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WO2024073700A2 - Méthodes de traitement de troubles auto-immuns à l'aide d'anticorps multimères anti-cd20/anti-cd3 - Google Patents

Méthodes de traitement de troubles auto-immuns à l'aide d'anticorps multimères anti-cd20/anti-cd3 Download PDF

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WO2024073700A2
WO2024073700A2 PCT/US2023/075566 US2023075566W WO2024073700A2 WO 2024073700 A2 WO2024073700 A2 WO 2024073700A2 US 2023075566 W US2023075566 W US 2023075566W WO 2024073700 A2 WO2024073700 A2 WO 2024073700A2
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seq
antibody
chain
igm
human
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WO2024073700A3 (fr
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Carrie SAULSBERY
Mary Beth HARLER
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IGM Biosciences Inc
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IGM Biosciences Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen

Definitions

  • the anti-CD20 antibody, rituximab which reduces the number of CD20+ B cells resulting in a reduction in pathogenic autoantibodies and leading to improvement in clinical outcomes has been approved the US FDA to treat the autoimmune disorders rheumatoid arthritis (RA), granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), pemphigus vulgaris (PV) and has been used for a variety of other autoimmune disorders o ⁇ -label. See, e.g., RITUXAN® Prescribing Information, Revised 12/2021 and Randall, Aust Prescr 2016;39:131–4.
  • rituximab does not result in substantial depletion of B cells in secondary lymphoid organs, which limit its e ⁇ ectiveness. See Kamburova, American Journal of Transplantation, 2013, 13:1503-1511.
  • Anti-CD20/anti-CD3 bispeci ⁇ c antibodies that engage T cells to attack CD20- expressing tumor cells have shown promise in treating various B cell malignancies but little or nothing is known about treating autoimmune disorders with such molecules. See, e.g., Budde, et al. Blood 2018;132(S1):399; Bannerji, et al. Blood 2019;134(S1):762.
  • Imvotamab also known as IGM-23273 is a bispeci ⁇ c antibody, based on an engineered pentameric IgM framework, which binds CD20, with a recombinant J-chain IGM Ref. No.001-061WO1 that is fused to an anti-CD3 scFv, and also to human serum albumin to increase serum half- life. See, e.g., WO 2022/109023 and U.S. Patent Nos. 10,787,520 and 10,618,978, which are incorporated herein by reference in their entireties.
  • imvotamab binds irreversibly to CD20-expressing cells and eliminates them through T cell-dependent cellular cytotoxicity (TDCC) and complement-dependent cytotoxicity (CDC).
  • T cell-dependent cellular cytotoxicity TDCC
  • CDC complement-dependent cytotoxicity
  • Most T cell engaging antibodies of the IgG isotype in preclinical and clinical evaluations are associated with toxicities primarily cytokine release syndrome (CRS).
  • CRS cytokine release syndrome
  • imvotamab did not induce signi ⁇ cant cytokine release in in vitro or in vivo preclinical studies and low rates of grade 2 or higher cytokine release syndrome (CRS), no ICANS, and minimal neutropenia.
  • a method of treating an autoimmune disorder comprising administering to a subject in need of treatment an e ⁇ ective amount of a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody, which comprises ⁇ ve bivalent binding units and a modi ⁇ ed J-chain, where each binding unit comprises two IgM heavy chains, each comprising a heavy chain variable region (VH) and an IgM constant region or multimerizing fragment or variant thereof, and two light chains, each comprising a light chain variable region (VL) and a light chain constant region, where the VH comprises three immunoglobulin complementarity determining regions: HCDR1, HCDR2, and HCDR3, and the VL comprises three immunoglobulin complementarity determining regions: LCDR1, LCDR2, and LCDR3, where the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise, respectively, the amino acid sequences SEQ ID NO: 58; SEQ ID NO: 59
  • the autoimmune disorder is a disorder where B lineage cells produce pathogenic autoantibodies and/or contribute to chronic in ⁇ ammation in the subject.
  • the autoimmune disorder is systemic lupus erythematosus (SLE), antiphospholipid syndrome (APS), idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (wAIHA), multiple sclerosis (MS), myasthenia gravis (MG), pemphigus vulgaris (PV), anti-neutrophil cytoplasmic autoantibody (ANCA) associated vasculitis (AAV), thyroid eye disease (TED), membranous glomerulonephritis (MGN), Neuromyelitis optica (NMO), Guillain-Barré syndrome (GBS), chronic in ⁇ ammatory demyelinating polyradiculoneuropathy (CIDP), IgA nephropathy, Goodpasture’s syndrome, gran
  • SLE systemic lupus erythematosus
  • APS anti
  • the autoimmune disorder is systemic lupus erythematosus (SLE), antiphospholipid syndrome (APS), idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (wAIHA), multiple sclerosis (MS), myasthenia gravis (MG), pemphigus vulgaris (PV), anti-neutrophil cytoplasmic autoantibody (ANCA) associated vasculitis (AAV), thyroid eye disease (TED), membranous glomerulonephritis (MGN), Neuromyelitis optica (NMO), Guillain-Barré syndrome (GBS), chronic in ⁇ ammatory demyelinating polyradiculoneuropathy (CIDP), IgA nephropathy, Goodpasture’s syndrome, or rheumatoid arthritis (RA).
  • SLE systemic lupus erythematosus
  • APS antiphospholipid syndrome
  • IDP idiopathic thrombocytopenic purpura
  • ITPP warm
  • the autoimmune disorder is SLE.
  • the SLE comprises lupus nephritis (LN).
  • the autoimmune disorder is RA.
  • the autoimmune disorder is MG.
  • the subject had previously been treated with a biologic autoimmune disorder treatment.
  • the subject had previously been treated with an anti-CD20 antibody, anti-B-lymphocyte stimulator (BlyS) antibody, or a TNF inhibitor.
  • the autoimmune disorder is SLE, and the subject had previously been treated with belimumab.
  • the autoimmune disorder is MS, and the subject had previously been treated with rituximab.
  • the autoimmune disorder is RA, and the subject had previously been treated with in ⁇ iximab, adalimumab, or etanercept.
  • the administration of the multimeric bispeci ⁇ c anti- CD20/anti-CD3 antibody comprises intravenous infusion.
  • the IGM Ref. No.001-061WO1 administration of the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody comprises subcutaneous injection.
  • the subject is a human.
  • the scFv comprises the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 43, SEQ ID NO: 48, or SEQ ID NO: 53. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 26.
  • the IgM heavy chain constant region or multimerizing fragment or variant thereof comprises a C ⁇ 4 region and a ⁇ tailpiece region. In some embodiments, the IgM heavy chain constant region or multimerizing fragment or variant thereof further comprises a C ⁇ 1 region, a C ⁇ 2 region, C ⁇ 3 region, or any combination thereof.
  • the IgM heavy chain constant region or multimerizing fragment or variant thereof is a human IgM constant region.
  • the modi ⁇ ed J-chain further comprises human serum albumin indirectly or directly fused to the J-chain or functional fragment or variant thereof.
  • the J-chain or functional fragment or variant thereof comprises SEQ ID NO: 4.
  • the modi ⁇ ed J-chain comprises SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 39, SEQ ID NO: 44, SEQ ID NO: 49, or SEQ ID NO: 54.
  • the modi ⁇ ed J-chain comprises SEQ ID NO: 27.
  • the modi ⁇ ed J-chain comprises SEQ ID NO: 29, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 46, SEQ ID NO: 51, or SEQ ID NO: 56. In some embodiments, the modi ⁇ ed J-chain comprises SEQ ID NO: 29. [0014] In some embodiments, the J-chain or functional fragment or variant thereof comprises an alanine (A) substitution at the amino acid position corresponding to amino acid Y102 of SEQ ID NO: 4. In some embodiments, the J-chain or functional fragment or variant thereof comprises the amino acid sequence SEQ ID NO: 5.
  • the modi ⁇ ed J-chain comprises SEQ ID NO: 28, SEQ ID NO: 35, SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 50, or SEQ ID NO: 55.
  • the modi ⁇ ed J- chain comprises SEQ ID NO: 30, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 47, SEQ ID NO: 52, or SEQ ID NO: 57.
  • the VH comprises SEQ ID NO: 64 and the VL comprises SEQ ID NO: 65.
  • the heavy chain comprises SEQ ID NO: 66 and the light chain comprises SEQ ID NO: 67.
  • the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody is imvotamab.
  • FIGS. 1A–1L show the percentage of circulating CD19+ B cell killing after treatment with various concentrations of imvotamab and rituximab in T-cell dependent cellular cytotoxicity (TDCC) assay on peripheral blood mononuclear cells (PBMCs) from biologic na ⁇ ve systemic lupus erythematosus (SLE) donors #1–#4 (FIGS.
  • TDCC T-cell dependent cellular cytotoxicity
  • FIGS.2A & 2B show the half maximal e ⁇ ective concentration (EC 50 ) (FIG.2A) or maximum percentage of circulating CD19+ B cell killing (FIG. 2B) determined from TDCC assays on PBMCs from 3–4 biologic na ⁇ ve SLE donors, 2 biologic experienced SLE donors, 2–3 MS donors, and 1–2 healthy donors.
  • FIGS. 3A–3H show the percentage of activated memory B cells of total remaining CD19+ cells after treatment with rituximab (FIGS. 3A, 3C, 3E, & 3G) or imvotamab (FIGS. 3B, 3D, 3F, and 3H) in a TDCC assay on PBMCs from 4 biologic na ⁇ ve SLE donors, (FIGS. 3A & 3B), 2 biologic experienced SLE donors (FIGS. 3C & 3D), 3 MS donors (FIGS.3E & 3F), or 3 healthy donors (FIGS.3G & 3H).
  • FIGS.4A & 4B show the percentage of activated memory B cells of total remaining CD19+ cells (FIG.
  • FIGS. 5A–5C shows the percentage of CD20+ B cells in spleen (FIG. 5A), mesenteric lymph nodes (MLN) (FIG. 5B), and bone marrow (FIG.
  • FIGS. 6A–6G show the percentage of circulating CD19+ B cell killing after treatment with various concentrations of imvotamab and rituximab in T-cell dependent cellular cytotoxicity (TDCC) assays on peripheral blood mononuclear cells (PBMCs) from biologic experienced SLE donors #3 & #4 (FIGS.
  • TDCC T-cell dependent cellular cytotoxicity
  • FIGS. 7A & 7B show the EC50 (FIG. 7A) or maximum percentage of circulating CD19+ B cell killing (FIG. 7B) determined from TDCC assays on PBMCs from 7–8 IGM Ref. No.001-061WO1 biologic na ⁇ ve SLE donors, 4 biologic experienced SLE donors, 2–3 MS donors, 2 RA donors, and 1–3 healthy donors.
  • FIGS. 7A & 7B show the EC50 (FIG. 7A) or maximum percentage of circulating CD19+ B cell killing (FIG. 7B) determined from TDCC assays on PBMCs from 7–8 IGM Ref. No.001-061WO1 biologic na ⁇ ve SLE donors, 4 biologic experienced SLE donors, 2–3 MS donors, 2 RA donors, and 1–3 healthy donors.
  • FIGS. 8A-8H show the percentage of activated memory B cells of total remaining CD19+ cells after treatment with rituximab (FIGS. 8A, 8C, 8E, and 8G) or imvotamab (FIGS. 8B, 8D, 8F, & 8H) in a TDCC assay on PBMCs from 2 RA donors (FIGS. 8A & 8B) 7 biologic na ⁇ ve SLE donors, (FIGS. 8C & 8D), 4 biologic experienced SLE donors (FIGS. 8E & 8F), or 3 healthy donors (1 healthy donor assayed in two separate TDCC assays) (FIGS.8G & 8H).
  • FIG.9 shows the total number of activated memory B cells after treatment with 90 nM rituximab or 100 nM imvotamab determined from TDCC assays on PBMCs from 3–4 biologic na ⁇ ve SLE donors, 2 biologic experienced SLE donors, 2 RA donors, and 1 healthy donor. [0025] FIGS.
  • FIGS.11A–11I show the percentage of circulating CD19+ B cell killing after treatment with various concentrations of imvotamab, bispeci ⁇ c IgG, and rituximab treated PBMCs from healthy donors (FIGS.10A-10C), biologic na ⁇ ve SLE donors (FIGS.10D & 10E), biologic experienced SLE donors (FIGS.10F–10H), and a RA donor (FIG 10I).
  • FIGS.11A–11I show the concentration of activated memory B cells after treatment with imvotamab or CD20 ⁇ CD3 IgG determined from TDCC assays on PBMCs from biologic na ⁇ ve SLE donors (FIGS.11A & 11B), biologic experienced SLE donors (FIGS.
  • FIGS.12A & 12B show the EC 50 (FIG.12A) or maximum percentage of circulating CD19+ B cell killing (FIG. 12B) by imvotamab, CD20 ⁇ CD3 IgG, or rituximab as determined from TDCC assays on PBMCs from biologic na ⁇ ve SLE donors, biologic experienced SLE donors, 2–3 MS donors, 2 RA donors, and 1–3 healthy donors.
  • FIGS.13A-13H show representative plots of IFN ⁇ (FIGS.13A-13B), IL-2 (FIGS. 13C-13D), IL-6 (FIGS. 13E-13F), and TNF ⁇ (FIGS. 13G-13H) concentrations in supernatants from PBMCs (healthy and autoimmune-disease donors) treated with various concentrations of imvotamab (FIGS.13A, 13C, 13E, & 13G) and CD20 ⁇ CD3 IgG (FIGS. 13B, 13D, 13F, & 13H) after 72 hours.
