CA2252468A1 - Peptide compositions which induce immune tolerance and methods of use - Google Patents

Abstract

Peptide compositions which inhibit the binding of one protein to another protein, and corresponding methods of use are disclosed. These peptide compositions include at least one peptide which binds to one protein, and at least one peptide which binds to the other protein. In the preferred embodiment, the peptide composition is composed of a combination of cyclic ICAM-1-based and LFA-1-based peptides which inhibit the binding of LFA-1 to ICAM-1. Such LFA-1/ICAM-1-based peptide compositions can be used to treat disease states such as rejection of transplanted organs, allergies, and autoimmune diseases.

Description

CA 022~2468 1998-10-23 PEPTIDE COMPOSITIONS WHICH INDUCE
IMMUNE TOLERANCE AND METHODS OF USE

S Related Application This application claims the benefit of provisional application Serial No.
60/ , filed April 26, 1996.

Sequence Listing A printed Sequence Listing accompanies this application, and also has been submitted with identical contents in the forrn of a computer-readable ASCII file on a floppy diskette.

BACKGROUND OF THE ~NVENTION
Field of the Invention 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 prefelled forrn of the invention relates to peptide compositions which inhibit the Iciinding 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 autoirnrnune diseases.

Description of the Prior Art Many biological phenomena involve the mutual recognition of proteins. For example, it has been known for decades that antibodies bind to antigens in order to protect the body against foreign substances (Carayannopoulos et al., 1993, Irnrnuno-globulins, Structure and Function, In: Fundarnental Irnrnunology, Paul, W.E., Ed., pp.
283-314, Raven Press). Also, Ras and Raf-l are proto-oncoproteins that transducegrowth and dirrele~ ion signals initiated by tyrosine kin~ec; 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, CA 022~2468 1998-10-23 - W O 97/41149 PCT~US97106799 activate the genes encoding enzymes of galactose utilization (Fields et al.,1989, Nature 340:245-246).
It is also widely known that the 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 (Cal~y~lopoulos 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 dirrele~ll areas of the same subunit as in the case of the a subunit of LFA-l (Edwards et al., 1995, J. Biol. Chem. 270:12635-12640; Stanley et al., 1994, EMBO 13:1790-1798).
Many autoimrnune diseases occur when 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 rheurnatoid arthritis, insulin-dependent diabetes mellitus, and multiple sclerosis.
Allograft rejection also results from T-cell attack.
Initiation of an immune response to an antigen involves interaction of a small subset of T-cells with the antigen, followed by activation and proliferation of those T-cell clones. Complete T-cell activation requires two signals: (1) interaction of the T-cell receptor with an al)plo~liate 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- I (CD11 a and CD18), to one of its counter-receptors such as ICAM-l (CD54) (Staunton et al., 1990, Cell 61:243-254). If the second signal is blocked, the antigen-specific T-cells are induced to die by apoptosis or to enter a state of cellular anergy. Blockage ofthis 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).
Previous studies have shown that peptide fragments from ICAM-l can inhibit T-cell-endothelial adherence (Ross et al., 1992, J. Biol. Chem. 267:8537-8543), HIV-1 replication in MT-2 cells (Fecondo et al., 1993 Aids Research and Human Retroviruses 9:733-740), homotypic adhesion of Raji cells, and cytotoxic cell-mediated killing of K562 cells. Peptides from the sequence of the a subunit of BFA-1 have also been shown to inhibit binding of T-cells to ICAM-I (Stanley et al., 1994, EMBO 13: 1790-1798). Additionally, short-chain peptides derived from active sites of ICAM-1 and LFA-1 also inhibit these types of interactions (Benedict et al., 1995, Tntern~tional Publication No. WO 95/28170, the te~ching~ of which are incorporated by reference .. . .

CA 022~2468 1998-10-23 - W O 97/41149 PCTrUS97/06799 herein). However, there are no reports in the prior art of peptide compositions which are effective in inducing immune tolerance in an org~ni.~m.

SUMMARY OF THE INVENTION
The present invention is predicated upon the idea that 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. In the case of the immnn~
response, inhibition of such protein binding can result in induction of immllne 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).
Specifically, 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. Preferably, the first protein is an integrin (e.g., the a and ~ subunits of LFA-l ) while the second protein is an integrin-binding protein (e.g., ICAM-1).
If the protein system is the LFA-1/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. Ideally, each ICAM-1-based peptide contains a sequence present in a sequence selected from the group con~i~ting of Sequence ID Nos. 1-14, contains a sequence selected from the group consisting of Sequence ID Nos. 1-14, or has asequence 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 con~ ting of Sequence ID Nos. 15-35, contains a sequence selected from thegroup con~i~ting of Sequence ID Nos. 11-35, or has a sequence selected from the group consisting of Sequence ID Nos. 11-35.
Advantageously, each peptide is not imml~nogenic, and has a molecular weight under 20 kilodaltons. Preferably, 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. Although the peptides described herein for the purposes of illustration are separate molecules, the present invention comprehends use of . . .

