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WO1993025572A1 - Facteurs d'induction presents sur la membrane des lymphocytes-t - Google Patents

Facteurs d'induction presents sur la membrane des lymphocytes-t Download PDF

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Publication number
WO1993025572A1
WO1993025572A1 PCT/US1993/005270 US9305270W WO9325572A1 WO 1993025572 A1 WO1993025572 A1 WO 1993025572A1 US 9305270 W US9305270 W US 9305270W WO 9325572 A1 WO9325572 A1 WO 9325572A1
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cells
tmip
cell
protein
purified
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Jean-Michel Dayer
Dan T. Stinchcomb
Michael J. Milhausen
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Amgen Boulder Inc
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Synergen Inc
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Priority to EP93914341A priority Critical patent/EP0652891A1/fr
Priority to AU44039/93A priority patent/AU4403993A/en
Priority to JP6501568A priority patent/JPH08501281A/ja
Publication of WO1993025572A1 publication Critical patent/WO1993025572A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/545—IL-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • This invention relates to the field of immunology and immunological diseases. More specifically, the invention provides therapeutically useful proteins for immune deficiency disorders.
  • T cells Many diseases are characterized by the suppression, dysfunction or deficiency of T cells.
  • Diseases which involve the suppression of T cells include HIV infection, diabetes mellitus, malaria, and cholera.
  • Diseases which involve the dysfunction of T cells include ataxia-telangiectasia, Hodgkin's disease, hypogammaglobulinemia, and chronic lympnocytic leukemia.
  • Diseases which are characterized by a deficiency of T cells include thymic aplasia, Di George's syndrome and lepromatous leprosy.
  • T cells One of the actions of T cells is to induce monocytes to produce cytokines or perform other inflammatory functions (Amento, E. P. et al., Proc. Natl. Acad. Sci. USA 79:5307 (1982); Amento, E. P. et al., J. Immunol. 134:350 (1985);
  • T cell surface molecules have been implicated in the induction of IL- l ⁇ production by monocytes. These include membrane-associated IL-l ⁇ (Dinarello, C.A., et al., J. Immunol. 139:1902 (1987)), membrane-associated TNF ⁇ (Weaver, C.T., et al., J. Immunol. 142:3469-3476 (1989)), CD2 (Webb, D.S., et al..
  • the present invention relates to the identification, characterization and purification of the T cell surface proteins which induce monocytes to produce cytokines and methods for purifying said T cell surface proteins.
  • the purified T cell-derived proteins which are designated as TMIP-1 and TMIP-2 have the following properties. They a) are naturally located on the plasma membrane of T cells; b) induce monocytes to produce cytokines; c) are substantially present on activated but not substantially present on unactivated T cells; d) are sensitive to trypsin; e) are solubilized by 3-( (3-cholamidopropyl)- dimethylammonio)-l-propanesulfonate (CHAPS); f) bind to Q-Sepharose resin at approximately pH 8.5 and can be eluted from the Q-Sepharose with
  • TMIP-1 36 to 37 kD by size exclusion chromatography (TMIP-2) .
  • TMIP-2 36 to 37 kD by size exclusion chromatography
  • substantially present means that the factor is present in sufficient quantity on the surface of T-cells to induce cytokine production from monocytes.
  • the cytokine measured for example, is IL-13.
  • activated T cells means T cells that have been treated with any of a variety of agents that are known to mimic the physiological activation of T cells through contact with antigen presenting cells. Such agents may include, but are not to be restricted to, PMA
  • T cell activating agents include phorbol-12-myristate 13-acetate, PHA (phytohemagglutinin) , other plant lectins, antibodies to T cell surface molecules such as CD3, CD28 or T cells treated with antigen presenting cells and a specific antigen or a non-specific superantigen. These agents are collectively referred to as "T cell activating agents”.
  • purified means that biochemical steps have been taken to separate the active proteins from other cellular constituents.
  • substantially pure means that a single peak of protein which is isolated from cell membranes is observed upon reverse phase HPLC analysis or a single band is observed upon SDS- polyacrylamide gel electrophoresis.
