RECOMBINANT pMHC CLASS II MOLECULES

A method of preparing a heterodimer comprising a first polypeptide, a second polypeptide, and an autoimmune disease-relevant antigen, wherein

• the first polypeptide and the second polypeptide meet at an interface, wherein the interface of the first polypeptide comprises an engineered protuberance which is positionable in an engineered cavity in the interface of the second polypeptide;
• wherein the engineered protuberance comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:
• 54,
• wherein the engineered cavity comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:
• 52;
• wherein
• (i) the first polypeptide comprises an MHC class II α1 domain, an MHC class II α2 domain, or a combination thereof;
• and the second polypeptide comprises an MHC class II β1 domain, an MHC class II β2 domain, or a combination thereof;
• or
• (ii) the first polypeptide comprises an MHC class II β1 domain, an MHC class II β2 domain, or a combination thereof;
• and the second polypeptide comprises an MHC class II α1 domain, an MHC class II α2 domain, or a combination thereof;
• wherein the autoimmune disease-relevant antigen is a polypeptide which is at least 11 amino acids in length connected to the MHC class II α1 domain or the MHC class II β1 domain by a flexible linker;
• and
• wherein neither the first polypeptide nor the second polypeptide comprises a leucine zipper domain, or an extraneous biotinylation site or purification tag;
• comprising the steps of:
• (i) culturing a host cell comprising a nucleic acid encoding the first polypeptide and second polypeptide including the interfaces thereof, wherein the nucleic acid encoding the interface of the first polypeptide has been altered from nucleic acid encoding an original interface of the first polypeptide to encode the protuberance, the nucleic acid encoding the interface of the second polypeptide has been altered from nucleic acid encoding an original interface of the second polypeptide to encode the cavity, or both, and wherein the culturing is such that the first polypeptide and second polypeptide are expressed;
• and
• (ii) recovering and purifying the heterodimer from the host cell culture.

A method of preparing a heterodimer comprising a first polypeptide, a second polypeptide, and an autoimmune disease-relevant antigen, wherein

• the first polypeptide and the second polypeptide meet at an interface, wherein the interface of the first polypeptide comprises an engineered protuberance which is positionable in an engineered cavity in the interface of the second polypeptide;
• wherein the engineered protuberance comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:
• 54,
• wherein the engineered cavity comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:
• 52;
• wherein
• (i) the first polypeptide comprises an MHC class II α1 domain, an MHC class II α2 domain, or a combination thereof;
• and the second polypeptide comprises an MHC class II β1 domain, an MHC class II β2 domain, or a combination thereof;
• or
• (ii) the first polypeptide comprises an MHC class II β1 domain, an MHC class II β2 domain, or a combination thereof;
• and the second polypeptide comprises an MHC class II α1 domain, an MHC class II α2 domain, or a combination thereof;
• wherein the autoimmune disease-relevant antigen is a polypeptide which is at least 11 amino acids in length connected to the MHC class II α1 domain or the MHC class II β1 domain by a flexible linker;
• and
• wherein neither the first polypeptide nor the second polypeptide comprises a leucine zipper domain, or an extraneous biotinylation site or purification tag;
• comprising the steps of:
• (i) culturing a first host cell comprising a nucleic acid encoding the first polypeptide;
• (ii) culturing a second host cell comprising a nucleic acid encoding the second polypeptide;
• (iii) recovering the first and second polypeptides from the host cell cultures;
• and
• (iv) forming the heterodimer by incubating the first and second polypeptides together;
• wherein the nucleic acid encoding the interface of the first polypeptide has been altered from nucleic acid encoding an original interface of the first polypeptide to encode the protuberance, the nucleic acid encoding the interface of the second polypeptide has been altered from nucleic acid encoding an original interface of the second polypeptide to encode the cavity, or both.