# US 2019/0169292 A1 · Covalently Bonded Diabodies Having Immunoreactivity with PD-1 and LAG-3, and Methods of Use Thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2019/0169292 A1 - Document ID: US20190169292A1 - Status: Published application - Kind: A1 - Application number: 16/189,071 - Filed: 2018-11-13 - Published: 2019-06-06 - Earliest claimed: 2014-06-26 - Assignees: MacroGenics, Inc. - Applicants: MacroGenics, Inc. - Inventors: Ezio Bonvini; Leslie S. Johnson; Kalpana Shah; Ross La Motte-Mohs; Paul A. Moore; Scott Koenig - Web page: https://patentgrep.com/patent/US20190169292A1 ## CPC - C07K 16/2803: {against the immunoglobulin superfamily} - A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics - A61P 31/04: Antibacterial agents - A61P 31/10: Antimycotics - A61P 31/12: Antivirals - A61P 35/00: Antineoplastic agents - A61P 35/02: specific for leukemia - C07K 16/2818: {against CD28 or CD152} - C07K 16/468: {Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies} - C07K 2317/31: multispecific - C07K 2317/626: Diabody or triabody - C07K 2317/74: Inducing 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 present invention is directed to bi-specific diabodies that comprise two or more polypeptide chains and which possess at least one Epitope-Binding Site that is immunospecific for an epitope of PD-1 and at least one Epitope-Binding Site that is immunospecific for an epitope of LAG-3 (i.e., a “PD-1×LAG-3 bi-specific diabody”). More preferably, the present invention is directed to bi-specific diabodies that comprise four polypeptide chains and which possess two Epitope-Binding Sites that are immunospecific for one (or two) epitope(s) of PD-1 and two Epitope-Binding Site that are immunospecific for one (or two) epitope(s) of LAG-3 (i.e., a “PD-1×LAG-3 bi-specific, tetra-valent diabody”). The present invention also is directed to such diabodies that additionally comprise an immunoglobulin Fc Domain (“bi-specific Fc diabodies and bi-specific, tetra-valent, Fc diabodies”). The diabodies of the present invention are capable of simultaneously binding to PD-1 and to LAG-3, particularly as such molecules are arrayed on the surfaces of human cells. The invention is directed to pharmaceutical compositions that contain such diabodies, and to methods involving the use of such diabodies in the treatment of cancer and other diseases and conditions. ## Claims ### Claim 1 A method of attenuating PD-1 and LAG-3-mediated immune system inhibition in a subject in need thereof, said method comprising administering to said subject a bi-specific Fc diabody capable of immunospecific binding to an epitope of PD-1 and to an epitope of LAG-3, wherein said diabody comprises a first, second, third and fourth polypeptide chains, each having an amino terminus and a carboxy terminus, and wherein: (A) said first and second polypeptide chains are covalently bonded to one another, said first and third polypeptide chains are covalently bonded to one another, and said third and fourth polypeptide chains are covalently bonded to one another; (B) said first and third polypeptide chains of said diabody each comprise, in the N-terminal to C-terminal direction, a Light Chain Variable Domain of an antibody that is immunospecific for PD-1 or LAG-3, a Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3 or PD-1, a Heterodimer-Promoting Domain and a CH2—CH3 Domain, wherein said Light Chain Variable Domains and said Heavy Chain Variable Domains are incapable of associating to form an Epitope-Binding Site capable of binding an epitope of PD-1 or an epitope of LAG-3; and (C) said second and said fourth polypeptide chains of said diabody each comprise, in the N-terminal to C-terminal direction, a Light Chain Variable Domain of an antibody that is immunospecific for PD-1 or LAG-3, a Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3 or PD-1, and a Heterodimer-Promoting Domain, wherein said Light Chain Variable Domains and said Heavy Chain Variable Domains are incapable of associating to form an Epitope-Binding Site capable of binding an epitope of PD-1 or an epitope of LAG-3; and wherein: (I) (1) said Light Chain Variable Domain of said first polypeptide chain and said Heavy Chain Variable Domain of said second polypeptide chain associate to form a first Epitope-Binding Site and said Heavy Chain Variable Domain of said first polypeptide chain and said Light Chain Variable Domain of said second polypeptide chain associate to form a second Epitope-Binding Site; and (2) said Light Chain Variable Domain of said third polypeptide chain and said Heavy Chain Variable