US20050100964A1 - Diagnostic methods for congestive heart failure - Google Patents

Diagnostic methods for congestive heart failure Download PDF

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US20050100964A1
US20050100964A1 US10/706,599 US70659903A US2005100964A1 US 20050100964 A1 US20050100964 A1 US 20050100964A1 US 70659903 A US70659903 A US 70659903A US 2005100964 A1 US2005100964 A1 US 2005100964A1
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glycophorin
antibody
chf
accordance
antigen
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George Jackowski
Tracy Van Lieshout
Brad Thatcher
Rulin Zhang
Jason Yantha
Michele Rasamoelisolo
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Nanogen Inc
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Assigned to NANOGEN, INC. reassignment NANOGEN, INC. SECURITY INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SYNX PHARMA INC.
Priority to CA002544838A priority patent/CA2544838A1/en
Priority to PCT/CA2004/001945 priority patent/WO2005045436A1/en
Priority to AU2004287908A priority patent/AU2004287908A1/en
Priority to EP04797198A priority patent/EP1692513B1/de
Priority to JP2006538618A priority patent/JP4646918B2/ja
Assigned to SYN X PHARMA, INC. reassignment SYN X PHARMA, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LIESHOUT, TRACY VAN, THATCHER, BRAD, ZHANG, RULIN, JACKOWSKI, GEORGE, YANTHA, JASON, RASAMOELISOLO, MICHELE
Publication of US20050100964A1 publication Critical patent/US20050100964A1/en
Assigned to NANOGEN POINT OF CARE INC. reassignment NANOGEN POINT OF CARE INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: SYN X PHARMA INC.
Assigned to NANOGEN INC. reassignment NANOGEN INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: NANOGEN POINT OF CARE INC.
Priority to US11/786,885 priority patent/US20090068676A1/en
Priority to US13/024,963 priority patent/US8263351B2/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure

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  • the instant invention relates generally to the field of immunology; particularly to the use of immunologic assays to diagnose abnormal or disease states and most particularly to a sandwich ELISA (enzyme-linked immunosorbent assay) assay for the quantification of a truncated glycophorin circulating in biological fluid which is diagnostic for congestive heart failure (CHF).
  • sandwich ELISA enzyme-linked immunosorbent assay
  • CHF congestive heart failure
  • CHF is a serious condition with a high mortality rate affecting approximately five million Americans (see U.S. Pat. No. 6,572,895 for a discussion of CHF). It is currently believed that CHF is not a distinct disease process in itself, but rather represents the effect of multiple abnormalities which interact together to ultimately produce the progressive loss of the ability of the heart to function as a circulatory pump.
  • Major pathophysiologic abnormalities which occur in CHF are activation of the hypothalmic-pituitary-adrenal axis, systemic endothelial dysfunction and myocardial re-structuring.
  • the progression of CHF can be initiated by an event such as myocardial infarction wherein the heart muscle is damaged or it can result from hypertension and/or cardiac malformations.
  • patients with certain conditions such as insulin resistance and Type II diabetes have a particularly high risk for heart failure and poor prognosis once they develop CHF (Solisser et al. European Heart Journal 20:789-795 1999).
  • Biopolymer markers are harbingers of disease and/or disease progression. Association of such biopolymer markers with abnormal and/or disease states provides new diagnostic avenues which may allow identification of patients in the early stages of disease or patients at risk for developing disease. Identification of biopolymer markers diagnostic for CHF is particularly advantageous considering the progressive pathophysiology involved in CHF. What is lacking in the art is an efficient, easy to perform diagnostic method capable of identifying an individual suffering from CHF.
  • the instant invention provides an efficient, easy to perform diagnostic method capable of identifying an individual suffering from CHF.
  • the method comprises a sandwich ELISA assay using mouse monoclonal antibodies (anti-glycophorins) to quantify elevated glycophorin in biological fluids.
  • Glycophorin is the major integral membrane protein of the mammalian red blood cell (RBC) and is highly glycosylated. The glycosylation of glycophorin is responsible for the overall negative charge of the RBC cellular surface leading to the normal electrostatic repulsion among red blood cells.
