Anti-BCMA treatment in autoimmune disorders

Abstract

The present invention relates to the treatment or management of autoimmune disorders, such as autoimmune disorders caused by autoreactive B-cell lineage cells, for example antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 975,663, filed February 12, 2020, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application incorporates by reference in its entirety a Sequence Listing in computer readable form (CRF) in ASCII text format. The Sequence Listing text file is named "14247-482-228_SEQ_LISTING", was created on February 11, 2021, and is 106,995 bytes in size.

[0003] FIELD OF THE INVENTION The present invention relates to the treatment or management of autoimmune disorders, such as autoimmune disorders caused by autoreactive B-cell lineage cells, for example antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). [Background technology]

[0004] background Autoimmune disorders occur when a subject's immune system attacks healthy tissues or organs of the subject's own body. In some cases, these disorders may result from abnormal recognition of antigens from the subject's own tissues ("autoantigens") by B cell lineage cells ("autoreactive B cell lineage cells"), such as memory B cells, plasmablasts, and / or plasma cells. In some cases, autoreactive plasmablasts and plasma cells may produce autoreactive antibodies ("autoantibodies") that recognize and / or attack healthy tissues or organs that express the autoantigens. Systemic lupus erythematosus (SLE) has been described as a typical autoimmune disorder (Fava, A. and Petri, M. (2019). Systemic lupus erythematosus: Diagnosis and clinical management. Journal of autoimmunity, 96, 1-13).

[0005] Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a severe autoimmune disorder caused by autoreactive B-cell lineage cells that is associated with considerable morbidity and mortality. Patients with AAV cycle between periods of active disease and remission of varying lengths. There is currently no cure for AAV, and 50% of patients die or suffer from severe complications during the active disease phase. AAV is characterized by destructive inflammation of small- to medium-sized blood vessels mediated by ANCA autoantibodies. Summary of the Invention [Problem to be solved by the invention]

[0006] Existing treatments for autoimmune disorders are not always effective in inducing or maintaining remission and / or may have undesirable side effects. Therefore, there is a need for additional therapies for the treatment or management of autoimmune disorders. [Means for solving the problem]

[0007] overview The present invention relates to BCMA and 1 or more Promotes T cell activation Resistance The present invention relates to methods of treating or managing a subject with an autoimmune disorder using multispecific (e.g., bispecific) antibodies that bind to an antigen (e.g., CD3).

[0008] In one embodiment, the present invention provides a method of treating or managing an autoimmune disorder, comprising administering to a subject (e.g., a human) in need of such treatment or management a multispecific (e.g., bispecific) antibody, wherein the multispecific antibody is capable of inhibiting BCMA and 1 or more Promotes T cell activation Resistance The method further provides a method for detecting a target antigen (e.g., CD3) that binds to a target antigen (e.g., CD4).

[0009] In other aspects, the present invention provides a method for treating or managing an autoimmune disorder in a subject (e.g., a human) comprising administering to a subject an antibody against BCMA and / or a nucleotide sequence encoding the antibody. 1 or more Promotes T cell activation ResistanceMultispecific (e.g., bispecific) antibodies that bind to an antigen (e.g., CD3) are provided.

[0010] In a preferred embodiment, the autoimmune disorder is caused by a B cell lineage cell (e.g., an autoreactive B cell lineage cell). In a preferred embodiment, the B cell lineage cell, e.g., an autoreactive B cell lineage cell, is a memory B cell, a plasmablast, and / or a plasma cell.

[0011] In some embodiments, the autoimmune disorder is selected from systemic lupus erythematosus, IgA nephropathy, IgG4-related disease, membranous nephropathy, myasthenia gravis, neuromyelitis optica, pemphigus vulgaris, anti-PAD4-activated rheumatoid arthritis, sensitized / pre-existing antibodies in solid organ transplantation, Guillain-Barré syndrome (acute inflammatory demyelinating polyneuropathy - AIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), immune thrombocytopenic purpura, rheumatoid arthritis, and ANCA-associated vasculitis (AAV). In preferred embodiments, the autoimmune disorder is not an IgG4-related disease. Preferably, the autoimmune disorder is ANCA-associated vasculitis (AAV), systemic lupus erythematosus (SLE), and / or rheumatoid arthritis. In preferred embodiments, the autoimmune disorder is ANCA-associated vasculitis (AAV) and / or rheumatoid arthritis.

[0012] In certain embodiments, the autoimmune disorder is newly diagnosed (e.g., newly diagnosed AAV, SLE, or rheumatoid arthritis). In certain embodiments, the autoimmune disorder is relapsed or refractory (e.g., relapsed or refractory AAV, SLE, or rheumatoid arthritis).

[0013] In some embodiments, the AAV comprises a disease selected from the group consisting of granulomatosis with polyangiitis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA), microscopic polyangiitis (MPA), and renal-confined ANCA-associated vasculitis. In some embodiments, the AAV is granulomatosis with polyangiitis (Wegener's granulomatosis), eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, or renal-confined ANCA-associated vasculitis.

[0014] In certain embodiments, the AAV affects one or more parts of the patient's body selected from the nervous system, eyes, nose, heart, kidneys, stomach, intestines, lungs, joints, muscles, and skin. In certain embodiments, the AAV is systemic with life-threatening or major organ-threatening manifestations, and the patient may also have diffuse alveolar hemorrhage (DAH). In other embodiments, the AAV localizes without any organ-threatening manifestations.

[0015] In some embodiments, the patient is in need of plasmablast reduction. In some embodiments, the patient is in need of induction of remission. In some embodiments, the patient is in need of maintenance of remission. In some embodiments, the method results in at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least a 95% or 100% reduction in plasmablasts in the patient compared to no treatment or a control treatment.

[0016] In some embodiments, the patient requires induction of remission. In some embodiments, the method is used to induce remission, optionally wherein the method results in induction of remission in the patient at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95% or 100% faster than the control treatment. In other embodiments, the patient requires maintenance of remission. In some embodiments, the method is used to maintain remission, optionally wherein the method results in maintenance of remission in the patient at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95% or 100% longer than the control treatment.

[0017] In some embodiments, the patient is at risk of developing cytokine release syndrome. In some embodiments, the method results in at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least a 95% or 100% reduction in the incidence of cytokine release syndrome in the patient compared to a control treatment.

[0018] In some embodiments, the patient is at risk of developing an infection. In some embodiments, the method results in at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least a 95% or 100% reduction in the incidence of infection in the patient compared to a control treatment.

[0019] In certain embodiments, the control treatment is treatment with a steroid (e.g., a glucocorticoid), cyclophosphamide, an anti-CD20 monoclonal antibody (e.g., rituximab), methotrexate, azathioprine, mycophenolic acid, mycophenolate mofetil, avacopan, an anti-TNF agent (e.g., infliximab, adalimumab, golimumab, etanercept), an anti-IL6R antibody (e.g., tocilizumab, sarilumab), costimulatory blockade (e.g., abatacept), a JAK inhibitor (e.g., tofacitinib, baricitinib), and / or belimumab, preferably wherein the control treatment is treatment with a steroid, cyclophosphamide, or rituximab.

[0020] In some embodiments, the multispecific (e.g., bispecific) antibody comprises an anti-BCMA antibody, or antigen-binding fragment thereof, that comprises a CDR1H, CDR2H, CDR3H, CDR1L, CDR2L, and CDR3L region combination selected from the group consisting of: a) the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 23, the CDR2L region of SEQ ID NO: 24 and the CDR3L region of SEQ ID NO: 20; b) the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 25, the CDR2L region of SEQ ID NO: 26 and the CDR3L region of SEQ ID NO: 20; c) the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 27, the CDR2L region of SEQ ID NO: 28 and the CDR3L region of SEQ ID NO: 20; d) the CDR1H region of SEQ ID NO: 29, the CDR2H region of SEQ ID NO: 30, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 31, the CDR2L region of SEQ ID NO: 32 and the CDR3L region of SEQ ID NO: 33; e) the CDR1H region of SEQ ID NO: 34, the CDR2H region of SEQ ID NO: 35, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 31, the CDR2L region of SEQ ID NO: 32 and the CDR3L region of SEQ ID NO: 33; f) the CDR1H region of SEQ ID NO: 36, the CDR2H region of SEQ ID NO: 37, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 31, the CDR2L region of SEQ ID NO: 32 and the CDR3L region of SEQ ID NO: 33; and g) The CDR1H region of SEQ ID NO: 15, the CDR2H region of SEQ ID NO: 16, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 18, the CDR2L region of SEQ ID NO: 19 and the CDR3L region of SEQ ID NO: 20.

[0021] In a particularly preferred embodiment, the anti-BCMA antibody or antigen-binding fragment thereof comprises a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ ID NO:22, a CDR3H region of SEQ ID NO:17, a CDR1L region of SEQ ID NO:27, a CDR2L region of SEQ ID NO:28 and a CDR3L region of SEQ ID NO:20.

[0022] In certain embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises a VH and a VL selected from the group consisting of: a) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 12; b) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13; c) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14; d) a VH region of SEQ ID NO: 38 and a VL region of SEQ ID NO: 12; e) a VH region of SEQ ID NO: 39 and a VL region of SEQ ID NO: 12; f) a VH region of SEQ ID NO: 40 and a VL region of SEQ ID NO: 12, or g) A VH region of SEQ ID NO: 9 and a VL region of SEQ ID NO: 11.

[0023] In one embodiment, the anti-BCMA antibody or antigen-binding fragment thereof a) a variable region VH comprising an amino acid sequence that is at least 90% identical, at least 95% identical, at least 99% identical or identical to the amino acid sequence of SEQ ID NO: 10 and a variable region VL comprising an amino acid sequence that is at least 90% identical, at least 95% identical, at least 99% identical or identical to the amino acid sequence of SEQ ID NO: 14; b) a variable region VH comprising an amino acid sequence that is at least 90% identical, at least 95% identical, at least 99% identical or identical to the amino acid sequence of SEQ ID NO: 10 and a variable region VL comprising an amino acid sequence that is at least 90% identical, at least 95% identical, at least 99% identical or identical to the amino acid sequence of SEQ ID NO: 13; or c) a variable region VH comprising an amino acid sequence that is at least 90% identical, at least 95% identical, at least 99% identical or identical to the amino acid sequence of SEQ ID NO: 9, and a variable region VL comprising an amino acid sequence that is at least 90% identical, at least 95% identical, at least 99% identical or identical to the amino acid sequence of SEQ ID NO: 11 Includes.

[0024] In a particularly preferred embodiment, the anti-BCMA antibody or antigen-binding fragment thereof comprises a VH region of SEQ ID NO:10 and a VL region of SEQ ID NO:14.

[0025] In one embodiment, 1 or more Promotes T cell activation ResistanceThe antigen is selected from the group consisting of CD3, TCRα, TCRβ, TCRγ, TCRζ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. 1 or more Promotes T cell activation Resistance Hara is CD3.

[0026] In a preferred embodiment, the multispecific (e.g., bispecific) antibody comprises an anti-CD3 antibody or antigen-binding fragment thereof comprising a variable domain VH comprising the heavy chain CDRs of SEQ ID NOs: 1, 2, and 3 as heavy chain CDR1H, CDR2H, and CDR3H, respectively, and a variable domain VL comprising the light chain CDRs of SEQ ID NOs: 4, 5, and 6 as light chain CDR1L, CDR2L, and CDR3L, respectively. In one embodiment, the anti-CD3 antibody or antigen-binding fragment thereof comprises the VH region of SEQ ID NO: 7 and the VL region of SEQ ID NO: 8.

[0027] In certain embodiments, the multispecific (e.g., bispecific) antibody comprises an anti-CD3 antibody, or antigen-binding fragment thereof, comprising a variable region VH comprising an amino acid sequence that is at least 90% identical, at least 95% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:7, and a variable region VL comprising an amino acid sequence that is at least 90% identical, at least 95% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:8.

[0028] In a particularly preferred embodiment, the multispecific (e.g. bispecific) antibody comprises an anti-BCMA antibody, or antigen-binding fragment thereof, comprising the VH region of SEQ ID NO: 10 and the VL region of SEQ ID NO: 14, and an anti-CD3 antibody, or antigen-binding fragment thereof, comprising the VH region of SEQ ID NO: 7 and the VL region of SEQ ID NO: 8.

[0029] In certain embodiments, the multispecific antibody is a bispecific antibody. In a preferred embodiment, the multispecific antibody is a bispecific antibody that binds to BCMA and CD3. In certain embodiments, the bispecific antibody is bivalent (e.g., in a 1+1 format). In certain embodiments, the bivalent bispecific antibody has the format: CD3 Fab-BCMA Fab (i.e., when Fc is absent). Alternatively, the bivalent bispecific antibody can have the format: Fc-CD3 Fab-BCMA Fab; Fc-BCMA Fab-CD3 Fab; or BCMA Fab-Fc-CD3 Fab (i.e., when Fc is present). In a preferred embodiment, the bivalent bispecific antibody has the format BCMA Fab-Fc-CD3 Fab. In certain embodiments, the bispecific antibody is trivalent (e.g., in a 2+1 format). In a preferred embodiment, the bispecific antibody is trivalent and comprises two Fab fragments of an anti-BCMA antibody, one Fab fragment of an anti-CD3 antibody, and one Fc portion. In certain embodiments, the trivalent bispecific antibody has the format: CD3 Fab-BCMA Fab-BCMA Fab; or BCMA Fab-CD3 Fab-BCMA Fab (i.e., when Fc is absent). Alternatively, the trivalent bispecific antibody may have the format: BCMA Fab-Fc-CD3 Fab-BCMA Fab; BCMA Fab-Fc-BCMA Fab-CD3 Fab; or CD3 Fab-Fc-BCMA Fab-BCMA Fab (i.e., when Fc is present). In a preferred embodiment, the trivalent bispecific antibody has the format BCMA Fab-Fc-CD3 Fab-BCMA Fab.

[0030] In one embodiment, the anti-CD3 Fab comprises a light chain and a heavy chain, wherein the light chain is a crossover light chain comprising a variable domain VH and a constant domain CL, and the heavy chain is a crossover heavy chain comprising a variable domain VL and a constant domain CH1.

[0031] In one embodiment, the CH1 domain of the anti-BCMA Fab fragment comprises the corresponding immunoglobulin light chain comprising a CL domain with the amino acid modifications K147E / D and K213E / D (numbering according to EU numbering) and the amino acid modifications E123K / R / H and Q124K / R / H (numbering according to Kabat).

[0032] In another embodiment, the CH1 domain of the anti-BCMA Fab fragment comprises the corresponding immunoglobulin light chain comprising a CL domain with the amino acid modifications A141W, L145E, K147T and Q175E (numbering according to EU numbering) or conservative substitutions thereof and the amino acid modifications F116A, Q124R, L135V and T178R (numbering according to Kabat) or conservative substitutions thereof.

[0033] In some embodiments, the multispecific (e.g., bispecific) antibody further comprises an Fc. In some embodiments, the Fc is an IgG1 Fc. In some embodiments, the (e.g., IgG1) Fc comprises a first Fc chain comprising a first constant domain, CH2, and CH3, and a second Fc chain comprising a second constant domain, CH2, and CH3, wherein: a) the first CH3 domain comprises the modifications T366S, L368A and Y407V or conservative substitutions thereof (numbering according to EU numbering); and b) The second CH3 domain contains the modification T366W or a conservative substitution thereof (numbering according to EU numbering).

[0034] In another embodiment, the (e.g., IgG1) Fc comprises a first Fc chain comprising first constant domains CH2 and CH3 and a second Fc chain comprising second constant domains CH2 and CH3, wherein: a) the first CH3 domain comprises the modifications T350V, L351Y, F405A and Y407V or conservative substitutions thereof (numbering according to EU numbering); and b) The second CH3 domain comprises the modifications T350V, T366L, K392L and T394W or conservative substitutions thereof (numbered according to EU numbering).

[0035] In one embodiment, the (e.g., IgG1) Fc is: a) the modifications L234A, L235A and P329G (numbering according to the EU numbering system); and / or b) Modifications D356E and L358M (numbered according to the EU numbering system) Includes.

[0036] In a further embodiment, the multispecific (e.g., bispecific) antibody of the invention comprises the following SEQ ID NO: i. 83A10-TCBcv:45, 46, 47(×2), 48 ii. 21-TCBcv:49, 50, 51(×2), 48 iii. 22-TCBcv:52, 53, 54(×2), 48 iv. 42-TCBcv:55, 56, 57(×2), 48 v. Mab101:58, 59, 60(×2), 48 vi. Mab102:61, 62, 63(×2), 48 vii. Mab103:64, 65, 66(×2), 48

[0037] In a preferred embodiment, the bispecific antibody of the invention is 42-TCBcv, Mab101 or Mab102. In a particularly preferred embodiment, the bispecific antibody of the invention is 42-TCBcv.

[0038] Aspects and embodiments of the invention are set out in the accompanying claims. These and other aspects and embodiments of the invention are also described herein. [Brief explanation of the drawings]

[0039] The present invention will now be described in more detail with reference to the accompanying drawings, in which: [Figure 1]Various formats of bispecific, bivalent antibodies for use in the present invention are described, including a Fab fragment that binds a T cell antigen (CD3 is exemplified) and BCMA in the format Fab BCMA-Fc-Fab CD3. The CD3 Fab may contain a VH-VL crossover to reduce light chain mispairing and side products. Amino acid substitutions ("RK / EE" is exemplified) may be introduced into CL-CH1 to reduce light chain mispairing / side products during manufacturing. The CD3 Fab and BCMA Fab may be linked to each other with a flexible linker.

[0040] [Figure 2] Various formats of bispecific trivalent antibodies for use in the present invention are described, including Fab fragments that bind to a T cell antigen (CD3 is exemplified) and BCMA in the following formats: Fab BCMA-Fc-Fab CD3-Fab BCMA (A, B); Fab BCMA-Fc-Fab BCMA-Fab CD3 (C, D). The CD3 Fab may contain a VH-VL crossover to reduce light chain mispairing and side products. Amino acid substitutions ("RK / EE" is exemplified) may be introduced into CL-CH1 to reduce light chain mispairing / side products during manufacturing. The CD3 Fab and BCMA Fab may be linked to each other with a flexible linker.

[0041] [Figure 3] Further formats of bispecific, bivalent antibodies for use in the present invention are shown, including Fab fragments that bind to a T cell antigen (CD3 is exemplified) and BCMA in the following formats: Fc-Fab CD3-Fab BCMA (A, B); Fc-Fab BCMA-Fab CD3 (C, D). The CD3 Fab may contain a VH-VL crossover to reduce light chain mispairing and side products. Amino acid substitutions ("RK / EE" is exemplified) may be introduced into CL-CH1 to reduce light chain mispairing / side products during manufacturing. The CD3 Fab and BCMA Fab may be linked to each other with a flexible linker.

[0042] [Figure 4]Figure 1 shows BMCA expression in plasmablasts (PB) from four normal healthy volunteers (NHV) compared with BCMA-expressing cancer cell lines (JEKO, RPMI-8226, and H929) as assessed by flow cytometry (A). Soluble BCMA levels are assessed by ELISA and shown in serum or plasma samples from NHV ('normal'), multiple myeloma ('MM'), or ANCA-associated vasculitis ('AAV') patients (B).

[0043] [Figure 5] Dose-response curves for T cell-mediated cell death (A) and T cell activation (B) are shown when JEKO cells were cultured with CD3+ T cells in a 1:2 target:effector (T:E) ratio and treated with anti-BCMA, anti-CD3 bispecific antibodies, i.e., BCMA T cell engagers (CC-93269, Mab101, or Mab102). T cell-mediated cytotoxicity of JEKO cells was assessed by Annexin V expression; the 20-hour time point is shown. T cell lineage and activation markers were analyzed by flow cytometry at 24 hours; %CD69 expression of CD8+ T cells is shown.

[0044] [Figure 6] Dose-response curves of T cell-mediated cell killing (A) and T cell activation (B) are shown when RPMI-8226 cells were co-cultured with NHV PBMCs at various target:effector (T:E) ratios and various concentrations of CC-93269.

[0045] [Figure 7]Dose-response curves for plasmablast death (A), T cell activation (C), and cytokine production (D) are shown for peripheral blood mononuclear cells (PBMCs) from healthy volunteers treated with various concentrations of BCMA T cell engagers, i.e., BCMA TCEs (Mab101, CC-93269, or Mab102), or a control 2+1 antibody, for 24 hours. Representative FACS plots show plasmablast differentiation as CD20(-) CD27(+) cells, gating on CD3(-) CD19(+) cells (B). Plasmablast death is assessed as a percentage of total CD19(+) cells (A). %CD69 expression of CD8+ T cells is shown (C).

[0046] [Figure 8] Dose-response curves for cytokine production (IFNγ, IL-6, IL-2, IL-10, granzyme B, and perforin) are shown when PBMC from healthy volunteers are treated with various concentrations of CC-93269 for 24 hours.

[0047] [Figure 9] Figure 1 shows the lineage distribution of B cells in PBMCs from healthy volunteers treated with various concentrations of CC-93269 for 24 hours. Naive B cells with CD20(+)CD27(-)IgD(+) expression (A), unswitched memory B cells with CD20(+)CD27(+)IgD(+) expression (B), and switched memory B cells with CD20(+)CD27(+)IgD(-) expression (C) are shown as a percentage of total CD19(+) cells.

[0048] [Figure 10] Dose-response curves for plasmablast death (A) and T cell activation (B) are shown for bone marrow (BM) mononuclear cells treated with CC-93269 for 24 hours compared to PBMCs suspended in medium or PBMCs suspended in BM supernatant. Plasmablast death is assessed as a percentage of total CD19(+) cells (A). % CD69 expression of CD8+ T cells is shown (B).

[0049] [Figure 11]Dose-response curves for plasmablast killing (A), T cell activation (C), and cytokine production (D) are shown for PBMCs from an AAV patient treated with various concentrations of BCMA TCE (Mab101, CC-93269, or Mab102) or control 2+1 antibody for 24 hours. Representative FACS plots show plasmablast differentiation as CD20(-) CD27(+) gating on CD3(-) CD19(+) cells (B). Plasmablast death is assessed as a percentage of total CD19(+) cells (A). %CD69 expression of CD8+ T cells is shown (C).

[0050] [Figure 12] Figure 1 shows T cell activation, assessed by staining for T cell lineage (CD4, CD8) and activation markers (CD69, CD25) in PBMCs from an AAV patient, AAV1, upon 24-hour incubation with BCMA TCE (Mab101 [A], CC-93269 [B], or Mab102 [C]). %CD69 or %CD25 expression levels in CD4+ or CD8+ cells are shown.

[0051] [Figure 13] Selective plasmablast (PB) depletion after incubation with various concentrations of CC-93269 (B, C) or control 2+1 antibody (A) is shown in PBMCs from an AAV patient, AAV-5, who last received rituximab 5 months prior.

[0052] [Figure 14] Baseline CD19(+) CD20(-) CD27(+) plasmablast targets (A) and CD4(+) / CD8(+) T cells are shown in AAV-2 subjects. PBMCs from AAV-2 were incubated with various concentrations of BCMA TCE, and after 24 hours of incubation, T cell activation was assessed by flow cytometry (C).

[0053] [Figure 15]Dose-response curves of T cell activation upon incubation of JEKO-1 cells with CC-93269 or a control 2+1 antibody at various T:E ratios are shown. JEKO-1 cells were cultured with PBMCs at a T:E ratio of 1:10 or 1:500, mimicking the T:E ratios observed with multiple myeloma (MM) or AAV, respectively. After 24 hours of incubation, cells were washed, and CD8(+) T cells were then assessed for CD69 (A) and CD25 (B) expression.

[0054] [Figure 16] PBMCs from healthy volunteers were incubated with various concentrations of BCMA TCE for 24 hours and then stimulated with ODN2006 (CpG, 10 μg / mL) or cultured with the growth factors IL-2 (20 U / mL), BAFF (200 ng / mL), and IL-21 (100 ng / mL) for 4 to 7 days. Plasmablast levels (A) and total IgG secretion (B) are shown.

[0055] [Figure 17] Dose-response curves for plasmablast death (A), T cell activation (B), and cytokine production (C) are shown when PBMCs from rheumatoid arthritis (RA) patients were treated with various concentrations of CC-93269 or control 2+1 antibody for 24 hours.

[0056] [Figure 18] The lineages of B cells after 24-hour incubation of PBMCs from RA with various concentrations of CC-93269 are shown. Naive B cells with CD20(+)CD27(-)IgD(+) expression (A), unswitched memory B cells with CD20(+)CD27(+)IgD(+) expression (B), and switched memory B cells with CD20(+)CD27(+)IgD(-) expression (C) are shown as a percentage of total CD19(+) cells.

[0057] [Figure 19]Selective IRF4+ plasmablast (PB) depletion (A) and minimal CD20(+) B cell depletion (B) and minimal increase in the frequency of activated CD69(+)CD8(+) T cells (C) are shown in PBMCs from cynomolgus monkeys after incubation with various concentrations of CC-93269 or control 2+1 antibody.

[0058] [Figure 20] Dose-response curves for plasmablast death (A) and T cell activation (B) are shown when PBMCs (n=5) from patients with systemic lupus erythematosus (SLE) were treated with various concentrations of CC-93269 for 24 hours.

[0059] [Figure 21] The effects of exogenous soluble BCMA (sBCMA) on plasmablast death (A) and T cell activation (B) are shown when PBMCs from normal healthy volunteers are treated with CC-93269 for 24 hours.

[0060] [Figure 22] Dose-response curves of T cell activation (A) and cytokine production (B) are shown when whole blood samples from normal healthy volunteers (n=4) or AAV patients (n=2) were treated with various concentrations of CC-93269 for 24 hours.

[0061] [Figure 23] A representative SEC chromatogram overlay of the 22-TCBcv molecule after storage under the conditions specified in Example 18.3.3 is shown.

[0062] [Figure 24] Figure 1 shows the thermal unfolding of bispecific antibodies Mab101, Mab102, 83A10-TCBcv, and 22-TCBcv. All bispecific antibodies showed similar onset temperatures of thermal unfolding (approximately 60°C). However, the Tmapp values ​​for the maximum transition were approximately 5°C higher for Mab101 and 83A10-TCBcv molecules, which contain common CDR regions.

[0063] [Figure 25]Figure 1 shows colloidal stability evaluation of bispecific antibodies Mab101, Mab102, 83A10-TCBcv, and 22-TCBcv. Evaluation was performed by PEG 6000 precipitation and carried out in pH 6 buffer. Molecules containing the CDR regions of 83A10 (i.e., Mab101 and 83A10-TCBcv) required approximately twice as much PEG 6000 to induce native-state precipitation due to the excluded volume effect. The solid line is a fit to Equation 1.

[0064] [Figure 26] Representative single-cycle kinetic SPR sensorgrams of the bispecific antibody 83A10-TCBcv are shown. The antibody was buffered at pH 6 and stored at 2-8°C for 2 weeks (A) or buffered at pH 8 and stored at 40°C for 2 weeks (B). The dashed line represents the mean of 10 independent measurements of the unstressed sample before storage, and the dotted lines indicate three standard deviations (SD) of this mean. The percent similarity score was calculated using the number of data points of the stressed sample that fell within the SD window in Equation 2.

[0065] [Figure 27] Sequence alignment of bispecific antibodies Mab101, Mab102, 83A10-TCBcv and 22-TCBcv is shown. CDR regions are shaded and percent sequence identity is indicated above. DETAILED DESCRIPTION OF THE INVENTION

[0066] Detailed Description As used herein, the articles "a" and "an" may mean one or more than one (e.g., at least one) of the article.

[0067] "About" generally refers to an acceptable degree of error for the quantity being measured, given the nature or accuracy of the measurement. Examples of degrees of error are within 20 percent (%), typically within 10%, and more usually within 5% of a value or range of values.

[0068] An embodiment described herein as "comprising" one or more features may also be considered to disclose corresponding embodiments that "consist" and / or "consist essentially of" such features.

[0069] Concentrations, amounts, volumes, percentages, and other numerical values ​​may be presented herein in a range format, with the understanding that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values ​​specified as the limits of the range, but also each individual numerical value or range included therein, as if each numerical value and range were expressly recited.

[0070] Treatment method The present invention relates, at least in part, to a method for treating a subject with an autoimmune disorder, 1 or more Promotes T cell activation Resistance The method is based on treatment or administration with a multispecific (e.g., bispecific) antibody that binds to both CD3 (e.g., CD3) and BCMA.

