WO1989002899A1 - Anticorps monoclonal reconnaissant un epitope de la chaine gamma de recepteurs de surface des lymphocytes t de l'homme - Google Patents

Anticorps monoclonal reconnaissant un epitope de la chaine gamma de recepteurs de surface des lymphocytes t de l'homme Download PDF

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Publication number
WO1989002899A1
WO1989002899A1 PCT/US1988/003123 US8803123W WO8902899A1 WO 1989002899 A1 WO1989002899 A1 WO 1989002899A1 US 8803123 W US8803123 W US 8803123W WO 8902899 A1 WO8902899 A1 WO 8902899A1
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WIPO (PCT)
Prior art keywords
monoclonal antibody
lymphocytes
tigammaa
human
cells
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Application number
PCT/US1988/003123
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English (en)
Inventor
Thierry Hercend
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Coulter Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Coulter Corporation filed Critical Coulter Corporation
Priority to KR1019890700940A priority Critical patent/KR890701630A/ko
Publication of WO1989002899A1 publication Critical patent/WO1989002899A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates generally to monoclonal antibodies and their interaction with specific antigenic determinants, and relates to a hybridoma cell line which produces a monoclonal antibody which specif ⁇ ically binds to the cell surface receptor of human T lymphocytes. More particularly, the monoclonal antibody embodying the invention binds a unique epitope of the human T cell surface receptor encoded by a T cell gamma rearranging gene.
  • Background Art In this technical field, it is well known that the human immune system is comprised of cellular and soluble elements in blood together with various organs of the lymphoreticular system. A foreign substance, called an antigen, entering the body system, when iden- tified, will result in an immune system response or • reaction.
  • the reaction can take the form of a local ⁇ ized inflammation or phagocytic reaction or may result in a specific humoral or cell mediated reaction.
  • These specific reactions require the interaction of lymphocytes to produce antibody against the foreign substance, which may be a cell, virus or bacteria, for instance.
  • the major elements in an immune response are T cells derived from the thymus and B cells derived from bone marrow. The T cells will interact with the B cells to create an antibody formation against the foreign substance in what is termed a cell mediated immunity.
  • the humoral immune response requires only the presence of a presensitized B lymphocyte together with a foreign substance. Their interaction causes the B cells to proliferate and begin synthesizing im- munoglobulins or antibody protein against the antigen.
  • Circulatory peripheral blood cellular components com- prise red blood cells or erythro ⁇ ytes, white blood cells or leukocytes and platelets.
  • the leukocytes are divided into five major populations called neutrophils, eosinophils, basophils, monocytes and lymphocytes.
  • the lymphocytes are further subdivided into thymus derived T cells and bone marrow derived B cells.
  • the T and B lymphocytes are further divided into numerous subsets, such as inducer cells, suppressor cells, killer cells, among others, with increasing numbers of subsets being identified in current research efforts.
  • monoclonal antibodies have been developed which bind solid tumor cells, glycoprotein surface molecules of red blood-"cells, and inflammatory sites of human tissues infected by virus, such as in AIDS dis ⁇ eases, just to name a few.
  • the effective study of hu- man cells, both normal and abnormal, for their function and impact on the human immune system increases spec ⁇ tacularly as new monoclonal antibodies are developed for diagnostic and therapeutic applications, both in vitro and in vivo.
  • the monoclonal antibody designated "Anti-T to T3" was able to block the ability of any functional T cell clone to recognize its target antigen. It was learned that each different mature T cell clone has a 5 unique protein molecular structure which enables each T cell to recognize its target antigen. However, the patent teaches that although T cell clones may differ in their so-called “recognition structure", they appear to have certain common characteristics.
  • T cell clones One common characteristic of all T cell clones is constituted by the presence of T3 glycoproteins and of a dimer composed of two different protein molecules as identified in the said patent.
  • the monoclonal antibody described in the patent recognized specifically the T
  • the monoclonal antibody embodying the invention recognizes a sub-population of human peripheral T cells which represents approximately 1-15% of circulating mononuclear blood cells and a mean -value of about 3% of
  • lymphocytes express a T cell surface receptor of the "second family", encoded by genes of the gamma class which rearrange specifically in the T cells.
  • the monoclonal antibody of the invention binds to a unique T cell surface receptor of the "second family", encoded by genes of the gamma class which rearrange specifically in the T cells.
  • the monoclonal antibody of the invention binds to a unique T cell surface receptor of the "second family", encoded by genes of the gamma class which rearrange specifically in the T cells.
  • the monoclonal antibody of the invention binds to a unique
  • the monoclonal antibody of the invention identi ⁇ fies, a novel sub-set of human lymphocytes with T cell 5 receptor gamma complex.
  • the monoclonal antibody is called "anti-TigammaA”.
  • This monoclonal antibody belongs to the IgG2 sub-class.
  • This monoclonal antibody can be labelled with a suitable marker such as radio label or a fluorescent marker for identifying and defining the sub-population of human T lymphocytes in circulating blood drawn from healthy individuals, ill individuals or individuals having been subjected to an organ graft or a bone marrow transplant.
