Methods and compositions for treating multiple sclerosis

Abstract

The present invention provides methods of treating and ameliorating symptoms of multiple sclerosis using an antibody or antigen-binding fragment thereof that specifically binds human CD19 (e.g., ocrelizumab).

Description

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

[0002] sequence list This application contains a sequence list, which is submitted electronically in XML file format and incorporated herein by reference in its entirety. The XML copy created on September 19, 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 mechanisms of the disease are not fully understood, MS is characterized by inflammation, demyelination, and neurodegeneration, as well as the formation of plaques or lesions within the CNS. Symptoms of MS are often varied and difficult to predict, but individuals with MS may experience pain, fatigue, cognitive difficulties, depression, limited mobility, lack of coordination, speech or swallowing difficulties, various visual disturbances, and many other symptoms. Although disease-modifying therapies have been approved for the treatment of MS, there is currently no known cure for MS.

[0004] 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 with MS undergoing long-term treatment may predictably lose most of a vital branch of the immune system, essential for effective and protective immune responses, over decades. The long-term health outcomes of maintaining B cell depletion for most of a person's lifespan are unknown, but it may make individuals less able to mount a protective response to vaccines and increase susceptibility to infections. Furthermore, when an individual chooses to discontinue anti-CD20 therapy, recovery to normal immune function may potentially take months, while B cells re-proliferate from the bone marrow (Margoni et al., 2022). Summary of the Invention

[0005] This invention provides a safer and more effective treatment for MS based on anti-CD19 antibodies, such as obexelimab. Among other things, the anti-CD19 antibody-based therapies described herein (e.g., obexelimab-based therapies) significantly reduce side effects and significantly benefit subjects with MS who require discontinuation of treatment and restoration of protective immune function due to the significant risk of immunosuppression (as highlighted by the COVID pandemic). Other anti-CD19 antibodies with the ability to reduce or suppress B cells without depletion are also anticipated for the treatment of MS.

[0006] In one aspect, the present invention provides a method for treating multiple sclerosis in a subject of need, the method comprising administering to the subject an anti-CD19 antibody comprising a light chain and a heavy chain, 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 wherein 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 wherein the heavy chain comprises an Fc region containing amino acid substitutions for 267E and L328F.

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

[0008] In some embodiments, the anti-CD19 antibody comprises a light chain containing a light chain variable region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing a heavy chain variable region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, wherein the light chain contains a light chain variable region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 11, and wherein the heavy chain contains a heavy chain variable region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a heavy chain and a light chain, wherein the heavy chain contains an amino acid sequence having at least 90% identity with SEQ ID NO: 10; and wherein the light chain contains an amino acid sequence having at least 90% identity with 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 implementations, the anti-CD19 antibody is obbelimumab.

[0010] In some implementations, administration of anti-CD19 antibodies can improve or alleviate one or more symptoms of MS or delay its onset.

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

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

[0013] In some implementations, anti-CD19 antibodies are administered subcutaneously.

[0014] In some implementations, obbelimumab is administered in the form of a liquid formulation comprising 125 mg / ml obbelimumab, 2.35 mg / ml sodium acetate trihydrate, 0.17 mg / ml acetic acid, 30 mg / ml L-proline, 0.1 mg / ml polysorbate 80, at a pH of 5.5.

[0015] In some implementations, anti-CD19 antibodies are administered using a prefilled syringe or an auto-injector. Attached Figure Description

[0016] The accompanying drawings are for illustrative purposes only and are not intended to be limiting.

[0017] Figure 1A-Figure 1B This study illustrates the prevention of disease induction in an experimental autoimmune encephalomyelitis (“EAE”) model. An obbelimumab alternative antibody was evaluated against a rituximab alternative antibody and a control vector in a preclinical EAE model. Figure 1A The prevention of disease with obbelimumab alternatives is shown, as measured by the standard clinical EAE score of surviving mice at each time point (the number of surviving mice in each treatment group at each time point is indicated below the x-axis). Figure 1B The results showed that, compared with the reduction measured in mice treated with rituximab alternatives, the obelimab alternative was not associated with a significant reduction in blood B cells (p<0.0001).

[0018] Figures 2A-2B This study illustrates the inhibition of ovalbumin (OVA) antigen (Ag) uptake and Ag-presenting cell (APC) function in mouse B cells. Figure 2A The inhibition of Ag-coated bead uptake by follicular (Fo) B cells by the mouse alternative (mObx; 2480E) compared to the anti-CD19 idiotype (2480F) is shown, as measured by flow cytometry. Internalized beads bound to the cell membrane and external beads 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 a single mouse (**p < 0.01, ****p < 0.0001). Figure 2B This demonstrates T cell activation via Ag-loaded B cells. In vitro Inhibited by mObx. OVA-sp T cells (purified OT-II TCR transgenic T cells) were labeled with Cell Tracker Violet (CTViolet) and co-cultured with B cells without Ag (with culture medium) or with beads loaded with anti-IgM biotinylated OVA (all other conditions) and with mObx or a control. Flow cytometry was used to identify proliferating T cells with diluted intracellular CTViolets.

[0019] Figures 3A-3FThis study illustrates the reduced 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 for 2 hours at 37°C and 5% CO2 with 2 μm goat anti-mouse IgM F(ab')2 and biotinylated OVA-conjugated beads. Cells were then pretreated for 1 hour at 37°C and 5% CO2 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). Figure 3A A fixable viable dye (FVD) is shown for distinguishing between live and dead cells before identifying B cell subsets. Figure 3B The use of FACS to identify Fo B cells as B220+, CD4-, CD11b-, IgMlo, IgDhi, CD21int, and CD23+ is shown. Figure 3C MZ B cells identified using FACS as B220+, CD4-, CD11b-, IgMhi, IgDlo, CD21hi, and CD23int are shown. Figure 3D B1 cells identified as B220+, CD4-, F4 / 80-, IgMhi, IgDlo, CD21-, CD23- are shown. Figure 3E Streptomycin staining, used to distinguish between internalized and extracellular beads, is shown. Figure 3F The mean percentage of FoB, MZ, and B1 cells in beads with internalized goat anti-mouse IgM F(ab')2 coating is shown. Each symbol represents data from cells isolated from a single mouse (n=4) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant).

[0020] Figures 4A-4F This study illustrates the reduced BCR-mediated antigen uptake by human FoB, MZ, and B1 B cell subsets. Primary human B cells were isolated from healthy donors and pretreated for 1 hour at 37°C and 5% CO2 with Obx (30 µg / ml) or cytochalasin D (30 µg / ml). Flow cytometry was then used to assess B cell phagocytosis. Figure 4A A fixable viable dye (FVD) is shown for distinguishing between live and dead cells before identifying B cell subsets. Figure 4BFo B cells identified using FACS as CD20+, CD4-, CD27-, CD43-, and CD70- are shown. Figure 4C MZ B cells identified using FACS as CD20+, CD4-, CD27+, IgDhi, and IgMhi are shown. Figure 4D B1-like cells identified as CD20+, CD4-, CD27+, CD43+, and CD70- are shown. Figure 4E Streptomycin staining, used to distinguish between internalized and extracellular beads, is shown. Figure 4F The mean percentage of FoB, MZ, and B1-like cells with internalized anti-human IgM-coated beads is shown. Each symbol represents data from cells isolated from a single donor (n=4) (*p < 0.05, ns not significant).

[0021] Figures 5A-5I This study illustrates that mObx-treated B cells exhibit 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 for 4 days at 37°C and 5% CO2 with OT-II T cells and beads conjugated with anti-mouse IgM biotinylated OVA. Prior to incubation with the beads, some cells were pretreated for 1 hour at 37°C and 5% CO2 with mObx (30 µg / ml) or its idiotype control (30 µg / ml). Figure 5A A flow cytometry scatter plot of B cells identified as CD45R+CD4- is shown. Figure 5B The histogram of CD80 is shown. Figure 5C The mean fluorescence intensity (MFI) of CD80 under different treatment conditions is shown. Figure 5D The histogram of CD86 is shown. Figure 5E The MFI of CD86 under different processing conditions is shown. Figure 5F The histogram of CD11b is shown. Figure 5G The MFI of CD11b under different processing conditions is shown. Figure 5H The histogram of CD11c is shown. Figure 5I MFI of CD11c under different treatment conditions is shown. Each symbol represents data from cells isolated from a single pair of mice (n=3) (*p < 0.05, **p < 0.01).

[0022] Figures 6A-6GThis study illustrates the reduced ability of mObx-treated B cells 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 for 4 days at 37°C and 5% CO2 with 2B-KIX B cells and beads conjugated with anti-mouse IgM biotinylated OVA. Prior to incubation with the beads, some cells were pretreated for 1 hour at 37°C and 5% CO2 with mObx (30 µg / ml) or its idiotype control (30 µg / ml). Figure 6A The image shows an identification as CD4. + CD45R - CD4 T cells. Figure 6B A representative flow plot showing the percentage of OT-II T cell proliferation stained with CTViolet is shown. Figure 6C The average percentage of total OT-II T cell proliferation is shown. Figure 6D The mean MFI of CD44 expressed on the surface of OT-II T cells is shown. Figure 6E It is shown in CD44 + The mean MFI of CD25 expressed on the upper surface of OT-II T cells. Figure 6F It is shown in CD44 + Representative histogram of MFI for CD25 expressed on the upper surface of OT-II T cells. Figure 6G The mean percentage of OT-II CD4 T cells in each proliferation cycle is shown. Each symbol represents data from cells isolated from a single pair of mice (n=3) (*p < 0.05, **p < 0.01, ***p < 0.001). Detailed Implementation

[0023] The present invention particularly provides a method for treating MS in a subject of need, the method comprising administering to the subject an anti-CD19 antibody comprising an Fc region modified by amino acid substitutions of S267E and L328F, wherein administration of the anti-CD19 antibody improves, alleviates, or delays one or more symptoms of MS or its onset. In some embodiments, the anti-CD19 antibody is obelizimab.

[0024] In some implementations, one or more symptoms of MS include pain, fatigue, cognitive difficulties, depression, limited mobility, lack of coordination, speech or swallowing difficulties, and various visual impairments, as well as many other symptoms.

