METHODS FOR MAKING CONJUGATES FROM DISULFIDE-CONTAINING PROTEINS

摘要

The invention provides methods to prepare protein conjugates from proteins having at least four accessible cysteine residues. In one embodiment, an antibody with four reducible disulfide linkages is reduced to provide four pairs of free cysteines. Each pair of free cysteines is reacted with a 1,3-dihaloacetone or similar reactant, linking the sulfur atoms of each pair together though a 3-carbon tether with a reactive ketone. A pair of these reactive ketones are linked together with and used to attach a single payload molecule, thus conjugating the antibody to two payload groups. This method gives a stabilized antibody conjugate with high selectivity for a ratio of two payloads per antibody.

主权项

1. A polypeptide-payload conjugate comprising: wherein the circle represents a polypeptide or polypeptide complex, which may comprise modifications other than the one shown; each S is a sulfur atom of a cysteine residue of the polypeptide or polypeptide complex; T1 and T2 each represent a tether that connects two sulfur atoms through 1-5 carbon atoms, wherein T1 also attaches to L1 and T2 also attaches to L2; L1 and L2 each represent a linking group connecting T1 or T2, respectively, to A; L3 is a linking group that connects A to R; A represents a group connecting L1, L2 and L3; and R represents a payload or a reactive functional group for attaching a payload.