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AssigneeProtein Engineering Corporation
InventorsRobert C. Ladner, Sonia K. Guterman, Bruce L. Roberts, William Markland, Arthur C. Ley, Rachel B. Kent
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In order to obtain a novel binding protein against a chosen target, DNA molecules, each encoding a protein comprising one of a family of similar potential binding domains and a structural signal calling for the display of the protein on the outer surface of a chosen bacterial cell, bacterial spore or phage (genetic package) are introduced into a genetic package. The protein is expressed and the potential binding domain is displayed on the outer surface of the package. The cells or viruses bearing the binding domains which recognize the target molecule are isolated and amplified. The successful binding domains are then characterized. One or more of these successful binding domains is used as a model for the design of a new family of potential binding domains, and the process is repeated until a novel binding domain having a desired affinity for the target molecule is obtained. In one embodiment, the first family of potential binding domains is related to bovine pancreatic trypsin inhibitor, the genetic package is M13 phage, and the protein includes the outer surface transport signal of the M13 gene III protein.
A method of obtaining a nucleic acid encoding a binding protein having a proteinaceous binding domain that binds a predetermined target material comprising:
In a process for developing novel binding proteins, other than single chain antibodies, with a desired binding activity against a particular target material, by mutagenesis of a gene encoding a known protein other than a single chain antibody, the improvement comprising displaying a proteinaceous potential binding domain on the outer surface of an amplifiable genetic package selected from the group consisting of cells, spores and viruses, each said genetic package being genetically alterable, said potential binding domain not being natively associated with the outer surface of said package, said package containing the gene encoding said binding domain and means for directing said domain to the outer surface of said package, contacting the package with the target material, and determining whether the package displaying the potential binding domain binds to said target material, with the proviso that when the target is an antibody,the predetermined parental protein is not an antigen specifically bound by that antibody.
A variegated population of replicable genetic packages, each package including a nucleic acid construct coding for a chimeric potential binding protein, each said construct comprising DNA encoding (i) a potential binding domain which is a mutant of a predetermined parental binding domain, and (ii) an outer surface transport signal for obtaining the display of the potential binding domain on the outer surface of the genetic package, wherein said initial binding domain is not a single chain antibody and is not identical to or substantially homologous with a binding domain natively associated with said transport signal, and wherein said variegated population of genetic packages collectively display a plurality of different potential binding domains, the differentiation among said plurality of different potential binding domains occurring through the at least partially random variation of one or more predetermined amino acid positions of said parental binding domain to randomly obtain at each said position an amino acid belonging to a predetermined set of two or more amino acids, the amino acids of said set occurring at said position in predetermined expected proportions.
A variegated population of DNA molecules encoding chimeric binding proteins, each said chimeric binding protein comprising (i) a binding domain, and (ii) at least a segment of an outer surface protein of a cell or virus, said segment acting to cause the display of the chimeric binding protein or a processed form thereof on the outer surface of the cell or virus, said binding domain being capable of binding to a target material to which said outer surface protein is not capable of preferentially binding, wherein said variegated population of DNA molecules encode chimeric binding proteins which collectively include a plurality of different binding domains, the differentiation among said plurality of different potential binding domains occurring through the at least partially random variation of one or more predetermined amino acid positions thereof to randomly obtain at each said position an amino acid belonging to a predetermined set of two or more amino acids, the amino acids of said set occurring at said position in predetermined expected proportions.
A process for developing novel binding proteins with a desired binding activity against a particular target material which comprises providing a library of phage which each displays on its surface, as a result of expression of a first phage gene, one or more copies of a particular chimeric coat protein, each chimeric coat protein comprising (a) a potential binding domain which is a mutant of a known protein domain foreign to said phage, as well as (b) at least a functional portion of a coat protein native to said phage, said library collectively displaying a plurality of potential binding domains, wherein the differentiation among said plurality of different potential binding domains occurs through the controlled random variation of one or more predetermined amino acid positions of said known domain to randomly obtain at each said position an amino acid belonging to a predetermined set of two or more amino acids, the amino acids of said set occurring at said position in predetermined expected proportions, contacting said library of phage with the target material, and separating the phage on the basis of their affinity for the target materials characterized in that said chimeric coat protein further comprises a linker peptide which is specifically cleavable by a site-specific proteases said linker peptide being positioned in between (a) said epitope or potential binding domain, and (b) said native coat protein sequence, whereby (a) may be freed from (b).
A process for developing novel binding proteins with a desired binding activity against a particular target material which comprises providing a library of phage which each displays on its surface, as a result of expression of a first phage gene, one or more copies of a particular chimeric coat protein, each chimeric coat protein comprising a mutant of a known protein domain foreign to said phage, said library collectively displaying a plurality of potential binding domains, wherein the differentiation among said plurality of different potential binding domains occurs through the controlled random variation of one or more predetermined amino acid positions of said known domain to randomly obtain at each said position an amino acid belonging to a predetermined set of two or more amino acids, the amino acids of said set occurring at said position in predetermined expected proportions, contacting said library of phage with the target material, and separating the phage on the basis of their affinity for the target material, characterized in that said potential binding domain has at least one intrachain covalent crosslink between a first amino acid position and a second amino acid position thereof, the amino acids at said first and second positions being invariant in all of the chimeric proteins displayed by said library, and where low affinity phage are removed from said target material first, and then high affinity phage are released or rendered more readily eluted from the target material by treating the phage with a reagent which cleaves the crosslink.
