WO1995029243A1 - Proteine du domaine-i derivee de mac-1 et utilisee dans le blocage de l'adhesion et de la migration des neutrophiles - Google Patents

Proteine du domaine-i derivee de mac-1 et utilisee dans le blocage de l'adhesion et de la migration des neutrophiles Download PDF

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WO1995029243A1
WO1995029243A1 PCT/US1995/004439 US9504439W WO9529243A1 WO 1995029243 A1 WO1995029243 A1 WO 1995029243A1 US 9504439 W US9504439 W US 9504439W WO 9529243 A1 WO9529243 A1 WO 9529243A1
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Prior art keywords
domain
protein
gst
gly
adhesion
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PCT/US1995/004439
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English (en)
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Robert L. Heinrikson
Donald C. Anderson
Che-Shen C. Tomich
Michael B. Fairbanks
Mary L. Bajt-Jaeschke
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Pharmacia & Upjohn Company
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Priority to JP7527670A priority Critical patent/JPH09512426A/ja
Priority to EP95917555A priority patent/EP0758393A1/fr
Priority to AU23558/95A priority patent/AU2355895A/en
Publication of WO1995029243A1 publication Critical patent/WO1995029243A1/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
    • C07K14/70546—Integrin superfamily
    • C07K14/70553—Integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Mac-1 1-Domain Protein Useful in Blocking Adhesion and Migration of Neutrophils BACKGROUND OF THE INVENTION
  • Cell adhesion molecules are essential in a number of cellular processes including immunity and inflammation, cell anchorage and migration, and cell growth and differentiation.
  • the leukocyte integrins are involved in mediating the adhesion and endothelial trans-migration of leukocytes into inflamed tissue.
  • These integrins are membrane-anchored proteins on the surface of the leukocytes, serving as receptors to various ligands.
  • These receptors are heterodimer proteins consisting of alpha and beta subunits, and since the 3 known leukocyte integrins share a common beta-2 subunit, they are also called beta-2 integrins.
  • the ⁇ subunit CD 18 interacts with the ⁇ subunit CD 11 in a noncovalent manner.
  • Both the ⁇ and ⁇ subunits of these integrins are large glycoproteins; the
  • CD 18 subunit is about 95 kd in molecular mass.
  • the 170 kd ⁇ subunit CD lib in Mac-1 contains an I-domain (or A-domain) which is also conserved in CD Ila, CD lie, and a few other integrins (Larson et al, J. Cell. Biol., 108:703-12 (1989)).
  • This I- domain of about 200 amino acid residues appears to be an insertion sequence; it is not found in some other integrins.
  • the I-domain exhibits sequence similarity to the domains involved in ligand binding of proteins such as von Willebrand factor, cartilage matrix protein, and complement proteins C2 and factor B.
  • the present invention demonstrates that producing recombinant proteins in E. coli as fusions to GST has some of the foUowing advantages: 1) GST itself is expressed at high levels and therefore should promote high level production of the fusion protein, 2) GST itself is highly soluble and can serve to faciUtate/stabilize the folding of the fusion protein, leading to a soluble and active product, and 3) GST can be used as a purification handle, i.e., one-step purification of the fusion protein by affinity chromatography on immobiUzed glutathione, using a glutathione Sepharose 4B column.
  • the present construct overcomes problems caused by the physical close proximity of the GST and I-domain, masking the accessibiUty of the cleavage site.
  • the present construct introduces residues such as glycine at the GST and I-domain fusion point to separate the two structures.
  • the present invention teaches a GST and I-domain fusion with a Factor X a cleavage site to compare the efficiency of thrombin and Factor X a proteolysis in such fusion products.
  • the different fusion junctions linking GST and I-domain are shown below.
  • the residues underlined are the recognition sequence for thrombin or Factor X a with the arrow(- T-) indicating the cleavage point.
  • Factor X a GST-Ile-Glu-Glv-Ar ? -T-Glv-Ile-Pro-Glv-Glv-(Serl33...Glu337)
  • the CDllb I-domain sequence is that defined by the serine at position #133 ending at the glutamic acid at #337.
  • their E. coli- ⁇ e ⁇ ved recombinant CDllb I-domain starts at glycine at #111 and ends with alanine at #318 (see Figure 1).
  • Michishita et al took advantage of the available restriction enzyme sites to clone a sequence coding for the region around the I-domain, thus shifting the I- domain towards the N-terminus of CDllb.
  • the addition of two glycine residues between the GST and I-domain in the subject construct does aUow thrombin processing of the fusion protein.
  • the construct with the Factor X a cleavage site also can be processed by Factor X a efficiently, giving a better yield of I-domain than that from the fusion with thrombin cleavage.
  • the subject invention provides ceU extracts prepared from the E. coli strain producing the fusion protein with the X a cleavage site for downstream isolation of the CDllb I-domain.
  • European Patent AppUcation 0365837 discloses the general background for interceUular adhesion molecules (ICAM-1) and their function derivatives which may be useful in the treatment of inflammation.
  • ICM-1 interceUular adhesion molecules
  • European Patent AppUcation 0391088 discloses the interceUular adhesion molecules (ICAM-1) and their function derivatives which may be useful in the treatment of viral infections.
  • ICM-1 interceUular adhesion molecules
  • European Patent AppUcation 0364690 discloses the leukocyte adhesion receptor Mac-1 alpha subunit and corresponding DNA and derivatives which may be useful in the treatment of inflammation.
  • European Patent AppUcation 0387668 discloses the interceUular adhesion molecules classified as ICAM-2 which are described to be involved in the process where lymphocytes migrate to inflammation sites. Diamond et al., "The I-domain is a Major Recognition Site on the Leukocyte
  • Integrin Mac-1 (CDllb/CD18) for Four Distinct Adhesion Ligands, J. of CeU Biology, 120, 4, 1031-1043 (1993) discloses that mAbs specific for the I-domain block Mac-1- dependent adhesion.
  • Kern et al. "The Role of the I Domain in Ligand Binding of the Human Integrin ⁇ 1 ⁇ 1 ", J. Biological Chemistry, 269, 36, 22811-22816 (September 9, 1994) report that the I Domain plays a central role in Ugand recognition for aU integrin ⁇ subunits containing this domain.