[0071] In some embodiments, the "subject" or "patient" is a human. In some embodiments, the "subject" or "patient" is under 18 years of age. In some embodiments, the "subject" or "patient" is 18 years of age or older. In some embodiments, the subject requires induction of remission. In some embodiments, the subject requires maintenance of remission. In some embodiments, the patient requires plasmablastopenia.

[0072] As used herein, the terms "treatment" or "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. Preferably, the effect is therapeutic, i.e., a partial or complete cure of a disease and / or adverse symptoms resulting from the disease. Alternatively, the pharmacological and / or physiological effect may be prophylactic, i.e., a partial or complete prevention of a disease or its symptoms.

[0073] As used herein, the terms "management" or "managing" and the like refer to inhibiting and / or slowing the progression and / or worsening of a disease and / or adverse symptoms resulting from a disease.

[0074] The present invention provides 1 or more Promotes T cell activation Resistance The present invention relates to the treatment or management of autoimmune disorders with multispecific (e.g., bispecific) antibodies that bind to both CD3 (e.g., CD3) and BCMA.

[0075] In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention can selectively bind to cells that cause an autoimmune disorder, e.g., BCMA-expressing cells that cause an autoimmune disorder.

[0076] In a preferred embodiment, the autoimmune disorder is caused by B cell lineage cells (e.g., BCMA-expressing B cell lineage cells). In certain embodiments, the B cell lineage cells (e.g., BCMA-expressing B cell lineage cells) are autoreactive B cell lineage cells. In certain embodiments, the B cell lineage cells (e.g., BCMA-expressing B cell lineage cells) drive autoimmunity, for example, by acting as antigen-presenting cells or by secreting pro-inflammatory cytokines.

[0077] As used herein, the term "autoreactive B cell lineage cells" refers to B cell lineage cells that can recognize antigens on a subject's own tissues ("autoantigens"). Autoreactive B cell lineage cells can be antibody-secreting cells and / or can secrete antibodies. In certain embodiments, autoreactive B cell lineage cells are plasmablasts, plasma cells, memory B cells, or any combination thereof. In preferred embodiments, autoreactive B cell lineage cells are plasmablasts, plasma cells, and memory B cells. In particularly preferred embodiments, autoreactive B cell lineage cells are plasmablasts.

[0078] In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention target autoreactive B β agonists that cause autoimmune disorders. In a preferred embodiment, the autoreactive B cell lineage cells are BCMA-expressing cells, such as memory B cells, plasmablasts and / or plasma cells.

[0079] Plasmablasts are precursors of plasma cells. They can be identified by a combination of one or more markers selected from the group consisting of CD19(+), CD20(-), CD27(+), CD38(+), BCMA(+), IgD(-), CD24(-), SLAMF7(+), and / or CD138(-). In one embodiment, plasmablasts are identified as cells that exhibit BCMA(+) and SLAMF7(+), optionally wherein the cells also exhibit one or more of the markers IgD(-), CD38(+), and / or CD138(-). In one embodiment, plasmablasts are identified as cells that exhibit CD19(+) CD20(-) CD27(+) BCMA(+) SLAMF7(+) IgD(-) CD38(+) CD138(-). In a particularly preferred embodiment, plasmablasts are identified as cells exhibiting the markers CD19(+) CD20(-) CD27(+), optionally wherein the cells also exhibit one or more of the markers BCMA(+) and / or CD38(+).

[0080] Plasma cells are antibody-secreting cells. As used herein, the term "plasma cells" can refer to short-lived and / or long-lived plasma cells. They can be identified by a combination of one or more markers selected from CD19(+), CD20(-), CD27(+), CD38(+), BCMA(+), IgD(-), CD138(+) CD24(-), and / or SLAMF7(+). In one embodiment, plasma cells are identified as cells that exhibit the markers BCMA(+) SLAMF7(+) CD138(+), optionally wherein the cells also exhibit one or more of the markers IgD(-) and / or CD38(+). In one embodiment, plasma cells are identified as cells that exhibit CD19(+) CD20(-) CD27(+) BCMA(+) SLAMF7(+) IgD(-) CD38(+) CD138(+). In a particularly preferred embodiment, plasma cells are identified as cells exhibiting the markers CD19(+) CD20(-) CD27(+), optionally wherein the cells also exhibit one or more of the markers BCMA(+) CD38(+) and / or CD138(+).

[0081] Memory B cells are B cells activated by antigen and T cell helpers in extrafollicular or GC reactions. They can be identified by a combination of one or more markers selected from CD19(+), CD20(+), CD27(+), CD38(-), BCMA(+ / -), IgD(-), and / or CD24(+). In one embodiment, memory B cells are identified as cells that exhibit the markers IgD(-) CD38(-) BCMA(+ / -). In one embodiment, memory B cells are identified as cells that exhibit the markers CD19(+) CD20(+) CD27(+) IgD(-) CD38(-) BCMA(+ / -). In a particularly preferred embodiment, memory B cells are identified as cells that exhibit the markers CD19(+) CD20(+) CD27(+), optionally wherein the cells also exhibit one or more markers selected from BCMA(+) and / or CD38(-).

[0082] The inventors have determined that the multispecific (e.g., bispecific) antibodies of the invention can be used to treat or manage patients with autoimmune disorders, where the amount of soluble BCMA in a blood sample from the patient is less than the amount of soluble BCMA in multiple myeloma (MM) patients and / or the amount of soluble BCMA is equivalent to the amount of soluble BCMA in normal healthy patients. Soluble BCMA in a blood sample (e.g., isolated serum or plasma) from a patient can be measured by ELISA, for example, using a bead-based immunoassay by Ampersand Biosciences (Lake Clear, NY).

[0083] In some embodiments, the amount of soluble BCMA in a blood sample from a patient with an autoimmune disorder is less than the amount of soluble BCMA in a patient with a B-cell malignancy (e.g., a BCMA-expressing cancer). In some embodiments, the amount of soluble BCMA in a blood sample from a patient with an autoimmune disorder is less than the amount of soluble BCMA in a patient with MM, preferably about 1.5-fold less, about 2-fold less, about 2.5-fold less, about 3-fold less, about 3.5-fold less, about 4-fold less, about 4.5-fold less, about 5-fold less, about 5.5-fold less, or about 6-fold less than the amount of soluble BCMA in a patient with an autoimmune disorder, as measured by ELISA, is less than about 150 ng / ml, less than about 100 ng / ml, less than about 75 ng / ml, less than about 50 ng / ml, less than about 40 ng / ml, less than about 35 ng / ml, or less than about 30 ng / ml. In certain embodiments, the amount of soluble BCMA in a blood sample from a patient with an autoimmune disorder is within about 40 ng / ml, about 30 ng / ml, about 20 ng / ml, about 15 ng / ml, about 10 ng / ml, or about 5 ng / ml of the amount of soluble BCMA in a normal healthy individual, as measured by ELISA. In certain embodiments, the amount of soluble BCMA in a blood sample from a patient with an autoimmune disorder is at least 5 ng / ml, at least 7.5 ng / ml, at least 10 ng / ml, at least 12.5 ng / ml, or at least 15 ng / ml, as measured by ELISA. In certain embodiments, the amount of soluble BCMA in a blood sample from a patient with an autoimmune disorder is about 5 ng / ml to 50 ng / ml, about 5 ng / ml to 40 ng / ml, about 5 ng / ml to 30 ng / ml, about 10 ng / ml to 50 ng / ml, about 10 ng / ml to 40 ng / ml, or about 10 ng / ml to 30 ng / ml, as measured by ELISA. Soluble BCMA in a blood sample (e.g., isolated serum or plasma) from a patient as measured by ELISA can be measured using a bead-based immunoassay by Ampersand Biosciences (Lake Clear, NY).The inventors have determined that the multispecific (e.g. bispecific) antibodies of the invention can be used to treat or manage patients with autoimmune disorders caused by plasmablasts and / or plasma cells, wherein the plasmablast BCMA surface receptor density of the patients is equivalent to the plasmablast BCMA surface receptor density of normal healthy volunteers.

[0084] In certain embodiments, the BCMA surface receptor density on plasmablasts of a patient in need of treatment or management of an autoimmune disorder is less than about 10,000 molecules, less than about 5000 molecules, less than about 2500 molecules, or less than about 2000 molecules, as measured using a flow cytometry system based on a standard curve generated with anti-BCMA antibody-coated beads.

[0085] In certain embodiments, the BCMA surface receptor density on plasmablasts of a patient in need of treatment or management of an autoimmune disorder is at least about 500 molecules, at least about 700 molecules, at least about 900 molecules, or at least about 1000 molecules, as measured using a flow cytometry system based on a standard curve generated with anti-BCMA antibody-coated beads.

[0086] In certain embodiments, the BCMA surface receptor density on plasmablasts in a patient in need of treatment or management of an autoimmune disorder is about 800 to about 2200 molecules, about 900 to 2100 molecules, or about 1000 to 2000 molecules using a flow cytometry system based on a standard curve generated with anti-BCMA antibody-coated beads.

[0087] In certain embodiments, the treatment or management methods of the present invention result in at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduction in plasmablasts in a patient compared to no treatment or a control treatment. In a preferred embodiment, the treatment or management methods of the present invention result in at least a 75% reduction in plasmablasts in a patient. In a particularly preferred embodiment, the treatment or management methods of the present invention result in at least a 90% reduction in plasmablasts in a patient.

[0088] In some embodiments, the treatment or management methods of the present invention result in at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduction in plasma cells in a patient compared to no treatment or a control treatment. In a preferred embodiment, the treatment or management methods of the present invention result in at least a 75% reduction in plasma cells in a patient. In a particularly preferred embodiment, the treatment or management methods of the present invention result in at least a 90% reduction in plasma cells in a patient.

[0089] In some embodiments, the treatment or management methods of the present invention result in at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduction in plasma cells and plasmablasts in a patient compared to no treatment or a control treatment. In a preferred embodiment, the treatment or management methods of the present invention result in at least a 75% reduction in plasma cells and plasmablasts in a patient. In a particularly preferred embodiment, the treatment or management methods of the present invention result in at least a 90% reduction in plasma cells and plasmablasts in a patient.

[0090] Existing treatments for autoimmune disorders such as AAV, SLE, or RA include the combination of cyclophosphamide and / or anti-CD20 monoclonal antibodies (e.g., rituximab) with high-dose steroids (e.g., glucocorticoids). As used herein, the term "control treatment" (e.g., for AAV) preferably refers to treatment with cyclophosphamide, rituximab, and / or steroids. Alternatively or additionally, "control treatment" (e.g., for SLE or RA) refers to treatment with anti-TNF agents (e.g., infliximab, adalimumab, golimumab, etanercept), anti-IL6R antibodies (e.g., tocilizumab, sarilumab), costimulatory blockade (e.g., abatacept), JAK inhibitors (e.g., tofacitinib, baricitinib), and / or belimumab. Additional treatments include cyclophosphamide, methotrexate, azathioprine, mycophenolic acid, mycophenolate mofetil, and / or avacopan. However, such existing treatments are not always effective and / or durable, and may have adverse side effects.

[0091] Without being bound by theory, it is predicted that the present invention selectively depletes B cell lineage cells, e.g., autoreactive B lineage cells, e.g., plasmablasts, plasma cells, and memory B cells, that cause autoimmune disorders. The treatment or management methods of the present invention are therefore predicted to induce faster remission and / or have fewer off-target effects compared to control treatments that do not selectively deplete B cell lineage cells that cause autoimmune disorders.

[0092] In some embodiments, the treatment methods are used to induce remission. In some embodiments, the treatment or management methods of the invention result in at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, or 100% faster induction of remission in patients compared to control treatment.

[0093] The treatment or management methods of the present invention may result in better maintenance of remission compared to control treatments. For example, after depletion of plasmablasts and plasma cells, the multispecific (e.g., bispecific) antibodies of the present invention may suppress and / or delay the recovery of plasmablasts and plasma cells from BCMA-negative precursors (e.g., as measured by FACS and / or IgG secretion), even after incubation with growth factors for regeneration. Furthermore, after depletion of plasmablasts and plasma cells, the multispecific (e.g., bispecific) antibodies of the present invention may suppress and / or delay the production of antibodies, such as autoantibodies that cause autoimmune disorders, even after stimulation with CpG.

[0094] In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention inhibit and / or delay the production of IgG antibodies, regardless of stimulation with IL-2, BAFF, and IL-21 or CpG (ODN2006) to induce plasmablast / plasma cell differentiation. Multispecific (e.g., bispecific) antibodies of the invention are administered to isolated peripheral blood mononuclear cells (PBMCs) for 24 hours and the EC of plasmablast lysis. 50 After incubation at 10 μg / mL, stimulation with CpG (ODN2006 10 μg / mL), IL-2 (20 U / mL), BAFF (200 ng / mL), and IL-21 (100 ng / mL) for 7 days, the IgG concentration may be less than about 4000 pg / mL, less than about 3500 pg / mL, less than about 3000 pg / mL, less than about 2500 pg / mL, or less than about 2000 pg / mL, as measured by ELISA. Multispecific (e.g., bispecific) antibodies of the invention may also be used to measure the EC of plasmablast lysis in isolated peripheral blood mononuclear cells (PBMCs) for 24 hours. 90 After incubation at a concentration of 10 μg / mL, the cells are stimulated with CpG (ODN2006 10 μg / mL), IL-2 (20 U / ml), BAFF (200 ng / ml), and IL-21 (100 ng / ml) for 7 days, after which the IgG concentration can be less than about 2000 pg / ml, less than about 1800 pg / ml, less than about 1000 pg / ml, or less than about 500 pg / ml, as measured by ELISA.

[0095] In some embodiments, the treatment method is used to induce and maintain remission. In some embodiments, the method is used to maintain remission. In some embodiments, the treatment or management method of the present invention results in at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95% or 100% longer maintenance of remission in patients compared to control treatment.

[0096] In certain embodiments, the autoimmune disorder is relapsing or refractory. As used herein, the term "relapsing" is intended to mean that the disorder or signs and symptoms of the disorder return after a period of improvement. As used herein, the term "refractory" is intended to mean that a particular disorder is resistant or unresponsive to treatment with a particular therapeutic agent. A disorder may be refractory to treatment with a particular therapeutic agent from the start of treatment with the particular therapeutic agent (i.e., unresponsive to initial exposure to the therapeutic agent) or as a result of developing resistance to the therapeutic agent during the first treatment period or during subsequent treatment periods with the therapeutic agent.

[0097] In certain embodiments, the autoimmune disorder is relapsing or is treated with cyclophosphamide, anti-CD20 monoclonal antibodies (e.g., rituximab), glucocorticoids (e.g., methylprednisolone, dexamethasone), antifolates (e.g., methotrexate), purine synthesis inhibitors (e.g., azathioprine, mycophenolate and / or mycophenolate mofetil), C5a inhibitors (e.g., avacopan), anti-CD19 antibodies, BAFF / APRI Refractory to L antagonists, proteasome inhibitors (e.g., bortezomib), anti-CD22 monoclonal antibodies, anti-TNF agents (e.g., infliximab, adalimumab, golimumab, etanercept), anti-IL6R antibodies (e.g., tocilizumab, sarilumab), costimulation blockade (e.g., abatacept), JAK inhibitors (e.g., tofacitinib, baricitinib), belimumab, and / or Bruton's tyrosine kinase (BTK) inhibitors.

[0098] In another embodiment, the autoimmune disorder is newly diagnosed.

[0099] Autoimmune disorders suitable for treatment with the multispecific (e.g., bispecific) antibodies of the invention include achalasia, Addison's disease, acute inflammatory demyelinating polyneuropathy - AIDP, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, anti-PAD4-activated rheumatoid arthritis, antiphospholipid syndrome, asthma, atopic dermatitis, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenia, autoimmune urticarial, axonal and neuronal neuropathy (AMAN), Baro's disease, Behcet's disease, benign mucosal leukemia, and rheumatoid arthritis. Pemphigoid, Bullous pemphigoid, Castleman's disease (CD), Celiac disease, Chagas' disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic relapsing multiple myelitis (CRMO), Churg-Strauss syndrome (CSS) or Eosinophilic granulomatosis (EGPA), Cicatricial pemphigoid, Cogan's syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, Crest syndrome, Crohn's disease, Dermatitis, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (Neuromyelitis optica), Diabetes, Discoid lupus, Dressler's syndrome, Endometriosis, Eosinophilic esophagitis (EoE), Eosinophilic fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evans' syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Goodpasture's syndrome, Granulomatosis with polyangiitis , Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, Hashimoto's thyroiditis, autoimmune hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes gestationis or pemphigoid of gestationis (PG), hidradenitis suppurativa (HS) (acne inversa), hypogammaglobulinemia, idiopathic membranous nephropathy, idiopathic thrombocytopenic purpura, IgA nephropathy, IgG4-related disease, IgG4-related sclerosing disease, IgG neuropathy , IgM polyneuropathy, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), inflammatory bowel disease (IBD), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear IgA disease (LAD), lupus, chronic Lyme disease, membranous nephropathy,Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Much-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Lombard syndrome Legue syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, pemphigus vulgaris, pemphigus foliaceus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome types I, II and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone-induced dermatitis, psoriasis, psoriatic joints inflammation, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's syndrome, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, juvenile rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, sensitization / pre-existing antibodies in solid organ transplantation, Sjogren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome (SPS), systemic lupus erythematosus These include, but are not limited to, systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura, thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, leukoplakia, Vogt-Koyanagi-Harada disease, and Wegener's disease. In a preferred embodiment, the autoimmune disorder is not an IgG4-related disease. In a preferred embodiment, the autoimmune disorder is AAV (e.g., relapsed or refractory AAV), SLE (e.g., relapsed or refractory SLE), or rheumatoid arthritis (e.g., relapsed or refractory rheumatoid arthritis). In particularly preferred embodiments, the autoimmune disorder is AAV (e.g., relapsed or refractory AAV) or rheumatoid arthritis (e.g., relapsed or refractory rheumatoid arthritis).

[0100] In some embodiments, the multispecific (e.g., bispecific) antibodies of the invention treat or manage AAV. In some embodiments, the multispecific (e.g., bispecific) antibodies of the invention treat or manage rheumatoid arthritis. In some embodiments, the multispecific (e.g., bispecific) antibodies of the invention treat or manage SLE. In some embodiments, the multispecific (e.g., bispecific) antibodies of the invention treat or manage AAV and rheumatoid arthritis. In some embodiments, the multispecific (e.g., bispecific) antibodies of the invention treat or manage AAV, SLE, and rheumatoid arthritis.

[0101] In one embodiment, the present invention provides a method of treating or managing AAV, comprising administering to a subject (e.g., a human) in need of such treatment or management a multispecific (e.g., bispecific) antibody, wherein the multispecific antibody is capable of targeting BCMA and 1 or more Promotes T cell activation Resistance The method further provides a method for detecting a target antigen (e.g., CD3) that binds to a target antigen (e.g., CD4).

[0102] In other aspects, the present invention provides a method for treating or managing AAV in a subject (e.g., a human) comprising administering to a subject an antibody against BCMA and 1 or more Multispecific (eg, bispecific) antibodies are provided that bind to antigens (eg, CD3) that promote T cell activation.

[0103] In one embodiment, the present invention provides a method of treating or managing rheumatoid arthritis (e.g., relapsed or refractory rheumatoid arthritis), comprising administering to a subject (e.g., a human) in need of such treatment or management a multispecific (e.g., bispecific) antibody, wherein the multispecific antibody is a BCMA and 1 or more Promotes T cell activation Resistance The method further provides a method for detecting a target antigen (e.g., CD3) that binds to a target antigen (e.g., CD4).

[0104] In other aspects, the present invention provides a method for treating or managing rheumatoid arthritis (e.g., relapsed or refractory rheumatoid arthritis) in a subject (e.g., a human) comprising administering to the patient a therapeutically effective amount of BCMA and / or a medicament for the treatment or management of rheumatoid arthritis (e.g., relapsed or refractory rheumatoid arthritis). 1 or more Promotes T cell activation Resistance Multispecific (e.g., bispecific) antibodies that bind to an antigen (e.g., CD3) are provided.

[0105] In one embodiment, the present invention provides a method of treating or managing SLE (e.g., relapsed or refractory SLE), comprising administering to a subject (e.g., a human) in need of such treatment or management a multispecific (e.g., bispecific) antibody, wherein the multispecific antibody is a BCMA and 1 or more Promotes T cell activation Resistance The method further provides a method for detecting a target antigen (e.g., CD3) that binds to a target antigen (e.g., CD4).

[0106] In other aspects, the present invention provides a method for treating or managing SLE (e.g., relapsed or refractory SLE) in a subject (e.g., a human) comprising administering to a subject an antibody against BCMA and / or IL-16, for use in the treatment or management of SLE (e.g., relapsed or refractory SLE). 1 or more Promotes T cell activation Resistance Multispecific (e.g., bispecific) antibodies that bind to an antigen (e.g., CD3) are provided.

[0107] T cell lysis, T cell activation and cytokine production Multispecific (e.g., bispecific) antibodies of the invention include: 1 or more Promotes T cell activation Resistance As used herein, the term "T cell antigen" refers to a T cell antigen that binds to a specific antigen (e.g., CD3). 1 or more Promotes T cell activation Resistance This refers to the original (e.g., CD3).

[0108] In a preferred embodiment, the T cell antigen (e.g., CD3) is a human T cell antigen (e.g., human CD3). In a preferred embodiment, the T cell antigen is CD3.

[0109] Thus, binding of a multispecific (e.g., bispecific) antibody of the invention to a T cell antigen (e.g., CD3) may enable recruitment of T cells to BCMA-expressing cells (e.g., plasmablasts, plasma cells, and / or memory B cells) followed by lysis of the BCMA-expressing cells. Thus, a multispecific (e.g., bispecific) antibody of the invention can induce selective depletion of BCMA-expressing cells by redirecting cytotoxic T cells to BCMA-expressing cells (e.g., plasmablasts, plasma cells, and / or memory B cells).

[0110] The inventors have determined that the multispecific (e.g. bispecific) antibodies of the invention can be administered in the treatment of disorders characterized by a low ratio of target BCMA-expressing cells to effector T cells (T:E ratio), e.g. autoimmune disorders, at lower doses than are required to treat diseases characterized by a high T:E ratio, e.g. B-cell malignancies such as multiple myeloma.

[0111] Thus, in certain embodiments, multispecific (e.g., bispecific) antibodies of the invention are administered to a subject with an autoimmune disorder, wherein the ratio of BCMA-expressing cells to effector T cells in the individual is specified to be less than about 1:15, less than about 1:30, less than about 1:50, less than about 1:100, or less than 1:500. The ratio of BCMA-expressing cells to effector T cells (T:E ratio) may be measured in an isolated body fluid sample from a subject with an autoimmune disorder, for example, a blood sample, bone marrow aspirate, or synovial fluid from a subject with an autoimmune disorder.

[0112] The inventors have determined that the multispecific (e.g., bispecific) antibodies of the invention can achieve therapeutic efficacy in treating the autoimmune disorders disclosed herein (e.g., autoimmune disorders caused by BCMA-expressing B-cell lineage cells) at lower doses than are required for treating B-cell malignancies (e.g., BCMA-expressing cancers such as multiple myeloma). In particular, the multispecific (e.g., bispecific) antibodies of the invention can lyse pathogenic cells of an autoimmune disorder (e.g., BCMA-expressing autoreactive B-cell lineage cells) at doses lower (e.g., 10- to 100-fold lower) than the doses required to lyse pathogenic cells of multiple myeloma (e.g., BCMA-expressing malignant cells).

[0113] Thus, in one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis. 1 or more Promotes T cell activation Resistance

[0013] Methods for treating or managing an autoimmune disorder with a multispecific (e.g., bispecific) antibody that binds to an antigen (e.g., CD3) and BCMA, wherein the treatment comprises administering the multispecific (e.g., bispecific) antibody at a dose of about 0.01 mg to about 1 mg. In embodiments where the multispecific (e.g., bispecific) antibody is the CC-93269 antibody disclosed herein, the dose can be about 0.01 mg to about 1 mg.

[0114] In certain embodiments of any of the aspects disclosed herein, the treatment comprises at least one dose of the multispecific (e.g., bispecific) antibody, e.g., at least two doses or at least three doses of the multispecific (e.g., bispecific) antibody. In another embodiment, up to three doses of the multispecific (e.g., bispecific) antibody are administered, e.g., up to two doses or a single dose of the multispecific (e.g., bispecific) antibody. In a preferred embodiment, a single dose of the multispecific (e.g., bispecific) antibody is administered.

[0115] In a particularly preferred embodiment, the treatment comprises a single dose of multispecific (eg, bispecific) antibody at a dose of about 0.01 mg to about 1 mg.

[0116] In certain embodiments, multispecific (e.g., bispecific) antibodies of the invention achieve plasmablast lysis when incubated with isolated PBMCs (e.g., isolated PBMCs from a patient with an autoimmune disorder) for 24 hours, wherein the multispecific (e.g., bispecific) antibodies of the invention are at a concentration lower than that required for cell killing of BCMA-expressing cancer cells (e.g., JEKO-1 or MM cell lines). PBMCs can be isolated from whole blood samples, for example, using a Ficoll gradient followed by resuspension in RPMI + 10% HI FBS. In certain embodiments, multispecific (e.g., bispecific) antibodies of the invention achieve plasmablast lysis when incubated with blood (e.g., a whole blood sample from a patient with an autoimmune disorder) for 24 hours, wherein the multispecific (e.g., bispecific) antibodies of the invention are at a concentration lower than that required for cell killing of BCMA-expressing cancer cells (e.g., JEKO-1 or MM cell lines). Plasmablast depletion is measured by FACS, whereby plasmablasts are identified as CD19(+) CD20(-) CD27(+) cells. In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention have a median effective concentration (EC) of less than about 1 nM, less than about 0.8 nM, less than about 0.6 nM, less than about 0.4 nM, less than about 0.3 nM, or less than about 0.25 nM, less than about 0.2 nM, less than about 0.1 nM, less than about 0.05 nM, less than about 0.02 nM, less than about 0.01 nM, or less than about 0.005 nM. 50) can achieve lysis of plasmablasts when incubated with isolated PBMCs (e.g., isolated PBMCs from a patient with an autoimmune disorder) for 24 hours. In a preferred embodiment, the multispecific (e.g., bispecific) antibodies of the invention can achieve 50% lysis of plasmablasts when incubated with isolated PBMCs (e.g., isolated PBMCs from a patient with an autoimmune disorder) for 24 hours at a concentration of about 1 nM or less, about 0.8 nM or less, about 0.6 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.25 nM or less, about 0.02 nM or less, about 0.01 nM or less, about 0.007 nM or less, or about 0.005 nM or less. Plasmablast depletion is measured by FACS, whereby plasmablasts are identified as CD19(+) CD20(-) CD27(+) cells. In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention have a 90% effective concentration (EC ) of less than about 1 nM, less than about 0.8 nM, less than about 0.6 nM, less than about 0.4 nM, less than about 0.3 nM, less than about 0.2 nM, less than about 0.1 nM, less than about 0.08 nM, or less than about 0.06 nM. 90 ) can achieve lysis of plasmablasts when incubated with isolated PBMCs (e.g., isolated PBMCs from a patient with an autoimmune disorder) for 24 hours. In a preferred embodiment, the multispecific (e.g., bispecific) antibodies of the invention can achieve 90% lysis of plasmablasts when incubated with isolated PBMCs (e.g., isolated PBMCs from a patient with an autoimmune disorder) for 24 hours at a concentration of less than about 1 nM, less than about 0.8 nM, less than about 0.6 nM, less than about 0.4 nM, less than about 0.3 nM, less than about 0.2 nM, less than about 0.1 nM, less than about 0.08 nM, or less than about 0.06 nM. Plasmablast depletion is measured by FACS, whereby plasmablasts are identified as CD19(+) CD20(-) CD27(+) cells.

[0117] In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention have a 99% effective concentration (EC ) of less than about 1 nM, less than about 0.9 nM, less than about 0.8 nM, less than about 0.7 nM, or less than about 0.6 nM. 99) can achieve lysis of plasmablasts when incubated with isolated PBMCs (e.g., isolated PBMCs from a patient with an autoimmune disorder) for 24 hours. In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention achieve 99% lysis of plasmablasts when incubated with isolated PBMCs (e.g., isolated PBMCs from a patient with an autoimmune disorder) for 24 hours at a concentration of less than about 1 nM, less than about 0.9 nM, less than about 0.8 nM, less than about 0.7 nM, or less than about 0.6 nM. Plasmablast depletion is measured by FACS, whereby plasmablasts are identified as CD19(+) CD20(-) CD27(+) cells.

