WO2004104028A1 - Agent anti-inflammatoires - Google Patents

Agent anti-inflammatoires Download PDF

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Publication number
WO2004104028A1
WO2004104028A1 PCT/GB2004/002163 GB2004002163W WO2004104028A1 WO 2004104028 A1 WO2004104028 A1 WO 2004104028A1 GB 2004002163 W GB2004002163 W GB 2004002163W WO 2004104028 A1 WO2004104028 A1 WO 2004104028A1
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icam
seq
cells
peptides
peptide
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Britta Engelhardt
John Greenwood
Peter Adamson
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Institute of Ophthalmology UCL
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Institute of Ophthalmology UCL
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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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70525—ICAM molecules, e.g. CD50, CD54, CD102
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to anti-inflammatory agents and compositions, in particular for the treatment of neuroinflammation.
  • T cells Interaction of T cells with the vascular endothelium is a critical step during T cell extravasation from blood into tissue in immunosurveillance and inflammation.
  • Cell adhesion molecules CAMs expressed on the T cell and endothelial cell surfaces play essential roles in this intercellular interaction. Lymphocyte interaction with the endothelium in vivo is initiated by selectin mediated rolling followed by activation of integrins, firm attachment of the lymphocyte and subsequent migration across the endothelium.
  • the intercellular adhesion molecules (ICAM)-l and ICAM-2, members of the immunoglobulin - superfamily, were both identified by their ability to bind the same B2-integrin ligand LFA-1, which is expressed on all leukocytes. Binding of T cells via LFA-1 to antigen presenting cells or target cells is necessary for T cell activation and T cell effector functions and for firm attachment and migration of leukocytes across the endothelium.
  • ICAM-1 and ICAM-2 are expressed at low levels on endothelial cells and ICAM-1 expression is strongly inducible by inflammatory cytokines. Strikingly, ICAM-1 or ICAM-2 deficient mice are both viable and show relatively mild defects in their immune responses. ICAM-1 deficient mice suffer from a moderate leukocytosis while activation and migration of leukocytes to places of inflammation are reduced resulting in impaired immune and inflammatory responses.
  • ICAM-1 interleukocyte adhesion-mediated signalling responses to endothelial cells. It has previously been demonstrated that the expression of the adhesion molecule ICAM- 1 on endothelial cells is pivotal in supporting lymphocyte migration across vascular endothelium in both peripheral tissue and the CNS.
  • Endothelial cells of the blood-central nervous system (CNS) barriers display characteristics that differentiate them from endothelium of other organs. In particular these cells express well-developed intercellular tight junctions and are responsible for exceptionally low paracellular permeability.
  • the endothelia which facilitates the passage of migratory cells across the vessel wall through the expression of cell adhesion molecules such as ICAM-1.
  • ICAM-1 may mediate leukocyte migration under inflammatory conditions whereas ICAM-1 may play the major role in mediating basal infiltration of leukocytes in immunosurveillance.
  • ICAM-1 and ICAM-2 appear to control different signaling responses in endothelial cells (Adamson et al., 1999: Thompson et al., 2002)
  • ICAM-1 brain endothelial cell ICAM-1 is capable of evoking signal transduction events, suggesting that endothelial cells actively respond to leukocyte adhesion (Etienne et al., 1998; Adamson et al., 1999; Durieu-Trautmann, 1994).
  • ICAM- 1 elicits these signals.
  • the short cytoplasmic domain of ICAM-1 is known to bind a variety of molecules such as ⁇ -actinin, ⁇ -tubulin, glyceraldehyde-3 -phosphate dehydrogenase and ezrin
  • ICAM-1 has no catalytic activity associated with this region. It lacks intrinsic kinase activity or known protein-protein interaction domains that could recruit downstream signalling components. Previous attempts to reduce transendothelial migration using ⁇ Interferon have produced less than promising results.
  • the cytoplasmic domain of ICAM-1 consists of 27 amino acids in rat and mouse and 28 amino acids in human.
