# US 9,040,258 B2 · Multi-chain eukaryotic display vectors and uses thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,040,258 B2 - Document ID: US9040258B2 - Status: Granted - Kind: B2 - Application number: 13/170,362 - Filed: 2011-06-28 - Published: 2011-11-24 - Granted: 2015-05-26 - Earliest claimed: 2001-10-01 - Assignees: Dyax Corp. - Applicants: Simon E. Hufton; Hendricus Renerus Jacobus Mattheus Hoogenboom - Inventors: Simon E. Hufton; Hendricus Renerus Jacobus Mattheus Hoogenboom - Web page: https://patentgrep.com/patent/US9040258B2 ## CPC - C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display} - C12N 15/85: for animal cells - C40B 40/02: Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors - G01N 33/554: the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells - C12N 2795/00043: viral genome or elements thereof as genetic vector - C12N 2800/108: episomal vectors - C12N 2830/002: inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor - C12N 2840/20: translation of more than one cistron - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract A eukaryotic expression vector capable of displaying a multi-chain polypeptide on the surface of a host cell is provided, such that the biological activity of the multi-chain polypeptide is exhibited at the surface of the host cell. Such a vector allows for the display of complex biologically active polypeptides, e.g., biologically active multi-chain polypeptides such as immunoglobulin Fab fragments. The present invention describes and enables the successful display of a multi-chain polypeptide on the surface of a eukaryotic host cell. Preferred vectors are described for expressing the chains of a multi-chain polypeptide in a host cell separately and independently (e.g., under separate vector control elements, and/or on separate expression vectors, thus forming a matched vector set). The use of such matched vector sets provides flexibility and versatility in the generation of eukaryotic display libraries, for example the ability to generate and to display multi-chain polypeptides by combining and recombining vectors that express variegations of the individual chains of a multi-chain polypeptide. Entire repertoires of novel chain combinations can be devised using such vector sets. ## Claims ### Claim 1 A method for selecting an antibody that binds to a target antigen by yeast display, comprising the step of: contacting yeast cells that display a library of multi-chain antibodies with a target antigen, selecting the yeast cell that binds to the target antigen; and identifying the antibody displayed by the selected yeast cell, wherein the multi-chain antibodies each comprise a heavy chain variable region (VH) and a light chain variable region (VL), which are on separate chains. ### Claim 2 (depends on 1) The method of claim 1, wherein the diversity of the library of antibodies is at least 1×106. ### Claim 3 (depends on 1) The method of claim 1, wherein the diversity of the library of antibodies is at least 1×107. ### Claim 4 (depends on 1) The method of claim 1, wherein the sequence of the antibody heavy or light chain variable region is fully human. ### Claim 5 (depends on 1) The method of claim 1, wherein the antibodies each comprise a VH and a VL, which are encoded respectively by a heavy chain variable region of a human immunoglobulin gene and a light-chain variable region of a human immunoglobulin gene. ### Claim 6 (depends on 1) The method of claim 1, wherein the antibodies each comprise a VH encoded by a heavy chain variable region of a first human immunoglobulin gene and a VL encoded by a light chain variable region of a second immunoglobulin gene different from the first human immunoglobulin gene. ### Claim 7 (depends on 1) The method of claim 1, wherein the antibody heavy or light chain variable region is encoded by a human IgG gene. ### Claim 8 (depends on 1) The method of claim 1, wherein the yeast cell that binds to the target antigen is selected by flow cytometric cell sorting. ### Claim 9 A library of yeast cells displaying a library of multi-chain antibodies on the surface of the yeast cells, comprising: yeast cells transformed with a library of yeast expression vectors encoding a library of antibodies each comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) wherein the VH and VL are on separate chains, wherein at least one yeast expression vector comprises a polynucleotide sequence encoding the VH or a polynucleotide sequence encoding the VL, which is inserted into the at least one yeast expression vector by homologous recombination, and also a sequence encoding a yeast surface anchoring protein subunit. ### Claim 10 (depends on 9) The library of claim 9, wherein the yeast cells are Saccharomyces cerevisiae cells. ### Claim 11 (depends on 9) The library of claim 9, wherein the yeast surface anchoring protein subunit is a-agglutinin or α-agglutinin. ### Claim 12 (depends on 9) The library of claim 9, wherein a region of the VH or VL is mutagenized. ### Claim 13 (depends on 9) The library of claim 9, wherein the diversity of the library of antibodies is at least 1×106. ### Claim 14 (depends on 9) The library of claim 9, wherein the diversity of the library of antibodies is at least 1×107. ### Claim 15 (depends on 9) The library of claim 9, wherein the sequence of the antibody heavy or light chain variable region is fully human. ### Claim 16 (depends on 9) The library of claim 9, wherein the VH and VL are encoded respectively by a heavy chain variable region of a human immunoglobulin gene and a light-chain variable region of a human immunoglobulin gene. ### Claim 17 (depends on 9) The library of claim 9, wherein the VH is encoded by a heavy-chain variable region of a first human immunoglobulin gene, and the VL is encoded by a light chain variable region of a second human immunoglobulin gene different from the first human immunoglobulin gene. ### Claim 18 (depends on 9) The library of claim 9, wherein the antibody heavy or light chain variable region is encoded by a human IgG gene.