  • the antibody is also useful for characterizing the state of differ ⁇ entiation of human T lymphocytes in circulating blood. Further, the antibody may be applied therapeutically by in-vitro, ex-vivo or in-vivo eventual manipulations of human T lymphocytes bearing the TigammaA epitope. Disclosure of Invention
  • a hybridoma cell line which produces a monoclonal antibody which specifically binds a unique epitope of the human T cell surface receptor of T lymphocytes in peripheral blood encoded by a T cell gamma rearranging gene.
  • the epitope is located in a glycoprotein struc ⁇ ture on the surface of human T lymphocytes in circulat ⁇ ing blood having a molecular mass of approximately 85,000 daltons determined under non-reducing conditions and of 41,000-44 . ,000 daltons determined under reducing conditions.
  • the monoclonal antibody of the invention is called "anti-TigammaA" and belongs to the IgG2 sub ⁇ class. This monoclonal antibody enables characteriza ⁇ tion of human T lymphocytes in circulating blood which express the TigammaA epitope or antigenic determinant. Best Mode for Carrying Out the Invention
  • the monoclonal antibody anti-TigammaA binds to an antigenic determinant of the human T lymphocyte recep ⁇ tor and specifically to the Ti complex of the Ti structure of the human T cell surface receptor.
  • the Ti molecular structure bears the gamma chain and the anti- TigammaA monoclonal antibody defines the gamma chain so that the invention enables detection, quantitation and thorough characterization of the Ti complex of the "second family" of the T cell surface receptor.
  • This epitope appears on a sub-population of T lymphocytes in human blood and in human lymphoid organs, such as the thymus, the spleen, the ganglions and the bone marrow.
  • the advantages capable of being derived from the diag ⁇ nostic and therapeutic applications of this monoclonal antibody will be amplified herein.
  • the anti-TigammaA monoclonal antibody of this in ⁇ vention was developed as hereinafter described.
  • the suspension is injected into a Biozzi mouse. Immunization is carried out by two first in- traperitoneal injections of 4 x 10° cells in a Freund adjuvant, followed by an intravenous injection of 4 x 10 cells without adjuvant. Three days later, the animal is killed, the spleen is drawn and the spleen cells are harvested.
  • Hybridization is then carried out by fusing the harvested spleen cells and a murine NS-1 myeloma cell line.
  • a cell fusing has been carried out accord- ing to a known process disclosed in "Moingeon P et al., 1986, Nature, Vol. 323, p. 638".
  • the hybrid cells ob ⁇ tained after fusing are cultivated in a HAT medium con ⁇ taining hypoxanthine, aminopterin, thymidine, 10% fetal bovine serum and 10% horse serum. The hybridomas are then selected.
  • the screening is carried out by testing the ability of hybridoma su- pernatants to block cytotoxic reactions exerted by the clone immunizing against a leukemia cell line maintained in the culture.
  • the hybridoma producing the monoclonal antibodies are cloned by techniques of limiting dilution. Final ⁇ ly, immune ascites are produced, wherein the antibodies can be taken.
  • the so-made anti-TigammaA antibodies belong to the IgG2 subclass and can fix complement protein.
  • the reactivity of the monoclonal antibody and the extent of its action have been determined with a sub- population of human lymphocytes by means of two color immunofluorescence analysis and reading with cytofluorometri ⁇ methods.
  • the cells of the sub ⁇ population to be tested have been isolated from the mononuclear portion of the peripheral blood taken from 30 healthy adult donors. This portion has been purified by a centrifugation technique on Fi ⁇ oll gradient. Samples of 3.0 x 10 ⁇ cells are analysed on a cytofluorometer, such as an "EPICS C" flow ⁇ ytometer manufactured by Coulter Corpo ⁇ ration of Hialeah, Florida, U.S.A.
  • the control reagents are FITC-IgG and PE-IgG.
  • the non-adherent lymphocytes are treated during 30 minutes with the monoclonal antibody of the invention, anti-TigammaA, provided with a fluorescent green markers, namely fluorescein, in presence of another known antibody which is also provided with a fluores ⁇ cent red marker, which is the phycoerythrin (PE).
  • the other known antibodies are the following: anti-CD2, -CD3, -CD4, -CD5, -CD8, NKH1, and those antibodies determining the molecules encoded by genes called the class II genes of the major complex of histocompat- ibility. These known antibodies are provided by the company Coultronics, France of Margency, France.
  • the results obtained from the antibody according to the invention allow to characterize human T lymphocytes expressing the TigammaA antigenic determinan .
  • TigammaA+ lymphocytes ex ⁇ press CD2 and CD3 glycoproteins.
  • a majority of Tigam- maA+ lymphocytes express CDS proteins. It is to be ob- served here that, where these CDS proteins are present, the density of the CD5 antigen per cell is lower in the TigammaA+ cell subset than in the population of conven ⁇ tional lymphocytes expressing the T a/ ⁇ receptor.
  • the expression of CDS in the TigammaA+ subpopulation varies widely from an individual to the other. In most of the individuals, only a minority of TigammaA+ lymphocytes express the CD8 protein with a weak antigenic density. At rest, the TigammaA+ lymphocytes do not express the CD4 proteins, NKH1 and molecules encoded by genes of the class II of the major complex of histo ⁇ ompatibility.
  • TigammaA+ sub-population represents a majority of circulating lymphocytes which express CD3 proteins but which do not express on their surface the conventional T a/ ⁇ receptor.
  • the TigammaA+ lymphocytes are pre-activated with a mitogen such as e.g. the phytohemagglutinin with a dose of about 2 mg/ml, the cultivated and stimulated with the anti-TigammaA monoclonal antibody coupled with sepharose beads activated by the cyanogen bromide, a proliferation of the T lymphocytes is clearly shown 48 hours after the stimulation.