A process for developing binding proteins with a desired binding activity against a particular target material which comprises providing a library of phage which each displays on its surface, as a result of expression of a first phage gene, one or more copies of a particular chimeric coat protein, each chimeric coat protein comprising a potential epitope, or a potential binding domain which is a mutant of a known protein domain foreign to said phage, said library collectively displaying a plurality of potential epitomes or binding domains, wherein the differentiation among said plurality of different potential binding domains occurs through the controlled random variation of one or more predetermined amino acid positions of said known domain to randomly obtain at each said position an amino acid belonging to a predetermined set of two or more amino acids, the amino acids of said set occurring at said position in predetermined expected proportions, contacting said library of phage with the target material, and separating the phage on the basis of their affinity for the target material, characterized in that the chimeric coat protein includes only an assemblable fragment of a coat protein of said phage, and not that portion of the coat protein which is responsible for pilus binding, and the phage also comprises a second phage gene encoding the cognate native coat protein of the phage.
A process for developing binding proteins with a desired binding activity against a particular target material which comprises providing a library of phage which each displays on its surface, as a result of expression of a first phage gene, one or more copies of a particular chimeric coat, protein, each chimeric coat protein comprising a potential binding domain which is a mutant of a known protein domain foreign to said phage, said library collectively displaying a plurality of potential binding domains, wherein the differentiation among said plurality of different potential binding domains occurs through the controlled random variation of one or more predetermined amino acid positions of said known domain to randomly obtain at each said position an amino acid belonging to a predetermined set of two or more amino acids, the amino acids of said set occurring at said position in predetermined expected proportions, contacting said library of phage with the target material, and separating the phage on the basis of their affinity for the target material, characterized in that the phage also comprises a second phage gene encoding the cognate native coat protein of the phage, and the initiation codon of the second phage gene is a Leucine codon.
A library of display phage which each displays on its surface, as a result of expression of a first phage gene, one or more copies of a particular chimeric coat protein, each chimeric coat protein comprising a potential binding domain which is a mutant of a known protein domain foreign to said phage, said library collectively displaying a plurality of potential binding domains, wherein the differentiation among said plurality of different potential binding domains occurs through the controlled random variation of one or more predetermined amino acid positions of said known domain to randomly obtain at each said position an amino acid belonging to a predetermined set of two or more amino acids, the amino acids of said set occurring at said position in predetermined expected proportions, contacting said library of phage with the target material, and separating the phage on the basis of their affinity for the binding protein target material, wherein the differentiation among said plurality of different potential binding domains occurs through the at least partially random variation of one or more predetermined amino acid positions of said known domain to randomly obtain at each said position an amino acid belonging to a predetermined set of two or more amino acids, the amino acids of said set occurring at said position in predetermined expected proportions, and in substantially all sets the ratio of the frequency of occurrence of the most favored amino acid to that for the least favored amino acid is less than 2.6, characterized in that, at at least one such position, the predetermined set consists of less than all twenty different genetically encodable amino acids, but includes three or more of the classes of genetically encodable amino acids.
A library of display phage which each displays on its surface, as a result of expression of a first phage gene, one or more copies of a particular chimeric coat protein, each chimeric coat protein comprising (a) a potential binding domain which is a mutant of a known protein domain foreign to said phage, as well as (b) at least a functional portion of a coat protein native to said phage, said library collectively displaying a plurality of potential binding domains, wherein said chimeric coat protein further comprises a linker peptide which is specifically cleavable by said site-specific protease, said linker peptide being positioned inbetween (a) said potential binding domain, and (b) said native coat protein sequence, whereby (a) may be freed from (b).
A process for developing novel binding proteins with a desired binding activity against a particular target material which comprises providing a library of phage which each displays on its surface, as a result of expression of a first phage gene, one or more copies of a particular chimeric coat protein, each chimeric coat protein comprising (a) a potential binding domain which is a mutant of a known protein domain foreign to said phage, as well as (b) at least a functional portion of a coat protein native to said phage, said library collectively displaying a plurality of potential binding domains, wherein the differentiation among said plurality of different potential binding domains occurs through the controlled random variation of one or more predetermined amino acid positions of said known domain to randomly obtain at each said position an amino acid belonging to a predetermined set of two or more amino acids, the amino acids of said set occurring at said position in predetermined expected proportions, contacting said library of phage with the target materials and separating the phage on the basis of their affinity for the target material, wherein the phage gene encoding said chimeric coat protein further comprises a cytoplasmic secretion signal sequence which codes for a signal peptide which directs the immediate expression product to the inner membrane of the bacterial host cell infected by said phage, where it is processed to remove said signal peptide, yielding a mature chimeric coat protein comprising the potential binding domain and at least a portion of a geneVIII like protein of the phage, said chimeric protein being assembled with wild-type coat protein into the phage coat, wherein the secretion signal is encoded by a signal sequence selected from the group consisting of the signal sequences of the phoA, bla and geneIII genes.
Dependencies are inferred from the claim text.