# US 10,118,970 B2 · Multispecific antibodies > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,118,970 B2 - Document ID: US10118970B2 - Status: Granted - Kind: B2 - Application number: 11/893,693 - Filed: 2007-08-17 - Published: 2008-03-20 - Granted: 2018-11-06 - Earliest claimed: 2006-08-30 - Assignees: Genentech, Inc. - Applicants: Germaine Fuh; Jenny M. Bostrom - Inventors: Germaine Fuh; Jenny M. Bostrom - Web page: https://patentgrep.com/patent/US10118970B2 ## CPC - C07K 16/32: against translation products of oncogenes - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - C07K 16/22: against growth factors {; against growth regulators} - C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95} - C07K 16/4283: {against an allotypic or isotypic determinant on Ig} - C07K 16/468: {Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies} - C07K 2317/55: Fab or Fab' - C07K 2317/56: variable (Fv) region, i.e. VH and/or VL - C07K 2317/622: Single chain antibody (scFv) - C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation - 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) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS ## Abstract The invention provides multispecific antibodies and methods of making and using such antibodies. In general, the multispecific antibodies are made by methods involving the steps of altering the nucleic acid sequence encoding the light chain variable domain (VL) of an antibody that binds a first epitope, and selecting a multispecific antibody capable of binding the first and a second epitope. ## Claims ### Claim 1 A method of making a multispecific antibody comprising a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL of the multispecific antibody pair together to form an antigen binding site that specifically binds to a first epitope and a second epitope, said method comprising the steps of: (1) providing an antibody that comprises a VH and VL, wherein the VH and VL pair together to form an antigen binding site that binds to the first epitope but not the second epitope; (2) altering the nucleic acid sequence encoding the VL of the antibody of step (1), wherein one or more solvent accessible amino acid residues are altered at one or more amino acid residue positions selected from the group consisting of positions 28, 29, 30-32, 50-53, 66-70, and 91-94, according to the Kabat numbering system, and wherein the one or more solvent accessible amino acid residues include an amino acid residue of position 30 or position 93, according to the Kabat numbering system; (3) expressing the VH and the altered nucleic acid sequence of the VL of step (2); and (4) following step (3), selecting for antibody binding to said first epitope and said second epitope, thereby selecting for a multispecific antibody by its ability to bind to both said first epitope and said second epitope, said multispecific antibody comprising the VH and the altered VL of step (3), wherein the VH and VL of the multispecific antibody pair together to form an antigen binding site that specifically binds to the first epitope and the second epitope. ### Claim 2 A method of making a multispecific antibody comprising a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL of the multispecific antibody pair together to form an antigen binding site that specifically binds to a first epitope and a second epitope, said method comprising the steps of: (1) providing an antibody that comprises a VH and VL, wherein the VH and VL pair together to form an antigen binding site that binds to the first epitope but not the second epitope; (2) holding the nucleic acid sequence encoding the VH constant and altering the nucleic acid sequence encoding the VL of the antibody of step (1), wherein one or more solvent accessible amino acid residues are altered; (3) expressing the nucleic acid sequence of the VH and the altered nucleic acid sequence of the VL of step (2); and (4) following step (3), selecting for antibody binding to said first epitope and said second epitope, thereby selecting for a multispecific antibody by its ability to bind to both said first epitope and said second epitope, said multispecific antibody comprising the VH and the altered VL of step (3), wherein the VH and the VL of the multispecific antibody pair together to form an antigen binding site that specifically binds to the first epitope and the second epitope. ### Claim 3 (depends on 1, 2) The method according to claim 1 or 2, wherein the nucleic acid sequences targeted for altering in step (2) encode a plurality of predetermined amino acid residues. ### Claim 4 (depends on 3) The method according to claim 3, wherein the nucleic acid sequence is altered based on the diversity of a plurality of naturally occurring light chain amino acid sequences. ### Claim 5 (depends on 3) The method according to claim 3, wherein the plurality of predetermined amino acid residues comprise amino acid residue positions 28-32, 50-53, and 91-94, according to the Kabat numbering system. ### Claim 6 (depends on 1, 2) The method according to claim 1 or 2, wherein the altered VL are displayed on phage with the VH during the selection of step (4). ### Claim 7 (depends on 1, 2) The method according to claim 1 or 2, wherein the antibody of step (1) comprises a CDR-H1 comprising IKDTYI (SEQ ID NO:1), a CDR-H2 comprising ARIYPTNGYTRY (SEQ ID NO:2), and a CDR-H3 comprising RWGGDGFY (SEQ ID NO:3). ### Claim 8 (depends on 7) The method according to claim 7, wherein the antibody of step (1) further comprises a CDR-L1 comprising DVSTAV (SEQ ID NO:4), CDR-L2 comprising SASF (SEQ ID