Polypeptides for co-engagement of type i and ii FC receptors to mediate Anti-inflammatory activities and use thereof

Abstract

This disclosure is based, at least in part, on an unexpected discovery that coexpression of both type I Fc receptor (e.g., FcyRIIB) and type II Fc receptor (e.g., DC-SIGN) augments the binding of IVIG and its active component, sialylated Fc. The disclosed Fc variants can achieve a significant enhancement of in vivo protection as compared to IVIG in several models of autoantibody mediated inflammation.

Description

[0001] Attorney Docket No: 070413.20840

[0002] POLYPEPTIDES FOR CO-ENGAGEMENT OF TYPE I AND II FC RECEPTORS TO MEDIATE ANTI-INFLAMMATORY ACTIVITIES AND USE THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application is entitled to priority pursuant to 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 650,447, filed on May 22, 2024. The content of the application is incorporated herein by reference in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under Grant No. R01 AI153441 awarded by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health and under Grant No. R01CA244327 awarded by the National Cancer Institute of the National Institutes of Health. The government has certain rights in the invention.

[0007] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0008] The contents of the electronic sequence listing (070413.20840SeqList.xml; Size: (35,761 bytes; and Date of Creation: May 21, 2025) is herein incorporated by reference in its entirety.

[0009] FIELD OF THE INVENTION

[0010] This disclosure generally relates to polypeptides for co-engagement of type I and II Fc receptors to mediate anti-inflammatory activities and use thereof.

[0011] BACKGROUND OF THE INVENTION

[0012] Inflammatory disorders, including autoimmune diseases, are disorders involving abnormal activation and subsequent migration of white blood cells to affected areas of the body. These conditions encompass a wide range of ailments that affect the lives of millions of people throughout the world. Although various treatments are presently available, many possess significant side effects or are not very effective in alleviating all symptoms. Thus, there are needs for anti-inflammatory agents for treating inflammatory disorders and needs for methods of identifying and evaluating such agents. Attorney Docket No: 070413.20840

[0013] Immunoglobulin G (IgG) has long been appreciated to mediate both pro- and antiinflammatory activities through interactions mediated by its Fc fragment. While Fc-FcyR interactions are responsible for the pro-inflammatory properties of immune complexes and cytotoxic antibodies, intravenous gamma globulin (IVIG) and its Fc fragments are antiinflammatory and are widely used to suppress inflammatory diseases. Intravenous immunoglobulin, an IgG mixture purified from the thousands of donors, has been an FDA approved therapeutic staple for the treatment of a wide variety of autoimmune indications for decades. However, the high dose requirement (1-2 g / kg), difficulty in administration, and availability has prompted studies to identify a readily available, recombinantly expressed, replacement product.

[0014] Studies in animal models have established the requirement of terminal a2,6 linked sialylation of the single N-linked glycan (N297) in the IgGl Fc-domain in mediating a major pathway of anti-inflammatory activity of IVIG. Furthermore, genetic knockout studies in murine models have implicated both type I (canonical, IgG superfamily members), and type II (C-type lectin) IgG Fc receptors (FcyRs) in the mechanism by which sialylated IgG mediates its activity in vivo. Recombinantly expressed sialylated IgGl Fc (sFc) or an Fc mutant that phenocopies the structural perturbation of the sialylated IgGl CH2 domain (F241A Fc), show in vivo potency at 100 mg / kg doses, a 10-20-fold enhancement in effective dose compared to IVIG in the same models. However, this 100 mg / kg dose remains economically burdensome and is considerably higher than that of the recent FDA approved high-affinity neonatal Fc receptor (FcRn) inhibitors, which are dosed at 10 mg / kg.

[0015] SUMMARY OF THE INVENTION

[0016] The disclosure addresses and meets the above-mentioned needs by identifying polypeptides which are at least 100-fold more potent than IVIG in reducing the inflammatory sequelae of antibody or T cell mediated autoimmune diseases, thus providing the basis for a new class of anti-inflammatory therapeutics. This disclosure relates to agents, such as polypeptides and antibodies, and methods for treating inflammatory disorders or diseases, e.g., autoimmune diseases.

[0017] One aspect of this disclosure features an isolated polypeptide comprising a modified IgG Fc region, wherein the polypeptide is capable of co-engaging a type I FcyR and a type II FcyR. In some embodiments, the type I FcyR is FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, Attorney Docket No: 070413.20840

[0018] FcyRIIIb, or FcyRIIb. In one embodiment, the type I FcyR is FcyRIIB. In some embodiments, the type II FcyR is DC-SIGN or CD23. In one embodiment, the polypeptide has an enhanced affinity to FcyRIIB. In one embodiment, the polypeptide exhibits selective binding to FcyRIIB.

[0019] In some embodiments, the modified IgG Fc region comprises one or more of: a) a P238D mutation; b) an E233D mutation; c) a G237D mutation; d) a H268D mutation; e) a P271 G mutation; f) an A33 OR mutation; g) an S267E mutation; and h) an L328F mutation.

[0020] In one embodiment, the modified IgG Fc region comprises G237D / P238D / H268D / P271G / A330R mutations. In one embodiment, the modified IgG Fc region comprises an F241A mutation.

[0021] In some embodiments, the modified IgG Fc region is at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence of SEQ ID NOs: 1-30 or comprises an amino acid sequence of SEQ ID NOs: 1-30.

[0022] In one embodiment, the IgG Fc glycan at position N297 is unmodified. In one embodiment, the IgG Fc glycan at position N297 is terminally galactosylated. In one embodiment, the IgG Fc glycan at position N297 is terminally sialylated and the sialic acid is attached in a 2,6 linkage to the penultimate galactose.

[0023] In some embodiments, provided is an antibody comprising of the above-described modified Fc region. In some embodiments, provided is an isolated nucleic acid molecule comprising a sequence encoding the above-described polypeptide or the above-described antibody. In one embodiment, provided is an expression vector comprising the abovedescribed nucleic acid molecule. In one embodiment, provided is a host cell comprising the above-described nucleic acid molecule.

[0024] In some embodiments, provided is a method of producing the above-described polypeptide or the above-described antibody thereof, comprising culturing the above Attorney Docket No: 070413.20840 described host cell in a medium under conditions permitting expression of a polypeptide encoded by the nucleic acid molecule, and isolating the polypeptide from the cultured cell or the medium of the cell.

[0025] In some embodiments, provided is a pharmaceutical composition comprising (i) the above-described polypeptide, the above-described antibody, or the above-described nucleic acid molecule, and (ii) optionally a pharmaceutically acceptable carrier.

[0026] In one embodiment, provided is a method of treating an inflammatory disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of the above-described pharmaceutical composition. In some embodiments, the method comprises administering the polypeptide at a dose of about 1 mg / kg to about 1000 mg / kg. In one embodiment, the inflammatory disease or disorder comprises an infection that is a viral infection or a bacterial infection. In one embodiment, the subject is a human. In one embodiment, the treatment results in at least 100-fold anti-inflammatory protection compared to intravenous immunoglobulin (IVIG).

[0027] In one embodiment, provided is a pharmaceutical composition as described above for use in treating an inflammatory disease or disorder. In one embodiment, the inflammatory disease or disorder comprises an infection that is a viral infection or a bacterial infection.

[0028] In one embodiment, provided is the use of the above-described polypeptide of a pharmaceutical composition in the manufacture of a medicament for treating an inflammatory disease or disorder. In one embodiment, the inflammatory disease or disorder comprises an infection that is a viral infection or a bacterial infection.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIGS. 1A-1C are graphs analyzing the results of human FcyRIIB and DC-SIGN expression in 293-T cells. FIG. 1A shows graphs of human FcyRIIB and DC-SIGN expression on 293-T cells transfected with plasmids encoding the full length FcyRIIB and DC-SIGN as determined by flow cytometry. Mock untransfected, FcyRIIB, or FcyRIIB DC-SIGN transfected cells were gated and plotted for FcyRIIB high (FcyRIIB+++) expressing cells. ***p, unpaired T-test. FIG. IB shows the binding of IVIG (10 ug / ml) to cells transfected with indicated type I and / or type II FcyR, measured by flow cytometry via staining cells with an anti-human IgGl secondary antibody. FIG. 1C shows the binding of Attorney Docket No: 070413.20840 glycoengineered human IgGl (Rituximab) containing galactosylated (G2F) or sialylated (S2G2F) glycans to transfected 293-T cells expressing FcyRIIB alone or in combination with the type II FcyRs mouse CD23 (mCD23), human CD23 (hCD23), or human DC- SIGN. Binding was measured using an anti-human IgGl secondary antibody **p, two-way ANOVA, multiple comparisons test.

[0031] FIGS. 2A-2C characterize the structural interface FcyRIIB and human IgGl Fc. FIG. 2A depicts the structural interface between FcyRIIB and human IgGl Fc. Glycans N106 and N187 on the ectodomain of FcyRIIB are highlighted. A glycan knock-out variant of the full-length FcyRIIB was generated by mutagenesis of N106 and N187 to alanine residues (N106A, N187A). 293-T cells were transfected with plasmids encoding wild-type FcyRIIB (WT) or glycan knockout FcyRIIB (A) alone or in combination with mCD23 or DC-SIGN. Total FcyRIIB cell surface expression (FIG. 2B) and FcyRIIB high expressing cells (FIG. 2C) were measured by flow cytometry. **p, two-way ANOVA, multiple comparisons test.

[0032] FIGS. 3A-3B characterize the sVl 1 variant. FIG. 3A is a western blot and protein gel analysis of hlgG-WT Fc and VI 1 Fc proteins expressed in 293-F cells alone or cotransfected with plasmids encoding Beta-l,4-galactosyltransferase 1 (B4GALT1) and Betagalactoside alpha-2, 6-sialyltransferase 1 (ST6GAL1). Western blotting with the a2,6 sialic acid specific lectin SNA confirms the generated of sialylated WT-Fc (B4ST6) and VI 1-Fc (B4ST6), hereafter referred to as WT sFc and VI 1 sFc. FIG. 3B shows surface plasmon analysis of the soluble ectodomain of human FcyRIIB (8000 nM to 62.5 nM) binding to protein-G immobilized WT sFc or VI 1 sFc.

[0033] FIGS. 4A-4D illustrate the results of treating humanized FcyR (hFcyR) mice with IVIG or sFc two hours prior to injection with anti-CD4 IgGl. FIG. 4A depicts the experiment scheme. hFcyR mice were treated with IVIG or sFc two hours prior to injection with anti-CD4 IgGl (afucosylated, 0.5 mg / kg, i.p (intraperitoneal). The depletion and rebound of circulating CD4+ T-cells was tracked on days 1, 2, 3, and 7 post anti-CD4 IgG infusion. Humanized FcyR mice were administered (FIG. 4B) IVIG (1000 mg / kg, i.v. (intravenous)), (FIG. 4C) WT sFc (25 mg / kg), or Vl l sFc (25 mg / kg) two hours prior to injection with anti-CD4. Circulating CD4 T-cells was measured by flow cytometry and plotted as a percent of baseline CD4 T-cell frequencies. FIG. 4D shows the percent CD4+ Attorney Docket No: 070413.20840

[0034] T-cells (of baseline) in blood measured two days post anti-CD4 depletion ***p value, oneway ANOVA, compared to anti-CD4 treated group.

[0035] FIGS. 5A-5D illustrate the results of hFcyR mice treated with WT sFc or VI 1 sFc one hour before injection with K / BxN sera. FIG. 5A depicts the experiment scheme. Humanized FcyR (hFcyR) mice were administered WT sFc or Vll sFc (10 mg / kg, i.v.) one hour before injection with K / BxN sera (200 pl, i.p.). FIG. 5B shows the development of arthritis related inflammation in the ankles and wrist joints was measured over 5 days using clinical scoring. FIG. 5C shows clinical scores, and FIG. 5D shows ankle thickness measured 5 days post K / BxN serum administration. ***p value, one-way ANOVA, compared to K / BxN alone group.

[0036] FIGS. 6A-6D illustrate the results of hFcyR mice treated with or VI 1 sFc one hour before K / BxN serum administration. FIG. 6A depicts the experiment scheme. hFcyR mice were administered IVIG (1000 mg / kg, i.v) or VI 1 sFc (10 mg / kg, 5 mg / kg, or 1 mg / kg, i.v.) one hour before K / BxN serum administration (100 pl, i.p). FIGS. 6B and 6C are graphs of the ankle thickness tracked over the course of 12 days and measured as a change from baseline (A ankle thickness). FIG. 6D is a graph of the area under curve analysis of kinetics analysis of the kinetics of ankle thickness change over time. ****p value, one-way ANOVA, compared to K / BxN alone group.

[0037] FIGS. 7A-7E illustrate that type I Fc receptor engagement is required for the antiinflammatory activity of sialylated IgG. FIG. 7A shows the structure of the fully processed Asn297 (N297) glycan in the human IgGl antibody Fc domain. The sugars galactose and a2,6 sialic acid are linked to the core glycan via the glycotransferases Beta- 1,4- galactosyltransferase 1 (B4GALT1) and Beta-galactoside alpha-2, 6-sialyltransferase 1 (ST6GAL1), respectively. FIG. 7B shows protein gel and lectin blots of recombinant produced hlgGl-WT Fc protein (WT Fc), expressed alone or in combination with B4GALT1 and ST6GAL1 expression plasmids, resulting in a sialylated Fc (WT sFc) protein. Treatment of WT sFc protein with neuraminidase cleaves sialic acid, generating an asialylated WT Fc. Terminal a2,6 sialic acid or galactose detected by the Sambucus Nigra (SNA) lectin and Erythrina Cristagalli (ECL) lectin, respectively. The left of FIG. 7C shows hFcyR mice (males, 5-6 / group) dosed with IVIG (2500 mg / kg), WT sFc (100 mg / kg) or neuraminidase treated WT sFc (100 mg / kg) one hour prior to injection of K / BxN serum. Attorney Docket No: 070413.20840

[0038] Right, average ankle thickness (mm) of mice 4 days post K / BxN serum transfer induced arthritis (STIA) (mean + / - SEM). FIG. 7D is a table of binding affinities (KD) of WT Fc WT sFc and GRLR ((G236R / L328R) sFc for type I FcyRs, determined by surface plasmon resonance (SPR). Fold change compared to WT Fc indicated. N.B.D, no binding detected. FIG. 7E is a graph of ankle thickness (mm) of hFcyR mice (male, 4 / group) dosed with 100 mg / kg of WT sFc or GRLR sFc one hour prior to K / BxN serum injection. One-way ANOVA, followed by Tukey’s multiple comparisons test for FIGS. 7C and 7E. P-values indicated above groups, significant values in bold.

[0039] FIGS. 8A-8C demonstrate Fc-engineering IgG to target FcyRIIB potently enhances the anti-inflammatory activity of sialylated IgG. FIG. 8A is a table of binding affinities (KD) of WT sFc, GA (G236A) sFc or VI 1 sFc to type I FcyRs, determined by SPR. Fold change in affinity compared to WT sFc indicated. FIG. 8B is a graph of ankle thickness (mm), measured daily for 8 days, of hFcyR mice (4 / group) dosed with WT sFc (100 mg / kg), WT sFc (10 mg / kg), GA sFc (10 mg / kg) or VI 1 sFc (10 mg / kg) one hour prior to injection of K / BxN serum. FIG. 8C is a graph of average ankle thickness (mm) of mice 4 days post K / BxN serum transfer induced arthritis (STIA). One-way ANOVA, followed by Tukey’s multiple comparisons test. P-values indicated above groups, significant values in bold.

