WO1989010938A1 - Micelles de proteine - Google Patents

Micelles de proteine Download PDF

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Publication number
WO1989010938A1
WO1989010938A1 PCT/US1989/001858 US8901858W WO8910938A1 WO 1989010938 A1 WO1989010938 A1 WO 1989010938A1 US 8901858 W US8901858 W US 8901858W WO 8910938 A1 WO8910938 A1 WO 8910938A1
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lfa
cells
protein
mlfa
micelles
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PCT/US1989/001858
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English (en)
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Michael L. Dustin
Timothy A. Springer
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Dana-Farber Cancer Institute
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Publication of WO1989010938A1 publication Critical patent/WO1989010938A1/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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70528—CD58
    • 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
    • C07K16/2806—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 CD2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • LFA-3 lymphocyte function associated antigen-3
  • CD2 cluster of differentiation 2
  • thymocyte binding of thymocyte to thymic epithelial cells requires CD2 on the thymocyte and LFA-3 on the thymic epithelial cell.
  • LFA-3 in purified form inhibits intercellular adhesion between T lymphocytes and erythrocytes and mediates aggregation of T lymphocytes.
  • the invention provides a micelle of an adhesion protein, e.g., LFA-3, which naturally includes a phosphatidylinositol lipid ("PI") anchor; the micelle (which preferably contains less than about ten molecules of the protein) is capable of binding multivalently to a plurality of target molecules on a cell surface.
  • LFA-3 an adhesion protein
  • the invention provides multimeric, purified LFA-3 which has an avidity for CD2-bearing cells such that the Kd of the LFA-3 for the cells is lower than the Kd of monome ic LFA-3, which has been determined to be about 4.00 nM. More preferably, the Kd is ⁇ 50 nM, and even more 1 preferably ⁇ 20 nM.
  • An adhesion protein is defined 3 herein: as any protein mediating the contact and union of two or more human cells, and preferably, is a protein present on the surface of a cell.
  • a target molecule is defined herein as a molecule to which an adhesion protein binds selectively, i.e., binds to a degree greater than the degree to which it binds to any other molecule, preferably, this binding is exclusive.
  • the micelles of the invention can be used to inhibit the adhesion of a first cell to a second cell where the first cell bears on its surface a PI anchor-bearing protein capable of binding to a molecule present in multiple form on the second cell.
  • the method includes contacting the second cell with a micelle, preferably of less than about 10 such protein molecules (or micelle-forming, binding fragments thereof), which is capable of binding multivalently to the multiple molecules on the second cell.
  • the adhesion protein micelles of the invention can further be used for the treatment of medical conditions characterized by the presence of an excess of activated T- ⁇ ells by administering LFA-3 micelles in a physiologically compatible buffer to a patient to achieve a bloodstream concentration of LFA-3 effective to. inhibit the binding of the activated T-cells to other cells in the patient; the resultant bloodstream " concentration of LFA-3 is preferably 0.4 to 40 nM of LFA-3.
  • T-cells Disease states characterized by the presence of an excess of activated T-cells include multiple sclerosis, sarcoidosis, juvenile type diabetes mellitus, systemic lupus eryth atosis, thyroiditis, rheumatoid arthritis, an yloses spondylitis, primary biliary cirrhosis, autoimmune hemolytic anemia, immune thrombocytopenia purpura, myesthenia gravis, allograft rejection and graft-versus-host disease.
  • Another aspect of the invention features a method of increasing the avidity and decreasing the dissociation constant of any adhesion protein which naturally includes a PI anchor.
  • the method includes solubilizing the adhesion protein in a detergent solution and diluting the detergent below its critical micelle concentration in such a way as to form adhesion protein micelles.
  • a detergent is defined herein as any small amphipathic molecule having hydrophobic and hydrophilic regions that are preferably spatially separated on the molecule, i.e., one end is charged or polar and the other end is apolar.
  • the critical micelle concentration (CMC) is the concentration of a specific detergent in solution at which micelles just begin to form in the solution. This CMC is different for different detergents.