  • FIG.13A-13B shows representative plots of IFN ⁇ (FIGS.13A-13B), IL-2 (FIGS. 13C-13D), IL-6 (FIGS. 13E-13F), and TNF ⁇ (FIGS. 13G-13H) concentrations in supernatants from PBMCs (healthy and autoimmune-disease donors) treated with various concentrations of imvotamab (FIGS.13A, 13C, 13E,
  • FIGS.15A & 15B show the activation of CD4+ T cells (FIG.15A) or CD8+ T cells (FIG.15B) from PBMCs from healthy or AI donors by imvotamab or CD20 ⁇ CD3 IgG at 200 nM after 72 hours.
  • FIG.16A shows a kinetic analysis from live cell imaging of complement dependent cytotoxicity mediated by imvotamab (closed circles) or rituximab (open circles) in the presence of SLE biologic na ⁇ ve patient serum.
  • FIG. 16B shows the maximum cell death achieved in the assay from 16A.
  • FIG.16C shows the area under the curve in the assay from 16A. A students T test was used for statistical analysis, **p ⁇ 0.001.
  • FIG.17A shows a histogram of CD20 expression gated on total CD19+ B cells from representative healthy donor and AI patient PBMCs.
  • SLE systemic lupus erythematosis
  • SLE BN systemic lupus erythematosis
  • SLE BE SLE biologic experienced
  • RA rheumatoid arthritis
  • a or “an” entity refers to one or more of that entity; for example, "a binding molecule,” is understood to represent one or more binding molecules.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • “and/or” where used herein is to be taken as speci ⁇ c disclosure of each of the two speci ⁇ ed features or components with or without the other.
  • the term and/or" as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).
  • polypeptide is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids and does not refer to a speci ⁇ c length of the product.
  • peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the de ⁇ nition of "polypeptide,” and the term “polypeptide” can be used instead of any of these terms.
  • polypeptide is also intended to refer to the products of post-expression modi ⁇ cations of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, and derivatization by known protecting/blocking groups, proteolytic cleavage, or modi ⁇ cation by non-naturally occurring amino acids.
  • a polypeptide can be derived from a biological source or produced by recombinant technology but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
  • a polypeptide as disclosed herein can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids.
  • Polypeptides can have a de ⁇ ned three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a de ⁇ ned three-dimensional structure are referred to as folded, and IGM Ref. No.001-061WO1 polypeptides which do not possess a de ⁇ ned three-dimensional structure, but rather can adopt many di ⁇ erent conformations and are referred to as unfolded.
  • glycoprotein refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein via an oxygen-containing or a nitrogen-containing side chain of an amino acid, e.g., a serine or an asparagine.
  • Asparagine (N)-linked glycans are described in more detail elsewhere in this disclosure.
  • an "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of puri ⁇ cation is required. For example, an isolated polypeptide can be removed from its native or natural environment.
  • Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially puri ⁇ ed by any suitable technique.
  • a non-naturally occurring polypeptide or any grammatical variants thereof, is a conditional de ⁇ nition that explicitly excludes, but only excludes, those forms of the polypeptide that are, or might be, determined or interpreted by a judge or an administrative or judicial body, to be “naturally-occurring.”
  • Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof.
  • fragment include any polypeptides which retain at least some of the properties of the corresponding native antibody or polypeptide, for example, speci ⁇ cally binding to an antigen. Fragments of polypeptides include, for example, proteolytic fragments, as well as deletion fragments, in addition to speci ⁇ c antibody fragments discussed elsewhere herein. Variants of, e.g., a polypeptide include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. In certain embodiments, variants can be non-naturally occurring. Non-naturally occurring variants can be produced using art- known mutagenesis techniques.
  • Variant polypeptides can comprise conservative or non- conservative amino acid substitutions, deletions, or additions.
  • Derivatives are polypeptides that have been altered to exhibit additional features not found on the original polypeptide. Examples include fusion proteins.
  • a "derivative" of a polypeptide can also refer to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Also included as “derivatives" are those polypeptides that contain one or more derivatives of the twenty standard amino acids. For example, 4- IGM Ref.
  • binding molecule refers in its broadest sense to a molecule that speci ⁇ cally binds to a receptor or target, e.g., an epitope or an antigenic determinant.
  • a binding molecule can comprise one of more “binding domains,” e.g., “antigen-binding domains” described herein.
  • binding molecule is an antibody or antibody-like molecule as described in detail herein that retains antigen-speci ⁇ c binding.
  • a “binding molecule” comprises an antibody or antibody-like or antibody-derived molecule as described in detail herein.
  • binding domain or “antigen-binding domain” (can be used interchangeably) refer to a region of a binding molecule, e.g., an antibody or antibody- like, or antibody-derived molecule, that is necessary and su ⁇ cient to speci ⁇ cally bind to a target, e.g., an epitope, a polypeptide, a cell, or an organ.
  • an “Fv,” e.g., a heavy chain variable region and a light chain variable region of an antibody, either as two separate polypeptide subunits or as a single chain, is considered to be a “binding domain.”
  • Other antigen-binding domains include, without limitation, a single domain heavy chain variable region (VHH) of an antibody derived from a camelid species, or six immunoglobulin complementarity determining regions (CDRs) expressed in a ⁇ bronectin sca ⁇ old.
  • a “binding molecule,” e.g., an “antibody” as described herein can include one, two, three, four, ⁇ ve, six, seven, eight, nine, ten, eleven, twelve, or more “antigen-binding domains.”
  • the terms "antibody” and "immunoglobulin” can be used interchangeably herein.
  • An antibody (or a fragment, variant, or derivative thereof as disclosed herein, e.g., an IgM- like antibody) includes at least the variable domain of a heavy chain (e.g., from a camelid species) or at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are relatively well understood.
  • antibody encompasses anything ranging from a small antigen-binding fragment of an antibody to a full sized antibody, e.g., an IgG antibody that includes two complete heavy chains and two complete light chains, an IgA antibody that includes four complete heavy chains and four complete light chains and includes a J-chain and/or a secretory component, or an IgM-derived IGM Ref.
  • No.001-061WO1 binding molecule e.g., an IgM antibody or IgM-like antibody, that includes ten or twelve complete heavy chains and ten or twelve complete light chains and optionally includes a J- chain or functional fragment or variant thereof.
  • immunoglobulin comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classi ⁇ ed as gamma, mu, alpha, delta, or epsilon, ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) with some subclasses among them (e.g., ⁇ 1- ⁇ 4 or ⁇ 1- ⁇ 2).
  • Each heavy chain class can be bound with either a kappa or lambda light chain.
  • the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disul ⁇ de linkages or non-covalent linkages when the immunoglobulins are expressed, e.g., by hybridomas, B cells or genetically engineered host cells.
  • the amino acid sequences run from an N-terminus at the forked ends of the Y con ⁇ guration to the C-terminus at the bottom of each chain.
  • binding unit is used herein to refer to the portion of a binding molecule, e.g., an antibody, antibody-like molecule, or antibody-derived molecule, antigen-binding fragment thereof, or multimerizing fragment thereof, which corresponds to a standard “H2L2” immunoglobulin structure, i.e., two heavy chains or fragments thereof and two light chains or fragments thereof.
  • binding molecule is a bivalent IgG antibody or antigen-binding fragment thereof
  • binding unit binding molecules are equivalent.
  • Such binding molecules are also referred to herein as “monomeric.”
  • the binding molecule is a “multimeric binding molecule,” e.g., a pentameric or hexameric IgM antibody, a pentameric or hexameric IgM-like antibody, or a pentameric or hexameric IgM-derived binding molecule or any derivative thereof
  • the binding molecule comprises two or more “binding units.” Two in the case of an IgA dimer, four in the case of an IgA tetramer, or IGM Ref.
  • binding unit need not include full-length antibody heavy and light chains, but will typically be bivalent, i.e., will include two “antigen-binding domains,” as de ⁇ ned above.
  • certain binding molecules provided in this disclosure are “pentameric” or “hexameric,” and include ⁇ ve or six bivalent binding units that include IgM constant regions or multimerizing fragments or variants thereof.
  • a binding molecule e.g., an antibody or antibody-like molecule or antibody-derived binding molecule, comprising two or more, e.g., two, ⁇ ve, or six binding units, is referred to herein as “multimeric.”
  • J-chain refers to the J-chain of IgM or IgA antibodies of any animal species, any functional fragment thereof, derivative thereof, and/or variant thereof, including a mature human J-chain, the amino acid sequence of which is presented as SEQ ID NO: 4.
  • Various J-chain variants and modi ⁇ ed J-chain derivatives are available, e.g., in U.S. Patent No. 10,899,835.
  • a functional fragment or “a functional variant” includes those fragments and variants that can associate with IgM heavy chain constant regions to form a pentameric IgM antibody.
  • modified J-chain is used herein to refer to a derivative of a J-chain polypeptide comprising a heterologous moiety, e.g., a heterologous polypeptide, e.g., an extraneous binding domain or functional domain introduced into or attached to the J-chain sequence.
  • the introduction can be achieved by any means, including direct or indirect fusion of the heterologous polypeptide or other moiety or by attachment through a peptide or chemical linker.
  • modified human J-chain encompasses, without limitation, a native sequence human J-chain comprising the amino acid sequence of SEQ ID NO: 4 or functional fragment thereof, or functional variant thereof, modi ⁇ ed by the introduction of a heterologous moiety, e.g., a heterologous polypeptide, e.g., an extraneous binding domain.
  • a heterologous moiety e.g., a heterologous polypeptide, e.g., an extraneous binding domain.
  • the heterologous moiety does not interfere with e ⁇ cient polymerization of IgM into a pentamer or IgA into a multimer, e.g., a dimer or tetramer, and binding of such polymers to a target.
  • valency refers to the number of binding domains, e.g., antigen-binding domains in a given antibody or in a given binding unit.
  • bivalent As such, the terms “bivalent”, “tetravalent”, and “hexavalent” in reference to a given antibody or multimerizing fragment thereof, denote the presence of two antigen-binding domains, four antigen-binding domains, and six antigen-binding IGM Ref. No.001-061WO1 domains, respectively.
  • a bivalent or multivalent binding molecule e.g., antibody or antibody-derived molecule
  • epitopes e.g., bind to di ⁇ erent epitopes on the same antigen, or bind to entirely di ⁇ erent antigens.
  • epitopes e.g., bind to di ⁇ erent epitopes on the same antigen, or bind to entirely di ⁇ erent antigens.
  • epitopes e.g., bind to di ⁇ erent epitopes on the same antigen, or bind to entirely di ⁇ erent antigens.
  • an epitope can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, can have three-dimensional structural characteristics, and or speci ⁇ c charge characteristics.
  • An epitope is a region of a target that is bound by an antigen- binding domain of an antibody.
  • target is used in the broadest sense to include substances that can be bound by an antibody or antibody-derived molecules, such as an scFv.
  • a target can be, e.g., a polypeptide, a nucleic acid, a carbohydrate, a lipid, or other molecule.
  • a “target” can, for example, be a cell, an organ, or an organism that comprises an epitope that can be bound by an antibody or antibody-derived molecules, such as an scFv.
  • a “target antigen” is a target molecule, e.g., a polypeptide, a nucleic acid, a carbohydrate, a lipid, or other molecule that can be bound by an antibody or antibody- derived molecules, such as an scFv as provided herein.
  • a target antigen can appear on the surface of a cell, e.g., a tumor cell.
  • tumor-speci ⁇ c antigen as used herein is a protein or other cell surface target antigen that is unique to tumor cells, at least at later stages of development of the organism.
  • a “tumor-associated antigen” is a protein or other cell surface target antigen that is not necessarily unique to tumor cells but is typically expressed much more abundantly and/or at higher density on tumor cells than on normal, healthy cells.
  • variable domains of both the variable light (VL) and variable heavy (VH) chain portions determine antigen recognition and speci ⁇ city.
  • constant region domains of the light chain (CL) and the heavy chain e.g., CH1, CH2, CH3, or CH4 confer biological properties such as secretion, transplacental mobility, Fc receptor binding, IGM Ref. No.001-061WO1 complement binding, and the like.
  • the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino- terminus of the antibody.
  • a “full length IgM antibody heavy chain” is a polypeptide that includes, in N- terminal to C terminal direction, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CM1 or C ⁇ 1), an antibody heavy chain constant domain 2 (CM2 or C ⁇ 2), an antibody heavy chain constant domain 3 (CM3 or C ⁇ 3), and an antibody heavy chain constant domain 4 (CM4 or C ⁇ 4) that can include a ⁇ tailpiece.
  • VH antibody heavy chain variable domain
  • CM1 or C ⁇ 1 an antibody heavy chain constant domain 1
  • CM2 or C ⁇ 2 an antibody heavy chain constant domain 2
  • CM3 or C ⁇ 3 an antibody heavy chain constant domain 4
  • variable region(s) allow an antibody or an antibody-derived molecule, such as an scFv to selectively recognize and speci ⁇ cally bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the antigen-binding domain. More speci ⁇ cally, an antigen-binding domain can be de ⁇ ned by three CDRs on each of the VH and VL chains. Certain antibodies form larger structures.
  • IgA can form a molecule that includes two H2L2 binding units and a J-chain covalently connected via disul ⁇ de bonds, which can be further associated with a secretory component
  • IgM can form a pentameric or hexameric molecule that includes ⁇ ve or six H2L2 binding units and optionally a J-chain covalently connected via disul ⁇ de bonds.
  • the six “complementarity determining regions” or “CDRs” present in an antibody antigen-binding domain are short, non-contiguous sequences of amino acids that are speci ⁇ cally positioned to form the antigen-binding domain as the antibody assumes its three-dimensional con ⁇ guration in an aqueous environment.
  • framework regions show less inter- molecular variability.
  • the framework regions largely adopt a ⁇ -sheet conformation and the CDRs form loops which connect, and in some cases form part of, the ⁇ -sheet structure.
  • framework regions act to form a sca ⁇ old that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
  • the antigen-binding domain formed by the positioned CDRs de ⁇ nes a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope.