[0118] Cytokine release syndrome (CRS) is one of the most frequent and serious adverse effects of T cell-directed immunotherapy for the treatment of cancers such as multiple myeloma (Shimabukuro-Vornhagen, A. et. al (2018) Cytokine release syndrome. J Immunother Cancer, 6(1) 56). Without being bound by theory, CRS may result in massive and / or rapid secretion of cytokines, for example, due to activation and / or proliferation of immune effector cells. Elevated levels of cytokines such as IFNγ, IL-1β, IL-6, IL-2, IL-10, and / or granzyme B have been observed after treatment of multiple myeloma with T cell-directed immunotherapy.

[0119] Because autoimmune disorders are associated with high cytokine levels even prior to treatment (see Kunz, M. and Ibrahim, S. (2009) Cytokines and Cytokine Profiles in Human Autoimmune Diseases and Animal Models of Autoimmunity. Mediators of Inflammation, Article ID 979258; and Andreakos et al. (2002), Cytokines and anti-cytokine biologicals in autoimmunity: present and future. Cytokine & Growth Factor Reviews, Issues 4-5, Pages 299-313), T cell-directed immunotherapy may not be considered attractive for treating autoimmune disorders. However, the present inventors have surprisingly determined that the multispecific (e.g., bispecific) antibodies of the present invention can be administered at therapeutically effective doses for the autoimmune disorders of the present invention without significant or minimal T cell activation and without significant or minimal cytokine production. Thus, the multispecific (e.g., bispecific) antibodies of the invention can treat or manage the autoimmune disorders of the invention with minimal risk of adverse events associated with T cell activation and / or cytokine production, e.g., CRS.

[0120] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: 1 or more Promotes T cell activation Resistance

[0003] Provided are methods of treating or managing an autoimmune disorder with a multispecific (e.g., bispecific) antibody that binds to an antigen (e.g., CD3) and BCMA, wherein the treatment comprises administering the multispecific (e.g., bispecific) antibody at a therapeutically effective dose without significant or little T cell activation.

[0121] As used herein, "no significant T cell activation or minimal T cell activation" refers to T cell activation being less than 20%, less than 15%, less than 10%, or less than 5% above baseline in isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) as measured by surface expression of the activation marker CD69. Preferably, "no significant T cell activation or minimal T cell activation" refers to T cell activation being less than 20% above baseline in isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) as measured by surface expression of the activation marker CD69. Preferably, T cell activation is described for CD3+ T cells (e.g., CD3+CD4+ T cells or CD3+CD8+ T cells or both).

[0122] As used herein, a "baseline" of T cell activation (e.g., a "baseline" of CD8(+) T cells expressing CD69) is defined as the percentage of relevant T cells (e.g., CD8(+) T cells) expressing a relevant activation marker (e.g., CD69) in a control sample of isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder). Preferably, the control sample is treated with a control anti-CD3 antibody, e.g., a control 2+1 anti-HEL anti-CD3 antibody. Preferably, T cell activation is described for CD3+ T cells (e.g., CD3+CD4+ T cells or CD3+CD8+ T cells, or both). In one embodiment, the multispecific (e.g., bispecific) antibodies of the invention are administered at a concentration equal to or greater than 50% effective concentration for plasmablast lysis (EC 50 When incubated with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) for 24 hours, there is little or no significant T cell activation. T cell activation can be measured by staining for T cell lineage (CD3, CD4, and CD8) and activation markers (CD69, CD25, and CD154). Preferably, T cell activation is described for CD3+ T cells (CD3+CD4+ T cells or CD3+CD8+ T cells or both).

[0123] In a preferred embodiment, the multispecific (e.g., bispecific) antibodies of the invention are administered at a concentration of 50% effective concentration for plasmablast lysis (EC 50 When incubated with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) for 24 hours, there is no significant increase in CD8(+) T cells expressing CD69 or there is a minimal increase in CD8(+) T cells expressing CD69. In a preferred embodiment, the multispecific (e.g., bispecific) antibodies of the invention are administered at a concentration equal to or greater than 50% effective concentration for plasmablast lysis (EC 50 ) with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) for 24 hours, less than 10%, less than 5%, less than 4%, less than about 3%, less than 2%, or less than 1% of CD8(+) T cells expressing CD69 are present above baseline. In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention are administered at a concentration of 50% effective concentration for plasmablast lysis (EC 50 ) with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) for 24 hours, there is no increase above baseline in the frequency of CD8(+) T cells expressing CD69.

[0124] In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention are administered at a concentration equal to 50% effective concentration for plasmablast lysis (EC 50 ) with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) for 24 hours, less than 10%, less than 5%, less than 4%, less than about 3%, less than 2%, or less than 1% of CD4(+) T cells expressing CD69 and CD8(+) T cells expressing CD69 are present above baseline. In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention are administered at a concentration greater than 50% effective concentration for plasmablast lysis (EC 50 When incubated with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) for 24 hours, there is no increase above baseline in CD69-expressing CD4(+) T cells and CD69-expressing CD8(+) T cells.

[0125] In one embodiment, the multispecific (e.g., bispecific) antibodies of the invention are administered at a concentration of 90% effective concentration for plasmablast lysis (EC 90 ) with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) for 24 hours, there is no significant or minimal T cell activation. T cell activation can be measured by staining for T cell lineage (CD3, CD4, and CD8) and activation markers (CD69, CD25, and CD154). Preferably, T cell activation is described for CD3+ T cells (CD3+CD4+ T cells or CD3+CD8+ T cells or both). In a preferred embodiment, the multispecific (e.g., bispecific) antibodies of the invention are administered at a 90% effective concentration (EC 90 When incubated with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) for 24 hours, there is no significant increase in CD8(+) T cells expressing CD69 or there is a minimal increase in CD8(+) T cells expressing CD69. In a preferred embodiment, the multispecific (e.g., bispecific) antibodies of the invention are administered at a 90% effective concentration (EC 90 ) with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) for 24 hours, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than about 3%, less than 2%, or less than 1% of CD8(+) T cells expressing CD69 are present above baseline.

[0126] In one embodiment, the multispecific (e.g., bispecific) antibodies of the invention are administered at a concentration of 90% effective concentration for plasmablast lysis (EC 90) with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) for 24 hours, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than about 3%, less than 2%, or less than 1% of CD4(+) T cells expressing CD69 and CD8(+) T cells expressing CD69 are present above baseline. In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention are administered at a 90% effective concentration (EC 90 When incubated with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) for 24 hours, there is no increase above baseline in CD69-expressing CD4(+) T cells and CD69-expressing CD8(+) T cells.

[0127] In one embodiment, the multispecific (e.g., bispecific) antibodies of the invention are administered at a 99% effective concentration (EC 99 ) with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood PBMCs from a patient with an autoimmune disorder) for 24 hours, fewer than 40%, 30%, 20%, 15%, 10%, or 5% of CD8(+) T cells expressing CD69 are present above baseline. Preferably, T cell activation is described for CD3+ T cells (CD3+CD4+ T cells or CD3+CD8+ T cells or both).

[0128] In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention can achieve greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than about 100% lysis of plasmablasts when incubated with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) for 24 hours at concentrations that result in less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% increase above baseline in the number of CD8(+) T cells expressing CD69.

[0129] In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention can achieve about 50%, about 90%, or about 99% lysis of plasmablasts when incubated with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood of a patient with an autoimmune disorder) for 24 hours at concentrations where the number of CD8(+) T cells expressing CD69 is less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% above baseline. 1 or more Promotes T cell activation Resistance

[0003] Methods of treating or managing an autoimmune disorder with a multispecific (e.g., bispecific) antibody that binds to an antigen (e.g., CD3) and BCMA, wherein treatment comprises administering the multispecific (e.g., bispecific) antibody at a therapeutically effective dose without significant or negligible cytokine production.

[0130] As used herein, "no significant or minimal cytokine production" refers to cytokine levels that are less than 20 pg / mL, less than 10 pg / mL, or less than 5 pg / mL above baseline in isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder). Preferably, "no significant or minimal cytokine production" refers to cytokine levels that are less than 5 pg / mL above baseline cytokine levels in the donor sample. Cytokine levels may be measured using the MSD Pro-inflammatory I assay.

[0131] As used herein, a "baseline" cytokine level (e.g., a "baseline" level of IFNγ) is defined as the level of the relevant cytokine (e.g., IFNγ) in a control sample of isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder). Preferably, the control sample is treated with a control anti-CD3 antibody, e.g., a control 2+1 anti-HEL anti-CD3 antibody. Cytokine levels may be measured using the MSD Pro-inflammatory I assay.

[0132] In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention are administered at a concentration equal to 50% effective concentration for plasmablast lysis (EC 50 ) with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) for 24 hours, there is no significant or minimal cytokine production. Production of the cytokines IFNγ, IL-6, IL-2, IL-10, IL-1β, granzyme B, and / or perforin is measured using the MSD Pro-inflammatory I assay. In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention are administered at a concentration of 50% effective concentration for plasmablast lysis (EC 50 ) with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) for 24 hours, the levels of pro-inflammatory cytokines (e.g., IFNγ, IL-6, IL-2, IL-10, IL-1β, granzyme B and / or perforin) above baseline are less than 50 pg / mL, less than 20 pg / mL, less than 10 pg / mL, or less than 5 pg / mL.

[0133] In a preferred embodiment, the multispecific (e.g., bispecific) antibodies of the invention are administered at a concentration of 50% effective concentration for plasmablast lysis (EC 50) can be incubated with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood of a patient with an autoimmune disorder) for 24 hours, there is no significant increase in IFNγ or a minimal increase in IFNγ. In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention are administered at a concentration equal to or greater than the 50% effective concentration for plasmablast lysis (EC 50 ) with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood of a patient with an autoimmune disorder) for 24 hours, the level of IFNγ above baseline is less than 50 pg / mL, less than 20 pg / mL, less than 10 pg / mL, or less than 5 pg / mL. In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention can achieve greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than about 100% lysis of plasmablasts when incubated with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood of a patient with an autoimmune disorder) for 24 hours at concentrations that result in an increase in IFNγ above baseline of less than about 50 pg / mL, less than about 10 pg / mL, or less than about 5 pg / mL.

[0134] Anti-IL-6 receptor therapy is used to treat CRS, given the central role of IL-6 in driving CRS (Shimabukuro-Vornhagen, A. et. al (2018) Cytokine release syndrome. J Immunother Cancer, 6(1) 56). In certain embodiments, the level of IL-6 is determined by administering a multispecific (e.g., bispecific) antibody of the invention to a patient at a 50% effective concentration for plasmablast lysis (EC 50 ) for 24 hours with isolated PBMCs or whole blood (e.g., isolated PBMCs or whole blood from a patient with an autoimmune disorder) is less than 10 pg / mL or less than 5 pg / mL above baseline.

[0135] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: 1 or more Promotes T cell activation Resistance

[0003] Methods of treating or managing an autoimmune disorder with a multispecific (e.g., bispecific) antibody that binds to an antigen (e.g., CD3) and BCMA, wherein treatment comprises administering the multispecific (e.g., bispecific) antibody at a therapeutically effective dose that minimizes CRS.

[0136] As used herein, "minimal CRS" refers to at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least a 95%, or 100% lower incidence of CTCAE v. 5.0 Grade 1 cytokine release syndrome (CRS) in patients compared to a control treatment. Preferably, "minimal CRS" refers to CTCAE v. 5.0 Grade 1 CRS, as defined by the Cancer Therapy Evaluation Program (CTEP), Division of Cancer Treatment and Diagnosis (DCTD), National Cancer Institute, NIH.

[0137] In certain embodiments, the methods of treatment or management of the invention result in at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least a 95% or 100% reduction in the incidence of cytokine release syndrome in a patient compared to a control treatment.

[0138] In certain embodiments, the methods of treatment or management of the invention result in at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least a 95% or 100% reduction in the incidence of infection in a patient compared to a control treatment.

[0139] multispecific antibodies The multispecific (e.g., bispecific) antibodies of the present invention are directed to BCMA and 1 or more Promotes T cell activation ResistanceThe terms "antibody to BCMA and an antigen (e.g., CD3) that promotes activation of one or more T cells" or "BCMA and 1 or more Promotes T cell activation Resistance An "antibody that binds to an antigen (e.g., CD3)" is an antibody that binds to BCMA and CD3 with sufficient affinity so that the antibody is useful as a therapeutic agent. 1 or more Promotes T cell activation Resistance This refers to a multispecific (e.g., bispecific) antibody that can bind to a first antibody or antigen-binding fragment that binds BCMA and a second antibody or antigen-binding fragment that binds to BCMA (e.g., CD3). 1 or more Promotes T cell activation Resistance This can be achieved by producing a molecule containing a second antibody or antigen-binding fragment that binds to the antigen (e.g., CD3). Such multispecific antibodies can be trispecific or bispecific. In a preferred embodiment, the multispecific antibody is a bispecific antibody.

[0140] As used herein, the term "BCMA" refers to human B-cell maturation antigen, also known as BCMA; TR17_HUMAN, TNFRSF17 (UniProt Q02223), a member of the tumor necrosis receptor superfamily that is preferentially expressed on differentiated plasma cells. The extracellular domain of BCMA consists of amino acids 1-54 (or 5-51) according to UniProt. As used herein, the terms "antibody against BCMA," "anti-BCMA antibody," or "antibody that binds to BCMA" refer to an antibody that specifically binds to the extracellular domain of BCMA.

[0141] The term "specifically binds to BCMA" refers to an antibody that can bind to a defined target with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting BCMA. In certain embodiments, an antibody that specifically binds BCMA does not bind to other antigens or does not bind to other antigens with sufficient affinity to produce a physiological effect.

[0142] In certain embodiments, the extent to which an anti-BCMA antibody binds to an unrelated, non-BCMA protein is about 10-fold, preferably >100-fold, less than the binding of the antibody to BCMA, as measured, for example, by surface plasmon resonance (SPR), e.g., Biacore®, enzyme-linked immunosorbent assay (ELISA), or flow cytometry (FACS). -8 M or less, preferably 10 -8 M~10 -13 M, preferably 10 -9 M~10 -13 It has a dissociation constant (Kd) of M.

[0143] In one embodiment, the anti-BCMA antibody binds to an epitope of BCMA that is conserved in BCMA from various species, preferably human and cynomolgus monkey and more preferably also mouse and rat BCMA.

[0144] Preferably, the anti-BCMA antibody specifically binds to a panel of BCMA consisting of human BCMA and BCMA of non-human mammalian origin, preferably BCMA from cynomolgus monkey, mouse, and / or rat. The anti-BCMA antibody is analyzed for binding to human BCMA by ELISA using plate-bound BCMA. For this assay, preferably 1.5 μg / mL of plate-bound BCMA and an amount of anti-BCMA antibody in the concentration range of 0.1 pM to 200 nM are used.

[0145] Multispecific (e.g., bispecific) antibodies of the invention include: 1 or more Promotes T cell activation Resistance In a preferred embodiment, the T cell antigen is a human T cell antigen. 1 or more Promotes T cell activation Resistance The antigen may be selected from the group consisting of CD3, TCRα, TCRβ, TCRγ, TCRζ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. 1 or more Promotes T cell activation Resistance The source is CD3, e.g., human CD3. Thus, in a preferred embodiment, the multispecific (e.g., bispecific) antibodies of the invention bind to CD3, e.g., human CD3.

[0146] The term "CD3" refers to the human CD3 protein multisubunit complex. The CD3 protein multisubunit complex is composed of six distinct polypeptide chains. Thus, the term refers to the CD3 gamma chain (SwissProt P09693), the CD3 delta chain (SwissProt P04234), two CD3 epsilon chains (SwissProt P07766), and one CD3 zeta chain homodimer (SwissProt 20963), which bind to the T cell receptor alpha and beta chains. The term includes "full-length," unprocessed CD3 as well as all CD3 variants, isoforms, and species homologs that are naturally expressed by cells (including T cells) or that can be expressed in cells transfected with genes or cDNAs encoding these polypeptides.

[0147] The term "specifically binds to CD3" refers to an antibody that can bind to a defined target with sufficient affinity so that the antibody is useful as a therapeutic agent in targeting CD3. In certain embodiments, an antibody that specifically binds to CD3 does not bind to other antigens or does not bind to other antigens with sufficient affinity to produce a physiological effect.

[0148] Multispecific (e.g., bispecific) antibodies of the invention can be analyzed for binding to CD3 by SPR, e.g., Biacore®. In one embodiment, the bispecific antibody exhibits a CD3 binding affinity of about 10, as determined by a surface plasmon resonance assay, preferably measured using a Biacore 8K at 25°C. -7 The dissociation constant (K D ), about 10 -8 K below M D , about 10 -9 K below M D , about 10-10 K below M D , about 10 -11 K below M D or about 10 -12 K below M D In a preferred embodiment, the multispecific (e.g., bispecific) antibody binds to human CD3 at a concentration of about 10 -8 The dissociation constant (K D ) binds to human CD3.

[0149] The term "antibody" as used herein encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific (e.g., bispecific) antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0150] A "heavy chain" comprises a heavy chain variable region (abbreviated herein as "VH") and a heavy chain constant region (abbreviated herein as "CH") The heavy chain constant region comprises heavy chain constant domains CH1, CH2, and CH3 (antibody classes IgA, IgD, and IgG) and optionally heavy chain constant domain CH4 (antibody classes IgE and IgM).

[0151] A "light chain" comprises a light chain variable domain (abbreviated herein as "VL") and a light chain constant domain (abbreviated herein as "CL"). The variable domains VH and VL can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The "constant domains" of the heavy and light chains are not directly involved in binding the antibody to its target, but exert various effector functions.

[0152] The binding between an antibody and its target antigen or epitope is mediated by complementarity-determining regions (CDRs). CDRs are regions of high sequence variability located within the variable regions of the heavy and light chains of an antibody that form the antigen-binding site. CDRs are the primary determinants of antigen specificity. Typically, an antibody heavy and light chain each contains three non-contiguous CDRs. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity / affinity of the antibodies of the present invention and therefore provide an embodiment of the present invention.

[0153] As used herein, the term "antigen-binding fragment" includes any naturally occurring or artificially constructed configuration of an antigen-binding polypeptide containing one, two, or three light chain CDRs and / or one, two, or three heavy chain CDRs, wherein the polypeptide is capable of binding to an antigen. Thus, the term refers to a molecule other than an intact antibody, including a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bispecific antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific (e.g., bispecific) antibodies formed from multiple antibody fragments.

[0154] The terms "Fab fragment" and "Fab" are used interchangeably herein and comprise a single light chain (i.e., constant domains CL and VL) and a single heavy chain (i.e., constant domains CH1 and VH). The heavy chain of a Fab fragment cannot form disulfide bonds with other heavy chains.

[0155] A "Fab' fragment" contains a single light chain and a single heavy chain, but in addition to CH1 and VH, it also contains the region of the heavy chain necessary for interchain disulfide bond formation between the CH1 and CH2 domains. Thus, two "Fab' fragments" can combine via disulfide bond formation to form an F(ab')2 molecule.

[0156] A "F(ab')2 fragment" contains two light chains and two heavy chains, each of which contains a portion of the constant region necessary for interchain disulfide bond formation between the two heavy chains.

[0157] An "Fv fragment" contains only the variable regions of the heavy and light chains. It does not contain the constant regions.

[0158] A "single domain antibody" is an antibody fragment comprising a single antibody domain unit (eg, VH or VL).

[0159] A "single-chain Fv" ("scFv") is an antibody fragment that contains the VH and VL domains of an antibody linked together to form a single chain. A polypeptide linker is generally used to connect the VH and VL domains of an scFv.

[0160] "Tandem scFv" is a Tandem Ab (登録商標) Also known as scFvs, they are single-chain Fv molecules formed by covalently linking two scFvs in a tandem orientation with a flexible peptide linker.

[0161] "Bispecific T cell engager" (BiTE (登録商標) ) is a fusion protein consisting of two single-chain variable fragments (scFv) on a single peptide chain. One of the scFvs binds to T cells via the CD3 receptor, and the other binds to a tumor cell antigen.

[0162] "Bispecific antibodies" are small bivalent and bispecific antibody fragments that contain a heavy (VH) chain variable domain (VH-VL) connected to a light chain variable domain (VL) on the same polypeptide chain by a peptide linker that is too short to allow pairing between the two domains on the same chain (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). This allows pairing with the complementary domains of another chain, promoting the formation of a dimeric molecule with two functional antigen-binding sites.

[0163] "DARPins" are dual-specific ankyrin repeat molecules. DARPins are derived from natural ankyrin proteins, which are found in the human genome and are one of the most abundant binding protein types. DARPin library modules are defined by natural ankyrin repeat protein sequences, using 229 ankyrin repeats for initial design and another 2200 for subsequent refinement. Modules serve as building blocks for the DARPin library. Library modules mimic human genome sequences. DARPins consist of 4-6 modules. Each module is approximately 3.5 kDa, resulting in an average DARPin size of 16-21 kDa. Binder selection is completely cell-free and performed by ribosome display as described in He M. and Taussig MJ., Biochem Soc Trans. 2007, Nov;35(Pt 5):962-5.

[0164] The sequences of CDRs can be identified using any numbering system known in the art, such as the Kabat system (Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)); the Chothia system (Chothia & Lesk, "Canonical Structures for the Hypervariable Regions of Immunoglobulins," J. Mol. Biol. 196, 901-917 (1987)); or the IMGT system (Lefranc et al., "IMGT Unique Numbering for Immunoglobulin and Cell Receptor Variable Domains and Ig superfamily V-like domains," Dev. Comp. Immunol. 27, 55-77 (2003)).

[0165] [Table 1]

[0166] For the heavy chain constant region amino acid positions described herein, numbering is according to the EU index first described in Edelman, GM, et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85. Edelman's EU numbering is also set forth in Kabat et al. (1991) (supra). Thus, the terms "EU index as set forth in Kabat," "EU index," "Kabat's EU index," or "EU numbering" in the context of heavy chains refer to the residue numbering system based on the human IgG1 EU antibody of Edelman et al. as set forth in Kabat et al. (1991). The numbering system used for light chain constant region amino acid sequences is similar to that set forth in Kabat et al. (supra). Thus, as used herein, "Kabat numbering" refers to Kabat as set forth in Kabat et al. (supra).

[0167] The antibodies and antigen-binding fragments of the present invention can be derived from any species by recombinant means. For example, the antibodies or antigen-binding fragments can be murine, rat, goat, horse, pig, cow, chicken, rabbit, camelid, donkey, human, or chimeric versions thereof. For use in human administration, non-human-derived antibodies or antigen-binding fragments can be genetically or structurally altered to render them less antigenic upon administration to a human patient.

[0168] Particularly preferred are human or humanized antibodies, especially as recombinant human or humanized antibodies.

[0169] The term "humanized antibody" refers to an antibody whose framework or "complementarity-determining regions" (CDRs) have been modified to contain CDRs from an immunoglobulin of different specificity compared to the parent immunoglobulin. For example, a murine CDR can be grafted into the framework region of a human antibody to prepare a "humanized antibody." See, for example, Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, MS, et al., Nature 314 (1985) 268-270. In certain embodiments, a "humanized antibody" is one whose constant region has been further modified or altered from the original antibody to produce the properties of the antibody of the present invention, particularly with respect to Clq binding and / or Fc receptor (FcR) binding.

[0170] The term "human antibody" is one having an amino acid sequence corresponding to that of an antibody produced from a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries.

[0171] The term "chimeric antibody" refers to an antibody, typically prepared by recombinant DNA technology, that contains a variable region, i.e., a binding region, from one source or species and at least a portion of a constant region from a different source or species. Chimeric antibodies containing a murine variable region and a human constant region are preferred. Other preferred forms of "chimeric antibodies" encompassed by the present invention include those in which the constant region has been modified or altered from that of the original antibody to produce the properties of the antibody of the present invention, particularly with respect to Clq binding and / or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as "class-switched antibodies." Chimeric antibodies are the product of expression of immunoglobulin genes containing DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies, including conventional recombinant DNA and gene transfection techniques, are well known in the art. See, e.g., Morrison, SL, et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Patents 5,202,238 and 5,204,244.

[0172] The terms "Fc region" and "Fc" are used interchangeably herein and refer to the portion of a native immunoglobulin formed by two Fc chains. Each "Fc chain" contains a constant domain CH2 and a constant domain CH3. Each Fc chain may also contain a hinge region. Native Fc regions are homodimers. In certain embodiments, the Fc region may contain modifications that allow Fc heterodimerization.

[0173] The term "Fc portion" refers to the portion of an antibody of the invention or an antigen-binding fragment thereof that corresponds to the Fc region.

[0174] There are five major classes of heavy chain constant regions, classified as IgA, IgG, IgD, IgE, and IgM, each with a characteristic effector function designated by its isotype. For example, IgG can be divided into four subclasses known as IgG1, IgG2, IgG3, and IgG4. Ig molecules interact with multiple classes of cellular receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγR), namely, FcγRI, FcγRII, and FcγRIII, which are specific for IgG class antibodies. It has been reported that sequences important for IgG binding to FcγR receptors are located in the CH2 and CH3 domains.

[0175] The antibody or antigen-binding fragment thereof of the present invention may be of any isotype, i.e., IgA, IgD, IgE, IgG, and IgM, as well as a synthetic multimer of a four-chain immunoglobulin (Ig) structure. In a preferred embodiment, the antibody or antigen-binding fragment thereof is an IgG isotype. The antibody or antigen-binding fragment thereof may be of any IgG subclass, such as an IgG1, IgG2, IgG3, or IgG4 isotype. In a preferred embodiment, the antibody or antigen-binding fragment thereof is an IgG1 isotype.

[0176] In some embodiments, the antibody comprises a heavy chain constant region of an IgG isotype. In some embodiments, the antibody comprises a portion of a heavy chain constant region of an IgG isotype. In some embodiments, the IgG constant region or portion thereof is an IgG1, IgG2, IgG3, or IgG4 constant region. Preferably, the IgG constant region or portion thereof is an IgG1 constant region.

[0177] The antibodies or antigen-binding fragments thereof of the present invention may comprise a lambda light chain or a kappa light chain.

[0178] In preferred embodiments, the antibody or antigen-binding fragment thereof comprises a light chain that is a kappa light chain. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain that comprises a light chain constant region (CL) that is a kappa constant region.

[0179] In one embodiment, the antibody comprises a light chain comprising a light chain variable region (VL) that is a kappa variable region. Preferably, the kappa light chain comprises a VL that is a kappa VL and a CL that is a kappa CL.

[0180] Alternatively, the antibody or antigen-binding fragment thereof may comprise a light chain that is a lambda light chain. In certain embodiments, the antibody or antigen-binding fragment comprises a light chain that comprises a light chain constant region (CL) that is a lambda constant region. In certain embodiments, the antibody comprises a light chain that comprises a light chain variable region (VL) that is a lambda variable region.

[0181] Engineered antibodies and antigen-binding fragments thereof include those in which framework residues within VH and / or VL have been modified. Such modifications may improve the properties of the antibody, for example, reduce the immunogenicity of the antibody and / or improve antibody production and purification.

[0182] The antibodies and antigen-binding fragments thereof disclosed herein can be further modified using conventional techniques known in the art, for example, by amino acid deletion, insertion, substitution, addition, and / or recombination, and / or any other modification known in the art, either alone or in combination. Methods for introducing such modifications into the underlying DNA sequence of the amino acid sequence of an immunoglobulin chain are well known to those skilled in the art.

[0183] The antibodies and antigen-binding fragments thereof of the present invention include derivatives that have been modified (e.g., by the covalent attachment of any type of molecule to the antibody) such that the covalent attachment does not prevent the antibody from binding to its epitope or otherwise impair the biological activity of the antibody. Examples of suitable derivatives include, but are not limited to, fucosylated antibodies, glycosylated antibodies, acetylated antibodies, pegylated antibodies, phosphorylated antibodies, and amidated antibodies.