CA 022~2468 1998-10-23 - W O 97/41149 PCTrUS97/06799 peptides which are attached to one another. In the most l)rerel.~,d embodiment of the invention, 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 ill~ldling 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, 10cIE-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-la-based peptides LAB, LAB-L, LAB-C, LAB-R, cLAB-L, cLAB-C, and cLAB-R, and the locations in the a subunit of LFA- 1 from which their sequences were derived;
15Fig. 4 is a diagrarn illustrating LFA-la-based peptides LAB.2, LAB.2-L, LAB.2-C, LAB.2-R, cLAB.2-L, cLAB.2-C, and cLAB.2-R, and the locations in the a subunit of LFA- 1 from which their sequences were derived;
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 ,B subunit of LFA-1 20from 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-a&esion assay wherein the cells were phorbol 12,13-dibutyrate (PDB)-stimulated;
25Fig. 8 is a photograph showing cells from a homotypic-a&esion assay wherein the cells were PDB-stimulated and treated with cLBE-R;
Fig. 9 is a bar graph illustrating the results of homotypic-a&esion assays wherein cells were unstimulated, PDB-stimulated, PDB-stimulated and treated withcIB-R, and PDB-stim~ 3ted and treated with a mixture of cIB-R, cLAB-L, cLAB.2-L,30and cLBE-C (4 cyclics);
Fig. 10 is a graph illustrating the arthritis score test results generated during the intlllt~ed 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.

,.

CA 022~2468 1998-10-23 Fig. 12 is a graph illustrating the data developed during the mouse skin transplant test of Example 3;
Fig. 13 is a bar graph illu~LI~Li~lg 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 illu~lldlillg 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 illu~lldlillg 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 ilh~ aling 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; and 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.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention is directed to compositions cont~ining two or more peptides which inhibit the binding of a first protein to a second protein. Specifically, at least one peptide binds to the first protein, and at least one peptide binds to the second protein. In general terms, these peptide compositions are made according to the following steps:
1. A protein system is selected in which a first protein binds to a second protein.

2. The contact sites within the proteins are identified.

3 . Peptides having sequences found within the contact sites are synth.o~

4. The peptides are assayed to de~ ine which peptides inhibit binding of - the two proteins.

5. Combinations of peptides co~ g 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.

, . . .

CA 022~2468 1998-10-23 - W O 97141149 PCT~US97/06799 6. The peptides and compositions are assayed in biological assay, e.g., a mixed lymphocyte reaction, inflllced arthritis study and/or skin transplant study.
In regard to 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.
In regard to 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.
These three methods are summarized as follows:
a. The first method has been previously described (Stanley et al., 1994, EMBO ~ 3 :1790-1798, the te~r11ing~ of which are incorporated by reference herein). In this method, 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 loc~li7~d b. 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 de~h1ced Those skilled in the art recognize that site-directed mutagenesis is a routine and widely-used technique. In fact, 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). ln this method, 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 CA 022~2468 1998-10-23 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. If the domains of the first protein and the second protein bind within the yeast cell, a functional hybrid transcription factor is formed from the ll~lsc~ ion-factor domains encoded by Plasmids A and B. This hybrid transcription factor results in the c .~iession of a reporter gene. Thus, ~k~ ion of the reporter gene indicates that the dom~in~ of the first and second proteins contain contact sites of Proteins A and B.
In regard to step 3, those skilled in the art would recognize that peptides can be commercially synth~ci7Pd by a variety of laboratories.
In regard to step 4, Example 1 below gives a detailed protocol for con(lllcting a homotypic-adhesion assay. This assay can be used to determine which peptides inhibit the binding of LFA-l to ICAM-1 if the LFA-I/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. However, those skilled in the art would understand that the antibody-binding assay can be used with other protein systems, and would know how to modify the assay accordingly.
In regard to step 5, combinations of peptides can be assayed as described in step 4, except that multiple peptides are tested simultaneously rather than a single peptide.
In regard to step 6, the examples below give details of mixed Iymphocyte reaction (MLR), rhe~lm~toid 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 ind~lçed proliferation. Thus, the MLR is a direct mea~ulelllent of allogeneic T cell responses. Moreover, 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 tre~tment of autoimm--ne diseases such asrhellm~tQid 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 prefell~d embodiment ofthe invention was the LFA-1/ICAM-l system. Noncontiguous domains of LFA- 1 and ICAM- 1 which were believed to be the contact sites of the two proteins CA 022~2468 l998-l0-23 - W O 97/41149 PCT~US97/06799 were selected. The sequences of LFA-1 and ICAM-I are known. Peptides based upon the sequences found in these contact sites were co~ cially synth~ l using standard techniques. Some of the peptides were cyclized. Homotypic-adhesion assays using the peptides were con~1uctecl to determine which LFA-l-based peptides and which ICAM-1 -based peptides inhibited the binding of LFA- 1 to ICA~I-l .
Five linear peptides (three LFA-l-based peptides and two ICAM-l-based peptides) that were individually effective in inhibiting protein binding were cycli~ed and combined. This combination was tested for its ability to inhibit the binding of LFA-l to ICAM-l using the homotypic-adhesion assay. Other peptides are constructed which individually and/or in combination inhibit the binding of LFA-l and ICAM-l .
These peptides and peptide combinations are injected into mice that have received skin allografts in order to ~l~terrnine which peptides or peptide combinations are effective in inhibiting the rejection of transplanted organs. Those of or~inal y 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 ~ e~es.