[0025] Various aspects of the invention are described in detail in the following sections. The use of sections is not intended to limit the invention. Each section can be applied to any aspect of the invention. In this application, unless otherwise stated, the use of "or" means "and / or".

[0026] definition This article describes several definitions. Such definitions are intended to cover syntactic equivalents.

[0027] Antibody The term "antibody" as used herein refers to a protein comprising one or more polypeptides substantially encoded by all or part of a recognized immunoglobulin gene. Recognized immunoglobulin genes (e.g., in humans) include the κ (K), λ (l), and heavy chain loci, which together constitute numerous variable region genes, and the constant region gene γ (y), which encodes isotypes of IgG (IgG1, IgG2, IgG3, and IgG4). The term "antibody" as used herein refers to both full-length antibodies and antibody fragments, and may refer to naturally occurring antibodies, engineered antibodies, or recombinant antibodies derived from any organism for experimental, therapeutic, or other purposes.

[0028] Effector Function As used herein, the term "effective function" refers to a biochemical event resulting from the interaction of the antibody's Fc region with an Fc receptor or ligand. Effective functions include FcγR-mediated effective functions (such as ADCC and ADCP) and complement-mediated effective functions (such as CDC). Furthermore, effective functions include FcγRIlb-mediated effective functions, such as inhibitory functions, such as downregulation, reduction, or suppression of B cell responses (e.g., humoral immune responses).

[0029] Fc or Fc area As used herein, the term "Fc" or "Fc region" refers to a polypeptide containing a constant region of an antibody that does not include the first constant region immunoglobulin domain and, in some cases, a portion of the hinge. Therefore, Fc can refer to the last two constant region immunoglobulin domains of IgG and the N-terminus of the flexible hinge of these domains. For IgG, Fc contains the immunoglobulin domains Cγ2 and Cγ3 (Cgamma2 and Cgamma3) and the hinge between Cγ1 (Cgamma1) and Cγ2. Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is generally defined as the residues C226 or P230 contained at its carboxyl terminus, numbered according to the EU index as described in Kabat. Fc can refer to this region as a separate region, or, in the case of an Fc polypeptide, to this region as described below.

[0030] Fc γ receptor or FcγR As used herein, the term “Fcγ receptor” or “FcγR” refers to any member of the protein family that binds to the Fc region of IgG antibodies 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 alloforms H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including alloforms V158 and F158) and FcγRIIIb (including alloforms FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, which is incorporated herein by reference in its entirety), and any undiscovered human FcγR or FcγR isoforms or alloforms. FcγR can 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 isoforms or allotypes.

[0031] Modification The term "modification" as used herein refers to an alteration in the physical, chemical, or sequence properties of a protein, polypeptide, antibody, or immunoglobulin. Modifications described herein include amino acid modifications (including amino acid substitutions) and glycoform modifications.

[0032] target antigen As used herein, the term "target antigen" refers to a molecule bound by a fusion partner of a variable region or Fc fusion of a given antibody. Target antigens can be proteins, carbohydrates, lipids, or other chemical compounds. Based on their affinity for the target antigen, an antibody or Fc fusion is said to be "specific" to a given target antigen. In some embodiments, the target antigen of obbelimumab is CD19.

[0033] target cells As used herein, the term “target cell” refers to a cell that expresses the target antigen.

[0034] treat As used in this article, the term “treatment” means to cover improving, alleviating or delaying the onset of one or more symptoms of a disease or condition.

[0035] Obelimab As used herein, the term "obelizumab" is an Fc-engineered humanized monoclonal antibody (mAb) that binds to the human B-cell restriction surface antigen CD19 and has enhanced Fc binding affinity. Receptor IIb (Fc) Fc binding of RIIb). The molecule is IgG1 immunoglobulin with a κ light chain and two amino acid substitutions in the constant portion of the heavy chain. Obelimab is a monoclonal antibody with an expected mass of approximately 147,426 Da based on its amino acid sequence. The heavy and light chains of obbelimab are given by SEQ ID NO: 10 and SEQ ID NO: 9, respectively.

[0036] Multiple sclerosis (MS) MS is a chronic and potentially disabling immune-mediated CNS disease characterized by progressive destruction of the myelin sheath. MS manifests as a variety of neurological symptoms, such as motor paralysis, sensory impairment, higher brain dysfunction, vision loss, and urinary difficulties, due to inflammation of the perineurium myelin proteins caused by the infiltration of autoreactive lymphocytes (e.g., T cells or B cells) into the brain, spinal cord, or optic nerve. Patients with MS experience transient and recurrent inflammation in different sites of the CNS. With each type of inflammation, the presentation of neurological symptoms depends on the site of inflammation.

[0037] Although the disease mechanisms are not fully understood, B cells play a role in the progression of MS. 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 not performed by B cells themselves, but by activated cells differentiated into plasma cells. High-affinity antibodies are crucial for protective pathogen-specific immune responses following infection or vaccination. However, while high-affinity autoantigen (Ag)-specific antibodies do contribute to the pathology of MS-related conditions NMOSD and MOGSD (Jain, RW et al., 2022. Nat. Rev. Immunol. 22: 513–524), antibody production is not the primary pathological contribution of B cells to MS. In fact, while therapeutic anti-CD20 antibodies effectively eliminate B cells, they do not eliminate antibody-producing plasma cells, nor do they reduce antibody levels over the time frame 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 sustained inflammatory response that underlies disease progression.

[0038] One of the leading candidate mechanisms for B cell-driven CNS autoimmunity is through the presentation of B cell-derived Ag to T cells. This, combined with any cytokines that B cells may simultaneously produce, can directly activate naive autoimmune T cells in lymphoid tissues and influence their differentiation into effector subsets. The Ag presentation process requires Ag-presenting cells (APCs) to collect and internalize protein Ag, then process it to load it onto the major histocompatibility complex (MHC). This peptide:MHC combination is then transported to the cell surface and exposed to migrating T cells that sample local APCs targeting homologous Ag via their specific T cell receptors (TCRs).

[0039] Although B cells are described as one of three “specialized” APCs (alongside dendritic cells and macrophages), their role as T cell activators is not well-known. In fact, their APC function is best understood in the context of high-affinity antibody responses, where B cell activation, rather than T cell activation, is the primary outcome. These events occur within specialized structures called germinal centers (GCs) within secondary lymphoid tissue. In this context, naive B cells encountering an Ag that binds to its specific B cell receptor (BCR) internalize the Ag (and any substances physically bound to it) and present it to activated T follicular helper (Tfh) cells that are specifically activated to the same antigen. These homologous interactions are essential for the full activation of B cells and form the basis of the mechanism for selecting high-affinity B cells in GCs and for differentiating them into plasma cells to produce antibodies (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).

[0040] B cells can present Ag to T cells, but other factors leading to T cell activation are less clear. Because B cells selectively acquire Ag via BCR binding, they are known to concentrate low concentrations of Ag more efficiently than other APCs, and for this reason, they have been involved in the initiation of some autoimmune T cell responses (Rodríguez-Pinto, D. 2005. Cell. Immunol. 238: 67–75). However, B cells specific to any given antigen are extremely rare, and B cells are usually physically separated from naive T cells in lymphoid tissues; therefore, it is unclear when or where such naive T cell:homogeneous B cell interactions might occur within the lymphoid microanatomy.

[0041] While activated T cells readily infiltrate demyelinating lesions of MS, B cells are relatively rare within the CNS parenchyma (Jain and Yong, 2022). Instead, a significant number of B cells can collect alongside T cells in the meninges, often 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 can sometimes become sufficiently ordered to resemble lymphoid tissue with separate T-cell regions and B-cell follicles, but most often they are a disordered mixture of these cells. Due to their association with the lesion and because their presence is associated with more severe disease (Jain and Yong, 2022), there is great interest in these clusters as potential sites where B cells can perform pathological functions. Because they can resemble secondary lymphoid tissue, these structures can maintain autoimmune antimyelin responses originating behind the blood-brain / meningeal barrier (Pipi, E. et al., 2018. Front. Immunol. 9). In this context, B cells can act as APCs to present locally acquired CNS-derived autoantigens to recently recruited effector T cells, leading to their reactivation and driving inflammation against oligodendrocytes in the local parenchyma. Therefore, B cells can function as APCs to drive autoimmune responses from multiple sites, including the peripheral lymphoid system and the meninges.

[0042] MS disease model MS only affects humans, but certain aspects 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 general term that encompasses many “artificially” generated antimyelin autoimmune responses that lead to immune-mediated demyelination in the CNS.

[0043] Exemplary EAE models and model Ag systems primarily based on the autoantigen myelin oligodendrocyte glycoprotein (MOG) are described in Table 1. Improved disease models are described in this application, particularly in the examples section.

[0044] Table 1: Mouse strains and model antigens. Anti-CD19 antibody According to the present invention, an anti-CD19 antibody is used to treat human patients with MS. In some embodiments, the anti-CD19 antibody has been enhanced to bind to the Fc of FcγRIIb. In some embodiments, the anti-CD19 antibody comprises amino acid modifications S267E and L328F, wherein the amino acid numbers are based on the EU index according to Kabat.

[0045] This article describes an exemplary anti-CD19 antibody.

[0046] Obelimab In some implementations, obelilimab is used to treat human patients with MS. In one aspect, the present invention provides a method of administering obelilimab for the treatment of MS. Obelilimab is a monoclonal antibody specific to CD19, the monoclonal antibody comprising: a light chain, the light chain including a variable region having: CDR1 containing RSSKSLQNVNGNTYLY (SEQ ID NO: 2), CDR2 containing RMSNLNS (SEQ ID NO: 3), and CDR3 containing MQHLEYPIT (SEQ ID NO: 4); and Heavy chain, which contains a variable region, the variable region having CDR1 containing SYVMH (SEQ ID NO: 5), CDR2 containing WIGYINPYNDGTKY (SEQ ID NO: 6), and CDR3 containing GTYYYGTRVFDY (SEQ ID NO: 7), Compared to SEQ ID NO: 8, the heavy chain contains amino acid substitutions S267E and L328F in the Fc region: The numbering is based on the EU index, as in Kabat.