  • red blood cell (RBC) membrane proteins including glycophorins
  • the instant inventors identified an abnormal, circulating glycophorin in the plasma of CHF patients. This glycophorin had a lower molecular weight than that of normal glycophorin, thus it is predicted to be a truncated fragment which has been cleaved from the RBC membrane surface during the disease process.
  • Three mouse monoclonal antibodies are used in the ELISA assay of the instant invention; 3F4, 6G4 and 5F4.
  • Monoclonal antibody 3F4 recognizes amino acid residues 5-25 of SEQ ID NO:2 and SEQ ID NO:4 (glycophorins A and B).
  • Monoclonal antibody 6G4 recognizes amino acid residues 39-45 of SEQ ID NO:2 (glycophorin A).
  • Monoclonal antibody 5F4 recognizes amino acid residues 107-119 of SEQ ID NO:2 (glycophorin A).
  • CHF congestive heart failure
  • FIG. 1 shows the data resulting from the sandwich ELISA using monoclonal antibody 3F4.
  • FIG. 2 shows the data resulting from the sandwich ELISA using monoclonal antibodies 6G4, 5F4 and 3F4.
  • FIG. 3 shows the data resulting from the direct ELISA evaluating the presence of an autoantibody to glycophorin.
  • FIG. 4 shows the results of immunoprecipitation of glycophorin from the plasma of CHF patients.
  • FIGS. 5 A-C show chromatograms; FIG. 5A shows captured glycophorin from CHF patients; FIG. 5B shows captured glycophorin from healthy patients and FIG. 5C shows captured purified glycophorin.
  • FIG. 6 shows chromatograms after deglycosylation treatment; the top chromatograph shows purified glycophorin; the middle chromatograph shows captured glycophorin from CHF patients and the bottom chromatograph is a control run without a glycophorin sample.
  • CHF congestive heart failure
  • GP glycophorin
  • glycophorin A As used herein, the abbreviation “GPA” refers to glycophorin A.
  • glycophorin B As used herein, the abbreviation “GPB” refers to glycophorin B.
  • GPAx2 refers to the dimerized form of glycophorin A.
  • GPBx2 refers to the dimerized form of glycophorin B.
  • ELISA enzyme-linked immumosorbent assay
  • red blood cell As used herein, the abbreviation “RBC” refers to red blood cell.
  • MoAb refers to monoclonal antibody.
  • MS mass spectrometry
  • SELDI refers to a mass spectrometric technique; surface enhanced laser desorption ionization.
  • PBS phosphate buffered saline
  • RBC red blood cell
  • erythrocyte erythrocyte
  • glycophorin refers to the major integral glycoprotein of the mammalian erythrocyte membrane. Glycophorin is highly glycosylated and occurs in isoforms A and B (see Concise Encyclopedia: Biochemistry and Molecular Biology, Third Edition, Revised and Expanded by Thomas A. Scott and E. Ian Mercer, Walter de Gruyter, Berlin-New York 1997, pages 201-202 and Instant Notes: BioChemistry, 2nd edition, B. D. Hames and N. M. Hooper, Springer-Verlag New York 2000, pages 125, 126 and 130 for an introduction to the RBC membrane and glycophorins).
  • the term “circulating, truncated glycophorin” refers to the abnormal glycophorin fragment identified by the assay of the instant invention in the serum of CHF patients.
  • the 3F4 mouse anti-glycophorin monoclonal antibody which recognizes the extracellular portion of glycophorin A and B binds to this circulating, truncated glycophorin.
  • This circulating, truncated glycophorin is structurally different from the normal soluble glycophorin and is theorized to be a fragment cleaved from the RBC surface during disease processes.
  • biological fluid refers to any bodily fluid.
  • Illustrative, albeit non-limiting examples are blood, blood products, urine, saliva, cerebrospinal fluid and lymphatic fluid.
  • subject refers to any mammalian organism.
  • a particularly preferred subject is a human.