The following examples are set forth by way of illustration only, and nothing therein shall be taken as a limitation upon the overall scope of the invention.

MATE~IALS AND METHODS
Peptides. Peptides were synth~si7Pd either at the La Jolla Cancer Res~arch Foundation (La Jolla, CA) or the University of Kansas, Department of Ph~nn~ceutical Chemistry (Lawrence, KS). Peptide synthesis was generally con.l~lcte(l acco~.lillg t~ the standard-phase protocols employing t-butyloxyc~l,~"ate amino acid chemistry as previously described (Atherton et al., 1989, Solid Phase Peptide Synthesis: A Practic;al Approach, I.R.L. Press). Linear peptides having a penicill~mine residue (Xaa in ~he Sequence Listing) or a cysteine residue at their amino tl ....i..~l ends and a cysteine residue at their carboxy-tennin~l ends were cyclized by forming a disulfide bridge between the terrnin~l residues by adding dropwise a 2% aqueous potassiurn ferricyal~ide solution to dilute peptide solutions.
Homotypic-Adhesion Assay. Fresh human tonsils were suspended in TC-PBS
co"~ 5% FCS and were minced. Cells in ~u~pension were se~ualal~d from tissue with a strainer, and the res~lltin~ cell suspension was used for T-cell separation. T-cells . . .. .

CA 022~2468 1998-10-23 WO 97/41149 PCT~US97/06799 were isolated from other cells using the comrnonly used E-rosette method (Chiratha-worn et al., 1995, J: Immunol. 155:5479-5482, the te~chings of which are incorporated by reference herein). Cells were counted and resuspended in RPMI 1640 growth medium co~ 5% FCS (hereafter referred to as RPMI) at 4X106 cells/ml and rested overnight at 37~C. Typically, greater than 98% of the purified cells were positive for the T-cell surface marker CD3, as assessed by flow cytometr~r.
96-well Falcon tissue culture plates were used for the homotypic-adhesion assay. pH neutralized, lyophili7P~l peptides were resuspended in RPMI at a concentra-tion of 5 mM. Aliquots of the peptides were added to each well at ~prupl;ate volumes to yield a final concentration of 1, 10, 100, 250, 500, or 1000 IlM in a total sample volume of 100 IlL. RPMI was added to each well to bring the total volume to 50 ~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 ~1 of cell suspension was then added to each ofthe wells cont~ining RPMI alone (unstimulated cells). To the rem~ininE cells, PDB was added to a final concentration of l o-8 M. After mixing well, 50 111 of this cell suspension was added to each of the rem~ining wells. The plate was mixed gently, then incubated at 37~C for 4 to 6 hours. After incubation, wells were photographed using an Olympus photomicroscope with a 40x m~gnification.
Photographed samples were later assessed for index of clumping relative to the unstimulated and PDB-induced samples.

