JPH03500363A - Monoclonal antibody that recognizes epitope of gamma chain of human T cell surface receptor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Monoclonal antibody that recognizes epitope of gamma chain of human T cell surface receptor Technical field This invention generally relates to monoclonal antibodies and their interactions with specific antigenic determinants. A monoclonal protein that specifically binds to cell surface receptors on human T lymphocytes. The present invention relates to hybridoma cell lines that produce synthetic antibodies. For more details, see Monoclonal antibodies embodying the invention are encoded by T cell gamma rearranged genes. It binds to a unique epitope of the human T cell surface receptor that is expressed.

Background technology In this technical field, the human immune system is involved in various organs of the lymphoreticular system as well as cells in the blood. It is well known that it consists of cells and soluble elements. Antigens that enter the body system When identified, a foreign substance called a (reaction). The reaction may be in the form of a local inflammatory or phagocytic response. or may cause specific humoral or cell-mediated reactions.

For example, it requires the production of antibodies against cells, viruses or bacteria.

The main elements in the immune response are T cells derived from the thymus and B cells derived from the bone marrow. be. T cells interact with B cells to protect against foreign substances in a manner called cell-mediated immunity. Perform antibody formation. The humoral immune response is caused by the presence of presensitized B lymphocytes together with foreign substances. All you need to do is Their interaction causes B cells to proliferate and respond to antigens. Begins to synthesize immunoglobulin or antibody proteins. Circulating peripheral blood cell components are , consisting of red blood cells, white blood cells, and platelets. White blood cells include neutrophils, eosinophils, basophils, Divided into five major populations called monocytes and lymphocytes.

Lymphocytes are further divided into thymus-derived T cells and bone marrow-derived B cells. T and B phosphorus Paracells further contain numerous sub-cells such as inducer cells, suppressor cells, and killer cells. Among others, recent research efforts have identified a number of subsets. is increasing.

Koehler and Milstein in 1975 monoclonal antibody technology, Nature, 25 6゜pp. 495-497 is a study on the functions of leukocytes and their relationship to human physiology. It gave a great stimulus to academic research. Monoclonal antibodies have different origins and Development of human T lymphocyte and B lymphocyte surface antigens or epitopes during maturation stage T and B cells are classified as leukocytes and lymphomas and T cells are graded.

Monoclonal antibodies that recognize various functionally related T or B cell surface molecular structures The effectiveness of Allow confirmation.

In addition, solidified tumor cells, erythrocyte glycoprotein surface molecules and binds to inflamed sites in human tissues infected with viruses, such as in the AIDS and AIDS diseases. Monoclonal antibodies were developed. Both normal and abnormal human immune system Valid research on cell function and impact can be tested for diagnostic and therapeutic applications. Dramatically increases the number of novel monoclonal antibodies generated both in vitro and in vivo. ing.

U.S. Patent No. 45 titled "Monoclonal Antibody Recognizing Human T Cells" Structures referred to as rTiJ'TaJ and "T," in the specification of No. 50086 Definition of surface molecules expressed on T lymphocytes and other cells, such as Attention is directed to nit1on).

A monoclonal clone called anti-T3 (ant, 1-Ts to Ta) J against rTs A functional antibody determines the ability of any functional T cell clone to recognize its target antigen. I was able to block it. Different mature T-cell clones, each T cell has its target antigen. Each protein has a unique molecular structure that allows it to recognize I was there for a while.

However, the patent states that T-cell clones differ in their so-called "recognized tissue structure." However, we teach that they appear to have certain common characteristics.

One common feature of all T cell clones is the T3 glycoprotein and the patent It consists of a dimer consisting of two different protein molecules identified in Before The monoclonal antibodies described in the patent specifically target T cell surface receptor complexes. I recognized it.

