Covalently Bonded Diabodies Having Immunoreactivity with PD-1 and LAG-3, and Methods of Use Thereof

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.