Active protease-resistant antibody FC mutants
The present invention relates to modified Fc-containing molecules including modified antibodies characterized by increased resistance to host and pathogen-derived proteases, ability to interact with FcγR receptors except with FcγRI, and lack of induction of IL-10 secretion by macrophages, and methods of using and making them.
1. An isolated modified Fc-containing molecule or a fragment thereof comprising a wild type human IgG1 Fc region of SEQ ID NO: 1 comprising a hinge, a CH2 domain and a CH3 domain, wherein
a) the sequence of E233-L234-L235-G236 in the hinge is replaced with P233-V234-A235 with G236 deleted; and
b) the CH2 domain comprises at least one substitution selected from S239D/1332E, K326A/E333A, H268F/S324T/1332E, F243L/R292P/Y300L, S239D/H268F/S324T/1332E, S267E/H268F/S324T/1332E, K326A/1332E/E333A, S239D/K326A/E333A, S267E/I332E and G237X/S239D/1332E where X is A, D, P, Q or S; wherein amino acid residues are numbered according to EU numbering.
2. The isolated modified Fc-domain containing molecule of claim 1 , wherein the molecule is resistant to proteolytic degradation by a protease that cleaves the wild type human IgG1 molecule between or at residues 222-237 (EU numbering).
3. The isolated modified Fc-domain containing molecule of claim 1 , wherein the molecule is capable of promoting antibody-dependent cellular phagocytosis (ADCP) measured in the presence of CD14 positive and/or CD11b positive human monocyte-derived macrophages, is capable of promoting antibody-dependent cell-mediated cytotoxicity (ADCC) measured in the presence of blood mononuclear cells, and/or is capable of promoting complement-dependent cytotoxicity (CDC) measured by cell lysis in the presence of complement.
4. The isolated modified Fc-domain containing molecule of claim 3 , wherein the molecule induces IL-10 secretion by human monocyte-derived macrophages by about no more than three times more when compared to the IL-10 secretion by the human monocyte-derived macrophages in the absence of the isolated modified Fc-containing molecule.
5. The isolated modified Fc-domain containing molecule of claim 4 , wherein the molecule has an IC 50 fold change ratio value of 0.04 or less measured in a competition assay with biotinylated human IgG1 to 0.2 μg/ml soluble human FcγRI, wherein the IC 50 fold change ratio is a ratio of an IC 50 value for a wild type human IgG1 to an IC 50 value for the isolated modified Fc-domain containing protein of claim 1 .
6. The isolated modified Fc-domain containing molecule of claim 5 , wherein the molecule at a concentration of 0.5 μg/ml induces interferon-gamma (IFNγ) secretion by pheripheral blood mononuclear cells (PBMCs) by at least three times more when compared to the IFNγ secretion by the PBMCs in the absence of the isolated modified Fc-domain containing molecule.
7. The isolated modified Fc-domain containing molecule of claim 1 , wherein the protease that cleaves the wild type IgG1 molecule between or at residues 222-237 is matrix metalloprotease (MMP) 2 (MMP-2), MMP-3, MMP-7, MMP-9, MMP-12, MMP-13, immunoglobulin degrading enzyme from Strep. Pyrongenes (IdeS), glutamyl endopeptidase I from Staph. aureus (GluV8), human neutrophil elastase (HNE), plasmin, cathepsin G, or pepsin.
8. The isolated modified Fc-domain containing molecule of claim 7 , wherein the protease that cleaves the wild type IgG1 molecule between or at residues 222-237 is MMP-3, MMP-7, MMP-12, MMP-13, IdeS, or GluV8.
9. The isolated modified Fc-domain containing molecule of claim 1 , wherein the Fc-containing molecule comprises the polypeptide sequence of SEQ ID NOs: 8, 10-15 or 18-20.
10. The isolated modified Fc-domain containing molecule of claim 1 , wherein the Fc-containing molecule is an antibody or an Fc fusion protein.
11. An isolated antibody or fragment thereof comprising a modified Fc-containing molecule comprising a wild type human IgG1 Fc region of SEQ ID NO: 1 comprising a hinge, a CH2 domain and a CH3 domain, wherein
a) the sequence of E233-L234-L235-G236 in the hinge is replaced with P233-V234-A235 with G236 deleted; and
b) the CH2 domain comprises at least one substitution selected from S239D/1332E, K326A/E333A, H268F/S324T/1332E, F243L/R292P/Y300L, S239D/H268F/S324T/1332E, S267E/H268F/S324T/1332E, K326A/1332E/E333A, S239D/K326A/E333A, S267E/I332E and G237X/S239D/1332E where X is A, D, P, Q or S; wherein amino acid residues are numbered according to EU numbering.
