Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 

 

 

Title:  Peptides binding to bone marrow stromal cell antigen

United States Patent:  6,414,113

Inventors:  Sato; Atsushi (Kamakura, JP); Jingami; Hisato (Kyoto, JP)

Assignee:   Biomolecular Engineering Research Institute (Osaka, JP)

Appl. No.:   300410

Filed:   April 27, 1999

Abstract

Peptides capable of binding to bone marrow stromal cell antigen-1 (BST-1), and peptides capable of binding to BST-1 and inhibiting ADP-ribosyl cyclase activity and cADP-ribose hydrolase activity thereof are provided. The peptides are useful for treating rheumatoid arthritis and multiple myeloma.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purpose of seeking an agent that binds to BST-1, human BST-1 was expressed in insect cells, and highly purified BST-1 was obtained in a large amount. Using purified BST-1, the inventors have selected from a phage display peptide library (Jikken Igaku Experimental Medicine, 11, No. 13, August, 95-100, 1993) two peptides consisting of 15 amino acid residues that bind to BST-1. One of them was identified to inhibit ADP-ribosyl cyclase activity. Details of the procedures are discussed below.

Human BST-1 may be prepared by, for example, recombinant DNA technology. Host cells for the expression may be selected from E. coli, yeast, insect, and animal cells. When insect cells are used, cDNA encoding human BST-1 (Kaisho T. et al, Proc. Natl. Acad. Sci. USA, 91, 5325-5329, 1994) is inserted downstream of a promoter which functions in insect cells, for instance, polyhedrine promoter, in conventional manner (King and Possee, The baculovirus expression system, Chapman & Hall, 1992). Purification of expressed products may be accomplished by salting out, ion-chromatography, centrifugation, and the like.

For obtaining peptides that bind to human BST-1, a peptide library method may be conveniently used as described below.

Random peptide phage library may be constructed by binding synthetic genes having random sequences to, for instance, coat protein genes (e.g. gene III or IIIV) of M13 phage. For this purpose, the method described in Science, 249, 386, 1990, or Proc. Natl. Acad. Sci., USA, 87, 6378, 1990, may be used. Size of the synthetic gene to be inserted is not limitative so far as the expressed peptide is stable. However, the gene having preferred size will be the one encoding from six (6) to fifteen (15) amino acid residues so that the resulting library may cover as many random sequences as possible and can bind to the target molecule, BST-1. Selection of phage capable of binding to BST-1 is accomplished by immobilizing purified BST-1 on a column or plate, directly or via a linker such as antibodies, contacting the library with the immobilized BST-1, and then washing out unbound phage. After washing, bound phage are eluted with acids, neutralized, and amplified by infecting E. coli cells. This procedure is repeated for three or four rounds to concentrate phage having affinity to BST-1. In order to obtain a single uniform phage, the concentrated phage are infected to E. coli cells, and a single colony is allowed to form on an agar plate containing antibiotics. The colony is cultured in a liquid medium, and the phage in supernatant is concentrated by precipitation with polyethylene glycol. Sequencing of the phage DNA reveals the amino acid sequence of the peptide bound to BST-1.

The peptide library containing random amino acid sequences may also be prepared by chemical synthesis by means of a method employing beads (Nature, 354, 82, 1991), liquid phase focusing (Nature, 354, 84, 1991) and micro plate method, and the like.

Large-scale production of the desired peptide may be carried out by chemical synthesis or recombinant DNA technology using E. coli, yeast, insect, or animal cells as a host. Conventional peptide synthesis may be used for the former, and solid phase synthesis is preferred. In this method, preparation of variant peptides in which one or more amino acid residues are altered may be readily accomplished (Saibo Kogaku (cell technology), extra number, Experimental protocol for anti-peptide antibody, p26-46, Shu-jun sha, 1994). As for the latter, it is an established technique that the DNA sequence is determined according to the amino acid sequence of the peptide bound to BST-1 on the basis of codon usage, and a DNA prepared according to the DNA sequence determined is incorporated into a host cell (Maniatis et al; Molecular Cloning, Appendix D1, Cold Spring Habor Laboratory, 1989). Amino acid residue(s) in the sequence can be substituted with other amino acid residue(s) by incorporation of mutation into the DNA sequence.

