Methods and compositions for treating multiple sclerosis

Abstract

The present invention provides a method for treating and improving the symptoms of multiple sclerosis using an antibody or its antigen-binding fragment (e.g., obexerimab) that specifically binds to human CD19. The present invention provides a safer and more effective treatment of MS based on anti-CD19 antibodies such as obexerimab. In particular, the anti-CD19 antibody-based therapies described herein (e.g., obexerimab-based therapies) can significantly reduce side effects and substantially benefit subjects with MS who need to discontinue treatment and restore protective immune function due to a significant risk of immunosuppression (highlighted by the COVID pandemic).

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the interests of U.S. Provisional Patent Application No. 63 / 587,401, filed on 2 October 2023, and U.S. Provisional Patent Application No. 63 / 595,992, filed on 3 November 2023, both of which are incorporated herein by reference.

[0002] (Sequence Listing) This application includes a sequence listing submitted electronically in XML file format, the entirety of which is incorporated herein by reference. The XML copy, created on 19 September 2024, is named ZEN-017WO1_SL and is 13,137 bytes in size. [Background technology]

[0003] Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS). While the exact cause and mechanism of the disease are unknown, MS is characterized by inflammation, demyelination, and neurodegeneration, along with the formation of plaques or lesions within the CNS. Symptoms of MS are often variable and difficult to predict, but individuals with MS may experience pain, fatigue, cognitive impairment, depression, limited mobility, lack of coordination, difficulty speaking or swallowing, and various visual impairments, as well as many other symptoms. Disease-modifying therapies are approved for the treatment of MS, but currently, there is no known cure for MS. Current anti-CD20-based therapies for MS indiscriminately deplete almost all B cells (Graf et al., 2021; Lee, DSW, et al., 2021. Nat. Rev. Drug Discov. 20: 179-199; and Margoni, M., et al., 2022. J. Neurol. 269: 1316-1334). Individuals undergoing long-term treatment for MS may lose a significant portion of the major branching of the immune system necessary for an effective and protective immune response for decades. While long-term health outcomes of maintaining B cell depletion over a significant portion of a person's lifespan are unknown, individuals may become unable to initiate a protective response to vaccines and may experience increased susceptibility to infection. Furthermore, if an individual chooses to discontinue anti-CD20 therapy, it may take months to return to normal immune function, while B cells regrow from the bone marrow (Margoni et al., 2022). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Lee, DSW, et al., 2021. Nat. Rev. Drug Discov. 20: 179-199 [Non-Patent Document 2] Margoni, M., et al., 2022. J. Neurol. 269: 1316-1334 [Overview of the project]

[0005] The present invention provides safer and more effective treatments for MS based on anti-CD19 antibodies such as obexerimab. In particular, the anti-CD19 antibody-based therapies described herein (e.g., obexerimab-based therapies) can significantly reduce side effects and substantially benefit subjects with MS who need to discontinue treatment and restore protective immune function due to the significant risk of immunosuppression (highlighted by the COVID pandemic). It is also intended that MS may be treated using other anti-CD19 antibodies that have the ability to reduce or inhibit B cells without depleting them.

[0006] In one embodiment, the present invention provides a method for treating multiple sclerosis in a subject requiring treatment for multiple sclerosis, comprising administering an anti-CD19 antibody comprising a light chain and a heavy chain to the subject, wherein the antibody comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 2, LCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 4, the heavy chain comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 7, and the heavy chain comprises an Fc region comprising amino acid substitutions 267E and L328F.

[0007] In some embodiments, the present invention provides a method for treating multiple sclerosis in a subject requiring treatment for multiple sclerosis, comprising administering an anti-CD19 antibody comprising a light chain and a heavy chain to the subject, wherein the light chain comprises a light chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 11, the heavy chain comprises a heavy chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, and the heavy chain comprises an Fc region which includes amino acid substitutions 267E and L328F. In some embodiments, the anti-CD19 antibody comprises a light chain which contains a light chain variable region which is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody comprises a heavy chain which contains a heavy chain variable region which is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 12 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 11.

[0008] In some embodiments, the anti-CD19 antibody includes a light chain containing a light chain variable region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody includes a heavy chain containing a heavy chain variable region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain containing a light chain variable region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11, and the heavy chain containing a heavy chain variable region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a heavy chain and a light chain, the heavy chain containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 10, and the light chain containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 10 and a light chain containing the amino acid sequence of SEQ ID NO: 9.

[0009] In some embodiments, the anti-CD19 antibody is obexerimab.

[0010] In some embodiments, administration of an anti-CD19 antibody improves, alleviates, or delays the onset of one or more symptoms of MS.

[0011] In some embodiments, the anti-CD19 antibody is administered intrathecally.

[0012] In some embodiments, the anti-CD19 antibody is administered intravenously.

[0013] In some embodiments, the anti-CD19 antibody is administered subcutaneously.

[0014] In some embodiments, obexerimab is administered as a liquid formulation containing 125 mg / mL of obexerimab, 2.35 mg / mL of sodium acetate trihydrate, 0.17 mg / mL of acetic acid, 30 mg / mL of L-proline, and 0.1 mg / mL of polysorbate 80 at pH 5.5.

[0015] In some embodiments, the anti-CD19 antibody is administered using a pre-filled syringe or an autoinjector. [Brief explanation of the drawing]

[0016] The drawings are for illustrative purposes only, not limiting purposes.