[0047] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8) In some embodiments, obbelimab comprises a light chain containing the following amino acid sequence: DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 9); And a heavy chain, which contains the following amino acid sequence: EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10).

[0048] Table 2: Heavy and light chain amino acid sequences of obbelimumab Table 3: Kabat heavy and light chain CDRs of obbelimumab. In some embodiments, obembelimab includes light chain variable regions and heavy chain variable regions as shown in Table 2. In some embodiments, obembelimab includes CDRs as shown in Table 3.

[0049] In some embodiments, the anti-CD19 antibody comprises a light chain containing the amino acid sequence of 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. In some embodiments, the anti-CD19 antibody comprises both a light chain and a heavy chain, wherein the light chain contains the amino acid sequence of SEQ ID NO: 9, and wherein the heavy chain contains the amino acid sequence of SEQ ID NO: 10.

[0050] In some embodiments, the anti-CD19 antibody comprises a light chain, wherein the light chain includes 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, wherein the heavy chain includes 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, wherein the light chain includes a light chain variable region containing the amino acid sequence of SEQ ID NO: 11, and wherein the heavy chain includes 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, wherein the heavy chain includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 12, and wherein the heavy chain includes 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, wherein the light chain comprises a light chain variable region containing the amino acid sequence of SEQ ID NO: 11, and wherein the heavy chain comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 10, and wherein the heavy chain comprises an Fc region containing amino acid substitutions S267E and L328F.

[0051] In some embodiments, the anti-CD19 antibody comprises a light chain containing LCDR1 with the amino acid sequence of SEQ ID NO: 2, LCDR2 with the amino acid sequence of SEQ ID NO: 3, and LCDR3 with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1 with the amino acid sequence of SEQ ID NO: 5, HCDR2 with the amino acid sequence of SEQ ID NO: 6, and HCDR3 with the amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1 with the amino acid sequence of SEQ ID NO: 5, HCDR2 with the amino acid sequence of SEQ ID NO: 6, and HCDR3 with the amino acid sequence of SEQ ID NO: 7, and wherein the heavy chain contains an Fc region containing amino acid substitutions for S267E and L328F. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, 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 wherein 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. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, 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 wherein 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 wherein the heavy chain comprises an Fc region containing amino acid substitutions for S267E and L328F.

[0052] Obeliximab works by modulating B-cell receptor (BCR) signaling via FcγRIIb. Obeliximab binds to CD19 of the BCR complex, and its Fc receptor is engineered to increase its affinity for the inhibitory FcγRIIb. Because CD19 associates with the BCR, obeliximab tethers CD19 and FcγRIIb to the same cell, thus inhibiting the BCR complex during antigen-induced BCR aggregation. Obeliximab utilizes the natural 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). Compared to B-cell depletion antibodies, obeliximab may also have an improved safety profile because it may not mediate B-cell killing.

[0053] variants In some embodiments, variants of obeliximab are CD19-specific immunoglobulins comprising: a light chain containing a variable region having a CDR1 containing RSSKSLQNVNGNTYLY, a CDR2 containing RMSNLNS, and a CDR3 containing MQHLEYPIT; and a heavy chain containing a variable region having a CDR1 containing SYVMH, a CDR2 containing WIGYINPYNDGTKY, and a CDR3 containing GTYYYGTRVFDY, wherein the heavy chain contains amino acid substitutions S267E and L328F in the Fc region compared to: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8) The numbering is based on the EU index, as in Kabat.

[0054] In some embodiments, variants of obeliximab include a heavy chain variable region (VH) and / or a light chain variable region (VL) comprising CDR1, CDR2, and CDR3, each differing 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 no more than 1, 2, 3, 4, or 5 amino acid residues. In some embodiments, variants of obeliximab include a heavy chain containing an Fc region with amino acid substitutions for S267E and L328F.

[0055] In some embodiments, the anti-CD19 antibody comprises a light chain containing LCDR1, LCDR2, and LCDR3, each differing from each of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 by no more than one residue. In some embodiments, the anti-CD19 antibody comprises a light chain containing LCDR1, LCDR2, and LCDR3, each differing from each of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 by no more than two residues. In some embodiments, the anti-CD19 antibody comprises a light chain containing LCDR1, LCDR2, and LCDR3, each differing from each of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 by no more than three residues. In some embodiments, the anti-CD19 antibody comprises a light chain containing LCDR1, LCDR2, and LCDR3, each differing from each of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 by no more than 4 residues. In some embodiments, the anti-CD19 antibody comprises a light chain containing LCDR1, LCDR2, and LCDR3, each differing from each of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 by no more than 5 residues.

[0056] In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1, HCDR2, and HCDR3, each differing from each of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 by no more than one residue. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1, HCDR2, and HCDR3, each differing from each of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 by no more than two residues. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1, HCDR2, and HCDR3, each differing from each of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 by no more than three residues. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1, HCDR2, and HCDR3, each differing from each of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 by no more than 4 residues. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1, HCDR2, and HCDR3, each differing from each of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 by no more than 5 residues.

[0057] In some embodiments, the anti-CD19 antibody comprises a heavy chain containing HCDR1, HCDR2, and HCDR3, each differing from each of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 by no more than one residue, and wherein the heavy chain contains an Fc region containing amino acid substitutions for 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 NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 by no more than two residues, and wherein the heavy chain contains an Fc region containing amino acid substitutions for 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 NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 by no more than 3 residues, and wherein the heavy chain contains an Fc region containing amino acid substitutions for 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 NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 by no more than 4 residues, and wherein the heavy chain contains an Fc region containing amino acid substitutions for 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 NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 by no more than 5 residues, and wherein the heavy chain contains an Fc region containing amino acid substitutions S267E and L328F.

[0058] In some embodiments, variants of obeliximab include a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the light chain variable regions identified in Table 2. In some embodiments, variants of obeliximab include a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the heavy chain variable regions identified in Table 2.

[0059] In some embodiments, the anti-CD19 antibody comprises a light chain containing a light chain variable region having at least 80% identity with SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody comprises a light chain containing a light chain variable region having at least 85% identity with SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody comprises a light chain containing a light chain variable region having at least 90% identity with SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody comprises a light chain containing a light chain variable region having at least 91% identity with SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody comprises a light chain containing a light chain variable region having at least 92% identity with SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody comprises a light chain containing a light chain variable region having at least 93% identity with SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody comprises a light chain containing a light chain variable region having at least 94% identity with SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody comprises a light chain containing a light chain variable region having at least 95% identity with SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody comprises a light chain containing a light chain variable region having at least 96% identity with SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody comprises a light chain containing a light chain variable region having at least 97% identity with SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody comprises a light chain containing a light chain variable region having at least 98% identity with SEQ ID NO: 11. In some embodiments, the anti-CD19 antibody comprises a light chain containing a light chain variable region having at least 99% identity with SEQ ID NO: 11.

[0060] In some embodiments, the anti-CD19 antibody comprises a heavy chain containing a light chain variable region having at least 80% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing a light chain variable region having at least 85% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing a light chain variable region having at least 90% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing a light chain variable region having at least 91% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing a light chain variable region having at least 92% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing a light chain variable region having at least 93% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing a light chain variable region having at least 94% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing a light chain variable region having at least 95% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing a light chain variable region having at least 96% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing a light chain variable region having at least 97% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing a light chain variable region having at least 98% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a heavy chain containing a light chain variable region having at least 99% identity with SEQ ID NO: 12.

[0061] In some embodiments, variants of obbelimumab comprise: a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the light chain variable regions identified in Table 2; and a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the heavy chain variable regions identified in Table 2, and further comprising, compared to SEQ ID NO: 8, amino acid substitutions S267E and L328F in the Fc region, wherein the numbering is based on the EU index.

[0062] In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, wherein the light chain contains a light chain variable region having at least 80% identity with SEQ ID NO: 11, and wherein the heavy chain contains a heavy chain variable region having at least 80% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, wherein the light chain contains a light chain variable region having at least 85% identity with SEQ ID NO: 11, and wherein the heavy chain contains a heavy chain variable region having at least 85% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, wherein the light chain contains a light chain variable region having at least 90% identity with SEQ ID NO: 11, and wherein the heavy chain contains a heavy chain variable region having at least 90% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, wherein the light chain contains a light chain variable region having at least 91% identity with SEQ ID NO: 11, and wherein the heavy chain contains a heavy chain variable region having at least 91% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, wherein the light chain contains a light chain variable region having at least 92% identity with SEQ ID NO: 11, and wherein the heavy chain contains a heavy chain variable region having at least 92% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, wherein the light chain contains a light chain variable region having at least 93% identity with SEQ ID NO: 11, and wherein the heavy chain contains a heavy chain variable region having at least 93% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, wherein the light chain contains a light chain variable region having at least 94% identity with SEQ ID NO: 11, and wherein the heavy chain contains a heavy chain variable region having at least 94% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, wherein the light chain contains a light chain variable region having at least 95% identity with SEQ ID NO: 11, and wherein the heavy chain contains a heavy chain variable region having at least 95% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, wherein the light chain contains a light chain variable region having at least 96% identity with SEQ ID NO: 11, and wherein the heavy chain contains a heavy chain variable region having at least 96% identity with SEQ ID NO: 12.In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, wherein the light chain contains a light chain variable region having at least 97% identity with SEQ ID NO: 11, and wherein the heavy chain contains a heavy chain variable region having at least 97% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, wherein the light chain contains a light chain variable region having at least 98% identity with SEQ ID NO: 11, and wherein the heavy chain contains a heavy chain variable region having at least 98% identity with SEQ ID NO: 12. In some embodiments, the anti-CD19 antibody comprises a light chain and a heavy chain, wherein the light chain contains a light chain variable region having at least 99% identity with SEQ ID NO: 11, and wherein the heavy chain contains a heavy chain variable region having at least 99% identity with SEQ ID NO: 12.

[0063] In some embodiments, variants of obeliximab comprise: a light chain containing an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with the light chains identified as shown in Table 2. In some embodiments, variants of obeliximab comprise: a heavy chain containing an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with the heavy chains identified as shown in Table 2.