  • a further aspect of the invention features a method of effecting activation or proliferation of peripheral blood mononuclear cells or T lymphocytes by contacting such cells with LFA-3 micelles together with an antibody to CD2.
  • Figure 1 is a graph depicting the results of gel filtration of two forms of LFA-3;
  • Figure 2 is a schematic of the transmembrane (TM) form of LFA-3;
  • Figure 3 is a schematic of an adhesion protein micelle
  • 5 Figure 4 is the DNA sequence and deduced amino acid sequence of human TM LFA-3 cDNA.
  • Figure 5 is a graph of the dissociation time-course for LFA-3 micelles from Jurkat cells
  • Figures 6a and 6b are graphs depicting l_ ⁇ " equilibrium binding of LFA-3 micelles to Jurkat and resting T cells and the same data analyzed by the Scatchard method, respectively;
  • Figures 7a and 7b are graphs showing the time-course .and dose-response for LFA-3 and CD2 15 monoclonal antibody (MAb)-induced proliferation;
  • Figure 8 is an emission spectra graph which shows the cytoplasmic mobilization of Ca +2 over time in Jurkat cells induced by LFA-3 and CD2 MAb;
  • Figure 9 is an emission spectra graph which 20 shows the cytoplasmic mobilization of Ca +2 over time in resting T cells induced by LFA-3 and CD2 MAb;
  • Figures 10a and 10b are an emission spectra graph and a set of histograms, respectively, that show the concentration dependence of Ca +2 i.nflux increase
  • PBMC peripheral blood mononuclear cells
  • Peripheral blood T lymphocytes were enriched by nylon wool (Polyscience, Warrington, PA) filtration and plastic adherence.
  • the Jurkat cell line was obtained from Dr.
  • LFA-3 was purified from Triton X-100 lysates of human erythrocytes (Dustin, M.L., et al. , J. Exp. Med. , lS5_:677 (1987)), or JY B lymphoblastoid cells ( allner, B.P.. et al., J. Exp.
  • PLC phosphatidylinositol-specific phospholipase C
  • HBSS Hank's buffered saline solution
  • LFA-3 from erythrocytes was eluted from the immunoaffinity column at pH 3 in the presence of 1% octylglucoside (OG) detergent.
  • LFA-3 from PIPLC supernatants was eluted at pH 3 in the 0 absence of detergent, fractions containing LFA-3 were pooled (4-6 ml), passed over a 1 ml phenyl-Sepharose column (Pharmacia) equilibrated with TSA. Some preparations were passed over a 1 ml protein A-Sepharose CL-4B column after diluting 1:1 with MAPS binding buffer (with 1% OG for LFA-3 from erythrocytes) (Biorad, Richmond, CA) .
  • the LFA—3 purified as above has two forms: the mLFA-3 form, isolated from erythrocytes or lymphoblastoid cells, and the sLFA-3 form, isolated from lymphoblastoid cells.
  • mLFA-3 has an intact phosphatidylinositol (PI) membrane anchor and is also referred to as the lipid-linked form.
  • sLFA-3 has had the EH membrane anchor cleaved by PIPLC.
  • onTy- ⁇ rEFA--3 can form micelles.
  • LFA-3 transmembrane LFA-3
  • TM LFA-3 transmembrane LFA-3
  • TM LFA-3 can form micelles and will be described in more detail below. First, sLFA-3 will be described.
  • the middle step was added to remove traces of membranous material or LFA-3 micelles which might be released during enzyme treatment.
  • the yield from 50g of cells was about lOO ⁇ g.
  • N-glycanase When this material was treated with N-glycanase, a single band of 25.5 kD was obtained which corresponds to the lipid-linked forms from JY cells and erythrocytes.
  • the structure of mLFA-3 in the form of micelles, as used in subsequent activation experiments, was studied by gel filtration. The results are shown graphically in Figure 1. The size of mLFA-3 in the presence and absence of 1% OG detergent was examined by HPLC gel filtration.