[0017] [Figure 1A] Figures 1A-1B: Showing prevention of disease induction in experimental autoimmune encephalomyelitis ("EAE") models. Obexerimab surrogate antibodies were evaluated against rituximab surrogate antibodies and vehicle controls in preclinical EAE models. Figure 1A shows disease prevention by obexerimab surrogates, measured by standard clinical EAE scores of surviving mice at each time point (number of surviving mice per treatment group at each time point, shown at the bottom of the x-axis). Figure 1B shows that obexerimab surrogates were not associated with a significant decrease in blood B cells compared to the decrease measured in mice treated with rituximab surrogates (p<0.0001). [Figure 1B] Figures 1A-1B: Showing prevention of disease induction in experimental autoimmune encephalomyelitis ("EAE") models. Obexerimab surrogate antibodies were evaluated against rituximab surrogate antibodies and vehicle controls in preclinical EAE models. Figure 1A shows disease prevention by obexerimab surrogates, measured by standard clinical EAE scores of surviving mice at each time point (number of surviving mice per treatment group at each time point, shown at the bottom of the x-axis). Figure 1B shows that obexerimab surrogates were not associated with a significant decrease in blood B cells compared to the decrease measured in mice treated with rituximab surrogates (p<0.0001). [Figure 2A]Figures 2A and 2B show the inhibition of ovalbumin (OVA) antigen (Ag) uptake and Ag-presenting cell (APC) function in mouse B cells. Figure 2A shows the inhibition of Ag-coated bead uptake by follicular (Fo) B cells by obexerimab mouse surrogate (mObx; 2480E) compared to anti-CD19 idiotype (2480F) as measured by flow cytometry. Internalized and external beads bound to the cell membrane were identified by secondary staining. The percentage of the total Fo B cell subset with internalized beads is shown. Each symbol represents a cell isolated from an individual mouse (**p<0.01, ****p<0.0001). Figure 2B shows that T cell activation by Ag-carrying B cells is inhibited ex vivo by mObx. OVA-sp T cells (purified OT-II TCR transgenic T cells) were labeled with Cell Tracker Violet (CTViolet) and co-cultured with Ag-free B cells (in culture medium) or with B cells loaded with anti-IgM biotinylated OVA-coated beads (under all other conditions), and further co-cultured with either mObx or a control. Proliferative T cells with diluted CTViolet inside the cells were identified using flow cytometry. [Figure 2B]Figures 2A to 2B show the uptake of ovalbumin (OVA) antigen (Ag) and the inhibition of antigen-presenting cell (APC) function in mouse B cells. Figure 2A shows the inhibition of follicular (Fo) B cell uptake of Ag-coated beads by the obexelimab mouse surrogate (mObx; 2480E), compared to the anti-CD19 idiotype (2480F) measured by flow cytometry. Internalized beads and external beads bound to the cell membrane were identified by secondary staining. The percentage of the total Fo B cell subset with internalized beads is shown. Each symbol represents cells isolated from an individual mouse (**p<0.01, ****p<0.0001). Figure 2B shows that T cell activation by Ag-bearing B cells is inhibited by mObx ex vivo. OVA-sp T cells (purified OT-II TCR transgenic T cells) were labeled with Cell Tracker Violet (CTViolet) and co-cultured with B cells containing no Ag (medium), or co-cultured with B cells loaded with anti-IgM biotinylated OVA-coated beads (all other conditions), and further co-cultured with either mObx or a control. Flow cytometry was used to identify proliferating T cells with diluted CTViolet inside the cells. [Figure 3A]Figures 3A–3F show the reduction in BCR-mediated antigen uptake by mouse Fo B2 (Fo B), marginal zone (MZ), and B1 B cell subsets. Mouse B cells were isolated from the spleen and peritoneal cavity of 2B-KIX mice and incubated with 2 μM goat anti-mouse IgM F(ab')2 and biotinylated OVA conjugate beads at 37°C and 5% CO2 for 2 hours. Cells were pretreated with mObx (30 μg / ml) (2480E), its idiotype control (30 μg / ml) (rat anti-mouse CD19 without Fc effect; 2480F), or cytochalasin D (30 μg / ml) at 37°C and 5% CO2 for 1 hour. Figure 3A shows fixable live cell dyes (FVDs) used to distinguish live and dead cells before identification of B cell subsets. Figure 3B shows that Fo B cells were identified as B220+, CD4-, CD11b-, IgMlo, IgDhi, CD21int, and CD23+ using FACS. Figure 3C shows MZ B cells identified as B220+, CD4-, CD11b-, IgMhi, IgDlo, CD21hi, and CD23int using FACS. Figure 3D shows B1 cells identified as B220+, CD4-, F4 / 80-, IgMhi, IgDlo, CD21-, and CD23-. Figure 3E shows streptavidin staining used to distinguish internalized beads from extracellular beads. Figure 3F shows the mean percentage of Fo B cells, MZ cells, and B1 cells with internalized goat anti-mouse IgM F(ab')2 coated beads. Each symbol represents data from cells isolated from individual mice (n=4) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns are not statistically significant). [Figure 3B]Figures 3A–3F show the reduction in BCR-mediated antigen uptake by mouse Fo B2 (Fo B), marginal zone (MZ), and B1 B cell subsets. Mouse B cells were isolated from the spleen and peritoneal cavity of 2B-KIX mice and incubated with 2 μM goat anti-mouse IgM F(ab')2 and biotinylated OVA conjugate beads at 37°C and 5% CO2 for 2 hours. Cells were pretreated with mObx (30 μg / ml) (2480E), its idiotype control (30 μg / ml) (rat anti-mouse CD19 without Fc effect; 2480F), or cytochalasin D (30 μg / ml) at 37°C and 5% CO2 for 1 hour. Figure 3A shows fixable live cell dyes (FVDs) used to distinguish live and dead cells before identification of B cell subsets. Figure 3B shows that Fo B cells were identified as B220+, CD4-, CD11b-, IgMlo, IgDhi, CD21int, and CD23+ using FACS. Figure 3C shows MZ B cells identified as B220+, CD4-, CD11b-, IgMhi, IgDlo, CD21hi, and CD23int using FACS. Figure 3D shows B1 cells identified as B220+, CD4-, F4 / 80-, IgMhi, IgDlo, CD21-, and CD23-. Figure 3E shows streptavidin staining used to distinguish internalized beads from extracellular beads. Figure 3F shows the mean percentage of Fo B cells, MZ cells, and B1 cells with internalized goat anti-mouse IgM F(ab')2 coated beads. Each symbol represents data from cells isolated from individual mice (n=4) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns are not statistically significant). [Figure 3C]Figures 3A–3F show the reduction in BCR-mediated antigen uptake by mouse Fo B2 (Fo B), marginal zone (MZ), and B1 B cell subsets. Mouse B cells were isolated from the spleen and peritoneal cavity of 2B-KIX mice and incubated with 2 μM goat anti-mouse IgM F(ab')2 and biotinylated OVA conjugate beads at 37°C and 5% CO2 for 2 hours. Cells were pretreated with mObx (30 μg / ml) (2480E), its idiotype control (30 μg / ml) (rat anti-mouse CD19 without Fc effect; 2480F), or cytochalasin D (30 μg / ml) at 37°C and 5% CO2 for 1 hour. Figure 3A shows fixable live cell dyes (FVDs) used to distinguish live and dead cells before identification of B cell subsets. Figure 3B shows that Fo B cells were identified as B220+, CD4-, CD11b-, IgMlo, IgDhi, CD21int, and CD23+ using FACS. Figure 3C shows MZ B cells identified as B220+, CD4-, CD11b-, IgMhi, IgDlo, CD21hi, and CD23int using FACS. Figure 3D shows B1 cells identified as B220+, CD4-, F4 / 80-, IgMhi, IgDlo, CD21-, and CD23-. Figure 3E shows streptavidin staining used to distinguish internalized beads from extracellular beads. Figure 3F shows the mean percentage of Fo B cells, MZ cells, and B1 cells with internalized goat anti-mouse IgM F(ab')2 coated beads. Each symbol represents data from cells isolated from individual mice (n=4) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns are not statistically significant). [Figure 3D]Figures 3A to 3F show the reduction of BCR-mediated antigen uptake by Fo B2 (Fo B), marginal zone (MZ), and B1 B cell subsets in mice. Mouse B cells were isolated from the spleen and peritoneal cavity of 2B-KIX mice, and incubated with 2 μM goat anti-mouse IgM F(ab')2 and biotinylated OVA-conjugated beads at 37°C and 5% CO2 for 2 hours. The cells were pretreated with mObx (30 μg / ml) (2480E), its idiotype control (30 μg / ml) (rat anti-mouse CD19 without Fc effect; 2480F), or cytochalasin D (30 μg / ml) at 37°C and 5% CO2 for 1 hour. Figure 3A shows the fixable viability dye (FVD) used to distinguish live cells from dead cells prior to identification of B cell subsets. Figure 3B shows that Fo B cells were identified as B220+, CD4-, CD11b-, IgMlo, IgDhi, CD21int, CD23+ using FACS. Figure 3C shows MZ B cells identified as B220+, CD4-, CD11b-, IgMhi, IgDlo, CD21hi, CD23int using FACS. Figure 3D shows B1 cells identified as B220+, CD4-, F4 / 80-, IgMhi, IgDlo, CD21-, CD23-. Figure 3E shows streptavidin staining used to distinguish internalized beads from extracellular beads. Figure 3F shows the average percentage of Fo B cells, MZ cells, and B1 cells that have taken up goat anti-mouse IgM F(ab')2-coated beads into the cells. Each symbol represents data from cells isolated from an individual mouse (n=4) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns is not significant). [Figure 3E]Figures 3A–3F show the reduction in BCR-mediated antigen uptake by mouse Fo B2 (Fo B), marginal zone (MZ), and B1 B cell subsets. Mouse B cells were isolated from the spleen and peritoneal cavity of 2B-KIX mice and incubated with 2 μM goat anti-mouse IgM F(ab')2 and biotinylated OVA conjugate beads at 37°C and 5% CO2 for 2 hours. Cells were pretreated with mObx (30 μg / ml) (2480E), its idiotype control (30 μg / ml) (rat anti-mouse CD19 without Fc effect; 2480F), or cytochalasin D (30 μg / ml) at 37°C and 5% CO2 for 1 hour. Figure 3A shows fixable live cell dyes (FVDs) used to distinguish live and dead cells before identification of B cell subsets. Figure 3B shows that Fo B cells were identified as B220+, CD4-, CD11b-, IgMlo, IgDhi, CD21int, and CD23+ using FACS. Figure 3C shows MZ B cells identified as B220+, CD4-, CD11b-, IgMhi, IgDlo, CD21hi, and CD23int using FACS. Figure 3D shows B1 cells identified as B220+, CD4-, F4 / 80-, IgMhi, IgDlo, CD21-, and CD23-. Figure 3E shows streptavidin staining used to distinguish internalized beads from extracellular beads. Figure 3F shows the mean percentage of Fo B cells, MZ cells, and B1 cells with internalized goat anti-mouse IgM F(ab')2 coated beads. Each symbol represents data from cells isolated from individual mice (n=4) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns are not statistically significant). [Figure 3F]Figures 3A–3F show the reduction in BCR-mediated antigen uptake by mouse Fo B2 (Fo B), marginal zone (MZ), and B1 B cell subsets. Mouse B cells were isolated from the spleen and peritoneal cavity of 2B-KIX mice and incubated with 2 μM goat anti-mouse IgM F(ab')2 and biotinylated OVA conjugate beads at 37°C and 5% CO2 for 2 hours. Cells were pretreated with mObx (30 μg / ml) (2480E), its idiotype control (30 μg / ml) (rat anti-mouse CD19 without Fc effect; 2480F), or cytochalasin D (30 μg / ml) at 37°C and 5% CO2 for 1 hour. Figure 3A shows fixable live cell dyes (FVDs) used to distinguish live and dead cells before identification of B cell subsets. Figure 3B shows that Fo B cells were identified as B220+, CD4-, CD11b-, IgMlo, IgDhi, CD21int, and CD23+ using FACS. Figure 3C shows MZ B cells identified as B220+, CD4-, CD11b-, IgMhi, IgDlo, CD21hi, and CD23int using FACS. Figure 3D shows B1 cells identified as B220+, CD4-, F4 / 80-, IgMhi, IgDlo, CD21-, and CD23-. Figure 3E shows streptavidin staining used to distinguish internalized beads from extracellular beads. Figure 3F shows the mean percentage of Fo B cells, MZ cells, and B1 cells with internalized goat anti-mouse IgM F(ab')2 coated beads. Each symbol represents data from cells isolated from individual mice (n=4) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns are not statistically significant). [Figure 4A]Figures 4A–4F show the reduced BCR-mediated antigen uptake by human Fo B cell subsets, MZ cell subsets, and B1 B cell subsets. Primary human B cells were isolated from healthy donors and pretreated with Obx (30 μg / ml) or cytochalasin D (30 μg / ml) at 37°C and 5% CO2 for 1 hour. B cell phagocytosis was then evaluated using flow cytometry. Figure 4A shows fixable live cell dyes (FVDs) used to distinguish live and dead cells before identification of B cell subsets. Figure 4B shows Fo B cells identified as CD20+, CD4-, CD27-, CD43-, and CD70- using FACS. Figure 4C shows MZ B cells identified as CD20+, CD4-, CD27+, IgDhi, and IgMhi using FACS. Figure 4D shows B1-like cells identified as CD20+, CD4-, CD27+, CD43+, and CD70-. Figure 4E shows streptavidin staining used to distinguish internalized beads from extracellular beads. Figure 4F shows the mean percentage of Fo B, MZ, and B1-like cells with internalized anti-human IgM coated beads. Each symbol represents data from cells isolated from individual donors (n=4) (*p<0.05, ns are not significant). [Figure 4B]Figures 4A–4F show the reduced BCR-mediated antigen uptake by human Fo B cell subsets, MZ cell subsets, and B1 B cell subsets. Primary human B cells were isolated from healthy donors and pretreated with Obx (30 μg / ml) or cytochalasin D (30 μg / ml) at 37°C and 5% CO2 for 1 hour. B cell phagocytosis was then evaluated using flow cytometry. Figure 4A shows fixable live cell dyes (FVDs) used to distinguish live and dead cells before identification of B cell subsets. Figure 4B shows Fo B cells identified as CD20+, CD4-, CD27-, CD43-, and CD70- using FACS. Figure 4C shows MZ B cells identified as CD20+, CD4-, CD27+, IgDhi, and IgMhi using FACS. Figure 4D shows B1-like cells identified as CD20+, CD4-, CD27+, CD43+, and CD70-. Figure 4E shows streptavidin staining used to distinguish internalized beads from extracellular beads. Figure 4F shows the mean percentage of Fo B, MZ, and B1-like cells with internalized anti-human IgM coated beads. Each symbol represents data from cells isolated from individual donors (n=4) (*p<0.05, ns are not significant). [Figure 4C]Figures 4A–4F show the reduced BCR-mediated antigen uptake by human Fo B cell subsets, MZ cell subsets, and B1 B cell subsets. Primary human B cells were isolated from healthy donors and pretreated with Obx (30 μg / ml) or cytochalasin D (30 μg / ml) at 37°C and 5% CO2 for 1 hour. B cell phagocytosis was then evaluated using flow cytometry. Figure 4A shows fixable live cell dyes (FVDs) used to distinguish live and dead cells before identification of B cell subsets. Figure 4B shows Fo B cells identified as CD20+, CD4-, CD27-, CD43-, and CD70- using FACS. Figure 4C shows MZ B cells identified as CD20+, CD4-, CD27+, IgDhi, and IgMhi using FACS. Figure 4D shows B1-like cells identified as CD20+, CD4-, CD27+, CD43+, and CD70-. Figure 4E shows streptavidin staining used to distinguish internalized beads from extracellular beads. Figure 4F shows the mean percentage of Fo B, MZ, and B1-like cells with internalized anti-human IgM coated beads. Each symbol represents data from cells isolated from individual donors (n=4) (*p<0.05, ns are not significant). [Figure 4D]Figures 4A–4F show the reduced BCR-mediated antigen uptake by human Fo B cell subsets, MZ cell subsets, and B1 B cell subsets. Primary human B cells were isolated from healthy donors and pretreated with Obx (30 μg / ml) or cytochalasin D (30 μg / ml) at 37°C and 5% CO2 for 1 hour. B cell phagocytosis was then evaluated using flow cytometry. Figure 4A shows fixable live cell dyes (FVDs) used to distinguish live and dead cells before identification of B cell subsets. Figure 4B shows Fo B cells identified as CD20+, CD4-, CD27-, CD43-, and CD70- using FACS. Figure 4C shows MZ B cells identified as CD20+, CD4-, CD27+, IgDhi, and IgMhi using FACS. Figure 4D shows B1-like cells identified as CD20+, CD4-, CD27+, CD43+, and CD70-. Figure 4E shows streptavidin staining used to distinguish internalized beads from extracellular beads. Figure 4F shows the mean percentage of Fo B, MZ, and B1-like cells with internalized anti-human IgM coated beads. Each symbol represents data from cells isolated from individual donors (n=4) (*p<0.05, ns are not significant). [Figure 4E]Figures 4A–4F show the reduced BCR-mediated antigen uptake by human Fo B cell subsets, MZ cell subsets, and B1 B cell subsets. Primary human B cells were isolated from healthy donors and pretreated with Obx (30 μg / ml) or cytochalasin D (30 μg / ml) at 37°C and 5% CO2 for 1 hour. B cell phagocytosis was then evaluated using flow cytometry. Figure 4A shows fixable live cell dyes (FVDs) used to distinguish live and dead cells before identification of B cell subsets. Figure 4B shows Fo B cells identified as CD20+, CD4-, CD27-, CD43-, and CD70- using FACS. Figure 4C shows MZ B cells identified as CD20+, CD4-, CD27+, IgDhi, and IgMhi using FACS. Figure 4D shows B1-like cells identified as CD20+, CD4-, CD27+, CD43+, and CD70-. Figure 4E shows streptavidin staining used to distinguish internalized beads from extracellular beads. Figure 4F shows the mean percentage of Fo B, MZ, and B1-like cells with internalized anti-human IgM coated beads. Each symbol represents data from cells isolated from individual donors (n=4) (*p<0.05, ns are not significant). [Figure 4F]Figures 4A–4F show the reduced BCR-mediated antigen uptake by human Fo B cell subsets, MZ cell subsets, and B1 B cell subsets. Primary human B cells were isolated from healthy donors and pretreated with Obx (30 μg / ml) or cytochalasin D (30 μg / ml) at 37°C and 5% CO2 for 1 hour. B cell phagocytosis was then evaluated using flow cytometry. Figure 4A shows fixable live cell dyes (FVDs) used to distinguish live and dead cells before identification of B cell subsets. Figure 4B shows Fo B cells identified as CD20+, CD4-, CD27-, CD43-, and CD70- using FACS. Figure 4C shows MZ B cells identified as CD20+, CD4-, CD27+, IgDhi, and IgMhi using FACS. Figure 4D shows B1-like cells identified as CD20+, CD4-, CD27+, CD43+, and CD70-. Figure 4E shows streptavidin staining used to distinguish internalized beads from extracellular beads. Figure 4F shows the mean percentage of Fo B, MZ, and B1-like cells with internalized anti-human IgM coated beads. Each symbol represents data from cells isolated from individual donors (n=4) (*p<0.05, ns are not significant). [Figure 5A]Figures 5A-5I show that B cells treated with mObx exhibited a reduced activation phenotype. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. 2B-KIX B cells were co-cultured with OT-II T cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 5A shows the flow cytometry scattering plot of B cells identified as CD45R+CD4-. Figure 5B shows the histogram of CD80. Figure 5C shows the mean fluorescence intensity (MFI) of CD80 under various treatment conditions. Figure 5D shows the histogram of CD86. Figure 5E shows the MFI of CD86 under various treatment conditions. Figure 5F shows the histogram of CD11b. Figure 5G shows the MFI of CD11b under various treatment conditions. Figure 5H shows the histogram of CD11c. Figure 5I shows the MFI of CD11c under various treatment conditions. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01). [Figure 5B]Figures 5A-5I show that B cells treated with mObx exhibited a reduced activation phenotype. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. 2B-KIX B cells were co-cultured with OT-II T cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 5A shows the flow cytometry scattering plot of B cells identified as CD45R+CD4-. Figure 5B shows the histogram of CD80. Figure 5C shows the mean fluorescence intensity (MFI) of CD80 under various treatment conditions. Figure 5D shows the histogram of CD86. Figure 5E shows the MFI of CD86 under various treatment conditions. Figure 5F shows the histogram of CD11b. Figure 5G shows the MFI of CD11b under various treatment conditions. Figure 5H shows the histogram of CD11c. Figure 5I shows the MFI of CD11c under various treatment conditions. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01). [Figure 5C]Figures 5A-5I show that B cells treated with mObx exhibited a reduced activation phenotype. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. 2B-KIX B cells were co-cultured with OT-II T cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 5A shows the flow cytometry scattering plot of B cells identified as CD45R+CD4-. Figure 5B shows the histogram of CD80. Figure 5C shows the mean fluorescence intensity (MFI) of CD80 under various treatment conditions. Figure 5D shows the histogram of CD86. Figure 5E shows the MFI of CD86 under various treatment conditions. Figure 5F shows the histogram of CD11b. Figure 5G shows the MFI of CD11b under various treatment conditions. Figure 5H shows the histogram of CD11c. Figure 5I shows the MFI of CD11c under various treatment conditions. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01). [Figure 5D]Figures 5A-5I show that B cells treated with mObx exhibited a reduced activation phenotype. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. 2B-KIX B cells were co-cultured with OT-II T cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 5A shows the flow cytometry scattering plot of B cells identified as CD45R+CD4-. Figure 5B shows the histogram of CD80. Figure 5C shows the mean fluorescence intensity (MFI) of CD80 under various treatment conditions. Figure 5D shows the histogram of CD86. Figure 5E shows the MFI of CD86 under various treatment conditions. Figure 5F shows the histogram of CD11b. Figure 5G shows the MFI of CD11b under various treatment conditions. Figure 5H shows the histogram of CD11c. Figure 5I shows the MFI of CD11c under various treatment conditions. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01). [Figure 5E]Figures 5A-5I show that B cells treated with mObx exhibited a reduced activation phenotype. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. 2B-KIX B cells were co-cultured with OT-II T cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 5A shows the flow cytometry scattering plot of B cells identified as CD45R+CD4-. Figure 5B shows the histogram of CD80. Figure 5C shows the mean fluorescence intensity (MFI) of CD80 under various treatment conditions. Figure 5D shows the histogram of CD86. Figure 5E shows the MFI of CD86 under various treatment conditions. Figure 5F shows the histogram of CD11b. Figure 5G shows the MFI of CD11b under various treatment conditions. Figure 5H shows the histogram of CD11c. Figure 5I shows the MFI of CD11c under various treatment conditions. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01). [Figure 5F]Figures 5A-5I show that B cells treated with mObx exhibited a reduced activation phenotype. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. 2B-KIX B cells were co-cultured with OT-II T cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 5A shows the flow cytometry scattering plot of B cells identified as CD45R+CD4-. Figure 5B shows the histogram of CD80. Figure 5C shows the mean fluorescence intensity (MFI) of CD80 under various treatment conditions. Figure 5D shows the histogram of CD86. Figure 5E shows the MFI of CD86 under various treatment conditions. Figure 5F shows the histogram of CD11b. Figure 5G shows the MFI of CD11b under various treatment conditions. Figure 5H shows the histogram of CD11c. Figure 5I shows the MFI of CD11c under various treatment conditions. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01). [Figure 5G]Figures 5A-5I show that B cells treated with mObx exhibited a reduced activation phenotype. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. 2B-KIX B cells were co-cultured with OT-II T cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 5A shows the flow cytometry scattering plot of B cells identified as CD45R+CD4-. Figure 5B shows the histogram of CD80. Figure 5C shows the mean fluorescence intensity (MFI) of CD80 under various treatment conditions. Figure 5D shows the histogram of CD86. Figure 5E shows the MFI of CD86 under various treatment conditions. Figure 5F shows the histogram of CD11b. Figure 5G shows the MFI of CD11b under various treatment conditions. Figure 5H shows the histogram of CD11c. Figure 5I shows the MFI of CD11c under various treatment conditions. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01). [Figure 5H]Figures 5A-5I show that B cells treated with mObx exhibited a reduced activation phenotype. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. 2B-KIX B cells were co-cultured with OT-II T cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 5A shows the flow cytometry scattering plot of B cells identified as CD45R+CD4-. Figure 5B shows the histogram of CD80. Figure 5C shows the mean fluorescence intensity (MFI) of CD80 under various treatment conditions. Figure 5D shows the histogram of CD86. Figure 5E shows the MFI of CD86 under various treatment conditions. Figure 5F shows the histogram of CD11b. Figure 5G shows the MFI of CD11b under various treatment conditions. Figure 5H shows the histogram of CD11c. Figure 5I shows the MFI of CD11c under various treatment conditions. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01). [Figure 5I]Figures 5A-5I show that B cells treated with mObx exhibited a reduced activation phenotype. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. 2B-KIX B cells were co-cultured with OT-II T cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 5A shows the flow cytometry scattering plot of B cells identified as CD45R+CD4-. Figure 5B shows the histogram of CD80. Figure 5C shows the mean fluorescence intensity (MFI) of CD80 under various treatment conditions. Figure 5D shows the histogram of CD86. Figure 5E shows the MFI of CD86 under various treatment conditions. Figure 5F shows the histogram of CD11b. Figure 5G shows the MFI of CD11b under various treatment conditions. Figure 5H shows the histogram of CD11c. Figure 5I shows the MFI of CD11c under various treatment conditions. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01). [Figure 6A]Figures 6A–6G show that B cells treated with mObx have a reduced ability to activate CD4 T cells. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. OT-II T cells were stained with CTViolet and co-cultured with 2B-KIX B cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 6A shows CD4 T cells identified as CD4+ CD45R-. Figure 6B shows a representative flow plot showing the percentage of CTViolet-stained OT-II T cell proliferation. Figure 6C shows the mean percentage of total OT-II T cell proliferation. Figure 6D shows the mean MFI of surface-expressed CD44 on OT-II T cells. Figure 6E shows the mean MFI of surface-expressed CD25 on CD44+ OT-II T cells. Figure 6F shows a representative histogram of MFI of surface-expressed CD25 on CD44+ OT-II T cells. Figure 6G shows the mean percentage of OT-II CD4 T cells in each proliferation cycle. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01, ***p<0.001). [Figure 6B]Figures 6A–6G show that B cells treated with mObx have a reduced ability to activate CD4 T cells. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. OT-II T cells were stained with CTViolet and co-cultured with 2B-KIX B cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 6A shows CD4 T cells identified as CD4+ CD45R-. Figure 6B shows a representative flow plot showing the percentage of CTViolet-stained OT-II T cell proliferation. Figure 6C shows the mean percentage of total OT-II T cell proliferation. Figure 6D shows the mean MFI of surface-expressed CD44 on OT-II T cells. Figure 6E shows the mean MFI of surface-expressed CD25 on CD44+ OT-II T cells. Figure 6F shows a representative histogram of MFI of surface-expressed CD25 on CD44+ OT-II T cells. Figure 6G shows the mean percentage of OT-II CD4 T cells in each proliferation cycle. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01, ***p<0.001). [Figure 6C]Figures 6A–6G show that B cells treated with mObx have a reduced ability to activate CD4 T cells. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. OT-II T cells were stained with CTViolet and co-cultured with 2B-KIX B cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 6A shows CD4 T cells identified as CD4+ CD45R-. Figure 6B shows a representative flow plot showing the percentage of CTViolet-stained OT-II T cell proliferation. Figure 6C shows the mean percentage of total OT-II T cell proliferation. Figure 6D shows the mean MFI of surface-expressed CD44 on OT-II T cells. Figure 6E shows the mean MFI of surface-expressed CD25 on CD44+ OT-II T cells. Figure 6F shows a representative histogram of MFI of surface-expressed CD25 on CD44+ OT-II T cells. Figure 6G shows the mean percentage of OT-II CD4 T cells in each proliferation cycle. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01, ***p<0.001). [Figure 6D]Figures 6A–6G show that B cells treated with mObx have a reduced ability to activate CD4 T cells. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. OT-II T cells were stained with CTViolet and co-cultured with 2B-KIX B cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 6A shows CD4 T cells identified as CD4+ CD45R-. Figure 6B shows a representative flow plot showing the percentage of CTViolet-stained OT-II T cell proliferation. Figure 6C shows the mean percentage of total OT-II T cell proliferation. Figure 6D shows the mean MFI of surface-expressed CD44 on OT-II T cells. Figure 6E shows the mean MFI of surface-expressed CD25 on CD44+ OT-II T cells. Figure 6F shows a representative histogram of MFI of surface-expressed CD25 on CD44+ OT-II T cells. Figure 6G shows the mean percentage of OT-II CD4 T cells in each proliferation cycle. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01, ***p<0.001). [Figure 6E]Figures 6A–6G show that B cells treated with mObx have a reduced ability to activate CD4 T cells. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. OT-II T cells were stained with CTViolet and co-cultured with 2B-KIX B cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 6A shows CD4 T cells identified as CD4+ CD45R-. Figure 6B shows a representative flow plot showing the percentage of CTViolet-stained OT-II T cell proliferation. Figure 6C shows the mean percentage of total OT-II T cell proliferation. Figure 6D shows the mean MFI of surface-expressed CD44 on OT-II T cells. Figure 6E shows the mean MFI of surface-expressed CD25 on CD44+ OT-II T cells. Figure 6F shows a representative histogram of MFI of surface-expressed CD25 on CD44+ OT-II T cells. Figure 6G shows the mean percentage of OT-II CD4 T cells in each proliferation cycle. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01, ***p<0.001). [Figure 6F]Figures 6A–6G show that B cells treated with mObx have a reduced ability to activate CD4 T cells. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. OT-II T cells were stained with CTViolet and co-cultured with 2B-KIX B cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 6A shows CD4 T cells identified as CD4+ CD45R-. Figure 6B shows a representative flow plot showing the percentage of CTViolet-stained OT-II T cell proliferation. Figure 6C shows the mean percentage of total OT-II T cell proliferation. Figure 6D shows the mean MFI of surface-expressed CD44 on OT-II T cells. Figure 6E shows the mean MFI of surface-expressed CD25 on CD44+ OT-II T cells. Figure 6F shows a representative histogram of MFI of surface-expressed CD25 on CD44+ OT-II T cells. Figure 6G shows the mean percentage of OT-II CD4 T cells in each proliferation cycle. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01, ***p<0.001). [Figure 6G]Figures 6A–6G show that B cells treated with mObx have a reduced ability to activate CD4 T cells. Mouse B cells were isolated from the spleen and lymph nodes of 2B-KIX and OT-II mice. OT-II T cells were stained with CTViolet and co-cultured with 2B-KIX B cells and anti-mouse IgM biotinylated OVA conjugate beads at 37°C and 5% CO2 for 4 days. Prior to incubation with the beads, some cells were pre-treated with mObx (30 μg / ml) or its idiotype control (30 μg / ml) for 1 hour at 37°C and 5% CO2. Figure 6A shows CD4 T cells identified as CD4+ CD45R-. Figure 6B shows a representative flow plot showing the percentage of CTViolet-stained OT-II T cell proliferation. Figure 6C shows the mean percentage of total OT-II T cell proliferation. Figure 6D shows the mean MFI of surface-expressed CD44 on OT-II T cells. Figure 6E shows the mean MFI of surface-expressed CD25 on CD44+ OT-II T cells. Figure 6F shows a representative histogram of MFI of surface-expressed CD25 on CD44+ OT-II T cells. Figure 6G shows the mean percentage of OT-II CD4 T cells in each proliferation cycle. Each symbol represents data from cells isolated from individual mouse pairs (n=3) (*p<0.05, **p<0.01, ***p<0.001). [Modes for carrying out the invention]