[0040] FIGS. 9A-9E show that VI 1 sFc phenocopies the anti-inflammatory activity in at least 100-fold lower doses and ameliorates neurological autoimmunity in a mouse model of multiple sclerosis. FIG. 9A is a graph of ankle (A) Ankle thickness (mm), measured daily for 12 days, of hFcyR mice (3 / group) dosed with IVIG sFc (1000 mg / kg), VI 1 sFc (10 mg / kg), VI 1 sFc (5 mg / kg) or VI 1 sFc (1 mg / kg) prior to K / BxN serum injection. FIG. 9B is a graph of average ankle thickness (mm) of mice 4 days post K / BxN serum transfer induced arthritis (STIA). FIG. 9C shows hFcyR mice (7-11 / group) that were immunized with MOG35-55 peptides emulsified in CFA on day 0, along with two subcutaneous injections of pertussis toxin (PTX) on days 0 and 1. Mice were then injected with PBS, WT sFc (10 mg / kg) or VI 1 sFc (10 mg / kg) on days 5, 10, 15, and 20. Daily clinical scoring to detect neurological symptoms started on day 7, mice were monitored until day 28. FIG. 9D is a graph of the area under the curve (AUC) analysis of experimental autoimmune encephalomyelitis (EAE) clinical scores in PBS, WT sFc, or VI 1 sFc treated mice. FIG. 9E Attorney Docket No: 070413.20840 is quantified relative expression of myelin basic protein (MBP) in spinal cord sections in untreated and VI 1 sFc treated mice.

[0041] FIGS. 10A-10L show that type II FcyRs contribute to Vl l sFc mediated antiinflammatory activity and synergize with type I FcyRs to bind IgG. FIG. 10A is a schematic of hFcyR mice (4 / group) were dosed with 10 mg / kg of VI 1 sFc one hour prior to K / BxN serum injection. Mice were then treated daily with an isotype control or a SIGN-R1 blocking antibody. FIG. 10B is a graph of average ankle thickness (mm). FIG. 10C is a graph of clinical scores of mice 4 days post K / BxN serum injection. FIG. 10D is a histogram of FcyRIIB expression in HEK 293-T cells transfected with FcyRIIB alone or in combination with DC-SIGN. FIG. 10E is a western blot analysis of cell lysates or anti- FLAG tag immunoprecipitants of293-F cells transfected with FLAG tagged FcyRIIB and / or HA-tagged DC-SIGN. Immunoblot (IB). FIG. 10F shows immobilized recombinant DC- SIGN binding to serial dilutions of recombinant FcyRIIB, measured by SPR. FIG. 10G shows the structure of IgGl Fc in complex with FcyRIIB (PBD 3WJJ). Putative N-linked glycosylation sites of FcyRIIB are N106, N180, and N187. FIG. 10H shows lectin blots and protein gel of recombinant wild-type (WT) FcyRIIB or single glycan knock-out mutants (N106Q, N180Q, and N187Q). FIG. 101 shows immobilized DC-SIGN binding to serial dilutions of single glycan mutant FcyRIIB, measured by SPR. FIGS. 10J-10L depict the results of fluorescently labeled IVIG (10 ug / ml), glycoengineered Rituximab-G2F (galactosylated), or Rituximab-S2G2F (sialylated) to 293-T cells expressing FcyRIIB alone or in combination with CD23 or DC-SIGN. One-way ANOVA, followed by Tukey’s multiple comparisons test for (FIGS. 10B, 10C, 10J, 10K, and 10L). P-values indicated above groups. All SPR experiments performed in presence of 2 mM CaCh.

[0042] FIGS. 11A and 11B illustrate Surface Plasmon Resonance (SPR) analysis of serial dilutions (1000 nM, 1 :2) of soluble Human FcyRs binding to immobilized WT, F241 A, Vl l or F241 A-Vl 1 sFc. FIG. HA shows graphs of the response units from SPR analysis. FIG. 11B is a table of the binding affinities of soluble human FcyRs to sFc variants, determined by SPR (KD, M). Affinities fold change in affinity compared to WT sFc indicated.

[0043] FIGS. 12A and 12B illustrate that F241A-V11 retains potent anti-inflammatory activity. FIG. 12A is a schematic of FcyR humanized (hFcyR) mice were dosed with Vl l sFc (10 mg / kg), F241 A-Vl 1 sFc (10 mg / kg), or neuraminidase treated F241 A-Vl 1 sFc (10 Attorney Docket No: 070413.20840 mg / kg) one hour prior to K / BxN serum injection. FIG. 12B is a graph of ankle measurements of mice measured 4 days post K / BxN serum injection. One-way ANOVA, followed by Tukey’s multiple comparisons test.

[0044] FIGS. 13A and 13B illustrates Surface Plasmon Resonance (SPR) analysis of serial dilutions (1000 nM, 1 :2) of soluble Human FcyRs binding to immobilized WT, VI 1, or SE sFc. FIG. 13A shows graphs of the response units from SPR analysis. FIG. 13B is a table of the binding affinities of soluble human FcyRs to sFc variants, determined by SPR (KD, M). Affinities Fold change in affinity compared to WT sFc indicated for each FcyR. Inhibitory / activating ratio determined as fold change in affinity for FcyRIIB compared to each activating FcyR.

[0045] FIGS. 14A and 14B demonstrate that enhanced affinities to FcyRIIB of Vl l and SE sFc drives anti-inflammatory activity at low doses. FIG. 14A is schematic of FcyR humanized (hFcyR) mice were dosed with Vl l sFc (10 mg / kg) or SE sFc (10 mg / kg) one hour prior to K / BxN serum injection. FIG. 14B is a graph of ankle measurements of mice measured 4 days post K / BxN serum injection. One-way ANOVA, followed by Tukey’s multiple comparisons test.

[0046] FIGS. 15A and 15B demonstrate that Vl l sFc, F241A-V11 sFc, and SE sFc all promote CD4 T-cell recovery in in vivo CD4 depletion models. FIG.15A is a schematic of FcyR humanized (hFcyR) mice were dosed with Vl l sFc (25 mg / kg), F241 A-Vl 1 sFc (25 mg / kg), or SE sFc (25 mg / kg) one hour prior to injection with an afucosylated anti-CD4 IgG (0.5 mg / kg). FIG. 15B is a graph of the frequencies (% of total CD3+cells) of CD4 T-cells in blood 2 days post injection with anti-CD4 IgG determined as a percent of baseline % CD4 T-cells.

[0047] FIGS. 16A-16C are tables that show the binding affinities of soluble human FcyRs to sFc variants, determined by SPR (KD, M). Affinities fold change in affinity compared to WT sFc. NBD, no binding detected. >1000 inhibitory / activating ratio is given when the indicated activating FcyR has null-Fc binding.

[0048] FIGS. 17A and 17B illustrate that neuraminidase treated Vl l sFc failed to protect mice from controlling the early onset of K / BxN serum induced inflammation, but significantly reduced inflammation by day six post K / BxN serum inj ection. FIG. 17A shows lectin blots and protein gels of IgGl Vl l Fc expressed alone or in combination with B4GALT1 and ST6GAL1 expression plasmids (Vl l sFc), as well as neuraminidase treated Attorney Docket No: 070413.20840

[0049] VI 1 sFc. Terminal a2,6 sialic acid or galactose detected by the Sambucus Nigra (SNA) lectin and Erythrina Cristagalli (ECL) lectin, respectively. FIG. 17B is a graph of ankle thickness (mm) of hFcyR mice (3 / group) dosed with Vl l sFc (10 mg / kg) or neuraminidase treated Vl l sFc (10 mg / kg) one hour prior to K / BxN serum injection, measured 3 and 6 days post K / BxN serum induced arthritis (STIA). One-way ANOVA, followed by Tukey’s multiple comparisons test. P-values indicated above groups, significant values in bold.

[0050] FIGS. 18A-18I demonstrate that VI 1 sFc conveys potent anti-inflammatory activity in antigen-independent manner at an equivalent effective dose (10 mg / kg) as high-affinity FcRn binding Fc proteins. FIG. 18A is a graph of the affinity of WT sFc for human FcRn determined by single-cycle kinetics SPR. FIG. 18B is a graph of the affinity of WT sFc for mouse FcRn determined by single-cycle kinetics SPR. Serial dilutions (1.56 - 25 nM) 880 of WT or VI 1 sFc were reacted to immobilized biotinylated hFcRn or mFcRn. FIG. 18C is a table of the binding affinities of hFcR and mFcRn against WT or Vl l sFc, with foldchange compared to WT sFc indicated. FIGS. 18D-18F demonstrate single cycle kinetics and binding affinities of WT or Vl l full-length sialylated IgG (slgG) against hFcRn or mFcRn. FIG. 18G illustrate serum half-life of WT sFc, Vl l sFc, or full-length Vl l slgG in humanized FcRn / hFcyR mice. Mice were injected with 200 pg of sFc or slgG protein on day 0, and serum concentrations were determined by ELISA on days, 1, 4, and 7. FIGS. 18H-18I show calculated half-life of WT sFc, Vl l sFc, or Vl l slgG. One-way ANOVA, followed by Tukey’s multiple comparisons test. P-values indicated above groups, significant values in bold.

[0051] DETAILED DESCRIPTION OF THE INVENTION

[0052] This disclosure is based, at least in part, on an unexpected discovery that the coengagement of both the inhibitory type I Fc receptor FcyRIIB and the type II Fc receptor (e.g., DC-SIGN or CD23) augments the binding of IVIG and its active component, sialylated Fc, to mediate anti-inflammatory activity. The disclosed modified Fc variants can achieve a significant enhancement of in vivo protection as compared to IVIG in several models of T-cell and autoantibody mediated inflammation. This discovery addresses a long- felt need of developing a new class of anti-inflammatory therapeutics. Attorney Docket No: 070413.20840

[0053] IgG and Fc Sialylation

[0054] IgG is the major serum immunoglobulin. It is a glycoprotein composed of two identical heavy chains and two light chains, which in turn are composed of variable and constant domains. IgG contains a single, N-linked glycan at Asn297in the CH2 domain on each of its two heavy chains. The covalently-linked, complex carbohydrate is composed of a core, biantennary penta-polysaccharide containing N-acetylglucosamine (GIcNAc) and mannose (man). Further modification of the core carbohydrate structure is observed in serum antibodies with the presence of fucose, branching GIcNAc, galactose (gal) and terminal sialic acid (sa) moieties variably found. Over 40 different glycoforms have thus been detected to be covalently attached to this single glycosylation site (Fujii etal., J. Biol. Chem. 265, 6009, 1990). Glycosylation of IgG has been shown to be essential for binding to all FcyRs by maintaining an open conformation of the two heavy chains. Jefferis and Lund, Immune. 1 Lett. 82, 57 (2002), Sondermann et al., J. Mol. Biol. 309, 737 (2001). It is believed that this IgG glycosylation for FcyR binding accounts for the inability of deglycosylated IgG antibodies to mediate in vivo triggered inflammatory responses, such as ADCC, phagocytosis and the release of inflammatory mediators. Nimmerjahn and Ravetch, Immunity 24, 19 (2006). Further observations that individual glycoforms of IgG may contribute to modulating inflammatory responses has been suggested by the altered affinities for individual FcyRs reported for IgG antibodies containing or lacking fucose and their consequential effects on cytotoxicity. Shields etal., J. Biol. Chem. 277, 26733 (2002), Nimmerjahn and Ravetch, Science 310, 1510 (2005). A link between autoimmune states and specific glycosylation patterns of IgG antibodies has been observed in patients with rheumatoid arthritis and several autoimmune vasculitis in which decreased galactosylation and sialylation of IgG antibodies have been reported. Parekh et al., Nature 316, 452 (1985), Rademacher etal., Proc. Natl. Acad. Sci. USA 91, 6123 (1994), Matsumoto etal., 128, 621 (2000), Holland etal., Biochim. Biophys. Acta December 27. Variations in IgG glycoforms have also been reported to be associated with aging and upon immunization, although the in vivo significance of these alterations has not been determined. Shikata et al., Glycoconj. J. 15, 683 (1998), Lastra, et al., Autoimmunity 28, 25 (1998).

[0055] Certain non-sialylated IgG Fc variants also confer anti-inflammatory activity. Such variants, including the F241 A variant, represent species within a larger genus of molecules Attorney Docket No: 070413.20840 that, by virtue of mimicking the structural and biological properties of sialylated Fc, but do not require sialylation, can also be developed as anti-inflammatory therapeutics.

[0056] Polypeptides Comprising Fc Variants

[0057] Polypeptides

[0058] As disclosed herein, this disclosure provides isolated polypeptides having sequences of variants of human IgG Fc that are sialylated (e.g., have glycans terminating in a2,6 sialic acids). In one embodiment, the sialyation is biantennary TV-linked glycan attached at Asn297, a site that is conserved in all the IgG subclasses and is attached in a 2,6 linkage to the penultimate galactose.

[0059] In some embodiments, provided are isolated polypeptides having sequences of variants of human IgG Fc that lack a polysaccharide chain having a terminal sialic acid connected to a galactose moiety through the a2,6 linkage at the aforementioned Asn297 position in the CH2 domain of the IgG Fc. Such non-sialylated IgG Fc variants may be either derived from a naturally occurring antibody or expressed in a cell line.

[0060] In some embodiments, provided are isolated polypeptides that comprise a modified IgG Fc region, wherein the polypeptide is capable of co-engaging a type I FcyR and a type II FcyR. In some embodiments, the type I FcyR is FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, FcyRIIIb, or FcyRIIb. In some embodiments, the type II FcyR is DC-SIGN or CD23 (Bournazos et al. Annu. Rev. Immunol. 2017. 35: 285-311 and Pincetic et al. Nature Immunology. 2014. 15(8): 707-716).

[0061] As used herein, the term “co-engaging” or “co-engagemenf ’ refers to simultaneous interaction of an Fc region with multiple (e.g., two or more) Fc receptors or Fc-binding partners. In some embodiments, an antibody or a protein comprising the polypeptide of the present disclosure can have simultaneous interaction of the Fc region with multiple (e.g., two or more) Fc receptors or Fc-binding partners.

[0062] In one embodiment, the Fc region includes one or more substitutions of the hlgGl amino acid sequence. Unless otherwise noted, all sequence numbering described herein is according to the EU index as in Kabat. Kabat et al. (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md.; see also FIGS. 3c-3f of U.S. Pat. App. Pub. No. 2008 / 0248028. Attorney Docket No: 070413.20840

[0063] While not limited thereto, exemplary IgGl Fc regions are provided as follows: Attorney Docket No: 070413.20840 Attorney Docket No: 070413.20840 Attorney Docket No: 070413.20840 Attorney Docket No: 070413.20840 Attorney Docket No: 070413.20840

[0064] Underline indicates hinge. Bold indicates mutation site. Bold and italics indicate F241A.

