Treatment of IgG4-related disease with anti-CD19 antibodies cross-linked to CD32B

Abstract

To provide immunoglobulins that bind FcγRIIb+ B cells and coengage CD19 on the cell's surface and an FcγRIIb on the cell's surface, methods for generation of the immunoglobulins, and methods for using the immunoglobulins for treatment of IgG4-related diseases.SOLUTION: An immunoglobulin comprises an Fc region, where the Fc region is an Fc variant of a parent Fc polypeptide, comprising a specific amino acid substitution.SELECTED DRAWING: Figure 29

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119 to U.S. Provisional Patent Application No. 62 / 347,419, filed June 8, 2016, entitled "METHODS AND COMPOSITIONS FOR INHIBITING CD32B EXPRESSING CELLS", U.S. Provisional Patent Application No. 62 / 399,896, filed September 26, 2016, entitled "METHODS AND COMPOSITIONS FOR INHIBITING CD32B EXPRESSING CELLS IN IGG4-RELATED DISEASES", and U.S. Provisional Patent Application No. 62 / 421,261, filed November 12, 2016, entitled "METHODS AND COMPOSITIONS FOR INHIBITING CD32B EXPRESSING CELLS IN IGG4-RELATED DISEASES". Sequence Listing

[0002] The Sequence Listing submitted with this disclosure is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates to immunoglobulins that bind to FcγRIIb+ B cells and coengage CD19 on the cell surface with FcγRIIb on the cell surface, methods for producing the immunoglobulins, and methods of using the immunoglobulins to treat IgG4-related diseases. [Background technology]

[0004] Antigen recognition by B cells is mediated by the B cell receptor (BCR), a surface-bound immunoglobulin in a complex of the signaling components CD79a (Igα) and CD79b (Igβ). Cross-linking of the BCR upon antigen binding results in phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on CD79a and CD79b, initiating a cascade of intracellular signaling events that recruit downstream molecules to the membrane and stimulate calcium mobilization. This induces a variety of B cell responses (e.g., cell survival, proliferation, antibody production, antigen presentation, differentiation, etc.) that result in humoral immune responses (DeFranco, AL, 1997, Curr. Opin. Immunol. 9, 296-308; Pierce, SK, 2002, Nat. Rev. Immunol. 2, 96-105; Ravetch, JV & Lanier, LL, 2000, Science 290, 84-89). Other components of the BCR coreceptor complex enhance (e.g., CD19, CD21, and CD81) or suppress (e.g., CD22 and CD72) BCR activation signals (Doody, GM et al., 1996, Curr. Opin. Immunol. 8, 378-382; Li, DH et al., 2006, J. Immunol. 176, 5321-5328). Thus, the immune system maintains multiple BCR regulatory mechanisms to ensure that B cell responses are tightly controlled.

[0005] When antibodies are produced against an antigen, circulating levels of immune complexes (e.g., antigen bound to an antibody) increase. These immune complexes are thought to downregulate antigen-induced B cell activation by co-engaging the cognate BCR with the low affinity inhibitory receptor FcγRIIb, the only IgG receptor on B cells (Heyman, B., 2003, Immunol. Lett. 88, 157-161). This negative feedback of antibody production is also driven by the F(ab') 2It is also believed that the interaction of FcγRIIb with the antibody Fc domain is required for immune complexes containing antibody fragments to be non-inhibitory (Chan, PL & Sinclair, NR, 1973, Immunology 24, 289-301). The intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) of FcγRIIb is required to inhibit intracellular signals induced by the BCR (Amigorena, S. et al., 1992, Science 256, 1808-1812; Muta, T., et al., 1994, Nature 368, 70-73). This inhibitory effect occurs through phosphorylation of FcγRIIb ITIMs, which recruits SH2-containing inositol polyphosphate 5-phosphatase (SHIP) to neutralize ITIM-induced intracellular calcium mobilization (Kiener, PA, et al., 1997, J. Biol. Chem. 272, 3838-3844; Ono, M., et al., 1996, Nature 383, 263-266; Ravetch, JV & Lanier, LL, 2000, Science 290, 84-89). In addition, FcγRIIb-mediated SHIP phosphorylation inhibits the downstream Ras-MAPK proliferation pathway (Tridandapani, S. et al., 1998, Immunol. 35, 1135-1146). Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides methods of using immunoglobulins to inhibit cells expressing FcγRIIb. +A cytostatic method includes treating IgG4-related disease (IgG4-RD) in a patient. The method includes administering an immunoglobulin that binds to FcγRIIb and CD19 on the surface of B cells, wherein the immunoglobulin comprises an Fc region, wherein the Fc region is an Fc variant of a parent Fc polypeptide that comprises at least one amino acid substitution selected from the group consisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N, 239D, 267D, 267E, 268E, 268D, 328F, and 328Y, where the numbering may be according to the EU index as in Kabat. In some embodiments, the at least one substitution may be selected from the group consisting of 267E and 328F. In other embodiments, the Fc variant may comprise at least two amino acid substitutions in the Fc region compared to the parent Fc polypeptide, wherein said at least two substitutions are selected from the group consisting of 235D / 267E, 235Y / 267E, 235D / S267D, 235I / 267E, 235I / 267D, 235Y / 267D, 236D / 267E, 236D / 267D, 267E / 328F, 267D / 328F, 268D / 267E, 268D / 267D, 268E / 267E, and 268E / 267D. In other embodiments, the at least two substitutions may be 267E / 328F. In other embodiments, the Fc region that binds FcγRIIb comprises SEQ ID NO:7 and SEQ ID NO:9.

[0007] Also disclosed herein is a method of alleviating at least one symptom associated with IgG4-RD in a subject. The method comprises administering an immunoglobulin that binds to FcγRIIb and CD19 on the surface of B cells, wherein said immunoglobulin comprises an Fc region, wherein said Fc region is an Fc variant of a parent Fc polypeptide comprising at least one amino acid substitution selected from the group consisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N, 239D, 267D, 267E, 268E, 268D, 328F, and 328Y, where the numbering is according to the EU index as in Kabat. In some embodiments, at least one substitution may be selected from the group consisting of 267E and 328F. In other embodiments, the Fc variant may comprise at least two amino acid substitutions in the Fc region compared to the parent Fc polypeptide, wherein said at least two substitutions are selected from the group consisting of 235D / 267E, 235Y / 267E, 235D / S267D, 235I / 267E, 235I / 267D, 235Y / 267D, 236D / 267E, 236D / 267D, 267E / 328F, 267D / 328F, 268D / 267E, 268D / 267D, 268E / 267E, and 268E / 267D. In other embodiments, the at least two substitutions may be 267E / 328F. In other embodiments, the Fc region that binds FcγRIIb comprises SEQ ID NO:7 and SEQ ID NO:9.

[0008] In some embodiments, at least one symptom associated with IgG4-RD may be alleviated within 7 days of administration of the immune globulin. In further embodiments, the at least one symptom associated with IgG4-RD is alleviated within 14 days of administration of the immune globulin. In other embodiments, the at least one symptom is present in an organ selected from lymph nodes, submandibular glands, parotid glands, lacrimal glands, kidneys, heart, pericardium, orbit, nasal cavity, lungs, bile ducts, salivary glands, and pancreas.

[0009] Also disclosed herein is a method of depleting plasmablasts in a subject suffering from IgG4-related disease. The method comprises administering an immunoglobulin that binds to FcγRIIb and CD19 on the surface of B cells, wherein said immunoglobulin comprises an Fc region, wherein said Fc region is an Fc variant of a parent Fc polypeptide comprising at least one amino acid substitution selected from the group consisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N, 239D, 267D, 267E, 268E, 268D, 328F, and 328Y, wherein the numbering is according to the EU index as in Kabat. In some embodiments, at least one substitution may be selected from the group consisting of 267E and 328F. In other embodiments, the Fc variant may comprise at least two amino acid substitutions in the Fc region compared to the parent Fc polypeptide, wherein said at least two substitutions are selected from the group consisting of 235D / 267E, 235Y / 267E, 235D / S267D, 235I / 267E, 235I / 267D, 235Y / 267D, 236D / 267E, 236D / 267D, 267E / 328F, 267D / 328F, 268D / 267E, 268D / 267D, 268E / 267E, and 268E / 267D. In other embodiments, the at least two substitutions may be 267E / 328F. In other embodiments, the Fc region that binds FcγRIIb comprises SEQ ID NO:7 and SEQ ID NO:9.

[0010] In some embodiments, depletion of plasmablasts is observed within 7 days after administration of an immunoglobulin that binds FcγRIIb and CD19 on the B cell surface. In other embodiments, plasmablasts may be depleted by at least 10% compared to the number of plasmablasts before administration of the immunoglobulin. In other embodiments, plasmablasts may be depleted by at least 20% compared to the number of plasmablasts before administration of the immunoglobulin. In other embodiments, plasmablasts may be depleted by at least 30% relative to baseline. In other embodiments, plasmablasts may be depleted by at least 40% compared to the number of plasmablasts before administration of the immunoglobulin. In other embodiments, plasmablasts may be depleted by at least 80% compared to the number of plasmablasts before administration of the immunoglobulin.

[0011] Further disclosed herein is a method of reducing CD4+SLAMF7+ CTL cell numbers in a subject suffering from IgG4-related disease, comprising administering an immunoglobulin that binds FcγRIIb and CD19 on the surface of B cells, wherein said immunoglobulin comprises an Fc region, wherein said Fc region is an Fc variant of a parent Fc polypeptide comprising at least one amino acid substitution selected from the group consisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N, 239D, 267D, 267E, 268E, 268D, 328F, and 328Y, where numbering is according to the EU index as in Kabat. In some embodiments, the at least one substitution may be selected from the group consisting of 267E and 328F. In other embodiments, the Fc variant may comprise at least two amino acid substitutions in the Fc region compared to the parent Fc polypeptide, wherein said at least two substitutions are selected from the group consisting of 235D / 267E, 235Y / 267E, 235D / S267D, 235I / 267E, 235I / 267D, 235Y / 267D, 236D / 267E, 236D / 267D, 267E / 328F, 267D / 328F, 268D / 267E, 268D / 267D, 268E / 267E, and 268E / 267D. In other embodiments, the at least two substitutions may be 267E / 328F. In other embodiments, the Fc region that binds FcγRIIb comprises SEQ ID NO:7 and SEQ ID NO:9.

[0012] In some embodiments, a reduction in CD4+SLAMF7+ CTL cell numbers can be observed within 24 days after administration of an immunoglobulin that binds to Fc RIIb and CD 19 on the B cell surface, in other embodiments, CD4+SLAMF7+ CTL cells are reduced by at least 10% compared to the CD4+SLAMF7+ CTL cell numbers prior to immunoglobulin administration.

[0013] Disclosed herein are methods of treating a disease in a subject. The method comprises administering an immunoglobulin that binds FcγRIIb and CD19 on the surface of a B cell, wherein said immunoglobulin comprises an Fc region, wherein said Fc region is an Fc variant of a parent Fc polypeptide comprising at least one amino acid substitution selected from the group consisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N, 239D, 267D, 267E, 268E, 268D, 328F, and 328Y, wherein the numbering is according to the EU index as in Kabat, and wherein the disease is selected from the group consisting of IgG4-related sialadenitis (chronic sclerosing sialadenitis, Kuttner's tumor, Mikulicz's disease), IgG4-related dacryoadenitis (Mikulicz's disease), IgG4-related eye disease (idiopathic orbital inflammatory disease, orbital pseudotumor), chronic sinusitis, eosinophilic angiocentric fibrosis, I IgG4-related hypophysitis (IgG4-related panhypophysitis, IgG4-related adenohypophysitis, IgG4-related infundibular pituitary inflammation, autoimmune hypophysitis), IgG4-related meningitis, IgG4-related leptomeningeal inflammation (idiopathic hypertrophic meningitis), IgG4-related pancreatitis (type 1 autoimmune pancreatitis, IgG4-related AIP, lymphoplasmacytic sclerosing pancreatitis, chronic pancreatitis associated with widespread irregular stenosis of the main pancreatic duct), IgG4-related lung disease (pulmonary inflammatory pseudotumor), IgG4-related rib disease pericarditis, IgG4-related liver disease, IgG4-related sclerosing cholangitis, IgG4-related cholecystitis, IgG4-related aortitis (inflammatory aortic aneurysm), IgG4-related periaortitis (chronic periaortitis), IgG4-related periarteritis, IgG4-related pericarditis, IgG4-related mediastinitis (fibrosing mediastinitis), IgG4-related retroperitoneal fibrosis (retroperitoneal fibrosis, Albarrán-Ormond syndrome, Ormond disease (retroperitoneal fibrosis fibrosis), perinephric fasciitis, Gerota fasciitis / syndrome, fibrous periureteritis, sclerosing lipogranuloma, sclerosing retroperitoneal granuloma, nonspecific retroperitoneal inflammation, sclerosing retroperitonitis, retroperitoneal vasculitis with perivascular fibrosis), IgG4-related mesenteritis (subtypes are: mesenteric panniculitis, mesenteric lipodystrophy and involutive mesenteritis) (sclerosing mesenteritis, generalized nodular subcutaneous panniculitis, liposclerosing mesenteritis, Weber-Christian disease of the mesenterium, mesenteric lipogranuloma, xanthogranulomatous mesenteritis), IgG4-related mastitis (sclerosing mastitis),IgG4-related kidney disease (IgG4-RKD), IgG4-related tubulointerstitial nephritis (IgG4-TIN), IgG4-related membranous glomerulonephritis (idiopathic tubulointerstitial nephritis), IgG4-related prostatitis, IgG4-related perivasal fibrosis (chronic scrotal pain), IgG4-related pseudopseudotumor, IgG4-related epididymo-orchitis (paratesticular fibrous pseudotumor, inflammatory pseudotumor of the spermatic cord, pseudosarcomatous myofibroblastic proliferation of the spermatic cord, proliferative spermatic cord inflammation, chronic proliferative and IgG4-related thyroid disease (Riedel's thyroiditis), eosinophilic angiocentric fibrosis (involving the orbit and upper respiratory tract), inflammatory pseudotumor, and multifocal fibrosclerosis. In some embodiments, the disease may be selected from the group consisting of autoimmune pancreatitis (lymphoplasmacytic sclerosing pancreatitis), eosinophilic angiocentric fibrosis (affecting the orbit and upper respiratory tract), fibrosing mediastinitis, idiopathic hypertrophic meningitis, idiopathic tubulointerstitial nephritis, inflammatory pseudotumor, Kuttner's tumor, Mikulicz's disease, fibrosclerosis, periaortitis, periarteritis, inflammatory multifocal aortic aneurysm, Ormond's disease (retroperitoneal fibrosis), Riedel's thyroiditis, and sclerosing mesenteritis. In some embodiments, at least one substitution may be selected from the group consisting of 267E and 328F. In other embodiments, the Fc variant may comprise at least two amino acid substitutions in the Fc region compared to the parent Fc polypeptide, wherein said at least two substitutions are selected from the group consisting of 235D / 267E, 235Y / 267E, 235D / S267D, 235I / 267E, 235I / 267D, 235Y / 267D, 236D / 267E, 236D / 267D, 267E / 328F, 267D / 328F, 268D / 267E, 268D / 267D, 268E / 267E, and 268E / 267D. In other embodiments, the at least two substitutions may be 267E / 328F. In other embodiments, the Fc region that binds FcγRIIb comprises SEQ ID NO:7 and SEQ ID NO:9.

[0014] Administration of immunoglobulin to a subject by any of the methods disclosed herein may also include a reduction in the subject's IgG4-RD responder index score (IgG4-RD RI score). In some embodiments, within 2 weeks after administration of the immunoglobulin, the subject's IgG4-RD RI score is reduced from the baseline score by at least 1. In other embodiments, the IgG4-RD RI score is reduced by ≧3 within 2 weeks after administration of the immunoglobulin.

[0015] Administering immunoglobulin to a subject by any of the methods disclosed herein can also include administering about 1 to about 10 mg / kg body weight of immunoglobulin to the subject. In some embodiments, about 5 mg / kg body weight of immunoglobulin is administered to the subject. In some embodiments, the immunoglobulin is administered to the subject every 14 days. In other embodiments, the immunoglobulin is administered to the subject in at least two doses every 14 days. In other embodiments, the immunoglobulin is administered to the subject in at least six doses every 14 days. In other embodiments, the immunoglobulin is administered to the subject in at least 12 doses every 14 days.

[0016] Administration of immune globulin to a subject by any of the methods disclosed herein may further include administration of standard of care treatments for IgG4-related disease, including anti-inflammatory analgesics (e.g., NSAIDs such as aspirin, ibuprofen, naproxen, or Celebrex), acetaminophen, steroids, glucocorticoids (i.e., prednisone), immunosuppressants (i.e., azathioprine, mycophenolate mofetil), and immunosuppressant biologics (i.e., rituximab, bortezomib). In further embodiments, any of the methods disclosed herein may further include tapering and / or discontinuing the use of steroids. In other embodiments, the subject of any of the methods disclosed herein is likely to relapse or has relapsed or has been refractory to rituximab.

[0017] The B cells of any of the methods disclosed herein may be selected from the group consisting of plasma cells and plasmablasts. In a further embodiment, the B cells may be plasmablasts. [Brief description of the drawings]

[0018] [Figure 1A] Alignment of the amino acid sequences of human IgG immunoglobulins IgG1, IgG2, IgG3, and IgG4. Figure 1A provides the sequences of the CH1 (Cγ1) and hinge domains, and Figure 1B provides the sequences of the CH2 (Cγ2) and CH3 (Cγ3) domains. Positions are numbered according to the EU index of the IgG1 sequence, and differences between IgG1 and the other immunoglobulins IgG2, IgG3, and IgG4 are shown in grey. Allotypic polymorphisms exist at many positions, so minor differences between the presented sequences and those of the prior art may exist. The start of the putative Fc region is labeled and defined herein as either EU position 226 or 230. [Figure 1B] Alignment of the amino acid sequences of human IgG immunoglobulins IgG1, IgG2, IgG3, and IgG4. Figure 1A provides the sequences of the CH1 (Cγ1) and hinge domains, and Figure 1B provides the sequences of the CH2 (Cγ2) and CH3 (Cγ3) domains. Positions are numbered according to the EU index of the IgG1 sequence, and differences between IgG1 and the other immunoglobulins IgG2, IgG3, and IgG4 are shown in grey. Allotypic polymorphisms exist at many positions, so minor differences between the presented sequences and those of the prior art may exist. The start of the putative Fc region is labeled and defined herein as either EU position 226 or 230.

[0019] [Diagram 2] Common haplotypes of human gamma 1 (Figure 2A) and gamma 2 (Figure 2B) chains.

[0020] [Diagram 3]A method of inhibiting B cell activation, where CR represents a co-receptor of the BCR complex, but can be any antigen expressed on any FcγRIIb+ cell.

[0021] [Figure 4] Biacore surface plasmon resonance sensorgrams showing binding of Fc variant anti-CD19 antibodies to human FcγRIIb.

[0022] [Figure 5A] Affinity of Fc variant antibodies to human FcγRs as measured by Biacore surface plasmon resonance. Graph shows -log(KD) for binding of anti-CD19 variant and WT IgG1 antibodies to human FcγRI(I), R131FcγRIIa(RIIa), H131FcγRIIa(HIIa), FcγRIIb(IIb), and V158FcγRIIIa(VIIIa). Binding of L235Y / S267E, G236D / S267E, and S267E / L328F to V158FcγRIIIa was undetectable. [Figure 5B] Affinity of Fc variant antibodies to human FcγRs as measured by Biacore surface plasmon resonance. Graph shows -log(KD) for binding of anti-CD19 variant and WT IgG1 antibodies to human FcγRI(I), R131FcγRIIa(RIIa), H131FcγRIIa(HIIa), FcγRIIb(IIb), and V158FcγRIIIa(VIIIa). Binding of L235Y / S267E, G236D / S267E, and S267E / L328F to V158FcγRIIIa was undetectable. [Figure 5C]Affinity of Fc variant antibodies to human FcγRs as measured by Biacore surface plasmon resonance. Graph shows -log(KD) for binding of anti-CD19 variant and WT IgG1 antibodies to human FcγRI(I), R131FcγRIIa(RIIa), H131FcγRIIa(HIIa), FcγRIIb(IIb), and V158FcγRIIIa(VIIIa). Binding of L235Y / S267E, G236D / S267E, and S267E / L328F to V158FcγRIIIa was undetectable. [Figure 5D] Affinity of Fc variant antibodies to human FcγRs as measured by Biacore surface plasmon resonance. Graph shows -log(KD) for binding of anti-CD19 variant and WT IgG1 antibodies to human FcγRI(I), R131FcγRIIa(RIIa), H131FcγRIIa(HIIa), FcγRIIb(IIb), and V158FcγRIIIa(VIIIa). Binding of L235Y / S267E, G236D / S267E, and S267E / L328F to V158FcγRIIIa was undetectable.

[0023] [Figure 6] Binding of Fc variant antibodies to human FcγRs matches that of WT IgG1 as measured by cell surface binding. Antibodies (variant and WT IgG1) were added to HEK293T cells transfected with FcγRIIb to assess cell surface binding. Binding curves were constructed by plotting MFI as a function of Fc variant concentration.

[0024] [Figure 7] Affinity of Fc variant antibodies to human FcγRs as measured by Biacore surface plasmon resonance. Graph shows -log(KD) for binding of anti-CD19 variant and WT IgG1 antibodies to human FcγRI(I), R131FcγRIIa (RIIa), H131FcγRIIa (HIIa), FcγRIIb (IIb), and V158FcγRIIIa (VIIIa).

[0025] [Figure 8] Here, ATP-dependent B cell viability assays demonstrating survival of primary human B cells upon BCR activation, performed by cross-linking with anti-mu (Figure 8A) or anti-CD79b (Figure 8B) antibodies.

[0026] [Figure 9] Inhibition of B cell proliferation by Fc variant anti-CD19 antibodies. Anti-RSV (respiratory syncytial virus) S267E / L328F is used as a control (RSV is not expressed on B cells). An ATP-dependent luminescence assay was used to measure B cell proliferation in the presence of 10 μg / ml of anti-CD79b activating antibody, and the effect of anti-CD19-S267E / L328F was compared to anti-CD19-IgG1 (natural IgG1 Fv control) and anti-RSV-S267E / L328F (non-CD19 Fc control). To assess the importance of CD19 and FcγRIIb co-binding, anti-RSV-S267E / L328F alone or in combination with anti-CD19-IgG1 was used.

[0027] [Figure 10] Anti-CD19-S267E / L328F inhibits the anti-apoptotic effect of BCR activation in primary human B cells. Inhibition of BCR-mediated survival signals by co-engagement of Fcγ RIIb and CD19 was examined using Annexin V staining in the presence of 10 μg / ml anti-CD79b. B cell apoptosis was stimulated by anti-CD19-S267E / L328F but not by anti-CD19-IgG1 (Fv control), anti-RSV-S267E / L328F (Fc control), or the combined two controls.

[0028] [Figure 11]Assessment of the ability of CD19 and FcγRIIb co-engagement to suppress human B cell activation in vivo. (FIG. 11A) Schematic representation of the experimental protocol. (FIG. 11B) Anti-tetanus toxoid (TT)-specific antibody titers in huPBL-SCID mice following TT immunization and treatment with vehicle (PBS), anti-CD19 IgG1WT, anti-CD19 with high FcγRIIb affinity (a-CD19 S267E / L328F), or anti-CD20 (rituximab).

[0029] [Figure 12] ATP-dependent luminescence assay measuring B cell proliferation in the presence of 1 μg / ml anti-CD79b-SN8-G236R / L328R antibody and varying concentrations of high FcγRIIb variant (S267E / L328F) or FcγR knockout variant (G236R / L328R or ^236R / L328R) versions of anti-CD20 (clone PRO70769), -CD52 (Campath), and -CD19 (HuAM4G7) antibodies.

[0030] [Figure 13] ATP-dependent luminescence assay measuring B cell proliferation in the presence of 1 μg / ml anti-CD79b-SN8-G236R / L328R antibody and varying concentrations of high FcγRIIb variant (S267E / L328F) or FcγR knockout variant (G236R / L328R or ^236R / L328R) versions of anti-CD19 antibodies (clones HD37, 21D4, or HuAM4G7).

[0031] [Figure 14A] Figure 14A lists the amino acid sequences of various variable regions, heavy chain constant regions, and full-length antibodies. Figure 14B is a continuation of the listing in Figure 14A. Figure 14C is a continuation of the listings in Figures 14A and 14B. Figure 14D is a continuation of the listings in Figures 14A-14C. [Figure 14B]Figure 14A lists the amino acid sequences of various variable regions, heavy chain constant regions, and full-length antibodies. Figure 14B is a continuation of the listing in Figure 14A. Figure 14C is a continuation of the listings in Figures 14A and 14B. Figure 14D is a continuation of the listings in Figures 14A-14C. [Figure 14C] Figure 14A lists the amino acid sequences of various variable regions, heavy chain constant regions, and full-length antibodies. Figure 14B is a continuation of the listing in Figure 14A. Figure 14C is a continuation of the listings in Figures 14A and 14B. Figure 14D is a continuation of the listings in Figures 14A-14C. [Figure 14D] Figure 14A lists the amino acid sequences of various variable regions, heavy chain constant regions, and full-length antibodies. Figure 14B is a continuation of the listing in Figure 14A. Figure 14C is a continuation of the listings in Figures 14A and 14B. Figure 14D is a continuation of the listings in Figures 14A-14C.

[0032] [Figure 15] Figure 15A is a diagram of the anti-CD19 antibody S267E / L328F, an immunoglobulin that contains an Fc domain that binds FcγRIIb and an Fv domain that binds CD19. Figure 15B is a diagram showing that the anti-CD19 antibody S267E / L328F binds FcγRIIb+ cells and co-engages CD19, thereby enhancing the natural regulatory role of FcγRIIb (inhibition of B cell activation).

[0033] [Figure 16] Number of affected organs at baseline. Median number was 4 (range 1-10).

[0034] [Figure 17] Active organs at baseline. Organ sites involved with a frequency of ≥50% included lymph nodes, submandibular glands, parotid glands, and lacrimal glands.

[0035] [Figure 18]IgG4-RD responder index over time. Twelve of 15 patients (80%) had an initial response to anti-CD19 antibody with modification S267E / L328F with a reduction in IgG4-RD RI of ≥ 2 points within 2 weeks of the first dose. Five patients achieved an IgG4-RD RI of 0 (no disease activity).

[0036] [Figure 19A] FIG. 19A demonstrates that administration of 2 mg / kg of anti-CD19 antibody S267E / L328F results in suppression of stimulated expression of CD86, which then slowly returns to baseline approximately 100 days after administration of anti-CD19 antibody S267E / L328F. FIG. 19B demonstrates that administration of 2 mg / kg of anti-CD19 antibody S267E / L328F results in a reversible reduction in peripheral B cell counts, which return to normal approximately 40-60 days after administration of anti-CD19 antibody S267E / L328F. FIG. 19C demonstrates that subjects challenged with tetanus antigen and then administered various doses of anti-CD19 antibody S267E / L328F ranging from 0.03-10 mg / kg show a detectable reduction in anti-tetanus IgG relative to placebo-treated subjects. [Figure 19B] FIG. 19A demonstrates that administration of 2 mg / kg of anti-CD19 antibody S267E / L328F results in suppression of stimulated expression of CD86, which then slowly returns to baseline approximately 100 days after administration of anti-CD19 antibody S267E / L328F. FIG. 19B demonstrates that administration of 2 mg / kg of anti-CD19 antibody S267E / L328F results in a reversible reduction in peripheral B cell counts, which return to normal approximately 40-60 days after administration of anti-CD19 antibody S267E / L328F. FIG. 19C demonstrates that subjects challenged with tetanus antigen and then administered various doses of anti-CD19 antibody S267E / L328F ranging from 0.03-10 mg / kg show a detectable reduction in anti-tetanus IgG relative to placebo-treated subjects. [Figure 19C]FIG. 19A demonstrates that administration of 2 mg / kg of anti-CD19 antibody S267E / L328F results in suppression of stimulated expression of CD86, which then slowly returns to baseline approximately 100 days after administration of anti-CD19 antibody S267E / L328F. FIG. 19B demonstrates that administration of 2 mg / kg of anti-CD19 antibody S267E / L328F results in a reversible reduction in peripheral B cell counts, which return to normal approximately 40-60 days after administration of anti-CD19 antibody S267E / L328F. FIG. 19C demonstrates that subjects challenged with tetanus antigen and then administered various doses of anti-CD19 antibody S267E / L328F ranging from 0.03-10 mg / kg show a detectable reduction in anti-tetanus IgG relative to placebo-treated subjects.

[0037] [Figure 20] Flow cytometry gating strategies for B cells and plasmablasts.

[0038] [Figure 21] Summary of B cell counts during treatment. The percentage of initial total B cell (CD79b+) counts is shown for 14 patients. Each line represents an individual patient (representing 14 patients).

[0039] [Figure 22A] Figure 22A shows the effect of antibody therapy on CD79+ plasmablast (CD79b+CD3-CD30-CD27hiCD38hi) counts. Figure 22B shows the effect of antibody therapy on total plasmablast (CD3-CD20-CD27hiCD38hi) counts. Each line represents an individual patient (14 patients represented). [Figure 22B] Figure 22A shows the effect of antibody therapy on CD79+ plasmablast (CD79b+CD3-CD30-CD27hiCD38hi) counts. Figure 22B shows the effect of antibody therapy on total plasmablast (CD3-CD20-CD27hiCD38hi) counts. Each line represents an individual patient (14 patients represented).

[0040] [Diagram 23] Baseline circulating plasmablasts. CD19+ plasmablasts per 104 CD19+ B cells are shown for 11 healthy donors and 14 pre-treatment IgG4-RD patients. Plasmablasts are elevated in active IgG4-RD patients compared to healthy donors.

[0041] [Figure 24] Flow cytometry gating strategy for CD4 CTLs.

[0042] [Diagram 25] Figure 25A shows the effect of anti-CD19 antibody S267E / L328F on the number of SLAMF7+ CD4 CTL (CD4+ CD45RO+ CD27- SLAMF7+). Figure 25B shows the effect of anti-CD19 antibody S267E / L328F on the number of effector CD4 CTL (CD4+ CD45RO+ CD27- SLAMF7+ CD57+ CD28-). Each line represents an individual patient.

[0043] [Figure 26] Flow cytometry gating strategies for apoptosis assays.

[0044] [Figure 27] Apoptosis assay. No significant apoptosis of B cells (Figures 27A-27D), CD4+ T cells (Figures 27E-27H), or CD8+ T cells (Figures 27I-27L) was observed in patients after anti-CD19 antibody S267E / L328F treatment at days 1, 8, 15, and 29.

[0045] [Figure 28]Anti-CD19 antibody S267E / L328F blocks BCR-linked signaling pathways. Phosphorylation of BTK, AKT, ERK, and SYK was examined before treatment with anti-CD19 antibody S267E / L328F (Figures 28A-28D), 2 hours after treatment (Figures 28E-28H), and 24 hours after treatment (Figures 28I-28L). Yellow represents FMO control; blue represents no treatment; and red represents F(ab)2(IgM+IgG) treatment.

[0046] [Figure 29] CD4+SLAMF7+ CTL numbers were increased in the peripheral blood of IgG4-RD patients compared to controls. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Detailed Description of the Invention Humoral immune responses (e.g., the result of diverse B cell responses) can be initiated when B cells are activated with antigen and subsequently differentiate into plasma cells. Engagement of the membrane-bound B cell receptor (BCR) on B cells by antigen activates an intracellular signal cascade, including calcium mobilization, which leads to cell proliferation and differentiation. Co-engagement of the inhibitory Fc receptor (FcγRIIb) with the cognate BCR suppresses B cell activation signals by a negative feedback loop.