Domain of said fourth polypeptide chain associate to form a third Epitope-Binding Site and said Heavy Chain Variable Domain of said third polypeptide chain and said Light Chain Variable Domain of said fourth polypeptide chain associate to form a fourth Epitope-Binding Site; wherein two of said formed Epitope-Binding Sites are capable of immunospecifically binding to an epitope of PD-1 and two of said formed Epitope-Binding Sites are capable of immunospecifically binding to an epitope of LAG-3; II. said Heterodimer-Promoting Domains of said first and second polypeptide chains differ, wherein one of said first and second polypeptide chains comprises an amino acid sequence of SEQ ID NO:16 and the other of said first and second polypeptide chains comprises an amino acid sequence of SEQ ID NO:17; and III. said CH2—CH3 Domains of said first and third polypeptide chains associate to form an Fc Domain. ### Claim 2 (depends on 1) The method of claim 1, wherein said CH2—CH3 Domains of said first and third polypeptide chains each have the amino acid sequence of SEQ ID NO:24. ### Claim 3 (depends on 1) The method of claim 1, wherein: (a) said Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3 comprises the amino acid sequence of SEQ ID NO:11, and wherein said Light Chain Variable Domain of an antibody that is immunospecific for LAG-3 comprises the amino acid sequence of SEQ ID NO:12. and/or (b) said Heavy Chain Variable Domain of an antibody that is immunospecific for PD-1 has the amino acid sequence of SEQ ID NO:2, and wherein said Light Chain Variable Domain of an antibody that is immunospecific for PD-1 has the amino acid sequence of SEQ ID NO:3. ### Claim 4 (depends on 2) The method of claim 2, wherein: (a) said Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3 comprises the amino acid sequence of SEQ ID NO:11, and wherein said Light Chain Variable Domain of an antibody that is immunospecific for LAG-3 comprises the amino acid sequence of SEQ ID NO:12; and/or (b) said Heavy Chain Variable Domain of an antibody that is immunospecific for PD-1 comprises the amino acid sequence of SEQ ID NO:2, and wherein said Light Chain Variable Domain of an antibody that is immunospecific for PD-1 comprises the amino acid sequence of SEQ ID NO:3. ### Claim 5 (canceled) ### Claim 6 (canceled) ### Claim 7 (canceled) ### Claim 8 (canceled) ### Claim 9 (canceled) ### Claim 10 (canceled) ### Claim 11 (canceled) ### Claim 12 (depends on 1) The method of claim 1, wherein: (A) said first and third polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for PD-1 and a Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3; and (B) said second and fourth polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for LAG-3 and a Heavy Chain Variable Domain of an antibody that is immunospecific for PD-1. ### Claim 13 (depends on 2) The method of claim 2, wherein: (A) said first and third polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for PD-1 and a Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3; and (B) said second and fourth polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for LAG-3 and a Heavy Chain Variable Domain of an antibody that is immunospecific for PD-1. ### Claim 14 (depends on 1) The method diabody of claim 1, wherein: (A) said first and third polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for LAG-3 and a Heavy Chain Variable Domain of an antibody that is immunospecific for PD-1; and (B) said second and fourth polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for PD-1 and a Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3. ### Claim 15 (depends on 2) The method diabody of claim 2, wherein: (A) said first and third polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for LAG-3 and a Heavy Chain Variable Domain of an antibody that is immunospecific for PD-1; and (B) said second and fourth polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for PD-1 and a Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3. ### Claim 16 (depends on 1) The method of claim 1, wherein said Heavy Chain Variable Domain is separated from said Heterodimer-Promoting Domain in each of said first, second, third and fourth polypeptide chains by a linker comprising the amino acid sequence of SEQ ID NO:18. ### Claim 17 (depends on 2) The method of claim 2, wherein said Heavy Chain Variable Domain is separated from said Heterodimer-Promoting Domain in each of said first, second, third and fourth polypeptide chains by a linker comprising the amino acid sequence of SEQ ID NO:18. ### Claim 18 (depends on 12) The method of claim 12, wherein