  • amino acids that make up the CDRs and the framework regions, respectively, can be readily identi ⁇ ed for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been de ⁇ ned in various IGM Ref. No.001-061WO1 di ⁇ erent ways (see, "Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entireties).
  • CDR complementarity determining region
  • Kabat numbering refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest” (1983). Unless use of the Kabat numbering system is explicitly noted, however, consecutive numbering is used for all amino acid sequences in this disclosure. [0060] The Kabat numbering system for the human IgM constant domain can be found in Kabat, et. al.
  • IgM constant regions can be numbered sequentially (i.e., amino acid #1 starting with the ⁇ rst amino acid of the constant region, or by using the Kabat numbering scheme.
  • a comparison of the numbering of two alleles of the human IgM constant region sequentially (presented herein as SEQ ID NO: 1 (allele IGHM*03) and SEQ ID NO: 2 (allele IGHM*04)) and by the Kabat system is set out below. The underlined amino acid residues are not accounted for in IGM Ref.
  • ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019.
  • a binding molecule e.g., an antibody or fragment, variant, or derivative thereof binds to an epitope via its antigen- binding domain, and that the binding entails some complementarity between the antigen- binding domain and the epitope.
  • a binding molecule e.g., antibody, antibody-like, or antibody-derived molecule
  • a binding molecule is said to "speci ⁇ cally bind" to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.
  • spei ⁇ city is used herein to qualify the relative a ⁇ nity by which a certain binding molecule binds to a certain epitope.
  • binding molecule "A” can be deemed to have a higher speci ⁇ city for a given epitope than binding molecule "B,” or binding molecule “A” can be said to bind to epitope "C” with a higher speci ⁇ city than it has for related epitope “D.”
  • the term "a ⁇ nity” refers to a measure of the strength of the binding of an individual epitope with one or more antigen-binding domains, e.g., of an IGM Ref. No.001-061WO1 immunoglobulin molecule. See, e.g., Green ⁇ eld, E.A. (ed.), Antibodies: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 2014.
  • the term "avidity” refers to the overall stability of the complex between a population of antigen- binding domains and an antigen. Avidity is related to both the a ⁇ nity of individual antigen- binding domains in the population with speci ⁇ c epitopes, and also the valencies of the immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity. An interaction between a bivalent monoclonal antibody with a receptor present at a high density on a cell surface would also be of high avidity.
  • Binding molecules e.g., antibodies or fragments, variants, or derivatives thereof as disclosed herein can also be described or speci ⁇ ed in terms of their cross-reactivity.
  • cross-reactivity refers to the ability of a binding molecule, e.g., an antibody or fragment, variant, or derivative thereof, speci ⁇ c for one antigen, to react with a second antigen; a measure of relatedness between two di ⁇ erent antigenic substances.
  • a binding molecule is cross reactive if it binds to an epitope other than the one that induced its formation.
  • Antigen-binding antibody fragments including single-chain antibodies or other antigen-binding domains can exist alone or in combination with one or more of the following: hinge region, CH1, CH2, CH3, or CH4 domains, J-chain, or secretory component. Also included are antigen-binding fragments that can include any combination of variable region(s) with one or more of a hinge region, CH1, CH2, CH3, or CH4 domains, a J-chain, or a secretory component.
  • Binding molecules e.g., antibodies, or antigen-binding fragments thereof can be from any animal origin including birds and mammals.
  • the antibodies can be human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.
  • the variable region can be condricthoid in origin (e.g., from sharks).
  • "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and can in some instances express endogenous immunoglobulins and some not, as described infra and, for example in, U.S. Pat.
  • an IgM antibody as provided IGM Ref. No.001-061WO1 herein can include an antigen-binding fragment of an antibody, e.g., a scFv fragment, so long as the IgM antibody is able to form a multimer, e.g., a hexamer or a pentamer as used herein such a fragment comprises a “multimerizing fragment.”
  • the term “heavy chain subunit” includes amino acid sequences derived from an immunoglobulin heavy chain, a binding molecule, e.g., an antibody, antibody-like, or antibody-derived molecule comprising a heavy chain subunit can include at least one of: a VH domain, a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain
  • a binding molecule e.g., an antibody, antibody-like, or antibody-derived molecule, or fragment, e.g., multimerizing fragment, variant, or derivative thereof can include without limitation, in addition to a VH domain:, a CH1 domain; a CH1 domain, a hinge, and a CH2 domain; a CH1 domain and a CH3 domain; a CH1 domain, a hinge, and a CH3 domain; or a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain.
  • a binding molecule e.g., an antibody, antibody-like, or antibody-derived molecule, or fragment, e.g., multimerizing fragment, variant, or derivative thereof can include, in addition to a VH domain, a CH3 domain and a CH4 domain; or a CH3 domain, a CH4 domain, and a J-chain.
  • a binding molecule e.g., an antibody, antibody-like, or antibody-derived molecule, for use in the disclosure can lack certain constant region portions, e.g., all or part of a CH2 domain.
  • an IgM antibody, IgM-like antibody, or other IgM-derived binding molecule as provided herein comprises su ⁇ cient portions of an IgM heavy chain constant region to allow the IgM antibody, IgM-like antibody, or other IgM-derived binding molecule to form a multimer, e.g., a hexamer or a pentamer.
  • the term “light chain subunit” includes amino acid sequences derived from an immunoglobulin light chain.
  • the light chain subunit includes at least a VL, and can further include a CL (e.g., C ⁇ or C ⁇ ) domain.
  • Binding molecules e.g., antibodies, antibody-like molecules, antibody-derived molecules, antigen-binding fragments, variants, or derivatives thereof, or multimerizing fragments thereof can be described or speci ⁇ ed in terms of the epitope(s) or portion(s) of a target, e.g., a target antigen that they recognize or speci ⁇ cally bind.
  • a target e.g., a target antigen that they recognize or speci ⁇ cally bind.
  • the portion of a target IGM Ref The portion of a target IGM Ref.
  • No.001-061WO1 antigen that speci ⁇ cally interacts with the antigen-binding domain of an antibody is an "epitope," or an "antigenic determinant.”
  • a target antigen can comprise a single epitope or at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen.
  • the term “chimeric antibody” refers to an antibody in which the immunoreactive region or site is obtained or derived from a ⁇ rst species and the constant region (which can be intact, partial, or modi ⁇ ed) is obtained from a second species.
  • the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.
  • multispeci ⁇ c antibody or “bispeci ⁇ c antibody” refer to an antibody, antibody-like, or antibody-derived molecule that has antigen-binding domains for two or more di ⁇ erent epitopes within a single antibody molecule.
  • Other binding molecules in addition to the canonical antibody structure can be constructed with two binding speci ⁇ cities. Epitope binding by bispeci ⁇ c or multispeci ⁇ c antibodies can be simultaneous or sequential.
  • Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispeci ⁇ c antibodies.
  • Bispeci ⁇ c antibodies can also be constructed by recombinant means. (Ströhlein and Heiss, Future Oncol.6:1387-94 (2010); Mabry and Snavely, IDrugs. 13:543-9 (2010)).
  • a bispeci ⁇ c antibody can also be a diabody.
  • engineered antibody refers to an antibody in which a variable domain, constant region, and/or J-chain is altered by at least partial replacement of one or more amino acids. In certain embodiments entire CDRs from an antibody of known speci ⁇ city can be grafted into the framework regions of a heterologous antibody.
  • CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived
  • CDRs can also be derived from an antibody of di ⁇ erent class, e.g., from an antibody from a di ⁇ erent species.
  • An engineered antibody in which one or more "donor" CDRs from a non-human antibody of known speci ⁇ city are grafted into a human heavy or light chain framework region is referred to herein as a "humanized antibody.”
  • not all of the CDRs are replaced with the complete CDRs from the donor variable region and yet the antigen- binding capacity of the donor can still be transferred to the recipient variable domains.
  • engineered includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, nucleic acids, or glycans, or some combination of these techniques).
  • in-frame fusion refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the translational reading frame of the original ORFs.
  • a recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments can be physically or spatially separated by, for example, in-frame linker sequence.
  • polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the "fused" CDRs are co-translated as part of a continuous polypeptide.
  • a "linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which amino acids that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
  • a portion of a polypeptide that is “amino-terminal” or “N-terminal” to another portion of a polypeptide is that portion that comes earlier in the sequential polypeptide chain.
  • a portion of a polypeptide that is “carboxy-terminal” or “C- terminal” to another portion of a polypeptide is that portion that comes later in the sequential polypeptide chain.
  • variable domain is “N-terminal” to the constant region
  • constant region is “C-terminal” to the variable domain.
  • expression refers to a process by which a gene produces a biochemical, for example, a polypeptide.
  • the process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into RNA, e.g., messenger RNA (mRNA), and the translation of such mRNA into polypeptide(s). If the ⁇ nal desired product is a biochemical, IGM Ref. No.001-061WO1 expression includes the creation of that biochemical and any precursors.
  • a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide that is translated from a transcript.
  • Gene products described herein further include nucleic acids with post transcriptional modi ⁇ cations, e.g., polyadenylation, or polypeptides with post translational modi ⁇ cations, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.
  • the “potency” of a binding molecule or a subunit thereof, e.g., an antibody or an individual binding domain of an antibody is a measure of the molecule’s activity expressed in terms of the amount required to produce a desired e ⁇ ect, either in vitro or in vivo.
  • a bivalent antibody speci ⁇ c for CD20 can have a speci ⁇ c potency for killing 50% of CD-20-expressing cells in an in vitro assay, expressed as an EC50 measured either by mass/volume (e.g., in ⁇ g/ml) or as a molar concentration.
  • Two di ⁇ erent anti-CD20 antibodies can have vastly di ⁇ erent potencies, meaning the EC50s of the molecules in a given assay di ⁇ er signi ⁇ cantly. Potency can also refer to the amount of a binding molecule required to achieve a desired result in a subject treated with the molecule, e.g., the amount of the molecule required to slow or stop progression of an autoimmune disorder or to reduce or remove signs or symptoms of an autoimmune disorder. [0077]
  • the use of a generic name of a biologic therapeutic is understood to include any and all biosimilars bearing the generic name, unless speci ⁇ cally stated otherwise.
  • rituximab would include e.g., rituximab, rituximab-pvvr, and rituximab- abbs.
  • Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, lessen the severity of symptoms of, and/or halt or slow the progression of an existing diagnosed pathologic condition or disorder.
  • Terms such as “prevent,” “prevention,” “avoid,” “deterrence,” “prophylactic,” and the like refer to prophylactic or preventative measures that prevent the development of an undiagnosed targeted pathologic condition or disorder.
  • “those in need of treatment” can include those already with the disorder.
  • “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any mammalian subject. In certain embodiments the subject is a subject for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on. IGM Ref.
  • a subject that would bene ⁇ t from therapy refers to a subset of subjects, from amongst all prospective subjects, which would bene ⁇ t from administration of a given therapeutic agent, e.g., a binding molecule such as an antibody, comprising one or more antigen-binding domains.
  • a given therapeutic agent e.g., a binding molecule such as an antibody, comprising one or more antigen-binding domains.
  • binding molecules e.g., antibodies
  • autoimmune disorder refers to a disease or disorder wherein the subject’s immune system pathogenically responds to one or more self-antigens as foreign antigens.
  • autoimmune disorders typically result in damage of otherwise healthy tissues and/or chronic in ⁇ ammation.
  • Methods of treating autoimmune disorders [0082]
  • a method of an autoimmune disorder in a subject e.g., an autoimmune disorder patient in need of treatment, the method comprising administering to the subject a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody, such as a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody disclosed herein.
  • the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody is a multimeric antibody comprising ⁇ ve bivalent binding units and a modi ⁇ ed J-chain.
  • the method comprises administering to the subject a therapeutically e ⁇ ective amount of a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody as provided herein.
  • a therapeutically e ⁇ ective dose or amount or “e ⁇ ective amount” is intended an amount of a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody that when administered brings about a positive therapeutic response with respect to treatment of subject.
  • E ⁇ ective doses of compositions for treatment of a disease or disorder vary depending upon many di ⁇ erent factors, including means of administration, target site, physiological state of the subject, whether the subject is human or an animal, other medications administered, and whether administration is prophylactic or therapeutic.
  • the subject is a human in need of treatment, but non-human mammals including transgenic mammals can also be treated.
  • the autoimmune disorder is a disorder where B lineage cells produce pathogenic autoantibodies and/or contribute to chronic in ⁇ ammation in the subject.
  • the autoimmune disorder is a disorder where T cell lineage cells that express CD20 contribute to the autoimmune disorder. See, e.g., Vlaming et al., 2021, Scienti ⁇ c Reports, 11, 20499.
  • the autoimmune disorder is IGM Ref.
  • the autoimmune disorder is systemic lupus erythematosus (SLE), antiphospholipid syndrome (APS), idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (wAIHA), multiple sclerosis (MS), myasthenia gravis (MG), pemphigus vulgaris (PV), anti-neutrophil cytoplasmic autoantibody (ANCA) associated vasculitis (AAV), thyroid eye disease (TED), membranous glomerulonephritis (MGN), Neuromyelitis optica (NMO), Guillain- Barré syndrome (GBS), chronic in ⁇ ammatory demyelinating polyradiculoneuropathy (CIDP), IgA nephropathy, Goodpasture’s syndrome, rheumatoid arthritis (RA), or scleroderma.
  • SLE systemic lupus erythematosus
  • APS antiphospholipid syndrome
  • IDP idiopathic thrombocytopenic pur
  • the autoimmune disorder is systemic lupus erythematosus (SLE).
  • the SLE comprises lupus nephritis (LN).
  • the autoimmune disorder is RA.
  • the autoimmune disorder is MG.