  • the mRNA encoding this form differs from that of the mLFA-3 form, but only in the transmembrane and cytoplasmic domains of the molecule.
  • the CD2 binding domain (the extracellular portion) of both forms of LFA-3 is identical.
  • TM LFA-3 has no lipid anchor, the cytoplasmic domain includes a hydrophobic amino acid sequence which remains attached to the purified protein. Micelle formation occurs by protein-protein interactions rather than lipid-lipid interactions but the result, micelle formation, is the same. TM LFA-3 and mLFA-3 micelles have comparable stability and target binding avidity.
  • mice of mLFA-3 and TM LFA-3 were prepared by- threes- cycles ⁇ f ultrafiltration using a Centricon 30 apparatus (Amicon, Danvers, MA.), adding 2 ml of phosphate buffered saline (PBS) and reducing the volume to 50 ⁇ l with each cycle. Because the final ultrafiltrate of the last step contained the same low molecular weight components as the final retentate but not any LFA-3, this ultrafiltrate was used as a control for effects of buffer components in subsequent experiments.
  • PBS phosphate buffered saline
  • LFA-3 micelles occurs spontaneously when detergent is removed or diluted below its critical micelle concentration.
  • Detergent removal can be achieved by several techniques. The major requirement is that detergent is removed or diluted such that: protein aggregation is the most favorable event rather; than protein adsorbtion to other materials—i.e., test: tube surfaces, etc.
  • One technique which is successful with mLFA-3 is ultrafiltration in which an mI___3_ ⁇ -3- solution in 1% octylglucoside detergent is concentrated from 2ml to 50 ⁇ l and diluted with detergent-free solution. This results in dilution of octylglucoside to below its critical micelle concentration (25mM) .
  • Micelles created according to the invention have their hydrophobic lipid or hydrophobic proteinaceous "tails" sequestered within the micelle and the globular hydrophilic domains at the perimeter facing outwards.
  • Figure 3 is a schematic of such a micelle containing five monomer units. Formation of Micelles From Recombinant LFA-3
  • Micelles can also be formed from recombinantly produced LFA-3.
  • the preferred method of producing recombinant LFA-3 is in a eukaryotic expression system that enzymatically attaches the PI anchor, e.g., Chinese Hamster Ovary (CHO) cells.
  • TM form of- LES-3 can be produced in CHO cells or other cell systems that are not capable of attaching the PI anchor, such as murine L cells (Flavell et al. 1987).
  • TM LFA-3 or mLFA-3 is isolated and caused to form micelles in the same manner as naturally occurring LFA-3, as described above. Competition between LFA-3 Micelles and CD2 MAb.
  • CD2 MAb's were tested for their ability to block binding of mLFA-3 to Jurkat cells or peripheral T cells at saturating concentrations of MAb (Table 1). CD2 MAb's which strongly inhibit E-rosetting were able to completely block mLFA-3 binding.
  • the binding domains of these proteins in the micelles participate in multiple interactions when the micelle contacts the surface of a cell, thereby greatly increasing the avidity of the proteins for their targets.
  • a single protein-target interaction has a tendency to rapidly dissociate whereas the micelle can cause up to approximately 5 to 10 such interactions to occur simultaneously.
  • the overall dissociation constant is greatly decreased for the micelle form of an adhesion protein due to this multivalent binding.
  • LFA-3 from JY cells and erythrocytes exhibit different degrees of glycosylation. Therefore, both mLFA-3 and sLFA-3 were isolated from JY cells to rule out differences in binding arising from differences in glycoslyation.
  • mLFA-3 micelles from JY cells were of similar size to mLFA-3 micelles from erythrocytes despite being a mixture of PI and TM forms of LFA-3 (Dustin et al., Nature, supra).
  • sLFA-3 was iodinated to 5 the same specific activity as mLFA-3, and after incubation with Jurkat leukemic T-cells for 90 min at 4°C cells were spun through an oil cushion. The level of JY mLFA-3 micelle binding was found to be 150 fold higher: than. JY sLFA-3 binding (Table 4) . Consistent
  • CD2.1 MAb demonstrating that mLFA-3 binds to CD2 in the absence of other cell surface components.