[0065] The above Fc variants VI 1, V9, SE, and SELF and other Fc variants that are suitable for this disclosure are described in U.S. Patent Nos. 10,894,835 and 9,587,025, the contents of which are incorporated herein by reference.

[0066] The above Fc variants F241, F243, and other Fc variants that are suitable for this disclosure are described in International Patent Application PCT / US2012 / 068718, the content of which is incorporated herein by reference.

[0067] The terms “peptide,” “polypeptide,” and “protein” are used herein interchangeably to describe the arrangement of amino acid residues in a polymer. A peptide, polypeptide, or protein can be composed of the standard 20 naturally occurring amino acid, in addition to rare amino acids and synthetic amino acid analogs. They can be any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation). The peptide, polypeptide, or protein “of this invention” include recombinantly or synthetically produced versions having the particular domains or portions that bind to DC-SIGN, FcyRIIA, and FcyRIIB. The term also encompasses polypeptides that have an added amino-terminal methionine (useful for expression in prokaryotic cells).

[0068] An “isolated” polypeptide or protein refers to a polypeptide or protein that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide / protein can constitute at least 10% (i.e., any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 85%, 90%, 95%, and 99%) by dry weight of the purified preparation. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. An isolated polypeptide / protein described in the disclosure can be purified from a natural source, produced by recombinant DNA techniques, or by chemical methods. A functional equivalent of IgG Fc refers to a polypeptide derivative of IgG Fc, e.g., a protein Attorney Docket No: 070413.20840 having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. It retains substantially the activity of the IgG Fc, i.e., the ability to bind to the respective receptor and trigger the respective cellular response.

[0069] A peptide or polypeptide “fragment” as used herein refers to a less than full-length peptide, polypeptide, or protein. For example, a peptide or polypeptide fragment can have at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40 amino acids in length, or single unit lengths thereof. For example, the fragment may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more amino acids in length. There is no upper limit to the size of a peptide fragment. However, in some embodiments, peptide fragments can be less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less than about 250 amino acids in length. Preferably the peptide fragment can elicit an immune response when used to inoculate an animal. A peptide fragment may be used to elicit an immune response by inoculating an animal with a peptide fragment in combination with an adjuvant, a peptide fragment that is coupled to an adjuvant, or a peptide fragment that is coupled to arsanilic acid, sulfanilic acid, an acetyl group, or a picryl group. A peptide fragment can include a non-amide bond and can be a peptidomimetic.

[0070] In some embodiments, the modified IgG Fc region comprises:

[0071] (1) P238D;

[0072] (2) E233D / P238D (VI);

[0073] (3) G237D / P238D (V2);

[0074] (4) P238D / H268D (V3);

[0075] (5) P238D / P271G (V4);

[0076] (6) P238D / A330R (V5);

[0077] (7) E233D / P238D / A330R (V6);

[0078] (8) E233D / P238D / P271G / A330R (V7);

[0079] (9) G237D / P238D / H268D / P271G (V8);

[0080] (10) G237D / P238D / P271 G / A33 OR ( V9);

[0081] (11) E233D / P238D / H268D / P271G / A330R (V10);

[0082] (12) G237D / P238D / H268D / P271G / A330R (VI 1);

[0083] (13) E233D / G237D / P238D / H268D / P271G / A330R (V12);

[0084] (14) S267E (SE);

[0085] (15) S267E / L328F (SELF);

[0086] (16) F241A / P238D;

[0087] (17) F241A / E233D / P238D (F241A-V1);

[0088] (18) F241A / G237D / P238D (F241A-V2);

[0089] (19) F241A / P238D / H268D (F241A-V3); Attorney Docket No: 070413.20840

[0090] (20) F241A / P238D / P271G (F241A-V4);

[0091] (21) F241A / P238D / A330R (F241A-V5);

[0092] (22) F241A / E233D / P238D / A330R (F241A-V6);

[0093] (23) F241A / E233D / P238D / P271G / A330R (F241A-V7);

[0094] (24) F241A / G237D / P238D / H268D / P271G (F241A-V8);

[0095] (25) F241A / G237D / P238D / P271G / A330R (F241A-V9);

[0096] (26) F241A / E233D / P238D / H268D / P271G / A330R (F241A-V10);

[0097] (27) F241A / G237D / P238D / H268D / P271G / A330R (F241A-V11);

[0098] (28) F241A / E233D / G237D / P238D / H268D / P271G / A330R (F241A-V12);

[0099] (29) F241A / S267E (F241A-SE); or

[0100] (30) F241A / S267E / L328F (F241A-SELF).

[0101] In some embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NOs: 1-30. In general, the functional equivalent is at least 75% (e.g., any number between 75% and 100%, inclusive, e.g., 75 %, 80%, 85%, 90%, 95%, and 99%) identical to an amino acid sequence of SEQ ID NOs: 1-30.

[0102] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 1, while maintaining the P238D mutation.

[0103] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 2. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 2, while maintaining the VI mutations (E233D / P238D).

[0104] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 3. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Attorney Docket No: 070413.20840

[0105] 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 3, while maintaining the V2 mutations (G237D / P238D).

[0106] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 4, while maintaining the V3 mutations (P238D / H268D).

[0107] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 5. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 5, while maintaining the V4 mutations (P238D / P271G).

[0108] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 6. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 6, while maintaining the V5 mutations (P238D / A330R).

[0109] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 7. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 7, while maintaining the V6 mutations (E233D / P238D / A330R). Attorney Docket No: 070413.20840

[0110] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 8. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 8, while maintaining the V7 mutations (E233D / P238D / P271G / A330R).

[0111] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 9. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 9, while maintaining the V8 mutations (G237D / P238D / H268D / P271G).

[0112] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 11, while maintaining the VI 1 mutations (G237D / P238D / H268D / P271G / A330R).

[0113] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 12. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 12, while maintaining the V10 mutations (E233D / P238D / H268D / P271G / A330R).

[0114] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 10. In Attorney Docket No: 070413.20840 some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 10, while maintaining the V9 mutations (G237D / P238D / P271G / A330R).

[0115] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 13. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 13, while maintaining the V12 mutations (E233D / G237D / P238D / H268D / P271G / A330R).

[0116] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 14. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 14, while maintaining the SE mutation (S267E).

[0117] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 15. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 15, while maintaining the SELF mutations (S267E / L328F).

[0118] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 16. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Attorney Docket No: 070413.20840

[0119] 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 16, while maintaining the F241 A and P238D mutations.

[0120] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 17. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 17, while maintaining the F241A and VI (E233D / P238D) mutations.

[0121] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 18. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 18, while maintaining the F241 A and V3 (G237D / P238D) mutations.

[0122] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 19. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 19, while maintaining the F241 A and V3 (P238D / H268D) mutations.

[0123] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 20. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 20, while maintaining the F241 A and V4 (P238D / P271G) mutations. Attorney Docket No: 070413.20840

[0124] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 21. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 21, while maintaining the F241 A and V5 (P238D / A330R) mutations.

[0125] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 22. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 22, while maintaining the F241 A and V6 (E233D / P238D / A330R) mutations.

[0126] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 23. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 23, while maintaining the F241 A and V7 (E233D / P238D / P271G / A330R) mutations.

[0127] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 24. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 24, while maintaining the F241 A and V8 (G237D / P238D / H268D / P271G) mutations.

[0128] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 25. In Attorney Docket No: 070413.20840 some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 25, while maintaining the F241 A and V9 (G237D / P238D / P271G / A330R) mutations.

[0129] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 26. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 26, while maintaining the F241A and V10 (E233D / P238D / H268D / P271G / A330R) mutations.

[0130] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 27. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 27, while maintaining the F241 A and VI 1 (G237D / P238D / H268D / P271G / A330R) mutations.

[0131] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 28. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 28, while maintaining the F241 A and V12 (E233D / G237D / H268D / P271G / A330R) mutations.

[0132] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 29. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Attorney Docket No: 070413.20840

[0133] 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 29, while maintaining the F241 A and SE (S267E) mutations.

[0134] In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 30. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% (e.g., 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid of SEQ ID NO: 30, while maintaining the F241 A and SELF (S267E / L328F) mutations.

[0135] In one embodiment, the polypeptide comprises a modified IgG Fc region, wherein the modified IgG Fc region comprises the G237D, F241A, P238D, H268D, P271G, A330R mutations.

[0136] The "percent identity" of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264- 68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength- 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the disclosure. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the disclosure. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0137] The amino acid composition of the polypeptide described herein may vary without disrupting the ability of the polypeptide to bind to the respective receptor and trigger the respective cellular response. For example, it can contain one or more conservative amino acid substitutions. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families Attorney Docket No: 070413.20840 include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), betabranched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in, e.g., SEQ ID NO: 1, is preferably replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of the sequences, such as by saturation mutagenesis, and the resultant mutants can be screened for the ability to bind to the respective receptor and trigger the respective cellular response to identify mutants that retain the activity as described below in the examples.

[0138] A polypeptide as described in this disclosure can be obtained as a recombinant polypeptide. To prepare a recombinant polypeptide, a nucleic acid encoding it can be linked to another nucleic acid encoding a fusion partner, e.g., glutathione-s-transf erase (GST), 6x- His epitope tag, or Ml 3 Gene 3 protein. The resultant fusion nucleic acid expresses in suitable host cells a fusion protein that can be isolated by methods known in the art. The isolated fusion protein can be further treated, e.g., by enzymatic digestion, to remove the fusion partner and obtain the recombinant polypeptide of this disclosure.

[0139] In some embodiments, the polypeptide as disclosed herein has at least 10-fold increased affinity for FcyRIIB compared to wild-type Fc (2xl0‘6M). (at least 10-fold, at least 15-fold, at least 20-fold, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50).

[0140] In some embodiments, the polypeptide as disclosed herein has at least 5-fold increased affinity for FcyRIIB compared to FcyRIIA-H131 (at least 10-fold, at least 15-fold, at least 20-fold, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50). In some embodiments, the polypeptide lacks binding to FcyRIIA-H131 while retaining binding to FcyRIIB.

[0141] In some embodiments, the polypeptide as disclosed herein has at least 5-fold increased affinity for FcyRIIB compared to FcyRIIA-Rl 31 (at least 10-fold, at least 15 -fold, at least 20-fold, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50). In Attorney Docket No: 070413.20840 some embodiments, the polypeptide lacks binding to FcyRIIA-R131 while retaining binding to Fey RUB.

[0142] In some embodiments, the polypeptide as disclosed herein has at least 5-fold increased affinity for FcyRIIB compared to FcyRIIIA-F158 (at least 10-fold, at least 15- fold, at least 20-fold, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50). In some embodiments, the polypeptide lacks binding to FcyRIIIA-F158 while retaining binding to FcyRIIB.

[0143] In some embodiments, the polypeptide as disclosed herein has at least 5-fold increased affinity for FcyRIIB compared to FcyRIIIA-V158 (at least 10-fold, at least 15- fold, at least 20-fold, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50). In some embodiments, the polypeptide lacks binding to FcyRIIIA-V158 while retaining binding to FcyRIIB.

[0144] Nucleic Acids, Vectors, and Cells

[0145] Another aspect of the disclosure features an isolated nucleic acid comprising a sequence that encodes the polypeptide or protein described above. A nucleic acid refers to a DNA molecule (e.g., a cDNA or genomic DNA), an RNA molecule (e.g., an mRNA), or a DNA or RNA analog. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An "isolated nucleic acid" refers to a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, z.e., a gene encoding a fusion protein. The nucleic acid described above can be used to express the fusion protein of this disclosure. For Attorney Docket No: 070413.20840 this purpose, one can operatively link the nucleic acid to suitable regulatory sequences to generate an expression vector.

[0146] A vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The vector can be capable of autonomous replication or integration into a host DNA. Examples of vectors include a plasmid, cosmid, or viral vector. The vector includes a nucleic acid in a form suitable for expression of the nucleic acid in a host cell. In some embodiments, the vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed.

[0147] A "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and / or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein or RNA desired, and the like. The expression vector can be introduced into host cells to produce a polypeptide of this disclosure. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency.

[0148] Any polynucleotide as mentioned above or a biologically equivalent polynucleotide available to the artisan for the same intended purpose may be inserted into an appropriate expression vector and linked with other DNA molecules to form "recombinant DNA molecules" expressing this receptor. These vectors may be comprised of DNA or RNA; for most cloning purposes DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage and cosmids, yeast artificial chromosomes and other forms of episomal or integrated DNA. It is well within the purview of the artisan to determine an appropriate vector for a particular use.

[0149] A variety of mammalian expression vectors may be used to express the above- mentioned IgG Fes in mammalian cells. As noted above, expression vectors can be DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic DNA in a variety of hosts, such as bacteria, blue green algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria- Attorney Docket No: 070413.20840 yeast or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. Commercially available mammalian expression vectors which may be suitable, include but are not limited to, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and IZD35 (ATCC 37565).

[0150] Also within the scope of this disclosure is a host cell that contains the abovedescribed nucleic acid. Examples include E. coli cells, insect cells (e.g., using baculovirus expression vectors), yeast cells, or mammalian cells. See e.g., Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. To produce a polypeptide of this disclosure, one can culture a host cell in a medium under conditions permitting expression of the polypeptide encoded by a nucleic acid of this disclosure, and purify the polypeptide from the cultured cell or the medium of the cell. Alternatively, the nucleic acid of this disclosure can be transcribed and translated in vitro, e.g., using T7 promoter regulatory sequences and T7 polymerase.

[0151] All of naturally occurring IgG Fcs, genetically engineered IgG Fcs, and chemically synthesized IgG Fes can be used to practice the disclosure disclosed therein. IgG Fc obtained by recombinant DNA technology may have the same amino acid sequence as SEQ ID NO: 12 or a functionally equivalent thereof. The term “IgG Fc” also covers chemically modified versions. Examples of chemically modified IgG Fc include IgG Fes subjected to conformational change, addition or deletion of a sugar chain, and IgG Fc to which a compound such as polyethylene glycol has been bound.

[0152] One can verify the efficacy of a polypeptide / protein thus-made using an animal model, such as a transgenic mouse, as described below. Any statistically significant increase Attorney Docket No: 070413.20840 in in vivo expression of IL-33 basophils or expression of the FcyRIIB receptor on effector macrophages indicates the polypeptide / protein is a candidate for treating the disorders mentioned below. In one embodiment, the above-described assays may be based on measurement of a binding to DC-SIGN protein or DC-SIGN(+)cells. The art is replete with various techniques available to the artisan that will be suitable for measuring the ability of a compound to a DC-SIGN or to DC-SIGN(+)cells and related changes in expression of a gene regulated by the DC-SING pathway, such as IL-33. The artisan will be capable of mixing and matching these various research tools without undue experimentation. Once purified and tested by standard methods or according to the assays and methods described in the examples below, non-sialylated IgG Fc variants can be included in pharmaceutical composition for treating inflammatory disorders.

