# US 12,180,279 B2 · Method for in vivo generation of multispecific antibodies from monospecific antibodies > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,180,279 B2 - Document ID: US12180279B2 - Status: Granted - Kind: B2 - Application number: 16/860,406 - Filed: 2020-04-28 - Published: 2020-08-13 - Granted: 2024-12-31 - Earliest claimed: 2017-10-30 - Assignees: Hoffmann-La Roche Inc. - Applicants: Hoffmann-La Roche Inc. - Inventors: Ulrich Brinkmann; Klaus Mayer; Steffen Dickopf - Web page: https://patentgrep.com/patent/US12180279B2 ## CPC - C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies - C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex} - C07K 16/065: {Purification, fragmentation} - C07K 16/461: {Igs containing Ig-regions, -domains or -residues form different species} - C07K 16/468: {Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies} - A61K 2039/505: {comprising antibodies} - C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature - C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered - C07K 2317/31: multispecific - C07K 2317/526: CH3 domain - C07K 2317/55: Fab or Fab' - C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody® - 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) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract Herein is reported a method for the generation of multispecific antibodies directly on the cell-surface at the site of action by a half-antibody exchange reaction between two 2/3-IgGs or two 2/3-BiFabs destabilized in one half by asymmetric perturbing mutations fostering the generation of correctly assembled full length bi- or multispecific antibodies. The method is performed in the absence hinge region disulfide bonds in the starting 2/3-IgGs or 2/3-BiFabs. ## Claims ### Claim 1 A multimeric polypeptide comprising a first polypeptide comprising i) in N- to C-terminal direction a) a first antibody variable domain selected from a pair of an antibody light and heavy chain variable domain specifically binding to a first target, and b) a first human immunoglobulin G CH3 domain, and ii) a pair of an antibody light and heavy chain variable domain specifically binding to a second target either N-terminal to the first antibody variable domain or C-terminal to the first CH3 domain, and a second polypeptide comprising in N- to C-terminal direction a) a second antibody variable domain selected from a pair of an antibody light and heavy chain variable domain specifically binding to a third target, and b) a second human immunoglobulin G CH3 domain, wherein the second polypeptide lacks a pair of an antibody light and heavy chain variable domain specifically binding to a target, wherein the second antibody variable domain is an antibody light chain variable domain if the first antibody variable domain is an antibody heavy chain variable domain; or the second antibody variable domain is an antibody heavy chain variable domain if the first antibody variable domain is an antibody light chain variable domain, and wherein the second CH3 domain comprises a perturbing mutation selected from the group of mutations consisting of D356K, E357K, K370E and K439E, wherein the first CH3 domain comprises a) the amino acid residue K at position 439 if the perturbing mutation is D356K, or b) the amino acid residue K at position 370 if the perturbing mutation is E357K, or c) the amino acid residue E at position 357 if the perturbing mutation is K370E, or d) the amino acid residue D at position 356 if the perturbing mutation is K439E, wherein all numbering is according to Kabat EU index. ### Claim 2 A multimeric polypeptide comprising a first polypeptide comprising i) in N- to C-terminal direction a) a first human immunoglobulin G CH3 domain, and b) a first antibody variable domain selected from a pair of an antibody light and heavy chain variable domain specifically binding to a first target, ii) a pair of an antibody light and heavy chain variable domain specifically binding to a second target either N-terminal to the first CH3 domain or C-terminal to the first variable domain, a second polypeptide comprising in N- to C-terminal direction a) a second human immunoglobulin G CH3 domain and b) a second antibody variable domain selected from a pair of an antibody light and heavy chain variable domain specifically binding to a third target, wherein the second antibody variable domain is an antibody light chain variable domain if the first antibody variable domain is an antibody heavy chain variable domain; or the second antibody variable domain is an antibody heavy chain variable domain if the first antibody variable domain is an antibody light chain variable domain, and wherein the second CH3 domain comprises a perturbing mutation selected from the group of mutations consisting of D356K, E357K, K370E and K439E, whereby the first CH3 domain comprises a) the amino acid residue K at position 439 if the perturbing mutations is D356K, or b) the amino acid residue K at position 370 if the perturbing mutations is E357K, or c) the amino acid residue E at position 357 if the perturbing mutations is K370E, or d) the amino acid residue D at position 356 if the perturbing mutations is K439E, wherein all numbering is according to Kabat EU index. ### Claim 3 (depends on 1) The multimeric polypeptide according to claim 1, wherein the first CH3 domain and the second CH3 domain comprise further mutations to foster heterodimer formation between said first CH3 domain and said second CH3 domain and that are different from the perturbing mutation. ### Claim 4 (depends on 1) The multimeric polypeptide according to claim 1, wherein the first CH3 domain comprises a) the mutation T366W, or b) the mutations T366S/L368A/Y407V, and the second CH3 domain comprises a) the mutations T366S/L368A/Y407V