US20210363268A1 - Antibodies That Bind CD39 and Uses Thereof - Google Patents

Antibodies That Bind CD39 and Uses Thereof Download PDF

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US20210363268A1
US20210363268A1 US16/980,271 US201916980271A US2021363268A1 US 20210363268 A1 US20210363268 A1 US 20210363268A1 US 201916980271 A US201916980271 A US 201916980271A US 2021363268 A1 US2021363268 A1 US 2021363268A1
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amino acid
acid sequence
antibody
heavy chain
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Scott Chappel
Andrew Lake
Michael Warren
Austin DULAK
Erik DEVEREAUX
Pamela M. Holland
Tauqeer ZAIDI
Matthew RAUSCH
Bianka Prinz
Nels P. Nielson
Sonia DAS
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Surface Oncology Inc
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Surface Oncology Inc
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    • C07—ORGANIC CHEMISTRY
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

Definitions

  • Cancers are able to grow by subverting immune suppressive pathways, to prevent the malignant cells as being recognized as dangerous or foreign. This mechanism prevents the cancer from being eliminated by the immune system and allows disease to progress from a very early stage to a lethal state.
  • Immunotherapies are newly developing interventions that modify the patient's immune system to fight cancer, by either directly stimulating rejection-type processes or blocking suppressive pathways.
  • Extracellular adenosine generated by the ectonucleotidases CD39 and CD73 is a newly recognized “immune checkpoint mediator” that interferes with anti-tumor immune responses.
  • Adenosine is an immunomodulatory metabolite within the tumor microenvironment (TME).
  • TME tumor microenvironment
  • extracellular adenosine accumulates and subsequently inhibits the function of immune cells, including T cells, dendritic cells (DC), and NK cells, thereby contributing to anti-tumor immune suppression and supporting tumor growth.
  • the ectonucleotidase CD39 hydrolyzes extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to generate adenosine, which binds to adenosine receptors and inhibits immune cells such as T-cells and natural killer (NK)-cells, thereby suppressing the immune system.
  • ATP adenosine triphosphate
  • ADP adenosine diphosphate
  • adenosine binds to adenosine receptors and inhibits immune cells such as T-cells and natural killer (NK)-cells, thereby suppressing the immune system.
  • adenosine pathway refers to the extracellular conversion of ATP to adenosine and the signaling of adenosine through the A2A/A2B adenosine receptors on immune cells.
  • CD39 Under normal conditions, CD39 works to maintain the balance of extracellular levels of immunosuppressive adenosine and immunostimulatory ATP. In healthy tissues, ATP is barely detectable in the extracellular environment because ATP is rapidly broken down by CD39 to generate adenosine monophosphate, or AMP, which is then converted to adenosine by CD73. Under conditions of cellular stress, including cancer, extracellular ATP levels rise significantly, but because ATP is rapidly broken down, leading to low levels of ATP coupled with high levels of adenosine, recognition of the tumor by the immune system, and thus the immune response against the tumor, is hindered.
  • the anti-CD39 antibodies disclosed are non-competitive, allosteric inhibitors of CD39.
  • the anti-CD39 antibodies allow substrate (ATP) binding, but prohibit its conversion to ADP and/or adenosine, thus maintaining or enhancing levels of ATP in the tumor microenvironment (TME) and/or preventing undesirable levels of adenosine in the TME.
  • Nucleic acid molecules encoding the antibody molecules, expression vectors, host cells and methods for making the antibody molecules are also provided.
  • Pharmaceutical compositions comprising the antibody molecules are also provided.
  • anti-CD39 antibodies, or antigen binding portions thereof, disclosed herein can be used (alone or in combination with other therapeutic agents or procedures) to treat, prevent and/or diagnose disorders, including immune disorders and cancer.
  • compositions and methods for treating and/or diagnosing various disorders, including cancer and immune disorders, using the anti-CD39 antibody molecules are disclosed herein.
  • the disclosure provides anti-CD39 antibodies, including antibodies, that bind to and antagonize human CD39, or an antigen binding portion thereof, wherein the antibody or antigen binding portion thereof exhibits one or more of the following properties:
  • (d) inhibits or reduces conversion by human CD39 of extracellular adenosine triphosphate (eATP) or extracellular adenosine diphosphate (eADP) to extracellular adenosine monophosphate (eAMP);
  • eATP extracellular adenosine triphosphate
  • eADP extracellular adenosine diphosphate
  • eAMP extracellular adenosine monophosphate
  • (j) increases or enhances secretion of one or more cytokines from dendritic cells
  • an isolated anti-CD39 antibody comprising, consisting, or consisting essentially of:
  • the anti-CD39 antibody comprises the structural features described in paragraph [0008] and one or more of the functional features described in paragraph [0007]. In some embodiments, the anti-CD39 antibody, or antigen binding fragment thereof, binds to substantially the same epitope as a reference antibody described in paragraphs [0007] or [0008].
  • the disclosure provides an anti-CD39 antibody, or antigen binding portion thereof, comprising or consisting of heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 27, 28, and 29, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 37, 38, and 39, respectively.
  • the disclosure provides an anti-CD39 antibody, or antigen binding portion thereof, comprising or consisting of a variable heavy chain at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33, and a variable light chain at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43.
  • the disclosure provides an anti-CD39 antibody, or antigen binding portion thereof, comprising or consisting of a heavy chain at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 49, and a light chain at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45.
  • the disclosure provides an anti-CD39 antibody that binds to and antagonizes human CD39 comprising or consisting of heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 27, 28, and 29, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 37, 38, and 39, respectively; or heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 4, 5, and 6, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 15, 16, and 17, respectively, wherein the antibody:
  • the anti-CD39 antibody, or antigen binding portion thereof comprises an IgG1, an IgG2, an IgG3, an IgG4, an IgM, an IgA1, an IgA2, ad IgD, or an IgE antibody.
  • the anti-CD39 antibody, or antigen binding portion thereof comprises a wild type or mutant IgG1 or IgG4 antibody. In some embodiments, the anti-CD39 antibody, or antigen binding portion thereof, comprises a mutant IgG4 heavy chain constant region, wherein the mutation i) reduces the ability of the IgG4 to form half-molecules; and/or ii) minimizes binding to Fc receptors.
  • the anti-CD39 antibody, or antigen binding portion thereof comprises a mutant IgG4 heavy chain constant region comprising an S228P mutation. In some embodiments, the anti-CD39 antibody, or antigen binding portion thereof, comprises a mutant IgG4 heavy chain constant region comprising S228P and L235E mutations.
  • the anti-CD39 antibody, or antigen binding portion thereof comprises the heavy chain CDR3 sequence set forth in SEQ ID NO: 3. In some embodiments, the anti-CD39 antibody, or antigen binding portion thereof, binds to substantially the same epitope as a reference antibody or antigen binding portion thereof comprising the heavy chain CDR3 sequence set forth in SEQ ID NO: 3.
  • CD39 is increased in subjects resistant to anti-PD1/anti-PD-L1 therapy. See, e.g., Hotson/Luke et al., Oral presentation at Society for Immunotherapy of Cancer (SITC) 32nd Annual Meeting (2017) [retrieved on 2019-03-13]. Retrieved from the Internet: ⁇ URL: https://www.corvuspharma.com/file.cfm/23/docs/SITC_2017_Slides.pdf>.
  • a method of treating human subjects that are resistant to anti-PD1 or anti-PD-L1 therapy comprising administering any one of the anti-CD39 antibodies described herein.
  • Subjects who are resistant to anti-PD1 or anti-PD-L1 include subject whose benefit from the anti-PD1 or anti-PD-L1 therapy remained diminished by at least one standard deviation as compared to a non-resistant control for greater than three months.
  • the disclosure provides an antibody or antigen binding portion thereof that binds to human recombinant CD39 and/or membrane-bound human CD39. In one aspect, the disclosure provides an antibody or antigen binding portion thereof that binds to human CD39 with an equilibrium dissociation constant (K D ) of less than 10 nM. In one aspect, the disclosure provides an antibody or antigen binding portion thereof that inhibits or reduces an enzymatic activity of human CD39. In one aspect, the disclosure provides an antibody or antigen binding portion thereof that binds to human recombinant CD39 and/or membrane-bound human CD39 with an equilibrium dissociation constant (K D ) of less than 10 nM and inhibits or reduces an enzymatic activity of human CD39. In some aspects, the enzymatic activity of human CD39 is the hydrolysis of eATP or eADP.
  • the antibody or antigen binding portion of the disclosure inhibits or reduces the conversion of eATP or eADP to eAMP. In some aspects, the antibody or antigen binding portion thereof binds to human recombinant CD39 and/or membrane-bound human CD39 with an equilibrium dissociation constant (K D ) of less than 10 nM, and inhibits or reduces the conversion of eATP or eADP to eAMP.
  • K D equilibrium dissociation constant
  • the antibody or antigen binding portion of the disclosure decreases or reduces a level of extracellular adenosine.
  • the antibody or antigen binding portion thereof binds to human recombinant CD39 and/or membrane-bound human CD39 with an equilibrium dissociation constant (K D ) of less than 10 nM, and decreases or reduces a level of extracellular adenosine.
  • K D equilibrium dissociation constant
  • the antibody or antigen binding portion of the disclosure maintains, increases or enhances an immunostimulatory level of eATP.
  • the antibody or antigen binding portion thereof binds to human recombinant CD39 and/or membrane-bound human CD39 with an equilibrium dissociation constant (K D ) of less than 10 nM, and maintains, increases or enhances an immunostimulatory level of eATP.
  • the antibody or antigen binding portion of the disclosure increases proliferation of a lymphocyte.
  • the antibody or antigen binding portion thereof binds to human recombinant CD39 and/or membrane-bound human CD39 with an equilibrium dissociation constant (K D ) of less than 10 nM, and increases proliferation of a lymphocyte.
  • the antibody or antigen binding portion thereof binds to human recombinant CD39 and/or membrane-bound human CD39 with an equilibrium dissociation constant (K D ) of less than 10 nM, inhibits or reduces an enzymatic activity of human CD39, and increases proliferation of a lymphocyte.
  • the antibody or antigen binding portion thereof binds to human recombinant CD39 and/or membrane-bound human CD39 with an equilibrium dissociation constant (K D ) of less than 10 nM, maintains, increases or enhances a level eATP, and increases proliferation of a lymphocyte. In some aspects, the antibody or antigen binding portion thereof binds to human recombinant CD39 and/or membrane-bound human CD39 with an equilibrium dissociation constant (K D ) of less than 10 nM, maintains, increases or enhances a level eATP, and/or decreases or reduces a level of adenosine, and increases proliferation of a lymphocyte.
  • the lymphocyte is a tumor-infiltrating lymphocyte. In some aspects, the lymphocyte is T cell. In some aspects, the T cell is a CD4+ T cell.
  • the antibody or antigen binding portion of the disclosure increases or enhances expression of one or more dendritic cell activation markers and/or increases or enhances secretion of one or more cytokines from dendritic cells.
  • the antibody or antigen binding portion thereof binds to human recombinant CD39 and/or membrane-bound human CD39 with an equilibrium dissociation constant (K D ) of less than 10 nM, and increases or enhances expression of one or more dendritic cell activation markers and/or increases or enhances secretion of one or more cytokines from dendritic cells.
  • the antibody or antigen binding portion thereof binds to human recombinant CD39 and/or membrane-bound human CD39 with an equilibrium dissociation constant (K D ) of less than 10 nM, inhibits or reduces an enzymatic activity of human CD39, and increases or enhances expression of one or more dendritic cell activation markers and/or increases or enhances secretion of one or more cytokines from dendritic cells.
  • K D equilibrium dissociation constant
  • the antibody or antigen binding portion thereof binds to human recombinant CD39 and/or membrane-bound human CD39 with an equilibrium dissociation constant (K D ) of less than 10 nM, maintains, increases or enhances a level eATP, and increases or enhances expression of one or more dendritic cell activation markers and/or increases or enhances secretion of one or more cytokines from dendritic cells.
  • K D equilibrium dissociation constant
  • the antibody or antigen binding portion thereof binds to human recombinant CD39 and/or membrane-bound human CD39 with an equilibrium dissociation constant (K D ) of less than 10 nM, maintains, increases or enhances a level eATP, and/or decreases or reduces a level of adenosine, and increases or enhances expression of one or more dendritic cell activation markers and/or increases or enhances secretion of one or more cytokines from dendritic cells.
  • the one or more dendritic cell activation markers is CD86, HLA-DR, or a combination thereof.
  • the one or more cytokines is IL-16, IL-12/IL-23p40, VEGFA, or any combination thereof.
  • the antibody or antigen binding portion of the disclosure causes antagonism of human CD39 in a tumor microenvironment of a tissue.
  • the antibody or antigen binding portion of the disclosure cross-reacts with cynomolgus CD39 and/or mouse CD39.
  • the antibody or antigen binding portion of the disclosure is selected from an IgG1, an IgG2, and IgG3, an IgG4, and IgM, and IgA1, and IgA2, and IgD, and an IgE antibody.
  • the antibody or antigen binding portion of the disclosure is an IgG1 antibody or an IgG4 antibody.
  • the antibody or antigen binding portion of the disclosure comprises a wild type IgG1 heavy chain constant region.
  • the antibody or antigen binding portion of the disclosure comprises a wild type IgG4 heavy chain constant region.
  • the antibody or antigen binding portion of the disclosure comprises an Fc domain comprising at least one mutation.
  • the antibody or antigen binding portion of the disclosure comprises a mutant IgG1 heavy chain constant region. In some aspects, the antibody or antigen binding portion of the disclosure comprises a mutant IgG4 heavy chain constant region. In some aspects, the antibody or antigen binding portion of the disclosure comprises a mutant IgG4 heavy chain constant region, wherein the mutant IgG4 heavy chain constant region comprises any one of the substitutions S228P, L235E, L235A, or a combination thereof, according to EU numbering. In some aspects, the antibody or antigen binding portion of the disclosure comprises a mutant IgG4 heavy chain constant region, wherein the mutant IgG4 heavy chain constant region comprises a S228P substitution.
  • the antibody or antigen binding portion of the disclosure comprises the heavy chain CDR3 sequence set forth in SEQ ID NO: 3. In some aspects, the antibody or antigen binding portion of the disclosure binds to substantially the same epitope as a reference antibody or antigen binding portion thereof comprising the heavy chain CDR3 sequence set forth in SEQ ID NO: 3. In some aspects, the antibody or antigen binding portion of the disclosure binds to at least one of the amino acid residues bound by a reference antibody or antigen binding portion thereof comprising the heavy chain CDR3 sequence set forth in SEQ ID NOs: 3.
  • the antibody or antigen binding portion of the disclosure wherein a mutation of the epitope bound by the antibody inhibits, reduces, or blocks binding to both the antibody and to a reference antibody or antigen binding portion thereof comprising the heavy chain CDR3 sequence set forth in SEQ ID NOs: 3.
  • the antibody or antigen binding portion of the disclosure binds to substantially the same epitope as a reference antibody or antigen binding portion thereof comprising heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 27, 28 and 29, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 38 and 39, respectively.
  • the antibody or antigen binding portion of the disclosure binds to at least one of the amino acid residues bound by a reference antibody or antigen binding portion thereof comprising heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 27, 28 and 29, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 38 and 39, respectively.
  • a mutation of the epitope bound by the antibody or antigen binding portion of the disclosure inhibits, reduces, or blocks binding to both the antibody and to a reference antibody or antigen binding portion thereof comprising heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 27, 28 and 29, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 38 and 39, respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to substantially the same epitope as a reference antibody or antigen binding portion thereof comprising heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 30, 31 and 32, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 41 and 42, respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to at least one of the amino acid residues bound by a reference antibody or antigen binding portion thereof comprising heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 30, 31 and 32, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 41 and 42, respectively.
  • a mutation of the epitope bound by the antibody or antigen binding portion thereof of the disclosure inhibits, reduces, or blocks binding to both the antibody or antigen binding portion thereof and to a reference antibody or antigen binding portion thereof comprising heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 30, 31 and 32, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 41 and 42, respectively.
  • the antibody or antigen binding portion of the disclosure binds to and antagonizes human CD39 and comprises heavy and light chain CDRs selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises heavy and light chain CDRs, wherein the heavy chain CDR1, CDR2 and CDR3 sequences are set forth in SEQ ID NOs: 27, 28 and 29, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are set forth in SEQ ID NOs: 37, 38 and 39, respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises heavy and light chain CDRs selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises heavy and light chain CDRs, wherein the heavy chain CDR1, CDR2 and CDR3 sequences are set forth in SEQ ID NOs: 30, 31 and 32, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are set forth in SEQ ID NOs: 40, 41 and 42, respectively;
  • the antibody or antigen binding portion of the disclosure binds to and antagonizes human CD39 and comprises heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 7, 59, 85 and 111; and wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 17, 69, 95 and 121.
  • the antibody or antigen binding portion thereof of the disclosure binds to an antagonizes human CD39 and comprises heavy and light chain variable regions comprising amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises heavy and light chain variable regions comprising amino acid sequences set forth in SEQ ID NO: 33 and 43, respectively.
  • the antibody or antigen binding portion of the disclosure binds to and antagonizes human CD39 and comprises heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 7, 59, 85 and 111; and wherein the light chain variable region comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 17, 69, 95 and 121.
  • the antibody or antigen binding portion of the disclosure binds to an antagonizes human CD39 and comprises heavy and light chain variable regions comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises heavy and light chain variable regions comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences set forth in SEQ ID NO: 33 and 43, respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 9, 61, 87 and 113; and wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 19, 71, 97 and 123.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 21, 73, 99, and 125; and wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 19, 71, 97 and 123.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 21, 73, 99 and 125; and wherein the light chain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 19, 71, 97 and 123.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 23, 75, 101 and 127; and wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 19, 71, 97 and 123.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 23, 75, 101 and 127; and wherein the light chain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 19, 71, 97 and 123.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 25, 77, 103 and 129; and wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 19, 71, 97 and 123.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 25, 77, 103 and 129; and wherein the light chain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 19, 71, 97 and 123.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences set forth in SEQ ID NO: 35 and 45, respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences set forth in SEQ ID NO: 35 and 45, respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences set forth in SEQ ID NO: 47 and 45, respectively; respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences set forth in SEQ ID NO: 47 and 45, respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences set forth in SEQ ID NO: 49 and 45, respectively; respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences set forth in SEQ ID NO: 49 and 45, respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences set forth in SEQ ID NO: 51 and 45, respectively; respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences set forth in SEQ ID NO: 51 and 45, respectively.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an isolated anti-CD39 antibody or antigen binding portion thereof of the disclosure, and a pharmaceutically acceptable carrier.
  • the disclosure provides a nucleic acid comprising a nucleotide sequence encoding the light chain, heavy chain, or both light and heavy chains of the isolated antibody, or antigen binding portion thereof, of the disclosure.
  • the disclosure provides an expression vector comprising the nucleic acid of the disclosure.
  • the disclosure provides a cell transformed with an expression vector of the disclosure.