  • mLFA-3 binding was defined as any binding inhibited by excess TS2/18 MAb. Binding of mLFA-3 was done for 1 hr. at 4°C in the presence of 0 control MAb (squares) or TS2/18 MAb (triangles). mLFA-3 bound saturably to Jurkat cells (filled symbols) and PBL-T (open symbols) (Fig. 6a). Scatchard plots showed that the K, for Jurkat CD2 was 1.7-2.2 nM, while the K d for PBL-T was 12-16 nM (Fig. 6b). This Kd value is 5 a measure of the avidity of the LFA-3 micelles for each of these cell types.
  • PBL-T in the presence of submitogenic concentrations of anti-CD2 MAb's.
  • the combination of mLFA-3 micelles (40 nM) with CD2.1 MAb was found to be strongly mitogenic for peripheral blood mononuclear cells (PBMC) from all donors tested (Table 7). This response was usually (9 of 10 donors) seen in the absence of exogenous IL-2, although phorbol myristate acetate (PMA) was still required for a maximal response.
  • PBMC peripheral blood mononuclear cells
  • Proliferation induced by the combination of mLFA-3 micelles and CD2.1 MAb was generally lower than that obtained with phytohemagglutanin (PHA), while the combination of mLFA-3, CD2.1 and PMA resulted in greater thymidine incorporation than PHA alone by up to two fold in some donors.
  • the combination of mLFA-3 and CD2.1 MAb was also mitogenic for nylon wool enriched T cells (Table 8).
  • the combination of sLFA-3 at concentrations up to 800 nM and CD2.1 MAb, with or without PMA, was not mitogenic for PBMC in two experiments for which sufficient amount of material could be obtained. Experiments with the same donors showed strong responses to mLFA-3 plus CD2.1 MAb with or without PMA.
  • Figures 7a and 7b are graphs showing the time-course and dose-response for LFA-3 and CD2.1 MAb induced proliferation.
  • PBMC peripheral blood mononuclear cells
  • the dose-responses for mLFA-3 and CD2.l MAb were determined in the presence of saturating concentrations of the other reagent.
  • PBMC were treated 5 with the indicated concentration of mLFA-3 with no addition (open squares), PMA (1 nM) (filled squares), CD2.1 (lO ⁇ g/ml), or CD2.1 and PMA (open circles).
  • Wells were pulsed on day 3 for 16 hr.
  • CD2.1 MAb 67 nM
  • maximal PBMC or PBL-T 0 proliferation was obtained with 4 nM mLFA-3 ( Figure 7b) . This is in the range of the K, for mLFA-3 binding to PBL-T of 12 nM.
  • CD2 molecules on different cells PBL-T or Jurkat cells were loaded with the fluorophore Indo-1 by incubation with, its precursor, Indo-1 acetoxymethyl ester, and relative [Ca ]. was determined by flow microfluorimetry using the ratio of Indo-1 emissions at different wavelengths (410 nm/480 nm) as an indication of [Ca +2 ] i> When mLFA-3 (up to 240 nM) was added to Jurkat cells (at 30-60 sec.) there was no change in the ratio within 15 min. ( Figure 9). Similarly, CD2.1 MAb (67 nM) alone had no effect on [Ca +2]. ( Figure 9).
  • FIG. 10b is a graph showing histograms of sections through activation time plots at 3 minutes: 0.04 nM (solid line), 0.4 nM (dashed line), 1.2 nM (dot-dash line), 4 nM (dot-dot-dash line) and
  • Protein micelles of the invention can be used therapeutically to competitively inhibit reactivity of specific surface antigens on target molecules.
  • LFA-3 micelles can be co- internalized with CD2, causing loss of CD2 from the cell surface. This irreversible internalization allows LFA-3 micelles to be used therapeutically below the Kd of monomeric LFA-3 Kd to down-regulate CD2.