  • SLE Systemic lupus erythematosus
  • LN Lupus nephritis
  • Antiphospholipid syndrome is an autoimmune disorder characterized by persistent antiphospholipid antibody that leads to thrombosis. Methods of diagnosing APS are known, such as those methods described in Garcia, 2018, N Engl J Med, 378:2010- 2021.
  • Idiopathic thrombocytopenic purpura is an autoimmune disorder where the immune system attacks platelets. ITP is typically characterized by a decrease in platelet counts and bleeding complications. Standard of care therapy is typically corticosteroids, IGM Ref. No.001-061WO1 but other therapy options are available.
  • Warm autoimmune hemolytic anemia is an autoimmune disorder characterized by the destruction of red blood cells (RBCs) by “warm” antibodies. Warm antibodies are polyclonal immunoglobulins that maximally bind RBCs at 37 °C. Methods of diagnosing wAIHA are known, such as those methods described in Jager, 2019, Blood Reviews, doi: 10.1016/j.blre.2019.100648.
  • MS Multiple sclerosis
  • Methods of diagnosing MS are known, such as those methods described in McGinley, 2021, JAMA, 325(8): 765-779.
  • MG Myasthenia gravis
  • Methods of diagnosing MS are known, such as those methods described in Rousse ⁇ , 2021, J Clin Med, 10(1736):1-16.
  • PV Pemphigus vulgaris
  • Methods of diagnosing PV are known, such as those methods described in Vietnamese, 2019, Experimental Therapeutic Medicine, 18(6): 5056-5060.
  • Anti-neutrophil cytoplasmic autoantibody (ANCA) associated vasculitis is characterized by autoantibodies against neutrophils and monocytes, which results in in ⁇ ammation of blood vessels.
  • Methods of diagnosing AAV are known, such as those methods described in Guchelaar, 2021, Autoimmunity Reviews, 20(1): 102716.
  • Thyroid eye disease (TED) is believed to be caused by autoantibodies directed against receptors seen on the extraocular muscles (EOMs) and soft tissues of the orbit. Methods of diagnosing TED are known, such as those methods described in Manjandavida, Huawei J Ophthalmol 2020; 32:10-26.
  • MGN Membranous glomerulonephritis
  • Methods of diagnosing MGN are known, such as those methods described in Maifata, 2019, Biomedicines, 7(86): 1-14.
  • NMO Neuromyelitis optica
  • Methods of diagnosing NMO are known, such as those methods described in Fujihara, 2019, Curr Opin Neurol, 32(3):385-394.
  • GBS Guillain-Barré syndrome
  • CIDP chronic in ⁇ ammatory demyelinating polyradiculoneuropathy
  • Methods of diagnosing CIDP are known, such as those methods described in Stino, 2020, Muscle Nerve, 63(2): 157-169.
  • IgA nephropathy is an autoimmune disorder characterized by the deposition of IgA in the glomerular mesangium. Methods of diagnosing CIDP are known, such as those methods described in Selvaskandan, 2019, Clinical Experimental Nephrology, 23: 577- 588.
  • Goodpasture’s syndrome also called anti-glomerular basement membrane (anti- GBM) or Goodpasture’s disease
  • anti- GBM anti-glomerular basement membrane
  • Goodpasture’s disease is an autoimmune disorder characterized by autoantibodies to type IV collagen antigens in the glomerular and alveolar basement membranes.
  • Methods of diagnosing Goodpasture’s syndrome are known, such as those methods described in Moiseev, et al., 2020, Autoimmunity Reviews, 19(9): 102618.
  • Rheumatoid arthritis (RA) is an autoimmune disorder a ⁇ ecting joints. Methods of diagnosing RA are known, such as those methods described in Lin et al., 2020, Cells, 9(880): 1-43.
  • Scleroderma is an autoimmune disorder characterized by production of autoantibodies, ⁇ broblast dysfunction, small vessel vasculopathy. Methods of diagnosing scleroderma are known, such as those methods described in van den Hoogen et al., 2013, Arthritis and Rheumatism, 65(11): 2737-2747.
  • GPA polyangiitis
  • MPA Microscopic polyangiitis
  • Sjögren syndrome is an autoimmune disorder characterized by lymphocytic in ⁇ ltrate of the exocrine glands. Methods of diagnosing Sjögren syndrome are known, such as those methods described in Thorne et al., 2017, British Journal of Hospital Medicine, 78(8): 438- 442.
  • Behcet’s disease is an autoimmune disorder characterized by blood vessel in ⁇ ammation.
  • Alopecia areata is an autoimmune disorder characterized by hair loss. Methods of diagnosing alopecia areata are known, such as those methods described in Finner, 2011, Dermatologic Therapy, 24: 348-354.
  • Immunoglobulin G4-related disease (IgG4-RD) is an autoimmune disorder characterized by elevated serum IgG4 levels.
  • Phospholipase A2 receptor-associated membranous nephropathy is an autoimmune disorder characterized by anti-PLA2R autoantibodies.
  • Methods of diagnosing PLA2R MN are known, such as those methods described in VanBeek et al., 2015, Clinical Nephrology, 84: 1-9.
  • Myositis is an autoimmune disorder characterized by muscle weakness and includes four subtypes: dermatomyositis (DM), polymyositis (PM), necrotizing myopathy (NM), and inclusion body myositis (IBM). Methods of diagnosing myositis are known, such as those methods described in Carstens et al., 2014, Clinical and Experimental Immunology, 175: 425-438.
  • Type 1 diabetes is an autoimmune disorder characterized by pancreatic ⁇ -cell loss resulting in insulin de ⁇ ciency. Methods of diagnosing type 1 diabetes are known, such as those methods described in Katsarou et al., Nature Reviews Disease Primers, 3(17016):1- 17.
  • Systemic sclerosis is an autoimmune disorder characterized by vascular abnormalities. Methods of diagnosing systemic are known, such as those methods described in Hudson et al., 2014, Journal of Autoimmunity, 48-49: 38-41. [0112] In some embodiments, the subject did not receive any prior autoimmune disorder therapies. In some embodiments, the subject had previously received a prior autoimmune disorder therapy. In some embodiments, the subject was resistant and/or refractory to the prior autoimmune disorder. In some embodiments, the prior autoimmune disorder therapy comprises a biologic. In some embodiments, the biologic is an anti-CD20 antibody, such as rituximab.
  • the prior autoimmune disorder therapy comprises an anti-B-lymphocyte stimulator (BlyS) antibody, e.g., belimumab.
  • Belimumab (sold under the tradename BENLYSTA®) is US FDA approved to treat SLE. See BENLYSTA® Prescribing Information, revised 7/2022.
  • the prior autoimmune disorder therapy comprises a TNF inhibitor such as in ⁇ iximab, adalimumab, or etanercept.
  • In ⁇ iximab (sold under the IGM Ref. No.001-061WO1 tradename REMICADE®) is US FDA approved to treat rheumatoid arthritis.
  • Adalimumab (sold under the tradename HUMIRA®) is US FDA approved to treat rheumatoid arthritis. See HUMIRA® Prescribing Information, revised 2/2021.
  • Etanercept (sold under the tradename ENBREL®) is US FDA approved to treat rheumatoid arthritis. See ENBREL ® Prescribing Information, revised 12/2012. [0114] In some embodiments, the subject previously received rituximab.
  • a method of treating an autoimmune disorder in a subject in need thereof comprises administering a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody disclosed herein to the subject, where the subject had previously been administered rituximab.
  • the administering of the multimeric bispeci ⁇ c anti- CD20/anti-CD3 antibody occurs at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 9 months, at least 1 year, or at least 2 years after the prior autoimmune disorder therapy was administered.
  • the administering of the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody occurs less than 2 years, e.g., less than 1 year, less than 9 months, less than 6 months, less than 3 months, less than two months, or less than 1 month after the prior autoimmune disorder therapy was administered. In some embodiments, the administering of the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody occurs 1 week to 2 years after the prior autoimmune disorder therapy was administered, such as 2 weeks to 1 year, 1 month to 1 year, 2 months to 1 year, 3 months to 1 year, 6 months to 1 year, or 9 months to 1 year.
  • administration of the multimeric bispeci ⁇ c anti-CD20/anti- CD3 antibody as employed herein to a subject is more potent than administration of an equivalent amount of a reference anti-CD20 antibody.
  • an equivalent amount is meant, e.g., an amount measured by molecular weight, e.g., in total milligrams, or alternatively, a molar equivalent, e.g., where equivalent numbers of molecules are administered.
  • the reference anti-CD20 antibody is rituximab.
  • the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody kills a greater percentage of B cells than the reference anti-CD20 antibody.
  • the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody kills a greater percentage of B cells than the reference anti-CD20 antibody in the blood and/or secondary or tertiary lymphoid organs including but not limited to spleen, mesenteric lymph node, bone marrow, or combinations thereof.
  • the multimeric bispeci ⁇ c IGM Ref. No.001-061WO1 anti-CD20/anti-CD3 antibody kills a greater percentage of activated memory B cells than the reference anti-CD20 antibody.
  • the multimeric bispeci ⁇ c anti- CD20/anti-CD3 antibody kills a greater percentage of activated memory B cells than the reference anti-CD20 antibody in the blood and/or secondary or tertiary lymphoid organs including but not limited to spleen, mesenteric lymph node, bone marrow, or combinations thereof.
  • a preparation to be administered to a subject is a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody as described herein administered in a conventional dosage form, which can be combined with a pharmaceutical excipient, carrier or diluent as described elsewhere herein, such as in a composition described herein.
  • a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody as described herein can be administered by any suitable method, e.g., parenterally, intraventricularly, orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • administration of the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody comprises intravenous infusion.
  • administration of the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody comprises subcutaneous administration.
  • a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody as described herein in the manufacture of a medicament to treat an autoimmune disorder.
  • a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody as described herein or a pharmaceutical composition comprising the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody as described herein, in a method disclosed herein.
  • the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody administered in the methods disclosed herein is part of a composition, e.g., a pharmaceutical composition.
  • a composition as provided herein can further include a pharmaceutically acceptable carrier and/or excipient and can be formulated so as to be suitable for a desired mode of administration.
  • Methods of preparing a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody employed in the methods as provided herein can be determined by a skilled person in view of this disclosure.
  • a suitable pharmaceutical composition can include a bu ⁇ er (e.g., acetate, IGM Ref.
  • a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody as provided herein can be administered in a pharmaceutically e ⁇ ective amount for the treatment of a subject in need thereof.
  • the multimeric bispeci ⁇ c anti-CD20/anti- CD3 antibody can be formulated so as to facilitate administration and promote stability of the active agent.
  • the “e ⁇ ective amount” of the provided multimeric bispeci ⁇ c anti- CD20/anti-CD3 antibody to treat an autoimmune disorder according to methods provided in this disclosure can be determined, e.g., by a healthcare provider upon assessing the severity and nature of the subject’s autoimmune disorder.
  • exemplary dosing and dosing schedules for treating B-cell-related cancers e.g., non-Hodgkin’s lymphoma with imvotamab (IGM-2323), can be found, e.g., in PCT Publication No. WO2022/109023, which is incorporated by reference herein in its entirety.
  • compositions accordingly can include a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic bu ⁇ ers, preservatives, and the like. Suitable formulations are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 23rd ed. (2020). [0125] Certain pharmaceutical compositions provided herein can be orally administered in an acceptable dosage form including, e.g., capsules, tablets, aqueous suspensions, or solutions. [0126] Certain pharmaceutical compositions also can be administered by nasal aerosol or inhalation.
  • compositions can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.
  • the pharmaceutical composition is administered by nasal aerosol.
  • the pharmaceutical composition is for administration by nasal aerosol.
  • the pharmaceutical composition such as a pharmaceutical composition for administration by nasal aerosol, comprises a pH adjuster, such as HCl; a bu ⁇ er; an emulsi ⁇ er, such as polysorbate or carbomer; sugar or mono- or polyol, such as a monosaccharide (e.g., glucose, dextrose, or fructose), disaccharide (e.g., sucrose, lactose, or maltose), ribose, glycerine, sorbitol, xylitol, inositol, propylene glycol, galactose, mannose, xylose, rhamnose, glutaraldehyde, ethanol, mannitol, polyethylene glycol, glycerol, chitosal, phenylethyl alcohol; a preservative; cellulose, such as microcrystalline cellulose or carboxymethylcellulose; or mixtures thereof.
  • a pH adjuster such as HCl
  • the pharmaceutical composition is administered by inhalation.
  • the pharmaceutical composition is for administration by inhalation.
  • the pharmaceutical composition such as a pharmaceutical composition for administration by inhalation, is a dry powder, such as for a dry powder inhaler, or a liquid, such as for a nebulizer, such as an airjet-compressor nebulizer or a mesh-based nebulizer.
  • the pharmaceutical composition such as a pharmaceutical composition for administration by inhalation, comprises sugar or mono- or polyol, such as lactose, trelose, mannitol, sorbitol; bu ⁇ er, such as histidine, proline, or arginine bu ⁇ er; saline; polysorbate; or mixtures thereof.
  • sugar or mono- or polyol such as lactose, trelose, mannitol, sorbitol
  • bu ⁇ er such as histidine, proline, or arginine bu ⁇ er
  • saline polysorbate
  • the amount of a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody that can be combined with carrier materials to produce a single dosage form will vary depending, e.g., upon the subject treated and the particular mode of administration.
  • the composition can be administered as a single dose, multiple doses or over an established period of time in an infusion.
  • a multimeric bispeci ⁇ c anti- CD20/anti-CD3 antibody as described herein can be administered to a subject in need of therapy in an amount su ⁇ cient to produce a therapeutic e ⁇ ect.
