH I FOERFARANDET ANVAENDBAR COMPOSITION. - Google Patents

Description

75435

Diagnostic method utilizing a novel monoclonal antibody specific for a human T cell antigen and a composition of matter useful in the method 5

Dividing separated from application 810026

The invention relates to a diagnostic method for detecting a deficiency or excess of 0KT11 + cells in humans and to a composition of matter useful in the method. The method utilizes a monoclonal antibody to a surface antigen found in virtually all normal human peripheral T cells and about 95% of normal human thymocytes.

The antibody is produced using a new hybrid cell line.

In 1975, Kohler and Milstein combined mouse myeloma cells with spleen cells from 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 homogenous (so-called "monoclonal") antibody. Since then, many attempts have been made to prepare various hybrid cells (so-called hybrid domains) and to try to apply the antibody produced by these hybridomas to scientific use. See, e.g., Current Topics in 25 Microbiology and Immunology, vol. 81 - "Lymphosyte Hybri-domas", edited by F. Melchers, M. Potter and N. Warner, Springer-Verlag 1978 and references cited therein; C.J. Barnstable et al., Cell 14 (May 1978) 9-20; P. Parham and W.F. Bodmer, Nature 276 (1978) 397-399; 30 Handbook of Experimental Immunology, 3rd Edition, Vol. 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 from us. Although the manufacturing method is in principle well known, there are many difficulties with 2,754,35 and each case is different. It cannot be guaranteed in advance that an attempt will be made to try to produce the desired hybrid land and that, if successful, the hybrid Doma 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 10 rare cases. See, e.g., Current Topics in Microbiology and Immunology, vol. 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 hybrid-15 Domas 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.

Recently, publications have been described depicting the preparation and testing of hybridomas that produce an antibody specific for certain T cell antigens. See, e.g., Reinherz, E.L., et al., J. Immunol. 123, 1312-1317 (1979); Reinherz, E.L., et al., Proc. Natl. Acad. Sci., 76, 4061-4065 (1979); and Kung, P.C., et al., Science, 25206, 347-349 (1979).

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 Ka-30 tea ear are termed "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 75435 (e.g., transplant rejection). 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 systems. The mechanism of this form of cell interaction is not yet fully understood.

The second group of lymphocytes (10 cells from the bone marrow, or B cells) includes those that secrete 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 Fabri-15 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-20 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 Experimen-25 or Medicine 145 (1977) 221-232 and L. Chess and S.F. Schloss-man - "Functional Analysis of Distinct Human T-cell Subsets Bearing Unique Differentiation Antigens", in "Contemporary Topics in Immunobiology", edited by O. Stutman, Plenum Press 1977, vol. 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 these two groups of lymphocytes 4,754,355. 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", 5 edited by S. Thierfelder 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, malignant disease states, and agammaglobulinemia-10 sa, T cell subsets have become unbalanced.

It has been suggested that in autoimmune diseases, "helper" T cells are generally present in excess and certain "hi-field" T cells are deficient, whereas agammaglobulemia is associated with an excess of "15" or "attenuator" T cells. helper "-T cell deficiency. In malignant disease states, "attenuator" T cells are usually present in excess. In some forms of leukemia, stagnant T cells are formed in excess. Diagnostic success may thus depend on whether this imbalance or excess can be detected or determined at which stage of development occurs in excess. See, e.g., J. Kersey et al., "Surface Markers Define Human Lymphoid Malignancies with Differing Prognoses," in Hematology and Blood Transfusion, vol. 20, references in Springer-Verlag 1977, 17-23 and Jul-25, and EL Reinberg et al., J. Clin. Invest. 64 (1979) 392-397.

Agammaglobulinemia, a disease state in which no immunoglobulin is formed, belongs to at least two different types. In type I, immunoglobulin is not formed due to the excess of attenuator-30 T cells, whereas in type II this is due to a lack of helper T cells. In both types, the patient's B cells, or lymphocytes, which are responsible for the actual secretion of the antibody, are completely normal and deficient, whereas the function of these B cells is either impaired or "not helped", which significantly reduces immunoglobulin production or suspend 5,754,35 production altogether. The type of agammaglobulinemia can thus be determined by the presence of an excess of attenuator T cells or a deficiency of helper T cells.

There are indications that when administering to patients with auto-immune disease or malignancies antibodies that are specific for the overexpressing T cell species, the antibodies may have a beneficial effect on the development of the disease. For example, helper T cell cancer (certain skin T cell lymphoma tumors and certain acute forms of T cell lymphoblastic leukemia) can be treated by administering to the patient an antibody specific for the helper T cell antigen. Autoimmune disease caused by an excess of helper cells can also be treated in the same manner. Such diseases lurk. malignant disease state-15 and or type I agammaglobulinemia) caused by an excess of attenuating T cells can be treated by administering to the patient an antibody specific for the attenuating T cell antigen.

