WO1997041149A1 - Compositions de petpides entraînant une tolerance immunitaire et leurs procedes d'utilisation - Google Patents

Compositions de petpides entraînant une tolerance immunitaire et leurs procedes d'utilisation Download PDF

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WO1997041149A1
WO1997041149A1 PCT/US1997/006799 US9706799W WO9741149A1 WO 1997041149 A1 WO1997041149 A1 WO 1997041149A1 US 9706799 W US9706799 W US 9706799W WO 9741149 A1 WO9741149 A1 WO 9741149A1
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Prior art keywords
protein
peptides
peptide
binding
group
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PCT/US1997/006799
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English (en)
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Stephen Benedict
Teruna J. Siahaan
Marcia A. Chan
Scott A. Tibbetts
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The University Of Kansas
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Priority claimed from US08/844,978 external-priority patent/US6075004A/en
Application filed by The University Of Kansas filed Critical The University Of Kansas
Priority to AU29246/97A priority Critical patent/AU2924697A/en
Priority to JP53900997A priority patent/JP2002515873A/ja
Priority to EP97923445A priority patent/EP0973797A4/fr
Publication of WO1997041149A1 publication Critical patent/WO1997041149A1/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
    • 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

Definitions

  • the present invention is broadly concerned with compositions of two or more peptides effective in inhibiting the binding of proteins, and corresponding methods of use. More particularly, the preferred form of the invention relates to peptide compositions which inhibit the binding of leukocyte function-associated antigen (LFA- 1) and intercellular adhesion molecule (ICAM-1); these peptide compositions can be used to treat disease states such as rejection of transplanted organs, allergies, and autoimmune diseases.
  • LFA-1 leukocyte function-associated antigen
  • IAM-1 intercellular adhesion molecule
  • Ras and Raf-1 are proto-oncoproteins that transduce growth and differentiation signals initiated by tyrosine kinases; ras binds to Raf-1 and thereby inhibits Ras-GAP activity (Zhang et al., 1993, Nature 364:308-313).
  • Yeast Cdc7 protein kinase and Dbf protein are both required for the initiation of DNA replication; these proteins bind to each other, and it is thought that Dbf4 is specific for the activation of Cdc7 kinase (Jackson et al., 1993, Mol Cell Biol. 13:2899-2908).
  • Yeast GAL4 protein consists of two protein subunits which, when bound together, activate the genes encoding enzymes of galactose utilization (Fields et al., 1989, Nature 340:245-246).
  • contact sites within proteins that bind to one another are noncontinuous domains of amino acids. These contact sites can be found in different subunits of a protein as in the case of the heavy and light chains of antibodies (Carayannopoulos et al., 1993; Perutz, 1992, Protein Structure, New Approaches to Disease and Therapy, pp. 41-76, W.H. Freeman and Co.). Alternatively, these contact sites can be found in different areas of the same subunit as in the case of the ⁇ subunit of LFA-1 (Edwards et al., 1995, J Biol. Chem. 270:12635-12640; Stanley et al., 1994, EMBO 13:1790-1798).
  • T-cells of an organism recognize and react to "self proteins. This recognition occurs when specific proteins on the surface of the T-cells bind to the corresponding self proteins. This type of reaction results in rheumatoid arthritis, insulin-dependent diabetes mellitus, and multiple sclerosis. Allograft rejection also results from T-cell attack.
  • T-cell activation requires two signals: (1) interaction of the T- cell receptor with an appropriate MHC-antigen complex, and (2) a second signal provided by adhesion molecules.
  • the second signal may be provided by binding of the adhesion receptor, LFA-1 (CDl la and CDl 8), to one of its counter-receptors such as ICAM-1 (CD54) (Staunton et al., 1990, Cell 61 :243-254).
  • the antigen-specific T-cells are induced to die by apoptosis or to enter a state of cellular anergy. Blockage of this interaction by monoclonal antibodies to LFA-1 and ICAM-1 results in increased survival time for mice receiving heart allografts (Isobe et al., 1992, Science 255:1125-1127).
  • compositions of short- chain peptides can inhibit the binding of one protein (a first protein) to another protein (a second protein).