RESULTS
Peptides. The table below shows peptides constructed in accordance with the present invention, corresponding Sequence ID Numbers, and corresponding clumping-index values obtained from homotypic-adhesion assays:

Peptide'Sequence ID Number Clumping Index2 n3 Control 010 14 PDB 100~0 14 IB4 1 56~7 14 IB-L 2 37~26 4 IB-C 3 68~12 6 , W O 97/41149 PCTrUS97106799 Peptide' Sequence ID Number Clumping Index2 n3 clB-L S 61 ~9 7 cIB-C 6 61+13 7 cIB-R 7 73+8 6 IE6 8 63+8 12 IE-C 10 83~3 2 cIE-L 12 ND
cIE-C 13 23+3 2 10 clE-R 14 ND
LAB' 15 75~5 14 LAB-L 16 70+10 6 LAB-C 17 77~11 6 l ScLAB-L 19 78+ 13 S
cLAB-C 20 80+ I S 3 cLAB-R 21 77+ 11 6 LAB.28 22 48+ 10 6 LAB.2-L 23 ND
20LAB.2-C 24 100+0 3 LAB.2-R 25 100+0 3 cLAB.2-L 26 50+3 2 cLAB.2-C 27 ND
cLAB.2-R 28 ND
25 LBE9 29 73+7 14 LBE-L 30 78~5 6 LBE-C 31 62+10 6 LBE-R 32 63+9 7 . .

CA 022~2468 l998-l0-23 W O97/41149 PCTrUS97/06799 Peptide'Sequence ID NumberClumping Index2 n3 cLBE-L 33 75+10 6 cLBE-C 34 82+7 5 cLBE-R 35 lOO~tO 2 EBL'~ 36 100~0 5 'Nomenclature of peptides is as follows: the first upper case letter jn~ljc~tec whether the peptide is derived from ICAM- I (I) or LFA- 1 (L); the second upper case letter in the name of each LFA- I -based peptide indicates whether the peptide is derived from the a (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 ofanother 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) segn~ent of parent peptide IB, IE, LAB, LAB.2, or LBE; a lower case "c" indicates that the peptide is cyclic.
2The clumping index refers to the relative degree to which cells were clumped in the homotypic-adhesion assay; clumping-index values of O 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 det~rmining the mean clumping index per test run +/- standard error.
3Number of test runs.
4IB and peptides derived therefrom are illustrated in Fig. 1.
5Not done.
6IE and peptides derived the.erlolll are illustrated in Fig. 2.
7LAB and peptides derived therefrom are illustrated in Fig. 3.
8LAB.2 and peptides derived therer~olll are illustrated in Fig. 4.

9LBE and peptides derived therefrom are illustrated in Fig. 5.
'~EBL, which has the reverse sequence of LBE and was used as a negative control, is illustrated in Fig. 5.

CA 022~2468 1998-10-23 W O 97/41149 PCTrUS97/06799 Homotypic-Adhesion Assay. The table above gives the results of the homotypic-adhesion assays using individual peptides. In this assay, PDB induces intercellular clumping of human T-cells by activating the LFA-1/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 clumpmg.
The degree to which PDB-stimulated cell clumping is inhibited by a peptide indicates the degree to which the peptide blocks the interaction b~w~en LFA-1 present on the surface of one T-cell and ICAM-1 present on the surface of another T-cell.
Those peptides which gave a clumping-index value of less than 100 all showed statistically significant inhibition of PDB-stimulated cell clumping, indicating that these peptides inhibit the binding of LFA-I to ICAM-1. Significantly, 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 lln~tim~ ted 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-l to ICAM-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-l 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. 9, 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-stim~ ted cells showed the greatest amount of clumping. The clumping-index values for unstimulated cells and 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-stimlll~ted cells.
Since the clurnping-index values of 0 and 10 were based upon test runs giving the least amount and greatest amount of cell clumping, re~eclively, multiple test runs using un~tim--l~ted cells resulted in a mean clumping-index value greater than 0, and multiple CA 022~2468 1998-10-23 - W O 97/41149 PCT~US97/06799 test runs using PDB-stim~ ted cells resulted in a mean clumping index less than 10.
As in the table above, clumping index was quantified by detçrmining the mean clumping index per test run +/- standard error.

In this example, commercially available female DBA/lJ 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.
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 libitNm. A control and three test groups (P l, P2 and P3) each consisted of ten mice.
Type Il 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 cont~ining 200 ~lg of Type II collagen was injected intrt~ lIy 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) cont~ining 200 llg of the Type II
collagen was injected intr~dçrm~lly.
The 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:

P1: cIB-R (SEQ ID No. 7~, 50 ~lg; cLBE-C (SEQ ID No. 34), 25 ~g; cLAB.2-L
(SEQ ID No. 26), 12.5 llg; and cLAB-L (SEQ ID No. 19), 12.5 ,ug.

