# US 2014/0155581 A1 · Methods For Making Multimeric Polypeptides > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0155581 A1 - Document ID: US20140155581A1 - Status: Published application - Kind: A1 - Application number: 14/126,757 - Filed: 2012-07-02 - Published: 2014-06-05 - Earliest claimed: 2011-07-06 - Applicants: Medimmune, LLC - Inventors: Changshou Gao; Nazzareno Dimasi - Web page: https://patentgrep.com/patent/US20140155581A1 ## CPC - C07K 16/468: {Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies} - C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof - C07K 16/2863: {against receptors for growth factors, growth regulators} - 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 Detailed herein are contiguous, multimeric, multispecific polypeptides, nucleic acids encoding such polypeptides, and methods for making such polypeptides and nucleic acids. ## Claims ### Claim 1 A nucleic acid molecule encoding a contiguous, multimeric polypeptide comprising at least two subunits, each of which includes at least a functional domain (FD) and an interaction domain (ID), wherein the nucleic acid molecule comprises: i) a nucleic acid portion comprising a nucleotide sequence that encodes a functional domain (FD1) that binds to a first binding site; ii) a nucleic acid portion comprising a nucleotide sequence that encodes an interaction domain (ID1); iii) a nucleic acid portion comprising a nucleotide sequence that encodes a functional domain (FD2) that binds to a second binding site; and iv) a nucleic acid portion comprising a nucleotide sequence that encodes an interaction domain (ID2); wherein the nucleic acid molecule further comprises at least one nucleic acid portion comprising a nucleotide sequence encoding a polypeptide linker, which polypeptide linker includes at least one protease cleavage site; wherein ID1 and ID2 are capable of associating with each other, and wherein the contiguous, multimeric polypeptide is multispecific. ### Claim at position 2 2-4. (canceled) ### Claim 5 (depends on 1) The nucleic acid molecule of claim 1, wherein the nucleic acid portion comprising the nucleotide sequence that encodes the ID1 and the nucleic acid portion comprising the nucleotide sequence that encodes the ID2 each comprises a nucleic acid segment comprising a nucleotide sequence that encodes a coil-coil dimerization motif; or the nucleic acid portion comprising the nucleotide sequence that encodes the ID1 comprises a nucleic acid segment comprising a nucleotide sequence that encodes a heavy constant domain 1 and the nucleic acid portion comprising the nucleotide sequence that encodes the ID2 comprises a nucleic acid segment comprising a nucleotide sequence that encodes a heavy constant domain 2. ### Claim 6 (canceled) ### Claim 7 (depends on 1) The nucleic acid molecule of claim 1, wherein the nucleic acid portion comprising the nucleotide sequence that encodes the FD1 and/or FD2 comprises a nucleic acid segment comprising a nucleotide sequence that encodes an antigen binding portion of an antibody selected from the group consisting of: an scFv, a tandem scFv, a diabody, a triabody and a Fab domain. ### Claim at position 6 8-10. (canceled) ### Claim 11 (depends on 1) The nucleic acid molecule of claim 1, wherein the nucleic acid portion comprising the nucleotide sequence that encodes FD1 and/or FD2 comprises a nucleic acid segment comprising a nucleotide sequence that encodes an antibody mimetic. ### Claim 12 A nucleic acid molecule encoding a contiguous polypeptide, wherein the nucleic acid molecule comprises: i) a nucleic acid segment comprising a nucleotide sequence that encodes an antibody light chain comprising a variable domain (VL1) and a light constant domain 1; ii) a nucleic acid segment comprising a nucleotide sequence that encodes a first polypeptide linker; iii) a nucleic acid segment comprising a nucleotide sequence that encodes an antibody heavy chain comprising a variable domain (VH1) and a heavy constant domain 1; iv) a nucleic acid segment comprising a nucleotide sequence that encodes a second polypeptide linker; v) a nucleic acid segment comprising a nucleotide sequence that encodes an antibody light chain comprising a variable domain (VL2) and a light constant domain 2; vi) a nucleic acid segment comprising a nucleotide sequence that encodes a third polypeptide linker; and vii) a nucleic acid segment comprising a nucleotide sequence that encodes an antibody heavy chain comprising a variable domain (VH2) and a heavy constant domain 2; wherein the contiguous polypeptide is a multispecific polypeptide, and wherein each of the three polypeptide linkers comprises at least one protease cleavage site. ### Claim 13 (depends on 12) The nucleic acid molecule of claim 12, wherein VL1 and VL2 are not the same antibody light chain variable domain and/or VH1 and VH2 are not the same antibody heavy chain variable domain. ### Claim 14 (depends on 5) The nucleic acid molecule of claim 5, wherein the heavy constant domain 1 comprises one or more of a CH1, CH2, and CH3 regions, and wherein the heavy constant domain 2 comprises one or more of a CH1, CH2, and CH3 regions. ### Claim 15 (depends on 14) The nucleic acid molecule of claim 14, wherein the heavy constant domain 1 further comprises a hinge region, and wherein the heavy constant domain 2 further comprises a hinge region. ### Claim 16 (depends on 1) The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises at least two nucleic acid segments each of which comprises a nucleotide sequence that encodes a polypeptide linker. ### Claim 17 (depends on 1) The nucleic acid molecule of any of claim 1, wherein at least one of the polypeptide linkers comprises two protease cleavage sites. ### Claim 18 (depends on 16) The nucleic acid molecule of claim 16, wherein at least one of the polypeptide linkers is different. ### Claim 19 (depends on 1) An expression vector comprising the nucleic acid molecule of any of claim 1 operably linked to a promoter. ### Claim 20 (depends on 19) A host cell comprising the expression vector of claim 19, and which host cell expresses the contiguous polypeptide. ### Claim 21 (depends on 20) A method of producing a contiguous polypeptide, comprising: i) providing a host cell of claim 20 in a culture media suitable for growth of the cell and production of the polypeptide; and ii) purifying the polypeptide from the cell or culture media. ### Claim 22 (depends on 21) The method of claim 21, further comprising adding, either before or after the step of purifying the polypeptide from the cell or culture media, a protease that cleaves the at least one protease cleavage site. ### Claim at position 19 23-37. (canceled) ### Claim 38 (depends on 12) The nucleic acid molecule of claim 12, wherein the heavy constant domain 1 comprises a hinge, CH1, CH2, and CH3 regions, and wherein the heavy constant domain 2 comprises a hinge, CH1, CH2, and CH3 regions. ### Claim 39 (depends on 12) The nucleic acid molecule of any of claim 12, wherein at least one of the polypeptide linkers comprises two protease cleavage sites. ### Claim 40 (depends on 12) The nucleic acid molecule of claim 12, wherein at least one of the polypeptide linkers is different. ### Claim 41 (depends on 12) An expression vector comprising the nucleic acid molecule of claim 12 operably linked to a promoter. ### Claim 42 (depends on 41) A host cell comprising the expression vector of claim 41, and which host cell expresses the contiguous polypeptide. ### Claim 43 (depends on 42) A method of producing a contiguous polypeptide, comprising: i) providing a host cell of claim 42 in a culture media suitable for growth of the cell and production of the polypeptide; and ii) purifying the polypeptide from the cell or culture media. ### Claim 44 (depends on 43) The method of claim 43, further comprising adding, either before or after the step of purifying the polypeptide from the cell or culture media, a protease that cleaves the at least one protease cleavage site. ### Claim 45 (depends on 12) A polypeptide encoded by the nucleic acid molecule of claim 12.