  • the mutual binding of a pair of proteins is responsible for signal transductions occurring in many biological processes.
  • inhibition of such protein binding can result in induction of immune tolerance. Therefore, the peptide compositions of the present invention can be used, for example, as a treatment for disease states such as rejection of transplanted organs, allergies, and autoimmune diseases (e.g., rheumatoid arthritis, insulin-dependent diabetes mellitus, and multiple sclerosis).
  • the present invention is directed to peptide compositions, and methods of using these compositions, wherein at least one peptide binds to the first protein, and at least one peptide binds to the second protein, whereby the first protein is inhibited from binding to the second protein.
  • the first protein is an integrin (e.g., the ⁇ and ⁇ subunits of LFA-1) while the second protein is an integrin- binding protein (e.g., ICAM-1).
  • the protein system is the LFA-l/ICAM-1 system, each peptide which binds to LFA-1 is derived from ICAM-1, while each peptide which binds to ICAM-1 is derived from LFA-1.
  • each ICAM-1 -based peptide contains a sequence present in a sequence selected from the group consisting of Sequence ID Nos. 1-14, contains a sequence selected from the group consisting of Sequence ID Nos. 1-14, or has a sequence selected from the group consisting of Sequence ID Nos. 1-14; furthermore, each LFA-1 -based peptide contains a sequence present in a sequence selected from the group consisting of Sequence ID Nos. 15-35, contains a sequence selected from the group consisting of Sequence ID Nos. 11-35, or has a sequence selected from the group consisting of Sequence ID Nos. 11-35.
  • each peptide is not immunogenic, and has a molecular weight under 20 kilodaltons.
  • each peptide contains at least one unnatural amino acid (i.e., an amino acid that is itself not of the 20 normally found amino acids, or one of the normal 20 amino acids in an abnormal location) and is cyclic in order to protect the peptide from degradation.
  • the peptides described herein for the purposes of illustration are separate molecules, the present invention comprehends use of peptides which are attached to one another.
  • the peptide composition includes a combination of cyclic peptides (e.g., peptides having the sequences of Sequence ID Nos. 7, 19, 26, and 34).
  • Figure 1 is a diagram illustrating ICAM-1 -based peptides IB, IB-L, IB-C, IB-R, cIB-L, cIB-C, and cIB-R, and the locations in ICAM-1 from which their sequences were derived (see footnote 1 of the table below for peptide nomenclature);
  • Fig. 2 is a diagram illustrating ICAM-1 -based peptides IE, IE-L, IE-C, IE-R, cIE-L, cIE-C, and cIE-R, and the locations in ICAM- 1 from which their sequences were derived;
  • Fig. 3 is a diagram illustrating LFA-1 ⁇ -based peptides LAB, LAB-L, LAB- C, LAB-R, cLAB-L, cLAB-C, and cLAB-R, and the locations in the ⁇ subunit of LFA-1 from which their sequences were derived;
  • Fig. 4 is a diagram illustrating LFA-l ⁇ -based peptides LAB.2, LAB.2-L,
  • Fig. 5 is a diagram illustrating LFA-1 ⁇ -based peptides LBE, LBE-L, LBE-C, LBE-R, cLBE-L, cLBE-C, and cLBE-R, and the locations in the ⁇ subunit of LFA-1 from which their sequences were derived;
  • Fig. 6 is a photograph showing cells from a homotypic-adhesion assay wherein the cells were unstimulated;
  • Fig. 7 is a photograph showing cells from a homotypic-adhesion assay wherein the cells were phorbol 12,13-dibutyrate (PDB)-stimulated
  • Fig. 8 is a photograph showing cells from a homotypic-adhesion assay wherein the cells were PDB-stimulated and treated with cLBE-R;
  • PDB phorbol 12,13-dibutyrate
  • Fig. 9 is a bar graph illustrating the results of homotypic-adhesion assays wherein cells were unstimulated, PDB-stimulated, PDB-stimulated and treated with cIB-R, and PDB-stimulated and treated with a mixture of cIB-R, cLAB-L, cLAB.2-L, and cLBE-C (4 cyclics);
  • Fig. 10 is a graph illustrating the arthritis score test results generated during the induced arthritis mouse test described in Example 2.