  • the disclosure provides a method for producing an antibody that binds human CD39, or an antigen binding portion thereof, the method comprising maintaining a cell according to the disclosure under conditions permitting expression of the antibody or antigen binding portion thereof. In some aspects, the method further comprises obtaining the antibody or antigen binding portion thereof.
  • the disclosure provides a method of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes human CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier.
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes human CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or pharmaceutical composition inhibits or reduces the enzymatic activity of human CD39 in a tumor microenvironment, thereby treating the cancer.
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes human CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of human CD39, wherein the inhibition or reduction of the enzymatic activity of CD39 inhibits or reduces the conversion of extracellular adenosine triphosphate (eATP) or extracellular adenosine diphosphate (eADP) to extracellular adenosine monophosphate (AMP) in a tumor microenvironment, thereby treating the cancer.
  • eATP extracellular adenosine triphosphate
  • eADP extracellular adenosine diphosphate
  • AMP extracellular adenosine monophosphate
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of human CD39, wherein the inhibition or reduction of the enzymatic activity of CD39 increases or enhances a level of extracellular adenosine triphosphate (eATP) in a tumor microenvironment, thereby treating the cancer.
  • eATP extracellular adenosine triphosphate
  • the anti-CD39 antibody is monoclonal.
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an isolated anti-CD39 antibody, or antigen binding portion thereof, that binds to and antagonizes human CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of human CD39, wherein the inhibition or reduction of the enzymatic activity of CD39 decreases or reduces a level of extracellular adenosine in a tumor microenvironment, thereby treating the cancer.
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes human CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of human CD39, wherein the inhibition or reduction of the enzymatic activity of human CD39 increases or enhances a level of extracellular adenosine triphosphate (eATP) and decreases or reduces a level of extracellular adenosine in a tumor microenvironment, thereby treating the cancer.
  • eATP extracellular adenosine triphosphate
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes human CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of human CD39, wherein the inhibition or reduction of the enzymatic activity of human CD39 maintains, increases or enhances an immunostimulatory level of extracellular adenosine triphosphate (eATP) in a tumor microenvironment, thereby treating the cancer.
  • eATP extracellular adenosine triphosphate
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes human CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of human CD39, wherein the inhibition or reduction of the enzymatic activity of human CD39 increases or enhances the proliferation of a lymphocyte in the tumor microenvironment, thereby treating the cancer.
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes human CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of human CD39, wherein the inhibition or reduction of the enzymatic activity of human CD39 enhances expression of one or more dendritic cell activation markers.
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes human CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of human CD39, wherein the inhibition or reduction of the enzymatic activity of human CD39 enhances secretion of one or more cytokines from dendritic cells.
  • the disclosure provides methods of treating cancer in a subject, wherein the cancer is selected from the group consisting of: lung cancer (e.g., non-small cell lung cancer), ovarian cancer, kidney cancer, testicular cancer, pancreas cancer, breast cancer (e.g., triple-negative breast cancer), melanoma, head and neck cancer (e.g., squamous head and neck cancer), colorectal cancer, bladder cancer, endometrial cancer, prostate cancer, thyroid cancer, hepatocellular carcinoma, gastric cancer, brain cancer, lymphoma or renal cancer (e.g., renal cell carcinoma).
  • lung cancer e.g., non-small cell lung cancer
  • ovarian cancer ovarian cancer
  • kidney cancer testicular cancer
  • pancreas cancer breast cancer (e.g., triple-negative breast cancer)
  • melanoma melanoma
  • head and neck cancer e.g., squamous head and neck cancer
  • colorectal cancer bladder cancer, endometrial cancer
  • the disclosure provides use of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes human CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, in stimulating an immune response in a subject or treating cancer in a subject, optionally in combination with one or more additional therapeutic agents or procedure.
  • the disclosure provides a kit comprising an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes human CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, and instructions for use in stimulating an immune response in a subject or treating cancer in a subject, optionally with instructions for use in combination with one or more additional therapeutic agents or procedure.
  • the disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes CD39, provided by the disclosure, in combination with one or more additional therapeutic agents or procedure.
  • the second therapeutic agent or procedure is selected from the group consisting of: a chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, surgical procedure, a radiation procedure, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, a vaccine, or a cellular immunotherapy, or a combination thereof.
  • the one or more additional therapeutic agents is a PD-1 antagonist, an adenosine A2AR antagonist, a CD73 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, chimeric antigen receptor (CAR) cell therapy, or a combination thereof.
  • the one or more additional therapeutic agents is a combination of a CD73 inhibitor and an A2AR antagonist. In some embodiments, the one or more additional therapeutic agents is a combination of a PD-1 antagonist and an adenosine A2AR antagonist. In some embodiments, the one or more additional therapeutic agents is a PD-1 antagonist.
  • the PD-1 antagonist is selected from the group consisting of: PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In some embodiments, the PD-1 antagonist is selected from the group consisting of: FAZ053, Atezolizumab, Avelumab, Durvalumab, and BMS-936559.
  • the one or more additional therapeutic agents is an adenosine A2AR antagonist.
  • the adenosine A2AR antagonist is selected from the group consisting of: NIR178, CPI-444, AZD4635, Vipadenant, GBV-2034, and AB928.
  • the adenosine A2AR antagonist is CPI-444.
  • the one or more additional therapeutic agents is a CD73 inhibitor.
  • the CD73 inhibitor is selected from the group consisting of: AB421, MEDI9447, and BMS-986179.
  • the one or more additional therapeutic agents is a CTLA-4 inhibitor.
  • the CTLA-4 inhibitor is Ipilimumab or Tremelimumab.
  • the one or more additional therapeutic agents is a TIM-3 inhibitor.
  • the TIM-3 inhibitor is MGB453 or TSR-022.
  • the one or more additional therapeutic agents is a LAG-3 inhibitor.
  • the LAG-3 inhibitor is selected from the group consisting of LAG525, BMS-986016, and TSR-033.
  • the one or more additional therapeutic agents is a chimeric antigen receptor (CAR) cell therapy.
  • the CAR cell therapy is CTL019.
  • the one or more additional therapeutic agents is an anthracycline.
  • the anthracycline is selected from doxorubicin, daunorubicin, epirubicin, idarubicin, and valrubicin.
  • the anthracycline is doxorubicin.
  • the disclosure provides a method of detecting CD39 in a biological sample or in a subject, comprising (i) contacting the sample or the subject (and optionally, a reference sample or subject) with any antibody in Table 1 under conditions that allow interaction of the antibody molecule and CD39 to occur, and (ii) detecting formation of a complex between the antibody molecule and the sample or the subject (and optionally, the reference sample or subject).
  • FIG. 1A provides a graph quantifying the expression of CD86 on dendritic cells treated with anti-CD39 antibodies or control antibodies, as indicated, in the presence or absence of ATP. Expression of CD86 was determined by flow cytometry analysis.
  • FIG. 1B provides a graph quantifying the expression of CD86 and HLA-DR on dendritic cells treated with anti-CD39 antibody or control antibody, as indicated, in the presence or absence of ATP. Expression of CD86 and HLA-DR was determined by flow cytometry analysis.
  • FIG. 1C provides a graph quantifying the secretion of cytokine IL-16 by dendritic cells treated with anti-CD39 antibody or control antibody, as indicated, in the presence or absence of ATP. Quantification of IL-16 was determined by MSD.
  • FIG. 1A provides a graph quantifying the expression of CD86 on dendritic cells treated with anti-CD39 antibodies or control antibodies, as indicated, in the presence or absence of ATP. Quantification of IL-16 was determined by MSD.
  • FIG. 2 provides a graph depicting the proliferation index of CD4+ T cells treated with anti-CD39 antibodies or control antibodies, as indicated, in the presence of ATP. Cells were stained with cell trace violet and proliferation was determined by flow cytometry analysis.
  • FIG. 3D provides a graph depicting the percent inhibition of CD39 activity on the surface of primary human monocytes treated with a range of concentrations of anti-CD39 antibodies or control antibodies, as indicated. Inhibition of ATP conversion was determined by a malachite green phosphate assay.
  • FIG. 4A provides a graph depicting the extent of anti-CD39 antibody binding or control antibody binding to the surface of SK-MEL-28 cells. Cells were treated with a range of concentrations of fluorescently-labeled anti-CD39 antibodies or control antibodies, as indicated. Extent of antibody binding was determined by flow cytometry analysis.
  • FIG. 4B provides a graph depicting the extent of anti-CD39 antibody binding or control antibody binding to the surface of MOLP-8 cells. Cells were treated with a range of concentrations of fluorescently-labeled anti-CD39 antibodies or control antibodies, as indicated. Extent of antibody binding was determined by flow cytometry analysis.
  • FIG. 5A provides a graph depicting tumor volume measurements in mice implanted with SK-MEL-28 cells and treated with an isotype control antibody.
  • FIG. 5B provides a graph depicting tumor volume measurements in mice implanted with SK-MEL-28 cells and treated with an anti-CD39 antibody, as indicated.
  • FIG. 7 provides a table showing affinities (K D ) measured by ForteBio and MSD analysis for exemplary anti-CD39 antibodies to recombinant human CD39.
  • FIG. 8A provides a graph depicting tumor volume measurements over time in mice implanted with MOLP-8 human multiple myeloma cells and treated with an anti-CD39 antibody (SRF367-A) alone or in combination with the anthracycline doxorubicin (Dox), as indicated.
  • Black arrows indicate treatment with antibody.
  • Grey arrows indicate treatment with doxorubicin.
  • Mice treated with an isotype control antibody or with doxorubicin alone were used as comparators.
  • FIG. 8B provides a graph depicting mean tumor volumes of mice treated as in FIG. 8A on day 19.
  • FIG. 9 provides a graph depicting tumor volume measurements over time in mice implanted with MOLP-8 human multiple myeloma cells and treated with an anti-CD39 antibody (SRF367-A) alone or in combination with an adenosine A2A receptor (A2AR) antagonist (CPI-444) as indicated. Mice treated with an isotype control antibody or with CPI-444 alone were used as comparators.
  • an “isolated” molecule is a molecule that has been removed from its natural milieu. As such, the term “isolated” does not necessarily reflect the extent to which the molecule has been purified.
  • extracellular adenosine triphosphate or “extracellular ATP” or “eATP” refers to adenosine 5′-triphosphate that is located in a tissue or tissue sample outside of cells in the tissue or tissue sample, and functions in purinergic signaling.
  • extracellular adenosine refers to adenosine that is located in a tissue or tissue sample outside of cells in the tissue or tissue sample, and functions in purinergic signaling.
  • the steady-state cytosolic (i.e., intracellular) concentration of ATP ranges from approximately 3 mM to approximately 10 mM, whereas the amount, concentration, or level of extracellular ATP is approximately 10 nM.
  • the level of extracellular ATP is maintained as a result of the activities of extracellular enzymes (e.g., ectonucleotidases, CD39, CD79) that metabolize or convert extracellular ATP into extracellular adenosine 5′-diphosphate (eADP), extracellular adenosine 5′-monophosphate (eAMP), and extracellular adenosine (Trautmann (2009) Sci Signal 2(56):pe6).
  • extracellular enzymes e.g., ectonucleotidases, CD39, CD79
  • Extracellular nucleosides e.g., extracellular adenosine
  • nucleotides e.g., extracellular ATP
  • purinergic signaling which is involved in mediating normal physiological cellular responses including, but not limited to, stimulation (or inhibition) of cell death, cell proliferation, migration, and/or differentiation, and secretion of growth factors and/or inflammatory mediators by cells.
  • Pathophysiological processes such as tissue homeostasis, wound healing, neurodegeneration, anti-tumor immunity, inflammation and cancer are also modulated by purinergic signaling (Bours et al., (2011) Front Biosci (Schol Ed) 3:1443-1456; Khakh et al., (2006) Nature 442:527-532; Idzko et al., (2014) 509:310-317; Antonioli et al., (2013) Rev Cancer 13:842-857).
  • a low concentration of extracellular ATP surrounding resting cells in a tissue signals the presence of neighboring living cells.
  • transient increases in extracellular ATP are associated with normal physiological signaling, for example, in the nervous and vascular systems.
  • extracellular ATP levels are relatively low (approximately 10 nM)
  • elevated levels of extracellular ATP at sites of tissue damage, inflammation and in the tumor microenvironment (TME) can reach concentrations of greater than 100p1V1 (Virgilio and Adinolfi (2017) Oncogene 36:293-303). Elevated levels of extracellular ATP in a damaged tissue or the TME has been shown to result in immunomodulatory and immunostimulatory effects (Vijayan et al., (2017) Nat Rev Cancer 17:709-724).
  • the term “immunostimulatory level of ATP” refers to an amount, quantity, concentration, abundance or level of extracellular ATP that induces, stimulates or enhances an immune response.
  • conditions within the TME can ultimately result in the accumulation of extracellular adenosine as a consequence of accelerated hydrolysis of elevated levels of extracellular ATP.
  • extracellular adenosine is known to induce immunosuppressive effects, for example, in the TME of some cancers (Virgilio (2012) Cancer Res 72(21):5441-5447).
  • the enzymes primarily responsible for the conversion of extracellular ATP to extracellular adenosine in the TME are the ectonucleotidases CD39 and CD73.
  • the immunosuppressive effects of adenosine have been shown to be mediated, at least in part, by expansion of regulatory T cells (Tregs), inhibition of effector T cell responses, and expansion of myeloid derived suppressor cells (MDSCs) (Allard et al., (2016) Curr Opin Pharmacol 29:7-16; Allard et al., (2016) Immunotherapy 8:145-163)
  • Tregs regulatory T cells
  • MDSCs myeloid derived suppressor cells
  • alanine scanning refers to a technique used to determine the contribution of a specific wild-type residue to the stability or function(s) (e.g., binding affinity) of given protein or polypeptide.
  • the technique involves the substitution of an alanine residue for a wild-type residue in a polypeptide, followed by an assessment of the stability or function(s) (e.g., binding affinity) of the alanine-substituted derivative or mutant polypeptide and comparison to the wild-type polypeptide.
  • Techniques to substitute alanine for a wild-type residue in a polypeptide are known in the art.
  • ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, e.g., cancer, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.
  • 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 refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a 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 refers 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.
  • Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes.
  • amino acid substitution refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (an amino acid sequence of a starting polypeptide) with a second, different “replacement” amino acid residue.
  • amino acid insertion refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, larger “peptide insertions,” can also be made, e.g. insertion of about three to about five or even up to about ten, fifteen, or twenty amino acid residues. The inserted residue(s) may be naturally occurring or non-naturally occurring as disclosed above.
  • amino acid deletion refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.
  • the term “amount” or “level” is used in the broadest sense and refers to a quantity, concentration or abundance of a substance (e.g., a metabolite, a small molecule, a protein, an mRNA, a marker).
  • a substance e.g., a metabolite, a small molecule, a protein, an mRNA, a marker.
  • a metabolite or small molecule e.g. adenosine triphosphate (ATP)
  • ATP adenosine triphosphate
  • the elevated level of a substance (e.g., ATP) in a sample refers to an increase in the amount of the substance of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., malachite green assay).
  • a substance e.g., ATP
  • Reduced levels refers to a decrease in the quantity, concentration or abundance of a substance (e.g., ATP) in an individual relative to a control, such as from an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control. In some embodiments, a reduced level is little or no detectable quantity, concentration or abundance.
  • a substance e.g., ATP
  • the reduced level of a substance (e.g., ATP) in a sample refers to a decrease in the amount of the substance of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., malachite green assay).
  • a substance e.g., ATP
  • the terms “level of expression” or “expression level” in general are used interchangeably and generally refer to a detectable amount of a protein, mRNA, or marker in a biological sample.
  • a detectable amount or detectable level of a protein, mRNA or a marker is associated with a likelihood of a response to an agent, such as those described herein.
  • “Expression” generally refers to the process by which information contained within a gene is converted into the structures (e.g., a protein marker, such as CD86) present and operating in the cell.
  • expression may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide) shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis.
  • “Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs).
  • “Elevated expression,” “elevated expression levels,” or “elevated levels” refers to an increased expression or increased levels of a substance within a sample relative to a control sample, such as an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control.
  • the elevated expression of a substance refers to an increase in the amount of the substance of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., FACS).
  • a substance e.g., a protein marker, such as CD86
  • Reduced expression refers to a decrease expression or decreased levels of a substance (e.g., a protein marker) in an individual relative to a control, such as an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control. In some embodiments, reduced expression is little or no expression.
  • a substance e.g., a protein marker
  • reduced expression is little or no expression.
  • the reduced expression of a substance refers to a decrease in the amount of the substance of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., FACS).
  • a substance e.g., a protein marker
  • an antibody that “antagonizes human CD39” is one that partially or fully blocks, inhibits, neutralizes, eliminates, or removes the enzymatic activity of the CD39 enzyme. In some embodiments, antagonizing activity is observed in a dose-dependent manner.
  • the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% lower than the signal measured with a negative control under comparable conditions. Also disclosed herein, are methods of identifying antagonists suitable for use in the methods of the disclosure.
  • these methods include, identifying antibodies that bind to an antagonize human CD39, such as, for example, binding assays such as enzyme-linked immuno-absorbent assay (ELISA), Forte Bio ⁇ systems, and radioimmunoassay (MA). Assays such as these can be used to determine the ability of an antibody to bind the polypeptide of interest (e.g., CD39) as well as the ability of the antibody to antagonize the polypeptide (e.g., CD39). Efficacy of an antagonist antibody can also be determined using functional assays, such as the ability of an antagonist to inhibit the function of the polypeptide or an agonist.
  • binding assays such as enzyme-linked immuno-absorbent assay (ELISA), Forte Bio ⁇ systems, and radioimmunoassay (MA).
  • Assays such as these can be used to determine the ability of an antibody to bind the polypeptide of interest (e.g., CD39) as well as the ability of the antibody to antagonize
  • a functional assay may comprise contacting a polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide.
  • the potency of an antagonist may be defined by its IC 50 value (concentration required to inhibit 50% of the agonist response). The lower the IC 50 value the greater the potency of the antagonist and the lower the concentration that is required to inhibit the maximum biological response.
  • anti-CD39 antagonist antibody refers to an antibody described herein, for example, in Table 1.
  • an anti-CD39 antibody binds to CD39 (e.g., human CD39) and antagonizes a CD39 biological activity and/or downstream pathway(s) mediated by CD39 signaling or other CD39-mediated function, e.g., enzymatic activity.
  • An anti-CD39 antagonist antibody encompasses antibodies that block, antagonize, suppress, inhibit, eliminate, or reduce CD39 biological activity (e.g., ligand binding, enzymatic activity), including downstream pathways mediated by CD39 signaling or function, such as receptor binding and/or elicitation of a cellular response to CD39 or its metabolites.
  • an anti-CD39 antibody specifically binds to CD39.
  • an anti-CD39 antagonist antibody binds to CD39 and prevents or inhibits CD39 binding to its cognate or normal ligand. In some embodiments, an anti-CD39 antagonist antibody binds to CD39 and inhibits or reduces the enzymatic conversion of adenosine triphosphate (ATP) to adenosine monophosphate (AMP). In some embodiments, an anti-CD39 antagonist antibody binds to CD39 and inhibits or reduces the enzymatic conversion of adenosine diphosphate (ADP) to adenosine monophosphate (AMP). In some embodiments, an anti-CD39 antagonist antibody binds to CD39 and maintains or increases an immunostimulatory amount of ATP.