[0048] The importance of FcγRIIb in downregulating B cell responses was demonstrated using FcγRIIb-deficient mice, which are unable to regulate humoral responses (Wernersson, S. et al., 1999, J. Immunol. 163, 618-622), are susceptible to collagen-induced arthritis (Yuasa, T. et al., 1999, J. Exp. Med. 189, 187-194), and develop lupus-like disease (Fukuyama, H. et al., JV, 2005, Nat. Immunol. 6, 99-106; McGaha, TL et al., 2005, Science 307, 590-593) and Goodpasture syndrome (Nakamura, A. et al., 2000, J. Exp. Med. 191, 592-593). FcγRIIb dysregulation has also been associated with human autoimmune diseases. For example, polymorphisms in the promoter (Blank, MC et al., 2005, Hum. Genet. 117, 220-227; Olferiev, M. et al., 2007, J. Biol. Chem. 282, 1738-1746) and transmembrane domain (Chen, JY et al., 2006, Arthritis Rheum. 54, 3908-3917; Floto, RA et al., Nat. Med. 11, 1056-1058; Li, X. et al., 2003, Arthritis Rheum. 48, 3242-3252) of FcγRIIb are associated with a high prevalence of systemic lupus erythematosus (SLE). SLE patients also exhibit low FcγRIIb surface expression on B cells (Mackay, M. et al., 2006, J. Exp. Med. 203, 2157-2164; Su, K. et al., 2007, J. Immunol. 178, 3272-3280) and, as a result, dysregulated calcium signaling (Mackay, M. et al., 2006, J. Exp. Med. 203, 2157-2164).The pivotal role of FcγRIIb in regulating B cells, supported by mouse models and clinical evidence, makes it an attractive therapeutic target for controlling autoimmune and inflammatory diseases (Pritchard, NR & Smith, KG, 2003, Immunology 108, 263-273; Ravetch, JV & Lanier, LL, 2000, Science 290, 84-89; Stefanescu, RN et al., 2004, J. Clin. Immunol. 24, 315-326).

[0049] Described herein are antibodies that mimic the inhibitory effect of co-engagement of FcγRIIb with cognate BCR on B cells. For example, described herein are variant anti-CD19 antibodies designed such that the Fc domain binds FcγRIIb with up to 430-fold higher affinity. Relative to native IgG1, the FcγRIIb-binding enhanced variant (IIbE) inhibits BCR-induced calcium mobilization and viability in primary human B cells. The inhibitory effect involves phosphorylation of SH2-containing inositol polyphosphate 5-phosphatase (SHIP), which is known to be involved in FcγRIIb-induced negative feedback of B cell activation. Co-engagement of BCR and FcγRIIb by the IIbE variant also eliminated the anti-apoptotic effect of BCR activation. The use of a single antibody to inhibit B cell function via co-engagement of cognate BCR and FcγRIIb may be a novel approach in the treatment of B cell-mediated diseases. Non-limiting examples of B cell mediated diseases include IgG4-related diseases.

[0050] Several definitions are set forth herein. Such definitions are intended to encompass grammatical equivalents.

[0051] As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibody on a target cell, resulting in subsequent lysis of the target cell.

[0052] As used herein, "ADCP" or "antibody-dependent cell-mediated phagocytosis" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibody on a target cell, followed by phagocytosis of the target cell.

[0053] As used herein, "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by all or a portion of a recognized immunoglobulin gene. For example, in humans, the recognized immunoglobulin genes include the kappa (k), lambda (λ), and heavy chain loci, which together constitute a myriad of variable region genes, as well as the constant region genes mu (υ), delta (δ), gamma (γ), sigma (σ), and alpha (α), which code for the IgM, IgD, IgG (IgG1, IgG2, IgG3, and IgG4), IgE, and IgA (IgA1 and IgA2) isotypes, respectively. Antibodies, as used herein, are intended to include full-length antibodies and antibody fragments, and may refer to natural antibodies from any organism, modified antibodies, or antibodies recombinantly produced for experimental, therapeutic, or other purposes.

[0054] As used herein, "amino acid" and "amino acid identity" refer to one of the 20 naturally occurring amino acids or any non-natural analogue, which may be present at a particular, defined position.

[0055] As used herein, "CD32b + cells" or "FcγRIIb + By "cell" is meant any cell or cell type that expresses CD32b (FcγRIIb). CD32b+ cells include, but are not limited to, B cells, plasma cells, dendritic cells, macrophages, neutrophils, mast cells, basophils, or eosinophils.

[0056] As used herein, "CDC" or "complement dependent cytotoxicity" refers to a reaction in which one or more complement protein components recognize bound antibody on a target cell, resulting in subsequent lysis of the target cell.

[0057] As defined herein, "constant region" refers to the region of an antibody that is encoded by one of the light or heavy chain immunoglobulin constant region genes. As used herein, "constant light chain" or "light chain constant region" refers to the region of an antibody that is encoded by the kappa (Cκ) or lambda (Cλ) light chain. The constant light chain typically comprises a single domain and refers to positions 108-214 of the Cκ or Cλ as defined herein, with numbering according to the EU index. As used herein, "constant heavy chain" or "heavy chain constant region" refers to the region of an antibody that is encoded by the mu, delta, gamma, alpha, or epsilon genes that define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. In a full-length IgG antibody, the constant heavy chain, as defined herein, refers to the N-terminus of the CH1 domain to the C-terminus of the CH3 domain and thus includes positions 118-447, with numbering according to the EU index.

[0058] As used herein, "effector function" refers to a biochemical event resulting from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include FcγR-mediated effector functions, such as ADCC and ADCP, and complement-mediated effector functions, such as CDC. Additionally, effector functions include FcγRIIb-mediated inhibitory functions, such as inhibitory functions (e.g., downregulating, reducing, inhibiting, etc., B cell responses, e.g., humoral immune responses).

[0059] As used herein, "effector cell" refers to a cell of the immune system that expresses one or more Fc and / or complement receptors and mediates one or more effector functions. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and γδ T cells, and can be from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey.

[0060] As used herein, "Fab" or "Fab region" refers to a polypeptide comprising the VH, CH1, VH, and CL immunoglobulin domains. Fab may refer to this region alone or in the context of a full-length antibody or antibody fragment.

[0061] As used herein, "Fc" or "Fc region" refers to a polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain and, in some cases, a portion of the hinge. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. In IgA and IgM, Fc may include the J chain. In IgG, Fc includes immunoglobulin domains C gamma 2 and C gamma 3 (Cγ2 and Cγ3), and the hinge between C gamma 1 (Cγ1) and C gamma 2 (Cγ2). Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl terminus, with numbering according to Kabat's EU index. Fc may refer to this region alone or in the context of an Fc polypeptide, described below.

[0062] As used herein, "Fc polypeptide" refers to a polypeptide that includes all or a portion of an Fc region. Fc polypeptides include antibodies, Fc fusions, isolated Fcs, and Fc fragments. An immunoglobulin may be an Fc polypeptide.

[0063] As used herein, "Fc fusion" refers to a protein in which one or more polypeptides are operatively linked to Fc. As used herein, Fc fusion is intended to be synonymous with the terms "immunoadhesin", "Ig fusion", "Ig chimera", and "receptor globulin" (sometimes with a dash) used in the prior art (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200, both of which are incorporated herein by reference in their entirety). Fc fusion combines the Fc region of an immunoglobulin with a fusion partner, which can generally be any protein, polypeptide, or small molecule. The role of the non-Fc portion of an Fc fusion, i.e., the fusion partner, is to mediate target binding and is thus functionally similar to the variable region of an antibody. Virtually any protein or small molecule can be linked to Fc to generate an Fc fusion. Protein fusion partners may include, but are not limited to, the target binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain. Small molecule fusion partners may include any therapeutic agent that directs the Fc fusion to a therapeutic target. Such a target may be any molecule, for example, an extracellular receptor involved in a disease.

[0064] As used herein, "Fc gamma receptor" or "FcγR" refers to any member of a family of proteins that bind the IgG antibody Fc region and are substantially encoded by the FcγR genes. In humans, this family includes, but is not limited to, FcγRI (CD64), including isotypes FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isotypes FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; FcγRIII (CD16), including isotypes FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, incorporated by reference in its entirety), as well as any undiscovered human FcγR or FcγR isotype or allotype. FcγR can be from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. Mouse FcγR includes, but is not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγR or FcγR isotype or allotype.

[0065] As used herein, "Fc ligand" or "Fc receptor" refers to a molecule, e.g., a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc ligand complex. Fc ligands include, but are not limited to, FcγR, FcγR, FcγR, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), a family of Fc receptors that are homologous to FcγR (Davis et al., 2002, Immunological Reviews 190:123-136). Fc ligands may also include undiscovered molecules that bind Fc.

[0066] As used herein, "full-length antibody" refers to the structure that constitutes the natural biological form of an antibody, including variable and constant regions. For example, in most mammals, including humans and mice, full-length antibodies of the IgG isotype are tetramers, consisting of two identical pairs of two immunoglobulin chains, each pair having one light chain and one heavy chain, each light chain containing immunoglobulin domains VL and CL, and each heavy chain containing immunoglobulin domains VH, Cγ1, Cγ2, and Cγ3. For example, in some mammals, such as camels and llamas, IgG antibodies may consist of only two heavy chains, each heavy chain containing a variable domain that is connected to the Fc region.

[0067] As used herein, "immunoglobulin" refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include, but are not limited to, antibodies (bispecific antibodies) and Fc fusions. Immunoglobulins can have several structural forms, including, but are not limited to, full-length antibodies, antibody fragments, and individual immunoglobulin domains.

[0068] As used herein, "immunoglobulin (Ig) domain" refers to a region of an immunoglobulin that exists as a separate structural entity as recognized by those skilled in the art of protein structure. Ig domains typically have a unique β-sandwich fold topology. The known Ig domains in the IgG isotype of antibody are VH Cγ1, Cγ2, Cγ3, VL, and CL.

[0069] As used herein, "IgG" or "IgG immunoglobulin" refers to a polypeptide belonging to the class of antibodies substantially encoded by the recognized immunoglobulin gamma genes. In humans, this class includes the subclasses or isotypes IgG1, IgG2, IgG3, and IgG4. As used herein, "isotype" refers to any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE.

[0070] As used herein, "modification" refers to alterations in the physical, chemical, or sequence properties of a protein, polypeptide, antibody, or immunoglobulin. Modifications as described herein include amino acid modifications and glycoform modifications.

[0071] As used herein, "amino acid modification" refers to the substitution, insertion, and / or deletion of an amino acid in a polypeptide sequence. As used herein, "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. For example, the substitution S267E refers to a variant polypeptide, in this case a constant heavy chain variant, in which the serine at position 267 is replaced with glutamic acid. As used herein, "amino acid insertion" or "insertion" refers to the addition of an amino acid at a particular position in a parent polypeptide sequence. As used herein, "amino acid deletion" or "deletion" refers to the removal of an amino acid at a particular position in a parent polypeptide sequence.

[0072] As used herein, "glycoform modification" or "modified glycoform" or "engineered glycoform" refers to a sugar composition that is covalently attached to a protein, e.g., an antibody, that is chemically different from the parent protein. A modified glycoform typically refers to a different sugar or oligosaccharide, thus, for example, an Fc variant can include a modified glycoform. Alternatively, a modified glycoform can refer to an Fc variant that includes a different sugar or oligosaccharide.

[0073] As used herein, "parent polypeptide", "parent protein", "parent immunoglobulin", "precursor polypeptide", "precursor protein", or "precursor immunoglobulin" refers to an unmodified polypeptide, protein, or immunoglobulin that is subsequently modified to generate a variant, e.g., any polypeptide, protein, or immunoglobulin, that serves as a template and / or basis for at least one amino acid modification as described herein. A parent polypeptide may be a naturally occurring polypeptide or a variant or engineered version of a naturally occurring polypeptide. A parent polypeptide may refer to the polypeptide itself, a composition that includes the parent polypeptide, or the amino acid sequence that encodes it. Thus, as used herein, "parent Fc polypeptide" refers to an Fc polypeptide that is modified to generate a variant Fc polypeptide, and as used herein, "parent antibody" refers to an antibody that is modified to generate a variant antibody (e.g., a parent antibody may include, but is not limited to, a protein that includes a naturally occurring Ig constant region).

[0074] As used herein, "position" refers to a position in the sequence of a protein. Positions may be numbered sequentially or according to established formats, such as the EU index as in Kabat. For example, position 297 is a position in human antibody IgG1.

[0075] As used herein, "polypeptide" or "protein" means at least two covalently linked amino acids, including proteins, polypeptides, oligopeptides, and peptides.

[0076] As used herein, "residue" refers to a position in a protein and its associated amino acid identity. For example, asparagine 297 (also called Asn297, also called N297) is a residue in human antibody IgG1.

[0077] As used herein, "target antigen" refers to the molecule bound by the variable region of a given antibody, or the fusion partner of an Fc fusion. A target antigen may be a protein, sugar, lipid, or other chemical compound. An antibody or Fc fusion is said to be "specific" for a given target antigen based on having affinity for the target antigen.

[0078] As used herein, "target cell" means a cell that expresses a target antigen.

[0079] As used herein, "variable region" refers to a region of an immunoglobulin that contains one or more Ig domains substantially encoded by either the Vk, Vlambda, and / or VH genes that constitute the kappa, lambda, and heavy chain immunoglobulin loci, respectively.

[0080] As used herein, "variant polypeptide," "polypeptide variant," or "variant" refers to a polypeptide sequence that differs from a parent polypeptide sequence by at least one amino acid modification. The parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide, or a modified version of a wild-type polypeptide. A variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or the amino acid sequence encoding it. In some embodiments, a variant polypeptide (e.g., a variant immunoglobulin) disclosed herein may have at least one amino acid modification compared to the parent polypeptide, e.g., about 1-10 amino acid modifications, about 1-5 amino acid modifications, etc., compared to the parent. As used herein, a variant polypeptide sequence may have at least about 80% homology, e.g., at least about 90% homology, 95% homology, etc., with the parent polypeptide sequence. Thus, as used herein, an "Fc variant" or "variant Fc" refers to an Fc sequence that differs from a parent Fc sequence by at least one amino acid modification. An Fc variant may encompass only the Fc region or may be present in the context of an antibody, an Fc fusion, an isolated Fc, an Fc fragment, or other polypeptide substantially encoded by an Fc. An Fc variant may refer to the Fc polypeptide itself, a composition comprising an Fc variant polypeptide, or the amino acid sequence encoding it. As used herein, an "Fc polypeptide variant" or "variant Fc polypeptide" refers to an Fc polypeptide that differs from a parent Fc polypeptide by at least one amino acid modification. As used herein, a "protein variant" or "variant protein" refers to a protein that differs from a parent protein by at least one amino acid modification. As used herein, an "antibody variant" or "variant antibody" refers to an antibody that differs from a parent antibody by at least one amino acid modification. As used herein, an "IgG variant" or "variant IgG" refers to an antibody that differs from a parent IgG by at least one amino acid modification.As used herein, "immunoglobulin variant" or "variant immunoglobulin" means an immunoglobulin sequence that differs from a parent immunoglobulin sequence by means of at least one amino acid modification.

[0081] As used herein, "wild type" or "WT" refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A wild-type protein, polypeptide, antibody, immunoglobulin, IgG, etc., has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0082] Immunoglobulins

[0083] As described herein, the immunoglobulin may be an antibody, an Fc fusion, an isolated Fc, an Fc fragment, or an Fc polypeptide. In one embodiment, the immunoglobulin is an antibody.

[0084] Antibodies are immunological proteins that bind specific antigens. In most mammals, including humans and mice, antibodies are constructed from paired heavy and light chain polypeptides. The light and heavy chain variable regions show significant sequence diversity among antibodies and are responsible for binding the target antigen. Each chain consists of individual immunoglobulin (Ig) domains, hence the collective term immunoglobulin is used for such proteins.

[0085] Traditional antibody structural units generally comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each chain having one "light" chain (usually having a molecular weight of about 25 kDa) and one "heavy" chain (usually having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, which define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA has several subclasses, including but not limited to IgA1 and IgA2. Thus, "isotype" as used herein refers to the class and subclass of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE.

[0086] Each of the light and heavy chains consists of two unique regions, designated as the variable and constant regions. The IgG heavy chain is composed of four immunoglobulins linked N-to-C-terminus in the order VH-CH1-CH2-CH3, designated as the heavy chain variable region, heavy chain constant region 1, heavy chain constant region 2, and heavy chain constant region 3, respectively (also designated as VH-Cγ1-Cγ2-Cγ3, designated as the heavy chain variable region, constant gamma 1 region, constant gamma 2 region, and constant gamma region 3, respectively). The IgG light chain is composed of two immunoglobulins linked N-to-C-terminus in the order VL-CL, designated as the light chain variable region and light chain constant region, respectively. The constant regions show less sequence diversity and are responsible for binding several natural proteins and eliciting important biochemical events. The distinguishing feature between these antibody classes is the constant region, although elusive chains may exist in the variable region.

[0087] The variable region of an antibody contains the antigen-binding determinants of the molecule and thus determines the specificity of the antibody for its target antigen. The variable region is so named because it is the most different in sequence from other antibodies within the same class. The amino-terminal portion of each chain contains about 100-110 or more variable regions that are primarily responsible for antigen recognition. In the variable region, three loops come together for each of the V domains of the heavy and light chains to form the antigen-binding site. Each of the loops is referred to as a complementarity determining region (hereinafter also referred to as "CDR") and has the most significant variation in amino acid sequence. There are six CDRs, three each for the heavy and light chains, denoted VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The variable regions outside the CDRs are referred to as framework (FR) regions. Although not as diverse as the CDRs, sequence variability also occurs in the FR regions between different antibodies. Overall, this characteristic structure of antibodies provides a stable scaffold (FR region) on which the vast antigen-binding diversity (CDR) can be explored by the immune system to obtain specificity for a wide array of antigens. Many well-characterized structures are available for various variable region fragments from different organisms, some unbound and some complexed with antigen. The sequences and structural features of antibody variable regions are disclosed, for example, in Morea et al., 1997, Biophys Chem 68:9-16; Morea et al., 2000, Methods 20:267-279, which are incorporated herein by reference in their entirety, and the conserved features of antibodies are disclosed, for example, in Maynard et al., 2000, Annu Rev Biomed Eng2:339-376, which are incorporated herein by reference in their entirety.

[0088] The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. As used herein, "immunoglobulin (Ig) domain" refers to a region of an immunoglobulin that has a defined tertiary structure. Of interest in the embodiments described herein are the heavy chain domains, including the constant heavy (CH) domain and the hinge region. In the context of IgG antibodies, each IgG isotype has three CH regions. Thus, in the context of IgG, the "CH" domains are as follows: "CH1" refers to positions 118-220 according to the Kabat EU index; "CH2" refers to positions 237-340 according to the Kabat EU index; and "CH3" refers to positions 341-447 according to the Kabat EU index.

[0089] Another important region of the heavy chain is the hinge region. As used herein, "hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" refers to a flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 220 and the IgG CH2 domain begins at residue EU position 237. Thus, in IgG, the antibody hinge is defined herein to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1), numbering according to the EU index of Kabat. In some embodiments, the lower hinge is included, e.g., in the context of the Fc region, generally "lower hinge" refers to positions 226 or 230 to 236.

[0090] The interest of the embodiments described herein is the Fc region. As used herein, "Fc" or "Fc region" refers to a polypeptide that includes the first constant region immunoglobulin domain and, in some cases, the constant region of an antibody, excluding a portion of the hinge. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. In IgA and IgM, Fc may include the J chain. In IgG, Fc includes the immunoglobulin domains C gamma 2 and C gamma 3 (Cγ2 and Cγ3), and the lower hinge region between C gamma 1 (Cγ1) and C gamma 2 (Cγ2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to the carboxyl terminus, where numbering is according to the EU index of Kabat. Fc may refer to this region alone or in conjunction with an Fc polypeptide, described below. As used herein, "Fc polypeptide" means a polypeptide comprising all or a portion of an Fc region. Fc polypeptide includes antibodies, Fc fusions, isolated Fcs, and Fc fragments.

[0091] The Fc region of an antibody interacts with several Fc receptors and ligands, conferring a number of important functional capabilities, referred to as effector functions. In IgG, the Fc region includes Ig domains Cγ2 and Cγ3, and an N-terminal hinge leading to Cγ2. An important family of Fc receptors in the IgG class are the Fcγ receptors (FcγR). These receptors mediate communication between antibodies and the cellular arm of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290, both of which are incorporated herein by reference in their entirety). In humans, this protein family includes FcγRI (CD64), which includes isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, incorporated herein by reference in its entirety). These receptors typically have an extracellular domain that mediates binding to Fc, a transmembrane region, and an intracellular domain that may mediate several signaling events within the cell. These receptors may be expressed on a variety of immune cells, including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and γγT cells. The formation of the Fc / FcγR complex recruits these effector cells to the site of bound antigen, typically resulting in intracellular signaling events and subsequent important immune responses, such as release of inflammatory mediators, activation of B cells, endocytosis, phagocytosis, and cytotoxic attack.The ability to mediate cytotoxic and phagocytic effector functions is a potential mechanism by which antibodies destroy target cells. The cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibodies on target cells and subsequently cause lysis of the target cells is called antibody-dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275-290, both of which are incorporated herein by reference in their entirety). The cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibodies on target cells and subsequently cause phagocytosis of the target cells is called antibody-dependent cell-mediated phagocytosis (ADCP).

[0092] Different IgG subclasses have different affinities for FcγR, with IgG1 and IgG3 typically binding substantially better to this receptor than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett 82:57-65, incorporated herein by reference in its entirety). FcγR binds to the IgG Fc region with different affinities. The extracellular domains of FcγRIIIa and FcγRIIIb are 96% identical, but FcγRIIIb does not have an intracellular signaling domain. Furthermore, FcγRI, FcγRIIa / c, and FcγRIIIa are positive regulators of immune complex-induced activity, characterized by having an intracellular domain with an immunoreceptor tyrosine-based activation motif (ITAM), whereas FcγRIIb has an immunoreceptor tyrosine-based inhibitory motif and is therefore inhibitory. Thus, the former are referred to as activating receptors, and FcγRIIb as an inhibitory receptor. Despite these differences in affinity and activation, all FcγRs bind to the same region on Fc, at the N-terminus of the Cγ2 domain and the aforementioned hinge. This interaction has been structurally well characterized (Sondermann et al., 2001, J Mol Biol 309:737-749, incorporated herein by reference in its entirety), and several structures of human Fc bound to the extracellular domain of human FcγRIIIb have been solved (pdb accession code 1E4K) (Sondermann et al., 2000, Nature 406:267-273, incorporated herein by reference in its entirety) (pdb accession codes 1IIS and 1IIX) (Radaev et al., 2001, J Biol Chem 276:16469-16477, incorporated herein by reference in its entirety).

[0093] An overlapping, but distinct, site on Fc serves as the interface for the complement protein C1q. In the same way that Fc / FcγR binding mediates ADCC, Fc / C1q binding mediates complement-dependent cytotoxicity (CDC). A site on Fc between the Cγ2 and Cγ3 domains mediates interaction with the neonatal receptor FcRn, a link that recirculates endocytosed antibodies from endosomes back into the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766, both of which are incorporated herein by reference in their entireties). This process, along with the prevention of renal filtration by large, full-length molecules, results in favorable antibody serum half-lives ranging from 1 to 3 weeks. Binding of Fc to FcRn also plays a major role in antibody transport. The binding site for FcRn on Fc is also the site at which bacterial proteins A and G bind. The tight binding of these proteins is typically exploited as a means to purify antibodies during protein purification by utilizing affinity chromatography of protein A or protein G. The fidelity of these regions, complement, and FcRn / protein A binding regions are important to both the clinical properties of antibodies and their development.

[0094] A key feature of the Fc region is the conserved N-linked glycosylation that occurs at N297. These sugars, sometimes referred to as oligosaccharides, play important structural and functional roles in antibodies and are one of the main reasons why antibodies must be produced using mammalian expression systems. Efficient Fc binding to FcγR and C1q requires this modification, and alterations in the composition of the N297 sugar, or its loss, affect binding to these proteins (Umana et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Mimura et al., 2001, J Biol Chem 276:45539-45547.; Radaev et al., 2001, J Biol Chem 276:16478-16483; Shields et al., 2001, J Biol Chem 276:6591-6604; Shields et al., 2002, J Biol Chem 277:26733-26740; Simmons et al., 2002, J Biol Chem 277:26733-26740). Immunol Methods 263:133-147, all of which are incorporated herein by reference in their entireties).

[0095] The immunoglobulins of the embodiments described herein may be antibody-like proteins also referred to as Fc fusions (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200, both of which are incorporated herein by reference in their entirety). As used herein, "Fc fusion" is intended to be synonymous with the terms "immunoadhesin", "Ig fusion", "Ig chimera", and "receptor globulin" (sometimes with a dash) used in the prior art (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200). An Fc fusion is a protein in which one or more polypeptides, referred to herein as "fusion partners", are operably linked to Fc. Fc fusions combine the Fc region of an antibody, and thus its favorable effector function and pharmacokinetics, with the target-binding region of a receptor, ligand, or some other protein, or protein domain. The role of the latter is to mediate target recognition, and thus is functionally similar to the variable region of an antibody. Because of the structural and functional overlap of Fc fusions with antibodies, the discussion of antibodies in this disclosure is also extended to Fc fusions.

[0096] Virtually any protein or small molecule can be linked to Fc to generate an Fc fusion. Protein fusion partners can include, but are not limited to, the variable region of any antibody, the target binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain. Small molecule fusion partners can include any agent that directs the Fc fusion to a target antigen. Such a target antigen can be any molecule, such as, for example, an extracellular receptor, involved in a disease. The Fc fusions of the embodiments described herein include CD32b. + Any antigen expressed on a cell can be targeted.

[0097] The fusion partner may be linked to any region of the Fc region, including the N- or C-terminus, or some residue between the termini. In one embodiment, the fusion partner is linked at the N- or C-terminus of the Fc region. A variety of linkers may find use in some embodiments described herein to covalently link the Fc region to the fusion partner. As used herein, "linker," "linker sequence," "spacer," "tether sequence," or grammatical equivalents thereof refer to a molecule or group of molecules (such as a monomer or polymer) that serves to link two molecules together and often arrange the two molecules into a structure. Linkers are known in the art, for example, homo- or heterobifunctional linkers are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, which is incorporated by reference in its entirety). Several approaches may be used to covalently link molecules together. These include, but are not limited to, polypeptide linkages between the N- and C-termini of a protein or protein domain, linkages via disulfide bonds, and linkages via chemical cross-linking reagents. In one aspect of this embodiment, the linker is a peptide bond, generated by recombinant techniques or peptide synthesis. The linker peptide comprises primarily the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should be of a suitable length to link two molecules in a manner that allows them to assume the appropriate conformation relative to each other so as to maintain the desired activity. A suitable length for this purpose comprises at least one and not more than 50 amino acid residues. In one embodiment, the linker is about 1-30 amino acids in length. In one embodiment, a linker of 1-20 amino acids in length may be used.Useful linkers include glycine-serine polymers (e.g., (GS), (GSGGS) (set forth as SEQ ID NO: 1), (GGGGS) (set forth as SEQ ID NO: 2), and (GGGS) (set forth as SEQ ID NO: 3), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers, as will be appreciated by those of skill in the art. Alternatively, a variety of non-proteinaceous polymers may find use as linkers, i.e., to link the Fc region to a fusion partner, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.

[0098] Fc polypeptides are also contemplated as fusion partners. Thus, the immunoglobulins described herein may be multimeric Fc polypeptides, comprising two or more Fc regions. The advantage of such molecules is that they provide multiple binding sites for Fc receptors in a single protein molecule. In one embodiment, the Fc regions may be linked using chemical engineering approaches. For example, Fab' and Fc' may be linked by thioether bonds derived from cysteine ​​residues in the hinge to generate molecules such as FabFc2. The Fc regions may be linked using disulfide engineering and / or chemical cross-linking. In one embodiment, the Fc regions may be genetically linked. In one embodiment, the Fc regions in the immunoglobulins are genetically linked to the generated tandemly linked Fc regions described in U.S. Patent Publication No. 2005 / 0249723, which is incorporated by reference in its entirety. The tandemly linked Fc polypeptide may comprise two or more Fc regions, e.g., one to three Fc regions, two Fc regions. It may be advantageous to explore many engineered structures to obtain homo- or hetero-tandemly linked Fc regions with the most suitable structural and functional properties. Tandemly linked Fc regions may be homo-tandemly linked Fc regions, i.e., an Fc region of one isotype may be genetically fused to another Fc region of the same isotype. It is expected that effector function and / or pharmacokinetics may be enhanced due to the presence of multiple FcγR, C1q, and / or FcRn binding sites on the tandemly linked Fc polypeptide. In an alternative embodiment, Fc regions from different isotypes may be tandemly linked, referred to as hetero-tandemly linked Fc regions. For example, immunoglobulins that bind both FcγR and FcαRI due to their ability to target FcγR and FcαRI receptors may provide clinical improvements.

[0099] The immunoglobulins of the embodiments disclosed herein may also be substantially encoded by immunoglobulin genes belonging to any of the antibody classes. In certain embodiments, the immunoglobulins disclosed herein find use in antibodies or Fc fusions that include sequences belonging to the IgG class of antibodies, including IgG1, IgG2, IgG3, or IgG4. Figure 1 provides an alignment of these human IgG sequences. In alternative embodiments, the immunoglobulins disclosed herein find use in antibodies or Fc fusions that include sequences belonging to the IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG, or IgM classes of antibodies. The immunoglobulins disclosed herein may include one or more protein chains, and may be antibodies or Fc fusions that are monomeric or oligomeric, including, for example, homo- or hetero-oligomers.

[0100] The immunoglobulins disclosed herein may be substantially encoded by genes from any organism, e.g., mammals (including but not limited to humans, rodents (including but not limited to mice and rats), lagomorphs (including but not limited to rabbits and hares), camelids (including but not limited to camels, llamas, and dromedaries), and non-human primates, including but not limited to prosimians, platyrrhines (New World monkeys), Cercopithecinae (Old World monkeys), and Hominoidea, including gibbons, lesser and greater apes. In certain embodiments, the immunoglobulins disclosed herein may be substantially human.

[0101] As is well known in the art, immunoglobulin polymorphisms exist in the human population. The Gm polymorphism is determined by the IGHG1, IGHG2 and IGHG3 genes, with alleles encoding allotypic antigenic determinants, termed G1m, G2m and G3m allotypes of markers of human IgG1, IgG2 and IgG3 molecules (no Gm allotypes have been found on the γ4 chain). Markers can be classified into "allotypes" and "isoallotypes". These are differentiated on different serological grounds, depending on the strong sequence homology between isotypes. Allotypes are antigenic determinants specified by the allelic type of Ig genes. Allotypes represent slight differences in the amino acid sequence of different individual heavy or light chains. Even a single amino acid difference can give rise to an allotypic determinant, but in many cases several amino acid substitutions have occurred. Allotypes are sequence differences between alleles of a subclass, where antisera only recognize the allelic difference. Isoallotypes are alleles in one isotype that generate epitopes shared with non-polymorphic homologous regions of one or more other isotypes, such that antisera react with both the relevant allotype and the relevant homologous isotype (Clark, 1997, IgG effector mechanisms, Chem Immunol. 65:88-110; Gorman & Clark, 1990, Semin Immunol 2(6):457-66, both of which are incorporated herein by reference in their entireties).