  • Monocytes are uninduced unless otherwise specified.
  • cytokine production is low or nil (e.g., measured IL-lj8 production less than about 10 pg/ml at monocyte cell concentration of about 2.5 x 10 5 cells/ml).
  • cytokine production is significant (e.g., measured IL-1/3 production greater than about 10 pg/ml at a monocyte cell concentration of about 2.5 x 10 5 cells/ml).
  • T cells One of the actions of T cells is to induce monocytes to produce cytokines or perform other inflammatory functions (Amento, E. P. et al., Proc. Natl. Acad. Sci. USA 79:5307 (1982); Amento, E. P. et al., J. Immunol. 134:350 (1985); Knight, E. Jr. et al., J. Immunol. 146:2280 (1991); Halpern, M.T. et al., J. Clin. Immunol. 11:1 (1991)).
  • T lymphocytes prior to the instant invention, the mechanism by which T lymphocytes induce monocytes by direct contact in a non-antigen dependent manner was unknown.
  • monocytes are induced to produce cytokines by direct cell-cell contact with activated T lymphocytes.
  • TMIPs T cell plasma membrane proteins
  • the present inventors have identified, characterized and purified two novel proteins on the surface of activated T cells that induce monocytes to express the inflammatory cytokine, IL-l ⁇ .
  • TMIP novel proteins
  • T cell-derived monocyte inducing proteins may prove to be useful therapeutics in patients suffering from dysfunctions or deficiencies of T cells, discussed above, for example.
  • TMIPs may be useful in the .in vitro induction of tumor infiltrating monocytes for treatment of cancer.
  • TMIP may also be useful in the treatment of listeriosis.
  • the present invention solves the problems discussed above by providing proteins useful to treat diseases which are characterized by suppressed, dysfunctional or deficient T cell action or to induce tumor infiltrating monocytes to destroy cancer cells.
  • T cells The effect of contact by T cells on monocyte cytokine production was investigated using double-chamber culture wells.
  • Adherent monocytes were cultured in the lower chambers of Transwell plates. The lower and upper chambers were separated by a 3- ⁇ m membrane which prevented physical contact between the cells but allowed diffusion of soluble factors.
  • T cells were seeded in either the upper or the lower chamber with or without the addition of a T cell activating substance. Cells were cultured and cytokine production was measured in the supernatants.
  • cytokine concentrations were six- to eight-fold higher than when unactivated T cells and uninduced monocytes were co- cultured in the absence of a separating membrane.
  • SUBSTITUTE SHEET cells before contact with monocytes Fixation was used to preserve the integrity of cell surface molecules while preventing the release of cell-derived lymphokines or other metabolic products of the T cells that could induce monocytes.
  • a monocyte cell line having a homogeneous population was used to facilitate the assay of monocyte cytokine induction.
  • the monocyte cell line was chosen for its very low baseline release of IL-l ⁇ , even when co-cultured with fixed unactivated T cells (Tsuchiya, S., et al. Int. J. Cancer 26:171 (1980)).
  • Freshly isolated T cells were activated with PMA and PHA and were fixed with paraformaldehyde. The fixed PMA/PHA-activated T cells strongly induced IL-l ⁇ production by monocytes.
  • the cell lines were unactivated or activated with PMA and PHA.
  • Serial dilutions of the plasma membranes purified from T cells were added to monocytes, and IL-l ⁇ was measured in the supernatants by ELISA.
  • Plasma membranes isolated from unactivated T cells failed to induce measurable IL-l ⁇ expression.
  • plasma membranes isolated from the activated T cells induced high levels of IL-l ⁇ expression by monocytes.
  • Trypsin treatment destroyed at least 70% of the inducing activity present in the plasma membranes isolated from induced T cells. Therefore, a protein component present on the plasma membranes of activated T cells is required for the induction of IL-l ⁇ secretion by monocytes.
  • CHAPS-solubilized plasma membrane protein prepared from activated T cells was applied to a Q Sepharose (Pharmacia, Upsalla, Sweden) column. Bound proteins were eluted with a gradient of NaCl in the presence of CHAPS and individual fractions were tested for their ability to induce monocyte IL- l ⁇ expression. The inducing activity eluted as two symmetric peaks; the first peak (peak I) eluted at 0.12 to 0.18 M NaCl and the second peak eluted at 0.28 to 0.36 M NaCl (peak II). Fractions representing each peak of activity were pooled separately, concentrated and applied to a Superdex 75 s ⁇ . exclusion column. Peak I eluted as a single, symmetric peak of 12 - 13 kilodaltons. Peak II eluted as a single, symmetric