[0184] Minor variations in the amino acid sequence of the antibodies of the invention are considered to be within the scope of the invention so long as the variations in amino acid sequence maintain at least 75%, more preferably at least 80%, at least 90%, at least 95% and most preferably at least 99% sequence identity with an antibody or antigen-binding fragment thereof of the invention as defined elsewhere herein.

[0185] The antibodies of the present invention may include variants in which an amino acid residue from one species is substituted with the corresponding residue from another species, either at a conserved or non-conserved position. In certain embodiments, an amino acid residue at a non-conserved position is substituted with a conserved or non-conserved residue. Conservative amino acid substitutions are particularly contemplated.

[0186] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the amino acid substitution is considered conservative. The inclusion of conservatively modified variants in the antibodies of the invention includes other forms of variants, including, but not limited to, polymorphic variants, interspecies homologs, and alleles.

[0187] "Non-conservative amino acid substitutions" include those in which (i) a residue having a positively charged side chain (e.g., Arg, His, or Lys) is substituted for or replaced by a negatively charged residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for or replaced by a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val), (iii) a cysteine ​​or proline is substituted for or replaced by any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) is substituted for or replaced by a residue having a small side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).

[0188] antibody format Multispecific (e.g., bispecific) antibody formats are known in the state of the art. For example, bispecific antibody formats are described in Kontermann RE, mAbs 4:2 1-16 (2012); Holliger P., Hudson PJ, Nature Biotech.23 (2005) 1126-1136, Chan AC, Carter PJ Nature Reviews Immunology 10, 301-316 (2010) and Cuesta AM et al., Trends Biotech 28 (2011) 355-362.

[0189] The multispecific (e.g., bispecific) antibodies of the present invention may be in any format. Multispecific (e.g., bispecific) antibody formats include, for example, multivalent single chain antibodies, bispecific antibodies, and triabodies, as well as antibodies having the constant domain structure of a full-length antibody to which additional antigen-binding domains (e.g., single chain Fvs, tandem scFvs, VH and / or VL domains, Fab or (Fab)2) are linked via one or more peptide-linkers, and antibody mimetics such as DARPins. In one embodiment, the multispecific (e.g., bispecific) antibodies of the present invention are fused to a bispecific T cell engager (BITE). (登録商標) In certain embodiments, the antibodies of the invention are single chain antibodies comprising a first domain that binds to BCMA, a second domain that binds to a T cell antigen (e.g., CD3), and a third domain comprising two polypeptide monomers, each comprising a hinge, a CH2 domain, and a CH3 domain, wherein the two polypeptide monomers are fused to each other via a peptide linker (e.g., (hinge-CH2-CH3-linker-hinge-CH2-CH3).

[0190] The "valency" of an antibody refers to the number of binding domains. That is, the terms "bivalent," "trivalent," and "multivalent" refer to the presence of two binding domains, three binding domains, and multiple binding domains, respectively. Multispecific (e.g., bispecific) antibodies of the present invention may have more than one binding domain capable of binding to each target antigen (i.e., the antibody is trivalent or multivalent). In preferred embodiments, multispecific (e.g., bispecific) antibodies of the present invention have more than one binding domain capable of binding to the same epitope on each target antigen. In certain embodiments, multispecific (e.g., bispecific) antibodies of the present invention have more than one binding domain capable of binding to different epitopes on each target antigen.

[0191] Multispecific (e.g., bispecific) antibodies of the invention can be bivalent, trivalent, or tetravalent. In a preferred embodiment, the multispecific (e.g., bispecific) antibody is trivalent, preferably where the trivalent antibody is bivalent for BCMA. Thus, the bispecific antibody can be trivalent, where the trivalent antibody is bivalent for BCMA.

[0192] Specific (e.g., bispecific) antibodies can be full-length from a single species or can be chimerized or humanized. For antibodies with more than two antigen-binding domains, some of the binding domains can be identical, as long as the protein has binding domains for two different antigens.

[0193] The multispecific (e.g., bispecific) antibodies of the present invention may have a bispecific heterodimeric format. In certain embodiments, the bispecific antibody comprises two different heavy chains and two different light chains. In other embodiments, the multispecific (e.g., bispecific) antibody comprises two identical light chains and two different heavy chains. In certain embodiments, in the multispecific (e.g., bispecific) antibodies of the present invention, one of the two pairs of heavy and light chains (HC / LC) specifically binds to CD3, and the other specifically binds to BCMA.

[0194] In embodiments in which the bispecific antibodies of the invention are bivalent, they may comprise one anti-BCMA antibody and one anti-CD3 antibody (referred to herein as a "1+1" format).

[0195] In embodiments where the BCMA and CD3 antibodies are Fabs, the bivalent bispecific antibody in a 1+1 format may have the format: CD3 Fab-BCMA Fab (i.e., when Fc is absent). Alternatively, the bispecific antibody may have the format: Fc-CD3 Fab-BCMA Fab; Fc-BCMA Fab-CD3 Fab; or BCMA Fab-Fc-CD3 Fab (i.e., when Fc is present). In a preferred embodiment, the bivalent bispecific antibody has the format BCMA Fab-Fc-CD3 Fab.

[0196] "CD3 Fab-BCMA Fab" means that the CD3 Fab is bound via its N-terminus to the C-terminus of the BCMA Fab.

[0197] "Fc-BCMA Fab-CD3 Fab" means that the BCMA Fab is bound via its C-terminus to the N-terminus of the Fc, and the CD3 Fab is bound via its C-terminus to the N-terminus of the BCMA Fab.

[0198] "Fc-CD3 Fab-BCMA Fab" means that CD3 Fab is bound via its C-terminus to the N-terminus of Fc, and BCMA Fab is bound via its C-terminus to the N-terminus of CD3 Fab.

[0199] "BCMA Fab-Fc-CD3 Fab" means that the BCMA and CD3 Fab fragments are linked via their C-termini to the N-terminus of Fc.

[0200] In embodiments in which the bispecific antibodies of the invention are trivalent, they may comprise two anti-BCMA antibodies and one anti-CD3 antibody (referred to herein as a "2+1" format).

[0201] In embodiments where the BCMA and CD3 antibodies are Fabs, the trivalent bispecific antibody in a 2+1 format may have the format: CD3 Fab-BCMA Fab-BCMA Fab; or BCMA Fab-CD3 Fab-BCMA Fab (i.e., when Fc is absent). Alternatively, the bispecific antibody may have the format: BCMA Fab-Fc-CD3 Fab-BCMA Fab; BCMA Fab-Fc-BCMA Fab-CD3 Fab; or CD3 Fab-Fc-BCMA Fab-BCMA Fab (i.e., when Fc is present). In a preferred embodiment, the trivalent bispecific antibody has the format BCMA Fab-Fc-CD3 Fab-BCMA Fab.

[0202] "CD3 Fab-BCMA Fab-BCMA Fab" means that a CD3 Fab is bound via its C-terminus to the N-terminus of a first BCMA Fab, and a first BCMA Fab is bound via its C-terminus to the N-terminus of a second BCMA Fab.

[0203] "BCMA Fab-CD3 Fab-BCMA Fab" means that a first BCMA Fab is bound via its C-terminus to the N-terminus of a CD3 Fab, and a CD3 Fab is bound via its C-terminus to the N-terminus of a second BCMA Fab.

[0204] "BCMA Fab-Fc-CD3 Fab-BCMA Fab means that a first BCMA Fab and a CD3 Fab are bound via their C-terminus to the N-terminus of the Fc, and a second BCMA Fab is bound via its C-terminus to the N-terminus of the CD3 Fab.

[0205] "BCMA Fab-Fc-BCMA Fab-CD3 Fab" means that a first BCMA Fab and a second BCMA Fab are bound via their C-terminus to the N-terminus of Fc, and a CD3 Fab is bound via its C-terminus to the N-terminus of the second BCMA Fab.

[0206] "CD3 Fab-Fc-BCMA Fab-BCMA Fab" means that the CD3 Fab and the first BCMA Fab are bound via their C-terminus to the N-terminus of the Fc, and the second BCMA Fab is bound via its C-terminus to the N-terminus of the first BCMA Fab.

[0207] In one embodiment, a bispecific antibody of the invention comprises no more than one BCMA Fab that specifically binds BCMA, no more than one CD3 Fab that specifically binds CD3, and no more than one Fc portion.

[0208] In certain embodiments, the bispecific antibody comprises no more than one CD3 Fab that specifically binds to CD3, no more than two BCMA Fabs that specifically bind to BCMA, and no more than one Fc portion. In certain embodiments, no more than one CD3 Fab and no more than one BCMA Fab are linked to the Fc portion, and the linkage is achieved via C-terminal attachment of the Fab to the hinge region of the Fc portion. In certain embodiments, the second BCMA Fab is attached via its C-terminus to the N-terminus of the CD3 Fab or to the hinge region of the Fc portion, and is thus between the Fc portion and the CD3 Fab of the bispecific antibody.

[0209] In embodiments comprising two BCMA Fabs, the BCMA Fabs are preferably derived from the same antibody and preferably have identical CDR sequences, variable domain sequences VH and VL and / or constant domain sequences CH1 and CL. Preferably, the amino acid sequences of the two BCMA Fabs are identical.

[0210] Bispecific antibodies of the invention can also comprise scFvs instead of Fabs. Thus, in certain embodiments, the bispecific antibody has any of the above formats, in which each Fab is replaced with a corresponding scFv.

[0211] The components of the bispecific antibody of the present invention, such as Fab fragments, can be chemically linked together using a suitable linker according to the state of the art. In a preferred embodiment, a (Gly4-Ser1)3 linker is used (Desplancq DK et al., Protein Eng. 1994 Aug;7(8):1027-33 and Mack M. et al., PNAS July 18, 1995 vol. 92 no. 15 7021-7025). As used herein, "chemically linked" (or "linked") means that the components are linked by a covalent bond. Since the linker is a peptidic linker, such covalent linkage is usually achieved by biochemical recombinant means. For example, linkage can be achieved using nucleic acids encoding the VL and / or VH domains of each Fab fragment, the linker, and, if the antibody contains an Fc, an Fc partial chain.

[0212] Where a linker is used, the linker may be of a length and sequence sufficient to ensure that each of the first and second domains can maintain their different binding specificities independently of each other.

[0213] Antibody sequence In some embodiments, the multispecific (e.g., bispecific) antibody comprises an anti-BCMA antibody, or antigen-binding fragment thereof, that comprises a CDR1H, CDR2H, CDR3H, CDR1L, CDR2L, and CDR3L region combination selected from the group consisting of: a) the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 23, the CDR2L region of SEQ ID NO: 24 and the CDR3L region of SEQ ID NO: 20; b) the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 25, the CDR2L region of SEQ ID NO: 26 and the CDR3L region of SEQ ID NO: 20; c) the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 27, the CDR2L region of SEQ ID NO: 28 and the CDR3L region of SEQ ID NO: 20; d) the CDR1H region of SEQ ID NO: 29, the CDR2H region of SEQ ID NO: 30, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 31, the CDR2L region of SEQ ID NO: 32 and the CDR3L region of SEQ ID NO: 33; e) the CDR1H region of SEQ ID NO: 34, the CDR2H region of SEQ ID NO: 35, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 31, the CDR2L region of SEQ ID NO: 32 and the CDR3L region of SEQ ID NO: 33; f) the CDR1H region of SEQ ID NO: 36, the CDR2H region of SEQ ID NO: 37, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 31, the CDR2L region of SEQ ID NO: 32 and the CDR3L region of SEQ ID NO: 33; and g) The CDR1H region of SEQ ID NO: 15, the CDR2H region of SEQ ID NO: 16, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 18, the CDR2L region of SEQ ID NO: 19 and the CDR3L region of SEQ ID NO: 20.

[0214] In any of the embodiments disclosed herein, the CDR1L region of SEQ ID NO: 18 may be replaced with the CDR1L region of SEQ ID NO: 67, and the CDR2L region of SEQ ID NO: 19 may be replaced with the CDR2L region of SEQ ID NO: 68. Thus, in some embodiments, a multispecific (e.g. bispecific) antibody may comprise an anti-BCMA antibody, or antigen-binding fragment thereof, comprising the CDR1H region of SEQ ID NO: 15, the CDR2H region of SEQ ID NO: 16, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 67, the CDR2L region of SEQ ID NO: 68, and the CDR3L region of SEQ ID NO: 20.

[0215] In any of the embodiments disclosed herein, the CDR1L region of SEQ ID NO: 27 may be replaced with the CDR1L region of SEQ ID NO: 71; and the CDR2L region of SEQ ID NO: 28 may be replaced with the CDR2L region of SEQ ID NO: 72. Thus, in some embodiments, a multispecific (e.g. bispecific) antibody may comprise an anti-BCMA antibody, or antigen-binding fragment thereof, comprising the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 71, the CDR2L region of SEQ ID NO: 72, and the CDR3L region of SEQ ID NO: 20.

[0216] In any of the embodiments disclosed herein, the CDR1L region of SEQ ID NO: 25 may be replaced with the CDR1L region of SEQ ID NO: 69; and the CDR2L region of SEQ ID NO: 26 may be replaced with the CDR2L region of SEQ ID NO: 70. Thus, in some embodiments, a multispecific (e.g. bispecific) antibody may comprise an anti-BCMA antibody, or antigen-binding fragment thereof, comprising the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 69, the CDR2L region of SEQ ID NO: 70, and the CDR3L region of SEQ ID NO: 20.

[0217] In a preferred embodiment, the multispecific (e.g., bispecific) antibody comprises an anti-BCMA antibody, or antigen-binding fragment thereof, comprising a CDR1H, CDR2H, CDR3H CDR1L, CDR2L, and CDR3L region combination selected from the following: a) the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 27, the CDR2L region of SEQ ID NO: 28 and the CDR3L region of SEQ ID NO: 20; b) the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 25, the CDR2L region of SEQ ID NO: 26 and the CDR3L region of SEQ ID NO: 20; or c) The CDR1H region of SEQ ID NO: 15, the CDR2H region of SEQ ID NO: 16, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 18, the CDR2L region of SEQ ID NO: 19 and the CDR3L region of SEQ ID NO: 20.

[0218] In particularly preferred embodiments, the multispecific (e.g., bispecific) antibody comprises an anti-BCMA antibody, or antigen-binding fragment thereof, comprising a VH region comprising the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, and the CDR3H region of SEQ ID NO: 17, and a VL region comprising the CDR1L region of SEQ ID NO: 27, the CDR2L region of SEQ ID NO: 28, and the CDR3L region of SEQ ID NO: 20. In certain embodiments, the multispecific (e.g., bispecific) antibody comprises an anti-BCMA antibody, or antigen-binding fragment thereof, comprising a VH and VL selected from the group consisting of: a) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 12; b) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13; c) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14; d) a VH region of SEQ ID NO: 38 and a VL region of SEQ ID NO: 12; e) a VH region of SEQ ID NO: 39 and a VL region of SEQ ID NO: 12; f) a VH region of SEQ ID NO: 40 and a VL region of SEQ ID NO: 12, or g) A VH region of SEQ ID NO: 9 and a VL region of SEQ ID NO: 11.

[0219] In certain embodiments, the multispecific (e.g., bispecific) antibody comprises an anti-BCMA antibody, or antigen-binding fragment thereof, comprising a VH and a VL selected from the group consisting of: a) a VH comprising an amino acid sequence that is at least 75% identical, at least 90% identical, at least 95% identical or identical to the amino acid sequence of SEQ ID NO: 10, and a VL comprising an amino acid sequence that is at least 90% identical, at least 95% identical or identical to the amino acid sequence of SEQ ID NO: 14; b) a VH comprising an amino acid sequence that is at least 75% identical, at least 90% identical, at least 95% identical or identical to the amino acid sequence of SEQ ID NO: 10 and a VL comprising an amino acid sequence that is at least 75% identical, at least 90% identical, at least 95% identical or identical to the amino acid sequence of SEQ ID NO: 13; or c) A VH comprising an amino acid sequence that is at least 75% identical, at least 90% identical, at least 95% identical or identical to the amino acid sequence of SEQ ID NO: 9, and a VL comprising an amino acid sequence that is at least 75% identical, at least 90% identical, at least 95% identical or identical to the amino acid sequence of SEQ ID NO: 11.

[0220] In certain embodiments, the multispecific (e.g., bispecific) antibody comprises an anti-BCMA antibody, or antigen-binding fragment thereof, comprising a VH and a VL selected from the group consisting of: a) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13; b) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14, or c) A VH region of SEQ ID NO: 9 and a VL region of SEQ ID NO: 11.

[0221] In a particularly preferred embodiment, the anti-BCMA antibody or antigen-binding fragment thereof comprises a VH region of SEQ ID NO:10 and a VL region of SEQ ID NO:14.

[0222] In certain embodiments, the multispecific (e.g., bispecific) antibody comprises an anti-CD3 antibody or an antigen-binding fragment thereof.

[0223] Examples of anti-CD3 antibodies include OKT3, TR66, APA 1 / 1, SP34, CH2527, WT31, 7D6, UCHT-1, Leu-4, BC-3, H2C, HuM291 (visilizumab), Hu291 (PDL), ChAglyCD3 (otelixizumab), hOKT3γ1(Ala-Ala) (teplizumab), and NI-0401 (foralumab).

[0224] The first anti-CD3 antibody produced was OKT3 (muromonab-CD3), a murine antibody that binds to the CD3 ε domain. Subsequent anti-CD3 antibodies include humanized or human antibodies and engineered antibodies, such as antibodies containing modified Fc regions.

[0225] Anti-CD3 antibodies can recognize epitopes on a single polypeptide chain, such as APA 1 / 1 or SP3 (Yang SJ, The Journal of Immunology (1986) 137; 1097-1100), or structural epitopes located on two or more subunits of CD3, such as WT31, 7D6, UCHT-1 (see WO2000041474), and Leu-44. Clinical trials using several anti-CD3 antibodies, including BC-3 (Anasetti et al., Transplantation 54: 844 (1992)) and H2C (WO2008119567A2), are underway. Anti-CD3 antibodies in clinical development include HuM291 (visilizumab) (Norman et al., Transplantation. 2000 Dec 27;70(12):1707-12.), Hu291 (PDL), ChAglyCD3 (otelixizumab) (H Waldmann), hOKT3γ1(Ala-Ala) (teplizumab) (J Bluestone and Johnson and Johnson), and (NI-0401) foralumab.

[0226] Any anti-CD3 antibody or antigen-binding fragment thereof may be suitable for use in the multispecific (e.g., bispecific) antibodies of the present invention. For example, the multispecific (e.g., bispecific) antibodies may include an anti-CD3 antibody selected from OKT3, TR66, APA 1 / 1, SP34, CH2527, WT31, 7D6, UCHT-1, Leu-4, BC-3, H2C, HuM291 (vidilizumab), Hu291 (PDL), ChAglyCD3 (otelixizumab), hOKT3γ1 (Ala-Ala) (teplizumab), and NI-0401 (foralumab). In some embodiments, the multispecific (e.g., bispecific) antibodies of the present invention include a humanized SP34 antibody or an antigen-binding fragment thereof.

[0227] In certain preferred embodiments, the anti-CD3 antibody or antigen-binding fragment thereof may be derived from SP34 and may have sequence similarity and the same properties with respect to epitope binding as antibody SP34.

[0228] In one embodiment, the multispecific (e.g., bispecific) antibody comprises an anti-CD3 antibody, or an antigen-binding fragment thereof, comprising a variable domain VH comprising the heavy chain CDRs of SEQ ID NOs: 1, 2, and 3 as heavy chain CDR1H, CDR2H, and CDR3H, respectively, and a variable domain VL comprising the light chain CDRs of SEQ ID NOs: 4, 5, and 6 as light chain CDR1L, CDR2L, and CDR3L, respectively.

[0229] In certain embodiments, the multispecific (e.g., bispecific) antibody comprises an anti-CD3 antibody, or antigen-binding fragment thereof, comprising the variable domains of SEQ ID NO: 7 (VH) and SEQ ID NO: 8 (VL). In certain embodiments, the multispecific (e.g., bispecific) antibody comprises an anti-CD3 antibody, or antigen-binding fragment thereof, comprising a variable region VH comprising an amino acid sequence that is at least 75% identical, at least 90% identical, at least 95% identical, or identical to the amino acid sequence of SEQ ID NO: 7, and a variable region VL comprising an amino acid sequence that is at least 75% identical, at least 90% identical, at least 95% identical, or identical to the amino acid sequence of SEQ ID NO: 8.

[0230] In certain embodiments, the multispecific (e.g., bispecific) antibody is an anti-BCMA antibody or antigen-binding fragment thereof that comprises a CDR1H, CDR2H, CDR3H, CDR1L, CDR2L, and CDR3L region combination selected from the group consisting of: a) the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 23, the CDR2L region of SEQ ID NO: 24 and the CDR3L region of SEQ ID NO: 20; b) the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 25, the CDR2L region of SEQ ID NO: 26 and the CDR3L region of SEQ ID NO: 20; c) the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 27, the CDR2L region of SEQ ID NO: 28 and the CDR3L region of SEQ ID NO: 20; d) the CDR1H region of SEQ ID NO: 29, the CDR2H region of SEQ ID NO: 30, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 31, the CDR2L region of SEQ ID NO: 32 and the CDR3L region of SEQ ID NO: 33; e) the CDR1H region of SEQ ID NO: 34, the CDR2H region of SEQ ID NO: 35, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 31, the CDR2L region of SEQ ID NO: 32 and the CDR3L region of SEQ ID NO: 33; f) the CDR1H region of SEQ ID NO: 36, the CDR2H region of SEQ ID NO: 37, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 31, the CDR2L region of SEQ ID NO: 32 and the CDR3L region of SEQ ID NO: 33; or g) the CDR1H region of SEQ ID NO: 15, the CDR2H region of SEQ ID NO: 16, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 18, the CDR2L region of SEQ ID NO: 19 and the CDR3L region of SEQ ID NO: 20; and an anti-CD3 antibody or antigen-binding fragment thereof comprising the CDR1H region of SEQ ID NO: 1, the CDR2H region of SEQ ID NO: 2, the CDR3H region of SEQ ID NO: 3, the CDR1L region of SEQ ID NO: 4, the CDR2L region of SEQ ID NO: 5, and the CDR3L region of SEQ ID NO: 6. Includes.

[0231] In a particularly preferred embodiment, multispecific (e.g., bispecific) antibodies are an anti-BCMA antibody or antigen-binding fragment thereof, comprising a VH region comprising the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, and the CDR3H region of SEQ ID NO: 17, and a VL region comprising the CDR1L region of SEQ ID NO: 27, the CDR2L region of SEQ ID NO: 28, and the CDR3L region of SEQ ID NO: 20; and An anti-CD3 antibody or antigen-binding fragment thereof comprising the CDR1H region of SEQ ID NO: 1, the CDR2H region of SEQ ID NO: 2, the CDR3H region of SEQ ID NO: 3, the CDR1L region of SEQ ID NO: 4, the CDR2L region of SEQ ID NO: 5, and the CDR3L region of SEQ ID NO: 6. Includes.

[0232] In certain embodiments, the multispecific (e.g., bispecific) antibody comprises an anti-BCMA antibody or antigen-binding fragment thereof comprising a VH and a VL selected from the group consisting of: a) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 12; b) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13; c) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14; d) a VH region of SEQ ID NO: 38 and a VL region of SEQ ID NO: 12; e) a VH region of SEQ ID NO: 39 and a VL region of SEQ ID NO: 12; f) a VH region of SEQ ID NO: 40 and a VL region of SEQ ID NO: 12; or g) a VH region of SEQ ID NO: 9 and a VL region of SEQ ID NO: 11, and An anti-CD3 antibody or antigen-binding fragment thereof comprising the VH region of SEQ ID NO: 7 and the VL region of SEQ ID NO: 8 Includes.

[0233] In a particularly preferred embodiment, the multispecific (e.g. bispecific) antibody comprises an anti-BCMA antibody, or antigen-binding fragment thereof, comprising the VH region of SEQ ID NO: 10 and the VL region of SEQ ID NO: 14, and an anti-CD3 antibody, or antigen-binding fragment thereof, comprising the VH region of SEQ ID NO: 7 and the VL region of SEQ ID NO: 8.

[0234] In some embodiments, the multispecific (e.g., bispecific) antibody comprises an anti-BCMA antibody, or antigen-binding fragment thereof, comprising the CDR3H, CDR3L, CDR1H, CDR2H, CDR1L, and CDR2L of one of GSK2857916, AMG-420, AMG-701, JNJ-957, JNJ-64007957, PF-06863135, REGN-5458, or TNB-383B. In some embodiments, the multispecific (e.g., bispecific) antibody comprises an anti-BCMA antibody, or antigen-binding fragment thereof, comprising the VH and VL of one of GSK2857916, AMG-420, AMG-701, JNJ-957, JNJ-64007957, PF-06863135, REGN-5458, or TNB-383B.

[0235] Fc Multispecific (e.g., bispecific) antibodies of the invention may or may not comprise an Fc. In a preferred embodiment, the multispecific (e.g., bispecific) antibodies of the invention comprise an Fc, preferably a human Fc.

[0236] In certain embodiments, the Fc is a variant Fc, e.g., an Fc sequence that has been modified (e.g., by amino acid substitution, deletion, and / or insertion) relative to the parent Fc sequence (e.g., the unmodified Fc polypeptide that is subsequently modified to produce the variant) to possess desirable structural properties and / or biological activity.

[0237] Thus, multispecific (e.g., bispecific) antibodies of the invention may typically comprise an Fc that comprises one or more modifications to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. The Fc may be linked to an anti-BCMA and / or anti-CD3 Fab fragment in the antibodies of the invention.

[0238] The presence of Fc has the advantage of extending the elimination half-life of the antibody. Multispecific (e.g., bispecific) antibodies of the invention can have an elimination half-life of greater than 12 hours, preferably greater than 3 days, in mice or cynomolgus monkeys, preferably cynomolgus monkeys. In certain embodiments, multispecific (e.g., bispecific) antibodies of the invention have an elimination half-life of about 1 to 12 days, allowing for administration at least once or twice weekly.

[0239] Reduced effector function Preferably, the multispecific (e.g., bispecific) antibodies of the present invention comprise an Fc region (e.g., of the IgG1 subclass) that includes modifications to avoid FcR and C1q binding and minimize ADCC / CDC. This provides the advantage that bispecific antibodies mediate their tumor cell killing efficacy purely through potent mechanisms of effector cells, e.g., T cell, redirection / activation. Thus, additional mechanisms of action, such as effects on the complement system and FcR-expressing effector cells, are avoided, and side effects, such as infusion-related reactions, are reduced.

[0240] In a preferred embodiment, the multispecific (eg, bispecific) antibodies of the invention comprise an Fc region of an IgG, particularly an IgG1, comprising the modifications L234A, L235A and P329G (numbering according to EU numbering).

[0241] Heterodimerization Multispecific (e.g., bispecific) antibodies of the invention may be heteromultimeric antibodies. Such heteromultimeric antibodies may contain modifications in regions involved in interactions between antibody chains to facilitate correct assembly of the antibody.

[0242] For example, a multispecific (e.g., bispecific) antibody of the invention can comprise an Fc with one or more modifications in the CH2 and CH3 domains to facilitate Fc heterodimerization. Alternatively or additionally, a multispecific (e.g., bispecific) antibody of the invention can comprise modifications in the CH1 and CL regions to promote preferential pairing of the heavy and light chains of the Fab fragments.

[0243] Several strategies exist for promoting heterodimerization. These strategies include the introduction of non-symmetric, complementary modifications into each of the two antibody chains so that both chains are compatible with each other and can therefore form heterodimers, but each chain cannot dimerize by itself. Such modifications can include insertions, deletions, conservative and non-conservative substitutions, and rearrangements.

[0244] Heterodimerization can be promoted by introducing charged residues to create favorable electrostatic interactions between the first and second antibody chains. For example, one or more positively charged amino acids can be introduced into the first antibody chain and one or more negatively charged amino acids can be introduced into the corresponding positions of the second antibody chain.

[0245] Alternatively or additionally, heterodimerization can be promoted by introducing steric hindrance between contacting residues: for example, one or more residues with bulky side chains can be introduced into the first antibody chain, and one or more residues that can accommodate the bulky side chains can be introduced into the second antibody chain.

[0246] Alternatively or additionally, heterodimerization can be promoted by introducing one or more modifications to hydrophilic and hydrophobic residues at the interface between the chains to entropically and enthalpically favor heterodimer formation over homodimer formation.