[0102] Allelic types of human immunoglobulins have been well characterized (WHO Review of the notation for the allotypic and related markers of human immunoglobulins. J Immunogen 1976,3:357-362; WHO Review of the notation for the allotypic and related markers of human immunoglobulins.1976,Eur.J.Immunol.6,599-601; Loghem E van,1986,Allotypic markers, Monogr Allergy 19:40-51, all of which are incorporated herein by reference in their entirety). In addition, other polymorphisms have been characterized (Kim et al.,2001,J.Mol.Evol.54:1-9, all of which are incorporated herein by reference in their entirety). Currently, 18 Gm allotypes are known: G1m(1, 2, 3, 17) or G1m(a, x, f, z), G2m(23) or G2m(n), G3m(5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m(b1, c3, b5, b0, b3, b4, s, t, g1, c5, u, v, g5) (Lefranc, et al., The human IgG subclasses: molecular analysis of structure, function and regulation. Pergamon, Oxford, pp. 43-78(1990); Lefranc, G. et al., 1979, Hum. Genet.: 50, 199-211, both of which are incorporated herein by reference in their entireties). Allotypes inherited in fixed combinations are called Gm haplotypes. Figure 2 shows common haplotypes of the gamma chain of human IgG1 (Figure 2A) and IgG2 (Figure 2A), showing the positions and associated amino acid substitutions. The immunoglobulins disclosed herein can be substantially encoded by any allotype, isoallotype, or haplotype of any immunoglobulin gene.

[0103] The immunoglobulins disclosed herein may comprise Fc polypeptides, including, but not limited to, antibodies, isolated Fc, Fc fragments, and Fc fusions. In one embodiment, the immunoglobulins disclosed herein are full-length antibodies that comprise the natural biological form of an antibody, including variable and constant regions. In the IgG isotype, full-length antibodies are tetramers and consist of two identical pairs of two immunoglobulin chains, each pair having one light chain and one heavy chain, each light chain comprising immunoglobulin domains VL and CL, and each heavy chain comprising immunoglobulin domains VH, Cγ1, Cγ2, and Cγ3. In another embodiment, the immunoglobulins disclosed herein are isolated Fc regions or Fc fragments.

[0104] The immunoglobulins disclosed herein may be of a variety of structures, including, but not limited to, antibody fragments, bispecific antibodies, miniantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments of each.

[0105] In one embodiment, the antibody is an antibody fragment. Particular antibody fragments include, but are not limited to, (i) Fab fragments consisting of VL, VH, CL, and CH1 domains, (ii) Fd fragments consisting of VH and CH1 domains, (iii) Fv fragments consisting of the VL and VH domains of a single antibody, (iv) dAb fragments consisting of a single variable, (v) isolated CDR regions, (vi) F(ab')2 fragments, which are bivalent fragments containing two linked Fab fragments, (vii) single chain Fv molecules (scFv), in which the VH and VL domains are linked by a peptide linker that allows the formation of an antigen-binding site by linking the two domains, (viii) bispecific single chain Fv dimers, and (ix) "diabodies" or "triabodies", which are multivalent or multispecific fragments constructed by gene fusion. Antibody fragments may be modified. For example, the molecule may be stabilized by the incorporation of disulfide bridges linking the VH and VL domains. Examples of antibody formats and structures are described in Holliger & Hudson, 2006, Nature Biotechnology 23(9):1126-1136 and Carter 2006, Nature Reviews Immunology 6:343-357 and references cited therein, all of which are expressly incorporated by reference.

[0106] In one embodiment, the antibodies disclosed herein may be multispecific antibodies, and in particular bispecific antibodies, sometimes referred to as "bispecific antibodies." These are antibodies that bind to two (or more) different antigens. Bispecific antibodies can be produced in a variety of ways known in the art, for example, chemically or prepared from hybrid hybridomas. In one embodiment, the antibody is a miniantibody. Miniantibodies are minimized antibody-like proteins that include an scFv linked to a CH3 domain. In some cases, the scFv can be linked to an Fc region and can include some or all of the hinge region. For a description of multispecific antibodies, see Holliger & Hudson, 2006, Nature Biotechnology 23(9):1126-1136, and references cited therein, all of which are expressly incorporated by reference.

[0107] Non-human, chimeric, humanized, and fully human antibodies

[0108] As is well known in the art, the variable region of an antibody can comprise sequences from various species. In some embodiments, the antibody variable region can be from a non-human source, including, but not limited to, mouse, rat, rabbit, camel, llama, and monkey. In some embodiments, the scaffold components can be a mixture of different species. Thus, the antibodies disclosed herein can be chimeric and / or humanized antibodies. In general, both "chimeric antibodies" and "humanized antibodies" refer to antibodies that combine regions from more than one species. For example, a "chimeric antibody" traditionally contains a variable region from mouse or other non-human species and a constant region from a human.

[0109] "Humanized antibody" generally refers to a non-human antibody with variable domain framework regions replaced with sequences found in human antibodies. Generally, in a humanized antibody, the entire antibody, except for the CDRs, is encoded by a polynucleotide of human origin or is identical to such an antibody except within its CDRs. Some or all of the CDRs encoded by nucleic acids from organisms other than human are grafted onto the beta-sheet framework of human antibody variable regions to generate an antibody with specificity determined by the grafted CDRs. The generation of such antibodies is described, for example, in WO 92 / 11018; Jones, 1986, Nature 321:522-525; Verhoeyen et al., 1988, Science 239:1534-1536. "Backmutation" of selected acceptor framework residues to the corresponding donor residues is often required to restore affinity lost in the initial grafted construct (U.S. Patent No. 5,693,762, incorporated by reference in its entirety). A humanized antibody optimally comprises at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus typically comprises a human Fc region. A humanized antibody can also be produced using a mouse with a genetically engineered immune system. Roque et al., 2004, Biotechnol. Prog. 20: 639-654. Various techniques and methods for humanizing and reconstructing non-human antibodies are well known in the art (see Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references cited therein). Humanization or other methods for reducing the immunogenicity of non-human antibody variable regions can include resurfacing methods, for example, as described in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91: 969-973. In one embodiment, the parent antibody has been affinity matured, as known in the art.Structure-based methods may be used in humanization and affinity maturation, for example, as described in US Patent Application No. 11 / 004,590. Selection-based methods may be used to humanize and / or affinity mature antibody variable regions, i.e., to increase the affinity of the variable regions for their target antigen. Other humanization methods may include grafting only a portion of the CDRs, including, but not limited to, the methods described in US Patent No. 09 / 810,502, Tan et al., 2002, J.Immunol.169:1119-1125, De Pascalis et al., 2002, J.Immunol.169:3076-3084. Structure-based methods may be used in humanization and affinity maturation, for example, as described in US Patent No. 7,117,096, and related applications, which are incorporated by reference in their entirety. In a particular variation, the immunogenicity of the antibody is reduced using the methods described in US Pat. No. 7,657,380, which is incorporated by reference in its entirety.

[0110] In one embodiment, the antibody is a fully human antibody with at least one modification as outlined herein. "Fully human antibody" or "complete human antibody" refers to a human antibody with the gene sequence of the antibody derived from a human chromosome, with the modifications as outlined herein. Fully human antibodies may be obtained, for example, using transgenic mice (Bruggemann et al., 1997, Curr Opin Biotechnol 8:455-458) or human antibody libraries in conjunction with selection methods (Griffiths et al., 1998, Curr Opin Biotechnol 9:102-108).

[0111] target antigen

[0112] In one embodiment, antigens may be targeted by the immunoglobulins disclosed herein, including but not limited to, targeting proteins, subunits, domains, motifs, and / or epitopes belonging to CD19.

[0113] Fc variants and Fc receptor binding properties

[0114] The immunoglobulins disclosed herein include Fc variants. The Fc variants include one or more amino acid modifications relative to a parent Fc polypeptide, which amino acid modifications provide one or more optimized properties. The Fc variants disclosed herein differ in amino acid sequence from their parent by at least one amino acid modification. Thus, the Fc variants disclosed herein have at least one amino acid modification compared to the parent. Alternatively, the Fc variants disclosed herein may have one or more amino acid modifications compared to the parent, such as about 2-50 amino acid modifications, such as about 2-10 amino acid modifications, about 2-5 amino acid modifications, etc. compared to the parent. Thus, the sequence of the Fc variant and the sequence of the parent Fc polypeptide are substantially homologous. For example, herein, the mutated Fc variant sequence has about 80% homology, such as at least about 90% homology, at least about 95% homology, at least 98% homology, at least about 99% homology, etc., with the parent Fc variant sequence. Modifications disclosed herein include amino acid modifications, including insertions, deletions, and substitutions. Modifications disclosed herein also include glycoform modifications. Modifications are generally performed using molecular biology, or may be performed enzymatically or chemically.

[0115] The Fc variants disclosed herein are defined by the amino acid modifications that compose them. Thus, for example, S267E is an Fc variant with substitution S267E relative to the parent Fc polypeptide. Similarly, S267E / L328F defines an Fc variant with substitutions S267E and L328F relative to the parent Fc polypeptide. The identity of wild-type amino acids may be unspecified, in which case the variant is referred to as 267E / 328F. It is noted that the order in which the substitutions are provided is arbitrary, i.e., for example, 267E / 328F is the same Fc variant as 328F / 267E, etc. Unless otherwise indicated, positions discussed herein are numbered according to the EU index as described in Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, incorporated herein by reference in its entirety). Briefly, EU is the name of the first antibody molecule whose entire amino acid sequence was determined (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, incorporated herein by reference in its entirety), and so that amino acid sequence became the standard numbering scheme for heavy chain constant regions. The EU protein became the standard reference for defining numbering. Kabat et al. listed the EU sequence for a set of indexes that aligned it with other antibody sequences, serving as a necessary tool for aligning antibodies against the EU numbering scheme. Thus, as will be appreciated by those skilled in the art, the standard way to refer to the EU numbering is to refer to the alignment of the sequences in Kabat et al. because it uses EU with respect to antibodies of other variable domain lengths. Thus, as used herein, "EU index as in Kabat" or "numbering is according to the EU index as in Kabat" refers to the numbering of EU antibodies as described in Kabat.

[0116] In certain embodiments, the Fc variants disclosed herein are based on human IgG sequences, and thus the human IgG sequence is used as a "base" sequence to which other sequences are compared, including, but not limited to, sequences from other organisms, such as rodent and primate sequences. Immunoglobulins also include sequences from other immunoglobulin classes, such as IgA, IgE, IgGD, IgG, etc. It is understood that while the Fc variants disclosed herein are engineered in the context of one parent IgG, the variants may also be associated with or "transferred" to another, second parent IgG. This is typically done by determining "equivalent" or "corresponding" residues and substitutions between the first and second IgG, based on sequence or structural homology between the sequences of the first and second IgG. To establish homology, the amino acid sequence of the first IgG outlined herein is directly compared to the sequence of the second IgG. After aligning the sequences, one or more homology alignment programs known in the art (e.g., using conserved residues between species) and making necessary insertions and deletions to maintain the alignment (i.e., avoiding elimination of conserved residues through arbitrary deletions and insertions) are used to define residues equivalent to a particular amino acid in the primary sequence of the first immunoglobulin. Alignment of conserved residues may preserve 100% of such residues. However, alignment of more than 75% or even as little as 50% of conserved residues is sufficient to define equivalent residues. Equivalent residues may be defined by determining the structural homology between the first and second IgG, which is at the level of the tertiary structure of the IgG whose structure has been determined. In this case, equivalent residues are defined as those where the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the parent or precursor (N on N, CA on CA, C on C, and O on O) are within about 0.13 nm after alignment. In another embodiment, equivalent residues are within about 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of the atomic coordinates of the non-hydrogen protein atoms of the protein.Regardless of how the equivalent or corresponding residues are determined, and regardless of the identity of the parent IgG from which the IgG is made, the discovered Fc variants disclosed herein may be engineered into any second parent IgG that has significant sequence or structural homology with the Fc variant. Thus, for example, if a variant antibody is generated where the parent antibody is a human IgG1, by using the methods described above for determining equivalent residues, or other methods, the variant antibody may be engineered into another IgG1 parent antibody that binds to a different antigen, a human IgG2 parent antibody, a human IgA parent antibody, a mouse IgG2a or IgG2b parent antibody, etc. Also, as noted above, the composition of the parent Fc variant does not affect the ability to transfer the Fc variants disclosed herein to other parent IgGs.

[0117] The Fc variants disclosed herein may be optimized for various Fc receptor binding properties. Fc variants engineered or predicted to exhibit one or more optimized properties are referred to herein as "optimized Fc variants". Properties that may be optimized include, but are not limited to, enhanced or reduced affinity for FcγR. In one embodiment, the Fc variants disclosed herein are optimized to have enhanced affinity for the inhibitory receptor FcγRIIb. In other embodiments, the immunoglobulins disclosed herein provide enhanced affinity for FcγRIIb but reduced affinity for one or more activating FcγRs, including, for example, FcγRI, FcγRIIa, FcγRIIIa, and / or FcγRIIIb. FcγR receptors may be expressed on cells from any organism, including, but not limited to, human, cynomolgus monkey, and mouse. The Fc variants disclosed herein may be optimized to have enhanced affinity for human FcγRIIb.

[0118] As used herein, "greater affinity" or "improved affinity" or "enhanced affinity" or "better affinity" than a parent Fc polypeptide means that the Fc variant has a significantly higher association equilibrium constant (K) than the parent Fc polypeptide when the amounts of variant and parent polypeptide in the binding assay are essentially the same. A or Ka), or a low dissociation equilibrium constant (K D or Kd) relative to the Fc receptor. For example, an Fc variant with improved Fc receptor binding affinity may exhibit about a 5-fold to about 1000-fold, e.g., about a 10-fold to about 500-fold improvement in Fc receptor binding affinity compared to the parent Fc polypeptide, as determined by one of skill in the art, for example, by the binding methods disclosed herein, including but not limited to, Biacore. Thus, as used herein, "reduced affinity" compared to the parent polypeptide means that the Fc variant binds to an Fc receptor with a significantly lower Kd than the parent Fc polypeptide. A or high K D It means that the Fc variants bind to the Fc receptor with an affinity of about 1.5 μM, or about 1500 nM. Greater or reduced affinity may also be defined relative to the absolute level of affinity. For example, according to the data herein, wild-type (natural) IgG1 binds to FcγRIIb with an affinity of about 1.5 μM, or about 1500 nM. Furthermore, some Fc variants described herein bind to FcγRIIb with an affinity that is about 10-fold greater than that of wild-type IgG1. As disclosed herein, greater or enhanced affinity refers to a K of less than about 100 nM, e.g., between about 10 nM and about 100 nM, between about 1 and about 100 nM, or less than about 1 nM. D It means having

[0119] In one embodiment, the Fc variant provides selectively enhanced affinity for FcγRIIb to one or more activating receptors. By selectively enhanced affinity, it is meant that either the Fc variant has improved affinity for FcγRIIb to the activating receptor compared to the parent Fc polypeptide, but has reduced affinity to the activating receptor compared to the parent Fc polypeptide, or the Fc variant has improved affinity to both FcγRIIb and the activating receptor compared to the parent Fc polypeptide, but the affinity improvement is greater for FcγRIIb than to the activating receptor. In alternative embodiments, the Fc variant reduces or ablates binding to one or more activating FcγRs, reduces or ablates binding to one or more complement proteins, reduces or ablates one or more FcγR-mediated effector functions, and / or reduces or ablates one or more complement-mediated effector functions.

[0120] The existence of different polymorphic forms of FcγR provides yet another parameter that influences the therapeutic utility of the Fc variants disclosed herein. The specificity and selectivity of a given Fc variant for different classes of FcγR significantly impacts the ability of the Fc variant to target a given antigen for the treatment of a given disease, but the specificity or selectivity of the Fc variant for different polymorphic forms of these receptors may in part determine which studies or preclinical experiments may be suitable for testing, and ultimately which patient populations are likely to respond to the treatment or not. Thus, the specificity or selectivity of the Fc variants disclosed herein for Fc receptor polymorphisms, including but not limited to FcγRIIa, FcγRIIIa, etc., may be used to guide the selection of effective studies and preclinical experiments, clinical trial design, patient selection, dose dependency, and / or other aspects involved in clinical trials.

[0121] Fc variants disclosed herein may include modifications that modulate interactions with Fc receptors other than FcγR, including but not limited to complement proteins, FcRn, and Fc receptor homologs (cRH), including but not limited to FcRH1, FcRH2, FcRH3, FcRH4, FcRH5, and FcRH6 (Davis et al., 2002, Immunol. Reviews 190:123-136).

[0122] The key parameter that determines the most beneficial selection of a given Fc variant for treating a given disease is the composition of the Fc variant. Thus, the Fc receptor selectivity or specificity of a given Fc variant provides different properties depending on whether it is composed of an antibody, an Fc fusion, or an Fc variant bound to a fusion partner. In one embodiment, the Fc receptor specificity of the Fc variant disclosed herein determines its therapeutic utility. The usefulness of a given Fc variant for therapeutic purposes depends on the epitope or form of the target antigen and the disease or indication being treated. In some targets and indications, greater FcγRIIb affinity and reduced activating FcγR-mediated effector function may be beneficial. In other target antigens and therapeutic applications, it may be beneficial to increase affinity to FcγRIIb, or to increase affinity to both FcγRIIb and activating receptors.

[0123] Inhibitory properties and CD32b + How to Inhibit Cells

[0124] The target antigen (CD19) of the immunoglobulin disclosed herein can be expressed on various cell types. In some embodiments, the immunoglobulin disclosed herein is specific for CD19 expressed on CD32b+ cells. The cell types that can be targeted by the immunoglobulin disclosed herein include, but are not limited to, B cells, plasma cells, dendritic cells, macrophages, neutrophils, mast cells, basophils, and eosinophils.

[0125] Disclosed herein is a method of inhibiting CD32b+ cells. Without being limiting, FIG. 3 is a schematic diagram of a proposed mechanism by which the immunoglobulins disclosed herein inhibit CD32b+ cells. Disclosed herein is thus a method of inhibiting CD32b+ cells, comprising contacting CD32b+ cells with an immunoglobulin comprising an Fc region with enhanced affinity for FcγRIIb. The immunoglobulin binds to at least two B cell proteins, e.g., at least two proteins that bind to the B cell surface. In one embodiment, the first of the B cell proteins is FcγRIIb. In another embodiment, the second of the B cell proteins is CD19. In some embodiments, the immunoglobulin inhibits calcium release from B cells upon their stimulation by the B cell receptor. In another embodiment, the immunoglobulins disclosed herein bind at least two B cell proteins on the same B cell surface (see FIG. 3).

[0126] Modifications to optimize inhibitory function

[0127] Disclosed herein are directed to immunoglobulins containing modifications, which alter the affinity for the FcγRIIb receptor and / or alter the ability of the immunoglobulin to mediate one or more FcγRIIb-mediated effector functions. The disclosed modifications include amino acid modifications and glycoform modifications.

[0128] As described herein, simultaneous high affinity co-engagement of cognate BCR and FcγRIIb can be used to inhibit FcγRIIb+ cells. Such co-engagement can occur through the use of immunoglobulins described herein, such as immunoglobulins used to co-engage both FcγRIIb via their Fc region, and target antigens (e.g., CD19) on the surface of FcγRIIb+ cells via their Fv region. Amino acid modifications at positions 234, 235, 236, 239, 267, 268, and 328 of the heavy chain constant region allow for modification of immunoglobulin FcγRIIb binding properties, effector functions, and potential clinical properties of the antibody.

[0129] In one embodiment, an immunoglobulin that binds to FcγRIIb+ cells and co-binds a target antigen on the cell surface with FcγRIIb on the cell surface as disclosed herein may be a variant immunoglobulin relative to a parent immunoglobulin. In one embodiment, the variant immunoglobulin comprises a variant Fc region, wherein the variant Fc region comprises one or more (e.g., two or more) modifications compared to the parent constant region, wherein the modification(s) are at a position selected from the group consisting of 234, 235, 236, 239, 267, 268, and 328, where the numbering is according to the EU index as in Kabat. In one embodiment, the variant Fc region comprises one or more (e.g., two or more) modifications compared to the parent constant region, wherein the modifications are at a position selected from the group consisting of 267 and 328, where the numbering is according to the EU index as in Kabat.

[0130] In one embodiment, the modification(s) is at least one substitution (e.g., one or more substitutions, two or more substitutions, etc.) selected from the group consisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N, 239D, 267D, 267E, 268E, 268D, 328F, and 328Y, where numbering is according to the EU index as in Kabat. In another embodiment, the modification(s) is at least one substitution selected from the group consisting of 235Y, 235R, 236D, 267D, 267E, and 328F. In one embodiment, the modification(s) is at least one substitution selected from the group consisting of 267E and 328F, where numbering is according to the EU index as in Kabat. In one embodiment, the modification(s) is at least one substitution selected from the group consisting of L234W, L235I, L235Y, L235R, L235D, G236D, G236N, S239D, S267D, S267E, H268E, H268D, L328F, and L328Y, where numbering is according to the EU index as in Kabat. In another embodiment, the modification(s) is at least one substitution selected from the group consisting of L235Y, L235R, G236D, S267D, S267E, and L328F. In one embodiment, the modification(s) is at least one substitution selected from the group consisting of S267E and L328F, where numbering is according to the EU index as in Kabat.

[0131] In another embodiment, the modification(s) is at least two modifications (e.g., a combination of modifications) at positions selected from the group consisting of 235 / 267, 236 / 267, 236 / 267, 267 / 328, and 268 / 267, where numbering is according to the EU index as in Kabat. In another embodiment, the modification(s) is at least two substitutions at positions 267 / 328, where numbering is according to the EU index as in Kabat. In a further embodiment, the modification(s) is at least two substitutions selected from the group consisting of 235D / 267E, 235Y / 267E, 235D / S267D, 235I / 267E, 235I / 267D, 235Y / 267D, 236D / 267E, 236D / 267D, 267E / 328F, 267D / 328F, 268D / 267E, 268D / 267D, 268E / 267E, and 268E / 267D, where the numbering is according to the EU index as in Kabat. In another embodiment, the modification(s) are at least two substitutions selected from the group consisting of 235D / 267E, 235Y / 267E, 235Y / 267D, 236D / 267E, 267E / 328F, 268D / 267E, 268E / 267E, and 268E / 267D, where numbering is according to the EU index as in Kabat. In one embodiment, the modification(s) are at least 267E / 328F, where numbering is according to the EU index as in Kabat. In another embodiment, the modification(s) are at least two substitutions selected from the group consisting of L235D / S267E, L235Y / S267E, L235D / S267D, L235I / S267E, L235I / S267D, L235Y / S267D, G236D / S267E, G236D / S267D, S267E / L328F, S267D / L328F, H268D / S267E, H268D / S267D, H268E / S267E, and H268E / S267D, where numbering is according to the EU index as in Kabat.In another embodiment, the modification(s) are at least two substitutions selected from the group consisting of L235D / S267E, L235Y / S267E, L235Y / S267D, G236D / S267E, S267E / L328F, H268D / S267E, H268E / S267E, and H268E / S267D, where numbering is according to the EU index as in Kabat. In one embodiment, the modification(s) is at least S267E / L328F, where numbering is according to the EU index as in Kabat.

[0132] In another embodiment, the modification(s) is at least three substitutions (e.g., combinations of modifications) at positions 236 / 267 / 328, where the numbering is according to the EU index as in Kabat. In another embodiment, the modification(s) is at least 236D / 267E / 328F. In another embodiment, the substitution means is at least G236D / S267E / L328F.

[0133] In some embodiments, the antibody may comprise an isotype modification, i.e., modification of the parent IgG to another IgG amino acid type. For example, as illustrated in FIG. 1, an IgG1 / IgG3 hybrid variant may be constructed by substituting IgG1 positions in the CH2 and / or CH3 regions with amino acids from IgG3 at positions where the two isotypes differ. Thus, a hybrid variant IgG antibody may be constructed that comprises one or more substitutions selected from the group consisting of 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 422I, 435R, and 436F. In other embodiments of the invention, an IgG1 / IgG2 hybrid variant may be constructed by substituting IgG2 positions in the CH2 and / or CH3 regions with amino acids from IgG1 at positions where the two isotypes differ. Thus, a hybrid variant gG antibody may be constructed that contains one or more modifications selected from the group consisting of 233E, 234L, 235L, -236G (referring to the insertion of a glycine at position 236), and 327A.

[0134] Means of Optimizing Effector Function

[0135] Immunoglobulins described herein include those comprising means for altering the affinity of an immunoglobulin to an Fc by modifying its ability to mediate one or more Fc receptors or for which the immunoglobulin has a γRIIb receptor or γRIIb-mediated effector function. The disclosed means include amino acid modifications (eg, optimized effector function position means, optimized effector function substitution means, etc.) and glycoform modifications (eg, glycoform modification means).

[0136] Amino acid modifications

[0137] As described herein, positional approaches to optimizing effector function include, but are not limited to, amino acid modifications at one or more of the heavy chain constant region positions 234, 235, 236, 239, 267, 268, and 328 that allow for modification of immunoglobulin FcγRIIb binding characteristics, effector function, and potential clinical properties.

[0138] In particular, substitution means that optimize FcγRIIb effector function, e.g., by altering affinity for FcγRIIb, include, but are not limited to, amino acid substitutions at one or more heavy chain constant region positions, e.g., one or more amino acid substitutions at heavy chain constant region positions 234, 235, 236, 239, 267, 268, and 328, where numbering is according to the EU index as in Kabat. In one embodiment, the substitution means(s) includes at least one substitution (e.g., one or more substitutions, two or more substitutions, etc.) compared to the parent Fc region, where the substitution is at a position selected from the group consisting of 267 and 328 according to the EU index as in Kabat. In one embodiment, the substitution means(s) is at least one substitution selected from the group consisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N, 239D, 267D, 267E, 268E, 268D, 328F, and 328Y, where the numbering is according to the EU index as in Kabat. In another embodiment, the substitution means(s) is at least one substitution selected from the group consisting of 235Y, 235R, 236D, 267D, 267E, and 328F, where the numbering is according to the EU index as in Kabat. In one embodiment, the substitution means(s) is at least one substitution selected from the group consisting of 267E and 328F, where the numbering is according to the EU index as in Kabat. In one embodiment, the substitution(s) is at least one substitution selected from the group consisting of L234W, L235I, L235Y, L235R, L235D, G236D, G236N, S239D, S267D, S267E, H268E, H268D, L328F, and L328Y, where the numbering is according to the EU index as in Kabat.In another embodiment, the substitution(s) is at least one substitution selected from the group consisting of L235Y, L235R, G236D, S267D, S267E, and L328F, where numbering is according to the EU index as in Kabat. In one embodiment, the substitution(s) is at least one substitution selected from the group consisting of S267E and L328F, where numbering is according to the EU index as in Kabat.

[0139] In another embodiment, the substitution means(s) are at least two substitutions (e.g., combinations of modifications) at positions 235 / 267, 236 / 267, 236 / 267, 267 / 328, and 268 / 267, where numbering is according to the EU index as in Kabat. In another embodiment, the substitution means(s) are at least two substitutions at positions 267 / 328, where numbering is according to the EU index as in Kabat. In a further embodiment, the substitution means or means is at least two substitutions at positions 235D / 267E, 235Y / 267E, 235D / S267D, 235I / 267E, 235I / 267D, 235Y / 267D, 236D / 267E, 236D / 267D, 267E / 328F, 267D / 328F, 268D / 267E, 268D / 267D, 268E / 267E, and 268E / 267D, where the numbering is according to the EU index as in Kabat. In another embodiment, the substitution(s) are at least two substitutions at positions 235D / 267E, 235Y / 267E, 235Y / 267D, 236D / 267E, 267E / 328F, 268D / 267E, 268E / 267E, and 268E / 267D, where numbering is according to the EU index as in Kabat. In one embodiment, the substitution(s) are at least 267E / 328F, where numbering is according to the EU index as in Kabat. In another embodiment, the substitution means or means is at least two substitutions at positions L235D / S267E, L235Y / S267E, L235D / S267D, L235I / S267E, L235I / S267D, L235Y / S267D, G236D / S267E, G236D / S267D, S267E / L328F, S267D / L328F, H268D / S267E, H268D / S267D, H268E / S267E, and H268E / S267D, where the numbering is according to the EU index as in Kabat.In another embodiment, the substitution(s) are at least two substitutions at positions L235D / S267E, L235Y / S267E, L235Y / S267D, G236D / S267E, S267E / L328F, H268D / S267E, H268E / S267E, and H268E / S267D, where numbering is according to the EU index as in Kabat. In one embodiment, the substitution(s) are at least S267E / L328F, where numbering is according to the EU index as in Kabat.

[0140] In another embodiment, the substitution means(s) is at least three substitutions (e.g., combinations of modifications) at positions 236 / 267 / 328, where numbering is according to the EU index as in Kabat. In another embodiment, the substitution means(s) is at least 236D / 267E / 328F, where numbering is according to the EU index as in Kabat. In another embodiment, the substitution means(s) is at least G236D / S267E / L328F, where numbering is according to the EU index as in Kabat.

[0141] Glycoform modification

[0142] Many polypeptides, including antibodies, undergo a variety of post-translational modifications, including glycosylation with oligosaccharides. There are several factors that can affect glycosylation. Species, tissues, and cell types have all been shown to be important in how glycosylation occurs. In addition, the extracellular environment, through alterations in culture conditions such as serum concentration, has a direct effect on glycosylation (Lifely et al., 1995, Glycobiology 5(8):813-822).

[0143] All antibodies contain sugars at conserved positions in the constant region of the heavy chain. Each antibody isotype has a distinct variety of N-linked sugar structures. Apart from the sugars attached to the heavy chain, up to 30% of human IgGs have glycosylated Fab regions. IgGs have a single linked biantennary sugar at Asn297 in the CH2 domain. Whether IgG is produced from serum or extracellularly in hybridomas or engineered cells, IgGs are heterogeneous with respect to the Asn297 linked sugar (Jefferis et al., 1998, Immunol. Rev. 163:59-76; Wright et al., 1997, Trends Biotech 15:26-32). In human IgG, the core oligosaccharide is usually GlcNAc with different numbers of outer residues. 2 Man 3 It consists of GlcNAc.

[0144] The carbohydrate moieties of the immunoglobulins disclosed herein are described with reference to the nomenclature commonly used to describe oligosaccharides. A review of carbohydrate chemistry using this nomenclature can be found in Hubbard et al. 1981, Ann. Rev. Biochem. 50:555-583. This nomenclature includes, for example, Man for mannose, GlcNAc for 2-N-acetylglucosamine, Gal for galactose, Fuc for fucose, and Glc for glucose. Sialic acids are described by the abbreviations NeuNAc for 5-N-acetylneuraminic acid, and NeuNGc for 5-glycolylneuraminic acid.

[0145] "Glycosylation" refers to the attachment of oligosaccharides (saccharides containing two or more monosaccharide linkages, e.g., 2 to about 12 monosaccharide linkages) to a glycoprotein. The oligosaccharide side chains are typically linked to the glycoprotein backbone through either N- or O-linkages. The oligosaccharides of the immunoglobulins disclosed herein are generally attached to the CH2 domain of the Fc region as N-linked oligosaccharides. "N-linked glycosylation" refers to the attachment of a sugar chain to an asparagine residue in the glycoprotein chain. One of skill in the art will recognize that, for example, each of mouse IgG1, IgG2a, IgG2b, and IgG3, and human IgG1, IgG2, IgG3, IgG4, IgA, and IgD CH2 domains have a single site for N-linked glycosylation at amino acid residue 297 (Kabat et al. Sequences of Proteins of Immunological Interest, 1991).