[0153] Antibodies

[0154] The term “antibody” as referred to herein includes whole antibodies and any antigenbinding fragment or single chains thereof. Whole antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2, and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain variable region CDRs and FRs are HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, HFR4. The light chain variable region CDRs and FRs are LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, LFR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Attorney Docket No: 070413.20840

[0155] The term “antigen-binding fragment or portion” of an antibody (or simply “antibody fragment or portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigenbinding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment or portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is essentially a Fab with part of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3rd ed. 1993)); (iv) a Fd fragment consisting of the VH and CHI domains; (v) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (vi) a dAb fragment (Ward et al.. (1989) Nature 341 :544-546), which consists of a VH domain; (vii) an isolated CDR; and (viii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv or scFv); see, e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment or portion” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0156] An “isolated antibody,” as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities. An isolated antibody can be substantially free of other cellular material and / or chemicals.

[0157] The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Attorney Docket No: 070413.20840

[0158] The term “human antibody” is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0159] The term “human monoclonal antibody” refers to antibodies displaying a single binding specificity, which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies can be produced by a hybridoma that includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0160] The term “recombinant human antibody,” as used herein, includes all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human Attorney Docket No: 070413.20840 germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0161] The term “isotype” refers to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes. The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.”

[0162] The term “human antibody derivatives” refers to any modified form of the human antibody, e.g., a conjugate of the antibody and another agent or antibody. The term “humanized antibody” is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences.

[0163] The term “chimeric antibody” is intended to refer to antibodies in which the variable region sequences are derived from one species, and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody, and the constant region sequences are derived from a human antibody. The term can also refer to an antibody in which its variable region sequence or CDR(s) is derived from one source (e.g., an IgAl antibody), and the constant region sequence or Fc is derived from a different source (e.g., a different antibody, such as an IgG, IgA2, IgD, IgE or IgM antibody).

[0164] In some embodiments, provided is an antibody thereof comprising the abovedescribed polypeptide. In some embodiments, the antibody or antigen-binding fragment thereof comprising the above-described polypeptide targets CD20 (such as rituximab) or CD4 (such as ibalizumab). In some embodiments, such antibody or antigen-binding fragment thereof is described in U.S. Pat. Nos. 5,736,137; 7,682,612; 10,577,588; or 5,871,732, the contents of which are incorporated herein by reference.

[0165] Compositions

[0166] Within the scope of this disclosure is a composition that contains a suitable carrier and one or more of the agents described above, such as the non-sialylated IgG Fc variants. Attorney Docket No: 070413.20840

[0167] The composition can be a pharmaceutical composition that contains a pharmaceutically acceptable carrier or a cosmetic composition that contains a cosmetically acceptable carrier.

[0168] The term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0169] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the composition, and is relatively non-toxic, z.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0170] As used herein, the term “pharmaceutically acceptable carrier” includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; diluent; granulating agent; lubricant; binder; disintegrating agent; wetting agent; emulsifier; coloring agent; release agent; coating agent; sweetening agent; flavoring agent; perfuming agent; preservative; antioxidant; plasticizer; gelling agent; thickener; hardener; setting agent; suspending agent; surfactant; humectant; carrier; stabilizer; and other non Attorney Docket No: 070413.20840 toxic compatible substances employed in pharmaceutical formulations, or any combination thereof. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of one or more components of this disclosure, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.

[0171] The above-described composition, in any of the forms described above, can be used for treating disorders characterized by inflammation. An effective amount refers to the amount of an active compound / agent that is required to confer a therapeutic effect on a treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on the types of diseases treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment.

[0172] A pharmaceutical composition of this disclosure can be administered parenterally, orally, nasally, rectally, topically, or buccally. The term “parenteral” as used herein refers to subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique.

[0173] A sterile injectable composition can be a solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Such solutions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer’s solution, and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acids, such as, but not limited to, oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as, but not limited to, olive oil or castor oil, polyoxyethylated versions thereof. These oil solutions or suspensions also can contain a long chain alcohol diluent or dispersant such as, but not limited to, carboxymethyl cellulose, or similar dispersing agents. Other commonly used surfactants, such as, but not limited to, TWEENS or SPANS or other similar emulsifying agents or bioavailability enhancers, which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms also can be used for the purpose of formulation. Attorney Docket No: 070413.20840

[0174] A composition for oral administration can be any orally acceptable dosage form including capsules, tablets, emulsions and aqueous suspensions, dispersions, and solutions. In the case of tablets, commonly used carriers include, but are not limited to, lactose and corn starch. Lubricating agents, such as, but not limited to, magnesium stearate, also are typically added. For oral administration in a capsule form, useful diluents include, but are not limited to, lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.

[0175] Pharmaceutical compositions for topical administration according to the described disclosure can be formulated as solutions, ointments, creams, suspensions, lotions, powders, pastes, gels, sprays, aerosols, or oils. Alternatively, topical formulations can be in the form of patches or dressings impregnated with active ingredient(s), which can optionally comprise one or more excipients or diluents. In some preferred embodiments, the topical formulations include a material that would enhance absorption or penetration of the active agent(s) through the skin or other affected areas. The topical composition is useful for treating inflammatory disorders in the skin, including, but not limited to eczema, acne, rosacea, psoriasis, contact dermatitis, and reactions to poison ivy.

[0176] A topical composition contains a safe and effective amount of a dermatologically acceptable carrier suitable for application to the skin. A “cosmetically acceptable” or “dermatologically-acceptable” composition or component refers a composition or component that is suitable for use in contact with human skin without undue toxicity, incompatibility, instability, allergic response, and the like. The carrier enables an active agent and optional component to be delivered to the skin at an appropriate concentration(s). The carrier thus can act as a diluent, dispersant, solvent, or the like to ensure that the active materials are applied to and distributed evenly over the selected target at an appropriate concentration. The carrier can be solid, semi-solid, or liquid. The carrier can be in the form of a lotion, a cream, or a gel, in particular one that has a sufficient thickness or yield point to prevent the active materials from sedimenting. The carrier can be inert or possess dermatological benefits. It also should be physically and chemically compatible with the active components described herein, and should not unduly impair stability, efficacy, or Attorney Docket No: 070413.20840 other use benefits associated with the composition. The topical composition may be a cosmetic or dermatologic product in the form known in the art for topical or transdermal applications, including solutions, aerosols, creams, gels, patches, ointment, lotion, or foam.

[0177] Treatment Methods

[0178] Provided are methods for treating a subject with inflammatory disorders. Inflammatory Disorder

[0179] Provided are methods for treating a subject who has an inflammatory disorder. The term “inflammatory disorder” or “inflammatory disorder” refers to a disorder or disease that is characterized by abnormal or unwanted inflammation, such as an autoimmune disease or an infection. Autoimmune diseases are disorders characterized by the chronic activation of immune cells under non-activating conditions. Examples include psoriasis, inflammatory bowel diseases (e.g., Crohn’s disease and ulcerative colitis), systemic lupus erythematosus (SLE), multiple sclerosis (MS), Guillain-Barre syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), rheumatoid arthritis (RA), psoriatic arthritis, multiple sclerosis, lupus, type I diabetes, primary biliary cirrhosis, and transplant. In one embodiment, the infection comprises a viral infection or a bacterial infection. In some embodiments, the infection comprises HIV or influenza. In some embodiments, the infection is due to Streptococcus pneumoniae, Cryptococcus neoformans, or Bacillus anthraci. In some embodiments, the infection comprises a viral disease that has an autoimmune component such as long-covid or post-viral autoimmunity.

[0180] Other examples of inflammatory disorders that can be treated by the methods of this disclosure include asthma, myocardial infarction, stroke, inflammatory dermatoses (e.g., dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria, necrotizing vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, eosinophilic myositis, polymyositis, dermatomyositis, and eosinophilic fasciitis), acute respiratory distress syndrome, fulminant hepatitis, hypersensitivity lung diseases (e.g., hypersensitivity pneumonitis, eosinophilic pneumonia, delayed-type hypersensitivity, interstitial lung disease (ILD), idiopathic pulmonary fibrosis, and ILD associated with rheumatoid arthritis), and allergic rhinitis. Additional examples also include myasthenia gravis (MG), juvenile onset diabetes, glomerulonephritis, autoimmune thyroiditis, ankylosing spondylitis, systemic sclerosis, acute and chronic inflammatory diseases (e.g., systemic anaphylaxia or Attorney Docket No: 070413.20840 hypersensitivity responses, drug allergies, insect sting allergies, allograft rejection, and graft-versus-host disease), and Sjogren’s syndrome.

[0181] In one embodiment, the treatment results in at least 5-fold (e.g., 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 fold or more) antiinflammatory protection compared to intravenous immunoglobulin (IVIG). In one embodiment, the treatment results in at least 100-fold anti-inflammatory protection compared to intravenous immunoglobulin (IVIG).

[0182] As used herein, the term “anti-inflammatory protection” refers to protection from the effects of inflammation such as chronic activation of immune cells or inhibiting the activation of such cells. Chronic activation of immune cells results in various effects, including, but not limited to pain, swelling discoloration of a body part, or the development of autoimmune diseases or disorders.

[0183] A “subject” refers to a human and a non-human animal. Examples of a non-human animal include all vertebrates, e.g., mammals, such as non-human mammals, non-human primates (particularly higher primates), dog, rodent e.g., mouse or rat), guinea pig, cat, and rabbit, and non-mammals, such as birds, amphibians, reptiles, etc. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental, non-human animal or animal suitable as a disease model.

[0184] A subject to be treated for an inflammatory disorder can be identified by standard diagnosing techniques for the disorder. Optionally, the subject can be examined for the level or percentage of one or more cytokines or cells in a test sample obtained from the subject by methods known in the art. If the level or percentage is at or below a threshold value (which can be obtained from a normal subject), the subject is a candidate for treatment described herein. To confirm the inhibition or treatment, one can evaluate and / or verify the level or percentage of one or more of the above-mentioned cytokines or cells in the subject after treatment.

[0185] “Treating” or “treatment” refers to administration of a compound or agent to a subject who has a disorder with the purpose to cure, alleviate, relieve, remedy, delay the onset of, prevent, or ameliorate the disorder, the symptom of the disorder, the disease state secondary to the disorder, or the predisposition toward the disorder. Attorney Docket No: 070413.20840

[0186] An “effective amount” or “therapeutically effective amount” refers to an amount of the compound or agent that is capable of producing a medically desirable result in a treated subject. The treatment method can be performed in vivo or ex vivo, alone or in conjunction with other drugs or therapy. A therapeutically effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

[0187] The agent can be administered in vivo or ex vivo, alone or co-administered in conjunction with other drugs or therapy, i.e., a cocktail therapy. As used herein, the term “co-administration” or “co-administered” refers to the administration of at least two agents or therapies to a subject. In some embodiments, the co-administration of two or more agents / therapies is concurrent. In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. Those of skill in the art understand that the formulations and / or routes of administration of the various agents / therapies used may vary.

[0188] In an in vivo approach, a compound or agent is administered to a subject. Generally, the compound or agent is suspended in a pharmaceutically-acceptable carrier (such as, for example, but not limited to, physiological saline) and administered orally or by intravenous infusion, or injected or implanted subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.

[0189] The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. In some embodiments, the treatment method comprises administering the above-described polypeptide at a dose of about 1 mg / kg to about 1000 mg / kg. For example, the dose can be about 1 mg / kg, about 10 mg / kg, about 50 mg / kg, about 100 mg / kg, about 150 mg / kg, about

[0190] 200 mg / kg, about 250 mg / kg, about 300 mg / kg, about 400 mg / kg, about 450 mg / kg. about

[0191] 500 mg / kg, about 550 mg / kg, about 600 mg / kg, about 650 mg / kg, about 700 mg / kg, about

[0192] 750 mg / kg, about 800 mg / kg, about 850 mg / kg, about 900 mg / kg, about 950 mg / kg, about

[0193] 1000 mg / kg, or any amount in-between. Variations in the needed dosage are to be expected in view of the variety of compounds / agents available and the different efficiencies of various routes of administration. For example, oral administration would be expected to require Attorney Docket No: 070413.20840 higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Encapsulation of the compound in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) can increase the efficiency of delivery, particularly for oral delivery.

[0194] Additional Definitions

[0195] To aid in understanding the detailed description of the compositions and methods according to the disclosure, a few express definitions are provided to facilitate an unambiguous disclosure of the various aspects of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0196] The term “recombinant,” as used herein, refers to antibodies or antigen-binding fragments thereof of the invention created, expressed, isolated, or obtained by technologies or methods known in the art as recombinant DNA technology, which include, e.g., DNA splicing and transgenic expression. The term refers to antibodies expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice), or a cell (e.g., CHO cells) expression system or isolated from a recombinant combinatorial human antibody library.

[0197] A “nucleic acid” or “polynucleotide” refers to a DNA molecule (for example, but not limited to, a cDNA or genomic DNA) or an RNA molecule (for example, but not limited to, an mRNA), and includes DNA or RNA analogs. A DNA or RNA analog can be synthesized from nucleotide analogs. The DNA or RNA molecules may include portions that are not naturally occurring, such as modified bases, modified backbone, deoxyribonucleotides in an RNA, etc. The nucleic acid molecule can be single-stranded or double-stranded.

[0198] As used herein, the term “affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its Attorney Docket No: 070413.20840 partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0199] The term “specifically binds,” or “binds specifically to,” or the like, refers to an antibody that binds to a single epitope, e.g., under physiologic conditions., but which does not bind to more than one epitope. Accordingly, an antibody that specifically binds to a polypeptide will bind to an epitope that is present on the polypeptide, but which is not present on other polypeptides. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1x1 O'8M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0200] The term “Kassoc” or “Ka,” as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term “Kdis” or “Kd,” as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term “KD,” as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (z.e., Kd / Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. A preferred method for determining the KD of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore™ system.

[0201] As used herein, the term “disease” is intended to be generally synonymous and is used interchangeably with the terms “disorder” and “condition” (as in medical condition), in that all reflect an abnormal condition (e.g., inflammatory disorder) of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.

[0202] As used herein, the term “treating” or “treatment” of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder (z.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment, “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the patient. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, Attorney Docket No: 070413.20840

[0203] (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, “treating” or “treatment” refers to preventing or delaying the onset or development or progression of the disease or disorder.

[0204] The terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

[0205] The terms “decrease,” “reduced,” “reduction,” “decrease,” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced,” “reduction,” “decrease,” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example, a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[0206] As used herein, the term “agent” denotes a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g, a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. The activity of such agents may render it suitable as a “therapeutic agent,” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.

[0207] As used herein, the terms “therapeutic agent,” “therapeutic capable agent,” or “treatment agent” are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally counteracting a disease, symptom, disorder or pathological condition. Attorney Docket No: 070413.20840

[0208] The term “therapeutic effect” is art-recognized and refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance.

