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Title:  Peptide Compositions which induce immune tolerance and methods of use

United States Patent:  6,653,282

Issued:  November 25, 2003

Inventors:  Benedict; Stephen H. (Dept. of Microbiology Univ. KS, Lawrence, KS 66045); Siaiiann; Teruna J. (2912 Iris La., Lawrence, KS 66047); Chan; Marcia A. (Dept. Molecular Bioscience, Univ. Kansas, Lawrence, KS 66045); Tibbetts; Scott A. (5636 Waterman Blvd., Apt. 33, St. Louis, MO 63112)

Appl. No.:  479378

Filed:  January 5, 2000

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.

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 immune response, 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).

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 .alpha. and .beta. subunits of LFA-1) 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 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. 15-35, or has a sequence selected from the group consisting of Sequence ID Nos. 11-35.

Advantageously, each peptide is not immunogenic, 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 peptides which are attached to one another. In the most preferred 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).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is directed to compositions containing 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 synthesized.

4. The peptides are assayed to determine which peptides inhibit binding of the two proteins.

5. 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.

6. The peptides and compositions are assayed in biological assay, e.g., a mixed lymphocyte reaction, induced 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 13:1790-1798, the teachings 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 localized.

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 deduced. 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. Calif.).

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, Calif.). In 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 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 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.

In regard to step 3, those skilled in the art would recognize that peptides can be commercially synthesized by a variety of laboratories.

In regard to step 4, 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-1/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-1/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 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. 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 treatment of autoimmune diseases such as rheumatoid arthritis, insulin-dependent diabetes and multiple sclerosis.

The protein system selected by the applicants to illustrate the preferred embodiment of the invention was the LFA1/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 ICAM-1-based peptides inhibited the binding of LFA-1 to ICAM-1.

Five linear peptides (three LFA-1-based peptides and two ICAM-1-based peptides) that were individually effective in inhibiting protein binding were cyclized and combined. This combination was tested for its ability to inhibit the binding of LFA-1 to ICAM-1 using the homotypic-adhesion assay. Other peptides are constructed which individually and/or in combination inhibit the binding of LFA-1 and ICAM-1. These 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.

Claim 1 of 17 Claims

We claim:

1. A method of inhibiting the binding of LFA-1 to ICAM-1 comprising administering a peptide composition including at least two peptides, wherein at least one peptide comprises a sequence present in ICAM-1 and binds to LFA-1 and at least one peptide comprises a sequence present in .alpha. or .beta. subunits of LFA-1 and binds to ICAM-1, whereby LFA-1 is inhibited from binding to ICAM-1.




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