  • a mitogen such as e.g. the phytohemagglutinin with a dose of about 2 mg/ml
  • T lymphocytes fur ⁇ ther can secrete their own Interleukin 2.
  • the cytotoxic activity of the lymphocytes has been observed more precisely by means of other experiments.
  • the cytotoxicity is measured during an experiment ree hours after marking of target cells by
  • the TigammaA+ cells determined by the monoclonal antibody of the invention appear, when at rest, as a very homogeneous population of granular lymphocytes. However, they mediate a little or no spontaneous cytotoxic activity against the conventional targets of the system called "natural cytotoxic system", as for example the K562 line which derives from a pleural ef ⁇ fusion of chronic myeloid leukemia.
  • this sub-population does not sig ⁇ nificantly contribute to the natural cytotoxic activity of the peripheral blood, and this is compatible with the absence of expression on the surface of the NKH1 marker, as explained previously.
  • TigammaA+ lymphocytes are activated by means of mitogens and by exogeneous Interleukin 2, they become very cytotoxic against numerous tumor cells, in particular those cells of the K562 line. This cytotoxicity can be induced within a very short time period from 3 to 5 days. The so ac ⁇ tivated TigammaA+ lymphocytes may then, participate to the phenomenon called "lymphokine activated killer function" (which corresponds to the British “LAK phenomenon").
  • the type of modification depends on the experimen ⁇ tal conditions used.
  • the action of the antibody results in a substantial increase of the cell cytotoxici y.
  • the anti-TigammaA antibody can. bind the F ⁇ fragment ex ⁇ pressed on certain target cells. More precisely, in such a binding, the antibody establishes a bridge be ⁇ tween effector and target cells and modifies the Tigam ⁇ maA antigenic determinant. Consequently, the lytic process is triggered.
  • target cells deprived of receptor for the Fc fragment of im- munoglobulins as e.g. the leukemia cell line called "jurkat"
  • the action of the antibody results in a vir ⁇ tual abrogation of the cytotoxic function.
  • the reaction of bridge binding disclosed above for the U 937 human histiocytic cell line does not occur.
  • the antibody interacts only with its specific antigenic determinant through the Fab fragment. This interaction induces a modulation of the CD3-TigammaA molecular com ⁇ plex, the CD3 and TigammaA molecules being non- covalently linked to the cell surface. This phenomenon results in an important decrease of the cell lysis. It is to be observed here that all the results previously described directly lead to the hypothesis that TigammaA represents a portion of a functional receptor involved in mechanisms of cell cytotoxicity which is not restricted by the genes of the major com ⁇ plex of histocompatibility.
  • the results of the electrophoretic analysis are the following.
  • the TigammaA molecule migrates and produces an homogeneous band at 85,000 daitons approximately under non-reducing conditions. With reducing conditions, the molecule appears as a major band at about 44,000 daltons and a minor band at about 41,000 daltons.
  • This immunoprecipitation pattern is very analogous from one cell to the other.
  • Minimal differences of elec ⁇ trophoretic mobility, from one cell to another, proba ⁇ bly correspond to differences of glycosylation.
  • the precipitated gene encoding the TigammaA protein on the cell surface is more precisely charac ⁇ terized by a series of experiments called "Southern and Northern blot" using several appropriate molecular probes such as probes of the C gamma, J gamma and V gamma type. The results of these experiments have led to the following conclusions.
  • the specific rearrangement resulting in the tran ⁇ scription of the RNA messenger which provides the TigammaA protein involves a unique junction between the V gamma 9 gene and the-J gamma P gene. This rear- rangement is further transcribed with the C gamma 1 constant region, because the protein expressed at the cell surface is a di er bound by disulphide bridges.
  • the anti-TigammaA monoclonal antibody recognizes a glycoprotein structure, encoded by a recombined gene asso ⁇ iating a V gamma 9 segment to a J gamma P segment.
  • the hybridoma cell line producing the anti- TigammaA antibody is deposited in the American Type Culture Collection (ATCC) at Rockville, Maryland, U.S.A. under ATCC No. 9528.
  • the monoclonal antibody according to the invention can be used to identify the human T lymphocyte cells bearing a receptor encoded by the gamma genes. It can also be used to characterize the state of differentia ⁇ tion of these T cells.
  • this monoclonal antibody can be applied therapeutically against auto-immune diseases, carcinoma and viral infections.
  • the monoclonal antibody used ex- vivo could allow the depletion of corresponding lymphocyte populations before.organ or bone marrow transplants. It is also contemplated to use the antibody after transplantation procedures in-vivo for mediating against either the graft rejection, or the graft versus host disease, in the event it is determined that the cells expressing TigammaA play a part in these biological phenomena.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention se rapporte à un nouvel anticorps monoclonal du type anti-TigammaA. Cet anticorps monoclonal reconnaît un récepteur unique des lymphocytes T de la ''seconde famille'' codés par des gènes appelés ''gamma'' qui se redisposent dans les lymphocytes T de façon spécifique. La présente invention peut être utilisée dans le diagnostic et le traitement de maladies auto-immunitaires, de carcinomes et d'infections virales, par exemple.
PCT/US1988/003123 1987-09-30 1988-09-09 Anticorps monoclonal reconnaissant un epitope de la chaine gamma de recepteurs de surface des lymphocytes t de l'homme WO1989002899A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1019890700940A KR890701630A (ko) 1987-09-30 1988-09-09 인간 t세포 표면 수용기들 중 감마 사슬의 에피토프를 인지하는 단일 클론 항체