[0146] For purposes herein, "mature core glycostructure" refers to a processed core glycostructure attached to an Fc region, typically consisting of the following glycostructure: GlcNAc(Fucose)-GlcNAc-Man-(Man-GlcNAc)2, typical of biantennary oligosaccharides. The mature core glycostructure is typically attached to an Fc region of a glycoprotein via an N-linkage to Asn297 of the CH2 domain of the Fc region. A "bisecting GlcNAc" is a GlcNAc residue that is attached to the β1,4 mannose of the mature core glycostructure. A bisecting GlcNAc can be enzymatically attached to the mature core glycostructure by the β(1,4)-N-acetylglucosaminyltransferase III enzyme (GnTIII). CHO cells do not normally express GnTIII (Stanley et al., 1984, J. Biol. Chem. 261:13370-13378), but can be engineered to do so (Umana et al., 1999, Nature Biotech. 17:176-180).

[0147] Fc variants including modified or engineered glycoforms are described herein. As used herein, "modified glycoform" or "engineered glycoform" refers to a sugar composition that is covalently attached to a protein, e.g., an antibody, that is chemically different from that of the parent protein. Engineered glycoforms may be useful for a variety of purposes, including, but not limited to, enhancing or reducing FcγR-mediated effector functions. In one embodiment, the immunoglobulins disclosed herein are modified to control the level of fucosylation and / or bisected oligosaccharides that are covalently attached to the Fc region.

[0148] Various methods for producing modified glycoforms are well known in the art (Umana et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473), (US 6,602,684, US Publication No. 2003 / 0157108; US Publication No. 2003 / 0003097, PCT WO 00 / 61739A1, PCT WO 01 / 29246A1, PCT WO 02 / 31140A1, PCT WO 02 / 30954A1), (Potelligent® technology [Biowa, Inc., Princeton, NJ], GlycoMAb® glycosylation engineering technology [GLYCART biotechnology AG, Zurich, Switzerland], all expressly incorporated by reference). These techniques control the levels of fucosylation and / or bisected oligosaccharides covalently attached to the Fc region, for example, by expressing IgG in engineered different organisms or cell lines (e.g., Lec-13 CHO cells, or rat hybridoma YB2 / 0 cells), or alternatively by modulating enzymes involved in the glycosylation pathway (e.g., FUT8 [α1,6-fucosyltransferase] and / or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by modifying the sugar(s) after IgG is expressed. Other methods for modifying glycoforms of the immunoglobulins disclosed herein include the use of glycoengineered yeast strains (Li et al., 2006, Nature Biotechnology 24(2):210-215), mosses (Nechansky et al., 2007, Mol Immunjol 44(7):1826-8), and plants (Cox et al., 2006, Nat Biotechnol 24(12):1591-7).The use of a particular method to generate a modified glycoform does not limit the embodiment to that method, rather, the embodiments disclosed herein encompass Fc variants with modified glycoforms regardless of how they are generated.

[0149] In one embodiment, the immunoglobulins disclosed herein are glycoengineered to alter the level of sialylation. High levels of sialylated Fc glucans in immunoglobulin G molecules can adversely affect functionality (Scallon et al., 2007, Mol Immunol. 44(7):1524-34), and differences in the level of Fc sialylation can result in altered anti-inflammatory activity (Kaneko et al., 2006, Science 313:670-673). Because antibodies can acquire anti-inflammatory properties upon sialylation of the Fc core polysaccharide, it can be beneficial to glycoengineer the immunoglobulins disclosed herein to increase or decrease the Fc sialic acid content.

[0150] An engineered glycoform typically refers to a different sugar or oligosaccharide, thus, for example, an immunoglobulin may comprise an engineered glycoform. Alternatively, an engineered glycoform may refer to an immunoglobulin that comprises a different sugar or oligosaccharide. In one embodiment, a composition disclosed herein comprises a glycosylated Fc variant having an Fc region, wherein about 51-100% of the glycosylated antibodies, e.g., about 80-100%, 90-100%, 95-100%, etc., of the antibodies in the composition comprise a mature core glycostructure lacking fucose. In another embodiment, an antibody in the composition both comprises a mature core glycostructure lacking fucose and further comprises at least one amino acid modification in the Fc region. In an alternative embodiment, the composition comprises a glycosylated Fc variant having an Fc region, wherein about 51-100% of the glycosylated antibodies, e.g., about 80-100% or 90-100% of the antibodies in the composition, comprise a mature core glycostructure lacking sialic acid. In another embodiment, the antibodies in the composition comprise a mature core glycostructure lacking sialic acid and further comprise at least one amino acid modification in the Fc region, or both. In yet another embodiment, the composition comprises a glycosylated Fc variant having an Fc region, wherein about 51-100% of the glycosylated antibodies, e.g., about 80-100% or 90-100% of the antibodies in the composition, comprise a mature core glycostructure containing sialic acid. In another embodiment, the antibodies in the composition comprise a mature core glycostructure containing sialic acid and further comprise at least one amino acid modification in the Fc region, or both. In another embodiment, the combination of engineered glycoforms and amino acid modifications provides the antibody with optimal Fc receptor binding properties.

[0151] Other Modifications

[0152] The immunoglobulins disclosed herein may contain one or more modifications that provide optimized properties that are not specifically related to FcγR or complement-mediated function itself. The modifications may be amino acid modifications or modifications that are enzymatically or chemically performed. Such modifications may provide some improvement to the immunoglobulin, such as, for example, enhancing its stability, solubility, or clinical use. Various improvements are disclosed herein that may be made by combining the immunoglobulins disclosed herein with further modifications.

[0153] In one embodiment, the variable regions of the antibodies disclosed herein can be affinity matured, i.e., amino acid modifications can be made in the VH and / or VL domains of the antibody to enhance the binding of the antibody to its target antigen. Such types of modifications can improve the association and / or dissociation kinetics of binding to the target antigen. Other modifications include those that improve selectivity for the target antigen over alternative targets. These include modifications that improve selectivity for antigens expressed on the target over non-target cells. Other improvements to the target recognition properties can be provided by further modifications. Such properties may include, but are not limited to, specific kinetic properties (i.e., association and dissociation kinetics), selectivity for a particular target over alternative targets, and selectivity for a particular target form over alternative forms. Examples include selectivity for full-length versus splice variants, soluble forms over cell surfaces, various polymorphic variants, or specific conformational forms of the target antigen. The immunoglobulins disclosed herein may include one or more modifications that provide reduced or enhanced internalization of the immunoglobulin.

[0154] In one embodiment, modifications are made to improve the biophysical properties of the immunoglobulins disclosed herein, including, but not limited to, stability, solubility, and oligomeric state. Modifications can include, for example, substitutions that provide more favorable intramolecular interactions of the immunoglobulin to provide higher stability, or replacement of exposed non-polar amino acids with polar amino acids for higher solubility. Numerous optimization goals and methods are described in U.S. Patent Publication No. 2004 / 0110226 (incorporated by reference in its entirety), which may find use in engineering additional modifications to further optimize the immunoglobulins disclosed herein. The immunoglobulins disclosed herein can also be combined with additional modifications that reduce the multimerization state or size to enhance tumor presentation or, if desired, increase in vivo clearance rate.

[0155] Other modifications to immunoglobulins disclosed herein include those that allow for the specific formation of homodimeric or homomultimeric molecules. Such modifications include, but are not limited to, engineered disulfides as well as chemical modification or aggregation methods that can provide mechanisms for generating covalent homodimeric or homomultimeric molecules. For example, methods and compositions for engineering such molecules are described in Kan et al., 2001, J. Immunol., 2001, 166: 1320-1326; Stevenson et al., 2002, Recent Results Cancer Res. 159: 104-12; US 5,681,566; Caron et al., 1992, J Exp Med 176:1191-1195 and Shopes, 1992, J Immunol 148(9):2918-22 (all of which are incorporated by reference in their entirety). Further modifications to the variants disclosed herein include those that allow specific formation of heterodimeric, heteromultimeric, bifunctional and / or multifunctional molecules. Such modifications include, but are not limited to, one or more amino acid substitutions in the CH3 domain, where the substitutions reduce homodimer formation and increase heterodimer formation. For example, methods and compositions for engineering such molecules are described in Atwell et al., 1997, J Mol Biol 270(1):26-35 and Carter et al., 2001, J Immunol Methods 248:7-15 (both of which are incorporated by reference in their entirety). Further modifications include modifications in the hinge and CH3 domain, where the modifications reduce the propensity to form dimers.

[0156] In further embodiments, the immunoglobulins disclosed herein include modifications that remove proteolytic sites. These may include, for example, protease sites that reduce production yields, as well as proteases that degrade the administered protein in vivo. In one embodiment, further modifications are made to remove covalent degradation sites, such as deamidation (i.e., deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues), oxidation, and proteolytic sites. Deamidation sites that are particularly useful for removal are those that enhance the propensity for deamidation, including, but not limited to, asparaginyl and glutamyl residues followed by glycine (NG and QG motifs, respectively). In such cases, substitution of either residue can significantly reduce the propensity for deamidation. Common oxidation sites include methionine and cysteine ​​residues. Other covalent modifications which may be introduced or removed include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the "-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983), incorporated by reference in its entirety), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group. Further modifications may also include post-translational modifications, such as, but not limited to, N- or O-linked glycosylation and phosphorylation.

[0157] Modifications may include those that improve expression and / or purification yields obtained from hosts or host cells commonly used in the production of biological products. These include, but are not limited to, various mammalian cell lines (e.g., CHO), yeast cell lines, bacterial cells, and plants. Further modifications include modifications that eliminate or reduce the ability of the heavy chains to form interchain disulfide bonds. Further modifications include modifications that eliminate or reduce the ability of the heavy chains to form intrachain disulfide bonds.

[0158] The immunoglobulins disclosed herein may include modifications including the use of unnatural amino acids, incorporated using techniques developed by Schultz and coworkers, including, but not limited to, those described in Cropp & Shultz, 2004, Trends Genet. 20(12):625-30; Anderson et al., 2004, Proc. Natl. Acad. Sci. USA 101(2):7566-71; Zhang et al., 2003, 303(5656):371-3; and Chin et al., 2003, Science 301(5635):964-7, all of which are incorporated by reference in their entirety. In some embodiments, these modifications allow for the manipulation of various functional, biophysical, immunological, or manufacturing properties as described above. In further embodiments, these modifications allow for further chemical modifications for other purposes. Other modifications are contemplated herein. For example, immunoglobulins may be linked to one of a variety of nonproteinaceous polymers, such as, for example, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. Further amino acid modifications may be made to allow for specific or nonspecific chemical or post-translational modifications of the immunoglobulin. Such modifications include, but are not limited to, pegylation and glycosylation. Specific substitutions that may be utilized to allow for pegylation include, but are not limited to, the introduction of a novel cysteine ​​residue or a non-natural amino acid such that efficient and specific coupling chemistries can be used to attach PEG or otherwise polymeric moieties. The introduction of a specific glycosylation site may be achieved by introducing a novel NXT / S sequence into the immunoglobulins disclosed herein.

[0159] Modifications to reduce immunogenicity may include modifications that reduce the binding of processed peptides derived from parental sequences to MHC proteins. For example, amino acid modifications may be engineered to have no or a minimal number of immune epitopes that are predicted to bind any common MHC alleles with high affinity. Several methods of identifying MHC-binding epitopes in protein sequences are known in the art and may be used to score epitopes for the antibodies disclosed herein. See, for example, U.S. Patent Publication Nos. 2002 / 0119492, 2004 / 0230380, and 2006 / 0148009, all of which are expressly incorporated by reference, and the references cited therein.

[0160] In some embodiments, the immunoglobulins disclosed herein may be combined with immunoglobulins that alter FcRn binding. Such variants may provide the immunoglobulin with improved pharmacokinetic properties. In particular, variants that increase Fc binding to FcRn include, but are not limited to, 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356, US11 / 102621, PCT / US2003 / 033037, PCT / US2004 / 011213, US10 / 822300, US10 / 687118, PCT / US2004 / 034440, US10 / 966673, all of which are incorporated by reference in their entireties), 256A, 272A, 286A, 305A, 307A, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al, Journal of Biological Chemistry, 2001, 276(9):6591-6604, US 10 / 982470, U.S. Patent No. 6,737,056, US 11 / 429793, US 11 / 429786, PCT / US2005 / 029511, US 11 / 208422, which are incorporated by reference in their entireties), 252F, 252T , 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S, 433R, 433S, 433 I, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 311 S(Dall Acqua et al. Journal of Immunology,2002,169:5171-5180, U.S. Patent No. 7,083,784, PCT / US97 / 03321, U.S. Patent No. 6,821,505, PCT / US01 / 48432, USNo. 11 / 397328, incorporated by reference in its entirety), 257C, 257M, 257L, 257N, 257Y, 279E, 279Q, 279Y, the insertion of Ser after 281, 283F, 284E, 306Y, 307V, 308F, 308Y, 311V, 385H, 385N, (PCT / US2005 / 041220, US No. 11 / 274065, US No. 11 / 436,266, incorporated by reference in their entireties), 204D, 284E, 285E, 286D, and 290E (PCT / US2004 / 037929, incorporated by reference in their entireties).

[0161] Covalent modifications of antibodies are included within the scope of the immunoglobulins disclosed herein and are generally, but not necessarily, carried out post-translationally. For example, some types of covalent modifications of antibodies are introduced into the molecule by reacting specific amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains, or with the N- or C-terminal residues.

[0162] In some embodiments, the covalent modification of the antibodies disclosed herein comprises the addition of one or more labels. The term "label group" refers to any detectable label. In some embodiments, the label group is attached to the antibody by a spacer arm of various lengths to reduce potential steric hindrance. Various methods of labeling proteins are known in the art and can be used to generate the immunoglobulins disclosed herein. Generally, labels are classified into various types depending on the assay in which they are detected: b) isotopic labels, which can be radioisotopes or heavy isotopes; c) redox-active moieties; d) optical dyes; enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylation groups; and f) predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.). In some embodiments, the labeling group is attached to the antibody by spacer arms of various lengths to reduce potential steric hindrance. A variety of methods for labeling proteins are known in the art and can be used to generate the immunoglobulins disclosed herein. Specific labels include optical dyes, including but not limited to chromophores, phosphorescent materials, and fluorophores, the latter being specific in many cases. Fluorophores can be either "small molecule" fluorophores or proteinaceous fluorophores. By "fluorescent label" is meant any molecule that can be detected by its inherent fluorescent properties.

[0163] Complex

[0164] In one embodiment, the immunoglobulins disclosed herein are antibody "fusion proteins", sometimes also referred to as "antibody conjugates". The fusion or conjugation partners can be proteinaceous or non-proteinaceous, the latter generally being generated using functional groups on the antibody and the conjugation partner. Conjugation and fusion partners can be any molecule, including small molecule chemical compounds and polypeptides. For example, various antibody conjugates and methods are described in Trail et al., 1999, Curr. Opin. Immunol. 11:584-588, which is incorporated by reference in its entirety. Possible conjugation partners include, but are not limited to, cytokines, cytotoxic agents, toxins, radioisotopes, chemotherapeutic agents, anti-angiogenic agents, tyrosine kinase inhibitors, and other therapeutically active agents. In some embodiments, the conjugation partner may be considered more of a payload, i.e., the purpose of the conjugate is targeted delivery of the conjugation partner to a target cell, e.g., a cancer cell or an immune cell, by the immunoglobulin. Thus, for example, conjugation of immunoglobulins with toxins targets the delivery of the toxins to cells expressing the target antigen.As will be understood by those skilled in the art, in practice, the concepts and definitions of fusion and conjugation overlap.The meaning of fusion or conjugation does not limit it to any particular embodiment disclosed herein.Rather, these terms are used loosely to convey the broad concept that any immunoglobulin disclosed herein may be genetically, chemically, or otherwise linked to one or more polypeptides or molecules to provide certain desired properties.

[0165] Suitable conjugates include, but are not limited to, drugs and cytotoxic agents, including, but not limited to, labels, cytotoxic drugs (e.g., chemotherapeutic agents), or toxins or active fragments of such toxins, as described below. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, and the like. Cytotoxic agents also include radiochemicals, made by conjugating a radioisotope to an antibody or by binding a radionuclide to a chelator that is covalently attached to an antibody. Further embodiments utilize calicheamicins, auristatins, geldanamycins, maytansines, and duocarmycins and analogs; regarding the latter, see US2003 / 0050331, incorporated by reference in its entirety.

[0166] In one embodiment, the immunoglobulins disclosed herein are fused or conjugated to cytokines. As used herein, "cytokine" is a general term for a protein released by one cell population that acts on another cell as an intercellular mediator. For example, cytokines can be fused to antibodies to provide a set of desired properties, as described in Penichet et al., 2001, J.Immunol.Methods 248:91-101, which is incorporated by reference in its entirety. Examples of such cytokines include lymphokines, monokines, and conventional polypeptide hormones. Growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; glycoprotein hormones such as parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle-stimulating hormone (TSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor α and β, Mullerian inhibitory factor, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrins, thrombopoietin (TPO), nerve growth factors such as NGF-β, platelet growth factor, transforming growth factors (TGF-α and TGF-β), Included among the cytokines are: GF, insulin-like growth factors I and II, erythropoietin (EPO), osteogenic factor, interferons such as interferon alpha, beta, and gamma, colony stimulating factors (CSF) such as macrophage CSF (M-CSF), granulocyte macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF), interleukins (IL) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, tumor necrosis factors such as TNF alpha or TNF beta, C5a, and other polypeptide factors including LIF and kit ligand (KL). As used herein, cytokines include proteins from natural sources or recombinant cell culture, and biologically active equivalents of the native sequence cytokines.

[0167] In another embodiment, the immunoglobulins disclosed herein are fused, conjugated or operably linked to toxins, including but not limited to small molecule toxins of bacterial, fungal, plant or animal origin and enzymatically active toxins, including fragments and / or variants thereof. For example, various immunotoxins and immunotoxin methods are described in Thrush et al., 1996, Ann. Rev. Immunol. 14:49-71, incorporated by reference in its entirety. Small molecule toxins include, but are not limited to, calicheamicin, maytansine (US 5,208,020, incorporated by reference in its entirety), trichothene and CC1065. In certain embodiments, the immunoglobulins disclosed herein may be conjugated to one or more maytansine molecules (e.g., about 1 to about 10 maytansine molecules per antibody molecule). Maytansine can be converted, for example, to May-SS-Me, which can be reduced to May-SH3 and reacted with a modified antibody (Chari et al., 1992, Cancer Research 52: 127-131, incorporated by reference in its entirety) to generate a maytansinoid-antibody. Other conjugates of interest include immunoglobulins linked to one or more calicheamicin molecules. The calicheamicin family of antibiotics has the ability to produce double-stranded DNA breaks at sub-picomolar concentrations. Calicheamicin structural analogs that can be used include γ- 1 1 , α 2 1 , α 3 , N-acetyl-γ 1 1 , PSAG, and θ 1 1(Hinman et al., 1993, Cancer Research 53:3336-3342; Lode et al., 1998, Cancer Research 58:2925-2928) (US 5,714,586; US 5,712,374; US 5,264,586; US 5,773,001, all of which are incorporated by reference in their entireties). Dolastatin 10 analogs such as auristatin E (AE) and monomethylauristatin E (MMAE) may find use as conjugates for the immunoglobulins disclosed herein (Doronina et al., 2003, Nat Biotechnol 21(7):778-84; Francisco et al., 2003 Blood 102(4):1458-65, both of which are incorporated by reference in their entireties). Useful enzymatically active toxins include, but are not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the trichothecenes. See, eg, PCT WO93 / 21232, which is incorporated by reference in its entirety.Embodiments further include conjugates between the immunoglobulins disclosed herein and a compound with nucleolytic activity (e.g., a ribonuclease, or a DNA endonuclease such as a deoxyribonuclease (DNase)).

[0168] In alternative embodiments, the immunoglobulins disclosed herein can be fused, conjugated or operably linked to radioisotopes to form radioconjugates.A variety of radioactive isotopes are available for producing radioconjugate antibodies.Examples include, but are not limited to, At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and Lu radioisotopes.See, for example, references.

[0169] In yet another embodiment, the immunoglobulins disclosed herein may be conjugated to a "receptor" (such as streptavidin) for use in tumor pretargeting. In that case, the immunoglobulin-receptor conjugate is administered to the patient, followed by the use of a clearing agent to remove unbound conjugate from the circulation, followed by administration of a "ligand" (such as avidin) conjugated to a cytotoxic agent (such as a radionucleotide). In another embodiment, the immunoglobulin is conjugated or operably linked to an enzyme for use in antibody-dependent enzyme-mediated prodrug therapy (ADEPT). ADEPT may be used by conjugating or operably linking the immunoglobulin to a prodrug-activating enzyme that converts a prodrug (such as a peptide chemotherapeutic agent, see PCT WO81 / 01145, which is incorporated by reference in its entirety) into an active anticancer drug. See, for example, PCT WO88 / 07378 and US4,975,278, both of which are incorporated by reference in their entirety. The enzyme component of an immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way as to convert it into its more active, cytotoxic form.Enzymes useful in the methods disclosed herein include alkaline phosphatases, useful for converting phosphate-containing prodrugs to free drugs; arylsulfatases, useful for converting sulfate-containing prodrugs to free drugs; cytosine deaminases, useful for converting the non-toxic 5-fluorocytosine to the anticancer drug 5-fluorouracil; proteases, such as Serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), useful for converting peptide-containing prodrugs to free drugs; Examples of suitable enzymes include, but are not limited to, D-alanyl carboxypeptidases, useful for converting prodrugs to free active drugs; carbohydrate cleaving enzymes, such as beta-galactosidase and neuramimidase, useful for converting glycosylated prodrugs to free drugs; beta-lactamases, useful for converting drugs derivatized with alpha-lactams to free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at the amine nitrogen with a phenoxyacetyl or phenylacetyl group, respectively, to free drugs. Alternatively, antibodies with enzymatic activity (also known in the art as "antibody enzymes") can be used to convert the prodrugs disclosed herein to free active drugs (see, e.g., Massey, 1987, Nature 328: 457-458, incorporated by reference in its entirety). Immunoglobulin-antibody enzyme conjugates can be prepared to deliver the antibody enzyme to tumor cell populations. A variety of additional conjugates are contemplated to the immunoglobulins disclosed herein. A variety of chemotherapeutic agents, antiangiogenic agents, tyrosine kinase inhibitors and other therapeutic agents are described below that find use as immunoglobulin conjugates.

[0170] The complex partner may be linked to any region of the immunoglobulin disclosed herein, including the N- or C-terminus, or some residue between the termini. A variety of linkers may find use with the immunoglobulins disclosed herein to covalently link the complex partner to the immunoglobulin. As used herein, "linker," "linker sequence," "spacer," "tether sequence," or grammatical equivalents thereof, refers to a molecule or group of molecules (such as a monomer or polymer) that serves to link two molecules together and often to arrange the two molecules into one structure. Linkers are known in the art, for example, homo- or heterobifunctional linkers are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, which is incorporated by reference in its entirety). Several methods can be used to covalently link molecules together. These include, but are not limited to, polypeptide linkages between the N- and C-terminus of a protein or protein domain, linkages via disulfide bonds, and linkages via chemical cross-linking reagents. In one aspect of this embodiment, the linker is a peptide bond, generated by recombinant technology or peptide synthesis. The linker peptide may primarily comprise the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should be of suitable length to link two molecules in a manner that allows them to assume the appropriate conformation relative to one another so as to maintain the desired activity. A suitable length for this purpose comprises at least one and not more than 50 amino acid residues. In one embodiment, the linker is about 1-30 amino acids in length, for example, the linker may be 1-20 amino acids in length. Useful linkers include glycine-serine polymers (e.g., including (GS), (GSGGS) (as depicted in SEQ ID NO: 1), (GGGGS) (as depicted in SEQ ID NO: 2), and (GGGS) (as depicted in SEQ ID NO: 3), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers, as will be appreciated by those of skill in the art.Alternatively, a variety of non-proteinaceous polymers may find use as linkers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.

[0171] Immunoglobulin production

[0172] Methods for producing and experimentally testing immunoglobulins are also disclosed herein. The disclosed methods do not limit the embodiments to any particular application or theory of operation. Rather, the methods provided generally exemplify and explain how one or more immunoglobulins can be produced and experimentally tested to obtain immunoglobulins. General methods for antibody molecular biology, expression, production, and screening are described in Antibody Engineering, edited by Duebel & Kontermann, Springer-Verlag, Heidelberg, 2001, and in Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; Antibodies: A Laboratory Manual by Harlow & Lane, New York: Cold Spring Harbor Laboratory Press, 1988, all of which are incorporated by reference in their entirety.

[0173] In one embodiment disclosed herein, nucleic acids are generated that encode immunoglobulins and can then be cloned, expressed, and measured in a host cell, if desired. Thus, nucleic acids, and in particular DNA, that encode each protein sequence can be generated. These practices are carried out using well-known procedures. For example, various methods that can find use in the production of immunoglobulins disclosed herein are described in Molecular Cloning - A Laboratory Manual, 3. rdEd. (Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001), and Current Protocols in Molecular Biology (John Wiley & Sons), both of which are incorporated by reference in their entirety. As will be appreciated by those skilled in the art, the generation of accurate sequences of libraries containing many sequences can be expensive and time-consuming. As used herein, a "library" refers to a collection of variants in any form, including, but not limited to, a list of nucleic acid or amino acid sequences, a list of nucleic acids or amino acids at various positions, a physical library containing nucleic acids encoding library sequences, or a physical library containing variant proteins in either purified or unpurified form. Thus, there are a variety of techniques that can be used to effectively generate the libraries disclosed herein. Methods that may find use for making the immunoglobulins disclosed herein are described or referenced in US6,403,312, US Publication No. 2002 / 0048772; US7,315,786; US Publication No. 2003 / 0130827; PCT WO01 / 40091; and PCT WO02 / 25588, all of which are incorporated by reference in their entirety. Such methods include, but are not limited to, gene assembly methods, PCR-based methods and methods using variations of PCR, ligase chain reaction-based methods, synthetic recombination, error-prone amplification methods, and mixed oligo methods such as methods using oligos with random mutations, classical site-directed mutagenesis, cassette mutagenesis, and other amplification and gene synthesis methods. As is known in the art, there are a variety of commercially available kits and methods for gene assembly, mutagenesis, vector subcloning, and the like, and such commercially available products find use in generating nucleic acids encoding immunoglobulins.

[0174] The immunoglobulins disclosed herein can be produced by culturing a host cell transformed with a nucleic acid, e.g., an expression vector, containing a nucleic acid encoding the immunoglobulin, under appropriate conditions that induce or result in expression of the protein. Appropriate conditions for expression will vary with the choice of expression vector and host cell, and will be readily ascertained by one of ordinary skill in the art through routine experimentation. A wide variety of suitable host cells may be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. For example, various cell lines that may find use in producing the immunoglobulins disclosed herein are described in the ATCC® cell line catalog available from the American Type Culture Collection.

[0175] In one embodiment, the immunoglobulins are expressed in a mammalian expression system, including systems in which the expression construct is introduced into mammalian cells using a virus, such as a retrovirus or adenovirus. Any mammalian cell can be used, such as human, mouse, rat, hamster, and primate cells. Suitable cells also include known research cells, including, but not limited to, Jurkat T cells, NIH3T3, CHO, BHK, COS, HEK293, PER C.6, HeLa, Sp2 / 0, NS0 cells, and variants thereof. In an alternative embodiment, the library proteins are expressed in bacterial cells. Bacterial expression systems are well known in the art and include E. coli, Bacillus subtilis, Streptococcus cremoris, and Streptococcus lividans. In alternative embodiments, immunoglobulins are produced in insect cells (e.g., Sf21 / Sf9, Saccharomyces cerevisiae Bti-Tn5b1-4) or yeast cells (e.g., Saccharomyces cerevisiae, Pichia, etc.). In alternative embodiments, immunoglobulins are expressed in vitro using cell-free translation systems. In vitro translation systems derived from both prokaryotic (e.g., E. coli) and eukaryotic (malt germ, rabbit reticulocyte) cells are available and may be selected based on the expression levels and functional properties of the protein of interest. For example, as will be appreciated by those skilled in the art, in vitro translation is required for some display technologies, such as, for example, ribosome display. In addition, immunoglobulins may be produced by chemical synthesis methods, as well as transformation expression systems from both animals (e.g., cow, sheep, or goat milk, embryonated chicken eggs, whole insect larvae, etc.) and plants (e.g., corn, tobacco, duckweed, etc.).

[0176] The nucleic acids encoding the immunoglobulins disclosed herein may be introduced into an expression vector to express the protein. A variety of expression vectors may be utilized for protein expression. The expression vector may include a self-replicating extrachromosomal vector or a vector that integrates into the host genome. The expression vector is constructed to be compatible with the host cell type. Thus, expression vectors that find use in the production of the immunoglobulins disclosed herein include, but are not limited to, those that allow protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems. As is known in the art, a variety of expression vectors are commercially or otherwise available that may find use in expressing the immunoglobulins disclosed herein.

[0177] An expression vector typically includes a protein operably linked to a control or regulatory sequence, a selectable marker, any fusion partner, and / or additional elements. As used herein, "operably linked" means that a nucleic acid is placed in a functional relationship with another nucleic acid sequence. Generally, these expression vectors include transcriptional and translational regulatory nucleic acids operably linked to the nucleic acid encoding an immunoglobulin, and are typically suitable for the host cell used to express the protein. In general, the transcriptional and translational regulatory sequences may include promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and transcriptional enhancer or activator sequences. Also, as known in the art, an expression vector typically includes a selection gene or marker that allows the selection of transformed host cells that contain the expression vector. Selection genes are well known in the art and vary depending on the host cell used.

[0178] The immunoglobulin may be operably linked to a fusion partner to allow for targeting, purification, screening, presentation, etc. of the expressed protein. The fusion partner may be linked to the immunoglobulin sequence via a linker sequence. The linker sequence generally comprises a small number of amino acids, typically less than 10 amino acids, although longer linkers may also be used. Typically, the linker sequence is selected to be flexible and resistant to degradation. As will be appreciated by those of skill in the art, any of a wide variety of sequences may be used as linkers. For example, a common linker sequence comprises the amino acid sequence GGGGS. The fusion partner may be a target or signal sequence that directs the immunoglobulin and any associated fusion partner to a desired cellular location or to the extracellular medium. As is known in the art, certain signaling sequences may target proteins to be secreted into either the growth medium or the periplasmic space located between the inner and outer membranes of the cell. The fusion partner may also be a sequence that encodes a peptide or protein, allowing for purification and / or screening. Such fusion partners include, but are not limited to, polyhistidine tags (His-tags) (e.g., H6 and H10 for use in immobilized metal affinity chromatography (IMAC) systems, or other tags (e.g., Ni +2 Examples of tags include those that are targeted by antibodies, such as antibodies that are targeted by antibodies against a specific antigen ... +2 The His tag may be used to generate antibodies against Ni by immobilizing them on an affinity column, and then after purification, the same His tag can be used to bind Ni to perform ELISA or other binding assays (as described below). +2The fusion partner may be used to immobilize the immunoglobulin on a coated plate. The fusion partner may allow the use of selection methods to screen the immunoglobulins (see below). Fusion partners that allow various selection methods are well known in the art. For example, phage display methods can be employed by fusing members of an immunoglobulin library to gene III proteins (Kay et al., Phage display of peptides and proteins: a laboratory manual, Academic Press, San Diego, CA, 1996; Lowman et al., 1991, Biochemistry 30: 10832-10838; Smith, 1985, Science 228: 1315-1317, which are incorporated by reference in their entirety). The fusion partner may allow labeling of the immunoglobulin. Alternatively, the fusion partner may be bound to a specific sequence on an expression vector, allowing the fusion partner and the associated immunoglobulin to be linked, covalently or non-covalently, to the nucleic acid that encodes them. The method of introducing exogenous nucleic acid into host cells is well known in the art and varies depending on the host cell used.Techniques include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, calcium chloride treatment, polybrene-mediated transfection, protoplast fusion, electroporation, viral or phage infection, encapsulation of oligonucleotides in liposomes, and direct microinjection of DNA into nucleus.For mammalian cells, transfection can be either transient or stable.