[0209] The term “effective amount,” “effective dose,” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve a desired effect. A “therapeutically effective amount” or “therapeutically effective dosage” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. A “prophylactically effective amount” or a “prophylactically effective dosage” of a drug is an amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or of suffering a recurrence of disease, inhibits the development or recurrence of the disease. The ability of a therapeutic or prophylactic agent to promote disease regression or inhibit the development or recurrence of the disease can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0210] Doses are often expressed in relation to body weight. Thus, a dose which is expressed as [g, mg, or other unit] / kg (or g, mg etc.) usually refers to [g, mg, or other unit] “per kg (or g, mg etc.) bodyweight,” even if the term “bodyweight” is not explicitly mentioned.

[0211] As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one component useful within the invention with other components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of one or more components of the invention to an organism.

[0212] “Combination” therapy, as used herein, unless otherwise clear from the context, is meant to encompass administration of two or more therapeutic agents in a coordinated fashion and includes, but is not limited to, concurrent dosing. Specifically, combination therapy encompasses both co-administration (e.g., administration of a co-formulation or Attorney Docket No: 070413.20840 simultaneous administration of separate therapeutic compositions) and serial or sequential administration, provided that administration of one therapeutic agent is conditioned in some way on the administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a prescribed period of time. See, e.g., Kohrt etal. (2011) Blood 117:2423.

[0213] As used herein, the term “co-administration” or “co-administered” refers to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the co-administration of two or more agents / therapies is concurrent. In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. Those of skill in the art understand that the formulations and / or routes of administration of the various agents / therapies used may vary.

[0214] As used herein, the term “zzz vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0215] As used herein, the term “zzz vivo" refers to events that occur within a multi-cellular organism, such as a non-human animal.

[0216] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[0217] As used herein, the terms “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted.

[0218] As used herein, the phrases “in one embodiment,” “in various embodiments,” “in some embodiments,” and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may unless the context dictates otherwise.

[0219] As used herein, the terms “and / or” or “ / ” means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0220] As used herein, the word “substantially” does not exclude “completely,” e.g., a composition that is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention. Attorney Docket No: 070413.20840

[0221] As used herein, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.

[0222] As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Unless indicated otherwise herein, the term “about” is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.

[0223] As disclosed herein, a number of ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0224] The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. Attorney Docket No: 070413.20840

[0225] All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. In regard to any of the methods provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, the steps may occur in any order, unless noted otherwise. In cases in which a method comprises a combination of steps, each and every combination or sub-combination of the steps is encompassed within the scope of the disclosure, unless otherwise noted herein.

[0226] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent that it is not inconsistent with the present disclosure. Publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0227] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

[0228] Examples

[0229] Example 1. Co-expression of DC-SIGN with FcyRIIB augments binding to IVIG or sialylated IgGl Fc in a sialylation dependent manner.

[0230] As cells which constitutively express type II FcyRs also express type I FcyRs (Table 1), it was determined binding of sialylated Fc or IVIG was dependent upon the presence of both receptor families. When co-transfecting 293-T cells with plasmids encoding FcyRIIB and DC-SIGN, a noticeable and significant enhancement was measured in the cell surface expression of FcyRIIB (FIG. 1A), suggesting co-expression of DC-SIGN with FcyRIIB results in stabilization of FcyRIIB on the cell surface. Furthermore, expression of either type I or type II FcyRs alone showed minimal binding activity to IVIG, while co-expression Attorney Docket No: 070413.20840 of the two receptor families significantly augmented binding to IVIG (FIG. IB) or sialylated IgGl Fc in a sialylation dependent manner using glycoengineered hlgGls expressing terminally galactosylated (G2F) or sialylated (S2G2F) glycans (FIG. 1C). Mutation of the glycosylation sites on FcyRIIB (FIG.2A) did not affect its overall cell surface expression (FIG. 2B) although it significantly reduced its ability to enhance its expression when coexpressed with type II FcyRs (FIG. 2C), suggesting a glycan dependent mechanism of type II FcyR mediated stabilization of type I FcyRs. This data supports recent reports which demonstrated other C-type lectins, such as Dectin- 1, interacting with FcyRIIB to stabilize its cell-surface expression and enhance binding to IVIG (Seeling, M. et al. Immunoglobulin G-dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin- 1. Immunity 56, 1046-1063. el047 (2023)).

[0231] Expression patterns of type I and type II FcyRs among leukocyte types are depicted in Table 1. Cells constitutively expressing type II FcyRs and FcyRIIB are in bold (§).

[0232] Table 1.

[0233] + : Constitutively Expressed

[0234] + / - : Cell subset restricted

[0235] I : Inducible

[0236] Example 2. Generation of the Vll sFc variant. Attorney Docket No: 070413.20840

[0237] Next, the WT and VI 1 sFc proteins, which has enhanced and selective affinity for FcyRIIB (Mimoto, F. et al. Engineered antibody Fc variant with selectively enhanced FcyRIIb binding over both FcyRIIa(Rl 31) and FcyRIIa(H131). Protein Eng Des Sei 26, 589- 598 (2013) and Liu, R., Oldham, R. J., Teal, E., Beers, S. A. & Cragg, M. S. Fc-Engineering for Modulated Effector Functions-Improving Antibodies for Cancer Treatment. Antibodies (Basel) 9 (2020)) were generated, and their affinity for FcyRIIB was characterized by SPR (FIG. 3B). The VI 1 sFc variant (G237D / P238D / H268D / P271G / A330R) contains a parental P238D mutation, which itself enhances binding to FcyRIIB while reducing or ablating binding to activating FcyRs. The VI 1 sFc variant was tested in mice humanized for type I FcyRs (hFcyR mice) and its activity was compared to IVIG and WT sFc mediated protection in multiple models of autoantibody-mediated diseases. In CD4 T-cell depletion models, a surrogate model for immune thrombocytopenia, pre-dosing mice with IVIG (2500 mg / kg) results in an enhanced rebound of CD4 T-cells two days post injection with an anti-CD4 afucosylated depleting antibody (FIG. 4A-B). Unexpectedly, VI 1 sFc dosed at 40-fold lower (25 mg / kg), compared to IVIG, recapitulated the same protected and enhanced rebound effect of IVIG, while no protection was observed when treated with WT sFc at the equivalent 25 mg / kg dose (FIGS. 4C-4D). These results indicate that targeting sFc to FcyRIIB through Fc-engineering enhances the anti-inflammatory activity of sialylated IgG.

[0238] Example 3. Vll sFc in the K / BxN serum transferred arthritis model.

[0239] The Vl l sFc was tested in the K / BxN serum transferred arthritis model (Christensen, A. D., Haase, C., Cook, A. D. & Hamilton, J. A. K / BxN Serum-Transfer Arthritis as a Model for Human Inflammatory Arthritis. Front Immunol 7, 213 (2016)), in which hFcyR mice are treated with WT sFc (10 mg / kg) or Vl l sFc (10 mg / kg) one hour before injection with arthritogenic K / BxN serum, and the development of arthritic inflammation was measured over five days by clinical scoring and measurement of ankle thickness (Anthony, R. M. et al. Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science 320, 373-376 (2008) and Anthony, R. M., Kobayashi, T., Wermeling, F. & Ravetch, J. V. Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway. Nature 475, 110-113 (2011)). (FIG. 5A). Vl l sFc administration, but not WT sFc, significantly ameliorated inflammation (FIGS. 5C-5D), demonstrating the protective efficacy of Vl l sFc in mouse models of arthritic inflammation. A dose titration was Attorney Docket No: 070413.20840 performed of VI 1 sFc (10, 5, and 1 mg / kg) and these doses were compared to IVIG (1000 mg / kg) in the K / BxN serum transfer inflammation model (FIG. 6A). Tracking inflammation over the course of twelve days, 10 mg / kg of VI 1 sFc recapitulated the effect of 1000 mg / kg IVIG, a 100-fold enhancement in the protective dose (FIGS. 6B-6D). Additionally, 5 mg / kg of Vl l sFc significantly reduced inflammation compared to K / BxN serum alone treated mice, a 200-fold improvement in dose compared to IVIG (1000 mg / kg).

[0240] Example 4. Materials and methods.

[0241] This Example describes the materials and methods used in Examples 5-7.

[0242] Recombinant protein generation

[0243] Recombinant human IgGl Fc proteins, consisting of the full length IgGl hinge, CHg2, and CHg3 domains, were produced via transient transfection of HEK 293-F cells (ThermoFisher, Cat no: A14635) and purified from cell-fire supernatants by affinity chromatography using Protein G Sepharose beads (GE Healthcare). To remove aggregates, Fc proteins were subsequently re-purified and dialyzed into phosphate-buffered saline (PBS) via size exclusion chromatography using a HiLoad® 16 / 600 Superdex® 200 pg resin column on an AKTA Pure (Cytivia) chromatography system. Monomeric Fc proteins were then concentrated with Amicon Ultra Centrifugal Filters (Millipore), filter-sterilized (0.22 pm), and quantified via BCA protein assay (ThermoFisher).

[0244] Sialylated Fc proteins were generated by co-transfecting HEK 293-F cells with Fc expression vectors and plasmids encoding open reading frames of Human Beta- 1,4- galactosyltransferase 1 (B4GALT1) (Invivogen, Cat no: punol-hb4galtl), and Human Betagalactoside alpha-2, 6-sialyltransferase 1 (ST6GAL1) (Invivogen, Cat no: punol-hst6gall). The human Fc variants GRLR (G236R / L328R), GA (G236A), or Vl l (G237D, P238D, H268D, P271G, A330R) were generated via site-directed mutagenesis or as synthetic genes (IDT), and expressed and purified similar to sialylated WT Fc proteins. Prior to in vivo experiments, the monomeric purity of the Fc proteins was reassessed via size exclusion chromatography with a Superdex™ 200 Increase 10 / 300 GL column. All Fc proteins were at least 95% monomeric protein. To generate asialylated Fc proteins, sialylated Fc proteins were treated with a2-3,6,8 Neuraminidase (NEB) overnight and re-purified via size exclusion chromatography. Attorney Docket No: 070413.20840

[0245] Recombinant human FcyRs were generated as His-tagged ectodomains derived from annotated (Uniprot) sequences, expressed as soluble proteins via transient transfection of 293-F cells, and purified by affinity chromatography using HisTrap HP resin columns, followed by size exclusion chromatography to isolate monomeric FcyRs.

[0246] Animal Studies

[0247] All in vivo experiments were performed in compliance with federal laws and institutional guidelines and approved by The Rockefeller University Institutional Animal Care and Use Committee (Protocol number 20029-H). Mice were bred and maintained in the Comparative Bioscience Center at The Rockefeller University. FcyR Humanized (hFcyR) mice (FcyRanull, hFcyRU, hFcyRIIAR131+, hFcyRIIB+, FcyRIIIAF158+, FcyRIIIB+) were generated on the C57BL / 6 background and have been extensively characterized in previous studies (J. Bayry et al., Intravenous Immunoglobulin: Mechanism of Action in Autoimmune and Inflammatory Conditions. J Allergy Clin Immunol Pract 11, 1688-1697 (2023); A. Epp etal., Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions. Journal of Allergy and Clinical Immunology 141, 399-402. e398 (2018)). K / BxN serum transfer induced arthritis was conducted as previously described (R. M. Anthony et al., Recapitulation of IVIG anti-inflammatory activity with a recombinant IgGFc. Science 320, 373-376 (2008)). Briefly, KRN TCR transgenic mice were bred with male NOD mice (strain 032445, Jackson Laboratory) to generate K / BxN mice which spontaneously develop arthritis (B. Chen et al., Humanised effector-null FcyRIIA antibody inhibits immune complex-mediated proinflammatory responses. Ann Rheum Dis 78, 228-237 (2019)). Serum from arthritic mice was collected, pooled together, and frozen. To induce arthritis, 100 pl K / BxN serum was injected intraperitoneally in seven-eight week old male hFcyR+ mice, and the development of arthritis and inflammation was evaluated through clinical scoring and measurement of ankle thickness using a digital caliper. To characterize the prophylactic anti-inflammatory activity of IVIG or Fc proteins, male hFcyR mice were injected intravenously with IVIG or Fc proteins at the indicated dose one hour prior to intraperitoneal injection with 100 pl of K / BxN serum. For ankle measurements, the thickness of each ankle was averaged together and represented as one data point per mouse. For clinical scoring, a score of 0-3 was given to each paw, a score of 0 being no inflammation Attorney Docket No: 070413.20840 and 3 representing severe swelling and inflammation. Scores from each paw were summed together to give an overall clinical score.

[0248] For SIGN-R1 blocking experiments, male hFcyR+mice were treated with 10 mg / kg of Vl l sFc intravenously one hour before intraperitoneal injection with 100 pl of K / BxN serum. To block SIGN-R1, mice were injected subcutaneously with 100 pg of an anti-SIGN- R1 antibody (clone 22D1, Bioxcell, Cat no: BE0220) 24 hours prior to sFc and K / BxN serum injection. Mice received two additional injections with anti-SIGN-Rl on days 1 and 3 post serum transfer. A control group of mice was injected with an isotype control (Bioxcell, Cat no: BE0091) at the same dose and schedule as anti-SIGN-Rl treatment.

[0249] Experimental autoimmune encephalomyelitis (EAE) was induced with 8-to-10 week old hFcyR+mice by immunizing mice subcutaneously with 200 pl of an emulsion consisting of MOG35-55 antigen (MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 31), Hooke Labs) emulsified in complete Freund’s adjuvant (Hooke Labs, Cat no: EK-2110). Mice were subsequently intraperitoneally injected with 100 ng of pertussis toxin (Hooke Labs) 4 and 24 hours post immunization. Development of disease was monitored daily according to the following criteria: 0, no clinical signs; 0.5, partial loss of tail tone; 1, paralyzed tail; 2, loss of coordinated movement, and hind limb paresis; 2.5 one hind limb completely paralyzed; 3, both hind limbs paralyzed; 3.5, hind limbs paralyzed and / or hunched back and disorientation; 4, severely hunched back and weakness in forelimbs; 4.5, forelimbs paralyzed; 5, moribund. To characterize the anti-inflammatory effect of VI 1 sFc in the EAE model, male hFcyR+mice were subcutaneously injected with 10 mg / kg of VI 1 sFc on days 5, 10, 15, and 20 post immunization.

[0250] For serum half-life studies, hFcyR / hFcRn mice were injected subcutaneously with 200 pg of WT sFc, VI 1 sFc, or VI 1 slgG. Sera from mice days 1, 4, and 7 post injection were collected and concentration sFc or slgG in sera was determined by ELISA, using WT sFc or VI 1 slgG for standard curves.

[0251] Tissue Imaging

[0252] To characterize inflammation in joints of Vl l sFc treated and untreated K / BxN serum induced arthritic mice, hind legs were collected from euthanized mice and fixed Attorney Docket No: 070413.20840 overnight in 10% Neutral Buffered Formalin (NBF). Hind legs were subsequently decalcified for a week (Decal, Statlab) before embedding in Tissue-Tek O.C.T and snap freezing to -80 °C. Frozen ankles were sectioned (10 um) on a Cryostat (Leica), dried overnight, and stained with a hematoxylin and eosin stain kit (Vector Labs).

