WO2024030956A2 - Cd39-specific binding agents and methods of using the same - Google Patents

Cd39-specific binding agents and methods of using the same Download PDF

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Publication number
WO2024030956A2
WO2024030956A2 PCT/US2023/071512 US2023071512W WO2024030956A2 WO 2024030956 A2 WO2024030956 A2 WO 2024030956A2 US 2023071512 W US2023071512 W US 2023071512W WO 2024030956 A2 WO2024030956 A2 WO 2024030956A2
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Prior art keywords
seq
bispecific antibody
heavy chain
chain according
scfv
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PCT/US2023/071512
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French (fr)
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WO2024030956A3 (en
Inventor
Courtney Crane
Susan JULIEN
Su Jung YANG
Kristine SWIDEREK
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Mozart Therapeutics, Inc.
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Publication of WO2024030956A2 publication Critical patent/WO2024030956A2/en
Publication of WO2024030956A3 publication Critical patent/WO2024030956A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C07K16/2815—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 CD8
    • 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
    • C07K16/2818—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 CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • C07K2317/70—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
    • C07K2317/75—Agonist effect on 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the immune system includes the innate immune and the adaptive immune system.
  • the adaptive immune system has a number of cell subtypes, including T cells subsets and B cell subsets.
  • T cell subsets include a variety of types of T cells, including na ⁇ ve T lymphocytes and effector T lymphocytes, such as cytotoxic T cells and helper T cells, and regulatory T cells.
  • the activity of these T cell types is achieved by a balance between the activity of effector T cells and regulation by regulatory T cells. While effector T cells promote inflammation, regulatory T cells (Tregs) are generally thought to control it. Therefore, Tregs play an important role in autoimmune pathogenesis by maintaining self- tolerance, limiting autoimmunity, and by controlling expansion and activation of autoreactive CD4+ T effector cells.
  • binding agents and their methods of use to modulate the activity of CD8+ regulatory T cells are bispecific or multi-specific and specifically bind to antigens expressed on the surface of the CD8+ Tregs.
  • the CD8+ Tregs express KIR.
  • the CD8+ Tregs do not express KIR.
  • the CD8+KIR+ Tregs are MHC class I restricted.
  • the CD8+KIR+ Tregs are not MHC Qa-1 restricted. Also provided are methods of using the binding agents for the treatment of an inflammatory disease or an autoimmune disease.
  • a binding agent is provided that comprises a first binding domain that specifically binds to CD39 expressed on the CD8+ Tregs, and a second binding domain that specifically binds to a T cell antigen expressed on the CD8+ Tregs.
  • the second binding domain specifically binds to an antigen selected from ICOS, CD8a, and PD-1.
  • the bispecific antibody is a CrossMab bispecific, a Bottle Opener bispecific, a scFV-Fc bispecific, or a DART-Fc bispecific comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb, anti-CD8a Mb1b IgG1r mAb, or anti-PD1 MK-3475 IgG1r mAb.
  • the bispecific antibody is a CrossMab bispecific comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb, anti-CD8a Mb1b IgG1r mAb, or anti-PD1 MK-3475 IgG1r mAb .
  • the bispecific antibody is a Bottle Opener bispecific antibody comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti- ICOS 422 H2L5 IgG1r mAb or anti-CD8a Mb1b IgG1r mAb.
  • the bispecific antibody is a "scFV-Fc" antibody comprising binding domains from (i) anti-CD39 27577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb.
  • the bispecific antibody is a "scFv" antibody comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb.
  • a bispecific anti-CD39/anti-ICOS CrossMab antibody is provided, based on the anti-CD39 bivalent monospecific antibody 27577 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:77 and 78, respectively) and the anti-ICOS bivalent monospecific antibody 422 H2L5 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:81 and 82, respectively).
  • the CrossMab antibody comprises an anti-CD39 (27577) light chain and heavy chain according to SEQ ID NOs:9 and 10, respectively; and an anti-ICOS (422 H2L5) light chain and heavy chain according to SEQ ID NOs: 42 and 43, respectively.
  • the first binding domain specifically binds to CD39.
  • the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL having amino acid sequences selected from the pairs of amino acid sequences set forth in the group consisting of: (i) SEQ ID NO:5 and SEQ ID NO:1, respectively; and (ii) SEQ ID NO:15 and SEQ ID NO:11, respectively.
  • the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy and light chain variable regions comprising hCDR1, hCDR1, and hCDR3, and lCDR1, lCDR2, and lCDR3, respectively, the CDRs having amino acid sequences selected from the sets of amino acid sequences set forth in the group consisting of: (i) SEQ ID NOs:6-8 and 2-4, respectively; and (ii) SEQ ID NOs:16-18 and 12-14, respectively.
  • the second binding domain specifically binds to ICOS.
  • the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL having amino acid sequences set forth in SEQ ID NO:38 and SEQ ID NO:34, respectively.
  • the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy and light chain variable regions comprising hCDR1, hCDR1, and hCDR3, and lCDR1, lCDR2, and lCDR3, respectively, the CDRs having amino acid sequences set forth in SEQ ID NOs: 39-41 and SEQ ID NOs:35-37, respectively.
  • the second binding domain specifically binds to CD8a.
  • the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL having amino acid sequences set forth in SEQ ID NO:25 and SEQ ID NO:21, respectively.
  • the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy and light chain variable regions comprising hCDR1, hCDR1, and hCDR3, and lCDR1, lCDR2, and lCDR3, respectively, the CDRs having amino acid sequences selected from the sets of amino acid sequences set forth in the group consisting of: (i) SEQ ID NOs:26-28 and 22-24, respectively; and (ii) SEQ ID NOs: 29-31 and 22-24, respectively.
  • the second binding domain specifically binds to PD-1.
  • the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL having amino acid sequences set forth in SEQ ID NO:48 and SEQ ID NO:44, respectively.
  • the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy and light chain variable regions comprising hCDR1, hCDR1, and hCDR3, and lCDR1, lCDR2, and lCDR3, respectively, the CDRs having amino acid sequences set forth in SEQ ID NOs:49-51 and 45-47, respectively.
  • the binding agent does not contain an Fc domain.
  • the binding agent includes an Fc domain.
  • the Fc domain is selected from an IgG1 and an IgG4 Fc domain.
  • the binding agent has substantially no effector function activity.
  • the Fc domain is an IgG1 Fc domain.
  • the Fc domain is an IgG1 Fc null.
  • a pharmaceutical composition comprising the binding agent of any of the embodiments described herein and a pharmaceutically acceptable carrier.
  • nucleic acids encoding the binding agent of any of the embodiments described herein.
  • a vector comprising any of the embodiments of nucleic acids described herein.
  • cell lines comprising any of the embodiments of nucleic acids or vectors described herein.
  • a method of treating an autoimmune disease comprising administering any of the embodiments of binding agents or pharmaceutical compositions described herein to a subject in need thereof in an amount effective to decrease the number or activity of pathogenic immune cells in the subject and thereby ameliorate a symptom of the autoimmune disease.
  • provided is a method of suppressing an immune response mediated by pathogenic immune cells comprising contacting CD8+ T regulatory cells (Tregs) with any of the embodiments of binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate the CD8+ Tregs (activated Tregs), whereby the number or activity of pathogenic immune cells is decreased.
  • a method of suppressing an immune response to an autoantigen comprising administering to a subject in need thereof any of the embodiments of binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate the CD8+ Tregs, whereby the number or activity of pathogenic immune cells that are responsive to the autoantigen is decreased.
  • a method of suppressing an immune response to an antigen comprising administering to a subject in need thereof any of the embodiments of binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate the CD8+ Tregs, whereby the number or activity of pathogenic immune cells that are responsive to the antigen is decreased.
  • the CD8+ Tregs are contacted with the binding agent in vivo.
  • the CD8+ Tregs are contacted with the binding agent ex vivo.
  • the activated CD8+ Tregs are administered in an effective amount to a subject in need thereof.
  • the pathogenic immune cells are autoreactive CD4+ T cells, autoantibody producing B cells or self antigen presenting dendritic cells. In some embodiments, the pathogenic immune cells are self antigen presenting cells. In some embodiments, the titer of autoantibodies is decreased in the subject. In some embodiments, the subject has an autoimmune disease.
  • the autoimmune disease is selected from the group consisting of celiac disease, Crohn’s disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus/pemphigoid, rheumatoid arthritis (RA), scleroderma/systemic sclerosis, Sjögren’s syndrome (SjS), systemic lupus erythematosus (SLE), and ulcerative colitis.
  • IBD inflammatory bowel disease
  • IDDM or type 1 diabetes insulin-dependent diabetes mellitus
  • lupus nephritis myasthenia gravis
  • myocarditis multiple sclerosis
  • MS multiple sclerosis
  • RA pemphigus/pemphigoid
  • RA rheumatoid
  • the binding agent specifically binds to CD39 and ICOS, CD8a, or PD-1 on CD8+ Tregs. In some embodiments, the binding agent specifically binds to CD39 and an inhibitory KIR protein on CD8+KIR+ Tregs. In some embodiments, the binding agent specifically binds to CD39 and ICOS on CD8+ Tregs. In some embodiments, the binding agent specifically binds to CD39 and CD8a on CD8+ Tregs. In some embodiments, the binding agent specifically binds to CD39 and PD-1 on CD8+ Tregs. In some embodiments, the CD8+ Tregs are MHC class I restricted.
  • the CD8+ Tregs are not MHC HLA E (Qa-1b) restricted.
  • the methods further include administering an immunosuppressive agent to the subject.
  • the administration of the binding agent to the subject results in an improved treatment outcome in the subject.
  • the improved treatment outcome is a reduced frequency or severity disease flares, reduced systemic inflammatory cytokines, or reduced self reporting of symptoms associated with the autoimmune disease.
  • the binding agent is administered intravenously. In some embodiments, the binding agent is administered subcutaneously.
  • the binding agent is administered in a dose of about 0.01 mg/kg to about 20 mg/kg. In some embodiments, the binding agent has substantially no effector function activity. In some embodiments, provided is the use of any of the embodiments of the binding agents or the pharmaceutical compositions described herein for the treatment of autoimmune disease in a subject by activating or stimulating CD8+ Tregs. In some embodiments, provided is the use of any of the embodiments of the binding agents or the pharmaceutical compositions described herein for the reduction of an immune response by pathogenic immune cells by activating or stimulating CD8+ Tregs.
  • FIG.1A to 1D show exemplary bispecific antibody structures.
  • FIG.2 shows binding of antibodies to cells expressing CD39.
  • the CD39+ICOS- SK- MEL 28 cell line was stained with a bispecific anti-CD39/anti-ICOS CrossMab antibody and the parental anti-CD39 bivalent monospecific antibody, followed by a secondary antibody.
  • the graph illustrates geometric mean fluorescence intensity (gMFI) of human IgG bound cells.
  • Results for the monospecific anti-CD39 antibody and the bispecific anti-CD39/anti- ICOS CrossMab antibody are shown on the graph with an open and a filled circle, respectively.
  • FIG.3 shows binding of antibodies to cells expressing ICOS.293T cells were transduced with an ICOS expression vector and used for ICOS binding assay. The points represent gMFI of human IgG bound cells.
  • Results for the monospecific anti-ICOS antibody and the bispecific anti-CD39/anti-ICOS CrossMab antibody are shown on the graph with an open and a filled circle, respectively.
  • FIG.4 shows binding of the bispecific anti-CD39/anti-ICOS CrossMab antibody to a CD39 overexpressing Sk-Mel cell line (Bastid et al, 2014 Cancer Immunol Res.3(3):254-65).
  • the gMFI of human IgG bound cells is shown, with results for the monospecific anti-ICOS antibody and the monospecific anti-CD39 antibody indicated with an open circle or an open diamond, respectively.
  • Results for the bispecific anti-CD39/anti-ICOS CrossMab antibody are shown as a filled circle.
  • FIG.5A to FIG.5E show binding of the antibodies in different immune cell populations.
  • the graph represents the gMFI of the monospecific anti-ICOS antibody, the monospecific anti-CD39 antibody, or the bispecific anti-CD39/anti-ICOS CrossMab antibody binding to PBMC within the CD4+, CD8+, CD14+ and CD20+ gated populations.
  • the bispecific anti-CD39/anti-ICOS CrossMab antibody is represented by filled circle.
  • the monospecific anti-ICOS antibody and the monospecific anti-CD39 antibody are represented by an open circle or an open diamond, respectively.
  • FIG.5A shows binding in CD4+ cells;
  • FIG.5B shows binding in CD8+;
  • FIG.5C shows binding in CD14+ cells;
  • FIG.5D shows binding in CD20+ cells; and
  • FIG.5E shows binding in CD8+ Treg cells.
  • FIG.6 shows direct killing of pathogenic CD4+ T cells by CD8+ Tregs in an IncuCyte-based cytotoxicity assay.
  • the fluorescence of a target SKW-GFP cell line over time in co-culture with cytotoxic CD8+ regulatory cells is shown.
  • the dotted line represents the level of fluorescence of the target cell with no antibodies added.
  • the monospecific anti-ICOS antibody and the monospecific anti-CD39 antibody are shown in the graph with an open circle and an open diamond, respectively.
  • the bispecific anti-CD39/anti-ICOS CrossMab antibody is shown as a filled circle.
  • FIG.7 shows suppression of CD4+ proliferation by CD8+ Tregs.
  • FIG.8 shows suppression of CD4+ proliferation by CD8+ Tregs in vitro. Percentages of divided CD4+ cells in gate G1 were used to quantify suppression.
  • FIGS.9A-9B show the effect of TGF- ⁇ or IL-15 and IL-7 on the phenotype of CD8+ Treg cells.
  • CD8+ Tregs isolated from PBMCs were cultured in TGF- ⁇ (FIG.9A) or in IL-15 and IL-7 (FIG.9B) and assessed for expression of CD8, CD39, CD103, PD-1, ICOS, CXCR3, and NKG2D by flow cytometry.
  • FIG.10 shows the effect of IL-15 on CD39 expression in CD3+CD8+KIR+ T cells.
  • CD3+CD8+KIR+ T cells isolated from PBMCs derived from three separate donors with celiac disease were cultured in different concentrations of IL-15 (0.05-5 ng/mL) for twelve (12) days and then re-evaluated for KIR expression by flow cytometry.
  • FIG.11 shows the effects of different combinations of ⁇ chain cytokines on CD39 expression in CD3+CD8+KIR+ T cells derived from donors with celiac disease and from healthy donors.
  • FIG.12 shows the effect of the anti-CD39/anti-ICOS CrossMAb antibody on CD39 expression in CD3+CD8+T cells.
  • a non-celiac (healthy donor) organoid was treated with anti-CD3 and anti-CD28 antibodies to activate the T cell population.
  • Flow cytometry was used to evaluate the expression of CD39 on CD3+CD8+ T cells. Treatment with monospecific anti-ICOS antibody alone was tested as a control.
  • FIG.13 shows the co-expression of KIR and CD39 in CD3+CD8+ T cells from patients with different autoimmune diseases.
  • the % of CD3+CD8+CD39+ T cells positive and negative for KIR expression are shown
  • FIGS.14A-14B show the contribution of the affinity of each of anti-CD39 and anti- ICOS to the overall avidity of the anti-CD39/anti-ICOS CrossMAb antibody. Affinity was tested using biolayer interferometry (BLI).
  • FIG.14A shows a co-binding sensorgram for the anti-CD39/anti-ICOS CrossMAb antibody (of Example 1, comprising anti-CD39 clone 27577 and anti-ICOS clone 422 H2L5).
  • FIG 14B shows a co-binding sensorgram for the anti-CD39/anti-ICOS CrossMAb antibody (of Example 1, comprising anti-CD39 clone 27577 and anti-ICOS clone 422 H2L5).
  • FIG.15 shows antibody affinity (KD), association rate (ka), and dissociation rate (kd) for two CrossMAbs including: (1) the anti-CD39/anti-ICOS CrossMAb antibody (of Example 1, comprising anti-CD39 clone 27577 and anti-ICOS clone 422 H2L5), and (2) a second anti-CD39/anti-ICOS CrossMAb antibody (comprising anti- CD39 clone 31815 and anti-ICOS clone 422 H2L5).
  • FIG.15 shows the ability of the bispecific anti-CD39/anti-ICOS CrossMAb antibody to reduce expansion of CD4+ T cells in vitro.
  • CD8+ Tregs isolated from PBMCs derived from donors with celiac disease were treated with the bispecific anti-CD39/anti-ICOS CrossMAb antibody and then incubated with autologous CTV-labeled CD4+ T cells that were previously activated with anti-CD3 and anti-CD28 stimulation.
  • CD4+ T cells were also incubated with untreated CD8+ Tregs, non-Tregs, and CD8+ Tregs treated with anti-CD39 or anti-ICOS antibody alone, as controls. Expansion of the CD4+ T cell population was evaluated by flow cytometry.
  • FIG.16 shows direct killing of pathogenic CD4+ T cells by CD8+ Tregs in an IncuCyte-based cytotoxicity assay.
  • the fluorescence of a target SKW-GFP cell line over time in co-culture with cytotoxic CD8+ regulatory cells is shown.
  • the dotted line represents the level of fluorescence of the target cell with no antibodies added.
  • the monospecific anti-ICOS antibody and the monospecific anti-CD39 antibody are shown in the graph with an closed circle and downward-facing closed triangle, respectively.
  • the bispecific anti-CD39/anti- ICOS CrossMab antibody is shown as a upward-facing closed triangle.
  • FIG.17 shows the ability of the bispecific anti-CD39/anti-ICOS CrossMAb antibody to decrease epithelial cell death in a non-celiac (healthy donor) organoid.
  • Organoid cultures were treated for 48 hours with the bispecific anti-CD39/anti-ICOS CrossMAb antibody prior to analysis by flow cytometry. Untreated organoid cultures and organoid cultures treated with anti-ICOS antibody alone were tested as controls. After 48 hours, organoid cultures were stained with Annexin-V live/dead stain and gated to include only the Epcam+MHCI+ epithelial cell population. DETAILED DESCRIPTION I. Glossary The following sections provide a detailed description of binding agents, particularly antibodies or antigen-binding fragments thereof, that target CD39, and related pharmaceutical compositions, methods of activating CD8+ regulatory T cells (CD8+ Treg cells), and methods of treating or preventing disease (e.g., an autoimmune disease).
  • composition consisting essentially of limits the scope of a claim to the specified materials or steps, or to those that do not materially affect the basic characteristics of a claimed invention.
  • a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from an isolation and purification method and would not exclude pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • a protein consists essentially of a particular amino acid sequence when the protein includes additional amino acids that contribute to at most 20% of the length of the protein and do not substantially affect the activity of the protein (e.g., alters the activity of the protein by no more than 50%).
  • Embodiments defined by each of these transitional terms are within the scope of this invention.
  • the term “about” means + 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms “a” and “an” as used herein includes “one” or “one or more” of the enumerated components unless stated otherwise. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives, and may be used synonymously with “and/or”. As used herein, the terms “include” and “have” are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ - carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ -carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.
  • the terms “peptide”, “polypeptide”, and “protein”, and variations of these terms refer to a molecule that comprises at least two amino acids joined to each other by a (normal or modified) peptide bond.
  • a peptide, polypeptide, or protein may comprise or be composed of a plurality of amino acids selected from the 20 amino acids defined by the genetic code or an amino acid analog or mimetic, each being linked to at least one other by a peptide bond.
  • a peptide, polypeptide, or protein can comprise or be composed of L-amino acids and/or D-amino acids (or analogs or mimetics thereof).
  • the terms "peptide”, “polypeptide”, and “protein” also include “peptidomimetics” which are defined as peptide analogs containing non-peptidic structural elements, which peptides are capable of mimicking or antagonizing the biological action(s) of a natural parent peptide.
  • a peptidomimetic lacks characteristics such as enzymatically scissile peptide bonds.
  • a peptide, polypeptide, or protein may comprise amino acids other than the 20 amino acids defined by the genetic code in addition to these amino acids, or it can be composed of amino acids other than the 20 amino acids defined by the genetic code.
  • a peptide, polypeptide, or protein in the context of the present disclosure can comprise amino acids that are modified by natural processes, such as post-translational maturation processes, or by chemical processes (e.g., synthetic processes), which are known in the art and include those described herein. Such modifications can appear anywhere in the polypeptide; e.g., in the peptide skeleton; in the amino acid chain; or at the carboxy- or amino-terminal ends.
  • a peptide or polypeptide can be branched, such as following an ubiquitination, or may be cyclic, with or without branching.
  • the terms "peptide”, “polypeptide”, and “protein” also include modified peptides, polypeptides and proteins.
  • peptide, polypeptide, or protein modifications can include acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of a nucleotide or of a nucleotide derivative, covalent fixation of a lipid or of a lipidic derivative, the covalent fixation of a phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfatation, or amino acid addition such as arginylation or ubiquitination.
  • variant proteins, peptides, and polypeptides of this disclosure comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to an amino acid sequence of a defined or reference amino acid sequence as described herein.
  • Protein and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • protein and “polypeptide” are used interchangeably herein when referring to an encoded gene product and fragments thereof. Additionally, as used herein, "(poly)peptide” and “protein” may be used interchangeably in reference to a polymer of amino acid residues, such as a plurality of amino acid monomers linked by peptide bonds.
  • Nucleic acid molecule or “polynucleotide” or “nucleic acid” refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring).
  • Purine bases include adenine, guanine, hypoxanthine, and xanthine
  • pyrimidine bases include uracil, thymine, and cytosine.
  • Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like.
  • Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, any of which may be single or double-stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense strand). Polynucleotides (including oligonucleotides), and fragments thereof may be generated, for example, by polymerase chain reaction (PCR) or by in vitro translation, or generated by any of ligation, scission, endonuclease action, or exonuclease action.
  • PCR polymerase chain reaction
  • a nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) may be removed through co- or post-transcriptional mechanisms. Different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing, or both. Variants of nucleic acid molecules of this disclosure are also contemplated.
  • Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68oC or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42oC. Nucleic acid molecule variants retain the capacity to encode a binding domain having a functionality described herein, such as specifically binding a target molecule.
  • sequence variant refers to any sequence having one or more alterations in comparison to a reference sequence, whereby a reference sequence is any published sequence and/or any of the sequences disclosed herein, i.e., SEQ ID NO:1 to SEQ ID NO:90.
  • sequence variant includes nucleotide sequence variants and amino acid sequence variants.
  • a sequence variant in the context of a nucleotide sequence the reference sequence is also a nucleotide sequence
  • the reference sequence is also an amino acid sequence.
  • sequence variant as used herein can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference sequence.
  • Percent sequence identity refers to a relationship between two or more sequences, as determined by comparing the sequences. Methods to determine sequence identity can be designed to give the best match between the sequences being compared. For example, the sequences may be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment).
  • sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to determine sequence identity and similarity can be found in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithm used in the BLAST programs can be found in Altschul et al., Nucleic Acids Res.25:3389-3402, 1997. Within the context of this disclosure, it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced.
  • Default values mean any set of values or parameters that originally load with the software when first initialized.
  • a "sequence variant" in the context of a nucleic acid (nucleotide) sequence has an altered sequence in which one or more of the nucleotides in the reference sequence is deleted or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides are referred to herein by the standard one-letter designation (A, C, G, or T).
  • a "sequence variant" of a nucleotide sequence can either result in a change in the respective reference amino acid sequence, i.e., in an amino acid "sequence variant” or not.
  • a nucleotide sequence variant does not result in an amino acid sequence variant (e.g., a silent mutation).
  • a nucleotide sequence variant that results in one or more "non-silent" mutation is contemplated.
  • a nucleotide sequence variant of the present disclosure encodes an amino acid sequence that is at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a reference amino acid sequence.
  • Nucleotide and amino sequences as disclosed herein refer also to codon-optimized versions of a reference or wild-type nucleotide or amino acid sequence.
  • a polynucleotide of the present disclosure may be codon-optimized for a host cell containing the polynucleotide (see, e.g., Scholten et al., Clin. Immunol.119:135-145 (2006). Codon optimization can be performed using known techniques and tools, e.g., using the GenScript® OptimumGeneTM tool, or the GeneArt Gene Synthesis Tool (Thermo Fisher Scientific). Codon-optimized sequences include sequences that are partially codon-optimized (i.e., at least one codon is optimized for expression in the host cell) and those that are fully codon-optimized.
  • a "sequence variant" in the context of an amino acid sequence has an altered sequence in which one or more of the amino acids is deleted, substituted, or inserted in comparison to a reference amino acid sequence.
  • a sequence variant has an amino acid sequence which is at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference amino acid sequence.
  • a variant sequence that has no more than 10 alterations i.e., any combination of deletions, insertions or substitutions, is "at least 90% identical" to the reference sequence.
  • a “conservative substitution” refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution found in one of the following groups: Group 1: Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gln or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (Ile or I), Leucine (Leu or L), Methionine (Met or M), Valine (Val or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or
  • amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing).
  • an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Val, Leu, and Ile.
  • Other conservative substitutions groups include: sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.
  • Amino acid sequence insertions can include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include the fusion to the N- or C-terminus of an amino acid sequence to a reporter molecule or an enzyme. In general, alterations in the sequence variants do not abolish or significantly reduce a desired functionality of the respective reference sequence.
  • a variant sequence of the present disclosure does not significantly reduce or completely abrogate the functionality of a sequence of an antibody, or antigen binding fragment thereof, to bind to the same epitope as compared to antibody or antigen binding fragment having (or encoded by) the reference sequence.
  • Guidance in determining which nucleotides and amino acid residues, respectively, may be substituted, inserted, or deleted without abolishing a desired structure or functionality can be found by using, e.g., known computer programs.
  • a nucleic acid sequence or an amino acid sequence "derived from" a designated nucleic acid, peptide, polypeptide, or protein refers to the origin of the nucleic acid, peptide, polypeptide, or protein.
  • a nucleic acid sequence or amino acid sequence that is derived from a particular sequence may have an amino acid sequence that is essentially identical to that sequence or a portion thereof, from which it is derived, whereby "essentially identical” includes sequence variants as defined above.
  • a nucleic acid sequence or amino acid sequence that is derived from a particular peptide or protein may be derived from the corresponding domain in the particular peptide or protein.
  • "corresponding” refers to possession of a same functionality or characteristic of interest. For example, an "extracellular domain” corresponds to another "extracellular domain” (of another protein), or a “transmembrane domain” corresponds to another “transmembrane domain” (of another protein).
  • nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be identical to the starting nucleic acid, peptide, polypeptide, or protein (from which it is derived).
  • nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may also have one or more mutations relative to the starting nucleic acid, peptide, polypeptide, or protein (from which it is derived), in particular a nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be a functional sequence variant as described above of the starting nucleic acid, peptide, polypeptide, or protein (from which it is derived). For example, in a peptide/protein, one or more amino acid residues may be substituted with other amino acid residues, or one or more amino acid residue insertions or deletions may occur.
  • mutation relates to a change in a nucleic acid sequence and/or in an amino acid sequence in comparison to a reference sequence, e.g., a corresponding genomic, wild type, or reference sequence.
  • a mutation e.g., in comparison to a reference genomic sequence, may be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation, e.g., induced by enzymes, chemicals or radiation, or a mutation obtained by site-directed mutagenesis (molecular biology methods for making specific and intentional changes in the nucleic acid sequence and/or in the amino acid sequence)
  • mutation or “mutating” shall be understood to also include physically making or inducing a mutation, e.g., in a nucleic acid sequence or in an amino acid sequence.
  • a mutation includes substitution, deletion, and insertion of one or more nucleotides or amino acids as well as inversion of several successive nucleotides or amino acids.
  • a mutation may be introduced into the nucleotide sequence encoding said amino acid sequence in order to express a (recombinant) mutated polypeptide.
  • a mutation may be achieved, for example, by altering (e.g., by site- directed mutagenesis) a codon (e.g., by altering one, two, or three nucleotide bases therein) of a nucleic acid molecule encoding one amino acid to provide a codon that encodes a different amino acid, or that encodes a same amino acid, or by synthesizing a sequence variant.
  • a codon e.g., by altering one, two, or three nucleotide bases therein
  • the term "introduced” in the context of inserting a nucleic acid molecule into a cell means “transfection", or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • a cell e.g., chromosome, plasmid, plastid, or mitochondrial DNA
  • transiently expressed e.g., transfected mRNA
  • recombinant refers to any molecule (antibody, protein, nucleic acid, or the like) which is prepared, expressed, created, or isolated by recombinant means, and which is not naturally occurring.
  • “Recombinant” can be used synonymously with “engineered” or “non-natural” and can refer to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention).
  • Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, fusion proteins, or enzymes, or other nucleic acid molecule additions, deletions, or substitutions or other functional disruption of a cell's genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene or operon.
  • heterologous or non-endogenous or “exogenous” refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or a subject, or any gene, protein, compound, nucleic acid molecule, or activity native to a host cell or a subject that has been altered.
  • Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered such that the structure, activity, or both is different as between the native and altered genes, proteins, compounds, or nucleic acid molecules.
  • heterologous, non-endogenous, or exogenous genes, proteins, or nucleic acid molecules may not be endogenous to a host cell or a subject, but instead nucleic acids encoding such genes, proteins, or nucleic acid molecules may have been added to a host cell by conjugation, transformation, transfection, electroporation, or the like, wherein the added nucleic acid molecule may integrate into a host cell genome or can exist as extra-chromosomal genetic material (e.g., as a plasmid or other self-replicating vector).
  • homologous or homolog refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain.
  • a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof.
  • a non-endogenous polynucleotide or gene, as well as the encoded polypeptide or activity may be from the same species, a different species, or a combination thereof.
  • the term “endogenous” or “native” refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or a subject.
  • the terms “cell,” “cell line, “ and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same or substantially the same function, phenotype, or biological activity as screened for in the originally transformed cell are included.
  • isolated refers in the case of a nucleic acid, polypeptide, or protein, to a nucleic acid, polypeptide, or protein separated from at least one other component (e.g., nucleic acid or polypeptide or protein) that is present with the nucleic acid, polypeptide, or protein as found in its natural source and/or that would be present with the nucleic acid, polypeptide, or protein when expressed by a cell, or secreted in the case of secreted polypeptides and proteins.
  • component e.g., nucleic acid or polypeptide or protein
  • a chemically synthesized nucleic acid, polypeptide, or protein, or one synthesized using in vitro transcription/translation, is considered “isolated.”
  • the terms “purified” or “substantially purified” refer to an isolated nucleic acid, polypeptide, or protein that is at least 95% by weight the subject nucleic acid, polypeptide, or protein, including, for example, at least 96%, at least 97%, at least 98%, at least 99%, or more.
  • pathogenic immune cells refer to immune cells that are reactive with an autoantigen or that induce a response to an autoantigen.
  • pathogenic immune cells examples include autoreactive CD4+ T cells, autoantibody producing B cells, self antigen presenting dendritic cells, and other self antigen presenting cells, as are known in the art.
  • CD39 is also known as ectonucleoside triphosphate diphosphohydrolase 1, SPG64 ATPDase, and NTPDase-1. It encodes plasma membrane protein that hydrolyzes extracellular ATP and ADP to AMP.
  • CD39 polypeptides include, but are not limited to, those having the amino acid sequences disclosed in NP_001307845.1, NP_001157651.1, NP_001157650.1, NP_001091645.1, NP_001767.3, NP_001299583.1, NP_001157655.1, NP_001157654.1, and NP_001157653.1; these amino acid sequences are incorporated by reference herein.
  • CD8alpha is a protein that is expressed on T cells, including regulatory T cells.
  • CD8alpha polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_001759.3, NP001139345.1, NP_741969.1, NP_001369627.1, NP_757362.1, NP_001171571.1, NP_742100.1, NP_742099.1, and NP_004922; these sequences are incorporated by reference herein.
  • ICOS, or inducible T cell costimulatory is also referred to as AILIM, CD278, and CVID1.
  • ICOS polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_036224.1; this amino acid sequence is incorporated by reference herein.
  • PD-1 is also referred to as programmed cell death protein 1.
  • PD-1 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_005009.2; this amino acid sequence is incorporated by reference herein.
  • KIR may refer to one or more of KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2, and KIR2DL3.
  • KIR3DL1 is a protein expressed on NK cells and on some T cells. It is also known as CD158E1, KIR, KIR2DL5B, KIR3DL1/S1, NKAT-3, NKAT3, NKB1, and NKB1B.
  • KIR3DL1 polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_037421.2 and NP_001309097.1; these sequences are incorporated by reference herein.
  • KIR3DL2 is a protein expressed on NK cells and on some T cells. It is also known as 3DL2, CD158K, KIR-3DL2, NKAT-4, NKAT4, NKAT4B, and p140.
  • KIR3DL2 polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_006728.2 and NP_001229796.1; these sequences are incorporated by reference herein.
  • KIR2DL1 is a protein expressed on NK cells and on some T cells.
  • KIR2DL1 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_055033.2; this sequence is incorporated by reference herein.
  • KIR2DL2 is a protein expressed on NK cells and on some T cells. It is also known as CD158B1, CD158b, NKAT-6, NKAT6, and p58.2.
  • KIR2DL2 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_055034.2; this sequence is incorporated by reference herein.
  • KIR2DL3 is a protein expressed on NK cells and on some T cells. It is also known as CD158B2, CD158b, GL183, KIR-023GB, KIR-K7b, KIR-K7c, KIR2DL, KIR2DS5, KIRCL23, NKAT, NKAT2, NKAT2A, NKAT2B, and p58.
  • KIR2DL3 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_056952.2; this sequence is incorporated by reference herein.
  • CD103, or integrin subunit alpha E (ITGAE) is also referred to as HUMINAE.
  • CD103 polypeptides include, but are not limited to, those having the amino acid sequence disclosed in NP_002199.3; this amino acid sequence is incorporated by reference herein.
  • CXCR3, or C-X-C motif chemokine receptor 3 is also referred to as GPR9, MigR, CD182, CD183, Mig-R, CKR-L2, CMKAR3, and IP10-R.
  • CXCR3 polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_001495.1 and NP_001136269.1; these amino acid sequences are incorporated by reference herein.
  • NKG2D, or Killer Cell Lectin Like Receptor K1 is also referred to as KLRK1, D12S2489E, NKG2-D, CD314, and KLR.
  • NKG2D polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_031386.2 and NP_001186734.1; these amino acid sequences are incorporated by reference herein.
  • II. Modulation of CD8+ Regulatory T cells Provided herein are binding agents comprising binding domains that specifically bind to antigens expressed on CD8+ regulatory T cells (Tregs).
  • the CD8+ Tregs express KIR (CD8+KIR+ Tregs).
  • the KIR expressed by the CD8+KIR+ Treg is an inhibitory KIR.
  • the inhibitory KIR is, for example, KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2, or KIR2DL3 or a combination thereof.
  • the CD8+ Tregs do not express KIR.
  • the CD8+ Tregs are MHC class I restricted.
  • the CD8+ Tregs are not MHC Qa-1 (HLA-E) restricted. Also provided are methods of using the binding agents for the treatment of autoimmune disease, infectious disease, and cancer.
  • the binding agent includes a first binding domain that specifically binds to CD39 expressed on the CD8+ Tregs, and a second binding domain that specifically binds to a T cell antigen expressed on the CD8+ Tregs.
  • the second binding domain specifically binds to an antigen selected from ICOS, CD8a, and PD-1.
  • the second antigen is selected from a functional antagonist to reduce function inhibition of CD8+ Tregs.