  • ATP adenosine triphosphate
  • AMP adenosine monophosphate
  • an anti-CD39 antagonist antibody binds to CD39 and maintains or increases an immunostimulatory amount of ATP.
  • an anti-CD39 antagonist antibody binds to CD39 reduces or decreases adenosine levels. In some embodiments, an anti-CD39 antagonist antibody binds to CD39 and stimulates or enhances an anti-tumor response. In some embodiments, the anti-CD39 antagonist antibody binds to CD39 with an affinity of about 5 nM-20 nM. In some embodiment, the anti-CD39 antagonist antibody binds to CD39 and comprises a wild type or mutant IgG1 or a wild type or mutant IgG4 heavy chain constant region. Examples of anti-CD39 antagonist antibodies are provided herein.
  • antibody refers to a whole antibody comprising two light chain polypeptides and two heavy chain polypeptides. Whole antibodies include different antibody isotypes including IgM, IgG, IgA, IgD, and IgE antibodies.
  • antibody includes a polyclonal antibody, a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a primatized antibody, a deimmunized antibody, and a fully human antibody.
  • the antibody can be made in or derived from any of a variety of species, e.g., mammals such as humans, non-human primates (e.g., orangutan, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice.
  • mammals such as humans, non-human primates (e.g., orangutan, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice.
  • the antibody can be an isolated, purified or a recombinant antibody.
  • antibody fragment refers to a fragment of an antibody that retains the ability to bind to a target antigen (e.g., CD39) and inhibit the activity of the target antigen, but is less than full length.
  • target antigen e.g., CD39
  • fragments include, e.g., a single chain antibody, a single chain Fv fragment (scFv), an Fd fragment, an Fab fragment, an Fab′ fragment, or an F(ab′)2 fragment.
  • scFv fragment is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived.
  • intrabodies, minibodies, triabodies, and diabodies are also included in the definition of antibody and are compatible for use in the methods described herein. See, e.g., Todorovska et al., (2001) J. Immunol. Methods 248(1):47-66; Hudson and Kortt, (1999) J. Immunol. Methods 231(1):177-189; Poljak, (1994) Structure 2(12):1121-1123; Rondon and Marasco, (1997) Annu. Rev. Microbiol. 51:257-283, the disclosures of each of which are incorporated herein by reference in their entirety.
  • antibody fragment also includes, e.g., single domain antibodies such as camelized single domain antibodies. See, e.g., Muyldermans et al., (2001) Trends Biochem. Sci. 26:230-235; Nuttall et al., (2000) Curr. Pharm. Biotech. 1:253-263; Reichmann et al., (1999) J. Immunol. Meth. 231:25-38; PCT application publication nos. WO 94/04678 and WO 94/25591; and U.S. Pat. No. 6,005,079, all of which are incorporated herein by reference in their entireties.
  • the disclosure provides single domain antibodies comprising two VH domains with modifications such that single domain antibodies are formed.
  • an antigen-binding fragment includes the variable region of a heavy chain polypeptide and the variable region of a light chain polypeptide. In some embodiments, an antigen-binding fragment described herein comprises the CDRs of the light chain and heavy chain polypeptide of an antibody.
  • Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.
  • recombinant host cells e.g. E. coli or phage
  • APC antigen presenting cell
  • T cells recognize this complex using T cell receptor (TCR).
  • APCs include, but are not limited to, B cells, dendritic cells (DCs), peripheral blood mononuclear cells (PBMC), monocytes (such as THP-1), B lymphoblastoid cells (such as C1R.A2, 1518 B-LCL) and monocyte-derived dendritic cells (DCs).
  • DCs dendritic cells
  • PBMC peripheral blood mononuclear cells
  • monocytes such as THP-1
  • B lymphoblastoid cells such as C1R.A2, 1518 B-LCL
  • DCs monocyte-derived dendritic cells
  • antigen presentation refers to the process by which APCs capture antigens and enables their recognition by T cells, e.g., as a component of an MHC-I and/or MHC-II conjugate.
  • apoptosis refers to the process of programmed cell death that occurs in multicellular organisms (e.g. humans).
  • the highly-regulated biochemical and molecular events that result in apoptosis can lead to observable and characteristic morphological changes to a cell, including membrane blebbing, cell volume shrinkage, chromosomal DNA condensation and fragmentation, and mRNA decay.
  • a common method to identify cells, including T cells, undergoing apoptosis is to expose cells to a fluorophore-conjugated protein (Annexin V). Annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine on the outer leaflet of the plasma membrane, which is an early indicator that the cell is undergoing the process of apoptosis.
  • B cell refers to a type of white blood cell of the lymphocyte subtype.
  • B cells function in the humoral immunity component of the adaptive immune system by secreting antibodies.
  • B cells also present antigen and secrete cytokines.
  • B cells unlike the other two classes of lymphocytes, T cells and natural killer cells, express B cell receptors (BCRs) on their cell membrane. BCRs allow the B cell to bind to a specific antigen, against which it will initiate an antibody response.
  • the term “binds to immobilized CD39,” refers to the ability of an antibody of the disclosure to bind to CD39, for example, expressed on the surface of a cell or which is attached to a solid support.
  • bispecific or “bifunctional antibody” refers to an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai & Lachmann, (1990) Clin. Exp. Immunol. 79:315-321; Kostelny et al., (1992) J. Immunol. 148:1547-1553.
  • bispecific antibodies are based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chain/light-chain pairs have different specificities (Milstein and Cuello, (1983) Nature 305:537-539).
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion of the heavy chain variable region is preferably with an immunoglobulin heavy-chain constant domain, including at least part of the hinge, CH2, and CH3 regions.
  • Bispecific antibodies also include cross-linked or heteroconjugate antibodies. Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • bispecific antibodies have been produced using leucine zippers. See, e.g., Kostelny et al. (1992) J Immunol 148(5):1547-1553.
  • the leucine zipper peptides from the Fos and Jun proteins may be linked to the Fab′ portions of two different antibodies by gene fusion.
  • the antibody homodimers may be reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • scFv single-chain Fv
  • the antibodies can be “linear antibodies” as described in, e.g., Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
  • Antibodies with more than two valencies are contemplated and described in, e.g., Tutt et al. (1991) J Immunol 147:60.
  • the disclosure also embraces variant forms of multi-specific antibodies such as the dual variable domain immunoglobulin (DVD-Ig) molecules described in Wu et al. (2007) Nat Biotechnol 25(11): 1290-1297.
  • DVD-Ig molecules are designed such that two different light chain variable domains (VL) from two different parent antibodies are linked in tandem directly or via a short linker by recombinant DNA techniques, followed by the light chain constant domain.
  • the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by the constant domain CH1 and Fc region.
  • Methods for making DVD-Ig molecules from two parent antibodies are further described in, e.g., PCT Publication Nos. WO 08/024188 and WO 07/024715.
  • the bispecific antibody is a Fabs-in-Tandem immunoglobulin, in which the light chain variable region with a second specificity is fused to the heavy chain variable region of a whole antibody.
  • Fabs-in-Tandem immunoglobulin in which the light chain variable region with a second specificity is fused to the heavy chain variable region of a whole antibody.
  • cancer antigen refers to (i) tumor-specific antigens, (ii) tumor-associated antigens, (iii) cells that express tumor-specific antigens, (iv) cells that express tumor-associated antigens, (v) embryonic antigens on tumors, (vi) autologous tumor cells, (vii) tumor-specific membrane antigens, (viii) tumor-associated membrane antigens, (ix) growth factor receptors, (x) growth factor ligands, and (xi) any other type of antigen or antigen-presenting cell or material that is associated with a cancer.
  • cancer-specific immune response refers to the immune response induced by the presence of tumors, cancer cells, or cancer antigens.
  • the response includes the proliferation of cancer antigen specific lymphocytes.
  • the response includes expression and upregulation of antibodies and T-cell receptors and the formation and release of lymphokines, chemokines, and cytokines. Both innate and acquired immune systems interact to initiate antigenic responses against the tumors, cancer cells, or cancer antigens.
  • the cancer-specific immune response is a T cell response.
  • carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
  • the anti-CD39 antibodies described herein can be used to treat patients who have, who are suspected of having, or who may be at high risk for developing any type of cancer, including renal carcinoma or melanoma, or any viral disease.
  • Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • carcinosarcomas which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • CD39 refers to the ectonucleoside triphosphate diphospholydrolase 1 polypeptide encoded in humans by the ENTPD1 gene.
  • Other names for CD39 include ENTPD1, ATPDase, NTPDase-1, and SPG64.
  • CD39 catalyzes the hydrolysis of ⁇ - and ⁇ -phosphate residues of extracellular nucleoside triphosphates (NTPs; e.g., adenosine triphosphate or ATP) and nucleoside diphosphates (NDPs; e.g., adenosine diphosphate or ADP), converting these molecules to the nucleoside monophosphate (NMP; e.g., adenosine monophosphate or AMP) derivative.
  • NTPs nucleoside triphosphates
  • NDPs nucleoside diphosphates
  • NMP nucleoside monophosphate
  • AMP nucleoside monophosphate
  • An exemplary amino acid sequence of CD39 is set forth in SEQ ID NO: 138, and also at NCBI Reference Sequence: NP 001767.3.
  • the present disclosure provides antibodies that bind and antagonize human CD39.
  • CD86 (B70/B7-2) refers to a cell surface protein of about 75 kD, which is a second ligand for CD28 and CTLA-4 and plays an important role in co-stimulation of T cells in early immune response (Azuma M. et al., 1993, Nature 366: 76; Nozawa Y. et al., 1993, J. Pathology 169: 309; Engle, P. et al. 1994, Blood 84: 1402; Engel, P. et al., CD86 Workshop Report. In: Leukocyte Typing V. Schlossman, S. F. et al. eds., 1994, Oxford University Press; Yang, X. F.
  • CDR means a complementarity-determining region.
  • hypervariable region or “HVR” is sometimes used in place of “CDR”, and both terms refer to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and/or form structurally defined loops (“hypervariable loops”) and/or contain the antigen-contacting residues (“antigen contacts”).
  • CDRs complementarity determining regions
  • antigen contacts antigen-contacting residues
  • antibodies comprise six HVRs/CDRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3).
  • chemotherapeutic agent refers to a chemical or pharmacological agent that is known to be of use in the treatment of cancer.
  • the term connotes those pharmacological agents that are generally cytotoxic, non-specific intracellular poisons, especially those that function to inhibit the process of cell division known as mitosis, and excludes pharmacological agents that more selectively target cellular components known to cause or contribute to the formation, development and/or maintenance of cancer.
  • Chemotherapeutic agents can induce one or more cell death modalities including immunogenic cell death which can lead to ATP release.
  • an assay e.g., a competitive binding assay; a cross-blocking assay
  • a test antigen-binding protein e.g., a test antibody
  • inhibits e.g., reduces or blocks
  • a reference antigen-binding protein e.g.,
  • a polypeptide or amino acid sequence “derived from” a designated polypeptide or protein refers to the origin of the polypeptide.
  • the polypeptide or amino acid sequence which is derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the sequence.
  • Polypeptides derived from another peptide may have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions.
  • a polypeptide can comprise an amino acid sequence which is not naturally occurring. Such variants necessarily have less than 100% sequence identity or similarity with the starting molecule. In certain embodiments, the variant will have an amino acid sequence from about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, more preferably from about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% to less than 100%, e.g., over the length of the variant molecule.
  • a polypeptide consists of, consists essentially of, or comprises an amino acid sequence selected from a sequence set forth in Table 1.
  • a polypeptide includes an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from a sequence set forth in Table 1.
  • a polypeptide includes a contiguous amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous amino acid sequence selected from a sequence set forth in Table 1.
  • the antibodies of the disclosure are encoded by a nucleotide sequence.
  • Nucleotide sequences of the invention can be useful for a number of applications, including cloning, gene therapy, protein expression and purification, mutation introduction, DNA vaccination of a host in need thereof, antibody generation for, e.g., passive immunization, PCR, primer and probe generation, and the like.
  • the nucleotide sequence of the invention comprises, consists of, or consists essentially of, a nucleotide sequence selected from a sequence set forth in Table 1.
  • a nucleotide sequence includes a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence selected from a sequence set forth in Table 1.
  • a nucleotide sequence includes a contiguous nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous nucleotide sequence selected from a sequence set forth in Table 1.
  • a nucleotide sequence includes a nucleotide sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers) contiguous nucleotides of a nucleotide sequence selected from a sequence set forth in Table 1.
  • antibodies suitable for use in the methods disclosed herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.
  • nucleotide or amino acid substitutions leading to conservative substitutions or changes at “non-essential” amino acid residues may be made.
  • Mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • the antibodies suitable for use in the methods disclosed herein may comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid
  • a nonessential amino acid residue in a binding polypeptide is preferably replaced with another amino acid residue from the same side chain family.
  • a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
  • mutations may be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into binding polypeptides of the invention and screened for their ability to bind to the desired target.
  • antigen cross-presentation refers to presentation of exogenous protein antigens to T cells via MHC class I and class II molecules on APCs.
  • cross-reacts refers to the ability of an antibody of the disclosure to bind to CD39 from a different species.
  • an antibody of the present disclosure which binds human CD39 may also bind another species of CD39.
  • cross-reactivity is measured by detecting a specific reactivity with purified antigen in binding assays (e.g., SPR, ELISA) or binding to, or otherwise functionally interacting with, cells physiologically expressing CD39.
  • Methods for determining cross-reactivity include standard binding assays as described herein, for example, by Biacore surface plasmon resonance (SPR) analysis using a Biacore 2000 SPR instrument (Biacore AB, Uppsala, Sweden), or flow cytometric techniques.
  • SPR surface plasmon resonance
  • cytotoxic T lymphocyte (CTL) response refers to an immune response induced by cytotoxic T cells. CTL responses are mediated primarily by CD8 + T cells.
  • DC dendritic cell
  • BM bone marrow
  • MHC major histocompatibility complex
  • DCs are heterogeneous, e.g. myeloid and plasmacytoid DCs; although all DCs are capable of antigen uptake, processing and presentation to naive T cells, the DC subtypes have distinct markers and differ in location, migratory pathways, detailed immunological function and dependence on infections or inflammatory stimuli for their generation.
  • Th1 polarized T-helper 1
  • Th2 polarized T-helper 1
  • dendritic cell activation refers to the transition from immature to mature dendritic cell; and the activated dendritic cells encompass mature dendritic cells and dendritic cells in the process of the transition, wherein the expression of CD80 and CD86 that induce costimulatory signals are elevated by the activating stimuli.
  • Mature human dendritic cells are cells that are positive for the expression of CD40, CD80, CD86, and HLA-class II (e.g., HLA-DR).
  • An immature dendritic cell can be distinguished from a mature dendritic cell, for example, based on markers selected from the group consisting of CD80 and CD86.
  • An immature dendritic cell is weakly positive and preferably negative for these markers, while a mature dendritic cell is positive. Discrimination of mature dendritic cells is routinely performed by those skilled in the art, and the respective markers described above and methods for measuring their expression are also well known to those skilled in the art.
  • EC 50 refers to the concentration of an antibody or an antigen-binding portion thereof, which induces a response, either in an in vitro or an in vivo assay, which is 50% of the maximal response, i.e., halfway between the maximal response and the baseline.
  • the term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect.
  • therapeutically effective dose is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts effective for this use will depend upon the severity of the disorder being treated and the general state of the patient's own immune system.
  • epitope or “antigenic determinant” refers to a site on an antigen to which an immunoglobulin or antibody specifically binds.
  • epitope mapping refers to a process or method of identifying the binding site, or epitope, of an antibody, or antigen binding fragment thereof, on its target protein antigen. Epitope mapping methods and techniques are provided herein. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation.
  • Methods for determining what epitopes are bound by a given antibody i.e., epitope mapping
  • epitope mapping include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from CD39 are tested for reactivity with the given anti-CD39 antibody.
  • Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology , Vol. 66, G. E. Morris, Ed. (1996)).
  • antibodies that bind to an epitope on CD39 which comprises all, or a portion of an epitope recognized by the particular antibodies described herein (e.g., the same or an overlapping region or a region between or spanning the region).
  • antibodies that bind the same epitope and/or antibodies that compete for binding to human CD39 with the antibodies described herein can be identified using routine techniques. Such techniques include, for example, an immunoassay, which shows the ability of one antibody to block the binding of another antibody to a target antigen, i.e., a competitive binding assay. Competitive binding is determined in an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen, such as CD39.
  • solid phase direct or indirect radioimmunoassay MA
  • solid phase direct or indirect enzyme immunoassay EIA
  • sandwich competition assay see Stahli et al., Methods in Enzymology 9:242 (1983)
  • solid phase direct biotin-avidin EIA see Kirkland et al., J. Immunol. 137:3614 (1986)
  • solid phase direct labeled assay solid phase direct labeled sandwich assay
  • solid phase direct label MA using I-125 label see Morel et al., Mol. Immunol.
  • Such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin.
  • Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin.
  • the test immunoglobulin is present in excess.
  • a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.
  • epitope mapping methods such as, x-ray analyses of crystals of antigen:antibody complexes which provides atomic resolution of the epitope and mass spectrometry combined with hydrogen/deuterium (H/D) exchange which studies the conformation and dynamics of antigen:antibody interactions.
  • H/D hydrogen/deuterium
  • Other methods monitor the binding of the antibody to antigen fragments or mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component.
  • computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library.
  • computational algorithms have also been developed which have been shown to map conformational discontinuous epitopes.
  • Fc-mediated effector functions or “Fc effector functions” refer to the biological activities of an antibody other than the antibody's primary function and purpose.
  • the effector functions of a therapeutic agnostic antibody are the biological activities other than the activation of the target protein or pathway.
  • antibody effect functions include C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); lack of activation of platelets that express Fc receptor; and B cell activation.
  • Many effector functions begin with Fc binding to an Fc ⁇ receptor.
  • the tumor antigen-targeting antibody has effector function, e.g., ADCC activity.
  • a tumor antigen-targeting antibody described herein comprises a variant constant region having increased effector function (e.g. increased ability to mediate ADCC) relative to the unmodified form of the constant region.
  • Fc receptor refers to a polypeptide found on the surface of immune effector cells, which is bound by the Fc region of an antibody.
  • the Fc receptor is an Fc ⁇ receptor.
  • Fc ⁇ RI CD64
  • Fc ⁇ RII CD32
  • Fc ⁇ RIII CD16
  • IgG isotypes IgG1, IgG2, IgG3 and IgG4
  • bind and activate Fc receptors Fc ⁇ RI, Fc ⁇ RIIA and Fc ⁇ RIIIA Fc ⁇ RIIB is an inhibitory receptor, and therefore antibody binding to this receptor does not activate complement and cellular responses.
  • Fc ⁇ RI is a high affinity receptor that binds to IgG in monomeric form
  • Fc ⁇ RIIA and Fc ⁇ RIIA are low affinity receptors that bind IgG only in multimeric form and have slightly lower affinity.