  • the human sequence is as follows:
  • the mouse sequence is as follows:
  • QRKIKKYRLQQAQ SEQ. ID. NO:3
  • mice this corresponds to the sequence
  • QRKIR ⁇ YKLQKAQ SEQ. ID. NO:4
  • Examples of representative peptide sequences effective against at least one of human, rat and mouse ICAM-1 are those having or containing the sequence: QRKJKKYRLQQAQ (SEQ. ID NO:3) QRKIR ⁇ YKLQKAQ (SEQ. ID NO:4) QRKKK(YP)RLQQAQ (SEQ. ID NO:5) QRKIRI(YP)KLQKAQ (SEQ. ID NO:6)
  • the present invention comprises an anti-inflammatory agent which reduces or inhibits transendothelial migration of T -lymphocytes.
  • the anti-inflammatory agent preferably mimics at least a part of the cytoplasmic domain of ICAM-1.
  • the anti-inflammatory agent may comprise a peptide consisting of, or containing the amino acid sequences described above (SEQ. ID NOS: 3 to 6), or sub-sequences contained therein or other amino acid sequence within the ICAM-1 cytoplasmic domain or sequences from binding partners that recognise the ICAM-1 cytoplasmic domain.
  • the peptide is preferably cell permeant and may be linked to a peptide, such as a penetratin peptide, consisting of or containing the amino acid sequence:
  • amino acid and any reference to a specific amino acid is meant to include naturally occurring proteogenic amino acids as well as non-naturally occurring amino acids such as amino acid analogues.
  • proteogenic amino acids include naturally occurring proteogenic (L) amino acids, chemically modified amino acids (including amino acid analogues such as penicillamine, 3-mercapto-D-valine), naturally occurring non- proteogenic amino acids such as norleucine and chemically synthesised compounds that have properties known in the art to be characteristic of an amino acid (e.g D-amino acids).
  • proteogenic indicates that the amino acid can be incorporated into a protein in cell through well-described metabolic pathways.
  • peptide is used in its broadest sense to refer to compounds containing amino acid equivalents or other non-amino acid groups, while still retaining the desired functional activity of a peptide.
  • Peptide equivalents can differ from the representative peptides specified above by the placement of one or more amino acids with related organic acids (such as PABA) amino acids or the like, or the substitution or modification of side chains or functional groups.
  • certain amino acids in the peptide may be phosphorylated.
  • Modifications can include for example additions, deletions or substitutions of amino acid residues with compounds that can mimic amino acid structure or functions, as well as the addition of chemical moieties such as amino or acetyl groups.
  • the modifications can be deliberate or accidental, and can be modifications of composition of the structure. Variants can be determined by simple experiments in the light of the present disclosure as a whole.
  • the inhibitor may comprise a peptidomimetic which functions in a manner equivalent to the peptide inhibitors specified above.
  • Peptidomimetic agents are compounds with a chemical structure which mimic the structure of peptide sequences.
  • the invention further comprises a pharmaceutical composition comprising an inhibitor as defined above, in particular for human or vetinary use.
  • the invention further comprises a method of inhibiting or reducing inflammation, for example neuroinflammation by administration of a defined peptide or peptidomimetic to a patient in need thereof.
  • a pharmaceutical composition comprising a peptide or peptidomimetic of the present invention can be administered to a subject having inflammation, by a variety of routes for example by an oral or intravenous route.
  • the immortalised Lewis rat brain microvascular endothelial cell (EC) line GP8/3.9 (22) was maintained in Ham's F-10 medium supplemented with 10% FCS, 2mM glutamine,
  • GP8/3.9 cells transfected with RSVpuro human ICAM-1 constructs were maintained in GP8/3.9 medium containing puromycin
  • Wild type human ICAM-1 and mutant ICAM-1 molecules were generated using RT-PCR from CHO cells overexpressing human ICAM-1 (a gift from Dr. J. Pearson, Kings College London, UK).