[0064] In some embodiments, the anti-CD19 antibody comprises a light chain amino acid sequence having at least 70% identity with SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody comprises a light chain amino acid sequence having at least 75% identity with SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody comprises a light chain amino acid sequence having at least 80% identity with SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody comprises a light chain amino acid sequence having at least 85% identity with SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody comprises a light chain amino acid sequence having at least 90% identity with SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody comprises a light chain amino acid sequence having at least 91% identity with SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody comprises a light chain amino acid sequence having at least 92% identity with SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody comprises a light chain amino acid sequence having at least 93% identity with SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody comprises a light chain amino acid sequence having at least 94% identity with SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody comprises a light chain amino acid sequence having at least 95% identity with SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody comprises a light chain amino acid sequence having at least 96% identity with SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody comprises a light chain amino acid sequence having at least 97% identity with SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody comprises a light chain amino acid sequence having at least 98% identity with SEQ ID NO: 9. In some embodiments, the anti-CD19 antibody comprises a light chain amino acid sequence having at least 99% identity with SEQ ID NO: 9.

[0065] In some embodiments, the anti-CD19 antibody comprises a heavy chain amino acid sequence having at least 70% identity with SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody comprises a heavy chain amino acid sequence having at least 75% identity with SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody comprises a heavy chain amino acid sequence having at least 80% identity with SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody comprises a heavy chain amino acid sequence having at least 85% identity with SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody comprises a heavy chain amino acid sequence having at least 90% identity with SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody comprises a heavy chain amino acid sequence having at least 91% identity with SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody comprises a heavy chain amino acid sequence having at least 92% identity with SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody comprises a heavy chain amino acid sequence having at least 93% identity with SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody comprises a heavy chain amino acid sequence having at least 94% identity with SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody comprises a heavy chain amino acid sequence having at least 95% identity with SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody comprises a heavy chain amino acid sequence having at least 96% identity with SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody comprises a heavy chain amino acid sequence having at least 97% identity with SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody comprises a heavy chain amino acid sequence having at least 98% identity with SEQ ID NO: 10. In some embodiments, the anti-CD19 antibody comprises a heavy chain amino acid sequence having at least 99% identity with SEQ ID NO: 10.

[0066] In some embodiments, the variant of obbelimumab comprises: a light chain containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the light chain sequence identified as in Table 2; and a heavy chain containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the heavy chain sequence identified as in Table 2, and, compared to SEQ ID NO: 8, the heavy chain of the variant contains amino acid substitutions S267E and L328F in the Fc region, wherein the numbering is based on the EU index, as in Kabat.

[0067] In some implementations, suitable variants of obélimumab bind to the same epitopes on human CD19 with antibodies comprising the light and heavy chains identified as shown in Table 2. Epitope binding can be determined using methods known in the art.

[0068] In some implementations, suitable variants of obbelimumab compete with antibodies containing the light and heavy chains identified as shown in Table 2 for binding to human CD19 under binning assays known in the art. As used herein, binning assays refer to any method that regionally maps the epitopes to which the antibody binds. Standard methods for characterizing such antibodies, also known as epitope binning, typically involve surface plasmon resonance (SPR) technology. Using SPR, monoclonal antibody candidates are screened in pairs for binding to the target protein. Other standard methods involve ELISA-based screening and may require the synthesis of overlapping peptide sets corresponding to the protein of interest.

[0069] In some embodiments, human CD19 comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-CD19 antibody binds to the extracellular domain of human CD19.

[0070] MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNR SSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARP VLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEED SEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR (SEQ ID NO: 1) Fc receptor binding properties The anti-CD19 antibodies disclosed herein (e.g., obbelimumab) contain an Fc variant with enhanced Fc binding to the inhibitory Fcγ receptor IIb (FcγRIIb). FcγRIIb is the only FcR on B cells, acting as an antibody-sensing downregulator of humoral immunity naturally mediated by immune complexes. When sufficient antibodies are generated against a given antigen, specific immune complexes form FcγRIIb and the B cell receptor (BCR) and co-bind with it with high affinity, selectively inhibiting only B cells that recognize homologous antigens. Additionally, FcγRIIb modulates the activity of other B cell stimulating factors, including interleukin (IL)-4, LPS, and BAFF, which amplifies BCR-driven proliferation and differentiation. By simultaneously binding CD19 and FcγRIIb, obbelimumab (and the variants described herein) mimics the action of antigen-antibody complexes and downregulates B cell activity.

[0071] The Fc variants disclosed herein can be optimized for various Fc receptor binding properties. Fc variants engineered or predicted to exhibit one or more optimized properties are referred to herein as “optimized Fc variants.” Optimizable properties include, but are not limited to, enhanced or reduced affinity for FcγR. In one embodiment, the Fc variants disclosed herein are optimized to have enhanced affinity for the inhibitory receptor FcγRIIb. In other embodiments, the immunoglobulins disclosed herein result in enhanced affinity for FcγRIIb but reduced affinity for one or more activating FcγRs, including, for example, FcγRI, FcγRIIa, FcγRIIIa, and / or FcγRIIIb. FcγR receptors can be expressed on cells from any organism, including but not limited to humans, cynomolgus monkeys, and mice. The Fc variants disclosed herein can be optimized to have enhanced affinity for human FcγRIIb.

[0072] The Fc variants contain one or more amino acid modifications relative to the parental Fc polypeptide, wherein the one or more amino acid modifications provide one or more optimized properties. The Fc variants disclosed herein differ from their parents in amino acid sequence 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 more than one amino acid modification compared to the parent, such as about 20 to 50 amino acid modifications, such as about 2 to 10 amino acid modifications, about 2 to about 5 amino acid modifications, etc. Therefore, the sequence of the Fc variant and the sequence of the parental Fc polypeptide are substantially homologous. For example, the sequence of the variant Fc variants disclosed herein will have about 80% homology with the sequence of the parental Fc variants, such as 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.

[0073] Genetic modification can be performed using molecular biology, or enzymatic or chemical modification can be performed.

[0074] The Fc variants disclosed herein are defined according to the amino acid modifications constituting them. Thus, for example, S267E is an Fc variant having the substitution of S267E relative to the parental Fc polypeptide. Similarly, S267E / L328F defines an Fc variant having the substitutions of S267E and L328F relative to the parental Fc polypeptide. The identity of the WT amino acids may be unspecified; in this case, the aforementioned variant is referred to as 267E / 328F. It should be noted that the order of substitutions provided is arbitrary, that is, for example, 267E / 328F and 328F / 267E are the same Fc variant, etc. Unless otherwise stated, the positions discussed herein are based on, for example, Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition). The EU numbering is based on the EU index described in the United States Public Health Service, National Institutes of Health, Bethesda (this document is incorporated herein by reference in its entirety). In short, an EU is the name of the first antibody molecule whose entire amino acid sequence is determined (Edelman et al., 1969, Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci USA) 63:78-85, which is incorporated herein by reference in its entirety), and its amino acid sequence has become the standard numbering scheme for heavy chain constant regions. EU proteins have become the standard reference for defining numbering. Kabat et al. listed a set of EU sequences in an index that aligned them with other antibody sequences, thus serving as a necessary tool for aligning antibodies to the EU numbering scheme. Therefore, as those skilled in the art will understand, the standard way to refer to EU numbering is to refer to the sequence alignment of Kabat et al., as this places EU in the context of antibodies with other variable domain lengths. Therefore, as used herein, "EU index as in Kabat" or "numbering is based on EU index as in Kabat" refers to the numbering of EU antibodies as described in Kabat.

[0075] In some embodiments, the Fc variants disclosed herein are based on human IgG sequences, and therefore the human IgG sequences are used as the “base” sequence for comparison with other sequences, including but not limited to sequences from other organisms, such as rodent and primate sequences. It is conceivable that while the Fc variants disclosed herein are engineered in the case of a parent IgG, the variants can be engineered or “transferred” to another second parent IgG. This is accomplished by determining “equivalent” or “corresponding” residues and substitutions between the first and second IgGs, typically based on sequence or structural homology between the sequences of the first and second IgGs. To establish homology, the amino acid sequence of the first IgG outlined herein is directly compared with the sequence of the second IgG. After sequence alignment, one or more homology alignment procedures known in the art (e.g., using conserved residues between species) are used, allowing necessary insertions and deletions to maintain the alignment (i.e., avoiding the elimination of conserved residues by arbitrary deletions and insertions), defining residues equivalent to specific amino acids in the primary sequence of the first immunoglobulin. Alignment of conserved residues can conserve 100% of such residues. However, alignments of more than 75% or as few as 50% of conserved residues are sufficient to define equivalent residues. Equivalent residues can also be defined by determining structural homology between a first and second IgG at the tertiary structural level of an IgG whose structure has been determined. In this case, equivalent residues are defined as those residues in the main chain atoms of specific amino acid residues (N on N, CA on CA, C on C, and O on O) of the parent or precursor after alignment, where the atomic coordinates are within about 0.13 nm. In another embodiment, equivalent residues are within about 0.1 nm after alignment. Alignment is performed after the optimal model has been oriented and positioned to give maximum overlap of the 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 used to prepare the IgG, it is intended to convey that the discovered Fc variants, as disclosed herein, can be engineered into any second parent IgG with significant sequence or structural homology to the Fc variant. Therefore, for example, if a variant antibody in which the parent antibody is human IgG1 is generated, then by using the methods described above or other methods for determining equivalent residues, this variant antibody can be engineered into another IgG1 parent antibody that binds to a different antigen, namely, a human IgG2 parent antibody, a human IgA parent antibody, a mouse IgG2a or IgG2b parent antibody, etc. Similarly, as stated above, the background of the parental Fc variant does not affect the ability to transfer the Fc variants disclosed herein to other parental IgGs.