if the first CH3 domain comprises the mutation T366W, or b) the mutation T366W if the first CH3 domain comprises the mutations T366S/L368A/Y407V, wherein all numbering is according to Kabat EU index. ### Claim 5 (depends on 1) The multimeric polypeptide according to claim 1, wherein the first polypeptide and the second polypeptide are a non-covalent dimer. ### Claim 6 (depends on 1) The multimeric polypeptide according to claim 1, wherein the first variable domain and the second variable domain form a non-functional binding site. ### Claim 7 (depends on 1) The multimeric polypeptide according to claim 1, wherein the first and the second polypeptide each comprise the amino acid sequence DKTHTSPPS (SEQ ID NO: 66) or DKTHT (SEQ ID NO: 94) or GGGS (SEQ ID NO: 69) or DKTHGGGGS (SEQ ID NO: 97) N-terminal to each of the first and second variable domains. ### Claim 8 (depends on 1) The multimeric polypeptide according to claim 1, wherein i) the first CH3 domain comprises the mutation T366W and the amino acid residue K at position 439, and the second CH3 domain comprises the perturbing mutation D356K and the mutations T366S/L368A/Y407V, or ii) the first CH3 domain comprises the mutation T366W and the amino acid residue K at position 370, and the second CH3 domain comprises the perturbing mutation E357K and the mutations T366S/L368A/Y407V, or iii) the first CH3 domain comprises the mutations T366S/L368A/Y407V and the amino acid residue E at position 357, and the second CH3 domain comprises the perturbing mutation K370E and the mutation T366W, or iv) the first CH3 domain comprises the mutations T366S/L368A/Y407V and the amino acid residue D at position 356, and the second CH3 domain comprises the perturbing mutation K439E and the mutation T366W, wherein all numbering is according to Kabat EU index. ### Claim 9 (depends on 1) The multimeric polypeptide according to claim 1, wherein the first, second and third target are different. ### Claim 10 (depends on 1) The multimeric polypeptide according to claim 1, wherein the first target and/or the third target is human CD3. ### Claim 11 (depends on 1) The multimeric polypeptide according to claim 1, wherein the pair of an antibody light and heavy chain variable domain specifically binding to the second target is an Fv, scFv, Fab, scFab, dsscFab, CrossFab, or bispecific Fab. ### Claim 12 (depends on 1) The multimeric polypeptide according to claim 1, wherein each of the first and the second polypeptide further comprises an immunoglobulin G CH2 domain directly N-terminal to the CH3 domain. ### Claim 13 (depends on 1) The multimeric polypeptide according to claim 1, wherein the human immunoglobulin G is human IgG1 or human IgG2 or human IgG3 or human IgG4. ### Claim 14 (depends on 1) A composition comprising a first multimeric polypeptide and a second multimeric polypeptide according to claim 1, wherein the second CH3 domain of the first multimeric polypeptide comprises the mutation D356K and the second CH3 domain of the second multimeric polypeptide comprises the mutation K439E, or the second CH3 domain of the first multimeric polypeptide comprises the mutation E357K and the second CH3 domain of the second multimeric polypeptide comprises the mutation K370E, and the first antibody variable domain of the first multimeric polypeptide and the first variable domain of the second multimeric polypeptide are a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically bind to the first target, and the second antibody variable domain of the first multimeric polypeptide and the second variable domain of the second multimeric polypeptide are a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically bind to the third target, wherein all numbering is according to Kabat EU index. ### Claim 15 (depends on 14) The composition according to claim 14, wherein for each of the first multimeric polypeptide and second multimeric polypeptide, the first CH3 domain of the first polypeptide comprises a) the mutation T366W, or b) the mutations T366S/L368A/Y407V, and the second CH3 domain of the second polypeptide comprises a) the mutations T366S/L368A/Y407V if the first CH3 domain comprises the mutation T366W, or b) the mutation T366W if the first CH3 domain comprises the mutations T366S/L368A/Y407V, wherein all numbering is according to Kabat EU index. ### Claim 16 (depends on 14) The composition according to claim 14, wherein the first CH3 domain of the first multimeric polypeptide and the second CH3 domain of the second multimeric polypeptide comprise the mutation T366W or the mutations T366S/L368A/Y407V, and the second CH3 domain of the first multimeric polypeptide comprises the mutation D356K and the second CH3 domain of the second multimeric polypeptide comprises the mutation K439E, or the second CH3 domain of the first multimeric polypeptide comprises the mutation E357K and the second CH3 domain of the second multimeric polypeptide comprises the mutation K370E, and the first antibody variable domain of the first multimeric polypeptide and the first variable domain of the second multimeric polypeptide are a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically bind to the first target, and the second antibody variable domain of the first multimeric polypeptide and the second variable domain of the second multimeric polypeptide are a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically bind to the third target, wherein all numbering is according to Kabat EU index. ### Claim 17 (depends on 14) The composition according to claim 14, wherein the first and/or the third target is human CD3. ### Claim 18 (depends on 14) The composition according to claim 14, wherein the composition is a pharmaceutical composition and optionally further comprises a pharmaceutically acceptable excipient.