  • LFA-3 micelles of the invention in a physiologically compatible carrier such as saline are preferably administered to yield a therapeutically effective concentration of LFA-3 in the blood of 0.04 to 4 nM.
  • a physiologically compatible carrier such as saline
  • 0.5 to 50 ⁇ g/kg of micelle/patient/day are administered intravenously to achieve this concentration.
  • the micelles function by effectively saturating CD2 receptor sites on the surfaces of T-cells and thereby inhibit binding of those T-cells to other cells.
  • This binding inhibition can ameliorate the effects of disease states in.which binding of T-cells to other cells is a continuing factor, e.g., autoimmune diseases such as rheumatoid arthritis; allograft rejection; and graft-versus-host disease.
  • Input counts were 100,00/10 cells. Bound and free were separated by centrifugation through a 15% BSA cushion. All t MAb except control and OKT3 are against CD2. Results are average of quadruplicates with standard deviation and are representative of two experiments.
  • 125 I-mLFA-3 was 200,000 cpm. Bound and free radioactivity were separated by centrifugation through a 15% BSA cushion. Control IgGl, TS2/18 and TS2/9 were added at lO ⁇ g/ml and cold mLFA-3 was added at 5 ⁇ g/ml. Results are averages of duplicates and are representative of three experiments.
  • PBMC peripheral blood mononuclear cells

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Abstract

Micelles d'une protéine d'adhésion comprenant naturellement un lipide de phosphatidylinositol d'ancrage, la micelle pouvant se lier de manière multivalente à plusieurs molécules cibles sur une surface cellulaire.
PCT/US1989/001858 1988-05-04 1989-05-02 Micelles de proteine WO1989010938A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US18997188A 1988-05-04 1988-05-04
US189,971 1988-05-04
US23892688A 1988-08-31 1988-08-31
US238,926 1988-08-31

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EP (1) EP0372056A4 (fr)
JP (1) JPH03500659A (fr)
AU (1) AU638210B2 (fr)
CA (1) CA1338079C (fr)
WO (1) WO1989010938A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991013981A1 (fr) * 1990-03-15 1991-09-19 Basf Aktiengesellschaft Nouvelle proteine, sa fabrication et son utilisation
EP0463157A1 (fr) * 1990-01-24 1992-01-02 Biogen, Inc. Lfa-3 utilise comme adjuvant de vaccins
EP0466806A1 (fr) * 1989-04-10 1992-01-22 Biogen, Inc. Liaison de proteines par phosphatidylinositole
EP0468257A1 (fr) * 1990-07-20 1992-01-29 Bayer Corporation Formes multimériques du récepteur du rhinovirus humain
US5589453A (en) * 1988-09-01 1996-12-31 Molecular Therapeutics, Inc. Human rhinovirus receptor protein (ICAM-1) that inhibits rhinovirus attachment and infectivity
US5674982A (en) * 1990-07-20 1997-10-07 Bayer Corporation Multimeric form of human rhinovirus receptor protein
US5686582A (en) * 1990-07-20 1997-11-11 Bayer Corporation Multimeric forms of human rhinovirus receptor protein
US6107461A (en) * 1990-07-20 2000-08-22 Bayer Corporation Multimeric forms of human rhinovirus receptor and fragments thereof, and method of use
US6143298A (en) * 1988-09-01 2000-11-07 Bayer Corporation Soluble truncated forms of ICAM-1

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US4169090A (en) * 1976-09-30 1979-09-25 General Foods, Limited Protein product and process for preparing same
US4578269A (en) * 1982-10-18 1986-03-25 Bror Morein Immunogenic protein or peptide complex, method or producing said complex and the use thereof as an immune stimulant and as a vaccine