  • such antigen is ICOS, CD8a, or PD-1.
  • the second antigen is a tethering moiety to enhance specificity of the binding agent to CD8+ Tregs.
  • such antigen is ICOS, CD8a, or PD-1.
  • CD8+KIR+ regulatory T cells are characterized by the phenotype of being CD8+ KIR+ and are typically MHC Class I restricted.
  • the CD8+KIR+ regulatory T cells express inhibitory KIR proteins.
  • the KIR proteins expressed by the cells can include one or more of the inhibitory KIR proteins, e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, and KIR3DL2; and may specifically include one or more of KIR2DL2, KIR2DL3, and KIR3DL1.
  • the CD8+KIR+ regulatory T cells are not HLA E (Qa-1b) restricted.
  • the CD8+KIR+ regulatory T cells can also be characterized as being CD44+, CD122+, and are not HLA E (Qa-1b) restricted.
  • the CD8+KIR+ regulatory T cells can also be characterized as being CD28-.
  • the CD8+KIR+ regulatory T cells can also be characterized as being CD28-, CD44+, and CD122+.
  • the CD8+KIR+ regulatory T cells can also be characterized as being CD28-, CD44+, and CD122+, and are not HLA E (Qa-1b) restricted.
  • the CD8+KIR+ Tregs express the following antigens: CD3, CD8, PD-1, CD16, CD122, CD39, CXCR3, ICOS, CD103, and inhibitory KIR proteins.
  • CD8+KIR+ Tregs express one or more of the following antigens: CD3, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG- 3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4, and 41BB.
  • CD8+KIR+ Tregs express one or more of the following antigens: CD3, CD5, CD16, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, KLRB1, KLRG1, LAG- 3/CD223, NKG2C, NKG2D, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4, and 41BB.
  • CD8+KIR+ Tregs express one or more of the following antigens: CD39, KLRB1, KLRG1, NKG2C, NKG2D, CXCR3, and CD122.
  • the present disclosure provides an antibody, or an antigen binding fragment thereof, that is capable of binding to CD39 and capable of binding to ICOS, CD8a, or PD-1. Such antibodies, or antigen binding fragments thereof, can bind to and modulate the activity of CD8+CD39+ regulatory T cells (Tregs).
  • the CD8+ Tregs are also KIR+.
  • Antibodies generally are comprised of a heavy chain and a light chain.
  • Each heavy chain is composed of a variable region (abbreviated as VH) and a constant region.
  • the heavy chain constant region may include three domains CH1, CH2, and CH3 and optionally a fourth domain, CH4. Each of these domains is referred to as an "Fc domain".
  • Fc domain When used herein, when a binding agent includes an Fc domain, it can include one or more Fc domains, or an entire Fc region, unless otherwise specified by context.
  • Each light chain is composed of a variable region (abbreviated as VL) and a constant region or constant domain.
  • the light chain constant region is a CL domain.
  • VH and VL regions may be further divided into hypervariable regions referred to as complementarity-determining regions (CDRs) and interspersed with conserved regions referred to as framework regions (FR).
  • CDRs complementarity-determining regions
  • FR framework regions
  • Each VH and VL region thus consists of three CDRs and four FRs that are arranged from the N terminus to the C terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. This structure is well known to those skilled in the art.
  • antibody refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter- connected by disulfide bonds (though it will be understood that heavy chain antibodies, which lack light chains, are still encompassed by the term “antibody”), as well as any antigen- binding portion or fragment of an intact antibody that has or retains the ability to bind to the antigen target molecule recognized by the intact antibody, such as, for example, a scFv, Fab, or F(ab')2 fragment.
  • antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen-binding (Fab) fragments, F(ab') 2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • antibody should be understood to encompass functional antibody fragments thereof.
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class thereof, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
  • antibody fragment As used herein, the terms “antigen binding fragment,” “fragment, “ and “antibody fragment” are used interchangeably to refer to any fragment of an antibody of the disclosure that retains the antigen-binding activity of the antibody.
  • antibody fragments include, but are not limited to, a single chain antibody, Fab, Fab’, F(ab')2, Fv, and scFv.
  • Human antibodies are known (e.g., van Dijk, M. A., and van de Winkel, J. G., Curr. Opin. Chem. Biol.5 (2001) 368-374).
  • Human antibodies can be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol.7 (1993) 3340).
  • transgenic animals e.g., mice
  • Human antibodies can also be produced in phage display libraries (Hoogenboom, H. R., and Winter, G., J. Mol. Biol.227 (1992) 381-388; Marks, J. D., et al., J. Mol. Biol.222 (1991) 581-597).
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); and Boerner, P., et al., J. Immunol.147 (1991) 86-95).
  • Human monoclonal antibodies may be prepared by using improved EBV-B cell immortalization as described in Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. (2004): An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med.10(8):871-5.
  • the term "human antibody” as used herein also comprises such antibodies which are modified, e.g., in the variable region, to generate properties according to the antibodies and antibody fragments of the present disclosure.
  • Antibodies according to the present disclosure can be of any isotype (e.g., IgA, IgG, IgM, IgE, IgD; i.e., comprising a ⁇ , ⁇ , ⁇ , or ⁇ heavy chain).
  • IgG immunoglobulin G
  • antibodies may be IgG1, IgG2, IgG3, or IgG4 subclass.
  • an antibody of the present disclosure is an IgG1 antibody.
  • Antibodies or antigen binding fragments provided herein may include a ⁇ or a ⁇ light chain.
  • variable region denotes each variable region polypeptide of the pair of light and heavy chains, which, in most instances, is involved directly in binding the antibody to the antigen.
  • VL variable region of a light chain
  • VH variable region of a heavy chain
  • the terms “VL” and “VH” refer to the variable binding region from an antibody light and heavy chain, respectively.
  • the variable binding regions are made up of discrete, well-defined sub-regions known as “complementarity-determining regions” (CDRs) and “framework regions” (FRs).
  • CDRs complementarity-determining regions
  • FRs framework regions
  • CDR complementarity-determining region
  • HVR hypervariable region
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 also referred to herein as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, respectively.
  • a "variant" of a CDR refers to a functional variant of a CDR sequence having up to 1-3 amino acid substitutions, deletions, or combinations thereof.
  • an antibody or antigen binding fragment of the present disclosure can comprise all or part of a heavy chain (HC), a light chain (LC), or both.
  • HC heavy chain
  • LC light chain
  • a full-length intact IgG antibody monomer typically includes a VH, a CH1, a CH2, a CH3, a VL, and a CL. Fc components are described further herein.
  • an antibody or antigen binding fragment of the present disclosure comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and a CDRL3 according to any one of the presently disclosed VH and VL sequences, respectively.
  • Fragments of the antibodies described herein can be obtained from the antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction.
  • fragments of the antibodies can be obtained by cloning and expression of part of the sequences of the heavy or light chains.
  • the present disclosure encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of an antibody as described herein, including, for example, an scFv comprising the CDRs from an antibody according to the present description, heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, as well as single chain antibodies, in which the heavy and light chain variable domains are joined by a peptide linker.
  • an antibody according to the present disclosure, or an antigen binding fragment thereof comprises a purified antibody, a monoclonal antibody, a single chain antibody, Fab, Fab’, F(ab')2, Fv or scFv.
  • binding proteins antibodies, antigen binding fragments thereof, and fusion proteins may individually or collectively (e.g., in any combination) be referred to as "binding proteins". Binding proteins according to the present disclosure may be provided in purified form. For example, an antibody may be present in a composition that is substantially free of other polypeptides, e.g., where less than 90% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides. Binding proteins according to the present disclosure may be immunogenic in human and/or in non-human (or heterologous) hosts; e.g., in mice. For example, an antibody may have an idiotope that is immunogenic in non-human hosts, but not in a human host.
  • Antibodies of the disclosure for human use include those that are not typically isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, or the like, and in some instances are not obtained by humanization or from xeno-mice. Also contemplated herein are variant forms of the disclosed antibodies, which are engineered so as to reduce known or potential immunogenicity and/or other potential liabilities, or to confer a desired structure and/or functionality of the antibody in a non-human animal, such as a mouse (e.g., a "murinized " antibody wherein one or more human amino acid residue, sequence, or motif is replaced by a residue, sequence, or motif that has reduced or abrogated immunogenicity or other liability, or has a desired structure and/or function, in a mouse; e.g., for model studies using a mouse).
  • Antibodies or antigen-binding fragments thereof such as those described herein, including but not limited to scFv, may, in certain embodiments, be comprised in a fusion protein that is capable of specifically binding to an antigen as described herein.
  • fusion protein refers to a protein that, in a single chain, has at least two distinct domains or motifs, wherein the domains or motifs are not naturally found together, or in the given arrangement, in a protein.
  • a polynucleotide encoding a fusion protein may be constructed using PCR, recombinantly engineered, or the like, or such fusion proteins can be synthesized.
  • Immunoglobulin sequences can be aligned to a numbering scheme (e.g., Kabat, EU, International Immunogenetics Information System (IMGT), and Aho), which can allow equivalent residue positions to be annotated and for different molecules to be compared using Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300).
  • a numbering scheme e.g., Kabat, EU, International Immunogenetics Information System (IMGT), and Aho
  • IMGT International Immunogenetics Information System
  • Aho Antigen receptor Numbering And Receptor Classification
  • binding protein e.g., an antibody or antigen binding fragment thereof
  • binding domain e.g., an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10 5 M -1 (which equals the ratio of the on-rate [Kon] to the off rate [Koff] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample.
  • Binding proteins or binding domains may be classified as "high-affinity" binding proteins or binding domains or as "low-affinity” binding proteins or binding domains.
  • High-affinity binding proteins or binding domains refer to those binding proteins or binding domains having a Ka of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 .
  • Low-affinity binding proteins or binding domains refer to those binding proteins or binding domains having a Ka of up to 10 7 M -1 , up to 10 6 M- 1 , or up to 10 5 M -1 .
  • affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10 -5 M to 10 -13 M).
  • binding and “specifically binding” and similar references do not encompass non-specific sticking. Binding of a binding protein can be determined or assessed using an appropriate assay, such as, for example, Surface Plasmon Resonance (SPR) methods, e.g., a BiacoreTM system; kinetic exclusion assays such as KinExA®; and BioLayer interferometry (e.g., using the ForteBio® Octet platform); isothermal titration calorimetry (ITC), or the like, an antigen- binding ELISA (e.g., direct or indirect) with imaging by, e.g., optical density at 450nm, or by flow cytometry, or the like.
  • SPR Surface Plasmon Resonance
  • BiacoreTM system kinetic exclusion assays
  • KinExA® kinetic exclusion assays
  • BioLayer interferometry e.g., using the ForteBio® Octet platform
  • ITC isothermal titration calorimetry
  • epitope or “antigenic epitope” includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin or other binding molecule, domain, or protein
  • Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • An epitope to which binding protein binds may be linear (continuous) or conformational (discontinuous).
  • a linear or a sequential epitope is an epitope that is recognized by an antibody according to its linear sequence of amino acids, or primary structure.
  • a conformational epitope may be recognized according to a three-dimensional shape and protein structure.
  • Multispecific Antibodies and binding fragments of the present disclosure may, in embodiments, be multispecific (e.g., bispecific, trispecific, tetraspecific, or the like), and may be provided in any multispecific format, as disclosed herein.
  • Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites or antigens.
  • an antibody or antigen binding fragment of the present disclosure is a multispecific antibody, such as a bispecific or trispecific antibody.
  • Bispecific T cell Engagers BiTEs
  • DARTs DARTs
  • Knobs-Into-Holes KH
  • scFv-CH3-KIH assemblies KIH Common Light-Chain antibodies
  • TandAbs Triple Bodies
  • TriBi Minibodies Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCabs, Intrabodies, CrossMabs, Dual Action Fabs (DAFs) (two-in-one or four-
  • Bispecific and multi-specific antibodies include the following: an scFv1-ScFv2, an ScFv12-Fc-scFv22, an IgG-scFv, a DVD-Ig, a triomab/quadroma, a two-in-one IgG, a scFv2-Fc, a TandAb, an scFv-HSA-scFv, an scFv-VHH, a Fab-scFv-Fc, a Fab-VHH-Fc, a dAb-IgG, an IgG-VHH, a Tandem scFv-Fc, a (scFv1)2-Fc-(VHH)2, a scFv-Fc, a one-armed tandem scFv-Fc, and a DART-Fc.
  • An IgG-scFv may be an IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, or IgG-2scFv.
  • Multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J.10: 3655 (1991)), and "knob-in-hole” engineering (see, e.g., U.S. Pat. No.5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat.
  • CrossMab antibodies are also included herein (see, e.g., WO 2009/080251, WO 2009/080252, WO2009/080253, WO2009/080254, and WO2013/026833).
  • Bottle Opener antibodies are also included herein.
  • one heavy chain of the antibody comprises a scFv and the other heavy chain comprises a standard FAb, comprising a variable heavy chain and a light chain.
  • This structure is sometimes referred to as the “bottle-opener” format, due to a rough visual similarity to a bottle-opener, or “triple F” format, referring to “scFv-FAb-Fc”.
  • the antibodies or antigen binding fragments disclosed herein comprise different antigen-binding sites, fused to one or the other of the two subunits of the Fc domain; thus, the two subunits of the Fc domain may be comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific molecules in recombinant production, it is advantageous to introduce in the Fc domain of the binding agent a modification promoting the association of the desired polypeptides.
  • a binding agent e.g., an antibody or antigen binding fragment thereof
  • a binding agent comprising (a) at least a first binding domain, (b) a second binding domain, and (c) a Fc domain composed of a first and a second subunit capable of stable association, wherein the Fc domain comprises a modification promoting the association of the first and second subunit of the Fc domain.
  • the site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain.
  • said modification is in the CH3 domain of the Fc domain.
  • the Fc modification is a so-called "knob-into-hole” modification, comprising a "knob” modification in one of the two subunits of the Fc domain and a "hole” modification in the other one of the two subunits of the Fc domain.
  • the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to Kabat EU index).
  • the knob-into-hole technology is described in, e.g., U.S. Pat.
  • the method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation.
  • Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). Accordingly, in some embodiments, in a CH3 domain of an Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain which is positionable in a cavity within a CH3 domain of a second Fc domain, and in the CH3 domain of the second Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second Fc domain within which the protuberance within the CH3 domain of the first Fc domain is positionable.
  • the protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis.
  • the threonine residue at position 366 is replaced with a tryptophan residue (T366W)
  • T366W tryptophan residue
  • Y407V valine residue
  • the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A).
  • the serine residue at position 354 is replaced with a cysteine residue (S354C)
  • the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C).
  • S354C cysteine residue
  • Y349C cysteine residue
  • the first Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering)
  • the second Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to Kabat EU index).
  • a modification promoting association of the first and the second Fc domains comprises a modification mediating electrostatic steering effects, for example, as described in PCT publication WO 2009/089004.
  • this method involves replacement of one or more amino acid residues at the interface of the two Fc domains by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.
  • a binding agent e.g., an antibody or antigen binding fragment thereof
  • scFv is a fusion protein of the variable regions of the heavy (VH) and light chain (VL) variable regions of an antibody, connected with a short linker peptide of ten to about 25 amino acids.
  • the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa.
  • This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker.
  • scFv antibodies are, described in, e.g., Houston, J. S., Methods in Enzymol.203 (1991) 46-96.
  • Binding agents e.g., an antibody or antigen binding fragment thereof
  • Binding agents that are scFv- Fcs have been described by Sokolowska-Wedzina et al., Mol. Cancer Res.15(8):1040-1050, 2017.
  • a binding agent e.g., an antibody or antigen binding fragment thereof
  • a bispecific T cell engager or BiTE
  • This approach utilizes two antibody variable domains arranged on a single polypeptide.
  • a single polypeptide chain can include two single chain Fv (scFv) fragments, each having a variable heavy chain (VH) and a variable light chain (VL) domain separated by a polypeptide linker of a length sufficient to allow intramolecular association between the two domains.
  • This single polypeptide further includes a polypeptide spacer sequence between the two scFv fragments.
  • Each scFv recognizes a different epitope, and these epitopes may be specific for different proteins, such that both proteins are bound by the BiTE.
  • the bispecific T cell engager may be expressed using any prokaryotic or eukaryotic cell expression system known in the art, e.g., a CHO cell line.
  • specific purification techniques see, e.g., EP1691833 may be necessary to separate monomeric bispecific T cell engagers from other multimeric species, which may have biological activities other than the intended activity of the monomer.
  • a solution containing secreted polypeptides is first subjected to a metal affinity chromatography, and polypeptides are eluted with a gradient of imidazole concentrations. This eluate is further purified using anion exchange chromatography, and polypeptides are eluted using with a gradient of sodium chloride concentrations. Finally, this eluate is subjected to size exclusion chromatography to separate monomers from multimeric species.
  • a binding agent that is a bispecific antibody is composed of a single polypeptide chain comprising two single chain FV fragments (scFV) fused to each other by a peptide linker.
  • a single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain.
  • Single-domain antibodies can be derived from the variable domain of the antibody heavy chain from camelids (e.g., nanobodies or VHH fragments).
  • the term single-domain antibody includes an autonomous human heavy chain variable domain (aVH) or VNAR fragments derived from sharks (see, e.g., Hasler et al., Mol. Immunol.75:28-37, 2016).
  • Techniques for producing single-domain antibodies are known in the art, as disclosed in, for example, Cossins et al. (2006, Prot Express Purif 51:253-259 and Li et al., Immunol.
  • Single-domain antibodies may be obtained, for example, from camels, alpacas or llamas by standard immunization techniques.
  • a VHH may have potent antigen-binding capacity and can interact with novel epitopes that are inacessible to conventional VH-VL pairs (see, e.g., Muyldermans et al., 2001).
  • Alpaca serum IgG contains about 50% camelid heavy chain only IgG antibodies (HCAbs) (see, e.g., Maass et al., 2007).
  • Alpacas may be immunized with antigens and VHHs can be isolated that bind to and neutralize the target antigen (see, e.g., Maass et al., 2007).
  • PCR primers that amplify alpaca VHH coding sequences have been identified and may be used to construct alpaca VHH phage display libraries, which can be used for antibody fragment isolation by standard biopanning techniques well known in the art (see, e.g., Maass et al., 2007).
  • a binding agent e.g., an antibody or antigen binding fragment thereof
  • IgG-scFv formats include IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, and IgG-2scFv.
  • the binding agent e.g., an antibody or antigen binding fragment thereof
  • is a scaffold antigen binding protein such as for example, fibronectin and designed ankyrin repeat proteins (DARPins) which have been used as alternative scaffolds for antigen-binding domains, see, e.g., Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008).
  • DARPins ankyrin repeat proteins
  • a scaffold antigen binding protein is selected from the group consisting of Lipocalins (Anticalin), a Protein A-derived molecule such as Z-domain of Protein A (Affibody), an A-domain (Avimer/Maxibody), a serum transferrin (trans-body); a designed ankyrin repeat protein (DARPin), a fibronectin (AdNectin), a C-type lectin domain (Tetranectin); a variable domain of a new antigen receptor beta-lactamase (VNAR fragments), a human gamma-crystallin or ubiquitin (Affilin molecules); a kunitz type domain of human protease inhibitors, and microbodies such as the proteins from the knottin family, peptide aptamers, and fibronectin (adnectin).
  • Lipocalins Lipocalins
  • Protein A-derived molecule such as Z-domain of Protein A (Affibody), an A-
  • Lipocalins are a family of extracellular proteins which transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. They have a rigid beta-sheet secondary structure with a number of loops at the open end of the conical structure which can be engineered to bind to different target antigens. Anticalins are between 160-180 amino acids in size, and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482: 337-350 (2000), U.S. Pat. No.7,250,297B1, and US20070224633.
  • DARPins Designed Ankyrin Repeat Proteins
  • Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton.
  • a single ankyrin repeat is a 33-residue motif consisting of two alpha-helices and a beta-turn. They can be engineered to bind different target antigens by randomizing residues in the first alpha-helix and a beta-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details, see J. Mol. Biol.332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol.
  • a binding protein e.g., antibody or an antigen binding fragment thereof
  • the Fc moiety may be derived from human origin, e.g., from human IgG1, IgG2, IgG3, and/or IgG4, or from another Ig class or isotype.
  • an antibody or antigen binding fragments can comprise an Fc moiety derived from human IgG1.
  • an Fc moiety refers to a sequence comprising, consisting, consisting essentially of, or derived from a portion of an immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site (e.g., residue 216 in native IgG, taking the first residue of heavy chain constant region to be 114) and ending at the C-terminus of the immunoglobulin heavy chain.
  • an Fc moiety may be a complete Fc moiety or a portion (e.g., a domain) thereof.
  • a complete Fc moiety comprises a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acid positions 216-446).
  • Amino acid positions within an Fc moiety can be numbered according to the EU numbering system of Kabat, see, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest", U.S. Dept. Health and Human Services, 1983 and 1987. Amino acid positions of an Fc moiety can also be numbered according to the IMGT numbering system (including unique numbering for the C-domain and exon numbering) and the Kabat numbering system.
  • an Fc moiety comprises at least one of: a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant, portion, or fragment thereof.
  • a hinge e.g., upper, middle, and/or lower hinge region
  • an Fc moiety comprises at least a hinge domain, a CH2 domain or a CH3 domain.
  • the Fc moiety is a complete Fc moiety.
  • the Fc moiety may also comprise one or more amino acid insertions, deletions, or substitutions relative to a naturally occurring Fc moiety. For example, at least one of a hinge domain, CH2 domain, or CH3 domain, or a portion thereof, may be deleted.
  • an Fc moiety may comprise or consist of: (i) hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), (ii) a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), (iii) a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), (iv) a hinge domain (or a portion thereof), (v) a CH2 domain (or a portion thereof), or (vi) a CH3 domain or a portion thereof.
  • An Fc moiety of the present disclosure may be modified such that it varies in amino acid sequence from the complete Fc moiety of a naturally occurring immunoglobulin molecule, while retaining or enhancing at least one desirable function conferred by the naturally occurring Fc moiety, and/or reducing an undesired function of a naturally occurring Fc moiety.
  • Such functions include, for example, Fc receptor (FcR) binding, antibody half-life modulation (e.g., by binding to FcRn), ADCC function, protein A binding, protein G binding, and complement binding. Portions of naturally occurring Fc moieties which are involved with such functions have been described in the art.
  • an Fc region or Fc domain has substantially no binding to at least one Fc receptor selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b).
  • an Fc region or domain exhibits substantially no binding to any of the Fc receptors selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b).
  • substantially no binding refers to weak to no binding to a selected Fcgamma receptor or receptors.
  • substantially no binding refers to a reduction in binding affinity (e.g., increase in Kd) to a Fc gamma receptor of at least 1000-fold.
  • an Fc domain or region is an Fc null.
  • an "Fc null” refers to an Fc region or Fc domain that exhibits weak to no binding to any of the Fcgamma receptors.
  • an Fc null domain or region exhibits a reduction in binding affinity (e.g., increase in Kd) to Fc gamma receptors of at least 1000-fold.
  • an Fc domain has reduced or substantially no effector function activity.
  • effector function activity refers to antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), and/or complement dependent cytotoxicity (CDC).
  • ADCC antibody dependent cellular cytotoxicity
  • ADCP antibody dependent cellular phagocytosis
  • CDC complement dependent cytotoxicity
  • an Fc domain exhibits reduced ADCC, ADCP, or CDC activity, as compared to a wildtype Fc domain.
  • an Fc domain exhibits a reduction in ADCC, ADCP, and CDC, as compared to a wildtype Fc domain.
  • an Fc domain exhibits substantially no effector function (i.e., the ability to stimulate ADCC, ADCP, or CDC).
  • substantially no effector function refers to a reduction in effector function activity of at least 1000-fold, as compared to a wildtype Fc domain.
  • an Fc domain has reduced or no ADCC activity.
  • reduced or no ADCC activity refers to a decrease in ADCC activity of an Fc domain by of a factor of at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500.
  • an Fc domain has reduced or no CDC activity.
  • reduced or no CDC activity refers to a decrease in CDC activity of an Fc domain by of a factor of at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks Fcgamma receptor (hence likely lacking ADCC activity).
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol.9:457-492 (1991).
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)).
  • non-radioactive assays methods may be employed (see, e.g., ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96TM non-radioactive cytotoxicity assay (Promega, Madison, Wis.).
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat’l Acad. Sci. USA 95:652-656 (1998).
  • C1q binding assays may also be carried out to confirm that an antibody or Fc domain or region is unable to bind C1q and hence lacks CDC activity or has reduced CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)).
  • an Fc domain has reduced or no ADCP activity.
  • reduced or no ADCP activity refers to a decrease in ADCP activity of an Fc domain by of a factor of at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500.
  • ADCP binding assays may also be carried out to confirm that an antibody or Fc domain or region lacks ADCP activity or has reduced ADCP activity. See, e.g., US20190079077 and US20190048078 and the references disclosed therein.
  • Antibodies with reduced effector function activity include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (see U.S. Pat. No. 6,737,056).
  • Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (see U.S. Pat. No.7,332,581).
  • Certain antibody variants with diminished binding to FcRs are also known. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol.
  • a binding agent comprises an Fc domain or region with one or more amino acid substitutions which diminish FcgammaR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues).
  • the substitutions are L234A and L235A (LALA).
  • the Fc domain further comprises D265A and/or P329G in an Fc region derived from a human IgG1 Fc region.
  • the substitutions are L234A, L235A, and P329G (LALA-PG) in an Fc region derived from a human IgG1 Fc region.
  • substitutions are L234A, L235A, and D265A (LALA-DA) in an Fc region derived from a human IgG1 Fc region.
  • alterations are made in the Fc region that result in altered (i.e., either diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No.6,194,551, WO 99/51642, and Idusogie et al. J. Immunol.164: 4178-4184 (2000).
  • CDC Complement Dependent Cytotoxicity
  • binding proteins e.g., antibody or an antigen binding fragment thereof
  • binding proteins can be produced in human, murine, or other animal-derived cells lines.
  • Recombinant DNA expression can be used to produce the binding agents. This allows the production of antibodies as well as a spectrum of antigen binding portions and other binding agents (including fusion proteins) in a host species of choice.
  • the production of antibodies, antigen binding portions thereof and other binding agents in bacteria, yeast, transgenic animals, and chicken eggs are also alternatives for cell-based production systems.
  • the main advantages of transgenic animals are potential high yields from renewable sources.
  • Nucleic acid molecules encoding the amino acid sequence of an antibody, or antigen binding portion thereof, as well as other binding agents can be prepared by a variety of methods known in the art.
  • oligonucleotide-mediated (or site-directed) mutagenesis, PCR- mediated mutagenesis, and cassette mutagenesis can be used to prepare nucleotide sequences encoding an antibody or antigen binding portion as well as other binding agents.
  • a nucleic acid sequence encoding at least an antibody, antigen binding portion thereof, binding agent, or a polypeptide thereof, as described herein, can be recombined with vector DNA in accordance with conventional techniques, such as, for example, blunt-ended or staggered- ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed, e.g., by Maniatis et al., Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab.
  • a VH polypeptide is encoded by a first nucleic acid.
  • a VL polypeptide is encoded by a second nucleic acid.
  • the VH and VL polypeptides are encoded by one nucleic acid.
  • a nucleic acid molecule, such as DNA, is said to be “capable of expressing” a polypeptide if it contains nucleotide sequences that contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences that encode the polypeptide.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed (e.g., an antibody or antigen binding portion thereof) are connected in such a way as to permit gene expression of a polypeptide(s) or antigen binding portions in recoverable amounts.
  • the precise nature of the regulatory regions needed for gene expression may vary from organism to organism, as is well known in the analogous art.
  • Suitable hosts include bacterial or eukaryotic hosts, including yeast, insects, fungi, bird, and mammalian cells either in vivo or in situ, or host cells of mammalian, insect, bird, or yeast origin.
  • the mammalian cell or tissue can be of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog, or cat origin, but any other mammalian cell may be used.
  • yeast ubiquitin hydrolase system in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be accomplished.
  • the fusion proteins so produced can be processed in vivo or purified and processed in vitro, allowing synthesis of an antibody or antigen binding portion thereof as described herein with a specified amino terminus sequence.
  • problems associated with retention of initiation codon-derived methionine residues in direct yeast (or bacterial) expression may be avoided.
  • Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeast are grown in medium rich in glucose can be utilized to obtain recombinant antibodies or antigen-binding portions thereof or other binding agents.
  • Known glycolytic genes can also provide very efficient transcriptional control signals.
  • the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.
  • the introduced nucleic acid sequence (encoding an antibody or antigen binding portion thereof or a polypeptide thereof or other binding agent) is incorporated into a plasmid or viral vector capable of autonomous replication in a recipient host cell.
  • a plasmid or viral vector capable of autonomous replication in a recipient host cell. Any of a wide variety of vectors can be employed for this purpose and are known and available to those of ordinary skill in the art. See, e.g., Ausubel et al., 1987-1993.
  • Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species.
  • Exemplary prokaryotic vectors known in the art include plasmids such as those capable of replication in E. coli.
  • Other gene expression elements useful for the expression of DNA encoding antibodies or antigen-binding portions thereof and other binding agents include, but are not limited to (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter, (Okayama et al., 3 Mol. Cell.
  • Rous sarcoma virus LTR Rous sarcoma virus LTR (Gorman et al., 79 PNAS 6777 (1982)), and Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)); (b) splice regions and polyadenylation sites such as those derived from the SV40 late region (Okayarea et al., 1983), and (c) polyadenylation sites such as in SV40 (Okayama et al., 1983).
  • Immunoglobulin-encoding DNA genes can be expressed as described by Liu et al., infra, and Weidle et al., 51 Gene 21 (1987), using as expression elements the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancers, SV40 late region mRNA splicing, rabbit S- globin intervening sequence, immunoglobulin and rabbit S-globin polyadenylation sites, and SV40 polyadenylation elements.
  • the transcriptional promoter can be, for example, human cytomegalovirus
  • the promoter enhancers can be cytomegalovirus and mouse/human immunoglobulin.
  • the transcriptional promoter can be a viral LTR sequence
  • the transcriptional promoter enhancers can be either or both the mouse immunoglobulin heavy chain enhancer and the viral LTR enhancer
  • the polyadenylation and transcription termination regions DNA sequences encoding other proteins are combined with the above-recited expression elements to achieve expression of the proteins in mammalian cells.
  • Each coding region or gene fusion is assembled in, or inserted into, an expression vector.
  • Recipient cells capable of expressing the variable region(s) or antigen binding portions thereof are then transfected singly with nucleotides encoding an antibody or an antibody polypeptide or antigen-binding portion thereof, or are co-transfected with a polynucleotide(s) encoding VH and a VL chain coding regions.
  • the transfected recipient cells are cultured under conditions that permit expression of the incorporated coding regions and the expressed antibody chains or intact antibodies or antigen binding portions are recovered from the culture.
  • the nucleic acids containing the coding regions encoding an antibody or antigen-binding portion thereof are assembled in separate expression vectors that are then used to co-transfect a recipient host cell. Each vector can contain one or more selectable genes.
  • two selectable genes are used, a first selectable gene designed for selection in a bacterial system and a second selectable gene designed for selection in a eukaryotic system, wherein each vector has a set of coding regions.
  • This strategy results in vectors which first direct the production, and permit amplification, of the nucleotide sequences in a bacterial system.
  • the DNA vectors so produced and amplified in a bacterial host are subsequently used to co-transfect a eukaryotic cell, and allow selection of a co-transfected cell carrying the desired transfected nucleic acids (e.g., encoding antibody heavy and light chains).
  • Non-limiting examples of selectable genes for use in a bacterial system are the gene that confers resistance to ampicillin and the gene that confers resistance to chloramphenicol.
  • Selectable genes for use in eukaryotic transfectants include the xanthine guanine phosphoribosyl transferase gene (designated gpt) and the phosphotransferase gene from Tn5 (designated neo).
  • the fused nucleotide sequences encoding VH and VL chains can be assembled on the same expression vector.
  • the recipient cell line can be a Chinese Hamster ovary cell line (e.g., DG44) or a myeloma cell.
  • Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by transfected immunoglobulin genes and possess the mechanism for glycosylation of the immunoglobulin.
  • the recipient cell is the recombinant Ig-producing myeloma cell SP2/0. SP2/0 cells only produce immunoglobulins encoded by the transfected genes.
  • Myeloma cells can be grown in culture or in the peritoneal cavity of a mouse, where secreted immunoglobulin can be obtained from ascites fluid.
  • An expression vector encoding an antibody or antigen-binding portion thereof or other binding agent can be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, protoplast fusion, calcium phosphate-precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection and microprojectile bombardment.
  • biochemical means as transformation, transfection, protoplast fusion, calcium phosphate-precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection and microprojectile bombardment.
  • DEAE diethylaminoethyl
  • Yeast provides certain advantages over bacteria for the production of immunoglobulin heavy and light chains. Yeasts carry out post-translational peptide modifications including glycosylation. A number of recombinant DNA strategies exist that utilize strong promoter sequences and high copy number plasmids which can be used for production of the desired proteins in yeast. Yeast recognizes leader sequences of cloned mammalian gene products and secretes polypeptides bearing leader sequences (i.e., pre-polypeptides). See, e.g., Hitzman et al., 11th Intl. Conf. Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).
  • Yeast gene expression systems can be routinely evaluated for the levels of production, secretion and the stability of antibodies, and assembled antibodies and antigen binding portions thereof.
  • Various yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeasts are grown in media rich in glucose can be utilized.
  • Known glycolytic genes can also provide very efficient transcription control signals.
  • the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized.
  • Another example is the translational elongation factor 1alpha promoter.
  • Bacterial strains can also be utilized as hosts for the production of the antibody molecules or antigen binding portions thereof or other binding agents described herein.
  • E. coli K12 strains such as E. coli W3110, Bacillus species, enterobacteria such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species can be used.
  • Plasmid vectors containing replicon and control sequences which are derived from species compatible with a host cell are used in connection with these bacterial hosts.
  • the vector carries a replication site, as well as specific genes which are capable of providing phenotypic selection in transformed cells.
  • a number of approaches can be taken for evaluating the expression plasmids for the production of antibodies and antigen binding portions thereof in bacteria (see Glover, 1985; Ausubel, 1987, 1993; Sambrook, 1989; Colligan, 1992-1996).
  • Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin molecules including leader peptide removal, folding and assembly of VH and VL chains, glycosylation of the antibody molecules, and secretion of functional antibody and/or antigen binding portions thereof.
  • Mammalian cells that can be useful as hosts for the production of antibody proteins include cells of fibroblast origin, such as Vero or CHO-K1 cells.
  • Exemplary eukaryotic cells that can be used to express immunoglobulin polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, CHO-K1, and DG44 cells; PERC6 TM cells (Crucell); and NSO cells.
  • a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains.
  • CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
  • one or more antibodies or antigen-binding portions thereof or other binding agents can be produced in vivo in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptides, according to any suitable method.
  • an antibody or antigen-binding portion thereof is produced in a cell-free system.
  • Non-limiting exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol.
  • VH and VL chains are available for the expression of the VH and VL chains in mammalian cells (see Glover, 1985).
  • Various approaches can be followed to obtain intact antibodies.
  • the co-expression can occur by using either the same or different plasmids in the same host.
  • Nucleic acids encoding the VH and VL chains or antigen binding portions thereof can be placed into the same plasmid, which is then transfected into cells, thereby selecting directly for cells that express both chains.
