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WO1991018011A1 - Inhibition d'adhesion cellulaire a l'aide de peptides analogues a la molecule-1-d'adhesion intercellulaire et/ou d'analogues de ces peptides - Google Patents

Inhibition d'adhesion cellulaire a l'aide de peptides analogues a la molecule-1-d'adhesion intercellulaire et/ou d'analogues de ces peptides Download PDF

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Publication number
WO1991018011A1
WO1991018011A1 PCT/AU1991/000205 AU9100205W WO9118011A1 WO 1991018011 A1 WO1991018011 A1 WO 1991018011A1 AU 9100205 W AU9100205 W AU 9100205W WO 9118011 A1 WO9118011 A1 WO 9118011A1
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Prior art keywords
icam
peptide
analogue
amino acid
polypeptide
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PCT/AU1991/000205
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English (en)
Inventor
John Vincent Fecondo
Andrew Wallace Boyd
Stephen Brian Henry Kent
Original Assignee
Swinburne Limited
The Walter And Eliza Hall Institute Of Medical Research
The California Institute Of Technology
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Application filed by Swinburne Limited, The Walter And Eliza Hall Institute Of Medical Research, The California Institute Of Technology filed Critical Swinburne Limited
Publication of WO1991018011A1 publication Critical patent/WO1991018011A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is directed to Intercellular Adhesion Molecule-1-like peptides, analogues thereof and antibodies thereto, to pharmaceutical compositions comprising same and the use thereof to inhibit or reduce cell adhesion in mammals.
  • the present invention also relates to a method for fingerprinting the functional domain of a polypeptide or protein.
  • ICAM-1 Intracellular fibroblasts
  • LFA-1 Leukocyte Function Associated antigen-1
  • ICAM-1 Intracellular cytoplasmic factor-1
  • LFA-1 Leukocyte Function Associated antigen-1
  • Cytokines involved in the "inflammatory" response IFN- ⁇ , TNF ⁇ and IL-1, induce expression of ICAM-1 (Pober et al, 1987: Dustin et al, 1988; Boyd et al, 1989a,b; Campbell et al, 1989).
  • ICAM-1 has been included as a member of the immunoglobulin supergene family (Staunton et al, 1988; Simmons et al, 1988; Williams and Barclay, 1988).
  • the receptor for ICAM-1, LFA-1 belongs to the structurally different integrin family of proteins.
  • the integrins are heterodimeric proteins, a number of which, bind via the tripeptide RGD motif in their ligands (Ruoslahti and Pierschbacher, 1987).
  • RGD sequence in ICAM-1 Staunton et al, 1988; Simmons et al 1988
  • GRGDSP peptide inhibit ICAM-l/LFA-1 binding (Marlin and Springer, 1987).
  • ICAM-l/LFA-1 linkage is of critical importance in the homotypic adhesion of haemopoietic cells (Makgoba et al, 1988; Boyd et al, 1989a), migration of lymphocytes (Dustin and Springer, 1988; Boyd et al, 1989c) and in cell-cell contact-mediated immune mechanisms such as T and B cell activation (Boyd et aj., 1988; Dougherty et al, 1988; Altmann et al .
  • ICAM-1 T-cell cytotoxicity and non-specific cytotoxic cell activity
  • This central role of ICAM-1 in cell-cell contact mediated-immune mechanisms and the identification of ICAM-1 as the major human rhinovirus receptor (Greve et al, 1989; Staunton et al, 1989a) and as a receptor for red blood cells infected with Plasmodium falciparum (Berendt et al, 1989) has raised the possibility that ICAM-1 might be a target for therapy. Blocking of ICAM-1 function may be beneficial in the treatment of diseases including autoimmune disorders, graft versus host disease, graft rejection, severe allergic reactions, the common cold and malaria.
  • ICAM-1 sequence possible binding domains within the ICAM-1 sequence were sought by comparing a hydrophobicity analysis (Kyte and Doolittle, 1982) of ICAM-1 with a dot matrix sequence comparison (Maizel and Lenk, 1981) of ICAM-1 with a structurally similar but functionally distinct protein, Myelin-Associated Glycoprotein (MAG).
  • MAG Myelin-Associated Glycoprotein
  • a unique region of minimal identity within the ICAM-1 sequence was determined.
  • a synthetic peptide based on this sequence mimics the effects of anti-ICAM-1 antibody in inhibiting ICAM-1/LFA- 1-mediated adhesion.