  • the binding of an antibody to an Fc receptor and/or C1q is governed by specific residues or domains within the Fc regions. Binding also depends on residues located within the hinge region and within the CH2 portion of the antibody.
  • the agonistic and/or therapeutic activity of the antibodies described herein is dependent on binding of the Fc region to the Fc receptor (e.g., Fc ⁇ R).
  • the agonistic and/or therapeutic activity of the antibodies described herein is enhanced by binding of the Fc region to the Fc receptor (e.g., Fc ⁇ R).
  • human antibody includes antibodies having variable and constant regions (if present) of human germline immunoglobulin sequences.
  • Human antibodies of the disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) (See, e.g., Lonberg et al., (1994) Nature 368(6474): 856-859); Lonberg, (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg & Huszar, (1995) Intern. Rev. Immunol. 13:65-93, and Harding & Lonberg, (1995) Ann. N.Y. Acad.
  • human antibody does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e. humanized antibodies).
  • a “heterologous antibody” is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal.
  • inducing an immune response and “enhancing an immune response” are used interchangeably and refer the stimulation of an immune response (i.e., either passive or adaptive) to a particular antigen.
  • induce as used with respect to inducing CDC or ADCC refer to the stimulation of particular direct cell killing mechanisms.
  • the term “immunogenic cell death” refers to a cell death modality wherein contact of a tumor cell with a chemical, biological, or pharmacological agent is associated with the activation of one or more signaling pathways that induces the pre-mortem expression and emission of damaged-associated molecular pattern (DAMPs) molecules (e.g., adenosine triphosphate, ATP) from the tumor cell, resulting in the increase of immunogenicity of the tumor cell and the death of the tumor cell in an immunogenic manner (e.g., by phagocytosis).
  • DAMPs damaged-associated molecular pattern
  • ICD is a form of cell death which induces endoplasmic reticulum (ER) stress and involves changes in the composition of the cell surface as well as the release of DAMPs that elevate the immunogenic potential of dying cells.
  • DAMPs include calreticulin, heat-shock proteins, secreted amphoterin (HMGB1) and ATP.
  • HMGB1 secreted amphoterin
  • ATP adenosine triphosphate
  • HSP70 and HSP90 are also translocated to the plasma membrane where they interact with antigen-presenting cell (APCs) and facilitate cross-presentation of tumor antigens with MHC class I molecules, resulting in a CD8+ T cell response.
  • APCs antigen-presenting cell
  • HMGB1 is released into the extracellular space where is binds Toll-like receptors on APCs and facilitates presentation of tumor antigens by dendritic cells (professional APCs) to T cells. ATP secretion recruit's monocytes to the site of cell death. Changes associated with ICD of tumor or cancer cells can induce an effective anti-tumor immune response through activation, maturation and enhanced antigen presentation of dendritic cells and activation of a specific T cell response in a subject.
  • immunogenic cell death-inducing agent refers to a chemical, biological, or pharmacological agent that induces an immunogenic cell death process, pathway, or modality.
  • the term “in combination,” as used in connection with a therapeutic treatment is understood to mean that two (or more) different treatments, for example, two (or more) therapeutic agents, are delivered to the subject during the course of the subject's affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time.
  • the delivery of one treatment ends before the delivery of the other treatment begins (e.g., the first treatment is prior to a second or third (a subsequent) treatment).
  • the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration.
  • the reduction of a symptom, or other parameter related to the disorder upon delivery of a combination therapy is greater than what would be observed with one treatment delivered in the absence of the other.
  • the terms “inhibits” or “blocks” e.g., referring to inhibition/blocking of human CD39-mediated conversion of a nucleoside triphosphate (e.g. adenosine triphosphate, ATP) or a nucleoside diphosphate (e.g. adenosine diphosphate, ADP) into a nucleoside monophosphate (e.g. adenosine monophosphate, AMP)
  • a nucleoside triphosphate e.g. adenosine triphosphate, ATP
  • a nucleoside diphosphate e.g. adenosine diphosphate, ADP
  • AMP nucleoside monophosphate
  • Inhibition and blocking are also intended to include any measurable decrease in the binding affinity of CD39 when in contact with an anti-CD39 antibody as compared to CD39 not in contact with an anti-CD39 antibody, e.g., inhibits binding of CD39 by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the anti-CD39 antibody inhibits the conversion of nucleoside triphosphate (e.g., ATP) by at least about 70%.
  • the anti-CD39 antibody inhibits the conversion of nucleotide triphosphate (e.g. ATP) by at least 80%.
  • the term “inhibits growth” is intended to include any measurable decrease in the growth of a cell, e.g., the inhibition of growth of a cell by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
  • a subject “in need of prevention,” “in need of treatment,” or “in need thereof,” refers to one, who by the judgment of an appropriate medical practitioner (e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment (such as treatment with a composition comprising an anti-CD39 antibody).
  • an appropriate medical practitioner e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals
  • in vivo refers to processes that occur in a living organism.
  • isolated antibody is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that binds to human CD39 is substantially free of antibodies that bind antigens other than CD39).
  • An isolated antibody that binds to an epitope may, however, have cross-reactivity to other CD39 proteins from different species. However, the antibody continues to display specific binding to human CD39 in a specific binding assay as described herein.
  • an isolated antibody is typically substantially free of other cellular material and/or chemicals.
  • a combination of “isolated” antibodies having different CD39 specificities is combined in a well-defined composition.
  • isolated nucleic acid molecule refers to nucleic acids encoding antibodies or antibody portions (e.g., VH, VL, CDR3) that bind to CD39, is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than CD39, which other sequences may naturally flank the nucleic acid in human genomic DNA.
  • a sequence selected from a sequence set forth in Table 1 corresponds to the nucleotide sequences comprising the heavy chain (VH) and light chain (VL) variable regions of anti-CD39 antibody antibodies described herein.
  • isotype refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.
  • a human antibody of the disclosure is of the IgG1 isotype.
  • a human antibody of the disclosure is of the IgG2 isotype.
  • a human antibody of the disclosure is of the IgG3 isotype.
  • a human antibody of the disclosure is of the IgG4 isotype.
  • the isotype is wildtype. In some embodiments, the isotype is mutant.
  • an antibody having an “A” designation has an IgG4 isotype comprising a S228P mutation, according to EU numbering.
  • an antibody having an “B” designation has an IgG4 isotype comprising a S228P and L235E mutation, according to EU numbering.
  • an antibody having an “C” designation has “C” designation has a wild type IgG1 isotype.
  • an antibody having an “D” designation has a wild type IgG4 isotype. See, e.g., Vidarsson et al. Front Immunol. (2014), 5: 520, incorporated by reference herein in its entirety, and at page 6, col.
  • isotype switching refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.
  • K D refers to the equilibrium dissociation constant of a binding reaction between an antibody and an antigen.
  • the value of K D is a numeric representation of the ratio of the antibody off-rate constant (kd) to the antibody on-rate constant (ka).
  • the value of K D is inversely related to the binding affinity of an antibody to an antigen. The smaller the K D value the greater the affinity of the antibody for its antigen. Affinity is the strength of binding of a single molecule to its ligand and is typically measured and reported by the equilibrium dissociation constant (K D ), which is used to evaluate and rank order strengths of bimolecular interactions.
  • kd or “k d ” (alternatively “koff” or “k off ”) is intended to refer to the off-rate constant for the dissociation of an antibody from an antibody/antigen complex.
  • the value of kd is a numeric representation of the fraction of complexes that decay or dissociate per second, and is expressed in units sec ⁇ 1 .
  • ka or “ka” (alternatively “kon” or “k on ”) is intended to refer to the on-rate constant for the association of an antibody with an antigen.
  • the value of ka is a numeric representation of the number of antibody/antigen complexes formed per second in a 1 molar (1M) solution of antibody and antigen, and is expressed in units M ⁇ 1 sec ⁇ 1 .
  • leukocyte refers to a type of white blood cell involved in defending the body against infective organisms and foreign substances. Leukocytes are produced in the bone marrow. There are 5 main types of white blood cells, subdivided between 2 main groups: polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) and mononuclear leukocytes (monocytes and lymphocytes).
  • lymphocytes refers to a type of leukocyte or white blood cell that is involved in the immune defenses of the body. There are two main types of lymphocytes: B-cells and T-cells.
  • the terms “linked,” “fused”, or “fusion”, are used interchangeably. These terms refer to the joining together of two more elements or components or domains, by whatever means including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art.
  • local administration refers to delivery that does not rely upon transport of the composition or agent to its intended target tissue or site via the vascular system.
  • the composition may be delivered by injection or implantation of the composition or agent or by injection or implantation of a device containing the composition or agent.
  • the composition or agent, or one or more components thereof may diffuse to the intended target tissue or site.
  • MHC molecules refers to two types of molecules, MHC class I and MHC class II.
  • MHC class I molecules present antigen to specific CD8+ T cells and MHC class II molecules present antigen to specific CD4+ T cells.
  • Antigens delivered exogenously to APCs are processed primarily for association with MHC class II.
  • antigens delivered endogenously to APCs are processed primarily for association with MHC class I.
  • human monoclonal antibody refers to an antibody which displays a single binding specificity and affinity for a particular epitope.
  • human monoclonal antibody refers to an antibody which displays a single binding specificity and which has variable and optional constant regions derived from human germline immunoglobulin sequences.
  • human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • monocyte refers to a type of leukocyte and can differentiate into macrophages and dendritic cells to effect an immune response.
  • NK cell refers to a type of cytotoxic lymphocyte. These are large, usually granular, non-T, non-B lymphocytes, which kill certain tumor cells and play an important in innate immunity to viruses and other intracellular pathogens, as well as in antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • naturally-occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
  • nonswitched isotype refers to the isotypic class of heavy chain that is produced when no isotype switching has taken place; the CH gene encoding the nonswitched isotype is typically the first CH gene immediately downstream from the functionally rearranged VDJ gene. Isotype switching has been classified as classical or non-classical isotype switching. Classical isotype switching occurs by recombination events which involve at least one switch sequence region in the transgene. Non-classical isotype switching may occur by, for example, homologous recombination between human ⁇ and human ⁇ ( ⁇ -associated deletion). Alternative non-classical switching mechanisms, such as intertransgene and/or interchromosomal recombination, among others, may occur and effectuate isotype switching.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., Biol. Chem. 260:2605-2608, 1985; and Cassol et al, 1992; Rossolini et al, Mol. Cell. Probes 8:91-98, 1994).
  • arginine and leucine modifications at the second base can also be conservative.
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • Polynucleotides used herein can be composed of any polyribonucleotide or polydeoxribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • a polynucleotide can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
  • parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrasternal injection and infusion.
  • patient is used interchangeably with “subject” and “individual” and includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
  • PD-1 antagonist refers to any chemical compound or biological molecule that inhibits the PD-1 signaling pathway or that otherwise inhibits PD-1 function in a cell (e.g. an immune cell).
  • a PD-1 antagonist blocks binding of PD-L1 to PD-1 and/or PD-L2 to PD-1.
  • the PD-1 antagonist binds PD-1.
  • the PD-1 antagonist binds PD-L1.
  • percent identity in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection.
  • sequence comparison algorithms e.g., BLASTP and BLASTN or other algorithms available to persons of skill
  • the “percent identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).
  • BLAST algorithm One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.
  • “pharmaceutically acceptable” refers 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, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • a “pharmaceutically acceptable carrier” refers to, and includes, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the compositions can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see, e.g., Berge et al. (1977) J Pharm Sci 66:1-19).
  • polypeptide As used herein, the terms “polypeptide,” “peptide”, and “protein” are used interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • the term “preventing” when used in relation to a condition refers to administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.
  • the term “purified” or “isolated” as applied to any of the proteins (antibodies or fragments) described herein refers to a polypeptide that has been separated or purified from components (e.g., proteins or other naturally-occurring biological or organic molecules) which naturally accompany it, e.g., other proteins, lipids, and nucleic acid in a prokaryote expressing the proteins.
  • a polypeptide is purified when it constitutes at least 60 (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99) %, by weight, of the total protein in a sample.
  • PD-1 Programmmed Cell Death Protein 1
  • PD-1 refers to the Programmed Cell Death Protein 1 polypeptide, an immune-inhibitory receptor belonging to the CD28 family and is encoded by the PDCD1 gene in humans.
  • Alternative names or synonyms for PD-1 include: PDCD1, PD1, CD279 and SLEB2.
  • PD-1 is expressed predominantly on previously activated T cells, B cells, and myeloid cells in vivo, and binds to two ligands, PD-L1 and PD-L2.
  • the term “PD-1” as used herein includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank Accession No. AAC51773.
  • the term “Programmed Death Ligand-1” or “PD-L1” is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1.
  • Alternative names and synonyms for PD-L1 include: PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H.
  • the term “PD-L1” as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank Accession No. Q9NZQ7.
  • PD-1 is known as an immune-inhibitory protein that negatively regulates TCR signals (Ishida, Y. et al. (1992) EMBO J. 11:3887-3895; Blank, C. et al. (Epub 2006 Dec. 29) Immunol. Immunother. 56(5):739-745).
  • the interaction between PD-1 and PD-L1 can act as an immune checkpoint, which can lead to a decrease in T-cell receptor mediated proliferation (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100).
  • Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2; the effect is additive when the interaction of PD-1 with PD-L2 is blocked as well (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).
  • tumor survival and proliferation is sustained by tumor-mediated immune checkpoint modulation.
  • This modulation can result in the disruption of anti-cancer immune system functions.
  • immune checkpoint receptors ligands such as PD-L1 or PD-L2
  • tumor cells can downregulate immune system activity in the tumor microenvironment and promote cancer immune evasion. particularly by suppressing T cells.
  • PD-L1 is abundantly expressed by a variety of human cancers (Dong et al., (2002) Nat Med 8:787-789).
  • the receptor for PD-L1, PD-1 is expressed on lymphocytes (e.g., activated T cells) and is normally involved in down-regulating the immune system and promoting self-tolerance, particularly by suppressing T cells.
  • lymphocytes e.g., activated T cells
  • PD-1 receptors expressed on T cells bind to cognate PD-L1 ligands on tumor cells, the resulting T cell suppression contributes to an impaired immune response against the tumor (e.g., a decrease in tumor infiltrating lymphocytes or the establishment of immune evasion by cancer cells).
  • PD-1 expression on tumor lymphocytes was found to mark dysfunctional T cells in breast cancer (Kitano et al., (2017) ESMO Open 2(2):e000150) and melanoma (Kleffel et al., (2015) Cell 162(6):1242-1256).
  • PD-1 antagonists such as those that affect the function of the PD-1/PD-L1/PD-L2 signaling axis and/or disrupt the interaction between PD-1 and PD-L1 and/or PD-L2, for example, have been developed and represent a novel class of anti-tumor inhibitors that function via modulation of immune cell-tumor cell interaction.
  • the term “rearranged” refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete VH or VL domain, respectively.
  • a rearranged immunoglobulin gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element.
  • the term “recombinant human antibody” includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
  • variable and constant regions that utilize particular human germline immunoglobulin sequences are encoded by the germline genes, but include subsequent rearrangements and mutations which occur, for example, during antibody maturation.
  • the variable region contains the antigen binding domain, which is encoded by various genes that rearrange to form an antibody specific for a foreign antigen.
  • the variable region can be further modified by multiple single amino acid changes (referred to as somatic mutation or hypermutation) to increase the affinity of the antibody to the foreign antigen.
  • the constant region will change in further response to an antigen (i.e., isotype switch).
  • the rearranged and somatically mutated nucleic acid molecules that encode the light chain and heavy chain immunoglobulin polypeptides in response to an antigen may not have sequence identity with the original nucleic acid molecules, but instead will be substantially identical or similar (i.e., have at least 80% identity).
  • reference antibody (used interchangeably with “reference mAb”) or “reference antigen-binding protein” refers to an antibody, or an antigen-binding fragment thereof, that binds to a specific epitope on CD39 and is used to establish a relationship between itself and one or more distinct antibodies, wherein the relationship is the binding of the reference antibody and the one or more distinct antibodies to the same epitope on CD39.
  • variable heavy (VH) and light chain (VL) amino acid sequences of an exemplary reference antibody are provided in Table 1 (VH, SEQ ID NO. 33; VL, SEQ ID NO. 43).
  • the terms “specific binding,” “selective binding,” “selectively binds,” and “specifically binds,” refer to antibody binding to an epitope on a predetermined antigen.
  • the antibody binds with an equilibrium dissociation constant (K D ) of approximately less than 10 ⁇ 6 M, such as approximately less than 10 ⁇ 7 , 10 ⁇ 8 M, 10 ⁇ 9 M or 10 ⁇ 10 M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE 2000 instrument using recombinant human CD39 as the analyte and the antibody as the ligand and binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.
  • a non-specific antigen e.g., BSA, casein
  • switch sequence refers to those DNA sequences responsible for switch recombination.
  • a “switch donor” sequence typically a ⁇ switch region, will be 5′ (i.e., upstream) of the construct region to be deleted during the switch recombination.
  • the “switch acceptor” region will be between the construct region to be deleted and the replacement constant region (e.g., ⁇ , ⁇ , etc.). As there is no specific site where recombination always occurs, the final gene sequence will typically not be predictable from the construct.
  • the term “subject” includes any human or non-human animal.
  • the methods and compositions of the present invention can be used to treat a subject with an immune disorder.
  • non-human animal includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.
  • nucleic acids For nucleic acids, the term “substantial homology” indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol.
  • the nucleic acid and protein sequences of the present disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences.
  • Such searches can be performed using the NBLAST and) (BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST See http://www.ncbi.nlm.nih.gov.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).
  • nucleic acid compositions of the present disclosure while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic or mixtures thereof may be mutated, in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations, may affect amino acid sequence as desired.
  • DNA sequences substantially homologous to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where “derived” indicates that a sequence is identical or modified from another sequence).
  • T cell refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of a T cell receptor on the cell surface.
  • T helper cells a.k.a.
  • TH cells or CD4+ T cells and subtypes, including TH1, TH2, TH3, TH17, TH9, and TFH cells, cytotoxic T cells (a.k.a TC cells, CD8+ T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memory T cells and subtypes, including central memory T cells (TCM cells), effector memory T cells (TEM and TEMRA cells), and resident memory T cells (TRM cells), regulatory T cells (a.k.a.
  • Treg cells or suppressor T cells and subtypes, including CD4+ FOXP3+ Treg cells, CD4+ FOXP3 ⁇ Treg cells, Tr1 cells, Th3 cells, and Treg17 cells, natural killer T cells (a.k.a. NKT cells), mucosal associated invariant T cells (MAITs), and gamma delta T cells ( ⁇ T cells), including V ⁇ 9/V ⁇ 2 T cells.
  • NKT cells natural killer T cells
  • MAITs mucosal associated invariant T cells
  • ⁇ T cells gamma delta T cells
  • Any one or more of the aforementioned or unmentioned T cells may be the target cell type for a method of use of the invention.
  • T cell-mediated response refers to any response mediated by T cells, including, but not limited to, effector T cells (e.g., CD8+ cells) and helper T cells (e.g., CD4+ cells).