[0018] The present invention provides, in particular, a method for treating MS in a subject requiring treatment for MS, comprising administering to the subject an anti-CD19 antibody comprising an Fc region modified by amino acid substitutions S267E and L328F, wherein the administration of the anti-CD19 antibody improves, alleviates, or delays the onset of one or more symptoms of MS. In some embodiments, the anti-CD19 antibody is obexerimab.

[0019] In some embodiments, one or more symptoms of MS include pain, fatigue, cognitive impairment, depression, limited mobility, lack of coordination, difficulty speaking or swallowing, and various visual disturbances, as well as many other symptoms.

[0020] Various aspects of the present invention will be described in detail in the following sections. The use of these sections is not intended to limit the present invention. Each section may be applied to any aspect of the present invention. In this application, unless otherwise stated, the use of "or" means "and / or".

[0021] definition Several definitions are provided herein. These definitions are intended to encompass grammatical equivalents.

[0022] Antibodies: The term “antibody” as used herein means a protein comprising one or more polypeptides substantially encoded by all or part of a recognized immunoglobulin gene. Recognized immunoglobulin genes include, for example in humans, kappa (K), lambda (l), and heavy chain loci, which together may include a multitude of variable region genes, as well as the constant region gene gamma (γ) encoding the IgG (IgG1, IgG2, IgG3, and IgG4) isotypes. Antibodies as used herein are intended to include full-length antibodies and antibody fragments, and may refer to naturally occurring antibodies of any organism, engineered antibodies, or antibodies recombinantly produced for experimental, therapeutic, or other purposes.

[0023] Effector Function: As used herein, the term “effector function” means a biochemical event resulting from the interaction of an antibody Fc region with an Fc receptor or ligand. Examples of effector functions include FcγR-mediated effector functions such as ADCC and ADCP, as well as complement-mediated effector functions such as CDC. Furthermore, effector functions include FcγRIlb-mediated effector functions such as inhibitory functions (e.g., downregulation, reduction, inhibition of B cell responses, e.g., humoral immune responses).

[0024] Fc or Fc Region: As used herein, the terms “Fc” or “Fc region” mean the polypeptide containing the first constant-region immunoglobulin domain and, optionally, part of the hinge, the constant-region of the antibody. Thus, Fc may refer to the last two immunoglobulin domains of the constant-region of IgG and the movable hinge region located on the N-terminal side of these domains. For IgG, Fc contains the immunoglobulin domains C-gamma 2 and C-gamma 3 (Cγ2 and Cγ3) and the hinge between C-gamma 1 (Cγ1) and C-gamma 2. While the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is typically defined as containing residue C226 or P230 at its carboxyl terminus, and numbering follows the EU index, as in Kabat. Fc may refer to this region alone or in relation to the Fc polypeptide, as described below.

[0025] Fc gamma receptor, or FcγR: As used herein, the terms “Fc gamma receptor” or “FcγR” mean any member of the protein family that binds to the Fc region of an IgG antibody and is substantially encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI(CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32) including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16) including isoforms FcγRIIIa (including allotypes V1 58 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, the whole is incorporated by reference), as well as any undiscovered human FcγR or FcγR isoform or allotype. FcγR may be derived from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγR includes, but is not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIII-2(CD16-2), as well as any undiscovered mouse FcγR or FcγR isoform or allotype.

[0026] Modification: As used herein, “modification” means a change in the physical, chemical, or sequence properties of a protein, polypeptide, antibody, or immunoglobulin. Modifications as described herein include amino acid modifications (including amino acid substitutions) and glycoform modifications.

[0027] Target antigen: As used herein, the term “target antigen” means the molecule to which a given antibody’s variable region is bound, or the fusion partner of an Fc fusion. The target antigen may be a protein, carbohydrate, lipid, or other chemical substance. The antibody or Fc fusion is said to be “specific” to a given target antigen based on having affinity for the target antigen. In some embodiments, the target antigen of obexerimab is CD19.

[0028] Target cells: As used herein, the term “target cells” means cells that express a target antigen.

[0029] Treatment: As used herein, the term “treatment” means to include the improvement, relief, or delay of the onset of one or more symptoms of a disease or disorder.

[0030] Obexerimab: As used herein, the term “obexerimab” refers to an Fc-modified humanized monoclonal antibody (mAb) that binds to the human B cell restriction surface antigen CD19 and has enhanced Fc binding to the Fcγ receptor IIb (FcγRIIb). The molecule is an IgG1 immunoglobulin with two amino acid substitutions in the constant portions of the kappa light chain and heavy chain. Obexerimab is a monoclonal antibody with a predicted mass of approximately 147,426 Da based on its amino acid sequence. The heavy and light chains of obexerimab are given by SEQ ID NOs: 10 and 9, respectively.

[0031] Multiple sclerosis (MS) MS is a chronic, potentially physically debilitating immune-mediated disorder affecting the central nervous system (CNS) characterized by the progressive destruction of myelin. MS presents with a variety of neurological symptoms, including motor paralysis, sensory impairment, higher-order brain dysfunction, visual impairment, and urinary dysfunction, due to inflammation targeting perineurinary myelin proteins caused by the infiltration of autoreactive lymphocytes (such as T cells or B cells) into the brain, spinal cord, or optic nerve. Transient and recurrent inflammation occurs in various parts of the CNS in MS patients. Each inflammation manifests with neurological symptoms depending on the site of inflammation.