Antisera that affect all human T-so-20 bones (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 T Cell Monitoring," Surgery 40 (1976) 155-163, and references 30-30, and Wechter et al., “Manufacture of Antithymocyte

Globulin for Clinical Trials ", Transplantation 28 (4) (1979) 303-307.

To date, spontaneous autoantibodies or antisera selective for human T cells, prepared by immunizing animals with human 6 75435 T cells, removing blood to obtain serum, and adsorbing the antiserum to autologous autologous methods, for example, have been used to identify and attenuate human T cell species and subspecies. . with non-allogeneic B cells to remove antibodies with unwanted reactivity. The preparation of these antisera 5 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 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 the 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 **.

See, for example, Chess and Schlossman's article cited above (page 365 onwards) and the article cited in The Chemical and Engineering News, which describe the shortcomings of antisera used to date and the advantages of the monoclonal antibody.

A new hybridoma (OKT 11) has now been discovered which is capable of producing a novel monoclonal antibody to a surface antigen found in virtually all normal human peripheral T cells and about 95% of normal human thymocytes, but which is not present. found in normal human B cells and not in null 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 antisera known to date (which naturally contain antibodies as impurities, which react well with many human antigens), and hitherto known monoclonal antibodies (which are not monospecific for a single human thymocyte antigen). In addition, this hybridoma can be cultured to produce antibody, so that the use of animals to produce antibody is not necessary, nor are the complex adsorption and purification steps required in previous methods for preparing crude antisera.

10 Both the present hybridoma and the antibody produced by it are referred to herein as "OKT11", the context being whichever is meant. The present hybridoma was deposited on December 13, 1979 with the American Type Culture Collection, 12301 15 Parklawn Drive, Rockville, Maryland 20852, and has been assigned ATCC Accession No. CRL 8027.

The preparation of the hybridoma and the antibody produced by it is described in FI patent application 810026.

The diagnostic method of the present invention using the above hybridoma and antibody is characterized by reacting a lymphocyte sample from a patient with a diagnostically effective amount of a mouse monoclonal antibody produced by a hybrid cell line having an ATCC accession number of 25. 8027 and which antibody reacts with virtually all normal human peripheral T cells and about 95% of normal human thymocytes but does not react with normal human B cells or null cells, and is determined against said antibody. the percentage of the population that reacted with.

Example 1 Characterization of OKT11 antibody reactivity A. Isolation of lymphocyte strains

Peripheral mononuclear blood cells were isolated from the blood of healthy volunteer blood donors (ages 15-35 to 40 years) by Ficoll-Hypaque density gradient centrifugation (Pharmacia Fine Chemicals, Piscataway, NH), as described by Boyum, Scand. J. Clin. Lab. Invest.

21 (suppl. 97) (1968) 77. Unfractionated mononuclear cells were separated into surface Ig (B) and surface Ig ~ (T and nol-5 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). 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-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 (zero-lu) population was separated from the Ficoll interface. This latter population was <5% E-positive and <2-% slg-positive. The surface Ig + (B) population was separated from the Sephadex-G-200 column by elution with normal 20 human gamma globulin as previously described. This population was> 95% surface Ig-positive and <5% E-positive.

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 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 Ficoll-Hypaque centrifuge and washed as described in A. The thymocytes thus obtained were> 95% viable and> 90% E-rosette positive.

9 75435

Example 2

Cell fluorographic analysis and cell separation

Cell fluorographic analysis of monoclonal antibodies and reaction products of all cell populations was performed by an indirect immunofluorescence method in which cells were stained with fluorescein-conjugated goat anti-mouse IgG (G / M FITC) (Meloy Laboratories).

g (Ortho Instruments). At this time, 1 x 10 cells were treated with 0.15 ml of antibody at a 1: 500 dilution of OKT5, 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 15 cells were then analyzed by cell fluorography, in which the fluorescence intensity induced by each cell was recorded on a pulse height analyzer. The reactivity pattern was similar at a dilution of 1: 10,000, but no reactions occurred at higher dilutions. 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-producing hybrid clone.

Figure 1 shows the fluorescence resistance spectrum obtained from a cell fluorograph for normal human thymocytes and peripheral E + and E cells reacted with OKT11 and other monoclonal antibodies (1: 500 dilution) and G / M FITC. Background fluorescence staining was determined by adding to each population a 1: 500 dilution of aqueous humor from a mouse injected with a non-producing clone and incubating the mixture.