  • RNA was extracted from ICAM-1 expressing CHO cells using an RNeasy® kit (Qiagen, West Wales UK) according to the manufacturer's instructions.
  • Reverse transcriptase was used to generate cDNA, from oligo(dT) ⁇ 5- ⁇ 8 primed RNA, which was used as a template for PCR.
  • PCR reactions were performed using the forward primer 5'-GGAAGCTTCTAGAATGGCTCCCAGCAGCCCCC-3' (SEQ. ID.
  • Hind III and Sail sites which were engineered into the forward and reverse PCR primers respectively were used to clone the PCR fragment into a Hind Ill/Xho I restricted pcDNA3/RSVpuro plasmid.
  • the identity of the PCR generated ICAM-1 inserts was confirmed by DNA sequencing.
  • GPI-ICAM-1 A GPI anchored human ICAM-1 construct (pCDM8 GPI-ICAM-1) in which both the C-terminal domain and transmembrane domain are deleted by truncating ICAM-1 at codon 480 and fusing this sequence to the GPI-anchor sequence of human LFA3
  • the GPI-ICAM-1 sequence was excised from pCDM8 using Hindlll and Xbal and directionally cloned into Hindlll/Xbal digested RSVpuro.
  • Penetratin peptides were N-terminally biotinylated and consisted of 16 residues of the penetratin sequence (RQIKIWFQNRRMKWKK (SEQ. LD. NO:7)).
  • the 13 C-terminal amino acids of human (h) or rat ICAM-1 (r) were synthesised distal to the penetratin sequence.
  • Peptides were HPLC purified before use. Sequences used were hICAM-1 (QRKTKKYRLQQAQ (SEQ. ID. NO:3)), YP-hICAM-1 (QRKIKK(YP)RLQQAQ (SEQ. ID. NO:5)), rICAM-1 (QRKJRIYKLQKAQ (SEQ. LD.
  • YP-rICAM-1 QRKIRI(YP)KLQKAQ (SEQ. ID. NO:6)
  • CKPMSNFRFGENH an irrelevant sequence from the soluble part of rat rod opsin
  • GP8/3.9 rat brain EC lines overexpressing human ICAM-1
  • Pre-confluent GP8/3.9 EC (approx. 0.5x10° cells) were transfected with 3-6 ⁇ g of each of the ICAM-1 constructs or with the pCDNA3/RSVpuro vector (no insert) using Fugene transfection reagent according to the manufacturer's instructions. After 24-48h, puromycin (20 ⁇ g/ml) was added to cultures to select for ICAM-1 expressing cells. In subsequent studies transfected cells were maintained in medium containing 20 ⁇ g/ml puromycin and removed prior to co-culture with T-lymphocytes.
  • Puromycin resistant GP8/3.9 brain EC clones were generated and assessed by flow cytometric analysis using a human specific anti -ICAM-1 antibody (clone BBA4) to demonstrate the presence of human ICAM-1 expression.
  • clone BBA4 mAb lO ⁇ g/ml
  • cell pellets were resuspended in lOO ⁇ l of FITC- conjugated goat- anti-mouse IgG (1/100 dilution) and incubated for a further 30 min prior to standard paraformaldehyde fixation and flow cytometric analysis. Data was quantified and rendered using Cellquest® software. A secondary isotype matched IgG control sample for each cell line was also acquired.
  • Adhesion assays and transendothelial migration assays were carried out as previously described using cells harvested from Lewis rat peripheral lymph nodes and MBP-antigen specific T-lymphocyte lines (Greenwood et al., 1995; Pryce et al., 1997; Greenwood and Calder 1993). The results are expressed as the means ⁇ SEM and significant differences between groups determined by Student's t-test.
  • ICAM-1 mediated support of transendothelial lymphocyte migration could be inhibited with cell permeant peptide mimicking the intracellular domain of ICAM-1.