[0076] As used herein, the terms “greater affinity,” “improved affinity,” “enhanced affinity,” or “better affinity” than the parental Fc peptide mean that, when the amounts of the variant and the parent peptide are substantially the same in a binding assay, the Fc variant binds to the Fc receptor with a significantly higher equilibrium association constant (KA or Ka) or a lower equilibrium dissociation constant (KD or Kd) than the parental Fc peptide. For example, an Fc variant with improved Fc receptor binding affinity can show an improvement of about 5 to about 1000 times, for example, about 10 to about 500 times, of Fc receptor binding affinity compared to the parental Fc peptide, wherein the Fc receptor binding affinity is determined by those skilled in the art, for example, through binding methods disclosed herein, including but not limited to Biacore. Thus, “reduced affinity” compared to the parental Fc peptide as used herein means that the Fc variant binds to the Fc receptor with a significantly lower KA or a higher KD than the parental Fc peptide. Greater or reduced affinity can also be defined relative to absolute affinity levels. For example, according to data presented herein, WT (natural) lgG1 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 approximately 10 times greater than that of WT lgG1. As disclosed herein, greater or enhanced affinity means a KD below about 100 nM, for example, between about 10 nM and about 100 nM, between about 1 and about 100 nM, or less than about 1 nM.

[0077] In one embodiment, the Fc variant provides selectively enhanced affinity for FcγRIIb relative to one or more activating receptors. Selectively enhanced affinity means that the Fc variant has improved affinity for FcγRIIb relative to one or more activating receptors compared to the parental Fc peptide, but reduced affinity for one or more activating receptors compared to the parental Fc peptide; or it means that the Fc variant has improved affinity for both FcγRIIb and activating receptors compared to the parental Fc peptide, however, the improvement in affinity for FcγRIIb is greater than the improvement in affinity for one or more activating receptors. In alternative embodiments, the Fc variant reduces or eliminates binding to one or more activating FcγRs, reduces or eliminates binding to one or more complement proteins, reduces or eliminates one or more FcγR-mediated effector functions, and / or reduces or eliminates one or more complement-mediated effector functions.

[0078] The presence of different polymorphic forms of FcγR provides another parameter influencing the therapeutic efficacy of the Fc variants disclosed herein. While the specificity and selectivity of a given Fc variant to different classes of FcγR significantly affects its ability to target a given antigen to treat a given disease, the specificity or selectivity of Fc variants to these different polymorphic forms of these receptors can partially determine which studies or preclinical trials may be suitable for testing, and ultimately which patient populations may or may not respond to treatment. Therefore, the specificity or selectivity of the Fc variants to Fc receptor polymorphisms (including but not limited to FcγRIIa, FcγRIIIa, etc.) disclosed herein can be used to guide effective studies and preclinical trials, clinical trial design, patient selection, dosing dependence, and / or other aspects of clinical trial selection.

[0079] The Fc variants disclosed herein may include modifications that regulate interactions with Fc receptors other than FcγR, including but not limited to complement proteins, FcRn, and Fc receptor homologs (FcRH). FcRH includes, but is not limited to, FcRFH, FcRH2, FcRH3, FcRH4, FcRH5, and FcRH6 (Davis et al., 2002, Immunol. Reviews 190:123-136).

[0080] A crucial parameter for determining the most beneficial selectivity of a given Fc variant for treating a given disease is the background of the Fc variant. Therefore, the Fc receptor selectivity or specificity of a given Fc variant will provide different properties depending on whether it constitutes an antibody, an Fc fusion, or an Fc variant with a coupled fusion partner. In one embodiment, the Fc receptor specificity of the Fc variant disclosed herein will determine its therapeutic utility. The utility of a given Fc variant for therapeutic purposes will depend on the epitope or form of the target antigen and the disease or indication being treated. For some targets and indications, greater FcγRIIb affinity and reduced activated FcγR-mediated effector function may be beneficial. For other target antigens and therapeutic applications, increased affinity for FcγRIIb or increased affinity for both FcγRIIb and the activated receptor may be beneficial.

[0081] Treatment of multiple sclerosis with anti-CD19 antibodies In some aspects, the present invention provides a method for treating MS in a subject of need using an anti-CD19 antibody or an antigen-binding fragment thereof.

[0082] In some embodiments, a method of treating MS in a subject in need includes 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 wherein 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 wherein the heavy chain comprises an Fc region containing amino acid substitutions for 267E and L328F.

[0083] In some respects, methods of treating MS in subjects in need include 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 having at least 90% identity with the amino acid sequence of SEQ ID NO: 11, and wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 12, and wherein the heavy chain comprises an Fc region comprising amino acid substitutions 267E and L328F.

[0084] In some implementations, treatment of MS includes administration of obelizumab.

[0085] The method of administration can 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 drug-loaded syringe or an auto-injector.

[0086] In some embodiments, administration of anti-CD19 antibodies improves or alleviates one or more symptoms of MS or delays its onset. In some embodiments, administration of anti-CD19 antibodies delays the onset of one or more symptoms of MS.

[0087] formulations and pharmaceutical compositions This invention provides pharmaceutical compositions and formulations of anti-CD19 antibodies (e.g., obembelimab). The pharmaceutical compositions, formulations, and related methods of this invention can be used to deliver anti-CD19 antibodies (e.g., obembelimab) and for the treatment of MS and related diseases. By combining said antibody with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, ...), ... Osol, A. (ed., 1980, which is incorporated herein by reference in its entirety) to prepare formulations of the anti-CD19 antibody disclosed herein for storage, in the form of lyophilized formulations or aqueous solutions.

[0088] In some embodiments, the pharmaceutical composition of interest comprises various concentrations of anti-CD19 antibodies (e.g., obélixumab). In some embodiments, a suitable formulation may comprise the antibody of interest at concentrations of up to about 250 mg / ml (e.g., up to about 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).

[0089] In some embodiments, suitable formulations may contain anti-CD19 antibodies at concentrations ranging from about 10 to 300 mg / ml (e.g., about 10 to 250 mg / ml, about 10 to 200 mg / ml, about 10 to 180 mg / ml, about 10 to 160 mg / ml, about 10 to 150 mg / ml, about 10 to 140 mg / ml, about 10 to 130 mg / ml, about 10 to 125 mg / ml, about 100 to 125 mg / ml, about 100 to 180 mg / ml, about 100 to 150 mg / ml, about 100 to 130 mg / ml, about 100 to 125 mg / ml, about 100 to 170 mg / ml, about 100 to 160 mg / ml, about 100 to 150 mg / ml, about 100 to 200 mg / ml, about 120 to 130 mg / ml).

[0090] In some embodiments, the formulation suitable for administration may contain an anti-CD19 antibody (e.g., obbelimab) at concentrations of approximately 100 mg / ml, 115 mg / ml, 120 mg / ml, 125 mg / ml, 130 mg / ml, 135 mg / ml, 140 mg / ml, 145 mg / ml, 150 mg / ml, 200 mg / ml, or 300 mg / ml.

[0091] 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 a 5 mM sodium phosphate solution, pH 7.0) and sugar-containing solutions (e.g., up to 3% (e.g., up to 2.4%, 2.0%, 1.5%, 1.0%) of sucrose in a 5 mM sodium phosphate solution, pH 7.0). In some embodiments, a suitable formulation composition is a brine solution (e.g., 150 mM NaCl in water).

[0092] Many therapeutic agents, and specifically the antibodies of this invention, require controlled pH and specific excipients to maintain their solubility and stability in the pharmaceutical compositions of this invention.

[0093] The pH of the pharmaceutical composition is another factor that can alter the solubility of anti-CD19 antibodies (e.g., obbelimab) in an aqueous pharmaceutical composition. In some embodiments, the pharmaceutical compositions of the present invention contain one or more buffer solutions. In some embodiments, the compositions according to the present invention contain an amount of buffer solution sufficient 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 solution contains up to 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) of sodium phosphate. Suitable buffer solutions include, for example, acetate, succinate, citrate, phosphate, other organic acids, and tris(hydroxymethyl)aminomethane (“Tris”).

[0094] Suitable buffer concentrations can be from about 1 mM to about 100 mM, or from about 3 mM to about 20 mM, depending on, for example, the desired isotonicity of the buffer and the 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.

[0095] In some embodiments, the formulation contains an isotonic agent to maintain the formulation's isotonicity. Exemplary isotonic agents include, but are not limited to, glycine, sorbitol, mannitol, sodium chloride, and arginine. In some embodiments, a suitable 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%).

[0096] In some embodiments, the formulation may contain a stabilizer to protect the antibody. Typically, suitable stabilizers are non-reducing sugars such as sucrose, raffinose, trehalose, or amino acids such as glycine, arginine, and methionine. The amount of stabilizer in the formulation is generally such that the formulation will be isotonic. However, hypertonic formulations may also be suitable. Additionally, the amount of stabilizer must not be so low that unacceptable degradation / aggregation of the antibody 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 optionally, from 0.1% to 15% by weight (e.g., 1% to 10%, 5% to 15%, 5% to 10%). 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 can 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, the stabilizer suitable for lyophilization is also a lyophilization protectant.

[0097] In some implementations, it is desirable to add surfactants to the formulation. Exemplary surfactants include nonionic surfactants such as polysorbates (e.g., polysorbate 20 or 80); poloxamer (e.g., poloxamer 188); Triton; sodium lauryl 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; lauroylaminopropyl-, cocamidopropyl-, linoleamide-, myristamidopropyl-, palmitamidopropyl-, or isostearamidopropyl-betaine (e.g., lauroylaminopropyl); myristamidopropyl-, palmitamidopropyl-, or isostearamidopropyl-dimethylamine; sodium methylcocoyl or disodium methylmercapto-taurine; and the MONAQUAT™ series (Mona Industries, Inc., Paterson, NJ), polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.). Typically, the amount of surfactant added is such that it reduces protein aggregation and minimizes particle or effervescent formation. For example, surfactants may be present in formulations at a concentration of about 0.001% to 0.5% (e.g., about 0.005% to 0.05% or 0.005% to 0.01%). In particular, surfactants may be present in formulations at concentrations of about 0.005%, 0.01%, 0.02%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%.

[0098] In some embodiments, suitable formulations may further include one or more fillers, particularly for lyophilization formylation. A “filler” is a compound that increases the mass of 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., a substantially uniform lyophilized cake). Suitable fillers include, but are not limited to, sodium chloride, lactose, mannitol, glycine, sucrose, trehalose, and hydroxyethyl starch. Exemplary concentrations of fillers are from 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%).