  • U.S. Patent 5,091,303 to Arnaout et al. discloses a 29kD neutrophiUc protein which binds to autoantibodies present in the sera of individuals afflicted with Wegener's granulomatosis and methods using these autoantibodies to diagnose individuals afflicted with Wegener's granulomatosis.
  • U.S. Patent 5,200,319 to Arnaout et al. discloses a 29kD neutrophiUc protein which is used in a method of diagnosing pauci-immune nectrotizing and/or crescentic glomerulnephritis in a patient.
  • the subject invention is a fusion protein of glutathione-S- transferase (GST) and I-Domain derived from human leukocyte B 2 -integrin Mac-1 in which the GST and the I-Domain are linked by a peptide segment containing a Factor X a cleavage site as set forth in ID SEQ NO: I.
  • This fusion protein provides an improved means for handling the I-Domain protein for synthesis and expression and a unique cleavage site which provides accessabiUty for cleavage by a Factor X a enzyme.
  • the invention is an I-Domain protein defined by the amino acid sequence Gly 226 through Glu 435, inclusive, as set forth in ID SEQ NO: I.
  • This sequence contains the I-Domain and the above mentioned special cleavage site.
  • the invention is a pharmaceutical composition comprising the recombinant I-Domain protein derived from human leukocyte B 2 - integrin Mac-1 defined by the amino acid sequence Gly 226 through Glu 435, inclusive, as set forth in ID SEQ NO: I; and a pharmaceuticaUy acceptable carrier or excipient.
  • the pharmaceutical composition can also consist of a fragment, analog or chemical derivative of the aforementioned I-Domain protein.
  • the invention is a method for treating inflammation comprising the administration to a patient suffering from an inflammatory condition a pharmaceuticaUy effective amount of an anti-inflammatory agent comprising a recombinant I-Domain protein derived from human leukocyte B 2 -integrin Mac-1 defined by the amino acid sequence Gly 226 through Glu 435, inclusive, as set forth in ID SEQ NO: I.
  • the method can also consist of a fragment, analog or chemical derivative of the aforementioned recombinant I-Domain protein.
  • the fusion protein, I-domain protein (which can contain the special cleavage segment) and the pharmaceutical compositions prepared therefrom are aU recombinant proteins substantiaUy free of contaminants or other biological impurities.
  • FIG. 1 Schematic representation of the A-domain described by Michishita et al., "A Novel Divalent Cation-Binding Site in the A Domain of the Beta-2 Integrin CR3 (CD11/CD18) Is Essential for Ligand Binding", CeU, 72, 857-867 (March 26, 1993) and the I-domain of the present invention. Note that the A-domain is about the same size as the I-domain, but begins about 20 amino acids upstream in the sequence of Mac-1 (Gly ⁇ ). Accordingly, it ends at Ala 31g , some 20 residues upstream of the C-terminal residue in the I-domain (Glu 337 ).
  • the A-domain has a C-terminal extension of Asn-Ser-Ser, introduced as part of the cloning strategy.
  • the I-domain is N-terminaUy extended by H 2 N-Gly-Ile-Pro-Gly-Gly-.. , a sequence required to promote cleavage by Factor X a .
  • the first Mac-1 residue in the I-domain construct is the serine corresponding to Ser 133 .
  • the present invention is an isolated and purified protein corresponding to the I-domain from the human leukocyte ⁇ 2 -integrin Mac-1, expressed in recombinant Escherichia coli as a soluble fusion protein with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • the I-Domain protein amino acids are numbered according to their location in the fusion protein.
  • the NH 2 -terminal portion of the molecule (residue 220) is glutathione-S-transferase (GST), a fusion partner which helps increase the level of soluble expression, and faciUtates purification by GSH-affinity chromatography.
  • Residues from 221-230 indicate the segment linking GST to the I-domain and the site of cleavage by Factor X a at Arg225 - Gly226 is dictated by the sequence; [Ile-Glu-Gly-Arg-i-Gly-Ile-Pro-].
  • the boldface Gly-Gly sequence represents a spacer to aUow accommodation of the proteinase to the site of cleavage at Arg-Gly.
  • the Ser at 231 foUowing the Gly-Gly sequence corresponds to the beginning of the I-domain, Ser 133 in processed Mac-1, and the construct ends with Glu 337 .
  • the I-domain begins with the pentapeptide sequence: Gly-Ue-Pro-Gly-Gly- required to aUow removal of the GST by Factor X a , foUowed by the I-domain of Mac-1 (residues 133 through 337).
  • This structure is schematicized in Fig. 1.
  • the isolated protein is useful for the treatment of inflammation and related conditions in human patients and other warm blooded animals by either parenteral or oral administration.
  • compositions of this invention may be prepared by combining the protein of the I-domain from the human leukocyte ⁇ 2 -integrin Mac-1 (SEQ ID NO: I), fragment, variant, analog or chemical derivative thereof of this invention with pharmaceuticaUy acceptable carrier, pharmaceuticaUy acceptable adjuvants or excipients employing standard and conventional techniques.
  • the pharmaceutical composition is prepared using conventional techniques in unit dosage form containing an anti-inflammatory effective or appropriate amounts of the active ingredient, protein, that is, I-domain from the human leukocyte ⁇ 2 -integrin Mac-1 (SEQ ID NO: I), fragment, variant, analog or chemical derivative thereof.
  • an anti-inflammatory effective or appropriate amounts of the active ingredient, protein that is, I-domain from the human leukocyte ⁇ 2 -integrin Mac-1 (SEQ ID NO: I), fragment, variant, analog or chemical derivative thereof.
  • the quantity of active component, according to this invention, in the pharmaceutical composition and unit dosage form thereof may be varied or adjusted widely depending upon the particular appUcation, the potency of the particular compound or the desired concentration.
  • the isolated and purified recombinant protein or pharmaceutical compositions thereof will be administered oraUy and/or parenteraUy at a dosage to obtain and maintain a concentration, that is, an amount, or blood- level of active component in the patient undergoing treatment which wiU be anti- inflammatoriaUy effective.
  • GeneraUy such pharmaceuticaUy effective amount of dosage of active component will be evidenced by monitoring of the inflammatory site.
