Methods of crosslinking IgG
This disclosure relates to dimeric immunotherapeutics that comprise an IgG that is crosslinked with a disulfide bond. The IgG may include two heavy chains including a cysteine mutation that forms the disulfide bond. The dimeric immunotherapeutics are formed by increasing the concentration of the IgG such that spontaneous dimerization via the formation of disulfide bonds between the IgG molecules occurs over time.
1 . A method to manufacture a dimeric immunotherapeutic, comprising:
providing a first immunotherapeutic, wherein (a) the first immunotherapeutic comprises a first IgG with two heavy chains that have a first amino acid sequence; (b) the first amino acid sequence includes a mutation of a native amino acid to a cysteine; and (c) the two heavy chains of the first immunotherapeutic are covalently crosslinked with a first one or more disulfide bond(s);
mixing the first immunotherapeutic with a phosphate-buffered saline; and
incubating the first immunotherapeutic and the phosphate-buffered saline under oxidizing conditions to form a disulfide bond between (a) the cysteine of the heavy chain from the first IgG of and (b) the cysteine of the heavy chain from another first IgG to result in the dimeric immunotherapeutic,
wherein:
the first IgG has a first VL CDR1 amino acid sequence, a first VL CDR2 amino acid sequence, a first VL CDR3 amino acid sequence, a first VH CDR1 amino acid sequence, a first VH CDR2 amino acid sequence, and a first VH CDR3 amino acid sequence;
the first IgG is a human IgG1;
the native amino acid is S444; and
the mutation is S444C,
wherein amino acid positions in the first amino acid sequence are defined by the EU numbering system.
2 . A method to manufacture a dimeric immunotherapeutic, comprising:
providing a first immunotherapeutic, wherein (a) the first immunotherapeutic comprises a first IgG with two heavy chains that have a first amino acid sequence; (b) the first amino acid sequence includes a mutation of a native amino acid to a cysteine; and (c) the two heavy chains of the first immunotherapeutic are covalently crosslinked with a first one or more disulfide bond(s);
mixing the first immunotherapeutic with a phosphate-buffered saline; and
incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline under oxidizing conditions to form a disulfide bond between (a) the cysteine of the heavy chain from the first IgG and (b) the cysteine of the heavy chain from another first IgG.
3 . The method of claim 2 , wherein:
the first IgG has a first VL CDR1 amino acid sequence, a first VL CDR2 amino acid sequence, a first VL CDR3 amino acid sequence, a first VH CDR1 amino acid sequence, a first VH CDR2 amino acid sequence, and a first VH CDR3 amino acid sequence;
the first IgG is a human IgG1;
the native amino acid is S444; and
the mutation is S444C,
wherein amino acid positions in the first amino acid sequence are defined by the EU numbering system.
4 . The method of claim 3 , wherein:
the first VL CDR1 amino acid sequence is identical to SEQ ID NO: 8;
the first VL CDR2 amino acid sequence is identical to SEQ ID NO: 9;
the first VL CDR3 amino acid sequence is identical to SEQ ID NO: 10;
the first VH CDR1 amino acid sequence is identical to SEQ ID NO: 5;
the first VH CDR2 amino acid sequence is identical to SEQ ID NO: 6; and
the first VH CDR3 amino acid sequence is identical to SEQ ID NO: 7.
5 . The method of claim 4 , wherein:
the first amino acid sequence has at least 90 percent sequence identity with SEQ ID NO: 3; and
the first IgG comprises two light chains that have a first light chain amino acid sequence that has at least 90 percent sequence identity with SEQ ID NO: 4.
6 . The method of claim 4 , wherein:
the first amino acid sequence has at least 95 percent sequence identity with SEQ ID NO: 3; and
the first IgG comprises two light chains that have a first light chain amino acid sequence that comprises SEQ ID NO: 4.
7 . The method of claim 2 , wherein:
the first IgG comprises two light chains that have a first light chain amino acid sequence.
8 . The method of claim 2 , wherein incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline comprises incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline at a pH of approximately 7.4.
9 . The method of claim 2 , wherein:
the first IgG is a human IgG4; and
the first IgG has a lysine at amino acid position 409 and a phenylalanine at amino acid position 405.
10 . The method of claim 2 , wherein:
the first IgG is a human IgG4; and
the first IgG has an arginine at amino acid position 409 and a leucine at amino acid position 405.
11 . The method of claim 2 , wherein incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline comprises incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline for at least 3 days after mixing the solution.
12 . The method of claim 2 , wherein incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline comprises incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline for at least 5 days after mixing the solution.
13 . The method of claim 2 , wherein incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline comprises incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline for at least 7 days after mixing the solution.
14 . The method of claim 2 , wherein incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline comprises incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline for at least 9 days after mixing the solution.
15 . The method of claim 2 , wherein incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline comprises incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline for at least 11 days after mixing the solution.
16 . The method of claim 2 , wherein incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline comprises incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline for at least 14 days after mixing the solution.
17 . The method of claim 2 , wherein incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline comprises incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline while constantly mixing the solution by rotating the solution at 150 rotations per minute.
18 . The method of claim 2 , wherein incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline comprises incubating a solution comprising the first immunotherapeutic and the phosphate-buffered saline at 37° C. for up to 14 days.
19 . The method of claim 2 , wherein:
the first IgG has a lysine at amino acid position 409 and a phenylalanine at amino acid position 405.
20 . The method of claim 2 , wherein:
the first IgG has an arginine at amino acid position 409 and a leucine at amino acid position 405.