  • the present invention provides ICAM-1-like peptides and analogues thereof having anti-adhesive properties. More particularly, the ICAM-1-like peptides and their analogues are capable of inhibiting or reducing cell adhesion in mammals.
  • the mammal is a human, ruminant animal or domestic animal. Most preferably, the mammal is a human.
  • ICAM-like peptides in accordance with this invention and as used in the present specification and claims, is meant a peptide, or a polypeptide, substantially similar to, or identical with, a region of ICAM-1 capable of binding, associating or otherwise interacting with LFA-1 and thereby inhibiting or reducing ICAM-1-dependent aggregation and/or inhibiting or reducing ICAM-1- dependent effector function of the immune response.
  • the ICAM-1-like peptides or their analogues therefore, are also capable of binding, associating or otherwise interacting with LFA-1.
  • An amino acid sequence substantially similar to a region of ICAM-1 includes sequences having greater than 70% homology and preferably greater than 80% homology with the selected ICAM-1 region.
  • the ICAM-1-like peptides may have an amino acid sequence identical with the corresponding sequence in ICAM-1 or may contain amino acid derivatives comprising single or multiple amino acid additions, deletions and/or substitutions compared to the amino acid sequence of the particular region of ICAM-1.
  • the peptides contemplated herein may be chemically synthesized such as by solid phase peptide synthesis or may be prepared by subjecting the ICAM-1 polypeptide to hydrolysis or other chemically disruptive processes whereby fragments of the molecule are produced. Alternatively, the peptides could be made in vitro or in vivo using recombinant DNA technology.
  • the peptides may need to be synthesized in combination with other proteins and then subsequently isolated by chemical cleavage or the peptides may be synthesized in multiple repeat units.
  • multiple antigen peptides could also be prepared according to Tam (1988). The selection of a method of producing the subject peptides will depend on factors such as the required type, quantity and purity of the peptides as well as ease of production and convenience.
  • analogues extends to any functional chemical equivalent of an ICAM- 1-like peptide characterized by its increased stability and/or efficacy in vivo or in vitro in respect of the ability to bind, associate or otherwise interact with LFA-1.
  • analogue is also used herein to extend to any amino acid derivative of the ICAM-1-like peptides as described above.
  • Analogues of ICAM-1-like peptides contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the peptides or their analogues.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ⁇ amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6, trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal- 5'-phosphate followed by reduction with NaBH -
  • modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ⁇ amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6, trinitrobenzene sulphonic acid (TNBS); acylation
  • the guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatisation, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuric-4- nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4- amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • peptides could be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids, introduction of double bonds between C ⁇ and C p atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between.a side chain and the N or C terminus.
  • the ICAM-1-like peptides or their analogues may single length or in tandem or multiple repeats.
  • a single type of peptide or analogue may form the repeats or the repeats may be composed of different molecules including suitable carrier molecules.
  • the present invention therefore, extends to peptides or polypeptides and amino acid and/or chemical analogues thereof corresponding to regions of ICAM-1 capable of, or responsible for, binding, associating or otherwise interacting with LFA-1 or parts thereof and thereby inhibit or reduce ICAM-1-dependent aggregation and/or ICAM-1-dependent effector function of the immune response.
  • Reference in the specification and claims herein to "ICAM-1-like peptide” includes all such amino acid and chemical analogues broadly described above.
  • the ICAM-1-like peptide corresponds to amino acids in one or more of the regions 330 to 430, 100 to 200 and/or 50-90 of ICAM-1. More preferably, the ICAM-1-like peptides correspond to amino acids in one or more of the regions 367 to 394, 114 to 141 and/or 54 to 82 of ICAM-1.
  • the foregoing amino acid positions are according to Simmons et al (1988).
  • the present invention provides the following ICAM-1-like peptides:
  • JF9 having the amino acid sequence: VLYGPRLDERDAPGNWTWPENSQQTPMC and its amino acid and chemical analogues;
  • JF10 having the amino acid sequence:
  • Peptide JF9 corresponds to positions 367 to 394 of ICAM-1 but Cys-378 is replaced by Ala.
  • the present invention extends to the latter peptide with Cys in position 378, i.e. without the Ala substitution.
  • the aforementioned peptides inhibit ICAM-1-dependent aggregation and ICAM-1-dependent effector function of the immune response, such as, but not limited to, the inhibition of T-cell mediated killing of the K562 erythroid cell line.