  • T cell mediated responses include, for example, T cell cytotoxicity and proliferation.
  • terapéuticaally effective amount or “therapeutically effective dose,” or similar terms used herein are intended to mean an amount of an agent (e.g., an anti-CD39 antibody or an antigen-binding fragment thereof) that will elicit the desired biological or medical response (e.g., an improvement in one or more symptoms of a cancer).
  • an agent e.g., an anti-CD39 antibody or an antigen-binding fragment thereof
  • treat refers to clinical intervention in an attempt to alter the natural course of the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
  • treatment as used herein refer to therapeutic or preventative measures.
  • treatment employ administration to a subject, in need of such treatment, a human antibody of the present disclosure, for example, a subject in need of an enhanced immune response against a particular antigen or a subject who ultimately may acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • a human antibody of the present disclosure for example, a subject in need of an enhanced immune response against a particular antigen or a subject who ultimately may acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • tumor microenvironment refers to the cellular environment or milieu in which the tumor or neoplasm exists, including surrounding blood vessels as well as non-cancerous cells including, but not limited to, immune cells, fibroblasts, bone marrow-derived inflammatory cells, and lymphocytes. Signaling molecules and the extracellular matrix also comprise the TME.
  • the tumor and the surrounding microenvironment are closely related and interact constantly. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of tumor cells.
  • V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment.
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the present disclosure provides antibodies, and antigen binding portions thereof, that bind to and antagonize CD39, in particular human CD39.
  • Provided herein are isolated antibodies or antigen binding portion thereof that bind to human CD39, comprising heavy and light chain CDRs and variable sequences as set forth in Table 1.
  • the adenosine pathway is a signaling system which acts to fine-tune immune cell functions, such as cell-to-cell interactions, cytokine and chemokine secretion, surface antigen shedding, intracellular pathogen removal, and generating reactive oxygen species (ROS).
  • ATP and ADP play a fundamental role in inflammation, immune system regulation and tissue homeostasis through the activation of receptors (Eltzschig et al., (2012) N Engl J Med 367:2322-2333).
  • Mediators, such as ATP and adenosine are released into the extracellular space in response to metabolic disturbances or other types of insults, and operate both as sensory and efferent signals to shape immune responses.
  • ATP is released either by cell lysis or by non-lytic mechanisms including: (i) exocytosis of ATP-containing vesicles, (ii) through nucleotide-permeable channels (connexin/pannexin hemichannels, maxi-anion channels, volume-regulated anion channels or P2X7 receptor channels), (iii) via transport vesicles that deliver proteins to the cell membrane, and (iv) via lysosomes.
  • CD39 hydrolyzes extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to generate adenosine.
  • the P1 adenosine receptor family encompasses the A1, A2A (the main adenosine receptor expressed by T cells), A2B, and A3 G-protein-coupled receptors.
  • the adenosine-A2A receptor axis provides an immunosuppressive mechanism that dampens inflammation and protects normal tissues from immune system-mediated damage Ohta et al., (2001) Nature 414:916-920). In some cancers, this immunosuppressive pathway is aberrantly activated and provides protection for cancer cells against the immune system (Sitkovsky et al., (2014) Cancer Immunol Res) 2:598-605).
  • CD39 also known as ecto-nucleoside triphosphate diphosphohydrolase 1, E-NTPDase1, and cluster of differentiation 39
  • CD73 also known as ecto-5′-nucleotidase, Ecto5′NTase, 5′-nucleotidase (5′-NT), and cluster of differentiation 73
  • the activity of CD39 and CD73 represents the primary source of extracellular adenosine.
  • CD39 hydrolyses extracellular ATP and ADP into adenosine monophosphate (AMP) (Deaglio et al., (2007) J.
  • AMP adenosine monophosphate
  • AMP is then processed into the anti-inflammatory adenosine, essentially by the ectonucleotidase CD73.
  • adenosine Upon binding to A2A receptors on T cells, adenosine induces the accumulation of intracellular cyclic AMP, thereby preventing TCR-induced CD25 upregulation and inhibiting effector T-lymphocyte proliferation and inflammatory cytokine secretion (Huang et al., (1997) Blood 90-1600-1610; Lokshin et al., (2006) Cancer Res 66:7758-7765).
  • Adenosine also blocks the cytotoxic activity and cytokine production of activated natural killer (NK) cells.
  • the isolated antibody, or antigen binding portion thereof binds to recombinant CD39 and/or membrane-bound CD39. In some embodiments, the isolated antibody, or antigen binding portion thereof, binds to recombinant human CD39 and/or membrane-bound human CD39.
  • the isolated antibody, or antigen binding portion thereof binds to CD39 with an equilibrium dissociation constant (KD) of less than 10 nM.
  • KD equilibrium dissociation constant
  • the isolated antibody, or antigen binding portion thereof inhibits or reduces an enzymatic activity of CD39.
  • the isolated antibody, or antigen binding portion thereof inhibits or reduces the enzymatic activity of CD39, wherein the enzymatic activity of CD39 is the hydrolysis of extracellular adenosine triphosphate (eATP) or extracellular adenosine diphosphate (eADP).
  • eATP extracellular adenosine triphosphate
  • eADP extracellular adenosine diphosphate
  • the isolated antibody, or antigen binding portion thereof inhibits or reduces the conversion of extracellular adenosine triphosphate (eATP) or extracellular adenosine diphosphate (eADP) to extracellular adenosine monophosphate (eAMP).
  • eATP extracellular adenosine triphosphate
  • eADP extracellular adenosine diphosphate
  • eAMP extracellular adenosine monophosphate
  • the isolated antibody, or antigen binding portion thereof increases or enhances a level of extracellular adenosine triphosphate (eATP).
  • eATP extracellular adenosine triphosphate
  • the isolated antibody, or antigen binding portion thereof decreases or reduces a level of extracellular adenosine.
  • the isolated antibody, or antigen binding portion thereof increases or enhances an immunostimulatory level of extracellular adenosine triphosphate (eATP).
  • eATP extracellular adenosine triphosphate
  • the isolated antibody, or antigen binding portion thereof increases proliferation of a lymphocyte.
  • the lymphocyte is a tumor-infiltrating lymphocyte.
  • the lymphocyte is T cell.
  • the T cell is a CD4+ T cell.
  • the isolated antibody, or antigen binding portion thereof enhances expression of one or more dendritic cell activation markers.
  • the one or more dendritic cell activation markers is CD86, HLA-DR, or a combination thereof.
  • the isolated antibody, or antigen binding portion thereof enhances secretion of one or more cytokines from dendritic cells.
  • the one or more cytokines is IL-16, IL-12/IL-23p40, VEGFA, or any combination thereof.
  • the isolated antibody, or antigen binding portion thereof increases or enhances macrophage infiltration in tumors.
  • the isolated antibody, or antigen binding portion thereof increases or enhances secretion of macrophage attracting chemokines.
  • the isolated antibody, or antigen binding portion thereof antagonizes human CD39 in a tumor microenvironment.
  • the isolated antibody, or antigen binding portion thereof cross-reacts with cynomolgus CD39 and/or mouse CD39.
  • the isolated antibody, or antigen binding portion thereof is selected from the group consisting of an IgG1, an IgG2, and IgG3, an IgG4, and IgM, and IgA1, and IgA2, and IgD, and an IgE antibody.
  • the isolated antibody, or antigen binding portion thereof is an IgG1 antibody of IgG4 antibody.
  • the isolated antibody, or antigen binding portion thereof comprises a wild type IgG1 heavy chain constant region.
  • the isolated antibody, or antigen binding portion thereof comprises a wild type IgG4 heavy chain constant region.
  • the isolated antibody, or antigen binding portion thereof comprises an Fc domain comprising at least one mutation.
  • the isolated antibody, or antigen binding portion thereof comprises a mutant IgG4 heavy chain constant region.
  • the mutant IgG4 heavy chain constant region comprises any one of the substitutions S228P, L235E, L235A, or a combination thereof, according to EU numbering.
  • antibody or antigen binding portion thereof the disclosure comprises the heavy chain CDR3 sequence set forth in SEQ ID NO: 3. In some aspects, the antibody or antigen binding portion of the disclosure binds to substantially the same epitope as a reference antibody or antigen binding portion thereof comprising the heavy chain CDR3 sequence set forth in SEQ ID NO: 3. In some aspects, the antibody or antigen binding portion of the disclosure binds to at least one of the amino acid residues bound by a reference antibody or antigen binding portion thereof comprising the heavy chain CDR3 sequence set forth in SEQ ID NOs: 3.
  • the antibody or antigen binding portion of the disclosure wherein a mutation of the epitope bound by the antibody inhibits, reduces, or blocks binding to both the antibody and to a reference antibody or antigen binding portion thereof comprising the heavy chain CDR3 sequence set forth in SEQ ID NOs: 3.
  • the antibody or antigen binding portion of the disclosure binds to substantially the same epitope as a reference antibody or antigen binding portion thereof comprising heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 27, 28 and 29, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 38 and 39, respectively.
  • the antibody or antigen binding portion of the disclosure binds to at least one of the amino acid residues bound by a reference antibody or antigen binding portion thereof comprising heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 27, 28 and 29, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 38 and 39, respectively.
  • a mutation of the epitope bound by the antibody or antigen binding portion of the disclosure inhibits, reduces, or blocks binding to both the antibody and to a reference antibody or antigen binding portion thereof comprising heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 27, 28 and 29, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 38 and 39, respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to substantially the same epitope as a reference antibody or antigen binding portion thereof comprising heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 30, 31 and 32, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 41 and 42, respectively.
  • a mutation of the epitope bound by the antibody or antigen binding portion thereof of the disclosure inhibits, reduces, or blocks binding to both the antibody and to a reference antibody or antigen binding portion thereof comprising heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 30, 31 and 32, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 41 and 42, respectively.
  • the antibody or antigen binding portion of the disclosure binds to and antagonizes human CD39 and comprises heavy and light chain CDRs selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises heavy and light chain CDRs, wherein the heavy chain CDR1, CDR2 and CDR3 sequences are set forth in SEQ ID NOs: 27, 28 and 29, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are set forth in SEQ ID NOs: 37, 38 and 39, respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises heavy and light chain CDRs selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises heavy and light chain CDRs, wherein the heavy chain CDR1, CDR2 and CDR3 sequences are set forth in SEQ ID NOs: 30, 31 and 32, respectively, and the light chain CDR1, CDR2 and CDR3 sequences are set forth in SEQ ID NOs: 40, 41 and 42, respectively;
  • the antibody or antigen binding portion of the disclosure binds to and antagonizes human CD39 and comprises heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 7, 59, 85 and 111; and wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 17, 69, 95 and 121.
  • the antibody or antigen binding portion thereof of the disclosure binds to an antagonizes human CD39 and comprises heavy and light chain variable regions comprising amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion of the disclosure binds to and antagonizes human CD39 and comprises heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 7, 59, 85 and 111; and wherein the light chain variable region comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 17, 69, 95 and 121.
  • the antibody or antigen binding portion of the disclosure binds to an antagonizes human CD39 and comprises heavy and light chain variable regions comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises heavy and light chain variable regions comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences set forth in SEQ ID NO: 33 and 43, respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 9, 61, 87 and 113; and wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 19, 71, 97 and 123.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 9, 61, 87 and 113; and wherein the light chain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% to the amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 19, 71, 97 and 123.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 21, 73, 99, and 125; and wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 19, 71, 97 and 123.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 21, 73, 99 and 125; and wherein the light chain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 19, 71, 97 and 123.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 23, 75, 101 and 127; and wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 19, 71, 97 and 123.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 23, 75, 101 and 127; and wherein the light chain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 19, 71, 97 and 123.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 25, 77, 103 and 129; and wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 19, 71, 97 and 123.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 25, 77, 103 and 129; and wherein the light chain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 19, 71, 97 and 123.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences selected from the group consisting of:
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences set forth in SEQ ID NO: 35 and 45, respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences set forth in SEQ ID NO: 35 and 45, respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences set forth in SEQ ID NO: 47 and 45, respectively; respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences set forth in SEQ ID NO: 47 and 45, respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences set forth in SEQ ID NO: 49 and 45, respectively; respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences set forth in SEQ ID NO: 49 and 45, respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences set forth in SEQ ID NO: 51 and 45, respectively; respectively.
  • the antibody or antigen binding portion thereof of the disclosure binds to and antagonizes human CD39 and comprises a heavy chain and a light chain comprising amino acid sequences at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences set forth in SEQ ID NO: 51 and 45, respectively.
  • the isolated antibody, or antigen binding portion thereof comprises a mutant IgG4 heavy chain constant region.
  • the mutant IgG4 heavy chain constant region comprises a S228P substitution.
  • the mutant IgG4 heavy chain constant region comprises a S228P substitution and an L235E substitution.
  • the mutant IgG4 heavy chain constant region comprises a S228P substitution and an L235A substitution. Numbering according to EU numbering.
  • methods for producing any of the anti-CD39 antibodies or antigen-binding fragments thereof described herein can include immunizing a subject (e.g., a non-human mammal) with an appropriate immunogen. Suitable immunogens for generating any of the antibodies described herein are set forth herein.
  • a skilled artisan can immunize a suitable subject (e.g., a non-human mammal such as a rat, a mouse, a gerbil, a hamster, a dog, a cat, a pig, a goat, a horse, or a non-human primate) with a full-length CD39 polypeptide such as a full-length human CD39 polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 138.
  • a suitable subject e.g., a non-human mammal such as a rat, a mouse, a gerbil, a hamster, a dog, a cat, a pig, a goat, a horse, or a non-human primate
  • a full-length CD39 polypeptide such as a full-length human CD39 polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 138.
  • a suitable subject e.g., a non-human mammal
  • the immunogen can be administered to a subject (e.g., a non-human mammal) with an adjuvant.
  • Adjuvants useful in producing an antibody in a subject include, but are not limited to, protein adjuvants; bacterial adjuvants, e.g., whole bacteria (BCG, Corynebacterium parvum or Salmonella minnesota ) and bacterial components including cell wall skeleton, trehalose dimycolate, monophosphoryl lipid A, methanol extractable residue (MER) of tubercle bacillus , complete or incomplete Freund's adjuvant; viral adjuvants; chemical adjuvants, e.g., aluminum hydroxide, and iodoacetate and cholesteryl hemisuccinate.
  • protein adjuvants e.g., whole bacteria (BCG, Corynebacterium parvum or Salmonella minnesota ) and bacterial components including cell wall skeleton, trehalose dimycolate, monophosphoryl lipid A, methanol extractable residue (MER) of tubercle bacillus , complete or incomplete Freund's adj
  • the methods include preparing a hybridoma cell line that secretes a monoclonal antibody that binds to the immunogen.
  • a suitable mammal such as a laboratory mouse is immunized with a CD39 polypeptide as described above.
  • Antibody-producing cells e.g., B cells of the spleen
  • B cells of the spleen of the immunized mammal can be isolated two to four days after at least one booster immunization of the immunogen and then grown briefly in culture before fusion with cells of a suitable myeloma cell line.
  • the cells can be fused in the presence of a fusion promoter such as, e.g., vaccinia virus or polyethylene glycol.
  • the hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected.
  • spleen cells of Balb/c mice immunized with a suitable immunogen can be fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag 14.
  • suitable culture medium which is supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells.
  • the obtained hybrid cells are then screened for secretion of the desired antibodies, e.g., an antibody that binds to human CD39 and
  • the desired antibodies e.g., an antibody that binds to human CD39
  • a skilled artisan can identify an anti-CD39 antibody from a non-immune biased library as described in, e.g., U.S. Pat. No. 6,300,064 (to Knappik et al.; Morphosys AG) and Schoonbroodt et al. (2005) Nucleic Acids Res 33(9):e81.
  • the methods described herein can involve, or be used in conjunction with, e.g., phage display technologies, bacterial display, yeast surface display, eukaryotic viral display, mammalian cell display, and cell-free (e.g., ribosomal display) antibody screening techniques (see, e.g., Etz et al. (2001) J Bacteriol 183:6924-6935; Cornelis (2000) Curr Opin Biotechnol 11:450-454; Klemm et al. (2000) Microbiology 146:3025-3032; Kieke et al. (1997) Protein Eng 10:1303-1310; Yeung et al.
  • phage display technologies e.g., phage display technologies, bacterial display, yeast surface display, eukaryotic viral display, mammalian cell display, and cell-free (e.g., ribosomal display) antibody screening techniques (see, e.g., Etz et al. (2001) J Bacteriol
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • phage can be utilized to display antigen-binding domains of antibodies, such as Fab, Fv, or disulfide-bond stabilized Fv antibody fragments, expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage used in these methods are typically filamentous phage such as fd and M13.
  • the antigen binding domains are expressed as a recombinantly fused protein to any of the phage coat proteins pIII, pVIII, or pIX.
  • phage display methods that can be used to make the immunoglobulins, or fragments thereof, described herein include those disclosed in Brinkman et al. (1995) J Immunol Methods 182:41-50; Ames et al. (1995) J Immunol Methods 184:177-186; Kettleborough et al. (1994) Eur J Immunol 24:952-958; Persic et al. (1997) Gene 187:9-18; Burton et al. (1994) Advances in Immunology 57:191-280; and PCT publication nos.
  • WO 90/02809 WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, and WO 95/20401.
  • Suitable methods are also described in, e.g., U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.
  • the cDNA coding for the variable regions of the heavy chain and light chain polypeptides of immunoglobulins are used to construct the phage display library. Methods for generating such a library are described in, e.g., Merz et al. (1995) J Neurosci Methods 62(1-2):213-9; Di Niro et al. (2005) Biochem J 388(Pt 3):889-894; and Engberg et al. (1995) Methods Mol Biol 51:355-376.
  • a combination of selection and screening can be employed to identify an antibody of interest from, e.g., a population of hybridoma-derived antibodies or a phage display antibody library.
  • Suitable methods are known in the art and are described in, e.g., Hoogenboom (1997) Trends in Biotechnology 15:62-70; Brinkman et al. (1995), supra; Ames et al. (1995), supra; Kettleborough et al. (1994), supra; Persic et al. (1997), supra; and Burton et al. (1994), supra.
  • a plurality of phagemid vectors each encoding a fusion protein of a bacteriophage coat protein (e.g., pIII, pVIII, or pIX of M13 phage) and a different antigen-combining region are produced using standard molecular biology techniques and then introduced into a population of bacteria (e.g., E. coli ).
  • Expression of the bacteriophage in bacteria can, in some embodiments, require use of a helper phage. In some embodiments, no helper phage is required (see, e.g., Chasteen et al., (2006) Nucleic Acids Res 34(21):e145).
  • Phage produced from the bacteria are recovered and then contacted to, e.g., a target antigen bound to a solid support (immobilized). Phage may also be contacted to antigen in solution, and the complex is subsequently bound to a solid support.
  • a subpopulation of antibodies screened using the above methods can be characterized for their specificity and binding affinity for a particular antigen (e.g., human CD39) using any immunological or biochemical based method known in the art.