[0099] Formulations according to the present invention can be evaluated based on product quality analysis, reconstitution time (if lyophilized), reconstitution quality (if lyophilized), high molecular weight, moisture content, and glass transition temperature. Typically, protein quality and product analysis includes analysis of the product degradation rate 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, turbidity determination, 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 appearance (liquid or cake appearance).

[0100] Typically, formulations (lyophilized or aqueous) can be stored at room temperature for extended periods. Storage temperatures can generally range from 0°C. Up to 45 (For example, 4) 20 25 45 (etc.). The formulation can be stored for periods ranging from several months to several years. Typical storage periods will be 24 months, 12 months, 6 months, 4.5 months, 3 months, 2 months, or 1 month. The formulation can be stored directly in the container intended for application, thus eliminating the need for transfer steps.

[0101] Formulations can be stored directly in lyophilized containers (if lyophilized), which can also be used as reconstitution containers, thereby eliminating transfer steps. Alternatively, lyophilized product formulations can be stored in small increments. Storage should generally avoid conditions that could lead to protein degradation, including but not limited to exposure to sunlight, UV radiation, other forms of electromagnetic radiation, overheating or overcooling, rapid thermal shock, and mechanical shock.

[0102] In some embodiments, the formulations according to the invention are in liquid or aqueous form. In some embodiments, the formulations of the invention are lyophilized. Such lyophilized formulations can be reconstituted by adding one or more diluents prior to administration to a patient. Suitable diluents include, but are not limited to, sterile water, antibacterial water for injection, and sterile saline solutions. Preferably, during reconstitution, the antibodies contained therein are stable, soluble, and tolerable when administered to a patient.

[0103] The pharmaceutical compositions of the present invention are characterized by their tolerability. As used herein, the terms "tolerable" and "well-tolerated" refer to the ability of the pharmaceutical compositions of the present invention to not cause adverse reactions in patients administered such compositions, or alternatively, not cause serious adverse reactions in patients administered such compositions. In some embodiments, the pharmaceutical compositions of the present invention are well-tolerated by patients administered such compositions.

[0104] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphates, citrates, acetates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; and hydrophilic agents. Polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrose; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; sweeteners and other flavorings; 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).

[0105] In some embodiments, pharmaceutical compositions comprising the antibodies disclosed herein may be in a water-soluble form, such as as pharmaceutically acceptable salts, meaning both acid and base addition salts. A "pharmaceutically acceptable acid addition salt" is a salt formed from inorganic and organic acids that retains the biological effectiveness of the free base and is undesirable non-biologically or otherwise. The inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; the organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

[0106] "Pharmaceutically acceptable base addition salts" include base addition salts 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, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable 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.

[0107] For in vivo The applied formulation can be sterile. In some embodiments, the formulation is sterilized by filtration through a sterile filter membrane.

[0108] Non-limiting examples of buffers include phosphates, citrates, acetates, glutamates, carbonates, tartrates, triethanolamine (TRIS), diglucopyranosides, histidine, glycine, lysine, arginine, and other organic acids. More specifically, non-limiting examples of buffers include sodium HEPES, MES, potassium phosphate, potassium thiocyanate, sterilizing agents, TAE, TBE, ammonium sulfate / HEPES, BuffAR, sodium acetate, sodium carbonate, sodium citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium phosphate. Additionally, buffers can be in various hydrate forms. For example, buffers can be monohydrates, dihydrates, trihydrates, tetrahydrates, pentahydrates, hexahydrates, heptahydrates, octahydrates, nonahydrates, decahydrates, undecahydrates, and dodecahydrates. Sometimes, the hydrate can be a fractional hydrate, such as a hemihydrate or sesquihydrate. Non-limiting examples of tension-modified buffers include sodium chloride, acetate, L-proline, dextran, mannitol, potassium chloride, glycerin, and glycerol. Non-limiting examples of solvents include water, propylene glycol, polyethylene glycol, ethanol, dimethyl sulfoxide, N-methyl-2-pyrrolidone, tetraethylene glycol, Solketal™, glyceryl formal, acetone, tetrahydrofurfuryl alcohol, diethylene glycol dimethyl ether, dimethyl isosorbide, and ethyl lactate. Non-limiting examples of solvents include polysorbates (e.g., polysorbate-20, polysorbate-80), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene trioleate (span 85), lecithin, and polyoxyethylene polyoxypropylene copolymers (Plannick, Plannick F-68).

[0109] The amounts of anti-CD19 antibody (e.g., obbelimumab), buffer, tension modifier, solvent, and surfactant can vary. In some embodiments, the anti-CD19 antibody (e.g., obbelimumab) is formulated at a concentration of 125 mg / ml obbelimumab, 2.35 mg / ml sodium acetate trihydrate, 0.17 mg / ml acetic acid (density 1.053 g / ml), 30 mg / ml L-proline, and 0.1 mg / ml polysorbate 80, at pH 5.5. In some embodiments, the anti-CD19 antibody (e.g., obbelimumab) is formulated at a concentration of 80-200 mg / ml obbelimumab, 1.5-3 mg / ml sodium acetate trihydrate, 0.1-0.2 mg / ml acetic acid (density 1.053 g / ml), 10-50 mg / ml L-proline, and 0.05-0.2 mg / ml polysorbate 80, at pH 5.0-6.0. In some embodiments, the anti-CD19 antibody (e.g., obbelimumab) is formulated with a concentration of 122-127 mg / ml obbelimumab, 2.0-2.5 mg / ml sodium acetate trihydrate, 0.15-0.19 mg / ml acetic acid (density 1.053 g / ml), 25-35 mg / ml L-proline, and 0.05-0.15 mg / ml polysorbate 80, at a pH of 5.0-6.0.

[0110] In some embodiments, the formulation comprises an anti-CD19 antibody (e.g., obbelimab), one or more buffers, one or more tension modifiers, one or more solvents, and one or more surfactants. In some embodiments, the buffer may be sodium acetate buffer. For example, the buffer may be sodium acetate trihydrate. In some embodiments, the tension modifier may be acetic acid, L-proline, and combinations thereof. In another embodiment, the solvent is water.

[0111] 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., obbelimumab), sodium acetate trihydrate, acetic acid and L-proline, water and polysorbate-80.

[0112] In some embodiments, the formulation comprises an anti-CD19 antibody (e.g., obbelimab) 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; up to about 1 ml of water; 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 an anti-CD19 antibody (e.g., obbelimab) in an amount of about 100 mg to about 250 mg / ml, sodium acetate trihydrate in an amount of about 1 to about 2.5 mg / ml, acetic acid and L-proline in an amount of about 20 to about 40 mg / ml, water in an amount of up to about 1 mg / ml, and polysorbate-80 in an amount of about 0.05 to about 0.2 mg / ml.

[0113] The anti-CD19 antibodies (e.g., obbelimumab) disclosed herein can also be formulated into immunoliposomes. Liposomes are small vesicles containing various types of lipids, phospholipids, and / or surfactants that can be used to deliver anti-CD19 antibodies (e.g., obbelimumab) to mammals. Liposomes containing anti-CD19 antibodies (e.g., obbelimumab) are prepared by methods known in the art. Liposomes with enhanced cycle times are disclosed in US 5,013,556, which is incorporated herein by reference in its entirety. In some embodiments, the anti-CD19 antibody (e.g., obbelimumab) is formulated in liposomes generated by a reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with defined pore sizes to produce liposomes with a desired diameter.

[0114] In some embodiments, an anti-CD19 antibody (e.g., obbelimab) is captured in microcapsules prepared by methods including, but not limited to, coagulation techniques, interfacial polymerization (e.g., using hydroxymethyl cellulose or gelatin-microcapsules or poly-(methyl methacrylate) 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. In Osol, A. (ed.), 1980, this reference is included in its entirety by way of citation.

[0115] In some embodiments, sustained-release formulations of anti-CD19 antibodies (e.g., obbelimab) can be prepared. Suitable examples of sustained-release formulations include semi-permeable matrices of solid hydrophobic polymers, which are in the form of molded articles, such as membranes or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methyl acrylate) or poly(vinyl alcohol)), polylactic acid (US 3,773,919, all incorporated herein by reference), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid esters, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron Depot® (which are injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate), poly(-)-3-hydroxybutyric acid, and ProLease® (commercially available from Alkermes), which are microsphere-based delivery systems consisting of desired bioactive molecules incorporated into a poly-DL-lactide-co-glycoside (PLG) matrix.

[0116] In some embodiments, the liquid pharmaceutical composition comprises 122-127 mg / ml anti-CD19 antibody (e.g., obbelimab), 2.0-2.5 mg / ml sodium acetate trihydrate (pH 5.0-6.0), 0.15-0.19 mg / ml acetic acid (density 1.053 g / ml), 25-35 mg / ml L-proline, and 0.05-0.15 mg / ml polysorbate 80.

[0117] In some embodiments, the liquid pharmaceutical composition comprises about 125 mg / ml of anti-CD19 antibody (e.g., obbelimab), 2.35 mg / ml of sodium acetate trihydrate, 0.17 mg / ml of acetic acid, 30 mg / ml of L-proline, 0.1 mg / ml of polysorbate 80, and a pH of 5.5.

[0118] In some embodiments, the liquid pharmaceutical composition comprises 122-127 mg / ml obbelimumab, 2.0-2.5 mg / ml sodium acetate trihydrate (pH 5.0-6.0), 0.15-0.19 mg / ml acetic acid (density 1.053 g / ml), 25-35 mg / ml L-proline, and 0.05-0.15 mg / ml polysorbate 80.

[0119] In some embodiments, the liquid pharmaceutical composition comprises about 125 mg / ml oxibizumab, 2.35 mg / ml sodium acetate trihydrate, 0.17 mg / ml acetic acid, 30 mg / ml L-proline, 0.1 mg / ml polysorbate 80, and a pH of 5.5.