[0179] In one embodiment, immunoglobulins are purified or isolated after expression. Proteins can be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques including ion exchange, hydrophobic interaction, affinity, size or gel filtration, and reverse phase, performed at atmospheric or elevated pressure using systems such as FPLC and HPLC. Purification methods also include electrophoretic, immunological, precipitation, dialysis, and isoelectric focusing. Ultrafiltration and diafiltration, in conjunction with protein concentration, are also useful. As is well known in the art, various natural proteins bind to Fc and antibodies, and these proteins can find use for purifying the immunoglobulins disclosed herein. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies, and of course, to the antibody's target antigen as well. Purification can often be made possible by specific fusion partners. For example, immunoglobulins can be purified by binding to glutathione resins when GST fusions are employed, Ni when His tags are employed, or to Fab when His tags are employed. +2 Purification may be performed using affinity chromatography, or, if a flag tag is used, immobilized anti-flag antibodies. For general guidance on suitable purification techniques, see, for example, Protein Purification: Principles and Practice, 3rd Edition, incorporated by reference in its entirety. rd Ed., Scopes, Springer-Verlag, NY, 1994. The degree of purification required varies depending on the screening or use of the immunoglobulin. In some cases, purification is not necessary. For example, in one embodiment, if the immunoglobulin is secreted, screening may be performed directly from the medium. As is well known in the art, some selection methods do not include purification of the protein. Thus, for example, if a library of immunoglobulins is made into a phage display library, protein purification may not be performed.

[0180] In vitro experiments

[0181] Immunoglobulins may be screened using a variety of methods, including, but not limited to, in vitro assays, in vivo and cell-based assays, and those using selection techniques. Automated high-throughput screening techniques may be utilized in the screening procedures. Screening may employ the use of fusion partners or labels. The use of fusion partners has been discussed above. As used herein, "labeled" means that the immunoglobulins disclosed herein have one or more elements, isotopes, or chemical compounds attached to them to allow for detection in screening. Generally, labels are classified into three categories: a) immunolabels, which may be epitopes incorporated as fusion partners recognized by antibodies; b) isotopic labels, which may be radioactive or heavy isotopes; and c) small molecule labels, which may include molecules such as biotin that allow for fluorescent and colorimetric dyes, or other labeling methods. Labels may be incorporated into the compound at any position, and may be incorporated in vitro or in vivo during protein expression.

[0182] In one embodiment, the functional and / or biophysical properties of immunoglobulins are screened in in vitro assays. In vitro assays may allow for a wide dynamic range of screening properties of interest. Immunoglobulin properties that may be screened include, but are not limited to, stability, solubility, and affinity for Fc ligands, such as FcγR. Multiple properties may be screened simultaneously or separately. Proteins may be purified or unpurified, depending on the requirements of the assay. In one embodiment, the screen is a qualitative or quantitative binding assay for the binding of immunoglobulins to proteins or non-protein molecules known or suspected to bind immunoglobulins. In one embodiment, the screen is a binding assay to measure binding to a target antigen. In an alternative embodiment, the screen is an assay for the binding of immunoglobulins to Fc ligands, including, but not limited to, the family of FcγRs, the neonatal receptor FcRn, the complement protein C1q, and bacterial proteins A and G. The Fc ligand may be from any organism. In one embodiment, the Fc ligand is from human, mouse, rat, rabbit, and / or monkey. Binding assays can be performed using a variety of methods known in the art, including, but not limited to, assays using FRET (fluorescence resonance energy transfer) and BRET (bioluminescence resonance energy transfer), AlphaScreen® (amplified luminescent proximity homogeneous assay), scintillation proximity assay, ELISA (enzyme-linked immunosorbent assay), SPR (surface plasmon resonance, also known as BIACORE®), isothermal titration calorimetry, differential scanning calorimetry, gel electrophoresis, and chromatography, including gel filtration. These and other methods may take advantage of certain fusion partners or labels of immunoglobulins. Assays may employ a variety of detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels.

[0183] The biophysical properties of immunoglobulins, such as stability and solubility, may be screened using a variety of methods known in the art. Protein stability may be determined by measuring the thermodynamic equilibrium between folded and unfolded states. For example, the immunoglobulins disclosed herein may be unfolded using chemical denaturants, heat, or pH, and this transition may be monitored using methods including, but not limited to, circular dichroism spectroscopy, fluorescence spectroscopy, absorbance spectroscopy, NMR spectroscopy, calorimetry, and proteolysis. As will be appreciated by those skilled in the art, the kinetic parameters of the folding and unfolding transitions may also be monitored using these and other techniques. The solubility and overall structural integrity of immunoglobulins may be quantitatively or qualitatively determined using a wide range of methods known in the art. Methods that may find use for characterizing the biophysical properties of the immunoglobulins disclosed herein include chromatography such as gel electrophoresis, isoelectric focusing, capillary electrophoresis, molecular sieve chromatography, ion exchange chromatography, and reversed-phase high performance liquid chromatography, peptide mapping, oligosaccharide mapping, mass spectrometry, ultraviolet absorption spectroscopy, fluorescence spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry, differential scanning calorimetry, analytical ultracentrifugation, dynamic light scattering, proteolysis and cross-linking, turbidity measurements, filter retardation assays, immunoassays, fluorescent dye binding assays, protein staining, microscopy, and detection of aggregates via ELISA or other binding assays. Structural analysis employing X-ray crystallographic techniques and NMR spectroscopy may also find use. In one embodiment, stability and / or solubility may be measured by determining the amount of protein solution after a given period of time. In this assay, the protein may or may not be exposed to certain extreme conditions, such as, for example, high temperature, low pH, or the presence of denaturants. Since function typically requires a stable, soluble, and / or well-folded structure of the protein, the functional and binding assays described above also provide a format for making such measurements.For example, a solution containing an immunoglobulin can be measured for its ability to bind a target antigen, then exposed to an elevated temperature for one or more predetermined time periods, and then measured again for antigen binding. Since unfolded and aggregated proteins are not expected to be capable of binding antigen, the amount of activity remaining provides a measure of the stability and solubility of the immunoglobulin.

[0184] In one embodiment, immunoglobulins are screened using one or more cell-based or in vitro assays. In such assays, purified or unpurified immunoglobulins are typically added exogenously so that cells are exposed to individual variants or groups of variants from a library. These assays are typically, but not necessarily, based on the biology of the ability of immunoglobulins to bind to target antigens and mediate certain biochemical events, such as cell lysis, phagocytosis, ligand / receptor binding inhibition, inhibition of growth and / or proliferation, apoptosis, etc. Such assays often include monitoring the response of cells to immunoglobulins, such as cell survival, cell phagocytosis, cell lysis, changes in cell morphology, or transcriptional activation, such as cell expression of natural or reporter genes. For example, such assays may measure the ability of immunoglobulins to induce ADCC, ADCP, or CDC. In some assays, additional cells or components may need to be added, i.e., in addition to the target cells, such as, for example, serum complement or effector cells such as peripheral blood monocytes (PBMCs), NK cells, macrophages, etc. Such additional cells may be from any organism, for example, human, mouse, rat, rabbit, and monkey. Crosslinking or monomeric antibodies may result in apoptosis of a particular cell line expressing the antibody's target antigen, or mediate attack of the target cells by immune cells added to the assay. Methods for monitoring cell death or survival are known in the art and include the use of dyes, fluorophores, immunochemistry, cytochemistry, and radioactive reagents. For example, caspase assays or annexin-fluoro conjugates may allow for the measurement of apoptosis, and uptake or release of radioactive substrates (e.g., chromium-51 release assays) or metabolic reduction of fluorescent dyes such as alamar blue may allow for the monitoring of cell growth, proliferation, or activity. In one embodiment, a cytotoxicity assay using DELFIA® EuTDA (Perkin Elmer, MA) is used.Alternatively, dead or damaged target cells can be monitored by measuring the release of one or more natural intracellular proteins, such as lactate dehydrogenase. Transcriptional activation also serves as a method for measuring the function of cell-based assays. In this case, the response can be monitored by measuring for natural genes or proteins that may be up-regulated or down-regulated, for example, the release of a particular interleukin can be measured, or alternatively, the readout can be via a luciferase or GFP reporter construct. Cell-based assays can also include measuring cell morphology changes in response to the presence of immunoglobulin. The cell type for such assays can be prokaryotic or eukaryotic, and various cell lines known in the art can be employed. Alternatively, cell-based screening is performed using cells that have been transformed or transfected with nucleic acids encoding immunoglobulins.

[0185] In vitro assays include, but are not limited to, binding assays, ADCC, CDC, cytotoxicity, proliferation, peroxide / ozone release, effector cell chemotaxis, inhibition of such analytes by reduced effector function antibodies, a range of activities such as greater than 100-fold improvement or greater than 100-fold reduction, a mixture of receptor activities, and assay results that are predicted from such receptor profiles.

[0186] In vivo experiments

[0187] The biological properties of the immunoglobulins disclosed herein may be characterized in cell, tissue, and whole organism experiments. As known in the art, drugs are often tested in animals, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and monkeys, to measure the effect of the drug in treating a disease or disease model, or to measure the pharmacokinetics, toxicity, and other properties of the drug. The animals may be referred to as disease models. With respect to the immunoglobulins disclosed herein, special challenges arise when using animal models to evaluate the potential efficacy of candidate polypeptides in humans. These are due, at least in part, to the fact that immunoglobulins that have a particular effect on affinity to human Fc receptors may not have the same affinity effect as orthologous animal receptors. These problems can be exacerbated by the inevitable uncertainty associated with the precise assignment of true orthologues (Mechetina et al., Immunogenetics, 2002 54:463-468, incorporated by reference in its entirety) and the fact that some orthologues simply do not exist in animals (e.g., humans have FcγRIIa, but mice do not). Therapeutic agents are often tested in mice, including but not limited to mouse strains NZB, NOD, BXSB, MRL / lpr, K / BxN, and transgenic (including knock-in and knock-out). Such mice can develop a variety of autoimmune conditions that resemble human organ-specific, systemic autoimmune or inflammatory disease pathologies, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). For example, immunoglobulins disclosed herein intended for autoimmune disease can be tested in such mouse models by treating the mice to determine the ability of the immunoglobulin to reduce or inhibit the development of disease lesions.Due to the incompatibility between mouse and human Fcγ receptor systems, an alternative approach uses mouse SCID models, where human PBL or PBMC (huPBL-SCID, huPBMC-SCID) are engrafted into immune-deficient mice to provide a semi-functional human immune system with human effector cells and Fc receptors. In such models, antigen inoculation (such as tetanus toxoid) activates B cells to differentiate into plasma cells and secrete immunoglobulins, thus reconstituting antigen-specific humoral immunity. Thus, the dual-targeted immunoglobulins disclosed herein that specifically bind to antigens (such as CD19 or CD79a / b) and FcγRIIb on B cells may be tested to examine their ability to specifically inhibit B cell differentiation. Such experimental methods may provide meaningful data for determining the potential of the immunoglobulins to be used as therapeutic agents. Other organisms, such as mammals, may also be used for testing. For example, monkeys are suitable as therapeutic models due to their genetic similarity to humans, and therefore can be used to test the efficacy, toxicity, pharmacokinetics, or other properties of the immunoglobulins disclosed herein. Testing of the immunoglobulins disclosed herein in humans will ultimately be required for approval as a drug, and therefore, of course, these experiments are intended. Thus, the immunoglobulins disclosed herein can be tested in humans to determine their therapeutic efficacy, toxicity, pharmacokinetics, and / or other clinical properties.

[0188] The immunoglobulins disclosed herein may provide superior performance to Fc-containing therapeutics in animal models or humans. The receptor binding profiles of such immunoglobulins described herein may be selected to target specific effector functions or effector cells, for example, to increase the potency of cytotoxic drugs or to improve the selectivity of drug action. Furthermore, receptor binding profiles may be selected that may reduce some or all effector functions and thus reduce the side effects or toxicity of such Fc-containing drugs. For example, immunoglobulins with reduced binding to FcγRIIIa, FcγRI and FcγRIIa may be selected to eliminate most cell-mediated effector functions, or immunoglobulins with reduced binding to C1q may be selected to limit complement-mediated effector functions. In some circumstances, such effector functions are known to have the potential for toxic effects. Thus, their elimination increases the safety of Fc-bearing drugs, and such improved safety may be characterized in animal models. In some situations, such effector functions are known to mediate desirable therapeutic activities, and thus their enhancement increases the activity or potency of Fc-bearing drugs, and such improved activity or potency can be characterized in animal models.

[0189] In some embodiments, the immunoglobulins disclosed herein can be evaluated for efficacy in clinically relevant animal models of various human diseases. Often, the relevant models include various transgenic animals for specific antigens and receptors.

[0190] Appropriate transgenic models, such as those expressing human Fc receptors (e.g., CD32b), can be used to evaluate and test immunoglobulins and Fc fusions in their effects. Evaluation of immunoglobulins in mice or other rodents by introduction of human genes that directly or indirectly mediate effector functions may allow physiological studies of effects in autoimmune diseases and RA. Human Fc receptors, such as FcγRIIb, have polymorphisms in the gene promoter (G to C-343), or in the transmembrane domain of the receptor 187 I or T, which would further allow the introduction of specificity and combinations of human polymorphisms into rodents. Various studies involving polymorphic specific FcRs are not limited to this section and include all discussions and uses of FcRs in general as specified throughout this specification. The immunoglobulins disclosed herein can provide superior activity to Fc-containing drugs in such transgenic models, and in particular variants with optimized binding profiles for human FcγRIIb-mediated activity can show superior activity in transgenic CD32b mice. Similar improvements in the efficacy of transgenic mice for other human Fc receptors, such as FcγRIIa, FcγRI, etc., can be observed for immunoglobulins with optimized binding profiles for each receptor. Transgenic mice with multiple human receptors will show improved activity for immunoglobulins with optimized binding profiles for the corresponding multiple receptors.

[0191] Due to the problems and uncertainties associated with the use of animal models to characterize the potential effect of candidate therapeutic antibodies in human patients, some variant polypeptides disclosed herein may find utility as surrogates to assess the evaluation in humans. Such surrogate molecules may mimic the FcR and / or complement biology of the corresponding candidate human immunoglobulin in an animal system. This mimicry is most likely manifested by the relative relative affinity between a particular immunoglobulin and the human receptor for the animal. For example, when using a mouse model to evaluate the potential human effect of an Fc variant with reduced affinity for the inhibitor human FcγRIIb, a suitable surrogate variant would have reduced affinity for mouse FcγRII. It should be noted that the surrogate Fc variant may be made in conjunction with a human Fc variant, an animal Fc variant, or both.

[0192] In one embodiment, testing of the immunoglobulin may include testing of efficacy in primates (e.g., cynomolgus monkey models) to facilitate evaluation of reduction and / or depletion of specific target cells bearing the target antigen. Additional primate models include, but are not limited to, the use of rhesus monkeys to evaluate Fc polypeptides in autoimmune, transplantation, and cancer therapeutic trials.

[0193] Toxicity studies are performed to determine antibody or Fc fusion-related effects that cannot be assessed with standard pharmacological profiles or that occur only after repeated administration of the drug. Most toxicity studies are performed in rodent and two non-rodent species to ensure that any unforeseen adverse effects are not overlooked before the new therapeutic entity is introduced into humans. Generally, these models can measure a variety of toxicities, including genotoxicity, chronic toxicity, immunogenicity, reproductive / developmental toxicity, and carcinogenesis. The aforementioned parameters include food consumption, body weight, antibody production, standard measurements of clinical chemistry, and gross and microscopic examination of standard organs / tissues (e.g., cardiotoxicity). Further parameters of measurement are injection site damage and neutralizing antibody measurements, if any. Traditionally, naked or conjugated monoclonal antibody therapeutics are evaluated for cross-reactivity with normal tissues of the radiolabeled species, immunogenicity / antibody production, conjugate or linker toxicity, and "bystander" toxicity. Nonetheless, such studies may be individualized to address specific issues and must follow the guidance provided in ICH S6 (Safety Testing for Biotechnology Products, discussed above). Thus, the general rule is that the product is sufficiently well characterized, impurities / contaminants are removed, test materials are comparable throughout development, and GLP compliance is maintained.

[0194] The pharmacokinetics (PK) of the immunoglobulins disclosed herein may be tested in various animal systems, most pertinently non-human primates such as cynomolgus and rhesus monkeys. Using plasma concentrations and clearance, single or repeated IV / SC administrations over a 6000-fold dose range (0.05-300 mg / kg) may be evaluated for half-life (days-weeks). Volume of distribution and systemic absorption levels at steady state may also be measured. Examples of parameters for such measurements generally include maximum observed plasma concentration (Cmax), time to reach Cmax (Tmax), area under the plasma concentration-time curve from time 0 to infinity [AUC(0-inf)], and apparent elimination half-life (T1 / 2). Further measurement parameters may include compartmental analysis of concentration-time data obtained after IV administration and bioavailability. Examples of pharmacology / toxicology studies using cynomolgus monkeys have been established for Rituxan and Zevalin, which are cross-reactive monoclonal antibodies against CD20. Biodistribution, dosimetry (for radiolabeled antibodies), and PK studies can also be performed in rodent models. Such studies will assess tolerability at all administered doses, local tissue toxicity, preferential localization in rodent xenograft animal models, reduction and / or depletion of target cells (e.g., CD20 positive cells).

[0195] The immunoglobulins disclosed herein may provide superior pharmacokinetics to Fc-containing therapeutics in animal systems or humans. For example, increased binding to FcRn may increase the half-life and exposure of Fc-containing drugs. Alternatively, decreased binding to FcRn may reduce the half-life and exposure of Fc-containing drugs when reduced exposure is preferred, such as when such drugs have side effects.

[0196] It is known in the art that multiple Fc receptors are differentially expressed on various immune cell types and in different tissues. Differential tissue distribution of Fc receptors may ultimately affect the pharmacodynamic (PD) and pharmacokinetic (PK) properties of the immunoglobulins disclosed herein. Because the immunoglobulins of the present invention have different affinities for multiple Fc receptors, further screening of the polypeptides for PD and / or PK properties may be very useful to determine the optimal balance of PD, PK, and therapeutic effects conferred by each candidate polypeptide.

[0197] Pharmacodynamics testing may include, but is not limited to, targeting specific cells, or blocking signal transduction mechanisms, measuring inhibition of antigen-specific antibodies, etc. The immunoglobulins disclosed herein can direct Fc-containing drugs to target specific effector cell populations, thereby improving the potential or eliciting specific activity to increase penetration into specific preferred physiological compartments. For example, neutrophil activity and localization can be targeted by immunoglobulins that target FcγRIIIb. Such pharmacodynamic effects can be demonstrated in animal models, or in humans.

[0198] Clinical use

[0199] The immunoglobulins disclosed herein may find use in a wide range of products. In one embodiment, the immunoglobulins disclosed herein are therapeutic, diagnostic, or research reagents. The immunoglobulins may find use in compositions that are monoclonal or polyclonal. The immunoglobulins disclosed herein may be used for therapeutic purposes. As will be appreciated by those skilled in the art, the immunoglobulins disclosed herein may be used for any therapeutic purpose for which antibodies and the like may be used. The immunoglobulins may be administered to patients to treat IgG4-related diseases.

[0200] A "patient" for the purposes disclosed herein includes both humans and other animals, e.g., other mammals. Thus, the immunoglobulins disclosed herein have both human therapeutic and veterinary uses. As disclosed herein, "treatment" or "treating" is meant to include therapeutic treatments for a disease or disorder, as well as preventative or suppressive measures. Thus, for example, administration of an immunoglobulin well before the onset of a disease results in treatment of the disease. As another example, administration of an optimized immunoglobulin well after the clinical presentation of a disease to combat the symptoms of the disease is included in the treatment of a disease. "Treatment" and "treating" also encompass administration of an optimized immunoglobulin well after the onset of a disease to eradicate the disease. Administration of an agent well after onset and after clinical symptoms have developed, with the expected reduction in clinical symptoms and the possibility of reversing the disease, is included in the treatment of a disease. These "in need of treatment" include mammals already with a disease or disorder, as well as those prone to have a disease or disorder, including those in which the disease or disorder is to be prevented.

[0201] The immunoglobulins disclosed herein can be used to treat IgG4-related disease (also referred to as "IgG4-RD"). IgG4-RD is also known and referred to as IgG4-related systemic disease (IgG4-RSD), IgG4-related sclerosing disease, IgG4-related systemic sclerosing disease, IgG4-related autoimmune disease, IgG4-related multifocal systemic fibrosis, IgG4-related disease, IgG4 syndrome, excess IgG4 disease, systemic IgG4-related plasma cell syndrome, IgG4-positive multiorgan lymphoproliferative syndrome, (IgG4-related multiorgan lymphoproliferative syndrome (IgG4-MOLPS), systemic IgG4-related sclerosing syndrome, multifocal fibrosclerosis, and multifocal idiopathic fibrosclerosis.

[0202] IgG4-RD is a systemic inflammatory condition that may affect any organ system. The immunoglobulins disclosed herein can also be used to treat symptoms of IgG4-RD or IgG4-RD-related disease. Non-limiting examples of symptoms related to IgG4-RD and / or IgG4-RD-related disease include: IgG4-related sialadenitis (chronic sclerosing sialadenitis, Kuttner's tumor, Mikulicz's disease), IgG4-related dacryoadenitis (Mikulicz's disease), IgG4-related eye disease (idiopathic orbital inflammatory disease, orbital pseudotumor), chronic sinusitis, eosinophilic angiocentric fibrosis, IgG4-related hypophysitis (IgG4-related panhypophysitis, IgG4-related adenohypophysitis, IgG4-related posterior infundibulohypophysitis, autoimmune hypophysitis), IgG4-related meningitis, IgG4-related leptomeningitis (idiopathic hypertrophic meningitis), IgG4-related pancreatitis (type 1). Autoimmune pancreatitis, IgG4-related AIP, lymphoplasmacytic sclerosing pancreatitis, chronic pancreatitis associated with widespread irregular stenosis of the main pancreatic duct), IgG4-related lung disease (pulmonary inflammatory pseudotumor), IgG4-related pleurisy, IgG4-related liver disease, IgG4-related sclerosing cholangitis, IgG4-related cholecystitis, IgG4-related aortitis (inflammatory aortic aneurysm), IgG4-related periaortitis (chronic periaortitis), IgG4-related periarteritis, IgG4-related pericarditis, IgG4-related mediastinitis (fibrosing mediastinitis), IgG4-related retroperitoneal fibrosis (retroperitoneal fibrosis, Albarrán-Ormond syndrome, Ormond disease (retroperitoneal fibrosis (tetroperitoneal fibrosis)fibrosis), perinephric fasciitis, Gerota fasciitis / syndrome, fibrous periureteritis, sclerosing lipogranuloma, sclerosing retroperitoneal granuloma, nonspecific retroperitoneal inflammation, sclerosing retroperitonitis, retroperitoneal vasculitis with perivascular fibrosis), IgG4-related mesenteritis (subtypes are: mesenteric panniculitis, mesenteric lipodystrophy and involutive mesenteritis) (sclerosing mesenteritis, generalized nodular subcutaneous panniculitis, liposclerosing mesenteritis, Weber-Christian disease of the mesenterium, mesenteric lipogranuloma, xanthogranulomatous mesenteritis), IgG4-related mastitis (sclerosing mastitis), IgG4-related kidney disease (IgG4-RKD), IgG4-related tubulointerstitial nephritis (IgG4-TIN), IgG4-related membranous glomerulonephritis (idiopathic cytoplasmic glomerulonephritis), tubular interstitial nephritis), IgG4-related prostatitis, IgG4-related perivasal fibrosis (chronic scrotal pain), IgG4-related pseudopseudotumor, IgG4-related epididymo-orchitis (paratesticular fibrous pseudotumor, inflammatory pseudotumor of the spermatic cord, pseudosarcomatous myofibroblastic proliferation of the spermatic cord, proliferative funiculitis, chronic proliferative perispermitis, fibromatous perispermitis, nodular perispermitis, reactive perispermitis, fibrous mesothelioma), IgG4-related lymphadenopathy, IgG4-related skin diseases (angiolymphocytosis with eosinophilia, cutaneous pseudolymphoma), IgG4-related perineural disease, and IgG4-related thyroid disease (Riedel's thyroiditis), eosinophilic angiocentric fibrosis (involving the orbit and upper respiratory tract), inflammatory pseudotumor, and multifocal fibrous sclerosis.

[0203] More specifically, in some embodiments, the immunoglobulins disclosed herein can be used to treat autoimmune pancreatitis (lymphoplasmacytic sclerosing pancreatitis), eosinophilic angiocentric fibrosis (affecting the orbit and upper respiratory tract), fibrosing mediastinitis, idiopathic hypertrophic meningitis, idiopathic tubulointerstitial nephritis, inflammatory pseudotumor, Kuttner's tumor, Mikulicz's disease, multifocal fibrosclerosis, periaortitis, periarteritis, inflammatory aortic aneurysm, Ormond's disease (retroperitoneal fibrosis), Riedel's thyroiditis, and sclerosing mesenteritis.

[0204] In further embodiments, the immunoglobulins disclosed herein can be used to treat autoimmune pancreatitis (lymphoplasmacytic sclerosing pancreatitis). In other embodiments, the immunoglobulins disclosed herein can be used to treat eosinophilic angiocentric fibrosis (affecting the orbit and upper airway). In other embodiments, the immunoglobulins disclosed herein can be used to treat fibrosing mediastinitis. In other embodiments, the immunoglobulins disclosed herein can be used to treat idiopathic hypertrophic meningitis. In other embodiments, the immunoglobulins disclosed herein can be used to treat idiopathic tubulointerstitial nephritis. In other embodiments, the immunoglobulins disclosed herein can be used to treat inflammatory pseudotumors. In other embodiments, the immunoglobulins disclosed herein can be used to treat Kuttner's tumors. In other embodiments, the immunoglobulins disclosed herein can be used to treat Mikulicz's disease. In other embodiments, the immunoglobulins disclosed herein can be used to treat In other embodiments, the immunoglobulins disclosed herein can be used to treat multifocal fibrosclerosis. In other embodiments, the immunoglobulins disclosed herein can be used to treat periaortitis. In other embodiments, the immunoglobulins disclosed herein can be used to treat periarteritis. In other embodiments, the immunoglobulins disclosed herein can be used to treat inflammatory aortic aneurysms. In other embodiments, the immunoglobulins disclosed herein can be used to treat Ormond's disease (retroperitoneal fibrosis). In other embodiments, the immunoglobulins disclosed herein can be used to treat Riedel's thyroiditis. In other embodiments, the immunoglobulins disclosed herein can be used to treat sclerosing mesenteritis.

[0205] Without wishing to be bound by any theory, the immunoglobulin disclosed for use in treating IgG4-RD co-binds FcγRIIb and CD19 expressed on B cells. Co-binding of FcγRIIb and CD19 inhibits B cell activation and prevents or limits antibody production. Specifically, co-binding of FcγRIIb and CD19 inhibits or reduces the production of antigen-specific antibodies.

[0206] As mentioned above, the immunoglobulins disclosed herein can be used to treat IgG4-RD, as well as symptoms and / or diseases and conditions associated with IgG4-RD (such as, but not limited to, those listed above). In some embodiments, treating can treat symptoms associated with IgG4-RD. In some embodiments, treating can prevent IgG4-RD. In some embodiments, treating can be a prophylactic treatment of IgG4-RD.

[0207] The immunoglobulins disclosed herein can be used to suppress B cells for the treatment of IgG4-RD. The antibody used to treat IgG4-RD can be an immunoglobulin that includes a domain that binds to FcγRIIb and a domain that binds to CD19. More specifically, the immunoglobulin used to treat IgG4-RD can be an antibody that includes an Fc domain that binds to FcγRIIb and an Fv domain that binds to CD19.

[0208] In some embodiments, the Fc domain that binds FcγRIIb comprises at least one modification selected from 234W, 235I, 235Y, 235R, 235D, 236D, 236N, 239D, 267D, 267E, 268E, 268D, 328F, and 328Y, where the numbering is according to the EU index as in Kabat. In another embodiment, the Fc domain that binds FcγRIIb comprises at least one modification selected from the group consisting of 235Y, 235R, 236D, 267D, 267E, and 328F. In a further embodiment, the Fc domain that binds FcγRIIb comprises at least one modification selected from 267E and 328F. In a further embodiment, the Fc domain that binds FcγRIIb comprises a modification selected from 235D / 267E, 235Y / 267E, 235D / 267D, 235I / 267E, 235I / 267D, 235Y / 267D, 236D / 267E, 236D / 267D, 267E / 328F, 267D / 328F, 268D / 267E, H268D / S267D, 268E / 267E, and 268E / 267D. In further embodiments, the Fc domain that binds FcγRIIb comprises a modification selected from 235D / 267E, 235Y / 267E, 235Y / 267D, 236D / 267E, 267E / 328F, 268D / 267E, 268E / 267E, and 268E / 267D. In further embodiments, the Fc domain that binds FcγRIIb comprises the modification 236D / 267E / 328F. In another embodiment, the Fc domain that binds FcγRIIb comprises the modification 267E / 328F, where the numbering is according to the EU index as in Kabat. In some embodiments, the Fc domain that binds FcγRIIb comprises SEQ ID NO:7 and SEQ ID NO:9. In some embodiments, the Fv domain that binds CD19 comprises one or more sequences selected from the group consisting of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40.

[0209] As mentioned above, immunoglobulins (such as antibodies) that co-bind FcγRIIb and CD19 can be used to treat IgG4-RD. In one embodiment, administration of an immunoglobulin that co-binds FcγRIIb and CD19 to treat IgG4-RD can increase or decrease the value of a pharmacodynamic marker. In a further embodiment, administration of an immunoglobulin that co-binds FcγRIIb and CD19 to treat IgG4-RD can result in a reduction in the severity and / or number of symptoms associated with IgG4-RD. Administration of an immunoglobulin that co-binds FcγRIIb and CD19 to treat IgG4-RD can also reduce the IgG4-RD responder index (IgG4-RD RI) score. IgG4-RD RI is described in more detail below. Using an antibody that co-binds FcγRIIb and CD19, the IgG4-RD RI score can be significantly reduced relative to baseline.

[0210] Pharmacodynamic markers

[0211] In some cases, administration of immunoglobulins that bind FcγRIIb and CD19 to treat IgG4-RD can result in changes in the values ​​of pharmacodynamic markers. Pharmacodynamic markers include, but are not limited to, B cell count, plasmablast count, CD4+SLAMF7+CTL ​​cell count, and CD19 receptor occupancy. In some cases, it can result in a decrease in the total B cell count. In other cases, it can result in a decrease in the total plasmablast count. In other cases, it can result in a decrease in the CD4+SLAMF7+CTL ​​cell count. In other cases, it can result in an increase in CD19 receptor occupancy.