  • Peptides comprising the membrane proximal part of the rat or human ICAM-1 sequence within the intracellular domain were used to antagonise the binding potential of ICAM-1 binding/signalling partners.
  • ICAM-1 C-terminal sequences were fused to the penetratin sequence to facilitate uptake of peptides into cells (Hall et al, 1996; Peck and Isacke 1998).
  • a penetratin peptide fused to the 13 amino acids of the intracellular domain of rat ICAM-1 immediately adjacent to the plasma membrane (rICAM-1 : QRKIRrYKLQKAQ (SEQ. ID. NO:4)) or an identical peptide in which the conserved tyrosine residue corresponding to codon 512 of the rat ICAM-1 sequence was phosphorylated (YP-rICAM-1: QRKTRI(YP)KLQKAQ (SEQ. ID.
  • both phosphorylated and non-phosphorylated human peptides were also effective in inhibiting transendothelial lymphocyte migration.
  • Human ICAM-1 intracellular domain peptides abolish the enhanced transendothelial migration of lymphocytes mediated through ectopic expression of hICAM-1 in rat brain EC without affecting lymphocyte adhesion
  • penetratin peptides containing the human ICAM-1 sequence were used to treat rat brain EC expressing WT-hICAM-1, both non-phosphorylated and tyrosyl- phosphopeptides were able to abolish the increase in transendothelial lymphocyte migration associated with the ectopic expression of WT-hICAM-1.
  • Rat brain EC expressing WT-hICAM-1 treated with penetratin peptides containing the human ICAM-1 sequence were unable to significantly reduce lymphocyte adhesion which again is consistent with those experiments conducted on rat brain EC expressing only endogenous ICAM-1.
  • ICAM-1 C-terminal peptides mimicking the ICAM-1 intracellular domain were shown to be effective in inhibiting transendothelial migration of lymphocytes. Furthermore, inclusion of a phosphorylated tyrosine residue at a position in the peptide corresponding to codon 512 of ICAM-1 was no more potent an inhibitor of transendothelial migration than the non-phosphorylated peptide, suggesting phosphorylation of the conserved tyrosine residue is not required for ICAM-1 mediated signal transduction enabling transendothelial migration of T-cells.
  • Endothelioma cell lines were established by infection of primary brain endothelial cells with a recombinant retrovirus coding for the Polyoma middle T oncogene (Kiefer et al, 1994) exactly as described before
  • the wild type line bEnd.5 served as a control cell line.
  • the ICAM-1 deficient brain endothelioma cell line bEndl 1.1 and its daughter cell line bEnd 11.1 -ICAM-1 re-expressing wild type ICAM-1 after retro viral transduction into bEndll.l were described in detail before (Reiss et al., 1998).
  • ICAM-r /" ICAM-2 " ⁇ brain endothelioma cell line bEndl 1/2.1 was established by retro viral transduction of primary brain endothelial cells derived form ICAM-l "/ TCAM-2 " " mice with the Polyoma middle T oncogene.
  • ICAM-1 " ' " ICAM ⁇ " mice were obtained by cross-breeding the ICAM-1- and the ICAM-2-def ⁇ cient mice
  • proteolipid protein peptide aa 139 - 153 -specific T cell lines SJL.PLP3 to SJL.PLP9 were established from draining lymph nodes of SJL/N mice previously immunized with PLP in CFA (Bomholtgard, Denmark) and have been described in great detail before (Engelhardt et al., 1998). They were used for experiments starting after the third round of antigen-specific stimulation, when proliferation only occured in response to the specific-antigen as measured by the incorporation of 3 H-thymidine. These PLP- specific T cell lines are Tm memory/effector T cells and were used in the assays at days 3 to 5 after antigen-specific restimulation. Generation of ICAM-1 mutants
  • a pBluescript plasmid clone coding for the full length open reading frame of murine ICAM-1 was used as a template to generate the ICAM-1 mutants by PCR-mutagenesis and PCR fragment amplification, which were subcloned into the retroviral vector pBABEpuro.