  • Fig. 11 is a graph depicting the arthritis incidence data generated during the induced arthritis mouse test described in Example 2.
  • Fig. 12 is a graph illustrating the data developed during the mouse skin transplant test of Example 3;
  • Fig. 13 is a bar graph illustrating the proliferation of responder cells in a mixed lymphocyte reaction assay, and the inhibitory effect of certain peptides in accordance with the invention on such proliferation;
  • Fig. 14 is a bar graph illustrating the proliferation of responder cells in a mixed lymphocyte reaction assay, and the inhibitory effect of certain peptides in accordance with the invention on such proliferation;
  • Fig. 15 is a bar graph illustrating the proliferation of responder cells in a mixed lymphocyte reaction assay, and the inhibitory effect of certain peptides in accordance with the invention on such proliferation;
  • Fig. 16 is a bar graph illustrating the proliferation of responder cells in a mixed lymphocyte reaction assay, and the inhibitory effect of certain peptides in accordance with the invention on such proliferation
  • Fig. 17 is a bar graph illustrating the proliferation of responder cells in a mixed lymphocyte reaction assay, and the inhibitory effect of certain peptides in accordance with the invention on such proliferation.
  • compositions containing two or more peptides which inhibit the binding of a first protein to a second protein are made according to the following steps:
  • a protein system is selected in which a first protein binds to a second protein.
  • Combinations of peptides containing at least one first protein-binding peptide (derived from the second protein) and at least one second protein-binding peptide (derived from the first protein) are assayed to determine which combinations are effective in inhibiting the binding of the two proteins.
  • the peptides and compositions are assayed in biological assay, e.g., a mixed lymphocyte reaction, induced arthritis study and/or skin transplant study.
  • step 1 the prior art abounds with examples of protein systems in which a first protein binds to a second protein. A few of these examples are described above.
  • step 2 there are three standard methods by which contact sites within proteins are identified. All three methods require that the gene encoding the protein is cloned and sequenced. Hundreds of such gene sequences have been reported in the prior art. If the gene encoding the protein of interest has not been cloned and sequenced, the state of molecular biology today makes clear that doing so is routine to a skilled artisan.
  • the first method has been previously described (Stanley et al., 1994, EMBO 13:1790- 1798, the teachings of which are incorporated by reference herein).
  • a domain of the first or second protein is removed by deleting the corresponding segment from the gene encoding the protein.
  • the altered gene is expressed in bacteria to produce an altered protein.
  • the altered protein is then assayed for its ability to bind to the other protein. If the altered protein does not bind to the other protein, the contact site was removed. The contact site is thereby localized.
  • the second method is employed once a contact site has been localized as described above.
  • This method involves site-directed mutagenesis of the gene of the protein to identify precisely which amino acids are involved in protein binding.
  • a specific nucleotide is changed in order to change a single amino acid in the contact site.
  • An altered protein generated in this manner is then assayed for its ability to bind to the other protein as described above. The importance of the specific amino acid to protein binding is thereby deduced.
  • site-directed mutagenesis is a routine and widely-used technique.
  • many site-directed mutagenesis kits are commercially available.
  • One such kit is the "Transformer Site Directed Mutagenesis Kit" sold by Clontech Laboratories (Palo Alto, CA). c.
  • the third method is the "Yeast Two-Hybrid System.” This method can be performed using commercially available kits.
  • One such kit is the "MATCHMAKER Two-Hybrid System” sold by Clontech Laboratories (Palo Alto, CA).
  • a domain of the first protein is subcloned into a plasmid (Plasmid A) and is expressed in yeast as a fusion protein.
  • This fusion protein contains a domain of a yeast transcription factor in addition to the domain of the first protein.
  • the gene of the second protein is also subcloned into a plasmid (Plasmid B) and is also expressed in yeast as a fusion protein; in this case, the fusion protein contains a different domain of the yeast transcription factor.
  • a functional hybrid transcription factor is formed from the transcription-factor domains encoded by Plasmids A and B. This hybrid transcription factor results in the expression of a reporter gene. Thus, expression of the reporter gene indicates that the domains of the first and second proteins contain contact sites of Proteins A and B.