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR8713532A FR2621127B1 (fr) 1987-09-30 1987-09-30 Anticorps monoclonal reconnaissant un epitope de la chaine gamma des recepteurs de surface des cellules t humaines, lignee de cellules d'hybridomes produisant cet anticorps, et utilisation de cet anticorps monoclonal
FR87/13532 1987-09-30

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WO1989002899A1 true WO1989002899A1 (fr) 1989-04-06

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EP (1) EP0393062A4 (fr)
JP (1) JPH03500363A (fr)
KR (1) KR890701630A (fr)
AU (1) AU615233B2 (fr)
FR (1) FR2621127B1 (fr)
WO (1) WO1989002899A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0289252A2 (fr) * 1987-04-25 1988-11-02 Kyowa Hakko Kogyo Co., Ltd. Anticorps monoclonal dirigé contre la chaine gamma du récepteur des cellules T
US5223426A (en) * 1988-12-15 1993-06-29 T Cell Sciences, Inc. Monoclonal antibodies reactive with defined regions of the t-cell antigen receptor
US5766947A (en) * 1988-12-14 1998-06-16 Astra Ab Monoclonal antibodies reactive with an epitope of a Vβ3.1 variable region of a T cell receptor

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2656532A1 (fr) * 1989-12-29 1991-07-05 Inst Nat Sante Rech Med Proteine antigenique presente sur des cellules sat, anticorps correspondants et leurs applications.