  • the term “corresponding” is used generally with reference to antibody-antigen binding complexes, for example, an antibody corresponding to an antigen will bind to the antigen under physiologic conditions.
  • the bound antibody-antigen is referred to as an antibody-antigen binding complex.
  • signal generating substance refers to any material which undergoes a measurable reaction. Illustrative, albeit non-limiting examples are fluorophores, enzymes and radioisotopes. A particularly preferred signal generating substance is peroxidase, the use of which is illustrated in the examples herein.
  • the term “congestive heart failure” refers to a progressive, debilitating condition wherein the heart loses its ability to function as a circulatory pump.
  • antibody refers to a protein secreted by B lymphocytes capable of binding specific molecules under physiologic conditions.
  • monoclonal antibody refers to an antibody having single epitope specificity.
  • polyclonal antibody refers to an antibody capable of binding with multiple epitopes.
  • the term “antigen” broadly refers to any substance which induces an immune reaction; more particularly the term “antigen” refers to the corresponding binding partner of an antibody.
  • auto-antibody refers to an antibody which recognizes self antigens, for example, antibodies produced by an organism which bind the organism's own proteins are referred to as auto-antibodies.
  • Specific antibodies can be used to quantify the amount of corresponding antigen in a biological sample.
  • the term “ELISA” refers to an enzyme-linked immunosorbent assay which can quickly detect and quantify minute amounts (less than a nanogram) of antigen in a biological sample.
  • the test antibody is bound to an inert polymer support, such as a plastic tray with molded wells, and then exposed to the biological sample. Unbound proteins are washed away and a second antibody that reacts with the antigen at a different epitope than the test antibody reacts with is added. This second antibody has an enzyme attached to it that converts a colorless or nonfluorescent substrate into a colored or fluorescent product.
  • the amount of second antibody bound, and hence the amount of protein antigen present in the original biological sample, is determined by the quantification of the intensity of color or fluorescence produced.
  • This ELISA assay is also referred to as an “indirect ELISA” or a “sandwich ELISA”. (see Instant Notes: BioChemistry, 2nd edition, B. D. Hames and N. M. Hooper, Springer-Verlag New York 2000, pages 112-114 for an introduction to the general principles of ELISA assays).
  • direct wherein the antigen is bound to an inert polymer support and exposed to a biological sample containing the corresponding antibody.
  • the immune system views these biopolymer markers as invading pathogens or foreign bodies whose threat must be neutralized.
  • auto-antibodies are formed to these biopolymer markers.
  • These auto-antibodies can be characterized as sequela which are indicative of the original damaging insult to the organism.
  • the presence of the auto-antibodies validates the theory that cellular damage acts as an initiator of an immune response leading to a cascade of auto-antibody production which ultimately manifests itself in a characteristic and often predictable disease state.
  • the appearance of these biopolymer markers and their associated auto-antibodies are harbingers of disease and/or disease progression and are useful for diagnostic purposes.
  • red blood cell membrane Damage to the red blood cell membrane is known to occur in disease processes such as diabetes and CHF.
  • enzyme production and/or activation neutril proteases, metalloproteases, sialidases and endopeptidases
  • red blood cell membrane proteins Gaczy ⁇ ska et al. Cytobios 75:7-11 1993; Venerando et al. Blood 99(3):1064-1070 2002; Wegner et al. Cardiovascular Research 31:891-898 1996; Piwowar et al. Clinical Chemistry Lab Medicine 38(12):1257-1261 2000 and Santos-Silva et al. Clinica Chimica Acta 320:29-35 2002).
  • erythrocyte (RBC) aggregability is increased in diabetes and in vascular atherosclerotic disease (Caimi et al. Thromb Haemost 83:516-517 2000; Demiroglu et al. Experimental Clinical Endocrinol Diabetes 107(1):35-39 1999; Martinez et al. Clinical Hemorheology and Microcirculation 18:253-258 1998 and Ziegler et al. Metabolism 43(9):1182-1186 1994). Alterations in RBC membrane phospholipids are associated with RBC aggregability (Mart ⁇ nez et al. Clinical Hemorheology and Microcirculation 18:253-258 1998).