  • Oligonucleotides used were as follows: RL01: cct gat gtc gac tea gcg gtt ata ac ata aga ggc (SEQ. ID. NO: 13), RL02: cgt gat ggc age tag ctt tgt ttt t (SEQ. ID.
  • RL03 gcc aga gaa aga tac gta tat tea age tgc aga agg etc agg
  • RL04 cgc aat taa ccc tea eta aag g
  • RL06 ggc agg agt cga etc cag cag get cag gg
  • RL11 ggt aca tac gtg tgc cat gc (SEQ. ID.
  • PCR mutagenesis of internal DNA sequences was based on a two-step PCR procedure using the proofreading VENT DNA-polymerase (Biolabs, Schwalbach, Germany) and combinations of mutagenesis primers with two peripheral primers. In the first round the mutagenesis primer and the opposite peripheral primer were used. After purification the generated PCR fragment was used as a megaprimer together with both peripheral primers for the second round of PCR.
  • the cytoplasmic deletion mutant pICAM-l(Ml) was generated by introducing a stop codon into the reading frame of the cDNA for murine ICAM-1 by PCR using the oligonucleotides RL01 and RL04. The resulting PCR fragment was cloned Eco RI/S ⁇ / 1 into pBluescript. In order to reduce the fragment size generated by PCR a Spe 1/Sal I insert from pBluescript was cloned into pBABEpuro (Spe 1/Sal I) to obtain a shuttle construct (pRL202). Finally, a Hind III fragment from pBABEpuro-ICAM-1 was substituted for the Hind HI fragment of pRL202.
  • the ICAM-1 mutant M2 where the tyrosines 507 and 509 were replaced by phenylalanines was derived by PCR mutagenesis using the oligonucleotides RL02, RL04 and RL06.
  • the PCR fragment was cloned into Hind 111/ Sal I cut pBluescript.
  • pICAM-l(Ml) was substituted for the corresponding insert of the construct created resulting in pBluescriptICAM-l(M2).
  • pICAM-l(M5) a PCR fragment was generated with use of the oligonucleotides RL11 and RL13 and as template pBluescript-ICAM-l(M2). After restriction digest of this PCR fragment with Nco USal I it was substituted for the corresponding insert of pBluescript ICAM-1 (M3). Full length mutated ICAM-1 was shifted into pBABEpuro Eco RUSal I to give the final pICAM-l(M5) retro viral construct. All fragments generated by PCR were sequenced to verify the D ⁇ A sequence.
  • GP+ E86 The retrovirus packaging cell line GP+ E86 (Markowitz et al, 1988) by CaPO 4 precipitation.
  • Stably transfected GP+E-86 clones were selected in the presence of puromycin (2 ⁇ g/ml) or in case of the ICAM-2 construct in the presence of hygromycin (50 ⁇ g/ml).
  • bEndll.l or bEndIl/2.1 were grown up to 2/3 confluency in 100 mm petri-dishes prior to infection.
  • the cells were incubated twice for 16 hours with supernatants from the different GP+E-86 transfectants supplemented with 8 ⁇ g/ml Polybrene (Hexadimethine Bromide; Sigma). Selection was started 24 hours after the second infection. Clones were grown to passage 5 and expression of ICAM-1 or ICAM-1 mutants or ICAM-2 in retransfected endothelioma cells was analyzed by FACS analysis and immunofiuorescence.
  • Penetratin peptides were synthesised at the Kennedy Institute of Rheumatology, UK. Peptides were ⁇ -terminally biotinylated and consisted of 16 residues of the penetratin sequence (RQIKiWFQ ⁇ RRMKWKK (SEQ. LD. ⁇ O:7)). 13 C-terminal amino acids of murine ICAM-1 were synthesised distal to the penetratin sequence. Peptides were HPLC purified before use. Sequences used were biotinylated protein (BP)-ICAM-lwt (QRKIRiYKLQKAQ (SEQ. ID. NO:4)), BP-ICAM-1PY (QRKTRI(YP)KLQKAQ (SEQ.