  • a particular antigen e.g., human CD39
  • specific binding of an antibody to CD39 may be determined for example using immunological or biochemical based methods such as, but not limited to, an ELISA assay, SPR assays, immunoprecipitation assay, affinity chromatography, and equilibrium dialysis as described above.
  • nucleic acids encoding the CDRs can be chemically synthesized as described in, e.g., Shiraishi et al. (2007) Nucleic Acids Symposium Series 51(1):129-130 and U.S. Pat. No. 6,995,259.
  • the region of the nucleic acid sequence encoding the CDRs can be replaced with the chemically synthesized nucleic acids using standard molecular biology techniques.
  • the 5′ and 3′ ends of the chemically synthesized nucleic acids can be synthesized to comprise sticky end restriction enzyme sites for use in cloning the nucleic acids into the nucleic acid encoding the variable region of the donor antibody.
  • the anti-CD39 antibodies described herein comprise an IgG4 heavy chain constant region.
  • the IgG4 heavy chain constant region is a wild type IgG4 heavy chain constant region.
  • the IgG4 constant region comprises a mutation, e.g., one or both of S228P and L235E or L235A, e.g., according to EU numbering (Kabat, E. A., et al., supra). Representative sequences for use in antibodies of the disclosure of wild-type and mutant IgG4 constant regions are set forth in Table 1.
  • the anti-CD39 antibodies described herein comprise an IgG1 constant region.
  • the IgG1 heavy chain constant region is a wild type IgG1 heavy chain constant region. In another embodiment, the IgG1 heavy chain constant region comprises a mutation. Representative sequences for use in antibodies of the disclosure of wild-type and mutant IgG4 constant regions are set forth in Table 1.
  • An altered constant region with altered FcR binding affinity and/or ADCC activity and/or altered CDC activity is a polypeptide which has either an enhanced or diminished FcR binding activity and/or ADCC activity and/or CDC activity compared to the unaltered form of the constant region.
  • An altered constant region which displays increased binding to an FcR binds at least one FcR with greater affinity than the unaltered polypeptide.
  • An altered constant region which displays decreased binding to an FcR binds at least one FcR with lower affinity than the unaltered form of the constant region.
  • Such variants which display decreased binding to an FcR may possess little or no appreciable binding to an FcR, e.g., 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of the binding to the FcR as compared to the level of binding of a native sequence immunoglobulin constant or Fc region to the FcR.
  • an altered constant region that displays modulated ADCC and/or CDC activity may exhibit either increased or reduced ADCC and/or CDC activity compared to the unaltered constant region.
  • the anti-CD39 antibody comprising an altered constant region can exhibit approximately 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of the ADCC and/or CDC activity of the unaltered form of the constant region.
  • An anti-CD39 antibody described herein comprising an altered constant region displaying reduced ADCC and/or CDC may exhibit reduced or no ADCC and/or CDC activity.
  • an anti-CD39 antibody described herein exhibits reduced or no effector function.
  • an anti-CD39 antibody comprises a hybrid constant region, or a portion thereof, such as a G2/G4 hybrid constant region (see e.g., Burton et al. (1992) Adv Immun 51:1-18; Canfield et al. (1991) J. Exp Med 173:1483-1491; and Mueller et al. (1997) Mol Immunol 34(6):441-452). See above.
  • an anti-CD39 antibody may contain an altered constant region exhibiting enhanced or reduced complement dependent cytotoxicity (CDC).
  • Modulated CDC activity may be achieved by introducing one or more amino acid substitutions, insertions, or deletions in an Fc region of the antibody. See, e.g., U.S. Pat. No. 6,194,551. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved or reduced internalization capability and/or increased or decreased complement-mediated cell killing. See, e.g., Caron et al. (1992) J.
  • the antibodies or antigen-binding fragments thereof described herein can be produced using a variety of techniques known in the art of molecular biology and protein chemistry.
  • a nucleic acid encoding one or both of the heavy and light chain polypeptides of an antibody can be inserted into an expression vector that contains transcriptional and translational regulatory sequences, which include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcription terminator signals, polyadenylation signals, and enhancer or activator sequences.
  • the regulatory sequences include a promoter and transcriptional start and stop sequences.
  • the expression vector can include more than one replication system such that it can be maintained in two different organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.
  • Several possible vector systems are available for the expression of cloned heavy chain and light chain polypeptides from nucleic acids in mammalian cells.
  • One class of vectors relies upon the integration of the desired gene sequences into the host cell genome.
  • Cells which have stably integrated DNA can be selected by simultaneously introducing drug resistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) Mol Appl Genet 1:327).
  • the selectable marker gene can be either linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77).
  • a second class of vectors utilizes DNA elements which confer autonomously replicating capabilities to an extrachromosomal plasmid.
  • These vectors can be derived from animal viruses, such as bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, polyoma virus (Deans et al. (1984) Proc Natl Acad Sci USA 81:1292), or SV40 virus (Lusky and Botchan (1981) Nature 293:79).
  • the expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid.
  • the method of introduction is largely dictated by the targeted cell type, discussed below.
  • Exemplary methods include CaPO4 precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.
  • Appropriate host cells for the expression of antibodies or antigen-binding fragments thereof include yeast, bacteria, insect, plant, and mammalian cells. Of particular interest are bacteria such as E. coli , fungi such as Saccharomyces cerevisiae and Pichia pastoris , insect cells such as SF9, mammalian cell lines (e.g., human cell lines), as well as primary cell lines.
  • an antibody or fragment thereof can be expressed in, and purified from, transgenic animals (e.g., transgenic mammals).
  • transgenic animals e.g., transgenic mammals
  • an antibody can be produced in transgenic non-human mammals (e.g., rodents) and isolated from milk as described in, e.g., Houdebine (2002) Curr Opin Biotechnol 13(6):625-629; van Kuik-Romeijn et al. (2000) Transgenic Res 9(2):155-159; and Pollock et al. (1999) J Immunol Methods 231(1-2):147-157.
  • the antibodies and fragments thereof can be produced from the cells by culturing a host cell transformed with the expression vector containing nucleic acid encoding the antibodies or fragments, under conditions, and for an amount of time, sufficient to allow expression of the proteins.
  • Such conditions for protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.
  • antibodies expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10:319-30).
  • an antibody or fragment thereof can be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample.
  • Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography.
  • an antibody can be purified using a standard anti-antibody column (e.g., a protein-A or protein-G column). Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes (1994) “Protein Purification, 3rd edition,” Springer-Verlag, New York City, N.Y. The degree of purification necessary will vary depending on the desired use. In some instances, no purification of the expressed antibody or fragments thereof will be necessary.
  • Methods for determining the yield or purity of a purified antibody or fragment thereof include, e.g., Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoretic methods (e.g., using a protein stain such as Coomassie Blue or colloidal silver stain).
  • the antibodies or antigen-binding fragments thereof can be modified following their expression and purification.
  • the modifications can be covalent or non-covalent modifications.
  • Such modifications can be introduced into the antibodies or fragments by, e.g., reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • Suitable sites for modification can be chosen using any of a variety of criteria including, e.g., structural analysis or amino acid sequence analysis of the antibodies or fragments.
  • the antibodies or antigen-binding fragments thereof can be conjugated to a heterologous moiety.
  • the heterologous moiety can be, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), or a detectable label such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag such as biotin or streptavidin.
  • Suitable heterologous polypeptides include, e.g., an antigenic tag (FLAG (DYKDDDDK (SEQ ID NO: 135)), polyhistidine (6-His; HHHHHH (SEQ ID NO: 136), hemagglutinin (HA; YPYDVPDYA (SEQ ID NO: 137)), glutathione-S-transferase (GST), or maltose-binding protein (MBP)) for use in purifying the antibodies or fragments.
  • FLAG DYKDDDDK
  • polyhistidine 6-His
  • HHHHHHHH SEQ ID NO: 136
  • hemagglutinin HA
  • YPYDVPDYA SEQ ID NO: 137
  • GST glutathione-S-transferase
  • MBP maltose-binding protein
  • Heterologous polypeptides also include polypeptides (e.g., enzymes) that are useful as diagnostic or detectable markers, for example, luciferase, a fluorescent protein (e.g., green fluorescent protein (GFP)), or chloramphenicol acetyl transferase (CAT).
  • Suitable radioactive labels include, e.g., 32P, 33P, 14C, 125I, 131I, 35S, and 3H.
  • Suitable fluorescent labels include, without limitation, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLightTM 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5, allophycocyanin, and Cy7.
  • Luminescent labels include, e.g., any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates.
  • suitable europium chelates include the europium chelate of diethylene triamine pentaacetic acid (DTPA) or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).
  • Enzymatic labels include, e.g., alkaline phosphatase, CAT, luciferase, and horseradish peroxidase.
  • Two proteins can be cross-linked using any of a number of known chemical cross linkers.
  • cross linkers are those which link two amino acid residues via a linkage that includes a “hindered” disulfide bond.
  • a disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by the action, for example, of reduced glutathione or the enzyme disulfide reductase.
  • SMPT 4-succinimidyloxycarbonyl- ⁇ -methyl- ⁇ (2-pyridyldithio) toluene
  • cross-linkers include, without limitation, reagents which link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amino group and a guanidinium group that is present in the side chain of arginine (e.g., p-azidophenyl glyoxal monohydrate).
  • reagents which link two amino groups e.g., N-5-azido-2-nitrobenzoyloxysuccinimide
  • two sulfhydryl groups e.g.,
  • a radioactive label can be directly conjugated to the amino acid backbone of the antibody.
  • the radioactive label can be included as part of a larger molecule (e.g., 125I in meta-[125I]iodophenyl-N-hydroxysuccinimide ([125I]mIPNHS) which binds to free amino groups to form meta-iodophenyl (mIP) derivatives of relevant proteins (see, e.g., Rogers et al. (1997) J Nucl Med 38:1221-1229) or chelate (e.g., to DOTA or DTPA) which is in turn bound to the protein backbone.
  • a larger molecule e.g., 125I in meta-[125I]iodophenyl-N-hydroxysuccinimide ([125I]mIPNHS) which binds to free amino groups to form meta-iodophenyl (mIP) derivatives of relevant proteins (see, e.g., Rogers
  • fluorophores can be conjugated to free amino groups (e.g., of lysines) or sulfhydryl groups (e.g., cysteines) of proteins using succinimidyl (NETS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophores.
  • NETS succinimidyl
  • TFP tetrafluorophenyl
  • the fluorophores can be conjugated to a heterobifunctional cross-linker moiety such as sulfo-SMCC.
  • Suitable conjugation methods involve incubating an antibody protein, or fragment thereof, with the fluorophore under conditions that facilitate binding of the fluorophore to the protein. See, e.g., Welch and Redvanly (2003) “Handbook of Radiopharmaceuticals: Radiochemistry and Applications,” John Wiley and Sons (ISBN 0471495603).
  • the antibodies or fragments can be modified, e.g., with a moiety that improves the stabilization and/or retention of the antibodies in circulation, e.g., in blood, serum, or other tissues.
  • the antibody or fragment can be PEGylated as described in, e.g., Lee et al. (1999) Bioconjug Chem 10(6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476 or HESylated (Fresenius Kabi, Germany; see, e.g., Pavisi ⁇ et al.
  • the stabilization moiety can improve the stability, or retention of, the antibody (or fragment) by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.
  • the antibodies or antigen-binding fragments thereof described herein can be glycosylated.
  • an antibody or antigen-binding fragment thereof described herein can be subjected to enzymatic or chemical treatment, or produced from a cell, such that the antibody or fragment has reduced or absent glycosylation.
  • Methods for producing antibodies with reduced glycosylation are known in the art and described in, e.g., U.S. Pat. No. 6,933,368; Wright et al. (1991) EMBO J 10(10):2717-2723; and Co et al. (1993) Mol Immunol 30:1361.
  • the invention provides for a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-CD39 antibody with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
  • acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.
  • the formulation material(s) are for s.c. and/or I.V. administration.
  • the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents;
  • amino acids
  • the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH 5.2, 9% Sucrose.
  • the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and/or rate of in vivo clearance of the anti-CD39 antibody.
  • the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
  • the saline comprises isotonic phosphate-buffered saline.
  • neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute therefore.
  • a composition comprising an anti-CD39 antibody can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, a composition comprising an anti-CD39 antibody can be formulated as a lyophilizate using appropriate excipients such as sucrose.
  • the pharmaceutical composition can be selected for parenteral delivery. In certain embodiments, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally.
  • the preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art.
  • the formulation components are present in concentrations that are acceptable to the site of administration.
  • buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
  • a therapeutic composition when parenteral administration is contemplated, can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising an anti-CD39 antibody, in a pharmaceutically acceptable vehicle.
  • a vehicle for parenteral injection is sterile distilled water in which an anti-CD39 antibody is formulated as a sterile, isotonic solution, and properly preserved.
  • the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection.
  • hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation.
  • implantable drug delivery devices can be used to introduce the desired molecule.
  • a pharmaceutical composition can be formulated for inhalation.
  • an anti-CD39 antibody can be formulated as a dry powder for inhalation.
  • an inhalation solution comprising an anti-CD39 antibody can be formulated with a propellant for aerosol delivery.
  • solutions can be nebulized. Pulmonary administration is further described in PCT application No. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins.
  • formulations can be administered orally.
  • an anti-CD39 antibody that is administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
  • a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.
  • at least one additional agent can be included to facilitate absorption of an anti-CD39 antibody.
  • diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.
  • a pharmaceutical composition can involve an effective quantity of an anti-CD39 antibody in a mixture with non-toxic excipients which are suitable for the manufacture of tablets.
  • suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules.
  • Sustained release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem.
  • sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al, Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.
  • the pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, this can be accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical composition once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.
  • kits are provided for producing a single-dose administration unit.
  • the kit can contain both a first container having a dried protein and a second container having an aqueous formulation.
  • kits containing single and multi-chambered pre-filled syringes e.g., liquid syringes and lyosyringes are included.
  • the effective amount of a pharmaceutical composition comprising an anti-CD39 antibody to be employed therapeutically will depend, for example, upon the therapeutic context and objectives.
  • the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which an anti-CD39 antibody is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient.
  • the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • the frequency of dosing will take into account the pharmacokinetic parameters of an anti-CD39 antibody in the formulation used.
  • a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them.
  • appropriate dosages can be ascertained through use of appropriate dose-response data.
  • the route of administration of the pharmaceutical composition is in accord with known methods, e.g. orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, subcutaneously, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices.
  • the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
  • individual elements of the combination therapy may be administered by different routes.
  • the composition can be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.
  • it can be desirable to use a pharmaceutical composition comprising an anti-CD39 antibody in an ex vivo manner. In such instances, cells, tissues and/or organs that have been removed from the patient are exposed to a pharmaceutical composition comprising an anti-CD39 antibody after which the cells, tissues and/or organs are subsequently implanted back into the patient.
  • an anti-CD39 antibody can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptides.
  • such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic.
  • the cells can be immortalized.
  • the cells in order to decrease the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues.
  • the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.
  • compositions described herein can be used in a number of diagnostic and therapeutic applications.
  • detectably-labeled antigen-binding molecules can be used in assays to detect the presence or amount of the target antigens in a sample (e.g., a biological sample).
  • the compositions can be used in in vitro assays for studying inhibition of target antigen function.
  • the compositions can be used as positive controls in assays designed to identify additional novel compounds that inhibit complement activity or otherwise are useful for treating a complement-associated disorder.
  • a CD39-inhibiting composition can be used as a positive control in an assay to identify additional compounds (e.g., small molecules, aptamers, or antibodies) that reduce or abrogate CD39 production.
  • additional compounds e.g., small molecules, aptamers, or antibodies
  • the compositions can also be used in therapeutic methods as elaborated on below.
  • the disclosure provides a method of detecting CD39 in a biological sample or in a subject, comprising (i) contacting the sample or the subject (and optionally, a reference sample or subject) with any antibody in Table 1 under conditions that allow interaction of the antibody molecule and CD39 to occur, and (ii) detecting formation of a complex between the antibody molecule and the sample or the subject (and optionally, the reference sample or subject).
  • the disclosure provides a kit comprising an anti-CD39 antibody as disclosed herein, and instructions for use.
  • the disclosure provides a kit comprising an isolated antibody that binds human CD39, or antigen binding portion thereof, such as those described herein or the pharmaceutical composition comprising the antibody, or antigen binding portion thereof, and instructions for use in stimulating an immune response in a subject, or treating cancer in a subject, optionally with instructions for use in combination with one or more additional therapeutic agents or procedures.
  • the one or more additional therapeutic agents or procedures is selected from the group consisting of: a chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, surgical procedure, a radiation procedure, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, a vaccine, or a cellular immunotherapy, or a combination thereof.
  • the kit provides instructions for use in combination with a PD-1 antagonist, an adenosine A2AR antagonist, a CD73 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, chimeric antigen receptor (CAR) cell therapy, an anthracycline, or a combination thereof.
  • the kit provides instructions for use in combination with a combination of a CD73 inhibitor and an A2AR antagonist. In some embodiments, the kit provides instructions for use in combination with a combination of a PD-1 antagonist and an adenosine A2AR antagonist.
  • the kit provides instruction for use in combination with a PD-1 antagonist.
  • the PD-1 antagonist is selected from the group consisting of: PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224.
  • the PD-1 antagonist is selected from the group consisting of: FAZ053, Atezolizumab, Avelumab, Durvalumab, and BMS-936559.
  • the kit provides instructions for use in combination with an adenosine A2AR antagonist.
  • the adenosine A2AR antagonist is selected from the group consisting of: NIR178, CPI-444, AZD4635, Vipadenant, GBV-2034, and AB928.
  • the adenosine A2AR antagonist is CPI-444.
  • the kit provides instructions for use in combination with a CD73 inhibitor.
  • the CD73 inhibitor is selected from the group consisting of: AB421, MEDI9447, and BMS-986179.
  • the kit provides instructions for use in combination with a CTLA-4 inhibitor.
  • the CTLA-4 inhibitor is Ipilimumab or Tremelimumab.
  • the kit provides instruction for use in combination with a TIM-3 inhibitor.
  • the TIM-3 inhibitor is MGB453 or TSR-022.
  • the kit provides instructions for use in combination with a LAG-3 inhibitor.
  • the LAG-3 inhibitor is selected from the group consisting of LAG525, BMS-986016, and TSR-033.
  • the kit provides instructions for use in combination with a chimeric antigen receptor (CAR) cell therapy.
  • CAR chimeric antigen receptor
  • the CAR cell therapy is CTL019.
  • the kit provides instructions for use in combination with an anthracycline.
  • anthracycline is selected from doxorubicin, daunorubicin, epirubicin, idarubicin, and valrubicin.
  • the anthracycline is doxorubicin.
  • kits can include an anti-CD39 antibody as disclosed herein, and instructions for use.
  • the kits may comprise, in a suitable container, an anti-CD39 antibody, one or more controls, and various buffers, reagents, enzymes and other standard ingredients well known in the art.
  • the disclosure provides a kit comprising an anti-CD39 antibody or antigen-binding portion as disclosed herein, and instructions for use in stimulating an immune response in a subject, or treating cancer in a subject, optionally with instructions for use in combination with one or more additional therapeutic agents or procedure as disclosed herein.