[0032] Although the disease mechanisms are not fully understood, B cells play a role in MS disease progression. B cell subsets play several roles in immunity, most of which are beneficial. For example, the B cell lineage is best known for antibody production, a function performed not by B cells themselves, but by activated cells differentiated into plasma cells. High-affinity antibodies are important for pathogen-specific protective immune responses against infection or after vaccination. However, while high-affinity autoantigen (Ag)-specific antibodies are involved in the pathology of MS-related disorders NMOSD and MOGSD (Jain, RW, et al., 2022. Nat. Rev. Immunol. 22: 513-524), antibody production is not the primary B-cell pathological contribution to MS. In fact, therapeutic anti-CD20 antibodies effectively eliminate B cells, but antibody-producing plasma cells are not eliminated, and antibody levels do not decrease during the treatment period following administration of anti-CD20-based drugs (Graft et al., 2021, Lee et al., 2021, and Margoni et al., 2022). Therefore, B cells must have other functions that contribute to the ongoing inflammatory response underlying disease progression.

[0033] One of the main candidate mechanisms by which B cells can drive autoimmunity in the CNS is through the presentation of autoantigens (Ag) to T cells. This, combined with any cytokines that B cells may simultaneously produce, could directly activate naive autoimmune T cells in lymphoid tissue and influence their differentiation into effector subsets. In the process of Ag presentation, Ag-presenting cells (APCs) need to collect and take up the protein Ag before processing it to load it into the major histocompatibility complex (MHC). This peptide:MHC combination is then transported to the cell surface and presented to migratory T cells that sample local APCs for their specific Ag via their T cell receptor (TCR).

[0034] Although B cells are described as one of the three "specialized" APCs (along with dendritic cells and macrophages), they are not well known as T cell activators. In fact, their APC function is best understood in relation to high-affinity antibody responses, where B cell activation, rather than T cell activation, is the primary outcome. These events occur in specialized structures called germinal centers (GCs) within secondary lymphoid tissue. In this case, naive B cells encountering Ag bound to their specific B cell receptor (BCR) take it (and physically linked to it) into their cells and present it to already activated T follicle helper (Tfh) cells that are specific to the same antigen. These allogeneic interactions are necessary for the full activation of B cells, which are the basis for selecting high-affinity B cells in GC and for differentiation into plasma cells for antibody production (Haberman, AM, et al., 2019. Immunol. Rev. 288: 10-27, Kerfoot, SM, et al., 2011. Immunity 34: 947-960, Jain, RW, et al., 2018., Cell Rep. 25: 3342-3355.e5, Parham, KA, et al., 2022. J. Immunol. 209: 1703-1712).

[0035] Other scenarios in which B cells may present Ag to T cells, resulting in T cell activation, are not well understood. B cells are known to be able to enrich low concentrations of Ag far more effectively than other APCs because they selectively take up Ag via BCR binding, and for this reason, they are involved in initiating some autoimmune T cell responses (Rodriguez-Pinto, D. 2005. Cell. Immunol. 238: 67-75). However, B cells specific to a particular antigen are very rare, and typically B cells are physically separated from naive T cells in lymphoid tissue, so it is unclear when or where such interactions between naive T cells and homologous B cells may occur within the lymphoid tissue microstructure.

[0036] Activated T cells readily infiltrate MS demyelinating lesions, while B cells are relatively rare within the CNS parenchyma (Jain and Yong, 2022). Instead, a considerable number of B cells may be collected alongside meningeal T cells, often immediately adjacent to the lesion (Reali, C., R. et al., 2020. Brain Pathol. 30: 779-793, Bell, L., et al., 2020. Front. Immunol. 10, Choi, SR, et al., 2012. Brain J. Neurol. 135: 2925-2937). These lymphocyte clusters may take on a morphology resembling well-organized lymphoid tissue with distinguishable T cell regions and B cell follicles, but more often than not, these cells are simply irregularly mixed together. Due to their association with lesions and their presence being associated with more serious diseases (Jain and Yong, 2022), these clusters are of considerable interest as potential sites where B cells can exert pathological function. Because they may resemble secondary lymphoid tissues, these structures may maintain autoimmune anti-myelin responses from behind the blood-brain / meningeal barrier (Pipi, E. et al., 2018. Front. Immunol. 9). In this scenario, B cells function as APCs, presenting locally acquired CNS autoantigens to newly recruited effector T cells, which are then reactivated and drive inflammation targeting oligodendrocytes in local parenchymal tissue. Thus, B cells can function as APCs to drive autoimmune responses from multiple sites, including peripheral lymphatic vessels and meninges.

[0037] Disease models of MS While MS is a human disease, specific manifestations of the disease can be modeled in mice and other species to study the underlying pathological mechanisms of the human disease. Experimental autoimmune encephalomyelitis (EAE) is a broad term encompassing a number of "artificially" generated antimyelin autoimmune responses that result in immune-mediated demyelination in the central nervous system (CNS).

[0038] Exemplary EAE models and model Ag systems, primarily based on myelin oligodendrocyte glycoprotein (MOG), which is an autoantigen, are described in Table 1. Improved disease models are described in this application, particularly in the Examples section. [Table 1-1] [Table 1-2] [Table 1-3]

[0039] Anti-CD19 antibody According to the present invention, an anti-CD19 antibody is used to treat human patients suffering from MS. In some embodiments, the anti-CD19 antibody enhances Fc binding to FcγRIIb. In some embodiments, the anti-CD19 antibody includes amino acid modifications S267E and L328F, and the amino acid numbering follows the EU index by Kabat.

[0040] Exemplary anti-CD19 antibodies are described herein.

[0041] Obexerimab: In some embodiments, obexerimab is used to treat human patients suffering from MS. In one embodiment, the present invention provides a method for administering obexerimab for the treatment of MS. Obexerimab is a CD19-specific monoclonal antibody, CDR1 containing RSSKSLQNVNGNTYLY (Sequence ID 2), CDR2 containing RMSNLNS (SEQ ID NO: 3), and A light chain containing a variable region containing CDR3 including MQHLEYPIT (SEQ ID NO: 4), Heavy chain including a variable region having the following: CDR1 containing SYVMH (SEQ ID NO: 5), CDR2 containing WIGYINPYNDGTKY (SEQ ID NO: 6), and A heavy chain containing a variable region containing CDR3 including GTYYYGTRVFDY (SEQ ID NO: 7), The heavy chain, compared to SEQ ID NO: 8, contains amino acid substitutions S267E and L328F within the Fc region: The numbering follows the EU index, similar to Kabat. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 8)

[0042] In some embodiments, obexerimab has the amino acid sequence: A light chain containing DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 9), Amino acid sequence: EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP It contains a heavy chain including APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10). [Table 2] [Table 3]

[0043] In some embodiments, obexerimab includes light-chain variable regions and heavy-chain variable regions as shown in Table 2. In some embodiments, obexerimab includes CDRs as shown in Table 3.

[0044] In some embodiments, the anti-CD19 antibody includes a light chain containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody includes a heavy chain containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody includes a light chain and a heavy chain, the light chain containing the amino acid sequence of SEQ ID NO: 9 and the heavy chain containing the amino acid sequence of SEQ ID NO: 10.

[0045] In some embodiments, the anti-CD19 antibody comprises a light chain, the light chain comprising a light chain variable region containing the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody comprises a heavy chain, the heavy chain comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising a light chain variable region containing the amino acid sequence of SEQ ID NO: 11, and the heavy chain comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody comprises a heavy chain, the heavy chain comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 12, and the heavy chain comprising an Fc region containing the amino acid substitutions S267E and L328F. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising a light chain variable region containing the amino acid sequence of SEQ ID NO: 11, the heavy chain comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 10, and the heavy chain comprising an Fc region containing the amino acid substitutions S267E and L328F.

[0046] In some embodiments, the anti-CD19 antibody comprises a light chain containing LCDR1 containing the amino acid sequence of SEQ ID NO: 2, LCDR2 containing the amino acid sequence of SEQ ID NO: 3, and LCDR3 containing the amino acid sequence of SEQ ID NO: 4. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1 containing the amino acid sequence of SEQ ID NO: 5, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1 containing the amino acid sequence of SEQ ID NO: 5, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7, and the heavy chain comprises an Fc region containing amino acid substitutions S267E and L328F. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 2, LCDR2 containing the amino acid sequence of SEQ ID NO: 3, and LCDR3 containing the amino acid sequence of SEQ ID NO: 4, and the heavy chain comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 5, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 2, LCDR2 containing the amino acid sequence of SEQ ID NO: 3, and LCDR3 containing the amino acid sequence of SEQ ID NO: 4, the heavy chain comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 5, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7, and the heavy chain comprising an Fc region containing the amino acid substitutions S267E and L328F.

[0047] Obexerimab acts by utilizing the modulation of B cell receptor (BCR) signaling by FcγRIIb. Obexerimab binds to CD19 on the BCR complex, and its Fc is manipulated to increase its affinity for inhibitory FcγRIIb. Since CD19 is associated with the BCR, tethering obexerimab to FcγRIIb on the same cell balances the BCR complex for inhibition during antigen-induced BCR aggregation. Obexerimab utilizes the innate inhibitory mechanism of FcγRIIb, the only Fc receptor expressed by B cells, which acts as a negative regulator under conditions of antigen overload and immune complex formation (Chu et al., 2014). Obexerimab may also have an improved safety profile compared to B cell depletion antibodies because it may not mediate B cell killing.

[0048] variant In some embodiments, a variant of obexerimab is a CD19-specific immunoglobulin comprising a light chain comprising a variable region having CDR1 containing RSSKSLQNVNGNTYLY, CDR2 containing RMSNLNS, and CDR3 containing MQHLEYPIT, and a heavy chain comprising a variable region having CDR1 containing SYVMH, CDR2 containing WIGYINPYNDGTKY, and CDR3 containing GTYYYGTRVFDY, wherein the heavy chain comprises amino acid substitutions S267E and L328F in the Fc region, compared to: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 8) (The numbering follows the EU index, similar to Kabat.)

[0049] In some embodiments, the obexerimab variant comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) including CDR1, CDR2, and CDR3, each of which differs from each of RSSKSLQNVNGNTYLY (SEQ ID NO: 2), RMSNLNS (SEQ ID NO: 3), MQHLEYPIT (SEQ ID NO: 4), SYVMH (SEQ ID NO: 5), WIGYINPYNDGTKY (SEQ ID NO: 6), and / or GTYYYGTRVFDY (SEQ ID NO: 7) by 1, 2, 3, 4, or 5 or fewer amino acid residues. In some embodiments, the obexerimab variant comprises a heavy chain, the heavy chain including an Fc region containing the amino acid substitutions S267E and L328F.

[0050] In some embodiments, the anti-CD19 antibody comprises a light chain containing LCDR1, LCDR2, and LCDR3, each differing from each of SEQ ID NOs. 2, SEQ ID NOs. 3, and SEQ ID NOs. 4. In some embodiments, the anti-CD19 antibody comprises a light chain containing LCDR1, LCDR2, and LCDR3, each differing from each of SEQ ID NOs. 2, SEQ ID NOs. 3, and SEQ ID NOs. 4. In some embodiments, the anti-CD19 antibody comprises a light chain containing LCDR1, LCDR2, and LCDR3, each differing from each of SEQ ID NOs. 2, SEQ ID NOs. 3, and SEQ ID NOs. 4. In some embodiments, the anti-CD19 antibody comprises a light chain containing LCDR1, LCDR2, and LCDR3, each differing from each of SEQ ID NOs. 2, SEQ ID NOs. 3, and SEQ ID NOs. 4. In some embodiments, the anti-CD19 antibody comprises a light chain containing LCDR1, LCDR2, and LCDR3, each of which differs from each of SEQ ID NOs. 2, 3, and 4 by five or fewer residues.

[0051] In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1, HCDR2, and HCDR3, each differing from each of SEQ ID NOs. 5, 6, and 7 by one or fewer residues. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1, HCDR2, and HCDR3, each differing from each of SEQ ID NOs. 5, 6, and 7 by two or fewer residues. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1, HCDR2, and HCDR3, each differing from each of SEQ ID NOs. 5, 6, and 7 by three or fewer residues. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1, HCDR2, and HCDR3, each differing from each of SEQ ID NOs. 5, 6, and 7 by four or fewer residues. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1, HCDR2, and HCDR3, each of which differs from each of SEQ ID NOs. 5, 6, and 7 by five or fewer residues.

[0052] In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1, HCDR2, and HCDR3, each differing from each of SEQ ID NOs. 5, 6, and 7 by one or fewer residues, and the heavy chain comprises an Fc region containing amino acid substitutions S267E and L328F. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1, HCDR2, and HCDR3, each differing from each of SEQ ID NOs. 5, 6, and 7 by two or fewer residues, and the heavy chain comprises an Fc region containing amino acid substitutions S267E and L328F. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1, HCDR2, and HCDR3, each differing from each of SEQ ID NOs. 5, 6, and 7 by three or fewer residues, and the heavy chain comprises an Fc region containing amino acid substitutions S267E and L328F. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1, HCDR2, and HCDR3, each of which differs from each of SEQ ID NOs. 5, 6, and 7 by four or fewer residues, and the heavy chain comprises an Fc region containing amino acid substitutions S267E and L328F.

[0053] In some embodiments, the obexerimab variant includes a light chain variable region containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the light chain variable regions specified in Table 2. In some embodiments, the obexerimab variant includes a heavy chain variable region containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain variable regions specified in Table 2.

[0054] In some embodiments, the anti-CD19 antibody includes a light chain containing a light chain variable region that is at least 80% identical to SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody includes a light chain containing a light chain variable region that is at least 85% identical to SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody includes a light chain containing a light chain variable region that is at least 90% identical to SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody includes a light chain containing a light chain variable region that is at least 91% identical to SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody includes a light chain containing a light chain variable region that is at least 92% identical to SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody includes a light chain containing a light chain variable region that is at least 93% identical to SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody includes a light chain containing a light chain variable region that is at least 94% identical to SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody includes a light chain containing a light chain variable region that is at least 95% identical to SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody includes a light chain containing a light chain variable region that is at least 96% identical to SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody includes a light chain containing a light chain variable region that is at least 97% identical to SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody includes a light chain containing a light chain variable region that is at least 98% identical to SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody includes a light chain containing a light chain variable region that is at least 99% identical to SEQ ID NO: 11.

[0055] In some embodiments, the anti-CD19 antibody includes a heavy chain containing a heavy chain variable region that is at least 80% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody includes a heavy chain containing a heavy chain variable region that is at least 85% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody includes a heavy chain containing a heavy chain variable region that is at least 90% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody includes a heavy chain containing a heavy chain variable region that is at least 91% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody includes a heavy chain containing a heavy chain variable region that is at least 92% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody includes a heavy chain containing a heavy chain variable region that is at least 93% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody includes a heavy chain containing a heavy chain variable region that is at least 94% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody includes a heavy chain containing a heavy chain variable region that is at least 95% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody includes a heavy chain containing a heavy chain variable region that is at least 96% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody includes a heavy chain containing a heavy chain variable region that is at least 97% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody includes a heavy chain containing a heavy chain variable region that is at least 98% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody includes a heavy chain containing a heavy chain variable region that is at least 99% identical to SEQ ID NO: 12.