  • SUBSTITUTESHEET peak of 36 - 37 kilodaltons Sizes were determined by comparison to molecular weight size standards applied to the same column; the estimates have a range of error of about 2 to 5 kilodaltons. All molecular weights stated herein are presumed to include this range of error.
  • T cell surface molecules have been implicated in the induction of IL-l ⁇ production by monocytes. These include membrane-associated IL-l ⁇ , membrane-associated TNF ⁇ , CD2 and CD69. We have shown that a specific inhibitor of IL-l ⁇ and IL-l ⁇ , an inhibitor of TNF ⁇ and TNF ⁇
  • lymphotoxin antibodies directed against CD2 and CD69 fail to inhibit the induction of monocyte IL-l ⁇ expression mediated by activated T cell plasma membrane proteins.
  • the factors purified from activated T cells are novel proteins capable of inducing monocytes to synthesize IL-l ⁇ .
  • TMIPs novel activated T cell-derived monocyte inducing proteins
  • TMIP-1 12 - 13 kDa in size
  • TMIP-2 36 - 37 kDa in size
  • monocyte inducing proteins may be of therapeutic use in the treatment of immunodeficiency diseases or for the induction of tumor-infiltrating monocytes.
  • the method for purifying the T-cell derived proteins is as follows:
  • Plasma membranes are prepared from the homogenized, activated T cells.
  • the plasma membranes may be isolated by differential centrifugation or on a density or velocity gradient. Density gradients which can be used include: sucrose, dextrose or dextran sulfate.
  • Plasma membrane proteins are solubilized in a zwitterionic detergent such as CHAPS ((3-[ (3-cholamidopropyl)-dimethylammonio]-l- propanesulfonate) ) , bound to an anionic exchange resin such
  • SUBSTITUTESHEET as Q-sepharose resin (Pharmacia, Uppsala, Sweden) and are eluted with a 0.0 to 0.5 linear gradient of NaCl.
  • Other zwitterionic detergents which may be used for this purpose include: CHAPSO (3-[ (3-cholamidopropyl)-dimethylammonio]-2- hydroxy-1-propanesulfonate) , N-decyl-N,N-dimethyl-3-ammonio-l- propanesulfonate, N-dodecyl-N,N-dimethyl-3-ammonio-l- propanesulfonate, N-hexadecyl-N,N-dimethyl-3-ammonio-l- propanesulfonate, N-octadecyl-N,N-dimethyl-3-ammonio-l- propanesulfonate, N-octyl-N,N-dimethyl-3-ammonio
  • anionic exchange resins which may used for this purpose include: DEAE (diethylamino)-sepharose, diethylaminoethyl cellulose, diethylamino sephadex, diethyl[2- hydroxypropy1]aminoethyl-sephadex, epichlorohydrin triethanolamine cellulose, polyethyleneimine cellulose, diethyl-[2-hydroxypropyl]aminoethyl cellulose.
  • TMIP-1 Active fractions eluting at 0.12 to 0.18 M NaCl (TMIP-1) and at 0.28 to 0.36 M NaCl (TMIP-2) from the Q-sepharose column are separately pooled, concentrated and applied to a Superdex 75 size exclusion column (Pharmacia, Uppsala, Sweden) .
  • Other size exclusion resins include Bio-Gel A (Biorad, Richmond, CA) , Bio-Gel P (Biorad, Richmond, CA) , Superose (Pharmacia) , and Sepharose (Pharmacia) .
  • TMIP-1 activity elutes as a peak with a mobility indicating a molecular weight of 12 - 13 kD.
  • Active TMIP-1 fractions can be pooled and applied to reverse phase HPLC.
  • the active peak may be collected and may be substantially pure at this stage.
  • TMIP-2 activity elutes from Superdex 75 as a peak of 36 - 37 kD.
  • Active TMIP-2 fractions may be pooled and applied to a reverse phase HPLC column. Because TMIP-2 is inactivated by the reverse phase solvents, TMIP-2 will be identified by correlating an isotopically labelled protein with the TIMP-2 activity (e.g., Hannum, C. H., et al.. Nature 343:336-340 (1990)). Once labelled, the protein can be collected from reverse phase HPLC as a peak of radioactivity. TMIP-2 may be substantially pure at this stage.
  • the purified protein of the present invention can be used in a pharmaceutical composition comprising a pharmaceutically effective amount of the purified protein and a pharmaceutically acceptable carrier.
  • a pharmaceutically effective amount is any amount of protein which is effective in treating an animal in need of treatment.
  • Pharmaceutically acceptable carriers include, but are not limited to physiological saline, phosphate buffered saline, water and emulsions, such as oil and water emulsions.
  • TMIP TMIP