  • Sphingomyelin is the main phospholipid of the outer membrane and has been shown to contain a greater percentage of saturated fatty acids in diabetic patients than in non-diabetic patients. This increase in saturation is thought to reduce electrostatic repulsion between red blood cells, which in turn increases their aggregability.
  • Glycophorin is the major RBC integral membrane glycoprotein.
  • the high sialylation of glycophorin is responsible for the negative surface charge which leads to the normal electrostatic repulsion between red blood cells (Eylar et al. The Journal of Biological Chemistry 237(6):1992-2000 1962).
  • the increase in enzyme production and/or enzyme activation in disease processes such as diabetes results in the loss of glycophorins from the RBC membrane. These glycophorin fragments are released into the bodily fluids where they stimulate the production of auto-antibodies.
  • the decrease in glycophorin in turn leads to a decrease in the normal negative charge of the RBC membrane surface and thus decreases the overall electrostatic repulsion between blood cells. Loss of the electrostatic repulsion between red blood cells results with the aggregation of red blood cells seen in diabetes.
  • the instant inventors propose that the circulating, truncated glycophorin identified in the plasma of CHF patients using the sandwich ELISA assay described herein is an extracellular glycophorin fragment which has been cleaved from the RBC membrane during the disease process.
  • This circulating, truncated glycophorin is structurally different from the normal soluble form of glycophorin.
  • the mouse anti-glycophorin 3F4 monoclonal antibody which recognizes amino acid residues 5-25 of SEQ ID NO:2 and SEQ ID NO:4 (glycophorins A and B) also recognizes the circulating, truncated glycophorin.
  • Homo sapiens (human) glycophorin A nucleic acid sequence is disclosed as SEQ ID NO:1 and translates into glycophorin A protein disclosed as amino acid sequence SEQ ID NO:2.
  • Homo sapiens (human) glycophorin B nucleic acid sequence is disclosed as SEQ ID NO:3 and translates into glycophorin B protein disclosed as amino acid sequence SEQ ID NO:4.
  • the mouse anti-glycophorin monoclonal antibodies used in the following experiments were purchased from BioAtlantic.
  • Monoclonal antibody 6G4 recognizes amino acid residues 39-45 of SEQ ID NO:2 (glycophorin A).
  • Monoclonal antibody 5F4 recognizes the intracellular portion of glycophorin A comprising amino acid residues 107-119 of SEQ ID NO:2.
  • Monoclonal antibody 3F4 recognizes the extracellular portion of glycophorins A and B amino acid residues 5-25 of SEQ ID NO:2 and SEQ ID NO:4.
  • the binding of the 3F4 antibody to its epitope is sugar-dependent whereas the binding of the 6G4 antibody is not.
  • the mouse anti-glycophorin 3F4 monoclonal antibody was deposited with the American Type Culture Collection (ATCC) on Apr. 23, 2000 as hybridoma NaM26-3F4D11A2 under Accession number PTA-5154.
  • the American Type Culture Collection (ATCC) is located at 10801 University Boulevard, Manassas, Va. 20110-2209.
  • FIG. 1 shows the result of the sandwich ELISA using the 3F4 monoclonal antibody.
  • the absorbance at 450 nm is shown on the Y axis.
  • the signal is significantly higher in CHF plasma than in controls (p ⁇ 0.001) calculated by an independent t-test indicating a higher amount of glycophorins in CHF plasma samples.
  • the 3F4 MoAb recognizes the common sequence on both glycophorins A and B (amino acid residues 5-25 of SEQ ID NO:2 and SEQ ID NO:4). This binding is sugar-dependent since this fragment of glycophorin is highly glycosylated.
  • the MoAbs 6G4 (recognizes amino acid residues 39-45 of SEQ ID NO:2) and 5F4 (recognizes amino acid residues 112-129 of SEQ ID NO:2) were used. Both bind to the glycophorin A backbone independently of the sugar chains.
  • the Y axis represents the absorbance read at 450 nm. Glycophorin captured from the plasma of CHF patients is shown on the left and the glycophorin captured from normal plasma is shown on the right in all three panels.