[0212] Changes in one or more pharmacodynamic markers can be observed within one day of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about day 1 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about day 2 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about day 3 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about day 4 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about day 5 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about day 6 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about day 7 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about day 8 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about day 9 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about day 10 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about day 11 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about day 12 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about day 13 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about day 14 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about month 1 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about month 2 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about month 3 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about month 4 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about month 5 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about month 6 of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about month 7 of administration.In some instances, a change in one or more pharmacodynamic markers may be observed at about 8 months of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about 9 months of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about 10 months of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about 11 months of administration. In some instances, a change in one or more pharmacodynamic markers may be observed at about 12 months of administration.

[0213] Administration of immunoglobulin that co-binds FcγRIIb and CD19 can reduce total B cell numbers in subjects with IgG4-RD.

[0214] The reduction in B cells can be observed at about 24 hours, about day 2, about day 3, about day 4, about day 5, about day 6, about day 7, about day 8, about day 9, about day 10, about day 11, about day 12, about day 13, about day 14, about week 3, about week 4, about week 5, about week 6, about 2 months, or about 3 months after administration of the immunoglobulin that co-binds FcγRIIb and CD 19. In particular, the reduction in B cells can be observed within 7 days after administration of the immunoglobulin that co-binds FcγRIIb and CD 19.

[0215] The reduction of B cells can be measured by methods known in the art. For example, the alleviation of signs or symptoms of IgG4-RD can be correlated with the reduction of B cells. In addition, the reduction of B cells can be measured by measuring the number of p B cells in a biological sample. The biological sample can be a tissue sample or a body fluid sample. For example, microscopy or flow cytometry can be used to measure the reduction of B cells.

[0216] B cells may be reduced by more than 10% relative to baseline, where baseline is the amount of B cells prior to the start of treatment. In some variations, B cells may be reduced by more than 15% relative to baseline. In some variations, B cells may be reduced by more than 20% relative to baseline. In some variations, B cells may be reduced by more than 25% relative to baseline. In some variations, pB cells may be reduced by more than 30% relative to baseline. In some variations, B cells may be reduced by more than 35% relative to baseline. In some variations, B cells may be reduced by more than 40% relative to baseline. In some variations, B cells may be reduced by more than 45% relative to baseline. In some variations, B cells may be reduced by more than 50% relative to baseline. In some variations, B cells may be reduced by more than 55% relative to baseline. In some variations, B cells may be reduced by more than 60% relative to baseline. In some variations, B cells may be reduced by more than 65% relative to baseline. In some variations, B cells may be reduced by more than 70% relative to baseline. In some variations, B cells may be reduced by more than 75% relative to baseline. In some variations, B cells may be reduced by more than 80% relative to baseline. In some variations, B cells may be reduced by more than 85% relative to baseline. In some variations, B cells may be reduced by more than 90% relative to baseline. In some variations, B cells may be reduced by more than 95% relative to baseline.

[0217] In addition, B cells can be reduced by more than 1.2-fold relative to baseline. In some variations, B cells may be reduced more than 1.5-fold relative to baseline. In some variations, B cells may be reduced more than 2-fold relative to baseline. In some variations, B cells may be reduced more than 2.5-fold relative to baseline. In some variations, B cells may be reduced more than 5-fold relative to baseline. In some variations, B cells may be reduced more than 10-fold relative to baseline. In some variations, B cells may be reduced more than 20-fold relative to baseline. In some variations, B cells may be reduced more than 25-fold relative to baseline. In some variations, B cells may be reduced more than 50-fold relative to baseline. In some variations, B cells may be reduced more than 75-fold relative to baseline. In some variations, B cells may be reduced more than 100-fold relative to baseline. In some variations, B cells may be reduced more than 200-fold relative to baseline. In some variations, B cells may be reduced more than 500-fold relative to baseline. In some variations, B cells may be reduced more than 1000-fold relative to baseline.

[0218] Administration of immunoglobulin that co-binds FcγRIIb and CD19 reduces or depletes the number of plasmablasts in subjects suffering from IgG4-RD. Subjects suffering from IgG4-RD may have plasmablast levels of more than 100 cells / mL. For example, subjects suffering from IgG4-RD may have plasmablast levels of more than 100 cells / mL, more than 200 cells / mL, more than 300 cells / mL, more than 400 cells / mL, more than 500 cells / mL, more than 600 cells / mL, more than 700 cells / mL, more than 800 cells / mL, more than 900 cells / mL, more than 1000 cells / mL, more than 2000 cells / mL, more than 3000 cells / mL, more than 4000 cells / mL, or more than 5000 cells / mL. Thus, immunoglobulin for the treatment of IgG4-RD may deplete or reduce the number of plasmablasts.

[0219] Reduction or depletion of B cells can be observed at about 24 hours, about day 2, about day 3, about day 4, about day 5, about day 6, about day 7, about day 8, about day 9, about day 10, about day 11, about day 12, about day 13, about day 14, about week 3, about week 4, about week 5, about week 6, about week 2, or about month 3 after administration of an immunoglobulin that co-binds FcγRIIb and CD19.

[0220] The reduction or depletion of plasmablasts can be measured by methods known in the art. For example, alleviation of signs or symptoms of IgG4-RD can be correlated with the reduction or depletion of plasmablasts. In addition, the reduction or depletion of plasmablasts can be measured by measuring the number of p plasmablasts in a biological sample. The biological sample can be a tissue sample or a bodily fluid sample. For example, microscopy or flow cytometry can be used to measure the reduction or depletion of plasmablasts. The plasmablasts can be reduced or depleted by more than 10% relative to baseline, where baseline is the amount of plasmablasts before the start of treatment. In some variations, the plasmablasts can be reduced or depleted by more than 15% relative to baseline. In some variations, the plasmablasts can be reduced or depleted by more than 20% relative to baseline. In some variations, the plasmablasts can be reduced or depleted by more than 25% relative to baseline. In some variations, the p plasmablasts can be reduced or depleted by more than 30% relative to baseline. In some variations, plasmablasts may be reduced or depleted by more than 35% relative to baseline. In some variations, plasmablasts may be reduced or depleted by more than 40% relative to baseline. In some variations, plasmablasts may be reduced or depleted by more than 45% relative to baseline. In some variations, plasmablasts may be reduced or depleted by more than 50% relative to baseline. In some variations, plasmablasts may be reduced or depleted by more than 55% relative to baseline. In some variations, plasmablasts may be reduced or depleted by more than 60% relative to baseline. In some variations, plasmablasts may be reduced or depleted by more than 65% relative to baseline. In some variations, plasmablasts may be reduced or depleted by more than 70% relative to baseline. In some variations, plasmablasts may be reduced or depleted by more than 75% relative to baseline. In some variations, plasmablasts may be reduced or depleted by more than 80% relative to baseline.In some variations, plasmablasts may be reduced or depleted by more than 85% relative to baseline. In some variations, plasmablasts may be reduced or depleted by more than 90% relative to baseline. In some variations, plasmablasts may be reduced or depleted by more than 95% relative to baseline.

[0221] In addition, plasmablasts may be reduced or depleted more than 1.2-fold relative to baseline. In some variations, plasmablasts may be reduced or depleted more than 1.5-fold relative to baseline. In some variations, plasmablasts may be reduced or depleted more than 2-fold relative to baseline. In some variations, plasmablasts may be reduced or depleted more than 2.5-fold relative to baseline. In some variations, plasmablasts may be reduced or depleted more than 5-fold relative to baseline. In some variations, plasmablasts may be reduced or depleted more than 10-fold relative to baseline. In some variations, plasmablasts may be reduced or depleted more than 20-fold relative to baseline. In some variations, plasmablasts may be reduced or depleted more than 25-fold relative to baseline. In some variations, plasmablasts may be reduced or depleted more than 50-fold relative to baseline. In some variations, plasmablasts may be reduced or depleted more than 75-fold relative to baseline. In some variations, plasmablasts may be reduced or depleted more than 100-fold relative to baseline. In some variations, plasmablasts may be reduced or depleted more than 200-fold relative to baseline. In some variations, plasmablasts may be reduced or depleted more than 500-fold relative to baseline. In some variations, plasmablasts may be reduced or depleted more than 1000-fold relative to baseline.

[0222] Administration of immunoglobulins that co-bind FcγRIIb and CD19 can reduce the number of CD4+ cytotoxic T lymphocyte cells (CD4+ CTL cells) in subjects suffering from IgG4-RD. More specifically, administration of immunoglobulins that co-bind FcγRIIb and CD19 can reduce the number of CD4+ SLAMF7+ CTL cells (effector CD4+ CTL cells) in subjects suffering from IgG4-RD. CD4+ SLAMF7+ CTL numbers are enhanced in the peripheral blood of IgG4-RD patients (see FIG. 29). Rituximab has been reported to reduce circulating CD4+ SLMF7+ CTL numbers in patients suffering from IgG4-RD after rituximab treatment but by less than one month after treatment (Hamid Mattoo, et al., J Allergy Clin. Immunol. 2016). The depletion of CD4+SLAMF7+ CTL following administration of immunoglobulins that co-bind FcγRIIb and CD19 occurs very rapidly, such that in some instances depletion can be observed within one day of administration.

[0223] The reduction in CD4+CTL cells and / or CD4+SLAMF7+CTL ​​cells can be observed at about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 2 months, or about 3 months after administration of an immunoglobulin that co-binds FcγRIIb and CD19. In particular, the reduction in CD4+CTL cells and / or CD4+SLAMF7+CTL ​​cells can be observed within 1 day after administration of an immunoglobulin that co-binds FcγRIIb and CD19. The reduction in CD4+CD4+SLAMF7+CTL ​​cells can be observed within 1 day after administration of an immunoglobulin that co-binds FcγRIIb and CD19.

[0224] The reduction of CD4+CTL cells and / or CD4+SLAMF7+CTL ​​cells can be measured by methods known in the art. For example, the alleviation of signs or symptoms of IgG4-RD can be correlated with the reduction of CD4+CTL cells and / or CD4+SLAMF7+CTL ​​cells. In addition, the reduction of CD4+CTL cells and / or CD4+SLAMF7+CTL ​​cells can be measured by measuring the quantity of pCD4+CTL cells and / or CD4+SLAMF7+CTL ​​cells in a biological sample. The biological sample can be a tissue sample or a body fluid sample. For example, microscopy or flow cytometry can be used to measure the reduction of CD4+CTL cells and / or CD4+SLAMF7+CTL ​​cells.

[0225] The CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 10% relative to baseline, where baseline is the amount of CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells prior to the start of treatment. In some variations, the CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 15% relative to baseline. In some variations, the CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 20% relative to baseline. In some variations, the CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 25% relative to baseline. In some variations, the pCD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 30% relative to baseline. In some variations, the CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 35% relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 40% relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 45% relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 50% relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 55% relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 60% relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 65% relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 70% relative to baseline.In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 75% relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 80% relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 85% relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 90% relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 95% relative to baseline.

[0226] In addition, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 1.2-fold relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 1.5-fold relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 2-fold relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 2.5-fold relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 5-fold relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 10-fold relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 20-fold relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 25-fold relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 50-fold relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 75-fold relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 100-fold relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 200-fold relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 500-fold relative to baseline. In some variations, CD4+ CTL cells and / or CD4+SLAMF7+ CTL cells may be reduced by more than 1000-fold relative to baseline.

[0227] Administration of immunoglobulin that co-binds FcγRIIb and CD19 can result in CD19 receptor occupancy in subjects suffering from IgG4-RD. Without wishing to be bound by any theory, the disclosed immunoglobulin for use in treating IgG4-RD co-binds FcRIIb and CD19 expressed on B cells. This co-binding results in the occupancy of CD19 receptor by immunoglobulin that co-binds FcγRIIb and CD19. CD19 receptor occupancy can be measured by methods known in the art, such as, but not limited to, flow cytometry.

[0228] CD19 receptor occupancy in a subject following administration of an immunoglobulin that co-binds FcγRIIb and CD19 can be about 30% to about 100%. In some embodiments, CD19 receptor occupancy can be 30% or more. In some embodiments, CD19 receptor occupancy can be 40% or more. In some embodiments, CD19 receptor occupancy can be 50% or more. In some embodiments, CD19 receptor occupancy can be 60% or more. In some embodiments, CD19 receptor occupancy can be 70% or more. In some embodiments, CD19 receptor occupancy can be 80% or more. In some embodiments, CD19 receptor occupancy can be 90% or more. In some embodiments, CD19 receptor occupancy can be 95% or more. In some embodiments, CD19 receptor occupancy can be 100%.

[0229] Relief of symptoms

[0230] In some examples, administration of an immunoglobulin that binds to FcγRIIb and CD19 can result in a reduction in symptoms associated with IgG4-RD. In some embodiments, the reduction can be a reduction in the severity (i.e., intensity) of at least one symptom. In other embodiments, the reduction can be a reduction in the number of symptoms. In further embodiments, the reduction can be a reduction in the number of affected organs or tissues. In other embodiments, the reduction is a combination of one or more of a reduction in the severity (i.e., intensity) of at least one symptom, a reduction in the number of affected organs or tissues, and a reduction in the number of affected organs or tissues.

[0231] Symptoms are generally based on the organs or tissues affected by IgG4-RD.Non-limiting examples of symptoms of IgG4-RD include fibrosis (scarring), organ dysfunction, organ failure, weight loss, inflammation, pain, swelling, mass lesions, jaundice, convulsions, paralysis or hemiparesis, cranial nerve palsy, sensorineural hearing loss, pituitary hormone deficiency, vision loss, stroke, constrictive pericarditis, heart block, aortic aneurysm rupture, aortic dissection, carotid artery dissection, angina pectoris, sudden cardiac death, airway obstruction, pleural effusion, esophageal obstruction, intestinal obstruction, renal failure, hydronephrosis, and testicular pain.In some embodiments, the reduction in symptoms is a reduction in the number of organs and / or tissues affected.In other embodiments, the reduction in symptoms is a reduction in the severity of symptoms in at least one organ or tissue.

[0232] A subject with IgG4-RD may exhibit symptoms in one or more organs. For example, a subject with IgG4-RD may exhibit symptoms in one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more organs. In various aspects, a subject with IgG4-RD exhibits symptoms in one, two, three, four, five, six, seven, eight, nine, or ten or more organs. In one embodiment, a subject with IgG4-RD may exhibit symptoms in four or more organs. Non-limiting examples of organs exhibiting IgG4-RD symptoms include, but are not limited to, lymph nodes, submandibular glands, parotid glands, lacrimal glands, kidneys, heart, pericardium, orbit, nasal cavity, lungs, bile ducts, salivary glands, and pancreas. In certain embodiments, symptomatic organs include lymph nodes, submandibular glands, parotid glands, and lacrimal glands.

[0233] The reduction in symptoms may be observed within 7 days of administration. In some instances, the reduction in symptoms may be observed at about day 7 of administration of the antibody. In some instances, the reduction in symptoms may be observed at about day 8. In some instances, the reduction in symptoms may be observed at about day 9. In some instances, the reduction in symptoms may be observed at about day 10. In some instances, the reduction in symptoms may be observed at about day 11. In some instances, the reduction in symptoms may be observed at about day 12. In some instances, the reduction in symptoms may be observed at about day 13. In some instances, the reduction in symptoms may be observed at about day 14.

[0234] In some instances, a reduction in symptoms may be observed in changes in salivary glands, lacrimal glands, extraocular muscle involvement, lymph nodes, kidneys, bile ducts, and / or pancreatic involvement. A reduction in symptoms in salivary glands, lacrimal glands, extraocular muscle lesions, lymph nodes, kidneys, bile ducts, and / or pancreatic lesions may be observed in about 7 days. In other instances, a reduction in symptoms in salivary glands, lacrimal glands, extraocular muscle lesions, lymph nodes, kidneys, bile ducts, and / or pancreatic lesions may be observed in about 8 days. In other instances, a reduction in symptoms in salivary glands, lacrimal glands, extraocular muscle lesions, lymph nodes, kidneys, bile ducts, and / or pancreatic lesions may be observed in about 9 days. In other instances, a reduction in symptoms in salivary glands, lacrimal glands, extraocular muscle lesions, lymph nodes, kidneys, bile ducts, and / or pancreatic lesions may be observed in about 10 days. In other examples, a reduction in symptoms in the salivary gland, lacrimal gland, extraocular muscle lesion, lymph node, kidney, bile duct, and / or pancreatic lesion may be observed in about 11 days. In other examples, a reduction in symptoms in the salivary gland, lacrimal gland, extraocular muscle lesion, lymph node, kidney, bile duct, and / or pancreatic lesion may be observed in about 12 days. In other examples, a reduction in symptoms in the salivary gland, lacrimal gland, extraocular muscle lesion, lymph node, kidney, bile duct, and / or pancreatic lesion may be observed in about 13 days. In other examples, a reduction in symptoms in the salivary gland, lacrimal gland, extraocular muscle lesion, lymph node, kidney, bile duct, and / or pancreatic lesion may be observed in about 14 days. In other examples, a reduction in symptoms in the salivary gland, lacrimal gland, extraocular muscle involvement, lymph node, kidney, bile duct, and / or pancreatic involvement may be observed after about 14 days.

[0235] IgG4-RD Responder Index

[0236] In some cases, administration of immunoglobulins that bind FcγRIIb and CD19 may result in a decrease in the IgG4-RD Responder Index (IgG4-RD RI) score. The IgG4-RD RI is a tool designed to detect any changes in disease activity and identify improvements and deteriorations in the same and / or different organ systems. For a general overview of IgG4-RI scoring methods, see Carruthers et al., International Journal of Rheumatology 2012, the disclosure of which is incorporated herein by reference in its entirety. Briefly, the IgG4-RD RI uses a scoring system for each organ system or site and asks the clinician to assess the degree of disease activity and damage at the time of the clinical encounter. The following organ sites are typically evaluated by the clinician: pachymeninges, pituitary, orbital and lacrimal glands, salivary glands, thyroid, lymph nodes, lungs, aorta and large vessels, retroperitoneum, mediastinum, and mesentery, pancreas, bile duct and liver, kidneys, skin, and other sclerosis / mass formations. Clinicians enter a score of 0-3 for each affected organ / site, indicating whether the organ site is symptomatic (yes / no), whether the disease activity of each organ / site requires urgent treatment (yes / no), and whether the organ / site dysfunction is related to injury rather than active disease (yes / no). For the purposes of this disclosure, a 0-3 scoring system was used. Specifically, the following scoring system for disease activity is used: 0-unaffected or resolved; 1-improving but persisting; 2-new or recurrent (at treatment cessation) or unchanged; and 3-worsening or new despite treatment. The sum of disease activity in all organ sites results in a total activity score. If an organ site is labeled as urgent ("yes"), the score for that site is doubled. IgG4-RD RI scores are then compared between assessments to determine disease activity over time, which can be used for clinical trial endpoints.

[0237] The IgG4-RD RI may take into account the subject's serum IgG4 concentration in some examples disclosed in Currthers et al.. In the context of this disclosure, the IgG4-RD RI does not take into account the subject's serum IgG4 concentration.

[0238] The IgG4-RD RI score may be maintained compared to baseline or may be reduced compared to baseline, where baseline is the IgG4-RD RI score before the start of treatment. When the IgG4-RD RI score is maintained, there is no change in the IgG4-RD RI score compared to baseline.

[0239] In some cases, a decrease in IgG4-RD RI score relative to baseline may be observed within 14 days of administration. In some cases, a decrease in IgG4-RD RI score may be observed at about day 14 of administration. In some cases, a decrease in IgG4-RD RI score may be observed at about 1 month of administration. In some cases, a decrease in IgG4-RD RI score may be observed at about 2 months of administration. In some cases, a decrease in IgG4-RD RI score may be observed at about 3 months of administration. In some cases, a decrease in IgG4-RD RI score may be observed at about 4 months of administration. In some cases, a decrease in IgG4-RD RI score may be observed at about 5 months of administration. In some cases, a decrease in IgG4-RD RI score may be observed at about 6 months of administration. In some cases, a decrease in IgG4-RD RI score may be observed at about 7 months of administration. In some cases, a decrease in IgG4-RD RI score may be observed at about 8 months of administration. In some instances, a decrease in IgG4-RD RI score may be observed at about 9 months of administration. In some instances, a decrease in IgG4-RD RI score may be observed at about 10 months of administration. In some instances, a decrease in IgG4-RD RI score may be observed at about 11 months of administration. In some instances, a decrease in IgG4-RD RI score may be observed at about 12 months of administration.

[0240] If the IgG4-RD-RI score is decreased compared to baseline, the score may be decreased by at least 1 compared to baseline. In some variations, the IgG4-RD-RI score may be decreased by at least 2 relative to baseline. In some variations, the IgG4-RD-RI score may be decreased by at least 3. In some variations, the IgG4-RD-RI score may be lowered by at least 4. In some variations, the IgG4-RD-RI score may be decreased by at least 5. In some variations, the IgG4-RD-RI score may be decreased by at least 6. In some variations, the IgG4-RD-RI score may be decreased by at least 7. In some variations, the IgG4-RD-RI score may be decreased by at least 8. In some variations, the IgG4-RD-RI score may be decreased by at least 9. In some variations, the IgG4-RD-RI score may be decreased by at least 10. In some variations, the IgG4-RD-RI score may be decreased by at least 11. In some variations, the IgG4-RD-RI score may be decreased by at least 12. In some variations, the IgG4-RD-RI score may be decreased by at least 13. In some variations, the IgG4-RD-RI score may be decreased by at least 14. In some variations, the IgG4-RD-RI score may be decreased by at least 15. In some variations, the IgG4-RD-RI score may be decreased by at least 16. In some variations, the IgG4-RD-RI score may be decreased by at least 17. In some variations, the IgG4-RD-RI score may be decreased by at least 18. In some variations, the IgG4-RD-RI score may be decreased by at least 19. In some variations, the IgG4-RD-RI score may be decreased by at least 20 relative to baseline.In certain embodiments, the IgG4-RD-RI score can be reduced to 0.

[0241] When the IgG4-RD-RI score is reduced relative to baseline, the score may be decreased by at least 1 relative to baseline within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may be decreased by at least 2 relative to baseline within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may be decreased by at least 3 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may be decreased by at least 4 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may be decreased by at least 5 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may be decreased by at least 6 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may be decreased by at least 7 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may be decreased by at least 8 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may be decreased by at least 9 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may decrease by at least 10 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may decrease by at least 11 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may decrease by at least 12 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may decrease by at least 13 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may decrease by at least 14 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may decrease by at least 15 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may decrease by at least 16 within 2 weeks of the first dose.In some variations, the IgG4-RD-RI score may be reduced by at least 17 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may be reduced by at least 18 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may be reduced by at least 19 within 2 weeks of the first dose. In some variations, the IgG4-RD-RI score may be reduced by at least 20 relative to baseline within 2 weeks of the first dose. In certain embodiments, the IgG4-RD-RI score may be reduced to 0 within 2 weeks of the first dose.

[0242] Reductions in other treatments and medications

[0243] The use of immunoglobulins that co-bind FcγRIIb and CD19 to treat IgG4-RD may result in the reduction or elimination of side effects associated with other therapies. Without wishing to be bound by any theory, it is believed that the mechanism of action of the antibody reduces off-target effects, As a result, side effects associated with other treatments are reduced.

[0244] Furthermore, targeted therapy of IgG4-RD reduces or eliminates the need for additional agents to treat IgG4-RD. For example, targeted therapy of IgG4-RD reduces or eliminates the need for anti-inflammatory analgesics (NSAIDs, such as aspirin, ibuprofen, naproxen, or Celebrex), acetaminophen, steroids, glucocorticoids (i.e., prednisone), immunosuppressants (i.e., azathioprine, mycophenolate mofetil), and immunosuppressive biologics (i.e., rituximab, bortezomib), which are typically used to treat IgG4-RD.

[0245] In a specific embodiment, targeted treatment of IgG4-RD using an antibody that co-binds FcγRIIb and CD19 can result in tapering and eventual cessation of steroids. Methods for tapering steroids are known in the art. For example, dose reduction can be performed every 2-3 days (e.g., 6 tablets on days 1 and 2, 5 tablets on days 3 and 4, 4 tablets on days 5 and 6, 3 tablets on days 7 and 8, 2 tablets on days 9 and 10, and 1 tablet on days 11 and 12). Alternatively, the dose can be reduced by half every 3 days.

[0246] Furthermore, in some examples, IgG4-RD can be treated by administering an antibody that binds FcγRIIb and CD19 to a subject that is refractory to immunosuppressive biological therapy (e.g., rituximab, bortezomib). A subject that is refractory to an administered immunosuppressive biological therapy does not respond or shows no therapeutic response to a given immunosuppressive biological therapy, such as rituximab or bortezomib, and then re-develops disease symptoms. In some examples, IgG4-RD can be treated by administering an antibody that binds FcγRIIb and CD19 to a subject that is refractory to rituximab. In some examples, IgG4-RD can be treated by administering an antibody that binds FcγRIIb and CD19 to a subject that is refractory to bortezomib.

[0247] In some examples, IgG4-RD can be treated by administering an antibody that co-binds FcγRIIb and CD19 to a subject who has relapsed after treatment with an immunosuppressive biological therapy. A relapsed subject has responded to treatment with an immunosuppressive biological therapy, but has re-developed symptoms of the disease. In some examples, the immunosuppressive biological therapy can be rituximab. In some examples, the immunosuppressive biological therapy can be bortezomib.

[0248] Patient polymorphism

[0249] Many receptors that may interact with the immunoglobulins disclosed herein exhibit polymorphisms in the human population. For a given patient or patient population, the efficacy of the immunoglobulins disclosed herein may be affected by the presence or absence of certain protein polymorphisms. For example, FcγRIIIA exhibits polymorphism at position 158, which is typically V (high affinity) or F (low affinity). Patients with the V / V homozygous genotype have been observed to exhibit better clinical responses to treatment with the anti-CD20 antibody Rituxan® (rituximab), presumably because these patients exhibit stronger NK responses (Dall'Ozzo et al (2004) Cancer Res 64:4664-9, incorporated by reference in its entirety). Additional polymorphisms include, but are not limited to, FcγRIIA R131 or H131, which are known to increase or decrease Fc binding and subsequent biological activity depending on the polymorphism. The immunoglobulins disclosed herein may bind exclusively to a particular receptor polymorphic type, e.g., FcγRIIIA 158V, or may bind with equivalent affinity to all polymorphisms at a particular position of the receptor (e.g., both 158V and 158F of FcγRIIIA). In one embodiment, the immunoglobulins disclosed herein with equivalent binding to the polymorphisms may be used to eliminate the differential efficacy observed in patients with different polymorphisms. Such properties may provide greater consistency in the therapeutic response and reduce the non-responder patient population. Such Fc variants that exhibit the same binding to the receptor polymorphisms may enhance biological activity, e.g., ADCC, CDC, or circulating half-life, or alternatively reduce activity through modulation of binding to the corresponding Fc receptor. In one embodiment, the immunoglobulins disclosed herein may bind with higher or lower affinity to one of the polymorphisms of a receptor, accentuating or attenuating existing differences in binding. Such properties can allow for the creation of therapeutic agents whose efficacy is specifically tailored for patient populations that have such polymorphisms.For example, a patient population carrying a polymorphism with greater affinity for an inhibitory receptor (e.g., FcγRIIB) could be administered a drug containing an antibody with reduced binding to that polymorphic receptor type, resulting in the development of a more effective drug.

[0250] In one embodiment, patients are screened for one or more polymorphisms to predict the efficacy of the immunoglobulins disclosed herein. This information can be used to select patients for inclusion or exclusion from clinical trials, for example, and to provide guidance to physicians and patients regarding appropriate dosage and treatment options once approved. For example, in patients homozygous or heterozygous for FcγRIIIA 158F, antibody drugs, such as the anti-CD20 mAb rituximab, show marginal efficacy (Carton 2002 Blood 99:754-758; Weng 2003 J Clin Oncol 21:3940-3947, both of which are incorporated by reference in their entirety). Such patients may show a much better clinical response to the antibodies disclosed herein. In one embodiment, if the patient's genotype indicates that they are likely to respond significantly better to the immunoglobulins disclosed herein when compared to one or more conventionally used immunoglobulin therapeutics, they are selected for inclusion in clinical trials for the immunoglobulins disclosed herein. In another embodiment, appropriate dosages and treatment regimens are determined using such genotype information. In another embodiment, patients are selected for inclusion in clinical trials or for receiving post-approval treatments based on their polymorphic genotypes. In this case, such treatments include immunoglobulins engineered to be particularly effective against such populations, or such treatments include Fc variants that do not exert differential activity against different polymorphic types.

[0251] Also included in the invention are diagnostic tests to identify patients likely to have a favorable clinical response to the immunoglobulins disclosed herein, or likely to respond much better when treated with the immunoglobulins disclosed herein versus one or more conventionally used immunoglobulin therapeutics. Any of a number of methods for determining human FcγR polymorphisms known in the art can be used.

[0252] Also disclosed are prognostic tests performed on clinical samples, such as blood and tissue samples. Such tests can be assayed for effector function activity (including, but not limited to, ADCC, CDC, phagocytosis and opsonization) or for the killing of cancer cells or other pathogenic cells (regardless of the mechanism). In one embodiment, an ADCC assay (such as those previously described) is used to predict the efficacy of certain immunoglobulins disclosed herein for a particular patient. Such information can be used to identify patients for inclusion or exclusion from clinical trials or to inform decisions regarding appropriate dosages and treatment strategies. Such information can also be used to select pharmaceuticals that contain specific immunoglobulins that show superior activity in such assays.

[0253] Prescription

[0254] Pharmaceutical compositions are contemplated in which the immunoglobulins disclosed herein and one or more therapeutically active agents are formulated. Formulations of the immunoglobulins disclosed herein are prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing the immunoglobulins having the desired purity with any pharma- ceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, A. Osol Ed., 1980, incorporated by reference in its entirety). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers (e.g., phosphate, citric acid, acetic acid, and other organic acids); antioxidants (including ascorbic acid and methionine); preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethionine chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens (e.g., methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (e.g., for example, polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrins); chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); sweeteners and other flavors: fillers (e.g., microcrystalline cellulose, lactose, corn and other starches); binders; additives; colorants; salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants (e.g., TWEEN™, PLURONICS™, or polyethylene glycol (PEG)). In one embodiment, the pharmaceutical compositions comprising the immunoglobulins disclosed herein may be in a water-soluble form, e.g., provided as a pharma- ceutically acceptable salt (which is meant to include both acid and base addition salts)."Pharmaceutically acceptable acid addition salts" refer to salts which retain the biological effectiveness of the free base and are biologically and otherwise desirable, formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.) and organic acids (e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.). "Pharmaceutically acceptable base addition salts" include salts derived from inorganic bases, such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, etc. Some embodiments include at least one of the ammonium, sodium, calcium, and magnesium salts. Salts derived from pharma- ceutically acceptable organic non-toxic bases include primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins (e.g., isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine). Formulations to be used for in vivo administration can be sterile, which can be readily accomplished by filtration through sterile filtration membranes or other methods.