  • Penetratin peptides were localised in cells using streptavidin-Cy3 (1:100, Jackson, USA) following either fixation of the cells in 1% formaldehyde in PBS or permeabilisation with cold (-20°C) methanol and rehydration with PBS.
  • FACS analysis was performed exactly as described before. Endothelioma cells were harvested by incubating with 5 mM EDTA in HBSS at 37°C, washed and cell populations (5xl0 5 cells/sample) were incubated with primary rat mAb for 30 minutes at 4°C, washed twice with FACS buffer (PBS supplemented with 1% bovine serum albumin (BSA) and 0.1% NaN followed by incubation with a Phycoerythrin-conjugated goat anti-rat IgG F(ab')2 mAb (Biosource, Camarillo, CA, USA) for 30 minutes at 4°C, washed twice with FACS buffer and fixed in 1% formaldehyde in PBS.
  • FACS buffer PBS supplemented with 1% bovine serum albumin (BSA) and 0.1% NaN
  • Adhesion and transmigration assays were performed exactly as described in detail before Briefly, adhesion assays have been carried out using 16-well glass chamber-slides (Nunc, Wiesbaden, Germany) and analyzed by video associated light microscopy (NIH Image software, NIH, USA). Bound cells per pre-defined field were determined by counting 5 fields per well. Assays were performed in triplicates for each value. Transmigration assays were performed using 6.5mm Transwells (Costar, Bodenheim, Germany) with 5 ⁇ m pore size. Migrated T lymphocytes were collected for cell counting (CASY, Scharfe- System, Reutlingen, Germany). Confluency of the endothelial monolayer was confirmed after each assay on formalin fixed and 2.6% Giemsa stained inserts. Assays were performed as triplicates for each value.
  • bEndIl/2.1 do not support transendothelial migration (TEM) of PLP-specific T cells transendothelial migration (TEM) of PLP-specific T cells across unstimulated ICAM-1 7" ICAM-2 "7" .
  • TEM transendothelial migration
  • bEndl 1/2.1 and wild-type bEnd5 were always investigated simultaneoulsy within the same experiments. During a 4 hour time period about 40 % of T cells spontaneously migrated across a monolayer of bEnd5, whereas migration of T cells across bEndIl/2.1 over the same time period was negligible (Fig. 3A).
  • ICAM-1 and ICAM-2 reconstitute TEM of PLP-specific T cells across bEndl 1/2.1
  • ICAM-1, ICAM-2 or both were retrovirally transduced into bEndIl/2.1 and derivative cell lines of bEndIl/2.1 expressing ICAM-1 (bEndIl/2.1-ICAM-l), ICAM-2 (bEndIl/2.1 -ICAM-2) or both ICAM-l+ICAM-2 (bEndl 1/2.1-IC AM- 1/2) were established.
  • ICAM-1 and ICAM-2 Surface expression of both, ICAM-1 and ICAM-2, was verified by FACS-analysis and immuno fluorescence staining using 3 different anti-ICAM-1 monoclonal antibodies and one anti-ICAM-2 monoclonal antibody. Additionally, the presence of ICAM-1 and ICAM-2 protein was confirmed by Western blot analysis. Ectopic expression of ICAM-1 and/or ICAM-2 neither affected expression of other endothelial cell surface molecules such as endoglin, the MECA-32 antigen, VE-cadherin nor the inducibihty of VCAM-1 as investigated by FACS analysis .
  • cell permeable peptides derived from the cytoplasmic part of ICAM-1 inhibited TEM of T cells in a dominant negative fashion indicating the involvement of the cytoplasmic part of endothelial ICAM-1 in TEM of T cells directly at the site of T cell endothelial interaction.
  • peptides derived from the cytoplasmic tail of ICAM-1 interfered with TEM in a dominant negative fashion indicating that endothelial ICAM-1 delivers signals via its cytoplasmic tail into endothelial cells, which are essential for TEM of T cells.