  • Antibodies that react with TMIP can be used for screening expression libraries in order to obtain the gene which encodes TMIP.
  • Synthetic peptides can be synthesized which correspond to regions of the sequence of TMIP using an Applied Biosystems automated protein synthesizer. Such peptides can be used to prepare antibodies according to procedures known in the art. Such antibodies would be useful for diagnosing autoimmune diseases, or inflammatory diseases involving activated T cells by means well known to those skilled in the art.
  • EXAMPLE 1 Effect of co-culture of activated T cells and monocytes on cytokine production.
  • the effect of cell contact between activated T cells and monocytes on cytokine production was investigated using double chamber culture wells. Buffy coats were prepared from healthy donors, diluted (1:4) with Hank's Buffered Salt Solution (HBSS) (Gibco, Paisley, Scotland) and the mononuclear cells isolated by centrifugation on Ficoll- Paque (Pharmacia, Uppsala, Sweden) . The cells were washed three times with Phosphate Buffered Saline (PBS) (Dulbecco's, Gibco) and then resuspended in culture medium.
  • HBSS Hank's Buffered Salt Solution
  • PBS Phosphate Buffered Saline
  • Monocytes were purified by plating mononuclear cells at 4 x 10 6 /ml in the lower chambers of Transwell plates (Costar, MA) at 37°C for 1 h. After attachment, the wells were washed vigorously with warm (37°C) PBS. Adherent cells were 90-95% peroxidase-
  • T cells were isolated from non-adherent mononuclear cells by passing the cells through a nylon wool column two times. In some experiments, cells were further treated with L-Leucine methyl ester to eliminate any remaining monocytes. Purified unactivated T cells were 94-98% CD2 positive, 83-94% CD3 positive and less than 2% CD14 positive, as measured by indirect immunofluorescence on an EPICS V flow cytometer (Coulter, FL) . T cells were seeded in the upper or lower chamber as shown.
  • IL-1 alpha and IL-1 beta were measured using an ELISA described previously (J. Grassi et al. J. Immunol. Methods 123:193 (1989)) or an IL-l ⁇ ELISA kit (R & D Systems, Minneapolis, MN) .
  • the sensitivity of the assays for both IL-1 alpha and IL-1 beta is 10 pg/ml.
  • TNF alpha was measured by a TNF alpha IRMA kit (Medgenix, Belgium) .
  • the sensitivity of this assay is 15 pg/ml. The results are shown in Table 1.
  • EXAMPLE 2 Fixed activated T cells induce THP-1 cells to produce IL-l ⁇ .
  • T cells isolated as described in Example 1, were activated at 4 X 10 s cells per ml with PMA (5ng/ml, Sigma Chemical (St. Louis, MO)) and PHA (1 ⁇ g/ml) for 72 hours at 37° and 5% C0 2 . The T cells were washed three times with PBS, then fixed with freshly
  • THP-1 a leukemic cell line derived from a patient with acute monocytic leukemia was obtained from the American Type Culture Collection (Rockville, MD) . All cells were cultured in RPMI 1640 medium (Gibco, Scotland or Mediatech, Washington, D.C.) supplemented with 10% heat induced fetal calf serum (Gibco, Scotland) or 10% defined fetal bovine serum (Hyclone Laboratories, Inc., Logan, Utah), 2 mM L-glutamine, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin (Gibco, Scotland or Irvine Scientific, Santa Ana, CA) . Some isolates grew faster with the addition of 5 x 10 "5 M ⁇ -mercaptoethanol (Sigma Chemical, St. Louis, MO).
  • EXAMPLE 3 Subcellular fractionation of the monocyte inducing factors. Subcellular fractions of 5 x 10° unactivated or activated T lymphocytes were prepared as described by A. A. Aderem et al. (1986) Proc. Nat . Acad. Sci. USA 83. 5817. Briefly, cells were suspended at 5 x 10 7 /ml in calcium and magnesium-free PBS containing 0.25 M sucrose, 0.1 M MgCl 2 , 10 mM Tris-HCl pH 7.4, 1 mM phenylmethylsulfonylfluoride, 250 Units of aprotinin and 15 mM EDTA (ethylenediaminetetraacetic acid, Sigma, St. Louis, MO) .
  • the cell suspension was sonicated with three 5-second bursts of 90 watts power.
  • the homogenate was centrifuged at 1,000 x g for 15 min. to isolate unbroken cells and nuclei.
  • the supernatant was centrifuged at 100,000 x g for 30 min. in a Beckman Airfuge to separate cytosol and membranes. All manipulations were done at 4°C.
  • Each fraction was cultured for 48 hours with 5 x 10 4 THP-1 cells.
  • IL-l ⁇ in the supernatants was measured by ELISA. The results are presented in Table 3, below. As can be seen, the inducing factors were found primarily in the membrane fraction of activated T cells.
  • EXAMPLE 4 The monocyte inducing factors present on T cell plasma membranes are sensitive to trypsin.