  • the dosages may vary depending upon the requirements of the patient, the severity of the inflammation being treated, and the particular component being used. Also, it is to be understood that the initial dosage administered may be increased beyond a predetermined upper level in order to rapidly achieve the desired blood-level or the initial dosage may be smaUer than the optimum and the daily dosage may be progressively increased during the course of treatment depending on the particular situation. If desired, the daily dose may also be divided into multiple doses for administration, e.g., two to four times per day.
  • the compositions of this invention can be administered parenteraUy, i.e., by injection, for example, by intravenous injection or by other parenteral routes of administration.
  • compositions for parenteral administration will generaUy contain a pharmaceuticaUy acceptable amount of the recombinant I- domain protein from the human leukocyte ⁇ -integrin Mac-1 (SEQ ID NO: I), fragment, variant, analog or chemical derivative thereof mixed in a pharmaceuticaUy acceptable Uquid carrier such as, for example, water-for-injection and a buffer to provide a suitably buffered isotonic solution.
  • a pharmaceuticaUy acceptable Uquid carrier such as, for example, water-for-injection and a buffer to provide a suitably buffered isotonic solution.
  • the active component wiU be admixed in the carrier in an amount sufficient to provide a pharmaceuticaUy acceptable injectable concentration.
  • the resulting Uquid pharmaceutical composition wiU be administered so as to obtain the above- mentioned anti-inflammatory effective amount of dosage.
  • the fusion protein construct of the subject invention utiUzes the GST partner to faciUtate high levels of expression of soluble protein, and to aUow purification by affinity chromatography on immobilized glutathione (GSH). It was also designed to contain an accessible Factor X a -sensitive site in the region Unking GST to the I- domain so as to permit removal of the N-te ⁇ ninaUy attached GST moiety. Affinity chromatography of E. coli extracts over a column of GSH-Sepharose aUowed separation of the bound GST/I-domain from contaminating proteins which passed directly through the column. Removal of the GST/I-domain was effected by washing the column with buffer containing GSH.
  • the fusion protein thus purified was hydrolyzed with Factor X a which cleaved specificaUy in the linker region, and the resultant GST and I-domain proteins were separated by ion-exchange chromatography on a column of S-Sepharose.
  • the final I-domain product was shown to be >99% pure by sequence and compositional analysis, and by electrospray ionization mass spectroscopy which gave a molecular weight in agreement with that expected for the I-domain (25,767).
  • the fact that the I-domain was resistant to trypsin, coupled with physical characterization by circular dichroism and two- dimensional NMR provided evidence that the recombinant I-domain has a folded and ordered three dimensional structure.
  • the I-domain represents a reasonable target; it is relatively smaU in size, has no disulfide cross links, only low-level of glycosylation and, most importantly, it appears to be functionaUy relevant.
  • the I-domain of Mac- 1 in E. coli was cloned and expressed. Two I-domain constructs were designed. One had an extension at the COOH-terminus of six histidines (6His tag) to faciUtate purification by immobiUzed metal ion affinity chromatography.
  • This construct begins with H 2 N-Ser 133 -Asp-Ile-Ala-Phe-Leu..., and ends with ..Ser- Gln-Glu 337 -Ile-Leu-Gly-Arg-l-His-His-His-His-His-COOH, where the numbers indicate residues in the Mac-1 ⁇ -subunit (SEQ ID NO: I, Fig. 1), and the arrow indicates a bond designed, by virture of the preceeding 4 amino acids, to be a cleavage point for Factor X a .
  • the I-domain is defined, as for the 6His construct, as the sequence from Ser 133 and Glu 337 . This construct, therefore, had two handles for purification, GST and the 6His tag. Unfortunately, this protein could not be cleaved with thrombin, Factor X a , or even trypsin. It appeared that the thrombin site was Umited in accessibiUty, due to hindrance in the inter-domain region.
  • Thrombin construct GST-Leu-Val-Pro-Arg4-Gly-Ser-Gly-Gly-Ser 133 ....Glu 337 - COOH Factor X a construct: GST-Ile-Glu-Gly-Arg-T-Gly- ⁇ e-Pro-Gly-Gly-Ser 133 ...Glu 337 - COOH
  • Two constructs were made for the expression of the CDllb I-domain as a fusion to GST in the cytoplasm of E. coli.
  • One construct has the recognition site for Factor X a cleavage at the fusion junction, while the other construct has the thrombin proteolysis site.
  • the I-domain can be recovered from the fusion protein by Factor X a or thrombin processing.
  • two glycine residues were introduced into the fusion segment. Due to the proteolytic recognition sequence it was necessary to introduce other residues in addition to the two glycine residues.
  • the CDllb I- domain After proteolytic processing, the CDllb I- domain has the N-terminus extension shown in boldface.
  • the CDllb I-domain contains amino acid residues from Serl33 to Glu337.
  • Factor X a cleavage product: Gly-D.e-Pro-Gly-Gly-(Serl33....Glu337)
  • Thrombin cleavage product Gly-Ser-Gly-Gly-(Serl33....Glu337)
  • the resulting expression plasmid vectors carrying the I-domain fused to GST are named pGST-X a - CDllb/I and pGST-Throm-CDllb/I, with the Factor X a cleavage site and thrombin process site, respectively.
  • the fusion protein is under the control of the strong tac promoter which can be induced by IPTG.
  • the plasmids also carry the lacfl sequence to repress the tac promoter activity in the absence of an inducer such as IPTG.
  • the presence of the repressor lacr 1 is important for minimizing background expression of the recombinant protein, especiaUy in cases where the recombinant protein is detrimental to the host ceU.
  • the lacfi repressor sequence is not present in the plasmid, then one would want to maintain the plasmid in a E. coli host carrying the repressor, usually on the F' episome. Since the above vectors carry lacfl, the E. coli DH1 used for plasmid construction and E. coli K12S used for expression do not contain the repressor sequence.
  • pGST-X a -CDllb/I and pGST-Throm-CDllb/I were transformed into the 3 E. coli hosts JM103, JM109 and K12S.