  • peptides having an amino acid sequence which is the same as or which mimics the amino acid sequence of a binding partner of the ICAM-1 C terminal domain also act in a dominant negative fashion to interfere with TEM of T cells. Examples of this are peptides having substantial sequence similarity with the binding partner SlOOAlO/pl 1.
  • a and C Migration of antigen specific (MBP) T cells through or (B and D) adhesion of [ H] -labelled activated rat peripheral lymph node lymphocytes to control rat brain EC monolayers or EC pretreated with rat (A and B) or human (C and D) penetratin peptides.
  • MBP antigen specific
  • Lymphocyte adhesion to and migration through rat brain EC monolayers expressing human ICAM-1 Effect of penetratin-human ICAM-1 peptides.
  • A Migration of antigen specific (MBP) T cells through or (B) adhesion of [ 3 H]-labelled activated rat peripheral lymph node lymphocytes to control EC monolayers, EC transfected with RSVpuro empty vector (rsvpuro) or EC expressing human wild type ICAM-1 (WT-hICAM-1).
  • WT-hICAM-1 were also pretreated with either penetratin peptide containing C-terminal 16 amino acids of human ICAM-1 (hICAM-1) or penetratin peptide containing C-terminal 16 amino acids of ICAM-1 in which the tyrosine residue is phosphorylated (YP-hICAM-1).
  • Endothelial ICAM-1 and ICAM-2 are required for TEM of T cells.
  • A: One representative experiment comparing TEM of the T cell line SJLB.PLP3 across unstimulated and stimulated bEnd5 and ICAM-1 7" ICAM-2 7" bEndIl/2.1 is shown. Bars represent mean +/- SD (n 3). TEM of T cells across bEndIl/2.1 is reduced to an extremely significant level (p ⁇ 0.0005) when compared to bEnd5. This assay was reproduced 7 times.

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Abstract

L'invention concerne des agents anti-inflammatoires inhibiteurs d'une partie du domaine C-terminal de la molécule d'adhésion ICAM-1, limitant ainsi la migration transendothéliale des lymphocytes. Lesdits agents peuvent comprendre une séquence d'acides aminés identique ou semblable à la partie spécifique dudit domaine. En variante, les agents peuvent s'interposer dans l'interaction de ce domaine avec un ou plusieurs de ses partenaires de liaison.
PCT/GB2004/002163 2003-05-20 2004-05-20 Agent anti-inflammatoires Ceased WO2004104028A1 (fr)

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GBGB0311576.3A GB0311576D0 (en) 2003-05-20 2003-05-20 Anti-inflammatory agents
GB0311576.3 2003-05-20

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2346616A (en) * 1998-11-13 2000-08-16 Cyclacel Ltd Delivery vectors comprising truncated and modified forms of penetratin
WO2001094570A2 (fr) * 2000-06-07 2001-12-13 Otogene Aktiengesellschaft Procede de transfection

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2346616A (en) * 1998-11-13 2000-08-16 Cyclacel Ltd Delivery vectors comprising truncated and modified forms of penetratin
WO2001094570A2 (fr) * 2000-06-07 2001-12-13 Otogene Aktiengesellschaft Procede de transfection

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DEROSSI D (REPRINT) ET AL: "Trojan peptides: the penetratin system for intracellular delivery", TRENDS IN CELL BIOLOGY, ELSEVIER SCIENCE LTD, XX, vol. 8, no. 2, February 1998 (1998-02-01), pages 84 - 87, XP002122131, ISSN: 0962-8924 *
FISCHER ET AL: "Structure-activity relationship of truncated and substituted analogues of the intracellular delivery vector penetratin", JOURNAL OF PEPTIDE RESEARCH, MUNKSGAARD INTERNATIONAL PUBLISHERS, COPENHAGEN, DK, vol. 55, no. 2, 2000, pages 163 - 172, XP002140331, ISSN: 1397-002X *

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