Table 1 shows the reactivity of the monoclonal antibodies OKT1, OKT3-6 and OKT8-11 with different human lymphoid cell populations. Among other things, this reactivity-35 formula allows the present antibody OKT11 to be detected and distinguished from other antibodies.

10 75435

Figure 1 shows the fluorescence spectrum obtained from a cell fluorograph for normal human thymocyte suspensions and peripheral E + and E cells reacted with antibodies 0KT11, OKTIO and 0KT8 (dilution 1: 500) and G / M FITC. . In contrast to OKT1 and OKT3 T cell antigens (which increase in number as thymocytes mature into peripheral T cells), the amount of 0KT11 antigen decreases concomitantly. Among other things, the reactivity formula of Figure 1 allows the present antibody OKT11 to be detected and distinguished from other antibodies.

Table 2 shows the antigen phenotypes of human T lymphocytes using OKT11 antibody and other monoclonal antibodies. Also with this phenotype pattern 15, the antibody OKT11 can be detected and distinguished from other antibodies.

Table 3 shows the relationship between peripheral T cell and T cell fraction concentrations and different disease states. These ratios can be used for diagnostic purposes (e.g., to detect acute infectious mononucleosis) by taking a blood sample from a patient suspected of having one of these diseases and determining T cell and T cell fraction levels from the sample. Also, using the ratios shown in Table 3, the antibody OKT11 25 can be detected and distinguished from other antibodies.

Diagnostic methods using other monoclonal antibody-producing hybridomas (OKT1, OKT3, OKT4 and OKT5 and OKT6, OKT8, OKT9 and OKTIO) produced by the same applicants are described in FI patent applications 30 84 4703, 84 4705, 84 4704 and 84 4706 and 84 4707, 85 0062, 85 0063 and 85 0061.

The OKT11 of this antibody 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 those of so-called from their "fixed" regions, although the structure of an antibody specific for a particular antigen has a so-called

11,75435 "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 subclasses IgG1 to IgG2a, IgG2b or IgG1, or to 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, for example, complement or anti-mouse antibodies.

The present antibody can be used to diagnose various disease states, as shown in Table 3. In this case, the OKT11 antibody alone or a combination of OKT11 and other antibodies can be used (e.g.

15 OCT3 - OCTOBER). Reactivity formulas obtained by reacting a variety of antibodies with T cells and T cell fractions allow certain disease states to be determined more accurately than using known diagnostic methods.

Diagnostic compositions consisting of effective amounts of OKT11 antibody and diagnostically acceptable carriers are also within the scope of the present invention.

12,754 35

Table 1 Distribution of OKT Antigens in Cells and Tissues Monoclonal Antibody Ig Type Peripheral Spleen Bone Marrow Laying Blood (15) x (4) (6) (15) 5 ________ E ~ 0KT1 IgGi 100 ** o 29 + 9 <5 18 +8 OKT3 IgG2a 100 0 31 + 8 <5 21 + 8 10 “O KT 4 IgG2b 64 ± 4 0 16 + 3 <5 75 + 10 OKT5 IgGi 25 + 4 0 15 + 4 <5 77 + 8 OKT6 IgGi 0 0 0 <5 68 + 8 15 OKT8 IgG2a 34 ± 3 0 17 + 4 <5 81 + 7 OKT9 IgGi 0 0 0 <5 4 + 3 OKTIO IgGi 5 + 3 13 + 4 10 + 5 16 + 4 96 + 4 OKTll ++ IgGi loo 0 NT + NT 95 + 3 20 x number of samples examined - IS.D.

+ not determined ++ five samples of peripheral blood and five samples of thymus were examined.

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Claims (2)

15 75435 A diagnostic method for detecting a deficiency or excess of OKT11 + cells in humans, characterized in that a lymphocyte sample taken from a patient is reacted with a diagnostically effective amount of a mouse monoclonal antibody produced by a hybrid disin cell line having ATCC accession number CRL 8027 and the antibody reacts with virtually all 10 normal human peripheral T cells and about 95% of normal human thymocytes, but does not react with normal human B cells or null cells, and determining the percentage of the population that reacts with said antibody. 2. A diagnostic composition for detecting an excess or deficiency of OKT11 + cells, characterized in that it consists of a diagnostically acceptable carrier and an amount of murine monoclonal antibody produced by a hybrid cell line produced by the ATCC to detect an excess or deficiency of 0KT11 + cells. deposit number is CRL 8027 and which antibody reacts with virtually all normal human peripheral T cells and about 95% of normal human thymocytes, but does not react with normal human B cells or null cells.