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US4169090A (en) * 1976-09-30 1979-09-25 General Foods, Limited Protein product and process for preparing same
US4578269A (en) * 1982-10-18 1986-03-25 Bror Morein Immunogenic protein or peptide complex, method or producing said complex and the use thereof as an immune stimulant and as a vaccine

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Title
BARBOSA, J. of Immun., 36, No. 8, 3085, 1986 "The Mapping and Somatic Cell Hybrid Analysis of the Role of Human Lymphecyte Function-Accociated Antigen-3 (LFA-3) in CTL-Target Cell Interaction". *
Francisco Sanchez-Madrid, Proc. Natl. Acad. Sci. 79, 7489-7493, 1982 "Three Distinct Antigens Associated with Human T-Lymphoyte-Mediated Cytosis: LFA-1, LFA-2, and LFA-3. *
L.S. WOLF, Clinical Research, 34, No. 2, 1986 "Monoclonal Antibodies to T11, LFA-2, and LFA-3 Antigens Inhibit Binding of Human Thymocytes to Autologous Thymic Epithelial Cells" see 647A, first Paragraph. *
PAUL J. MARTIN, J. of Immun., 131, No. 1 180-185, 1983 "Identification and Functional Characterization of Two Distinct Epitopes on the Human T-Cell Surface Protein Tp 50". *
See also references of EP0372056A4 *
STEN HAMMERSTROM, Scand. J. Immunol., 2, 53-56, 1973 "Binding of Helix Pomatia A Hemagglutinin to Human Erythrocytes and Their Cells" see the Abstract. *
STEPHEN SHAW, Nature, 323, 262, 1986 "Two Antigen-Independent Pathway used by Human Cytotoxic T-Cell Clones" see the Paragraph Bridging pages 263 to 264. *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6143298A (en) * 1988-09-01 2000-11-07 Bayer Corporation Soluble truncated forms of ICAM-1
US5589453A (en) * 1988-09-01 1996-12-31 Molecular Therapeutics, Inc. Human rhinovirus receptor protein (ICAM-1) that inhibits rhinovirus attachment and infectivity
EP0466806A1 (fr) * 1989-04-10 1992-01-22 Biogen, Inc. Liaison de proteines par phosphatidylinositole
EP0466806A4 (en) * 1989-04-10 1992-08-12 Biogen, Inc. Phosphatidylinositol linking of proteins
EP0463157A1 (fr) * 1990-01-24 1992-01-02 Biogen, Inc. Lfa-3 utilise comme adjuvant de vaccins
EP0463157A4 (en) * 1990-01-24 1992-06-03 Biogen, Inc. Lfa-3 as a vaccine adjuvant
WO1991013981A1 (fr) * 1990-03-15 1991-09-19 Basf Aktiengesellschaft Nouvelle proteine, sa fabrication et son utilisation
US5686581A (en) * 1990-07-20 1997-11-11 Bayer Corporation Multimeric form of human rhinovirus receptor protein
US5674982A (en) * 1990-07-20 1997-10-07 Bayer Corporation Multimeric form of human rhinovirus receptor protein
US5686582A (en) * 1990-07-20 1997-11-11 Bayer Corporation Multimeric forms of human rhinovirus receptor protein
US5871733A (en) * 1990-07-20 1999-02-16 Bayer Corporation Multimeric forms of human rhinovirus receptor protein
EP0987329A2 (fr) * 1990-07-20 2000-03-22 Bayer Corporation Formes multimériques du récepteur du rhinovirus humain
US6096862A (en) * 1990-07-20 2000-08-01 Bayer Corporation Multimeric antiviral agent
US6107461A (en) * 1990-07-20 2000-08-22 Bayer Corporation Multimeric forms of human rhinovirus receptor and fragments thereof, and method of use
EP0468257A1 (fr) * 1990-07-20 1992-01-29 Bayer Corporation Formes multimériques du récepteur du rhinovirus humain
EP0987329A3 (fr) * 1990-07-20 2004-03-03 Bayer Corporation Formes multimériques du récepteur du rhinovirus humain

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EP0372056A1 (fr) 1990-06-13
EP0372056A4 (en) 1991-05-15
CA1338079C (fr) 1996-02-20
AU638210B2 (en) 1993-06-24
JPH03500659A (ja) 1991-02-14
AU3731089A (en) 1989-11-29

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