  • step 3 those skilled in the art would recognize that peptides can be commercially synthesized by a variety of laboratories.
  • Example 1 below gives a detailed protocol for conducting a homotypic-adhesion assay.
  • This assay can be used to determine which peptides inhibit the binding of LFA-1 to ICAM-1 if the LFA-l/ICAM-1 system was selected in step 1. If a different protein system was selected, an antibody-binding assay can be used to carry out step 4.
  • This assay, as applied to the LFA-l/ICAM-1 system is described in Benedict et al., Modulation of T Cell Morphology and Induction of Homotypic Adhesion by a Protein Tyrosine Kinase Inhibitor, Cellular Immunology, 162:001-010 (1995), incorporated by reference herein.
  • the antibody-binding assay can be used with other protein systems, and would know how to modify the assay accordingly.
  • step 5 combinations of peptides can be assayed as described in step
  • MLR mixed lymphocyte reaction
  • RA rheumatoid arthritis
  • skin graft studies give details of mixed lymphocyte reaction (MLR), rheumatoid arthritis (RA) and skin graft studies.
  • MLR is the normally best predictor of in vivo activity to aid in selection of inhibiting peptides. This is because the MLR measures a biological readout, namely cell interaction followed by induced proliferation. Thus, the MLR is a direct measurement of allogeneic T cell responses.
  • the MLR directly models the events involved in bone marrow transplantation, and indirectly predicts the biological effectiveness of the peptides in solid organ transplantation and in the treatment of autoimmune diseases such as rheumatoid arthritis, insulin-dependent diabetes and multiple sclerosis.
  • a detailed protocol of the MLR is set forth in Example 4 below.
  • the protein system selected by the applicants to illustrate the preferred embodiment of the invention was the LFA-l/ICAM-1 system.
  • Noncontiguous domains of LFA-1 and ICAM-1 which were believed to be the contact sites of the two proteins were selected.
  • the sequences of LFA-1 and ICAM-1 are known. Peptides based upon the sequences found in these contact sites were commercially synthesized using standard techniques. Some of the peptides were cyclized. Homotypic-adhesion assays using the peptides were conducted to determine which LFA-1 -based peptides and which IC AM- 1 -based peptides inhibited the binding of LFA- 1 to IC AM- 1.
  • peptides and peptide combinations are injected into mice that have received skin allografts in order to determine which peptides or peptide combinations are effective in inhibiting the rejection of transplanted organs.
  • Those of ordinary skill in the art understand that the results of in vitro and in vivo experiments described herein are predictive of the efficacy of such peptides and peptide combinations in inhibiting rejection of transplanted organs in humans and ameliorating autoimmune diseases.
  • Peptides were synthesized either at the La Jolla Cancer Research
  • 96-well Falcon tissue culture plates were used for the homotypic-adhesion assay. pH neutralized, lyophilized peptides were resuspended in RPMI at a concentra ⁇ tion of 5 mM. Aliquots of the peptides were added to each well at appropriate volumes to yield a final concentration of 1, 10, 100, 250, 500, or 1000 ⁇ M in a total sample volume of 100 ⁇ L. RPMI was added to each well to bring the total volume to 50 ⁇ L.
  • Freshly purified, resting cells were used for the assay. A quantity of cells sufficient for use in the assay was aliquoted from the stock. 50 ⁇ l of cell suspension was then added to each of the wells containing RPMI alone (unstimulated cells). To the remaining cells, PDB was added to a final concentration of 10 "8 M. After mixing well,
  • RESULTS Peptides The table below shows peptides constructed in accordance with the present invention, corresponding Sequence ID Numbers, and corresponding dumping- index values obtained from homotypic-adhesion assays:
  • Nomenclature of peptides is as follows: the first upper case letter indicates whether the peptide is derived from ICAM-1 (I) or LFA-1 (L); the second upper case letter in the name of each LFA-1 -based peptide indicates whether the peptide is derived from the ⁇ (A) or ⁇ (B) subunit of LFA-1 ; the second upper case letter in the name of each ICAM-based peptide and the third upper case letter in the name of each LFA-1 -based peptide indicates whether the peptide blocked (B) or enhanced (E) the binding of another peptide to either LFA-1 or ICAM-1 in previous studies; the upper case letter following a hyphen indicates whether the peptide is derived from the left (L), center (C), or right (R) segment of parent peptide IB, IE, LAB, LAB.2, or LBE; a lower case "c" indicates that the peptide is cyclic.