  • ceUs Using the E. coli strain K12S(pGST-X a -CDllb/I) which produces the I- domain fusion to GST with the Factor X a cleavage site, more than 25 Uters of ceUs can be grown in shake-flasks with lxlO "5 M IPTG to induce expression. From these ceUs, ceU extracts are prepared by sonication and supernatant extracts free of inclusion bodies can be obtained by centrifugation to remove the insoluble material. These supernatant extracts can be purified for the I-domain. Over 10 mg of purified I-domain can be obtained from 1 Uter of ceUs. This amount of production is significant, since shake-flasks are used to grow E. coli cultures, which reach a ceU density of only about 1.5 A550.
  • the unlabeled Celtone-N medium was dUuted with unlabeled M9 salts medium at a ratio of 1:1, 1:2 and 1:3 and used to culture strain K12S(pGST-X a - CDllb I) with lxlO "5 M IPTG for induction of expression.
  • Celtone medium itself appears to give lower expression levels (24% of total ceU protein) than the Luria Broth (30% of total ceU protein). Dilution of the Celtone medium with 1 part or 2 parts of M9 salts medium does not significantly decrease expression, while dilution with 3 parts of M9 did lower expression. Under these culture conditions, about 60% of the fusion product remains in the soluble state.
  • the recombinant I-domain of the ⁇ -subunit CDllb of human leukocyte integrin Mac-1 was produced in E. coli as a fusion to GST (glutathione S- transferase), under the control of the tac promoter in a pBR-based vector background.
  • High level expression leads to the accumulation of the fusion product to at least 30% of total ceU protein.
  • Induction of the tac promoter with high levels of IPTG results in the majority of the fusion product in insoluble, aggregated inclusion body form.
  • IPTG level By lowering the IPTG level to lxlO "5 M, over 60% of the fusion protein produced remains in a soluble form in the cytoplasm of E. coli.
  • Two fusion proteins were produced, each with a specific proteolytic cleavage site (Factor X a or thrombin) at the fusion junction to aUow the recovery of the I- domain from the fusion product by digestion with either Factor X a or thrombin.
  • Factor X a or thrombin a specific proteolytic cleavage site
  • glycine residues were introduced into the fusion junction to separate the two polypeptide structures. After cleavage, the resulting I-domain has 4-5 extra residues at the N-terminus.
  • multi-Uters of E. coli ceUs were cultured in shake-flasks, and inclusion body-free ceU extracts prepared.
  • AU enzymes were used according to the manufacturer's instructions.
  • AU plasmids used carry the ampicillin-resistance marker and were constructed and maintained in E. coli DH1 in the presence of 100 ⁇ g/ml ampicilUn in either Luria broth or on Antibiotic Medium #2 (Difco) agar plates. Isolation of DNA fragments, transformation, smaU and large scale plasmid preparation, and other basic molecular biology techniques were according to those described by
  • Plasmid pGEX-3X carries the Factor X a cleavage recognition site and pGEX-2T the thrombin cleavage site in the polylinker region downstream from the GST sequence.
  • These vectors contain the tac promoter [inducible by IPTG (isopropylthiogalactoside)] to control GST expression, the lacjfl repressor sequence to minimize the tac promoter activity in the absense of IPTG, a polyUnker region for gene cloning as a fusion to GST, the ampicillin-resistance marker, and the repUcation origin derived from pBR322.
  • the CDllb I-domain sequence was cloned from a plasmid containing human
  • CDllb cDNA which was originaUy constructed by Corbi et al, J. Biol. Chem., 263:12403-11 (1988).
  • oUgonucleotides named KAC250 and KAC251 were used to clone the I-domain sequence for fusion with GST in pGEX-3X, whUe KAC249 and KAC251 were used to clone the I-domain for fusion with GST in pGEX-2T.
  • the sequences of these oUgonucleotide primers are shown below. The underUned portions of the primers overlap with the I-domain sequence (see, ID SEQ NO: I).
  • KAC249 and KAC250 provide the N-terminus extension sequence for the I-domain and the restriction enzyme BamHI site for cloning of the I-domain into the vector pGEX-3X or pGEX-2T.
  • the unUned portion in KAC251 provides a protein synthesis stop codon TAG to terminate the I-domain sequence at glu337 and the restriction enzyme EcoRI site for cloning into the vectors.
  • KAC249 5' GGCTCGGATCCGGTGGCAGTGACATTGCCTTCTTGATTGATGGCTCT
  • KAC250 5' GGCTCGGGATCCCCGGTGGCAGTGACATTGCCTTCTTGATTGATGGC
  • KAC251 5' GAGCCTGAATTCTATTCCTGAGACATCTCATGCTCAAAGGAGCT
  • the PCR reaction mixture in a final volume of 100 ul, was composed of 68 ul water, 10 ul lOx reaction buffer (100 mM Tris-HCl, pH 8.3, 500 mM KC1, 15 mM MgCl 2 , 0.01% gelatin), 16 ul of a mixture of the four deoxynucleotide triphosphates (each at 1.25 mM), 1 uM each of the appropriate oUgonucleotide primer, 1 ng pET- CDllb/I(His) 6 plasmid DNA, and 1 ul of Taq polymerase.
  • the reaction was carried out by appUcation of the GeneAmp PCR system (Cetus) in the foUowing manner: The sample was heated at 94°C , then treated for 30 cycles of 94 C for 30 seconds, 65 C for 30 seconds, and 72°C for 45 seconds, foUowed by a 5 minute anneal at 65 C and a 5 minute extension at 72°C, and then cooled to 4°C. An aUquot of the PCR reaction mixture was analyzed by 1.5% agarose gel electrophoresis to confirm that the size of the DNA fragment is about 660 bp. The DNA fragment was then purified by phenol extraction and precipitated from the PCR reaction mixture by the addition of 100 ul of 0.3 M sodium acetate and 400 ul ethanol.
  • This 660 bp DNA fragment was treated with BamHI and EcoRI, purified by 1.5% agarose gel electrophoresis, purified by phenol extraction and ethanol precipitation, and stored at -20°C in 10 mM Tris-HCl, pH 7.4 and 1 mM EDTA until use for cloning into expression vectors.
  • Expression of the CDllb I-domain Fusion to GST was treated with BamHI and EcoRI, purified by 1.5% agarose gel electrophoresis, purified by phenol extraction and ethanol precipitation, and stored at -20°C in 10 mM Tris-HCl, pH 7.4 and 1 mM EDTA until use for cloning into expression vectors.
