Patent ID: 8892364

Claim:
A method for producing a stabilized antibody or antigen binding fragment thereof, which comprises a stabilized V H domain and a stabilized V L domain derived from a V H domain and a V L domain of a wild-type candidate antibody, wherein said method comprises: (a) providing a curated reference set of amino acid sequences which belong to immunoglobulin superfamily polypeptides; (b) aligning said reference set of amino acid sequences to generate an aligned set; (c) calculating covariation between two or more amino acid positions in the aligned set to generate a covariation dataset; (d) identifying non-covarying amino acids in said wild-type candidate antibody V H domain or said wild-type candidate antibody V L domain, wherein said non-covarying amino acids fail to satisfy one or more covariations observed in the covariation dataset; (e) substituting one or more of said non-covarying amino acids in the wild-type candidate antibody V H domain or wild-type candidate antibody V L domain with one or more replacement amino acids, wherein each replacement amino acid is an amino acid found at the corresponding amino acid position in the aligned set which satisfies one or more covariations in the covariation dataset, and wherein the replacement amino acid establishes one or more covariations in the candidate antibody; (f) expressing in a host cell the antibody or antigen binding fragment thereof comprising at least one substitution, and (g) testing at least one biophysical property of the antibody or antigen binding fragment thereof comprising at least one substitution to determine whether at least one biophysical property is improved with respect to the wild-typed candidate antibody, thereby making a stabilized antibody and antigen binding fragment thereof with an improved biophysical property.