  • Amino acid analogues of JF9 contemplated herein include, but are not necessarily limited to, the following amino acid sequences shown in Table 1:
  • AMINO ACID SEQUENCE ICAM-1 SEQUENCE POSITION ACCORDING TO SIMMONS (1988)
  • [X] is A or C Amino acid analogues of JF10 contemplated herein include but are not limited to the following sequences shown in Table 2:
  • AMINO ACID SEQUENCE ICAM-1 SEQUENCE POSITION ACCORDING TO SIMMONS (1988)
  • Amino acid analogues of JF14 contemplated herein include but are not limited to the following sequences shown in Table 3:
  • JF14 YELSNVQEDSQPMCYSNCPDGQSTAKTFL [54-82]
  • JF14J NVQEDSQPMC [58-67]
  • JF14K QEDSQPMCYSNCPDGQS [60-76]
  • Peptide JF9 corresponds to a sequence located within the predicted fourth Ig-like domain (D4) on the ICAM-1 molecule (Staunton et al. 1988). Staunton et al, (1989b) have suggested that the LFA-1 binding region of ICAM-1 is located within domains 1 and 2 (Dl and D2) using domain deletion and systematic amino acid substitution techniques.
  • the recently defined homologous ICAM-2 molecule has only three Ig-like domains corresponding to Dl, D2 and D3 of the ICAM-1 molecule (Staunton et al, 1989b) and lacks the unique region in ICAM-1 defined by the present studies. This incongruity reflects the likelihood that there is more than one binding domain within the ICAM-1 molecule mediating the interaction with LFA-1.
  • the inhibitory properties of JF9 show that the region of the ICAM-1 molecule identified herein is an important component of the binding process.
  • putative binding domains in JF9 and JF10 to LFA-1 may be different to other LFA-1 binding domains, such as those involved in human immunodeficiency virus infection (Fecondo et al, 1991).
  • the present invention provides a method for inhibiting ICAM-1-dependent cell adhesion and/or ICAM-1- mediated immune mechanisms in a mammal comprising administering to said mammal an effective amount of an ICAM-1-like peptide or analogue thereof for a time and under conditions sufficient to inhibit or reduce ICAM-1 mediated cell adhesion.
  • the mammal is a human or a domestic or ruminant animal. Most preferably, the mammal is a human.
  • the subject method will be particularly useful in the treatment of, inter alia.
  • graft versus host disease tissue graft rejection, allergic reaction, inflammatory condition such as labyrinthitis, cancers such as arising from the immune system and possibly infectious agents such as rhinovirus and/or other pathogenic organisms, eg. malaria parasite, where the pathogen interacts with the ICAM-1 target portion of ICAM-1.
  • the method of administration will vary depending on the circumstances. Examples of such administration would include intravenous injection or infusion. Depending on the particular ICAM-1-like peptide or analogue used, administration by other routes, such as intranasal, oral, intraperitoneal, subcutaneous, rectal or by any means whereby the active molecules can be put in contact with target cells may be possible. Administration may also be topical.
  • the peptides or analogues may have to be modified or co-administered with other molecules to prevent their breakdown or to prolong their half life or to facilitate entry into the bloodstream or target area.
  • microorganisms such as normal flora (e.g. gut organisms) may be engineered to express and release the ICAM-1 peptides or their analogues of the present invention.
  • the microorganisms may be mutated such that they remain viable but unable to grow.
  • a single length peptide may be secreted or in tandem or multiple repeats.
  • a number of different peptides may also be fused together or fused with a carrier molecule. Examples of suitable organisms include Escherichia coli.
  • the ICAM-1-like peptide may also be co-administered or sequentially administered with one or more active agents.
  • active agents include but are not limited to cytokines, anti-inflammatory agents and analogues of ICAM-1-like peptides.
  • the present invention extends to mixtures of two or more different ICAM-1-like peptides and/or their analogues.
  • the ICAM-1-like peptides may be part of a larger molecule where the larger molecule may or may not be related to native ICAM-1. Conveniently, however, the larger molecule is a larger fragment of ICAM-1.
  • the effective amount of ICAM-1-like peptide or its analogue will be that required to reduce or inhibit ICAM- 1-mediated binding, association or other interaction with LFA-1 involved in ICAM-l/LFA-1 cell adhesion and must not be at cytotoxic levels or at least cause only clinically acceptable cytotoxicity.