  • the clumping index refers to the relative degree to which cells were clumped in the homotypic-adhesion assay; clumping-index values of 0 and 100 represent the mean clumping-index values of unstimulated cells and PDB-stimulated cells, respectively; the degree to which each peptide inhibited PDB-stimulated cell clumping is indicated by the magnitude of the respective clumping index; the closer the clumping index is to 0, the greater the degree of inhibition of cell clumping effected by the respective peptide.
  • Clumping index was quantified by determining the mean clumping index per test run +/- standard error.
  • EBL which has the reverse sequence of LBE and was used as a negative control
  • Fig. 5 Homotypic- Adhesion Assay.
  • the table above gives the results of the homotypic-adhesion assays using individual peptides.
  • PDB induces intercellular clumping of human T-cells by activating the LFA-l/ICAM-1 binding interaction between the T-cells.
  • Test runs which do not include PDB do not result in significant cell clumping, while test runs including only PDB result in maximal clumping.
  • the degree to which PDB-stimulated cell clumping is inhibited by a peptide indicates the degree to which the peptide blocks the interaction between LFA-1 present on the surface of one T-cell and ICAM-1 present on the surface of another T-cell.
  • cIE-C inhibited PDB- stimulated cell clumping to a much greater extent than its linear parent IE-C, suggesting that cyclic fragments of IE may inhibit the binding of LFA-1 to ICAM-1 to a greater extent than the parent peptide IE.
  • Fig. 6 illustrates that unstimulated cells in the homotypic-adhesion assay remained unclumped.
  • Fig. 7 illustrates that cells treated with PDB only became clumped.
  • Fig. 8 illustrates that PDB-stimulated cells treated cLBE-R were clumped to a lesser extent relative to cells treated with PDB alone, indicating that cLBE-R inhibited the binding of LFA- 1 to IC AM- 1.
  • Fig. 9 illustrates that a combination of cIB-R, cLAB-L, cLAB.2-L, and cLBE-C was much more effective than cIB-R alone in inhibiting PDB-stimulated cell clumping, indicating that this combination of peptides was more effective than cIB-R in inhibiting the binding of LFA-1 to ICAM-1.
  • the results of homotypic-adhesion assays shown in Fig. 9 were represented using clumping-index values that ranged from 0 to 10 instead of using clumping-index values that ranged from 0 to 100 as shown in the table above. With respect to Fig.
  • a clumping-index value of 0 was obtained from the test run in which unstimulated cells showed the least amount of clumping.
  • a clumping-index value of 100 was obtained from the test run in which PDB-stimulated cells showed the greatest amount of clumping.
  • PDB-stimulated cells are not reported exactly as 0 and 10, respectively, because more than one test run was conducted using unstimulated cells and PDB-stimulated cells. Since the clumping-index values of 0 and 10 were based upon test runs giving the least amount and greatest amount of cell clumping, respectively, multiple test runs using unstimulated cells resulted in a mean clumping-index value greater than 0, and multiple test runs using PDB-stimulated cells resulted in a mean clumping index less than 10. As in the table above, clumping index was quantified by determining the mean clumping index per test run +/- standard error.
  • mice commercially available female DBA/1 J mice (the Jackson Laboratory) were treated to induce arthritis and a number of the mice were dosed with peptides in accordance with the present invention.
  • mice The female mice were housed in an air conditioned room and quarantined for one week before use. The mice were given standard laboratory chow and tap water ad libitum. A control and three test groups (Pl, P2 and P3) each consisted often mice.
  • Type II collagen (Sigma Chemical) was dissolved overnight in 0.05 N acetic acid at a concentration of 4 mg/ml, after which the solution was emulsified in an equal volume of complete Freund's adjuvant (Sigma Chemical). An aliquot of this emulsion containing 200 ⁇ g of Type II collagen was injected intradermally at the base of the tails of the test groups of mice. Twenty-one days later, an emulsion of Type II collagen and incomplete Freund's adjuvant (Sigma Chemical) containing 200 ⁇ g of the Type II collagen was injected intradermally.