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4550086A (en) * 1983-02-16 1985-10-29 Dana-Farber Cancer Institute, Inc. Monoclonal antibodies that recognize human T cells
US5340921A (en) * 1986-07-03 1994-08-23 T Cell Sciences, Inc. Γ, δT cell receptor and methods and detection
GB8626413D0 (en) * 1986-11-05 1986-12-03 Gilliland L K Antibodies
US5169938A (en) * 1987-04-25 1992-12-08 Kyowa Hakko Kogyo Co., Ltd. Anti-T cell receptor γ-chain monoclonal antibody
FI891226A (fi) * 1988-04-28 1989-10-29 Univ Leland Stanford Junior Reseptordeterminanter i anti-t-celler foer behandling av autoimmunsjukdom.

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Journal of Experimental Medicine, Volume 166, issued October 1987, S. JITSULAWA et al, "A Novel Subset of Human Lymphocytes with a T Cell Receptor-Gamma Complex", see pages 1192 and 1193. *
Journal of Experimental Medicine, Volume 167, issued February 1988, F. TRIEBEL et al, "A Unique V-J-C- Rearranged Gene Encodes a Gamma Protein Expressed on the Majority of CD3+T Cell Receptor-alpha-beta-Circulating Lymphocytes", see pages 698 and 699. *
Natrue, Volume 325, issued 19 February 1987, P. MOINGEON et al, "A gamma-Chain Complex Forms a Functional Receptor on Cloned Human Lymphoctes with Natural Killer-like Activity", see Abstract, page 723. *
Proceedings of the National Academy of Sciences, U.S.A., Volume 84, issued June 1987, IOANNIDES, C.G. et al, "Identification of a Second T-Cell Antigen Receptor in Human and Mouse by an Anti-Peptide Gamma-Chain-Specific Monoclonal Antibody", see pages 4244, 4247 and 4248. *
See also references of EP0393062A4 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0289252A2 (fr) * 1987-04-25 1988-11-02 Kyowa Hakko Kogyo Co., Ltd. Anticorps monoclonal dirigé contre la chaine gamma du récepteur des cellules T
EP0289252A3 (en) * 1987-04-25 1990-02-14 Kyowa Hakko Kogyo Co., Ltd. Anti-t cell receptor, y-chain monoclonal antibody
US5169938A (en) * 1987-04-25 1992-12-08 Kyowa Hakko Kogyo Co., Ltd. Anti-T cell receptor γ-chain monoclonal antibody
US5766947A (en) * 1988-12-14 1998-06-16 Astra Ab Monoclonal antibodies reactive with an epitope of a Vβ3.1 variable region of a T cell receptor
US5223426A (en) * 1988-12-15 1993-06-29 T Cell Sciences, Inc. Monoclonal antibodies reactive with defined regions of the t-cell antigen receptor
US5976533A (en) * 1988-12-15 1999-11-02 Astra Ab Monoclonal antibodies reactive with defined regions of the T cell antigen receptor
US5980892A (en) * 1988-12-15 1999-11-09 Astra Ab Monoclonal antibodies reactive with defined regions of the T cell antigen receptor
US6048526A (en) * 1988-12-15 2000-04-11 Astra Ab Monoclonal antibodies reactive with defined regions of the T cell antigen receptor
US6171799B1 (en) 1988-12-15 2001-01-09 Astra Ab Monoclonal antibodies reactive with defined regions of the T cell antigen receptor

Also Published As

Publication number Publication date
FR2621127B1 (fr) 1992-01-31
AU2526788A (en) 1989-04-18
KR890701630A (ko) 1989-12-21
FR2621127A1 (fr) 1989-03-31
EP0393062A4 (en) 1990-11-28
EP0393062A1 (fr) 1990-10-24
JPH03500363A (ja) 1991-01-31
AU615233B2 (en) 1991-09-26

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