  • the HUT 78 or JURKAT cell lines were activated with 5 ng/ l PMA and 1 pg/ml PHA, at 1 x 10 6 cells/ml for 24 hours at 37°C.
  • the activated cells were harvested by centrifugation and washed three times in phosphate buffered saline (PBS) (Dulbe ⁇ co's, with Ca +2 and Mg +2 ) .
  • PBS phosphate buffered saline
  • the cells were resuspended at 3 x 10 6 to 1 x 10 7 cells/ml in buffer A (20 mM Tris; pH7.5, 10 mM NaCl, 0.1 mM MgCl 2 ) with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.5 ⁇ g/ml Dnase I added prior to resuspension.
  • buffer A 20 mM Tris; pH7.5, 10 mM NaCl, 0.1 mM MgCl 2
  • PMSF phenylmethylsulfonyl fluoride
  • Dnase I 0.5 ⁇ g/ml Dnase I added prior to resuspension.
  • Cells were disrupted by three ten-second bursts with a Polytron homogenizer (Brinkman) at setting five (12,000 rpm) . Efficient breakage was assessed by microscopic examination.
  • SUBSTITUTESHEET cells by modification of the method of Maeda, et al. and were resuspended in serum-free growth medium. The plasma membranes then were incubated with 33 ⁇ g/ml trypsin for 30 minutes at 37°C. After incubation, 66 ⁇ g/ml soybean trypsin inhibitor and 10% (v/v) fetal bovine serum (FBS) were added. Control plasma membranes were incubated first with the soybean trypsin inhibitor; trypsin and FBS were added after incubation. The results shown in Table 4 indicate that much of the T cell plasma membrane-bound inducing factors are sensitive to trypsin.
  • the monocyte inducing proteins can be solubilized by 1% CHAPS.
  • the following procedure efficiently solubilized the inducing protein(s) without apparent loss of activity: Plasma membranes prepared as described above were spun at 100,000 x g for 30 minutes. The resulting pellet was resuspended in the desired volume of medium or buffer containing 0.25 - 1% 3-((3- cholamidopropyl)-dimethylammonio)-1-propanesulfonate (CHAPS) and 1 mM fresh PMSF. Vigorous resuspension was required to evenly distribute the plasma membrane pellet. Plasma membrane protein concentrations up to 1 mg/ml were used. The detergent-treated membranes were incubated on ice for one hour
  • T-cell derived monocyte inducing proteins bind Q Sepharose and can be eluted as distinct peaks.
  • Three liters of HUT-78 cells were grown and induced (as described above) in a 10-chamber Cell Factory (Nunc; Roshilde, Denmark) . Cells were disrupted using a sucrose gradient, although this could also be accomplished by the use of a homogenizer. Plasma membranes were prepared and were solubilized by treatment with 20 mM
  • SUBSTITUTESHEET Tris pH 8.5; 0.25% CHAPS and 1 mM PMSF.
  • CHAPS was selected for use in this experiment, it is expected that any zwitterionic detergent would be effective for solubilization.
  • the insoluble material was pelleted by centrifugation (100,000 x g, 30 minutes, 4°C) .
  • the soluble supernatant was filtered through a 0.2 ⁇ Acrodisc filter (Gelman Sciences, Ann Arbor, MI) .
  • Approximately 1 - 2 mg of solubilized plasma membrane protein was applied to an anion exchange resin column.
  • An 8 ml Q Sepharose column equilibrated with the above buffer was used herein.
  • Bound proteins were eluted with a 0 to 0.4 M NaCl gradient. Monocyte inducing activity was measured as described in Example 2, diluting each fraction at least eight-fold in the assay to prevent any effect of the buffers on cell viability or IL-l ⁇ production.
  • TMIP-1 elutes from the Q Sepharose resin at 0.12 to 0.18 M NaCl
  • TMIP-2 elutes from the Q Sepharose resin at 0.28 to 0.36 M NaCl.
  • TMIP-1 induces IL-l ⁇ expression without any detectable change in morphology.
  • TMIP-2 induces both IL-l ⁇ expression and striking morphological differentiation when added to THP-1 cells.
  • EXAMPLE 7 Size exclusion chromatography of TMIP-1 and TMIP-2. Active fractions that eluted from the Q Sepharose column were pooled and concentrated 3- to 8-fold using Centricon 10 filter devices (Amicon; Beverly, MA) . The material containing TMIP-1 was applied to a Superdex 75 size exclusion column (Pharmacia LKB Biotechnology, Uppsala, Sweden) and was eluted with PBS + 1% CHAPS + l mM PMSF equilibrated with the same buffer. A portion of each fraction was then assayed for the ability to induce THP-1 cells to produce IL-l ⁇ . The inducing protein elutes as a single, symmetric peak. The TMIP-1 protein is about 12 - 13 kilodaltons by comparison with molecular weight
  • TMIP-2 elutes as a single symmetric peak of approximately 36 - 37 kilodaltons.

