Heterodimeric receptor libraries using phagemids
AssigneeThe Scripps Research Institute
InventorsCarlos Barbas, Angray Kang, Richard A. Lerner
Life of this patent
Estimating the term…
Abstract
Filamentous phage comprising a matrix of cpVIII proteins encapsulating a genome encoding first and second polypeptides of an antogenously assembling receptor, such as an antibody, and a receptor comprised of the first and second polypeptides surface-integrated into the matrix via a filamentous phage coat protein membrane anchor domain fused to at least one of the polypeptides.
Claims36
A filamentous phage encapsulating a genome encoding a ligand-binding heterodimeric antibody that comprises first and second polypeptides wherein said first polypeptide is fused to a carboxy-terminal filamentous phage membrane cpIII anchor domain.
A ligand-binding heterodimeric antibody comprising first and second polypeptides wherein said first polypeptide is fused to a carboxy-terminal filamentous phage membrane cpIII anchor domain.
A vector for expressing first and second fusion proteins, said vector comprising:
- a) a first cassette for expressing a first fusion protein that includes first upstream and downstream translatable DNA sequences operatively linked via a sequence of nucleotides adapted for directional ligation of a first insert DNA, said first upstream sequence encoding a prokaryotic secretion signal, said downstream sequence encoding a carboxy-terminal filamentous phage membrane cpIII anchor domain, said translatable DNA sequences operatively linked to a set of DNA expression signals for expression of said translatable DNA sequences as portions of said first fusion protein;
- and
- b) a second cassette for expressing a second fusion protein that includes a second upstream translatable DNA sequence encoding a second prokaryotic secretion signal operatively linked via a sequence of nucleotides adapted for directional ligation of a second insert DNA, said second translatable DNA sequence operatively linked to a set of DNA expression signals for expression of said second translatable DNA sequence as a portion of said second fusion protein.
A method for changing the diversity of a library of filamentous phage particles comprising the steps of:
- a) providing a library of filamentous phage particles according to 26 or 12;
- b) contacting said library with a preselected ligand under conditions sufficient for members of the library to bind to the ligand and form a ligand-phage particle complex;
- and
- c) isolating phage particles in said complex away from non-bound library members to form a ligand-enriched library comprising isolated phage particles having binding specificity for said preselected ligand.
A method of producing a library of dicistronic DNA molecules, each dicistronic DNA molecule comprising first and second cistrons for expressing first and second polypeptides of a heterodimeric antibody on the surface of a filamentous phage, which method comprises:
- (a) forming a first ligation admixture by combining in a ligation buffer:
- (I) a repertoire of first polypeptide genes in the form of linear dsDNA, each having cohesive termini adapted for directional ligation, and
- (ii) a plurality of DNA expression vectors in linear form, each vector having upstream and downstream first cohesive termini that are (a) adapted for directionally receiving one of said first polypeptide genes in a common reading frame, and (b) operatively linked to respective upstream and downstream translatable DNA sequences, said upstream translatable DNA sequence encoding a prokaryotic secretion signal, said downstream translatable DNA sequence encoding a carboxy-terminal filamentous phage membrane cpIII anchor domain, and said translatable DNA sequences operatively linked to respective upstream and downstream DNA expression control sequences;
- and
- (b) subjecting said admixture to ligation conditions for a time period sufficient to operatively link said first polypeptide genes to said vectors and produce a plurality of circular DNA molecules each having said first cistron for expressing said first polypeptide;
- (c) producing a plurality of DNA expression vectors in linear form, each linear vector having second upstream and downstream cohesive termini (I) adapted for directionally receiving one of a repertoire of second polypeptide genes in a common reading frame, and (ii) operatively linked to respective upstream and downstream DNA sequences, said upstream DNA sequence being a translatable sequence encoding a prokaryotic secretion signal, said downstream DNA sequence having at least one stop codon in said reading frame, and said translatable DNA sequence operatively linked to a DNA expression control sequence, by treating said plurality of circular DNA molecules to restriction endonucleolytic conditions sufficient to cleave said circular DNA molecules and form said second cohesive termini;
- (d) forming a second ligation admixture by combining in a ligation buffer:
- (I) said plurality of DNA expression vectors formed in step (c), and
- (ii) said repertoire of second polypeptide genes in the form of dsDNA, each having cohesive termini adapted for directional ligation to said plurality of linear DNA vectors;
- and
- (e) subjecting said second admixture to ligation conditions for a time period sufficient to operatively link said second polypeptide genes to said vectors and produce a plurality of circular DNA molecules each having said second cistron for expressing said second polypeptide thereby forming said library.
Dependencies are inferred from the claim text.