# US 12,760,299 B2 · Binding members with altered diversity scaffold domains > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,760,299 B2 - Document ID: US12760299B2 - Status: Granted - Kind: B2 - Application number: 16/066,638 - Filed: 2017-01-09 - Published: 2019-08-22 - Granted: 2026-10-13 - Earliest claimed: 2016-01-08 - Assignees: Maxion Therapeutics Limited - Applicants: Maxion Therapeutics Limited - Inventors: Aneesh Karatt Vellatt; John McCafferty; Sachin Surade; Tim Luetkens; Edward Masters; Michael Dyson - Web page: https://patentgrep.com/patent/US12760299B2 ## CPC - C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display} - C07K 16/4208: {against an idiotypic determinant on Ig} - C07K 16/46: Hybrid immunoglobulins (hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00) - C12N 15/62: DNA sequences coding for fusion proteins - C12N 15/85: for animal cells - C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding - 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 - C40B 40/10: Libraries containing peptides or polypeptides, or derivatives thereof - C40B 50/14: Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support - C07K 2317/565: Complementarity determining region [CDR] - C07K 2317/567: Framework region [FR] - C07K 2318/20: Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics - C12N 2015/8518: {expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - 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) - 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) ## Abstract This invention relates to libraries of binding members that each comprise a fusion protein which contains a donor diversity scaffold domain, such as a cysteine rich protein, inserted within a recipient diversity scaffold domain, such as an antibody constant or variable domain. Libraries and methods of generating libraries are provided, along with screening methods, binding members and methods of using the binding members. ## Claims ### Claim 1 A library of binding members comprising: a diverse population of fusion proteins, each fusion protein comprising a donor diversity scaffold domain inserted into a recipient diversity scaffold domain, wherein the donor diversity scaffold domain comprises a donor scaffold and a donor interaction sequence and the recipient diversity scaffold domain comprises a recipient scaffold and a recipient interaction sequence, wherein the donor diversity scaffold domain is a knottin, and wherein the recipient diversity scaffold domain is an antibody variable domain and the donor diversity scaffold domain is a replacement for all or part of one of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 or VL CDR3 of the antibody variable domain, wherein the donor diversity scaffold domain: a) is a peptide that consists of 100 or fewer amino acids, and b) comprises two or more disulphide bridges formed by four or more cysteine residues within the donor scaffold, wherein the donor diversity scaffold is located 8 Angstroms to 13 Angstroms from the recipient diversity scaffold domain, calculated from the end of a preceding framework of the antibody variable domain of the recipient diversity scaffold domain to a first cysteine residue of the donor diversity scaffold domain wherein one or both of the donor interaction sequence and the recipient interaction sequence are diverse in said diverse population of fusion proteins. ### Claim 2 A fusion protein comprising a donor diversity scaffold domain inserted into a recipient diversity scaffold domain, wherein the donor diversity scaffold domain comprises a donor scaffold and a donor interaction sequence and the recipient diversity scaffold domain comprises a recipient scaffold and a recipient interaction sequence, wherein the donor diversity scaffold domain is a knottin, and wherein the N terminal and C terminal of the donor diversity scaffold domain are linked to the recipient diversity scaffold domain without linkers or with linkers of up to 4 amino acids, wherein the recipient diversity scaffold domain is an antibody variable domain and the donor diversity scaffold domain is a replacement for all or part of one of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 or VL CDR3 of the antibody variable domain, wherein the donor diversity scaffold domain: a) is a peptide that consists of 100 or fewer amino acids and b) comprises two or more disulphide bridges formed by four or more cysteine residues within the donor scaffold, and wherein the donor diversity scaffold is located 8 Angstroms to 13 Angstroms from the recipient diversity scaffold domain, calculated from the end of a preceding framework of the antibody variable domain of the recipient diversity scaffold domain to a first cysteine residue of the donor diversity scaffold domain. ### Claim 3 (depends on 2) A binding member comprising a fusion protein according to claim 2 and a partner domain. ### Claim 4 (depends on 2) The fusion protein of claim 2, wherein the recipient diversity scaffold domain is an antibody light chain variable (VL) domain. ### Claim 5 (depends on 4) The fusion protein of claim 4, which is associated with a partner domain, wherein the partner domain is an antibody heavy chain variable (VH) domain. ### Claim 6 (depends on 5) The fusion protein of claim 5, wherein the VL domain and the VH domain are connected by a linker. ### Claim 7 (depends