P2: cIB-R (SEQ ID No. 7), 50 ~g; cLBE-C (SEQ ID No. 34), 25 ,ug; and cLAB.2-L
(SEQ ID No. 26), 25 ~lg.

P3: cIB-R (SEQ ID No. 7), 50 ,ug; cLBE-C (SEQ ID No. 34), 25 llg; and cLAB-L
(SEQ ID No. 19), 25 ~g.
The clinical symptoms of arthritis in all four limbs of the mice were evaluated using a visual scoring system. Arthritic lesion of a limb was graded on a scale of 0-3 (0 = no change, 1 = swelling and erythema of the digit, 2 = mild swelling and erythema of the limb, 3 = gross swelling and erythema of the limb). The arthritis score of each mouse was the sum of the score for each of the four limbs, the maximum score thus CA 022~2468 1998-10-23 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.
Referring to Figs. 11 and 12, it will be seen that 90% of the mice in the control S group developed arthritis. The P 1 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 inciflPn~e after the eighth week. However, the arthritis score of this group was higher than that of the control.

In this example, the effect of peptides in accordance with the invention on the rate of skin graft rejection in mice was studied. The technique was essentially as previously described (Rosenberg, 1991, Skin allograft rejection, In: Current Protocols in Immunology, Coico, R., Ed., Unit 4.4, Supplement 2, Current Protocols, the te~ ing~ of which are incorporated by reference herein).
In the first step, 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 ofthe tail down its entire length and the skin was peeled off. The skin was placed, intemal surface down, on the bottom of a plastic petri dish conf~ining enough TC-PBS to keep the tissue moist.
The recipient mice (1 control and 1 test BALB/c Lwhite] 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~ 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.
100 ,ul normal saline (control mouse) or 100 ~l saline ct-nt~ining particles (test mouse) were ~l1rnini~tered i.p. (intraperitoneal) irmnediately prior to the surgery and once per day for four days following surgery. 175 ~lg each of cIB-R (SEQ ID No. 7), cLAB.2-L (SEQ ID No. 26), cLAB-L (SEQ ID No. 19), and cLBE-C (SEQ ID No. 34) were injected in a final volume of 100 111 normal saline in each peptide injection. The bandage was removed on day 7 following the operation and the grafts were graded daily by three separate people. A completely intact graft received a grade of 100%. A graft that had completely been rejected (scabbed over and fell off) was given a grade of 0%.

CA 022~2468 1998-10-23 This test (Fig. 12) demon~ d that peptide tre~ nt was able to significantly (p~.05) delay the rejection of the skin graft for two days.

S In this exarnple, an MLR assay is pelrol,.. ed 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 0 ~unction, 3.12.1-3.12.14 (1991), incorporated by reference). Specifically, human tonsil T cells were suspended at a concentration of 4 x 106 cell/ml in cell culture medium (RPMI), for use as responders. Stim~ tor cells were human mononuclear cells isolated from first Ficoll separation of peripheral blood (buffy coat) and were suspended at a concentration of 4 x 106 cell/ml in RPMI culture medium. The stimulator cells were inactivated before use by treatment with 3,000 rad of ionizing radiation from ~-l37Cs source. 100 ~11 of each cell suspension were combined in each well of 96-well plate in the absence or presence of varying concentrations of test peptides, as detailed below. Cell proliferation was ~c~çcsed after 72 hours, by 3H-thymidine addition to each well (1 ~Ci/well, 67 Ci/mmol) with incubation for six hours. The cells were harvested using a PHD Cell Harvester (Cambridge Technology, Inc., Watertown, MA); the incol~oldled 3H was assessed using a liquid scintillation counter (Pacl~ard Instrument, Inc., Donner's Grove, IL).
In Figs. l 3- 17, Y-axis values are mean cpm ~ SE (counts per minute). PBL[rad]
refers to i~adiated buffy coat cells, and 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-l interaction.
Referring to Fig. 13, the MLR assay was perforrned in the presence or absence of IE peptide (SEQ ID No. 8) or cyclic fr~mPnt~ of IE (SEQ ID Nos. 8, 12, 13 and 14).
Single peptides were tested at 250, 500, 750 and 1000 ,~M.
As shown in Fig. 14, 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 ofthe five peptides (m5) at 250, 500, 750 and 1000 ~lM. The combina-tion of the five peptides at 250 ~lM (cIB-L, cIE-L at 31.25 ~M each and cLAB-L, CA 022~2468 1998-10-23 - W O 97141149 PCT~US97/06799 cLAB.2-L and cLBE-L at 62.5 IlM each) was more effective for inhibiting the proliferative response than any of the single peptides at 250 IlM.
Fig. 15 illustrates MLR assay results using single peptides tested at 50,150, 250 and 500 IlM (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 concenkation levels.
The combined peptide trç~tment at each concenkation 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.
26)). These are cyclic fr~Ement~ of the parent peptides IB and LBE (Fig. 16) and LAB
and LAB.2 (Fig. 17). The above MLR assays demonskate that the test peptides inhibited proliferation of the responder cells to a greater or lesser extent.