  • test groups were dosed with peptide mixtures suspended in normal saline intravenously once a day for five days after the first collagen injection, as follows:
  • Pl cIB-R (SEQ ID No. 7), 50 ⁇ g; cLBE-C (SEQ ID No. 34), 25 ⁇ g; cLAB.2-L (SEQ ID No. 26), 12.5 ⁇ g; and cLAB-L (SEQ ID No. 19), 12.5 ⁇ g.
  • P2 cIB-R (SEQ ID No. 7), 50 ⁇ g; cLBE-C (SEQ ID No. 34), 25 ⁇ g; and cLAB.2-L
  • the arthritis score of each mouse was the sum of the score for each of the four limbs, the maximum score thus being 12. Arthritic incidence was also recorded. The incidence and arthritis score measurements were made over a period of five weeks, beginning at week 3 after initiation of the experiment.
  • Figs. 11 and 12 it will be seen that 90% of the mice in the control group developed arthritis.
  • the Pl and P2 groups showed inhibition of incidence, and the arthritis score was lower than the control between weeks 5-8.
  • the P3 group exhibited an inhibition of incidence after the eighth week.
  • the arthritis score of this group was higher than that of the control.
  • the donor skin tissue for the transplant was prepared.
  • the entire tail was removed from a C3H (black) mouse. An incision was made on the dorsum of the tail down its entire length and the skin was peeled off. The skin was placed, internal surface down, on the bottom of a plastic petri dish containing enough TC-PBS to keep the tissue moist.
  • mice (1 control and 1 test BALB/c [white] mouse) were anesthetized with a ketamine/xylazine cocktail. A circumferential band was shaved around the thorax and abdomen from the shoulder joints to the hip joints of the mice. A 1 cm 2 graft bed was cut, and the skin was peeled off, leaving the panniculus carnosus intact. The graft tissue was then applied to each mouse, and the animals were then bandaged and held in separate housing for the duration of the study.
  • EXAMPLE 4 In this example, an MLR assay is performed using certain peptides in accordance with the invention, in order to demonstrate that the peptides inhibit the biological effectiveness of the involved T cells.
  • the one-way mixed lymphocyte reaction has been previously described (Kruisbeek et al., Current Protocols in Immunology, Proliferative Assays for T Cell Function, ⁇ 3.12.1-3.12.14 (1991), inco ⁇ orated by reference).
  • human tonsil T cells were suspended at a concentration of 4 x IO 6 cell/ml in cell culture medium (RPMI), for use as responders.
  • Stimulator cells were human mononuclear cells isolated from first Ficoll separation of peripheral blood (buffy coat) and were suspended at a concentration of 4 x IO 6 cell/ml in RPMI culture medium.
  • the stimulator cells were inactivated before use by treatment with 3,000 rad of ionizing radiation from ⁇ "137 Cs source.
  • Y-axis values are mean cpm ⁇ SE (counts per minute).
  • PBLjrad refers to irradiated buffy coat cells
  • tonsil-T refers to T-lymphyocytes derived from tonsil.
  • Mix refers to non-treated, mixed lymphocyte sample, which is the positive control (represented by dashed line). Inhibition of proliferation by anti-CDl la antibody was used as a control for inhibition of the LFA-1 :ICAM-1 interaction.
  • the MLR assay was performed in the presence or absence of IE peptide (SEQ ID No. 8) or cyclic fragments of IE (SEQ ID Nos. 8, 12, 13 and 14). Single peptides were tested at 250, 500, 750 and 1000 ⁇ M.
  • MLR was performed in the absence or presence of certain other peptides, namely cIB-L (SEQ ID No. 5), cIE-L (SEQ ID No. 12), cLBE-L (SEQ ID No. 33), cLAB-L (SEQ ID No. 19), cLAB.2-L (SEQ ID No. 26) at 250 ⁇ M, and combinations of the five peptides (m5) at 250, 500, 750 and 1000 ⁇ M.