NO:5) and a CDR-L3 comprising HYTTPP (SEQ ID NO:6). ### Claim 9 (depends on 1, 2) The method according to claim 1 or 2, wherein the antigen binding site of the multispecific antibody of step (4) binds the first epitope and second epitope mutually exclusively. ### Claim 10 (depends on 1, 2) The method according to claim 1 or 2, wherein the first epitope is from one biological molecule and the second epitope is from a second biological molecule. ### Claim 11 (depends on 10) The method according to claim 10, wherein the first biological molecule and second biological molecule are selected from the group consisting of VEGF/HER2, VEGF-A/HER2, HER2/DR5, VEGF-A/PDGF, HER1/HER2, CD20/BR3, VEGF-A/VEGF-C, VEGF-C/VEGF-D, TNFalpha/TGF-beta, TNFalpha/IL-2, TNF alpha/IL-3, TNFalpha/IL-4, TNFalpha/IL-5, TNFalpha/IL6, TNFalpha/IL8, TNFalpha/IL-9, TNFalpha/IL-10, TNFalpha/IL-11, TNFalpha/IL-12, TNFalpha/IL-13, TNFalpha/IL-14, TNFalpha/IL-15, TNFalpha/IL-16, TNFalpha/IL-17, TNFalpha/IL-18, TNFalpha/IL-19, TNFalpha/IL-20, TNFalpha/IFNalpha, TNFalpha/CD4, VEGF/IL-8, VEGF/MET, VEGFR/MET receptor, HER2/Fc, HER2/HER3; EGFR/HER2, EGFR/HER3, EGFR/HER4, TNFalpha/IL-3, TNFalpha/IL-4, IL-13/CD40L, IL4/CD40L, TNFalpha/ICAM-1, TNFR1/IL-1R, TNFR1/IL-6R, and TNFR1/IL-18R. ### Claim 12 (depends on 11) The method according to claim 11, wherein said first biological molecule and second biological molecule are VEGF/HER2. ### Claim 13 (depends on 1) The method according to claim 1, wherein: (a) the one or more solvent accessible amino acid residues include an amino acid residue of position 30, and the method further comprises altering the nucleic acid sequence encoding the VL of the antibody of step (1) at a solvent accessible amino acid residue at a position selected from the group consisting of position 28, 29, 31, 32, 50, 51, 53, 91, 92, 93, and 94 of the VL; or (b) the one or more solvent accessible amino acid residues include an amino acid residue of position 93, and the method further comprises altering the nucleic acid sequence encoding the VL of the antibody of step (1) at a solvent accessible amino acid residue at a position selected from the group consisting of position 28, 29, 30, 31, 32, 50, 51, 53, 91, 92, and 94 of the VL. ### Claim 14 (depends on 10) The method according to claim 10, wherein the first biological molecule and the second biological molecule are proteins that have less than 60% amino acid identity to each other. ### Claim 15 (depends on 2) The method according to claim 2, wherein the one or more solvent accessible amino acid residues are altered at one or more amino acid residue positions selected from the group consisting of positions 28, 29, 30-32, 50-53, 66-70, and 91-94, according to the Kabat numbering system. ### Claim 16 (depends on 1, 2) The method according to claim 1 or 2, wherein the VH and VL of the multispecific antibody pair together to form an antigen binding site that specifically binds to the first epitope and the second epitope with at least 104 M affinity. ### Claim 17 (depends on 16) The method according to claim 16, wherein the VH and VL of the multispecific antibody pair together to form an antigen binding site that specifically binds to the first epitope with at least 10−6 M affinity and the second epitope with at least 10−4 M affinity. ### Claim 18 (depends on 15) The method according to claim 15, wherein the one or more solvent accessible amino acid residues include an amino acid residue of position 30, according to the Kabat numbering system. ### Claim 19 (depends on 15) The method according to claim 15, wherein the one or more solvent accessible amino acid residues include an amino acid residue of position 93, according to the Kabat numbering system. ### Claim 20 (depends on 17) The method according to claim 17, wherein the VH and VL of the multispecific antibody pair together to form an antigen binding site that specifically binds to the first epitope and the second epitope with at least 10−6 M affinity. ### Claim 21 (depends on 1, 2) The method according to claim 1 or 2, wherein the method further comprises the step of subjecting the multispecific antibody selected in step (4) to affinity maturation. ### Claim 22 (depends on 17) The method according to claim 17, wherein the VH and VL of the multispecific antibody pair together to form an antigen binding site that specifically binds to the first epitope with at least 10−7 M affinity and the second epitope with at least 10−5 M affinity. ### Claim 23 (depends on 20) The method according to claim 20, wherein the VH and VL of the multispecific antibody pair together to form an antigen binding site that specifically binds to the first epitope with at least 10−7 M affinity and the second epitope with at least 10−6 M affinity. ### Claim 24 (depends on 17) The method according to claim 17, wherein the VH and VL of the multispecific antibody pair together to form an antigen binding site that specifically binds to the first epitope with at least 10−8 M affinity and the second epitope with at least 10−4 M affinity. ### Claim 25 (depends on 15) The method according to claim 15, wherein the one or more solvent accessible amino acid residues are altered at one or more amino acid residue positions selected from the group consisting of positions 28, 29, 30, 31, 32, 50, 51, 53, 91, 92, 93, and 94, according to the Kabat numbering system. ### Claim 26 (depends on 1, 25) The method according to claim 1 or 25, wherein the one or more solvent accessible amino acid residues include an amino acid residue of positions 30 and 93, according to the Kabat numbering system.