  • cells can be transfected first with a plasmid encoding one chain, for example the VL chain, followed by transfection of the resulting cell line with a VH chain plasmid containing a second selectable marker.
  • Cell lines producing antibodies, antigen-binding portions thereof or other binding agents via either route could be transfected with plasmids encoding additional copies of peptides, VH, VL, or VH plus VL chains in conjunction with additional selectable markers to generate cell lines with enhanced properties, such as higher production of assembled antibodies or antigen binding portions thereof or enhanced stability of the transfected cell lines.
  • plants have emerged as a convenient, safe, and economical alternative expression system for recombinant antibody production, which are based on large scale culture of microbes or animal cells.
  • Antibodies or antigen binding portions can be expressed in plant cell culture, or plants grown conventionally. The expression in plants may be systemic, limited to sub-cellular plastids, or limited to seeds (endosperms).
  • variable regions (VH and VL) of the antibodies are typically linked to at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells, such as immortalized B-cells (see, e.g., WO 87/02671; which is incorporated by reference herein in its entirety).
  • An antibody can contain both light chain and heavy chain constant regions.
  • the heavy chain constant region can include CH1, hinge, CH2, CH3, and, sometimes, CH4 regions.
  • the CH2 domain can be deleted or omitted.
  • techniques described for the production of single chain antibodies see, e.g., U.S. Pat. No.4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci.
  • Single chain antibodies are formed by linking the heavy and light chain variable regions of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Techniques for the assembly of functional Fv fragments in E. coli can also be used (see, e.g., Skerra et al., Science 242:1038-1041 (1988); which is incorporated by reference herein in its entirety).
  • Intact (e.g., whole) antibodies, their dimers, individual light and heavy chains, or antigen binding portions thereof can be recovered and purified by known techniques, e.g., immunoadsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination of these. See generally, Scopes, Protein Purification (Springer-Verlag, N.Y., 1982). Substantially pure antibodies or antigen binding portions thereof of at least about 90% to 95% homogeneity are advantageous, as are those with 98% to 99% or more homogeneity, particularly for pharmaceutical uses.
  • an intact antibody or antigen binding portions thereof can then be used therapeutically or in developing and performing assay procedures, immunofluorescent staining, and the like. See generally, Vols. I & II Immunol. Meth. (Lefkovits & Pernis, eds., Acad. Press, NY, 1979 and 1981).
  • Anti-CD39 Antibodies and Antigen-Binding Fragments Thereof Antibodies to CD39 have been described in, for example, Published US Patent Application Nos.20190062448, 20130273062, and 20100303828.
  • a bispecific antibody targeting CD39 is prepared from these parental anti-CD39 antibodies.
  • a bispecific antibody targeting CD39 is provided, prepared from the anti-CD39 bivalent monospecific antibody 27577 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:77 and 78, respectively). In some embodiments, a bispecific antibody targeting CD39 is provided, prepared from the anti-CD39 bivalent monospecific antibody 31895 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:79 and 80, respectively).
  • a bispecific antibody targeting CD39 and ICOS is provided, prepared from the anti-ICOS bivalent monospecific antibody 422 H2L5 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:81 and 82, respectively).
  • a bispecific antibody targeting CD39 and CD8a is provided, prepared from the anti-CD8a bivalent monospecific antibody Mb1b IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:83 and 84, respectively).
  • a bispecific antibody targeting CD39 and PD-1 is provided, prepared from the anti-PD-1 bivalent monospecific antibody MK-3475 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:85 and 86, respectively).
  • the bispecific antibody is a CrossMab bispecific (4 chain assembly), a Bottle Opener bispecific (3 chain assembly), a scFV-Fc bispecific (2 chain assembly), or a DART-Fc bispecific (2 chain assembly).
  • the bispecific antibody is a CrossMab, in which the Fab and scFv are attached to an IgG1 hinge-CH2-CH3, and the CH3 domain is engineered to contain the "knobs-into-holes” mutations to enforce correct association of the two heterodimeric heavy chains.
  • the "knob” heavy chain includes mutations S354C and T366W
  • the "hole” heavy chain includes mutations Y349C, T366S, L368A, and Y407V.
  • the bispecific antibody is a CrossMab bispecific, a Bottle Opener bispecific, a scFV-Fc bispecific, or a DART-Fc bispecific comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb, anti-CD8a Mb1b IgG1r mAb, or anti-PD1 MK-3475 IgG1r mAb.
  • the bispecific antibody is a CrossMab bispecific comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb, anti-CD8a Mb1b IgG1r mAb, or anti-PD1 MK-3475 IgG1r mAb (FIG.1A).
  • the bispecific antibody is a Bottle Opener bispecific antibody comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti- ICOS 422 H2L5 IgG1r mAb or anti-CD8a Mb1b IgG1r mAb (FIG.1B).
  • the bispecific antibody is a "scFV-Fc" antibody comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb (FIG.1C).
  • the bispecific antibody is a "scFv" antibody comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb (FIG.1D).
  • a bispecific anti-CD39/anti-ICOS CrossMab antibody is provided, based on the anti-CD39 bivalent monospecific antibody 27577 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:77 and 78, respectively) and the anti-ICOS bivalent monospecific antibody 422 H2L5 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:81 and 82, respectively).
  • the CrossMab antibody comprises an anti-CD39 (27577) light chain and heavy chain according to SEQ ID NOs:9 and 10, respectively; and an anti-ICOS (422 H2L5) light chain and heavy chain according to SEQ ID NOs: 42 and 43, respectively.
  • the binding agents e.g., antibodies and antigen binding fragments thereof
  • the binding agents relate to compositions comprising active ingredients (i.e., including a binding agent as described herein or a nucleic acid encoding an antibody or antigen-binding portion thereof or other binding agent as described herein).
  • the composition is a pharmaceutical composition.
  • the term "pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier accepted for use in the pharmaceutical industry.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on any particular formulation.
  • compositions are prepared as injectable either as liquid solutions or suspensions; however, solid forms suitable for rehydration, or suspensions, in liquid prior to use can also be prepared.
  • a preparation can also be emulsified or presented as a liposome composition.
  • An antibody or antigen binding portion thereof or other binding agent can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
  • a pharmaceutical composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance or maintain the effectiveness of the active ingredient (e.g., an antibody or antigen binding portion thereof or other binding agent).
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance or maintain the effectiveness of the active ingredient (e.g., an antibody or antigen binding portion thereof or other binding agent).
  • the pharmaceutical compositions as described herein can include pharmaceutically acceptable salts of the components therein.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of a polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine, procaine, and the like.
  • Physiologically tolerable carriers are well known in the art.
  • Exemplary liquid carriers are sterile aqueous solutions that contain the active ingredients (e.g., an antibody and/or antigen binding portions thereof or other binding agent) and water, and may contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline.
  • aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
  • Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
  • the amount of an active agent that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • a pharmaceutical composition comprising an antibody or antigen-binding portion thereof or other binding agent or a nucleic acid encoding an antibody or antigen-binding portion thereof or other binding agent as described herein can be a lyophilisate.
  • a syringe comprising a therapeutically effective amount of a binding agent, or a pharmaceutical composition described herein is provided.
  • the binding agents e.g., antibodies and antigen binding fragments thereof
  • the autoimmune disease is autoimmune-induced hepatitis, Addison’s Disease, Alopecia Areata, Alport’s Syndrome, Ankylosing Spondylitis, Anti-phospholipid Syndrome, Arthritis, Ascariasis, Aspergillosis Atopic Allergy, Atopic Dermatitis, Atopic Rhinitis, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune Myositis, Behcet’s Disease, Bird-Fancier’s Lung, Bronchial Asthma, Caplan’s Syndrome, Cardiomyopathy, celiac Disease, Chagas’ Disease, Chronic Glomerulonephritis, Chronic Graft versus Host Disease, Cogan’s Syndrome,
  • the autoimmune disease is autoimmune hepatitis, celiac disease, Crohn’s disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus/pemphigoid, primary biliary cirrhosis/cholangitis, rheumatoid arthritis (RA), scleroderma/systemic sclerosis, Sjögren’s syndrome (SjS), systemic lupus erythematosus (SLE), or ulcerative colitis.
  • IBD inflammatory bowel disease
  • IDDM or type 1 diabetes insulin-dependent diabetes mellitus
  • lupus nephritis myasthenia gravis
  • myocarditis multiple sclerosis (MS), pemphigus/pemphigoid, primary
  • the autoimmune disease is selected from autoimmune hepatitis, celiac disease, Crohn’s disease, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), multiple sclerosis (MS), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or ulcerative colitis.
  • the binding agents e.g., antibodies and antigen binding fragments thereof
  • the binding agents as described herein can be used in a method(s) comprising administering a binding agent or a pharmaceutical composition as described herein to suppress an immune response mediated by pathogenic immune cells in a subject.
  • the binding agents e.g., antibodies and antigen binding fragments thereof
  • the binding agents e.g., antibodies and antigen binding fragments thereof
  • the binding agent or pharmaceutical composition is administered to suppress, reduce, or prevent an immune response to a virus.
  • the immune response that is suppresed, reduced, or prevented is an immune response to a virus, or antigenic portions thereof.
  • the virus is a viral vector
  • the administration of the binding agent suppresses, reduces, or prevents the induction of undesired immune responses associated with vector-mediated delivery of genetic material.
  • viral vectors such as adeno- associated virus (AAV) vectors
  • AAV adeno- associated virus
  • the host may produce neutralizing antibodies against the vector capsids based on exposure to the wild-type virus, blocking gene delivery.
  • the host may also produce neutralizing antibodies against the vector capsid that limit the effectiveness of re- administration of the vector in therapies requiring repeated dosing.
  • hosts can mount a cytotoxic T lymphocyte (CTL)-mediated cytotoxicity that clears transduced cells.
  • CTL cytotoxic T lymphocyte
  • binding agents e.g., antibodies and antigen binding fragments thereof
  • the binding agents can be used in a method(s) comprising administering a binding agent or a pharmaceutical composition as described herein to a subject to suppress, reduce, or prevent an immune response to a viral vector.
  • an immune response to a virus or “an immune response to a viral vector” may refer to any immune response to a virus, a viral vector, or antigenic portions thereof, e.g., viral proteins or fragments thereof.
  • the immune response may be activation or proliferation of CD4+ T cells.
  • the immune response may be characterized by, for example, pro-inflammatory cytokine (e.g., IFN- ⁇ ) production by CD4+ T cells.
  • the viral vector has been, is, or will be administered to the subject.
  • the immune response to the viral vector is induced by administration of a viral vector to the subject.
  • the virus or viral vector is a retrovirus, adenovirus, parvovirus, coronavirus, ortho-myxovirus, rhabdovirus, paramyxovirus, picornavirus, alphavirus, herpesvirus, poxvirus, Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, or hepatitis virus.
  • the virus or viral vector is an adeno-associated virus (AAV) vector, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVrh.43, AAVrh.74, or AAVhu.37, or a variant thereof.
  • AAV adeno-associated virus
  • the virus or viral vector is an adenovirus vector.
  • the virus or viral vector is a lentivirus vector.
  • CD8+ Tregs refers to an increase of the regulatory T cell functions of such cells, such as the ability to suppress an immune response.
  • Activation or stimulation of CD8+ Tregs may include removal of a suppressive effect on such cells, so as to restore the CD8+ Tregs (e.g., restore balance to the immune system or restore balanced immune activity in the subject prior to receiving a viral vector).
  • Activation or stimulation of CD8+ Tregs may also include results of such activation or stimulation, including removal of a CD4+ cells, B cells, or other cells mediating an immune response, such as by elimination, for example, cytolysis, of such cells.
  • the CD8+ Tregs are contacted with the binding agent in vivo. In some embodiments, the CD8+ Tregs are contacted with the binding agent ex vivo.
  • the activated CD8+ Tregs can then be administered in an effective amount to a subject in need thereof.
  • the activated CD8+ Tregs exert a suppressive effect on other immune cells, such as CD4+ T cells, antibody producing B cells, antigen presenting dendritic cells, or antigen presenting cells.
  • the activated CD8+ Tregs exert a suppressive effect on other immune cells, such as CD4+ T cells, antibody producing B cells, and antigen presenting dendritic cells.
  • the activated CD8+ Tregs deplete other immune cells, such as CD4 T cells, antibody producing B cells, and antigen presenting dendritic cells.
  • the activated CD8+ Tregs modulate the activity of undesired immune cells and decrease the titer of antibodies in the subject.
  • the activated CD8+ Tregs decrease the titer of antibodies in the subject.
  • the CD8+ Tregs are CD39+ and KIR+.
  • the CD8+ Tregs are CD39- and KIR+.
  • the CD8+ Tregs are CD39+ and KIR-.
  • the CD8+ Tregs are CD39- and KIR-.
  • a binding agent or a pharmaceutical composition comprising any of the binding agents described herein is administered with an immunosuppressive agent, such as a corticosteroid.
  • the immunosuppressive agent is one or more of: a calcineurin inhibitor, e.g., a cyclosporin or an ascomycin, e.g., cyclosporin A (NEORAL®), FK506 (tacrolimus), pimecrolimus, an mTOR inhibitor, e.g., rapamycin or a derivative thereof, e.g., sirolimus (RAPAMUNE®), everolimus (Certican®), temsirolimus, zotarolimus, biolimus-7, biolimus-9, a rapalog, e.g., ridaforolimus, azathioprine, campath 1H, a S1P receptor modulator, e.g., fingolimod or an ana calcineurin inhibitor,
  • a binding agent or a pharmaceutical composition of any of the binding agents described herein is administered with an anti-inflammatory agent, such as a corticosteroid.
  • the anti-inflammatory agent is one or more of: methotrexate, dexamethasone, dexamethasone alcohol, dexamethasone sodium phosphate, fluromethalone acetate, fluromethalone alcohol, lotoprendol etabonate, medrisone, prednisolone acetate, prednisolone sodium phosphate, difluprednate, rimexolone, hydrocortisone, hydrocortisone, lodoxamide tromethamine, aspirin, ibuprofen, suprofen, piroxicam, meloxicam, flubiprofen, naproxan, ketoprofen, tenoxicam, diclofenac sodium, ketotifen fumarate, diclofenac sodium, nepafenac, bromfenac, flurbiprofen sodium, suprofen, celecoxib, naproxen, rofecoxib, glucocortic
  • the anti-inflammatory agent is one or more nonsteroidal anti- inflammatory drugs (NSAIDs), such as naproxen sodium (Anaprox), celecoxib (Celebrex), sulindac (Clinoril), oxaprozin (Daypro ), salsalate (Disalcid), diflunisal (Dolobid), piroxicam (Feldene), indomethacin (Indocin), etodolac (Lodine), meloxicam (Mobic), naproxen (Naprosyn), nabumetone (Relafen), ketorolac tromethamine (Toradol), naproxen/ esomeprazole (Vimovo), and diclofenac (Voltaren), and combinations thereof.
  • NSAIDs nonsteroidal anti- inflammatory drugs
  • the binding agents e.g., antibodies and antigen binding fragments thereof
  • the binding agents are for use in the aforementioned methods, or are used in manufacture of a medicament for use in the aforementioned methods.
  • EXEMPLARY EMBODIMENTS 1.
  • a bispecific antibody, or an antigen binding fragment thereof, comprising: a first binding arm that specifically binds to CD39; and a second binding arm that specifically binds to ICOS, CD8a, or PD-1. 2.
  • the first binding arm comprises a light chain variable region (VL) comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:1; and comprises a heavy chain variable region (VH) comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:5. 4.
  • the bispecific antibody of embodiment 3, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:1.
  • the bispecific antibody of embodiment 3, wherein the VH of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:5.
  • the bispecific antibody of embodiment 3, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:1; and the VH comprises or consists of the amino acid sequence according to SEQ ID NO:5. 7.
  • the first binding arm comprises a VL comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:11; and comprises a VH comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:15.
  • the bispecific antibody of embodiment 8, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:11. 10. The bispecific antibody of embodiment 8, wherein the VH of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:15. 11. The bispecific antibody of embodiment 8, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:11; and the VH comprises or consists of the amino acid sequence according to SEQ ID NO:15. 12. The bispecific antibody of any one of embodiments 1-11, wherein the second binding arm specifically binds to ICOS. 13.
  • the bispecific antibody of embodiment 12, wherein the second binding arm comprises CDRL1, CDRL2, CDRL3 amino acid sequences according to SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37; and comprises CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41.
  • the second binding arm comprises a VL comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:34; and comprises a VH comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:38.
  • the bispecific antibody of embodiment 14, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:34. 16.
  • the bispecific antibody of embodiment 14, wherein the VH of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:38. 17.
  • the bispecific antibody of embodiment 14, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:34; and the VH comprises or consists of the amino acid sequence according to SEQ ID NO:38. 18.
  • the bispecific antibody of any one of embodiments 1-11, wherein the second binding arm specifically binds to CD8a. 19.
  • the bispecific antibody of embodiment 18, wherein the second binding arm comprises CDRL1, CDRL2, CDRL3 amino acid sequences according to SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24; and comprises CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.
  • the second binding domain comprises CDRL1, CDRL2, CDRL3 amino acid sequences according to SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24; and comprises CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. 21.
  • the bispecific antibody of embodiment 21, wherein the VH of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:25. 24.
  • the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:44.
  • the bispecific antibody of embodiment 27, wherein the VH of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:48. 30.
  • 31. The bispecific antibody of any one of embodiments 1-30, wherein the bispecific antibody comprises or is a CrossMab, a Bottle Opener bispecific, a diabody, an antibody Fc fusion, an scFv1-ScFv2, an ScFv12-Fc-scFv22, an IgG-scFv, a DVD-Ig, a triomab/quadroma, a two-in-one IgG, a scFv2-Fc, a TandAb, an scFv-HSA-scFv, an scFv- VHH, a Fab-scFv-Fc, a Fab-VHH-Fc, a dAb-IgG,
  • bispecific antibody of any one of embodiments 1-31, wherein the bispecific antibody is a CrossMab bispecific antibody.
  • 33. The bispecific antibody of any one of embodiments 1-31, wherein the bispecific antibody is a Bottle Opener bispecific antibody.
  • 34. The bispecific antibody of any one of embodiments 1-31, wherein the bispecific antibody is a scFv-Fc bispecific antibody.
  • 35. The bispecific antibody of any one of embodiments 1-31, wherein the bispecific antibody is a DART-Fc bispecific antibody. 36.
  • the bispecific antibody of embodiment 1 comprising an anti-CD39 light chain according to SEQ ID NO:9, an anti-CD39 heavy chain according to SEQ ID NO:10, an anti- ICOS light chain according to SEQ ID NO:42, and an anti-ICOS heavy chain according to SEQ ID NO:43.
  • the bispecific antibody of embodiment 38, wherein the bispecific antibody is a CrossMab bispecific antibody.
  • the bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:58, and an anti-ICOS scFv heavy chain according to SEQ ID NO:56. 47.
  • the bispecific antibody of embodiment 1, comprising an anti-CD39 light chain according to SEQ ID NO:19, an anti-CD39 heavy chain according to SEQ ID NO:20, and an anti-ICOS scFv heavy chain according to SEQ ID NO:55. 59.
  • the bispecific antibody of embodiment 1, comprising an anti-CD39 light chain according to SEQ ID NO:19, an anti-CD39 heavy chain according to SEQ ID NO:20, and an anti-ICOS scFv heavy chain according to SEQ ID NO:56.
  • the bispecific antibody of embodiment 1 comprising an anti-CD39 light chain according to SEQ ID NO:19, an anti-CD39 heavy chain according to SEQ ID NO:20, an anti- CD8a light chain according to SEQ ID NO:32, and an anti-CD8a heavy chain according to SEQ ID NO:33.
  • the bispecific antibody of embodiment 1, comprising an anti-CD39 light chain according to SEQ ID NO:9, an anti-CD39 heavy chain according to SEQ ID NO:10, and an anti-CD8a scFv heavy chain according to SEQ ID NO:54.
  • the bispecific antibody of embodiment 86, wherein the bispecific antibody is a Bottle Opener bispecific antibody. 88.
  • the bispecific antibody of embodiment 1 comprising an anti-CD39 light chain according to SEQ ID NO:9, an anti-CD39 heavy chain according to SEQ ID NO:10, an anti- PD-1 light chain according to SEQ ID NO:52, and an anti-PD-1 heavy chain according to SEQ ID NO:53.
  • the bispecific antibody of embodiment 90 wherein the bispecific antibody is a Cross- Mab bispecific antibody.
  • a pharmaceutical composition comprising the bispecific antibody of any one of embodiments 1-91 and a pharmaceutically acceptable carrier.
  • 94. A vector comprising the nucleic acid of embodiment 93.
  • 95. A cell line comprising the vector of embodiment 94.
  • 96. A method of treating an autoimmune disease, comprising administering the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 to a subject in need thereof in an amount effective to decrease the number or activity of pathogenic immune cells in the subject. 97.
  • the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 for use in treating an autoimmune disease in a subject comprises administering the bispecific antibody or the pharmaceutical composition to a subject in need thereof in an amount effective to decrease the number or activity of pathogenic immune cells in the subject.
  • Use of the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 in the manufacture of a medicament for use in treating an autoimmune disease in a subject wherein the use comprises administering the bispecific antibody or the pharmaceutical composition to a subject in need thereof in an amount effective to decrease the number or activity of pathogenic immune cells in the subject 99.
  • a method of suppressing an immune response mediated by pathogenic immune cells comprising contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs).
  • bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 in the manufacture of a medicament for use in suppressing an immune response mediated by pathogenic immune cells in a subject, wherein the use comprises contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody or the pharmaceutical composition in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs). 102.
  • a method of suppressing an immune response to an antigen, such as an autoantigen comprising administering to a subject in need thereof the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby the number or activity of pathogenic immune cells that are responsive to the antigen or autoantigen is decreased.
  • an antigen such as an autoantigen
  • the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 for use in suppressing an immune response to an antigen, such as an autoantigen, in a subject wherein the use comprises administering to a subject in need thereof the bispecific antibody or pharmaceutical composition in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby the number or activity of pathogenic immune cells that are responsive to the antigen or autoantigen is decreased.
  • bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 in the manufacture of a medicament for use in suppressing an immune response to an antigen, such as an autoantigen, in a subject, wherein the use comprises administering to a subject in need thereof the bispecific antibody or pharmaceutical composition in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby the number or activity of pathogenic immune cells that are responsive to the antigen or autoantigen is decreased.
  • autoimmune disease is celiac disease, Crohn’s disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), lupus, lupus nephritis, cutaneous lupus, discoid lupus, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus/pemphigoid, rheumatoid arthritis (RA), scleroderma/systemic sclerosis, Sjögren’s syndrome (SjS), systemic lupus erythematosus (SLE), or ulcerative colitis.
  • IBD inflammatory bowel disease
  • IDDM or type 1 diabetes insulin-dependent diabetes mellitus
  • lupus lupus nephritis
  • cutaneous lupus cutaneous lupus
  • discoid lupus myasthenia gravis
  • autoimmune disease is celiac disease, Crohn’s disease, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), , multiple sclerosis (MS), rheumatoid arthritis (RA), scleroderma/systemic sclerosis, Sjögren’s syndrome (SjS), lupus, lupus nephritis, cutaneous lupus, discoid lupus, systemic lupus erythematosus (SLE), or ulcerative colitis.
  • autoimmune disease is celiac disease, Crohn’s disease, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), , multiple sclerosis (MS), rheumatoid arthritis (RA), scleroderma/systemic sclerosis, Sjögren’s syndrome (SjS), lupus, lupus nephritis, cutaneous lup
  • a method of reducing or preventing onset of graft versus host disease (GVHD) following a transplant comprising administering the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92, wherein the bispecific antibody has substantially no effector function activity, to a subject in need thereof in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs) and thereby reduce or ameliorate at least one symptom of GVHD. 112.
  • the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 for use in reducing or preventing onset of graft versus host disease (GVHD) in a subject following a transplant wherein the use comprises administering the bispecific antibody or the pharmaceutical composition, wherein the bispecific antibody has substantially no effector function activity, to a subject in need thereof in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs) and thereby reduce or ameliorate at least one symptom of GVHD. 113.
  • bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 in the manufacture of a medicament for use in reducing or preventing onset of graft versus host disease (GVHD) in a subject following a transplant, wherein the use comprises administering the bispecific antibody or the pharmaceutical composition, wherein the bispecific antibody has substantially no effector function activity, to a subject in need thereof in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs) and thereby reduce or ameliorate at least one symptom of GVHD.
  • GVHD graft versus host disease
  • a method of treating a subject who has received a transplant comprising contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92, wherein the bispecific antibody has substantially no effector function activity, in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby GVHD is reduced or suppressed.
  • CD8+ T regulatory cells CD8+ Tregs
  • the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 for use in treating a subject who has received a transplant wherein the use comprises contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody or the pharmaceutical composition, wherein the bispecific antibody has substantially no effector function activity, in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby GVHD is reduced or suppressed.
  • CD8+ T regulatory cells CD8+ Tregs
  • the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 in the manufacture of a medicament for use in treating a subject who has received a transplant, wherein the use comprises contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody or the pharmaceutical composition, wherein the bispecific antibody has substantially no effector function activity, in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby GVHD is reduced or suppressed.
  • CD8+ Tregs CD8+ T regulatory cells
  • the bispecific antibody has substantially no effector function activity, in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby GVHD is reduced or suppressed.
  • a method of suppressing, reducing, or preventing an immune response to a viral vector in a subject comprising administering to the subject the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92that binds to CD8+ T regulatory cells (CD8+ Tregs).
  • CD8+ Tregs CD8+ T regulatory cells
  • CD8+ Tregs CD8+ T regulatory cells
  • the CrossMab antibody comprised an anti- CD39 (27577) light chain and heavy chain according to SEQ ID NOs:9 and 10, respectively; and an anti-ICOS (422 H2L5) light chain and heavy chain according to SEQ ID NOs: 42 and 43, respectively.
  • the SK-MEL-28 cell line ATCC, Manassas, VA
  • ICOS 422 H2L5 light chain and heavy chain according to SEQ ID NOs: 42 and 43, respectively.
  • SK-MEL-28 cell line ATCC, Manassas, VA
  • SK-MEL-28 cells were stained with Live/Dead stain (Biolegend, San Diego, CA) and seeded in a well of 96-well V bottom plate.
  • Each bispecific binder was serially diluted in FACS buffer (PBS, 2 mM EDTA, and 1% FBS) covering 3200 – 0.012nM and added to the corresponding wells in a volume of 25 uL, followed by incubation on ice for 30 min. Cells were washed two times with FACS buffer and then incubated with secondary antibody (APC goat anti-human IgG, Jackson ImmunoResearch, West Grove, PA) at 1:120 for 30 min at room temperature. Cells were washed and fixed with IC fixation buffer (ThermoFisher, Waltham, MA) overnight.
  • FACS buffer PBS, 2 mM EDTA, and 1% FBS
  • Example 2 Binding of Bispecific CD39/ICOS-Targeting Molecule to Donor-Derived PBMC
  • PBMC were activated with ImmunoCult T cell activator (Stemcell, Vancouver, Canada) for 5 days and then used for binding assays. Prior to seeding, cells were stained with Live/Dead (Biolegend) and washed with FACS buffer.
  • 1.4X10 5 cells/well were plated in 96-well V bottom plates and incubated with serially diluted Bispecific (3200 – 0.024 nM) for 30 min on ice. Cells were washed two times with FACS buffer and then incubated with secondary antibody (1:120, APC goat anti-human IgG, Jackson ImmunoResearch) together with CD4, CD8, CD14, and CD20 antibodies (Biolegend) for 30 min at room temperature. Cells were washed and fixed using IC fixation buffer overnight. Cells were washed next day and resuspended in FACS buffer for flow cytometry on BD Symphony. Data was analyzed in FlowJo to examine live cells and graphs plotted using GraphPad Prism.
  • the parental anti-CD39 antibody did not reach saturation at the highest concentration, suggesting poor binding to whole PBMC (FIG.4).
  • the bispecific anti-CD39/anti- ICOS CrossMab antibody and the parental anti-ICOS parental antibody bound well to PBMC, suggesting that the binding of the bispecific anti-CD39/anti-ICOS CrossMab antibody may have been driven by ICOS arm (FIG.4). Binding was explored in different cell populations, including CD20+ B cells and CD14+ monocytes (FIGS.5A-5E).
  • the parental anti-CD39 antibody alone showed low binding to all cell types tested (FIGS.5A to 5E).
  • the parental anti-ICOS antibody bound to both CD4+, CD8+, and CD8+ Treg cells (FIGS.5A, 5B, and 5E).
  • the bispecific anti- CD39/anti-ICOS CrossMab antibody showed greatest binding to CD8+ Treg cells, followed by CD4+, CD20+, CD14+, and CD8+ cells (FIGS.5A to 5E), implying that despite the low- affinity binding of the anti-CD39 arm, the anti-ICOS arm of the bispecific antibody induced preferred binding to CD8+ over other cell types.
  • Example 3 Direct Killing of Pathogenic CD4+ T cells by CD8+ Treg in an IncuCyte-Based Cytotoxicity Assay The ability of the bispecific anti-CD39/anti-ICOS CrossMAb antibody from Example 1 to enhance the direct killing of pathogenic gliadin-specific CD4+ T cells by CD8+ Treg was investigated.
  • CD8 + CD45RC low CD39 high cells were isolated from HLA-DQ2.5 + celiac patients PBMC by flow sorting using a BD Fusion cytometer (Beckton-Dickinson).
  • CD8+ cells were co-cultured with autologous T-cell depleted PBMC at a ratio of 1:1 in human T cell media (X-VIVO (Lonza, Basel, Switzerland), 5% human serum, 1% pen/strep and 1% glutamax) with cytokines IL-15 and IL-7 (10 ng/mL each, Biolegend). After one week, a further 25 ul/mL ImmunoCult was added, and cells incubated for a further week to expand.
  • human T cell media X-VIVO (Lonza, Basel, Switzerland), 5% human serum, 1% pen/strep and 1% glutamax
  • cytokines IL-15 and IL-7 10 ng/mL each, Biolegend
  • CD8+ Treg cells were then resuspended in human T cell media with IL-7 (2 ng/mL), IL-15 (10 ng/mL), and IL-2 (20 U/mL), and used as CD8+ Treg cells for IncuCyte killing assays.
  • CD8+ Treg cells at 1x10 4 /well were plated in a 96 well plate (Corning Inc, Corning, NY), followed by addition of the bispecific and parental antibodies, which were diluted out to have two final concentrations of 1 and 10 ug/mL in triplicates.
  • An SKW-GFP cell line expressing a gliadin-specific TCR (5x10 4 cells/well, ratio of 1 Treg to 5 SKW cells) were added into each well.
  • Autologous T-cell depleted PBMC were used as antigen presenting cells (APC) and incubated with four gliadin peptides (QLQPFPQPELPY (SEQ ID NO:87), PQPELPYPQPE (SEQ ID NO:88), QQPFPQPEQPFP (SEQ ID NO:89), FPQPEQPFPWQP (SEQ ID NO:90)) at 6.25 ug/mL at 37oC for an hour before being added to wells at 2.5x10 4 cells/well.
  • the final ratio of CD8+ Treg to SKW cell to APC was 1:5:2.5.
  • CD4+ T cells were purified by negative or positive selection and stained with CTV (CellTrace TM Violet, 1 uM, ThermoFisher) at 37oC for 10 min, followed by washing with FBS.
  • Autologous CD8 Treg cells were prepared as in Example 3, and stained with eF670 (1 uM, ThermoFisher) at 37oC for 10 mins.
  • CTV labelled CD4+ cells were added at 4x10 4 cells / well to a 96 well U bottom plates.
  • eF670-stained CD8+ Treg cells were then added to wells at a ratio of 1:2 or 1:16 to CD4.
  • Antibodies were prepared at 10ug/ml final concentration and added to the wells.
  • bispecific anti-CD39/anti-ICOS CrossMab antibody showed a lower percentage of CD4+ T cells proliferating compared to wells receiving no antibody or receiving anti-CD39 or anti-ICOS parental monospecific antibodies (FIG.7). This suggests that the bispecific anti-CD39/anti-ICOS CrossMab antibody was able to potentiate the function of CD8+ Treg in a manner that neither the anti- CD39 nor the anti-ICOS antibodies are able to replicate.
  • the effect of bispecific anti- CD39/anti-ICOS CrossMab antibody is quantified in FIG.8.
  • CD8+ Treg Phenotypes Can be Influenced by In vitro Culture Conditions
  • a culture system containing cytokines was designed. Isolated CD8+ Tregs were cultured in: (1) TGF- ⁇ or in (2) IL-15 and IL-7 ( ⁇ chain cytokines). Flow cytometry was used assess surface expression of CD8, CD39, CD103, PD-1, ICOS, CXCR3, and NKG2D, following culture in the cytokines.
  • CD8+ Tregs cultured in TGF- ⁇ versus IL-15 and IL-7 exhibited different cell surface phenotypes based on surface protein expression (FIGS.9A-9B).
  • Example 6 Cellular Expansion in the Presence of ⁇ Chain Cytokines Increases CD39 Expression on KIR+CD8+ T cells
  • ⁇ chain cytokines Increases CD39 Expression on KIR+CD8+T cells
  • in vitro assays were performed.
  • the effect of IL-15 on CD39 expression in CD3+CD8+KIR+ T cells was evaluated.
  • CD3+CD8+KIR+ T cells were isolated from PBMCs derived from three separate donors with celiac disease using flow cytometry-based cell-sorting.
  • the isolated CD3+CD8+KIR+ T cells were cultured in the presence of different concentrations of IL-15 (0.05-5 ng/mL) for twelve (12) days and then re-evaluated for KIR expression by flow cytometry (FIG.10).
  • the results showed that CD3+CD8+KIR+ T cells can gain CD39 expression following culture in IL-15 and that the increase in expression is dose-dependent.
  • the effects of different combinations of ⁇ chain cytokines on CD39 expression in CD3+CD8+KIR+ T cells were evaluated in cells derived from donors with celiac disease and from healthy donors.
  • PBMCs from donors with celiac disease were cultured in the presence of: (1) IL-7 and IL-15 or (2) IL-2 and IL-15 for twelve (12) days and then re-evaluated for CD39 expression by flow cytometry (FIG.11).
  • PBMCs from healthy normal donors were tested as a control. The results showed that increased CD39 expression in CD3+CD8+KIR+ T cells in response to ⁇ chain cytokines may be impaired in celiac donors as compared with normal healthy donors.
  • Example 7 CD39 Expression is Increased on KIR+CD8+ T Cells in Patients with Autoimmune Disorders
  • an in vitro assay was performed. A non-celiac (healthy donor) organoid was treated with anti-CD3 and anti-CD28 antibodies to activate the T cell population. Treatment with monospecific anti-ICOS antibody alone was tested as a control. Flow cytometry was used to evaluate the expression of CD39 on CD3+CD8+ T cells (FIG.12).
  • Example 8 Affinity Testing of Two Bispecific CD39/ICOS-Targeting Molecules The contribution of the affinity of each of anti-CD39 and anti-ICOS to the overall avidity of the anti-CD39/anti-ICOS CrossMAb antibody from Example 1 was tested using a biolayer interferometry (BLI) system from Octet (ForteBio) for measuring antibody affinity.
  • FIG.14A shows a co-binding sensorgram for the anti-CD39/anti-ICOS CrossMAb antibody (of Example 1, comprising anti-CD39 clone 27577 and anti-ICOS clone 422 H2L5).