[0056] In some embodiments, the obexerimab variants include a light chain variable region containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the light chain variable region specified in Table 2, and a heavy chain variable region containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain variable region specified in Table 2, and further having amino acid substitutions S267E and L328F in the Fc region compared to SEQ ID NO: 8, with numbering following the EU index.

[0057] In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising a light chain variable region that is at least 80% identical to SEQ ID NO: 11, and the heavy chain comprising a heavy chain variable region that is at least 80% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising a light chain variable region that is at least 85% identical to SEQ ID NO: 11, and the heavy chain comprising a heavy chain variable region that is at least 85% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising a light chain variable region that is at least 90% identical to SEQ ID NO: 11, and the heavy chain comprising a heavy chain variable region that is at least 90% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising a light chain variable region that is at least 91% identical to SEQ ID NO: 11, and the heavy chain comprising a heavy chain variable region that is at least 91% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising a light chain variable region that is at least 92% identical to SEQ ID NO: 11, and the heavy chain comprising a heavy chain variable region that is at least 92% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising a light chain variable region that is at least 93% identical to SEQ ID NO: 11, and the heavy chain comprising a heavy chain variable region that is at least 93% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising a light chain variable region that is at least 94% identical to SEQ ID NO: 11, and the heavy chain comprising a heavy chain variable region that is at least 94% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising a light chain variable region that is at least 95% identical to SEQ ID NO: 11, and the heavy chain comprising a heavy chain variable region that is at least 95% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising a light chain variable region that is at least 96% identical to SEQ ID NO: 11, and the heavy chain comprising a heavy chain variable region that is at least 96% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising a light chain variable region that is at least 97% identical to SEQ ID NO: 11, and the heavy chain comprising a heavy chain variable region that is at least 97% identical to SEQ ID NO: 12.In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising a light chain variable region that is at least 98% identical to SEQ ID NO: 11, and the heavy chain comprising a heavy chain variable region that is at least 98% identical to SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, the light chain comprising a light chain variable region that is at least 99% identical to SEQ ID NO: 11, and the heavy chain comprising a heavy chain variable region that is at least 99% identical to SEQ ID NO: 12.

[0058] In some embodiments, the obexerimab variant includes a light chain containing an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the light chains specified in Table 2. In some embodiments, the obexerimab variant includes a heavy chain containing an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the heavy chains specified in Table 2.

[0059] In some embodiments, the anti-CD19 antibody contains a light chain amino acid sequence that is at least 70% identical to SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody contains a light chain amino acid sequence that is at least 75% identical to SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody contains a light chain amino acid sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody contains a light chain amino acid sequence that is at least 85% identical to SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody contains a light chain amino acid sequence that is at least 90% identical to SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody contains a light chain amino acid sequence that is at least 91% identical to SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody contains a light chain amino acid sequence that is at least 92% identical to SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody contains a light chain amino acid sequence that is at least 93% identical to SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody contains a light chain amino acid sequence that is at least 94% identical to SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody contains a light chain amino acid sequence that is at least 95% identical to SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody contains a light chain amino acid sequence that is at least 96% identical to SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody contains a light chain amino acid sequence that is at least 97% identical to SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody contains a light chain amino acid sequence that is at least 98% identical to SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody contains a light chain amino acid sequence that is at least 99% identical to SEQ ID NO: 9.

[0060] In some embodiments, the anti-CD19 antibody contains a heavy chain amino acid sequence that is at least 70% identical to SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody contains a heavy chain amino acid sequence that is at least 75% identical to SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody contains a heavy chain amino acid sequence that is at least 80% identical to SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody contains a heavy chain amino acid sequence that is at least 85% identical to SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody contains a heavy chain amino acid sequence that is at least 90% identical to SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody contains a heavy chain amino acid sequence that is at least 91% identical to SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody contains a heavy chain amino acid sequence that is at least 92% identical to SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody contains a heavy chain amino acid sequence that is at least 93% identical to SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody contains a heavy chain amino acid sequence that is at least 94% identical to SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody contains a heavy chain amino acid sequence that is at least 95% identical to SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody contains a heavy chain amino acid sequence that is at least 96% identical to SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody contains a heavy chain amino acid sequence that is at least 97% identical to SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody contains a heavy chain amino acid sequence that is at least 98% identical to SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody contains a heavy chain amino acid sequence that is at least 99% identical to SEQ ID NO: 10.

[0061] In some embodiments, the variant of obexerimab comprises a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the light chain sequences specified in Table 2, and a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain sequences specified in Table 2, wherein the heavy chain of the variant contains amino acid substitutions at S267E and L328F in the Fc region compared to SEQ ID NO: 8, and the numbering follows the EU index as in Kabat.

[0062] In some embodiments, preferred variants of obexerimab are antibodies comprising light and heavy chains as specified in Table 2, which bind to the same epitope on human CD19. Epitope binding may be determined by methods known in the art.

[0063] In some embodiments, suitable variants of obexerimab compete for binding to human CD19 as antibodies containing light and heavy chains, as identified in Table 2, under binning assays known in the art. As used herein, a binning assay refers to any method for locally mapping the epitopes to which an antibody binds. A standard method for characterizing such antibodies, also known as epitope binning, typically involves surface plasmon resonance (SPR) techniques. Using SPR, monoclonal antibody candidates are screened in pairs for binding to a target protein. Other standard methods include ELISA-based screening, which may require the synthesis of a set of duplicate peptides corresponding to the protein of interest.

[0064] In some embodiments, human CD19 contains the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-CD19 antibody binds to the extracellular domain of human CD19. (Sequence ID 1)

[0065] Fc receptor binding properties The anti-CD19 antibodies disclosed herein (e.g., obexerimab) include Fc variants having enhanced Fc binding to inhibitory Fcγ receptor IIb (FcγRIIb). FcγRIIb, the sole FcR on B cells, functions as an antibody-sensing downregulator of humoral immunity, which is spontaneously associated by immune complexes. Once sufficient antibody is produced against a given antigen, a specific immune complex is formed, co-engaging FcγRIIb with the B cell receptor (BCR) with high avidity to selectively suppress only B cells that recognize the congener antigen. Furthermore, FcγRIIb modulates the activity of other B cell stimulants, including interleukin (IL)-4, LPS, and BAFF, which amplify BCR-driven proliferation and differentiation. By simultaneously binding to CD19 and FcγRIIb, obexerimab (and the variants described herein) mimic the action of antigen-antibody complexes and downregulate B cell activity.

[0066] The Fc variants disclosed herein can be optimized for various Fc receptor binding properties. An Fc variant that is manipulated or predicted to exhibit one or more optimized properties is referred to herein as an “optimized Fc variant.” Properties that can be optimized include, but are not limited to, enhanced or reduced affinity for FcγR. In one embodiment, the Fc variant disclosed herein is optimized to have enhanced affinity for the inhibitory receptor FcγRIIb. In other embodiments, the immunoglobulin disclosed herein provides enhanced affinity for FcγRIIb, but reduces affinity for one or more activated FcγRs, including, for example, FcγRI, FcγRIIa, FcγRIIIa, and / or FcγRIIIb. FcγR receptors can be expressed on cells of any organism, including, but not limited to, human, cynomolgus monkey, and mouse. The Fc variants disclosed herein can be optimized to have enhanced affinity for human FcγRIIb.

[0067] An Fc variant comprises one or more amino acid modifications to the parent Fc polypeptide, and the amino acid modifications provide one or more optimized properties. The Fc variants disclosed herein have different amino acid sequences from their parent due to at least one amino acid modification. Therefore, the Fc variants disclosed herein have at least one amino acid modification compared to the parent. Alternatively, the Fc variants disclosed herein may have two or more amino acid modifications compared to the parent, for example, about 2 to 50 amino acid modifications compared to the parent, for example, about 2 to 10 amino acid modifications, about 2 to about 5 amino acid modifications, etc. Therefore, the sequences of the Fc variants and the sequences of the parent Fc polypeptides are substantially homologous. For example, the Fc variant sequences described herein have about 80% homology to the parent Fc variant sequence, for example, at least about 90% homology, at least about 95% homology, at least about 98% homology, at least about 99% homology, etc. The modifications disclosed herein include amino acid modifications including insertions, deletions, and substitutions. The modifications disclosed herein also include glycoform modifications.

[0068] The modifications may be genetically synthesized using molecular biology, or they may be synthesized enzymatically or chemically.

[0069] The Fc variants disclosed herein are defined according to the amino acid modifications that constitute them. For example, S267E is an Fc variant having the substitution S267E on the parent Fc polypeptide. Similarly, S267E / L328F defines an Fc variant having the substitutions S267E and L328F on the parent Fc polypeptide. The identity of the WT amino acids is not required; in this case, the aforementioned variant is referred to as 267E / 328F. Note that the order in which substitutions are provided is arbitrary, i.e., for example, 267E / 328F is the same Fc variant as 328F / 267E. Unless otherwise stated, the locations considered herein are numbered according to the EU index described in Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, fully incorporated here by reference). Briefly, EU is the name of the first antibody molecule whose entire amino acid sequence has been determined (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, fully incorporated here by reference), and its amino acid sequence has become the standard numbering scheme for the heavy chain constant region. EU proteins serve as the standard reference defining the numbering. Kabat et al. list EU sequences in a set of indices for aligning EU sequences with other antibody sequences as a tool necessary for aligning antibodies to the EU numbering scheme. Therefore, as will be understood by those skilled in the art, the standard way to refer to EU numbering is to refer to Kabat et al.'s sequence alignment to place EU in relation to other antibodies with variable domain lengths. Thus, as used herein, “EU index as Kabat” or “numbering follows EU index as Kabat” refers to the numbering of EU antibodies as described in Kabat.

[0070] In certain embodiments, the Fc variants disclosed herein are based on human IgG sequences, and therefore the human IgG sequence is used as a “base” sequence on which other sequences are compared, including but not limited to sequences from other organisms, such as rodent sequences and primate sequences. The Fc variants disclosed herein are manipulated in the context of one parent IgG, but the variants are intended to be manipulated in the context of another second parent IgG, or to be “transmitted” to them. This is typically done by determining “equivalent” or “corresponding” residues and substitutions between the first and second IgG sequences based on sequence or structural homology between the first and second IgG sequences. To establish homology, the amino acid sequence of the first IgG outlined herein is directly compared to the sequence of the second IgG. After the sequences are aligned, residues corresponding to specific amino acids in the primary sequence of the first immunoglobulin are defined using one or more homologous alignment programs known in the art (e.g., using interspecies-conserved residues) to allow insertions and deletions necessary to maintain the alignment (i.e., avoiding the removal of conserved residues by any deletions and insertions). Alignment of conserved residues can preserve 100% of these residues. However, even if the sequence of conserved residues is 75% or more, or even around 50%, it is sufficient to define equivalent residues. Equivalent residues may also be defined by determining the structural homology between the first and second IgG at the tertiary structure level of the structurally determined IgG. In this case, equivalent residues are defined as those in which the atomic coordinates (N on N, CA on CA, C on C, and O on O) of two or more back-chain atoms of a particular amino acid residue of the parent or precursor are within approximately 0.13 nm after alignment. In another embodiment, equivalent residues are within approximately 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to yield the maximum overlap of atomic coordinates of the non-hydrogen protein atoms of the protein.Regardless of how equivalent or corresponding residues are determined, and regardless of the identity of the parent IgG from which the IgG is produced, the intended transfer is that the Fc variant discovered as disclosed herein can be manipulated into any second parent IgG having significant sequence or structural homology to the Fc variant. Therefore, for example, if a variant antibody whose parent antibody is human IgG1 is produced using the method described above or other methods for identifying equivalent residues, that variant antibody can be incorporated into another IgG1 parent antibody, human IgG2 parent antibody, human IgA parent antibody, mouse IgG2a or IgG2b parent antibody, etc., that binds to a different antigen. Again, as stated above, the context of the parent Fc variant does not affect the ability to transfer the Fc variant disclosed herein to other parent IgGs.

[0071] As used herein, the terms “greater affinity” or “improved affinity,” or “enhanced affinity” or “better affinity” compared to the parent Fc polypeptide mean that the Fc variant binds to the Fc receptor with a significantly higher equilibrium binding constant (KA or Ka) or a lower equilibrium dissociation constant (KD or Kd) than the parent Fc polypeptide when the amounts of the variant and parent polypeptide in the binding assay are essentially the same. For example, an Fc variant with improved Fc receptor binding affinity may show an improvement of about 5 to 1000 times, e.g., about 10 to 500 times, compared to the parent Fc polypeptide, and the Fc receptor binding affinity is determined, for example, by a binding method disclosed herein, including, but not limited to, Biacore, as can be seen by those skilled in the art. Thus, as used herein, “reduced affinity” compared to the parent Fc polypeptide means that the Fc variant binds to the Fc receptor with a significantly lower KA or higher KD than the parent Fc polypeptide. Greater or reduced affinity can also be defined in comparison to an absolute level of affinity. For example, according to the data herein, WT (natural) IgG1 binds to FcγRIIb with an affinity of about 1.5 mM, or about 1500 nM. Furthermore, some Fc variants described herein bind to FcγRIIb with an affinity about 10 times higher than WT IgG1. As disclosed herein, greater or enhanced affinity means having a KD of less than about 100 nM, for example, about 10 nM to about 100 nM, about 1 to about 100 nM, or less than about 1 nM.

[0072] In one embodiment, the Fc variant provides selectively enhanced affinity for FcγRIIb compared to one or more activating receptors. Selectively enhanced affinity means that the Fc variant has improved affinity for FcγRIIb to the activating receptor compared to the parental Fc polypeptide, but reduced affinity for the activating receptor compared to the parental Fc polypeptide, or that the Fc variant has improved affinity for both FcγRIIb and the activating receptor compared to the parental Fc polypeptide, but the improvement in affinity is greater for FcγRIIb than for the activating receptor. In an alternative embodiment, the Fc variant reduces or cleaves binding to one or more activating FcγRs, reduces or cleaves binding to one or more complement proteins, reduces or cleaves the function of one or more FcγR-mediated effector, and / or reduces or cleaves the function of one or more complement-mediated effector.