  • a molecule or cells as provided herein can be administered to the subject in a conventional dosage form prepared by combining the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody described herein with a conventional pharmaceutically acceptable carrier or diluent according to known techniques.
  • Multimeric Bispeci ⁇ c Anti-CD20/Anti-CD3 Antibodies [0130] Antibodies and antibody-like molecules that can multimerize, such as IgM and IgA antibodies, have emerged as promising drug candidates, e.g., in the ⁇ elds of immuno- oncology and infectious diseases, allowing for improved speci ⁇ city, improved avidity, and the ability to bind to multiple binding targets. See, e.g., U.S. Patent Nos.
  • each binding unit comprises two IgM heavy chains, each comprising a heavy chain variable region (VH) and an IgM constant region or multimerizing fragment or variant thereof, and two light chains, each comprising a light chain variable region (VL) and a light chain constant region.
  • VH heavy chain variable region
  • VL light chain variable region
  • each binding unit comprises the VH is situated amino terminal to the heavy chain constant region.
  • the VL is situated amino terminal to the light chain constant region, e.g., a kappa or lambda constant region.
  • the VH and VL combine to form a CD20 binding domain.
  • the VH comprises three immunoglobulin complementarity determining regions: HCDR1, HCDR2, and HCDR3, and the VL comprises three immunoglobulin complementarity determining regions: LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise, respectively, the amino acid sequence of SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; and SEQ ID NO: 63.
  • the VH comprises SEQ ID NO: 64.
  • the VL comprises SEQ ID NO: 65.
  • the modi ⁇ ed J-chain comprises SEQ ID NO: 29.
  • the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody is imvotamab. [0133] Imvotamab is an IgM-based bispeci ⁇ c antibody with ten CD20 binding domains and one CD3 binding domain. Imvotamab triggers a cytotoxic T-lymphocyte (CTL) response and complement-dependent cytotoxicity (CDC) against CD20-expressing cells.
  • CTL cytotoxic T-lymphocyte
  • CDC complement-dependent cytotoxicity
  • Imvotamab has been shown to be active in non-Hodgkin’s lymphoma (NHL) patients with a highly favorable safety pro ⁇ le, including low rates of grade 2 or higher cytokine release syndrome (CRS), no ICANS, and minimal neutropenia.
  • CNS cytokine release syndrome
  • CCS cytokine release syndrome
  • minimal neutropenia See Budde, et al., “A Phase 1 Dose Escalation Study of IGM-2323, a Novel Anti-CD20 x Anti-CD3 IgM T Cell Engager (TCE) in Patients with Advanced B-Cell Malignancies,” 63rd American Society of Hematology (ASH) Annual Meeting and Exposition – December 11-14, 2021 – Session: 623. Mantle Cell, Follicular, and Other B-Cell Lymphomas.
  • IgM is the ⁇ rst immunoglobulin produced by B cells in response to stimulation by antigen. IgM is naturally present at around 1.5 mg/ml in serum with a half-life of about 5 days. IgM is typically a pentameric or hexameric molecule and thus includes ⁇ ve or six IGM Ref. No.001-061WO1 binding units. An IgM binding unit typically includes two light and two heavy chains.
  • IgG heavy chain constant region contains three heavy chain constant domains (CH1, CH2 and CH3)
  • the heavy ( ⁇ ) constant region of IgM additionally contains a fourth constant domain (CH4) and includes a C-terminal “tailpiece.”
  • the human IgM constant region typically comprises the amino acid sequence SEQ ID NO: 1 (identical to, e.g., GenBank Accession Nos. pir
  • the human C ⁇ 1 region ranges from about amino acid 5 to about amino acid 102 of SEQ ID NO: 1 or SEQ ID NO: 2; the human C ⁇ 2 region ranges from about amino acid 114 to about amino acid 205 of SEQ ID NO: 1 or SEQ ID NO: 2, the human C ⁇ 3 region ranges from about amino acid 224 to about amino acid 319 of SEQ ID NO: 1 or SEQ ID NO: 2, the C ⁇ 4 region ranges from about amino acid 329 to about amino acid 430 of SEQ ID NO: 1 or SEQ ID NO: 2, and the tailpiece ranges from about amino acid 431 to about amino acid 453 of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the precursor form of the human J-chain is presented as SEQ ID NO: 3.
  • the signal peptide extends from amino acid 1 to about amino acid 22 of SEQ ID NO: 3
  • the mature human J-chain extends from about amino acid 23 to amino acid 159 of SEQ ID NO: 3.
  • the mature human J-chain includes the amino acid sequence SEQ ID NO: 4. [0137] Exemplary variant and modi ⁇ ed J-chains are provided elsewhere herein. Without the J-chain, an IgM antibody or IgM-like antibody typically assembles into a hexamer, comprising up to twelve antigen-binding domains.
  • an IgM antibody or IgM- like antibody typically assembles into a pentamer, comprising up to ten antigen-binding domains, or more, if the J-chain is a modi ⁇ ed J-chain comprising one or more heterologous polypeptides comprising additional antigen-binding domain(s).
  • the assembly of ⁇ ve or six IgM binding units into a pentameric or hexameric IgM antibody or IgM-like antibody is IGM Ref. No.001-061WO1 thought to involve the C ⁇ 4 and ⁇ tailpiece domains. See, e.g., Braathen, R., et al., J. Biol. Chem.
  • a pentameric or hexameric IgM antibody typically includes at least the C ⁇ 4 and ⁇ tailpiece domains.
  • a “multimerizing fragment” of an IgM heavy chain constant region thus includes at least the C ⁇ 4 and ⁇ tailpiece domains.
  • An IgM heavy chain constant region can additionally include a C ⁇ 3 domain or a fragment thereof, a C ⁇ 2 domain or a fragment thereof, a C ⁇ 1 domain or a fragment thereof, and/or other IgM heavy chain domains.
  • a multimeric bispeci ⁇ c anti- CD20/anti-CD3 antibody as described herein comprises a complete IgM heavy ( ⁇ ) chain constant domain, e.g., SEQ ID NO: 1 or SEQ ID NO: 2, or a variant, derivative, or analog thereof, e.g., as provided herein.
  • the two IgM heavy chain constant regions included in each binding unit are human heavy chain constant regions.
  • the heavy chains are glycosylated.
  • the heavy chains can be mutated to a ⁇ ect glycosylation.
  • the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody employed in the methods provided herein comprises a modi ⁇ ed J-chain comprising a J-chain or functional fragment or variant thereof associated with, e.g., directly or indirectly fused to an scFv molecule that speci ⁇ cally binds to CD3.
  • the modi ⁇ ed J-chain of a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody employed in the methods provided herein comprises a naturally occurring J-chain, such as a mature human J-chain sequence (e.g., SEQ ID NO: 4).
  • the modi ⁇ ed J-chain of a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody employed in the methods provided herein can comprise a variant J-chain, such as a variant sequence described herein with reduced glycosylation or reduced binding to one or more polymeric Ig receptors (e.g., pIgR, Fc alpha-mu receptor (Fc ⁇ R), or Fc mu receptor (Fc ⁇ R)).
  • polymeric Ig receptors e.g., pIgR, Fc alpha-mu receptor (Fc ⁇ R), or Fc mu receptor (Fc ⁇ R)
  • the modi ⁇ ed J-chain of a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody employed in the methods provided herein can comprise a functional fragment of a naturally occurring or variant J-chain.
  • a functional fragment or a “functional variant” in this context includes those fragments and variants that can associate with binding units to form a pentameric IgM antibody and/or can associate with certain immunoglobulin receptors, e.g., pIgR. IGM Ref.
  • the modi ⁇ ed J-chain can be mutated or otherwise engineered to a ⁇ ect, e.g., enhance, the serum half-life of the multimeric bispeci ⁇ c anti- CD20/anti-CD3antibody.
  • the J-chain can be mutated to a ⁇ ect glycosylation.
  • the modi ⁇ ed J-chain of the multimeric bispeci ⁇ c anti- CD20/anti-CD3 antibody employed in the methods provided herein comprises a functional variant J-chain that includes one or more single amino acid substitutions, deletions, or insertions relative to a reference J-chain identical to the variant J-chain except for the one or more single amino acid substitutions, deletions, or insertions.
  • certain J- chain amino acid substitutions, deletions, or insertions can result in a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody exhibiting an increased serum half-life upon administration to a subject animal relative to a reference multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody that is identical except for the one or more single amino acid substitutions, deletions, or insertions in the variant J-chain, and is administered using the same method to the same animal species.
  • the variant J-chain can include one, two, three, or four single amino acid substitutions, deletions, or insertions relative to the reference J-chain.
  • the modi ⁇ ed J-chain of a multimeric bispeci ⁇ c anti- CD20/anti-CD3 antibody employed in the methods provided herein comprises a variant J- chain comprising an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J-chain (SEQ ID NO: 4).
  • an amino acid corresponding to amino acid Y102 of the mature wild-type human J-chain is meant the amino acid in the sequence of the J-chain, which is homologous to Y102 in the human J-chain.
  • US Patent No. 10,899,835 which is incorporated herein by reference in its entirety.
  • the position corresponding to Y102 in SEQ ID NO: 4 is conserved in the J-chain amino acid sequences of at least 43 other species. See FIG. 4 of U.S. Patent No.9,951,134, which is incorporated by reference herein. Certain mutations at the position corresponding to Y102 of SEQ ID NO: 4 can inhibit the binding of IgM pentamers comprising the variant J-chain to certain immunoglobulin receptors, e.g., the human or murine Fc ⁇ receptor, the murine Fc ⁇ receptor, and/or the human or murine polymeric Ig receptor (pIgR).
  • immunoglobulin receptors e.g., the human or murine Fc ⁇ receptor, the murine Fc ⁇ receptor, and/or the human or murine polymeric Ig receptor (pIgR).
  • a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody comprising a J-chain mutation at the amino acid corresponding to Y102 of SEQ ID NO: 4 has an improved serum half-life when administered to an animal than a corresponding multimeric bispeci ⁇ c anti- IGM Ref. No.001-061WO1 CD20/anti-CD3 antibody that is identical except for the substitution, and which is administered to the same species in the same manner.
  • the amino acid corresponding to Y102 of SEQ ID NO: 4 can be substituted with any amino acid.
  • the amino acid corresponding to Y102 of SEQ ID NO: 4 can be substituted with alanine (A), serine (S) or arginine (R).
  • the amino acid corresponding to Y102 of SEQ ID NO: 4 can be substituted with alanine.
  • the J-chain or functional fragment or variant thereof is a variant human J-chain referred to herein as “J*,” and comprises the amino acid sequence SEQ ID NO: 5.
  • Wild-type J-chains typically include one N-linked glycosylation site.
  • a variant J-chain or functional fragment thereof of a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody described herein includes a mutation within the asparagine(N)-linked glycosylation motif N-X1-S/T, e.g., starting at the amino acid position corresponding to amino acid 49 (motif N6) of the mature human J-chain (SEQ ID NO: 4) or J* (SEQ ID NO: 5), where N is asparagine, X1 is any amino acid except proline, and S/T is serine or threonine, and where the mutation prevents glycosylation at that motif.
  • N asparagine
  • X1 is any amino acid except proline
  • S/T is serine or threonine
  • J-chain mutations preventing glycosylation at this site can result in a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody exhibiting an increased serum half-life upon administration to a subject animal relative to a reference multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody that is identical except for the mutation or mutations preventing glycosylation in the variant J-chain and is administered in the same way to the same animal species.
  • the variant J-chain or functional fragment thereof can include an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of SEQ ID NO: 4 or SEQ ID NO: 5, provided that the amino acid corresponding to S51 is not substituted with threonine (T), or where the variant J-chain comprises amino acid substitutions at the amino acid positions corresponding to both amino acids N49 and S51 of SEQ ID NO: 4 or SEQ ID NO: 5.
  • T threonine
  • the position corresponding to N49 of SEQ ID NO: 4 or SEQ ID NO: 5 is substituted with any amino acid, e.g., alanine (A), glycine (G), threonine (T), serine (S) or aspartic acid (D).
  • the position corresponding to N49 of SEQ ID NO: 4 or SEQ ID NO: 5 can be substituted with alanine (A).
  • the position corresponding to N49 of SEQ ID NO: 4 or SEQ ID NO: 5 can be substituted with aspartic acid (D).
  • the position corresponding to S51 of SEQ ID NO: 4 or IGM Ref is substituted with any amino acid, e.g., alanine (A), glycine (G), threonine (T), serine (S) or aspartic acid (D).
  • the position corresponding to N49 of SEQ ID NO: 4 or SEQ ID NO: 5 can be substituted with alanine (A).
  • the modi ⁇ ed J-chain of the multimeric bispeci ⁇ c anti- CD20/anti-CD3 antibody employed in the methods provided herein comprises an scFv molecule that speci ⁇ cally binds to CD3 and can further comprise a heterologous moiety, e.g., a polypeptide, without interfering with the ability of the antibody to assemble and bind CD20. See U.S. Patent Nos.
  • the scFv and/or heterologous moiety such as a polypeptide of the modi ⁇ ed J-chain of the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody employed in the methods provided herein is introduced, e.g., fused or chemically conjugated, into the J-chain or fragment or variant thereof.
  • the heterologous moiety is a heterologous polypeptide.
  • the scFv and/or the heterologous polypeptide can be fused to the N-terminus of the J-chain or functional fragment or variant thereof, the C-terminus of the J-chain or functional fragment or variant thereof, or to both the N-terminus and C-terminus of the J-chain or functional fragment or variant thereof.
  • the scFv and/or the heterologous polypeptide can be fused internally within the J-chain or functional fragment or variant thereof.
  • the scFv and/or the heterologous polypeptide can be introduced into the J- chain at or near a glycosylation site.