  • Expression vectors pGST-X a -CDllb/I and pGST-Throm-CDllb/I were transformed into 3 different E. coli hosts (JM103 and JM109 are commerciaUy avaflable; K12S was isolated to test for expression of the fusion protein). Induction of the tac promoter to express the fusion protein was by the addition of IPTG (isopropylthiogalactoside) at lxlO "3 M or as specified. The E. coli ceUs were grown at 37°C with aeration in growth medium (Luria Broth with 100 ⁇ g/ml ampiciUin). SpecificaUy, an overnight culture of E.
  • coli ceUs was diluted 50-100 fold with growth medium to A550 of about 0.1, aUowed to grow until mid-log phase (A550 0.6-0.8), and then induced by the addition of IPTG.
  • ceUs were coUected by centrifugation, resupended to A550 of 20 with 10 mM Tris-HCl, pH 7.4 and 1 mM EDTA, and stored at -20 C until further use.
  • Sonicated ceU extracts were prepared by sonication of the ceU suspension in a Branson Sonifier.
  • sonicated ceU extracts were analyzed by SDS-PAGE and the gel with the Coomassie stained protein bands was scanned by a Shimadzu densitometer. The amount of the fusion protein in the ceU extract was calculated as a percent of total ceU protein.
  • SDS-PAGE was carried out using a gel system where the cross Unker is N,N'-diaUytartar diamide (Morse et al, Anatomy of herpes simplex virus DNA, J. Virol., 26:389-410 (1978) at a polyacrylamide concentration of 17%. Protein molecular weight standards were purchased from
  • the soluble fusion protein found in the cytoplasm of the E. coli ceUs was used as the starting material for downstream purification of the I-domain.
  • E. coli ceUs to produce N- and 15 N 13 C-labeled I-domain were cultured in medium purchased form Martek Bioscience Corp., Columbia, Maryland. The medium was dfluted with M9 salts medium, Sambrook et al. (cited, above). To test the approriate dilution ratio, the Martek non-labeled medium Celtone-U was used with M9 salts medium without supplement of amino acids. AmpicilUn at 100 ⁇ g/ml was used and ceU growth was at 37°C.
  • ceUs were grown in Martek Celtone-N medium (>98% N) dfluted with 2 parts of M9 salts medium in which 2 gm/1 of N-ammonium chloride was used as the sole ammonium source.
  • ceUs were grown in Martek Celtone-CN medium (>98% 13 C and 15 N) dUuted with 2 parts of M9 salts medium in which 2 gm/1 of N-ammonium chloride and 4 gm/1 of C-glucose were used as the sole ammonium and carbon source.
  • the subject I-domain generated from this fusion protein by cleavage with Factor X a , corresponds exactly to residues 133 through 337 of mature Mac-1, plus a pentapeptide N-terminal extension, Gly-Ile-Pro-Gly-Gly, required to enable the proteolytic cleavage.
  • a schematic comparison of the I-domain structure with that pubUshed by Michishita et al. (cited, above) for the A-domain is given in Figure 1.
  • the subject protein begins and ends about 20-residues downstream from the A- domain (Fig. 1). This is a significant difference, and can account for differences which might be seen in the function of these proteins.
  • the subject I- domain has an N-terminal extension, whereas the A-domain has a COOH-terminal segment, neither of which belong to the protein in question.
  • Purification of I-domain Glutathione Sepharose Chromatography Glutathione-Sepharose and S-Sepharose Fast Flow were purchased from
  • the column was loaded at 1.0 ml/min and protein in the effluent was monitored at 280 nm.
  • the column was washed with 4 column volumes (CV) of equiUbration buffer and step-eluted with 2 CV of 50 mM Tris buffer, pH 8.0 containing 20 mM reduced glutathione.
  • the eluent was coUected in 9-ml fractions.
  • AU process fractions were subjected to SDS-PAGE under non- reducing conditions (NR).
  • Fractions containing GST/I-domain were pooled and stored at 4 °C.
  • Factor X a Cleavage Purified fusion protein was subjected to digestion with Factor X a (1% by weight) for 16-24 hours at 23°C. SDS-PAGE was always run to confirm that digestion was complete.
  • the protein digest was loaded directly onto an S-Sepharose Fast Flow column (50-ml; 1.6cm i.d. x 26cm length) pre-equiUbrated with 50 mM sodium phosphate buffer, pH 6.5.
  • the column was run at 1.0 ml/min and the effluent monitored at 280 nm.
  • the column was washed with 4 CV of equiUbration buffer, and eluted with a 2 CV linear salt gradient run from 0-1.0 M NaCl in the presence of 50 mM sodium phosphate, pH 6.5.
  • Column fractions were subjected to SDS-PAGE (NR). Purified I- domain was observed as a single band at approximately 25 kD, and eluted from the column at approximately 150 mM NaCl.
  • I-domain was concentrated to 20 mg/ml using an Amicon stirred-ceU ultrafiltration module containing a YM05 membrane. Nitrogen pressure was maintained at 60 psi during the concentration. Characterization of I-domain: Analytical Methods
  • Proteins were sequenced by automated Edman degradation in an AppUed Biosystems Model 470 Gas Phase Sequencer fitted with an on-Une HPLC analyzer (Model 120A) for identification and quantitation of phenylthiohydantoin (PTH) amino acids. Integration of the peaks from the HPLC was performed with a Nelson Analytical 3000 Series chromatography data system connected in paraUel with the recorder to the output of the Model 120A HPLC system.
  • Reversed-Phase (RP) HPLC was performed on an HP 1090 Uquid chromatograph with a 4.6 x 250 mm Vydac C4 column.
  • Gradient mobfle phases were water and acetonitrile, each containing 0.15% trifluoroacetic acid (TFA).
  • the gradient was run from 0-70% acetonitrile in 70 minutes with a flow rate of 1.0 ml/min.
  • the effluent was monitored simultaneously at 220 nm and 280 nm.
  • the I- domain eluted as a single peak, and was coUected in 2-ml polypropylene microfuge tubes, dried in a Speed Vac, and analyzed.