  • the container can include at least one vial, well, test tube, flask, bottle, syringe, or other container means, into which an anti-CD39 antibody may be placed, and in some instances, suitably aliquoted.
  • the kit can contain additional containers into which this component may be placed.
  • the kits can also include a means for containing an anti-CD39 antibody and any other reagent containers in close confinement for commercial sale.
  • Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • Containers and/or kits can include labeling with instructions for use and/or warnings.
  • compositions of the present invention have numerous in vitro and in vivo utilities involving the detection and/or quantification of CD39 and/or the antagonism of CD39 function.
  • the disclosure provides methods and uses of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier.
  • the disclosure provides methods and uses of treating a cancer in a subject, the method comprising administering to the subject an effective amount an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier.
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or pharmaceutical composition inhibits or reduces the enzymatic activity of CD39 in a tumor microenvironment, thereby treating the cancer.
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of CD39, wherein the inhibition or reduction of the enzymatic activity of CD39 inhibits or reduces the conversion of extracellular adenosine triphosphate (eATP) or extracellular adenosine diphosphate (eADP) to extracellular adenosine monophosphate (AMP) in a tumor microenvironment, thereby treating the cancer.
  • eATP extracellular adenosine triphosphate
  • eADP extracellular adenosine diphosphate
  • AMP extracellular adenosine monophosphate
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of CD39, wherein the inhibition or reduction of the enzymatic activity of CD39 increases or enhances a level of extracellular adenosine triphosphate (eATP) in a tumor microenvironment, thereby treating the cancer.
  • eATP extracellular adenosine triphosphate
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of CD39, wherein the inhibition or reduction of the enzymatic activity of CD39 decreases or reduces a level of extracellular adenosine in a tumor microenvironment, thereby treating the cancer.
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of CD39, wherein the inhibition or reduction of the enzymatic activity of CD39 increases or enhances a level of extracellular adenosine triphosphate (eATP) and decreases or reduces a level of extracellular adenosine in a tumor microenvironment, thereby treating the cancer.
  • eATP extracellular adenosine triphosphate
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of CD39, wherein the inhibition or reduction of the enzymatic activity of CD39 maintains, increases or enhances an immunostimulatory level of extracellular adenosine triphosphate (eATP) in a tumor microenvironment, thereby treating the cancer.
  • eATP extracellular adenosine triphosphate
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of CD39, wherein the inhibition or reduction of the enzymatic activity of CD39 increases or enhances the proliferation of a lymphocyte in the tumor microenvironment, thereby treating the cancer.
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of CD39, wherein the inhibition or reduction of the enzymatic activity of CD39 enhances expression of one or more dendritic cell activation markers.
  • the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody, or antigen binding portion thereof, that binds to and antagonizes CD39, provided by the disclosure, or a pharmaceutical composition comprising the antibody or antigen binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding portion thereof, or the pharmaceutical composition inhibits or reduces the enzymatic activity of CD39, wherein the inhibition or reduction of the enzymatic activity of CD39 enhances secretion of one or more cytokines from dendritic cells.
  • the disclosure provides methods of treating cancer in a subject, wherein the cancer is selected from the group consisting of: lung cancer (e.g., non-small cell lung cancer), ovarian cancer, kidney cancer, testicular cancer, pancreas cancer, breast cancer (e.g., triple-negative breast cancer), melanoma, head and neck cancer (e.g., squamous head and neck cancer), colorectal cancer, bladder cancer, endometrial cancer, prostate cancer, thyroid cancer, hepatocellular carcinoma, gastric cancer, brain cancer, lymphoma or renal cancer (e.g., renal cell carcinoma).
  • lung cancer e.g., non-small cell lung cancer
  • ovarian cancer ovarian cancer
  • kidney cancer testicular cancer
  • pancreas cancer breast cancer (e.g., triple-negative breast cancer)
  • melanoma melanoma
  • head and neck cancer e.g., squamous head and neck cancer
  • colorectal cancer bladder cancer, endometrial cancer
  • compositions are useful in, inter alia, methods for treating or preventing a variety of cancers in a subject.
  • the compositions can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of administration.
  • the route can be, e.g., intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, intramuscular injection (IM), or intrathecal injection (IT).
  • IV intravenous injection or infusion
  • SC subcutaneous injection
  • IP intraperitoneal
  • IM intramuscular injection
  • IT intrathecal injection
  • the injection can be in a bolus or a continuous infusion.
  • Administration can be achieved by, e.g., local infusion, injection, or by means of an implant.
  • the implant can be of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • the implant can be configured for sustained or periodic release of the composition to the subject. See, e.g., U.S. Patent Application Publication No. 20080241223; U.S. Pat. Nos. 5,501,856; 4,863,457; and 3,710,795; EP488401; and EP 430539, the disclosures of each of which are incorporated herein by reference in their entirety.
  • composition can be delivered to the subject by way of an implantable device based on, e.g., diffusive, erodible, or convective systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based systems, or electromechanical systems.
  • an implantable device based on, e.g., diffusive, erodible, or convective systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based systems, or electromechanical systems.
  • an anti-CD39 antibody or antigen-binding fragment thereof is therapeutically delivered to a subject by way of local administration.
  • a suitable dose of an antibody or fragment thereof described herein, which dose is capable of treating or preventing cancer in a subject can depend on a variety of factors including, e.g., the age, sex, and weight of a subject to be treated and the particular inhibitor compound used. For example, a different dose of a whole anti-CD39 antibody may be required to treat a subject with cancer as compared to the dose of a CD39-binding Fab′ antibody fragment required to treat the same subject. Other factors affecting the dose administered to the subject include, e.g., the type or severity of the cancer. For example, a subject having metastatic melanoma may require administration of a different dosage of an anti-CD39 antibody than a subject with glioblastoma.
  • Other factors can include, e.g., other medical disorders concurrently or previously affecting the subject, the general health of the subject, the genetic disposition of the subject, diet, time of administration, rate of excretion, drug combination, and any other additional therapeutics that are administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject will also depend upon the judgment of the treating medical practitioner (e.g., doctor or nurse). Suitable dosages are described herein.
  • a pharmaceutical composition can include a therapeutically effective amount of an anti-CD39 antibody or antigen-binding fragment thereof described herein.
  • Such effective amounts can be readily determined by one of ordinary skill in the art based, in part, on the effect of the administered antibody, or the combinatorial effect of the antibody and one or more additional active agents, if more than one agent is used.
  • a therapeutically effective amount of an antibody or fragment thereof described herein can also vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody (and one or more additional active agents) to elicit a desired response in the individual, e.g., reduction in tumor growth.
  • a therapeutically effective amount of an anti-CD39 antibody can inhibit (lessen the severity of or eliminate the occurrence of) and/or prevent a particular disorder, and/or any one of the symptoms of the particular disorder known in the art or described herein.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.
  • Suitable human doses of any of the antibodies or fragments thereof described herein can further be evaluated in, e.g., Phase I dose escalation studies. See, e.g., van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1):523-531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10): 3499-3500.
  • the composition contains any of the antibodies or antigen-binding fragments thereof described herein and one or more (e.g., two, three, four, five, six, seven, eight, nine, 10, or 11 or more) additional therapeutic agents such that the composition as a whole is therapeutically effective.
  • a composition can contain an anti-CD39 antibody described herein and an alkylating agent, wherein the antibody and agent are each at a concentration that when combined are therapeutically effective for treating or preventing a cancer (e.g., melanoma) in a subject.
  • Toxicity and therapeutic efficacy of such compositions can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be used, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • An antibody or antigen-binding fragment thereof that exhibits a high therapeutic index is preferred. While compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue and to minimize potential damage to normal cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such antibodies or antigen-binding fragments thereof lies generally within a range of circulating concentrations of the antibodies or fragments that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the antibody which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • cell culture or animal modeling can be used to determine a dose required to achieve a therapeutically effective concentration within the local site.
  • the methods can be performed in conjunction with other therapies for cancer.
  • the composition can be administered to a subject at the same time, prior to, or after, radiation, surgery, targeted or cytotoxic chemotherapy, chemoradiotherapy, hormone therapy, immunotherapy, gene therapy, cell transplant therapy, precision medicine, genome editing therapy, or other pharmacotherapy.
  • compositions described herein can be used to treat a variety of cancers such as but not limited to: Kaposi's sarcoma, leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblasts promyelocyte myelomonocytic monocytic erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma and marginal zone B cell lymphoma, Polycythemia vera Lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, sarcomas, and carcinomas, fibrosarcoma, myxosarcom
  • Priority indications may be selected based on a variety of protein expression patterns: 1) negligible CD39 expression on normal tissue with upregulation in tumor tissue (lung cancer, ovarian cancer, pancreatic cancer, kidney cancer, testicular cancer), 2) maintenance of CD39 expression on hematopoietic cancers (B-cell lymphomas, acute myelogenous leukemia, acute myeloid leukemia) and 3) positive CD39 expression on myeloid- or Treg-rich cancers (breast cancer, gastric cancer, head and neck cancer, esophageal cancer).
  • an anti-CD39 antibody, or antigen binding portion thereof, provided by the disclosure can be combined with one or more additional therapeutics or treatments, e.g., another therapeutic or treatment for a cancer.
  • the anti-CD39 antibody, or antigen binding portion thereof can be administered to a subject (e.g., a human patient) in combination with one or more additional therapeutics, wherein the combination provides a therapeutic benefit to a subject who has, or is at risk of developing, cancer.
  • an anti-CD39 antibody, or antigen binding portion thereof, and the one or more additional therapeutics are administered at the same time (e.g., simultaneously). In other embodiments, the anti-CD39 antibody, or antigen binding portion thereof, is administered first in time and the one or more additional therapeutics are administered second in time (e.g., sequentially). In some embodiments, the one or more additional therapeutics are administered first in time and the anti-CD39 antibody is administered second in time.
  • An anti-CD39 antibody or an antigen-binding fragment thereof described herein can replace or augment a previously or currently administered therapy.
  • administration of the one or more additional therapeutics can cease or diminish, e.g., be administered at lower levels.
  • administration of the previous therapy can be maintained.
  • a previous therapy will be maintained until the level of the anti-CD39 antibody reaches a level sufficient to provide a therapeutic effect.
  • the disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated anti-CD39 antibody, or antigen binding portion thereof, that binds to and antagonizes CD39, provided by the disclosure, in combination with one or more additional therapeutic agents or procedure, wherein the second therapeutic agent or procedure is selected from the group consisting of: a chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, surgical procedure, a radiation procedure, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, a vaccine, or a cellular immunotherapy, or a combination thereof.
  • the second therapeutic agent or procedure is selected from the group consisting of: a chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, surgical procedure, a radiation procedure, an activator of a costimulatory molecule,
  • the one or more additional therapeutic agents is a PD-1 antagonist, an adenosine A2AR antagonist, a CD73 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, chimeric antigen receptor (CAR) cell therapy, an anthracycline, or a combination thereof.
  • the one or more additional therapeutic agents is a combination of a CD73 inhibitor and an A2AR antagonist. In some embodiments, the one or more additional therapeutic agents is a combination of a PD-1 antagonist and an adenosine A2AR antagonist. In some embodiments, the one or more additional therapeutic agents is a PD-1 antagonist.
  • the PD-1 antagonist is selected from the group consisting of: PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In some embodiments, the PD-1 antagonist is selected from the group consisting of: FAZ053, Atezolizumab, Avelumab, Durvalumab, and BMS-936559.
  • the one or more additional therapeutic agents is an adenosine A2AR antagonist.
  • the adenosine A2AR antagonist is selected from the group consisting of: NIR178, CPI-444, AZD4635, Vipadenant, GBV-2034, and AB928.
  • the adenosine A2AR antagonist is CPI-444.
  • the one or more additional therapeutic agents is a CD73 inhibitor.
  • the CD73 inhibitor is selected from the group consisting of: AB421, MEDI9447, and BMS-986179.
  • the one or more additional therapeutic agents is a CTLA-4 inhibitor.
  • the CTLA-4 inhibitor is Ipilimumab or Tremelimumab.
  • the one or more additional therapeutic agents is a TIM-3 inhibitor.
  • the TIM-3 inhibitor is MGB453 or TSR-022.
  • the one or more additional therapeutic agents is a LAG-3 inhibitor.
  • the LAG-3 inhibitor is selected from the group consisting of LAG525, BMS-986016, and TSR-033.
  • the one or more additional therapeutic agents is a chimeric antigen receptor (CAR) cell therapy.
  • the CAR cell therapy is CTL019.
  • the one or more additional therapeutic agents is an anthracycline.
  • the anthracycline is selected from doxorubicin, daunorubicin, epirubicin, idarubicin, and valrubicin.
  • the anthracycline is doxorubicin.
  • a chemotherapeutic agent is used in combination with an anti-CD39 antibody described herein.
  • chemotherapeutic agents include, but are not limited to, anthracyclines (e.g., doxorubicin, idarubicin, daunorubicin, cytarabine, epirubicin, valrubicin and mitoxantrone) (see e.g., Minotti et al., (2004) Pharmacol Rev 56(2):185-229), topoisomerase inhibitors (e.g., topotecan; Hycamtin, camptothecin, etoposide) (see e.g., Pommier et al., (2010) Chem Biol 17(5):421-433; which is incorporated herein by reference in its entirety), bleomycin (Kimura et al., (1972) Cancer 29(1):58-60), gemcitabine (Plunkett et al., (1995) Semin Oncol 22(4 Supp
  • cyclophosphamide, bendamustine (Leoni et al., (2008) Clin Cancer Res 14(1):309-317), CHOP (drug combination of cyclophosphamide, doxorubicin hydrochloride, vincristine and prednisone) (Dunleavy (2014) Hematology Am Soc Hematol Educ Program 2014(1):107-112), and fluorouracil and derivatives thereof (Alvarez et al., (2012) Expert Opin Ther Pat 22(2):107-123, which is incorporated herein by reference in its entirety).
  • chemotherapeutic agents e.g. anthracyclines
  • ICD immunogenic cell death
  • the agent that induces ICD is an anthracycline.
  • the anthracycline is selected from doxorubicin, daunorubicin, epirubicin, idarubicin, and valrubicin.
  • the anthracycline is doxorubicin.
  • the agent that induces ICD is a platinum derivative.
  • the platinum derivative is selected from oxaliplatin, carboplatin, and cisplatin. In some embodiments, the platinum derivative is oxaliplatin.
  • chemotherapeutic agents suitable for combination and/or co-administration with compositions of the present invention include, for example: taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxyanthrancindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.
  • antimetabolites e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine
  • alkylating agents e.g.
  • anthracycline
  • the anti-CD39 antibodies, or antigen binding portions thereof, provided by the disclosure are combined (e.g., administered in combination) with one or more PD-1 antagonist that binds to human PD-1 or PD-L1 and inhibits PD-1/PD-L1 biological activity and/or downstream pathway(s) and/or cellular processed mediated by human PD-1/PD-L1 signaling or other human PD-1/PD-L1-mediated functions.
  • one or more PD-1 antagonist that binds to human PD-1 or PD-L1 and inhibits PD-1/PD-L1 biological activity and/or downstream pathway(s) and/or cellular processed mediated by human PD-1/PD-L1 signaling or other human PD-1/PD-L1-mediated functions.
  • PD-1 antagonists that directly or allosterically block, antagonize, suppress, inhibit or reduce PD-1/PD-L1 biological activity, including downstream pathways and/or cellular processes mediated by PD-1/PD-L1 signaling, such as receptor binding and/or elicitation of a cellular response to PD-1/PD-L1. Also provided herein are PD-1 antagonists that reduce the quantity or amount of human PD-1 or PD-L1 produced by a cell or subject.
  • the PD-1 antagonist inhibits PD-1 signaling or function. In some embodiments, the PD-1 antagonist blocks binding of PD-1 to PD-L1, PD-L2, or to both PD-L1 and PD-L2. In some embodiments, the PD-1 antagonist blocks binding of PD-1 to PD-L1. In some embodiments, the PD-1 antagonist blocks binding of PD-1 to PD-L2. In some embodiments, the PD-1 antagonist blocks the binding of PD-1 to PD-L1 and PD-L2. In some embodiments, the PD-1 antagonist binds PD-1. In some embodiments, the PD-1 antagonist binds PD-L1. In some embodiments, the PD-1 antagonist binds PD-L2.
  • the PD-1 antagonist inhibits the binding of PD-1 to its cognate ligand. In some embodiments, the PD-1 antagonist inhibits the binding of PD-1 to PD-L1, PD-1 to PD-L2, or PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 antagonist does not inhibit the binding of PD-1 to its cognate ligand.
  • the PD-1 antagonist is an isolated antibody (mAb), or antigen binding fragment thereof, which binds to PD-1 or PD-L1. In some embodiments, the PD-1 antagonist is an antibody or antigen binding fragment thereof that binds to human PD-1. In some embodiments, the PD-1 antagonist is an antibody or antigen binding fragment thereof that binds to human PD-L1. In some embodiments, the PD-1 antagonist is an antibody or antigen binding fragment that binds to human PD-L1 and inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-1 antagonist is an antibody or antigen binding fragment that binds to human PD-1 and inhibits the binding of PD-L1 to PD-1.
  • mAb isolated antibody
  • the PD-1 antagonist is an antibody or antigen binding fragment thereof that binds to human PD-1. In some embodiments, the PD-1 antagonist is an antibody or antigen binding fragment thereof that binds to human PD-L1. In some embodiments, the PD-1 antagonist
  • the PD-1 antagonist is pembrolizumab. In some embodiments, the PD-1 antagonist is nivolumab.
  • anti-human PD-L1 antibodies, or antigen binding fragments thereof, that may comprise the PD-1 antagonist in any of the compositions, methods, and uses provided by the disclosure include, but are not limited to: BAVENCIO® (avelumab, MSB0010718C, see WO2013/79174, which is incorporated herein by reference in its entirety; Merck/Pfizer), IMFINZI® (durvalumab, MEDI4736), TECENTRIQ® (atezolizumab, MPDL3280A, RG7446; see WO2010/077634, which is incorporated herein by reference in its entirety; Roche), MDX-1105 (BMS-936559, 12A4; see U.S. Pat. No.
  • the PD-1 antagonist is avelumab.
  • the PD-1 antagonist is durvalumab.
  • the PD-1 antagonist is atezolizumab.
  • the PD-1/PD-L1 antagonist is a small molecule, a nucleic acid, a peptide, a peptide mimetic, a protein, a carbohydrate, a carbohydrate derivative, or a glycopolymer.
  • exemplary small molecule PD-1 inhibitors are described in Zhan et al., (2016) Drug Discov Today 21(6):1027-1036.
  • an anti-CD39 antibody, or antigen binding portion thereof, provided by the disclosure is combined (e.g., administered in combination) with a CD73 inhibitor.
  • CD73 inhibitors include AB421 (Arcus), an antibody, or antigen binding portion thereof, that binds to CD73 such as MEDI9447 (Medimmune), BMS-986179 (Bristol Meyers Squibb), or such as described in US2018/0009899 (Corvus), which is incorporated herein by reference in its entirety.