[0073] The existence of different polymorphic forms of FcγR provides yet another parameter that influences the therapeutic utility of the Fc variants disclosed herein. While the specificity and selectivity of a given Fc variant to different classes of FcγR significantly influences the ability of the Fc variant to target a given antigen for the treatment of a given disease, the specificity or selectivity of an Fc variant to different polymorphic forms of these receptors can in part determine which studies or preclinical experiments may be appropriate for testing, and ultimately which patient populations may or may not respond to treatment. Thus, the specificity or selectivity of an Fc variant to the Fc receptor polymorphisms disclosed herein, including but not limited to FcγRIIa, FcγRIIIa, etc., can be used to guide the selection of effective studies and preclinical experiments, clinical trial designs, patient selection, dose dependence, and / or other aspects of clinical trials.

[0074] The Fc variants disclosed herein may include, but are not limited to, modifications that modulate interactions with Fc receptors other than FcγR, including complement proteins, FcRn, and Fc receptor homologs (FcRH). Examples of FcRH include, but are not limited to, FcRFH, FcRH2, FcRH3, FcRH4, FcRH5, and FcRH6 (Davis et al., 2002, Immunol. Reviews 190:123-136).

[0075] A key parameter determining the most beneficial selectivity of a given Fc variant for treating a given disease is the context of the Fc variant. Therefore, the selectivity or specificity of a given Fc variant to the Fc receptor will provide different properties depending on whether the antibody, Fc fusion, or Fc variant is composed with a binding fusion partner. In one embodiment, the Fc receptor specificity of an Fc variant disclosed herein determines its therapeutic utility. The utility of a given Fc variant for a therapeutic purpose will depend on the epitope or morphology of the target antigen and the disease or indication being treated. For some targets and indications, a higher FcγRIIb affinity and reduced activated FcγR-mediated effector function may be beneficial. For other target antigens and therapeutic applications, increasing affinity to FcγRIIb, or increasing affinity to both FcγRIIb and the activating receptor, may be beneficial.

[0076] Treatment of multiple sclerosis using anti-CD19 antibodies In some embodiments, the present invention provides a method for treating MS in subjects requiring treatment for MS using an anti-CD19 antibody or its antigen-binding fragment.

[0077] In some embodiments, a method for treating MS in a subject requiring treatment for MS, comprising administering an anti-CD19 antibody comprising a light chain and a heavy chain to the subject, wherein the light chain comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 2, LCDR2 containing the amino acid sequence of SEQ ID NO: 3, and LCDR3 containing the amino acid sequence of SEQ ID NO: 4, and the heavy chain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 5, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7, and the heavy chain comprises an Fc region containing amino acid substitutions 267E and L328F.

[0078] In some embodiments, a method for treating MS in a subject requiring treatment for MS, comprising administering an anti-CD19 antibody comprising a light chain and a heavy chain to the subject, wherein the light chain comprises a light chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 11, and the heavy chain comprises a heavy chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, and the heavy chain comprises an Fc region which comprises amino acid substitutions 267E and L328F.

[0079] In some embodiments, a method for treating MS includes administering obexerimab.

[0080] The method of administration may vary. Suitable routes of administration include various methods known in the art. In some embodiments, the anti-CD19 antibody is administered by injection. In some embodiments, the anti-CD19 antibody is administered intrathecally. In some embodiments, the anti-CD19 antibody is administered intravenously. In some embodiments, the anti-CD19 antibody is administered subcutaneously. In some embodiments, the anti-CD19 antibody is administered using a pre-filled syringe or an autoinjector.

[0081] In some embodiments, administration of an anti-CD19 antibody improves, alleviates, or delays the onset of one or more symptoms of MS. In some embodiments, administration of an anti-CD19 antibody improves or alleviates one or more symptoms of MS. In some embodiments, administration of an anti-CD19 antibody delays the onset of one or more symptoms of MS.

[0082] Pharmaceutical preparations and pharmaceutical compositions The present invention provides pharmaceutical compositions and formulations of an anti-CD19 antibody (e.g., obexerimab). The pharmaceutical compositions, formulations, and related methods of the present invention are useful for administering an anti-CD19 antibody (e.g., obexerimab) and for the treatment of MS and related diseases. The anti-CD19 antibody formulations disclosed herein are prepared for storage by mixing the antibody, having the desired purity, in the form of a lyophilized formulation or aqueous solution with any pharmaceutically acceptable carrier, excipient, or stabilizer (as fully incorporated by reference to Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980).

[0083] In some embodiments, the target pharmaceutical composition contains various concentrations of anti-CD19 antibody (e.g., obexerimab). In some embodiments, a suitable formulation may contain the target antibody at concentrations up to approximately 250 mg / ml (e.g., up to approximately 225 mg / ml, up to 200 mg / ml, up to 150 mg / ml, up to 140 mg / ml, up to 130 mg / ml, up to 125 mg / ml, up to 120 mg / ml, up to 115 mg / ml, up to 110 mg / ml, up to 105 mg / ml, up to 100 mg / ml, up to 90 mg / ml, up to 80 mg / ml, up to 70 mg / ml, up to 60 mg / ml, up to 50 mg / ml, up to 40 mg / ml, up to 30 mg / ml, up to 25 mg / ml, up to 20 mg / ml, up to 10 mg / ml).

[0084] In some embodiments, a suitable formulation may contain anti-CD19 antibody at a concentration in the range of about 10-300 mg / ml (for example, about 10-250 mg / ml, about 10-200 mg / ml, about 10-180 mg / ml, about 10-160 mg / ml, about 10-150 mg / ml, about 10-140 mg / ml, about 10-130 mg / ml, about 10-125 mg / ml, about 100-125 mg / ml, about 100-180 mg / ml, about 100-150 mg / ml, about 100-130 mg / ml, about 100-125 mg / ml, about 100-170 mg / ml, about 100-160 mg / ml, about 100-150 mg / ml, about 100-200 mg / ml, about 120-130 mg / ml).

[0085] In some embodiments, the formulation suitable for administration may contain an anti-CD19 antibody (e.g., obexerimab) at a concentration of approximately 100 mg / ml, approximately 115 mg / ml, approximately 120 mg / ml, approximately 125 mg / ml, approximately 130 mg / ml, approximately 135 mg / ml, approximately 140 mg / ml, approximately 145 mg / ml, approximately 150 mg / ml, approximately 200 mg / ml, or approximately 300 mg / ml.

[0086] In some embodiments, isotonic solutions are used. In some embodiments, slightly hypertonic solutions (e.g., up to 300 mM (e.g., up to 250 mM, 200 mM, 175 mM, 150 mM, 125 mM) of sodium chloride in 5 mM sodium phosphate at pH 7.0) and sugar-containing solutions (e.g., up to 2.4%, 2.0%, 1.5%, 1.0%) of sucrose in 5 mM sodium phosphate at pH 7.0) are used. In some embodiments, a preferred formulation composition is physiological saline (e.g., 150 mM NaCl in water).

[0087] Many therapeutic agents, particularly the antibodies of the present invention, require a controlled pH and specific excipients to maintain their solubility and stability in the pharmaceutical composition of the present invention.

[0088] The pH of the pharmaceutical composition is an additional factor that can alter the solubility of the anti-CD19 antibody (e.g., obexerimab) in the aqueous pharmaceutical composition. In some embodiments, the pharmaceutical composition of the present invention contains one or more buffers. In some embodiments, the composition according to the present invention contains a sufficient amount of buffer to maintain the optimal pH of the composition at about 4.0–8.0, about 5.0–7.5, about 5.5–7.0, about 6.0–7.0, and about 6.0–7.5. In other embodiments, the buffer contains sodium phosphate in a maximum of about 50 mM (e.g., up to about 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM). Suitable buffers include, for example, acetates, succinates, citrates, phosphates, other organic acids, and tris(hydroxymethyl)aminomethane ("Tris").

[0089] A suitable buffer concentration may be, for example, about 1 mM to about 100 mM, or about 3 mM to about 20 mM, depending on the desired isotonicity of the buffer and formulation. In some embodiments, suitable buffers are present at concentrations of about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM.

[0090] In some embodiments, the formulation contains an isotonic agent to maintain the formulation in an isotonic state. Exemplary isotonic agents include, but are not limited to, glycine, sorbitol, mannitol, sodium chloride, and arginine. In some embodiments, a preferred isotonic agent may be present in the formulation at a concentration of about 0.01 to 5% by weight (e.g., 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0, 4.0, or 5.0%).

[0091] In some embodiments, the formulation may contain a stabilizer to protect the antibody. Typically, preferred stabilizers are non-reducing sugars such as sucrose, raffinose, and trehalose, or amino acids such as glycine, arginine, and methionine. The amount of stabilizer in the formulation is generally such that the formulation is isotonic. However, hypertonic formulations may also be preferred. Furthermore, the amount of stabilizer should not be too low so that an unacceptable amount of antibody degradation / aggregation occurs. Exemplary stabilizer concentrations in the formulation may range from about 1 mM to about 400 mM (e.g., about 30 mM to about 300 mM, and about 50 mM to about 100 mM), or alternatively, 0.1% to 15% (e.g., 1% to 10%, 5% to 15%, 5% to 10%) (by weight). In some embodiments, the mass ratio of stabilizer to therapeutic agent is about 1:1. In other embodiments, the mass ratio of the stabilizer to the therapeutic agent may be about 0.1:1, 0.2:1, 0.25:1, 0.4:1, 0.5:1, 1:1, 2:1, 2.6:1, 3:1, 4:1, 5:1, 10:1, or 20:1. In some embodiments, a stabilizer suitable for freeze-drying is also a lyoprotectant.

[0092] In some embodiments, it is desirable to add a surfactant to the formulation. Exemplary surfactants include nonionic surfactants such as polysorbate (e.g., polysorbate 20 or 80); poloxamer (e.g., poloxamer 188); triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristalnidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium cocoyl methyl taurate, or disodium oleyl methyl taurate; and the MONAQUAT™ series (Mona Examples include polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics PF68). Typically, the amount of surfactant added is such that it reduces protein aggregation and minimizes particle or foam formation. For example, surfactants may be present in the formulation at concentrations of about 0.001–0.5% (e.g., about 0.005–0.05%, or 0.005–0.01%). In particular, surfactants may be present in the formulation at concentrations such as about 0.005%, 0.01%, 0.02%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%.

[0093] In some embodiments, a preferred formulation may further include one or more fillers, particularly for lyophilization and formylation. A “filler” is a compound that adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized cake. For example, a filler can improve the appearance of the lyophilized cake (e.g., an essentially homogeneous lyophilized cake). Preferred fillers include, but are not limited to, sodium chloride, lactose, mannitol, glycine, sucrose, trehalose, and hydroxyethyl starch. Exemplary concentrations of fillers are about 1% to about 10% (e.g., 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, and 10.0%).

[0094] The formulations according to the present invention can be evaluated based on product quality analysis, reconstitution time (if lyophilized), reconstitution quality (if lyophilized), molecular weight, moisture content, and glass transition temperature. Typically, protein quality and product analysis include product degradation rate analysis using methods including, but not limited to, size exclusion HPLC (SE-HPLC), cation exchange HPLC (CEX-HPLC), X-ray diffraction (XRD), modulated differential scanning calorimetry (mDSC), reversed-phase HPLC (RP-HPLC), multi-angle light scattering (MALS), fluorescence, ultraviolet absorption, turbidimetry, capillary electrophoresis (CE), SDS-PAGE, and combinations thereof. In some embodiments, evaluation of the product according to the present invention may include a step of evaluating its appearance (either liquid or cake appearance).

[0095] Generally, formulations (lyophilized or aqueous) can be stored for extended periods at room temperature. Storage temperatures can typically range from 0°C to 45°C (e.g., 4°C, 20°C, 25°C, 45°C, etc.). Formulations may be stored for periods ranging from several months to several years. Storage durations are commonly 24 months, 12 months, 6 months, 4.5 months, 3 months, 2 months, or 1 month. Formulations can be stored directly in the containers used for administration, eliminating the need for transfer.

[0096] The formulation can be stored directly in a lyophilized container (if lyophilized), which also serves as a reconstitution container, potentially eliminating the need for transfer. Alternatively, the lyophilized product formulation may be measured into smaller increments for storage. Storage should generally avoid situations that could lead to protein degradation, including but not limited to exposure to sunlight, UV radiation, other forms of electromagnetic radiation, excessive heat or cold, rapid thermal shock, and mechanical shock.

[0097] In some embodiments, the formulations according to the present invention are in liquid or aqueous form. In some embodiments, the formulations according to the present invention are lyophilized. Such lyophilized formulations may be reconstituted by adding one or more diluents to them before administration to a patient. Suitable diluents include, but are not limited to, sterile water, bacteriostatic water for injection, and sterile saline. Preferably, upon reconstitution, the antibodies contained therein are stable, soluble, and tolerable upon administration to a patient.

[0098] The pharmaceutical compositions of the present invention are characterized by their tolerability. As used herein, the terms “tolerable” and “tolerability” refer to the ability of the pharmaceutical compositions of the present invention to not cause an adverse reaction in a patient to whom such composition is administered, or, alternatively, to not cause a serious adverse reaction in a patient to whom such composition is administered. In some embodiments, the pharmaceutical compositions of the present invention are well accepted by the patient to whom such composition is administered.

[0099] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dose and concentration used and include buffers such as phosphates, citrates, acetates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; polyvinyl This material includes hydrophilic polymers such as lupyrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, sweeteners and other flavoring agents, fillers such as microcrystalline cellulose, lactose, corn, and other starches, binders, additives, colorants, salt-forming counterions such as sodium, metal complexes (e.g., Zn protein complexes), and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0100] In some embodiments, the antibody-containing pharmaceutical compositions disclosed herein may be in a water-soluble form, such as existing as a pharmaceutically acceptable salt, meaning that they contain both an acid and a base addition salt. A “pharmaceutically acceptable acid addition salt” means a salt that retains the biological efficacy of the free base and is not biologically or otherwise undesirable, and is formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid.

[0101] Examples of "pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Some embodiments include at least one of ammonium salts, potassium salts, sodium salts, calcium salts, and magnesium salts. Examples of pharmaceutically acceptable salts derived from organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.

[0102] The formulation used for in vivo administration may be sterile. In some embodiments, the formulation is sterilized by filtration through a sterile filtration membrane.