  • AU electrospray ionization (ESI) mass spectra were recorded on the Vestec 201A mass spectrometer. AUquots (5 ul) of a solution of the I-domain in acetonitrile/water/0.1% TFA (about 0.05 to 0.5 ug of protein isolated by HPLC in the acetonitrile TFA gradient system) were injected via a loop injector into the ion source. The mass spectrometer was scanned from m/z 500 to 2000 at 2 sec/scan. The data were acquired with the Teknivent Vector 2 data system. Ten scans were averaged and transfered to the Harris 800 computer for further processing. The average molecular weights (av.
  • M.W. were determined using programs developed in-house. For single components, the centroid program and the deconvolution program were used. For mixtures, the deconvolution program yielded better results. The experimentaUy obtained average M.W. were then compared with the theoretical av. M.W. of the various samples of I-domain.
  • the circular dichroism (CD) spectrum of I-domain ( 1.0 mg/ml) was measured at room temperature (20-22 °C) on a Jasco Model J-720 CD spectropolarimeter from 260-190 nm in a 0.086 mm ceU. The spectropolarimeter was caUbrated at 290 nm with D-10-camphorsulfonic acid. Molar intensities were computed from the concentration of the protein sample and a mean residue molecular weight of 113.2. The secondary structure was calculated using the method of Compton and Johnson against a data base of 16 proteins. Purification of I-domain
  • the E. coli expression system with plasmid pG-3x-CDllb provided reasonable levels of GST/I-domain fusion protein.
  • Purification of the GST/I-domain construct over immobilized GSH proved to be very straightforward, as good yields were observed with extremely high purities.
  • the only contaminant observed upon SDS-PAGE analysis appeared to be a low level ( ⁇ 5%) of free GST, which would be expected to co-purify with the fusion protein.
  • RP-HPLC of the protein recovered from the GSH-column yielded a single peak which, upon N-terminal sequence analysis yielded a single sequence corresponding to the N-terminus of GST.
  • Factor X a is a serine protease which, by design of the construct, should specificaUy cleave the fusion protein at the Arg- Gly bond leading into the Gly-Ile-Pro-Gly-Gly- N-terminal extension of the I-domain (SEQ. ID. NO. I , Fig. 1).
  • Factor X a was added directly to the purified fusion protein to a final concentration of 1% (by weight), and incubation was carried out at room temperature.
  • the Factor X a digest was loaded directly onto the cation exchange resin S- Sepharose Fast Flow under conditions that would aUow only I-domain to bind.
  • the I-domain eluted as a single homogeneous peak at around 150 mM NaCl in the gradient, resulting in its recovery in physiologic-type buffer. Efficiency of binding was monitored by RP-HPLC, which resolves GST from I-domain.
  • the S-Sepharose load showed two peaks upon RP-HPLC (corresponding to GST and I-domain), whUe the flowthrough, wash, and pooled fractions each contained predominantly one peak corresponding to GST, GST, and I-domain, respectively. Characterization of I-domain
  • N-terminal sequence analysis and amino acid analysis performed on the final product resulted in very high correlations to expected results with no hint of contamination or degradation.
  • Electrospray ionization mass spectrometry was used to further assess the integrity of the I-domain preparations.
  • Purified I-domain (2-3 nmoles) was coUected from RP-HPLC, lyophilized, and reconstituted in a smaU volume of 50% acetonitrile prior to appUcation to the spectrometer probe.
  • a series of molecular ions corresponding to mass per unit charge (m/z) was obtained, from which the average molecular weight can be determined.
  • AU Factor X a -derived I-domain preparations yielded essentiaUy identical results. The spectra identified two species with distinct molecular weights.
  • the molecular weight of the major species (80-95%) was determined to be 23,767, which matches the theoretical value exactly, whUe the minor species yielded a mass 164 daltons lower. Generation of this species does not appear to be due to C-terminal heterogeneity or typical post-translational modification based on this mass difference.
  • the monoclonal antibody (mAb) 3H5 directed against CDllb was generated, isolated and characterized.
  • Fibrinogen was purchased from Kabi Pharmacia (FrankUn, Ohio) and was fibronectin depleted utiUzing gelatin-Sepharose 4B (Pharmacia; Uppsala, Sweden).
  • PMNs Polymorphonuclear leukocytes
  • Plates were washed once with 200 ul of PBS containing 1 mM CaCl 2 and 1 mM MgCl 2 (PBS/Ca 2+ /Mg 2+ ) foUowed by the addition of 200 ul of human serum (dfluted 1:3 in PBS/Ca 2+ /Mg 2+ . Plates were washed three times with 200 ul of PBS containing 0.05% Tween 20 and 0.01% thimerosal.
  • 96 weU microtiter plates were coated with 50 ul of fibrinogen (100 ⁇ g/ml) for 2 hours at 37°C. The remaining sites on the plastic were blocked by addition of 1% gelatin in PBS. Plates were washed three times with PBS/Ca 2+ /Mg2 + . Fifty ⁇ l of mAb 3H5 (20 ⁇ g/ml) or I-domain protein (0.5, 1, 5, 10, or 20 ⁇ M) was added to some of the weUs before addition of ceUs. fMLP stimulated labeled neutrophUs were added to the weUs and incubated for 20 minutes at 37°C, and ceU adherence was determined as described above.
  • this protein was immunopurified from 70 grams of human placenta using a modification of a procedure as described (Mariin and Springer, Purified interceUular adhesion molecule-l(ICAM-l), CeU., 51:813-19 (1987)).
  • the anti ICAM-1 mAb 8.4 (*) was used for these affinity chromatography procedures.
  • microtiter plates were coated with purified ICAM-1 dfluted 1:15 (vol./vol.) in PBS/Ca 2+ /Mg 2+ . After a 2 hour incubation at 37°C, ICAM-1 substrates were blocked by addition of 1% gelatin in PBS.
  • adhesion of chemotacticaUy stimulated neutrophUs to Mac-1 substrates was assayed in Smith-HoUers chambers as previously described (Anderson et al., Abnormal mobiUty of neonatal polymorphonucler leukocytes, J. CUn. Invest. 68:863-74 (1981)).
  • Use of these adhesion chambers faciUtates analysis of adhesion in the absence of shear stress, conditions which enhance ⁇ 2 integrin-dependence (Lawrence et al., Effect of venous shear stress on CD- 18..., Blood, 75:227-37 (1989)).