The monoclonal antibodies embodying this invention recognize subpopulations of human peripheral T cells. , the subpopulation represents an average of about 1-15% of circulating mononuclear blood cells and about 3% of these lymphocytes. shows. This subpopulation of lymphocytes is a gamma class that specifically rearranges in T cells. encoded by the genes of “second family J” Expresses T cell surface receptors. The monoclonal antibodies of this invention can be used under non-reducing conditions. Approximately 85,000 Daltons measured below and 41,000-44 measured under reducing conditions Glycoprotein molecules on the surface of human T lymphocytes with a molecular mass of 000 daltons binds to a unique epitope of

The unique epitope identified by this monoclonal antibody is unlike anything previously achieved. Human T-phosphorus with much improved definity This allows for the confirmation of subpopulations of Pabules.

The monoclonal antibodies of this invention are human lymphocytes with T cell receptor gamma complexes. Identifying a new subset of Pa spheres. This monoclonal antibody is “anti-Ti gamma A. It is called (anti-TigammaA) J. This monoclonal antibody is I Belongs to the gGz subclass. This monoclonal antibody can be used in healthy, diseased or organ transplant patients. Human T cells in the circulating blood taken from individuals who have undergone explant or bone marrow transplantation. Use appropriate markers such as radiolabels or fluorescent markers to identify and define subpopulations. It can be marked with a car. The antibody inhibits the differentiation of human T lymphocytes in the circulating blood. It is also useful for checking the status. Furthermore, the antibody has a Ti gamma A epitope. In vitro, in vitro, or in vivo final handling of human T lymphocytes with It can be applied to

Disclosure of invention Human T lymphocytes in peripheral blood encoded by T cell gamma rearrangement genes monoclonal antibodies that specifically bind to unique epitopes on T cell surface receptors. Producing hybridoma cell line. The epitope was measured under non-reducing conditions. Approximately 85,000 Daltons and 41,000-44,000 Daltons measured under reducing conditions A glycoprotein on the surface of human T lymphocytes in the circulating blood with a molecular mass of located within the structure. The monoclonal antibody of this invention is called anti-Ti gamma A, It belongs to the 1gG2 subclass. This monoclonal antibody targets the Ti gamma A epitope. Enables identification of circulating human T lymphocytes expressing antigenic determinants. do.

BEST MODE FOR CARRYING OUT THE INVENTION The monoclonal antibody anti-Ti gamma A is an antigenic determinant of the human T lymphocyte receptor. , and specifically the Ti complex of the TsTi structure of the human T cell surface receptor. Applicable The Ti molecular structure has a gamma chain, and the anti-Ti gamma A monoclonal antibody has a gamma chain. Therefore, this invention describes the “second family” of T cell surface receptors. ” enables the detection, quantification and confirmation of Ti complexes. This epitope is found in human blood T-lymph in fluid and in human lymphoid organs such as thymus, spleen, ganglia and bone marrow Appears in subpopulations of spheres. The diagnostic and therapeutic uses of this monoclonal antibody The potential benefits are wide-ranging.

The anti-Ti gamma A monoclonal antibody of this invention was developed as described below. .

CD3′″, cell suspension of human lymphocyte clones of WT3F phenotype (Norwi Nowi et al., “Natural Clones Derived from Fetal (25 Weeks) Blood” J, J, Exp, Med, 63:16011986) was prepared, but the Cells do not express Tα/β receptors on their surface.

These cells express a functional activity called "natural cytotoxic activity."

The suspension is injected into Biozzi mice.

Immunization with two initial intraperitoneal injections of 4X106 cells in Freund's adjuvant. Subsequently (performed by intravenous injection of 4X10' cells without adjuvant. After 3 days , kill the animal and remove the spleen to obtain spleen cells.

Crossbreeding was achieved by fusing the obtained spleen cells with the murine N5-1 myeloma cell line. I do. The cell fusion is described by Moingeon P et al. It was carried out according to the known method described in Nature, Vol. 323, p. 638, 1986. fusion The resulting hybrid cells were treated with hyboxanthin, aminopterin, thymidine, and 10% horseradish. The cells are cultured in HAT medium containing fetal calf serum and 10% horse serum.