[0247] A further strategy to promote heterodimerization is rearrangement of portions of antibody chains such that each chain is compatible only with chains containing the corresponding rearrangement. For example, CrossMAb technology is based on crossover of antibody domains to allow precise chain pairing. (i) CrossMAb in which VH and VL are swapped and CH1 and CL are swapped. Fab (ii) CrossMAb in which VH and VL are exchanged VH-VL and (iii) CrossMAb in which CH1 and CL are swapped. CH1-CL There are three main CrossMAb formats (Klein et al., 2016. MABS, 8(6):1010-1020).

[0248] In certain embodiments, multispecific (e.g., bispecific) antibodies of the invention may comprise swapped VH and VL. In certain embodiments, multispecific (e.g., bispecific) antibodies of the invention may comprise swapped CH1 and CL. In certain embodiments, multispecific (e.g., bispecific) antibodies of the invention may comprise swapped VH and VL and swapped CH1 and CL.

[0249] In a preferred embodiment, the multispecific (eg, bispecific) antibodies of the invention comprise swapped VH and VL.

[0250] Other approaches to promote heterodimerization include the use of strand-exchange engineered domains (SEEDs) (Davis et al., 2010. Protein Eng Des Sel, 23 (4); 195-202).

[0251] A combination of the above strategies may be used to maximize the efficiency of conjugation while minimizing the impact on antibody stability.

[0252] Fc heterodimerization In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention may have a heterodimeric Fc, for example, comprising one heavy chain from an anti-BCMA antibody and one heavy chain from an anti-CD3 antibody.

[0253] The multispecific (e.g., bispecific) antibodies of the present invention may comprise a heterodimeric Fc comprising one or more modifications that promote pairing of the first CH2 and / or CH3 domain with the second CH2 and / or CH3 domain. In a preferred embodiment, the one or more modifications promote pairing of the first CH3 domain with the second CH3 domain, for example, by providing an asymmetric modification to the CH3 domain. The one or more modifications may include modifications selected from amino acid insertions, deletions, conservative and non-conservative substitutions and rearrangements, and combinations thereof.

[0254] Typically, the first CH3 domain and the second CH3 domain are both engineered in a complementary manner such that each CH3 domain (or the heavy chain containing it) can no longer homodimerize with itself, but rather is forced to heterodimerize with the other complementary engineered CH3 domain (thus, the first and second CH3 domains heterodimerize, and homodimers between the two first or two second CH3 domains are not formed).

[0255] Multispecific (e.g., bispecific) antibodies of the invention can comprise an Fc having one or more "knob-into-hole" modifications, some examples of which are detailed in, for example, WO96 / 027011; Ridgway, JB, et al., Protein Eng. 9 (1996) 617-621; Merchant, AM et al., Nat. Biotechnol. 16 (1998) 677-68; and WO98 / 050431.

[0256] In this method, the interaction surfaces of the two CH3 domains are altered to increase heterodimerization of both Fc chains containing these two CH3 domains. One of the two CH3 domains (of the two Fc chains) can be the "knob" and the other the "hole."

[0257] Thus, a multispecific (e.g., bispecific) antibody of the present invention may comprise two CH3 domains, wherein the first CH3 domain of a first Fc chain and the second CH3 domain of a second Fc chain each meet at an interface that comprises the original interface between antibody CH3 domains, where the interface has been modified to facilitate antibody formation.

[0258] In one embodiment: (i) the CH3 domain of one Fc chain is modified so that, within the original interface of the CH3 domain of one Fc chain that meets the original interface of the CH3 domain of the other Fc chain, amino acid residues are replaced with amino acid residues having a large side chain volume, thereby producing protrusions at the interface of the CH3 domain of one Fc chain that can be placed into cavities within the interface of the CH3 domain of the other Fc chain; and ii) The CH3 domain of one Fc chain is modified so that, within the original interface of the CH3 domain of the other Fc chain that meets the original interface of the CH3 domain of the other Fc chain, amino acid residues are replaced with amino acid residues having a small side chain volume, thereby creating a cavity at the interface of the CH3 domain of the other Fc chain, into which a protrusion within the interface of the CH3 domain of the one Fc chain can be placed.

[0259] Preferably, the amino acid residues having large side chain volumes are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0260] In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention comprise a first CH3 domain comprising modifications at positions T366, L368 and Y407, e.g., T366S, L368A and Y407V (numbering according to EU numbering).

[0261] In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention comprise a second CH3 domain comprising a modification at position T366 (a "knob modification"), e.g., T366W (numbering according to EU numbering).

[0262] In a particularly preferred embodiment, the multispecific (e.g., bispecific) antibody of the invention comprises a first CH3 domain comprising the modifications T366S, L368A and Y407V or a conservative substitution thereof and a second CH3 domain comprising the modification T366W or a conservative substitution thereof (numbering according to EU numbering).

[0263] In certain embodiments, a multispecific (eg, bispecific) antibody of the invention comprises a first CH3 domain comprising a modification shown in Table 2 and a second CH3 domain comprising a modification shown in Table 2.

[0264] [Table 2]

[0265] Multispecific (eg, bispecific) antibodies of the invention may include one or more of the modifications set forth in US Pat. No. 9,562,109 and US Pat. No. 9,574,010 (incorporated herein by reference).

[0266] In certain embodiments, multispecific (e.g., bispecific) antibodies of the invention comprise a first CH3 domain comprising one or more modifications at positions T350, L351, F405 and / or Y407 (numbering according to EU numbering), e.g., T350V, L351Y, F405A and / or Y407V. In certain embodiments, multispecific (e.g., bispecific) antibodies of the invention comprise a first CH3 domain comprising modifications at positions T350, L351, F405 and Y407 (numbering according to EU numbering), e.g., T350V, L351Y, F405A and Y407V.

[0267] In certain embodiments, multispecific (e.g., bispecific) antibodies of the invention comprise a second CH3 domain comprising one or more modifications at positions T350, T366, K392 and / or T394 (numbering according to EU numbering), e.g., T350V, T366L, K392L and / or T394W. In certain embodiments, multispecific (e.g., bispecific) antibodies of the invention comprise a second CH3 domain comprising modifications at positions T350, T366, K392 and T394 (numbering according to EU numbering), e.g., T350V, T366L, K392L and T394W.

[0268] In a preferred embodiment, the multispecific (e.g. bispecific) antibodies of the invention comprise a first CH3 domain comprising one or more modifications at positions T350, L351, F405 and / or Y407 (e.g. T350V, L351Y, F405A and / or Y407V) and a second CH3 domain (numbered according to EU numbering) comprising one or more modifications at positions T350, T366, K392 and / or T394 (e.g. T350V, T366L, K392L and / or T394W).

[0269] In a particularly preferred embodiment, the multispecific (e.g. bispecific) antibody of the invention comprises a first CH3 domain comprising modifications at positions T350, L351, F405 and Y407 (e.g. T350V, L351Y, F405A and Y407V) and a second CH3 domain comprising modifications at positions T350, T366, K392 and T394 (e.g. T350V, T366L, K392L and T394W) (numbering according to EU numbering).

[0270] The one or more modifications may modify charge, hydrophobic / hydrophilic interactions and / or steric hindrance between side chains.

[0271] In a particularly preferred embodiment, the multispecific (e.g. bispecific) antibody of the invention comprises a first CH3 domain comprising the modifications T350V, L351Y, F405A and Y407V or conservative substitutions thereof and a second CH3 domain comprising the modifications T350V, T366L, K392L and T394W or conservative substitutions thereof (numbering according to EU numbering).

[0272] In certain embodiments, a multispecific (eg, bispecific) antibody of the invention comprises a first CH3 domain comprising a modification shown in Table 3 and a second CH3 domain comprising a modification shown in Table 3.

[0273] [Table 3]

[0274] Other techniques for CH3 modification to effect heterodimerization are contemplated as alternatives of the present invention, including, for example, those described in WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO2013 / 157954, WO2013 / 157953 and WO2013 / 096291.

[0275] In one embodiment, the bispecific antibodies of the invention are of the IgG2 isotype and the heterodimerization approach described in WO2010 / 129304 may be used.

[0276] Other Fc modifications In one embodiment, a bispecific antibody of the present invention may comprise an Fc in which both CH3 domains have been modified by introducing cysteine ​​(C) as the amino acid at corresponding positions so that disulfide bridges can be formed between the two CH3 domains. The cysteine ​​may be introduced at position 349 in one of the CH3 domains and at position 354 in the other CH3 domain (EU numbering).

[0277] Preferably, the introduced cysteine ​​at position 354 is in the first CH3 domain and the introduced cysteine ​​at position 349 is in the second CH3 domain (numbering according to EU numbering).

[0278] The Fc may contain modifications such as D356E, L358M, N384S, K392N, V397M and V422I (numbering according to EU numbering). Preferably, both CH3 domains contain D356E and L358M (numbering according to EU numbering).

[0279] Light and heavy chain heterodimerization In the multispecific (e.g., bispecific) antibodies of the present invention, one or more of the immunoglobulin heavy and light chains can contain one or more modifications, such as amino acid modifications, that can promote preferential pairing of a specific heavy chain with a specific light chain when the heavy and light chains are coexpressed or coproduced. Such modifications can significantly improve production / purification without altering biological properties such as binding to BCMA. In particular, the introduction of one or more modifications, such as amino acid exchanges, can significantly reduce light chain mispairing and by-product formation during production, thereby increasing yield and facilitating purification.

[0280] The one or more modifications may promote preferential heterodimer pairing by introducing steric hindrance, substituting charged amino acids with opposite charges, and / or hydrophobic or hydrophilic interactions. In a preferred embodiment, the one or more modifications promote preferential heterodimer pairing by introducing steric hindrance and substituting charged amino acids with opposite charges.

[0281] Amino acid exchanges can be substitutions of charged amino acids with opposite charges (e.g., at the CH1 / CL interface), which reduces light chain mispairing, e.g., Bence-Jones type by-products.

[0282] In a preferred embodiment, the one or more modifications that favor light and heavy chain heterodimerization are amino acid modifications in the light and heavy chains outside of the CDRs.

[0283] The one or more modifications may be present in the anti-BCMA antibody or antigen-binding fragment thereof. Alternatively, the one or more modifications may be present in the anti-CD3 antibody or antigen-binding fragment thereof. In a preferred embodiment, the one or more modifications are present in the anti-BCMA antibody or antigen-binding fragment thereof.

[0284] In certain embodiments, a multispecific (e.g., bispecific) antibody of the invention comprises an immunoglobulin heavy chain comprising a CH1 domain with the amino acid modifications K147E / D and K213E / D (EU numbering) and a corresponding immunoglobulin light chain comprising a CL domain with the amino acid modifications E123K / R / H and Q124K / R / H (Kabat numbering). Preferably, the CH1 domain comprises the amino acid modifications K147E and K213E (EU numbering) or conservative substitutions thereof, and the corresponding CL domain comprises the amino acid modifications E123R and Q124K or conservative substitutions thereof (Kabat numbering). Such multispecific (e.g., bispecific) antibodies can be produced in high yield and easily purified.

[0285] In certain embodiments, the amino acid modifications described in Table 4 may be in a BCMA antibody or a CD3 antibody.

[0286] In one embodiment, the bispecific antibodies of the invention are bivalent and comprise one anti-BCMA antibody or antigen-binding fragment thereof and one anti-CD3 antibody or antigen-binding fragment thereof ("1+1" format), wherein: (a) a BCMA antibody or antigen-binding fragment thereof (e.g., a BCMA Fab) comprising a CH1 domain with an amino acid modification shown in Table 4 and a corresponding CL domain with an amino acid modification in Table 4; or (b) A CD3 antibody or antigen-binding fragment thereof (e.g., CD3 Fab) comprising a CH1 domain having an amino acid modification shown in Table 4 and a corresponding CL domain having an amino acid modification in Table 4.

[0287] In one embodiment, the bispecific antibodies of the invention are trivalent and comprise two anti-BCMA antibodies or antigen-binding fragments thereof and one anti-CD3 antibody or antigen-binding fragment thereof ("2+1" format), wherein: (a) one or both of the BCMA antibodies or antigen-binding fragments thereof (e.g., BCMA Fab) comprise a CH1 domain with an amino acid modification shown in Table 4 and a corresponding CL domain with an amino acid modification in Table 4; or (b) A CD3 antibody (eg, CD3 Fab) comprises a CH1 domain with an amino acid modification shown in Table 4 and a corresponding CL domain with an amino acid modification in Table 4.

[0288] In particular, each BCMA antibody (e.g., BCMA Fab) may comprise a CH1 domain with an amino acid modification shown in Table 4 and a CL domain with the corresponding amino acid modification.

[0289] [Table 4]

[0290] In preferred embodiments, multispecific (e.g., bispecific) antibodies of the invention comprise the modifications shown in Table 4 in combination with the modifications shown in Table 2. Thus, in certain embodiments, bispecific antibodies of the invention are bivalent and (a) one anti-BCMA antibody or antigen-binding fragment thereof and one anti-CD3 antibody or antigen-binding fragment thereof ("1+1" format), where (i) the BCMA antibody or antigen-binding fragment thereof (e.g., BCMA Fab) comprises a CH1 domain comprising the amino acid modifications K147E and K213E and a corresponding CL domain comprising the amino acid modifications E123R and Q124K (i.e., the modifications shown in Table 4), or (ii) the CD3 antibody or antigen-binding fragment thereof (e.g., CD3 Fab) comprises a CH1 domain comprising the amino acid modifications K147E and K213E and a corresponding CL domain comprising the amino acid modifications E123R and Q124K (i.e., the modifications shown in Table 4); and (b) a first CH3 domain comprising the modifications T366S, L368A, and Y407V and a second CH3 domain comprising the modification T366W (i.e., the modifications shown in Table 2). Includes.

[0291] In one embodiment, the bispecific antibodies of the invention are trivalent, (a) two anti-BCMA antibodies or antigen-binding fragments thereof and one anti-CD3 antibody or antigen-binding fragment thereof ("2+1" format), wherein (i) one or both of the BCMA antibodies or antigen-binding fragments thereof (e.g., BCMA Fab) comprise a CH1 domain comprising the amino acid modifications K147E and K213E and a corresponding CL domain comprising the amino acid modifications E123R and Q124K (i.e., the modifications shown in Table 4), or (ii) the CD3 antibody or antigen-binding fragment thereof (e.g., CD3 Fab) comprises a CH1 domain comprising the amino acid modifications K147E and K213E and a corresponding CL domain comprising the amino acid modifications E123R and Q124K (i.e., the modifications shown in Table 4); and (b) a first CH3 domain comprising the modifications T366S, L368A, and Y407V and a second CH3 domain comprising the modification T366W (i.e., the modifications shown in Table 2). Includes.

[0292] In particular, each BCMA antibody (e.g., BCMA Fab) may comprise a CH1 domain with the amino acid modifications shown in Table 4 and a corresponding CL domain with the amino acid modifications in Table 4. In a preferred embodiment, a first Fc chain is attached at the N-terminus of the Fc to the C-terminus of the first anti-BCMA antibody, and a second Fc chain is attached at the N-terminus of the Fc to the C-terminus of the anti-CD3 antibody.

[0293] In certain embodiments, multispecific (e.g., bispecific) antibodies of the invention comprise an immunoglobulin heavy chain comprising a CH1 domain with an amino acid modification at one or more of A141, L145, K147, Q175 (numbering according to EU numbering) and a corresponding immunoglobulin light chain comprising a CL domain with an amino acid modification at one or more of F116, Q124, L135, T178 (numbering according to Kabat). Preferably, the CH1 domain comprises the amino acid modification A141W, L145E, K147T, Q175E or a conservative substitution thereof (numbering according to EU numbering), and the corresponding CL domain comprises the amino acid modification F116A, Q124R, L135V, T178R or a conservative substitution thereof (numbering according to Kabat).

[0294] In certain embodiments, a multispecific (e.g., bispecific) antibody of the invention comprises a CH1 domain with an amino acid modification shown in Table 5 and a corresponding immunoglobulin light chain comprising a CL domain with an amino acid modification shown in Table 5. In embodiments where a multispecific (e.g., bispecific) antibody of the invention comprises an anti-BCMA antibody, or antigen-binding fragment thereof, of the invention and an anti-CD3 antibody, or antigen-binding fragment thereof, of the invention, the amino acid modification described in Table 5 can be in the BCMA antibody or the CD3 antibody.

[0295] In one embodiment, the bispecific antibodies of the invention are bivalent and comprise one anti-BCMA antibody and one anti-CD3 antibody ("1+1" format), wherein: (a) a BCMA antibody (e.g., a BCMA Fab) comprising a CH1 domain with an amino acid modification shown in Table 5 and a corresponding CL domain with an amino acid modification in Table 5; or (b) A CD3 antibody (eg, a CD3 Fab) comprises a CH1 domain with an amino acid modification shown in Table 5 and a corresponding CL domain with an amino acid modification in Table 5.

[0296] In one embodiment, the bispecific antibodies of the invention are trivalent and comprise two anti-BCMA antibodies and one anti-CD3 antibody ("2+1" format), wherein: (a) one or both of the BCMA antibodies (e.g., BCMA Fab) comprise a CH1 domain with an amino acid modification shown in Table 5 and a corresponding CL domain with an amino acid modification in Table 5; or (b) A CD3 antibody (eg, a CD3 Fab) comprises a CH1 domain with an amino acid modification shown in Table 5 and a corresponding CL domain with an amino acid modification in Table 5.

[0297] In particularly preferred embodiments, each BCMA antibody (e.g., BCMA Fab) may comprise a CH1 domain with an amino acid modification as shown in Table 5 and a corresponding CL domain with an amino acid modification in Table 5.

[0298] [Table 5]

[0299] In preferred embodiments, multispecific (e.g., bispecific) antibodies of the invention comprise an amino acid modification shown in Table 5 in combination with an amino acid modification shown in Table 3. Thus, in certain embodiments, bispecific antibodies of the invention are bivalent and (a) one anti-BCMA antibody and one anti-CD3 antibody ("1+1" format), where (i) the BCMA antibody (e.g., BCMA Fab) comprises a CH1 domain comprising the amino acid modifications A141W, L145E, K147T, and Q175E and a corresponding CL domain comprising the amino acid modifications F116A, Q124R, L135V, and T178R (i.e., the modifications shown in Table 5), or (ii) the CD3 antibody (e.g., CD3 Fab) comprises a CH1 domain comprising the amino acid modifications A141W, L145E, K147T, and Q175E and a corresponding CL domain comprising the amino acid modifications F116A, Q124R, L135V, and T178R (i.e., the modifications shown in Table 5); and (b) a first CH3 domain comprising the modifications T350V, L351Y, F405A, and Y407V and a second CH3 domain comprising the modifications T350V, T366L, K392L, and T394W (i.e., the modifications shown in Table 3). Includes.

[0300] In a preferred embodiment, the first Fc chain is attached at the N-terminus of the Fc to the C-terminus of the anti-BCMA antibody, and the second Fc chain is attached at the N-terminus of the Fc to the C-terminus of the anti-CD3 antibody.

[0301] In one embodiment, the bispecific antibodies of the invention are trivalent, (a) (i) two anti-BCMA antibodies and one anti-CD3 antibody (a "2+1" format), wherein one or both of the BCMA antibodies (e.g., BCMA Fab) comprise a CH1 domain comprising the amino acid modifications A141W, L145E, K147T, and Q175E and a corresponding CL domain comprising the amino acid modifications F116A, Q124R, L135V, and T178R (i.e., the modifications shown in Table 5), or (ii) the CD3 antibody (e.g., CD3 Fab) comprises a CH1 domain comprising the amino acid modifications A141W, L145E, K147T, and Q175E and a corresponding CL domain comprising the amino acid modifications F116A, Q124R, L135V, and T178R (i.e., the modifications shown in Table 5); and (b) a first CH3 domain comprising the modifications T350V, L351Y, F405A, and Y407V and a second CH3 domain comprising the modifications T350V, T366L, K392L, and T394W (i.e., the modifications shown in Table 3). Includes.

[0302] In particular, each BCMA antibody (e.g., BCMA Fab) comprises a CH1 domain with the amino acid modifications shown in Table 5 and a corresponding CL domain with the amino acid modifications in Table 5. In a preferred embodiment, a first Fc chain is attached at the N-terminus of the Fc to the C-terminus of the first anti-BCMA antibody, and a second Fc chain is attached at the N-terminus of the Fc to the C-terminus of the anti-CD3 antibody.

[0303] Alternatively, the CH1 domain may comprise an amino acid modification at position Q175 (EU numbering) and the corresponding CL domain may comprise an amino acid modification at one or more of F116, Q124, L135, T178 (Kabat numbering). The CH1 domain may comprise an amino acid modification Q175K (EU numbering) or a conservative substitution thereof, and the corresponding CL domain may comprise an amino acid modification F116A, Q124R, L135V, T178R (Kabat numbering) or a conservative substitution thereof.

[0304] In another embodiment, the CH1 domain may comprise an amino acid modification at position Q175 (EU numbering), and the corresponding CL domain may comprise an amino acid modification at one or more of Q124, L135, Q160, T180 (Kabat numbering). The CH1 domain may comprise an amino acid modification Q175K (EU numbering), or a conservative substitution thereof, and the corresponding CL domain may comprise amino acid modifications Q124E, L135W, Q160E, and T180E, or conservative substitutions thereof (Kabat numbering).

[0305] The multispecific (e.g. bispecific) antibodies of the invention may further comprise an amino acid substitution at position 49 of the VL region selected from the group consisting of the amino acids tyrosine (Y), glutamic acid (E), serine (S) and histidine (H) and / or an amino acid substitution at position 74 of the VL region with threonine (T) or alanine (A).

[0306] CrossMAb The multispecific (e.g., bispecific) antibodies of the present invention may include CrossMAb technology. CrossMAb technology is based on crossover of antibody domains to allow precise chain pairing. It is used to facilitate the formation of multispecific (e.g., bispecific) antibodies. (i) CrossMAb in which VH and VL are swapped and CH1 and CL are swapped. Fab (ii) CrossMAb in which VH and VL are exchanged VH-VL and (iii) CrossMAb in which CH1 and CL are swapped. CH1-CL There are three main CrossMAb formats (Klein et al., 2016. MABS, 8(6):1010-1020).

[0307] CrossMAb technology is known to be state of the art. Bispecific antibodies in which the variable domains VL and VH or the constant domains CL and CH1 are exchanged are described in WO2009080251 and WO2009080252.

[0308] In one or more of the antibodies or antigen-binding fragments within a multispecific (e.g., bispecific) antibody of the invention, the variable domains VL and VH or the constant domains CL and CH1 may be swapped. In certain embodiments, a multispecific (e.g., bispecific) antibody of the invention may comprise a swap of VH and VL and a swap of CH1 and CL. Thus, a multispecific (e.g., bispecific) antibody of the invention may comprise a crossover light chain and a crossover heavy chain. As used herein, a "crossover light chain" is a light chain that may comprise VH-CL, VL-CH1, or VH-CH1. As used herein, a "crossover heavy chain" is a heavy chain that may comprise VL-CH1, VH-CL, or VL-CL.

[0309] In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention (a) the light and heavy chains of an antibody that specifically binds to CD3; and (b) Light and heavy chains of an antibody that specifically binds to BCMA wherein the variable domains VL and VH and / or the constant domains CL and CH1 are exchanged with (i) an anti-BCMA antibody; and / or (ii) an anti-CD3 antibody.

[0310] In one embodiment, the variable domains VL and VH or the constant domains CL and CH1 of an anti-CD3 antibody or antigen-binding fragment thereof are exchanged. More preferably, the variable domains VL and VH of an anti-CD3 antibody or antigen-binding fragment thereof are exchanged.

[0311] In embodiments where the bispecific antibody in a 1+1 format has the formats: CD3 Fab-BCMA Fab (i.e., when Fc is absent); Fc-CD3 Fab-BCMA Fab; Fc-BCMA Fab-CD3 Fab; or BCMA Fab-Fc-CD3 Fab, the bispecific antibody may also be in a CrossMAb format, e.g., CrossMAb Fab , CrossMAb VH-VL or CrossMAb CH1-CL The BCMA Fab may be in a CrossMAb format, e.g., CrossMAbFab , CrossMAb VH-VL or CrossMAb CH1-CL Alternatively, the CD3 Fab may be in a CrossMAb format, e.g., CrossMAb Fab , CrossMAb VH-VL or CrossMAb CH1-CL In a preferred embodiment, the CD3 Fab of the bispecific antibody may have the following structure: CrossMAb VH-VL Includes format.

[0312] It is particularly preferred that bispecific antibodies of the invention having a 2+1 format comprise CrossMAb technology. Thus, in embodiments where a trivalent bispecific antibody in a 2+1 format has the formats: CD3 Fab-BCMA Fab-BCMA Fab; BCMA Fab-CD3 Fab-BCMA Fab (i.e., when Fc is absent); BCMA Fab-Fc-CD3 Fab-BCMA Fab; BCMA Fab-Fc-BCMA Fab-CD3 Fab; or CD3 Fab-Fc-BCMA Fab-BCMA Fab, the bispecific antibody may be in a CrossMAb format, e.g., CrossMAb technology. Fab , CrossMAb VH-VL or CrossMAb CH1-CL The BCMA Fab may be in a CrossMAb format, e.g., CrossMAb Fab , CrossMAb VH-VL or CrossMAb CH1-CL Alternatively, the CD3 Fab may be in a CrossMAb format, e.g., CrossMAb Fab , CrossMAb VH-VL or CrossMAb CH1-CL In a preferred embodiment, the CD3 Fab of the bispecific antibody may have the following structure: CrossMAb VH-VL Includes format.

[0313] In one embodiment, the bispecific antibodies of the invention having a 1+1 format do not comprise CrossMAb technology, i.e. neither the anti-BCMA antibody nor the anti-CD3 antibody have exchanged variable domains VL and VH or constant domains CL and CH1.

[0314] Exemplary Embodiments Exemplary embodiments are shown in Figures 1-3.

[0315] In one embodiment, the bispecific antibody of the invention is a bivalent, bispecific antibody comprising one Fab fragment of an anti-CD3 antibody, one Fab fragment of an anti-BCMA antibody, and one Fc portion according to the format BCMA Fab-Fc-CD3 Fab. The anti-BCMA Fab fragment comprises the amino acid modifications shown in Table 4 or Table 5. The anti-CD3 Fab fragment comprises a light chain and a heavy chain, wherein the light chain is a crossover light chain comprising the variable domain VH and the constant domain CL, and the heavy chain is a crossover heavy chain comprising the variable domain VL and the constant domain CH1. This embodiment is shown in Figure 1A with the amino acid modifications shown in Table 4.

[0316] In one embodiment, a bispecific antibody of the invention is a bivalent, bispecific antibody comprising one Fab fragment of an anti-CD3 antibody, one Fab fragment of an anti-BCMA antibody, and one Fc portion according to the format BCMA Fab-Fc-CD3 Fab. The anti-CD3 Fab fragment comprises (a) a light chain and a heavy chain, wherein the light chain is a crossover light chain comprising a variable domain VH and a constant domain CL, and the heavy chain is a crossover heavy chain comprising a variable domain VL and a constant domain CH1; and also (b) an amino acid modification as shown in Table 4 or Table 5. This embodiment is shown in Figure 1B with the amino acid modifications shown in Table 4.

[0317] In one embodiment, the bispecific antibody of the invention is a trivalent bispecific antibody comprising one Fab fragment of an anti-CD3 antibody, two Fab fragments of an anti-BCMA antibody, and an Fc portion according to the format BCMA Fab-Fc-CD3 Fab-BCMA Fab. Each anti-BCMA Fab fragment comprises the amino acid modifications shown in Table 4 or Table 5. The anti-CD3 Fab fragment comprises a light chain and a heavy chain, wherein the light chain is a crossover light chain comprising a variable domain VH and a constant domain CL, and the heavy chain is a crossover heavy chain comprising a variable domain VL and a constant domain CH1. This embodiment is shown in Figure 2A with the amino acid modifications shown in Table 4.

[0318] In one embodiment, a bispecific antibody of the invention is a trivalent bispecific antibody comprising one Fab fragment of an anti-CD3 antibody, two Fab fragments of an anti-BCMA antibody, and an Fc portion according to the format BCMA Fab-Fc-CD3 Fab-BCMA Fab. The anti-CD3 Fab fragment comprises (a) a light chain and a heavy chain, wherein the light chain is a crossover light chain comprising a variable domain VH and a constant domain CL, and the heavy chain is a crossover heavy chain comprising a variable domain VL and a constant domain CH1; and also (b) an amino acid modification shown in Table 4 or Table 5. This embodiment is shown in Figure 2B with the amino acid modifications shown in Table 4.

