Antigen-Binding Polypeptide Constructs Comprising Kappa and Lambda Light Chains and Uses Thereof

An antigen-binding polypeptide construct comprising a first heterodimer and a second heterodimer,

• the first heterodimer (H1L1) comprising a first immunoglobulin heavy chain polypeptide sequence (H1), and an immunoglobulin lambda light chain polypeptide sequence (L1) that form a first Fab region that specifically binds to a first antigen;
• and the second heterodimer (H2L2) comprising a second immunoglobulin heavy chain polypeptide sequence (H2), and an immunoglobulin kappa light chain polypeptide sequence (L2) that form a second Fab region that specifically binds to a second antigen,
• wherein:
• H1 is distinct from H2, and H1 and H2 each comprise a heavy chain variable domain (VH domain) and a heavy chain constant domain 1 (CH1 domain);
• L1 comprises a lambda light chain variable (VL-lambda) domain and a lambda light chain constant (CL-lambda) domain, and L2 comprises a kappa light chain variable (VL-kappa) domain and a kappa light chain constant (CL-kappa) domain;
• and
• one or more of H1, H2, L1, and L2 comprises amino acid modifications as compared to corresponding wild type H1, H2, L1, and L2 polypeptide sequences, that promote preferential pairing of H1 with L1 as compared to L2, and/or that promote preferential pairing of H2 with L2 as compared to L1, wherein the amino acid modifications do not remove a naturally occurring cysteine residue.