Hybridomas are then selected. Screening involves leukemia kept in culture Hybridoma supernatant produced by clones immunizing against diseased cell lines This is done by testing the ability of cells to block cytotoxic responses.

Hybridomas producing monoclonal antibodies are cloned using limiting dilution techniques. be done. Finally, an immune ascites is created in which antibodies can be taken. child The anti-Ti gamma A antibody developed as described above belongs to the IgGz subclass and is a complement Can bind to proteins.

Monoclonal antibody reactivity and its range of action were determined by human lymphocytosis. . The subpopulation of cells tested was derived from peripheral blood from 30 healthy adult blood donors. isolated from the mononuclear portion of the fluid.

This part was purified by Ficoll gradient centrifugation technique. 3.0XIG' cell sample Coulter-Corporay, Hialeah, Florida, USA. Cell fluorescence measurement devices such as the rEPiC3CJ flow cell measurement device manufactured by will be analyzed. Control reagents are FITC-TgG and PE-rgG.

Other known antibodies target non-adherent lymphocytes with a fluorescent red marker, algal pigment (PE). The present invention comprises a fluorescent green marker, i.e. fluorescein, in the presence of Treat with monoclonal antibody, anti-Ti gamma A for 30 minutes. Other known antibodies are: It is: anti-CD2. -CD3. -CD4. -CD5. -CD8.　 NK) II and classes of major complexes of histocompatibility ■ By genes called genes. Those antibodies determine which molecules are encoded. These known antibodies are France of Margency Courtesy Supplied by Coutronics.

The results obtained with the antibodies according to this invention are Allow to check for T lymphocytes.

The majority of Tj gamma A''' lymphocytes express CD2 and CD3 glycoproteins. Ru. The majority of Ti gamma A1 lymphocytes express the CD5 protein. in this case , where these CD5 proteins are present, the density of CD5 antigen per cell normal cells that express Tα/β receptors in the Ti gamma A+ cell subset. A lower in the lymphocyte population is observed. Ti gamma A+ subpopulation CD80 expression varies widely from one individual to another. most pieces In the body, only a small number of Ti gamma A1 lymphocytes have a weak antigen density. Express proteins.

At rest, Ti gamma A1 lymphocytes contain CD4 protein, NKHI and tissue The main complex class of conjugates does not express molecules encoded by genes.

Determination of the reactivity of anti-Ti gamma A monoclonal antibodies performed by fluorescent antibody technology Other comparative analyzes included available T, specific monoclonal antibodies and BMAO31 monoclonal antibodies. Using a loaner body (supplied by Behring Co.) cells expressing CD3 protein on the one hand and Tα/β receptors on the other hand. This made it possible to detect cells expressing . These specific antibodies are It was chosen because it prevents optimal signal in indirect fluorescent antibody analysis.

The results show that Ti gamma A' subpopulations express CD3 protein or on their surface. show that a large number of circulating lymphocytes do not express normal Tα/β receptors. .

On the other hand, lymphocytes expressing antigenic determinants corresponding to anti-Ti gamma A monoclonal antibodies Some functional effects induced by the interaction between the sphere and the sphere were investigated.

In the presence of exogenous interleukin-2 in an amount of approximately 50υ/JNI) The interaction of antigenic antibodies with antigenic determinants leads to the activation and proliferation of uncultured circulating lymphocytes. wake up

Ti gamma A" lymphocytes are treated with a drug such as phytoclinogen at a dose of about 2 μ/ml. Cephalometric cells preactivated with cytogens, cultured and activated with cyanogen bromide. When stimulated with anti-Ti gamma A monoclonal antibody in combination with base beads, Proliferation of 1923 spheres is clearly demonstrated 48 hours after stimulation. These 1923 balls are Furthermore, they can secrete their own interleukin-2.

The cytotoxic activity of lymphocytes was observed more precisely by other experiments.

Cytotoxicity is measured during the 3-hour run after labeling of target cells with s'Cr.

