Anchor libraries and identification of peptide binding...

Details Anchor libraries and identification of peptide binding... Anchor libraries and identification of peptide binding...

1995-06-07

2002-11-05

Gitomer, Ralph (Department: 1627)

Chemistry: analytical and immunological testing

Involving an insoluble carrier for immobilizing immunochemicals

C530S300000

Reexamination Certificate

active

06475806

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to anchor libraries and to methods of using anchor libraries to identify peptide sequences that bind to a target molecule.
BACKGROUND OF THE INVENTION
The identification of peptides which bind to target molecules which are involved in various physiological functions, can have significant implications for the diagnosis and/or treatment of various abnormal or diseased conditions. For example, a binding peptide might modulate the original activity of the target molecule and therefore be useful as a drug.
The use of standard libraries to identify peptide sequences which specifically bind to target molecules is generally limited to pre-existing natural sequences from the organism which is the source of the DNA. More recently, libraries have been described which have clones containing short synthetic random coding sequences. See, e.g., Scott and Smith, Science 249:386-390 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1990); Devlin et al., Science 249:404-406 (1990). These libraries are mixtures of filamentous phage clones, each displaying a random peptide sequence on the virion surface. In these types of libraries, the random amino acids are contiguous. The size of the peptides that can be screened for binding peptides in such contiguous random amino acid libraries is limited, in that as the size of the peptides increases, at some point it is not feasible to adequately search such a library since there are too many clones required to cover all possible permutations of the random amino acids in the peptides.
SUMMARY OF THE INVENTION
It is an object of the invention to identify peptide sequences that bind to specific target molecules.
It is another object of the invention to identify amino acid residues in a peptide that are important contacts between the peptide and a target molecule.
It is another object of the invention to determine where amino acid residues in a peptide that are important contacts between the peptide and a target molecule, are best positioned within the peptide.
It is another object of the invention to use an anchor library in which the random amino acid residues of the library are not continuous, for identifying amino acid residues in a peptide that are important contacts between the peptide and a target molecule.
It is another object of the invention to use an anchor library in which the random amino acid residues of the library are distributed throughout a much larger peptide domain consisting of random glycine and/or alanine residues, for identifying amino acid residues in a peptide that are important contacts between the peptide and a target molecule.
It is another object of the invention to search large peptide phage display libraries of, e.g., 16 mers, for a reduced number of essential amino acid residue contacts, e.g., four, between the peptide and a target molecule.
It is another object of the invention to identify a consensus sequence of a defined number of amino acid residues in any configuration of spacer amino acids, that are important contacts between a peptide and a target molecule.
It is yet another object of the invention to use a known core binding sequence on a peptide which binds to a target molecule, and identify surrounding amino acid residues which are additional important contacts between the peptide and the target molecule.
Still another object of the invention is to identify cysteine residues on a peptide which can form disulfide bridges and thereby increase the binding affinity of the peptide with a target molecule.
According to the invention, an anchor library is provided. The anchor library comprises a collection of recombinant vectors, e.g., viruses, phage, e.g., filamentous phage, plasmids or cosmids. Each of the vectors has a nucleic acid sequence inserted in a gene, e.g., a coat protein gene, e.g., gene III or gene VIII, thioredoxin, staphnuclease, lac repressor, gal4 or an antibody. The nucleic acid sequence encodes a displayed peptide sequence, e.g., displayed on the surface of a virion, cell, spore or gene product, which comprises:
X
1
(Y
1
)
c
1
X
2
(Y
2
)
c
2
X
3
(Y
3
)
c
3
X
4
wherein each X
1
, X
2
, X
3
and X
4
is an amino acid residue and any of X
1,
X
2
, X
3
and X
4
can be the same or different from any one other, wherein each Y
1
, Y
2
and Y
3
is alanine or glycine or a combination of alanine and glycine that is respectively, c
1
, c
2
and c
3
amino acid residues long and any of Y
1
, Y
2
and Y
3
if present can be the same or different from any one other, wherein each of c
1
, c
2
and c
3
preferably is 0 to about 20, more preferably is 0 to about 10, even more preferably is 0 to about 6, or most preferably is 0 to about 4, wherein X
1
and X
4
are each attached to an amino acid residue that flanks the displayed peptide sequence. In certain embodiments, at least about 10
5
to about 10
8
permutations of all possible permutations of the displayed peptide sequence are present in the anchor library. In other embodiments, the library does not contain more than about 10%, or more than about 1%, or more than about 0.1%, of displayed peptide sequences different from the first mentioned displayed peptide sequences.
Another aspect of the invention is where each Y
1
, Y
2
and Y
3
is any specified amino acid or combination of specified amino acids, e.g., alanine or cysteine or a combination of alanine and cysteine; or glycine or cysteine or a combination of glycine and cysteine.
In certain embodiments, the displayed peptide sequence further has at least one core binding sequence which is preferably about 1 to about 20 amino acid residues in length, more preferably about 4 to about 10, and most preferably is 6. The core binding sequence can be in addition to, or a replacement for, other amino acids in the displayed peptide sequence. Variations include the presence of more than one core binding sequence in the displayed peptide sequence, where, e.g., the core binding sequences can be adjacent, or not adjacent, to each other, and where they can be, e.g., identical or not identical to each other.
In other embodiments, the displayed peptide sequence further has at least one constraint, e.g., a crosslink, e.g., a disulfide bond, e.g., from the presence of a cysteine residue; a stacking interaction; a positive or negative charge; hydrophobicity; hydrophilicity; a structural motif, e.g., a zinc finger formation, a leucine zipper, or a &bgr;-turn structure, e.g., from the presence of the sequence asp gly or pro gly; or combinations thereof. Cysteine residues can be in addition to, or a replacement for, other amino acids in the displayed peptide sequence.
Another aspect of the invention is a method of making an anchor library. A collection of nucleic acid sequences is synthesized. The nucleic acid sequences are inserted into vectors to give recombinant vectors and the recombinant vectors are introduced into a host. The host having the recombinant vectors is propagated so as to result in a collection of recombinant vectors, each of which has a nucleic acid sequence from the collection of nucleic acid sequences which encodes a displayed peptide sequence comprising:
X
1
(Y
1
)
c
1
X
2
(Y
2
)
c
2
X
3
(Y
3
)
c
3
X
4
.
Another aspect of the invention is a method of using an anchor library to identify a peptide sequence that binds to a target. An anchor library having a collection of recombinant vectors is provided. Each of the recombinant vectors has a nucleic acid sequence which encodes a displayed peptide sequence comprising:
X
1
(Y
1
)
c
1
X
2
(Y
2
)
c
2
X
3
(Y
3
)
c
3
X
4
.
Expression and display of the peptide sequence is permitted. The anchor library is contacted with the target, e.g., proteinaceous or non-proteinaceous molecules, e.g., ligands, receptors, hormones, cytokines, antibodies, antigens, enzymes, enzyme substrates or viruses, under conditions in which the displayed peptide sequence binds to the target, and the displayed peptide sequence which binds to the target is identified, e.g., by sequencing the nucleic acid sequence on the recombinant vector wh

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