Title: Peptides binding to bone marrow stromal cell
United States Patent: 6,414,113
Inventors: Sato; Atsushi (Kamakura, JP); Jingami; Hisato
Assignee: Biomolecular Engineering Research Institute
Appl. No.: 300410
Filed: April 27, 1999
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
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.
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