  • the actual concentration selected will vary depending on the exigencies of the clinical situation but will generally be greater than 0.5 ⁇ g/ml and preferably in the range 0.005 ⁇ M to 200 ⁇ M.
  • the inclusion of agonists to the ICAM-1-like peptides or their analogues in any therapeutic programme or the inclusion of other molecules capable, for example, of suppressing immune responses, may result in less ICAM-1- like peptide or analogue being required. In such a case, 5nM to lOO ⁇ M of ICAM-1-like peptide or analogue may be sufficient.
  • compositions comprising an ICAM-1-like peptide and/or its analogue and one or more pharmaceutically acceptable carriers and/or diluents.
  • suitable carrier vehicles and their formulation inclusive of other human proteins, eg. human serum albumin, is described in Remington's Pharmaceutical Sciences 17th ed., Mack Publishing Co., edited by Osol et al., which is hereby incorporated by reference.
  • the pharmaceutical compositions may also include additional molecules to stabilize the active agent or which act as agonists or otherwise assist the ICAM-1- peptide and/or its analogue to perform the desired function.
  • the ICAM-1-peptide and/or its analogue will be in combination with another active agent which may inhibit cell adhesion and/or immune response mechanisms.
  • the ICAM-1-like peptide or analogue thereof may be used in the manufacture of a medicament for, the treatment of diseases arising from ICAM-l/LFA-1 cell adhesion such as, but not limited to, graft versus host disease, tissue graft rejection, cancer, severe allergic reaction and/or infection by pathogenic agents whose infectivity depends upon the function or integrity of ICAM-1, examples are the malaria parasites and rhinoviruses.
  • the present invention also extends to the use of ICAM-1- like peptides and/or analogues thereof to quantitatively or qualitatively detect or screen for LFA-1 receptors on cells in the blood stream or other body fluids.
  • a range of techniques is available which could use the above molecules directly or indirectly via antibodies to same, such as ELISA or radioimmuno assay.
  • the ICAM-1-like peptide and/or its analogue may also be used in immobilized form to isolate specific LFA-1 expressing cells.
  • the present invention also extends to monoclonal or polyclonal antibodies to the ICAM-1-like peptides or their analogues. Further in accordance with the present invention, there is, provided a method for determining functional binding domains in polypeptides or proteins.
  • the ICAM-1-like peptide JF9 was identified using this method. It is not the intention, however, to limit JF9 in any way to this method, nor should the applicability of this method be in any way limited to JF9 or ICAM-1-like peptides.
  • this aspect of the present invention is directed to a method of fingerprinting one or more functional domains in a polypeptide or protein, comprising identifying one or more sequences of amino acids in the hydrophilic portion of said polypeptide or protein which are substantially absent in a functionally distinct but structurally similar, second polypeptide or protein.
  • fingerprinting is used in its widest sense and includes characterising, identifying or otherwise determining a particular region of a polypeptide or protein.
  • the term is particularly used herein to refer to a target sequence of amino acids.
  • functional domain is meant the one or more continuous sequences of amino acids in a polypeptide or protein which, either separately or in combination with the remainder of the polypeptide or protein, including amino acid sequence and/or tertiary structure, are responsible for or contribute to a particular activity of function associated with the polypeptide or protein or parts thereof.
  • the functional domain of a protein ligand may be its receptor binding site.
  • the functional domains of interest in respect of the ICAM-1 molecule are those involved in adhesion of ICAM-1 to LFA-1.
  • a “functionally distinct" polypeptide or protein means one with a similar amino acid sequence but with a different function relative to another polypeptide or protein.
  • the hydrophilic portions of the polypeptide or protein may be determined by a number of known techniques. One convenient method is the Kyte-Doolittle algorithm for predicting potential hydrophilic binding regions (Kyte and Doolittle, 1982). In relation to membrane or cell wall associated or embedded polypeptides or proteins, these polar regions are likely to be surface exposed. Hydrophilic regions have negative hydropathy indices.
  • a functional domain is putatively identified in the hydrophilic region when said hydrophilic region contains at least one continuous sequence of amino acids substantially absent in a functionally distinct second polypeptide or protein under consideration.
  • Such a comparison of amino acid sequences may be accomplished by any number of procedures such as, but not limited to a dot-matrix sequence comparison which shows simple identity versus non- identity scoring of amino acids.
  • a functional domain is, therefore, putatively identified by finding an amino acid sequence in the polypeptide or protein of interest with essentially no identity in the functionally distinct polypeptide or protein.