[0319] In one embodiment, the bispecific antibody of the invention is a trivalent bispecific antibody comprising one Fab fragment of an anti-CD3 antibody, two Fab fragments of an anti-BCMA antibody, and an Fc portion according to the format BCMA Fab-Fc-BCMA Fab-CD3 Fab. Each anti-BCMA Fab fragment comprises the amino acid modifications shown in Table 4 or Table 5. The anti-CD3 Fab fragment comprises a light chain and a heavy chain, wherein the light chain is a crossover light chain comprising the variable domain VH and the constant domain CL, and the heavy chain is a crossover heavy chain comprising the variable domain VL and the constant domain CH1. This embodiment is shown in Figure 2C with the amino acid modifications shown in Table 4.

[0320] In one embodiment, a bispecific antibody of the invention is a trivalent bispecific antibody comprising one Fab fragment of an anti-CD3 antibody, two Fab fragments of an anti-BCMA antibody, and an Fc portion according to the format BCMA Fab-Fc-BCMA Fab-CD3 Fab. The anti-CD3 Fab fragment comprises (a) a light chain and a heavy chain, wherein the light chain is a crossover light chain comprising a variable domain VH and a constant domain CL, and the heavy chain is a crossover heavy chain comprising a variable domain VL and a constant domain CH1; and also (b) an amino acid modification as shown in Table 4 or Table 5. This embodiment is shown in Figure 2D with the amino acid modifications shown in Table 4.

[0321] In one embodiment, the bispecific antibody of the invention is a bivalent, bispecific antibody comprising one Fab fragment of an anti-CD3 antibody, one Fab fragment of an anti-BCMA antibody, and one Fc portion according to the format Fc-CD3 Fab-BCMA Fab. The anti-BCMA Fab fragment comprises the amino acid modifications shown in Table 4 or Table 5. The anti-CD3 Fab fragment comprises a light chain and a heavy chain, wherein the light chain is a crossover light chain comprising the variable domain VH and the constant domain CL, and the heavy chain is a crossover heavy chain comprising the variable domain VL and the constant domain CH1. This embodiment is shown in Figure 3A with the amino acid modifications shown in Table 4.

[0322] In one embodiment, a bispecific antibody of the invention is a bivalent, bispecific antibody comprising one Fab fragment of an anti-CD3 antibody, one Fab fragment of an anti-BCMA antibody, and one Fc portion according to the format Fc-CD3 Fab-BCMA Fab. The anti-CD3 Fab fragment comprises (a) a light chain and a heavy chain, wherein the light chain is a crossover light chain comprising the variable domain VH and the constant domain CL, and the heavy chain is a crossover heavy chain comprising the variable domain VL and the constant domain CH1; and also (b) an amino acid modification as shown in Table 4 or Table 5. This embodiment is shown in Figure 3B with the amino acid modifications as shown in Table 4.

[0323] In one embodiment, the bispecific antibody of the invention is a bivalent, bispecific antibody comprising one Fab fragment of an anti-CD3 antibody, one Fab fragment of an anti-BCMA antibody, and one Fc portion according to the format Fc-BCMA Fab-CD3 Fab. The anti-BCMA Fab fragment comprises the amino acid modifications shown in Table 4 or Table 5. The anti-CD3 Fab fragment comprises a light chain and a heavy chain, wherein the light chain is a crossover light chain comprising the variable domain VH and the constant domain CL, and the heavy chain is a crossover heavy chain comprising the variable domain VL and the constant domain CH1. This embodiment is shown in Figure 3C with the amino acid modifications shown in Table 4.

[0324] In one embodiment, a bispecific antibody of the invention is a bivalent, bispecific antibody comprising one Fab fragment of an anti-CD3 antibody, one Fab fragment of an anti-BCMA antibody, and one Fc portion according to the format Fc-BCMA Fab-CD3 Fab. The anti-CD3 Fab fragment comprises (a) a light chain and a heavy chain, wherein the light chain is a crossover light chain comprising the variable domain VH and the constant domain CL, and the heavy chain is a crossover heavy chain comprising the variable domain VL and the constant domain CH1; and also (b) an amino acid modification as shown in Table 4 or Table 5. This embodiment is shown in Figure 3D with the amino acid modifications as shown in Table 4.

[0325] In certain embodiments, the antibody shown in Figure 2 further comprises a modification shown in Table 2 or Table 3. For example, the antibody shown in Figure 2 may comprise a modification shown in Table 4 in combination with a modification shown in Table 2. Alternatively, the antibody shown in Figure 2 may comprise a modification shown in Table 5 in combination with a modification shown in Table 3.

[0326] In one embodiment, the bispecific antibodies of the invention are trivalent bispecific antibodies comprising one Fab fragment of an anti-CD3 antibody, two Fab fragments of an anti-BCMA antibody, and one Fc portion according to the format BCMA Fab-Fc-CD3 Fab-BCMA Fab. The anti-CD3 Fab fragment comprises a light chain and a heavy chain, where the light chain is a crossover light chain comprising a variable domain VH and a constant domain CL, and the heavy chain is a crossover heavy chain comprising a variable domain VL and a constant domain CH1. Each anti-BCMA Fab fragment comprises a light chain and a heavy chain, where the heavy chain comprises a CH1 domain comprising the amino acid modifications K147E and K213E (EU numbering), and the light chain comprises a corresponding CL domain comprising the amino acid modifications E123R and Q124K (Kabat numbering) (i.e., the modifications shown in Table 4). The Fc portion comprises a first Fc chain and a second Fc chain, wherein the first Fc chain comprises a first constant domain CH2 and a first constant domain CH3, and the second Fc chain comprises a second constant domain CH2 and a second constant domain CH3. The first Fc chain binds at the N-terminus of the Fc to the C-terminus of a first anti-BCMA Fab, and the second Fc chain binds at the N-terminus of the Fc to the C-terminus of an anti-CD3 Fab. The first CH3 domain comprises the modifications T366S, L368A, and Y407V ("hole modification"), and the second CH3 domain comprises the modification T366W ("knob modification") (numbered according to EU numbering) (i.e., the modifications shown in Table 2). Additionally, both Fc chains further comprise the modifications L234A, L235A, and P329G, and optionally D356E and L358M (numbered according to EU numbering). Optionally, the first CH3 domain further comprises the amino acid modification S354C and the second CH3 domain further comprises the amino acid modification Y349C (numbering according to EU numbering) such that a disulfide bridge is formed between both CH3 domains.

[0327] In other embodiments, the bispecific antibodies of the invention are trivalent bispecific antibodies comprising one Fab fragment of an anti-CD3 antibody, two Fab fragments of an anti-BCMA antibody, and one Fc portion, according to the format BCMA Fab-Fc-CD3 Fab-BCMA Fab. The anti-CD3 Fab fragment comprises a light chain and a heavy chain, wherein the light chain is a crossover light chain comprising a variable domain VH and a constant domain CL, and the heavy chain is a crossover heavy chain comprising a variable domain VL and a constant domain CH1. Each anti-BCMA Fab fragment comprises a light chain and a heavy chain, wherein the heavy chain comprises a CH1 domain comprising the amino acid modifications A141W, L145E, K147T, and Q175E (EU numbering), and the light chain comprises a corresponding CL domain comprising the amino acid modifications F116A, Q124R, L135V, and T178R (Kabat numbering) (i.e., the modifications shown in Table 5). The Fc portion comprises a first Fc chain and a second Fc chain, wherein the first Fc chain comprises a first constant domain CH2 and a first constant domain CH3, and the second Fc chain comprises a second constant domain CH2 and a second constant domain CH3. The first CH3 domain comprises the modifications T350V, L351Y, F405A, and Y407V, and the second CH3 domain comprises the modifications T350V, T366L, K392L, and T394W (numbered according to EU numbering) (i.e., the modifications shown in Table 3). In addition, both Fc chains further comprise the modifications L234A, L235A, and P329G, and optionally D356E and L358M (numbered according to EU numbering).

[0328] In one embodiment, the anti-BCMA Fab fragment comprises a CDR1H, CDR2H, CDR3H, CDR1L, CDR2L and CDR3L region combination selected from the group consisting of: a) the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 23, the CDR2L region of SEQ ID NO: 24 and the CDR3L region of SEQ ID NO: 20; b) the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 25, the CDR2L region of SEQ ID NO: 26 and the CDR3L region of SEQ ID NO: 20; c) the CDR1H region of SEQ ID NO: 21, the CDR2H region of SEQ ID NO: 22, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 27, the CDR2L region of SEQ ID NO: 28 and the CDR3L region of SEQ ID NO: 20; d) the CDR1H region of SEQ ID NO: 29, the CDR2H region of SEQ ID NO: 30, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 31, the CDR2L region of SEQ ID NO: 32 and the CDR3L region of SEQ ID NO: 33; e) the CDR1H region of SEQ ID NO: 34, the CDR2H region of SEQ ID NO: 35, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 31, the CDR2L region of SEQ ID NO: 32 and the CDR3L region of SEQ ID NO: 33; f) the CDR1H region of SEQ ID NO: 36, the CDR2H region of SEQ ID NO: 37, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 31, the CDR2L region of SEQ ID NO: 32 and the CDR3L region of SEQ ID NO: 33; g) the CDR1H region of SEQ ID NO: 15, the CDR2H region of SEQ ID NO: 16, the CDR3H region of SEQ ID NO: 17, the CDR1L region of SEQ ID NO: 18, the CDR2L region of SEQ ID NO: 19, and the CDR3L region of SEQ ID NO: 20 and The anti-CD3 Fab fragment comprises the CDR1H region of SEQ ID NO:1, the CDR2H region of SEQ ID NO:2, the CDR3H region of SEQ ID NO:3, the CDR1L region of SEQ ID NO:4, the CDR2L region of SEQ ID NO:5 and the CDR3L region of SEQ ID NO:6.

[0329] In one embodiment, the anti-BCMA Fab fragment has a VH and VL selected from the group: a) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 12; b) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13; c) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14; d) a VH region of SEQ ID NO: 38 and a VL region of SEQ ID NO: 12; e) a VH region of SEQ ID NO: 39 and a VL region of SEQ ID NO: 12; f) a VH region of SEQ ID NO: 40 and a VL region of SEQ ID NO: 12, or g) the VH region of SEQ ID NO: 9 and the VL region of SEQ ID NO: 11 and The anti-CD3 Fab fragment comprises the VH region of SEQ ID NO:7 and the VL region of SEQ ID NO:8.

[0330] In further embodiments, the multispecific (e.g., bispecific) antibodies of the invention comprise the following SEQ ID NOs (as set forth in Tables 6A, 7B and 7C below): 83A10-TCBcv: 45, 46, 47 (x2), 48 (Figure 2A) 21-TCBcv: 49, 50, 51 (x2), 48 (Figure 2A) 22-TCBcv: 52, 53, 54 (x2), 48 (Figure 2A) 42-TCBcv: 55, 56, 57 (x2), 48 (Figure 2A) Mab101: 58, 59, 60 (x2), 48 (except that instead of the described "RK / EE" substitutions, there are alternative amino acid substitutions in CL-CH1 to reduce light chain mispairing / by-products: A141W, L145E, K147T, Q175E ("WETE") and F116A, Q124R, L135V, T178R ("ARVR"), Figure 2A) Mab102: 61, 62, 63 (x2), 48 (except that instead of the described "RK / EE" substitutions, there are alternative amino acid substitutions in CL-CH1 to reduce light chain mispairing / by-products: A141W, L145E, K147T, Q175E ("WETE") and F116A, Q124R, L135V, T178R ("ARVR"), Figure 2A) Mab103: 64, 65, 66 (x2), 48 (except that instead of the described "RK / EE" substitutions, there are alternative amino acid substitutions in CL-CH1 to reduce light chain mispairing / by-products: A141W, L145E, K147T, Q175E ("WETE") and F116A, Q124R, L135V, T178R ("ARVR") Figure 2A).

[0331] As used herein, the term "83A10-TCBcv" refers to a bispecific antibody that specifically binds to BCMA and CD3, identified by the heavy and light chain combinations of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 (2x) and SEQ ID NO:48, shown in Figure 2A, and described in EP14179705.

[0332] As used herein, the terms "21-TCBcv, 22-TCBcv, 42-TCBcv" refer to the respective bispecific antibodies Mab21, identified by the heavy and light chain combinations of SEQ ID NOs: 48, 49, 50, and 51 (2x), Mab22, identified by the heavy and light chain combinations of SEQ ID NOs: 48, 52, 53, and 54 (2x), and Mab42, identified by the heavy and light chain combinations of SEQ ID NOs: 48, 55, 56, and 57 (2x), as shown in Figure 2A and described in WO2017 / 021450.

[0333] As used herein, the term "Mab101" refers to a bispecific antibody that specifically binds BCMA and CD3, identified by the heavy and light chain combinations of SEQ ID NO:48, SEQ ID NO:58, SEQ ID NO:60 (2x), and SEQ ID NO:59, and shown in Figure 2A (but with additional amino acid substitutions in CL-CH1 to reduce light chain mispairing / by-products: A141W, L145E, K147T, Q175E ("WETE") and F116A, Q124R, L135V, T178R ("ARVR"), rather than the "RK / EE" substitutions described). As used herein, the term "Mab102" refers to a bispecific antibody that specifically binds BCMA and CD3, identified by the heavy and light chain combinations of SEQ ID NO:48, SEQ ID NO:61, SEQ ID NO:63 (2x), and SEQ ID NO:62, and shown in Figure 2A (but with additional amino acid substitutions in CL-CH1 to reduce light chain mispairing / by-products: A141W, L145E, K147T, Q175E ("WETE") and F116A, Q124R, L135V, T178R ("ARVR"), rather than the "RK / EE" substitutions described). As used herein, the term "Mab103" refers to a bispecific antibody that specifically binds BCMA and CD3, identified by the heavy and light chain combinations of SEQ ID NO:48, SEQ ID NO:64, SEQ ID NO:66 (2x), and SEQ ID NO:65, and shown in Figure 2A (but with additional amino acid substitutions in CL-CH1 to reduce light chain mispairing / by-products: A141W, L145E, K147T, Q175E ("WETE") and F116A, Q124R, L135V, T178R ("ARVR"), rather than the "RK / EE" substitutions described).

[0334] In a preferred embodiment, the bispecific antibody of the invention is 42-TCBcv. As used herein, the term "CC-93269" refers to the bispecific antibody 42-TCBcv.

[0335] Antibodies with improved stability Provided herein are multispecific (e.g., bispecific) antibodies against BCMA and a T cell antigen (e.g., CD3) having one or more amino acid modifications that provide improved stability (e.g., improved physiochemical properties) compared to the antibody without the modifications. Also provided are nucleic acid molecules, vectors, host cells, and pharmaceutical compositions comprising same, and uses thereof, including methods of production and treatment.

[0336] In some embodiments of the present invention, a multispecific BCMA-binding antibody and a T cell antigen are provided, wherein the multispecific antibody comprises (i) an anti-BCMA antibody, or antigen-binding fragment thereof; (ii) an anti-T cell antigen antibody, or antigen-binding fragment thereof; and (iii) an Fc, wherein the anti-BCMA antibody, or antigen-binding fragment thereof: a) a VH domain comprising a CDR1H region of SEQ ID NO: 21, a CDR2H region of SEQ ID NO: 22, a CDR3H region of SEQ ID NO: 17, and a VL domain comprising a CDR1L region of SEQ ID NO: 27, a CDR2L region of SEQ ID NO: 28, and a CDR3L region of SEQ ID NO: 20; b) a VH domain comprising a CDR1H region of SEQ ID NO: 21, a CDR2H region of SEQ ID NO: 22, a CDR3H region of SEQ ID NO: 17, and a VL domain comprising a CDR1L region of SEQ ID NO: 25, a CDR2L region of SEQ ID NO: 26, and a CDR3L region of SEQ ID NO: 20; or c) a VH domain comprising the CDR1H region of SEQ ID NO: 15, the CDR2H region of SEQ ID NO: 16, and the CDR3H region of SEQ ID NO: 17, and a VL domain comprising the CDR1L region of SEQ ID NO: 18, the CDR2L region of SEQ ID NO: 19, and the CDR3L region of SEQ ID NO: 20 wherein the multispecific antibody comprises a CH1 domain and a CL domain, wherein the CH1 domain comprises amino acid modifications at positions A141, L145, K147 and Q175 (numbering according to EU numbering) and the CL domain comprises amino acid modifications at positions F116, Q124, L135 and T178 (numbering according to Kabat); and wherein the Fc comprises a first Fc chain comprising first constant domains CH2 and CH3 and a second Fc chain comprising second constant domains CH2 and CH3, wherein the first CH3 domain comprises modifications at amino acid positions T350, L351, F405 and Y407 (numbering according to EU numbering) and the second CH3 domain comprises modifications at amino acid positions T350, T366, K392 and T394 (numbering according to EU numbering), optionally wherein the T cell antigen is CD3.

[0337] In one embodiment, the CH1 domain comprises two or more (e.g., all) of the modifications A141W, L145E, K147T, and Q175E, or conservative substitutions thereof (numbering according to EU numbering), and the CL domain comprises two or more (e.g., all) of the modifications F116A, Q124R, L135V, and T178R, or conservative substitutions thereof (numbering according to Kabat).

[0338] The CH1 and CL domains comprising the amino acid modifications may be derived from an anti-BCMA antibody or antigen-binding fragment thereof or an anti-T cell antigen antibody or antigen-binding fragment thereof. In a preferred embodiment, the anti-BCMA antibody or antigen-binding fragment thereof comprises a CH1 and CL domain comprising the amino acid modifications.

[0339] In some embodiments, the first CH3 domain comprises one or more of the modifications T350V, L351Y, F405A, and Y407V, or conservative substitutions thereof (numbered according to EU numbering); and the second CH3 domain comprises one or more of the modifications T350V, T366L, K392L, and T394W, or conservative substitutions thereof (numbered according to EU numbering). In a preferred embodiment, the first CH3 domain comprises the modifications T350V, L351Y, F405A, and Y407V, or conservative substitutions thereof (numbered according to EU numbering); and the second CH3 domain comprises the modifications T350V, T366L, K392L, and T394W, or conservative substitutions thereof (numbered according to EU numbering).

[0340] In one embodiment, a multispecific (e.g., bispecific) antibody of the invention comprises an anti-CD3 antibody or antigen-binding fragment thereof, wherein the VH domain of the anti-CD3 antibody comprises the CDRs of SEQ ID NOs: 1, 2, and 3 as CDRH1, CDRH2, and CDRH3, respectively, and the VL domain of the anti-CD3 antibody comprises the CDRs of SEQ ID NOs: 4, 5, and 6 as light chain CDRL1, CDRL2, and CDRL3, respectively.

[0341] In a preferred embodiment, the multispecific (eg, bispecific) antibody of the invention comprises an anti-CD3 antibody or antigen-binding fragment thereof comprising a VH of SEQ ID NO:7 and a VL of SEQ ID NO:8.

[0342] In certain embodiments, the multispecific (e.g., bispecific) antibodies of the invention comprise an IgG1 Fc, optionally wherein the Fc is a) the modifications L234A, L235A and P329G (numbering according to the EU numbering system); and / or b) Modifications D356E and L358M (numbered according to the EU numbering system) Includes.

[0343] In one embodiment, the multispecific antibody of the invention is a bispecific trivalent antibody comprising two Fab fragments of an anti-BCMA antibody and one Fab fragment of an anti-CD3 antibody, optionally wherein the antibodies are in the format BCMA Fab-Fc-CD3 Fab-BCMA Fab.

[0344] In one embodiment of the invention, a multispecific antibody that binds to BCMA and CD3 is provided, wherein the multispecific antibody is in the format of a trivalent bispecific antibody, wherein the multispecific antibody comprises one Fab fragment of an anti-CD3 antibody, two Fab fragments of an anti-BCMA antibody and an Fc according to the format BCMA Fab-Fc-CD3 Fab-BCMA Fab, wherein: a) an anti-CD3 Fab fragment comprising a light chain and a heavy chain, wherein the light chain is a crossover light chain comprising a variable domain VH and a constant domain CL, and the heavy chain is a crossover heavy chain comprising a variable domain VL and a constant domain CH1; b) each anti-BCMA Fab fragment comprises a light chain and a heavy chain, wherein the light chain comprises a variable domain VL and a constant domain CL, and the heavy chain comprises a variable domain VH and a constant domain CH1, wherein: (i) the variable domain VH comprises heavy chain CDRs 1 to 3 of SEQ ID NOs: 21, 22, and 17, respectively, and the variable domain VL comprises light chain CDRs 1 to 3 of SEQ ID NOs: 27, 28, and 20, respectively; (ii) the variable domain VH comprises heavy chain CDRs 1 to 3 of SEQ ID NOs: 21, 22, and 17, respectively, and the variable domain VL comprises light chain CDRs 1 to 3 of SEQ ID NOs: 25, 26, and 20, respectively; or (iii) the variable domain VH comprises heavy chain CDRs 1 to 3 of SEQ ID NOs: 15, 16, and 17, respectively, and the variable domain VL comprises light chain CDRs 1 to 3 of SEQ ID NOs: 18, 19, and 20, respectively; c) the CH1 domain of each anti-BCMA Fab fragment comprises the modifications A141W, L145E, K147T and Q175E or conservative substitutions thereof (numbering according to EU numbering), and the corresponding CL domain of each anti-BCMA Fab fragment comprises the modifications F116A, Q124R, L135V, T178R or conservative substitutions thereof (numbering according to Kabat); d) the Fc comprises a first Fc chain and a second Fc chain, the first Fc chain comprising first constant domains CH2 and CH3 and the second Fc chain comprising second constant domains CH2 and CH3, wherein the first Fc chain is bound at the N-terminus of the Fc to the C-terminus of one anti-BCMA Fab fragment and the second Fc chain is bound at the N-terminus of the Fc to the C-terminus of an anti-CD3 Fab fragment, wherein: (i) the first CH3 domain comprises the modifications T350V, L351Y, F405A, and Y407V, and the second CH3 domain comprises the modifications T350V, T366L, K392L, and T394W (numbering according to EU numbering); and (ii) both Fc chains contain the modifications L234A, L235A and P329G and optionally the modifications D356E and L358M (numbering according to EU numbering);

[0345] In a further aspect of the invention, a trivalent bispecific antibody that binds BCMA and CD3 is provided, wherein the trivalent bispecific antibody has the following SEQ ID NO: i. Mab101:58, 59, 48 and 2x60 ii. Mab102:61, 62, 48 and 2x63 iii. Mab103:64, 65, 48 and 2x66 Includes.

[0346] Each molecule, Mab101, Mab102, and Mab103, is in the 2+1 bispecific format shown in Figure 2A, except that rather than the "RK / EE" substitutions described, there are alternative amino acid substitutions in CL-CH1, A141W, L145E, K147T, Q175E ("WETE") and F116A, Q124R, L135V, T178R ("ARVR"), to reduce light chain mispairing / by-products.

[0347] In a further aspect, a pharmaceutical composition is provided comprising a multispecific (e.g., bispecific) antibody of the invention and a pharmaceutically acceptable excipient. In a related aspect, a pharmaceutical composition is provided comprising a trivalent bispecific antibody of the invention and a pharmaceutically acceptable excipient.

[0348] In a further embodiment, the multispecific (e.g. bispecific) antibody of the invention, the trivalent bispecific antibody of the invention or the pharmaceutical composition of the invention is for use as a medicament.

[0349] In a related embodiment, a method of treating a subject is provided, comprising administering to a subject (e.g., a human) in need of such treatment a multispecific (e.g., bispecific) antibody of the invention, a trivalent bispecific antibody of the invention, or a pharmaceutical composition of the invention.

[0350] In a preferred embodiment, the multispecific (e.g., bispecific) antibody of the invention, the trivalent bispecific antibody of the invention, or the pharmaceutical composition of the invention is for use as a medicament for treating a plasma cell disorder. In certain embodiments, the plasma cell disorder is cancer. In a preferred embodiment, the cancer is multiple myeloma or plasma cell leukemia.

[0351] Pharmaceutical Composition The multispecific (e.g., bispecific) antibodies of the invention can be administered to a subject as a pharmaceutical composition. Accordingly, the invention also provides pharmaceutical compositions comprising a multispecific (e.g., bispecific) antibody of the invention and a pharmaceutically acceptable excipient.

[0352] As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopeia equivalents for use in animals, and more specifically, in humans.

[0353] The pharmaceutical compositions disclosed herein are for use in, but not limited to, diagnosing, detecting, or monitoring a disorder, preventing, treating, managing, or ameliorating a disorder or one or more of its symptoms, and / or studying the disorder. The pharmaceutical compositions disclosed herein may be suitable for veterinary or human pharmaceutical use.

[0354] Examples of suitable additives include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and the like, and any combination thereof. In many cases, it is preferable to include an isotonic agent, such as a sugar, a polyalcohol, or sodium chloride, in the composition. Particularly relevant examples of suitable additives include (1) Dulbecco's phosphate-buffered saline, pH about 7.4, with or without about 1 mg / mL to 25 mg / mL human serum albumin, (2) 0.9% saline (0.9% w / v sodium chloride (NaCl)), and (3) 5% (w / v) dextrose, and may also contain antioxidants such as tryptamine and Tween 20. (登録商標) It may also contain stabilizers such as

[0355] Those skilled in the art will appreciate that the appropriate choice of one or more excipients for use with the multispecific (eg, bispecific) antibodies of the invention will depend on the desired properties of the pharmaceutical composition.

[0356] The pharmaceutical compositions or multispecific (e.g., bispecific) antibodies of the present invention can be administered to a subject by any suitable systemic or local administration route. For example, administration can be oral, buccal, sublingual, ocular, intranasal, intratracheal, pulmonary, topical, transdermal, genitourinary, rectal, subcutaneous, intravenous, intraarterial, intraperitoneal, intramuscular, intracranial, intrathecal, epidural, intraventricular, or intratumor. In some embodiments, the pharmaceutical compositions or multispecific (e.g., bispecific) antibodies are administered intravenously or subcutaneously. In a preferred embodiment, the pharmaceutical compositions or multispecific (e.g., bispecific) antibodies are administered intravenously.

[0357] The pharmaceutical compositions of the present invention can be formulated for administration by any suitable means, such as epidermal or transdermal patches, ointments, lotions, creams, or gels; nebulizers, vaporizers, or inhalers; injections or infusions; or in the form of capsules, tablets, solutions or suspensions in water or non-aqueous media, drops, suppositories, enemas, sprays, or powders. The most suitable route of administration in any given case will depend on the physical and mental condition of the patient, the nature and severity of the disease, and the desired properties of the formulation.

[0358] Monotherapy and combination treatments In certain embodiments, the treatment comprises administering to the subject a multispecific (e.g., bispecific) antibody of the invention as monotherapy.

[0359] In certain embodiments, the treatment comprises administering to a subject a multispecific (e.g., bispecific) antibody of the invention as a combination therapy, wherein the combination therapy comprises the administration of a multispecific (e.g., bispecific) antibody of the invention and one or more additional therapeutic agents. The term "combination therapy" is intended to include treatments that involve the administration of multiple selected therapeutic agents to a single patient, where these agents are administered by the same or different routes of administration or at the same or different times.

[0360] In certain embodiments, the one or more additional therapeutic agents are selected from the group consisting of an antifolate (e.g., methotrexate), a purine synthesis inhibitor (e.g., azathioprine, mycophenolic acid, and / or mycophenolate mofetil), a C5a inhibitor (e.g., avacopan), an anti-CD19 antibody, an anti-CD20 antibody (e.g., rituximab), a steroid, a Bruton's tyrosine kinase (BTK) inhibitor, and / or a BAFF / APRIL antagonist (e.g., an anti-BAFF antibody).

[0361] The inventors have determined that there is minimal need for steroids in remission induction and / or maintenance. Thus, in certain embodiments, the one or more additional therapeutic agents is not a steroid, e.g., a glucocorticoid.

[0362] In certain embodiments, the one or more additional therapeutic agents are selected from the group consisting of thalidomide and its immunotherapeutic derivatives, anti-CD38 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, gamma secretase inhibitors (GSIs), anti-BCMA antibody drug conjugates, and anti-BCMA CAR T-cell therapy.