  • cIB-L SEQ ID No. 5
  • cIE-L SEQ ID No. 12
  • cLBE-L SEQ ID No. 33
  • cLAB-L SEQ ID No. 19
  • cLAB.2-L SEQ ID No. 26
  • Fig. 15 illustrates MLR assay results using single peptides tested at 50, 150, 250 and 500 ⁇ M (IB (SEQ ID No. 1), cLBE-C (SEQ ID No. 34), cLAB-L (SEQ ID No. 19) and cLAB.2-L (SEQ No. 26)), as well as combinations at the same concentration levels.
  • IB SEQ ID No. 1
  • cLBE-C SEQ ID No. 34
  • cLAB-L SEQ ID No. 19
  • cLAB.2-L SEQ No. 26
  • the combined peptide treatment at each concentration was more effective in inhibiting the proliferative response than any of the single peptides at the same concentration.
  • Figs. 16 and 17 illustrate an MLR wherein single peptides were tested at 250, 500 and 750 ⁇ M (cIB-R (SEQ ID No. 7), cIB-L (SEQ ID No. 5), cLBE-C (SEQ ID No. 34), cLBE-L (SEQ ID No. 33), cLAB-L (SEQ ID No. 19) and cLAB.2-L (SEQ ID No.
  • MOLECULE TYPE peptide (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : l :
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID NO:6 :

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Abstract

La présente invention a pour objet des compositions de peptide qui empêchent la fixation d'une protéine avec une autre, ainsi que les procédés d'utilisation correspondants. Lesdites compositions de peptide contiennent au moins un peptide se fixant à une protéine, et au moins un peptide se fixant à l'autre protéine. Dans la forme de réalisation préférée, la composition de peptide est constituée d'une combinaison de peptides cycliques à base d'ICAM-1 et de LFA-1 qui empêchent la fixation de LFA-1 à ICAM-1. Ces compositions de peptides à base de LFA-1/ICAM-1 peuvent servir à traiter des états malades tels que rejet d'organes transplantés, allergies et maladies auto-immunes.
PCT/US1997/006799 1996-04-26 1997-04-24 Compositions de petpides entraînant une tolerance immunitaire et leurs procedes d'utilisation WO1997041149A1 (fr)

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AU29246/97A AU2924697A (en) 1996-04-26 1997-04-24 Peptide compositions which induce immune tolerance and methods of use
JP53900997A JP2002515873A (ja) 1996-04-26 1997-04-24 免疫寛容を誘起するペプチド組成物およびその利用方法
EP97923445A EP0973797A4 (fr) 1996-04-26 1997-04-24 Compositions de petpides entra nant une tolerance immunitaire et leurs procedes d'utilisation

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US08/844,978 US6075004A (en) 1996-04-26 1997-04-23 Peptide compositions which induce immune tolerance and methods of use
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Cited By (3)

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Publication number Priority date Publication date Assignee Title
EP1194157A1 (fr) * 1999-04-21 2002-04-10 Hisamitsu Pharmaceutical Co., Inc. Polypeptide de forme d induisant une immunotolerance et techniques d'utilisation
EP1311280A2 (fr) * 2000-08-01 2003-05-21 The University Of Kansas Conjugues peptides-medicaments integres dans des cellules
US7481999B2 (en) 1998-05-05 2009-01-27 Adherex Technologies, Inc. Compounds and methods for modulating OB-cadherin-mediated function

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See also references of EP0973797A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7481999B2 (en) 1998-05-05 2009-01-27 Adherex Technologies, Inc. Compounds and methods for modulating OB-cadherin-mediated function
EP1194157A1 (fr) * 1999-04-21 2002-04-10 Hisamitsu Pharmaceutical Co., Inc. Polypeptide de forme d induisant une immunotolerance et techniques d'utilisation
EP1194157A4 (fr) * 1999-04-21 2002-08-14 Hisamitsu Pharmaceutical Co Polypeptide de forme d induisant une immunotolerance et techniques d'utilisation
EP1311280A2 (fr) * 2000-08-01 2003-05-21 The University Of Kansas Conjugues peptides-medicaments integres dans des cellules
EP1311280A4 (fr) * 2000-08-01 2005-02-23 Univ Kansas Conjugues peptides-medicaments integres dans des cellules

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CA2252468A1 (fr) 1997-11-06

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