  • FIG. 14B shows a co-binding sensorgram for the anti-CD39/anti-ICOS CrossMAb antibody (of Example 1, comprising anti-CD39 clone 27577 and anti-ICOS clone 422 H2L5).
  • FIG. 1 shows antibody affinity (KD), association rate (ka), and dissociation rate (kd) for two CrossMAbs including: (1) the anti-CD39/anti-ICOS CrossMAb antibody from Example 1 (comprising anti-CD39 clone 27577 and anti-ICOS clone 422 H2L5), which includes a low affinity anti-CD39 antibody, and (2) a second anti-CD39/anti-ICOS CrossMAb antibody (comprising anti-CD39 clone 31815 and anti-ICOS clone 422 H2L5), which includes a high affinity anti-CD39 antibody.
  • the anti-CD39/anti-ICOS CrossMAb antibody from Example 1 comprising anti-CD39 clone 27577 and anti-ICOS clone 422 H2L5
  • a second anti-CD39/anti-ICOS CrossMAb antibody comprising anti-CD39 clone 31815 and anti-ICOS clone 422 H2L5
  • Example 9 Bispecific CD39/ICOS-Targeting Molecule Increases Killing of CD4+ T cells and Reduces Cell Death in Cells from Disease Tissues
  • the ability of the bispecific anti-CD39/anti-ICOS CrossMAb antibody from Example 1 to reduce expansion of CD4+ T cells was evaluated using an in vitro assay.
  • CD8+ Tregs were isolated from PBMCs derived from donors with celiac disease and treated with the bispecific anti-CD39/anti-ICOS CrossMAb antibody.
  • CD8+Tregs were then incubated with autologous CTV-labeled CD4+ T cells that were previously activated with anti-CD3 and anti-CD28 stimulation.
  • CD4+ T cells were also incubated with untreated CD8+ Tregs, non- Tregs, and CD8+ Tregs treated with anti-CD39 or anti-ICOS antibody alone, as controls.
  • Expansion of the CD4+ T cell population was evaluated by flow cytometry five (5) days later (FIG.15). Results showed that the bispecific anti-CD39/anti-ICOS CrossMAb antibody from Example 1 was the most effective at reducing expansion of CD4+ T cells as compared to all control groups.
  • bispecific anti-CD39/anti-ICOS CrossMAb antibody from Example 1 was tested.
  • An IncuCyte-based cytotoxicity assay was used to determine the fluorescence of a target SKW-GFP cell line over time in co-culture with CD8+ Tregs. The dotted line represents the level of fluorescence of the target cell with no antibodies added.
  • Monospecific anti-ICOS antibody and monospecific anti-CD39 antibody were tested as controls. Results showed that the bispecific anti-CD39/anti-ICOS CrossMAb antibody from Example 1 enhanced killing of pathogenic CD4+ T cells by CD8+ Tregs (FIG.16).
  • the ability of the bispecific anti-CD39/anti-ICOS CrossMAb antibody from Example 1 to decrease epithelial cell death in a celiac donor-derived organoid stimulated with gliadin peptides was tested. Organoid cultures were treated for 48 hours with a gliadin peptide cocktail and the bispecific anti-CD39/anti-ICOS CrossMAb antibody prior to analysis by flow cytometry (FIG.17). Untreated organoid cultures and organoid cultures treated with anti-ICOS antibody were tested as controls. After 48 hours, organoid cultures were stained with Annexin-V live/dead stain and gated to include only the Epcam+MHCI+ epithelial cell population.
  • CDRs complementarity-determining regions
  • VL Variable Light
  • VH Region QVQLVQSGAEVKKPGSSVKVSCKASGGAFSSYAIGWVRQAPGQGLEWMGGIIPTFGTANYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGGGYQH

Abstract

Binding agents (e.g., antibodies or antibody binding fragments thereof) that specifically bind to CD39 expressed on CD8+ T regulatory cells, and their use in the treatment of diseases or disorders, such as an inflammatory disease or an autoimmune disease, are provided.

Description

CD39-SPECIFIC BINDING AGENTS AND METHODS OF USING THE SAME REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing 670151_408WO_SEQUENCE_LISTING.xml; Size: 115,235 bytes; and Date of Creation: July 24, 2023) is herein incorporated by reference in its entirety. BACKGROUND The immune system includes the innate immune and the adaptive immune system. The adaptive immune system has a number of cell subtypes, including T cells subsets and B cell subsets. T cell subsets include a variety of types of T cells, including naïve T lymphocytes and effector T lymphocytes, such as cytotoxic T cells and helper T cells, and regulatory T cells. The activity of these T cell types is achieved by a balance between the activity of effector T cells and regulation by regulatory T cells. While effector T cells promote inflammation, regulatory T cells (Tregs) are generally thought to control it. Therefore, Tregs play an important role in autoimmune pathogenesis by maintaining self- tolerance, limiting autoimmunity, and by controlling expansion and activation of autoreactive CD4+ T effector cells. Disruption of the balance between effector and regulatory T cells can lead to an inappropriate activation or suppression of an immune response, the loss of self- tolerance, autoimmune disorders, and cancer. There is a need for therapeutic agents that can rebalance immune cell activities and treat or prevent such diseases. BRIEF SUMMARY Provided herein are binding agents and their methods of use to modulate the activity of CD8+ regulatory T cells (Tregs). The binding agents are bispecific or multi-specific and specifically bind to antigens expressed on the surface of the CD8+ Tregs. In some embodiments the CD8+ Tregs express KIR. In some embodiments, the CD8+ Tregs do not express KIR. In some embodiments, the CD8+KIR+ Tregs are MHC class I restricted. In some embodiments, the CD8+KIR+ Tregs are not MHC Qa-1 restricted. Also provided are methods of using the binding agents for the treatment of an inflammatory disease or an autoimmune disease. In some embodiments, a binding agent is provided that comprises a first binding domain that specifically binds to CD39 expressed on the CD8+ Tregs, and a second binding domain that specifically binds to a T cell antigen expressed on the CD8+ Tregs. In some embodiments, the second binding domain specifically binds to an antigen selected from ICOS, CD8a, and PD-1. In some embodiments, the bispecific antibody is a CrossMab bispecific, a Bottle Opener bispecific, a scFV-Fc bispecific, or a DART-Fc bispecific comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb, anti-CD8a Mb1b IgG1r mAb, or anti-PD1 MK-3475 IgG1r mAb. In some embodiments, the bispecific antibody is a CrossMab bispecific comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb, anti-CD8a Mb1b IgG1r mAb, or anti-PD1 MK-3475 IgG1r mAb . In some embodiments, the bispecific antibody is a Bottle Opener bispecific antibody comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti- ICOS 422 H2L5 IgG1r mAb or anti-CD8a Mb1b IgG1r mAb. In some embodiments, the bispecific antibody is a "scFV-Fc" antibody comprising binding domains from (i) anti-CD39 27577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb. In some embodiments, the bispecific antibody is a "scFv" antibody comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb. In some embodiments, a bispecific anti-CD39/anti-ICOS CrossMab antibody is provided, based on the anti-CD39 bivalent monospecific antibody 27577 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:77 and 78, respectively) and the anti-ICOS bivalent monospecific antibody 422 H2L5 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:81 and 82, respectively). In some embodiments, the CrossMab antibody comprises an anti-CD39 (27577) light chain and heavy chain according to SEQ ID NOs:9 and 10, respectively; and an anti-ICOS (422 H2L5) light chain and heavy chain according to SEQ ID NOs: 42 and 43, respectively. In some embodiments, the first binding domain specifically binds to CD39. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL having amino acid sequences selected from the pairs of amino acid sequences set forth in the group consisting of: (i) SEQ ID NO:5 and SEQ ID NO:1, respectively; and (ii) SEQ ID NO:15 and SEQ ID NO:11, respectively. In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy and light chain variable regions comprising hCDR1, hCDR1, and hCDR3, and lCDR1, lCDR2, and lCDR3, respectively, the CDRs having amino acid sequences selected from the sets of amino acid sequences set forth in the group consisting of: (i) SEQ ID NOs:6-8 and 2-4, respectively; and (ii) SEQ ID NOs:16-18 and 12-14, respectively. In some embodiments, the second binding domain specifically binds to ICOS. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL having amino acid sequences set forth in SEQ ID NO:38 and SEQ ID NO:34, respectively. In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy and light chain variable regions comprising hCDR1, hCDR1, and hCDR3, and lCDR1, lCDR2, and lCDR3, respectively, the CDRs having amino acid sequences set forth in SEQ ID NOs: 39-41 and SEQ ID NOs:35-37, respectively. In some embodiments, the second binding domain specifically binds to CD8a. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL having amino acid sequences set forth in SEQ ID NO:25 and SEQ ID NO:21, respectively. In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy and light chain variable regions comprising hCDR1, hCDR1, and hCDR3, and lCDR1, lCDR2, and lCDR3, respectively, the CDRs having amino acid sequences selected from the sets of amino acid sequences set forth in the group consisting of: (i) SEQ ID NOs:26-28 and 22-24, respectively; and (ii) SEQ ID NOs: 29-31 and 22-24, respectively. In some embodiments, the second binding domain specifically binds to PD-1. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL having amino acid sequences set forth in SEQ ID NO:48 and SEQ ID NO:44, respectively. In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy and light chain variable regions comprising hCDR1, hCDR1, and hCDR3, and lCDR1, lCDR2, and lCDR3, respectively, the CDRs having amino acid sequences set forth in SEQ ID NOs:49-51 and 45-47, respectively. In some embodiments, the binding agent does not contain an Fc domain. In some embodiments, the binding agent includes an Fc domain. In some embodiments, the Fc domain is selected from an IgG1 and an IgG4 Fc domain. In some embodiments, the binding agent has substantially no effector function activity. In some embodiments, the Fc domain is an IgG1 Fc domain. In some embodiments, the Fc domain is an IgG1 Fc null. Also provided is a pharmaceutical composition comprising the binding agent of any of the embodiments described herein and a pharmaceutically acceptable carrier. Also provided are nucleic acids encoding the binding agent of any of the embodiments described herein. Further provided is a vector comprising any of the embodiments of nucleic acids described herein. Further also provided are cell lines comprising any of the embodiments of nucleic acids or vectors described herein. In some embodiments, provided is a method of treating an autoimmune disease, comprising administering any of the embodiments of binding agents or pharmaceutical compositions described herein to a subject in need thereof in an amount effective to decrease the number or activity of pathogenic immune cells in the subject and thereby ameliorate a symptom of the autoimmune disease. In some embodiments, provided is a method of suppressing an immune response mediated by pathogenic immune cells, comprising contacting CD8+ T regulatory cells (Tregs) with any of the embodiments of binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate the CD8+ Tregs (activated Tregs), whereby the number or activity of pathogenic immune cells is decreased. In some embodiments, provided is a method of suppressing an immune response to an autoantigen, comprising administering to a subject in need thereof any of the embodiments of binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate the CD8+ Tregs, whereby the number or activity of pathogenic immune cells that are responsive to the autoantigen is decreased. In some embodiments, provided is a method of suppressing an immune response to an antigen, comprising administering to a subject in need thereof any of the embodiments of binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate the CD8+ Tregs, whereby the number or activity of pathogenic immune cells that are responsive to the antigen is decreased. In some embodiments of these methods of treating autoimmune disease or suppressing an immune response, the CD8+ Tregs are contacted with the binding agent in vivo. In some embodiments, the CD8+ Tregs are contacted with the binding agent ex vivo. In some embodiments, the activated CD8+ Tregs are administered in an effective amount to a subject in need thereof. In some embodiments, the pathogenic immune cells are autoreactive CD4+ T cells, autoantibody producing B cells or self antigen presenting dendritic cells. In some embodiments, the pathogenic immune cells are self antigen presenting cells. In some embodiments, the titer of autoantibodies is decreased in the subject. In some embodiments, the subject has an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of celiac disease, Crohn’s disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus/pemphigoid, rheumatoid arthritis (RA), scleroderma/systemic sclerosis, Sjögren’s syndrome (SjS), systemic lupus erythematosus (SLE), and ulcerative colitis. In some embodiments of the methods of treating autoimmune disease or suppressing an immune response, the binding agent specifically binds to CD39 and ICOS, CD8a, or PD-1 on CD8+ Tregs. In some embodiments, the binding agent specifically binds to CD39 and an inhibitory KIR protein on CD8+KIR+ Tregs. In some embodiments, the binding agent specifically binds to CD39 and ICOS on CD8+ Tregs. In some embodiments, the binding agent specifically binds to CD39 and CD8a on CD8+ Tregs. In some embodiments, the binding agent specifically binds to CD39 and PD-1 on CD8+ Tregs. In some embodiments, the CD8+ Tregs are MHC class I restricted. In some embodiments, the CD8+ Tregs are not MHC HLA E (Qa-1b) restricted. In some embodiments of the methods of treating autoimmune disease or suppressing an immune response, the methods further include administering an immunosuppressive agent to the subject. In some embodiments, the administration of the binding agent to the subject results in an improved treatment outcome in the subject. In some embodiments, the improved treatment outcome is a reduced frequency or severity disease flares, reduced systemic inflammatory cytokines, or reduced self reporting of symptoms associated with the autoimmune disease. In some embodiments of the methods of treating autoimmune disease or suppressing an immune response, the binding agent is administered intravenously. In some embodiments, the binding agent is administered subcutaneously. In some embodiments, the binding agent is administered in a dose of about 0.01 mg/kg to about 20 mg/kg. In some embodiments, the binding agent has substantially no effector function activity. In some embodiments, provided is the use of any of the embodiments of the binding agents or the pharmaceutical compositions described herein for the treatment of autoimmune disease in a subject by activating or stimulating CD8+ Tregs. In some embodiments, provided is the use of any of the embodiments of the binding agents or the pharmaceutical compositions described herein for the reduction of an immune response by pathogenic immune cells by activating or stimulating CD8+ Tregs. In some embodiments, provided is the use of any of the embodiments of the binding agents or the pharmaceutical compositions described herein for the reduction of autoantibody titer in a subject by activating or stimulating CD8+ Tregs. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1A to 1D show exemplary bispecific antibody structures. FIG.2 shows binding of antibodies to cells expressing CD39. The CD39+ICOS- SK- MEL 28 cell line was stained with a bispecific anti-CD39/anti-ICOS CrossMab antibody and the parental anti-CD39 bivalent monospecific antibody, followed by a secondary antibody. The graph illustrates geometric mean fluorescence intensity (gMFI) of human IgG bound cells. Results for the monospecific anti-CD39 antibody and the bispecific anti-CD39/anti- ICOS CrossMab antibody are shown on the graph with an open and a filled circle, respectively. FIG.3 shows binding of antibodies to cells expressing ICOS.293T cells were transduced with an ICOS expression vector and used for ICOS binding assay. The points represent gMFI of human IgG bound cells. Results for the monospecific anti-ICOS antibody and the bispecific anti-CD39/anti-ICOS CrossMab antibody are shown on the graph with an open and a filled circle, respectively. FIG.4 shows binding of the bispecific anti-CD39/anti-ICOS CrossMab antibody to a CD39 overexpressing Sk-Mel cell line (Bastid et al, 2014 Cancer Immunol Res.3(3):254-65). The gMFI of human IgG bound cells is shown, with results for the monospecific anti-ICOS antibody and the monospecific anti-CD39 antibody indicated with an open circle or an open diamond, respectively. Results for the bispecific anti-CD39/anti-ICOS CrossMab antibody are shown as a filled circle. FIG.5A to FIG.5E show binding of the antibodies in different immune cell populations. The graph represents the gMFI of the monospecific anti-ICOS antibody, the monospecific anti-CD39 antibody, or the bispecific anti-CD39/anti-ICOS CrossMab antibody binding to PBMC within the CD4+, CD8+, CD14+ and CD20+ gated populations. The bispecific anti-CD39/anti-ICOS CrossMab antibody is represented by filled circle. The monospecific anti-ICOS antibody and the monospecific anti-CD39 antibody are represented by an open circle or an open diamond, respectively. FIG.5A shows binding in CD4+ cells; FIG.5B shows binding in CD8+; FIG.5C shows binding in CD14+ cells; FIG.5D shows binding in CD20+ cells; and FIG.5E shows binding in CD8+ Treg cells. FIG.6 shows direct killing of pathogenic CD4+ T cells by CD8+ Tregs in an IncuCyte-based cytotoxicity assay. The fluorescence of a target SKW-GFP cell line over time in co-culture with cytotoxic CD8+ regulatory cells is shown. The dotted line represents the level of fluorescence of the target cell with no antibodies added. The monospecific anti-ICOS antibody and the monospecific anti-CD39 antibody are shown in the graph with an open circle and an open diamond, respectively. The bispecific anti-CD39/anti-ICOS CrossMab antibody is shown as a filled circle. FIG.7 shows suppression of CD4+ proliferation by CD8+ Tregs. The CTV (CellTraceTM Violet)-fluorescence of stained CD4+ T cells after incubation with two different ratios to the cytotoxic CD8 cells, 1:2 and 1:16. Absence (No Ab) or presence of parental monospecific antibodies and bispecific (10 ug/mL) are shown, followed by addition of T cell activator beads. Proportion of cells that have divided are shown in gate G1. FIG.8 shows suppression of CD4+ proliferation by CD8+ Tregs in vitro. Percentages of divided CD4+ cells in gate G1 were used to quantify suppression. Suppression (%) was calculated by 100* [(G1 percentage from CD4 with activator beads, no CD8 Treg) – (G1 percentage from CD4 co-cultured with CD8 Treg)] / (G1 percentage from CD4 with activator beads, no CD8 Treg). FIGS.9A-9B show the effect of TGF-β or IL-15 and IL-7 on the phenotype of CD8+ Treg cells. CD8+ Tregs isolated from PBMCs were cultured in TGF-β (FIG.9A) or in IL-15 and IL-7 (FIG.9B) and assessed for expression of CD8, CD39, CD103, PD-1, ICOS, CXCR3, and NKG2D by flow cytometry. FIG.10 shows the effect of IL-15 on CD39 expression in CD3+CD8+KIR+ T cells. CD3+CD8+KIR+ T cells isolated from PBMCs derived from three separate donors with celiac disease were cultured in different concentrations of IL-15 (0.05-5 ng/mL) for twelve (12) days and then re-evaluated for KIR expression by flow cytometry. FIG.11 shows the effects of different combinations of γ chain cytokines on CD39 expression in CD3+CD8+KIR+ T cells derived from donors with celiac disease and from healthy donors. PBMCs from donors with celiac disease were cultured in the presence of: (1) IL-7 and IL-15 or (2) IL-2 and IL-15 for twelve (12) days and then re-evaluated for CD39 expression by flow cytometry. PBMCs from healthy normal donors were tested as a control. FIG.12 shows the effect of the anti-CD39/anti-ICOS CrossMAb antibody on CD39 expression in CD3+CD8+T cells. A non-celiac (healthy donor) organoid was treated with anti-CD3 and anti-CD28 antibodies to activate the T cell population. Flow cytometry was used to evaluate the expression of CD39 on CD3+CD8+ T cells. Treatment with monospecific anti-ICOS antibody alone was tested as a control. FIG.13 shows the co-expression of KIR and CD39 in CD3+CD8+ T cells from patients with different autoimmune diseases. The % of CD3+CD8+CD39+ T cells positive and negative for KIR expression are shown, FIGS.14A-14B show the contribution of the affinity of each of anti-CD39 and anti- ICOS to the overall avidity of the anti-CD39/anti-ICOS CrossMAb antibody. Affinity was tested using biolayer interferometry (BLI). FIG.14A shows a co-binding sensorgram for the anti-CD39/anti-ICOS CrossMAb antibody (of Example 1, comprising anti-CD39 clone 27577 and anti-ICOS clone 422 H2L5). FIG 14B. shows antibody affinity (KD), association rate (ka), and dissociation rate (kd) for two CrossMAbs including: (1) the anti-CD39/anti-ICOS CrossMAb antibody (of Example 1, comprising anti-CD39 clone 27577 and anti-ICOS clone 422 H2L5), and (2) a second anti-CD39/anti-ICOS CrossMAb antibody (comprising anti- CD39 clone 31815 and anti-ICOS clone 422 H2L5). FIG.15 shows the ability of the bispecific anti-CD39/anti-ICOS CrossMAb antibody to reduce expansion of CD4+ T cells in vitro. CD8+ Tregs isolated from PBMCs derived from donors with celiac disease were treated with the bispecific anti-CD39/anti-ICOS CrossMAb antibody and then incubated with autologous CTV-labeled CD4+ T cells that were previously activated with anti-CD3 and anti-CD28 stimulation. CD4+ T cells were also incubated with untreated CD8+ Tregs, non-Tregs, and CD8+ Tregs treated with anti-CD39 or anti-ICOS antibody alone, as controls. Expansion of the CD4+ T cell population was evaluated by flow cytometry. FIG.16 shows direct killing of pathogenic CD4+ T cells by CD8+ Tregs in an IncuCyte-based cytotoxicity assay. The fluorescence of a target SKW-GFP cell line over time in co-culture with cytotoxic CD8+ regulatory cells is shown. The dotted line represents the level of fluorescence of the target cell with no antibodies added. The monospecific anti-ICOS antibody and the monospecific anti-CD39 antibody are shown in the graph with an closed circle and downward-facing closed triangle, respectively. The bispecific anti-CD39/anti- ICOS CrossMab antibody is shown as a upward-facing closed triangle. FIG.17 shows the ability of the bispecific anti-CD39/anti-ICOS CrossMAb antibody to decrease epithelial cell death in a non-celiac (healthy donor) organoid. Organoid cultures were treated for 48 hours with the bispecific anti-CD39/anti-ICOS CrossMAb antibody prior to analysis by flow cytometry. Untreated organoid cultures and organoid cultures treated with anti-ICOS antibody alone were tested as controls. After 48 hours, organoid cultures were stained with Annexin-V live/dead stain and gated to include only the Epcam+MHCI+ epithelial cell population. DETAILED DESCRIPTION I. Glossary The following sections provide a detailed description of binding agents, particularly antibodies or antigen-binding fragments thereof, that target CD39, and related pharmaceutical compositions, methods of activating CD8+ regulatory T cells (CD8+ Treg cells), and methods of treating or preventing disease (e.g., an autoimmune disease). Prior to setting forth this disclosure in more detail, definitions of certain terms to be used herein are provided. Additional definitions are set forth throughout this disclosure. Unless the context requires otherwise, throughout the present specification and claims, the word "comprise" and variations thereof, such as "comprises" and "comprising," are to be construed in an open, inclusive sense, that is, as "including, but not limited to". "Consisting of" shall mean excluding more than trace elements of other ingredients and substantial method steps disclosed herein, and in the case of an amino acid or nucleic acid sequence, excluding additional amino acids or nucleotides, respectively. The term "consisting essentially of" limits the scope of a claim to the specified materials or steps, or to those that do not materially affect the basic characteristics of a claimed invention. For example, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from an isolation and purification method and would not exclude pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. Similarly, a protein consists essentially of a particular amino acid sequence when the protein includes additional amino acids that contribute to at most 20% of the length of the protein and do not substantially affect the activity of the protein (e.g., alters the activity of the protein by no more than 50%). Embodiments defined by each of these transitional terms are within the scope of this invention. In the present description, the term "about" means + 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms "a" and "an" as used herein includes "one" or "one or more" of the enumerated components unless stated otherwise. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives, and may be used synonymously with "and/or". As used herein, the terms "include" and "have" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting. The word "substantially" does not exclude "completely"; e.g., a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from definitions provided herein. "Optional" or "optionally" means that the subsequently described element, component, event, or circumstance may or may not occur, and that the description includes instances in which the element, component, event, or circumstance occurs and instances in which they do not. As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. As used herein, the terms "peptide", "polypeptide", and "protein", and variations of these terms, refer to a molecule that comprises at least two amino acids joined to each other by a (normal or modified) peptide bond. For example, a peptide, polypeptide, or protein may comprise or be composed of a plurality of amino acids selected from the 20 amino acids defined by the genetic code or an amino acid analog or mimetic, each being linked to at least one other by a peptide bond. A peptide, polypeptide, or protein can comprise or be composed of L-amino acids and/or D-amino acids (or analogs or mimetics thereof). The terms "peptide", "polypeptide", and "protein" also include "peptidomimetics" which are defined as peptide analogs containing non-peptidic structural elements, which peptides are capable of mimicking or antagonizing the biological action(s) of a natural parent peptide. In certain embodiments, a peptidomimetic lacks characteristics such as enzymatically scissile peptide bonds. A peptide, polypeptide, or protein may comprise amino acids other than the 20 amino acids defined by the genetic code in addition to these amino acids, or it can be composed of amino acids other than the 20 amino acids defined by the genetic code. In certain embodiments, a peptide, polypeptide, or protein in the context of the present disclosure can comprise amino acids that are modified by natural processes, such as post-translational maturation processes, or by chemical processes (e.g., synthetic processes), which are known in the art and include those described herein. Such modifications can appear anywhere in the polypeptide; e.g., in the peptide skeleton; in the amino acid chain; or at the carboxy- or amino-terminal ends. A peptide or polypeptide can be branched, such as following an ubiquitination, or may be cyclic, with or without branching. The terms "peptide", "polypeptide", and "protein" also include modified peptides, polypeptides and proteins. For example, peptide, polypeptide, or protein modifications can include acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of a nucleotide or of a nucleotide derivative, covalent fixation of a lipid or of a lipidic derivative, the covalent fixation of a phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfatation, or amino acid addition such as arginylation or ubiquitination. Such modifications have been described in the literature (see Proteins Structure and Molecular Properties (1993) 2nd Ed., T. E. Creighton, New York; Post- translational Covalent Modifications of Proteins (1983) B. C. Johnson, Ed., Academic Press, New York; Seifter et al. (1990) Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol.182: 626-646 and Rattan et al., (1992) Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci, 663: 48-62). Accordingly, the terms "peptide", "polypeptide", "protein" can include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, and the like. Variants of proteins, peptides, and polypeptides of this disclosure are also contemplated. In certain embodiments, variant proteins, peptides, and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to an amino acid sequence of a defined or reference amino acid sequence as described herein. "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to an encoded gene product and fragments thereof. Additionally, as used herein, "(poly)peptide" and "protein" may be used interchangeably in reference to a polymer of amino acid residues, such as a plurality of amino acid monomers linked by peptide bonds. "Nucleic acid molecule" or "polynucleotide" or "nucleic acid" refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, any of which may be single or double-stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense strand). Polynucleotides (including oligonucleotides), and fragments thereof may be generated, for example, by polymerase chain reaction (PCR) or by in vitro translation, or generated by any of ligation, scission, endonuclease action, or exonuclease action. A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) may be removed through co- or post-transcriptional mechanisms. Different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing, or both. Variants of nucleic acid molecules of this disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68ºC or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42ºC. Nucleic acid molecule variants retain the capacity to encode a binding domain having a functionality described herein, such as specifically binding a target molecule. As used herein, the term "sequence variant" refers to any sequence having one or more alterations in comparison to a reference sequence, whereby a reference sequence is any published sequence and/or any of the sequences disclosed herein, i.e., SEQ ID NO:1 to SEQ ID NO:90. Thus, the term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. In certain embodiments, a sequence variant in the context of a nucleotide sequence, the reference sequence is also a nucleotide sequence, whereas in certain embodiments for a sequence variant in the context of an amino acid sequence, the reference sequence is also an amino acid sequence. A "sequence variant" as used herein can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference sequence. "Percent sequence identity" refers to a relationship between two or more sequences, as determined by comparing the sequences. Methods to determine sequence identity can be designed to give the best match between the sequences being compared. For example, the sequences may be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment). Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to determine sequence identity and similarity can be found in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithm used in the BLAST programs can be found in Altschul et al., Nucleic Acids Res.25:3389-3402, 1997. Within the context of this disclosure, it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. "Default values" mean any set of values or parameters that originally load with the software when first initialized. A "sequence variant" in the context of a nucleic acid (nucleotide) sequence has an altered sequence in which one or more of the nucleotides in the reference sequence is deleted or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides are referred to herein by the standard one-letter designation (A, C, G, or T). Due to the degeneracy of the genetic code, a "sequence variant" of a nucleotide sequence can either result in a change in the respective reference amino acid sequence, i.e., in an amino acid "sequence variant" or not. In certain embodiments, a nucleotide sequence variant does not result in an amino acid sequence variant (e.g., a silent mutation). In some embodiments, a nucleotide sequence variant that results in one or more "non-silent" mutation is contemplated. In some embodiments, a nucleotide sequence variant of the present disclosure encodes an amino acid sequence that is at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a reference amino acid sequence. Nucleotide and amino sequences as disclosed herein refer also to codon-optimized versions of a reference or wild-type nucleotide or amino acid sequence. In any of the embodiments described herein, a polynucleotide of the present disclosure may be codon-optimized for a host cell containing the polynucleotide (see, e.g., Scholten et al., Clin. Immunol.119:135-145 (2006). Codon optimization can be performed using known techniques and tools, e.g., using the GenScript® OptimumGeneTM tool, or the GeneArt Gene Synthesis Tool (Thermo Fisher Scientific). Codon-optimized sequences include sequences that are partially codon-optimized (i.e., at least one codon is optimized for expression in the host cell) and those that are fully codon-optimized. A "sequence variant" in the context of an amino acid sequence has an altered sequence in which one or more of the amino acids is deleted, substituted, or inserted in comparison to a reference amino acid sequence. As a result of the alterations, such a sequence variant has an amino acid sequence which is at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference amino acid sequence. For example, per 100 amino acids of the reference sequence a variant sequence that has no more than 10 alterations, i.e., any combination of deletions, insertions or substitutions, is "at least 90% identical" to the reference sequence. A "conservative substitution" refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution found in one of the following groups: Group 1: Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gln or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (Ile or I), Leucine (Leu or L), Methionine (Met or M), Valine (Val or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Val, Leu, and Ile. Other conservative substitutions groups include: sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company. Amino acid sequence insertions can include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include the fusion to the N- or C-terminus of an amino acid sequence to a reporter molecule or an enzyme. In general, alterations in the sequence variants do not abolish or significantly reduce a desired functionality of the respective reference sequence. For example, it is preferred that a variant sequence of the present disclosure does not significantly reduce or completely abrogate the functionality of a sequence of an antibody, or antigen binding fragment thereof, to bind to the same epitope as compared to antibody or antigen binding fragment having (or encoded by) the reference sequence. Guidance in determining which nucleotides and amino acid residues, respectively, may be substituted, inserted, or deleted without abolishing a desired structure or functionality can be found by using, e.g., known computer programs. As used herein, a nucleic acid sequence or an amino acid sequence "derived from" a designated nucleic acid, peptide, polypeptide, or protein refers to the origin of the nucleic acid, peptide, polypeptide, or protein. A nucleic acid sequence or amino acid sequence that is derived from a particular sequence may have an amino acid sequence that is essentially identical to that sequence or a portion thereof, from which it is derived, whereby "essentially identical" includes sequence variants as defined above. A nucleic acid sequence or amino acid sequence that is derived from a particular peptide or protein, may be derived from the corresponding domain in the particular peptide or protein. In this context, "corresponding" refers to possession of a same functionality or characteristic of interest. For example, an "extracellular domain" corresponds to another "extracellular domain" (of another protein), or a "transmembrane domain" corresponds to another “transmembrane domain” (of another protein). "Corresponding" parts of peptides, proteins, and nucleic acids are thus easily identifiable to one of ordinary skill in the art. Likewise, a sequence "derived from" another (e.g., "source") sequence can be identified by one of ordinary skill in the art as having its origin in the source sequence. A nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be identical to the starting nucleic acid, peptide, polypeptide, or protein (from which it is derived). However, a nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may also have one or more mutations relative to the starting nucleic acid, peptide, polypeptide, or protein (from which it is derived), in particular a nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be a functional sequence variant as described above of the starting nucleic acid, peptide, polypeptide, or protein (from which it is derived). For example, in a peptide/protein, one or more amino acid residues may be substituted with other amino acid residues, or one or more amino acid residue insertions or deletions may occur. As used herein, the term "mutation" relates to a change in a nucleic acid sequence and/or in an amino acid sequence in comparison to a reference sequence, e.g., a corresponding genomic, wild type, or reference sequence. A mutation, e.g., in comparison to a reference genomic sequence, may be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation, e.g., induced by enzymes, chemicals or radiation, or a mutation obtained by site-directed mutagenesis (molecular biology methods for making specific and intentional changes in the nucleic acid sequence and/or in the amino acid sequence) Thus, the terms "mutation" or "mutating" shall be understood to also include physically making or inducing a mutation, e.g., in a nucleic acid sequence or in an amino acid sequence. A mutation includes substitution, deletion, and insertion of one or more nucleotides or amino acids as well as inversion of several successive nucleotides or amino acids. To achieve a mutation in an amino acid sequence, a mutation may be introduced into the nucleotide sequence encoding said amino acid sequence in order to express a (recombinant) mutated polypeptide. A mutation may be achieved, for example, by altering (e.g., by site- directed mutagenesis) a codon (e.g., by altering one, two, or three nucleotide bases therein) of a nucleic acid molecule encoding one amino acid to provide a codon that encodes a different amino acid, or that encodes a same amino acid, or by synthesizing a sequence variant. The term "introduced" in the context of inserting a nucleic acid molecule into a cell, means "transfection", or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA). The term "recombinant", as used herein (e.g., a recombinant antibody, a recombinant protein, a recombinant nucleic acid, or the like, refers to any molecule (antibody, protein, nucleic acid, or the like) which is prepared, expressed, created, or isolated by recombinant means, and which is not naturally occurring. "Recombinant" can be used synonymously with "engineered" or "non-natural" and can refer to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, fusion proteins, or enzymes, or other nucleic acid molecule additions, deletions, or substitutions or other functional disruption of a cell's genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene or operon. As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or a subject, or any gene, protein, compound, nucleic acid molecule, or activity native to a host cell or a subject that has been altered. Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered such that the structure, activity, or both is different as between the native and altered genes, proteins, compounds, or nucleic acid molecules. In certain embodiments, heterologous, non-endogenous, or exogenous genes, proteins, or nucleic acid molecules may not be endogenous to a host cell or a subject, but instead nucleic acids encoding such genes, proteins, or nucleic acid molecules may have been added to a host cell by conjugation, transformation, transfection, electroporation, or the like, wherein the added nucleic acid molecule may integrate into a host cell genome or can exist as extra-chromosomal genetic material (e.g., as a plasmid or other self-replicating vector). The term "homologous" or "homolog" refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof. A non-endogenous polynucleotide or gene, as well as the encoded polypeptide or activity, may be from the same species, a different species, or a combination thereof. As used herein, the term "endogenous" or "native" refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or a subject. As used herein, the terms "cell," "cell line, " and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same or substantially the same function, phenotype, or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context. The terms "isolated" or "partially purified" as used herein refer in the case of a nucleic acid, polypeptide, or protein, to a nucleic acid, polypeptide, or protein separated from at least one other component (e.g., nucleic acid or polypeptide or protein) that is present with the nucleic acid, polypeptide, or protein as found in its natural source and/or that would be present with the nucleic acid, polypeptide, or protein when expressed by a cell, or secreted in the case of secreted polypeptides and proteins. A chemically synthesized nucleic acid, polypeptide, or protein, or one synthesized using in vitro transcription/translation, is considered "isolated." The terms "purified" or "substantially purified" refer to an isolated nucleic acid, polypeptide, or protein that is at least 95% by weight the subject nucleic acid, polypeptide, or protein, including, for example, at least 96%, at least 97%, at least 98%, at least 99%, or more. As used herein, pathogenic immune cells refer to immune cells that are reactive with an autoantigen or that induce a response to an autoantigen. Examples of such pathogenic immune cells include autoreactive CD4+ T cells, autoantibody producing B cells, self antigen presenting dendritic cells, and other self antigen presenting cells, as are known in the art. CD39 is also known as ectonucleoside triphosphate diphosphohydrolase 1, SPG64 ATPDase, and NTPDase-1. It encodes plasma membrane protein that hydrolyzes extracellular ATP and ADP to AMP. CD39 polypeptides include, but are not limited to, those having the amino acid sequences disclosed in NP_001307845.1, NP_001157651.1, NP_001157650.1, NP_001091645.1, NP_001767.3, NP_001299583.1, NP_001157655.1, NP_001157654.1, and NP_001157653.1; these amino acid sequences are incorporated by reference herein. CD8alpha (or CD8a) is a protein that is expressed on T cells, including regulatory T cells. CD8alpha polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_001759.3, NP001139345.1, NP_741969.1, NP_001369627.1, NP_757362.1, NP_001171571.1, NP_742100.1, NP_742099.1, and NP_004922; these sequences are incorporated by reference herein. ICOS, or inducible T cell costimulatory, is also referred to as AILIM, CD278, and CVID1. ICOS polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_036224.1; this amino acid sequence is incorporated by reference herein. PD-1 is also referred to as programmed cell death protein 1. PD-1 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_005009.2; this amino acid sequence is incorporated by reference herein. As used herein, “KIR” may refer to one or more of KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2, and KIR2DL3. KIR3DL1 is a protein expressed on NK cells and on some T cells. It is also known as CD158E1, KIR, KIR2DL5B, KIR3DL1/S1, NKAT-3, NKAT3, NKB1, and NKB1B. KIR3DL1 polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_037421.2 and NP_001309097.1; these sequences are incorporated by reference herein. KIR3DL2 is a protein expressed on NK cells and on some T cells. It is also known as 3DL2, CD158K, KIR-3DL2, NKAT-4, NKAT4, NKAT4B, and p140. KIR3DL2 polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_006728.2 and NP_001229796.1; these sequences are incorporated by reference herein. KIR2DL1 is a protein expressed on NK cells and on some T cells. It is also known as CD158A, KIR-K64, KIR221, KIR2DL3, NKAT, NKAT-1, NKAT1, and p58.1. KIR2DL1 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_055033.2; this sequence is incorporated by reference herein. KIR2DL2 is a protein expressed on NK cells and on some T cells. It is also known as CD158B1, CD158b, NKAT-6, NKAT6, and p58.2. KIR2DL2 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_055034.2; this sequence is incorporated by reference herein. KIR2DL3 is a protein expressed on NK cells and on some T cells. It is also known as CD158B2, CD158b, GL183, KIR-023GB, KIR-K7b, KIR-K7c, KIR2DL, KIR2DS5, KIRCL23, NKAT, NKAT2, NKAT2A, NKAT2B, and p58. KIR2DL3 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_056952.2; this sequence is incorporated by reference herein. CD103, or integrin subunit alpha E (ITGAE), is also referred to as HUMINAE. CD103 polypeptides include, but are not limited to, those having the amino acid sequence disclosed in NP_002199.3; this amino acid sequence is incorporated by reference herein. CXCR3, or C-X-C motif chemokine receptor 3, is also referred to as GPR9, MigR, CD182, CD183, Mig-R, CKR-L2, CMKAR3, and IP10-R. CXCR3 polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_001495.1 and NP_001136269.1; these amino acid sequences are incorporated by reference herein. NKG2D, or Killer Cell Lectin Like Receptor K1, is also referred to as KLRK1, D12S2489E, NKG2-D, CD314, and KLR. NKG2D polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_031386.2 and NP_001186734.1; these amino acid sequences are incorporated by reference herein. II. Modulation of CD8+ Regulatory T cells Provided herein are binding agents comprising binding domains that specifically bind to antigens expressed on CD8+ regulatory T cells (Tregs). In some embodiments, the CD8+ Tregs express KIR (CD8+KIR+ Tregs). In some embodiments the KIR expressed by the CD8+KIR+ Treg is an inhibitory KIR. In some embodiments the inhibitory KIR is, for example, KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2, or KIR2DL3 or a combination thereof. In some embodiments, the CD8+ Tregs do not express KIR. In some embodiments the CD8+ Tregs are MHC class I restricted. In some embodiments, the CD8+ Tregs are not MHC Qa-1 (HLA-E) restricted. Also provided are methods of using the binding agents for the treatment of autoimmune disease, infectious disease, and cancer. In some embodiments, the binding agent includes a first binding domain that specifically binds to CD39 expressed on the CD8+ Tregs, and a second binding domain that specifically binds to a T cell antigen expressed on the CD8+ Tregs. In some embodiments, the second binding domain specifically binds to an antigen selected from ICOS, CD8a, and PD-1. In some embodiments, the second antigen is selected from a functional antagonist to reduce function inhibition of CD8+ Tregs. In some embodiments, such antigen is ICOS, CD8a, or PD-1. In some embodiments, the second antigen is a tethering moiety to enhance specificity of the binding agent to CD8+ Tregs. In some embodiments, such antigen is ICOS, CD8a, or PD-1. III. CD8+KIR+ Regulatory T Cells CD8+KIR+ regulatory T cells are characterized by the phenotype of being CD8+ KIR+ and are typically MHC Class I restricted. In humans, the CD8+KIR+ regulatory T cells express inhibitory KIR proteins. In some embodiments, the KIR proteins expressed by the cells can include one or more of the inhibitory KIR proteins, e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, and KIR3DL2; and may specifically include one or more of KIR2DL2, KIR2DL3, and KIR3DL1. In some embodiments, the CD8+KIR+ regulatory T cells are not HLA E (Qa-1b) restricted. (See, e.g., Lohwasser et al., International Immunology 13:321-327 (2001) and Sarantopoulos et al., J. Clin. Invest.114(9):1218-1221 (2004) for a general explanation of murine Qa-1b and human HLA E restriction.) In some embodiments, the CD8+KIR+ regulatory T cells can also be characterized as being CD44+, CD122+, and are not HLA E (Qa-1b) restricted. In some embodiments, the CD8+KIR+ regulatory T cells can also be characterized as being CD28-. In some embodiments, the CD8+KIR+ regulatory T cells can also be characterized as being CD28-, CD44+, and CD122+. In some embodiments, the CD8+KIR+ regulatory T cells can also be characterized as being CD28-, CD44+, and CD122+, and are not HLA E (Qa-1b) restricted. In some embodiments, the CD8+KIR+ Tregs express the following antigens: CD3, CD8, PD-1, CD16, CD122, CD39, CXCR3, ICOS, CD103, and inhibitory KIR proteins. In some embodiments, CD8+KIR+ Tregs express one or more of the following antigens: CD3, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG- 3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4, and 41BB. In some embodiments, CD8+KIR+ Tregs express one or more of the following antigens: CD3, CD5, CD16, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, KLRB1, KLRG1, LAG- 3/CD223, NKG2C, NKG2D, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4, and 41BB. In some embodiments, CD8+KIR+ Tregs express one or more of the following antigens: CD39, KLRB1, KLRG1, NKG2C, NKG2D, CXCR3, and CD122. IV. Antibodies and Antigen-Binding Fragments In one aspect, the present disclosure provides an antibody, or an antigen binding fragment thereof, that is capable of binding to CD39 and capable of binding to ICOS, CD8a, or PD-1. Such antibodies, or antigen binding fragments thereof, can bind to and modulate the activity of CD8+CD39+ regulatory T cells (Tregs). In some embodiments, the CD8+ Tregs are also KIR+. Antibodies generally are comprised of a heavy chain and a light chain. Each heavy chain is composed of a variable region (abbreviated as VH) and a constant region. The heavy chain constant region may include three domains CH1, CH2, and CH3 and optionally a fourth domain, CH4. Each of these domains is referred to as an "Fc domain". As used herein, when a binding agent includes an Fc domain, it can include one or more Fc domains, or an entire Fc region, unless otherwise specified by context. Each light chain is composed of a variable region (abbreviated as VL) and a constant region or constant domain. The light chain constant region is a CL domain. The VH and VL regions may be further divided into hypervariable regions referred to as complementarity-determining regions (CDRs) and interspersed with conserved regions referred to as framework regions (FR). Each VH and VL region thus consists of three CDRs and four FRs that are arranged from the N terminus to the C terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. This structure is well known to those skilled in the art. As used herein, and unless the context clearly indicates otherwise, "antibody" refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter- connected by disulfide bonds (though it will be understood that heavy chain antibodies, which lack light chains, are still encompassed by the term "antibody"), as well as any antigen- binding portion or fragment of an intact antibody that has or retains the ability to bind to the antigen target molecule recognized by the intact antibody, such as, for example, a scFv, Fab, or F(ab')2 fragment. Thus, the term "antibody" herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class thereof, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. As used herein, the terms "antigen binding fragment," "fragment, " and "antibody fragment" are used interchangeably to refer to any fragment of an antibody of the disclosure that retains the antigen-binding activity of the antibody. Examples of antibody fragments include, but are not limited to, a single chain antibody, Fab, Fab’, F(ab')2, Fv, and scFv. Human antibodies are known (e.g., van Dijk, M. A., and van de Winkel, J. G., Curr. Opin. Chem. Biol.5 (2001) 368-374). Human antibodies can be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol.7 (1993) 3340). Human antibodies can also be produced in phage display libraries (Hoogenboom, H. R., and Winter, G., J. Mol. Biol.227 (1992) 381-388; Marks, J. D., et al., J. Mol. Biol.222 (1991) 581-597). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); and Boerner, P., et al., J. Immunol.147 (1991) 86-95). Human monoclonal antibodies may be prepared by using improved EBV-B cell immortalization as described in Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. (2004): An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med.10(8):871-5. The term "human antibody" as used herein also comprises such antibodies which are modified, e.g., in the variable region, to generate properties according to the antibodies and antibody fragments of the present disclosure. Antibodies according to the present disclosure can be of any isotype (e.g., IgA, IgG, IgM, IgE, IgD; i.e., comprising a α, γ, µ, ɛ, or δ heavy chain). Within the IgG isotype, for example, antibodies may be IgG1, IgG2, IgG3, or IgG4 subclass. In specific embodiments, an antibody of the present disclosure is an IgG1 antibody. Antibodies or antigen binding fragments provided herein may include a κ or a λ light chain. As used herein, the term "variable region" (variable region of a light chain (VL), variable region of a heavy chain (VH)) denotes each variable region polypeptide of the pair of light and heavy chains, which, in most instances, is involved directly in binding the antibody to the antigen. The terms "VL" and "VH" refer to the variable binding region from an antibody light and heavy chain, respectively. The variable binding regions are made up of discrete, well-defined sub-regions known as "complementarity-determining regions" (CDRs) and "framework regions" (FRs). The terms "complementarity-determining region" and "CDR" are synonymous with "hypervariable region" or "HVR," and are known in the art to refer to sequences of amino acids within TCR or antibody variable regions, which confer antigen specificity and/or binding affinity and are separated by framework sequence. In general, there are three CDRs in each variable region of an immunoglobulin binding protein; e.g., for antibodies, the VH and VL regions comprise six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 (also referred to herein as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, respectively). As used herein, a "variant" of a CDR refers to a functional variant of a CDR sequence having up to 1-3 amino acid substitutions, deletions, or combinations thereof. It will be understood that in certain embodiments, an antibody or antigen binding fragment of the present disclosure can comprise all or part of a heavy chain (HC), a light chain (LC), or both. For example, a full-length intact IgG antibody monomer typically includes a VH, a CH1, a CH2, a CH3, a VL, and a CL. Fc components are described further herein. In certain embodiments, an antibody or antigen binding fragment of the present disclosure comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and a CDRL3 according to any one of the presently disclosed VH and VL sequences, respectively. Fragments of the antibodies described herein can be obtained from the antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, fragments of the antibodies can be obtained by cloning and expression of part of the sequences of the heavy or light chains. The present disclosure encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of an antibody as described herein, including, for example, an scFv comprising the CDRs from an antibody according to the present description, heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, as well as single chain antibodies, in which the heavy and light chain variable domains are joined by a peptide linker. In certain embodiments, an antibody according to the present disclosure, or an antigen binding fragment thereof, comprises a purified antibody, a monoclonal antibody, a single chain antibody, Fab, Fab’, F(ab')2, Fv or scFv. Throughout this disclosure, antibodies, antigen binding fragments thereof, and fusion proteins may individually or collectively (e.g., in any combination) be referred to as "binding proteins". Binding proteins according to the present disclosure may be provided in purified form. For example, an antibody may be present in a composition that is substantially free of other polypeptides, e.g., where less than 90% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides. Binding proteins according to the present disclosure may be immunogenic in human and/or in non-human (or heterologous) hosts; e.g., in mice. For example, an antibody may have an idiotope that is immunogenic in non-human hosts, but not in a human host. Antibodies of the disclosure for human use include those that are not typically isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, or the like, and in some instances are not obtained by humanization or from xeno-mice. Also contemplated herein are variant forms of the disclosed antibodies, which are engineered so as to reduce known or potential immunogenicity and/or other potential liabilities, or to confer a desired structure and/or functionality of the antibody in a non-human animal, such as a mouse (e.g., a "murinized " antibody wherein one or more human amino acid residue, sequence, or motif is replaced by a residue, sequence, or motif that has reduced or abrogated immunogenicity or other liability, or has a desired structure and/or function, in a mouse; e.g., for model studies using a mouse). Antibodies or antigen-binding fragments thereof such as those described herein, including but not limited to scFv, may, in certain embodiments, be comprised in a fusion protein that is capable of specifically binding to an antigen as described herein. As used herein, "fusion protein" refers to a protein that, in a single chain, has at least two distinct domains or motifs, wherein the domains or motifs are not naturally found together, or in the given arrangement, in a protein. A polynucleotide encoding a fusion protein may be constructed using PCR, recombinantly engineered, or the like, or such fusion proteins can be synthesized. Immunoglobulin sequences can be aligned to a numbering scheme (e.g., Kabat, EU, International Immunogenetics Information System (IMGT), and Aho), which can allow equivalent residue positions to be annotated and for different molecules to be compared using Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300). As used herein, "specifically binds" or "specific for" refers to an association or union of a binding protein (e.g., an antibody or antigen binding fragment thereof) or a binding domain to a target molecule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 105 M-1 (which equals the ratio of the on-rate [Kon] to the off rate [Koff] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample. Binding proteins or binding domains may be classified as "high-affinity" binding proteins or binding domains or as "low-affinity" binding proteins or binding domains. "High-affinity" binding proteins or binding domains refer to those binding proteins or binding domains having a Ka of at least 107 M-1, at least 108 M-1, at least 109 M-1, at least 1010 M-1, at least 1011 M-1, at least 1012 M-1, or at least 1013 M-1. "Low-affinity" binding proteins or binding domains refer to those binding proteins or binding domains having a Ka of up to 107 M-1, up to 106 M- 1, or up to 105 M-1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10-5 M to 10-13 M). The terms "binding" and "specifically binding" and similar references do not encompass non- specific sticking. Binding of a binding protein can be determined or assessed using an appropriate assay, such as, for example, Surface Plasmon Resonance (SPR) methods, e.g., a Biacore™ system; kinetic exclusion assays such as KinExA®; and BioLayer interferometry (e.g., using the ForteBio® Octet platform); isothermal titration calorimetry (ITC), or the like, an antigen- binding ELISA (e.g., direct or indirect) with imaging by, e.g., optical density at 450nm, or by flow cytometry, or the like. The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin or other binding molecule, domain, or protein Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics. An epitope to which binding protein binds may be linear (continuous) or conformational (discontinuous). A linear or a sequential epitope is an epitope that is recognized by an antibody according to its linear sequence of amino acids, or primary structure. A conformational epitope may be recognized according to a three-dimensional shape and protein structure. In the case of a conformational epitope (3D structure), the amino acid sequence typically forms a 3D structure as epitope and, thus, the amino acids forming the epitope may be or may be not located in adjacent positions of the primary structure (i.e., maybe or may be not consecutive amino acids in the amino acid sequence). Multispecific Antibodies and Binding Proteins Antibodies and antigen binding fragments of the present disclosure may, in embodiments, be multispecific (e.g., bispecific, trispecific, tetraspecific, or the like), and may be provided in any multispecific format, as disclosed herein. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites or antigens. In certain embodiments, an antibody or antigen binding fragment of the present disclosure is a multispecific antibody, such as a bispecific or trispecific antibody. Formats for bispecific antibodies are disclosed in, for example, Spiess et al., Mol. Immunol.67(2):95 (2015), and in Brinkmann and Kontermann, mAbs 9(2):182-212 (2017), which bispecific formats and methods of making the same are incorporated herein by reference and include, for example, Bispecific T cell Engagers (BiTEs), DARTs, Knobs-Into-Holes (KIH) assemblies, scFv-CH3-KIH assemblies, KIH Common Light-Chain antibodies, TandAbs, Triple Bodies, TriBi Minibodies, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCabs, Intrabodies, CrossMabs, Dual Action Fabs (DAFs) (two-in-one or four-in-one), DutaMabs, DT-IgG, Charge Pairs, Fab-arm Exchange, SEEDbodies, Triomabs, LUZ-Y assemblies, Fcabs, κλ-bodies, orthogonal Fabs, DVD-IgGs, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, and DVI-IgG (four-in-one). Bispecific and multi-specific antibodies include the following: an scFv1-ScFv2, an ScFv12-Fc-scFv22, an IgG-scFv, a DVD-Ig, a triomab/quadroma, a two-in-one IgG, a scFv2-Fc, a TandAb, an scFv-HSA-scFv, an scFv-VHH, a Fab-scFv-Fc, a Fab-VHH-Fc, a dAb-IgG, an IgG-VHH, a Tandem scFv-Fc, a (scFv1)2-Fc-(VHH)2, a scFv-Fc, a one-armed tandem scFv-Fc, and a DART-Fc. An IgG-scFv may be an IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, or IgG-2scFv. Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J.10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No.5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. No.4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol.147: 60 (1991). Engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies", are also included herein (see, e.g., US 2006/0025576A1). Antibodies or antigen binding fragments disclosed herein also include a "Dual Acting Fab" or "DAF" comprising an antigen binding site that binds to two different antigens (see, e.g., US 2008/0069820 and Bostrom et al., 2009, Science 323:1610-14). "CrossMab” antibodies are also included herein (see, e.g., WO 2009/080251, WO 2009/080252, WO2009/080253, WO2009/080254, and WO2013/026833). “Bottle Opener” antibodies are also included herein. In this embodiment, one heavy chain of the antibody comprises a scFv and the other heavy chain comprises a standard FAb, comprising a variable heavy chain and a light chain. This structure is sometimes referred to as the “bottle-opener” format, due to a rough visual similarity to a bottle-opener, or “triple F” format, referring to “scFv-FAb-Fc”. In some embodiments, the antibodies or antigen binding fragments disclosed herein comprise different antigen-binding sites, fused to one or the other of the two subunits of the Fc domain; thus, the two subunits of the Fc domain may be comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific molecules in recombinant production, it is advantageous to introduce in the Fc domain of the binding agent a modification promoting the association of the desired polypeptides. Accordingly, in particular aspects relates to a binding agent (e.g., an antibody or antigen binding fragment thereof) comprising (a) at least a first binding domain, (b) a second binding domain, and (c) a Fc domain composed of a first and a second subunit capable of stable association, wherein the Fc domain comprises a modification promoting the association of the first and second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one aspect said modification is in the CH3 domain of the Fc domain. In a specific aspect, the Fc modification is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other one of the two subunits of the Fc domain. In a particular aspect, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to Kabat EU index). The knob-into-hole technology is described in, e.g., U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). Accordingly, in some embodiments, in a CH3 domain of an Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain which is positionable in a cavity within a CH3 domain of a second Fc domain, and in the CH3 domain of the second Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second Fc domain within which the protuberance within the CH3 domain of the first Fc domain is positionable. The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis. In a specific embodiment, in the CH3 domain of the first Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In another embodiment, in the second Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A). In some embodiments, in the first Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C), and in the second Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C). Introduction of these two cysteine residues results in the formation of a disulfide bridge between the two Fc domains that further stabilizes the dimer (Carter (2001), J Immunol Methods 248, 7-15). In some embodiments, the first Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to Kabat EU index). In some embodiments, a modification promoting association of the first and the second Fc domains comprises a modification mediating electrostatic steering effects, for example, as described in PCT publication WO 2009/089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domains by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable. In some embodiments, a binding agent (e.g., an antibody or antigen binding fragment thereof) comprises one or more scFvs or “single-chain variable fragments”. An scFv is a fusion protein of the variable regions of the heavy (VH) and light chain (VL) variable regions of an antibody, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. scFv antibodies are, described in, e.g., Houston, J. S., Methods in Enzymol.203 (1991) 46-96. Methods for making scFv molecules and designing suitable peptide linkers are described in, for example, U.S. Pat. No.4,704,692; U.S. Pat. No.4,946,778; Raag and Whitlow, FASEB 9:73-80 (1995) and Bird and Walker, TIBTECH, 9: 132-137 (1991). Binding agents (e.g., an antibody or antigen binding fragment thereof) that are scFv- Fcs have been described by Sokolowska-Wedzina et al., Mol. Cancer Res.15(8):1040-1050, 2017. In some embodiments, a binding agent (e.g., an antibody or antigen binding fragment thereof) is a “bispecific T cell engager” or BiTE (see, e.g., WO2004/106381, WO2005/061547, WO2007/042261, and WO2008/119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain can include two single chain Fv (scFv) fragments, each having a variable heavy chain (VH) and a variable light chain (VL) domain separated by a polypeptide linker of a length sufficient to allow intramolecular association between the two domains. This single polypeptide further includes a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes may be specific for different proteins, such that both proteins are bound by the BiTE. As it is a single polypeptide, the bispecific T cell engager may be expressed using any prokaryotic or eukaryotic cell expression system known in the art, e.g., a CHO cell line. However, specific purification techniques (see, e.g., EP1691833) may be necessary to separate monomeric bispecific T cell engagers from other multimeric species, which may have biological activities other than the intended activity of the monomer. In one exemplary purification scheme, a solution containing secreted polypeptides is first subjected to a metal affinity chromatography, and polypeptides are eluted with a gradient of imidazole concentrations. This eluate is further purified using anion exchange chromatography, and polypeptides are eluted using with a gradient of sodium chloride concentrations. Finally, this eluate is subjected to size exclusion chromatography to separate monomers from multimeric species. In some embodiments, a binding agent that is a bispecific antibody is composed of a single polypeptide chain comprising two single chain FV fragments (scFV) fused to each other by a peptide linker. A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. Single-domain antibodies can be derived from the variable domain of the antibody heavy chain from camelids (e.g., nanobodies or VHH fragments). Furthermore, the term single-domain antibody includes an autonomous human heavy chain variable domain (aVH) or VNAR fragments derived from sharks (see, e.g., Hasler et al., Mol. Immunol.75:28-37, 2016). Techniques for producing single-domain antibodies (DABs or VHH) are known in the art, as disclosed in, for example, Cossins et al. (2006, Prot Express Purif 51:253-259 and Li et al., Immunol. Lett.188:89-95, 2017). Single-domain antibodies may be obtained, for example, from camels, alpacas or llamas by standard immunization techniques. (See, e.g., Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75, 2003; and Maass et al., J Immunol Methods 324:13-25, 2007.) A VHH may have potent antigen-binding capacity and can interact with novel epitopes that are inacessible to conventional VH-VL pairs (see, e.g., Muyldermans et al., 2001). Alpaca serum IgG contains about 50% camelid heavy chain only IgG antibodies (HCAbs) (see, e.g., Maass et al., 2007). Alpacas may be immunized with antigens and VHHs can be isolated that bind to and neutralize the target antigen (see, e.g., Maass et al., 2007). PCR primers that amplify alpaca VHH coding sequences have been identified and may be used to construct alpaca VHH phage display libraries, which can be used for antibody fragment isolation by standard biopanning techniques well known in the art (see, e.g., Maass et al., 2007). In some embodiments, a binding agent (e.g., an antibody or antigen binding fragment thereof) is a IgG-scFV. IgG-scFv formats include IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, and IgG-2scFv. These and other bispecific antibody formats and methods of making them have been described in for example, Brinkmann and Kontermann, MAbs 9(2):182-212 (2017); Wang et al., Antibodies, 2019, 8, 43; Dong et al., 2011, MAbs 3:273-88; Natsume et al., J. Biochem.140(3):359-368, 2006; Cheal et al., Mol. Cancer Ther. 13(7):1803-1812, 2014; and Bates and Power, Antibodies, 2019, 8, 28. Igg-like dual-variable domain antibodies (DVD-Ig) have been described by Wu et al., 2007, Nat Biotechnol 25:1290-97; Hasler et al., Mol. Immunol.75:28-37, 2016 and in WO 08/024188 and WO 07/024715. Triomabs have been described by Chelius et al., MAbs 2(3):309-319, 2010.2-in-1- IgGs have been described by Kontermann et al., Drug Discovery Today 20(7):838-847, 2015. Tandem antibody or TandAb have been described by Kontermann et al., Drug Discovery Today 20(7):838-847, 2015. ScFv-HSA-scFv antibodies have also been described by Kontermann et al., Drug Discovery Today 20(7):838-847, 2015. In some embodiments, the binding agent (e.g., an antibody or antigen binding fragment thereof) is a scaffold antigen binding protein, such as for example, fibronectin and designed ankyrin repeat proteins (DARPins) which have been used as alternative scaffolds for antigen-binding domains, see, e.g., Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008). In some embodiments, a scaffold antigen binding protein is selected from the group consisting of Lipocalins (Anticalin), a Protein A-derived molecule such as Z-domain of Protein A (Affibody), an A-domain (Avimer/Maxibody), a serum transferrin (trans-body); a designed ankyrin repeat protein (DARPin), a fibronectin (AdNectin), a C-type lectin domain (Tetranectin); a variable domain of a new antigen receptor beta-lactamase (VNAR fragments), a human gamma-crystallin or ubiquitin (Affilin molecules); a kunitz type domain of human protease inhibitors, and microbodies such as the proteins from the knottin family, peptide aptamers, and fibronectin (adnectin). Lipocalins are a family of extracellular proteins which transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. They have a rigid beta-sheet secondary structure with a number of loops at the open end of the conical structure which can be engineered to bind to different target antigens. Anticalins are between 160-180 amino acids in size, and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482: 337-350 (2000), U.S. Pat. No.7,250,297B1, and US20070224633. Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha-helices and a beta-turn. They can be engineered to bind different target antigens by randomizing residues in the first alpha-helix and a beta-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details, see J. Mol. Biol.332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol.369, 1015-1028 (2007), and US20040132028A1. Fc Domain Modifications In some embodiments, a binding protein (e.g., antibody or an antigen binding fragment thereof) of the present disclosure comprises an Fc moiety. In certain embodiments, the Fc moiety may be derived from human origin, e.g., from human IgG1, IgG2, IgG3, and/or IgG4, or from another Ig class or isotype. In specific embodiments, an antibody or antigen binding fragments can comprise an Fc moiety derived from human IgG1. As used herein, the term "Fc moiety" refers to a sequence comprising, consisting, consisting essentially of, or derived from a portion of an immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site (e.g., residue 216 in native IgG, taking the first residue of heavy chain constant region to be 114) and ending at the C-terminus of the immunoglobulin heavy chain. Accordingly, an Fc moiety may be a complete Fc moiety or a portion (e.g., a domain) thereof. In certain embodiments, a complete Fc moiety comprises a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acid positions 216-446). An additional lysine residue (K) is sometimes present at the extreme C-terminus of the Fc moiety, but is often cleaved from a mature antibody. Amino acid positions within an Fc moiety can be numbered according to the EU numbering system of Kabat, see, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest", U.S. Dept. Health and Human Services, 1983 and 1987. Amino acid positions of an Fc moiety can also be numbered according to the IMGT numbering system (including unique numbering for the C-domain and exon numbering) and the Kabat numbering system. In some embodiments, an Fc moiety comprises at least one of: a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant, portion, or fragment thereof. In some embodiments, an Fc moiety comprises at least a hinge domain, a CH2 domain or a CH3 domain. In further embodiments, the Fc moiety is a complete Fc moiety. The Fc moiety may also comprise one or more amino acid insertions, deletions, or substitutions relative to a naturally occurring Fc moiety. For example, at least one of a hinge domain, CH2 domain, or CH3 domain, or a portion thereof, may be deleted. For example, an Fc moiety may comprise or consist of: (i) hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), (ii) a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), (iii) a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), (iv) a hinge domain (or a portion thereof), (v) a CH2 domain (or a portion thereof), or (vi) a CH3 domain or a portion thereof. An Fc moiety of the present disclosure may be modified such that it varies in amino acid sequence from the complete Fc moiety of a naturally occurring immunoglobulin molecule, while retaining or enhancing at least one desirable function conferred by the naturally occurring Fc moiety, and/or reducing an undesired function of a naturally occurring Fc moiety. Such functions include, for example, Fc receptor (FcR) binding, antibody half-life modulation (e.g., by binding to FcRn), ADCC function, protein A binding, protein G binding, and complement binding. Portions of naturally occurring Fc moieties which are involved with such functions have been described in the art. In some embodiments, an Fc region or Fc domain has substantially no binding to at least one Fc receptor selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). In some embodiments, an Fc region or domain exhibits substantially no binding to any of the Fc receptors selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). As used herein, "substantially no binding" refers to weak to no binding to a selected Fcgamma receptor or receptors. In some embodiments, "substantially no binding" refers to a reduction in binding affinity (e.g., increase in Kd) to a Fc gamma receptor of at least 1000-fold. In some embodiments, an Fc domain or region is an Fc null. As used herein, an "Fc null" refers to an Fc region or Fc domain that exhibits weak to no binding to any of the Fcgamma receptors. In some embodiments, an Fc null domain or region exhibits a reduction in binding affinity (e.g., increase in Kd) to Fc gamma receptors of at least 1000-fold. In some embodiments, an Fc domain has reduced or substantially no effector function activity. As used herein, "effector function activity" refers to antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), and/or complement dependent cytotoxicity (CDC). In some embodiments, an Fc domain exhibits reduced ADCC, ADCP, or CDC activity, as compared to a wildtype Fc domain. In some embodiments, an Fc domain exhibits a reduction in ADCC, ADCP, and CDC, as compared to a wildtype Fc domain. In some embodiments, an Fc domain exhibits substantially no effector function (i.e., the ability to stimulate ADCC, ADCP, or CDC). As used herein, "substantially no effector function" refers to a reduction in effector function activity of at least 1000-fold, as compared to a wildtype Fc domain. In some embodiments, an Fc domain has reduced or no ADCC activity. As used herein reduced or no ADCC activity refers to a decrease in ADCC activity of an Fc domain by of a factor of at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500. In some embodiments, an Fc domain has reduced or no CDC activity. As used herein reduced or no CDC activity refers to a decrease in CDC activity of an Fc domain by of a factor of at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of ADCC and/or CDC activity. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks Fcgamma receptor (hence likely lacking ADCC activity). The primary cells for mediating ADCC, NK cells, express FcgammaRIII only, whereas monocytes express FcgammaRI, FcgammaRII, and FcgammaRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, e.g., ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96TM non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that an antibody or Fc domain or region is unable to bind C1q and hence lacks CDC activity or has reduced CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). In some embodiments, an Fc domain has reduced or no ADCP activity. As used herein reduced or no ADCP activity refers to a decrease in ADCP activity of an Fc domain by of a factor of at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500. ADCP binding assays may also be carried out to confirm that an antibody or Fc domain or region lacks ADCP activity or has reduced ADCP activity. See, e.g., US20190079077 and US20190048078 and the references disclosed therein. Antibodies with reduced effector function activity include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (see U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (see U.S. Pat. No.7,332,581). Certain antibody variants with diminished binding to FcRs are also known. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem.9(2): 6591-6604 (2001).) In certain embodiments, a binding agent comprises an Fc domain or region with one or more amino acid substitutions which diminish FcgammaR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). In some embodiments, the substitutions are L234A and L235A (LALA). In some embodiments, the Fc domain further comprises D265A and/or P329G in an Fc region derived from a human IgG1 Fc region. In some embodiments, the substitutions are L234A, L235A, and P329G (LALA-PG) in an Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2012/130831). In some embodiments, the substitutions are L234A, L235A, and D265A (LALA-DA) in an Fc region derived from a human IgG1 Fc region. In some embodiments, alterations are made in the Fc region that result in altered (i.e., either diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No.6,194,551, WO 99/51642, and Idusogie et al. J. Immunol.164: 4178-4184 (2000). Production of Binding Proteins In various embodiments, binding proteins (e.g., antibody or an antigen binding fragment thereof) can be produced in human, murine, or other animal-derived cells lines. Recombinant DNA expression can be used to produce the binding agents. This allows the production of antibodies as well as a spectrum of antigen binding portions and other binding agents (including fusion proteins) in a host species of choice. The production of antibodies, antigen binding portions thereof and other binding agents in bacteria, yeast, transgenic animals, and chicken eggs are also alternatives for cell-based production systems. The main advantages of transgenic animals are potential high yields from renewable sources. Nucleic acid molecules encoding the amino acid sequence of an antibody, or antigen binding portion thereof, as well as other binding agents can be prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation of synthetic nucleotide sequences encoding of an antibody, antigen binding portion or other binding agent(s). In addition, oligonucleotide-mediated (or site-directed) mutagenesis, PCR- mediated mutagenesis, and cassette mutagenesis can be used to prepare nucleotide sequences encoding an antibody or antigen binding portion as well as other binding agents. A nucleic acid sequence encoding at least an antibody, antigen binding portion thereof, binding agent, or a polypeptide thereof, as described herein, can be recombined with vector DNA in accordance with conventional techniques, such as, for example, blunt-ended or staggered- ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed, e.g., by Maniatis et al., Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons), 1987-1993, and can be used to construct nucleic acid sequences and vectors that encode an antibody or antigen binding portion thereof or a VH and/or VL polypeptide thereof. Where the binding agent comprises antibodies or antigen binding portions thereof, in some embodiments, a VH polypeptide is encoded by a first nucleic acid. In some embodiments, a VL polypeptide is encoded by a second nucleic acid. In some embodiments, the VH and VL polypeptides are encoded by one nucleic acid. A nucleic acid molecule, such as DNA, is said to be “capable of expressing” a polypeptide if it contains nucleotide sequences that contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences that encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed (e.g., an antibody or antigen binding portion thereof) are connected in such a way as to permit gene expression of a polypeptide(s) or antigen binding portions in recoverable amounts. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, as is well known in the analogous art. See, e.g., Sambrook et al., 1989; Ausubel et al., 1987-1993. Accordingly, the expression of an antibody or antigen-binding portion thereof or other binding agent as described herein can occur in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including yeast, insects, fungi, bird, and mammalian cells either in vivo or in situ, or host cells of mammalian, insect, bird, or yeast origin. The mammalian cell or tissue can be of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog, or cat origin, but any other mammalian cell may be used. Further, by use of, for example, the yeast ubiquitin hydrolase system, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be accomplished. The fusion proteins so produced can be processed in vivo or purified and processed in vitro, allowing synthesis of an antibody or antigen binding portion thereof as described herein with a specified amino terminus sequence. Moreover, problems associated with retention of initiation codon-derived methionine residues in direct yeast (or bacterial) expression may be avoided. (See, e.g., Sabin et al., 7 Bio/Technol.705 (1989); Miller et al., 7 Bio/Technol.698 (1989).) Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeast are grown in medium rich in glucose can be utilized to obtain recombinant antibodies or antigen-binding portions thereof or other binding agents. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized. Production of antibodies or antigen-binding portions thereof and other binding agents in insects can be achieved, for example, by infecting an insect host with a baculovirus engineered to express a polypeptide by methods known to those of ordinary skill in the art. See Ausubel et al., 1987-1993. In some embodiments, the introduced nucleic acid sequence (encoding an antibody or antigen binding portion thereof or a polypeptide thereof or other binding agent) is incorporated into a plasmid or viral vector capable of autonomous replication in a recipient host cell. Any of a wide variety of vectors can be employed for this purpose and are known and available to those of ordinary skill in the art. See, e.g., Ausubel et al., 1987-1993. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species. Exemplary prokaryotic vectors known in the art include plasmids such as those capable of replication in E. coli. Other gene expression elements useful for the expression of DNA encoding antibodies or antigen-binding portions thereof and other binding agents include, but are not limited to (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter, (Okayama et al., 3 Mol. Cell. Biol.280 (1983)), Rous sarcoma virus LTR (Gorman et al., 79 PNAS 6777 (1982)), and Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)); (b) splice regions and polyadenylation sites such as those derived from the SV40 late region (Okayarea et al., 1983), and (c) polyadenylation sites such as in SV40 (Okayama et al., 1983). Immunoglobulin-encoding DNA genes can be expressed as described by Liu et al., infra, and Weidle et al., 51 Gene 21 (1987), using as expression elements the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancers, SV40 late region mRNA splicing, rabbit S- globin intervening sequence, immunoglobulin and rabbit S-globin polyadenylation sites, and SV40 polyadenylation elements. For immunoglobulin encoding nucleotide sequences, the transcriptional promoter can be, for example, human cytomegalovirus, the promoter enhancers can be cytomegalovirus and mouse/human immunoglobulin. In some embodiments, for expression of DNA coding regions in rodent cells, the transcriptional promoter can be a viral LTR sequence, the transcriptional promoter enhancers can be either or both the mouse immunoglobulin heavy chain enhancer and the viral LTR enhancer, and the polyadenylation and transcription termination regions. In other embodiments, DNA sequences encoding other proteins are combined with the above-recited expression elements to achieve expression of the proteins in mammalian cells. Each coding region or gene fusion is assembled in, or inserted into, an expression vector. Recipient cells capable of expressing the variable region(s) or antigen binding portions thereof are then transfected singly with nucleotides encoding an antibody or an antibody polypeptide or antigen-binding portion thereof, or are co-transfected with a polynucleotide(s) encoding VH and a VL chain coding regions. The transfected recipient cells are cultured under conditions that permit expression of the incorporated coding regions and the expressed antibody chains or intact antibodies or antigen binding portions are recovered from the culture. In some embodiments, the nucleic acids containing the coding regions encoding an antibody or antigen-binding portion thereof are assembled in separate expression vectors that are then used to co-transfect a recipient host cell. Each vector can contain one or more selectable genes. For example, in some embodiments, two selectable genes are used, a first selectable gene designed for selection in a bacterial system and a second selectable gene designed for selection in a eukaryotic system, wherein each vector has a set of coding regions. This strategy results in vectors which first direct the production, and permit amplification, of the nucleotide sequences in a bacterial system. The DNA vectors so produced and amplified in a bacterial host are subsequently used to co-transfect a eukaryotic cell, and allow selection of a co-transfected cell carrying the desired transfected nucleic acids (e.g., encoding antibody heavy and light chains). Non-limiting examples of selectable genes for use in a bacterial system are the gene that confers resistance to ampicillin and the gene that confers resistance to chloramphenicol. Selectable genes for use in eukaryotic transfectants include the xanthine guanine phosphoribosyl transferase gene (designated gpt) and the phosphotransferase gene from Tn5 (designated neo). Alternatively, the fused nucleotide sequences encoding VH and VL chains can be assembled on the same expression vector. For transfection of the expression vectors and production of the antibodies or antigen binding portions thereof or other binding agents, the recipient cell line can be a Chinese Hamster ovary cell line (e.g., DG44) or a myeloma cell. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by transfected immunoglobulin genes and possess the mechanism for glycosylation of the immunoglobulin. For example, in some embodiments, the recipient cell is the recombinant Ig-producing myeloma cell SP2/0. SP2/0 cells only produce immunoglobulins encoded by the transfected genes. Myeloma cells can be grown in culture or in the peritoneal cavity of a mouse, where secreted immunoglobulin can be obtained from ascites fluid. An expression vector encoding an antibody or antigen-binding portion thereof or other binding agent can be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, protoplast fusion, calcium phosphate-precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection and microprojectile bombardment. Johnston et al., 240 Science 1538 (1988), as known to one of ordinary skill in the art. Yeast provides certain advantages over bacteria for the production of immunoglobulin heavy and light chains. Yeasts carry out post-translational peptide modifications including glycosylation. A number of recombinant DNA strategies exist that utilize strong promoter sequences and high copy number plasmids which can be used for production of the desired proteins in yeast. Yeast recognizes leader sequences of cloned mammalian gene products and secretes polypeptides bearing leader sequences (i.e., pre-polypeptides). See, e.g., Hitzman et al., 11th Intl. Conf. Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982). Yeast gene expression systems can be routinely evaluated for the levels of production, secretion and the stability of antibodies, and assembled antibodies and antigen binding portions thereof. Various yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeasts are grown in media rich in glucose can be utilized. Known glycolytic genes can also provide very efficient transcription control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. Another example is the translational elongation factor 1alpha promoter. A number of approaches can be taken for evaluating optimal expression plasmids for the expression of immunoglobulins in yeast. See II DNA Cloning 45, (Glover, ed., IRL Press, 1985) and, e.g., U.S. Publication No. US 2006/0270045 A1. Bacterial strains can also be utilized as hosts for the production of the antibody molecules or antigen binding portions thereof or other binding agents described herein. E. coli K12 strains such as E. coli W3110, Bacillus species, enterobacteria such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species can be used. Plasmid vectors containing replicon and control sequences which are derived from species compatible with a host cell are used in connection with these bacterial hosts. The vector carries a replication site, as well as specific genes which are capable of providing phenotypic selection in transformed cells. A number of approaches can be taken for evaluating the expression plasmids for the production of antibodies and antigen binding portions thereof in bacteria (see Glover, 1985; Ausubel, 1987, 1993; Sambrook, 1989; Colligan, 1992-1996). Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin molecules including leader peptide removal, folding and assembly of VH and VL chains, glycosylation of the antibody molecules, and secretion of functional antibody and/or antigen binding portions thereof. Mammalian cells that can be useful as hosts for the production of antibody proteins, in addition to the cells of lymphoid origin described above, include cells of fibroblast origin, such as Vero or CHO-K1 cells. Exemplary eukaryotic cells that can be used to express immunoglobulin polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, CHO-K1, and DG44 cells; PERC6TM cells (Crucell); and NSO cells. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells. In some embodiments, one or more antibodies or antigen-binding portions thereof or other binding agents can be produced in vivo in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptides, according to any suitable method. In some embodiments, an antibody or antigen-binding portion thereof is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol.498: 229-44 (2009); Spirin, Trends Biotechnol.22: 538-45 (2004); and Endo et al., Biotechnol. Adv.21: 695-713 (2003). Many vector systems are available for the expression of the VH and VL chains in mammalian cells (see Glover, 1985). Various approaches can be followed to obtain intact antibodies. As discussed above, it is possible to co-express VH and VL chains and optionally the associated constant regions in the same cells to achieve intracellular association and linkage of VH and VL chains into complete tetrameric H2L2 antibodies or antigen-binding portions thereof. The co-expression can occur by using either the same or different plasmids in the same host. Nucleic acids encoding the VH and VL chains or antigen binding portions thereof can be placed into the same plasmid, which is then transfected into cells, thereby selecting directly for cells that express both chains. Alternatively, cells can be transfected first with a plasmid encoding one chain, for example the VL chain, followed by transfection of the resulting cell line with a VH chain plasmid containing a second selectable marker. Cell lines producing antibodies, antigen-binding portions thereof or other binding agents via either route could be transfected with plasmids encoding additional copies of peptides, VH, VL, or VH plus VL chains in conjunction with additional selectable markers to generate cell lines with enhanced properties, such as higher production of assembled antibodies or antigen binding portions thereof or enhanced stability of the transfected cell lines. Additionally, plants have emerged as a convenient, safe, and economical alternative expression system for recombinant antibody production, which are based on large scale culture of microbes or animal cells. Antibodies or antigen binding portions can be expressed in plant cell culture, or plants grown conventionally. The expression in plants may be systemic, limited to sub-cellular plastids, or limited to seeds (endosperms). See, e.g., U.S. Patent Pub. No.2003/0167531; U.S. Pat. No.6,080,560; U.S. Pat. No.6,512,162; and WO 0129242. Several plant-derived antibodies have reached advanced stages of development, including clinical trials (see, e.g., Biolex, N.C.). For intact antibodies, the variable regions (VH and VL) of the antibodies are typically linked to at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells, such as immortalized B-cells (see, e.g., WO 87/02671; which is incorporated by reference herein in its entirety). An antibody can contain both light chain and heavy chain constant regions. The heavy chain constant region can include CH1, hinge, CH2, CH3, and, sometimes, CH4 regions. In some embodiments, the CH2 domain can be deleted or omitted. Alternatively, techniques described for the production of single chain antibodies (see, e.g., U.S. Pat. No.4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989); which are incorporated by reference herein in their entireties) can be adapted to produce single chain antibodies that specifically bind to the desired antigen. Single chain antibodies are formed by linking the heavy and light chain variable regions of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli can also be used (see, e.g., Skerra et al., Science 242:1038-1041 (1988); which is incorporated by reference herein in its entirety). Intact (e.g., whole) antibodies, their dimers, individual light and heavy chains, or antigen binding portions thereof can be recovered and purified by known techniques, e.g., immunoadsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination of these. See generally, Scopes, Protein Purification (Springer-Verlag, N.Y., 1982). Substantially pure antibodies or antigen binding portions thereof of at least about 90% to 95% homogeneity are advantageous, as are those with 98% to 99% or more homogeneity, particularly for pharmaceutical uses. Once purified, partially or to homogeneity as desired, an intact antibody or antigen binding portions thereof can then be used therapeutically or in developing and performing assay procedures, immunofluorescent staining, and the like. See generally, Vols. I & II Immunol. Meth. (Lefkovits & Pernis, eds., Acad. Press, NY, 1979 and 1981). Anti-CD39 Antibodies and Antigen-Binding Fragments Thereof Antibodies to CD39 have been described in, for example, Published US Patent Application Nos.20190062448, 20130273062, and 20100303828. In some embodiments, a bispecific antibody targeting CD39 is prepared from these parental anti-CD39 antibodies. In some embodiments, a bispecific antibody targeting CD39 is provided, prepared from the anti-CD39 bivalent monospecific antibody 27577 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:77 and 78, respectively). In some embodiments, a bispecific antibody targeting CD39 is provided, prepared from the anti-CD39 bivalent monospecific antibody 31895 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:79 and 80, respectively). In some embodiments, a bispecific antibody targeting CD39 and ICOS is provided, prepared from the anti-ICOS bivalent monospecific antibody 422 H2L5 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:81 and 82, respectively). In some embodiments, a bispecific antibody targeting CD39 and CD8a is provided, prepared from the anti-CD8a bivalent monospecific antibody Mb1b IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:83 and 84, respectively). In some embodiments, a bispecific antibody targeting CD39 and PD-1 is provided, prepared from the anti-PD-1 bivalent monospecific antibody MK-3475 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:85 and 86, respectively). In some embodiments, the bispecific antibody is a CrossMab bispecific (4 chain assembly), a Bottle Opener bispecific (3 chain assembly), a scFV-Fc bispecific (2 chain assembly), or a DART-Fc bispecific (2 chain assembly). In some embodiments, the bispecific antibody is a CrossMab, in which the Fab and scFv are attached to an IgG1 hinge-CH2-CH3, and the CH3 domain is engineered to contain the "knobs-into-holes" mutations to enforce correct association of the two heterodimeric heavy chains. The "knob" heavy chain includes mutations S354C and T366W, and the "hole" heavy chain includes mutations Y349C, T366S, L368A, and Y407V. In some embodiments, the bispecific antibody is a CrossMab bispecific, a Bottle Opener bispecific, a scFV-Fc bispecific, or a DART-Fc bispecific comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb, anti-CD8a Mb1b IgG1r mAb, or anti-PD1 MK-3475 IgG1r mAb. In some embodiments, the bispecific antibody is a CrossMab bispecific comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb, anti-CD8a Mb1b IgG1r mAb, or anti-PD1 MK-3475 IgG1r mAb (FIG.1A). In some embodiments, the bispecific antibody is a Bottle Opener bispecific antibody comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti- ICOS 422 H2L5 IgG1r mAb or anti-CD8a Mb1b IgG1r mAb (FIG.1B). In some embodiments, the bispecific antibody is a "scFV-Fc" antibody comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb (FIG.1C). In some embodiments, the bispecific antibody is a "scFv" antibody comprising binding domains from (i) anti-CD3927577 IgG1r mAb or 31895 IgG1r mAb; and (ii) anti-ICOS 422 H2L5 IgG1r mAb (FIG.1D). In some embodiments, a bispecific anti-CD39/anti-ICOS CrossMab antibody is provided, based on the anti-CD39 bivalent monospecific antibody 27577 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:77 and 78, respectively) and the anti-ICOS bivalent monospecific antibody 422 H2L5 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:81 and 82, respectively). In some embodiments, the CrossMab antibody comprises an anti-CD39 (27577) light chain and heavy chain according to SEQ ID NOs:9 and 10, respectively; and an anti-ICOS (422 H2L5) light chain and heavy chain according to SEQ ID NOs: 42 and 43, respectively. V. Pharmaceutical Compositions or Formulations In some aspects, the binding agents (e.g., antibodies and antigen binding fragments thereof) disclosed herein relate to compositions comprising active ingredients (i.e., including a binding agent as described herein or a nucleic acid encoding an antibody or antigen-binding portion thereof or other binding agent as described herein). In some embodiments, the composition is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to the active agent in combination with a pharmaceutically acceptable carrier accepted for use in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on any particular formulation. Typically, such compositions are prepared as injectable either as liquid solutions or suspensions; however, solid forms suitable for rehydration, or suspensions, in liquid prior to use can also be prepared. A preparation can also be emulsified or presented as a liposome composition. An antibody or antigen binding portion thereof or other binding agent can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, a pharmaceutical composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance or maintain the effectiveness of the active ingredient (e.g., an antibody or antigen binding portion thereof or other binding agent). The pharmaceutical compositions as described herein can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of a polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine, procaine, and the like. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain the active ingredients (e.g., an antibody and/or antigen binding portions thereof or other binding agent) and water, and may contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of an active agent that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In some embodiments, a pharmaceutical composition comprising an antibody or antigen-binding portion thereof or other binding agent or a nucleic acid encoding an antibody or antigen-binding portion thereof or other binding agent as described herein can be a lyophilisate. In some embodiments, a syringe comprising a therapeutically effective amount of a binding agent, or a pharmaceutical composition described herein is provided. VI. Methods of Treatment and Related Uses In some aspects, the binding agents (e.g., antibodies and antigen binding fragments thereof) as described herein can be used in a method(s) comprising administering a binding agent or a pharmaceutical composition as described herein to a subject having an inflammatory disease. In some aspects, the binding agents (e.g., antibodies and antigen binding fragments thereof) as described herein can be used in a method comprising administering a binding agent or a pharmaceutical composition as described herein to a subject having an autoimmune disease. In some embodiments, the autoimmune disease is autoimmune-induced hepatitis, Addison’s Disease, Alopecia Areata, Alport’s Syndrome, Ankylosing Spondylitis, Anti-phospholipid Syndrome, Arthritis, Ascariasis, Aspergillosis Atopic Allergy, Atopic Dermatitis, Atopic Rhinitis, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune Myositis, Behcet’s Disease, Bird-Fancier’s Lung, Bronchial Asthma, Caplan’s Syndrome, Cardiomyopathy, celiac Disease, Chagas’ Disease, Chronic Glomerulonephritis, Chronic Graft versus Host Disease, Cogan’s Syndrome, Cold Agglutinin Disease, CREST Syndrome, Crohn’s Disease, Cryoglobulinemia, Cushing’s Syndrome, Dermatomyositis, Discoid Lupus, Dressier’ s Syndrome, Eaton-Lambert Syndrome, Encephalomyelitis, Endocrine ophthalmopathy, Erythematosus, Evan’s Syndrome, Felty’s Syndrome, Fibromyalgia, Fuch’s Cyclitis, Gastric Atrophy, Gastrointestinal Allergy, Giant Cell Arteritis, Glomerulonephritis, Goodpasture’s Syndrome, Graft v. Host Disease, Graves’ Disease, Guillain-Barre Disease (Syndrome), Hashimoto’s Thyroiditis, Hemolytic Anemia, Henoch- Schonlein Purpura, Hyperviscosity Syndrome, Idiopathic Adrenal Atrophy, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenic Purpura, IgA Nephropathy, Inflammatory Bowel Disease (Syndrome), Insulin-Dependent Diabetes Mellitus (IDDM or Type I), Juvenile Arthritis, Juvenile Idiopathic Arthritis, Juvenile Diabetes Mellitus (Type I), Lambert-Eaton Syndrome Laminitis, Lichen Planus, Lupoid Hepatitis, Lupus, Lupus Nephritis, Lymphopenia, Macroglobulinemia, Meniere’s Disease, Mixed Connective Tissue Disease, Monoclonal Gammopathy of Undermined Origin, Multiple Sclerosis, Myasthenia Gravis, Myocarditis, Pemphigus/Pemphigoid, Pernicious Anemia, POEMS syndrome, Polyglandular Syndromes, Polyarteritis Nodosa, Polymyositis, Presenile Dementia, Primary Agammaglobulinemia, Primary Biliary Cirrhosis/Cholangitis, Psoriasis, Psoriatic Arthritis, Raynauds Phenomenon, Reiter’s Syndrome, Rheumatic Fever, Rheumatoid Arthritis, Sampter’s Syndrome, Schmidt’s Syndrome, Scleroderma/Systemic Sclerosis, Shulman’s Syndrome, Sjörgen’s Syndrome, Stiff-Man Syndrome, Sympathetic Ophthalmia, Systemic Lupus Erythematosus, Takayasu’s Arteritis, Temporal Arteritis, Thyroiditis, Thrombocytopenia, Thyrotoxicosis, Toxic Epidermal Necrolysis, Type B Insulin Resistance, Type I Diabetes Mellitus, Ulcerative Colitis, Uveitis, Vitiligo, Waldenstrom’s Macroglobulinemia, and/or Wegener’s Granulomatosis. In some embodiments the autoimmune disease is autoimmune hepatitis, celiac disease, Crohn’s disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus/pemphigoid, primary biliary cirrhosis/cholangitis, rheumatoid arthritis (RA), scleroderma/systemic sclerosis, Sjögren’s syndrome (SjS), systemic lupus erythematosus (SLE), or ulcerative colitis. In some embodiments, the autoimmune disease is selected from autoimmune hepatitis, celiac disease, Crohn’s disease, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), multiple sclerosis (MS), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or ulcerative colitis. In some aspects, the binding agents (e.g., antibodies and antigen binding fragments thereof) as described herein can be used in a method(s) comprising administering a binding agent or a pharmaceutical composition as described herein to suppress an immune response mediated by pathogenic immune cells in a subject. In some aspects, the binding agents (e.g., antibodies and antigen binding fragments thereof) as described herein can be used in a method of treating complications of a transplant associated with graft versus host disease (GVHD), comprising administering a binding agent or a pharmaceutical composition as described herein to a subject. In some aspects, the binding agents (e.g., antibodies and antigen binding fragments thereof) as described herein can be used in a method(s) comprising administering a binding agent or a pharmaceutical composition as described herein to a subject to modulate an immune response to a virus in a subject. In some embodiments, the binding agent or pharmaceutical composition is administered to suppress, reduce, or prevent an immune response to a virus. In some embodiments, the immune response that is suppresed, reduced, or prevented is an immune response to a virus, or antigenic portions thereof. In some embodiments, the virus is a viral vector, and the administration of the binding agent suppresses, reduces, or prevents the induction of undesired immune responses associated with vector-mediated delivery of genetic material. The use of viral vectors, such as adeno- associated virus (AAV) vectors, to deliver genes of interest is currently an important tool for therapeutic approaches involving gene replacement, gene silencing, gene addition, and gene editing. However, host immune responses can limit the effectiveness of these approaches (Wang et al., Nat Rev Drug Discov 18, 358–378 (2019), https://doi.org/10.1038/s41573-019- 0012-9). For example, the host may produce neutralizing antibodies against the vector capsids based on exposure to the wild-type virus, blocking gene delivery. The host may also produce neutralizing antibodies against the vector capsid that limit the effectiveness of re- administration of the vector in therapies requiring repeated dosing. Additionally, hosts can mount a cytotoxic T lymphocyte (CTL)-mediated cytotoxicity that clears transduced cells. In a subset of 'reactive patients', AAV mediated gene delivery can be associated with inflammatory side effects and toxicities mediated by pathogenic CD4+ T cells. Accordingly, the binding agents (e.g., antibodies and antigen binding fragments thereof) as described herein can be used in a method(s) comprising administering a binding agent or a pharmaceutical composition as described herein to a subject to suppress, reduce, or prevent an immune response to a viral vector. As used herein, "an immune response to a virus" or "an immune response to a viral vector" may refer to any immune response to a virus, a viral vector, or antigenic portions thereof, e.g., viral proteins or fragments thereof. In some embodiments, the immune response may be activation or proliferation of CD4+ T cells. The immune response may be characterized by, for example, pro-inflammatory cytokine (e.g., IFN-ɣ) production by CD4+ T cells. In some embodiments, the viral vector has been, is, or will be administered to the subject. In some embodiments, the immune response to the viral vector is induced by administration of a viral vector to the subject. In some embodiments, the virus or viral vector is a retrovirus, adenovirus, parvovirus, coronavirus, ortho-myxovirus, rhabdovirus, paramyxovirus, picornavirus, alphavirus, herpesvirus, poxvirus, Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, or hepatitis virus. In some embodiments, the virus or viral vector is an adeno-associated virus (AAV) vector, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVrh.43, AAVrh.74, or AAVhu.37, or a variant thereof. In some embodiments, the virus or viral vector is an adenovirus vector. In some embodiments, the virus or viral vector is a lentivirus vector. As used herein, to "activate or stimulate" CD8+ Tregs, or activated CD8+ Tregs, refers to an increase of the regulatory T cell functions of such cells, such as the ability to suppress an immune response. Activation or stimulation of CD8+ Tregs may include removal of a suppressive effect on such cells, so as to restore the CD8+ Tregs (e.g., restore balance to the immune system or restore balanced immune activity in the subject prior to receiving a viral vector). Activation or stimulation of CD8+ Tregs may also include results of such activation or stimulation, including removal of a CD4+ cells, B cells, or other cells mediating an immune response, such as by elimination, for example, cytolysis, of such cells. In some embodiments, the CD8+ Tregs are contacted with the binding agent in vivo. In some embodiments, the CD8+ Tregs are contacted with the binding agent ex vivo. The activated CD8+ Tregs can then be administered in an effective amount to a subject in need thereof. In some embodiments, the activated CD8+ Tregs exert a suppressive effect on other immune cells, such as CD4+ T cells, antibody producing B cells, antigen presenting dendritic cells, or antigen presenting cells. In some embodiments, the activated CD8+ Tregs exert a suppressive effect on other immune cells, such as CD4+ T cells, antibody producing B cells, and antigen presenting dendritic cells. In some embodiments, the activated CD8+ Tregs deplete other immune cells, such as CD4 T cells, antibody producing B cells, and antigen presenting dendritic cells. In some embodiments, the activated CD8+ Tregs modulate the activity of undesired immune cells and decrease the titer of antibodies in the subject. In some embodiments, the activated CD8+ Tregs decrease the titer of antibodies in the subject. In some embodiments, the CD8+ Tregs are CD39+ and KIR+. In some embodiments, the CD8+ Tregs are CD39- and KIR+. In some embodiments, the CD8+ Tregs are CD39+ and KIR-. In some embodiments, the CD8+ Tregs are CD39- and KIR-. In some embodiments, a binding agent or a pharmaceutical composition comprising any of the binding agents described herein, is administered with an immunosuppressive agent, such as a corticosteroid. In some embodiments, the immunosuppressive agent is one or more of: a calcineurin inhibitor, e.g., a cyclosporin or an ascomycin, e.g., cyclosporin A (NEORAL®), FK506 (tacrolimus), pimecrolimus, an mTOR inhibitor, e.g., rapamycin or a derivative thereof, e.g., sirolimus (RAPAMUNE®), everolimus (Certican®), temsirolimus, zotarolimus, biolimus-7, biolimus-9, a rapalog, e.g., ridaforolimus, azathioprine, campath 1H, a S1P receptor modulator, e.g., fingolimod or an analogue thereof, an anti-IL-8 antibody, mycophenolic acid or a salt thereof, e.g., sodium salt, e.g., mycophenolate mofetil (CELLCEPT®), OKT3 (ORTHOCLONE OKT3®), Prednisone, ATGAM®, THYMOGLOBULIN®, Brequinar Sodium, OKT4, T10B9.A-3A, 33B3.1, 15- deoxyspergualine, tresperimus, leflunomide ARAVA®, CTLAI-Ig, anti-CD25, anti-IL2R , basiliximab (SIMULECT®), Daclizumab (ZENAPAX®), mizorbine, methotrexate, dexamethasone, ISAtx-247, SDZ ASM 981 (pimec rolimus, Elidel®), CTLA4lg (Abatacept), belatacept, LFA3lg, etanercept (sold as Enbrel® by Immunex), adalimumab (Humira®), infliximab (Remicade®), an anti-LFA-1 antibody, natalizumab (Antegren®), Enlimomab, gavilimomab, antithymocyte immunoglobulin, siplizumab, alefacept efalizumab, pentase, mesalazine, asacol, codeine phosphate, benorylate, fenbufen, naprosin, diclofenac, etodolac, and indomethacin, tocilizumab (Actemra), siltuximab (Sylvant), secukibumab (Cosentyx), ustekinumab (Stelara), risankizumab, sifalimumab, aspirin, ibuprofen, imlifidase, a proteasome inhibitor, arsenic trioxide, and rabbit anti-thymocyte globulin. See, e.g., Chu et al., Frontiers in Immunology 12:658038 (2021). In some embodiments, a binding agent or a pharmaceutical composition of any of the binding agents described herein, is administered with an anti-inflammatory agent, such as a corticosteroid. In some embodiments, the anti-inflammatory agent is one or more of: methotrexate, dexamethasone, dexamethasone alcohol, dexamethasone sodium phosphate, fluromethalone acetate, fluromethalone alcohol, lotoprendol etabonate, medrisone, prednisolone acetate, prednisolone sodium phosphate, difluprednate, rimexolone, hydrocortisone, hydrocortisone, lodoxamide tromethamine, aspirin, ibuprofen, suprofen, piroxicam, meloxicam, flubiprofen, naproxan, ketoprofen, tenoxicam, diclofenac sodium, ketotifen fumarate, diclofenac sodium, nepafenac, bromfenac, flurbiprofen sodium, suprofen, celecoxib, naproxen, rofecoxib, glucocorticoids, diclofenac, and any combination thereof. In some embodiments, the anti-inflammatory agent is one or more nonsteroidal anti- inflammatory drugs (NSAIDs), such as naproxen sodium (Anaprox), celecoxib (Celebrex), sulindac (Clinoril), oxaprozin (Daypro ), salsalate (Disalcid), diflunisal (Dolobid), piroxicam (Feldene), indomethacin (Indocin), etodolac (Lodine), meloxicam (Mobic), naproxen (Naprosyn), nabumetone (Relafen), ketorolac tromethamine (Toradol), naproxen/ esomeprazole (Vimovo), and diclofenac (Voltaren), and combinations thereof. In some aspects, the binding agents (e.g., antibodies and antigen binding fragments thereof) disclosed herein are for use in the aforementioned methods, or are used in manufacture of a medicament for use in the aforementioned methods. EXEMPLARY EMBODIMENTS 1. A bispecific antibody, or an antigen binding fragment thereof, comprising: a first binding arm that specifically binds to CD39; and a second binding arm that specifically binds to ICOS, CD8a, or PD-1. 2. The bispecific antibody of embodiment 1, wherein the first binding arm comprises CDRL1, CDRL2, CDRL3 amino acid sequences according to SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; and comprises CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. 3. The bispecific antibody of embodiment 1 or embodiment 2, wherein the first binding arm comprises a light chain variable region (VL) comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:1; and comprises a heavy chain variable region (VH) comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:5. 4. The bispecific antibody of embodiment 3, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:1. 5. The bispecific antibody of embodiment 3, wherein the VH of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:5. 6. The bispecific antibody of embodiment 3, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:1; and the VH comprises or consists of the amino acid sequence according to SEQ ID NO:5. 7. The bispecific antibody of embodiment 1, wherein the first binding arm comprises CDRL1, CDRL2, CDRL3 amino acid sequences according to SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14; and comprises CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18. 8. The bispecific antibody of embodiment 1 or embodiment 7, wherein the first binding arm comprises a VL comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:11; and comprises a VH comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:15. 9. The bispecific antibody of embodiment 8, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:11. 10. The bispecific antibody of embodiment 8, wherein the VH of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:15. 11. The bispecific antibody of embodiment 8, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:11; and the VH comprises or consists of the amino acid sequence according to SEQ ID NO:15. 12. The bispecific antibody of any one of embodiments 1-11, wherein the second binding arm specifically binds to ICOS. 13. The bispecific antibody of embodiment 12, wherein the second binding arm comprises CDRL1, CDRL2, CDRL3 amino acid sequences according to SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37; and comprises CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41. 