[0363] The term "anti-CD38 antibody" as used herein refers to an antibody that specifically binds to human CD38. In one embodiment of the present invention, the anti-CD38 antibody is daratumumab (US20150246123). In one embodiment of the present invention, the anti-CD38 antibody is isatuximab (SAR650984, US8877899). In one embodiment of the present invention, the anti-CD38 antibody is MOR202 (WO2012041800). In one embodiment of the present invention, the anti-CD38 antibody is Ab79 (US8362211). In one embodiment of the present invention, the anti-CD38 antibody is Ab19 (US8362211). The administration of such anti-CD38 antibodies is carried out according to the state of the art and is described in the respective prescribing information. For example, the dosage of daratumumab is typically 16 mg / kg (www.ema.europa.eu).

[0364] The term "thalidomide compound" or "thalidomide and immunotherapeutic derivatives," as used herein, refers to 2-(2,6-dioxopiperidin-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione and its immunotherapeutic derivatives. In certain embodiments of the present invention, the thalidomide compound is selected from the group including, but not limited to, thalidomide (CAS Registry Number 50-35-1), lenalidomide (CAS Registry Number 191732-72-6), pomalidomide (CAS Registry Number 19171-19-8), CC122 (CAS Registry Number 1398053-45-6), and CC-220 (CAS Registry Number 1323403-33-3), and their respective salts (preferably 1:1 HCl salts). The chemical formula of CC-122 is 2,6-piperidinedione, 3-(5-amino-2-methyl-4-oxo-3(4H-quinazolinyl), hydrochloride (1:1), and the chemical formula of CC-220 is 2,6-piperidinedione, 3-[1,3-dihydro-4-[[4-(4-morpholinylmethyl)phenyl]methoxy]-1-oxo-2H-isoindol-2-yl]-, (3S)-, hydrochloride (1:1). Methods for producing CC-220 are disclosed, for example, in US20110196150, which is incorporated herein by reference in its entirety.

[0365] Administration of thalidomide compounds is performed according to the state of the art and is described in the respective prescribing information. For example, the REVLIMID® (lenalidomide) dosage is typically 25 mg orally once daily on days 1-21 of repeated 28-day cycles (www.revlimid.com), and the Pomalyst® (pomalidomide) dosage for the treatment of multiple myeloma is typically 4 mg orally daily on days 1-21 of repeated 28-day cycles (www.celgene.com). In one embodiment, 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione is administered in an amount of about 5 to about 50 mg per day.

[0366] In some embodiments, CC-122 and CC-220 are administered in an amount of about 5 to about 25 mg per day. In other embodiments, CC-122 and CC-220 are administered in an amount of about 5 mg, 10 mg, 15 mg, 25 mg, 30 mg, or 50 mg per day. In other embodiments, 10 mg or 25 mg of CC-122 and CC-220 are administered per day. In some embodiments, CC-122 and CC-220 are administered twice daily.

[0367] As used herein, the term "anti-PD-1 antibody" refers to an antibody that specifically binds to human PD-1. Such antibodies are described, for example, in WO2015026634 (MK-3475, pembrolizumab), US7521051, US8008449, and US8354509. Pembrolizumab (Keytruda®, MK-3475) is also described in WO2009 / 114335, Poole, RM Drugs (2014) 74: 1973; Seiwert, T., et al., J. Clin. Oncol. 32,5s (suppl;abstr 6011). In one embodiment of the invention, the PD-1 antibody is MK-3475 (WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013)), which comprises the heavy and light chain amino acid sequences set forth in Figure 6 of WO2015026634. The amino acid sequence of pembrolizumab is set forth in WO2008156712 (light chain CDR SEQ ID NOs: 15, 16, and 17 and heavy chain CDR SEQ ID NOs: 18, 19, and 20). In one embodiment of the invention, the PD-1 antibody is nivolumab (BMS-936558, MDX 1106; amino acid sequences set forth in WHO Drug Information, Vol. 27, No. 1, pages 68-69 (2013), WO2006 / 121168, WO2015026634). In one embodiment of the invention, the PD-1 antibody is pidilizumab (CT-011, also known as hBAT or hBAT-1; see amino acid sequence WO2003 / 099196; WO2009 / 101611, Fried I. et al.; Neuro Oncol (2014) 16 (suppl 5): v111-v112.). In one embodiment of the invention, the PD-1 antibody is MEDI-0680 (AMP-514, WO2010 / 027423, WO2010 / 027827, WO2010 / 027828, Hamid O. et al.; J Clin Oncol 33, 2015 (suppl; abstr TPS3087)).In one embodiment of the invention, the PD-1 antibody is PDR001 (Naing A. et al.; J Clin Oncol 34, 2016 (suppl; abstr 3060). In one embodiment of the invention, the PD-1 antibody is REGN2810 (Papadopoulos KP et al.; J Clin Oncol 34, 2016 (suppl; abstr 3024). In one embodiment of the invention, the PD-1 antibody is lambrolizumab (WO2008 / 156712). In one embodiment of the invention, the PD-1 antibody is h409All, h409A16, or h409A17, as described in WO2008 / 156712. Administration of such anti-PD-1 antibodies is performed according to the state of the art and is described in the respective prescribing information. For example, Keytruda® is typically administered at a concentration of 2 mg / kg body weight every three weeks (http: / / ec.europa.eu / health / documents).

[0368] As used herein, the term "anti-PD-L1 antibody" refers to an antibody that specifically binds to human PD-L1. Such antibodies are described, for example, in WO2015026634, WO2013 / 019906, WO2010 / 077634, and US8383796. In one embodiment of the invention, the PD-L1 antibody is MPDL3280A (atezolizumab, YW243.55.S70, WO2010 / 077634, McDermott DF. Et al., JCO March 10, 2016 vol. 34 no. 8 833-842). In one embodiment of the invention, the PD-L1 antibody is MDX-1105 (BMS-936559, WO2007 / 005874, Patrick A. Ott PA et al., DOI: 10.1158 / 1078-0432, Clinical Cancer Research-13-0143). In one embodiment of the invention, the PD-L1 antibody is MEDI4736 (durvalumab, WO2016 / 040238, Gilbert J. et al., Journal for ImmunoTherapy of Cancer 20153(Suppl 2):P152). In one embodiment of the invention, the PD-L1 antibody is MSB0010718C (avelumab, Disis ML. et al., Journal of Clinical Oncology, Vol 33, No 15_suppl (May 20 Supplement), 2015: 5509). In one embodiment of the invention, the PD-L1 antibody is an anti-PD-L1 antibody comprising the VH sequence of SEQ ID NO: 16 and the VL sequence of SEQ ID NO: 17 described in WO2016007235. Administration of such anti-PD-L1 antibodies is performed in accordance with the state of the art and is described in the respective prescribing information. For example, atezolizumab is typically administered at a concentration of 1200 mg as a 60-minute intravenous infusion every three weeks (www.accessdata.fda.gov).

[0369] As used herein, the term "gamma secretase" refers to any protein or protein complex that binds to a substrate having a gamma secretase cleavage sequence at a gamma secretase cleavage site, exhibits gamma secretase activity to catalyze cleavage of the gamma secretase cleavage sequence, and produces a substrate cleavage product. In some embodiments, the gamma secretase is a protein complex that includes one or more of the following subunits: presenilin, nicastrin, gamma-secretase subunit APH-1, and gamma-secretase subunit PEN-2.

[0370] As used herein, the term "gamma secretase inhibitor" or "GSI" refers to any molecule that can inhibit or reduce the expression and / or function of gamma secretase. In some embodiments, the GSI reduces the expression and / or function of a subunit of gamma secretase (e.g., presenilin, nicastrin, APH-1, or PEN-2). Any form of "gamma secretase inhibitor," such as a salt, cocrystal, crystalline form, or prodrug, is included within the scope of this term. In some embodiments, the GSI is selected from an antibody or antigen-binding fragment, a small molecule, a protein or peptide, and a nucleic acid.

[0371] Adverse events In some embodiments, the patient develops or is at risk of developing adverse events associated with the administration of multispecific (e.g., bispecific) antibodies.The adverse events can be cytokine-driven toxicity (e.g., cytokine release syndrome (CRS)), infusion-related reaction (IRR), infection, macrophage activation syndrome (MAS), neurotoxicity, severe tumor lysis syndrome (TLS), neutropenia, thrombocytopenia, elevated liver enzymes, and / or central nervous system (CNS) toxicity.In a specific embodiment, the adverse event is CRS.

[0372] If the patient develops or is at risk of developing an adverse event associated with administration of a multispecific (e.g., bispecific) antibody, treatment can further include administration of an agent that can treat, prevent, delay, reduce, or attenuate the development of the adverse event or the risk of developing the adverse event. The agent can be administered to the patient prior to initiating treatment with the multispecific (e.g., bispecific) antibody (e.g., as a prophylaxis to prevent or reduce the risk of the adverse event) or during treatment with the multispecific (e.g., bispecific) antibody (e.g., in response to the development of an adverse event).

[0373] In some embodiments, the agent comprises a steroid, such as a corticosteroid. As used herein, "corticosteroid" refers to any naturally occurring or synthetic steroid hormone derived from cholesterol and characterized by a hydrogenated cyclopentanoperhydrophenanthrene ring system. Naturally occurring corticosteroids are generally produced by the adrenal cortex. Synthetic corticosteroids may be halogenated. Functional groups required for activity include Δ4, C3 ketone, and C20 ketone double bonds. Corticosteroids may have glucocorticoid and / or mineralocorticoid activity. Illustrative corticosteroids include prednisolone, methylprednisolone, prednisone, triamcinolone, betamethasone, budesonide, and dexamethasone.

[0374] In some embodiments, the agent comprises an antagonist of a cytokine receptor or cytokine selected from GM-CSF, IL-10, IL-10R, IL-6, IL-6 receptor (IL-6R), IFNy, IFNGR, IL-2, IL-2R / CD25, MCP-1, CCR2, CCR4, MIPIβ, CCR5, TNFalpha, TNFR1, IL-1, and IL-1Ralpha / IL-1beta, wherein the antagonist is selected from an antibody or antigen-binding fragment, a small molecule, a protein or peptide, and a nucleic acid. The antagonist may be an anti-IL-6 antibody and / or an anti-IL6R antibody. For example, the antagonist can be selected from tocilizumab, siltuximab, clazakizumab, sarilumab, olokizumab, elsilimomab, ALD518 / BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX-109, lenzilumab, FE301, and FM101. In certain embodiments, the antagonist is tocilizumab and / or siltuximab.

[0375] In some embodiments, the agent comprises a molecule that reduces regulatory T cell (Treg) population.The agent that reduces (for example, depletes) the number of Treg cells is known in the art, and includes, for example, CD25 depletion, cyclophosphamide administration, anti-CTLA4 antibody and regulating glucocorticoid-induced TNLR family related gene (GITR) function.GITR is a member of the TNLR superfamily, which is upregulated by T cell activation and enhances the immune system.In some embodiments, the treatment comprises administering cyclophosphamide.

[0376] As noted above, the inventors have observed that there is no or minimal significant cytokine release following treatment with the bispecific antibodies of the invention. Thus, in certain embodiments where the adverse event is cytokine-driven toxicity (e.g., CRS), the treatment does not further include administration of an agent capable of treating, preventing, delaying, reducing, or attenuating the onset of the adverse event or the risk of onset of the adverse event, such as an antagonist of a cytokine receptor or cytokine.

[0377] Disclaimer The above-described embodiments should be understood as illustrative examples. Additional embodiments are contemplated. It should also be understood that any feature described in connection with one embodiment can be used alone or in combination with other features described, and can be used in combination with one or more features of any other embodiment or any combination of any other embodiment. Furthermore, equivalents and modifications not described above may be used without departing from the scope of the invention, as defined by the appended claims.

[0378] Other examples and variations of the antibodies and methods described herein in the context of the present invention will be apparent to those of skill in the art and are within the scope of the invention as set forth in the appended claims.

[0379] All documents cited herein are hereby incorporated by reference in their entirety, including all data, tables, figures, and text present in each such document.

[0380] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] Note: SEQ ID NO: 20 and SEQ ID NO: 33 are identical

[0381] [Table 14]

[0382] [Table 15]

[0383] [Table 16]

[0384] [Table 17] [Example]

[0385] Example 1: BCMA surface expression on plasmablasts and plasma cells and soluble BCMA levels in samples from normal healthy volunteers (NHV) and AAV patients Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected from four normal healthy volunteers (NHVs) using a Ficoll gradient. BMCA expression was assessed on plasmablasts (PBs) by flow cytometry. Plasmablasts were identified as CD19(+) CD20(-) CD27(+) CD38(+). Anti-BCMA antibody-coated fluorescent beads were used to generate a standard curve comparing mean fluorescence intensity versus BCMA surface receptor density. BCMA-expressing cancer cell lines (JEKO, RPMI-8226, and H929) were profiled for comparison (Figure 4A). BCMA surface expression was significantly lower in plasmablasts from NHVs than in the multiple myeloma cell lines RPMI-8226 and H929 for all NHVs tested. Soluble BCMA levels were assessed by ELISA in serum or plasma samples from NHV ('normal'), multiple myeloma ('MM'), or ANCA-associated vasculitis ('AAV') patients (Figure 4B). Soluble BCMA levels were much lower in NHV samples than in MM samples, indicating lower levels of BCMA cell surface expression on plasmablasts in NHV compared to MM patients. Soluble BCMA levels in both serum and plasma (PR3+) samples from AAV were comparable to NHV samples and much lower than MM samples. In light of this correlation, it is inferred that BCMA surface expression on plasmablasts and plasma cells in AAV is comparable to that in NHV.

[0386] Example 2: Production of T cell bispecific antibodies Anti-BCMA anti-CD3 bispecific antibodies were produced having the format first BCMA Fab-Fc-CD3 Fab-second BCMA Fab (referred to herein as a "2+1" format). Methods for producing anti-BCMA anti-CD3 bispecific antibodies can be found in WO2017 / 021450, which is incorporated herein by reference. HD1 is: - the CH3 domain of the Fc comprises one chain with the amino acid substitution T366W and one chain with the amino acid substitutions T366S, L368A and Y407V (as shown in Table 2); and - Both BCMA Fabs contain amino acid substitutions K147E and K213E in the CH1 domain and amino acid substitutions E123R and Q124K in the CL domain (as shown in Table 4) Indicates that; HD2 is: - the CH3 domain of CH2-Fc comprises one chain with the amino acid substitutions T350V, L351Y, F405A, Y407V and one chain with the amino acid substitutions T350V, T366L, K392L, T394W (as shown in Table 3); - Both BCMA Fabs contain amino acid substitutions A141W, L145E, K147T, Q175E in the CH1 domain and F116A, Q124R, L135V, T178R in the CL domain (as shown in Table 5) indicates that (Heavy chain constant region amino acid positions numbered according to EU numbering; light chain constant region amino acid positions numbered according to Kabat). In addition to the above modifications described under HD1 and HD2, the Fc of the bispecific antibody in this example contains the amino acid substitutions P329G, L234A and L235A (positions numbered according to EU numbering). The bispecific antibody of this example also includes "CrossMAb" technology, in which the VH and VL of the CD3 Fab are swapped. A summary of the antibodies produced in this example is provided in Table 8. [Table 18]

[0387] Example 3: Dose-dependent killing of BCMA-expressing cells by BCMA T cell engagers occurs in conjunction with T cell activation JEKO cells JEKO cells were cultured with CD3+ T cells (rested overnight) at a 1:2 target:effector (T:E) ratio and various concentrations of anti-BCMA, anti-CD3 bispecific antibody (BCMA T cell engager). T cell-mediated killing of JEKO cells was assessed by Annexin V expression measured over 24 hours, with images captured every 2 hours. Annexin V+ cell counts were determined using Incucyte ZOOM software. Data and EC calculated at 20 hours. 50 The values ​​are shown in Figure 5A and Table 9. [Table 19] This data demonstrates that BCMA T cell engagers (BCMA TCEs) can kill BCMA-expressing cells at levels comparable to plasmablasts from normal healthy volunteers. T cell activation was analyzed by flow cytometry at 24 hours. Cells were washed and then stained for T cell lineage markers (CD3, CD4, and CD8) and activation markers (CD69, CD25, and CD154). Flow cytometry samples were acquired on a BD LSRFortessa and analyzed using Treestar FlowJo X software. Cell imaging was performed on an Incucyte Live Cell Analysis Imaging System. Data were plotted and EC 50 Values ​​were calculated using Graphpad Prism 7 software. FIG. 5B shows T cell activation as indicated by CD69 expression on CD8+ T cells. These data demonstrate that the 50% effective concentration (EC ) of BCMA T cell engagers for killing of JEKO cells. 50 ) shows an increase in the frequency of activated T cells.

[0388] MM cells The multiple myeloma cell line RPMI-8226 was NHV PBMCs were co-cultured with various target:effector (T:E) ratios and various concentrations of CC-93269. T cell-mediated killing of RPMI-8226 cells was assessed by Annexin V expression measured over a 96-hour period, with images recorded hourly. Annexin V cell counts were determined using Incucyte ZOOM software; the 25- and 72-hour time points are shown in Figure 6A. T cell activation was analyzed by flow cytometry at 25 and 72 hours. After 25 hours, cells were washed and then stained for CD69 expression as a marker of CD8 T cell lineage and T cell activation (Figure 6B). These data indicate that significant T cell activation occurs at sublethal doses of CC-93269 that are sublethal to MM cells.

[0389] Example 4: Dose-dependent killing of BCMA-expressing plasmablasts from healthy volunteers by BCMA T cell engagers occurs in the absence of significant T cell activation Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected from healthy volunteers using a Ficoll gradient resuspended in RPMI + 10% HI FBS. PBMCs were treated with various concentrations of BCMA TCE or control 2+1 anti-HEL anti-CD3 antibodies. After 24 hours of incubation, plasmablast killing, T cell activation, and cytokine production were assessed. Plasmablast killing was assessed by FACS, whereby plasmablasts were identified as CD19(+) CD20(-) CD27(+) cells and are shown as a percentage of total CD19(+) cells normalized to untreated controls. CD19(+) CD20(-) CD27(+) cells were confirmed to obtain additional known markers of plasmablasts: BCMA(+) SLAMF7(+) IgD(-) CD38(+) CD138(-). Figure 7A shows a representative dose-response curve, and Figure 7B shows a representative FACS plot gated on CD3(-) CD19(+) cells. The 50% effective concentration (EC ) of CC-93269 for plasmablast killing in healthy volunteers (n=12) was calculated. 50 ) is 0.005 nM. Plasmablast killing occurs at lower BCMA TCE concentrations than are required for killing of BCMA-expressing cancer cell lines (e.g., JEKO cells).

[0390] For T cell activation, cells were washed and then stained for T cell lineage (CD3, CD4, and CD8) and activation markers (CD69, CD25, and CD154). Data shown in Figure 7C are CD69 expression on CD8(+) T cells. Culture supernatants were analyzed for cytokine production (IFNγ, IL-6, IL-2, IL-10, granzyme B, and perforin) using the MSD Pro-inflammatory I assay (Figures 7D and 8). Data in Figure 8 are for CC-93269. Taken together, these data demonstrate the median effective concentration (EC) of BCMA TCE for killing plasmablasts from PBMCs of healthy volunteers. 50) shows little increase in the frequency of activated T cells (i.e., less than 20% above baseline) and little cytokine production (i.e., less than 20 pg / mL above baseline).

[0391] Table 10 summarizes the data for CC-93269 and shows the 90% effective concentration (EC 90 ) barely increases the frequency of activated T cells. CC-93269 demonstrates profound depletion (>90%) of plasmablasts in PBMCs from healthy volunteers in the absence of significant T cell activation in vitro. [Table 20]

[0392] Example 5: Effects on other B cell populations after culture with CC-93269 in PBMCs from healthy volunteers CC-93269-treated PBMC samples from Example 4 were stained for B cell lineage markers (CD20, CD27, and IgD). Memory B cells were identified as cells displaying the markers CD19(+) CD20(+) CD27(+) and then further confirmed with the markers IgD(-) CD38(-) BCMA(+ / -). Data are shown in Figures 9A-C, presented as a percentage of total CD19(+) CD20(+) cells. The data demonstrate that CC-93269 achieved a 90% effective concentration (EC 90 ) and show that it does not significantly deplete naive, unswitched memory B cell, or switched memory B cell populations in PBMCs from healthy volunteers.

[0393] Example 6: Dose-dependent BCMA TCE-mediated killing of plasmablasts and minimal T cell activation in engineered bone marrow Bone marrow (BM) mononuclear cells were isolated from the bone marrow of healthy volunteers using a Ficoll gradient and then treated with various concentrations of BCMA TCE or control 2+1 anti-HEL anti-CD3 antibodies. After 24 hours of incubation, plasmablast killing (FIG. 10A, Table 11) and T cell activation (FIG. 10B) were assessed by flow cytometry as in Example 4. PBMCs isolated from healthy volunteers were suspended in medium or bone marrow (BM) supernatant and then treated with BCMA TCE or control 2+1 anti-HEL anti-CD3 antibodies for 24 hours for comparison. [Table 21] These data indicate that BCMA TCE induces killing of plasmablasts from bone marrow at concentrations similar to those from PBMCs suspended in culture medium, with minimal T cell activation, which is important because long-lived plasmablasts and plasma cells are preferentially found in the bone marrow.

[0394] Example 7: AAV-mediated plasmablast dose-dependent BCMA TCE-mediated killing with minimal T cell activation Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll gradient from whole blood collected from AAV patients, including those with relapsed or refractory AAV to rituximab, methotrexate, and folic acid. They were then treated with various concentrations of BCMA TCE (CC-93269, Mab101, or Mab102) or control 2+1 anti-HEL anti-CD3 antibodies. After 24 hours of incubation, plasmablast killing (Figures 11A-11B), T cell activation (Figures 11C, 12A-12C), and cytokine production (Figure 11D) were assessed. Plasmablast killing was assessed by FACS, whereby plasmablasts were identified as CD19(+) CD20(-) CD27(+) cells and are shown as a percentage of total CD19(+) cells normalized to untreated controls (Figure 11A). CD19(+) CD20(-) CD27(+) cells were confirmed to obtain additional known markers of plasmablasts: BCMA(+) SLAMF7(+) IgD(-) CD38(+) CD138(-). Figure 11B shows a representative FACS plot gated on CD3(-) CD19(+) cells. The 50% effective concentration (EC) of CC-93269 for plasmablast killing in AAV was calculated. 50 ) is 0.007 nM. AAV plasmablast killing occurs at a lower BCMA TCE concentration than that required to kill the JEKO cancer cell line, despite similar BCMA expression levels.

[0395] For T cell activation, cells were washed and then stained for T cell lineage (CD3, CD4, and CD8) and activation markers (CD69, CD25, and CD154). Data shown in Figure 11C are CD69 expression on CD8(+) T cells. Data shown in Figures 12A-C are CD69 or CD25 expression levels on CD4(+) or CD8(+) T cells. Culture supernatants were analyzed for cytokine production (IFNγ, IL-6, TNFα, IL-1β, granzyme A, granzyme B, and perforin) using the MSD Pro-inflammatory I assay. Data for IFNγ are shown in Figure 11D. Taken together, these data demonstrate that at AAV plasmablast-killing competent doses of BCMA TCE, there was no significant increase in activated T cell frequency (i.e., <20% above baseline) or cytokine production (i.e., <20 pg / mL above baseline).

[0396] Table 12 summarizes the data from Examples 4 and 7, highlighting the window of BCMA TCE concentrations for plasmablast (PB) killing in the absence of T cell activation, where adverse events associated with T cell activation or excessive cytokine production, such as cytokine release syndrome (CRS), are unlikely to occur. [Table 22]

[0397] Example 8: Dose-dependent CC-93269-mediated killing of plasmablasts from AAV patients treated with rituximab Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient from whole blood from an AAV patient, AAV-5, who had last received rituximab 5 months prior, and resuspended in RPMI + 10% HI FBS. PBMCs were treated with increasing concentrations of CC-93269 (Figures 13B-13C) or a control 2+1 anti-HEL anti-CD3 antibody (Figure 13A) for 24 hours and then evaluated by flow cytometry. Cells were gated for viability and singletness to arrive at a viable cell FACS plot (Figure 13). Plasmablasts are defined as CD19+CD27+CD20-. Plasmablasts were also confirmed to be CD38+, BCMA+, and CD138- (data not shown). From the CD19+CD20+ gate, naive B cells are defined as CD27-IgD+, unswitched memory B cells as CD27+IgD+, and switched memory B cells as CD27+ and IgD-. Representative dot plots from a normal healthy volunteer are shown. A lack of CD20(+) B cells but high CD20(-) CD27(+) plasmablast counts was observed in controls (Figure 13A), indicating that rituximab depleted B cells but not plasmablasts. CC-93269 induced selective depletion of plasmablasts at subnanomolar concentrations, with minimal B cell depletion (Figure 13B). CC-93269 rapidly depleted plasmablasts from PBMCs of AAV patients, even when the patients were treated with immunosuppressants such as rituximab.

[0398] Example 9: Target-free BCMA-TCE does not increase the frequency of activated T cells in AAV patient PBMCs PBMCs were isolated from an AAV patient, AAV-2, who had previously been treated with rituximab, mycophenolate, dexamethasone, and methylprednisolone. Figure 14A shows a FACS plot demonstrating the lack of CD19(+) CD20(-) CD27(+) plasmablasts and plasma cell targets at baseline in AAV-2 subjects compared to AAV-1 subjects. The plot is gated on CD3(-) CD19(+) cells. Figure 14B shows a FACS plot demonstrating the appropriate presence of CD4(+) and CD8(+) T cells in AAV-2 subjects. The plot is gated on CD3(+) cells. PBMCs from AAV-2 were treated with various concentrations of BCMA TCE, and after 24 hours of incubation, T cell activation was assessed by flow cytometry as in Example 7. Figure 14C shows the frequency of CD69(+) or CD25(+) on CD4(+) or CD8(+) T cells. These data indicate that BCMA-TCE does not induce T cell activation in the absence of target plasmablasts / plasma cells.

[0399] Example 10: T cell activation is low when BCMA-expressing cancer cells are killed at an effector:target ratio similar to AAV JEKO-1 cells (2500 cells / well) were cultured with PBMCs at a target:effector (T:E) ratio of 1:10 or 1:500 to mimic the BCMA+ cell:T cell ratio observed with multiple myeloma (MM) or AAV, respectively. After 24 hours of incubation with CC-93269 or control 2+1 anti-HEL anti-CD3 antibodies, cells were washed, and CD69 (Figure 15A) and CD25 (Figure 15B) expression on CD8(+) T cells was assessed. These data indicate that a greater T:E ratio (BCMA+ cells:T cells) results in greater T cell activation. Notably, the frequency of activated T cells is lower when the T:E ratio is comparable to the ratio of BCMA-expressing plasmablasts to T cells seen in healthy volunteers and AAV patients than when the T:E ratio mimics MM patients.

[0400] Example 11: BCMA-TCE abolishes the ability of IgG-producing plasmablasts and plasma cells to regenerate despite adequate plasmablast / plasma cell growth factor stimulation Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected from healthy volunteers using a Ficoll gradient and treated with various concentrations of BCMA TCE (CC-93269, Mab101, or Mab102) or control 2+1 anti-HEL anti-CD3 antibodies. Patient D214 achieved a 90% effective concentration (EC 90 ) and treated with BCMA TCE. After 24 hours of incubation, PBMCs were cultured with the growth factors IL-2 (20 U / ml), BAFF (200 ng / ml), and IL-21 (100 ng / ml) for 4–7 days to induce plasmablast / plasmacytic differentiation from BCMA-negative precursors. Some cultures were also stimulated with CpG (ODN2006 10 μg / ml) during this period. After culture, plasmablasts and plasma cells (CD19(+) CD20(-) CD27(+)) or CD20 + B cells were measured by flow cytometry (Figure 16A). BCMA-TCE specifically demonstrated a 90% effective concentration (EC 90 ) and inhibits plasmablast and plasma cell recovery after depletion despite adequate growth factors for regeneration. Culture supernatants were collected and total IgG secretion was measured by ELISA (Figure 16B). BCMA-TCE significantly inhibited the activity of ECs, particularly for plasmablast killing. 90 At concentrations above 1000kJ / mL, it suppresses IgG antibody production, regardless of stimulation with CpG, suggesting that 90% depletion of plasmablasts in vivo can suppress IgG autoantibody production.