[0103] Non-limiting examples of buffers include phosphates, citrates, acetates, glutamates, carbonates, tartrates, triethanolamine (TRIS), glycylglycine, histidine, glycine, lysine, arginine, and other organic acids. More specifically, non-limiting examples of buffers include sodium HEPES, MES, potassium phosphate, potassium thiocyanate, sterilizers, TAE, TBE, ammonium sulfate / HEPES, BuffAR, sodium acetate, sodium carbonate, sodium citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium phosphate. Furthermore, buffers may be in various hydrate forms. For example, buffers may be monohydrates, dihydrates, trihydrates, tetrahydrates, pentahydrates, hexahydrates, heptahydrates, octahydrates, nunahydrates, decahydrates, elevenhydrates, and dodecahydrates. In some cases, the hydrate may be partial, such as hemihydrate or 1.5hydrate. Non-limiting examples of tonicity modifiers include sodium chloride, acetic acid, L-proline, dextrose, mannitol, potassium chloride, glycerin, and glycerol. Non-limiting examples of solvents include water, propylene glycol, polyethylene glycol, ethanol, dimethyl sulfoxide, N-methyl-2-pyrrolidone, Glycoflol, Solketal®, glycerol formal, acetone, tetrahydrofurfuryl alcohol, diglym, dimethyl isosorbide, and ethyl lactate. Non-limiting examples of solvents include polysorbates (e.g., polysorbate-20, polysorbate-80), polyoxyethylene sorbitan monooleate (Tween® 80), sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween® 20), sorbitan trioleate (Span 85), lecithin, and polyoxyethylene polyoxypropylene copolymers (Pluronics, Pluronic® F-68).

[0104] The amounts of anti-CD19 antibody (e.g., obexerimab), buffer, isotonic modifier, solvent, and surfactant may vary. In some embodiments, the anti-CD19 antibody (e.g., obexerimab) is formulated at a concentration of 125 mg / mL of obexerimab, 2.35 mg / mL of sodium acetate trihydrate, 0.17 mg / mL of acetic acid (density 1.053 g / mL), 30 mg / mL of L-proline, and 0.1 mg / mL of polysorbate 80, at a pH of 5.5. In some embodiments, the anti-CD19 antibody (e.g., obexerimab) is formulated at a concentration of 80-200 mg / mL of obexerimab, 1.5-3 mg / mL of sodium acetate trihydrate, 0.1-0.2 mg / mL of acetic acid (density 1.053 g / mL), 10-50 mg / mL of L-proline, and 0.05-0.2 mg / mL of polysorbate 80, with a pH of 5.0-6.0. In some embodiments, the anti-CD19 antibody (e.g., obexerimab) is formulated at a concentration of 122-127 mg / mL of obexerimab, 2.0-2.5 mg / mL of sodium acetate trihydrate, 0.15-0.19 mg / mL of acetic acid (density 1.053 g / mL), 25-35 mg / mL of L-proline, and 0.05-0.15 mg / mL of polysorbate 80, with a pH of 5.0-6.0.

[0105] In certain embodiments, the formulation comprises an anti-CD19 antibody (e.g., obexerimab), one or more buffers, one or more tonic modifiers, one or more solvents, and one or more surfactants. In some embodiments, the buffer may be a sodium acetate buffer. For example, the buffer may be sodium acetate trihydrate. In one embodiment, the tonic modifier may be acetic acid, L-proline, or a combination thereof. In another embodiment, the solvent is water.

[0106] In some embodiments, the surfactant is polysorbate. In some embodiments, the polysorbate is polysorbate-80. In some embodiments, the formulation comprises an anti-CD19 antibody (e.g., obexerimab), sodium acetate trihydrate, acetic acid and L-proline, water, and polysorbate-80.

[0107] In some embodiments, the formulation contains an anti-CD19 antibody (e.g., obexerimab) in an amount of about 1 mg to about 500 mg / ml, or about 50 mg to about 250 mg / ml, or about 100 mg to about 250 mg / ml, sodium acetate trihydrate in an amount of about 1 to about 10 mg / ml, or about 1 to about 5 mg / ml, or about 1 to about 2.5 mg / ml, acetic acid and L-proline in an amount of about 5 to about 50 mg / ml, or about 10 to about 50 mg / ml, or about 20 to about 40 mg / ml, water up to a maximum of about 1 ml, and polysorbate 80 in an amount of about 0.01 mg to about 1 mg / ml, or about 0.01 to about 0.5 mg / ml, or about 0.05 to about 0.2 mg / ml. Specifically, the formulation contains approximately 100 mg to 250 mg / ml of anti-CD19 antibody (e.g., obexerimab), approximately 1 to 2.5 mg / ml of sodium acetate trihydrate, approximately 20 to 40 mg / ml of acetic acid and L-proline, up to approximately 1 mg / ml of water, and approximately 0.05 to 0.2 mg / ml of polysorbate-80.

[0108] The anti-CD19 antibodies (e.g., obexerimab) disclosed herein may also be formulated as immunoliposomes. Liposomes are vesicles containing various types of lipids, phospholipids, and / or surfactants useful for the delivery of anti-CD19 antibodies (e.g., obexerimab) to mammals. Liposomes containing anti-CD19 antibodies (e.g., obexerimab) are prepared by methods known in the art. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556, which is fully incorporated by reference. In some embodiments, the anti-CD19 antibody (e.g., obexerimab) is formulated in liposomes produced by reverse-phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with a defined pore size to obtain liposomes with a desired diameter.

[0109] In some embodiments, an anti-CD19 antibody (e.g., obexerimab) is encapsulated in microcapsules prepared by methods including, but not limited to, coacervation techniques, interfacial polymerization (e.g., using hydroxymethylcellulose or gelatin microcapsules, or poly-(methyl metasylate) microcapsules), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), and macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed., 1980, and are fully incorporated by reference.

[0110] In some embodiments, sustained-release preparations of anti-CD19 antibodies (e.g., obexerimab) may be prepared. Suitable examples of sustained-release preparations include a semipermeable matrix of a solid hydrophobic polymer, the matrix of which may be in the form of a molded article, such as a film, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (US3,773,919, the whole being incorporated by reference), L-glutamic acid and gamma-ethyl-L-glutamate copolymers, non-degradable ethylene vinyl acetate, degradable lactate-glycolic acid copolymers such as Lupron Depot® (an injectable microsphere composed of lactate-glycolic acid copolymer and leuprolide acetate), poly-D-(-)-3-hydroxybutyrate, and ProLease® (commercially available from Alkermes), which is a microsphere-based delivery system composed of desired bioactive molecules incorporated into a poly-DL-lactide-co-glycolide (PLG) substrate.

[0111] In some embodiments, the liquid pharmaceutical composition contains 122-127 mg / ml of anti-CD19 antibody (e.g., obexerimab) (pH 5.0-6.0), 2.0-2.5 mg / ml of sodium acetate trihydrate, 0.15-0.19 mg / ml of acetic acid (density 1.053 g / ml), 25-35 mg / ml of L-proline, and 0.05-0.15 mg / ml of polysorbate 80.

[0112] In some embodiments, the liquid pharmaceutical composition is pH 5.5 and contains approximately 125 mg / ml of anti-CD19 antibody (e.g., obexerimab), 2.35 mg / ml of sodium acetate trihydrate, 0.17 mg / ml of acetic acid, 30 mg / ml of L-proline, and 0.1 mg / ml of polysorbate 80.

[0113] In some embodiments, the liquid pharmaceutical composition contains 122-127 mg / ml of obexerimab, 2.0-2.5 mg / ml of sodium acetate trihydrate (pH 5.0-6.0), 0.15-0.19 mg / ml of acetic acid (density 1.053 g / ml), 25-35 mg / ml of L-proline, and 0.05-0.15 mg / ml of polysorbate 80.

[0114] In some embodiments, the liquid pharmaceutical composition is pH 5.5 and contains approximately 125 mg / ml of obexerimab, 2.35 mg / ml of sodium acetate trihydrate, 0.17 mg / ml of acetic acid, 30 mg / ml of L-proline, and 0.1 mg / ml of polysorbate 80.

[0115] Listed embodiments Embodiment 1. A method for treating MS in a subject requiring treatment for MS, comprising administering to the subject an anti-CD19 antibody comprising an Fc region modified by amino acid substitutions S267E and L328F, wherein the anti-CD19 antibody comprises HCDR1 comprising the amino acid sequence SYVMH (SEQ ID NO: 5), HCDR2 comprising the amino acid sequence WIGYINPYNDGTKY (SEQ ID NO: 6), HCDR3 comprising the amino acid sequence GTYYYGTRVFDY (SEQ ID NO: 7), and light chain LCDR1 comprising the amino acid sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 2), LCDR2 comprising the amino acid sequence RMSNLNS (SEQ ID NO: 3), and LCDR3 comprising the amino acid sequence MQHLEYPIT (SEQ ID NO: 4), wherein the administration of the anti-CD19 antibody improves, alleviates, or delays the onset of one or more symptoms of MS.

[0116] Embodiment 2. A method for treating MS in a subject requiring treatment for MS, comprising administering to the subject an anti-CD19 antibody comprising an Fc region modified by amino acid substitutions S267E and L328F, wherein the anti-CD19 antibody comprises a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 12 and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 11, and the administration of the anti-CD19 antibody improves, reduces or delays the onset of one or more symptoms of MS.

[0117] Embodiment 3. The method according to Embodiment 1 or 2, wherein the anti-CD19 antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 12 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 11.

[0118] Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein the anti-CD19 antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 10 and a light chain containing the amino acid sequence of SEQ ID NO: 9.

[0119] Embodiment 5. The method according to any one of Embodiments 1 to 4, wherein the anti-CD19 antibody is obexerimab.

[0120] Embodiment 6. The method according to any one of Embodiments 1 to 5, wherein an anti-CD19 antibody is administered intrathecally.

[0121] Embodiment 7. The method according to any one of Embodiments 1 to 6, wherein an anti-CD19 antibody is administered intravenously. [Examples]

[0122] Example 1: Prevention of disease activity induction in the EAE model Surrogate antibodies against obexerimab (Obx) and rituximab (anti-CD20 antibody) were evaluated in an EAE model using MOG protein / Bordetella pertussis. Briefly, huFcγRIIb transgenic mice (2B-KIX mice) were treated twice weekly with a mouse surrogate for Obx (mObx), a mouse surrogate for rituximab, or a vehicle control (phosphate-buffered saline). Mice treated with Obx or a surrogate for rituximab were administered 10 mg / kg intraperitoneally. One week after the initial treatment, mice were immunized twice with a combination of human MOG protein and pertussis toxin according to a standard protocol. Disease severity was assessed daily for each individual mouse on a scale of 0 to 5 for 27 days, with an increase in clinical score corresponding to an increase in observed disease severity. Mice with a clinical score of 4 were euthanized. All mice in the vehicle control group were euthanized within 15 days of MOG injection. Two mice in the obexerimab surrogate group were euthanized 11 days after MOG injection. One mouse in the rituximab surrogate group was euthanized 13 days after MOG injection. The individual clinical scores of the mice are shown in Table 4 below. [Table 4]

[0123] After 27 days, mObx demonstrated prevention of disease as measured by standard clinical EAE scores in surviving mice at each time point (Figure 1A). Mice treated with mObx had a 33% EAE incidence rate of (2 / 6), while mice treated with a rituximab surrogate had an 86% EAE incidence rate of (6 / 7), and mice treated with a vehicle control had a 100% EAE incidence rate of (3 / 3). On day 5, after the initial treatment but before immunization with human MOG, immunocytological analysis of whole blood was performed in mice. Mice treated with mObx showed a similar percentage of blood B cells as mice treated with a vehicle control, but mice treated with a rituximab surrogate showed a significantly reduced percentage of blood B cells in total lymphocytes (Figure 1B). The ability of mObx to suppress disease induction in the MOG-induced EAE model suggests the potential efficacy of Obx in preventing the onset of MS without depleting B cells in the blood.

[0124] Example 2: Mouse obexerimab substitute reduces mouse BCR-mediated phagocytosis. In this example, we investigate whether BCR-mediated phagocytosis is inhibited by treatment with 30 μg / ml of mObx. B cells isolated from 2B-KIX mice were treated with mObx (Chu, SY, et al., 2021. J. Transl. Autoimmun. 4: 100075) to elucidate how different stages of B cell APC function and interaction with T cells are affected in the standard immune response to exogenous Ag. As shown in Figures 2A and 2B, mObx suppresses BCR-mediated Ag uptake and APC function and activates T cells ex vivo.

[0125] Splenocytes were isolated from 2B-KIX mice and treated for 1 hour with cytochalasin D (an actin polymerization inhibitor that blocks Ag uptake), medium alone, mObx (2480E), or anti-CD19 (2480F) that does not engage with hFcRIIb as an idiotype control. After treatment, 2m fluorescent beads coated with anti-mouse IgM and biotinylated OVA were added to the medium, and the cells were incubated at 37°C in 5% CO2 for a further 2 hours. Bead uptake was analyzed by flow cytometry. Fo B cells were identified as CD19+, CD4-, CD11b-, IgMlo, IgDhi, CD23hi, and CD21int. Cells that took up the beads were identified as Bead+, but were not stained with fluorescent streptavidin, indicating that OVA-bio was taken up into the cells and was not available for staining. The percentage of total Fo B cells in the taken-up beads is shown (Figure 2A). Each symbol represents a cell isolated from an individual mouse (**p<0.01, ****p<0.0001).

[0126] B cells were isolated from lymphoid tissue collected from 2B-KIX mice using negative selection beads and then treated with medium alone (no medium or treatment), mObx (2480E), or idiotype control (2480F) for 1 hour at 37°C with 5% CO2. Similarly, OVA-sp T cells were collected from lymphoid tissue of OT-II mice, labeled with CTViolet, and then added to B cell cultures with no Ag (medium) or the anti-IgM / OVA-bio beads described above (all other groups). The cells were co-cultured for 4 days, and then T cell proliferation was analyzed by flow cytometry. Proliferated T cells were identified as CD4+CD45R- cells using diluted CTViolet. One pilot example is shown (Figure 2B).