[0255] The immunoglobulins disclosed herein can also be formulated as immunoliposomes. Liposomes are vesicles containing various types of lipids, phospholipids and / or surfactants, which are useful for the delivery of therapeutic agents to mammals. Liposomes containing immunoglobulins are known in the art and are prepared, for example, by methods described in Epstein et al. 1985, Proc Natl Acad Sci USA 82:3688; Hwang et al. 1980, Proc Natl Acad Sci USA 77:4030; US4,485,045; US4,544,545; and PCTWO97 / 38731 (incorporated by reference in their entirety). Liposomes with extended circulation time are disclosed in US5,013,556 (incorporated by reference in their entirety). The components of the liposome are typically arranged in a bilayer structure, similar to the lipid arrangement of biological membranes. Particularly useful liposomes can be generated by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters with specific pore sizes to obtain liposomes with the desired diameter. Chemotherapeutic agents or other therapeutically active agents are optionally contained within the liposomes (Gabizon et al. 1989, J National Cancer Inst 81:1484, incorporated by reference in its entirety).

[0256] Immunoglobulins and other therapeutically active agents are also incorporated into microcapsules prepared by methods including, but not limited to, coacervation techniques, surface polymerization (e.g., using hydroxymethylcellulose or gelatin-microcapsules, or poly-(methyl methacrylate) microcapsules), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), and microemulsions. Such techniques are described in (Remington's Pharmaceutical Sciences 16th edition, A. Osol Ed., 1980, incorporated by reference in its entirety). Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers, the matrices having the form of shaped articles, e.g., films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919, incorporated by reference in its entirety), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron Depot®, which are injectable microspheres containing lactic acid-glycolic acid copolymer and leuprolide acetate, poly-D-(-)-3-hydroxybutyric acid, and ProLease® (commercially available from Alkermes), which is a microsphere-based delivery system containing the desired bioactive molecule entrapped in a matrix of poly-DL-lactide-coglycolide (PLG).

[0257] In some embodiments, the immunoglobulins disclosed herein are formulated for intravenous (IV) or subcutaneous (SC) administration. In general, formulations for IV or SC administration include immunoglobulins, one or more buffers, one or more tonicity adjusters, one or more solvents, and one or more surfactants. Non-limiting examples of buffers include phosphate, citrate, acetate, glutamate, carbonate, tartrate, triethanolamine (TRIS), glycylglycine, histidine, glycine, lysine, arginine, and other organic acids. More specifically, non-limiting examples of buffers include sodium HEPES, MES, potassium phosphate, potassium thiocyanate, sterilizing agent, TAE, TBE, ammonium sulfate / HEPES, BuffAR, sodium acetate, sodium carbonate, sodium citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium phosphate. In addition, buffers may be in various hydrate forms. For example, the buffer may be monohydrate, dihydrate, trihydrate, tetrahydrate, pentahydrate, hexahydrate, heptahydrate, octahydrate, nonahydrate, decahydrate, eleven-hydrate, and dodecahydrate. Sometimes the hydrate may be a fraction, such as a hemihydrate or 1.5-hydrate. Non-limiting examples of tonicity adjusters include sodium chloride, acetic acid, L-proline, dextrose, mannitol, potassium chloride, glycerin, and glycerol. Non-limiting examples of solvents include water, propylene glycol, polyethylene glycol, ethanol, dimethyl sulfoxide, N-methyl-2-pyrrolidone, glycofurol, Solketal™, glycerol formal, acetone, tetrahydrofurfuryl alcohol, diglyme, dimethyl isosorbide, and ethyl lactate.Non-limiting examples of solvents include polysorbates (e.g., polysorbate-20, polysorbate-80), polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20), sorbitan trioleate (span 85), lecithin, and polyoxyethylene polyoxypropylene copolymer (Pluronics, Pluronic F-68).

[0258] In certain embodiments, the IV formulation comprises an immunoglobulin, one or more buffers, one or more tonicity adjusters, one or more solvents, and one or more surfactants. Specifically, the buffer may be one or more sodium phosphate buffer solutions. For example, the buffer may be sodium phosphate monobasic monohydrate, sodium phosphate dibasic heptahydrate, or a combination thereof. In one embodiment, the tonicity adjuster is sodium chloride. In another embodiment, the solvent is water. In a further embodiment, the surfactant is a polysorbate. For example, the polysorbate is polysorbate-20. Specifically, the IV formulation comprises an immunoglobulin, sodium phosphate monobasic monohydrate, sodium phosphate dibasic heptahydrate, sodium chloride, water, and polysorbate-20. The amounts of the immunoglobulin, buffers, tonicity adjusters, solvents, and surfactants may vary. The amount of the immunoglobulin may be about 1 mg to about 500 mg per mL, about 1 mg to about 100 mg per mL, or about 1 mg to about 50 mg per mL. The amount of the buffer may be about 1 to about 10 mg per mL, about 1 to about 5 mg per mL, or about 1 to about 2.5 mg per mL. The amount of the tonicity adjusting agent may be about 5 to about 15 mg per mL, about 5 to about 10 mg per mL, or about 8 to about 10 mg per mL. The amount of the surfactant may be about 0.01 mg to about 1 mg per mL, about 0.01 to about 0.5 mg per mL, or about 0.05 to about 0.2 mg per mL. Specifically, the IV formulation includes immunoglobulin in an amount of about 1 mg to about 500 mg per mL, about 1 mg to about 100 mg per mL, or about 1 mg to about 50 mg per mL, sodium phosphate monobasic monohydrate and sodium phosphate dibasic heptahydrate in an amount of about 1 to about 10 mg per mL, about 1 to about 5 mg per mL, or about 1 to about 2.5 mg per mL, sodium chloride in an amount of about 1 to about 10 mg per mL, about 1 to about 5 mg per mL, or about 1 to about 2.5 mg per mL, up to about 1 mL of water, and polysorbate-20 in an amount of about 0.01 mg to about 1 mg per mL, about 0.01 to about 0.5 mg per mL, or 0.05 to about 0.2 mg per mL.Specifically, the IV formulation includes immune globulin in an amount of about 1 mg to about 50 mg per mL, sodium phosphate monobasic monohydrate and sodium phosphate dibasic heptahydrate in an amount of about 1 to about 2.5 mg per mL, sodium chloride in an amount of about 1 to about 2.5 mg per mL, water up to about 1 mL, and polysorbate-20 in an amount of 0.05 to about 0.2 mg per mL.

[0259] In certain embodiments, the SC formulation comprises an immunoglobulin, one or more buffers, one or more tonicity modifiers, one or more solvents, and one or more surfactants. Specifically, the buffer may be a sodium acetate buffer solution. For example, the buffer may be sodium acetate trihydrate. In certain embodiments, the tonicity modifier may be acetic acid, L-proline, and combinations thereof. In another embodiment, the solvent is water. In a further embodiment, the surfactant is a polysorbate. For example, the polysorbate is polysorbate-80. Specifically, the SC formulation comprises an immunoglobulin, sodium acetate trihydrate, acetic acid and L-proline, water, and polysorbate-80. The amounts of the immunoglobulin, buffers, tonicity modifiers, solvents, and surfactants may vary. The amount of the immunoglobulin may be about 1 mg to about 500 mg per mL, about 50 mg to about 250 mg per mL, or about 100 mg to about 250 mg per mL. The amount of the buffer may be about 1 to about 10 mg per mL, about 1 to about 5 mg per mL, or about 1 to about 2.5 mg per mL. The amount of the tonicity adjusting agent may be about 5 to about 50 mg per mL, about 10 to about 50 mg per mL, or about 20 to about 40 mg per mL. The amount of the surfactant may be about 0.01 mg to about 1 mg per mL, about 0.01 to about 0.5 mg per mL, or about 0.05 to about 0.2 mg per mL. Specifically, the SC formulation includes immunoglobulin in an amount of about 1 mg to about 500 mg per mL, about 50 mg to about 250 mg per mL, or about 100 mg to about 250 mg per mL, sodium acetate trihydrate in an amount of about 1 to about 10 mg per mL, about 1 to about 5 mg per mL, or about 1 to about 2.5 mg per mL, acetic acid and L-proline in an amount of about 5 to about 50 mg per mL, about 10 to about 50 mg per mL, or about 20 to about 40 mg per mL, water up to about 1 mL, and polysorbate-80 in an amount of about 0.01 mg to about 1 mg per mL, about 0.01 to about 0.5 mg per mL, or 0.05 to about 0.2 mg per mL.Specifically, the SC formulation includes immunoglobulin in an amount of about 100 mg to about 250 mg per mL, sodium acetate trihydrate in an amount of about 1 to about 2.5 mg per mL, acetic acid and L-proline in an amount of about 20 to about 40 mg per mL, up to about 1 mL of water, and polysorbate-80 in an amount of 0.05 to about 0.2 mg per mL.

[0260] Administration

[0261] Administration of pharmaceutical compositions (e.g., sterile aqueous solutions) comprising the immunoglobulins disclosed herein can be accomplished in a variety of ways, including, but not limited to, oral, subcutaneous, intravenous, intranasal, intraaural, transdermal, topical (e.g., gels, ointments, lotions, creams, etc.), intraperitoneal, intramuscular, intrapulmonary, intravaginal, parenteral, rectal, or intraorbital. In some cases, e.g., for treatment of wounds, inflammation, etc., the immunoglobulins are directly applied as a solution or spray. As is known in the art, pharmaceutical compositions may be formulated accordingly depending on the mode of delivery.

[0262] Subcutaneous administration is used in circumstances where the patient can self-administer the pharmaceutical composition. Many protein therapeutics are not potent enough to allow the formulation to contain a therapeutically effective dose in the maximum volume that can be tolerated for subcutaneous administration. This problem can be partially solved by using a protein formulation that includes arginine-HCl, histidine and polysorbate (see WO04091658, which is incorporated by reference in its entirety). The antibodies disclosed herein are amenable to subcutaneous administration due to increased potency, improved serum half-life or enhanced solubility.

[0263] As is known in the art, protein therapeutics are often delivered by intravenous infusion or bolus. The antibodies disclosed herein may also be delivered in such a manner. For example, administration may be performed intravenously by intravenous infusion with 0.9% sodium chloride as the infusion vehicle.

[0264] Pulmonary delivery can be performed using a formulation that includes an inhaler or nebulizer and an aerosolizing agent. For example, AERx® inhalable technology available from Aradigm or Inhance™ pulmonary delivery system available from Nektar Therapeutics can be used. The antibodies disclosed herein are more amenable to pulmonary delivery. FcRn is present in the lungs and can facilitate transport from the lungs to the bloodstream (see, for example, WO04004798; Bitonti et al. 2004, Proc Natl Acad Sci 101:9763-8, which are incorporated by reference in their entirety). Thus, antibodies that bind more efficiently to FcRn in the lungs or are more efficiently released in the bloodstream may have improved bioavailability after pulmonary administration. The antibodies disclosed herein are also more amenable to pulmonary administration, for example, due to improved solubility or altered isoelectric point.

[0265] Furthermore, the immunoglobulins disclosed herein are more amenable to oral administration, for example, due to improved stability at gastric pH and increased resistance to proteolysis.Furthermore, FcRn appears to be expressed in the epithelium of the adult intestine (Dickinson et al. 1999, J Clin Invest 104:903-11, incorporated by reference in its entirety), and therefore, the antibodies disclosed herein with improved FcRn interaction profiles may exhibit enhanced bioavailability after oral administration.FcRn-mediated transport of antibodies may also occur in other mucosa, such as the mucosa of the gastrointestinal, respiratory and reproductive tracts (Yoshida et al. 2004, Immunity 20:769-83, incorporated by reference in its entirety).

[0266] Furthermore, numerous delivery systems are known in the art, any of which may be used to administer the antibodies disclosed herein. Examples of the above include, but are not limited to, liposomal encapsulation, microparticles, microspheres (e.g., PLA / PGA microspheres), and the like. Alternatively, implants of porous, nonporous, or gelatinous materials (including membranes or fibers) may be used. Suitable sustained release systems may include polymeric substances or matrices, such as polyesters, hydrogels, poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, ethylene vinyl acetate, lactic acid-glycolic acid copolymers (e.g., Lupron Depot®), poly-D-(−)-3-hydroxybutyric acid. Nucleic acids encoding the immunoglobulins disclosed herein may also be administered, for example, by retroviral infection, direct injection, or coating with lipids, cell surface receptors, or other transfection agents. In all cases, a controlled release system can be used to release the immunoglobulin at or near the desired site of action.

[0267] Dosage

[0268] Dosage and frequency of administration are, in one embodiment, selected to be therapeutically or prophylactically effective. Adjustments for age, weight, general health, sex, diet, time of administration, drug interactions, and severity of symptoms, as well as adjustments for proteolysis, systemic or local delivery, and de novo protease synthesis, as known in the art, may be necessary and ascertainable by one of ordinary skill in the art using routine experimentation.

[0269] The concentration of the therapeutically active immunoglobulin in the formulation may vary from about 0.1 to 100% by weight. In one embodiment, the concentration of the immunoglobulin ranges from 0.003 to 1.0 molar. To treat a patient, a therapeutically effective dose of the immunoglobulin disclosed herein may be administered. By "therapeutically effective dose" herein is meant a dose that produces the desired effect upon administration. The exact dose will depend on the purpose of the treatment and can be ascertained by one of skill in the art using known techniques. Dosages may range from 0.0001 to 100 mg / kg body weight or more, for example, 0.1, 1, 5, 10, or 50 mg / kg body weight. In one embodiment, dosages may range from about 1 to about 10 mg / kg. In another embodiment, dosages are about 5 mg / kg.

[0270] In some embodiments, the antibody used to treat IgG4-related disease may be administered at a dose of 0.2 mg / kg or more. For example, the antibody used to treat IgG4-related disease may be administered at a dose of 0.1 mg / kg or more, 0.5 mg / kg or more, 1 mg / kg or more, 2 mg / kg or more, 5 mg / kg or more, 10 mg / kg or more, 15 mg / kg or more, 20 mg / kg or more, or 25 mg / kg or more. Alternatively, the antibody used to treat IgG4-related disease may be administered at a dose of 25 mg / kg or more, 50 mg / kg or more, 75 mg / kg or more, 100 mg / kg or more, 125 mg / kg or more, 150 mg / kg or more, 175 mg / kg or more, or 200 mg / kg or more. In other embodiments, the antibody used to treat IgG4-related disease may be administered at a dose of about 0.2 mg / kg. For example, antibodies used to treat IgG4-related diseases may be administered at a dose of about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, or about 25 mg / kg. Alternatively, antibodies used to treat IgG4-related diseases may be administered at a dose of about 25 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, or about 200 mg / kg.

[0271] In some embodiments, only one administration of immunoglobulin is used. In other embodiments, multiple administrations of immunoglobulin are performed. The time lapse between administrations can be less than 1 hour, about 1 hour, about 1-2 hours, about 2-3 hours, about 3-4 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 2-4 days, about 4-6 days, about 7 days, about 14 days, or more than 14 days. In certain embodiments, the immunoglobulin is administered every 14 days. In some embodiments, the immunoglobulin is administered in at least one dose. In other embodiments, the immunoglobulin is administered in at least two doses. In other embodiments, the immunoglobulin is administered in at least three doses. In other embodiments, the immunoglobulin is administered in at least four doses. In other embodiments, the immunoglobulin is administered in at least five doses. In other embodiments, the immunoglobulin is administered in at least six doses. In other embodiments, the immunoglobulin is administered in at least seven doses. In other embodiments, the immunoglobulin is administered in at least 8 doses. In other embodiments, the immunoglobulin is administered in at least 9 doses. In other embodiments, the immunoglobulin is administered in at least 10 doses. In other embodiments, the immunoglobulin is administered in at least 11 doses. In other embodiments, the immunoglobulin is administered in at least 12 doses. In other embodiments, the immunoglobulin is administered in more than two doses. In one particular embodiment, the immunoglobulin is administered every 14 days for a total of 12 doses.

[0272] In other embodiments, the antibodies disclosed herein are administered in a metronomic dosing regimen, by continuous infusion or frequent dosing without long rest periods. Such metronomic dosing can include dosing at regular intervals without rest periods. Typically, such regimens include chronic low-dose dosing, or continuous infusion for extended periods, e.g., 1-2 days, 1-2 weeks, 1-2 months, or 6 months or more. The use of lower doses minimizes side effects and the need for rest periods.

[0273] In certain embodiments, the immunoglobulins disclosed herein and one or more other prophylactic or therapeutic agents are administered to a patient in a cyclical manner. Cycling therapy involves administering a first agent at a first time point, a second agent at a second time point, optionally administering an additional agent at an additional time point, optionally a rest period, followed by one or more repetitions of this sequence. The number of repetitions is typically 2-10. Cycling therapy may reduce the development of resistance to one or more agents, minimize side effects, or improve therapeutic efficacy.

[0274] Combination therapy

[0275] The antibodies disclosed herein can be given simultaneously with one or more other therapies or therapeutic agents. The additional therapies or therapeutic agents can be used to improve the efficacy or safety of the immunoglobulin. Furthermore, the additional therapies or therapeutic agents can be used to treat the disease or co-morbidities rather than altering the action of the immunoglobulin. For example, the immunoglobulins disclosed herein can be administered in conjunction with chemotherapy, radiation therapy, or both chemotherapy and radiation therapy. The immunoglobulins disclosed herein can be administered in combination with one or more other prophylactic or therapeutic agents, including, but not limited to, cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitors, antihormones, kinase inhibitors, antiangiogenic agents, cardioprotectants, immunostimulants, immunosuppressants, agents that promote the proliferation of hematological cells, angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitors, another antibody, FcγRIIb or other Fc receptor inhibitors, or other therapeutic agents.

[0276] "In combination with" and "co-administration" are not limited to administration of the prophylactic or therapeutic agents at exactly the same time. Instead, they are administered in sequence and within a time interval such that the immunoglobulins disclosed herein and other agents can act together to provide benefits over treatment with only the immunoglobulins disclosed herein or the other agents. In some embodiments, the immunoglobulins disclosed herein and the other agent(s) act additively, and sometimes synergistically. Such molecules are suitably present together in amounts that are effective for the intended purpose. A skilled medical practitioner can determine the appropriate timing and method of administration, as well as the appropriate dose of each therapeutic agent, empirically or by considering the pharmacokinetics and dynamics of action of the agent.

[0277] In some embodiments, the antibodies disclosed herein for the treatment of IgG4-related diseases can be used in combination with standard therapies for IgG4-RD, including, but not limited to, anti-inflammatory analgesics (e.g., NSAIDs such as aspirin, ibuprofen, naproxen, or Celebrex), acetaminophen, steroids, glucocorticoids (i.e., prednisone), immunosuppressants (i.e., azathioprine, mycophenolate mofetil), and immunosuppressant biologics (i.e., rituximab, bortezomib).

[0278] In some embodiments, the antibody disclosed herein is administered with one or more additional molecules comprising antibody or Fc.The antibody disclosed herein can be administered simultaneously with one or more other antibodies that are effective in treating the same disease or another co-morbidity.For example, two antibodies that recognize two antigens that are overexpressed in a certain type of cancer, or two antigens that mediate the development of autoimmunity or infectious disease can be administered.

[0279] Examples of anti-cancer antibodies that can be co-administered include, but are not limited to, anti-17-1A cell surface antigen antibodies, such as Panorex™ (edrecolomab); anti-4-1BB antibodies; anti-4Dc antibodies; anti-A33 antibodies, such as A33 and CDP-833; anti-α4β1 integrin antibodies, such as natalizumab; anti-α4β7 integrin antibodies, such as LDP-02; anti-αVβ1 integrin antibodies, such as F-200, M-200, and SJ-749; anti-αVβ3 integrin antibodies, such as abciximab, CNTO-95, Mab-17E6, and Vitaxin™; anti-complement factor 5 (C5) antibodies, such as 5G1.1; anti-CA125 antibodies, such as OvaRex® (oregovomab); anti-CD3 antibodies, such as Nuvion® (vicilimab) and lexomab; anti-CD4 antibodies, such as IDEC-151, MDX-CD4, OKT4A; anti-CD6 antibodies, such as oncolicin B and oncolicin CD6; anti-CD7 antibodies, such as HB2; anti-CD19 antibodies, such as B43, MT-103, and oncolicin B; anti-CD20 antibodies, such as 2H7, 2H7.v16, 2H7.v114, 2H7.v115, Bexxar® (tositumomab, I-131 labeled anti-CD20), Rituxan® (rituximab), and Zevalin® (ibritumab tiuxetan tiuxetan) Y-90 labeled anti-CD20; anti-CD22 antibodies such as Lymphocide™ (epratuzumab, Y-90 labeled anti-CD22); anti-CD23 antibodies such as IDEC-152; anti-CD25 antibodies such as basiliximab and Zenapax® (daclizumab); anti-CD30 antibodies such as AC10, MDX-060 and SGN-30; anti-CD33 antibodies such as Mylotarg® (gemtuzumab ozogamicin), Oncolicin M and Smart M195; anti-CD38 antibodies; anti-CD40 antibodies such as SGN-40 and toralizumab; anti-CD40L antibodies such as 5c8, Antova™ and IDEC-131; anti-CD44 antibodies, such as bivatuzumab; anti-CD46 antibodies; anti-CD52 antibodies, such as Campath® (alemtumab); anti-CD55 antibodies, such as SC-1; anti-CD56 antibodies, such as huN901-DM1; anti-CD64 antibodies, such as MDX-33; anti-CD66e antibodies, such as XR-303; anti-CD74 antibodies, such as IMMU-110; anti-CD80 antibodies, such as galiximab and IDEC-114; anti-CD89 antibodies, such as MDX-214; anti-CD123 antibodies; anti-CD138 antibodies, such as B-B4-DM1; anti-CD146 antibodies, such as AA-98; anti-CD148 antibodies; anti-CEA antibodies, such as cT84.66, labetuzumab, and Pentacea™; anti-CTLA-4 antibodies, such as MDX-101; anti-CXCR4 antibodies; anti-EGFR antibodies, such as ABX-EGF, Erbitux® (cetuximab), IMC-C225, and Merck Mab 425; anti-EpCAM antibodies, such as Crucell's anti-EpCAM, ING-1, and IS-IL-2; anti-ephrin B2 / EphB4 antibodies; anti-Her2 antibodies, such as Herceptin®, MdX-210; anti-FAP (fibroblast stimulatory protein) antibodies, such as sibrotuzumab; anti-ferritin antibodies, such as NXT-211; anti-FGF-1 antibodies; anti-FGF-3 antibodies; anti-FGF-8 antibodies; anti-FGFR antibodies, anti-fibrin antibodies; anti-G250 antibodies, such as WX-G250 and Rencarex®; anti-GD2 ganglioside antibodies, such as EMD-273063 and TriGem; anti-GD3 ganglioside antibodies, such as BEC2, KW-2871 and mitumomab; anti-gpIIb / IIIa antibodies, such as ReoPro; anti-heparinase antibodies; anti-Her2 / ErbB2 antibodies, such as Herceptin® (trastuzumab), MDX-210 and pertuzumab; anti-HLA antibodies, such as Oncolym®, Smart 1D10; anti-HM1.24 antibodies; anti-ICAM antibodies, such as ICM3; anti-IgA receptor antibodies; anti-IGF-1 antibodies, such as CP-751871 and EM-164; anti-IGF-1R antibodies, such as IMC-A12; anti-IL-6 antibodies, such as CNTO-328 and elsilimomab; anti-IL-15 antibodies, such as HuMax™-IL15; anti-KDR antibodies; anti-laminin antibodies; anti-Lewis Y antigen antibodies, such as Hu3S193 and IGN-311; anti-MCAM antibodies; anti-Muc1 antibodies, such as BravaRex and TriAb; anti-NCAM antibodies, such as ERIC-1 and ICRT; anti-PEM antigen antibodies, such as Theragyn and Therex; anti-PSA antibodies; anti- PSCA antibodies, such as IG8; anti-Ptk antibodies; anti-PTN antibodies; anti-RANKL antibodies, such as AMG-162; anti-RLIP76 antibodies; anti-SK-1 antigen antibodies, such as MonopharmC; anti-STEAP antibodies; anti-TAG72 antibodies, such as CC49-SCA and MDX-220; anti-TGF-β antibodies, such as CAT-152; anti-TNF-α antibodies, such as CDP571, CDP870, D2E7, Humira® (adalibumab), and Remicade® (infliximab); anti-TRAIL-R1 and TRAIL-R2 antibodies; anti-VE-cadherin-2 antibodies; and anti-VLA-4 antibodies, such as Antegren™. Additionally, anti-idiotypic antibodies can be used, including but not limited to GD3 epitope antibody (BEC2) and gp72 epitope antibody (105AD7). Additionally, bispecific antibodies can be used, including but not limited to anti-CD3 / CD20 antibody (Bi20).

[0280] Examples of antibodies that may be co-administered to treat autoimmune or inflammatory diseases, transplant rejection, GVHD, and the like include, but are not limited to, anti-α4β7 integrin antibodies (e.g., LDP-02), anti-β2 integrin antibodies (e.g., LDP-01), anti-complement (C5) antibodies (e.g., 5G1.1), anti-CD2 antibodies (e.g., BTI-322, MEDI-507), anti-CD3 antibodies (e.g., OKT3, SMART anti-CD3), anti-CD4 antibodies (e.g., IDEC-151, MDX-CD4, OKT4A), anti-CD11a antibodies, anti-CD14 antibodies (e.g., IC14), anti-CD18 antibodies, anti-CD23 antibodies (e.g., IDEC152), anti-CD25 antibodies (e.g., Zenapax), anti-CD40L antibodies (e.g., 5c8, Antova, IDEC-131), anti-CD64 antibodies (e.g., MDX-33), anti-CD80 antibodies (e.g. IDEC-114), anti-CD147 antibodies (e.g. ABX-CBL), anti-E-selectin antibodies (e.g. CDP850), anti-gpIIb / IIIa antibodies (e.g. ReoPro / Abcixima), anti-ICAM-3 antibodies (e.g. ICM3), anti-ICE antibodies (e.g. VX-740), anti-FcγR1 antibodies (e.g. MDX-33), anti-IgE antibodies (e.g. Examples of Fc-containing molecules that can be co-administered to treat autoimmune or inflammatory diseases, transplant rejection, GVHD, and the like include, but are not limited to, p75 TNF receptor / Fc fusions, Enbrel® (etanercept), and Regeneron's IL-1 trap.

[0281] Examples of antibodies that can be co-administered to treat infections include, but are not limited to, anti-anthrax antibodies (e.g., ABthrax), anti-CMV antibodies (e.g., CytoGam and Sevirumab), anti-Cryptosporidium antibodies (e.g., CryptoGAM, Sporidin-G), anti-Helicobacter antibodies (e.g., Pyloran), anti-Hepatitis B antibodies (e.g., HepeX-B, Nabi-HB), anti-HIV antibodies (e.g., HRG-214), anti-RSV antibodies (e.g., Felvizumab, HNK-20, Palivizumab, RespiGam), and anti-Staphylococcus antibodies (e.g., Aurexis, Aurograb, BSYX-A110, and SE-Mab).

[0282] Alternatively, the antibodies disclosed herein can be co-administered with one or more molecules that compete for binding to one or more Fc receptors. For example, co-administration of an inhibitor of the inhibitory receptor FcγRIIb can enhance effector function. Similarly, co-administration of an inhibitor of an activating receptor (e.g., FcγRIIIa) can minimize undesirable effector function. Fc receptor inhibitors include, but are not limited to, Fc molecules engineered to act as competitive inhibitors of binding to other immunoglobulins with FcγRIIb, FcγRIIIa or other Fc receptors, and in particular a treatment called IVIg (intravenous immunoglobulin). In one embodiment, the inhibitor is administered and allowed to act before administering the immunoglobulin. Another way to achieve the effect of sequential administration would be to provide a rapid release formulation of the Fc receptor inhibitor followed by a sustained release formulation of the immunoglobulin disclosed herein. The rapid release and controlled release formulations could be administered separately or combined into one unit dosage form. Administration of FcγRIIb inhibitors can also be used to limit undesirable immune responses, such as anti-Factor VIII antibody responses following administration of Factor VIII to hemophilia patients.

[0283] In some embodiments, the antibodies disclosed herein are administered with a chemotherapeutic agent. As used herein, "chemotherapeutic agent" refers to a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to: alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates, such as busulfan, improsulfan, and piposulfan; androgens, such as calsterone, dromostanol propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane; anti-androgens, such as furuta. amide, nilutamide, bicalutamide, leuprolide and goserelin; antibiotics such as aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, Nogalamycin, Olivomycin, Peplomycin, Potfilomycin, Puromycin, Queramycin, Lodorubicin, Streptonigrin, Streptozocin, Tubercidin, Ubenimex, Zinostatin, Zorubicin; Antiestrogens, such as Tamoxifen, Raloxifene, Aromatase Inhibitor 4(5)-Imidazole, 4-Hydroxytamoxifen, Trioxyphene, LY117018, Onapristone and Toremifene (Fareston); Antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylmelamines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide and trimethylolmelamine); folic acid supplements such as frolinic acid;Nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, fensterin, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; platinum analogs, such as cisplatin and carboplatin. platinum; proteins such as arginine deiminase and asparaginase; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; taxanes such as paclitaxel (TAXOL®, Bristol-Meyers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTER®, Rhone-Polenc Rorber, Antony, France);Topoisomerase inhibitors RFS2000;Thymidylate synthase inhibitors (e.g. Tomudex);Further chemotherapy agents including;Aceglatone;Aldophosphamide glycosides;Aminolevulinic acid;Amsacrine;Bestravcil;Bisantrene;Edatraxate;Dofofamine;Demecolcine;Diaziquone;Difluoromethylornithine (DMFO);Elformitine;Elliptinium acetate;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidamine;Mitoguazone;Mitoxantrone;Mopidamol;Nitracrine;Pentostatin;Fenameth;Pirarubicin;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK (registered trademark);Razoxane;Schizofuran;Spirogermanium;Tenuazonic acid (tenuazonic acid);triaziquone;2,2',2"-trichlorotriethylamine;urethane;vindesine;dacarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gacytosine;arabinoside ("Ara-C");cyclophosphamide;thiotepa;Chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; retinoic acid; esperamicin; capecitabine. Pharmaceutically acceptable salts, acids or derivatives of any of the above may also be used.;

[0284] Chemotherapeutic agents or other cytotoxic drugs can be administered as prodrugs. As used herein, "prodrug" refers to a precursor or derivative form of a medicamentously active substance that is less cytotoxic to tumor cells compared to the parent drug and is enzymatically activated or converted into a more active parent form. See, for example, Wilman, 1986, Biochemical Society Transactions, 615th Meeting Belfast, 14:375-382; Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery; and Borchardt et al. (ed): 247-267, Humana Press, 1985 (incorporated by reference in their entirety). Prodrugs that may be useful in the context of the present invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that may be converted to the more active cytotoxic free drug. Examples of cytotoxic drugs that may be derivatized into a prodrug form for use with the antibodies disclosed herein include, but are not limited to, any of the chemotherapeutic agents described above.