[0401] Example 12: Dose-dependent BCMA TCE-mediated killing of plasmablasts from rheumatoid arthritis with minimal T cell activation PBMCs were isolated from rheumatoid arthritis (RA) patients and treated with various concentrations of CC-93269. After 24 hours of incubation, plasmablast killing (FIG. 17A), T cell activation (FIG. 17B), and cytokine secretion (FIG. 17C) were assessed by flow cytometry as in Example 4. The 50% effective concentration (EC) of CC-93269 for plasmablast killing in RA 50 ) is 0.001 nM. Therefore, RA plasmablast killing occurs at BCMA TCE concentrations lower than those required for T cell activation or cytokine secretion.

[0402] Example 13: Effects on other B cell populations in PBMCs from RA patients after culture with CC-93269 CC-93269-treated PBMC samples from Example 12 were stained for B cell lineage markers (CD20, CD27, and IgD). Data shown in Figures 18A-C are presented as percent of total CD19(+)CD20(+) cells. Data demonstrate that in vitro, CC-93269 significantly increased the EC 90 These results show that at concentrations of 100 mg / mL IgG4, ...

[0403] Example 14: Dose-dependent BCMA TCE-mediated killing of plasmablasts from patients with systemic lupus erythematosus occurs with minimal T cell activation PBMCs were isolated from systemic lupus erythematosus (SLE) patients and treated with various concentrations of CC-93269 or a control 2+1 antibody. After 24 hours of incubation, plasmablast killing (FIG. 20A) and T cell activation (FIG. 20B) were assessed as in Example 4. The 50% effective concentration (EC) of CC-93269 for plasmablast lethality in SLE 50) is 0.01 nM (n=5). Therefore, SLE plasmablast killing occurs at BCMA TCE concentrations lower than those required for T cell activation.

[0404] Example 15: Selective depletion of plasmablasts by CC-93269 in cynomolgus monkeys PBMCs were isolated from whole blood of cynomolgus monkeys. Plasmablasts and CD20(+) B cells were treated with various concentrations of CC-93269 or control 2+1 anti-HEL anti-CD3 antibody for 24 hours. Plasmablast killing was assessed by FACS, whereby plasmablasts were identified as CD19(+)IRF4(+) and expressed as a percentage of total CD19(+) cells (Figure 19A). CD20(+) B cell killing was also assessed by FACS, whereby CD19(+)CD20(+) cells were expressed as a percentage of total CD19(+) cells (Figure 19B). IRF4+ plasmablast killing allows selective depletion of IRF4+ plasmablasts without CD20(+) B cell killing, extensive CD20(+) B cell depletion. T cell activation was assessed by FACS, whereby CD69(+)CD8(+) T cells are expressed as a percentage of total CD8(+) T cells (Figure 19C). Collectively, these data demonstrate selective killing of plasmablasts and minimal elevation of activated T cell frequencies by CC-93269 at concentrations that deplete IRF4+ plasmablasts without depleting CD20(+) B cells in an acceptable pharmacokinetic-pharmacodynamic (PK / PD) model that can be used to predict the pharmacological and toxicological effects of BCMA-TCE in vivo.

[0405] Example 16: Effect of exogenous soluble BCMA on CC-93269-mediated killing of plasmablasts from healthy volunteers PBMCs were isolated from normal healthy volunteers and spiked with various concentrations of exogenous soluble BCMA (sBCMA). The highest concentration of sBCMA, 67.6 ng / mL, was selected as representing twice the upper limit of sBCMA levels in autoimmune patients (data not shown). Soluble BCMA levels in serum or plasma from donor patients with autoimmune disorders were assessed by a bead-based immunoassay by Ampersand Biosciences (Lake Clear, NY). Samples were then treated with increasing concentrations of CC-93269 (0–50 nM) or a control 2+1 antibody for 24 hours, and plasmablast killing (Figure 21A) and T cell activation (Figure 21B) were assessed. Plasmablast killing was assessed by FACS, whereby plasmablasts were identified as CD19(+) CD20(-) CD27(+) cells and is shown as a percentage of total CD19(+) cells normalized to untreated controls (Figure 21A). Plasmablast killing in the presence of sBCMA occurs at lower concentrations of CC-93269 than are lethal for BCMA-expressing cancer cell lines (e.g., JEKO cells), even in the presence of sBCMA at concentrations higher than those in autoimmune patients. For T cell activation, cells were washed and then stained for T cell lineage (CD4 and CD8) and activation markers (CD69, CD25). The data shown in Figure 21B show the frequency of CD69(+) on CD4(+) or CD8(+) T cells.

[0406] Table 13 summarizes data from CC-93269 and shows the 90% effective concentration (EC) of BCMA TCE for in vitro depletion of plasmablasts in PBMCs from healthy volunteers. 90 ) showing that increasing sBCMA levels result in a minimal increase in the frequency of activated T cells (i.e., less than 20% above baseline). [Table 23]

[0407] Example 17: Minimal CC-93269-mediated T cell activation and cytokine secretion in whole blood samples from healthy volunteers and AAV patients Whole blood samples from normal healthy volunteers (n=4) and AAV patients (n=2) were treated with various concentrations of CC-93269 or control 2+1 antibody. After 24 hours of incubation, T cell activation was assessed as in Example 4. The data shown in Figure 22A show CD69 expression on CD8(+) T cells in whole blood samples from normal healthy volunteers (NHV) and AAV patients. Culture supernatants were analyzed for cytokine production (IFNγ, IL-1β, IL-6, IL-2, IL-10, and granzyme B) using the MSD Pro-inflammatory I assay (Figure 22B). Taken together, these data demonstrate that treatment of whole blood samples from normal healthy volunteers with CC-93269 resulted in minimal increases in the frequency of activated T cells (i.e., less than 20% above baseline) and minimal cytokine production (i.e., less than 20 pg / mL above baseline).

[0408] Example 18: Antibodies with improved stability The physicochemical properties of four BCMAxCD3 molecules were evaluated: Mab101, Mab102, 83A10-TCBcv, and 22-TCBcv. Mab101 and 83A10-TCBcv contain BCMA-binding domains that include the CDRs of antibody 83A10, while Mab102 and 22-TCBcv contain BCMA-binding domains that include the CDRs of antibody Mab22. All four variants have the same CD3-binding domain. A sequence alignment of the four BCMAxCD3 molecules is shown in Figure 27. Each molecule is in a 2+1 bispecific format as shown in Figure 2A, except that rather than the "RK / EE" substitutions described, there are alternative amino acid substitutions in CL-CH1 to reduce light chain mispairing / by-products: A141W, L145E, K147T, Q175E ("WETE") and F116A, Q124R, L135V, T178R ("ARVR").

[0409] The terms "HD1" and "HD1 platform" are used in these examples to refer to bispecific antibodies that contain "knob-into-hole" mutations. In particular, as used herein, the terms "HD1 format" and "HD1 platform" refer to bispecific antibodies of the format BCMA Fab-Fc-CD3 Fab-BCMA Fab, where: (i) an anti-CD3 Fab fragment comprises a light chain and a heavy chain, wherein the light chain is a crossover light chain comprising a variable domain VH and a constant domain CL, and the heavy chain is a crossover heavy chain comprising a variable domain VL and a constant domain CH1; (ii) anti-BCMA Fab contains amino acid substitutions 123R and 124K in the CL domain and amino acid substitutions 147E and 213E in the corresponding CH1 domain; (iii) the CH3 domain of the first Fc comprises the modification T366W and the CH3 domain of the second Fc comprises the modifications T366S, L368A and Y407V.

[0410] The terms "HD2 format" and "HD2 platform" are used in these examples to refer to bispecific antibodies comprising heterodimerization mutations of the invention. In particular, as used herein, the terms "HD2 format" and "HD2 platform" refer to bispecific antibodies of the format BCMA Fab-Fc-CD3 Fab-BCMA Fab, wherein: (i) an anti-CD3 Fab fragment comprises a light chain and a heavy chain, wherein the light chain is a crossover light chain comprising a variable domain VH and a constant domain CL, and the heavy chain is a crossover heavy chain comprising a variable domain VL and a constant domain CH1; (ii) the anti-BCMA Fab contains amino acid substitutions A141W, L145E, K147T, and Q175E in the CH1 domain and amino acid substitutions F116A, Q124R, L135V, and T178R in the corresponding CL domain; (iii) the CH3 domain of the first Fc comprises the modifications T350V, L351Y, F405A and Y407V, and the CH3 domain of the second Fc comprises the modifications T350V, T366L, K392L and T394W (numbering according to EU numbering).

[0411] As shown in Table 14, bispecific antibodies Mab101 and Mab102 contain HD2 mutations of the invention, while bispecific antibodies 83A10-TCBcv and 22-TCBcv contain "knob-into-hole" (HD1) mutations. "Knob-into-hole" modifications are described in detail, with some examples, in, for example, WO96 / 027011; Ridgway, JB, et al., Protein Eng. 9 (1996) 617-621; Merchant, AM et al., Nat. Biotechnol. 16 (1998) 677-68; and WO98 / 050431. These modifications consist of a first CH3 domain containing the modification T366W (the "knob modification") and a second CH3 domain containing the modifications T366S, L368A, and Y407V (the "hole modifications") (numbering according to the EU numbering system of Kabat). [Table 24]

[0412] The inventors have shown that the binding capacity of bispecific antibodies in the format of the invention (i.e., Mab101 and Mab102), as determined by surface plasmon resonance, is less affected by chemical stress (i.e., exposure to low pH, high pH, ​​and tert-butyl peroxide) than the binding capacity of bispecific antibodies containing the corresponding BCMA-binding domain in the HD1 format (i.e., 83A10-TCBcv and 22-TCBcv), thereby demonstrating that the HD2 format contributes to an overall increase in stability.

[0413] We demonstrate that the use of the HD2 format compensates for the reduced physical stability caused by the CDRs of Mab22. Notably, measurement of protein concentration by size exclusion chromatography (SEC) showed a clear decrease in protein concentration of HD1 bispecific 22-TCBcv after both agitation and low pH exposure, which was not observed with the HD2 platform equivalent, Mab102. The use of the HD2 platform in the equivalent Mab102 molecule therefore reduces the negative impact of the Mab22 CDRs on antibody stability.

[0414] Additionally, an overall stability score for each molecule was calculated based on a combined analysis of data generated by multiple physical and chemical stability assays (see Table 18). In this analysis, both HD2 platform molecules, Mab101 and Mab102, scored higher than their respective HD1 platform counterparts, 83A10-TCBcv and 22-TCBcv, suggesting that both bispecific molecules are more stable in the HD2 format.

[0415] Example 18.1: Chemical Stability Chemical stability evaluation consisted of a low pH (pH 4) to accelerate aspartate isomerization and fragmentation reactions, and a high pH (pH 8) to accelerate asparagine deamidation, oxidation reactions, and thioether formation. Tert-butyl peroxide (TBP) was also added to the pH 6 platform buffer to promote oxidation of solvent-exposed methionine residues.

[0416] The primary methods used to assess chemical stability were surface plasmon resonance (SPR) using the sensorgram comparison method described here and size exclusion chromatography (SEC) when chemical modifications may affect physical stability or lead to low molecular weight (LMW) clipping. Peptide mapping was also used on selected samples, depending on the SPR binding results.

[0417] Example 18.1.1 Analysis of Changes in BCMA and CD3 Binding by Surface Plasmon Resonance SPR experiments were performed using a Biacore T200 system (GE Healthcare, Uppsala, Sweden) with analytical and sample compartment temperatures set at 25°C and 7°C, respectively. Anti-human IgG (from the Anti-Human IgG Capture Kit) was amine-coupled to the surface of a CM5 chip according to the manufacturer's instructions. Bispecific antibodies diluted to 1 μg / mL in running buffer were captured onto the chip surface in a 60-second injection. Following single-cycle kinetic analysis, antigen was injected five times (30 seconds per injection) at increasing concentrations at a flow rate of 30 μL / min. Antigen concentrations ranged from 0.08 to 50 nM for BCMA and 12.7 to 1000 nM for CD3. A 300-second dissociation time was added after the last antigen injection. After each experiment, all flow cells were regenerated using a 30-second injection of 3M MgCl2. Prior to analysis, data were double-controlled by first subtracting data from a control flow cell and then subtracting a blank cycle in which buffer was injected instead of antigen. Sensorgram comparison analysis was performed using Biacore T200 (software version 3.0). All data were normalized to a response where 100% reflects the maximum binding obtained during the injection and 0% reflects the baseline. The mean and ±3 standard deviation (SD) sensorgrams were calculated from 10 or more independent sensorgrams of unstressed material (standard) for each molecule. To quantify the binding similarity of stressed samples to the corresponding unstressed standard, sample sensorgrams were simultaneously evaluated with the standard sensorgram. The degree of similarity was calculated based on how many sample data points fell within or outside the ±3 standard deviation limits according to Equation 2, where SSQ is the sum of squares (Karlsson, R., Pol, E., and Frostell, A. (2016); Analytical Biochemistry 502, 53-63). All materials for the SPR analysis are listed in Table 15. [Table 25] Representative SPR sensorgrams comparing variant 83A10-TCBcv stored at 2-8°C (pH 6) and 40°C (pH 8) for 2 weeks are shown in Figures 26A and 26B, respectively.

number

[0418] As shown in Table 18, 22-TCBcv (containing the HD1 platform mutation) showed the greatest overall decrease in CD3 and BCMA binding across all stress conditions except for tert-butyl peroxide exposure, and had the lowest overall chemical stability score. Conversely, Mab101 (containing the HD2 platform mutation of the present invention) showed the least change in binding across all stress conditions. Both bispecific antibodies containing HD2 mutations (i.e., Mab101 and Mab102) had higher overall chemical stability scores than the corresponding HD1 molecules containing the same CDR regions, suggesting that the HD2 platform contributes to the chemical stability of the antibodies. The SPR binding data therefore suggest that the binding capacity of bispecific antibodies containing HD2 mutations (i.e., Mab101 and Mab102) is less affected by chemical stress (i.e., low pH maintenance, high pH maintenance, and tert-butyl peroxide exposure) than the binding capacity of bispecific antibodies containing the corresponding BCMA-binding domain with HD1 mutations (i.e., 83A10-TCBcv and 22-TCBcv). Thus, these data indicate that the HD2 mutations of the invention, when used with CD3xBCM bispecific antibodies containing the CDRs of 83A10 or Mab22, improve the stability of the bispecific antibodies over HD1 mutations.

[0419] Example 18.1.2 Reduced Peptide Mapping To further differentiate the chemical stability of the bispecific antibodies, reduced peptide mapping was performed on all four bispecific antibodies. The molecules were stored for 2 weeks at 40 °C in the presence of TBP, buffered at pH 8 and pH 6. As a control, a pH 6 sample stored at 2-8 °C for 2 weeks was also analyzed. All samples were buffer exchanged into 50 mM acetic acid, pH 5.0 buffer using a 10 kDa MWCO filter. Samples were then digested according to the manufacturer's recommended AccuMAP protocol (Promego, Madison, WI). Briefly, samples were denatured with GdHCl, reduced with TCEP, and alkylated with iodoacetamide. A 1-hour predigestion was performed with LysC, followed by dilution of GdHCl to less than 1 M and addition of methionine to a concentration of 15 mM. The trypsin / LysC mixture was then digested for a final 3 hours at 37°C. All steps were performed at pH 5. Digestion was stopped by adding TFA at 2% of the final composition. Approximately 30 μg of digested sample was injected onto a Waters CSH C18 column (2.1 × 150 mm, 130 Å pore radius, 1.7 μm bead diameter) in series with an Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific, Waltham, MA). LC solvent A was 0.1% formic acid (FA) in water, and solvent B was 0.1% FA in ACN. The separation gradient started at 1% B and increased linearly to 27% B over 110 min, followed by a 5-min linear gradient from 30% to 40% B. The column temperature was set to 70 °C. Mass spectra were acquired using top-10 data-dependent acquisition. MS1 was performed on the Orbitrap set to 400 m / z and a resolving power of 60,000. CIDMS / MS was analyzed on the ion trap set to rapid scan. Dynamic exclusion was set to 15 s and a 10 ppm mass window. The raw data were analyzed with the Protein Metrics Byonic and Byologic software package. The modification ratio was calculated using the XIC intensities as follows: modified pep intensity / (modified pep intensity+unmodified pep intensity)×100%.

[0420] Tables 16 and 17 show modifications of residues in or near the BCMA and CD3 CDRs, respectively. Consistent with the SPR data, 22-TCBcv showed the greatest propensity for chemical modification in the BCMA and CD3 CDR regions, particularly for methionine and tryptophan oxidation. In contrast, the only modification observed at significant levels in the Mab101 molecule was M34 in the BCMA HC and HHC after TBP treatment (Table 16). [Table 26]

[0421] [Table 27]

[0422] Example 18.2: Physical Stability Physical stability assessment consisted of measuring thermal stability by differential scanning calorimetry (DSC) and colloidal stability by polyethylene glycol (PEG) precipitation in a platform pH 6 buffer. Physical stability was also assessed after agitation and freeze-thaw (F / T) stress in a pH 6 platform buffer. Finally, a short low pH hold at room temperature was used to mimic viral inactivation. This process often results in non-native aggregation of less structurally stable protein biologics.

[0423] Example 18.2.1 Evaluation of Thermal Stability by Differential Scanning Calorimetry Differential scanning calorimetry was performed on a TA Instruments NanoDSC (New Castle, Del.) using NanoAnalyze software. BCMAxCD3 samples were diluted to 1 mg / mL in 100 mM histidine pH 6.0 buffer (Table 19). Prior to analysis, samples and their corresponding protein-free buffers were degassed for 30 minutes. Samples and buffers were heated from 10°C to 100°C for 1 minute. -1After acquisition, the corresponding buffer was subtracted from each of the samples, and the data were normalized to kcal mol -1 °C -1 The results are reported as the onset temperature (°C) of the first unfolding transition. As shown in Figure 24, all four molecules had similar thermal unfolding onset temperatures (approximately 60°C), but the unfolding transition of the majority of the endothermic domain was approximately 5°C higher for Mab101 and the 83A10-TCBcv molecule (containing the BCMA CDRs of 83A10). app It has a value.

[0424] Example 18.2.2 Colloidal Stability Assessment by PEG Precipitation Colloidal stability evaluation of four BCMAxCD3 molecules was performed by PEG 6000 precipitation. PEG precipitation experiments were performed by preparing 160 μL solutions consisting of 1.0 g / mL BCMAxCD3 molecules buffered to pH 6.0 with 100 mM histidine in increasing concentrations of PEG-6000 from a 40% (w / v) stock solution (Table 19). The solutions were left standing overnight at 4°C and then stirred for 60 minutes at 25,000 RCF. The amount of protein remaining in the supernatant was then measured by absorbance at 280 nm using an Agilent Cary UV-8454 (Agilent, Palo Alto, CA). The data were fitted to an empirical four-parameter sigmoidal equation (Equation 1) to determine and report the percentage (w / v) of PEG-6000 required to precipitate half of the starting amount of protein (Cm). The parameters b, m and r represent the curve base, peak and velocity, respectively.

number

[0425] Example 18.2.3 Size Exclusion Chromatography Assessment of Physical Stability After Agitation and Freezing / Thawing Physical stability was also assessed by size exclusion chromatography after agitation and freeze-thaw (F / T) stress in pH 6 platform buffer.

[0426] Freeze / thaw 400 μL of each 1 mg / mL BCMAxCD3 molecule (Table 19) was dispensed into 0.5 mL freestanding Fisherbrand screw-cap cryotubes (part #02-707-357), stored in a single-row sample box with tube dividers, and frozen at -80°C. A total of five freeze-thaw (F / T) cycles were performed, each cycle consisting of freezing the sample for at least 1 hour, followed by thawing at room temperature and gentle mixing before the next F / T cycle. Samples were analyzed by SEC after the fifth F / T cycle.

[0427] Mixing 400 μL of each 1 mg / mL BCMAxCD3 molecule (Table 19) was transferred to a 1.5 mL standard Eppendorf tube (part # 022364111) and placed in a temperature-controlled VWR microplate shaker. The shaker was set at 1500 rpm at 25° C. for 24 hours. After agitation, the samples were analyzed by SEC.

[0428] Size exclusion chromatography Size-exclusion chromatography (SEC) was performed on an Agilent 1260 HPLC system (Agilent, Palo Alto, California) equipped with a single TSKgel G3000SWxl 7.8 x 300 mm, 5 μm column. The mobile phase consisted of 100 mM KH2PO4, 250 mM NaCl, pH 6.8. Twenty microliters of each sample was injected onto the column at 25°C and eluted isocratically for 30 minutes at a flow rate of 0.5 mL / min. Elution was monitored by UV detection at 280 nm, and protein concentrations were calculated using the integrated total curve area, flow rate, injection volume, flow cell path length, and permeability coefficient of each BCMAxCD3 molecule. Two values ​​are reported for the SEC analysis; % monomer loss and concentration for a pH 6 2-week sample maintained at 2-8° C. This method reduces the time and material requirements by eliminating a "time zero" sample, while minimizing the potential for chromatographic variation (e.g., column performance) by analyzing all samples within the same chromatographic sequence.

[0429] To account for potential dilution variability (as well as integration and injection variability), ten independent 1:10 dilutions from the same 10 mg / mL standard mAb solution were also prepared and analyzed using the same SEC sequence as the BCMAxCD3 stability samples. The monomer percentage and concentration reduction are reported relative to two standard deviations (2σ) of the mean percent monomer (2σM) and concentration (2σC) of these diluted standards, respectively. The results of the SEC evaluation are provided in Table 15, and the percent main peak monomer and the difference in concentration between each of the pH 6 2k 2-8°C (control) and stressed samples are shown in Table 18. Consistent with the SPR data, the SEC results showed a clear decrease in protein concentration of the HD1 platform molecule 22-TCBcv after stirring at pH 3, which was not observed for other molecules. Therefore, the use of the HD2 platform in the equivalent Mab101 molecule minimizes the negative impact of the Mab22 CDR on stability. Representative chromatograms of the 22-TCBcv variants are shown in Figure 26.

[0430] Scoring Molecules were scored according to the acceptance criteria for each method's response, indicating physical and chemical stability (light gray shaded area in Table 18), and assigned a score of 0, 1, or 2 based on how the experimental results (dark gray shaded area) fell within these criteria. The percent monomer and concentration reduction measured by SEC were scored relative to two standard deviations of the mean percent monomer (2σM) and concentration (2σC) determined from 10 independent 1:10 dilutions of a standard 10 mg / mL mAb solution; for the current study, 2σM and 2σC were 0.33 and 0.14, respectively. The sum of the physical and chemical stability scores was divided by the number of responses (8 for physical and 16 for chemical), resulting in scores ranging from 0 to 2 for both physical and chemical stability. The total score for each variant is thus the sum of the physical and chemical stability scores and therefore ranges from 0 to 4 (Table 18). The responses in Table 18 are equally weighted.

[0431] Table 18 shows that in the physical stability evaluation, after stirring and maintaining pH 3, the protein concentration of the 22-TCBcv molecule clearly decreased, which was not observed for the other molecules. 22-TCBcv also performed poorly in the chemical stability assessment, showing a significant loss of percent monomer after oxidation with TBP and a significant loss of CD3 and BCMA binding after low and high pH. As with the physical stability portion of the assessment, Mab101 showed the least change in concentration, percent monomer, and binding after all chemical stresses. Both HD2-platform molecules, Mab101 and Mab102, scored higher than their respective HD1-platform counterparts (Table 18). Therefore, these data clearly demonstrate that the HD2-platform format resulted in more stable molecules than the HD1-platform format for both the 83A10 and Mab22 bispecific antibodies.

[0432] Table 28

[0433] Table 29 Table 30

[0434] Table 31

Claims

1. 1. A pharmaceutical composition comprising a bispecific antibody that binds to B-cell maturation antigen (BCMA) and one or more antigens that promote activation of T cells for the treatment or management of an autoimmune disorder, the antigen that promotes activation of the one or more T cells is CD3; the bispecific antibody is a trivalent bispecific antibody comprising two Fab fragments of an anti-BCMA antibody, one Fab fragment of an anti-CD3 antibody, and one Fc portion, and the bispecific antibody is of the format BCMA Fab-Fc-CD3 Fab-BCMA Fab; the bispecific antibody comprises a variable region VH comprising an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 10 and a variable region VL comprising an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 14; the anti-CD3 antibody comprises a variable region V having an amino acid sequence identical to the amino acid sequence of SEQ ID NO:7 and a variable region VL comprising an amino acid sequence identical to the amino acid sequence of SEQ ID NO:8; and 1. A pharmaceutical composition, wherein the autoimmune disorder is selected from the group consisting of IgA nephropathy, membranous nephropathy, myasthenia gravis, neuromyelitis optica, pemphigus vulgaris, anti-PAD4 activated rheumatoid arthritis, sensitizing / pre-existing antibodies in solid organ transplantation, Guillain-Barre syndrome (acute inflammatory demyelinating polyneuropathy - AIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), immune thrombocytopenic purpura, rheumatoid arthritis (RA), and antineutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV).

2. 2. The pharmaceutical composition of claim 1, wherein the autoimmune disorder is AAV or RA.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the autoimmune disorder is AAV.

4. 4. The pharmaceutical composition of claim 1, wherein the bispecific antibody comprises a set of heavy and light chains consisting of the polypeptides of SEQ ID NO: 48, SEQ ID NO: 55, SEQ ID NO: 56, and two copies of SEQ ID NO:

57.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the AAV is used to treat a disease selected from the group consisting of granulomatosis with polyangiitis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA), microscopic polyangiitis (MPA), and renal-confined ANCA-associated vasculitis.

6. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the autoimmune disorder is refractory or recurrent, and / or the autoimmune disorder is newly diagnosed.

7. 7. The pharmaceutical composition of any of claims 1 to 6, wherein the AAV affects one or more parts of the patient's body selected from the nervous system, eyes, nose, heart, kidneys, stomach, intestines, lungs, joints, muscles, and skin.

8. 8. The pharmaceutical composition of any one of claims 1 to 7, wherein the AAV is systemic with life-threatening or major organ-threatening symptoms.

9. 9. The pharmaceutical composition of claim 8 for the treatment or management of patients with disseminated alveolar hemorrhage (DAH).

10. 8. The pharmaceutical composition of any one of claims 1 to 7, wherein the AAV is localized without any signs of organ threat.

11. (a) the patient requires plasmablastopenia; (b) the patient is at risk of developing cytokine release syndrome; (c) the patient is at risk of developing an infection; (d) the patient requires induction of remission, and / or (e) the patient requires maintenance of remission; The pharmaceutical composition of any one of claims 1 to 10.

12. Treatment or management results in: (a) results in at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least a 95% or 100% reduction in plasmablasts in the patient compared to no treatment or a control treatment; (b) results in at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least a 95% or 100% reduction in the incidence of cytokine release syndrome in a patient compared to a control treatment; (c) results in at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least a 95% or 100% reduction in the incidence of infection in patients compared to a control treatment; (d) results in at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95% or 100% faster induction of remission in patients compared to a control treatment; and / or (e) resulting in at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95% or 100% longer maintenance of remission in patients compared to control treatment; The pharmaceutical composition of any one of claims 1 to 11.

13. 13. The pharmaceutical composition of claim 12, wherein the control treatment is treatment with steroids, cyclophosphamide, anti-CD20 monoclonal antibodies, methotrexate, azathioprine, mycophenolic acid, mycophenolate mofetil, avacopan, anti-TNF agents, anti-IL6R antibodies, costimulatory blockade, JAK inhibitors, and / or belimumab.

14. (a) the steroid is a glucocorticoid; (b) the anti-CD20 monoclonal antibody is rituximab; (c) the anti-TNF agent is infliximab, adalimumab, golimumab, or etanercept; (d) the anti-IL6R antibody is tocilizumab or sarilumab; (e) the costimulatory blockade is abatacept; and / or (f) the JAK inhibitor is tofacitinib or baricitinib; 14. The pharmaceutical composition of claim 13.

15. 15. The pharmaceutical composition of claim 13 or 14, wherein the control treatment is treatment with a steroid, cyclophosphamide, or rituximab.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the treatment or management is aimed at inducing remission or maintaining remission.

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