[0253] To collect brains and spinal cords in EAE experiments, mice were euthanized and perfused via intra-cardiac puncture with cold PBS prior to perfusion with 10% Neutral Buffered Formalin and central nervous system tissues were harvested and stored in 10% NBF overnight. Following cryoprotection by incubation in a series of 15% then 30% sucrose / PBS solution overnight at 4 °C, the brain and spinal cords were then embedded in Tissue-Tek O.C.T. compound (Sakura) and cut on a cryostat. Serial sections (10-20 pm thickness, 7 series) were collected on positively charged microscope slides (VWR) dried overnight and stored at -80 °C until further processing.

[0254] For immunofluorescence imaging, spinal cord sections were stained with a Myelin Basic Protein (MBP) antibody (1 : 150) overnight before washing and staining with a fluorescent secondary antibody for one hour followed by mounting with ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, P36931) All sample acquisition and analysis were conducted using the AKOYA PhenoCycler®-Fusion 2.0 and QuPath v0.5.1.

[0255] Western Blotting

[0256] Recombinant Fc proteins (5 pg) were resolved on SDS-PAGE gels in non-reducing conditions, transferred to PVDF membranes (BioRad) and blocked for one hour with PBS + 1% BSA. Blots were then incubated overnight with biotinylated SNA lectin (2 pg / ml, Vector Labs, Cat no: B-1305-2) or ECL (5 pg / ml, Vector Labs, Cat no: B-l 145-5), followed by incubation with Peroxidase Streptavidin (1 :50,000, Jackson ImmunoResearch, Cat no: 016-030-084). Blots were developed with a chemiluminescent substrate (Clarity ECL, BioRad) and imaged on a ChemiDoc MP imaging system (BioRad). Parallel to SNA and ECL blots, 5 pg of Fc proteins were resolved via non-reducing SDS-PAGE gels and stained with SimplyBlue™ SafeStain (ThermoFisher) to visualize total protein. Gels were imaged on a ChemiDoc MP system. Blots and protein gels were analyzed on ImageJ software. To estimate the degree of sialylation or galactosylation, SNA and ECL band intensities were normalized protein gel loading controls and plotted as relative intensities. Attorney Docket No: 070413.20840

[0257] Immunoprecipitation

[0258] HEK 293-F cells were transiently transfected with FLAG-tagged FcyRIIB (Sino, Cat no: HG10260-NF), FLAG-tagged FcyRIIA, (Sino, HG10374-NF) and / or HA-tagged DC- SIGN (Sino, Cat no: HG10200-CY) full-length open reading frame expression vectors. Two days post transfection, cells were lysed with IP -lysis buffer (Pierce) with EDTA-free protease inhibitors added (Roche) and clarified cell lysates were immunoprecipitated (IP) using anti-FLAG tag magnetic agarose (ThermoFisher, Cat no: A36797). Cell lysates and IP elutions were resolved under reducing conditions on SDS-PAGE gels, transferred to PVDF membranes, and blocked for one hour with TBS + 1% BSA. Blots were subsequently incubated overnight with a fluorescently labeled anti-FLAG tag antibody (ThermoFisher, Cat no: MA1-91878-D650) or a biotinylated anti -HA tag antibody (ThermoFisher, Cat no: 26183-BTIN). FLAG-tag immunoblots were fluorescently imaged on a Chemidoc MP imaging system. HA-tag immunoblots were washed and incubated with Peroxidase Streptavidin (1 :50,0000, Jackson ImmunoResearch) before chemiluminescent imaging.

[0259] Surface Plasmon Resonance (SPR)

[0260] All experiments were performed with a Biacore™ T200 SPR System (Cytivia) at 25 °C. Fc proteins were immobilized on Series S Protein G sensor chips (Cytivia) in HBS-EP+ Buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% (v / v) surfactant P20). Serial dilutions of recombinant human FcyRs were injected to the flow cells at 30 pl min-1, with concentrations ranging from 8000 nM to 125 nM (1 :2 serial dilutions) or 300 nM to 4.69 nM (1 :2 serial dilutions) for the high affinity FcyRIA. FcyRs were allowed to associate for 120 seconds with Protein-G immobilized Fc proteins before a 900 second dissociation step. At the end of each cycle, the sensor chip was regenerated with glycine HCL buffer (10 mM, pH 1.5, 50 pl min-1, 30 seconds). Background binding to blank immobilized flow cells was subtracted and affinity constants (steady-state affinity, or 1 : 1 fit for FcyRIA) was calculated using Biacore™ T200 evaluation software (Cytivia).

[0261] Recombinant DC-SIGN (AcroBiosystems, Cat no: CD9-H5246-100pg) was immobilized to a Series S CM5 chip (Cytivia) using an amine couple kit (Cytivia) in HBS- P+ Buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20) supplemented with 2 mM CaCh and 2 mM MgCh. Serial dilutions of recombinant native Attorney Docket No: 070413.20840 or single glycan mutant FcyRIIB were injected into flow cells and associated against immobilized DC-SIGN for 120 seconds, followed by a 900 second dissociation step, and subsequent regeneration with 10 mM glycine HCL buffer (pH 1.5). A similar experimental setup was performed for native or PNGaseF treated (NEB, Cat no: P0706S) human FcyRIIAR131. For EDTA chelation, HBS-P+ Buffer + 2 mM CaCh + 2 mM MgCh was supplemented with an additional 5 mM EDTA. Background binding to non-immobilized flow cells was subtracted and affinity constants were calculated using Biacore™ T200 evaluation software (Two- State fit).

[0262] For FcRn binding assays, biotinylated human or mouse FcRn (Aero Biosystems, Cat no: FCM-H82W7-25pg, FCM-M82W5-25pg) was immobilized to a Series S sensor chip CAP (Biotin capture) in pH 6.0 HBS-EP+ buffer (HEPES, pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20 + 3 mM EDTA). Using single-cycle kinetics, serial dilutions of recombinant WT sFc, VI 1 sFc, WT slgG, or VI 1 slgG (25 to 1.56 nM, 1 :2 dilutions) were reacted against immobilized human or mouse FcRn. Background binding to nonimmobilized flow cells was subtracted and affinity constants were calculated using Biacore™ T200 evaluation software (1 : 1 fit).

[0263] Flow cytometry

[0264] Expi293 cell lines transiently transfected to express full length FcyRIIB, FcyRIIAH131, human CD23, or human DC-SIGN alone or in combination were harvested and stained with fluorescently labeled anti-human FcyRIIB (1 pg / ml, clone 2B6), antihuman FcyRIIA (1 pg / ml clone IV.3), anti-DC-SIGN (1 :200, BD, clone DCN46), antihuman CD23 (1 :200, BD, clone M-L233), and a fixable live / dead stain (ThermoFisher). Staining was performed at 4 °C for 30 minutes. Cells were washed twice and analyzed on an Attune NxT flow cytometry (Thermofisher) using Attune NxT software and data was analyzed using FlowJo software.

[0265] For IgG binding studies, IVIG or glycoengineered Rituximab G2F or S2G2F (D. Pinto et al., Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody. Nature 583, 290-295 (2020)) were fluorescently labeled with DyLight™ antibody labeling kits (ThermoFisher) and incubated overnight at 4 °C (10 pg / ml) with transfected FcyR expressing 293 cell lines in 10 mM Hepes, 150 mM NaCl, 10 mM CaCh, 10 mM Attorney Docket No: 070413.20840

[0266] MgCh, 0.5% BSA, pH 7.4 buffer. Cells were immediately fixed in 2% paraformaldehyde for 20 minutes at 4 °C before washing and staining with fluorescent anti-FcyR antibodies and a fixable live / dead stain. Cells were subsequently washed twice and analyzed on an Attune NxT flow cytometer as before, and IgG binding was determined through gating on live FcyR+ cells and measuring the median fluorescent intensity (MFI) (IVIG) or percent IgGl+cells (Rituximab G2F and S2G2F IgGl).

[0267] Example 5. Requirements of sialylation and type I FcyR engagement in hFcyR mouse models of IVIG mediated anti-inflammatory activity.

[0268] It was determined if IVIG and recombinant sialylated wild-type IgGl-Fc (WT sFc) mediate anti-inflammatory activity in hFcyR mice, and if this activity is sialylation dependent. To generate recombinant WT sFc proteins, HEK 293 -F cells were co-transfected with WT IgGl Fc expression vectors and plasmids encoding the genes for Beta-1, 4- galactosyltransferase 1 (B4GALT1) and Beta-galactoside alpha-2, 6-sialyltransferase 1 (ST6GAL1), which attach galactose and a2-6 linked sialic acid, respectively, to the terminal N-Acetylglucosamine (GlcNAc) on the complex, biantennary N297 glycan (FIG. 7A). This method of co-expressing B4GALT1 and ST6GAL1 with antibody or Fc expression vectors has been shown to result in an 80-90% sialylated product (S. L. Sneed etal., An engineered immunomodulatory IgGl Fc suppresses autoimmune inflammation through pathways shared with i.v. immunoglobulin. J Clin Invest, 134, (2024); C. Raymond etal., Production of a2,6-sialylated IgGl in CHO cells. mAbs 7, 571-583 (2015)). The glycan profile of this product was validated by probing with lectins specific for terminal galactose (ECL, Erythrina Cristagalli Lectin) or a2-6 linked sialic acid (SNA, Sambucus Nigra Lectin) (FIG. 7B) (C. Raymond etal., Production of a2,6-sialylated IgGl in CHO cells. mAbs 7, 571-583 (2015)). Treating the resulting sFc with neuraminidase cleaves sialic acid from the glycan, resulting in a terminally galactosylated, but non- sialylated Fc.

[0269] To characterize the in vivo anti-inflammatory activity of IVIG or recombinantly generated sFc in hFcyR mice, the K / BxN serum transfer induced arthritis (STIA) model was used. K / BxN mice spontaneously develop arthritis and swollen joints, and the transfer of serum derived from these mice to naive hosts results in the development of transient arthritis which can be evaluated through clinical scoring, histological evaluation of the joint, and Attorney Docket No: 070413.20840 measurement of ankle thickness due to inflammation (A. D. Christensen et al. K / BxN Serum-Transfer Arthritis as a Model for Human Inflammatory Arthritis. Front Immunol 7, 213 (2016)). Previous studies have demonstrated that high-dose IVIG (1-2.5 g / kg) suppresses K / BxN serum-mediated inflammation in a sialylation-dependent manner, and this activity can be phenocopied using recombinantly produced WT sFc at a 10-30 fold lower dose compared to IVIG (Y. Kaneko, F. Nimmerjahn, J. V. Ravetch, Antiinflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313, 670- 673 (2006); R. M. Anthony el al.. Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science 320, 373-376 (2008); R. M. Anthony, T. Kobayashi, F. Wermeling, J. V. Ravetch, Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway. Nature 475, 110-113 (2011)).

[0270] FcyR humanized mice were dosed with IVIG (2.5 g / kg), WT sFc (100 mg / kg), or neuraminidase treated WT sFc (100 mg / kg), prepared as described above, one hour before administration of K / BxN serum, and ankle thickness was measured four days later at the time of peak disease. Prophylactic IVIG treatment protected hFcyR mice from developing arthritis, and this effect was phenocopied with WT sFc at a 25-fold lower dose (100 mg / kg) (FIG. 7C), which translates to an approximately 9-fold lower effective molar dose for WT sFc (1.667 umol / kg) compared to IVIG (15.625 umol / kg). Notably, neuraminidase treated sFc was unable to protect mice from inflammation at an equivalent dose, demonstrating that sialylation of the WT Fc glycan is required for anti-inflammatory activity in hFcyR mice in this disease model.

[0271] It was next determined if type I FcyR engagement by sFc is required to drive antiinflammatory activity in hFcyR mice. A common method of knocking out type I FcyR binding to IgGl Fc is by cleaving the hIgGl-N297 glycan, either through mutagenesis or treatment with glycosidases, as this glycan is required for FcyR binding (M. Nose, H. Wigzell, Biological significance of carbohydrate chains on monoclonal antibodies. Proc Natl Acad Sci U S A 80, 6632-6636 (1983); G. P. Subedi, A. W. Barb, The Structural Role of Antibody N-Glycosylation in Receptor Interactions. Structure 23, 1573-1583 (2015)). However, this would not allow characterization of sialylated IgG in the absence of type I FcyR binding. Instead, a well-studied pair of mutations (G236R / L328R, GRLR) were used, which ablates type I FcyR engagement, leaves the N297 glycan intact, and has the same Attorney Docket No: 070413.20840 half-life and thermostability as WT IgGl (30-32). Sialylated hlgGl-GRLR Fc (GRLR sFc) was generated and its sialylation status was characterized by western blotting with SNA and ECL, and confirmed its lack of binding to type I FcyRs by surface plasmon resonance (SPR) (FIG. 7D) Next, hFcyR mice were dosed with 100 mg / kg of WT sFc or GRLR sFc one hour prior to administration of K / BxN serum and determined disease severity four days later. Unlike WT sFc treated mice, GRLR sFc treatment failed to protect mice from arthritis (FIG. 7E), indicating that type I FcyR engagement, along with sialylation, are both required to mediate the anti-inflammatory activity of IgG in FcyR humanized mice.

[0272] Sialylation of mouse IgG subclasses, notably mouse IgGl and mouse IgG2b, has been shown to decrease the affinities of activating mouse FcyRs while maintaining native affinities for the inhibitory receptor FcyRIIB, resulting in a shift towards engagement of inhibitory over activating FcyRs (Y. Kaneko, F. Nimmerjahn, J. V. Ravetch, Antiinflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313, 670- 673 (2006)). SPR was performed to measure binding affinities of human FcyRs to Protein G immobilized sFc or neuraminidase treated sFc. Unlike mouse IgG, sialylation of human IgGl Fc did not significantly impact its binding to type I human FcyRs and thus cannot account for the enhanced anti-inflammatory activity of sFc compared to neuraminidase treated sFc, consistent with a role for other receptors, such as the type II FcyRs, in the antiinflammatory activity of sialylated IgG.

[0273] Example 6. Targeting FcyRIIB through Fc-engineering enhances the antiinflammatory activity of sialylated IgG.

[0274] Canonical type I FcyRs are a group of activating and inhibitory IgG binding receptors expressed on a diverse array of immune cells. Engagement of ITAM-signaling activating FcyR pathways, such as FcyRIA (CD64), FcyRIIA (CD32a), and FcyRIIIA (CD 16a) conveys classical Fc-effector functions such as antibody dependent cellular phagocytosis (ADCP), antibody dependent cellular cytotoxicity (ADCC), and innate immune cell activation. Conversely, the ITIM-signaling FcyRIIB (CD32b) acts as a negative regulator of the immune response, safe-guarding against hyper-immune activation. FcyRIIB is required for IVIG to mediate anti-inflammatory activity in vivo, as mice in which FcyRIIB has been blocked or genetically knocked out are unresponsive to IVIG therapy in Attorney Docket No: 070413.20840 multiple models of autoimmunity. Most innate immune cells expressing activating FcyRs co-express FcyRIIB, and the balance of signaling between activating and inhibitory receptors determines a threshold for cell activation. Furthermore, a mechanism has been described in which infusion of IVIG or sFc induces the up-regulation of FcyRIIB on innate immune cells, raising the threshold for FcyR-mediated pro-inflammatory responses (R. M. Anthony, T. Kobayashi, F. Wermeling, I. V. Ravetch, Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway. Nature 475, 110-113 (2011)). This up-regulation of FcyRIIB in response to IVIG therapy has also been observed in patients with CIDP, a disease in which IVIG serves as a first-line therapy (B. Tackenberg et al., Impaired inhibitory Fcgamma receptor IIB expression on B cells in chronic inflammatory demyelinating polyneuropathy. Proc Natl Acad Sci U S A 106, 4788-4792 (2009)).