[0120] List of implementation plans Implementation Scheme 1. A method of treating multiple sclerosis (MS) in a subject of need, comprising administering to the subject an anti-CD19 antibody comprising an Fc region modified by amino acid substitutions of S267E and L328F, wherein the anti-CD19 antibody comprises a heavy chain HCDR1 containing the amino acid sequence SYVMH (SEQ ID NO: 5), an HCDR2 containing the amino acid sequence WIGYINPYNDGTKY (SEQ ID NO: 6), and an HCDR3 containing the amino acid sequence GTYYYGTRVFDY (SEQ ID NO: 7), and a light chain LCDR1 containing RSSKSLQNVNGNTYLY (SEQ ID NO: 2), an LCDR2 containing the amino acid sequence RMSNLNS (SEQ ID NO: 3), and an LCDR3 containing the amino acid sequence MQHLEYPIT (SEQ ID NO: 4), wherein administration of the anti-CD19 antibody improves, alleviates, or delays one or more symptoms of MS or its onset.

[0121] Implementation Scheme 2. A method of treating multiple sclerosis (MS) in a subject of need, comprising administering to the subject an anti-CD19 antibody comprising an Fc region modified by amino acid substitutions of S267E and L328F, wherein the anti-CD19 antibody comprises a heavy chain variable region containing an amino acid sequence having at least 90% identity with SEQ ID NO: 12; and a light chain variable region containing an amino acid sequence having at least 90% identity with SEQ ID NO: 11, wherein administration of the anti-CD19 antibody improves, alleviates, or delays one or more symptoms of MS or its onset.

[0122] Implementation Scheme 3. The method as described in Implementation Scheme 1 or 2, wherein the anti-CD19 antibody comprises a heavy chain variable region containing the amino acid sequence SEQ ID NO: 12; and a light chain variable region containing the amino acid sequence SEQ ID NO: 11.

[0123] Implementation Scheme 4. The method of any one of the preceding implementation schemes, 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.

[0124] Implementation Scheme 5. The method as described in any of the preceding implementation schemes, wherein the anti-CD19 antibody is obelidomide.

[0125] Implementation Scheme 6. The method as described in any of the preceding implementation schemes, wherein the anti-CD19 antibody is administered intrathecally.

[0126] Implementation Scheme 7. The method as described in any of the preceding implementation schemes, wherein the anti-CD19 antibody is administered intravenously.

[0127] Example Example 1: Induction of disease activity prevention in an EAE model Evaluate alternative antibodies against obiblimumab (Obx) and rituximab (anti-CD20) in a MOG protein / pertussis EAE model. Briefly, huFcγRIIb transgenic mice (2B-KIX mice) were treated twice weekly with a mouse alternative to Obx (mObx), a mouse alternative to rituximab, or a vector control (phosphate-buffered saline). Mice treated with Obx or a rituximab alternative received 10 mg / kg via intraperitoneal injection. One week after initial treatment, mice were immunized twice daily with human MOG protein and pertussis toxin according to a standard protocol. Over the following 27 days, disease severity in individual mice was assessed twice daily on a scale of 0 to 5, with progressively increasing clinical scores corresponding to observed increases in disease severity. Mice achieving a clinical score of 4 were euthanized. All mice in the vector control group were euthanized within 15 days of MOG injection. On day 11 following MOG injection, two mice in the olibulimab alternative group were euthanized. On day 13 following MOG injection, one mouse in the rituximab alternative group was euthanized. The clinical scores of the mice are shown in Table 4 below.

[0128] Table 4. Clinical scores of individual mice observed in the EAE mouse model. *According to IACUC requirements, animals are euthanized when the disease score is 4 due to severe EAE.

[0129] After 27 days, mObx showed disease prevention, as measured by standard clinical EAE scores of surviving mice at each time point. Figure 1A Mice treated with mObx experienced an EAE rate of 33% (2 / 6), while mice treated with the rituximab alternative experienced an EAE rate of 86% (6 / 7), and mice treated with the mediator control experienced an EAE rate of 100% (3 / 3). On day -5, after initial treatment but before immunization with human MOG, whole blood immune cell analysis was performed. Mice treated with mObx showed a blood B-cell percentage comparable to those treated with the mediator control, while mice treated with the rituximab alternative showed a significantly reduced blood B-cell percentage with total lymphocytes. Figure 1BThe ability of mObx to inhibit disease induction in a MOG-induced EAE model indicates the potential efficacy of Obx in preventing the onset of MS without depleting B cells in the blood.

[0130] Example 2: Mouse obbelimumab alternative reduces BCR-mediated phagocytosis in mice. This example investigated whether treatment with 30 µg / ml mObx inhibited BCR-mediated phagocytosis. mObx was used to treat B cells isolated from 2B-KIX mice (Chu, SY et al., 2021. J. Transl. Autoimmun. 4: 100075) to understand how different stages of B cell APC function and their interaction with T cells are affected in the standard immune response to exogenous Ag. Figure 2A and Figure 2B As shown, mObx restricts BCR-mediated Ag uptake and APC function to in vitro Activate T cells.

[0131] Spleen cells were isolated from 2B-KIX mice and treated for 1 hour with cytochalasin D (an actin polymerization inhibitor that blocks Ag uptake), culture medium alone, mObx (2480E), or anti-CD19 not conjugated with hFcγRIIb (2480F) as an idiotype control. Following treatment, 2 μm fluorescent beads coated with anti-mouse IgM and biotinylated OVA were added to the culture medium, and the cells were incubated at 37°C and 5% CO2 for another 2 hours. Bead uptake was analyzed by flow cytometry. FoB cells were identified as CD19+, CD4-, CD11b-, IgMlo, IgDhi, CD23hi, and CD21int. Cells with internalized beads were identified as bead+, but were not stained with fluorescent streptavidin, indicating that OVA-biotin was internalized and could not be used for staining. The percentage of total FoB cells in the internalized beads is shown. Figure 2A Each symbol represents a cell isolated from a single mouse (**p < 0.01, ****p < 0.0001).

[0132] B cells were isolated from lymphoid tissue harvested from 2B-KIX mice using negative selection beads and then treated for 1 hour at 37°C and 5% CO2 with either culture medium (medium and no treatment), mObx (2480E), or a idiotype control (2480F). OVA-sp T cells were similarly harvested from lymphoid tissue of OT-II mice and labeled with CTViolet before being added to B cell cultures without Ag (with culture medium) or with the aforementioned anti-IgM / OVA-bio beads (all other groups). Cells were co-cultured for 4 days, and T cell proliferation was then analyzed by flow cytometry. Proliferating T cells were identified as CD4+CD45R- cells using diluted CTViolet. A pilot example is shown ( Figure 2B ).

[0133] Also used in vitro Phagocytosis of mouse B cells was assessed. Splenic and peritoneal cells were isolated from wild-type 2B-KIX mice and incubated with 2 µm beads and mObx for 2 hours. Phagocytosis was subsequently evaluated by flow cytometry. Figures 3A-3D The bead targets were coated with anti-mouse IgM for targeting the BCR in an antigen-agnostic manner and biotinylated OVA to allow differentiation between internalized and extracellular beads using streptoacidin staining. B cell phagocytosis was observed, and Fo B2, MZ, and B1 B cells were all capable of phagocytosing the anti-IgM-coated beads. This ability was completely eliminated by treatment with cytochalasin D (a potent inhibitor of phagocytosis). Treatment with mObx at a concentration of 30 µg / ml significantly reduced the phagocytic uptake of these beads compared to co-culture with isotypical controls. Figure 3E and Figure 3F This indicates that mObx reduces BCR-mediated antigen uptake in mice via B cells.

[0134] Example 3: Obelimab inhibits human BCR-mediated antigen uptake and phagocytosis.

[0135] This embodiment uses in vitro The human B cell phagocytosis assay investigated whether treatment with 30 µg / ml Obx inhibited human BCR-mediated phagocytosis.

[0136] Human blood was drawn from healthy donors (n=4), and B cells were isolated and incubated for 2 hours with anti-human IgM conjugated beads and Obx. B cell phagocytosis was subsequently assessed by flow cytometry. Figures 4A-4DThe bead targets were coated with anti-human IgM for targeting the BCR in an antigen-agnostic manner and biotinylated OVA to allow differentiation between internalized and extracellular beads using streptoacidin staining. Human Fo B2, MZ, and B1 B cells were observed to phagocytose the anti-human IgM-coated beads. This ability was reduced by treatment with cytochalasin D (a potent inhibitor of phagocytosis). Treatment with 30 µg / ml Obx significantly reduced the phagocytic uptake of these beads compared to the untreated control. Figure 4E and Figure 4F This indicates that Obx reduces antigen uptake mediated by human BCR in B cells.

[0137] Example 4: Reduction of B cell and T cell activation in response to antigen stimulation in the presence of an obemizole alternative (mObx) Following B cell antigen acquisition, B cells directly interact with T cells, with B cells acting as antigen-presenting cells (APCs) and presenting the acquired antigens to T cells, leading to B cell activation. To assess whether treatment with mObx reduced B cell and T cell activation in response to B cell-T cell-dependent interactions, B cells (5 × 10⁻⁶) from 2B-KIX mice were used. 5 ) in OT-II CD4 T cells stained with CTViolet (5 × 10 5 ) and beads conjugated with anti-mouse IgM biotinylated OVA (5 × 10) 5 Four days before co-culturing (each bead), the cells were pretreated with 30 µg / ml mObx for 1 hour. Since T / B interactions are essential for B cell activation into T cell-dependent antigens, flow cytometry was used to analyze the expression of activation markers on B cells (…). 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 upregulation compared to untreated B cells. Figures 5B-5E When compared to idiotype controls, B cells co-cultured with mObx expressed less CD80 and CD86 on their surface, indicating that mObx-treated B cells are less susceptible to co-stimulation 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, this increase was reduced when B cells were also co-cultured with mObx compared to untreated B cells and idiotype-controlled B cells. Figures 5F-5I In summary, these findings suggest that treatment with mObx at a concentration of 30 µg / ml can reduce T cell-mediated B cell activation.