  • the scFv and/or the heterologous polypeptide can be introduced into the J-chain within about 10 amino acid residues from the C-terminus, or within about 10 amino acids from the N-terminus. In certain embodiments, the scFv and/or the heterologous polypeptide can be introduced into the mature human J-chain of SEQ ID NO: 4 between cysteine residues 92 and 101 of SEQ ID NO: 4, or an equivalent location in a J-chain sequence, e.g., a J-chain variant or functional fragment of a J-chain.
  • the scFv and/or heterologous polypeptide can be introduced into the mature human J-chain of SEQ ID NO: 4 at or near a glycosylation site. In a further embodiment, the scFv and/or heterologous polypeptide can be introduced into the mature human J-chain of SEQ ID NO: 4 within about 10 amino acid residues from the C-terminus, or within about 10 amino acids from the N-terminus.
  • the scFv and/or the heterologous polypeptide of the modi ⁇ ed J-chain of the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody employed in the methods provided herein is fused in frame to the J-chain or chemically conjugated to IGM Ref. No.001-061WO1 the J-chain or fragment or variant thereof.
  • the scFv and/or heterologous polypeptide is fused to the J-chain or functional fragment thereof via a peptide linker.
  • Any suitable linker can be used, for example the peptide linker can include at least 5 amino acids, at least ten amino acids, and least 20 amino acids, at least 30 amino acids or more, and so on.
  • the peptide linker includes least 5 amino acids, but no more than 25 amino acids. In certain embodiments the peptide linker can consist of 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, or 25 amino acids. In certain embodiments, the peptide linker consists of GGGGS (SEQ ID NO: 6), GGGGSGGGGS (SEQ ID NO: 7), GGGGSGGGGSGGGGS (SEQ ID NO: 8), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 9), or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 10).
  • Heterologous moieties to be attached to a J-chain can include, without limitation, a binding moiety, e.g., an antibody or antigen-binding fragment thereof, e.g., a single chain Fv (scFv) molecule, a stabilizing peptide that can increase the half-life of the binding molecule, e.g., human serum albumin (HSA) or an HSA binding molecule, or a heterologous chemical moiety such as a polymer.
  • the binding domain, e.g., scFv fragment can speci ⁇ cally bind to CD3.
  • the binding domain e.g., scFv fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), where the VH and VL comprise six immunoglobulin complementarity determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the CDRs of an antibody comprising the VH and VL of SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, or SEQ ID NO: 23 and SEQ ID NO: 24, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • the VH and VL comprise six immunoglobulin complementarity determining regions HCDR1,
  • the scFv comprises an antibody VH and a VL, where the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, or SEQ ID NO: 23 and SEQ ID NO: 24, respectively.
  • the scFv comprises the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 43, SEQ ID NO: 48, or SEQ ID NO: 53. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 26.
  • the modi ⁇ ed J-chain comprises the scFv, which comprises the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 43, SEQ ID NO: 48, or SEQ ID NO: 53 and a J chain comprising SEQ ID NO: 4 or SEQ ID NO: 5.
  • the modi ⁇ ed J-chain comprises SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 54, or SEQ ID NO: 55.
  • the modi ⁇ ed J-chain comprises SEQ ID NO: 27.
  • the modi ⁇ ed J-chain of a multimeric bispeci ⁇ c anti- CD20/anti-CD3 antibody employed in the methods provided herein comprises a half-life extending moiety.
  • the half-life extending moiety is an albumin, such as a human serum albumin (HSA).
  • HSA human serum albumin
  • the modi ⁇ ed J-chain comprises SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, or SEQ ID NO: 57.
  • the modi ⁇ ed J-chain comprises SEQ ID NO: 29.
  • IgM heavy chain constant regions of a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody employed in the methods provided herein can be engineered to confer certain desirable properties to the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibodies described herein.
  • IgM heavy chain constant regions can be engineered to confer enhanced serum half-life to multimeric bispeci ⁇ c anti-CD20/anti- CD3 antibodies as described herein.
  • Exemplary IgM heavy chain constant region mutations that can enhance serum half-life of multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibodies are disclosed in see US Patent No. 10,899,835, which is incorporated by reference herein in its entirety.
  • a variant IgM heavy chain constant region of a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody described herein can include an amino acid substitution at a position corresponding to amino acid S401, E402, E403, R344, and/or E345 of a wild-type human IgM constant region (e.g., SEQ ID NO: 1 or SEQ ID NO: 2).
  • an amino acid corresponding to amino acid S401, E402, E403, R344, and/or E345 of a wild-type human IgM constant region is meant the amino acid in the sequence of the IgM constant region of any species which is homologous to S401, E402, E403, R344, and/or E345 in the human IgM constant region.
  • the amino acid corresponding to S401, E402, E403, R344, and/or E345 of SEQ ID NO: 1 or SEQ ID NO: 2 can be substituted with any amino acid, e.g., alanine.
  • the IgM heavy chain constant regions of a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody employed in the methods provided herein can be engineered to exhibit reduced complement-dependent cytotoxicity (CDC) activity to cells in the presence of complement, relative to a reference multimeric bispeci ⁇ c anti- CD20/anti-CD3 antibody with corresponding reference human IgM constant regions identical, except for the mutations conferring reduced CDC activity.
  • CDC complement-dependent cytotoxicity
  • corresponding reference human IgM constant region is meant a human IgM constant region that is identical to the variant IgM constant region except for the modi ⁇ cation or modi ⁇ cations in the constant region a ⁇ ecting CDC activity.
  • the variant human IgM constant region includes one or more amino acid substitutions, e.g., in the C ⁇ 3 domain, relative to a wild-type human IgM constant region as described, e.g., in US Patent No.11,401,337, which is incorporated herein by reference in its entirety.
  • Assays for measuring CDC are well known to those of ordinary skill in the art, and exemplary assays are described e.g., in US Patent No.11,401,337.
  • a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position L310, P311, P313, and/or K315 of SEQ ID NO: 1 (human IgM constant region allele IGHM*03) or SEQ ID NO: 2 (human IgM constant region allele IGHM*04).
  • a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position P311 of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the variant IgM constant region as provided herein contains an amino acid substitution corresponding to the wild-type human IgM constant region at position P313 of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the variant IgM constant region as provided herein contains a combination of substitutions corresponding to the wild-type human IgM constant region at positions P311 of SEQ ID NO: 1 or SEQ ID NO: 2 and P313 of SEQ ID NO: 1 or SEQ ID NO: 2.
  • These proline residues can be independently substituted IGM Ref. No.001-061WO1 with any amino acid, e.g., with alanine, serine, or glycine.
  • a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position K315 of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the lysine residue can be independently substituted with any amino acid, e.g., with alanine, serine, glycine, or aspartic acid.
  • a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position K315 of SEQ ID NO: 1 or SEQ ID NO: 2 with aspartic acid.
  • Human and certain non-human primate IgM constant regions typically include ⁇ ve (5) naturally occurring asparagine (N)-linked glycosylation motifs or sites.
  • N-linked glycosylation motif comprises or consists of the amino acid sequence N-X1- S/T, where N is asparagine, X1 is any amino acid except proline (P), and S/T is serine (S) or threonine (T).
  • the glycan is attached to the nitrogen atom of the asparagine residue. See, e.g., Drickamer K, Taylor ME (2006), Introduction to Glycobiology (2nd ed.). Oxford University Press, USA.
  • N-linked glycosylation motifs occur in the human IgM heavy chain constant regions of SEQ ID NO: 1 or SEQ ID NO: 2 starting at positions 46 (“N1”), 209 (“N2”), 272 (“N3”), 279 (“N4”), and 440 (“N5”). These ⁇ ve motifs are conserved in non- human primate IgM heavy chain constant regions, and four of the ⁇ ve are conserved in the mouse IgM heavy chain constant region. Accordingly, in some embodiments, IgM heavy chain constant regions of a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody employed in the methods provided herein comprise 5 N-linked glycosylation motifs: N1, N2, N3, N4, and N5.
  • At least three of the N-linked glycosylation motifs (e.g., N1, N2, and N3) on each IgM heavy chain constant region are occupied by a complex glycan.
  • at least one, at least two, at least three, or at least four of the N- X1-S/T motifs can include an amino acid insertion, deletion, or substitution that prevents glycosylation at that motif.
  • the IgM-derived multimeric binding molecule can include an amino acid insertion, deletion, or substitution at motif N1, motif N2, motif N3, motif N5, or any combination of two or more, three or more, or all four of motifs N1, N2, N3, or N5, where the amino acid insertion, deletion, or substitution prevents glycosylation at that motif.
  • the IgM constant region comprises one or more substitutions relative to a wild-type human IgM constant region at positions 46, 209, 272, or 440 of SEQ ID NO: 1 (human IgM constant region allele IGHM*03) or SEQ ID NO: 2 (human IgM constant region allele IGHM*04). See, e.g., PCT Publication No.
  • the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibodies used in methods disclosed herein can be produced from a host cell comprising one or more vectors encoding the polypeptide subunits of a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody described herein using techniques known in the art.
  • One vector can comprise nucleotide sequences encoding the heavy chain, light chain, and J-chain.
  • two or three vectors can encode any combination of the heavy chain, light chain, and J-chain, such as one vector encoding the heavy chain and light chain, and a second vector encoding the J-chain or three vectors, one encoding the heavy chain, one encoding the light chain, and one encoding the J-chain.
  • This disclosure employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Green and Sambrook, ed.
  • Embodiment 1 A method of treating an autoimmune disorder comprising administering to a subject in need of treatment an e ⁇ ective amount of a multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody, which comprises ⁇ ve bivalent binding units and a modi ⁇ ed J-chain, wherein each binding unit comprises two IgM heavy chains, each comprising a heavy chain variable region (VH) and an IgM constant region or multimerizing fragment or variant thereof, and two light chains, each comprising a light chain variable region (VL) and a light chain constant region, wherein the VH comprises three immunoglobulin complementarity determining regions: HCDR1, HCDR2, and HCDR3, and the VL comprises three immunoglobulin complementarity determining regions: LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR
  • Embodiment 2 The method of embodiment 1, wherein the autoimmune disorder is a disorder wherein B lineage cells produce pathogenic autoantibodies and/or contribute to chronic in ⁇ ammation in the subject.
  • Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the autoimmune disorder is systemic lupus erythematosus (SLE), antiphospholipid syndrome (APS), idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (wAIHA), multiple sclerosis (MS), myasthenia gravis (MG), pemphigus vulgaris (PV), anti-neutrophil cytoplasmic autoantibody (ANCA) associated vasculitis (AAV), thyroid IGM Ref.
  • SLE systemic lupus erythematosus
  • APS antiphospholipid syndrome
  • ITP idiopathic thrombocytopenic purpura
  • wAIHA warm autoimmune hemolytic anemia
  • MS myasthenia gravis
  • MG
  • No.001-061WO1 eye disease TED
  • membranous glomerulonephritis MGN
  • Neuromyelitis optica NMO
  • Guillain-Barré syndrome GSS
  • chronic in ⁇ ammatory demyelinating polyradiculoneuropathy CIDP
  • IgA nephropathy Goodpasture’s syndrome
  • GPA granulomatosis with polyangiitis
  • MPA microscopic polyangiitis
  • Sjögren syndrome Sjögren syndrome
  • Behcet’s disease alopecia areata
  • immunoglobulin G4-related disease IgG4-RD
  • PKA2R MN phospholipase A2 receptor-associated membranous nephropathy
  • myositis type 1 diabetes, systemic sclerosis, or rheumatoid arthritis (RA).
  • Embodiment 4 The method of any one of embodiments 1 to 3, wherein the autoimmune disorder is SLE.
  • Embodiment 5. The method of embodiment 4, wherein the SLE comprises lupus nephritis (LN).
  • Embodiment 6. The method of any one of embodiments 1 to 3, wherein the autoimmune disorder is RA.
  • Embodiment 7. The method of any one of embodiments 1 to 3, wherein the autoimmune disorder is MG.
  • Embodiment 8. The method of any one of embodiments 1 to 3, wherein the autoimmune disorder is myositis.
  • Embodiment 10 The method of any one of embodiments 1 to 8, wherein the subject had previously been treated with a biologic autoimmune disorder treatment.
  • Embodiment 10 The method of any one of embodiments 1 to 8, wherein the subject had previously been treated with an anti-CD20 antibody, anti-B-lymphocyte stimulator (BlyS) antibody, or a TNF inhibitor.
  • Embodiment 11 The method of any one of embodiments 1 to 10, wherein the autoimmune disorder is SLE, and the subject had previously been treated with belimumab.
  • Embodiment 12 The method of any one of embodiments 1 to 3, wherein the autoimmune disorder is MS, and the subject had previously been treated with rituximab.
  • Embodiment 13 The method of any one of embodiments 1 to 8, wherein the subject had previously been treated with rituximab.
  • Embodiment 14 The method of any one of embodiments 1 to 13, wherein the administration of the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody comprises intravenous infusion. IGM Ref. No.001-061WO1
  • Embodiment 15 The method of any one of embodiments 1 to 13, wherein the administration of the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody comprises subcutaneous injection.
  • Embodiment 16 The method of any one of embodiments 1 to 15, wherein the subject is a human.
  • Embodiment 17 The method of any one of embodiments 1 to 16, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 43, SEQ ID NO: 48, or SEQ ID NO: 53.
  • Embodiment 18 The method of embodiment 17, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 26.
  • Embodiment 19 The method of any one of embodiments 1 to 18, wherein the IgM heavy chain constant region or multimerizing fragment or variant thereof comprises a C ⁇ 4 region and a ⁇ tailpiece region.
  • Embodiment 21 The method of any one of embodiments 1 to 20, wherein the IgM heavy chain constant region or multimerizing fragment or variant thereof is a human IgM constant region.