14. The bispecific antibody of embodiment 12 or embodiment 13, wherein the second binding arm comprises a VL comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:34; and comprises a VH comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:38. 15. The bispecific antibody of embodiment 14, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:34. 16. The bispecific antibody of embodiment 14, wherein the VH of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:38. 17. The bispecific antibody of embodiment 14, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:34; and the VH comprises or consists of the amino acid sequence according to SEQ ID NO:38. 18. The bispecific antibody of any one of embodiments 1-11, wherein the second binding arm specifically binds to CD8a. 19. The bispecific antibody of embodiment 18, wherein the second binding arm comprises CDRL1, CDRL2, CDRL3 amino acid sequences according to SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24; and comprises CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28. 20. The bispecific antibody of embodiment 18, wherein the second binding domain comprises CDRL1, CDRL2, CDRL3 amino acid sequences according to SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24; and comprises CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. 21. The bispecific antibody of any one of embodiments 18-20, wherein the second binding domain comprises a VL comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:21; and comprises a VH comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:25. 22. The bispecific antibody of embodiment 21, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:21. 23. The bispecific antibody of embodiment 21, wherein the VH of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:25. 24. The bispecific antibody of embodiment 21, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:21; and the VH comprises or consists of the amino acid sequence according to SEQ ID NO:25. 25. The bispecific antibody of any one of embodiments 1-11, wherein the second binding arm specifically binds to PD-1. 26. The bispecific antibody of embodiment 25, wherein the second binding domain comprises CDRL1, CDRL2, CDRL3 amino acid sequences according to SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47; and comprises CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51. 27. The bispecific antibody of embodiment 25 or 26, wherein the second binding domain comprises a VL comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:44; and comprises a VH comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:48. 28. The bispecific antibody of embodiment 27, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:44. 29. The bispecific antibody of embodiment 27, wherein the VH of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:48. 30. The bispecific antibody of embodiment 27, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:44; and the VH comprises or consists of the amino acid sequence according to SEQ ID NO:48. 31. The bispecific antibody of any one of embodiments 1-30, wherein the bispecific antibody comprises or is a CrossMab, a Bottle Opener bispecific, a diabody, an antibody Fc fusion, an scFv1-ScFv2, an ScFv12-Fc-scFv22, an IgG-scFv, a DVD-Ig, a triomab/quadroma, a two-in-one IgG, a scFv2-Fc, a TandAb, an scFv-HSA-scFv, an scFv- VHH, a Fab-scFv-Fc, a Fab-VHH-Fc, a dAb-IgG, an IgG-VHH, a Tandem scFv-Fc, a (scFv1)2-Fc-(VHH)2, a BiTe, a DART, a scFv-Fc, a one-armed tandem scFv-Fc, a DART- Fc, an anticalin, an affibody, an avimer, a DARPin, or an adnectin. 32. The bispecific antibody of any one of embodiments 1-31, wherein the bispecific antibody is a CrossMab bispecific antibody. 33. The bispecific antibody of any one of embodiments 1-31, wherein the bispecific antibody is a Bottle Opener bispecific antibody. 34. The bispecific antibody of any one of embodiments 1-31, wherein the bispecific antibody is a scFv-Fc bispecific antibody. 35. The bispecific antibody of any one of embodiments 1-31, wherein the bispecific antibody is a DART-Fc bispecific antibody. 36. The bispecific antibody of embodiment 1, comprising an anti-CD39 light chain according to SEQ ID NO:9, an anti-CD39 heavy chain according to SEQ ID NO:10, an anti- ICOS light chain according to SEQ ID NO:42, and an anti-ICOS heavy chain according to SEQ ID NO:43. 37. The bispecific antibody of embodiment 36, wherein the bispecific antibody is a CrossMab bispecific antibody. 38. The bispecific antibody of embodiment 1, comprising an anti-CD39 light chain according to SEQ ID NO:19, an anti-CD39 heavy chain according to SEQ ID NO:20, an anti- ICOS light chain according to SEQ ID NO:42, and an anti-ICOS heavy chain according to SEQ ID NO:43. 39. The bispecific antibody of embodiment 38, wherein the bispecific antibody is a CrossMab bispecific antibody. 40. The bispecific antibody of embodiment 1, comprising an anti-CD39 light chain according to SEQ ID NO:9, an anti-CD39 heavy chain according to SEQ ID NO:10, and an anti-ICOS scFv heavy chain according to SEQ ID NO:55. 41. The bispecific antibody of embodiment 1, comprising an anti-CD39 light chain according to SEQ ID NO:9, an anti-CD39 heavy chain according to SEQ ID NO:10, and an anti-ICOS scFv heavy chain according to SEQ ID NO:56. 42. The bispecific antibody of embodiment 40 or 41, wherein the bispecific antibody is a Bottle Opener bispecific antibody. 43. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:57, and an anti-ICOS scFv heavy chain according to SEQ ID NO:55. 44. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:57, and an anti-ICOS scFv heavy chain according to SEQ ID NO:56. 45. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:58, and an anti-ICOS scFv heavy chain according to SEQ ID NO:55. 46. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:58, and an anti-ICOS scFv heavy chain according to SEQ ID NO:56. 47. The bispecific antibody of any one of embodiments 43-46, wherein the bispecific antibody is an scFV-Fc bispecific antibody. 48. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:59, and an anti-ICOS scFv heavy chain according to SEQ ID NO:61. 49. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:59, and an anti-ICOS scFv heavy chain according to SEQ ID NO:62. 50. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:60, and an anti-ICOS scFv heavy chain according to SEQ ID NO:61. 51. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:60, and an anti-ICOS scFv heavy chain according to SEQ ID NO:62. 52. The bispecific antibody of any one of embodiments 48-51, wherein the bispecific antibody is an scFV-Fc bispecific antibody. 53. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:63, and an anti-ICOS scFv heavy chain according to SEQ ID NO:65. 54. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:63, and an anti-ICOS scFv heavy chain according to SEQ ID NO:66. 55. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:64, and an anti-ICOS scFv heavy chain according to SEQ ID NO:65. 56. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:64, and an anti-ICOS scFv heavy chain according to SEQ ID NO:66. 57. The bispecific antibody of any one of embodiments 53-56, wherein the bispecific antibody is an scFV-Fc bispecific antibody. 58. The bispecific antibody of embodiment 1, comprising an anti-CD39 light chain according to SEQ ID NO:19, an anti-CD39 heavy chain according to SEQ ID NO:20, and an anti-ICOS scFv heavy chain according to SEQ ID NO:55. 59. The bispecific antibody of embodiment 1, comprising an anti-CD39 light chain according to SEQ ID NO:19, an anti-CD39 heavy chain according to SEQ ID NO:20, and an anti-ICOS scFv heavy chain according to SEQ ID NO:56. 60. The bispecific antibody of embodiment 58 or embodiment 59, wherein the bispecific antibody is a Bottle Opener bispecific antibody. 61. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:67, and an anti-ICOS scFv heavy chain according to SEQ ID NO:55. 62. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:67, and an anti-ICOS scFv heavy chain according to SEQ ID NO:56. 63. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:68, and an anti-ICOS scFv heavy chain according to SEQ ID NO:55. 64. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:68, and an anti-ICOS scFv heavy chain according to SEQ ID NO:56. 65. The bispecific antibody of any one of embodiments 61-64, wherein the bispecific antibody is an scFV-Fc bispecific antibody. 66. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:69, and an anti-ICOS scFv heavy chain according to SEQ ID NO:61. 67. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:69, and an anti-ICOS scFv heavy chain according to SEQ ID NO:62. 68. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:70, and an anti-ICOS scFv heavy chain according to SEQ ID NO:61. 69. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:70, and an anti-ICOS scFv heavy chain according to SEQ ID NO:62. 70. The bispecific antibody of any one of embodiments 66-69, wherein the bispecific antibody is an scFV-Fc bispecific antibody. 71. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:71, and an anti-ICOS scFv heavy chain according to SEQ ID NO:65. 72. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:71, and an anti-ICOS scFv heavy chain according to SEQ ID NO:66. 73. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:72, and an anti-ICOS scFv heavy chain according to SEQ ID NO:65. 74. The bispecific antibody of embodiment 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:72, and an anti-ICOS scFv heavy chain according to SEQ ID NO:66. 75. The bispecific antibody of any one of embodiments 71-74, wherein the bispecific antibody is an scFV-Fc bispecific antibody. 76. The bispecific antibody of embodiment 1, comprising an anti-CD39 heavy chain according to SEQ ID NO:73, and an anti-ICOS heavy chain according to SEQ ID NO:74. 77. The bispecific antibody of embodiment 76, wherein the bispecific antibody is a DART-Fc bispecific antibody. 78. The bispecific antibody of embodiment 1, comprising an anti-CD39 heavy chain according to SEQ ID NO:75, and an anti-ICOS heavy chain according to SEQ ID NO:76. 79. The bispecific antibody of embodiment 78, wherein the bispecific antibody is a DART-Fc bispecific antibody. 80. The bispecific antibody of embodiment 1, comprising an anti-CD39 light chain according to SEQ ID NO:9, an anti-CD39 heavy chain according to SEQ ID NO:10, an anti- CD8a light chain according to SEQ ID NO:32, and an anti-CD8a heavy chain according to SEQ ID NO:33. 81. The bispecific antibody of embodiment 80, wherein the bispecific antibody is a Cross- Mab bispecific antibody. 82. The bispecific antibody of embodiment 1, comprising an anti-CD39 light chain according to SEQ ID NO:19, an anti-CD39 heavy chain according to SEQ ID NO:20, an anti- CD8a light chain according to SEQ ID NO:32, and an anti-CD8a heavy chain according to SEQ ID NO:33. 83. The bispecific antibody of embodiment 82, wherein the bispecific antibody is a Cross- Mab bispecific antibody. 84. The bispecific antibody of embodiment 1, comprising an anti-CD39 light chain according to SEQ ID NO:9, an anti-CD39 heavy chain according to SEQ ID NO:10, and an anti-CD8a scFv heavy chain according to SEQ ID NO:54. 85. The bispecific antibody of embodiment 84, wherein the bispecific antibody is a Bottle Opener bispecific antibody. 86. The bispecific antibody of embodiment 1, comprising an anti-CD39 light chain according to SEQ ID NO:19, an anti-CD39 heavy chain according to SEQ ID NO:20, and an anti-CD8a scFv heavy chain according to SEQ ID NO:54. 87. The bispecific antibody of embodiment 86, wherein the bispecific antibody is a Bottle Opener bispecific antibody. 88. The bispecific antibody of embodiment 1, comprising an anti-CD39 light chain according to SEQ ID NO:9, an anti-CD39 heavy chain according to SEQ ID NO:10, an anti- PD-1 light chain according to SEQ ID NO:52, and an anti-PD-1 heavy chain according to SEQ ID NO:53. 89. The bispecific antibody of embodiment 88, wherein the bispecific antibody is a Cross- Mab bispecific antibody. 90. The bispecific antibody of embodiment 1, comprising an anti-CD39 light chain according to SEQ ID NO:19, an anti-CD39 heavy chain according to SEQ ID NO:20, an anti- PD-1 light chain according to SEQ ID NO:52, and an anti-PD-1 heavy chain according to SEQ ID NO:53. 91. The bispecific antibody of embodiment 90, wherein the bispecific antibody is a Cross- Mab bispecific antibody. 92. A pharmaceutical composition comprising the bispecific antibody of any one of embodiments 1-91 and a pharmaceutically acceptable carrier. 93. A nucleic acid encoding the bispecific antibody of any one of embodiments 1-91. 94. A vector comprising the nucleic acid of embodiment 93. 95. A cell line comprising the vector of embodiment 94. 96. A method of treating an autoimmune disease, comprising administering the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 to a subject in need thereof in an amount effective to decrease the number or activity of pathogenic immune cells in the subject. 97. The bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 for use in treating an autoimmune disease in a subject, wherein the use comprises administering the bispecific antibody or the pharmaceutical composition to a subject in need thereof in an amount effective to decrease the number or activity of pathogenic immune cells in the subject. 98. Use of the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 in the manufacture of a medicament for use in treating an autoimmune disease in a subject, wherein the use comprises administering the bispecific antibody or the pharmaceutical composition to a subject in need thereof in an amount effective to decrease the number or activity of pathogenic immune cells in the subject 99. A method of suppressing an immune response mediated by pathogenic immune cells, comprising contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs). 100. The bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 for use in suppressing an immune response mediated by pathogenic immune cells in a subject, wherein the use comprises contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody or the pharmaceutical composition in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs). 101. Use of the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 in the manufacture of a medicament for use in suppressing an immune response mediated by pathogenic immune cells in a subject, wherein the use comprises contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody or the pharmaceutical composition in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs). 102. A method of suppressing an immune response to an antigen, such as an autoantigen, comprising administering to a subject in need thereof the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby the number or activity of pathogenic immune cells that are responsive to the antigen or autoantigen is decreased. 103. The bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 for use in suppressing an immune response to an antigen, such as an autoantigen, in a subject, wherein the use comprises administering to a subject in need thereof the bispecific antibody or pharmaceutical composition in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby the number or activity of pathogenic immune cells that are responsive to the antigen or autoantigen is decreased. 104. Use of the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 in the manufacture of a medicament for use in suppressing an immune response to an antigen, such as an autoantigen, in a subject, wherein the use comprises administering to a subject in need thereof the bispecific antibody or pharmaceutical composition in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby the number or activity of pathogenic immune cells that are responsive to the antigen or autoantigen is decreased. 105. The method, bispecific antibody or pharmaceutical composition for use, or use in manufacture of any one of embodiments 99-104, wherein the CD8+ Tregs are CD8+KIR+ Tregs. 106. The method, bispecific antibody or pharmaceutical composition for use, or use in manufacture of any one of embodiments 99-105, wherein the activated CD8+ Tregs are administered in an effective amount to a subject in need thereof. 107. The method, bispecific antibody or pharmaceutical composition for use, or use in manufacture of any one of embodiments 96-106, wherein the pathogenic immune cells are autoreactive CD4+ T cells, autoantibody producing B cells, or self antigen presenting dendritic cells. 108. The method, bispecific antibody or pharmaceutical composition for use, or use in manufacture of any one of embodiments 96-107, wherein the subject has an autoimmune disease. 109. The method, bispecific antibody or pharmaceutical composition for use, or use in manufacture of embodiment 108, wherein the autoimmune disease is celiac disease, Crohn’s disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), lupus, lupus nephritis, cutaneous lupus, discoid lupus, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus/pemphigoid, rheumatoid arthritis (RA), scleroderma/systemic sclerosis, Sjögren’s syndrome (SjS), systemic lupus erythematosus (SLE), or ulcerative colitis. 110. The method, bispecific antibody or pharmaceutical composition for use, or use in manufacture of embodiment 108, wherein the autoimmune disease is celiac disease, Crohn’s disease, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), , multiple sclerosis (MS), rheumatoid arthritis (RA), scleroderma/systemic sclerosis, Sjögren’s syndrome (SjS), lupus, lupus nephritis, cutaneous lupus, discoid lupus, systemic lupus erythematosus (SLE), or ulcerative colitis. 111. A method of reducing or preventing onset of graft versus host disease (GVHD) following a transplant, comprising administering the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92, wherein the bispecific antibody has substantially no effector function activity, to a subject in need thereof in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs) and thereby reduce or ameliorate at least one symptom of GVHD. 112. The bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 for use in reducing or preventing onset of graft versus host disease (GVHD) in a subject following a transplant, wherein the use comprises administering the bispecific antibody or the pharmaceutical composition, wherein the bispecific antibody has substantially no effector function activity, to a subject in need thereof in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs) and thereby reduce or ameliorate at least one symptom of GVHD. 113. Use of the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 in the manufacture of a medicament for use in reducing or preventing onset of graft versus host disease (GVHD) in a subject following a transplant, wherein the use comprises administering the bispecific antibody or the pharmaceutical composition, wherein the bispecific antibody has substantially no effector function activity, to a subject in need thereof in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs) and thereby reduce or ameliorate at least one symptom of GVHD. 114. A method of treating a subject who has received a transplant, comprising contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92, wherein the bispecific antibody has substantially no effector function activity, in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby GVHD is reduced or suppressed. 115. The bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 for use in treating a subject who has received a transplant, wherein the use comprises contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody or the pharmaceutical composition, wherein the bispecific antibody has substantially no effector function activity, in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby GVHD is reduced or suppressed. 116. Use of the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92 in the manufacture of a medicament for use in treating a subject who has received a transplant, wherein the use comprises contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody or the pharmaceutical composition, wherein the bispecific antibody has substantially no effector function activity, in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby GVHD is reduced or suppressed. 117. The method of any one of embodiments 111-116, wherein the CD8+ Tregs are CD8+KIR+ Tregs. 118. A method of suppressing, reducing, or preventing an immune response to a viral vector in a subject, the method comprising administering to the subject the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92that binds to CD8+ T regulatory cells (CD8+ Tregs). 119. A binding agent that binds to CD8+ T regulatory cells (CD8+ Tregs) for use in a method of suppressing, reducing, or preventing an immune response to a viral vector in a subject, wherein the use comprises administering to the subject the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92. 120. Use of the bispecific antibody of any one of embodiments 1-91 or the pharmaceutical composition of embodiment 92in the manufacture of a medicament for use in a method of suppressing, reducing, or preventing an immune response to a viral vector in a subject, wherein the use comprises administering to the subject a binding agent that binds to CD8+ T regulatory cells (CD8+ Tregs). 121. The method of any one of embodiments 118-120, wherein the viral vector has been, is, or will be administered to the subject. 122. The method of any one of embodiments 118-121, wherein the immune response to the viral vector is induced by administration of the viral vector to the subject. 123. The method of any one of embodiments 118-122, wherein the CD8+ Tregs are CD8+KIR+ Tregs. EXAMPLES Example 1: Binding of Bispecific CD39/ICOS-Targeting Molecule to Cells Expressing CD39 or ICOS A bispecific anti-CD39/anti-ICOS CrossMab antibody was produced, based on the anti-CD39 bivalent monospecific antibody 27577 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:77 and 78, respectively) and the anti-ICOS bivalent monospecific antibody 422 H2L5 IgG1r mAb (parental antibody light chain and heavy chain sequences SEQ ID NOs:81 and 82, respectively). The CrossMab antibody comprised an anti- CD39 (27577) light chain and heavy chain according to SEQ ID NOs:9 and 10, respectively; and an anti-ICOS (422 H2L5) light chain and heavy chain according to SEQ ID NOs: 42 and 43, respectively. To determine CD39 target binding, the SK-MEL-28 cell line (ATCC, Manassas, VA), which expresses CD39 but not ICOS, was used.6x104 SK-MEL-28 cells were stained with Live/Dead stain (Biolegend, San Diego, CA) and seeded in a well of 96-well V bottom plate. Each bispecific binder was serially diluted in FACS buffer (PBS, 2 mM EDTA, and 1% FBS) covering 3200 – 0.012nM and added to the corresponding wells in a volume of 25 uL, followed by incubation on ice for 30 min. Cells were washed two times with FACS buffer and then incubated with secondary antibody (APC goat anti-human IgG, Jackson ImmunoResearch, West Grove, PA) at 1:120 for 30 min at room temperature. Cells were washed and fixed with IC fixation buffer (ThermoFisher, Waltham, MA) overnight. The next day, cells were washed and resuspended in FACS buffer and analyzed by flow cytometry on BD Symphony (Becton-Dickinson, Franklin Lakes, NJ). Both the bispecific anti-CD39/anti-ICOS CrossMab antibody and the parental anti- CD39 antibody showed binding to CD39 (FIG.2). To determine ICOS target binding, 293T cells were transfected with ICOS expression plasmid (OriGene, Rockville, MD). Two days later, the cells were used for ICOS binding assays.293 cells stained with Live/Dead stain (6X104/well) were plated in 96 well V bottom plates and incubated with serially diluted bispecific or parental Mab (3200 – 0.024 nM) on ice for 30 min. Following two washes, APC goat-anti-human IgG at 1:120 were added into each well for 30 min incubation at room temperature. Cells were washed, resuspended in FACS buffer, and subjected to flow analysis on BD Symphony. Data were analysed in FlowJo to examine live cells and graphs plotted using GraphPad Prism. Both the bispecific anti-CD39/anti-ICOS CrossMab antibody and the parental anti- ICOS antibody showed binding to ICOS (FIG.3). Example 2: Binding of Bispecific CD39/ICOS-Targeting Molecule to Donor-Derived PBMC In order to determine the binding of the bispecific anti-CD39/anti-ICOS CrossMAb antibody from Example 1 to primary cells, PBMC were activated with ImmunoCult T cell activator (Stemcell, Vancouver, Canada) for 5 days and then used for binding assays. Prior to seeding, cells were stained with Live/Dead (Biolegend) and washed with FACS buffer. 1.4X105 cells/well were plated in 96-well V bottom plates and incubated with serially diluted Bispecific (3200 – 0.024 nM) for 30 min on ice. Cells were washed two times with FACS buffer and then incubated with secondary antibody (1:120, APC goat anti-human IgG, Jackson ImmunoResearch) together with CD4, CD8, CD14, and CD20 antibodies (Biolegend) for 30 min at room temperature. Cells were washed and fixed using IC fixation buffer overnight. Cells were washed next day and resuspended in FACS buffer for flow cytometry on BD Symphony. Data was analyzed in FlowJo to examine live cells and graphs plotted using GraphPad Prism. The parental anti-CD39 antibody did not reach saturation at the highest concentration, suggesting poor binding to whole PBMC (FIG.4). However, the bispecific anti-CD39/anti- ICOS CrossMab antibody and the parental anti-ICOS parental antibody bound well to PBMC, suggesting that the binding of the bispecific anti-CD39/anti-ICOS CrossMab antibody may have been driven by ICOS arm (FIG.4). Binding was explored in different cell populations, including CD20+ B cells and CD14+ monocytes (FIGS.5A-5E). The parental anti-CD39 antibody alone showed low binding to all cell types tested (FIGS.5A to 5E). The parental anti-ICOS antibody bound to both CD4+, CD8+, and CD8+ Treg cells (FIGS.5A, 5B, and 5E). The bispecific anti- CD39/anti-ICOS CrossMab antibody showed greatest binding to CD8+ Treg cells, followed by CD4+, CD20+, CD14+, and CD8+ cells (FIGS.5A to 5E), implying that despite the low- affinity binding of the anti-CD39 arm, the anti-ICOS arm of the bispecific antibody induced preferred binding to CD8+ over other cell types. Example 3: Direct Killing of Pathogenic CD4+ T cells by CD8+ Treg in an IncuCyte-Based Cytotoxicity Assay The ability of the bispecific anti-CD39/anti-ICOS CrossMAb antibody from Example 1 to enhance the direct killing of pathogenic gliadin-specific CD4+ T cells by CD8+ Treg was investigated. CD8+CD45RClowCD39high cells were isolated from HLA-DQ2.5+ celiac patients PBMC by flow sorting using a BD Fusion cytometer (Beckton-Dickinson). These isolated CD8+ cells were co-cultured with autologous T-cell depleted PBMC at a ratio of 1:1 in human T cell media (X-VIVO (Lonza, Basel, Switzerland), 5% human serum, 1% pen/strep and 1% glutamax) with cytokines IL-15 and IL-7 (10 ng/mL each, Biolegend). After one week, a further 25 ul/mL ImmunoCult was added, and cells incubated for a further week to expand. The purified, expanded CD8+ cells were then resuspended in human T cell media with IL-7 (2 ng/mL), IL-15 (10 ng/mL), and IL-2 (20 U/mL), and used as CD8+ Treg cells for IncuCyte killing assays. CD8+ Treg cells at 1x104/well were plated in a 96 well plate (Corning Inc, Corning, NY), followed by addition of the bispecific and parental antibodies, which were diluted out to have two final concentrations of 1 and 10 ug/mL in triplicates. An SKW-GFP cell line expressing a gliadin-specific TCR (5x104cells/well, ratio of 1 Treg to 5 SKW cells) were added into each well. Autologous T-cell depleted PBMC were used as antigen presenting cells (APC) and incubated with four gliadin peptides (QLQPFPQPELPY (SEQ ID NO:87), PQPELPYPQPE (SEQ ID NO:88), QQPFPQPEQPFP (SEQ ID NO:89), FPQPEQPFPWQP (SEQ ID NO:90)) at 6.25 ug/mL at 37ºC for an hour before being added to wells at 2.5x104 cells/well. The final ratio of CD8+ Treg to SKW cell to APC was 1:5:2.5. The plates were then placed in IncuCyte (Essen Bioscience, Ann Arbor, MI) and images were set to be taken every three hours for three days. The green fluorescence intensity of each time point were measured from the images and all signals were normalized to CD8+ Treg only (no bispecific treated) control (shown by dotted line in FIG.6). Wells treated with either the anti-ICOS or anti-CD39 parental antibodies showed a lower signal (FIG.6), suggesting fewer SKW cells were present in the culture, due to increased killing by CD8+ Treg. Wells treated with the bispecific anti- CD39/anti-ICOS CrossMab antibody showed further reduced levels of SKW cells compared to both the anti-ICOS and anti-CD39 parental antibodies (FIG.6), suggesting that the bispecific antibody enhanced CD8+ Treg-derived killing of SKW cell line relative to both monospecific antibodies. Example 4: Suppression of CD4+ Proliferation by CD8+ Treg To examine the ability of the bispecific anti-CD39/anti-ICOS CrossMab antibody from Example 1 to increase CD8+ Treg-driven reduction of CD4+ T cell proliferation, a direct suppression assay was performed. CD4+ T cells were purified by negative or positive selection and stained with CTV (CellTraceTM Violet, 1 uM, ThermoFisher) at 37ºC for 10 min, followed by washing with FBS. Autologous CD8 Treg cells were prepared as in Example 3, and stained with eF670 (1 uM, ThermoFisher) at 37ºC for 10 mins. CTV labelled CD4+ cells were added at 4x104 cells / well to a 96 well U bottom plates. eF670-stained CD8+ Treg cells were then added to wells at a ratio of 1:2 or 1:16 to CD4. Antibodies were prepared at 10ug/ml final concentration and added to the wells. Human T-Activator CD3/CD28 Dynabeads (ThermoFisher) were added at a ratio of 1:20 to cells in corresponding wells. Flow cytometry for CTV dilution was then performed 4 days later. In this assay, division of CD4+ T cells results in a reduction in the intensity of CTV staining. CD4+ T cells with T-activator beads showed dilution of the CTV stain, suggesting that at least one division had occurred in the majority of cells during the incubation. Addition of CD8+ Treg at a 1:2 ratio to CD4+ showed a lower percentage of CD4+ T cells dividing compared to the "no Treg" control, suggesting CD8+ Treg-driven suppression. At both 1:2 and 1:16 CD8+ Treg:CD4+ ratios, wells treated with the bispecific anti-CD39/anti-ICOS CrossMab antibody showed a lower percentage of CD4+ T cells proliferating compared to wells receiving no antibody or receiving anti-CD39 or anti-ICOS parental monospecific antibodies (FIG.7). This suggests that the bispecific anti-CD39/anti-ICOS CrossMab antibody was able to potentiate the function of CD8+ Treg in a manner that neither the anti- CD39 nor the anti-ICOS antibodies are able to replicate. The effect of bispecific anti- CD39/anti-ICOS CrossMab antibody is quantified in FIG.8. Example 5: CD8+ Treg Phenotypes Can be Influenced by In vitro Culture Conditions To examine the ability of cytokines to influence CD8+ Treg phenotypes, a culture system containing cytokines was designed. Isolated CD8+ Tregs were cultured in: (1) TGF-β or in (2) IL-15 and IL-7 (γ chain cytokines). Flow cytometry was used assess surface expression of CD8, CD39, CD103, PD-1, ICOS, CXCR3, and NKG2D, following culture in the cytokines. CD8+ Tregs cultured in TGF-β versus IL-15 and IL-7 exhibited different cell surface phenotypes based on surface protein expression (FIGS.9A-9B). The expression of multiple surface proteins was modulated, including the expression of CD39, in response to both culture conditions. Example 6: Cellular Expansion in the Presence of γ Chain Cytokines Increases CD39 Expression on KIR+CD8+ T cells To examine the effect of γ chain cytokines on CD39 expression in KIR+CD8+T cells, in vitro assays were performed. The effect of IL-15 on CD39 expression in CD3+CD8+KIR+ T cells was evaluated. CD3+CD8+KIR+ T cells were isolated from PBMCs derived from three separate donors with celiac disease using flow cytometry-based cell-sorting. The isolated CD3+CD8+KIR+ T cells were cultured in the presence of different concentrations of IL-15 (0.05-5 ng/mL) for twelve (12) days and then re-evaluated for KIR expression by flow cytometry (FIG.10). The results showed that CD3+CD8+KIR+ T cells can gain CD39 expression following culture in IL-15 and that the increase in expression is dose-dependent. The effects of different combinations of γ chain cytokines on CD39 expression in CD3+CD8+KIR+ T cells were evaluated in cells derived from donors with celiac disease and from healthy donors. PBMCs from donors with celiac disease were cultured in the presence of: (1) IL-7 and IL-15 or (2) IL-2 and IL-15 for twelve (12) days and then re-evaluated for CD39 expression by flow cytometry (FIG.11). PBMCs from healthy normal donors were tested as a control. The results showed that increased CD39 expression in CD3+CD8+KIR+ T cells in response to γ chain cytokines may be impaired in celiac donors as compared with normal healthy donors. Example 7: CD39 Expression is Increased on KIR+CD8+ T Cells in Patients with Autoimmune Disorders To examine the effect of the anti-CD39/anti-ICOS CrossMAb antibody from Example 1 on CD39 expression in CD3+CD8+T cells, an in vitro assay was performed. A non-celiac (healthy donor) organoid was treated with anti-CD3 and anti-CD28 antibodies to activate the T cell population. Treatment with monospecific anti-ICOS antibody alone was tested as a control. Flow cytometry was used to evaluate the expression of CD39 on CD3+CD8+ T cells (FIG.12). The results show that treatment with the anti-ICOS antibody alone (clone 422 H2L5) increased CD39 expression, while treatment with the anti-CD39/anti-ICOS CrossMAb antibody produced an even greater increase in expression of CD39. The co-expression of CD39 and KIR in CD3+CD8+ T cells from patients with different autoimmune diseases was also measured (FIG.13). Patients with autoimmune diseases such as systemic lupus erythematosus (SLE), psoriatic arthritis, ankylosing spondylitis, and Sjögren's syndrome were found to have a higher frequency of CD3+CD8+CD39+KIR+ T cells relative to healthy donors tested. Example 8: Affinity Testing of Two Bispecific CD39/ICOS-Targeting Molecules The contribution of the affinity of each of anti-CD39 and anti-ICOS to the overall avidity of the anti-CD39/anti-ICOS CrossMAb antibody from Example 1 was tested using a biolayer interferometry (BLI) system from Octet (ForteBio) for measuring antibody affinity. FIG.14A shows a co-binding sensorgram for the anti-CD39/anti-ICOS CrossMAb antibody (of Example 1, comprising anti-CD39 clone 27577 and anti-ICOS clone 422 H2L5). FIG. 14B. shows antibody affinity (KD), association rate (ka), and dissociation rate (kd) for two CrossMAbs including: (1) the anti-CD39/anti-ICOS CrossMAb antibody from Example 1 (comprising anti-CD39 clone 27577 and anti-ICOS clone 422 H2L5), which includes a low affinity anti-CD39 antibody, and (2) a second anti-CD39/anti-ICOS CrossMAb antibody (comprising anti-CD39 clone 31815 and anti-ICOS clone 422 H2L5), which includes a high affinity anti-CD39 antibody. The results showed decreased affinity (KD) for ICOS and increased affinity for CD39 relative to CD39 or ICOS alone for the anti-CD39/anti-ICOS CrossMAb antibody from Example 1. Example 9: Bispecific CD39/ICOS-Targeting Molecule Increases Killing of CD4+ T cells and Reduces Cell Death in Cells from Disease Tissues The ability of the bispecific anti-CD39/anti-ICOS CrossMAb antibody from Example 1 to reduce expansion of CD4+ T cells was evaluated using an in vitro assay. CD8+ Tregs were isolated from PBMCs derived from donors with celiac disease and treated with the bispecific anti-CD39/anti-ICOS CrossMAb antibody. The CD8+Tregs were then incubated with autologous CTV-labeled CD4+ T cells that were previously activated with anti-CD3 and anti-CD28 stimulation. CD4+ T cells were also incubated with untreated CD8+ Tregs, non- Tregs, and CD8+ Tregs treated with anti-CD39 or anti-ICOS antibody alone, as controls. Expansion of the CD4+ T cell population was evaluated by flow cytometry five (5) days later (FIG.15). Results showed that the bispecific anti-CD39/anti-ICOS CrossMAb antibody from Example 1 was the most effective at reducing expansion of CD4+ T cells as compared to all control groups. The ability of the bispecific anti-CD39/anti-ICOS CrossMAb antibody from Example 1 to increase direct killing of pathogenic CD4+ T cells by cytotoxic CD8+ Tregs was tested. An IncuCyte-based cytotoxicity assay was used to determine the fluorescence of a target SKW-GFP cell line over time in co-culture with CD8+ Tregs. The dotted line represents the level of fluorescence of the target cell with no antibodies added. Monospecific anti-ICOS antibody and monospecific anti-CD39 antibody were tested as controls. Results showed that the bispecific anti-CD39/anti-ICOS CrossMAb antibody from Example 1 enhanced killing of pathogenic CD4+ T cells by CD8+ Tregs (FIG.16). The ability of the bispecific anti-CD39/anti-ICOS CrossMAb antibody from Example 1 to decrease epithelial cell death in a celiac donor-derived organoid stimulated with gliadin peptides was tested. Organoid cultures were treated for 48 hours with a gliadin peptide cocktail and the bispecific anti-CD39/anti-ICOS CrossMAb antibody prior to analysis by flow cytometry (FIG.17). Untreated organoid cultures and organoid cultures treated with anti-ICOS antibody were tested as controls. After 48 hours, organoid cultures were stained with Annexin-V live/dead stain and gated to include only the Epcam+MHCI+ epithelial cell population. The results showed a reduction in the number of Epcam+MHC I+Annexin-V+ cells in the group treated with the bispecific anti-CD39/anti-ICOS CrossMAb antibody as compared to the untreated and anti-ICOS antibody treated groups. While specific embodiments have been illustrated and described, it will be readily appreciated that the various embodiments described above can be combined to provide further embodiments, and the various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, including U.S. Provisional Patent Application Nos.63/370,353 filed August 3, 2022, and 63/380,879 filed October 25, 2022, are incorporated herein by reference, in their entirety, unless explicitly stated otherwise. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments. These and other changes can be made to the embodiments in light of the above- detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