  • Glycophorin is known to be highly immunogenic due to the presence of a high amount of sugar chains. Once found in plasma it may induce an immune response generating anti-glycophorin auto-antibody.
  • the Y axis represents the absorbance read at 450 nm.
  • FIG. 3 shows the presence of auto-antibodies in CHF; independent to the blood group (M or N) and the heavy sialic acids on glycophorin.
  • the eluate was concentrated on CentriVap Concentrator (Labconco), resuspended in 50 ul of SDS-PAGE sample buffer, boiled 5 minutes at 100° C. and then loaded on 10% SDS-PAGE gel.
  • proteins were transferred onto a nitrocellulose membrane and stained with 3F4 MoAb anti-GPA+B followed by a peroxidase labeled goat polyclonal anti-mouse IgG (H+L) diluted ⁇ fraction (1/50,000) ⁇ in PBST (Jackson ImmunoResearch).
  • the immunoblot was then developed using ECL (Amersham Pharmacia). To control the cross-reactivity of the secondary antibody to the 3F4 eluted from the column, the blot was incubated with the secondary antibody alone.
  • glycophorins found in CHF plasma have a molecular weight of 75, 45 and 40 kDa (lane 2, blot incubated with 3F4).
  • glycophorins run at 80-70-40-37 and 20 kDa as dimer form of GPA, dimer GPA/GPB, dimer form of GPB, monomer form of GPA and monomer form of GPB, respectively as shown on lane 1 loaded with normal glycophorin purified from normal red blood cell membrane.
  • glycophorins found in the plasma of CHF patients have different molecular weights as compared to the normal glycophorin purified from RBC membranes.
  • the immunoblot was incubated with the secondary antibody alone (control) or with the 3F4 antibody and then the secondary antibody. Lane 1 (in both blots) shows glycophorin purified from RBC membranes and Lane 2 (both blots) shows glycophorin from CHF patient plasma. Protein markers from 25 to 200 kDaltons are shown on the far left.
  • the IgG identified in control and 3F4 blots is the mouse monoclonal 3F4 used for the immunoprecipitation and released from the column.
  • a band with a high MW>200 kDa is also detected. The instant inventors are not sure about the nature of this band.
  • the band may be a complex form of IgM or IgG autoantibodies and the glycophorins.
  • the method of the instant invention can be carried out using the techniques of mass spectrometry.
  • the PS20 chip (Ciphergen) was washed with pure Acetonitrile-190 (ACN) (Caledon) and allowed to air dry.
  • ACN Acetonitrile-190
  • 50 ⁇ g of Protein G (Pierce) was dissolved in 50 ⁇ l UF water and 1 ul was loaded to each spot containing 1 ⁇ l of ACN.
  • the mixture was incubated 1 hour in a humidity chamber and then the sp ot was blocked with 10 ⁇ l of 0.5M Tris-HCl pH 7.4 (Caledon) for 15 minutes.
  • the chip was then washed with UF water and allowed to air dry.
  • MoAb Monoclonal antibody
  • BioAtlantic Monoclonal antibody
  • 3F4 Monoclonal antibody
  • BioAtlantic was diluted 1 ⁇ 3 in PBS containing 0.1% TRITON X (Sigma) and 3 ⁇ l of the MoAb solution was loaded per spot and incubated for 1 hour in a humidity chamber. Unbound MoAb was washed away from the chip by washing with PBS.
  • the glycophorin at 1 mg/ml was diluted 1 ⁇ 5 in PBS; CHF and normal plasma samples were diluted 1 ⁇ 5 in PBS containing 0.05% Tween 20, and 2 ⁇ l of each were loaded per spot.
  • the chip was then incubated for 1 hour in a humidity chamber and washed twice with UF water.
  • the captured glycophorin was then treated with Endoproteinase GluC (Roche Diagnostics).
  • Endoproteinase GluC Endoproteinase GluC
  • FIG. 5A shows data resulting from the on-chip treatment of the captured glycophorin from CHF.
  • FIG. 5B shows data resulting from the on-chip treatment of the normal plasma samples.