12. The isolated antibody of claim 11 , wherein the antibody is resistant to proteolytic degradation by a protease that cleaves the wild type human IgG1 molecule between or at residues 222-237.
13. The isolated antibody of claim 11 , wherein the antibody is capable of promoting antibody-dependent cellular phagocytosis (ADCP) measured in the presence of CD14 positive and/or CD11b positive human monocyte-derived macrophages, is capable of promoting antibody-dependent cell-mediated cytotoxicity (ADCC) measured in the presence of blood mononuclear cells, or is capable of promoting complement-dependent cytotoxicity (CDC) measured by cell lysis in the presence of complement.
14. The isolated antibody of claim 11 , wherein the antibody is capable of promoting antibody-dependent cellular phagocytosis (ADCP) measured in the presence of CD14 positive and/or CD11b positive human monocyte-derived macrophages, is capable of promoting antibody-dependent cell-mediated cytotoxicity (ADCC) measured in the presence of blood mononuclear cells, and is capable of promoting complement-dependent cytotoxicity (CDC) measured by cell lysis in the presence of complement.
15. The isolated antibody of claim 13 or 14 , wherein the antibody induces IL-10 secretion by human monocyte-derived macrophages by about no more than three times more when compared to the IL-10 secretion by the human monocyte-derived macrophages in the absence of the isolated antibody.
16. The isolated antibody of claim 14 , wherein the antibody has an IC 50 fold change ratio value of 0.04 or less measured in a competition assay with biotinylated human IgG1 to 0.2 μg/ml soluble human FcγRI, wherein the IC 50 fold change ratio is a ratio of an IC 50 value for a wild type human IgG1 to an IC 50 value for the isolated antibody of claim 11 .
17. The isolated antibody of claim 16 , wherein the antibody at a concentration of 0.5 μg/ml induces interferon-gamma (IFNγ) secretion by pheripheral blood mononuclear cells (PBMCs) by at least three times more when compared to the IFNγ secretion by the PBMCs in the absence of the isolated antibody.
18. The isolated antibody of claim 12 , wherein the protease that cleaves the wild type IgG1 molecule between or at residues 222-237 is matrix metalloprotease (MMP) 2 (MMP-2), MMP-3, MMP-7, MMP-9, MMP-12, MMP-13, immunoglobulin degrading enzyme from Strep. Pyrongenes (IdeS), or glutamyl endopeptidase I from Staph. aureus (GluV8), human neutrophil elastase (HNE), plasmin, cathepsin G, or pepsin.
19. The isolated antibody of claim 18 , wherein the protease that cleaves the wild type IgG1 molecule between or at residues 222-237 is MMP-3, MMP-7, MMP-12, MMP-13, IdeS, or GluV8.
20. The isolated antibody of claim 11 , wherein the antibody comprises the polypeptide sequence of SEQ ID NOs: 8, 10-15 or 18-20.
21. The isolated antibody of claim 11 , wherein the antibody binds to an antigen on a tumor cell, tumor matrix, or tumor vasculature.
22. The antibody of claim 21 , wherein the antibody binds CD20, ErbB1, ErbB2, ErbB3, VEGF, RON, or tissue factor.
23. A pharmaceutical composition comprising the isolated modified Fc-domain containing molecule of claim 1 or the isolated antibody of claim 11 .
24. A method for treating a disease characterized by unwanted proliferation or migration of cells, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 23 to a patient in need thereof for a time sufficient to treat the disease characterized by unwanted proliferation or migration of cells.
25. The method of claim 24 , wherein the isolated modified Fc-domain containing molecule or the isolated antibody comprises at least one substitution selected from 1322E, S239D/1332E, H268F/S324T/1332E, S239D/H268F/S324T/1332E, G237X/S239D/1332E where X is A or S, K326A/1332E/E333A, S239D/K326A/E333A, S267E/H268F/S324T/1332E and S267E/I332E.
26. A method for treating an infection, comprising administering a therapeutically effective amount of a pharmaceutical composition of claim 23 to a patient in need thereof for a time sufficient to treat the infection.
27. The method of claim 24 , wherein the isolated modified Fc-domain containing molecule or the isolated antibody comprises at least one substitution selected from S267E/H268F/S324T/1332E, K326A/E333A, K326A/1332E/E333A, S239D/K326A/E333A and S267E/I332E.
28. The isolated antibody of claim 11 , wherein the CH2 domain comprises the substitution S239D/K326A/E333A having the polypeptide sequence of SEQ ID NO: 19.
29. The isolated antibody of claim 28 , wherein the antibody binds CD20, ErbB1, ErbB2, ErbB3, VEGF, RON, or tissue factor.
30. The isolated antibody of claim 29 , wherein the antibody binds CD20 or tissue factor.
31. The isolated antibody of claim 30 , wherein the antibody binds tissue factor.