  • Embodiment 22 The method of any one of embodiments 1 to 21, wherein the modi ⁇ ed J-chain further comprises human serum albumin indirectly or directly fused to the J-chain or functional fragment or variant thereof.
  • Embodiment 23 The method of any one of embodiments 1 to 21, wherein the modi ⁇ ed J-chain further comprises human serum albumin indirectly or directly fused to the J-chain or functional fragment or variant thereof.
  • Embodiment 24 The method of embodiment 23, wherein the modi ⁇ ed J-chain comprises SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 39, SEQ ID NO: 44, SEQ ID NO: 49, or SEQ ID NO: 54.
  • Embodiment 25 The method of embodiment 24, wherein the modi ⁇ ed J-chain comprises SEQ ID NO: 27.
  • Embodiment 26 The method of embodiment 24, wherein the modi ⁇ ed J-chain comprises SEQ ID NO: 27.
  • Embodiment 28 wherein the J-chain or functional fragment or variant thereof comprises the amino acid sequence SEQ ID NO: 5.
  • Embodiment 30 The method of embodiment 29, wherein the modi ⁇ ed J-chain comprises SEQ ID NO: 28, SEQ ID NO: 35, SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 50, or SEQ ID NO: 55.
  • Embodiment 31 The method of embodiment 30, wherein the modi ⁇ ed J-chain comprises SEQ ID NO: 30, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 47, SEQ ID NO: 52, or SEQ ID NO: 57.
  • Embodiment 32 Embodiment 32.
  • Embodiment 33 The method of any one of embodiments 1 to 32, wherein the heavy chain comprises SEQ ID NO: 66, the light chain comprises SEQ ID NO: 67, and the modi ⁇ ed J-chain comprises SEQ ID NO: 29.
  • Embodiment 34 The method of any one of claims 1 to 27 or 32 to 33, wherein the multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody is imvotamab. [0198] The following examples are o ⁇ ered by way of illustration and not by way of limitation.
  • T cell Dependent Cellular Cytotoxicity [0199] 2.5 ⁇ 10 5 peripheral blood mononuclear cells (PBMCs) from various healthy and autoimmune disorder patient samples (3 healthy donors, 3 patients with multiple sclerosis (MS) that have not previously received anti-CD20 therapy, 4 biologic na ⁇ ve systemic lupus erythematosus (SLE) patients, and 2 biologic experienced SLE patients from Discovery Life Sciences and Sanguine Bio) were incubated in the presence of imvotamab, at a starting concentration of 200 ⁇ g/mL, followed by 5-fold serial dilutions from 100 to 0.16 ⁇ g/mL, 2-fold serial dilutions from 0.16 to 0.005 ⁇ g/mL, and another 5-fold serial dilutions from 0.005 to 4.0 ⁇ 10 -5 ⁇ g/mL or rituximab from 200 to 3.2 ⁇ 10 -6 ⁇ g/mL in 200 ⁇ L of RPMI- 1640 medium (Gi
  • the plates were centrifuged at 300 ⁇ g at 4 °C and incubated for 15 minutes at room temperature with Fixable Viability Stain 780 (BD Biosciences).
  • the plates were centrifuged at 300 ⁇ g at 4 °C and washed twice in FACS stain bu ⁇ er (Phosphate Bu ⁇ ered-Saline, heat-inactivated FBS, and EDTA) and then incubated with Fc Block (Thermo Fisher Scienti ⁇ c) for 30 min at 4 °C.
  • the plates were stained with the following BD Biosciences antibodies: R719 anti-human CD19, BV711 anti-human CD56, PE anti-human CD69, BUV737 anti-human CD38, BUV395 anti-human CD24, BV659 anti-human CD27, BV421 anti-human CD20, APC anti-human CD4, V500 anti-human CD8, PerCPCy5.5 anti-human CD3, PE-CF594 anti-human CD21, BV786 anti-human IgD, and BB515 anti- human CD25 in Brilliant Stain bu ⁇ er for 30 min at 4 °C.
  • BD Biosciences antibodies R719 anti-human CD19, BV711 anti-human CD56, PE anti-human CD69, BUV737 anti-human CD38, BUV395 anti-human CD24, BV659 anti-human CD27, BV421 anti-human CD20, APC anti-human CD4, V500 anti-human CD8, PerCPCy5.5 anti-human CD3, PE-CF594
  • FIGS. 1A–1L The percent of TDCC killing of CD19+ B cells with various concentration of imvotamab or rituximab treated PBMCs from biologic na ⁇ ve SLE donors #1-#4, biologic experienced SLE donors #1-#2, MS donors #1-3, and healthy donors #1-#3 are shown in FIGS. 1A–1L, respectively.
  • the half maximal e ⁇ ective concentration (EC50) calculated for each condition (where applicable) are shown in FIG.2A and the maximum percentage IGM Ref. No.001-061WO1 of circulating CD19+ B cell killing for each condition are shown in FIG. 2B.
  • the average EC50 for treatment with imvotamab was signi ⁇ cantly lower as determined by t-test than the average EC50 for treatment with rituximab indicating that on average imvotamab is more potent at killing CD19+ B cells than rituximab in vitro.
  • the percentage of activated memory B cells of total remaining CD19+ cells after treatment with rituximab or imvotamab in a TDCC assay on PBMCs from 4 biologic na ⁇ ve SLE donors, 2 biologic experienced SLE donors, 3 MS donors, or 3 healthy donors are shown in FIGS.
  • PBMCs from SLE biologic na ⁇ ve donors treated with rituximab shown in FIG.3A PBMCs from SLE biologic na ⁇ ve donors treated with imvotamab shown in FIG. 3B
  • PBMCs from SLE biologic experienced donors treated with rituximab shown in FIG.3C PBMCs from SLE biologic experienced donors treated with imvotamab shown in FIG. 3D
  • PBMCs from MS donors treated with rituximab shown in FIG. 3E PBMCs from MS donors treated with imvotamab shown in FIG.3F
  • FIG. 4A The percentage of activated memory B cells of total remaining CD19+ cells for each donor after treatment with rituximab or imvotamab are shown FIG. 4A.
  • the averages for both activated memory B cell frequency and total cell number were signi ⁇ cantly lower as determined by t-test for donor PBMCs treated with imvotamab than with rituximab.
  • activated memory B cells do not themselves produce autoantibodies, they can maintain the "memory” to produce autoantibodies when they di ⁇ erentiate into plasmablasts, and therefore, are likely to be a critical B cell population to target in an autoimmune disorder.
  • Example 2 Multimeric Anti-CD20/Anti-CD3 Antibody Penetration of Lymphoid Tissues
  • No.001-061WO1 vehicle 5 mg/kg Antibody 1, or 25 mg/kg Antibody 1 via intravenous infusion on Days 1, 4, 7, and 10.
  • Scheduled necropsies were conducted on Day 11 with six monkeys (3 males, 3 females) and Day 38 with 4 recovery monkeys (2 males, 2 females) per group.
  • Spleen, mesenteric lymph node (MLN) and bone marrow (sternum) were extracted on Days 11 and 38 from all monkeys, ⁇ xed with 10% neutral bu ⁇ ered formalin for approximately 48 hours, transferred to 70% ethanol, and para ⁇ n embedded.
  • the formalin ⁇ xed para ⁇ n embedded (FFPE) tissues were sectioned with a microtome.
  • Immunohistochemistry (IHC) staining was performed using the Thermo Fisher Scienti ⁇ c system (IHC) and Sakura DRS Autostainer (depara ⁇ nization, counterstaining and dehydration).
  • IHC Immunohistochemistry
  • a mouse anti-human intracellular CD20 (L26) antibody 0.5 ⁇ g/mL, Agilent catalog# M0755
  • a mouse IgG isotype control 0.5 ⁇ g/mL, Vector Laboratories catalog# I-2000
  • CD20+ and CD19+ B cells in spleen, MLN and bone marrow, as well as immune-rich areas including the white pulp in spleen and follicles in MLN were quanti ⁇ ed by arti ⁇ cial intelligence image analysis using HALO software (Indica Laba, Albuquerque, NM).
  • HALO software Indica Laba, Albuquerque, NM.
  • FIGS. 5A–5C The multimeric bispeci ⁇ c anti-CD20/anti-CD3 antibody Antibody 1 penetrated lymphoid tissues and depleted greater than 90% of resident CD20+ B cells in cynomolgus monkeys.
  • mice carrying human CD3E/CD20 are randomized into eight groups of 8 mice per group.
  • Experimental autoimmune encephalomyelitis (EAE) is generated in the mice utilizing myelin oligodendrocyte protein IGM Ref. No.001-061WO1 (MOG).
  • a standard emulsion of MOG is injected subcutaneously into the hind ⁇ anks (100 ⁇ L/ ⁇ ank) on day 0 and is followed with an intraperitoneal injection of pertussis toxin 2 hours post-MOG injection and again 48 hours later.
  • Groups 1-4 are treated prophylactically daily for three days prior to initiation of disease (Day 0) while groups 5-8 are treated therapeutically beginning the ⁇ rst signs of disease, approximately day 7-10. Animals are assessed for body weight and clinical score daily throughout the course of the disease model, approximately day 0-29.
  • a description of treatment and dosing schedule is listed in Table 2.
  • TDCC T cell Dependent Cellular Cytotoxicity
  • PBMCs were incubated in the presence of imvotamab at a starting concentration of 200 ⁇ g/mL, followed by 10-fold serial dilutions from 20 to 4.0 ⁇ 10 -5 ⁇ g/mL.
  • imvotamab For comparison, healthy and autoimmune (AI) IGM Ref. No.001-061WO1 PBMCs were treated with rituximab from 320 to 3.2 ⁇ 10 -7 ⁇ g/mL in 200 ⁇ L of RPMI-1640 medium (Gibco, supplemented with heat-inactivated FBS, Glutamax, MEM amino acids solution, sodium pyruvate and penicillin-streptomycin) per well in a 96-well round bottom plate.
  • RPMI-1640 medium Gibco, supplemented with heat-inactivated FBS, Glutamax, MEM amino acids solution, sodium pyruvate and penicillin-streptomycin
  • the plates were centrifuged at 300 ⁇ g at 4 °C and incubated for 15 minutes at room temperature with Fixable Viability Stain 780 (BD Biosciences). The plates were centrifuged at 300 ⁇ g at 4 °C and washed twice in FACS stain bu ⁇ er (Phosphate Bu ⁇ ered-Saline, heat-inactivated FBS, and EDTA) and then incubated with Fc Block (Thermo Fisher Scienti ⁇ c) for 30 min at 4 °C.
  • FACS stain bu ⁇ er Phosphate Bu ⁇ ered-Saline, heat-inactivated FBS, and EDTA
  • Fc Block Thermo Fisher Scienti ⁇ c
  • the plates were stained with the following BD Biosciences antibodies: R719 anti-human CD19, BV711 anti-human CD56, BUV737 anti-human CD69, BV421 anti-human CD38, BUV395 anti-human CD24, BV650 anti-human CD27, PE anti-human CD20, APC anti-human CD4, V500 anti-human CD8, PerCPCy5.5 anti- human CD3, PE-CF594 anti-human CD21, BV786 anti-human IgD, and BB515 anti- human CD25 in Brilliant Stain bu ⁇ er for 30 min at 4 °C.
  • BD Biosciences antibodies R719 anti-human CD19, BV711 anti-human CD56, BUV737 anti-human CD69, BV421 anti-human CD38, BUV395 anti-human CD24, BV650 anti-human CD27, PE anti-human CD20, APC anti-human CD4, V500 anti-human CD8, PerCPCy5.5 anti- human CD3, PE-CF594 anti-
  • Percent killing of CD19+ B cells was quanti ⁇ ed using the following equation: 100% - (% CD19 positive B cells in treatment sample ⁇ % CD19 positive B cells in untreated sample) ⁇ 100 [0212]
  • the killing percentage numbers were exported and plotted in GraphPad Prism and ⁇ tted with a 4-parameter logistic model. Maximum killing percentage and EC 50 were calculated based on the ⁇ tting results. If a curve did not reach plateau, the killing percentage at the highest antibody concentration tested was reported as the maximum killing percentage, while the EC50 was unavailable and excluded from the calculation of average EC50 between di ⁇ erent donors.
  • FIGS. 6A–6G The percent of TDCC killing of CD19+ B cells with various concentrations of imvotamab or rituximab-treated PBMCs from biologic experienced SLE donors #3 & #4, RA donors #1& #2, and biologic na ⁇ ve SLE donors #5–#7 are shown in FIGS. 6A–6G, respectively.
  • the half maximal e ⁇ ective concentration (EC 50 ) calculated for each condition (where applicable) are shown in FIG. 7A and the maximum percentage of circulating CD19+ B cell killing for each condition are shown in FIG. 7B.
  • the average EC 50 for treatment with imvotamab was signi ⁇ cantly lower as determined by t-test than the average EC 50 for treatment with rituximab indicating that on average imvotamab is more potent at killing CD19+ B cells than rituximab in vitro.
  • the percentage of activated memory B cells of total remaining CD19+ cells after treatment with rituximab or imvotamab in a TDCC assay on PBMCs from 2 RA donors, 7 biologic na ⁇ ve SLE donors, 4 biologic experienced SLE donors, or 4 healthy donors are shown in FIGS. 8A–8H.
  • FIG.8A PBMCs from RA donors treated with rituximab are shown in FIG.8B
  • PBMCs from RA donors treated with imvotamab are shown in FIG.8B
  • PBMCs from SLE biologic na ⁇ ve donors treated with rituximab are shown in FIG. 8C
  • PBMCs from SLE biologic na ⁇ ve donors treated with imvotamab are shown in FIG. 8D
  • FIG. 8E PBMCs from biologic experienced SLE donors treated with imvotamab are shown in FIG.