SEQUENCES Underlined sequences are complementarity-determining regions (CDRs) SEQ ID NO:1 Anti-CD3927577 Variable Light (VL ) Region EIVMTQSPAILSVSPGERATLSCRASQSVGSNLAWYQQKPGQAPRLLIYGASTRATGIPARF SGSGSGTEFTLTISSLQSEDFAVYYCQQLTKWPLTFGGGTKVEIK SEQ ID NO:2 Anti-CD3927577 Light Chain CDR1 RASQSVGSNLA SEQ ID NO:3 Anti-CD3927577 Light Chain CDR2 GASTRAT SEQ ID NO:4 Anti-CD3927577 Light Chain CDR3 QQLTKWPLT SEQ ID NO:5 Anti-CD3927577 Variable Heavy (VH ) Region QVQLVQSGAEVKKPGSSVKVSCKASGGAFSSYAIGWVRQAPGQGLEWMGGIIPTFGTANYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGGGYQHHYFDLWGRGTLVTVSS SEQ ID NO:6 Anti-CD3927577 Heavy Chain CDR1 SYAIG SEQ ID NO:7 Anti-CD3927577 Heavy Chain CDR2 SEQ ID NO:8 Anti-CD3927577 Heavy Chain CDR3 DGGGYQHHYFDL SEQ ID NO:9 Anti-CD3927577 Light Chain EIVMTQSPAILSVSPGERATLSCRASQSVGSNLAWYQQKPGQAPRLLIYGASTRATGIPARF SGSGSGTEFTLTISSLQSEDFAVYYCQQLTKWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEK LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:10 Anti-CD3927577 Heavy Chain QVQLVQSGAEVKKPGSSVKVSCKASGGAFSSYAIGWVRQAPGQGLEWMGGIIPTFGTANYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGGGYQHHYFDLWGRGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK SEQ ID NO:11 Anti-CD3931895 Variable Light (VL ) Region EIVLIQSPGILSLSPGERATLSCRASQSVASSYLAWYQQKPGQAPRLLIYGASNRHTGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYHNAITFGGGTKVEIK SEQ ID NO:12 Anti-CD3931895 Light Chain CDR1 RASQSVASSYLA SEQ ID NO:13 Anti-CD3931895 Light Chain CDR2 GASNRHT SEQ ID NO:14 Anti-CD3931895 Light Chain CDR3 QQYHNAIT SEQ ID NO:15 Anti-CD3931895 Variable Heavy (VH ) Region QVQLVQSGAEVKKPGASVKVSCKASGYTFKSYEMHWVRQAPGQGLEWMGRINPSVGSTWYAQ KFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARGKREGGTEYLRKWGQGTLVTVSS SEQ ID NO:16 Anti-CD3931895 Heavy Chain CDR1 SYEMH SEQ ID NO:17 Anti-CD3931895 Heavy Chain CDR2 RINPSVGSTWYAQKFQG SEQ ID NO:18 Anti-CD3931895 Heavy Chain CDR3 GKREGGTEYLRKW SEQ ID NO:19 Anti-CD3931895 Light Chain EIVLIQSPGILSLSPGERATLSCRASQSVASSYLAWYQQKPGQAPRLLIYGASNRHTGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYHNAITFGGGTKVEIKRTVAAPSVFIFPPSDEK LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:20 Anti-CD3931895 Heavy Chain QVQLVQSGAEVKKPGASVKVSCKASGYTFKSYEMHWVRQAPGQGLEWMGRINPSVGSTWYAQ KFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARGKREGGTEYLRKWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK SEQ ID NO:21 Anti-CD8a Mb1b Variable Light (VL ) Region DVQITQSPSSLSASVGDRVTITCRTSRSISQYLAWYQQKPGKVPKLLIYSGSTLQSGVPSRF SGSGSGTDFTLTISSLQPEDVATYYCQQHNENPLTFGGGTKVEIK SEQ ID NO:22 Anti-CD8a Mb1b Light Chain CDR1 RTSRSISQYLA SEQ ID NO:23 Anti-CD8a Mb1b Light Chain CDR2 SGSTLQS SEQ ID NO:24 Anti-CD8a Mb1b Light Chain CDR3 QQHNENPLT SEQ ID NO:25 Anti-CD8a Mb1b Variable Heavy (VH ) Region Variable heavy (VH) region – Other CDR EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQAPGKGLEWIGRIDPANDNTLYAS KFQGKATISADTSKNTAYLQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSS Variable heavy (VH) region – Kabat CDR EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQAPGKGLEWIGRIDPANDNTLYAS KFQGKATISADTSKNTAYLQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSS SEQ ID NO:26 Anti-CD8a Mb1b Heavy Chain CDR1 GFNIKDT SEQ ID NO:27 Anti-CD8a Mb1b Heavy Chain CDR2 RIDPANDNT SEQ ID NO:28 Anti-CD8a Mb1b Heavy Chain CDR3 GYYVFDH SEQ ID NO:29 Anti-CD8a Mb1b Heavy Chain CDR1 Kabat DTYIH SEQ ID NO:30 Anti-CD8a Mb1b Heavy Chain CDR2 Kabat RIDPANDNTLYASKFQG SEQ ID NO:31 Anti-CD8a Mb1b Heavy Chain CDR3 Kabat GYGYYVFDH SEQ ID NO:32 Anti-CD8a Mb1b Light Chain DVQITQSPSSLSASVGDRVTITCRTSRSISQYLAWYQQKPGKVPKLLIYSGSTLQSGVPSRF SGSGSGTDFTLTISSLQPEDVATYYCQQHNENPLTFGGGTKVEIKSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO:33 Anti-CD8a Mb1b Heavy Chain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQAPGKGLEWIGRIDPANDNTLYAS KFQGKATISADTSKNTAYLQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSSASVAAP SVFIFPPSDEELKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAEGAPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSC AVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK SEQ ID NO:34 Anti-ICOS 422 H2L5 Variable Light (VL ) Region EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFS GSGSGTDYTLTISSLEPEDFAVYYCFQGSGYPYTFGQGTKLEIK SEQ ID NO:35 Anti-ICOS 422 H2L5 Light Chain CDR1 SASSSVSYMH SEQ ID NO:36 Anti-ICOS 422 H2L5 Light Chain CDR2 DTSKLAS SEQ ID NO:37 Anti-ICOS 422 H2L5 Light Chain CDR3 FQGSGYPYT SEQ ID NO:38 Anti-ICOS 422 H2L5 Variable Heavy (VH ) Region QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWMGLISIYSDHTNYNQ KFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGQGTTVTVSS SEQ ID NO:39 Anti-ICOS 422 H2L5 Heavy Chain CDR1 DYAMH SEQ ID NO:40 Anti-ICOS 422 H2L5 Heavy Chain CDR2 LISIYSDHTNYNQKFQG SEQ ID NO:41 Anti-ICOS 422 H2L5 Heavy Chain CDR3 NNYGNYGWYFDV SEQ ID NO:42 Anti-ICOS 422 H2L5 Light Chain EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFS GSGSGTDYTLTISSLEPEDFAVYYCFQGSGYPYTFGQGTKLEIKSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO:43 Anti-ICOS 422 H2L5 Heavy Chain QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWMGLISIYSDHTNYNQ KFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGQGTTVTVSSASV AAPSVFIFPPSDEELKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAEGAPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSRDELTKNQVS LSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK SEQ ID NO:44 Anti-PD1 MK-3475 Variable Light (VL ) Region EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGV PARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK SEQ ID NO:45 Anti-PD1 MK-3475 Light Chain CDR1 RASKGVSTSGYSYLH SEQ ID NO:46 Anti-PD1 MK-3475 Light Chain CDR2 LASYLES SEQ ID NO:47 Anti-PD1 MK-3475 Light Chain CDR3 QHSRDLPLT SEQ ID NO:48 Anti-PD1 MK-3475 Variable Heavy (VH ) Region QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNE KFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS SEQ ID NO:49 Anti-PD1 MK-3475 Heavy Chain CDR1 NYYMY SEQ ID NO:50 Anti-PD1 MK-3475 Heavy Chain CDR2 GINPSNGGTNFNEKFKN SEQ ID NO:51 Anti-PD1 MK-3475 Heavy Chain CDR3 RDYRFDMGFDY SEQ ID NO:52 Anti-PD1 MK-3475 Light Chain EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGV PARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO:53 Anti-PD1 MK-3475 Heavy Chain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNE KFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASVA APSVFIFPPSDEELKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAEGAPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSL SCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK SEQ ID NO:54 Anti-CD8a Mb1b LH scFv Heavy Chain DVQITQSPSSLSASVGDRVTITCRTSRSISQYLAWYQQKPGKVPKLLIYSGSTLQSGVPSRF SGSGSGTDFTLTISSLQPEDVATYYCQQHNENPLTFGGGTKVEIKGSTSGGGSGGGSGGGGS SEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQAPGKGLEWIGRIDPANDNTLYA SKFQGKATISADTSKNTAYLQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSSEPKSS DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL VSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* SEQ ID NO:55 Anti-ICOS 422 H2L5 LH scFv Heavy Chain EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFS GSGSGTDYTLTISSLEPEDFAVYYCFQGSGYPYTFGQGTKLEIKGSTSGGGSGGGSGGGGSS QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWMGLISIYSDHTNYNQ KFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGQGTTVTVSSEPK SSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQ PREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:56 Anti-ICOS 422 H2L5 HL scFv Heavy Chain QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWMGLISIYSDHTNYNQ KFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGQGTTVTVSSGST SGGGSGGGSGGGGSSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLI YDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCFQGSGYPYTFGQGTKLEIKEPK SSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQ PREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:57 Anti-CD3927577 LH scFv Heavy Chain EIVMTQSPAILSVSPGERATLSCRASQSVGSNLAWYQQKPGQAPRLLIYGASTRATGIPARF SGSGSGTEFTLTISSLQSEDFAVYYCQQLTKWPLTFGGGTKVEIKGGGGSGGGGSGGGGSAS QVQLVQSGAEVKKPGSSVKVSCKASGGAFSSYAIGWVRQAPGQGLEWMGGIIPTFGTANYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGGGYQHHYFDLWGRGTLVTVSSEPK SSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQ PREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:58 Anti-CD3927577 HL scFv Heavy Chain QVQLVQSGAEVKKPGSSVKVSCKASGGAFSSYAIGWVRQAPGQGLEWMGGIIPTFGTANYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGGGYQHHYFDLWGRGTLVTVSSGGG GSGGGGSGGGGSASEIVMTQSPAILSVSPGERATLSCRASQSVGSNLAWYQQKPGQAPRLLI YGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQLTKWPLTFGGGTKVEIKEPK SSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQ PREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:59 Anti-CD3927577 LH scFv Heavy Chain IgG2 Hinge EIVMTQSPAILSVSPGERATLSCRASQSVGSNLAWYQQKPGQAPRLLIYGASTRATGIPARF SGSGSGTEFTLTISSLQSEDFAVYYCQQLTKWPLTFGGGTKVEIKGGGGSGGGGSGGGGSAS QVQLVQSGAEVKKPGSSVKVSCKASGGAFSSYAIGWVRQAPGQGLEWMGGIIPTFGTANYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGGGYQHHYFDLWGRGTLVTVSSERK CCVECPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:60 Anti-CD3927577 HL scFv Heavy Chain IgG2 Hinge QVQLVQSGAEVKKPGSSVKVSCKASGGAFSSYAIGWVRQAPGQGLEWMGGIIPTFGTANYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGGGYQHHYFDLWGRGTLVTVSSGGG GSGGGGSGGGGSASEIVMTQSPAILSVSPGERATLSCRASQSVGSNLAWYQQKPGQAPRLLI YGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQLTKWPLTFGGGTKVEIKERK CCVECPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:61 Anti-ICOS 422 H2L5 LH scFv Heavy Chain IgG2 Hinge EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFS GSGSGTDYTLTISSLEPEDFAVYYCFQGSGYPYTFGQGTKLEIKGSTSGGGSGGGSGGGGSS QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWMGLISIYSDHTNYNQ KFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGQGTTVTVSSERK CCVECPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:62 Anti-ICOS 422 H2L5 HL scFv Heavy Chain IgG2 Hinge QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWMGLISIYSDHTNYNQ KFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGQGTTVTVSSGST SGGGSGGGSGGGGSSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLI YDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCFQGSGYPYTFGQGTKLEIKERK CCVECPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:63 Anti-CD3927577 LH scFv Heavy Chain IgG4 Hinge EIVMTQSPAILSVSPGERATLSCRASQSVGSNLAWYQQKPGQAPRLLIYGASTRATGIPARF SGSGSGTEFTLTISSLQSEDFAVYYCQQLTKWPLTFGGGTKVEIKGGGGSGGGGSGGGGSAS QVQLVQSGAEVKKPGSSVKVSCKASGGAFSSYAIGWVRQAPGQGLEWMGGIIPTFGTANYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGGGYQHHYFDLWGRGTLVTVSSESK YGPPCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:64 Anti-CD3927577 HL scFv Heavy Chain IgG4 Hinge QVQLVQSGAEVKKPGSSVKVSCKASGGAFSSYAIGWVRQAPGQGLEWMGGIIPTFGTANYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGGGYQHHYFDLWGRGTLVTVSSGGG GSGGGGSGGGGSASEIVMTQSPAILSVSPGERATLSCRASQSVGSNLAWYQQKPGQAPRLLI YGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQLTKWPLTFGGGTKVEIKESK YGPPCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:65 Anti-ICOS 422 H2L5 LH scFv Heavy Chain IgG4 Hinge EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFS GSGSGTDYTLTISSLEPEDFAVYYCFQGSGYPYTFGQGTKLEIKGSTSGGGSGGGSGGGGSS QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWMGLISIYSDHTNYNQ KFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGQGTTVTVSSESK YGPPCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:66 Anti-ICOS 422 H2L5 HL scFv Heavy Chain IgG4 Hinge QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWMGLISIYSDHTNYNQ KFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGQGTTVTVSSGST SGGGSGGGSGGGGSSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLI YDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCFQGSGYPYTFGQGTKLEIKESK YGPPCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:67 Anti-CD3931895 LH scFv Heavy Chain EIVLIQSPGILSLSPGERATLSCRASQSVASSYLAWYQQKPGQAPRLLIYGASNRHTGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYHNAITFGGGTKVEIKGGGGSGGGGSGGGGSAS QVQLVQSGAEVKKPGASVKVSCKASGYTFKSYEMHWVRQAPGQGLEWMGRINPSVGSTWYAQ KFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARGKREGGTEYLRKWGQGTLVTVSSEPK SSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQ PREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:68 Anti-CD3931895 LH scFv Heavy Chain QVQLVQSGAEVKKPGASVKVSCKASGYTFKSYEMHWVRQAPGQGLEWMGRINPSVGSTWYAQ KFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARGKREGGTEYLRKWGQGTLVTVSSGGG GSGGGGSGGGGSASEIVLIQSPGILSLSPGERATLSCRASQSVASSYLAWYQQKPGQAPRLL IYGASNRHTGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYHNAITFGGGTKVEIKEPK SSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQ PREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:69 Anti-CD3931895 LH scFv Heavy Chain IgG2 Hinge EIVLIQSPGILSLSPGERATLSCRASQSVASSYLAWYQQKPGQAPRLLIYGASNRHTGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYHNAITFGGGTKVEIKGGGGSGGGGSGGGGSAS QVQLVQSGAEVKKPGASVKVSCKASGYTFKSYEMHWVRQAPGQGLEWMGRINPSVGSTWYAQ KFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARGKREGGTEYLRKWGQGTLVTVSSERK CCVECPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:70 Anti-CD3931895 HL scFv Heavy Chain IgG2 Hinge QVQLVQSGAEVKKPGASVKVSCKASGYTFKSYEMHWVRQAPGQGLEWMGRINPSVGSTWYAQ KFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARGKREGGTEYLRKWGQGTLVTVSSGGG GSGGGGSGGGGSASEIVLIQSPGILSLSPGERATLSCRASQSVASSYLAWYQQKPGQAPRLL IYGASNRHTGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYHNAITFGGGTKVEIKERK CCVECPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:71 Anti-CD3931895 LH scFv Heavy Chain IgG4 Hinge EIVLIQSPGILSLSPGERATLSCRASQSVASSYLAWYQQKPGQAPRLLIYGASNRHTGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYHNAITFGGGTKVEIKGGGGSGGGGSGGGGSAS QVQLVQSGAEVKKPGASVKVSCKASGYTFKSYEMHWVRQAPGQGLEWMGRINPSVGSTWYAQ KFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARGKREGGTEYLRKWGQGTLVTVSSESK YGPPCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:72 Anti-CD3931895 HL scFv Heavy Chain IgG4 Hinge QVQLVQSGAEVKKPGASVKVSCKASGYTFKSYEMHWVRQAPGQGLEWMGRINPSVGSTWYAQ KFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARGKREGGTEYLRKWGQGTLVTVSSGGG GSGGGGSGGGGSASEIVLIQSPGILSLSPGERATLSCRASQSVASSYLAWYQQKPGQAPRLL IYGASNRHTGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYHNAITFGGGTKVEIKESK YGPPCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:73 Anti-CD3927577 VL-Mx-Anti-ICOS 422 H2L5 VH DART IgG1r-Fc C220S Heavy Chain Knob EIVMTQSPAILSVSPGERATLSCRASQSVGSNLAWYQQKPGQAPRLLIYGASTRATGIPARF SGSGSGTEFTLTISSLQSEDFAVYYCQQLTKWPLTFGGGTKVEIKGGGGSGGGGQVQLVQSG AEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWMGLISIYSDHTNYNQKFQGRVTI TADKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGQGTTVTVSSGCPPCPAPEAE GAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRDELT KNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:74 Mx-Anti-ICOS 422 H2L5 VL-Anti-CD3927577 VH DART IgG1r-Fc C220S Heavy Chain Hole EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFS GSGSGTDYTLTISSLEPEDFAVYYCFQGSGYPYTFGQGTKLEIKGGGSGGGGQVQLVQSGAE VKKPGSSVKVSCKASGGAFSSYAIGWVRQAPGQGLEWMGGIIPTFGTANYAQKFQGRVTITA DESTSTAYMELSSLRSEDTAVYYCARDGGGYQHHYFDLWGRGTLVTVSSGCPPCPAPEAEGA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSRDELTKN QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:75 Anti-CD3931895 VL-Mx-Anti-ICOS 422 H2L5 VH DART IgG1r-Fc C220S Heavy Chain Knob EIVLIQSPGILSLSPGERATLSCRASQSVASSYLAWYQQKPGQAPRLLIYGASNRHTGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYHNAITFGGGTKVEIKGGGGSGGGGQVQLVQSG AEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWMGLISIYSDHTNYNQKFQGRVTI TADKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGQGTTVTVSSGCPPCPAPEAE GAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRDELT KNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:76 Mx- Anti-ICOS 422 H2L5 VL- Anti-CD3931895 VH DART IgG1r-Fc C220S Heavy Chain Hole EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFS GSGSGTDYTLTISSLEPEDFAVYYCFQGSGYPYTFGQGTKLEIKGGGSGGGGQVQLVQSGAE VKKPGASVKVSCKASGYTFKSYEMHWVRQAPGQGLEWMGRINPSVGSTWYAQKFQGRVIMIR DTSTSTVYMELSSLRSEDTAVYYCARGKREGGTEYLRKWGQGTLVTVSSGCPPCPAPEAEGA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSRDELTKN QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:77 Anti-CD3927577 IgG1r mAb Light Chain EIVMTQSPAILSVSPGERATLSCRASQSVGSNLAWYQQKPGQAPRLLIYGASTRATGIPARF SGSGSGTEFTLTISSLQSEDFAVYYCQQLTKWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:78 Anti-CD3927577 IgG1r mAb Heavy Chain QVQLVQSGAEVKKPGSSVKVSCKASGGAFSSYAIGWVRQAPGQGLEWMGGIIPTFGTANYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGGGYQHHYFDLWGRGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK SEQ ID NO:79 Anti-CD3931895 IgG1r mAb Light Chain EIVLIQSPGILSLSPGERATLSCRASQSVASSYLAWYQQKPGQAPRLLIYGASNRHTGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYHNAITFGGGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:80 Anti-CD3931895 IgG1r mAb Heavy Chain QVQLVQSGAEVKKPGASVKVSCKASGYTFKSYEMHWVRQAPGQGLEWMGRINPSVGSTWYAQ KFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARGKREGGTEYLRKWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK SEQ ID NO:81 Anti-ICOS 422 H2L5 IgG1r mAb Light Chain EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFS GSGSGTDYTLTISSLEPEDFAVYYCFQGSGYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:82 Anti-ICOS 422 H2L5 IgG1r mAb Heavy Chain QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWMGLISIYSDHTNYNQ KFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGQGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK SEQ ID NO:83 Anti-CD8a Mb1b IgG1r mAb Light Chain DVQITQSPSSLSASVGDRVTITCRTSRSISQYLAWYQQKPGKVPKLLIYSGSTLQSGVPSRF SGSGSGTDFTLTISSLQPEDVATYYCQQHNENPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:84 Anti-CD8a Mb1b IgG1r mAb Heavy Chain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQAPGKGLEWIGRIDPANDNTLYAS KFQGKATISADTSKNTAYLQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID NO:85 Anti-PD1 MK-3475 IgG1r mAb Light Chain EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGV PARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:86 Anti-PD1 MK-3475 IgG1r mAb Heavy Chain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNE KFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK SEQ ID NO:87 gliadin peptide QLQPFPQPELPY SEQ ID NO:88 gliadin peptide PQPELPYPQPE SEQ ID NO:89 gliadin peptide QQPFPQPEQPFP SEQ ID NO:90 gliadin peptide FPQPEQPFPWQP

Claims

CLAIMS 1. A bispecific antibody, or an antigen binding fragment thereof, comprising: a first binding arm that specifically binds to CD39; and a second binding arm that specifically binds to ICOS, CD8a, or PD-1.
2. The bispecific antibody of claim 1, wherein the first binding arm comprises CDRL1, CDRL2, CDRL3 amino acid sequences according to SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; and comprises CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
3. The bispecific antibody of claim 1 or claim 2, wherein the first binding arm comprises a light chain variable region (VL) comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:1; and comprises a heavy chain variable region (VH) comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:5.
4. The bispecific antibody of claim 3, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:1.
5. The bispecific antibody of claim 3, wherein the VH of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:5.
6. The bispecific antibody of claim 3, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:1; and the VH comprises or consists of the amino acid sequence according to SEQ ID NO:5.
7. The bispecific antibody of claim 1, wherein the first binding arm comprises CDRL1, CDRL2, CDRL3 amino acid sequences according to SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14; and comprises CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.
8. The bispecific antibody of claim 1 or claim 7, wherein the first binding arm comprises a VL comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:11; and comprises a VH comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:15.
9. The bispecific antibody of claim 8, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:11.
10. The bispecific antibody of claim 8, wherein the VH of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:15.
11. The bispecific antibody of claim 8, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:11; and the VH comprises or consists of the amino acid sequence according to SEQ ID NO:15.
12. The bispecific antibody of any one of claims 1-11, wherein the second binding arm specifically binds to ICOS.
13. The bispecific antibody of claim 12, wherein the second binding arm comprises CDRL1, CDRL2, CDRL3 amino acid sequences according to SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37; and comprises CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41.
14. The bispecific antibody of claim 12 or claim 13, wherein the second binding arm comprises a VL comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:34; and comprises a VH comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:38.
15. The bispecific antibody of claim 14, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:34.
16. The bispecific antibody of claim 14, wherein the VH of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:38.
17. The bispecific antibody of claim 14, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:34; and the VH comprises or consists of the amino acid sequence according to SEQ ID NO:38.
18. The bispecific antibody of any one of claims 1-11, wherein the second binding arm specifically binds to CD8a.
19. The bispecific antibody of claim 18, wherein the second binding arm comprises CDRL1, CDRL2, CDRL3 amino acid sequences according to SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24; and comprises CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.
20. The bispecific antibody of claim 18, wherein the second binding domain comprises CDRL1, CDRL2, CDRL3 amino acid sequences according to SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24; and comprises CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.
21. The bispecific antibody of any one of claims 18-20, wherein the second binding domain comprises a VL comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:21; and comprises a VH comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:25.
22. The bispecific antibody of claim 21, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:21.
23. The bispecific antibody of claim 21, wherein the VH of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:25.
24. The bispecific antibody of claim 21, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:21; and the VH comprises or consists of the amino acid sequence according to SEQ ID NO:25.
25. The bispecific antibody of any one of claims 1-11, wherein the second binding arm specifically binds to PD-1.
26. The bispecific antibody of claim 25, wherein the second binding domain comprises CDRL1, CDRL2, CDRL3 amino acid sequences according to SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47; and comprises CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51.
27. The bispecific antibody of claim 25 or 26, wherein the second binding domain comprises a VL comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:44; and comprises a VH comprising at least 90% identity to the amino acid sequence according to SEQ ID NO:48.
28. The bispecific antibody of claim 27, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:44.
29. The bispecific antibody of claim 27, wherein the VH of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:48.
30. The bispecific antibody of claim 27, wherein the VL of the first binding arm comprises or consists of the amino acid sequence according to SEQ ID NO:44; and the VH comprises or consists of the amino acid sequence according to SEQ ID NO:48.
31. The bispecific antibody of any one of claims 1-30, wherein the bispecific antibody comprises or is a CrossMab, a Bottle Opener bispecific, a diabody, an antibody Fc fusion, an scFv1-ScFv2, an ScFv12-Fc-scFv22, an IgG-scFv, a DVD-Ig, a triomab/quadroma, a two-in- one IgG, a scFv2-Fc, a TandAb, an scFv-HSA-scFv, an scFv-VHH, a Fab-scFv-Fc, a Fab- VHH-Fc, a dAb-IgG, an IgG-VHH, a Tandem scFv-Fc, a (scFv1)2-Fc-(VHH)2, a BiTe, a DART, a scFv-Fc, a one-armed tandem scFv-Fc, a DART-Fc, an anticalin, an affibody, an avimer, a DARPin, or an adnectin.
32. The bispecific antibody of any one of claims 1-31, wherein the bispecific antibody is a CrossMab bispecific antibody.
33. The bispecific antibody of any one of claims 1-31, wherein the bispecific antibody is a Bottle Opener bispecific antibody.
34. The bispecific antibody of any one of claims 1-31, wherein the bispecific antibody is a scFv-Fc bispecific antibody.
35. The bispecific antibody of any one of claims 1-31, wherein the bispecific antibody is a DART-Fc bispecific antibody.
36. The bispecific antibody of claim 1, comprising an anti-CD39 light chain according to SEQ ID NO:9, an anti-CD39 heavy chain according to SEQ ID NO:10, an anti-ICOS light chain according to SEQ ID NO:42, and an anti-ICOS heavy chain according to SEQ ID NO:43.
37. The bispecific antibody of claim 36, wherein the bispecific antibody is a CrossMab bispecific antibody.
38. The bispecific antibody of claim 1, comprising an anti-CD39 light chain according to SEQ ID NO:19, an anti-CD39 heavy chain according to SEQ ID NO:20, an anti-ICOS light chain according to SEQ ID NO:42, and an anti-ICOS heavy chain according to SEQ ID NO:43.
39. The bispecific antibody of claim 38, wherein the bispecific antibody is a CrossMab bispecific antibody.
40. The bispecific antibody of claim 1, comprising an anti-CD39 light chain according to SEQ ID NO:9, an anti-CD39 heavy chain according to SEQ ID NO:10, and an anti-ICOS scFv heavy chain according to SEQ ID NO:55.
41. The bispecific antibody of claim 1, comprising an anti-CD39 light chain according to SEQ ID NO:9, an anti-CD39 heavy chain according to SEQ ID NO:10, and an anti-ICOS scFv heavy chain according to SEQ ID NO:56.
42. The bispecific antibody of claim 40 or 41, wherein the bispecific antibody is a Bottle Opener bispecific antibody.
43. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:57, and an anti-ICOS scFv heavy chain according to SEQ ID NO:55.
44. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:57, and an anti-ICOS scFv heavy chain according to SEQ ID NO:56.
45. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:58, and an anti-ICOS scFv heavy chain according to SEQ ID NO:55.
46. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:58, and an anti-ICOS scFv heavy chain according to SEQ ID NO:56.
47. The bispecific antibody of any one of claims 43-46, wherein the bispecific antibody is an scFV-Fc bispecific antibody.
48. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:59, and an anti-ICOS scFv heavy chain according to SEQ ID NO:61.
49. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:59, and an anti-ICOS scFv heavy chain according to SEQ ID NO:62.
50. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:60, and an anti-ICOS scFv heavy chain according to SEQ ID NO:61.
51. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:60, and an anti-ICOS scFv heavy chain according to SEQ ID NO:62.
52. The bispecific antibody of any one of claims 48-51, wherein the bispecific antibody is an scFV-Fc bispecific antibody.
53. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:63, and an anti-ICOS scFv heavy chain according to SEQ ID NO:65.
54. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:63, and an anti-ICOS scFv heavy chain according to SEQ ID NO:66.
55. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:64, and an anti-ICOS scFv heavy chain according to SEQ ID NO:65.
56. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:64, and an anti-ICOS scFv heavy chain according to SEQ ID NO:66.
57. The bispecific antibody of any one of claims 53-56, wherein the bispecific antibody is an scFV-Fc bispecific antibody.
58. The bispecific antibody of claim 1, comprising an anti-CD39 light chain according to SEQ ID NO:19, an anti-CD39 heavy chain according to SEQ ID NO:20, and an anti-ICOS scFv heavy chain according to SEQ ID NO:55.
59. The bispecific antibody of claim 1, comprising an anti-CD39 light chain according to SEQ ID NO:19, an anti-CD39 heavy chain according to SEQ ID NO:20, and an anti-ICOS scFv heavy chain according to SEQ ID NO:56.
60. The bispecific antibody of claim 58 or claim 59, wherein the bispecific antibody is a Bottle Opener bispecific antibody.
61. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:67, and an anti-ICOS scFv heavy chain according to SEQ ID NO:55.
62. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:67, and an anti-ICOS scFv heavy chain according to SEQ ID NO:56.
63. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:68, and an anti-ICOS scFv heavy chain according to SEQ ID NO:55.
64. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:68, and an anti-ICOS scFv heavy chain according to SEQ ID NO:56.
65. The bispecific antibody of any one of claims 61-64, wherein the bispecific antibody is an scFV-Fc bispecific antibody.
66. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:69, and an anti-ICOS scFv heavy chain according to SEQ ID NO:61.
67. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:69, and an anti-ICOS scFv heavy chain according to SEQ ID NO:62.
68. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:70, and an anti-ICOS scFv heavy chain according to SEQ ID NO:61.
69. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:70, and an anti-ICOS scFv heavy chain according to SEQ ID NO:62.
70. The bispecific antibody of any one of claims 66-69, wherein the bispecific antibody is an scFV-Fc bispecific antibody.
71. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:71, and an anti-ICOS scFv heavy chain according to SEQ ID NO:65.
72. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:71, and an anti-ICOS scFv heavy chain according to SEQ ID NO:66.
73. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:72, and an anti-ICOS scFv heavy chain according to SEQ ID NO:65.
74. The bispecific antibody of claim 1, comprising an anti-CD39 scFv heavy chain according to SEQ ID NO:72, and an anti-ICOS scFv heavy chain according to SEQ ID NO:66.
75. The bispecific antibody of any one of claims 71-74, wherein the bispecific antibody is an scFV-Fc bispecific antibody.
76. The bispecific antibody of claim 1, comprising an anti-CD39 heavy chain according to SEQ ID NO:73, and an anti-ICOS heavy chain according to SEQ ID NO:74.
77. The bispecific antibody of claim 76, wherein the bispecific antibody is a DART-Fc bispecific antibody.
78. The bispecific antibody of claim 1, comprising an anti-CD39 heavy chain according to SEQ ID NO:75, and an anti-ICOS heavy chain according to SEQ ID NO:76.
79. The bispecific antibody of claim 78, wherein the bispecific antibody is a DART-Fc bispecific antibody.
80. The bispecific antibody of claim 1, comprising an anti-CD39 light chain according to SEQ ID NO:9, an anti-CD39 heavy chain according to SEQ ID NO:10, an anti-CD8a light chain according to SEQ ID NO:32, and an anti-CD8a heavy chain according to SEQ ID NO:33.
81. The bispecific antibody of claim 80, wherein the bispecific antibody is a Cross-Mab bispecific antibody.
82. The bispecific antibody of claim 1, comprising an anti-CD39 light chain according to SEQ ID NO:19, an anti-CD39 heavy chain according to SEQ ID NO:20, an anti-CD8a light chain according to SEQ ID NO:32, and an anti-CD8a heavy chain according to SEQ ID NO:33.
83. The bispecific antibody of claim 82, wherein the bispecific antibody is a Cross-Mab bispecific antibody.
84. The bispecific antibody of claim 1, comprising an anti-CD39 light chain according to SEQ ID NO:9, an anti-CD39 heavy chain according to SEQ ID NO:10, and an anti-CD8a scFv heavy chain according to SEQ ID NO:54.
85. The bispecific antibody of claim 84, wherein the bispecific antibody is a Bottle Opener bispecific antibody.
86. The bispecific antibody of claim 1, comprising an anti-CD39 light chain according to SEQ ID NO:19, an anti-CD39 heavy chain according to SEQ ID NO:20, and an anti-CD8a scFv heavy chain according to SEQ ID NO:54.
87. The bispecific antibody of claim 86, wherein the bispecific antibody is a Bottle Opener bispecific antibody.
88. The bispecific antibody of claim 1, comprising an anti-CD39 light chain according to SEQ ID NO:9, an anti-CD39 heavy chain according to SEQ ID NO:10, an anti-PD-1 light chain according to SEQ ID NO:52, and an anti-PD-1 heavy chain according to SEQ ID NO:53.
89. The bispecific antibody of claim 88, wherein the bispecific antibody is a Cross-Mab bispecific antibody.
90. The bispecific antibody of claim 1, comprising an anti-CD39 light chain according to SEQ ID NO:19, an anti-CD39 heavy chain according to SEQ ID NO:20, an anti-PD-1 light chain according to SEQ ID NO:52, and an anti-PD-1 heavy chain according to SEQ ID NO:53.
91. The bispecific antibody of claim 90, wherein the bispecific antibody is a Cross-Mab bispecific antibody.
92. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-91 and a pharmaceutically acceptable carrier.
93. A nucleic acid encoding the bispecific antibody of any one of claims 1-91.
94. A vector comprising the nucleic acid of claim 93.
95. A cell line comprising the vector of claim 94.
96. A method of treating an autoimmune disease, comprising administering the bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92 to a subject in need thereof in an amount effective to decrease the number or activity of pathogenic immune cells in the subject.
97. The bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92 for use in treating an autoimmune disease in a subject, wherein the use comprises administering the bispecific antibody or the pharmaceutical composition to a subject in need thereof in an amount effective to decrease the number or activity of pathogenic immune cells in the subject.
98. Use of the bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92 in the manufacture of a medicament for use in treating an autoimmune disease in a subject, wherein the use comprises administering the bispecific antibody or the pharmaceutical composition to a subject in need thereof in an amount effective to decrease the number or activity of pathogenic immune cells in the subject
99. A method of suppressing an immune response mediated by pathogenic immune cells, comprising contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92 in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs).
100. The bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92 for use in suppressing an immune response mediated by pathogenic immune cells in a subject, wherein the use comprises contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody or the pharmaceutical composition in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs).
101. Use of the bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92 in the manufacture of a medicament for use in suppressing an immune response mediated by pathogenic immune cells in a subject, wherein the use comprises contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody or the pharmaceutical composition in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs).
102. A method of suppressing an immune response to an antigen, such as an autoantigen, comprising administering to a subject in need thereof the bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92 in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby the number or activity of pathogenic immune cells that are responsive to the antigen or autoantigen is decreased.
103. The bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92 for use in suppressing an immune response to an antigen, such as an autoantigen, in a subject, wherein the use comprises administering to a subject in need thereof the bispecific antibody or pharmaceutical composition in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby the number or activity of pathogenic immune cells that are responsive to the antigen or autoantigen is decreased.
104. Use of the bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92 in the manufacture of a medicament for use in suppressing an immune response to an antigen, such as an autoantigen, in a subject, wherein the use comprises administering to a subject in need thereof the bispecific antibody or pharmaceutical composition in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby the number or activity of pathogenic immune cells that are responsive to the antigen or autoantigen is decreased.
105. The method, bispecific antibody or pharmaceutical composition for use, or use in manufacture of any one of claims 99-104, wherein the CD8+ Tregs are CD8+KIR+ Tregs.
106. The method, bispecific antibody or pharmaceutical composition for use, or use in manufacture of any one of claims 99-105, wherein the activated CD8+ Tregs are administered in an effective amount to a subject in need thereof.
107. The method, bispecific antibody or pharmaceutical composition for use, or use in manufacture of any one of claims 96-106, wherein the pathogenic immune cells are autoreactive CD4+ T cells, autoantibody producing B cells, or self antigen presenting dendritic cells.
108. The method, bispecific antibody or pharmaceutical composition for use, or use in manufacture of any one of claims 96-107, wherein the subject has an autoimmune disease.
109. The method, bispecific antibody or pharmaceutical composition for use, or use in manufacture of claim 108, wherein the autoimmune disease is celiac disease, Crohn’s disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), lupus, lupus nephritis, cutaneous lupus, discoid lupus, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus/pemphigoid, rheumatoid arthritis (RA), scleroderma/systemic sclerosis, Sjögren’s syndrome (SjS), systemic lupus erythematosus (SLE), or ulcerative colitis.
110. The method, bispecific antibody or pharmaceutical composition for use, or use in manufacture of claim 108, wherein the autoimmune disease is celiac disease, Crohn’s disease, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), , multiple sclerosis (MS), rheumatoid arthritis (RA), scleroderma/systemic sclerosis, Sjögren’s syndrome (SjS), lupus, lupus nephritis, cutaneous lupus, discoid lupus, systemic lupus erythematosus (SLE), or ulcerative colitis.
111. A method of reducing or preventing onset of graft versus host disease (GVHD) following a transplant, comprising administering the bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92, wherein the bispecific antibody has substantially no effector function activity, to a subject in need thereof in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs) and thereby reduce or ameliorate at least one symptom of GVHD.
112. The bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92 for use in reducing or preventing onset of graft versus host disease (GVHD) in a subject following a transplant, wherein the use comprises administering the bispecific antibody or the pharmaceutical composition, wherein the bispecific antibody has substantially no effector function activity, to a subject in need thereof in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs) and thereby reduce or ameliorate at least one symptom of GVHD.
113. Use of the bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92 in the manufacture of a medicament for use in reducing or preventing onset of graft versus host disease (GVHD) in a subject following a transplant, wherein the use comprises administering the bispecific antibody or the pharmaceutical composition, wherein the bispecific antibody has substantially no effector function activity, to a subject in need thereof in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs) and thereby reduce or ameliorate at least one symptom of GVHD.
114. A method of treating a subject who has received a transplant, comprising contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92, wherein the bispecific antibody has substantially no effector function activity, in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby GVHD is reduced or suppressed.
115. The bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92 for use in treating a subject who has received a transplant, wherein the use comprises contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody or the pharmaceutical composition, wherein the bispecific antibody has substantially no effector function activity, in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby GVHD is reduced or suppressed.
116. Use of the bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92 in the manufacture of a medicament for use in treating a subject who has received a transplant, wherein the use comprises contacting CD8+ T regulatory cells (CD8+ Tregs) with the bispecific antibody or the pharmaceutical composition, wherein the bispecific antibody has substantially no effector function activity, in an amount effective to activate or stimulate CD8+ Tregs (activated CD8+ Tregs), whereby GVHD is reduced or suppressed.
117. The method of any one of claims 111-116, wherein the CD8+ Tregs are CD8+KIR+ Tregs.
118. A method of suppressing, reducing, or preventing an immune response to a viral vector in a subject, the method comprising administering to the subject the bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92 that binds to CD8+ T regulatory cells (CD8+ Tregs).
119. A binding agent that binds to CD8+ T regulatory cells (CD8+ Tregs) for use in a method of suppressing, reducing, or preventing an immune response to a viral vector in a subject, wherein the use comprises administering to the subject the bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92.
120. Use of the bispecific antibody of any one of claims 1-91 or the pharmaceutical composition of claim 92 in the manufacture of a medicament for use in a method of suppressing, reducing, or preventing an immune response to a viral vector in a subject, wherein the use comprises administering to the subject a binding agent that binds to CD8+ T regulatory cells (CD8+ Tregs).
121. The method of any one of claims 118-120, wherein the viral vector has been, is, or will be administered to the subject.
122. The method of any one of claims 118-121, wherein the immune response to the viral vector is induced by administration of the viral vector to the subject.
123. The method of any one of claims 118-122, wherein the CD8+ Tregs are CD8+KIR+ Tregs.
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