[0127] An ex vivo mouse B cell phagocytosis assay was also used. Spleen and peritoneal cells were isolated from wild-type 2B-KIX mice, incubated with 2 μm beads and mObx for 2 hours, and then B cell phagocytosis was evaluated by flow cytometry (Figures 3A-3D). The bead target particles were coated with both anti-mouse IgM, which targets the BCR in an antigen-independent manner, and biotinylated OVA to allow differentiation of internalized and extracellular beads using streptavidin staining. Observation of B cell phagocytosis revealed that Fo B2, MZ, and B1 B cells were all able to phagocytose the anti-IgM coated beads. This ability was completely abolished by treatment with cytochalasin D, a potent inhibitor of phagocytosis. Treatment with mObx at a concentration of 30 μg / ml significantly reduced phagocytic uptake of these beads compared to co-culture with an idiotype control (Figures 3E and 3F), demonstrating that mObx reduces mouse BCR-mediated antigen uptake by B cells.

[0128] Example 3: Obexerimab inhibits human BCR-mediated antigen uptake and phagocytosis. This example uses an ex vivo human B cell phagocytosis assay to investigate whether human BCR-mediated phagocytosis is inhibited by Obx treatment at 30 μg / ml.

[0129] Human blood was collected from healthy donors (n=4), B cells were isolated, and incubated with anti-human IgM conjugated beads and Obx for 2 hours. B cell phagocytosis was then evaluated by flow cytometry (Figures 4A–4D). The bead targets were coated with both anti-human IgM targeting the BCR, in an antigen-independent manner, and biotinylated OVA to distinguish between internalized and extracellular beads using streptavidin staining. Human Fo B2, MZ, and B1 B cells were all observed to be able to phagocytose the anti-human IgM coated beads. This ability was reduced by treatment with cytochalasin D, a potent inhibitor of phagocytosis. Treatment with Obx at a concentration of 30 μg / ml significantly reduced phagocytic uptake of these beads compared to untreated controls (Figures 4E and 4F), indicating that Obx reduces human BCR-mediated antigen uptake by B cells.

[0130] Example 4: Activation of B cells and T cells in response to antigen stimulation is reduced in the presence of an obexerimab surrogate (mObx). After B cells take up an antigen, they form a direct interaction with T cells, acting as antigen-presenting cells (APCs) that present the acquired antigen to T cells, leading to B cell activation. To evaluate whether treatment with mObx reduced B cell and T cell activation in response to B cell and T cell-dependent interactions, B cells (5 × 10) from 2B-KIX mice were used. 5 ) were pretreated with 30 μg / ml mObx for 1 hour, and then CTViolet-stained OT-II CD4 T cells (5 × 10) were processed. 5 ) and anti-mouse IgM biotinylated OVA conjugate beads (5 × 10 5B cells were co-cultured with beads for 4 days. Since T / B interaction is essential for B cell activation with T cell-dependent antigens, B cell expression of activation markers was analyzed using flow cytometry (Figure 5A). B cells co-cultured with bead-bound antigens and antigen-specific T cells showed increased expression of CD80 and CD86 on their surface. mObx treatment significantly reduced this upstimulation compared to untreated B cells (Figures 5B-5E). Compared to idiotype controls, B cells co-cultured with mObx showed less CD80 and CD86 expression on their surface, demonstrating that mObx-treated B cells are less co-stimulated and therefore less activated. B cells co-cultured with bead-bound antigens and antigen-specific T cells showed increased expression of CD11b and CD11c on their surface. Similarly, when B cells were co-cultured with mObx, this increase was reduced compared to untreated B cells and idiotype control-treated B cells (Figures 5F-5I). Overall, these findings suggest that treatment with mObx at a concentration of 30 μg / ml may reduce T cell-mediated B cell activation.

[0131] T cell activation markers were also evaluated using flow cytometry (Figure 6A). Antigen-specific CD4 T cells were observed to proliferate when co-cultured with antigen-binding beads and B cells. However, CD4 T cells cultured with B cells treated with 30 μg / ml mObx showed a significant decrease in CD4 T cell proliferation compared to co-culture with untreated B cells and B cells treated with idiotype controls (Figures 6B, 6C). Furthermore, CD4 T cells combined with mObx-treated B cells showed a significant decrease in surface-level CD44 expression compared to co-culture with untreated and idiotype-treated B cells (Figure 6D), confirming the decreased CD4 T cell activation and supporting the reduced proliferation. Looking at activated T cells (CD4+ CD44+), activated T cells from co-culture with mObx-treated B cells showed significantly reduced surface CD25 expression compared to untreated B cells. CD25 expression was also reduced compared to the idiotype control, but not statistically (Figure 6E, Figure 6F). Therefore, the activation status of proliferating CD4 T cells was also lower in the presence of mObx. However, proliferating B cells treated with mObx showed a similar number of divisions to untreated or idiotype-treated B cells (Figure 6G), indicating that proliferation itself was not affected. In summary, these data suggest that B cells treated with mObx have a reduced ability to activate CD4 T cells.

[0132] Example 5: Evaluation of B cell inhibition and depletion as approaches to limit systemic and local B cell function Some models of EAE require B cells to initiate. For example, B cells are present when the number of MOG-sp B cells is significantly increased in lymphoid tissue, and 2D2 IgH MOGIn spontaneously occurring EAE (sEAE) models, it is involved in the initiation of the autoimmune T cell response (possibly as APC) (Dang, AK, et al., Front. Immunol. 6: 470, Bettelli et al., 2006, and Krishnamoorthy, G., H. et al., 2006. J. Clin. Invest. 116: 2385-2392). In C57BL / 6 mice, human MOG (or bMOG) tag Diseases induced by humanized MOGs (such as proteins) depend on the production of anti-MOG antibodies for initiation (Lyons, J.-A., et al., 2002. Eur. J. Immunol. 32: 1905-1913, and Lu, Y., et al., 2022. J. Immunol. 209: 2083-2092). The production of high-affinity antibodies depends on the germinal center response, which is MHC class II dependent but not equivalent to its function as a specialized APC activating naive T cells. (Newly described PLP) ECD The induction model is unique in that it requires B-cell APC function (rather than antibody formation) to induce and maintain disease progression (Boyden, AW, et al., 2020. Sci. Rep. 10: 5011, and Wilhelm, CR, et al., 2023. J. Immunol. Ji2200721). mMOG tag In other EAE induction models involving antigen recognition by both T cells and B cells, such as inducible EAEs, B cells are not essential for disease development, but MOG-sp B cells appear to contribute to more severe disease (Dang, A. K et al., 2015. J. Neuroimmunol. 278C: 73-84).

[0133] Recent studies on B cell APC function do not distinguish, or cannot distinguish, between B cell APC function that may occur in lymphoid tissue and B cells that may occur in inflamed meninges. Targeting B cells in this region is difficult due to the blood-brain barrier / meninges barrier. In fact, a recent study of anti-CD20 treatment in sEAE mice showed that intravenously administered (iv) anti-CD20 rapidly bound to B cells in lymphoid tissue, but was excluded primarily from meningeal B cells, even as inflammation that “disrupts” the blood-brain barrier progressed (Tesfagiorgis, Y., E. et al., 2023. Systemic Administration of Anti-CD20 Indirectly Reduces B Cells in the Inflamed Meninges in a Chronic Model of Central Nervous System Autoimmunity). Lehmann-Horn et al. attempted to directly target CNS and meningeal B cells using intrathecal (it) administration of anti-CD20 (Lehmann-Horn, KS, et al., 2014. Ann. Clin. Transl. Neurol. 1: 490-496). However, these experiments showed that peripheral B cells were depleted to a similar extent as when anti-CD20 was administered intravenously (iv), indicating that intrathecal (it) administration is not limited to the CNS.

[0134] This example evaluates obexerimab (Obx), a bispecific monoclonal antibody that specifically binds to inhibitory FcγRIIb on CD19-expressing B cells and inhibits its function without destroying the cells in a mouse model of MS (Chu, SY, et al., 2008. Mol. Immunol. 45: 3926-3933). To determine whether inhibition of peripheral B cell function can prevent disease induction and progression, anti-CD20 or mObx was intravenously administered to PLPECD-induced EAE mice (this model has been shown to be dependent only on B cells, not antibodies, in disease progression (Boyden et al., 2020, and Wilhelm et al., 2023)) one day before PLPECD immunization, or 14 days after immunization, which corresponds to the time of disease onset. The disease course was tracked, and the mice were euthanized when they reached the chronic phase of the disease (2 weeks after onset). Blood and lymph nodes will be analyzed by flow cytometry to quantify B cell counts, confirm binding of anti-CD20 antibody or mObx, and assay T cell activation and differentiation into effector subsets. Spinal cord will be collected for immunofluorescence histochemistry of T and B cell inflammation in the meninges and white matter, as well as for demyelination, as previously described (Dang, AK, et al., 2015. Front. Immunol. 6: 470, Dang, AK, et al., 2015. J. Neuroimmunol. 278C: 73-84, and Tesfagiorgis, Y., et al., 2017. J. Immunol. 199: 449-457). Separate flow cytometry experiments on spinal cord cells will be used to quantify T and B cell counts in the tissue and determine the degree of B cell binding by intravenously administered anti-CD20 or mObx. This experiment will be conducted separately in male and female mice to determine whether there are sex differences in the therapeutic response to mObx.

[0135] Example 6: Inhibition of meningeal B cell function and disease progression Intrameningeal (it) administration of monoclonal antibodies bypasses the blood-brain barrier and blood-meningeal barrier, but does not prevent leakage of it-administered antibodies to the periphery. Therefore, intravenous administration may be used to treat B cells outside the CNS, while intrameningeal administration targets B cells in both the CNS / meninges and the periphery.

[0136] PLP ECD EAE mice induced by [method / technology] are treated with anti-CD20 or mObx pre-disease (1 day after immunization), acute (5 days after onset), or chronic (2 weeks after onset) phases. The disease course is followed, and at the end of the experiment, spinal cord is collected for analysis of the local pathology and inflammation described above. Meningeal cluster tissue, T cell-to-B cell ratio, and physical interactions between these cells within the cluster tissue are evaluated because B cell inhibition may alter T:B interactions and localization. Flow cytometry is used to confirm that B cells are bound to the intrathecally administered drug, and the number of infiltrating B and T cells, as well as their activation status, is quantified.

[0137] Example 7: Effects of B cell inhibition on T cell:B cell interactions in the meninges Meningeal B cells can influence local inflammation through local APC function and reactivate newly recruited autoimmune T cells by presenting locally acquired autoantigens (Pipi et al., 2018). Actual presentation of Ag arises from highly coordinated direct physical interactions between cells. This embodiment evaluates the effect of mObx inhibition of B cell function on T:B interactions in lymph nodes or meninges. Interactions between MOG-sp T cells and B cells are directly visualized using in vivo microscopy in the early stages of the anti-MOG response in lymph nodes. MOG-specific B cells form germinal centers by presenting Ag to anti-MOG T cells in the early stages of the post-immunization response using mMOGtag (Jain et al., 2018, Parham et al., 2022).

[0138] Activated MOG-sp RFP+ 2D2 T cells are transferred into 2B-KIX mice at the acute phase of the disease in mMOG tag -induced or PLP ECD -induced EAE. At the same time, mice are treated i.t. with mObx or isotype control. 2 or 7 days after treatment, spinal cords are harvested for immunofluorescent histology, thereby determining whether the association between RFP+ T cells and B cells is reduced, the number of T cells is decreased, or the number of these T cells present in the parenchyma or meninges is reduced in the treated mice.

[0139] As described above, in vivo microscopy of the inflamed spinal cord is used to directly visualize T cell interactions with meningeal B cells after mObx treatment. Transplanted RFP + The interaction time between 2D2 T cells and endogenous B cells (B cell-specific conditional GFP mice (CD19cre×ROSA26-EGFP mice as recipients)) is quantified. Furthermore, imaging at the base of the meningeal clusters is performed to assess whether autoreactive T cells cross the underlying white matter boundary after interaction with B cells following treatment with mObx.

[0140] Other Embodiments While several embodiments of the present invention are described herein, the present disclosure and examples may be modified to provide other methods and configurations of the present invention. It will therefore be understood that the scope of the present invention is to be defined by the appended claims, in addition to the specific embodiments presented by way of example. All references cited herein are hereby incorporated by reference.

Claims

1. A method for treating multiple sclerosis (MS) in a person requiring treatment for multiple sclerosis (MS), The procedure involves administering an anti-CD19 antibody containing light and heavy chains to the subject, The light chain comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 2, LCDR2 containing the amino acid sequence of SEQ ID NO: 3, and LCDR3 containing the amino acid sequence of SEQ ID NO:

4. The heavy chain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 5, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO:

7. A method wherein the heavy chain includes an Fc region containing amino acid substitutions 267E and L328F.

2. A method for treating multiple sclerosis (MS) in a person requiring treatment for multiple sclerosis (MS), The procedure involves administering an anti-CD19 antibody containing light and heavy chains to the subject, The light chain includes a light chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO:

11. The heavy chain includes a heavy chain variable region which is at least 90% identical to the amino acid sequence of SEQ ID NO:

12. A method wherein the heavy chain includes an Fc region containing amino acid substitutions 267E and L328F.

3. The method according to claim 1 or 2, wherein the anti-CD19 antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 12 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

11.

4. The anti-CD19 antibody comprises a heavy chain and a light chain, The heavy chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of Sequence ID No.

10. The method according to any one of claims 1 to 3, wherein the light chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

9.

5. The aforementioned anti-CD19 antibody A heavy chain containing the amino acid sequence of SEQ ID NO: 10, and The method according to any one of claims 1 to 4, comprising a light chain containing the amino acid sequence of SEQ ID NO:

9.

6. The method according to any one of claims 1 to 5, wherein the anti-CD19 antibody is obexerimab.

7. The method according to any one of claims 1 to 6, wherein the administration of the anti-CD19 antibody improves, alleviates, or delays the onset of one or more symptoms of MS.

8. The method according to any one of claims 1 to 7, wherein the anti-CD19 antibody is administered intrathecally.

9. The method according to any one of claims 1 to 8, wherein the anti-CD19 antibody is administered intravenously.

10. The method according to any one of claims 1 to 9, wherein the anti-CD19 antibody is administered subcutaneously.

11. The method according to any one of claims 1 to 10, wherein the anti-CD19 antibody is administered at pH 5.5 in a liquid formulation containing 125 mg / ml of anti-CD19 antibody, 2.35 mg / ml of sodium acetate trihydrate, 0.17 mg / ml of acetic acid, 30 mg / ml of L-proline, and 0.1 mg / ml of polysorbate 80.

12. The method according to any one of claims 1 to 11, wherein the anti-CD19 antibody is administered using a pre-filled syringe or an autoinjector.

Images