[0285] A variety of other therapeutic agents may be useful for administration together with the antibodies disclosed herein. In one embodiment, the immunoglobulin is administered together with an anti-angiogenic agent. As used herein, "anti-angiogenic agent" refers to a compound that blocks or impedes to some degree the development of blood vessels. For example, an anti-angiogenic factor is a small molecule or protein, such as an antibody, Fc fusion, or cytokine, that binds to a growth factor or growth factor receptor required to promote angiogenesis. In one embodiment, the anti-angiogenic factor can be an antibody that binds to vascular endothelial growth factor (VEGF). Other agents that inhibit signaling through VEGF can also be used, such as RNA-based therapeutic agents that reduce the expression levels of VEGF or VEGF-R, VEGF-toxin fusions, Regeneron's VEGF-trap, and antibodies that bind to VEGF-R. In yet another embodiment, the antibody is administered together with a therapeutic agent that induces or enhances an adaptive immune response, such as an antibody that targets CTLA-4.Additional anti-angiogenic agents include, but are not limited to, angiostatin (plasminogen fragment), antithrombin III, angiozyme, ABT-627, Bay 12-9566, benefin, bevacizumab, bisphosphonates, BMS-275291, cartilage-derived inhibitor (CDI), CAI, CD95 complement fragment, CEP-7055, Col 3, combretastatin A-4, endostatin (collagen XVIII fragment), farnesyltransferase inhibitor, fibronectin fragment, gro-beta, halofuginone, heparinase, heparin hexasaccharide fragment, HMV833, human chorionic gonadotropin (hCG), IM-862, interferon alpha, interferon beta, interferon gamma, interferon-inducible protein 10 (IP-10), interleukin-12, kringle 5 (plasminogen fragment), marimastat, metalloproteinase inhibitors (e.g. TIMPs), 2-methoestradiol, MMI 270 (CGS27023A), plasminogen activator (PAI), platelet factor-4 (PF4), prinomastat, prolactin 16 kDa fragment, proliferin-related protein (PRP), PTK787 / ZK222594, retinoids, solimasstat, squalamine, SS3304, SU5416, SU6668, SU11248, tetrahydrocortisol-S, tetrathiomolybdate, thalidomide, thrombospondin-1 (TSP-1), TNP-470, transforming growth factor beta (TGF-β), vasculostatin, vasostatin (calreticulin fragment), ZS6126, and ZD6474.

[0286] In one embodiment, the immune globulin is administered with a tyrosine kinase inhibitor. As used herein, "tyrosine kinase inhibitor" refers to a molecule that inhibits to some extent the tyrosine kinase activity of a tyrosine kinase. Examples of such inhibitors include (but are not limited to): quinazolines, e.g., PD153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrinidines, e.g., CGP59326, CGP60261, and CGP62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo(2,3-d)pyrimidine; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to ErbB-encoding nucleic acids); quinoxalines (US 5,804,396); tryphostin (US 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis / Scering AG); pan-ErbB inhibitors, such as C1-1033 (Pfizer); Afinitaq (ISIS3521; Isis / Lilly); Imatinib mesylate (STI571, Gleevec®; Novartis); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); C1-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis / Scering AG); INC-1C11 (Imclone); or those described in the following patent publications: US 5,804,396; PCT WO99 / 09016 (American Cyanamid); PCT WO98 / 43960 (American Cyanamid); PCT WO97 / 38983(Warner-Lambert);PCT WO99 / 06378(Warner-Lambert);PCT WO99 / 06396(Warner-Lambert);PCT WO96 / 30347(Pfizer, Inc);PCT WO96 / 33978 (AstraZeneca); PCT WO963397 (AstraZeneca); PCT WO96 / 33980 (AstraZeneca), gefitinib (IRESSA™, ZD1839, AstraZeneca), and OSI-774 (Tarceva™, OSI Pharmaceuticals / Genentech) (all patent documents are incorporated by reference in their entirety).

[0287] In another embodiment, the immunoglobulin is administered with one or more immunomodulatory agents that can increase or decrease one or more cytokines, upregulate or downregulate self-antigen presentation, mask MHC antigens, or promote proliferation, differentiation, migration, or activation of one or more types of immune cells. Immunomodulatory agents include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofed, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketoralac, oxaprozin, nabumentone, sulindac, tolmentin, rofecoxib, naproxen, ketoprofen, and nabumetone; steroids (e.g., glucocorticoids, dexamethasone, cortisone, hydroxycortisone, methylprednisolone, prednisone, prednisolone, trimcinolone, azulfidine eicosanoids, such as prostaglandins, thromboxanes, and leukotrienes; the above as well as topical steroids (e.g., anthralin, calcipotriene, clobetasol, and tazarotene); cytokines, such as TGFb, IFNa, IFNb, IFNg, IL-2, IL-4, IL-10;Cytokine, chemokine, or receptor antagonists, including antibodies, soluble receptors, and receptor-Fc fusions against: BAFF, B7, CCR2, CCR5, CD2, CD3, CD4, CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28, CD40, CD40L, CD44, CD45, CD52, CD64, CD80, CD86, CD147, CD152, complement factors (C5, D) CTLA4, eotaxin, Fas, ICAM, ICOS, IFNα, IFNβ, IFNγ, IFNAR, IgE, IL-1, IL-2, I L-2R, IL-4, IL-5R, IL-6, IL-8, IL-9, IL-12, IL-13, IL-13R1, IL-15, IL-18R, IL-23, Integrin, LFA-1, LFA-3, MHC, Sele cytidine, TGFβ, TNFα, TNFβ, TNF-R1, T cell receptors), (including Enbrel® (etanercept), Humira® (adalibumab) and Remicada® (infliximab); heterogeneous antilymphocyte globulin; other immunomodulatory molecules such as 2-amino-6-aryl-5 substituted pyrimidines, anti-idiotypic antibodies against MHC binding peptides and MHC fragments, azathioprine, brequinar, bromocriptine, cyclophosphamide, cyclosporine A, D-penicillamine, deoxyspergualin, FK506, glutaraldehyde, gold, hydroxychloroquine, leflunomide, malononitriloamide (e.g. leflunomide), methotrexate, minocycline, mizoribine, mycophenolate mofetil, rapamycin and sulfasasazine.

[0288] In another embodiment, the immunoglobulins disclosed herein are administered with cytokines. As used herein, "cytokine" refers to a general term for proteins released by one cell population that act on another cell as intercellular regulators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include, among others: growth hormones, such as human growth hormone, human N-methionyl growth hormone, and bovine growth hormone; parathyroid hormone; tyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factors; fibroblast growth factors; prolactin; placental lactogen; tumor necrosis factors-alpha and -beta; Mullerian inhibitory substance; mouse gonadotropin-related peptide; inhibitors; activins; vascular endothelial growth factors: integrins; thrombopoietin (TPO); nerve growth factors, such as NGF-beta; platelet growth factors; transforming growth factors (TGFs). such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteogenic factor; interferons, such as interferon-alpha, -beta and -gamma; colony-stimulating factors (CSF), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-alpha, IL-2, IL-3, IL-4, IL-5R, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15; tumor necrosis factors, such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources and from recombinant cell culture, as well as biologically active equivalents of the native sequence cytokines.

[0289] In one embodiment, a cytokine or other substance that stimulates cells of the immune system is co-administered with the immunoglobulins disclosed herein. Such therapeutic aspects can enhance desired effector functions. For example, a substance that stimulates NK cells can be co-administered, including, but not limited to, IL-2. In another embodiment, a substance that stimulates macrophages can be co-administered, including, but not limited to, C5a, formyl peptides such as N-formyl-methionyl-leucyl-phenylalanine (Beigier-Bompadre et al. 2003, Scand J Immunol 57:221-8, incorporated by reference in its entirety). Furthermore, a substance that stimulates neutrophils can be administered, including, but not limited to, G-CSF, GM-CSF, and the like. Furthermore, a substance that promotes the release of such immune stimulating cytokines can be used. Furthermore, another substance can promote one or more effector functions, including, but not limited to, interferon gamma, IL-3, and IL-7.

[0290] In yet another embodiment, cytokines or other substances that inhibit effector function are co-administered with the immunoglobulins disclosed herein. Such therapeutic modalities can limit undesirable effector function.

[0291] In yet another embodiment, the immune globulin is administered with one or more antibiotics, including, but not limited to, aminoglycosides (e.g., apramycin, arbekacin, bambermycin, butirosin, dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, ribostamycin, sisomicin, spectrinomycin), aminocyclitols (e.g., sprictinomycin), amphenicols (e.g., azidamphenicol, chloramphenicol, florfrnicol, and thiamphemicol), ansamycins (e.g., rifamide and rifampicin), carbapenems (e.g., imioenem, meropenem, panipenem); cephalosporins (e.g., cefaclor, cefadroxil, cefamandole, cefatrizine ... fazedon, cefozopran, cefpimizole, cefpiramide, cefpirome, cefprozil, cefuroxine, cefixime, cephalexin, cephradine), cephamycins (cefbuperazone, cefoxitin, cefminox, cefmetazole and cefotetan); lincosamides (e.g. clindamycin, lincomycin); macrolides (e.g. azithromycin, brefeldin A, clarithromycin, erythromycin, roxithromycin, tobramycin, monobactams (e.g. aztreonam, carumonam and tigernoam); mupirocin; oxacephems (e.g. flomoxef, latamoxef and moxalactam); penicillins (e.g. amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin, bexadilpenicillinate, benzylpenicillin sodium, epicillin, fenbenicillin, floxacillin, penamecillin, penesamate hydriodide, penicillin o-benethamin, penicillin O, penicillin V, penicillin V benzoate, penicillin V hydrabamine, penimepicycline and fencicycline potassium); polypeptides (e.g. bacitracin, colistin, polymyxin B, teicoplanin, vancomycin);Quinolones (amifloxacin, cinoxacin, ciprofloxacin, enoxacin, enrofloxacin, feroxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin, pipemidic acid acid), losoxacin, rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; rifampin; streptogramins (e.g. quinupristin, dalfopristin); sulfonamides (sulfanilamide, sulfamethoxazole); tetracyclines (chlorotetracycline, demeclocycline hydrochloride, demethylchlorotetracycline, doxycycline, duramycin, minocycline, neomycin, oxytetracycline, streptomycin, tetracycline, vancomycin).

[0292] Antifungal agents such as amphotericin B, ciclopirox, clotrimazole, econazole, fluconazole, flucytosine, itraconazole, ketoconazole, niconazole, nystatin, terbinafine, terconazole, and tioconazole may also be used.

[0293] Antiviral agents, including protease inhibitors, reverse transcriptase inhibitors, and others (including type I interferons, viral fusion inhibitors, and neuraminidase inhibitors), may also be used. Examples of antiviral agents include, but are not limited to, acyclovir, adefovir, amantadine, amprenavir, clevazine, enfuvirtide, entecavir, foscarnet, gangciclovir, idoxuridine, indinavir, lopinavir, pleconaril, ribavirin, rimantadine, ritonavir, saquinavir, trifluridine, vidarabine, and zidovudine may be used.

[0294] The immunoglobulins disclosed herein can be combined with other therapeutic strategies. For example, in certain embodiments, patients to be treated with the immunoglobulins disclosed herein can also undergo radiation therapy. Radiation therapy can be performed according to protocols commonly used in the art and known to those skilled in the art. Such therapies include, but are not limited to, cesium, iridium, iodine, or cobalt irradiation. Radiation therapy can be total body irradiation or can be locally directed to a specific site or tissue in or on the body (e.g., lung, bladder, or prostate). Typically, radiation therapy is administered in pulses over a period of about 1 to 2 weeks. However, radiation therapy can be administered for longer periods. For example, radiation therapy can be administered for about 6 to 7 weeks to patients with head and neck cancer. In some cases, radiation therapy can be administered as a single dose or as continuous multiple doses. A skilled medical practitioner can empirically determine the appropriate dose of radiation therapy useful herein. According to another embodiment, the immunoglobulins disclosed herein and one or more other anti-cancer agents can be used to treat cancer cells ex vivo. Such ex vivo therapy may be useful in bone marrow transplantation, and particularly in autologous bone marrow transplantation. For example, treatment of cells or tissues containing cancer cells with immunoglobulin and one or more other anti-cancer therapies (such as those described above) may be used to remove or substantially remove the cancer cells prior to transplantation into a recipient patient.

[0295] It is, of course, contemplated that the antibodies disclosed herein may also be utilized in combination with other therapeutic techniques, such as surgery or phototherapy. EXAMPLES

[0296] The examples presented below are for illustrative purposes only and are not meant to bind any of the embodiments disclosed herein to any particular application or theory of operation.

[0297] Example 1. FcγRIIb + How to Inhibit Cells

[0298] FcγRIIb is expressed on various immune cells, including B cells, plasma cells, dendritic cells, monocytes, and macrophages, where it plays an important role in immune regulation. In its normal function on B cells, FcγRIIb functions as a feedback mechanism to regulate B cell activity through the B cell receptor (BCR). Co-engagement of the B cell antigen receptor (BCR) by immune complex antigens on mature B cells activates intracellular signaling cascades, including calcium mobilization, leading to cell proliferation and differentiation. However, when IgG antibodies with specificity for antigens are produced, the associated immune complexes (ICs) can cross-link BCR with FcγRIIb, but the activity of BCR is inhibited by the associated intracellular signaling pathways that interfere with the binding of FcγRIIb and the down-regulation pathway of BCR activity.

[0299] B cells are not only responsible for producing antibodies and cytokines that control immune responses, but they are also antigen-presenting cells (APCs). Internalization of antigens by the BCR into B cells may play a role in presentation to and activation of T cells. Regulation of B cell activation through the BCR is also potentially regulated by antibody binding of FcγRIIb. Other APCs, such as dendritic cells, macrophages, and monocytes, are capable of internalizing antibody-bound antigens through activating receptors, such as FcγRIIa, FcγRIIIa, and FcγRI. Expression of FcγRIIb on these cell types, especially dendritic cells, can inhibit the activation of these cell types and their subsequent presentation to and activation of T cells (Desai et al., 2007, J Immunol).

[0300] A strategy to inhibit the activation of the aforementioned cell types is to use a single immunoglobulin that co-binds FcγRIIb with a surface antigen present on FcγRIIb+ cells. In the case of B cells, based on natural biological actions, this would potentially involve dual targeting of FcγRIIb and BCR with the goal of mimicking immune complex-mediated inhibition of B cell activation. Figure 3 illustrates one such possible mechanism, where an antibody is used to co-bind both FcγRIIb via its Fc region and a target antigen (CD19 in this example) associated with the BCR complex via its Fv region.

[0301] Example 2. Engineered immunoglobulins with selectively enhanced affinity for FcγRIIb

[0302] Under physiological conditions, cross-linking of FcγRIIb to the BCR and subsequent B cell inhibition occurs via immune complexes of IgGs and cognate antigens. The design strategy was to replicate this effect using a single cross-linking antibody. Human IgG binds human FcγRIIb with weak affinity (approximately 1 μM for IgG1), and FcγRIIb-mediated inhibition occurs in response to immune complexes, but not monomeric IgG. It was speculated that enhanced Fc affinity for this receptor is required for maximal inhibition of B cell activation. Protein engineering methods were used to design and screen Fc variants for enhanced FcγRIIb binding.

[0303] Using the solved structure of the human Fc / FcγRIIIb complex (and the sequences of human FcγRs), structural and sequence analyses were used to identify FcγR positions that contribute to the affinity and selectivity of FcγRIIb for activating receptors. The design strategy employed two steps. First, FcγR positions that are determinants of FcγRIIb and FcγRIIIa binding selectivity were identified by focusing on their proximity to the FcγR / Fc interface and amino acid differences between FcγRIIb and FcγRIIIa. Second, sequence positions in the Fc region proximal to these FcγR positions were identified. Fc variants were designed incorporating substitutions at these positions.

[0304] A library of Fc variants was generated and screened to explore amino acid modifications at these positions (see U.S. Patent No. 8,063,187 to Chu et al., incorporated by reference in its entirety). The variants were first generated in the context of an antibody targeting the antigen CD19, a regulatory component of the BCR coreceptor complex. The Fv region of this antibody is a humanized, affinity matured version of the antibody 4G7, referred to herein as HuAM4G7. The amino acid sequence of this antibody is provided in Figures 14A-D. The Fv gene of this antibody was subcloned into the mammalian expression vector pTT5 (National Research Council Canada). Mutations in the Fc domain were introduced using site-directed mutagenesis (QuikChange, Stratagene, Cedar Creek, TX). In addition, controlled knockout variants with truncated affinity for the Fc receptor were generated, including the substitution L328R, and either a G236R substitution or an Arg inserted after position 236. These variants (G236R / L328R and ^236R / L328R) are referred to as Fc-KO or FcγR knockout. To serve as non-CD19 Fc isotype controls, anti-respiratory syncytial virus (RSV) and anti-FITC antibodies were used with the appropriate V L and V H The region is divided into C with Fc changes. LKappa and C H The heavy and light chain constructs were co-transfected into HEK293E cells for expression, and the antibodies were purified using Protein A affinity chromatography (Pierce Biotechnology, Rockford, IL).

[0305] Human Fc receptor proteins FcγRI and FcγRIIb for binding and competition studies were purchased from R&D Systems (Minneapolis, MN). Genes encoding FcγRIIa and FcγRIIIa receptor proteins were purchased from Mammalian Gene Collection (ATCC) and subcloned into pTT5 vectors (National Research Council Canada) containing 6X His and GST tags. Allelic forms of the receptors (H131 and R131 for FcγRIIa and V158, and F158 for FcγRIIIa) were generated using QuikChange mutagenesis. Receptor-encoding vectors were transfected into HEK293T cells and proteins were purified using nickel affinity chromatography.

[0306] Variants were screened for receptor affinity using Biacore technology, a surface plasmon resonance (SPR)-based technology for testing biomolecular interactions in real time, also referred to herein as Biacore. SPR measurements were performed using a Biacore 3000 instrument (Biacore, Piscataway, NJ). Protein A / G (Pierce Biotechnology) CM5 biosensor chips (Biacore) were generated using a standard primary amine coupling protocol. All measurements were performed using HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% vol / vol surfactant P20, Biacore). Antibodies at 20 nM or 50 nM containing HBS-EP buffer were immobilized on the Protein A / G surface and FcγR was injected. After each cycle, the surface was regenerated by injecting glycine buffer (10 mM, pH 1.5). Data were processed by subtracting the appropriate non-specific signal (response of control channel and injection of running buffer) to zero the time and response before injection of FcγR. Kinetic analysis was performed by global fitting of the binding data with a 1:1 Langmuir binding model using BIAevaluation software (Biacore).

[0307] A representative series of sensorgrams of binding of select variant anti-CD19 antibodies to FcγRIIb is shown in FIG.

[0308] A useful quantity for variant analysis is their affinity fold to WT IgG1, which is derived by dividing the Kd for binding of WT IgG1 by the Kd for binding of the variant to each receptor. These affinity fold results are provided in Figures 5A-5D. Many variants had more than 2 log enhancement of FcγRIIb binding, and substantially reduced or eliminated affinity to activating receptors. In particular, S267E (single substitution), as well as L235Y / S267E, G236D / S267E, S239D / S267E, S267E / H268E, and S267E / L328F (double substitutions) have remarkably high affinity to FcγRIIb. In addition, these variants have affinity to the activating receptor FcγRIIIa that is comparable to native IgG1, slightly enhanced, or even significantly reduced.

[0309] The variant with the highest affinity for FcγRIIb, S267E / L328F, showed more than two orders of magnitude improvement in affinity for FcγRIIb and significantly decreased affinity for activating receptors including FcγRIIIa, FcγRI, and H131FcγRIIa.

[0310] To validate the Biacore data and evaluate the receptor binding of the variants on the cell surface, binding of selected antibodies to cells expressing FcγRIIb was measured. Because HEK293T cells do not express CD19 or FcγRs, transfection of these cells with FcγRIIb allowed the analysis of antibody binding to Fc receptors on the cell surface in isolation. HEK293T cells in DMEM with 10% FBS were transfected with human FcγRIIb cDNA in the pCMV6 expression vector (Origene Technologies, Rockville, MD), cultured for 3 days, harvested, washed twice with PBS, resuspended in PBS with 0.1% BSA (PBS / BSA), and plated at 2 × 10 per well in 96-well microtiter plates. 5The Fc variant antibodies were serially diluted in PBS / BSA and then added to the cells and incubated for 1 h at room temperature with mixing. After extensive washing with PBS / BSA, phycoerythrin (PE)-labeled anti-human Fab specific goat F(ab')2 fragments were added for detection. The cells were incubated for 30 min at room temperature, washed, and resuspended in PBS / BSA. Binding was assessed using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA), and mean fluorescence intensity (MFI) was plotted as a function of antibody concentration using GraphPad Prism software (GraphPad Software, San Diego, CA), from which half-maximal binding (EC50) values ​​were determined by sigmoidal dose-response modeling.

[0311] Receptor expression levels were assessed prior to antibody binding, and half-maximal effective concentration (EC50) values ​​of MFI were measured at various antibody concentrations. Figure 6 shows the results of this experiment. The EC50 values ​​of the tested variants showed a similar rank order to the Biacore results. Cell surface binding confirmed that the S267E / L328F variant tested had the highest affinity for FcγRIIb, with an EC50 of approximately 320-fold higher than WT IgG1. The strong agreement between these cell surface and Biacore binding data supports the accuracy of the affinity measurements.

[0312] Although the variants were screened against human IgG1, it was contemplated that the variants could be used against other antibody isotypes, including, for example, but not limited to, human IgG2, human IgG3, and human IgG4 (FIG. 1). To investigate the mobility of the variants against other antibody isotypes, the S267E / L328F variant was constructed and tested against the IgG1 / 2 ELLGG antibody, which is a variant of the IgG2 Fc region (US Publication No. 2006 / 0134105, incorporated by reference in its entirety). The mutations were constructed, the antibodies were purified, and binding data were performed as previously described. FIG. 7 shows the affinity of the IgG1 and IgG1 / 2 variant antibodies for human FcγRs as measured by Biacore. The data show that the greatly enhanced FcγRIIb affinity and overall FcγR binding profile are maintained in the variant IgG2 Fc region, thereby supporting the use of the variants in other isotype contexts.

[0313] Taken together, the above data indicate that variants with specific substitutions provide the desired properties, i.e., enhanced affinity for FcγRIIb and selectively enhanced FcγRIIb affinity for activating receptors FcγRI, FcγRIIa, and FcγRIIIa. Substitutions that increase affinity for FcγRIIb include: 234, 235, 236, 239, 267, 268, and 328.

[0314] Non-limiting combinations of positions for substitution to increase affinity to FcγRIIb include: 235 / 267, 236 / 267, 236 / 267, 267 / 328, and 268 / 267. Substitutions for increasing affinity to FcγRIIb include: L234W, L235I, L235Y, L235R, L235D, G236D, G236N, S239D, S267D, S267E, H268E, H268D, L328F, and L328Y. Substitution combinations for increasing affinity for FcγRIIb include: L235D / S267E, L235Y / S267E, L235D / S267D, L235I / S267E, L235I / S267D, L235Y / S267D, G236D / S267E, G236D / S267D, S267E / L328F, S267D / L328F, H268D / S267E, H268D / S267D, H268E / S267E, H268E / S267D, and G236D / S267E / L328F.

[0315] Example 3. Immunoglobulin inhibits BCR-mediated primary human B cell survival.

[0316] Although conventional B cells have a long in vivo half-life of about 5 weeks, their life span is greatly shortened in vitro. BCR stimulation with cross-linking antibodies, such as anti-IgM or anti-CD79b, overcomes this in vitro tendency toward apoptosis, leading to B cell activation and enhanced B cell survival. To demonstrate this, an ATP-dependent B cell survival study was performed. Human peripheral blood mononuclear cells (PBMCs) were purified from leukapheresis of anonymous healthy volunteers (HemaCare, Van Nuys, CA) using a Ficoll-Paque Plus density gradient (Amersham Biosciences, Newark, NJ). Primary human B cells were purified from PBMCs using a B cell enrichment kit (StemCell Technologies, Vancouver, British Columbia). Mouse anti-human CD79b (clone SN8) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-mu F(ab') 2 was purchased from Jackson Immunoresearch Lab (West Grove, PA). Serial dilutions of anti-mu or anti-CD79b antibodies were performed in triplicate in 96-well microtiter plates containing RPMI 1640 containing 10% FBS. Purified primary human B cells (5–7.5 × 10 per well) were cultured at 10 °C for 1 h. 4) were added to a final volume of 100 μl and incubated at 37° C. for 3 days. ATP-dependent luminescence was quantified to determine cell viability (Cell Titer-Glo Cell Viability Assay, Promega, Madison, WI), and a Topcount luminometer (PerkinElmer, Waltham, MA) was used for data acquisition. Figures 15A and 15B show the results of an assay demonstrating the viability of primary human B cells upon BCR activation, performed here by cross-linking with anti-mu (A) or anti-CD79b (B) antibodies. Such activation in vivo must occur via immune complexed antigens, which can be, for example, infectious agents, and in the cause of autoimmune or allergic reactions, can be autoimmune antigens or allergens.

[0317] To investigate whether BCR activation-mediated viability of primary human B cells can be inhibited by anti-CD19 antibodies with high Fc affinity for FcγRIIb, an ATP-dependent luminescence assay was used. The previous experiment was repeated, except that serial antibody dilutions of WT, variant, and control antibodies were performed in triplicate in 96-well microtiter plates in RPMI1640 containing 10% FBS plus 1 μg / ml anti-CD79b to stimulate the BCR. The results are shown in FIG. 9. Again, B cells had low viability in the absence of BCR crosslinking, and the addition of 10 μg / ml anti-CD79b antibody stimulated viability by approximately 6-fold (anti-CD79b to cells alone). Anti-CD19-S267E / L328F, the variant with the highest FcγRIIb affinity, inhibited BCR-stimulated viability in a dose-dependent manner. In contrast, control antibodies including anti-CD19-IgG1 (Fv control) and anti-RSV-S267E / L328F (Fc control) minimally inhibited viability. To assess whether this inhibitory effect required co-binding of CD19 and FcγRIIb rather than simultaneous binding of each receptor by different antibodies, the anti-CD19-S267E / L328F variant was compared to the combination of anti-CD19-IgG1 and anti-RSV-S267E / L328F control at the same concentration. These antibody combinations should simultaneously bind ...

Claims

1. 1. A pharmaceutical composition for reducing peripheral blood plasmablasts in a subject with IgG4-related disease (IgG4-RD), comprising an immunoglobulin that binds to Fc RIIb on the surface of B cells and co-binds with CD19, wherein the immunoglobulin comprises a heavy chain having the amino acid sequence of SEQ ID NO:9 and a light chain having the amino acid sequence of SEQ ID NO:

7.

2. 1. A pharmaceutical composition for reducing CD4+ SLAMF7+ CTL cells in a subject with IgG4-related disease (IgG4-RD), comprising an immunoglobulin that binds to Fc RIIb on the surface of B cells and co-binds with CD19, the immunoglobulin comprising a heavy chain having the amino acid sequence of SEQ ID NO:9 and a light chain having the amino acid sequence of SEQ ID NO:

7.

3. 3. The pharmaceutical composition of claim 2, wherein the decrease in CD4+SLAMF7+ CTL cells is observed within 24 days after administration of the immunoglobulin.

4. The pharmaceutical composition of claim 2, wherein the CD4+SLAMF7+ CTL cells are reduced by at least 10% compared to the number of CD4+SLAMF7+ CTL cells before administration of immunoglobulin.

5. The IgG4-related disease is IgG4-related sialadenitis (chronic sclerosing sialadenitis, Kuttner's tumor, Mikulicz's disease), IgG4-related dacryoadenitis (Mikulicz's disease), IgG4-related eye disease (idiopathic orbital inflammatory disease, orbital pseudotumor), chronic sinusitis, eosinophilic angiocentric fibrosis, IgG4-related hypophysitis (IgG4-related panhypophysitis, IgG4-related adenohypophysitis, IgG4-related posterior infundibulohypophysitis, autoimmune hypophysitis), IgG4-related meningitis, IgG4-related leptomeningeal inflammation (idiopathic hypertrophic meningitis), IgG4-related pancreatitis (type 1 autoimmune pancreatitis, IgG4-related AIP, lymphoproliferative IgG4-related pulmonary disease (pulmonary inflammatory pseudotumor), IgG4-related pleurisy, IgG4-related liver disease, IgG4-related sclerosing cholangitis, IgG4-related cholecystitis, IgG4-related aortitis (inflammatory aortic aneurysm), IgG4-related periaortitis (chronic periaortitis), IgG4-related periarteritis, IgG4-related pericarditis, IgG4-related mediastinitis (fibrosing mediastinitis), IgG4-related retroperitoneal fibrosis (retroperitoneal fibrosis, Albarran-Ormond syndrome, Ormond disease (retroperitoneal fibrosisfibrosis), perinephric fasciitis, Gerota fasciitis / syndrome, fibrous periureteritis, sclerosing lipogranuloma, sclerosing retroperitoneal granuloma, nonspecific retroperitoneal inflammation, sclerosing retroperitonitis, retroperitoneal vasculitis with perivascular fibrosis), IgG4-related mesenteritis (subtypes are: mesenteric panniculitis, mesenteric lipodystrophy and involutive mesenteritis) (sclerosing mesenteritis, systemic Nodular panniculitis, liposclerosing mesenteritis, Weber-Christian disease of the mesenteric, lipogranuloma of the mesenteric, xanthogranulomatous mesenteritis), IgG4-related mastitis (sclerosing mastitis), IgG4-related kidney disease (IgG4-RKD), IgG4-related tubulointerstitial nephritis (IgG4-TIN), IgG4-related membranous glomerulonephritis (idiopathic tubulointerstitial nephritis), IgG4-related prostate 5. The pharmaceutical composition according to claim 1, wherein the therapeutic agent is selected from the group consisting of epididymo-orchitis, IgG4-related perivasal fibrosis (chronic scrotal pain), IgG4-related pseudopseudotumor, IgG4-related epididymo-orchitis (paratesticular fibrous pseudotumor, inflammatory pseudotumor of spermatic cord, pseudosarcomatous myofibroblastic proliferation of spermatic cord, proliferative funiculitis, chronic proliferative pericylitis, fibromatous pericylitis, nodular pericylitis, reactive pericylitis, fibrous mesothelioma), IgG4-related lymphadenopathy, IgG4-related skin diseases (angiolymphocytosis with eosinophilia, cutaneous pseudolymphoma), IgG4-related perineural diseases, and IgG4-related thyroid disease (Riedel's thyroiditis), eosinophilic angiocentric fibrosis (affecting the orbit and upper respiratory tract), inflammatory pseudotumor, and multifocal fibrous sclerosis.

6. 6. The pharmaceutical composition of claim 5, wherein the IgG4-related disease is selected from the group consisting of autoimmune pancreatitis (lymphoplasmacytic sclerosing pancreatitis), eosinophilic angiocentric fibrosis (affecting the orbit and upper respiratory tract), fibrosing mediastinitis, idiopathic hypertrophic meningitis, idiopathic tubulointerstitial nephritis, inflammatory pseudotumor, Kuttner's tumor, Mikulicz's disease, fibrosclerosis, periaortitis, periarteritis, inflammatory multifocal aortic aneurysm, Ormond's disease (retroperitoneal fibrosis), Riedel's thyroiditis, and sclerosing mesenteritis.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the subject's IgG4-RD responder index score (IgG4-RD RI score) is reduced by at least 1 from the baseline score within two weeks after administration of the immune globulin.

8. The pharmaceutical composition of claim 7, wherein the IgG4-RD RI score is reduced by at least 3 within two weeks after administration of the immune globulin.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, further comprising an agent for treating an IgG4-related disease selected from the group consisting of anti-inflammatory analgesics (e.g., an NSAID such as aspirin, ibuprofen, naproxen, or Celebrex), acetaminophen, immunosuppressants, and immunosuppressant biologics.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the subject has recurrent disease or is refractory to rituximab.

11. The pharmaceutical composition of claim 1, wherein at least 5 mg / kg of body weight of immunoglobulin is administered to the subject.

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