Ti gamma 8'' cells measured with the monoclonal antibody of this invention In some cases, it appears as a highly homogeneous population of granular lymphocytes. But they are e.g. For example, strain 562 derived from pleural effusions of chronic myeloid leukemia [Natural mediate little spontaneous cytotoxic activity against the usual targets of the so-called "cytotoxic system". exist or not intervene.

Therefore, this subpopulation does not significantly contribute to the natural cytotoxic activity of peripheral blood. This is also due to the absence of surface expression of the NKHI label, as explained earlier. and match.

On the contrary, Ti gamma A+ lymphocytes contain mitogens and exogenous interleukin 2. When activated by a large number of tumor cells, especially those of the 562 lineage It is extremely cytotoxic to these cells. This cytotoxicity is extremely important for 3-5B. It can be induced within a short time. Then, the thus activated Ti gun MaA1 lymphocytes are known for their ability to activate killer functions (Lymph talent in activation killer function) It is involved in a phenomenon called "elephant").

Furthermore, between activated Ti gamma A1 lymphocytes and anti-Ti gamma monoclonal antibodies, Significant modifications of cytotoxic function are observed following the interaction of

The type of modification depends on the experimental conditions used.

For example, Fc receptors for immunoglobulins, such as the U937 human histiocytic lineage. When used with relatively resistant target cells expressing resulting in a considerable increase in sex.

The above results indicate that anti-Ti gamma A antibody is an Fc fragment expressed on some target cells. This is explained by the fact that it can be combined with

More specifically, in said binding, the antibody forms a bridge between the effector and the target cell. Create a bridge and modify the Ti gamma A antigenic determinant. Therefore, the lytic process The trigger is pulled.

On the other hand, for example, a leukemia cell line called "Jurkat" Targets deprived of receptors for Fc fragments of immunoglobulins, such as When selecting cells, the action of the antibody amounts to the virtual abolition of cytotoxic function.

In fact, if the target cells do not express Fc receptors, U937 human The cross-linking reaction described above for the histiocytic cell lineage does not occur. In this case, the antibody is Only that specific antigenic determinant is interacted with by the Fab fragment. This interaction The drug induces regulation of the CD3-Ti gamma A molecular complex. CD3 and Ti gamma The A molecule binds non-covalently to the cell surface. This phenomenon is caused by cell lysis. sjs).

In this case, all the above results suggest that Ti gamma A is the main complex gene of histocompatibility. Represents some of the functional receptors involved in the cytotoxic mechanism not restricted by It is observed that this directly leads to the hypothesis that

Some immunoprecipitation experiments performed with anti-Ti gamma A antibodies are confirmed by antibodies. The structure of the receptor is actually a molecular complex related to the CD3 protein; It was shown that the bodies are connected by disulfide bonds.

Furthermore, these immunoprecipitation experiments were performed with antigenic determination defined by anti-Ti gamma A antibodies. makes it possible to identify the molecular weight of the group.

The experiments are performed on a fetal human cell line known by the name F6C7. These F 6C7 cells have been described by Moingeon Pea et al., 1986, Nature, vol. 323, 6. +251 by using the standard lactoperoxidase method described on page 38. labeled on their surface.

Some prepurification steps are carried out on the Staphylococcus A suspension and also on other antibodies. . The precipitation reaction was then carried out with anti-Ti gamma A alone at a temperature of 4°C for about 4-6 hours. This is done using a magnetic antibody in combination with sepharose-protein beads. 5 DS-PAGE electrophoresis was performed on a 10% polyacrylamide gel, while non-reducing On the other hand, after addition of 5% of 2-MB.

The results of electrophoretic analysis are as follows.

Under non-reducing conditions, Ti gamma A molecules migrate to a uniform band of 85,000 daltons. Create an index. Under reducing conditions, the molecule is approximately 44,000 daltons, with a main band of approximately 4 He will appear as a small Nokundo at 1000 Daltons. This immunoprecipitation pattern is Extremely similar from one cell to another. of electrophoretic mobility by cells. The extremely small difference is probably due to differences in glycosylation. It will probably be dealt with.