said Heavy Chain Variable Domain is separated from said Heterodimer-Promoting Domain in each of said first, second, third and fourth polypeptide chains by a linker comprising the amino acid sequence of SEQ ID NO:18. ### Claim 19 (depends on 13) The method of claim 13, wherein said Heavy Chain Variable Domain is separated from said Heterodimer-Promoting Domain in each of said first, second, third and fourth polypeptide chains by a linker comprising the amino acid sequence of SEQ ID NO:18. ### Claim 20 (depends on 14) The method of claim 14, wherein said Heavy Chain Variable Domain is separated from said Heterodimer-Promoting Domain in each of said first, second, third and fourth polypeptide chains by a linker comprising the amino acid sequence of SEQ ID NO:18. ### Claim 21 (depends on 15) The method of claim 15, wherein said Heavy Chain Variable Domain is separated from said Heterodimer-Promoting Domain in each of said first, second, third and fourth polypeptide chains by a linker comprising the amino acid sequence of SEQ ID NO:18. ### Claim 22 A bi-specific Fc diabody capable of immunospecific binding to an epitope of PD-1 and to an epitope of LAG-3, wherein said diabody comprises first, second, third and fourth polypeptide chains, each having an amino terminus and a carboxy terminus, and wherein: (A) said first and second polypeptide chains are covalently bonded to one another, said first and third polypeptide chains are covalently bonded to one another, and said third and fourth polypeptide chains are covalently bonded to one another; (B) said first and third polypeptide chains of said diabody each comprise, in the N-terminal to C-terminal direction, a Light Chain Variable Domain of an antibody that is immunospecific for PD-1 or LAG-3, a Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3 or PD-1, a Heterodimer-Promoting Domain and a CH2—CH3 Domain, wherein said Light Chain Variable Domains and said Heavy Chain Variable Domains are incapable of associating to form an Epitope-Binding Site capable of binding an epitope of PD-1 or an epitope of LAG-3; and (C) said second and said fourth polypeptide chains of said diabody each comprise, in the N-terminal to C-terminal direction, a Light Chain Variable Domain of an antibody that is immunospecific for PD-1 or LAG-3, a Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3 or PD-1, and a Heterodimer-Promoting Domain, wherein said Light Chain Variable Domains and said Heavy Chain Variable Domains are incapable of associating to form an Epitope-Binding Site capable of binding an epitope of PD-1 or an epitope of LAG-3; and wherein: (I) (1) said Light Chain Variable Domain of said first polypeptide chain and said Heavy Chain Variable Domain of said second polypeptide chain associate to form a first Epitope-Binding Site and said Heavy Chain Variable Domain of said first polypeptide chain and said Light Chain Variable Domain of said second polypeptide chain associate to form a second Epitope-Binding Site; and (2) said Light Chain Variable Domain of said third polypeptide chain and said Heavy Chain Variable Domain of said fourth polypeptide chain associate to form a third Epitope-Binding Site and said Heavy Chain Variable Domain of said third polypeptide chain and said Light Chain Variable Domain of said fourth polypeptide chain associate to form a fourth Epitope-Binding Site; wherein two of said formed Epitope-Binding Sites are capable of immunospecifically binding to an epitope of PD-1 and two of said formed Epitope-Binding Sites are capable of immunospecifically binding to an epitope of LAG-3; II. said Heterodimer-Promoting Domains of said first and second polypeptide chains differ, wherein one of said first and second polypeptide chains comprises an amino acid sequence of SEQ ID NO: 14 and the other of said first and second polypeptide chains comprises an amino acid sequence of SEQ ID NO: 15; and III. said CH2—CH3 Domains of said first and third polypeptide chains associate to form an Fc Domain. ### Claim 23 (depends on 22) The bi-specific Fc diabody of claim 22, wherein said Heavy Chain Variable Domain is separated from said Heterodimer-Promoting Domain in each of said first, second, third and fourth polypeptide chains by a linker comprising the amino acid sequence of SEQ ID NO:18. ### Claim 24 (depends on 22) A pharmaceutical composition that comprises the bi-specific Fc diabody of claim 22, and a pharmaceutically acceptable carrier. ### Claim 25 (depends on 23) A pharmaceutical composition that comprises the bi-specific Fc diabody of claim 23, and a pharmaceutically acceptable carrier.