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.

Directed evolution of novel binding proteins

A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof, by virtue of the sbp member or a polypeptide component thereof being expressed as a fusion with a capsid component of the rgdp. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected rgdps for expression of the selected sbp members. Antibody sbp members may be thus obtained, with the different chains thereof expressed, one fused to the capsid component and the other in free form for association with the fusion partner polypeptide. A phagemid may be used as an expression vector, with said capsid fusion helping to package the phagemid DNA. Using this method libraries of DNA encoding respective chains of such multimeric sbp members may be combined, thereby obtaining a much greater genetic diversity in the sbp members than could easily be obtained by conventional methods.

Methods for producing members of specific binding pairs

Nucleotide sequences encoding proteins of interest are isolated from DNA libraries using bacteriophage to link the protein to the sequence which encodes it. DNA libraries are prepared from cells encoding the protein of interest and inserted into or adjacent to a coat protein of a bacteriophage vector, or into a sequence encoding a protein which may be linked by means of a ligand to a phage coat protein. By employing affinity purification techniques the phage particles containing sequences encoding the desired protein may be selected and the desired nucleotide sequences obtained therefrom. Thus, for example, novel proteins such as monoclonal antibodies may be produced and conventional hybridoma technology avoided.

Recombinant library screening methods

Methods are disclosed for the production of anti-self antibodies and antibody fragments, being antibodies or fragments of a particular species of mammal which bind self-antigens of that species. Methods comprise providing a library of replicable genetic display packages (rgdps), such as filamentous phage, each rgdp displaying at its surface a member of a specific binding pair which is an antibody or antibody fragment, and each rgdp containing nucleic acid sequence derived from a species of mammal. The nucleic acid sequence in each rgdp encodes a polypeptide chain which is a component part of the sbp member displayed at the surface of that rgdp. Anti-self antibody fragments are selected by binding with a self antigen from the said species of mammal. The displayed antibody fragments may be scFv, Fd, Fab or any other fragment which has the capability of binding antigen. Nucleic acid libraries used may be derived from a rearranged V-gene sequences of unimmunised mammal. Synthetic or artificial libraries are described and shown to be useful.

Production of anti-self antibodies from antibody segment repertoires and displayed on phage

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.

Heterodimeric receptor libraries using phagemids

Novel DNA-binding proteins, especially repressors of gene expression, are obtained by variegation of genes encoding known binding proteins and selection for proteins binding the desired target DNA sequence. A novel selection vector is used to reduce artifacts. Heterooligomeric proteins which bind to a target DNA sequence which need not be palindromic are obtained by a variety of methods, e.g., variegation to obtain proteins binding symmetrized forms of the half-targets and heterodimerization to obtain a protein binding the entire asymmetric target.

Generation and selection of novel DNA-binding proteins and polypeptides

Compositions comprising non-naturally occurring serum albumin binding moieties are described, together with methods of use thereof, e.g., for detecting or isolating serum albumin molecules in a solution, for blood circulation imaging, and for linking therapeutics or other molecules to albumin. Preferred serum albumin binding peptides having a high affinity for human serum albumin are particularly disclosed.

Serum albumin binding moieties

The distinctive separation attributes of mixed-mode resins and their application in monoclonal antibody downstream purification process.

We generated a series of libraries having variants of the first Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1, also known as tissue-factor pathway inhibitor-I) displayed on bacteriophage M13 as pIII-fusions. We varied LACI-DI iteratively in two regions: the P1 region (positions 10-21) and the "second loop", (positions 31-39), which together form one end of the domain. Display-phage library Lib#1 allows 31 200 amino-acid sequences in P1 region (residues 13, 16-19). Preliminary, we screened Lib#1 against human plasmin (PLA, EC 3.4.21.7) immobolized on agarose to enrich for phage displaying variants with PLA affinity. We introduced a 1600-fold increase in second-loop diversity (residues 31, 32, 34, 39) into the population of selectants from Lib#1, yielding Lib#2. Lib#2 (allowing approximately 50 million amino-acid sequences) was screened against PLA-agarose to isolate highest affinity binders. Protein EPI-P211, derived from the best isolate of Lib#2, inhibits PLA with Ki = 2 nM (at least 500-fold better than LACI-D1) and with high specificity. We used amino-acid sequences of PLA-binding selectants to design a PLA-biased library (Lib#3) which we screened against PLA. The protein EPI-P302 (derived from the best binder obtained from Lib#3) has Ki for PLA inhibition of 87 pM, which is 25-fold better than the first-round best binder and > or = 12 500-fold better than LACI-D1. EPI-P302 also shows very high specificity for PLA vs other human proteases and is resistant to inactivation by oxidants and extremes of temperature or pH. Thus, one can use selectants from one library to design target-tailored combinatorial libraries and obtain quite stable, highly specific, very high-affinity binding molecules while maintaining an essentially human framework.

Arthur C. Ley

Papers

Directed evolution of novel binding proteins

Generation and selection of novel DNA-binding proteins and polypeptides

Serum albumin binding moieties

The distinctive separation attributes of mixed-mode resins and their application in monoclonal antibody downstream purification process.

Iterative optimization of high-affinity proteases inhibitors using phage display. 1. Plasmin.