These experiments demonstrated that 44,000 and 41 Two bands of 000 daltons are fully and partially glycosylated and T cell gamma Indicates that it corresponds to a protein encoded by a rearranged gene.

The precipitating genes encoding the Ti gamma A protein on the cell surface are C gamma, J Using some suitable molecular probes such as gamma and v gamma type probes, This was confirmed more precisely by a series of experiments called the Southern and Northern methods. . The results of these experiments led to the following conclusions.

A specific protein that generates transcription of the RNA messenger containing the Ti gamma A protein. The rearrangement involves a unique linkage between the V gamma 9 gene and the J gamma P gene. this The rearrangement causes proteins expressed on the cell surface to be linked by disulfide bridges. Since it is a dimer, it is further transcribed in the C gamma 1 constant region. Therefore, anti-Ti The gamma A monoclonal antibody is a recombinant antibody that associates the V gamma 9 fragment with the J gamma P fragment. Recognizes glycoproteins encoded by synthetic genes.

All the experiments and results described above are based on anti-Ti gamma A monoclonal antibodies and therefore T is associated with the CD3 protein and is involved in cellular functions, especially cytotoxic functions. Confirm the receptor structure of lymphocyte cells.

The hybridoma cell line producing anti-Ti gamma A antibody is ATCCN (LHB Located in Rockville, Maryland, USA under 9528 American type culture collection (the american) Type Culture Co11ectionXATCC) Ru.

The monoclonal antibodies according to this invention are directed against receptors encoded by the gamma gene. can be used to identify human T lymphocyte cells that have -. Also, this can also be used to confirm the differentiation status of these T cells.

Additionally, this monoclonal antibody has therapeutic potential against autoimmune diseases, cancer, and viral infections. It can be applied therapeutically. Monoclonal antibodies used in vitro can be used in organs or bone marrow. It may be possible to allow depletion of the corresponding lymphocyte population prior to transplantation. transplant rejection and Cells expressing Ti gamma A play a role in the physiological phenomenon of graft-versus-host disease If it is determined that It is also conceivable to use transplanted hindlimb antibodies in vivo for this purpose.

Copy and translation of amendment) Submission (Article 184-8 of the Patent Law) March 30, 1990 Director General of the Patent Office Yoshi 1) Takeshi Moon 1. Display of 8 patent applications PCT/US 88103123 2. Name of the invention Monoclonal antibody that recognizes epitope of gamma chain of human T cell surface receptor 3. Patent applicant Name: Coulter Corporation 4, Managing Director Address: 7th floor, Kasumiya Building, 3-2-4 Kasumikaseki, Chiyoda-ku, Tokyo Telephone (58) 1) Number 2241 (representative) address: same place October 3, 1989 Amended scope of claims 1. (After correction) Monoclonal antibodies are activated by a gene called “gamma” ``Family 2'' T lymphocyte receptors encoded in the V or variable region of the macrogene An anti-Ti gamma type A monoclonal antibody characterized by recognizing a protein.

2. The monoclonal antibody belongs to the 1gG2 subclass according to claim 1. Monoclonal antibodies.

3. Monoclonal antibodies (after correction) were analyzed under non-reducing conditions by SDS-PAGE electrophoresis. on the surface of human T lymphocytes with a molecular weight of approximately 85,000 daltons, as measured under conditions. The monoclonal protein according to claim 1 or 2, which recognizes the glycoprotein structure of antibody.

4. (After correction) Monoclonal antibodies were analyzed under reducing conditions by SDS-PAGE electrophoresis. A human T-lion with a molecular weight of about 41,000 to 44,000 Daltons as measured below. Claim 1 or 2 that recognizes a glycoprotein structure on the surface of lymphocytes monoclonal antibodies.