When the peptide is expressed in E. coli cells, it is preferred that the resulting DNA is linked with a promoter sequence, such as tryptophan synthetase operon (Trp) promoter or lactose operon (lac) promoter, a ribosome-binding sequence, such as Shine-Dalgarno sequence, and a transcription terminator recognition site, is added thereto. The resulting expression vector may be inserted into E. coli cells according to the methods described in the afore-mentioned Molecular Cloning. Expressed products may be purified by, for example, various kinds of chromatography.

The fact that the peptide thus obtained inhibits ADP-ribosyl cyclase activity of BST-1 may be identified by comparing the ADP-ribosyl cyclase activity when measured in the absence of the peptide with the activity in the presence of the peptide. As previously mentioned, NAD is converted to cADP-ribose by ADP-ribosyl cyclase activity of BST-1, and therefore, the activity may be measured by allowing to react NAD with BST-1 and then quantitatively determining NAD and cADP-ribose after separation of them by anion-exchange chromatography (FEBS letters, 356, 244, 1994). Alternatively, nicotinamide guanine dinucleotide (NGD) may be used as a substrate instead of NAD, which is converted to cyclic guanosine-5'-diphosphate-ribose cGDP-ribose that can be fluorimetrically measured with an excitation wavelength of 300 nm and an emission wavelength of 410 nm, whereby the velocity of the formation of cGDP-ribose reflects ADP-ribosyl cyclase activity (J. Biol. Chem., 48, 30260, 1994).

The present invention has enabled those skilled in the art to obtain peptides that bind to BST-1, and additional peptides that bind to BST-1 and yet specifically inhibit ADP-ribosyl cyclase activity thereof. The peptides may be used for treating rheumatoid arthritis and multiple myeloma. In addition, the peptides may be immobilized on a carrier and used as a component of a medical extraperfusion apparatus for removing BST-1 from body fluid.

In more detail, the first object of the present invention is to provide peptides capable of binding to BST-1, which comprise an amino acid sequence (1) depicted in SEQ ID NO: 1 or an amino acid sequence (2) obtained by making deletion, substitution, or insertion of one or more amino acid residues to the amino acid sequence (1). As preferred embodiments are provided peptides having the amino acid sequence (2) which contains deletion, substitution, or insertion of amino acid residue(s) at the positions of 1, 3, 6, 13, and/or 14 of the amino acid sequence of SEQ ID NO: 1.

The second object of the invention is to provide peptides capable of binding to BST-1, which comprise an amino acid sequence (3) depicted in SEQ ID NO: 2 or an amino acid sequence (4) obtained by making deletion, substitution, or insertion of one or more amino acid residues to the amino acid sequence (3).

The third object of the invention is to provide peptides which bind to BST-1 and yet specifically inhibit ADP-ribosyl cyclase activity thereof.

The fourth object of the invention is to provide peptides which bind to BST-1 and yet specifically inhibit cADP-ribose hydrolase activity thereof.

The fifth embodiment of the invention is to provide a pharmaceutical formulation comprising as an essential component at least one of the peptides defined in the preceding objects.

The sixth object of the invention is to provide a diagnostic agent for detecting BST-1, which comprises as an essential component at least one of the peptides defined in the preceding objects.

The seventh object of the invention is to provide an adsorbing agent comprising at least one of the peptides defined in the preceding objects, said peptide(s) being immobilized on a carrier.

The eighth object of the invention is to provide a method for purification of BST-1 using the adsorbing agent defined above.

The ninth object of the invention is to provide a medical extraperfusion apparatus which contains as one of the components at least one of the peptides defined in the preceding objects, said peptides capable of inhibiting enzymatic activities of BST-1.

The tenth object of the invention is to provide a method of screening a substance capable of interacting with BST-1, which employs at least one of the peptides defined in the preceding objects.

Claim 1 of 12 Claims

What is claimed is:

1. A peptide which binds to bone marrow stromal cell antigen-1, said peptide comprising an amino acid sequence of SEQ ID No. 1 or an amino acid sequence which comprises deletion, substitution, and/or insertion of one or more amino acid residues at the positions 1, 3, 6, 13, 14 and/or 15 of SEQ ID No.1.

 


____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]