Antibodies comprising chimeric constant domains
Antibodies, antigen-binding proteins and Fc-fusion proteins that comprise recombinant polypeptides containing a chimeric heavy chain constant region sequence are provided that bind to certain Fc receptors however have reduced effector functions. Methods of making constructs for expression of such chimeric Fc-containing antibodies, antigen-binding proteins and Fc-fusion proteins in cell systems, and methods of producing and isolating the chimeric Fc-containing proteins are provided.
1. A nucleic acid molecule fully encoding a recombinant heavy chain constant (CH) region comprising, from N-terminus to C-terminus, a CH1 domain, a chimeric hinge, a CH2 domain, and a CH3 domain wherein:
(a) the CH1 domain comprises the amino acid sequence DKKV or DKRV from positions 212 to 215 (EU numbering),
(b) the chimeric hinge comprises a human IgG1 or a human IgG4 upper hinge amino acid sequence from positions 216 to 227 (EU numbering) and a human IgG2 lower hinge amino acid sequence PCPAPPVA (SEQ ID NO: 3) from positions 228 to 236 (EU numbering),
(c) the CH2 domain comprises a human IgG4 CH2 domain amino acid sequence from positions 237 to 340 (EU numbering), and
(d) the CH3 domain comprises a human IgG1 or a human IgG4 CH3 domain sequence from positions 341 to 447 (EU numbering).
2. The nucleic acid molecule of claim 1 , wherein the recombinant CH region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, and SEQ ID NO: 31, SEQ ID NO: 37, and SEQ ID NO: 38.
3. The nucleic acid molecule of claim 1 having a nucleotide sequence with greater than 99% sequence identity to the nucleotide sequence of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 36.
4. The nucleic acid molecule of claim 2 comprising a nucleotide sequence selected the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 32, and SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 36.
5. A vector comprising a nucleic acid molecule of claim 2 .
6. The vector of claim 5 , wherein the nucleic acid molecule is operatively linked to an expression control sequence suitable for expression in a host cell.
7. The vector of claim 6 , wherein the expression control sequence comprises a promoter selected from the group consisting of SV40, CMV, CMV-IE, CMV-MIE, UbC, RSV, SL3-3, MMTV, Ubi and HIV LTR.
8. The vector of claim 6 , wherein the expression control sequence comprises an expression cassette selected from the group consisting of TetR-ER LBD T2 fusion gene driven by the CMV-MIE promoter, blasticidin resistance gene driven by the SV40 promoter, and Arc-ER LBD T2 fusion gene driven by the CMV-MIE promoter.
9. The vector of claim 7 , wherein the promoter is a CMV-MIE/TetO or CMV-MIE/Arc hybrid promoter.
10. The vector of claim 7 , comprising one or more selectable marker genes selected from the group consisting of bla, bls, BSD, bsr, Sh ble, hpt, tetR, tetM, npt, kanR and pac.
11. A cell comprising a nucleic acid of claim 1 .
12. A cell comprising the vector of claim 5 .
13. The cell of claim 11 , wherein the nucleic acid is integrated into the genome of the cell.
14. The cell of claim 13 comprising a nucleic acid encoding a protein expression enhancer.
15. The cell of claim 14 comprising a nucleic acid encoding an XBP polypeptide.
16. The cell of claim 11 , wherein the cell is a eukaryotic cell.
17. The cell of claim 11 , wherein the cell is an animal cell.
18. The cell of claim 11 , wherein the cell is a mammalian cell.
19. The cell of claim 11 , wherein the cell is a CHO cell.
20. The cell of claim 11 , wherein the cell is a CHO-K1 cell.
21. The nucleic acid molecule of claim 1 , wherein the CH1 domain comprises the amino acid sequence DKKV (SEQ ID NO: 4), and the chimeric hinge comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 8).
22. The nucleic acid molecule of claim 1 , wherein the CH1 domain comprises the amino acid sequence DKRV (SEQ ID NO: 5), and the chimeric hinge comprises the amino acid sequence ESKYGPPCPPCPAPPVA (SEQ ID NO: 9).
23. The nucleic acid molecule of claim 1 , wherein the CH2 domain comprises the amino acid sequence SEQ ID NO: 10.
24. The nucleic acid molecule of claim 1 , wherein the CH3 domain comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 41, and SEQ ID NO: 42.