CA 022~2468 l998-l0-23 W O97/41149 PCTrUS97/06799 S~Qu~N~ LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Benedict, Stephen Siahaan, Teruna Chan, Marcia Tibbetts, Scott (ii) TITLE OF INVENTION: Peptide Compositions Which Induce Immune Tolerance and Methods of Use (iii) NUMBER OF SEQUENCES: 36 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: HOVEY, WILLIAMS, TIMMONS & COLLINS
(B) STREET: 2405 Grand Boulevard, Suite 400 (C) CITY: Kansas City (D) STATE: MO
(E) COVNTRY: USA
(F) ZIP: 64108 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #l.o/ Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
3 5 (viii) AllO~/AGENT INFORMATION:
(A) NAME: Collins, John M.
(B) REGISTRATION NUMBER: 26262 (C) REFERENCE/DOCKET NUMBER: 24769-A
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (816)474-9050 (B) TELEFAX: 816)474-9057 (C) TELEX: 434-363 (2) INFORMATION FOR SEQ ID NO:l:
(i) Shyu~ CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide CA 022~2468 1998-10-23 - W O 97/41149 PCTrUS97/06799 (xi) S~u~N~ DESCRIPTION: SEQ ID NO:l:
Gln Thr Ser Val Ser Pro Ser Lys Val Ile Leu Pro Arg Gly Gly Ser l 5 l0 15 Val Leu Val Thr Gly (3) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Xaa Gln Thr Ser Val Ser Pro Ser Lys Val Ile Cys l 5 l0 ( 4) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Xaa Pro Ser Lys Val Ile Leu Pro Arg Gly Gly Cys l 5 l0 (5) INFORMATION FOR SEQ ID NO:4:
(i) S~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 12 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Cys Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys l 5 l0 CA 022~2468 l998-l0-23 - W O 97/41149 PCTrUS97/06799 (6) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Xaa Gln Thr Ser Val Ser Pro Ser Lys Val Ile Cys (7) INFORMATION FOR SEQ ID NO:6:
( i ) S~QU~N~ CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) MOLECULE TYPE: peptide (xi ) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Xaa Pro Ser Lys Val Ile Leu Pro Arg Gly Gly Cys (8) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular ~ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
- Cys Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys ( 9 ) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi~ SEQUENCE DESCRIPTION: SEQ ID NO: a CA 022~2468 l998-l0-23 Asp Gln Pro Lys Leu Leu Gly Ile Glu Thr Pro Leu Pro Lys Lys Glu l 5 10 15 Leu Leu Leu Pro Gly Asn Asn Arg Lys (10) INFORMATION FOR SEQ ID NO:9:
(i) S~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Xaa Asp Gln Pro Lys Leu Leu Gly Ile Glu Thr Cys l 5 10 (11) INFORMATION FOR SEQ ID NO:10:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Xaa Glu Thr Pro Leu Pro Lys Lys Glu Leu Leu Cys ( 12) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) S~yu~N~ DESCRIPTION: SEQ ID NO:11:~5 Xaa Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Cys (13) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (3) TYPE: amino acid (D) TOPOLOGY: circular CA 022~2468 l998-l0-23 W O 97/41149 PCTrUS97/06799 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Xaa Asp Gln Pro Lys Leu Leu Gly Ile Glu Thr Cys (14) INFORMATION FOR SEQ ID NO:13:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Xaa Glu Thr Pro Leu Pro Lys Lys Glu Leu Leu Cys (15) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Xaa Glu Leu Leu Leù Pro Gly Asn Asn Arg Lys Cys lo (16) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Ile Thr Asp Gly Glu Ala Thr Asp Ser Gly Asn Ile Asp Ala Ala Lys Asp Ile Ile Tyr Ile Ile Gly Ile (17) INFORMATION FOR SEQ ID NO:16:

CA 022~2468 1998-10-23 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
0 Xaa Ile Thr Asp Gly Glu Ala Thr Asp Ser Gly Cys (18) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Xaa Asp Ser Gly Asn Ile Asp Ala Ala Lys Asp Cys (19) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Xaa Ala Lys Asp Ile Ile Tyr Ile Ile Gly Ile Cys 1 5 lo (20) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Xaa Ile Thr Asp Gly Glu Ala Thr Asp Ser Gly Cys , . _ CA 022~2468 l998-l0-23 (21) INFORMATION FOR SEQ ID NO:20:
(i) S~Q~NC~ CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) MOLECULE TYPE: peptide (xi) S~Q~N~ DESCRIPTION: SEQ ID NO:20:
Xaa Asp Ser Gly Asn Ile Asp Ala Ala Lys Asp Cys (22) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Xaa Ala Lys Asp Ile Ile Tyr Ile Ile Gly Ile Cys (23) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) ~Qu~ DESCRIPTION: SEQ ID NO:22:
Gly Val Asp Val Asp Gln Asp Gly Glu Thr Glu Leu Ile Gly Ala Pro l 5 10 15 Leu Phe Tyr Gly Glu Gln Arg Gly (24) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear , . . _ . .

CA 022~2468 l998-l0-23 - W O 97/41149 PCTrUS97/06799 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Xaa Gly Val Asp Val Asp Gln Asp Gly Glu Thr Cys (25) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Xaa Gly Glu Thr Glu Leu Ile Gly Ala Pro Leu Cys (26) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) S~QU~:N~'~ DESCRIPTION: SEQ ID NO:25:
Xaa Ala Pro Leu Phe Tyr Gly Glu Gln Arg Gly Cys (27) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Xaa Gly Val Asp Val Asp Gln Asp Gly Glu Thr Cys ( 28) INFORMATION FOR SEQ ID NO:27:
(i) S~OU~N~ CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid CA 022~2468 l998-l0-23 W O 97/41149 PCTrUS97/06799 (D) TOPOLOGY: circular (ii) MOLECULE TYPE: peptide (xi ) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Xaa Gly Glu Thr Glu Leu Ile Gly Ala Pro Leu Cys ~29) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Xaa Ala Pro Leu Phe Tyr Gly Glu Gln Arg Gly Cys (30) INFORMATION FOR SEQ ID NO:29:
(i) S~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 25 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Asp Leu Ser Tyr Ser Leu Asp Asp Leu Arg Asn Val Lys Lys Leu Gly Gly Asp Leu Leu Arg Ala Leu Asn Glu (31) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Xaa Asp Leu Ser Tyr Ser Leu Asp Asp Leu Arg Cys CA 022~2468 l998-l0-23 - W O 97141149 PCT~US97/06799 (32) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Xaa Asp Leu Arg Asn Val Lys Lys Leu Gly Gly Cys (33) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Xaa Gly Gly Leu Leu Arg Ala Leu Asn Glu Cys l 5 10 (34) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) MOLECULE TYPE: peptide (xi) S~:~u~N~ DESCRIPTION: SEQ ID NO:33:
Xaa Asp Leu Ser Tyr Ser Leu Asp Asp Leu Arg Cys (35) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

W O 97/41149 PCTrUS97/06799 Xaa Asp Leu Arg Asn Val Lys Lys Leu Gly Gly Cys l 5 l0 ~36) INFORMATION FOR SEQ ID NO:35:

(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: ll amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Xaa Gly Gly Leu Leu Arg Ala Leu Asn Glu Cys l 5 l0 (37) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids (B) TYPE: a~ino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Glu Asn Leu Ala Arg Leu Leu Asp Gly Gly Leu Lys Lys Val Asn Arg l 5 l0 15 Leu Asp Asp Leu Ser Tyr Ser Leu Asp

Claims (40)

We claim:

1. A peptide composition effective in inhibiting the binding of a first protein to a second protein, wherein the composition comprises at least two peptides, wherein at least one peptide binds to the first protein and at least one peptide binds to the second protein.

2. The peptide composition of claim 1, wherein the first protein is an integrin and the second protein is an integrin-binding protein.

3. The peptide composition of claim 2, wherein the first protein is selected from the group consisting of the a subunit of LFA-1 and the .beta. subunit of LFA-1, and the second protein is ICAM-1.

4. The peptide composition of claim 2, wherein at least one of the first protein-binding peptides contains a sequence present in an integrin-binding protein, and at least one of the second protein-binding peptides contains a sequence present in an integrin.