5. (After correction) Monoclonal antibody associates V gamma 9 fragment with J gamma P fragment A claim that specifically recognizes the glycoprotein structure encoded by the recombinant gene Monoclonal antibody according to scope 1 or 2.

6. Monoclonal antibodies were deposited with ATCC under accession number JB 9528. An anti-Ti gamma type A monomer characterized by being produced by hybridoma cell lines. Clonal antibodies.

7. (After amendment) Claim that the monoclonal antibody has a detectable label conjugated thereto The monoclonal antibody according to any one of Items 1 to 6.

8. (After amendment) Claim 7, wherein the label is a radioactive or fluorescent marker. monoclonal antibodies.

9. (After correction) The hybridoma cell line is caused by a gene called gamma. The second family of T lymphocyte receptors encoded in the V or variable region of the human gene. It is possible to produce an anti-Ti gamma A type monoclonal antibody that recognizes the titer. A hybridoma cell line produced by hybridoma technology characterized by.

10. (After correction) The hybridoma cell line is anti-Ti gamma type A monoclonal anti- American type culture collection number HB 95 that produces the body A hybridoma characterized in that it has the essential characteristics of a sample contributing in 28 Hybridoma cell lines produced by the technique.

11. (After correction) Molecule with T receptor structure associated with CD3 protein as claimed in any one of claims 1 to 6 for immunoprecipitation of Use of monoclonal antibodies.

12, (after correction) 41,000 to 4 measured under reducing and non-reducing conditions, respectively. Immunoprecipitate membrane molecules with molecular weights of 4000 Daltons and 85000 Daltons. A monoclonal clone according to any one of claims 1 to 6 for the purpose of Use of sexual antibodies.

13. (Delete) international search report PCT/US88103123

Claims (13)

[Claims] 1. Monoclonal antibodies have a gene called “gamma” that rearranges specifically within T cells. Recognizing the receptors of the “second family” of T lymphocytes that are encoded by genes. Characteristic anti-Ti gamma A monoclonal antibody. 2. Claim 1, wherein the monoclonal antibody belongs to the IgG2 subclass. Monoclonal antibodies. 3. Monoclonal antibodies were determined by SDS-PAGE electrophoresis under non-reducing conditions. A glycoprotein on the surface of human T lymphocytes with a molecular weight of approximately 85,000 Daltons. 3. The monoclonal antibody according to claim 1 or 2, which recognizes a structural structure. 4. Monoclonal antibodies were determined by SDS-PAGE electrophoresis under reducing conditions. The surface of human T lymphocytes with a molecular weight of about 41,000 to 44,000 daltons The monoclone according to claim 1 or 2, which recognizes the above glycoprotein structure. sexual antibodies. 5. The monoclonal antibody has a recombination gene that associates the V gamma 9 fragment with the J gamma P fragment. Claim 1: Specifically recognizing a glycoprotein structure encoded by a gene or the monoclonal antibody according to item 2. 6. A hybrid monoclonal antibody deposited with the ATCC under accession number HB9528. Anti-Ti gamma type A monoclonal clone characterized by being produced by the Doma cell line anti-inflammatory antibodies. 7. Any of claims 1 to 6 labeled with a detectable label. Monoclonal antibodies according to one section. 8. 8. The radiolabeled monoclonal antibody of claim 7. 9. The monoclonal antibody according to claim 7, comprising a fluorescent label or marker. . 10. The monoclonal antibody according to claim 1 can be produced. Characterized hybridoma cell lines. 11. A hybrid characterized by having been deposited with the ATCC under accession number HB9528. -ma cell lineage. 12. Immunoprecipitation of molecules with T receptor structure associated with CD3 protein A monoclonal clone according to any one of claims 1 to 6 for the purpose of Use of sexual antibodies. 13. 41,000 to 44,000 Da measured under reducing and non-reducing conditions, respectively. to immunoprecipitate membrane molecules with a molecular weight of 85,000 Daltons and 85,000 Daltons. Use of the monoclonal antibody according to any one of the claims 1 to 6 for.