[0275] To determine if targeting inhibitory or activating type I FcyR pathways by sFc mediates anti-inflammatory activity, sialylated versions of two well defined IgGl Fc variants were generated, the VI 1 (G237D, P238D, H268D, P271G, A330R) (F. Mimoto et al., Engineered antibody Fc variant with selectively enhanced FcgammaRIIb binding over both FcgammaRIIa(R131) and FcgammaRIIa(H131). Protein Eng Des Sei 26, 589-598 (2013)) and GA (G236A) (S. Boumazos, D. Corti, H. W. Virgin, I. V. Ravetch, Fc- optimized antibodies elicit CD8 immunity to viral respiratory infection. Nature 588, 485- 490 (2020)) sFc proteins which have enhanced affinity for inhibitory or activating type I FcyRs, respectively. Compared to WT sFc, GA sFc has an approximately 10-fold enhanced affinity for the activating FcyRIIA while having comparable affinities for FcyRIIB and FcyRIIIA, while the VI 1 sFc has a 37-fold enhancement in affinity for the inhibitory FcyRIIB (FIG. 8A). Additionally, VI 1 sFc binds to the high affinity FcyRIA with 193-fold weaker affinity compared to WT sFc, and is completely unable to bind the Hl 31 variant of FcyRIIA as well as both variants (Fl 58 and VI 58) of FcyRIIIA (F. Mimoto et al., Engineered antibody Fc variant with selectively enhanced FcgammaRIIb binding over both FcgammaRIIa (R131) and FcgammaRIIa (H131). Protein Eng Des Sei 26, 589-598 (2013)).

[0276] As both GA and Vl 1 sFc have enhanced affinities for activating or inhibitory FcyRs, respectively, it was theorized they may mediate anti-inflammatory activity at a lower dose compared to WT sFc. Thus, hFcyR mice were dosed with 100 mg / kg or 10 mg / kg of WT sFc, 10 mg / kg of GA sFc, or 10 mg / kg of Vl l sFc before administration of K / BxN serum. Attorney Docket No: 070413.20840

[0277] Mice were then monitored and scored over the next eight days to characterize onset, peak, and resolution of inflammation. Treating mice with 10 mg / kg of VI 1 sFc mediated potent anti-inflammatory activity, comparable to a 10-fold higher dose of WT sFc (100 mg / kg), indicating that targeting the inhibitory FcyRIIB pathway drives anti-inflammatory activity (FIGS. 8B and 8C). Histological analysis of ankle joints in VI 1 sFc treated mice show a marked reduction in immune cell infiltration and increased joint space compared to untreated mice. Unlike VI 1 sFc, GA sFc was ineffective in controlling inflammation at an equivalent dose, indicating that targeting or blocking the activating FcyRIIA pathway does not significantly contribute to the anti-inflammatory activity of sialylated IgG. Furthermore, the lack of binding of VI 1 sFc to the activating FcyRIIIA indicates that targeting or blocking this receptor is unnecessary to drive anti-inflammatory activity either.

[0278] To determine if GA sFc reduces inflammation at higher doses, hFcyR mice were treated with 100 mg / kg of WT sFc or GA sFc before administering K / BxN serum. At this dose, GA sFc did significantly reduce ankle swelling, compared to untreated mice, but this protection was markedly weaker compared to WT sFc. The weakened protection of GA sFc, compared to WT sFc, indicates that the anti-inflammatory activity of sialylated IgG is not driven by the blockade of activating FcyRs, as GA and WT sFc differ only in their affinities for FcyRIIA, while having similar affinities for FcyRIIB and FcyRIIIA. These studies further indicate that the inhibitory FcyRIIB pathway is critical for sialylated IgG to ameliorate inflammation, and Fc-engineering IgG to target this pathway, as seen with the VI 1 sFc, results a potent mediator of anti-inflammatory activity.

[0279] Interestingly, the anti-inflammatory activity of Vl l sFc was found to be partially independent of sialylation. Neuraminidase treated Vl l sFc failed to protect mice from controlling the early onset of K / BxN serum induced inflammation, but significantly reduced inflammation by day six post K / BxN serum injection (FIGS. 17A and 17B). However, this protection was significantly inferior to Vl l sFc, indicating that sialylation of Vl l Fc enhances, but may not be strictly required to mediate, anti-inflammatory activity. The increased affinity for FcyRIIB may account for the sialylation independent activity of the VI 1 Fc, though it is notable that some mutations in the VI 1 Fc (G237D, P238D) are located in a similar region as the F241A Fc variant, which structurally phenocopies sFc and promotes anti-inflammatory activity in a sialylation independent manner (A. A. Ahmed et Attorney Docket No: 070413.20840 al., Structural characterization of anti-inflammatory immunoglobulin G Fc proteins. J Mol Biol 426, 3166-3179 (2014)).

[0280] It was next determined if VI 1 sFc retains activity at lower doses than 10 mg / kg. A titration study was performed in which hFcyR mice were dosed with 10 mg / kg, 5 mg / kg, or 1 mg / kg of VI 1 sFc one hour before K / BxN serum inj ection. As a comparison, an additional group of mice was dosed with a standard therapeutic dose (1000 mg / kg) of IVIG. Treating mice with 10 mg / kg of VI 1 sFc again provided potent anti-inflammatory activity, blunting both the magnitude and duration of inflammation, and phenocopying the effects of IVIG at a 100-fold lower dose (FIGS. 9A and 9B), translating to an approximately 38-fold lower effective molar dose for VI 1 sFc (0.166 umol / kg) compared to IVIG (6.25 umol / kg). However, this activity is lost when VI 1 sFc is dosed at 5 or 1 mg / kg, though treatment with 5 mg / kg of VI 1 sFc did result in a modest improvement to the resolution of inflammation. These studies demonstrate the effective dose of Vl l sFc to be at least 10 mg / kg in FcyR humanized mice, a 100-fold lower dose compared to IVIG. This highly translatable dose is comparable to recent FDA approved engineered IgGl-Fc based therapeutics, which function as a blockade of the neonatal Fc receptor (FcRn) mediated antibody recycling pathway. These Fc proteins, which bind FcRn at high affinities, out-compete endogenous antibodies in accessing the antibody recycling pathway, resulting in a transient depletion of both pathogenic and non-pathogenic antibodies in patients, an immunosuppressive mechanism that is distinct from sialylated IgG (S. L. Sneed et al., An engineered immunomodulatory IgGl Fc suppresses autoimmune inflammation through pathways shared with i.v. immunoglobulin. J Clin Invest 134, (2024)). Indeed, both WT and Vl l sFc, as well as full length WT and Vl l sialylated IgG (slgG), have similar affinities for human and mouse FcRn, suggesting Vl l sFc does not function as a competitor for the antibody recycling pathway.

[0281] Next, the half-life of WT sFc and Vl l sFc was characterized in FcyR / FcRn humanized mice, which recapitulates the expression and function of both human FcyRs and FcRn (S. Borghi et al., FcRn, but not FcyRs, drives maternal-fetal transplacental transport of human IgG antibodies. Proc Natl Acad Sci U S A 117, 12943-12951 (2020)). As Fc proteins are known to have shorter half-life than their full-length IgG counterparts, the pharmacokinetics of Vl l sFc protein were compared to that of a full-length Vl l slgG Attorney Docket No: 070413.20840 specific for the SARS-CoV-2 RBD domain (S309) (D. Pinto et al., Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody. Nature 583, 290-295 (2020)). As expected, full-length VI 1 slgG has a significantly longer half-life in FcyR / FcRn humanized mice, compared to both WT and VI 1 sFc. Interestingly, VI 1 sFc has a modest, but significantly increased half-life compared to WT sFc, perhaps due its preferential capture and internalization by FcyRIIB, which has been shown to engage non-degradative antigen recycling pathways. While the full-length VI 1 slgG displays an extended half-life compared to VI 1 sFc, it is unable to protect mice from K / BxN serum induced inflammation at an equivalent dose (10 mg / kg) as VI 1 sFc. This is likely due to the molar differences in dosing as the molecular weight of full-length VI 1 slgG (-160 kDa) is about 2.6 larger than that of Vl l sFc (60 kDa), indicating a higher dose (-26 mg / kg) of Vl l slgG would be needed to match molarities. Thus, Vl l sFc conveys potent anti-inflammatory activity in antigen-independent manner at an equivalent effective dose (10 mg / kg) as high-affinity FcRn binding Fc proteins (FIGS. 18A-18I).

[0282] As IVIG is a first line therapy for several neurological autoimmune diseases, such as CIDP, Guillain-Barre syndrome (GBS), and multifocal motor neuropathy (MMN), it was next determined if Vl l sFc could control inflammation in mouse models of autoimmune neuropathy (J. D. Lunemann, I. Quast, M. C. Dalakas, Efficacy of Intravenous Immunoglobulin in Neurological Diseases. Neurotherapeutics 13, 34-46 (2016)). IVIG has been demonstrated to protect mice from developing neurological inflammation in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, primarily through promoting the expansion of T-regulatory cells. To determine if Vl l sFc protects mice from neuro-inflammation, EAE was induced by immunizing hFcyR mice with a myelin oligodendrocyte glycoprotein (MOG) derived peptide MOG35-55 emulsified in complete Freund’s adjuvant (CFA) along with pertussis toxin (PTX). Mice then received four doses of 10 mg / kg of WT or Vl l sFc, starting on day five, spaced five days apart. Treatment with 10 mg / kg Vl l sFc significantly lowered clinical scores in mice, compared to untreated controls and WT sFc treated animals, while WT sFc treatment failed to control neuro-inflammation (FIGS. 9C and 9D). Additionally, immunofluorescence and histochemical analysis was performed on spinal cord sections of untreated and Vl l sFc treated mice to determine the degree of demyelination, measured by immunofluorescent Attorney Docket No: 070413.20840 based detection of myelin-basic protein (MBP), as well as inflammation and cell destruction, measured by H&E staining. It was found VI 1 sFc treatment prevented cell destruction in spinal cords, and retained high levels of MBP staining, which was significantly reduced in untreated mice (FIG. 9E). The ability of VI 1 sFc to mediate anti-inflammatory activity in multiple models of both passively transferred and endogenously induced autoimmune diseases highlights its potential as a potent anti-inflammatory therapeutic with broad applications.

[0283] Example 7. Type I and II FcyRs synergize to bind IgG and drive anti-inflammatory activity.

[0284] A distinct class of Fc receptors, type II FcyRs are multimeric C-type lectin related receptors found on diverse subsets of immune cells including B-cells and myeloid-lineage derived innate immune cells, all of which co-express type I FcyRs. The type II FcyR DC- SIGN (CD209), or its mouse homolog SIGN-R1 (CD209b), is required for IVIG and sFc to mediate anti-inflammatory activity, as mice lacking these receptors fail to control inflammation in multiple models of autoimmunity. Additionally, engagement of the type II FcyR pathway by sFc has been shown to result in the up-regulation of FcyRIIB, release of the cytokine IL-33, and the expansion of T -regulatory cells. More recently, sialylated IgG was shown to repress nuclear-factor kB (NF-kB) driven responses in influenza infection models through the induction of the transcription factor repressor element- 1 silencing transcription factor (REST), protecting mice from severe lung inflammation, in a mechanism also dependent on the type II FcyR SIGN-R1 (S. Chakraborty et al., Sialylated IgG induces the transcription factor REST in alveolar macrophages to protect against lung inflammation and severe influenza disease. Immunity). However, the role of type II FcyRs in mediating anti-inflammatory activity has been challenged by other groups, with some arguing that DC-SIGN does not interact with human IgG or sialylated glycans.

[0285] To characterize the requirement of type II FcyRs in the anti-inflammatory activity of Vl l sFc, mice were administered VI 1 sFc prior to K / BxN serum injection, and mice received daily injections of a SIGN-R1 blocking antibody (Y.-S. Kang et al., The C-type lectin SIGN-R1 mediates uptake of the capsular polysaccharide of Streptococcus pneumoniae in the marginal zone of mouse spleen. Proceedings of the National Academy Attorney Docket No: 070413.20840 of Sciences 101, 215-220 (2004)) or an isotype control (FIG. 10A). Blocking SIGN-R1 resulted in the loss of anti-inflammatory activity of Vl l sFc, while isotype control treated mice were protected from severe inflammation (FIGS. 10B and 10C), demonstrating that Vl l sFc resolves inflammation in a type II FcyR dependent mechanism similar to IVIG (S. L. Sneed et al., An engineered immunomodulatory IgGl Fc suppresses autoimmune inflammation through pathways shared with i.v. immunoglobulin. J Clin Invest 134, (2024)). Recently, the C-type lectin Dectin- 1 was shown to interact with FcyRIIB to stabilize its cellsurface expression and enhance ability to bind IVIG (M. Seeling et al., Immunoglobulin G- dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin-1. Immunity 56, 1046-1063. el047 (2023)), indicating C-type lectins such as type II FcyRs may directly interact with type I FcyRs on the cell surface. Interestingly, it was found that co-expressing DC-SIGN and FcyRIIB in HEK 293-T cells enhance the expression level of FcyRIIB on the cell surface (FIG. 10D), indicating DC-SIGN may directly interact with FcyRIIB. To determine if DC-SIGN physically interacts with FcyRIIB, coimmunoprecipitation experiments were performed in which FLAG-tagged FcyRIIB and HA-tagged DC-SIGN were expressed alone or in combination in HEK 293 cells. One day post transfection, cells were lysed and FcyRIIB was immunoprecipitated (IP) with anti- FLAG tag beads. Cell lysates and IP elutions were blotted to detect FcyRIIB (anti-FLAG tag) or DC-SIGN (anti-HA). DC-SIGN co-immunoprecipitated with FcyRIIB in cells expressing both receptors, demonstrating a direct physical interaction between DC-SIGN and FcyRIIB, although non-specific binding of DC-SIGN itself to anti-FLAG beads was observed as well (FIG. 10E). Furthermore, immobilized recombinant DC-SIGN directly bound recombinant FcyRIIB when measured by SPR (in the presence of CaCh), further indicating direct interactions occurring between DC-SIGN and FcyRIIB (FIG. 10F).

