IL-2 Conjugates and Methods of Use Thereof

A method of making an IL-2 conjugate, comprising:

• reacting an IL-2 polypeptide, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, comprising an unnatural amino acid of formula
• wherein the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:
• 1, Position X−1 indicates the point of attachment to the preceding amino acid residue, Position X+1 indicates the point of attachment to the following amino acid residue, and Position X indicates the position of the amino acid for which the unnatural amino acid substitutes, wherein Position X is K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:
• 1,
• with an mPEG-DBCO of formula
• wherein n is an integer such that the mPEG-DBCO comprises a PEG having a molecular weight of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, or 50 kDa, to form the IL-2 conjugate.

A method of making an IL-2 conjugate, comprising:

• reacting an IL-2 polypeptide, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, comprising an unnatural amino acid of formula
• wherein the IL-2 polypeptide comprises an amino acid sequence of SEQ ID NO:
• 3 wherein Position X is P64 in reference to the amino acid positions within SEQ ID NO:
• 3, Position X−1 indicates the point of attachment to the preceding amino acid residue, Position X+1 indicates the point of attachment to the following amino acid residue,
• with an mPEG-DBCO of formula
• wherein n is an integer such that the mPEG-DBCO comprises a PEG having a molecular weight of about 30 kDa,
• to form the IL-2 conjugate, wherein the IL-2 conjugate is of Formula (XVI) or (XVII):
• wherein:
• m is 2;
• and
• the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO:
• 3 that are not replaced.