  • the order by which the steps of the subject of method is performed may be altered such that the continuous amino acid sequences in a polypeptide or protein of interest may first be identified and then a hydrophobicity plot conducted to locate which of these sequences correspond to a predicted hydrophilic (e.g. polar surface) region.
  • a hydrophobicity plot conducted to locate which of these sequences correspond to a predicted hydrophilic (e.g. polar surface) region.
  • another aspect of the present invention relates to a method for fingerprinting a functional domain in a polypeptide or protein which method comprises first identifying one or more continuous non-identical amino acid sequences in said first polypeptide or protein absent in a second functionally distinct but structurally similar polypeptide or protein, identifying likely surface exposed regions of said first polypeptide or protein and then determining whether said one or more amino acid sequences occurs in said surface exposed regions.
  • the approach used in accordance with the present invention has general application for the identification of functional domains in structurally similar proteins which do not share the same biological activity.
  • the subject method relies on two assumptions: (a) the functional domain(s) are likely to be hydrophilic in nature and (b) there is no significant sequence identity in the domain to other homologous but functionally distinct proteins.
  • Figure 1 is a graphical representation showing a structural and comparative analysis of ICAM-1.
  • the lines extending from the dot matrix plot to the hydropathy plot indicate the correspondence between the region of non-identity within the ICAM-1 sequence to a predicted polar and thus likely surface-exposed region of the ICAM-1 molecule.
  • Figure 2 is a photographic representation depicting inhibition studies of ICAM-l/LFA-1 dependent homotypic aggregation of Raji cells.
  • Raji cells with no peptide present (a) Raji cells with no peptide present; (b) inhibition of Raji cell aggregation with 20 ⁇ g/ml of purified WEHI-CAM-1 antibody; (c) inhibition of Raji cell aggregation by 80 ⁇ g/ml JF9 peptide; and (d) Raji cells in the presence of a control peptide, 500 ⁇ g/ml JF7B.
  • Figure 3 is a graphical representation showing inhibition of cytotoxic T cell activity by the JF9 peptide.
  • T cell- mediated killing of K562 cells was measured by the release of • ⁇ 1 Cr into the medium as previously described.
  • T cells were activated in a two way mixed lymphocyte response between two normal, unrelated donors.
  • the cells (10 6 /ml) were mixed in a 1:1 ratio and cultured in RPMI 1640/10% (v/v) FCS for 4 days.
  • Recombinant interleukin 2 (Cetus Corporation) was added (200 U/ml) and the cultures continued for a further 3 days.
  • Cells were harvested and added to a constant number (10* cells/well) of 51 Cr- labelled K562 cells in 96 well microtitre tray. 51 Cr release was measured after 4 hours.
  • the following example relates to the identification of functional binding regions in ICAM-1, the synthesis of peptides based thereon and their use to inhibit ICAM-1- mediated adhesion to cells which express LFA-1.
  • Peptides were synthesised using a highly optimised protocol (Clark-Lewis and Kent, 1989), coupling t-Boc amino acids to 4-methylbenzhydrylamine resin or 4- (oxymethyl) phenylacetamidomethyl-t-Boc-L-Pro resin with an average coupling efficiency of greater than 99.3% per cycle as determined by quantitative ninhydrin analysis (Sarin et ai, 1981). Following standard HF cleavage from the resins, all peptides were purified by reverse phase HPLC, using an Aquapore C8 100 x 10 mm 20 micron Prep 10 cartridge column and Aquapore RP-300 30 x 4.6 mm 7 micron cartridge columns (Applied Biosysterns, Santa Clara, CA).
  • the ICAM-1 sequence was analysed using the Kyte-Doolittle (1982) algorithm for predicting potential hydrophilic binding regions.
  • Figure 1(a) it can be seen that there are a number of predicted hydrophilic regions with hydropathy indices as low as -3.4, and clustering of both positive and negative charges. These polar regions are likely to be surface exposed.
  • the next step was to define which of these regions may be functional binding sites.
  • unique regions were predicted by a dot-matrix sequence comparison of ICAM-1 with its functionally distinct homologue, MAG. Simple identity versus non-identity scoring of amino acids is shown in the dot matrix plot in Figure 1(b).
  • a detailed analysis of the resulting plot revealed a region within the ICAM-1 sequence which had essentially no identity with MAG. This region spanned amino acid residues 369 to 391 and is indicated by a virtual absence of dots or stings of dots on the plot shown in Figure 1(b).