[0286] C-type lectins, such as DC-SIGN, are known to bind glycans in a calcium dependent manner, which can be blocked by the chelator EDTA. Type I FcyRs, like FcyRIIB, have multiple N-linked glycans on their ectodomains, consisting primarily of complex type glycans, which may potentially interact with DC-SIGN. Indeed, while DC-SIGN binds FcyRIIB in the presence of CaCh, this binding is completely abrogated in the presence of EDTA, indicating that the interaction between DC-SIGN and FcyRIIB is glycan dependent. The ectodomain of FcyRIIB contains three putative N-linked glycosylation sites (N106, Attorney Docket No: 070413.20840

[0287] N180, and N187) (F. Cambay et al., Glycosylation of Fey receptors influences their interaction with various IgGl glycoforms. Molecular Immunology 121, 144-158 (2020)) (FIG. 10G) A full-length variant of FcyRIIB lacking these core glycosylation sites (FcyRIIBD) (N106Q, N180Q, N187Q) was generated and co-transfection experiments were performed with DC-SIGN. Knocking out glycans on FcyRIIB resulted in the loss of enhanced cell surface expression when co-expressed with DC-SIGN or another major type II FcyR, CD23 (FceRII). To determine if DC-SIGN binds to a specific glycan on FcyRIIB, single glycan knockouts of FcyRIIB (N106Q, N180Q, andN187Q) were generated and their binding to DC-SIGN was determined. Both N106Q, and N187Q mutants of FcyRIIB had a slightly smaller molecular weight compared to wild-type FcyRIIB, however, the N180Q mutant appeared as the same size, indicating that the N180 site is likely non-glycosylated (FIG. 10H) (Z. Abdoollah, D. E. Marrero Roche, C. H. Pavan, E. Moore, K. B. Chandler, Site-Specific Glycosylation Analysis of Human and Murine Fey Receptor II Family Members Reveals Variant-Specific N-Glycosylation. Journal of Proteome Research 23, 3469-3483 (2024)). Additionally, both wild-type and the single glycan knockout mutants all react against ECL and SNA lectins, indicating they all retain complex-type N-linked glycans with a mixture of terminal galactosylated and sialylated glycoforms. Interesting, while the N180Q and N187Q mutants retained binding to DC-SIGN, binding to the N106Q mutant was almost completely abrogated (FIG. 101), which suggests the N106 glycan is critical in the interaction between DC-SIGN and FcyRIIB.

[0288] As the cell surface expression of FcyRIIB is enhanced upon co-expression with DC- SIGN, it was next determined if this enhanced expression leads to enhanced binding of IgG. Fluorescently labeled IVIG was incubated with FcyRIIB and / or DC-SIGN expressing cells and binding was measured by flow cytometry. As expected, FcyRIIB+DC-SIGN+cells have an enhanced ability to bind fluorescently labeled monomeric IVIG compared to FcyRIIB only expressing cells (FIG. 10J). Notably, DC-SIGN only expressing cells did not bind IVIG at this concentration, indicating that DC-SIGN functions to augment FcyRIIB binding to IgG when both are expressed together. Next, the anti-CD20 monoclonal antibody Rituximab was fluorescently labeled, and glycoengineered to be homogenously galactosylated (G2F) or sialylated (S2G2F) (T. Li et al., Modulating IgG effector function by Fc glycan engineering. Proceedings of the National Academy of Sciences 114, 3485- Attorney Docket No: 070413.20840

[0289] 3490 (2017)). Labeled IgGs were incubated with cells expressing FcyRIIB alone or in combination with CD23 or DC-SIGN and found that only the sialylated form of Rituximab exhibited enhanced binding to type I and II FcyR co-expressing cells, demonstrating that co-expression of type I and II FcyRs may drive the binding of sialylated IgG (FIGS. 10K- 10L)

[0290] Thus, these studies describe a novel function of DC-SIGN, and by extension other type II FcyRs, in which type II FcyRs directly interact with type I FcyRs to augment their binding to IgG, and this interaction is critical for mediating the anti-inflammatory activity of IVIG or sialylated IgG. The direct interaction between tetrameric DC-SIGN and FcyRIIB stabilizes the cell-surface expression of FcyRIIB, enhancing its expression level and thus binding and signaling capacity for sFc. The selectively enhanced binding of sialylated IgG to cells co-expressing DC-SIGN and FcyRIIB suggests DC-SIGN may contribute to the binding of both FcyRIIB as well as the sialylated glycan of IgG which further facilitates the binding and signaling of IgG through FcyRIIB. This finding may explain studies challenging the role of DC-SIGN interacting with sialylated IgG or IVIG, as these studies only characterized the binding of IgG by DC-SIGN alone or type I FcyR expressing cells, not in combination (A. R. Temming et al., Human DC-SIGN and CD23 do not interact with human IgG. Scientific Reports 9, 9995 (2019)), as type II FcyRs are expressed on cells which also express type I FcyRs, and these experiments suggest type I and II FcyRs form clusters on the cell surface and signal IgG (S. Bournazos, T. T. Wang, R. Dahan, J. Maamary, J. V. Ravetch, Signaling by Antibodies: Recent Progress. Annu Rev Immunol 35, 285-311 (2017); M. Seeling et al., Immunoglobulin G-dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin-1. Immunity 56, 1046-1063. el047 (2023)).

[0291] This data further indicates that FcyRIIB contributes to the anti-inflammatory activity of sialylated IgG through its ITIM domain. FcyRIIA, an ITAM containing Type I FcyR, is 92% homologous to FcyRIIB in its extracellular domain and is capable of associating with DC-SIGN, similar to FcyRIIB. However, increasing FcyRIIA binding by the GA Fc variant did not result in enhanced anti-inflammatory activity when this variant was sialylated (FIGS. 8B and 8C), while enhancing FcyRIIB binding alone, even in the absence of sialylation, resulted in a partial anti-inflammatory phenotype. Attorney Docket No: 070413.20840

[0292] These findings demonstrate that sialylated IgG engineered to have enhanced affinity to the inhibitory FcyRIIB has potent in vivo anti-inflammatory activity, effective at a dose at least 100-fold lower than conventional IVIG therapy in multiple mouse models of autoimmunity. Additionally, it was shown that this activity is dependent on the type II FcyR DC-SIGN and its mouse ortholog SIGN-R1 and describe a new function of type II FcyRs, which is to directly interact with type I FcyRs, augmenting their ability to bind and signal sialylated IgG. This data indicates a model in which VI 1 sFc engages in similar pathways as IVIG and sFc mediating anti-inflammatory activity, and through enhancing the affinity sFc for FcyRIIB, the effective therapeutic dose of sFc can be reduced at least 100-fold as compared to IVIG. Recombinantly produced VI 1 sFc thus represents an attractive replacement for IVIG therapy for a wide array of autoimmune disorders currently being treated with IVIG, clinically effective at substantially lower doses compared to IVIG. Its anti-inflammatory properties do not result in the depletion of endogenous IgG, as seen with FcRn blockade or B cell depletion therapies, thereby retaining the protective, anti-microbial properties of serum IgG, while mitigating the hyper-inflammatory sequalae of autoimmune diseases.

[0293] Example 8. Characterization of F241A-V11 Fc protein.

[0294] This example characterizes the F241A-V11 sFc variant (G237D, F241A, P238D, H268D, P271G, A33 OR), which has not been previously described. The binding of F241 A- VI 1 sFc to type I FcyRs was examined and it was found that F241A-V11 has almost exclusive binding to FcyRIIB (FIGS. 11A and 11B), which was unexpected as F241A sFc alone does not show this selective binding. However, the affinity of F241A-V11 sFc for FcyRIIB (1.8 xlO'6M) is similar to WT sFc (1.7 xlO'6M) and thus lacks the enhanced FcyRIIB affinity observed in the VI 1 sFc variant.

[0295] F241A-V11 Fc was next tested in vivo to determine if it retains anti-inflammatory activity. As it was previously demonstrated that non- sialylated F241 A Fc recapitulates the effects of sialylated IgG Fc in vivo, it was also determined the sialylation dependence of F241A-V11 Fc. Similar to the VI 1 sFc, both sialylated and non- sialylated F241A-V11 Fc significantly suppressed inflammation in the K / BxN serum transfer arthritis model at a 10 mg / kg dose, demonstrating F241A-V11 retains potent anti-inflammatory activity (FIGS. Attorney Docket No: 070413.20840

[0296] 12A and 12B) This data suggests that sFc proteins with selective binding, though not necessarily enhanced affinity, to FcyRIIB drives anti-inflammatory activity of IgG.

[0297] Example 9: Characterization of SE sFc protein

[0298] Next, the anti-inflammatory activity was determined of the other family of Fc mutants with enhanced affinities for FcyRIIB, the S267E (SE) Fc mutant. Sialylated SE sFc was generated. Its high affinity for FcyRIIB by SPR was confirmed (FIGS. 13A and 13B). Both VI 1 sFc and SE sFc, when dosed at 10 mg / kg, suppressed the development of arthritis in mice injected with K / BxN serum, indicating that the enhanced affinities to FcyRIIB of VI 1 and SE sFc drives anti-inflammatory activity at low doses (FIGS. 14A and 14B). Moreover, VI 1 sFc, F241 A-Vl 1 sFc, and SE sFc (dosed at 25 mg / kg) all promote CD4 T- cell recovery in in vivo CD4 depletion models (FIGS. 15A and 15B).

[0299] Table 2 summarizes the tested Fc variants with enhanced and / or selective binding to FcyRIIB, and its effectiveness in vivo to mediate anti-inflammatory activity.

[0300] Table 2. Fc variants with enhanced or selective binding to FcyRIIB, tested for in vivo antiinflammatory activity. NB, no binding. TBD, to be determined.

[0301] Promotes

[0302] FcyR Binding Affinity CD4 T-

[0303] Suppresses cell

[0304] K / BxN rebound inflammation at 25 Sialylation

[0305] Variant FcyRIIA FcyRIIB FcyRIIIA at 10 mg / kg? mg / kg? Dependent

[0306] WT ++ + ++ No No Yes

[0307] GA ++++ + / - + No No Yes

[0308] VI 1 ++ ++++ NB Yes Yes Yes / No

[0309] F?41 A- NB + NB Yes Yes No

[0310] SE ++++ ++++ + Yes Yes TBD

[0311] These data indicate that sFc proteins with enhanced affinities for FcyRIIB (at least 10-fold enhanced binding compared to WT sFc), or sFc proteins which selectively bind FcyRIIB over activating FcyRs (FcyRIIA, FcyRIIIA), have enhanced anti-inflammatory activity in vivo. Furthermore, for some Fc variants, this activity may be sialylation independent. A summary of the tested Fc variants is shown in FIGS. 16A-16C. Attorney Docket No: 070413.20840

[0312] Example 10: Determination of the anti-inflammatory activity of sFc proteins

[0313] Modified sialylated Fc proteins as encoded by SEQ ID NOs: 1-11, 13, 15-26, and 28-30 are generated and tested for their affinities to type I FcyRs by SPR to determine enhanced or selective FcyRIIB binding. Next, the anti-inflammatory activity of the modified sFc proteins are assessed in vivo as described in the above example. In the K / BxN model, hFcyR mice are dosed at 10 mg / kg before injection with K / BxN serum, and inflammation in ankles is assessed four days post serum injection. In CD4 depletion studies, hFcyR mice are dosed at 25 mg / kg of modified sFc before injection with an afucosylated anti-CD4 hlgGl antibody. Two days post injection, CD4 T-cells in blood is determined by flow and calculated as a percent of baseline to determine the rebound effect of the modified sFc protein. The sialylation dependence of the modified sFc proteins is determined by treatment with neuraminidase before testing in the K / BxN and CD4 depletion models.

[0314] The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entireties.

Claims

Attorney Docket No: 070413.20840CLAIMSWhat is claimed is:

1. An isolated polypeptide comprising a modified IgG Fc region, wherein the polypeptide is capable of co-engaging a type I FcyR and a type II FcyR.

2. The isolated polypeptide of claim 1, wherein the type I FcyR is FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, FcyRIIIb, or FcyRIIb.

3. The isolated polypeptide of claim 1 or 2, wherein the type I FcyR is FcyRIIB.

4. The isolated polypeptide of any one of claims 1-3, wherein the type II FcyR is DC- SIGN or CD23.

5. The isolated polypeptide of any of claims 1-4, wherein the polypeptide has an enhanced affinity to FcyRIIB.

6. The isolated polypeptide of any of claims 1-4, wherein the polypeptide exhibits selective binding to FcyRIIB.

7. The isolated polypeptide of any one of claims 1-6, wherein the modified IgG Fc region comprises one or more of a) a P238D mutation; b) an E233D mutation; c) a G237D mutation; d) a H268D mutation; e) a P271 G mutation; f) an A33 OR mutation; g) an S267E mutation; and h) an L328F mutation.

8. The isolated polypeptide of any one of claims 1-7, wherein the modified IgG Fc region comprises G237D / P238D / H268D / P271G / A330R mutations.Attorney Docket No: 070413.208409. The isolated polypeptide of any one of claims 1-8, wherein the modified IgG Fc region comprises an F241 A mutation.

10. The isolated polypeptide of any one of claims 1-9, wherein the modified IgG Fc region is at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence of SEQ ID NOs: 1-30 or comprises an amino acid sequence of SEQ ID NOs: 1-30.

11. The isolated peptide of any of claims 1-10, wherein the IgG Fc glycan at position N297 is unmodified.

12. The isolated peptide of any of claims 1-10, wherein the IgG Fc glycan at position N297 is terminally galactosylated.

13. The isolated peptide of any of claims 1-10, wherein the IgG Fc glycan at position N297 is terminally sialylated, and wherein the sialic acid is attached in a 2,6 linkage to the penultimate galactose.

14. An antibody or modified IgG Fc region thereof comprising the polypeptide of any one of claims 1-13.

15. An isolated nucleic acid molecule comprising a sequence encoding the polypeptide of any one of claims 1-14.

16. An expression vector comprising the nucleic acid molecule of claim 15.

17. A host cell comprising the nucleic acid molecule of claim 16.

18. A method of producing the polypeptide of any one of claims 1-14, comprising culturing the host cell of claim 17 in a medium under conditions permitting expression of a polypeptide encoded by the nucleic acid molecule, and isolating the polypeptide from the cultured cell or the medium of the cell.Attorney Docket No: 070413.2084019. A pharmaceutical composition comprising (i) the polypeptide of any one of claims 1- 14, or the nucleic acid molecule of claim 15, and (ii) optionally a pharmaceutically acceptable carrier.

20. A method of treating an inflammatory disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 19.

21. The method of claim 20, comprising administering the polypeptide at a dose of about 1 mg / kg to about 1000 mg / kg.

22. The method of claim 20 or 21, wherein the inflammatory disease or disorder comprises an infection that is a viral infection or a bacterial infection.

23. The method of any one of claims 20-22, wherein the subject is a human.

24. The method of any one of claims 20-23, wherein the treatment results in at least 100- fold anti-inflammatory protection compared to intravenous immunoglobulin (IVIG).

25. A pharmaceutical composition according to claim 19 for use in treating an inflammatory disease or disorder.

26. The pharmaceutical composition of claim 25, wherein the inflammatory disease or disorder comprises an infection that is a viral infection or a bacterial infection.

27. Use of a polypeptide of a pharmaceutical composition according to claim 19 in the manufacture of a medicament for treating an inflammatory disease or disorder.

28. The use of claim 27, wherein the inflammatory disease or disorder comprises an infection that is a viral infection or a bacterial infection.

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