  • NeutrophUs (10 7 /ml) were preincubated in suspensions containing fMLP (10 nM) and I-domain (2.5 - 25 ⁇ M) or in the CDllb mAb 3H5 (10 ⁇ g/ml) for 5 minutes at 21°C prior to incorporation in adhesion chambers (0.5 x 10 ).
  • KLH (1 mg/ml) or fibrinogen (10 mg/ml) were incubated on glass coversUps for 30 min at 37°C and washed prior to assay.
  • the iC3b substrates were prepared by incubating a human polyclonal IgG (75 ⁇ g/ml) - BSA (170 ⁇ g/ml) mixture for 1 hour at 37°C on glass coversUps prior to adding human serum (dUutes 1:3 in PBS) and incubating for an additional 1 hour at 37°C.
  • the percentages of adherent neutrophUs contacting protein substrates were calculated as described (Anderson et ⁇ Z,(1981), cited above). Neutrophil-endotheUal adhesion and migration assays (Table 5. 6)
  • CDllb I-domain Factor X a -cleaved GST fusion protein
  • GST GST
  • CDllb mAb 3H5 10 ⁇ g/ml
  • the percentage of adherent neutrophUs contacting HUVEC and the percentage of ceUs exhibiting transendotheUal migration were determined as previously described (Smith, C.W. et al., J. CUn. Invest. 82:1746-1756, (1988).
  • ICAM-1 was purified from 70 grams of human placenta by a modified procedure described (Marlin, S., Springer, T.A., CeU, 51:813-19 (1987)).
  • the anti- ICAM-1 monoclonal antibody 8.4 was used for the affinity chromatography purification.
  • Fluorescently labeled neutrophUs and I-domain constructs were added to microtiter weUs that had been coated with iC3b as described under "Materials and Methods". FoUowing a 20 minute incubation, nonadherent neutrophUs were removed by flicking and the microtiter weUs were washed three times with assay buffer. Adherent ceUs were quantitated by measuring the fluorescent intensity using a Pandex fluorescence concentration analyzer. The adhesion of neutrophUs was also determined in the presence of mAb 3H5 (anti-CD lib). Each value (expressed as fluorescence intensity) represents the mean ⁇ standard error of three determinations. Results are representative of three experiments.
  • the 6-His I-Domain is an extension at the COOH-terminus of six histidines (6His tag) to facUitate purification by immobiUzed metal ion affinity chromatography.
  • This construct begins with H N-Ser 133 -Asp-Ile-Ala-Phe-Leu..., and ends with ..Ser-Gln-Glu 337 -Ile-Leu-Gly-Arg--l-His-His-His-His-His-His-COOH, where the numbers indicate residues in the Mac-1 ⁇ -subunit (from, Michishita et al, CeU, 72:857-67 (1993), correspondes to Ser 231-Glu 435, SEQ ID NO: 1), and the arrow indicates a bond designed, by virture of the proceeding 4 amino acids, to be a cleavage point for Factor X .
  • the 6-His peptide is a control for the 6-His I-Domain starting Ser-Gbi-Glu-Ile-Leu-Gly-Arg-i-His-His-His-His-His-COOH to assure that the observed activity of the 6-His I-Domain is not an artifact.
  • Fluorescently labeled neutrophUs and I-domain constructs were added to microtiter weUs that had been coated with purified placental ICAM-1. FoUowing a 20-minute incubation, nonadherent neutrophUs were removed by flicking and the microtiter weUs were washed three times with assay buffer. Adherent ceUs were quantitated by measuring the fluorescent intensity using a Pandex fluorescence concentration analyzer. The adhesion of neutrophUs was also determined in the presence of mAb 3H5 (anti-CDllb). Each value (fluorescence intensity) represents the mean ⁇ standard error of three determinations.
  • Tables 1-3 show that CDllb I-Domain proteins have differential effects on neutrophU adhesion.
  • the Factor X I-Domain inhibits neutrophU adhesion to fibrinogen and ICAM-1 in a dose-dependent manner, but has no inhibitory effect on neutrophU adhesion to iC3b.
  • Half maximal inhibition to fibrinogen and ICAM-1 is observed at a concentration of 1 micromolar for both proteins.
  • the 6- His tag I-domain protein blocks neutrophU adhesion to Ugands iC3b and ICAM-1 in a dose-dependent manner such that half maximal inhibition occurs at concentrations of 1 and 3 micromolar, respectively.
  • a control peptide containing hexahistidine had no effect on neutrophU adhesion.
  • CDllb I-Domain Factor X a cleaved GST fusion protein
  • 3H5 mAb (10 ⁇ g/ml) 21 71 + KLH (1 mg ml) or fibrinogen (10 ⁇ g ml) were incubated on glass coverslips for 30 min. at 37°C and washed prior to assay.
  • the iC3b substrates were prepared by incubating an IgG (75 ⁇ g/ml) - BSA (170 ⁇ g ml) mixture for 1 hr. at 37°C on glass coverslips prior to adding a 1:3 dilution of human serum and incubating an additional 1 hr. at 37°C.
  • NeutrophUs were preincubated in fMLP and I-domain or mAb for 5 min. at 21°C prior to incorporation into Smith-Hollers adhesion chambers. Final concentrations of proteins are designated.
  • CDllb I-Domain Factor X a -cleaved GST fusion protein
  • NeutrophUs were preincubated with I-domain or mAb for 5 min. at 21°C prior to incorporation into Smith-Hollers adhesion chambers. Final concentrations of proteins are desginated.
  • results of the representative experiment shown above demonstrate the capacity of CDl lb - I-Domain to inhibit the adhesion of unstimulated neutrophUs to endotheUal monolayers pretreated with TNF to maximalUy induce expression of ICAM-1 and other neutrophU adhesion ligands.
  • final I-Domain concentrations of 5 ⁇ M or 25 ⁇ M inhibited adhesion (as compared to stimulated controls) by 37% or 24%, respectively, while the positive control 3H5 mAb inhibited adhesion by 56 when used at saturating concentrations.
  • the remaining adhesion (unblocked by 3H5 mAb) represents the contribution of E-selectin and other endotheUal ligands elicited by TNF and is not expected to be impacted by I-Domain protein in this assay.