  • FIG.8G PBMCs from healthy donors treated with rituximab
  • FIG.8H PBMCs from healthy donors treated with imvotamab
  • FIG.9 The total numbers of activated memory B cells of remaining CD19+ cells for each donor after treatment with rituximab or imvotamab are shown FIG.9.
  • Example 5 T cell Dependent Cellular Cytotoxicity (TDCC) [0217] 2.5 ⁇ 10 5 PBMCs from various healthy and autoimmune disorder patient samples (3 healthy donors, 3 patients with multiple sclerosis (MS) that have not previously received anti-CD20 therapy, 4 biologic na ⁇ ve systemic lupus erythematosus (SLE) patients, and 2 biologic experienced SLE patients from Discovery Life Sciences and Sanguine Bio) were incubated in the presence of imvotamab, at a starting concentration of 200 ⁇ g/mL, followed by a 2-fold dilution to 100 ⁇ g/mL, a 5-fold dilution to 20 ⁇ g/mL, and subsequent 10-fold dilutions from 2 ⁇ g/mL to 2.0 ⁇ 10 -5 ⁇ g/mLti, or a bispeci ⁇ c IgG having the same anti-CD20 and anti-CD3 binding domains as imvotamab at a starting concentration of 200 ⁇ g/mL, followed by a 2-fold dilution
  • RPMI-1640 medium Gibco, supplemented with heat-inactivated FBS, Glutamax, MEM amino acids solution, sodium pyruvate and penicillin-streptomycin
  • the plates were centrifuged at 300 ⁇ g at 4 °C and incubated for 15 minutes at room temperature with Fixable Viability Stain 780 (BD Biosciences). The plates were centrifuged at 300 ⁇ g at 4 °C and washed twice in FACS stain bu ⁇ er (Phosphate Bu ⁇ ered-Saline, heat-inactivated FBS, and EDTA) and then incubated with Fc Block (Thermo Fisher Scienti ⁇ c) for 30 min at 4 °C.
  • FACS stain bu ⁇ er Phosphate Bu ⁇ ered-Saline, heat-inactivated FBS, and EDTA
  • Fc Block Thermo Fisher Scienti ⁇ c
  • the plates were stained with the following BD Biosciences antibodies: R719 anti-human CD19, BV711 anti-human CD56, PE anti-human CD69, BUV737 anti-human CD38, BUV395 anti-human CD24, BV659 anti-human CD27, BV421 anti-human CD20, APC anti-human CD4, V500 anti-human CD8, PerCPCy5.5 anti-human CD3, PE-CF594 anti- human CD21, BV786 anti-human IgD, and BB515 anti-human CD25 in Brilliant Stain bu ⁇ er for 30 min at 4 °C.
  • BD Biosciences antibodies R719 anti-human CD19, BV711 anti-human CD56, PE anti-human CD69, BUV737 anti-human CD38, BUV395 anti-human CD24, BV659 anti-human CD27, BV421 anti-human CD20, APC anti-human CD4, V500 anti-human CD8, PerCPCy5.5 anti-human CD3, PE-CF594
  • the percent killing from PBMCs from biologic experienced SLE donors are shown in FIGS.10F–10H.
  • the percent killing from IGM Ref. No.001-061WO1 PBMCs from RA donors are shown in FIG. 10I.
  • the percent killing from PBMCs from healthy donors are shown in FIGS. 10A–10C.
  • the EC50 values were calculated for each condition (where applicable) as described in Example 4 above and are shown in FIG.12A.
  • the maximum percentage of circulating CD19+ B cell killing for each condition are shown in FIG.12B. Both EC50 and maximum % of B cell killing are comparable between groups.
  • T cell Dependent Cellular Cytotoxicity [0222] Autoimmune disorder and healthy patient samples were analyzed in a TDCC assay to compare imvotamab to rituximab and a CD20 ⁇ CD3 IgG antibody comprising SEQ ID NOs: 68-71 (prepared using a commercial vendor), which are the sequences listed in the National Institutes of Health (NIH) National Center for Advancing Translational Sciences (NCATS) Global Substance Registration System (GSRS) for record UNII LDJ89SS0YG, record version 29.
  • NASH National Institutes of Health
  • NCATS National Center for Advancing Translational Sciences
  • GSRS Global Substance Registration System
  • PBMCs from patient samples were incubated in the presence of various concentrations of imvotamab (6 healthy patients, 3 MS patients, 8 biologic na ⁇ ve SLE patients, 4 biologic experienced SLE patients, and 2 RA patients), rituximab (6 healthy patients, 3 MS patients, 7 biologic na ⁇ ve SLE patients, 4 biologic experienced SLE patients, and 2 RA patients), or CD20 ⁇ CD3 IgG (3 healthy patients, 2 biologic na ⁇ ve SLE patients, 3 biologic experienced SLE patients, and 1 RA patient) in 200 ⁇ L of RPMI-1640 medium (Gibco, supplemented with heat-inactivated FBS, Glutamax, MEM amino acids solution, sodium pyruvate and penicillin-streptomycin) per well in a 96-well round bottom plate.
  • imvotamab 6 healthy patients, 3 MS patients, 8 biologic na ⁇ ve SLE patients, 4 biologic experienced SLE patients, and 2 RA patients
  • rituximab (6 healthy patients, 3 MS patients,
  • the plates were stained with the following BD Biosciences antibodies: R719 anti-human CD19, BV711 anti-human CD56, BUV737 anti- human CD69, BV421 anti-human CD38, BUV395 anti-human CD24, BV650 anti-human CD27, PE anti-human CD20, APC anti-human CD4, V500 anti-human CD8, PerCPCy5.5 anti-human CD3, PE-CF594 anti-human CD21, BV786 anti-human IgD, and BB515 anti- human CD25 in Brilliant Stain bu ⁇ er for 30 min at 4 °C. IGM Ref.
  • Example 7 Cytokine Release [0227] Supernatants from TDCC assays performed in Example 6 were assayed for a panel of cytokines including IL-2, IL-6, IL-10, TNF ⁇ , and IFN ⁇ with Meso Scale Discovery V- PLEX Proin ⁇ ammatory Panel 1 human kit (K15049D-4) according to manufacturer’s protocol .
  • FIGS. 13A & 13B Representative plots of cytokine concentrations in supernatants from healthy or autoimmune-disease donor PBMCs treated with various concentrations of imvotamab or CD20 ⁇ CD3 IgG after 72 hours are shown in FIGS. 13A & 13B, respectively for IFN ⁇ , FIGS. 13C & 13D, respectively for IL-2, FIGS. 13E & 13F, respectively for IL-6, and FIGS. 13G & 13H, respectively for TNF ⁇ .
  • IFN ⁇ was the most elevated in healthy subjects and AI patients donors treated wth either imvotamab or the CD20 ⁇ CD3 bispeci ⁇ c IgG.
  • Imvotamab and the CD20 ⁇ CD3 induced similar IL-2, IL-6, and TNF ⁇ levels in the healthy and AI patient cohorts, except for IL-6 productions which was higher in the healthy subjects compared to AI patients.
  • AI patient donor treated with either imvotamab or CD20 ⁇ CD3 IgG IL-2, IL-6, and TNF ⁇ (cytokines strongly implicated in cytokine release syndrome) were released at very low IGM Ref. No.001-061WO1 levels ( ⁇ 500 pg/mL).
  • the maximum IFN ⁇ release for each condition are shown in FIG.14.
  • IFN ⁇ is released in response to both imvotamab and CD20 ⁇ CD3 IgG.
  • IFN ⁇ release is comparable between imvotamab and CD20 ⁇ CD3 IgG.
  • Example 8 T Cell Activation [0228] The ability of imvotamab to enhance T cell activation compared to CD20 ⁇ CD3 IgG was assessed as follows. 250 ⁇ 10 3 healthy or autoimmune-disease donor PBMCs were cultured in the presence of serial dilutions of imvotamab or CD20 ⁇ CD3 IgG in 200 ⁇ L total volume of culture media (RPMI-1640+ 10% heat inactivated FBS) for 72 hours.
  • the frequencies of activated CD4+ T cells (CD69+) for each PBMC type treated with 200 nM (highest dose) of imvotamab or CD20 ⁇ CD3 IgG after 72 hours are shown in FIG.15A.
  • CD69+ The frequencies of activated CD8+ T cells (CD69+) for each PBMC type treated with 200 nM (highest dose) of imvotamab or CD20 ⁇ CD3 IgG after 72 hours are shown in FIG. 15B.
  • imvotamab displayed similar activation of both CD4+ and CD8+ T cells. Hyper-activation of CD4+ T cells by imvotamab was not detected in either the healthy or AI patient samples.
  • Example 9 Comparative CDC Kinetics [0229] To determine if imvotamab has faster complement dependent cytotoxicity (CDC) killing kinetics compared to rituximab in the presence of systemic lupus erythematosus (SLE) patient serum, a series of CDC imaging experiments were performed. The Complement C3 level in the SLE patient serum was quanti ⁇ ed using the Human Complement C3 ELISA Kit (Abcam). The patient serum C3 concentration was 444 ⁇ g/mL, whereas the human C3 serum concentration normally runs in the range of 750–1750 ⁇ g/mL. [0230] 1 ⁇ 10 6 primary B cells from a healthy donor were centrifuged for 5 min.
  • CDC complement dependent cytotoxicity
  • FIG. 16A shows the percentage of dead cells over time in the imvotamab- and rituximab-treated samples. The kinetic analysis demonstrates faster killing kinetics by imvotamab compared to rituximab.
  • FIG. 16B notes that the degree of cell killing with imvotamab-triggered CDC plateaued at ⁇ 90% over the 30 min. assay, while rituximab- triggered CDC plateaued at ⁇ 25%.
  • FIG.16C notes that the total area under the FIG. 16A imvotamab curve was 5122 a.u., while the total area under the FIG. 16A rituximab curve was 1316 a.u.
  • Example 10 CD20 Expression on B Cell Subsets
  • 1x10 6 PBMCs from various healthy and autoimmune disorder patient samples (4 healthy donors, 4 patients with SLE biologic na ⁇ ve, 2 donors with SLE biologic experienced, and 2 donors with RA patients from Discovery Life Sciences and Sanguine Bio) were plated on 96-well round bottom plates. The plates were centrifuged at 300 ⁇ g at 4 °C and incubated for 15 minutes at room temperature with Fixable Viability Stain 780 (BD Biosciences).
  • the plates were centrifuged at 300 ⁇ g at 4 °C and washed twice in FACS stain bu ⁇ er (BD Biosciences stain buffer) and then incubated with Fc Block (Thermo Fisher Scienti ⁇ c) for 30 min at 4 °C.
  • the plates were stained with the following BD Biosciences antibodies: R719 anti-human CD19, BV711 anti-human CD56, BUV737 anti-human CD69, BUV421 anti-human CD38, BUV395 anti- human CD24, BV659 anti-human CD27, PE anti-human CD20, APC anti-human CD4, V500 anti-human CD8, PerCPCy5.5 anti-human CD3, PE-CF594 anti-human CD21, IGM Ref. No.001-061WO1 BV786 anti-human IgD, and BB515 anti-human CD25 in Brilliant Stain bu ⁇ er for 30 min at 4 °C.
  • the plates were then centrifuged, washed, and resuspended in 100 ⁇ L of FACS bu ⁇ er for acquisition on a BD FACSYMPHONYTM A5 Cell Analyzer (BD Biosciences). In the duplicate well, 100 ⁇ L of QUANTIBRITE TM PE beads (BD Biosciences) were added. B cell subsets were classified as outlined in Table 3. Quantitation of CD20 molecules per cell was performed as described by the manufacturer.
  • FIG.17A shows CD20 expression gated on total CD19+ B cells from representative healthy donor and AI patient PBMCs
  • SLE systemic lupus erythematosis
  • SLE BE SLE biologic experienced
  • RA rheumatoid arthritis

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Abstract

La présente divulgation concerne des méthodes d'un trouble auto-immun comprenant l'administration à un sujet ayant besoin d'un traitement, d'une quantité efficace d'un anticorps anti-CD20/anti-CD3 bispécifique multimère, qui comprend des unités de liaison bivalentes et une chaîne J modifiée. Par exemple, le trouble auto-immun est, dans certains modes de réalisation, un trouble dans lequel des cellules de lignée B produisent des auto-anticorps pathogènes et/ou contribuent à une inflammation chronique chez le sujet.
PCT/US2023/075566 2022-09-30 2023-09-29 Méthodes de traitement de troubles auto-immuns à l'aide d'anticorps multimères anti-cd20/anti-cd3 Ceased WO2024073700A2 (fr)

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WO2025137344A1 (fr) 2023-12-20 2025-06-26 Bristol-Myers Squibb Company Anticorps ciblant le récepteur bêta de l'il-18 (il-18rβ) et procédés associés
US12486336B2 (en) 2015-09-30 2025-12-02 Igm Biosciences, Inc. Binding molecules with modified J-chain

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IL297997A (en) * 2015-03-04 2023-01-01 Igm Biosciences Inc Cd20 binding molecules and uses thereof
CA3113268A1 (fr) * 2018-10-23 2020-04-30 Igm Biosciences, Inc. Molecules de liaison a base d'igm et d'iga-fc multivalentes
PE20211867A1 (es) * 2018-11-01 2021-09-21 Shandong New Time Pharmaceutical Co Ltd Anticuerpos biespecificos y su uso
US20240002526A1 (en) * 2020-11-17 2024-01-04 Igm Biosciences, Inc. Uses of effector cell engaging molecules with moieties of differing potencies

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12486336B2 (en) 2015-09-30 2025-12-02 Igm Biosciences, Inc. Binding molecules with modified J-chain
WO2025137344A1 (fr) 2023-12-20 2025-06-26 Bristol-Myers Squibb Company Anticorps ciblant le récepteur bêta de l'il-18 (il-18rβ) et procédés associés

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