  • FIG. 5C shows data resulting from the on-chip treatment of purified glycophorin.
  • a (glyco)peptide with a m/z of 2361+H is found in both CHF and glycophorin demonstrating that the (glyco)protein captured from CHF corresponds probably to the glycophorin.
  • the chromatograms FIGS. 5 A-C
  • FIG. 6 shows on-chip deglycosylation treatment of the glycopeptides captured from either purified glycophorin or CHF plasma using the 3F4 monoclonal antibody coated on a PS20 chip.
  • FIG. 6 shows on-chip deglycosylation treatment of the glycopeptides captured from either purified glycophorin or CHF plasma using the 3F4 monoclonal antibody coated on a PS20 chip.
  • FIG. 6 shows on-chip deglycosylation treatment of the glycopeptides captured from either purified glycophorin or CHF plasma using the 3F4 monoclonal antibody coated on a PS20 chip.
  • FIG. 6 shows on-chip deglycosylation treatment of the glycopeptides captured from either purified glycophorin or CHF plasma using the 3F4 monoclonal antibody coated on a PS20 chip.
  • FIG. 6 shows on-chip deglycosylation treatment of the glycopeptides captured from either purified glycophorin or CHF plasma using the 3F4 monoclonal antibody coated on a PS20 chip.
  • the instant invention provides a sandwich ELISA assay for quantification of a truncated, glycophorin circulating in biological fluid which is diagnostic for CHF. It is important to note that glycophorin has not been previously recognized as a marker for congestive heart failure (CHF).
  • CHF congestive heart failure
  • the instant inventors are the first to document glycophorin as a marker for CHF and the assay described herein provides an efficient, easy to perform diagnostic method capable of identifying an individual suffering from CHF.

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US10/706,599 US20050100964A1 (en) 2003-11-11 2003-11-11 Diagnostic methods for congestive heart failure
JP2006538618A JP4646918B2 (ja) 2003-11-11 2004-11-10 鬱血性心不全の診断法
EP04797198A EP1692513B1 (de) 2003-11-11 2004-11-10 Diagnoseverfahren für kongestives herzversagen
PCT/CA2004/001945 WO2005045436A1 (en) 2003-11-11 2004-11-10 Diagnostic methods for congestive heart failure
AU2004287908A AU2004287908A1 (en) 2003-11-11 2004-11-10 Diagnostic methods for congestive heart failure
CA002544838A CA2544838A1 (en) 2003-11-11 2004-11-10 Diagnostic methods for congestive heart failure
US11/786,885 US20090068676A1 (en) 2003-11-11 2007-04-13 Diagnostic methods for congestive heart failure
US13/024,963 US8263351B2 (en) 2003-11-11 2011-02-10 Diagnostic methods for congestive heart failure

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CN108129554A (zh) * 2010-08-10 2018-06-08 洛桑聚合联合学院 红细胞结合性治疗剂
US9517257B2 (en) 2010-08-10 2016-12-13 Ecole Polytechnique Federale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US10953101B2 (en) 2014-02-21 2021-03-23 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US10046056B2 (en) 2014-02-21 2018-08-14 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US10946079B2 (en) 2014-02-21 2021-03-16 Ecole Polytechnique Federale De Lausanne Glycotargeting therapeutics
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US11253579B2 (en) 2017-06-16 2022-02-22 The University Of Chicago Compositions and methods for inducing immune tolerance
EP3826676A4 (de) * 2018-10-31 2022-05-04 I-Mab Biopharma US Limited Neuartige cd47-antikörper und verfahren zur verwendung davon

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EP1692513B1 (de) 2012-06-06
US20090068676A1 (en) 2009-03-12
EP1692513A4 (de) 2008-02-20
JP4646918B2 (ja) 2011-03-09
US8263351B2 (en) 2012-09-11
WO2005045436A1 (en) 2005-05-19
JP2007510915A (ja) 2007-04-26
CA2544838A1 (en) 2005-05-19
EP1692513A1 (de) 2006-08-23
US20110183436A1 (en) 2011-07-28

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