# US 10,309,969 B2 · Protein combination-based FV library, and preparation method therefor > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,309,969 B2 - Document ID: US10309969B2 - Status: Granted - Kind: B2 - Application number: 14/760,996 - Filed: 2014-01-24 - Published: 2016-01-28 - Granted: 2019-06-04 - Earliest claimed: 2013-01-24 - Assignees: AbTLAS CO., LTD. - Applicants: AbTLAS CO., LTD. - Inventors: Byeong Doo Song; Jee Sun Yun; Song Yi Lee; Hye In Kim; Hyo Jung Choi; Jong Rip Choi - Web page: https://patentgrep.com/patent/US10309969B2 ## CPC - G01N 33/6845: {Methods of identifying protein-protein interactions in protein mixtures} - C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies - C07K 16/46: Hybrid immunoglobulins (hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00) - G01N 33/6857: {Antibody fragments} - C07K 2317/56: variable (Fv) region, i.e. VH and/or VL - C07K 2317/565: Complementarity determining region [CDR] - C07K 2317/62: comprising only variable region components - 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) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract The present invention relates to a method for constructing an Fv library based on a combination of proteins, a method of screening a desired antibody using the constructed Fv library, an Fv antibody screened by the screening method, and an Fv library constructed by the Fv library construction method. The Fv library of the present invention is based on a combination of proteins so that members thereof can be individually analyzed for their function. Moreover, the Fv library enables a desired Fv antibody to be screened without needing a target antigen preparation. In addition, the protein combination based Fv library makes it possible to significantly reduce the number of protein purification processes to thereby reduce costs and time, compared to conventional DNA-based libraries. ## Claims ### Claim 1 A method for constructing an Fv library comprising: (a) expressing and purifying individual heavy-chain variable region (VH) domain proteins from cells; (b) expressing and purifying light-chain variable region (VL) domain proteins from cells separate from the cells in (a); and (c) pairing the purified VH domain proteins and the purified VL domain proteins to one another, wherein the VH domain proteins are fusion proteins, wherein the VL domain proteins are fusion proteins, and wherein VH domain proteins derive from the same VH domain and a mutation is introduced into the VH domain proteins such that the VH domain proteins comprise amino acid sequences that differ from each other; or wherein VL domain proteins derive from the same VL domain and a mutation is introduced into the VL domain proteins such that the VL domain proteins comprise amino acid sequences that differ from each other. ### Claim 2 A method for constructing an Fv library comprising: (a) expressing and purifying individual heavy-chain variable region (VH) domain proteins from cells; (b) expressing and purifying light-chain variable region (VL) domain proteins from cells separate from the cells in (a); and (c) pairing the purified VH domain proteins and the purified VL domain proteins to one another, wherein the VH domain proteins are fusion proteins, wherein the VL domain proteins are fusion proteins, and wherein VH domain proteins derive from the same VH domain and a mutation is introduced into the VH domain proteins such that the VH domain proteins comprise amino acid sequences that differ from each other; and wherein VL domain proteins derive from the same VL domain and a mutation is introduced into the VL domain proteins such that the VL domain proteins comprise amino acid sequences that differ from each other. ### Claim 3 (depends on 1) The method of claim 1, wherein the VH domain proteins comprise complementarity determining regions (CDRs) and framework regions (FRs) and the VL domain proteins comprise complementarity determining regions (CDRs) and framework regions (FRs). ### Claim 4 (depends on 3) The method of claim 3 wherein CDRs of the VH domain proteins comprise amino acid sequences that differ from each other; CDRs of the VL domain proteins comprise amino acid sequences that differ from each other; FRs of the VH domain proteins comprise amino acid sequences that differ from each other; or FRs of the VL domain proteins comprise amino acid sequences that differ from each other. ### Claim 5 (depends on 4) The method of claim 4, wherein CDRs of the VH domain proteins comprise amino acid sequences that differ from each other; and CDRs of the VL domain proteins comprise amino acid sequences that differ from each other. ### Claim 6 (depends on 4) The method of claim 4 wherein FRs of the VH domain proteins comprise amino acid sequences that differ from each other; and FRs of the VL domain proteins comprise amino acid sequences that differ from each other. ### Claim 7 (depends on 5) The method of claim 5 wherein FRs of the VH domain proteins comprise amino acid sequences that differ from each other; and FRs of the VL domain proteins comprise amino acid sequences that differ from each other. ### Claim 8 (depends on 1) The method of claim 1, wherein step (c) comprises pairing the VH domain proteins and the VL domain proteins by random pairing or target pairing. ### Claim 9 (depends on 1) The method of claim 1, wherein the pairing of the VH domain proteins with the VL domain proteins in step (c) is performed by a method comprising: (i) pairing between wild-type VH domain proteins and the VL domain proteins; (ii) pairing by disulfide bonds between cysteine residues introduced into each of the VH domain proteins and the VL domain proteins; (iii) pairing by coiled-coil domains introduced into each of the VH domain proteins and the VL domain proteins; or (iv) pairing by protein-protein interaction between each of the VH domain proteins and the VL domain proteins. ### Claim 10 A method for constructing an Fv library comprising: (a) expressing and purifying individual heavy-chain variable region (VH) domain proteins from cells; (b) expressing and purifying light-chain variable region (VL) domain proteins from cells separate from the cells in (a); (c) pairing the purified VH domain proteins and the purified VL domain proteins to one another; and (d) storing the paired VH domain proteins and VL domain proteins of step (c) in individual compartments, wherein the VH domain proteins are fusion proteins, wherein the VL domain proteins are fusion proteins, and wherein the individual compartments are assigned a unique identification (ID) number, and wherein the compartments comprise plate wells, test tubes, microfluidic channels, or chips. ### Claim 11 (depends on 10) The method of claim 10, wherein the VH domain proteins comprise complementarity determining regions (CDRs) and framework regions (FRs) and the VL domain proteins comprise complementarity determining regions (CDRs) and framework regions (FRs). ### Claim 12 (depends on 11) The method of claim 11, wherein CDRs of the VH domain proteins comprise amino acid sequences that differ from each other; CDRs of the VL domain proteins comprise amino acid sequences that differ from each other; FRs of the VH domain proteins comprise amino acid sequences that differ from each other; or FRs of the VL domain proteins comprise amino acid sequences that differ from each other. ### Claim 13 (depends on 12) The method of claim 12, wherein CDRs of the VH domain proteins comprise amino acid sequences that differ from each other; and CDRs of the VL domain proteins comprise amino acid sequences that differ from each other. ### Claim 14 (depends on 12) The method of claim 12, wherein FRs of the VH domain proteins comprise amino acid sequences that differ from each other; and FRs of the VL domain proteins comprise amino acid sequences that differ from each other. ### Claim 15 (depends on 13) The method of claim 13, wherein FRs of the VH domain proteins comprise amino acid sequences that differ from each other; and FRs of the VL domain proteins comprise amino acid sequences that differ from each other. ### Claim 16 (depends on 2) The method of claim 2, wherein step (c) comprises pairing the VH domain proteins and the VL domain proteins by random pairing or target pairing. ### Claim 17 (depends on 2) The method of claim 2, wherein the pairing of the VH domain proteins with the VL domain proteins in step (c) is performed by a method comprising: (i) pairing between wild-type VH domain proteins and the VL domain proteins; (ii) pairing by disulfide bonds between cysteine residues introduced into each of the VH domain proteins and the VL domain proteins; (iii) pairing by coiled-coil domains introduced into each of the VH domain proteins and the VL domain proteins; or (iv) pairing by protein-protein interaction between each of the VH domain proteins and the VL domain proteins.