  • Results of the representative experiment shown above illustrate the capacity of CDllb - I-Domain to inhibit both neutrophU adhesion to and migration through endotheUal monolayers prestimulated with TNF.
  • An inverse relationship between I-domain concentration and degree of inhibition of adhesion is seen.
  • the optimal inhibitory concentration is 1 ⁇ M and in this experiment a 25 ⁇ M concentration is non inhibitory for adhesion.
  • a 1 ⁇ M concentration of I-Domain inhibits adhesion to the same extent as seen with the 3H5 mAb when used at saturating concentrations.
  • the GST (control) fusion protein fragment is non inhibitory.
  • ADDRESSEE The Upjohn Company, Intellectual Property Law (B) STREET: 301 Henrietta
  • Leu Lys Ser Ser Lys Tyr lie Ala Trp Pro Leu Gin Gly Trp Gin Ala 195 200 205
  • Gly Ser lie lie Pro His Asp Phe Arg Arg Met Lys Glu Phe Val Ser 245 250 255
  • Gly lie Pro Gly Gly 1 5

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Abstract

Protéine isolée et purifiée du Domaine-I dérivée de l'intégrine β2 Mac-1 des leucocytes humains, exprimée dans Escherichia coli recombiné, sous forme d'une protéine de fusion, avec la transférase glutathione S-(TGS). La protéine, un dérivé fonctionnel, un fragment, un analogue ou un dérivé chimique de ce fragment, sont utilisés dans le traitement des inflammations par interférence avec le mécanisme d'adhésion cellulaire afin de bloquer l'adhésion et la migration des neutrophiles.
PCT/US1995/004439 1994-04-26 1995-04-19 Proteine du domaine-i derivee de mac-1 et utilisee dans le blocage de l'adhesion et de la migration des neutrophiles WO1995029243A1 (fr)

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JP7527670A JPH09512426A (ja) 1994-04-26 1995-04-19 好中球の接着および移動の遮断に有用なＭａｃ−１ Ｉ−ドメイン蛋白質
EP95917555A EP0758393A1 (fr) 1994-04-26 1995-04-19 Proteine du domaine-i derivee de mac-1 et utilisee dans le blocage de l'adhesion et de la migration des neutrophiles
AU23558/95A AU2355895A (en) 1994-04-26 1995-04-19 Mac-1 i-domain protein useful in blocking adhesion and migration of neutrophils

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999006545A3 (fr) * 1997-08-01 1999-08-05 Max Planck Gesellschaft Composition et nouveau procede pour depister les maladies dues a la formation d'agregats de proteines ou de fibrilles du type amyloide
US6663863B2 (en) 2000-03-17 2003-12-16 Millennium Pharmaceuticals, Inc. Method of inhibiting stenosis and restenosis
CZ298260B6 (cs) * 1996-05-03 2007-08-08 Abbott Laboratories Antiangiogenní peptidy, zpusob jejich prípravy, polynukleotidy je kódující
CZ298612B6 (cs) * 1995-12-13 2007-11-21 The Children's Medical Center Corporation Inhibitor proliferace endothelových bunek a jeho použití
US7595199B1 (en) 1998-07-31 2009-09-29 Max-Planck-Gesellschaft zur Förderung der Wissenchaften e.V. Method of detecting amyloid-like fibrils or protein aggregates
WO2013159082A1 (fr) 2012-04-20 2013-10-24 Adhaere Pharmaceuticals, Inc. Composés et méthodes pour la régulation d'intégrines

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0364690A2 (fr) * 1988-08-23 1990-04-25 Dana Farber Cancer Institute Sous-unité du récepteur d'adhésion de leukocyte Mac-1

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0364690A2 (fr) * 1988-08-23 1990-04-25 Dana Farber Cancer Institute Sous-unité du récepteur d'adhésion de leukocyte Mac-1

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
D.B. SMITH AND K.S. JOHNSON: "Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase", GENE, vol. 67, no. 1, ELSEVIER SCIENCE PUBLISHERS,B.V.,AMSTERDAM,NL;, pages 31 - 40 *
M. MICHISHITA ET AL.: "A novel divalent cation-binding site in the A domain of the beta2 Integrin CR3 (CD11b/CD18) is essential for ligand binding", CELL, vol. 72, no. 6, 26 March 1993 (1993-03-26), CELL PRESS,CAMBRIDGE,MA,US;, pages 857 - 867 *
M.S. DIAMOND AND T.A. SPRINGER: "A subpopulation of Mac-1 (CD11b/CD18) molecules mediates neutrophilic adhesion to ICAM-1 and fibrinogen", J. CELL BIOL., vol. 120, no. 2, ROCKEFELLER UNIVERSITY PRESS,NY,US;, pages 545 - 556 *
M.S. DIAMOND ET AL.: "The I domain is a major recognition site on the leukocyte integrin Mac-1 (CD11b/CD18) for four distinct adhesion ligands", J. CELL BIOL., vol. 120, no. 4, ROCKEFELLER UNIVERSITY PRESS,NY,US;, pages 1031 - 1043 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CZ298612B6 (cs) * 1995-12-13 2007-11-21 The Children's Medical Center Corporation Inhibitor proliferace endothelových bunek a jeho použití
CZ298260B6 (cs) * 1996-05-03 2007-08-08 Abbott Laboratories Antiangiogenní peptidy, zpusob jejich prípravy, polynukleotidy je kódující
WO1999006545A3 (fr) * 1997-08-01 1999-08-05 Max Planck Gesellschaft Composition et nouveau procede pour depister les maladies dues a la formation d'agregats de proteines ou de fibrilles du type amyloide
US7078191B1 (en) 1997-08-01 2006-07-18 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Composition and method for the detection of diseases associated with amyloid-like fibril or protein aggregate formation
US7595199B1 (en) 1998-07-31 2009-09-29 Max-Planck-Gesellschaft zur Förderung der Wissenchaften e.V. Method of detecting amyloid-like fibrils or protein aggregates
US6663863B2 (en) 2000-03-17 2003-12-16 Millennium Pharmaceuticals, Inc. Method of inhibiting stenosis and restenosis
WO2013159082A1 (fr) 2012-04-20 2013-10-24 Adhaere Pharmaceuticals, Inc. Composés et méthodes pour la régulation d'intégrines

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