  • an anti-CD39 antibody, or antigen binding portion thereof, provided by the disclosure is combined (e.g., administered in combination) with an adenosine A2A receptor (A2AR) antagonist.
  • A2AR antagonists include Preladenant/SCH 420814 (Merck/Schering, CAS Registry Number: 377727-87-2), which is described in Hodgson et al., (2009) J Pharmacol Exp Ther 330(1):294-303 and incorporated herein by reference in its entirety; ST-4206 (Leadiant Biosciences), which is described in U.S. Pat. No.
  • an anti-CD39 antibody, or antigen binding portion thereof, provided by the disclosure is combined (e.g., administered in combination) with a CTLA-4 inhibitor.
  • the CTLA-4 inhibitor is an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.
  • the CTLA-4 inhibitor is Ipilimumab (Yervoy®, Bristol-Myers Squibb).
  • the CTLA-4 inhibitor is Tremelimumab (Pfizer).
  • the antibody Ipilimumab and other anti-CTLA-4 antibodies are disclosed in U.S. Pat. No. 6,984,720, which is incorporated herein by reference in its entirety.
  • the antibody Tremelimumab and other anti-CTLA-4 antibodies are disclosed in U.S. Pat. No. 7,411,057, which is incorporated herein by reference in its entirety.
  • an anti-CD39 antibody, or antigen binding portion thereof, provided by the disclosure is combined (e.g., administered in combination) with a TIM-3 inhibitor.
  • the TIM-3 inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.
  • the TIM-3 inhibitor is chosen from MGB453 (Novartis), TSR-022 (Tesaro), or LY3321367 (Eli Lilly).
  • the anti-CD39 antibody, or antigen binding portion thereof is administered in combination with MGB453.
  • the anti-CD39 antibody, or antigen binding portion thereof is administered in combination with TSR-022.
  • an anti-CD39 antibody, or antigen binding portion thereof, provided by the disclosure is combined (e.g., administered in combination) with a LAG-3 inhibitor.
  • the LAG-3 inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • the LAG-3 inhibitor is chosen from LAG525 (Novartis), BMS-986016 (Bristol-Myers Squibb), TSR-033 (Tesaro), MK-4280 (Merck & Co), or REGN3767 (Regeneron).
  • an anti-CD39 antibody, or antigen binding portion thereof is combined (e.g. administered in combination) with one or more additional therapeutics, wherein the one or more additional therapeutics comprises a cell, e.g., an immune effector cell, comprising an chimeric antigen receptor (CAR).
  • the CAR comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain.
  • the intracellular signaling domain comprises one or both of a primary signaling domain and a costimulatory domain.
  • the CAR may further comprise a leader sequence, optionally, a hinge sequence.
  • the antigen binding domain binds to a tumor antigen.
  • the antigen binding domain comprising the CAR can be any domain that binds to an antigen of, including but not limited, to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment or portion thereof, including but not limited to a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen binding domain.
  • the antigen binding domain of the CAR is a scFv antibody fragment.
  • the CAR comprises an antigen binding domain that binds to a tumor antigen selected from the group consisting of: CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDG1cp(1-i)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kina
  • the antigen binding domain of the CAR binds to CD19.
  • An exemplary CAR that binds to CD19 is described in US2015/0283178 (e.g., CTL019), which is incorporated herein by reference in its entirety.
  • the CD19 CAR comprises an amino acid sequence shown in US2015/0283178 or a sequence substantially identical thereto (e.g., a sequence having at least about 85%, 90%, or 95% sequence identity thereto).
  • the CAR comprises a transmembrane domain derived from a protein selected from the group consisting of: the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • the CAR comprises an intracellular signaling domain derived from a protein selected from the group consisting of: a MHC class I molecule, a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD5, ICAM-1, LFA-1 (CDL 1a/CD18), 4-1BB (CD137), B7-H3, CD5, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma
  • the cell comprising a CAR comprises a nucleic acid encoding the CAR.
  • the nucleic acid encoding the CAR is a lentiviral vector.
  • the nucleic acid encoding the CAR is introduced into the cells by lentiviral transduction.
  • the nucleic acid encoding the CAR is an RNA, e.g., an in vitro transcribed RNA.
  • the nucleic acid encoding the CAR is introduced into the cells by electroporation.
  • immune effector cells include T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • T cells e.g., alpha/beta T cells and gamma/delta T cells
  • B cells natural killer (NK) cells
  • natural killer T (NKT) cells e.g., myeloid-derived phagocytes.
  • Monitoring a subject for an improvement in a cancer, as defined herein, means evaluating the subject for a change in a disease parameter, e.g., a reduction in tumor growth.
  • the evaluation is performed at least one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration.
  • the subject can be evaluated in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered.
  • Evaluation can include evaluating the need for further treatment, e.g., evaluating whether a dosage, frequency of administration, or duration of treatment should be altered. It can also include evaluating the need to add or drop a selected therapeutic modality, e.g., adding or dropping any of the treatments for a cancer described herein.
  • an anti-CD39 antibody or an antigen-binding fragment thereof described herein can be employed in methods of detection and/or quantification of human CD39 in a biological sample.
  • CD39 has been identified as a potential diagnostic, prognostic and progression biomarker of diseases (Pulte et al., (2011) Clin Lymphoma Myeloma Leuk 11:367-372; Fan et al., (2017) Biomark Med 11:107-116; Zhao et al., (2017) Front Immunol 8:727)
  • CD39 antigens (recombinant CD39; R&D systems cat #4397-EN) were biotinylated using the EZ-Link Sulfo-NHS-Biotinylation Kit from Pierce.
  • Goat F(ab′)2 anti-human kappa-FITC (LC-FITC), ExtrAvidin-PE (EA-PE) and Streptavidin-AF633 (SA-633) were obtained from Southern Biotech, Sigma, and Molecular Probes, respectively. Streptavidin MicroBeads and MACS LC separation columns were purchased from Miltenyi Biotec.
  • Goat anti-human IgG-PE Human-PE was obtained from Southern Biotech.
  • wash buffer phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA)
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • Light chain batch diversification protocol Heavy chain plasmids from a naive selection output were extracted from the yeast via smash and grab, propagated in and subsequently purified from E. coli , and transformed into a light chain library with a diversity of 5 ⁇ 10 6 . Selections were performed with one round of MACS and three rounds of FACS as described in the na ⁇ ve discovery. In the different FACS rounds the libraries were looked at for PSR binding, species cross-reactivity, and affinity pressure by antigen titration. Sorting was performed in order to obtain a population with the desired characteristics.
  • CDRH1 and CDRH2 selection The CDRH3 of a single antibody was recombined into a premade library with CDRH1 and CDRH2 variants of a diversity of 1 ⁇ 10 8 and selections were performed with one round of MACS and four rounds of FACS as described in the na ⁇ ve discovery. In the different FACS rounds the libraries were looked at for PSR binding, species cross-reactivity, and affinity pressure by titration, and sorting was performed in order to obtain a population with the desired characteristics.
  • Yeast clones were grown to saturation and then induced for 48 h at 30° C. with shaking. After induction, yeast cells were pelleted and the supernatants were harvested for purification. IgGs were purified using a Protein A column and eluted with acetic acid, pH 2.0. Fab fragments were generated by papain digestion and purified over KappaSelect (GE Healthcare LifeSciences).
  • ForteBio affinity measurements were performed on an Octet RED384 generally as previously described (see, e.g., Estep et al, High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs 5(2), 270-278 (2013)). Briefly, ForteBio affinity measurements were performed by loading IgGs on-line onto AHC sensors. Sensors were equilibrated off-line in assay buffer for 30 min and then monitored on-line for 60 seconds for baseline establishment. Sensors with loaded IgGs were exposed to 100 nM antigen for 3 minutes, and afterwards were transferred to assay buffer for 3 min for off-rate measurement. All kinetics were analyzed using the 1:1 binding model.
  • Epitope binning/ligand blocking was performed using a standard sandwich format cross-blocking assay. Control anti-target IgG was loaded onto AHQ sensors and unoccupied Fc-binding sites on the sensor were blocked with an irrelevant human IgG1 antibody. The sensors were then exposed to 100 nM target antigen followed by a second anti-target antibody or ligand. Additional binding by the second antibody or ligand after antigen association indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (competitor or ligand blocking).
  • CD86 a transmembrane protein that provides a co-stimulatory signal necessary for T cell activation and survival.
  • Monocytes from 3 healthy donors were treated with GM-CSF (50 ng/mL) (R&D Systems) and IL-4 (10 ng/mL) (R&D Systems) for 4 days at 37C in RPMI-1640+10% FBS+1% Penicillin-streptomycin (R10) (Life Technologies) to generate immature dendritic cells.
  • Dendritic cells were washed with and resuspended in R10 supplemented with GM-CSF (50 ng/mL) and IL-4 (10 ng/mL).
  • Dendritic cells were added to each well of a 96-well U-bottom plate. Dendritic cells were incubated with isotype control antibody or anti-CD39 antibodies (10 ug/mL), as indicated in FIG. 1A , for 1 h at 37 C. After 1 h, R10 with or without ATP supplementation was added to the cells and further incubated for 24 h. Dendritic cells were stained with antibodies against CD86 and CD11c (Biolegend). Cells were acquired using a LSRFortessa X-20 (BD Biosciences) and analyzed with FlowJo software (Tree Star). Quantitation of CD86 expression (GeoMean) on dendritic cells derived from each donor is shown.
  • the expression level of both CD86 and the human leukocyte antigen-antigen D related (HLA-DR), an MEW class II cell surface receptor was determined essentially as described above. Dendritic cells were treated with the anti-CD39 antibody SRF367-A or an isotype control antibody (DNP-A) in the presence or absence of ATP, as indicated in FIG. 1B .
  • HLA-DR human leukocyte antigen-antigen D related
  • DNP-A isotype control antibody
  • cytokine secretion To determine the effect of anti-CD39 antibodies on cytokine secretion, the secretion of cytokines IL-16, IL-12/IL-23p40 and VEGFA from the same dendritic cells as in FIG. 1B was determined. Supernatants from treated dendritic cell cultures were diluted 1:2 and a Meso Scale Discovery (MSD) U-plex kit protocol was followed per manufacturer's instructions. Secretion of VEGF-A, IL-12/IL-23p40 and IL-16 were quantitated using MSD software according to manufacturer's instructions.
  • MSD Meso Scale Discovery
  • FIGS. 1C, 1D, and 1E treatment of dendritic cells with the anti-CD39 antibody SRF367-A in the presence of ATP resulted in an increase in secretion of IL-16 ( FIG. 1C ), IL-12/IL-23p40 ( FIG. 1D ) and IL-16 ( FIG. 1E ) that was higher than with treatment the isotype control antibody DNP-A.
  • FIGS. 1C, 1D, and 1E demonstrate that treatment of dendritic cells with the anti-CD39 antibody SRF367-A enhances the ATP-induced secretion of cytokines IL-16, IL-12/IL-23p40 and VEGFA, respectively.
  • CD4+ T cells To determine an effect of treatment with anti-CD39 antibodies on CD4+ T cells, the amount of proliferation of CD4+ T cells in vitro in response to treatment with a range of concentrations of anti-CD39 antibodies (SRF367-A, SRF367-B, and SRF370-A) or isotype control antibodies (DNP-A and DNP-B) was determined.
  • CD4+ cells from freshly PBMCs from human donor blood were stained with cell trace violet stain prior to seeding in 96-well plates. Cells were incubated with 250 ⁇ M ATP, anti-CD3/CD28 beads to stimulate the T-cells, and anti-CD39 or isotype control antibodies, as indicated in FIG.
  • CD39 is a membrane bound ectonucleosidase that converts adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to adenosine monophosphate (AMP).
  • ATP adenosine triphosphate
  • ADP adenosine diphosphate
  • AMP adenosine monophosphate
  • % INH Normalized percent inhibition
  • time zero control was a well with all of the reagents where the reaction is stopped immediately to mimic conditions where no phosphate is generated and CD39 is completely inhibited.
  • the ‘no antibody control’ is a well where all of the reagents and cells are added but no antibodies are present. This well mimics conditions where the maximal amount of phosphate is released and there is no inhibition of CD39.
  • percent inhibition the ‘no antibody control’ value is subtracted from the assay value and divided by the ‘no antibody control’ value subtracted from the ‘time zero control’ value.
  • MOLP-8 human multiple myeloma cell line
  • SK-MEL-28 primary human B cells (isolated from whole blood), or primary human monocytes (isolated from whole blood) were used in this assay.
  • MOLP-8 cells a human multiple myeloma cell line
  • MOLP-8 cells a human multiple myeloma cell line
  • a range of concentrations of anti-CD39 antibodies SRF360-A, SRF360-B, SRF365-A, SRF367-A, SRF367-B, and SRF370-A
  • ATP a control antibody
  • Inhibition of CD39 activity was determined by the extent of inorganic phosphate released and expressed as % inhibition (% INH).
  • MOLP-8 or SK-MEL-28 cells were treated with a range of concentrations of fluorescently-labeled anti-CD39 antibodies (SRF360-A, SRF360-B, SRF365-A, SRF367-A, SRF367-B, and SRF370-A) or isotype control antibodies (DNP-A and DNP-B), as indicated in FIGS. 4A and 4 B.
  • the extent of binding to cells was determined and expressed as mean fluorescent intensity (MFI). Cells were washed with FACS Buffer (2 mM EDTA, 2% FBS) and pelleted by centrifugation.
  • the cells were resuspended in FACS buffer containing a dose range of anti-CD39 or isotype control antibodies directly labelled with fluorophore Alexa Fluor 488 (AF488) and incubated for 30 minutes at room temperature. Cells were then washed twice with FACS buffer followed by fixation in 4% paraformaldehyde (PFA) and resuspended in FACS buffer and analyzed on a FACS Canto II analyzer (BD Biosciences).
  • FACS buffer containing a dose range of anti-CD39 or isotype control antibodies directly labelled with fluorophore Alexa Fluor 488 (AF488) and incubated for 30 minutes at room temperature. Cells were then washed twice with FACS buffer followed by fixation in 4% paraformaldehyde (PFA) and resuspended in FACS buffer and analyzed on a FACS Canto II analyzer (BD Biosciences).
  • PFA paraformaldehyde
  • Anti-CD39 antibodies SRF365-A, SRF367-A, SRF367-B, and SRF370-A bound to SK-MEL-28 to a lesser extent relative to SRF360-A and SRF360-B, but to a greater extent relative to the isotype control antibodies, consistent with the results shown in FIG. 4A .
  • anti-CD39 antibodies were tested for anti-tumor activity against established tumors in vivo.
  • Mice bearing SK-MEL-28 tumors were treated with either SRF367-B or an isotype control antibody, as indicated in FIGS. 5A and 5B , and tumor growth was measured.
  • the SK-MEL-28 human xenograft model was chosen because it expresses high amounts of CD39.
  • V the tumor volume
  • L the long diameter
  • W the short diameter.
  • All antibodies were dosed intraperitoneally (i.p.) at 400 ⁇ g/mouse BIW in 100 ⁇ l of PBS for a total of 5 injections (Days 1, 5, 8, 12, and 15).
  • PBMCs were isolated from the whole blood of 5 separate human donors. The PBMCs were incubated in complete cell culture media (10% FBS, RPMI or complete cell culture media supplemented with 10 ug/ml anti-PD-1 antibody Nivolumab) for 96 hours. Cells were washed, stained and fixed for flow cytometry analysis to determine the percentage of CD39 positive cells. Lineage markers were used to discriminate Tregs (CD3, CD4 and FoxP3); Monocytes (CD14); and, B-cells (CD19). As shown in FIG.
  • increased expression of CD39 in subjects treated with anti-PD1 (or anti-PD-L1) may be the mechanism by which subjects become resistant to anti-PD1 therapy. Treatment of these subjects with anti-CD39 antibodies disclosed herein is provided.
  • binding affinities of anti-CD39 antibodies were determined by measuring their kinetic constants (k a , k d , K D ) using a ForteBio Octet RED384 (Pall Forte Bio Corporation, Menlo Park, Calif.) generally as previously described (Estep et al. (2013) Mabs 5(2):270-278, which is incorporated herein by reference in its entirety).
  • Carrier free human CD39-His lacking transmembrane domains was biotinylated and used as the antigen (R&D Systems Cat: 4397-EN-010). Fortebio and MSD affinity measurements for the anti-CD39 antibodies are provided in FIG. 7 .
  • SCID severe combined immunodeficient mice. Briefly, 6-8 week-old SCID mice (Charles River Labs) were inoculated by subcutaneous injection into right flank with 1 ⁇ 107 MOLP-8 tumor cells in 0.1 mL of PBS mixed with Matrigel (1:1) and randomized into 4 treatment groups when tumors reached a mean volume of approximately 100 mm3.
  • mice were treated intraperitoneally (i.p) with an isotype control antibody (DNP-A), an anti-CD39 antibody (SRF367-A) alone (400 ⁇ g or 20 mg/kg) twice a week for 3 weeks, Aldoxorubicin alone (a doxorubicin prodrug that releases free doxorubicin in the tumor environment) (200 ⁇ g or 10 mg/kg) once a week for 3 weeks or both SRF367-A and Aldoxorubicin in combination. All antibodies tested were formulated in PBS (Gibco). Aldoxorubicin stock solution (100 mg/mL) was prepared in DMSO and diluted to 1 mg/mL in PBS.
  • DNP-A isotype control antibody
  • SRF367-A anti-CD39 antibody
  • Aldoxorubicin alone a doxorubicin prodrug that releases free doxorubicin in the tumor environment
  • All antibodies tested were formulated in PBS (Gibco).
  • Anti-tumor activity was determined, in part, by measuring tumor size (length and width) using a Vernier caliper and tumor volume was calculated using the following formula: (L*W*W)/2. Body weight (data not shown) and tumor volumes were determined twice weekly until day 19. For tumor volume analysis, a one-way Anova analysis was performed to test statistical significance for each group compared to control (p ⁇ 0.005).
  • FIG. 8A shows mean tumor volumes in mice over time following treatment as indicated. Black arrows indicate treatment with SRF367-A and grey arrows indicate treatment with Aldoxorubicin.
  • FIG. 8B shows the mean tumor volumes in mice on day 19 following treatment as indicated.
  • the anti-tumor activity of the anti-CD39 antibody SRF367-A in combination with the A2AR antagonist CPI-444 was evaluated in a subcutaneous xenograft MOLP-8 human multiple myeloma model in severe combined immunodeficient (SCID) mice. Briefly, 6-8-week-old SCID mice (Charles River Labs) were inoculated by subcutaneous injection into right flank with 1 ⁇ 107 MOLP-8 tumor cells in 0.1 mL of PBS mixed with Matrigel (1:1) and randomized into 4 treatment groups when tumors reached a mean volume of approximately 100 mm3.
  • Anti-tumor activity was determined, in part, by measuring tumor size (length and width) using a Vernier caliper and tumor volume was calculated using the following formula: (L*W*W)/2. Body weight (data not shown) and tumor volumes were determined thrice weekly until day 28. For tumor volume analysis, a one-way Anova was performed to test statistical significance for each group compared to control and to the single agent alone treatment arms. (p ⁇ 0.005).

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