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Abstract

L'invention se rapporte à deux nouvelles protéines se trouvant sur la surface de lymphocytes-T activés, qui induisent l'expression de cytokines dans les monocytes, ainsi qu'à des procédés de purification desdites protéines de surface de lymphocytes-T.
PCT/US1993/005270 1992-06-05 1993-06-04 Facteurs d'induction presents sur la membrane des lymphocytes-t Ceased WO1993025572A1 (fr)

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Application Number Priority Date Filing Date Title
EP93914341A EP0652891A1 (fr) 1992-06-05 1993-06-04 Facteurs d'induction presents sur la membrane des lymphocytes-t
AU44039/93A AU4403993A (en) 1992-06-05 1993-06-04 T-cell monocyte inducing proteins (TMIPS)
JP6501568A JPH08501281A (ja) 1992-06-05 1993-06-04 Ｔ細胞膜誘発因子

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US89385192A 1992-06-05 1992-06-05
US07/893,851 1992-06-05

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EP0666987A4 (fr) * 1992-10-30 1997-08-27 T Cell Diagnostics Inc Evaluation de la quantite totale d'une molecule dans un echantillon et procedes bases sur celle-ci.

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ITMI20022118A1 (it) * 2002-10-04 2004-04-05 Abiogen Pharma Spa Procedimento per la coltura su larga scala di t-linfociti in un sistema omogeneo.

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WO1988007549A1 (fr) * 1987-03-26 1988-10-06 Yale University Proteine de membrane de cellules t
US4977245A (en) * 1986-10-10 1990-12-11 Board Of Regents, The University Of Texas System Methods and compositions for inducing monocyte cytotoxicity
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Publication number Priority date Publication date Assignee Title
WO1984001504A1 (fr) * 1982-10-20 1984-04-26 Dana Farber Cancer Inst Inc Facteur de croissance de lymphocyte
US4977245A (en) * 1986-10-10 1990-12-11 Board Of Regents, The University Of Texas System Methods and compositions for inducing monocyte cytotoxicity
US5112948A (en) * 1986-10-10 1992-05-12 Jones C Michael Methods and compositions for inducing monocyte cytotoxicity
WO1988007549A1 (fr) * 1987-03-26 1988-10-06 Yale University Proteine de membrane de cellules t

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Title
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0666987A4 (fr) * 1992-10-30 1997-08-27 T Cell Diagnostics Inc Evaluation de la quantite totale d'une molecule dans un echantillon et procedes bases sur celle-ci.

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JPH08501281A (ja) 1996-02-13
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CA2135397A1 (fr) 1993-12-23

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