5. The peptide composition of claim 3, wherein at least one of the first protein-binding peptides contains a sequence present in a sequence selected from the group consisting of Sequences ID Nos. 1 through 14.

6. The peptide composition of claim 3, wherein at least one of the first protein-binding peptides contains a sequence selected from the group consisting of Sequences ID Nos. 1 through 14.

7. The peptide composition of claim 3, wherein at least one of the first protein-binding peptides has a sequence selected from the group consisting of Sequences ID Nos. 1 through 14.

8. The peptide composition of claim 3, wherein at least one of the second protein-binding peptides contains a sequence present in a sequence selected from the group consisting of Sequences ID Nos. 15 through 35.

9. The peptide composition of claim 3, wherein at least one of the second protein-binding peptides contains a sequence selected from the group consisting of Sequences ID Nos. 15 through 35.

10. The peptide composition of claim 3, wherein at least one of the second protein-binding peptides has a sequence selected from the group consisting of Sequences ID Nos. 15 through 35.

11. The peptide composition of claim 3, wherein the composition comprises peptides having the sequences of sequence ID Nos. 7, 19, 26, and 34.

12. The peptide composition of claim 1, wherein at least one of the peptides is not immunogenic.

13. The peptide composition of claim 1, wherein at least one of the peptides has a molecular weight under 20 kilodaltons.

14. The peptide composition of claim 1, wherein at least one of the peptides contains at least one unnatural amino acid.

15. The peptide composition of claim 1, wherein at least one of the peptides is cyclic.

16. The peptide composition of claim 1, wherein at least one of the peptides is not covalently attached to another peptide.

17. The peptide composition of claim 1, wherein the peptide composition is effective in treating a disease state.

18. The peptide composition of claim 17, wherein the disease state is selected from the group consisting of rejection of a transplanted organ, an allergy, and an autoimmune disease.

19. The peptide composition of claim 18, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, insulin-dependent diabetes mellitus, and multiple sclerosis.

20. A method of inhibiting the binding of a first protein to a second protein comprising administering to a locus for the first and second proteins a peptide composition including at least two peptides, 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.

21. The method of claim 20, wherein the locus is selected from the group consisting of an in vitro system and an organism.

22. The method of claim 21, wherein the organism is a mammal.

23. The method of claim 20, wherein the first protein is an integrin and the second protein is an integrin-binding protein.

24. The method of claim 23, wherein the first protein is selected from the group consisting of the a subunit of LFA-1 and the .beta. subunit of LFA-1, and the second protein is ICAM-1.

25. The method of claim 23, wherein at least one of the first protein-binding peptides contains a sequence present in a integrin-binding protein, and at leastone of the second protein-binding peptides contains a sequence present in an integrin.

26. The method of claim 24, wherein at least one of the first protein-binding peptides contains a sequence present in a sequence selected from the group consisting of Sequences ID Nos. 1 through 14.

27. The method of claim 24, wherein at least one of the first protein-binding peptides contains a sequence selected from the group consisting of Sequences ID Nos. 1 through 14.

28. The method of claim 24, wherein at least one of the first protein-binding peptides has a sequence selected from the group consisting of Sequences ID
Nos. 1 through 14.

29. The method of claim 24, wherein at least one of the second protein-binding peptides contains a sequence present in a sequence selected from the group consisting of Sequences ID Nos. 15 through 35.

30. The method of claim 24, wherein at least one of the second protein-binding peptides contains a sequence selected from the group consisting of Sequences ID Nos. 15 through 35.

31. The method of claim 24, wherein at least one of the second protein-binding peptides has a sequence selected from the group consisting of Sequences ID Nos. 15 through 35.

32. The method of claim 24, wherein the composition comprises peptides having the sequences of sequence ID Nos. 7, 19, 26, and 34.

33. The method of claim 20, wherein at least one of the peptides is not immunogenic.

34. The method of claim 20, wherein at least one of the peptides has a molecular weight under 20 kilodaltons.

35. The method of claim 20, wherein at least one of the peptides contains at least one unnatural amino acid.

36. The method of claim 20, wherein at least one of the peptides is cyclic.

37. The method of claim 20, wherein at least one of the peptides is not covalently attached to another peptide.

38. The method of claim 20, wherein the method is effective in treating a disease state.

39. The method of claim 38, wherein the disease state is selected from the group consisting of rejection of a transplanted organ, an allergy, and an autoimmune disease.

40. The method of claim 39, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, insulin-dependent diabetes mellitus, and multiple sclerosis.