[0138] Flow cytometry was also used to evaluate T cell activation markers. Figure 6A It was observed that antigen-specific CD4 T cells proliferated, as did B cells, when co-cultured with antigen-bound beads. However, CD4 T cells co-cultured with B cells treated with 30 µg / ml mObx showed a significant reduction in CD4 T cell proliferation compared to co-cultured with untreated B cells and B cells treated with idiotype controls. Figure 6B , Figure 6C Furthermore, when co-cultured with untreated and idiotype-treated B cells, CD4 T cells bound to mObx-treated B cells showed a significantly reduced level of surface CD44 expression. Figure 6D This confirmed that reduced CD4 T cell activation was corroborated by reduced proliferation. When observing activated T cells (CD4+CD44+), it was observed that activated T cells co-cultured with mObx-treated B cells showed significantly reduced CD25 expression on their surface compared to untreated B cells. CD25 expression was also reduced compared to idiotype controls, but not significantly. Figure 6E , Figure 6F Therefore, even proliferating CD4 T cells showed lower activation levels in the presence of mObx. However, mObx-treated proliferating B cells experienced a similar number of divisions as untreated or idiotype-treated B cells. Figure 6G The data indicate that proliferation itself is not affected. In summary, this data suggests that B cells treated with mObx have a reduced ability to activate CD4 T cells.

[0139] Example 5: Evaluation of B cell suppression and exhaustion as a method for limiting systemic and local B cell function B cells are necessary to initiate several EAE models. For example, in 2D2 IgH MOG In the spontaneous EAE (sEAE) model, B cells are involved in initiating an autoimmune T cell response (possibly as APCs), with the number of MOG-sp B cells significantly increased in lymphoid tissues (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 MOGs (or humanized MOGs, such as bMOG) are involved. tagDisease induced by the protein (MOG) depends on the generation of anti-MOG antibodies to initiate (Lyons, J.-A. et al., 2002. Eur. J. Immunol. 32: 1905–1913 and Lu, Y. et al., 2022. J. Immunol. 209: 2083–2092). The generation of high-affinity antibodies depends on a germinal center response, and although this germinal center response is MHC class II dependent, it is not equivalent to the function of specialized APCs activating naive T cells. The newly described PLP... ECD The induced model is unique because it requires B cell APC function (rather than antibody formation) to induce the disease and maintain its progression (Boyden, AW et al., 2020. Sci. Rep. 10: 5011 and Wilhelm, CR et al., 2023. J. Immunol. Ji2200721). Other induced EAE models that incorporate T cell and B cell recognition of antigens (such as mMOG) are also relevant. tag Induced EAEs do not rely on B cells to initiate disease, but MOG-sp B cells appear to contribute to more severe disease (Dang, A. K et al., 2015. J. Neuroimmunol. 278C: 73–84).

[0140] Most current studies on B-cell APC function do not or cannot distinguish between lymphoid tissues. and Potential B-cell APC function may occur in inflamed meninges. Targeting B cells in this location is difficult, largely due to the blood-brain / meningeal barrier. In fact, recent studies of anti-CD20 treatment in sEAE mice have shown that intravenous ( iv Anti-CD20, when administered intrathecally, rapidly binds to B cells in lymphoid tissues but is largely excluded from meningeal B cells, even in the presence of persistent inflammation described as “disrupting” the blood-blood barrier (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. used intrathecal anti-CD20... it.) was administered in an attempt to directly target B cells in the CNS and meninges (Lehmann-Horn, KS et al., 2014. Ann. Clin. Transl. Neurol. 1: 490–496). However, in these experiments, peripheral B cells were also depleted to the same level as Intravenous Similar levels of administration of anti-CD20 indicate Intrasheath Application is not limited to CNS.

[0141] This example evaluates obiblimab (Obx) in a mouse MS model, which is a repressive Fc inhibitor that specifically binds to CD19-expressing B cells. RIIb is a bispecific monoclonal antibody that inhibits B cell function without relying on B cell destruction (Chu, SY et al., 2008. Mol. Immunol. 45: 3926–3933). To determine whether inhibition of peripheral B cell function could prevent disease induction or progression, PLP was targeted. ECD Induced EAE mice (a model that has shown disease progression uniquely dependent on B cells rather than antibodies (Boyden et al., 2020 and Wilhelm et al., 2023)) were treated with PLP ECD Anti-CD20 or mObx was administered intravenously one day before immunization or alternatively 14 days after immunization (corresponding to disease onset). Disease progression was tracked, and mice were sacrificed once they reached the chronic phase of the disease (2 weeks after onset). Blood and lymph nodes were analyzed by flow cytometry to quantify B cell counts, confirm binding to anti-CD20 or mObx, and determine T cell activation and differentiation into effector subsets. Spinal cord was collected for immunofluorescence histological analysis of T cell and B cell inflammation and demyelination in the meninges and white matter, 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 are used to quantify the number of T cells and B cells in the tissue and determine... Intravenous The degree to which the administered anti-CD20 or mObx binds to B cells. This experiment was performed on male and female mice separately to determine whether there were sex differences in the treatment response to mObx.

[0142] Example 6. Inhibition of meningeal B cell function and disease progression Intrathecal monoclonal antibodies itThe administration bypassed the blood-brain and blood-meningeal barriers, but did not prevent [the spread of the virus]. sheath Inside The applied antibody leaks into the periphery. Therefore, Intravenous Application can be used to treat B cells outside the CNS, while Intrasheath Administer treatment targeting B cells in the CNS / meninges and peripheral regions.

[0143] PLP ECD EAE-induced mice were administered anti-CD20 or mObx before disease symptoms appeared (1 day after immunization), during the acute phase (5 days after onset), or during the chronic phase (2 weeks after onset). Intrasheath Treatment. Disease progression was tracked, and at the end of the experiment, spinal cord was harvested for analysis of local pathology and inflammation as described above. The tissue composition of the meningeal clusters, the T-cell to B-cell ratio, and the physical interactions between these cells within the clusters were evaluated, as B-cell inhibition may alter T:B interactions and localization. Flow cytometry was used to confirm the interaction between B cells and... Intrasheath The administered drugs are combined and used to quantify the number of infiltrating B cells and T cells and their activation status.

[0144] Example 7. Effects of B cell inhibition on T cell:B cell interactions in the meninges B cells in the meninges may influence local inflammation through local APC function, presenting locally acquired autoantigens and thereby reactivating newly recruited autoimmune T cells (Pipi et al., 2018). Actual Ag presentation occurs through highly coordinated direct physical interactions between cells. This example evaluates the effect of mObx inhibition of B cell function on T:B interactions in lymph nodes or the meninges. In the early stages of the anti-MOG response in lymph nodes, the interaction between MOG-sp T cells and B cells was directly observed using in vivo microscopy. MOG-specific B cells form germinal centers by presenting Ag to anti-MOG T cells in the early stages of the response following mMOGtag immunization (Jain et al., 2018; Parham et al., 2022).

[0145] Activate MOG-sp RFP + 2D2 T cells were transferred to mMOG cells in 2B-KIX mice in the acute phase of the disease. tag Induced or PLP ECD Induced EAE. Simultaneously, mice were treated with mObx or an isotype control. Intrasheath Treatment. Spinal cord was collected 2 or 7 days after treatment for immunofluorescence histology to determine RFP in treated mice. +Is the association between T cells and B cells weaker, is the number of T cells reduced, or are these T cells less abundant in the parenchyma or meninges?

[0146] Vial microscopy of the inflamed spinal cord was used to directly observe the interaction between T cells and meningeal B cells after mObx treatment as described above. This was also applied to metastatic RFP. + The duration of interaction between 2D2 T cells and endogenous B cells (B cell-specific conditional GFP mice (CD19cre x ROSA26-EGFP mice as acceptors) was quantified. Furthermore, imaging of the base of the meningeal clusters was performed after mObx treatment to evaluate whether autoreactive T cells crossed the boundary to reach the underlying white matter after interacting with B cells.

[0147] Other implementation plans While many embodiments of the invention have been described herein, changes may be made to this disclosure and embodiments to provide other methods and compositions of the invention. Therefore, it should be understood that the scope of this invention, in addition to the specific embodiments already illustrated by way of example, is defined by the appended claims. All references cited herein are hereby incorporated by way of reference.

Claims

1. A method for treating multiple sclerosis (MS) in a subject of need, comprising: The subjects were administered an anti-CD19 antibody containing both light and heavy chains. 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. The heavy chain contains an Fc region containing amino acid substitutions for 267E and L328F.

2. A method for treating multiple sclerosis (MS) in a subject of need, comprising: The subjects were administered an anti-CD19 antibody containing both light and heavy chains. The light chain contains a light chain variable region having at least 90% identity with the amino acid sequence of SEQ ID NO: 11, and The heavy chain contains a heavy chain variable region having at least 90% identity with the amino acid sequence of SEQ ID NO: 12, and The heavy chain contains an Fc region containing amino acid substitutions for 267E and L328F.

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

11.

4. The method as described in any of the preceding claims, wherein the anti-CD19 antibody comprises a heavy chain and a light chain. The heavy chain contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 10; and The light chain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:

9.

5. The method of any of the preceding claims, wherein the anti-CD19 antibody comprises: Heavy chains containing the amino acid sequence of SEQ ID NO: 10; and A light chain containing the amino acid sequence of SEQ ID NO:

9.

6. The method as described in any of the preceding claims, wherein the anti-CD19 antibody is obbelimab.

7. The method of any of the preceding claims, wherein the administration of the anti-CD19 antibody improves, alleviates or delays one or more symptoms of MS.

8. The method of any of the preceding claims, wherein the anti-CD19 antibody is administered intrathecally.

9. The method of any of the preceding claims, wherein the anti-CD19 antibody is administered intravenously.

10. The method of any of the preceding claims, wherein the anti-CD19 antibody is administered subcutaneously.

11. The method of any of the preceding claims, wherein the anti-CD19 antibody is administered in the form of a liquid formulation comprising 125 mg / ml anti-CD19 antibody, 2.35 mg / ml sodium acetate trihydrate, 0.17 mg / ml acetic acid, 30 mg / ml L-proline, 0.1 mg / ml polysorbate 80, and a pH of 5.

5.

12. The method of any of the preceding claims, wherein the anti-CD19 antibody is administered using a drug-loaded syringe or an autoinjector.

Images