Material and Methods for Improved Bioengineered Pairing of Antigen-Binding Variable Regions

An antigen-binding molecule, comprising

• a first light chain polypeptide comprising, in amino-terminus to carboxy-terminus order:
• VL1-LD1, wherein VL1 is a first light chain variable domain and LD1 is a first light chain dimerization domain;
• and
• a first heavy chain polypeptide comprising, in amino-terminus to carboxy-terminus order:
• VH1-HD1, wherein VH1 is a first heavy chain variable domain and HD1 is a first heavy chain dimerization domain,
• wherein
• a) i) LD1 comprises a beta-2 microglobulin (B2M) domain and HD1 comprises a HLA-E A3 (EA3) domain, or LD1 comprises an EA3 domain and HD1 comprises a B2M domain, wherein the B2M domain and the EA3 domain bind to each other to form a dimer;
• or
• ii) LD1 comprises a first intracellular adhesion molecule-1 (ICAM-1) D1 domain and HD1 comprises a second ICAM-1 D1 domain, wherein the first and second ICAM-1 D1 domains bind to each other to form a dimer;
• b) i) at least one of the B2M domain or EA3 domain differs from the wild-type B2M domain (SEQ ID NO:2) or wild-type EA3 domain (SEQ ID NO:33), respectively, and
• ii) the first ICAM-1 D1 domain differs from the second ICAM-1 D1 domain;
• and
• c) VL1 and VH1 form a first paratope.