  • this sequence was analysed in conjunction with the hydrophobicity plot data, there was a striking correspondence to a predicted polar surface region of the ICAM-1 sequence.
  • JF7B NAQTSVSPSKVILPRGGSVLVTC
  • JF9 VLYGPRLDERDAPGNWTWPENSQQTPMC JF10: GGAPRANLTWLLRPGKELKREPAVGEP
  • JF13A NRQRKIKKYRLQQAQKGTPMKPNTQATPP

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Abstract

La présente invention se rapporte à des peptides analogues à la Molécule-1 d'Adhésion Intercellulaire, à des analogues et à des anticorps de ces peptides, ainsi qu'à des compositions pharmaceutiques comprenant ces peptides et leur utilisation pour inhiber ou réduire l'adhésion des cellules chez les mammifères. L'invention se rapporte aussi à une méthode d'empreinte génétique du domaine fonctionnel d'un polypeptide ou d'une protéine.
PCT/AU1991/000205 1990-05-15 1991-05-14 Inhibition d'adhesion cellulaire a l'aide de peptides analogues a la molecule-1-d'adhesion intercellulaire et/ou d'analogues de ces peptides WO1991018011A1 (fr)

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Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004568A1 (fr) * 1991-12-18 1994-03-03 Centocor, Inc. Inhibiteurs peptidiques d'inflammation presentant une mediation par les selectines
WO1994027638A1 (fr) * 1993-05-21 1994-12-08 Dana-Farber Cancer Institute, Inc. Anticorps monoclonaux bloquant l'adhesion du ligand sur le recepteur cd22 dans les lymphocytes b matures
DE4335273A1 (de) * 1993-10-15 1995-04-20 Univ Ludwigs Albert Peptide zur Tumortherapie
WO1995028170A1 (fr) * 1994-04-19 1995-10-26 The University Of Kansas Peptides a chaine courte a base d'icam-1/lfa-1 et leur methode d'utilisation
US5525487A (en) * 1992-01-27 1996-06-11 Icos Corporation DNA encoding I-CAM related protein
US5532127A (en) * 1992-01-27 1996-07-02 Icos Corporation Assay for 1-CAM related protein expression
US5589453A (en) * 1988-09-01 1996-12-31 Molecular Therapeutics, Inc. Human rhinovirus receptor protein (ICAM-1) that inhibits rhinovirus attachment and infectivity
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US5871733A (en) * 1990-07-20 1999-02-16 Bayer Corporation Multimeric forms of human rhinovirus receptor protein
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US5891841A (en) * 1991-06-11 1999-04-06 The Center For Blood Research, Inc. Methods of using intercellular adhesion molecule-3 (ICAM-3), antibodies thereto, and soluble fragments thereof
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US6100383A (en) * 1992-01-27 2000-08-08 Icos Corporation Fusion proteins comprising ICAM-R polypeptides and immunoglobulin constant regions
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US5622700A (en) * 1992-08-21 1997-04-22 Genentech, Inc. Method for treating a LFA-1-mediated disorder
US5484892A (en) * 1993-05-21 1996-01-16 Dana-Farber Cancer Institute, Inc. Monoclonal antibodies that block ligand binding to the CD22 receptor in mature B cells
WO1994027638A1 (fr) * 1993-05-21 1994-12-08 Dana-Farber Cancer Institute, Inc. Anticorps monoclonaux bloquant l'adhesion du ligand sur le recepteur cd22 dans les lymphocytes b matures
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AU698406B2 (en) * 1994-04-19 1998-10-29 University Of Kansas, The ICAM-1/LFA-1 short-chain peptides and method of using same
US6001809A (en) * 1994-07-11 1999-12-14 Elan Pharmaceuticals, Inc. Inhibitors of leukocyte adhesion
US5914112A (en) * 1996-01-23 1999-06-22 Genentech, Inc. Anti-CD18 antibodies in stroke
US7655230B2 (en) 1996-01-23 2010-02-02 Genentech, Inc. Co-administration of a tissue plasminogen activator, anti-CD11b antibody and anti-CD18 antibody in stroke
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US6075004A (en) * 1996-04-26 2000-06-13 The University Of Kansas Peptide compositions which induce immune tolerance and methods of use
US6653282B1 (en) 1996-04-26 2003-11-25 Stephen H. Benedict Peptide Compositions which induce immune tolerance and methods of use
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