on 4) The fusion protein of claim 4, wherein the donor diversity scaffold domain is a replacement for the entirety of one the CDR1 or CDR2 of the antibody VL domain. ### Claim 8 (depends on 2) The fusion protein of claim 2, wherein the donor diversity scaffold domain is Ecballium elaterium trypsin inhibitor-II (EETI-II). ### Claim 9 (depends on 2) The fusion protein of claim 2, wherein a native N terminal and a native C terminal of the donor diversity scaffold domain are each linked to the recipient diversity scaffold domain. ### Claim 10 (depends on 2) The fusion protein of claim 2, wherein an artificial N terminal and an artificial C terminal generated by cyclisation and linearization of the donor diversity scaffold domain are each linked to the recipient diversity scaffold domain. ### Claim 11 (depends on 1) The library of claim 1, wherein the binding members in the library comprise; (i) diverse donor interaction sequences and the same recipient interaction sequence, (ii) diverse recipient interaction sequences and the same donor interaction sequence, or (iii) diverse recipient and diverse donor interaction sequences. ### Claim 12 (depends on 11) The library of claim 11, wherein the binding members in the library have diverse linker sequences. ### Claim 13 (depends on 1) The library of claim 1, wherein the binding members in the library have the same recipient interaction sequences, the same donor interaction sequences, and diverse linker sequences. ### Claim 14 (depends on 1) The library of claim 1, wherein the binding members in the library further comprise partner domains. ### Claim 15 (depends on 1) The library of claim 1, wherein each binding member in the library is displayed on the surface of a ribosome which comprises nucleic acid encoding the binding member, a cell which comprises nucleic acid encoding the binding member, or a virus which comprises nucleic acid encoding the binding member. ### Claim 16 (depends on 1) The library of claim 1, wherein the binding members are displayed on the surface of a bacteriophage or the surface of a eukaryotic cell. ### Claim 17 (depends on 1) The library of claim 1, wherein the fusion protein of the binding member is fused to a coat protein of a filamentous phage. ### Claim 18 (depends on 3) The binding member of claim 3, wherein the recipient diversity scaffold domain of the fusion protein is associated with a partner domain through a covalent or non-covalent bond. ### Claim 19 (depends on 3) The binding member of claim 3, wherein the binding member further comprises multiple partner domains. ### Claim 20 (depends on 3) The binding member of claim 3, wherein the fusion protein and the partner domain bind to the same target molecule, or wherein one of the fusion protein and the partner domain binds to a first target molecule and the other of the fusion protein and the partner domain binds to a second target molecule. ### Claim 21 (depends on 20) The binding member of claim 20, wherein the same target molecule, first target molecule, or second target molecule is an integral membrane protein. ### Claim 22 (depends on 21) The binding member of claim 21, wherein the integral membrane protein is an ion channel, a G protein-coupled receptor (GPCR), or Type I receptor. ### Claim 23 (depends on 2) The fusion protein of claim 2, wherein the recipient diversity scaffold domain is an antibody heavy chain variable (VH) domain. ### Claim 24 (depends on 23) The fusion protein of claim 23, wherein the partner domain is an antibody light chain variable (VL) domain. ### Claim 25 (depends on 2) The fusion protein of claim 2, wherein the interaction sequence of the donor diversity scaffold domain and one or more CDRs of the antibody variable domain interact with the same epitope on a target molecule. ### Claim 26 (depends on 25) The fusion protein of claim 25, wherein the one or more CDRs of the antibody variable domain that interact with the same epitope on the target molecule is a VH CDR3 or a VL CDR3. ### Claim 27 (depends on 2) The fusion protein of claim 2, wherein the donor diversity scaffold domain consists of at least 15 amino acids. ### Claim 28 (depends on 1) The library of binding members of claim 1, wherein the linkers are diverse in said population. ### Claim 29 (depends on 1) The library of binding members of claim 1, wherein the linkers are diverse in said population; and wherein one or both of the donor interaction sequence and the recipient interaction sequence are diverse in said population. ### Claim 30 (depends on 1) The library of binding members of claim 1, wherein the fusion proteins are associated with binding partners to form heterodimers. ### Claim 31 (depends on 1) The library of binding members of claim 1, wherein the donor diversity scaffold domain is a replacement for all or part of one of VH CDR1, VH CDR2, VL CDR1, or VL CDR2 of the antibody variable domain. ### Claim 32 (depends on 2) The fusion protein of claim 2, wherein the donor diversity scaffold domain is a replacement for all or part of one of VH CDR1, VH CDR2, VL CDR1, or VL CDR2 of the antibody variable domain. ### Claim 33 (depends on 1) The library of binding members of claim 1, wherein the N terminal and C terminal of the donor diversity scaffold domain are linked to the recipient diversity scaffold domain without linkers or with linkers of up to 4 amino acids.