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WO2020069382A1 - Anti-cd137 binding molecules having engineered fc domains and therapeutic uses thereof - Google Patents

Anti-cd137 binding molecules having engineered fc domains and therapeutic uses thereof Download PDF

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Publication number
WO2020069382A1
WO2020069382A1 PCT/US2019/053558 US2019053558W WO2020069382A1 WO 2020069382 A1 WO2020069382 A1 WO 2020069382A1 US 2019053558 W US2019053558 W US 2019053558W WO 2020069382 A1 WO2020069382 A1 WO 2020069382A1
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Prior art keywords
substitution
seq
binding molecule
cdl37
antibody
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PCT/US2019/053558
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French (fr)
Inventor
Jieyi Wang
Yi Wu
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Lyvgen Biopharma Co., Ltd.
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Priority to CN201980064263.XA priority Critical patent/CN113039201A/en
Publication of WO2020069382A1 publication Critical patent/WO2020069382A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • 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
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • CD137 Cluster of differentiation 137
  • 4-1BB and TNFRSF9 tumor necrosis factor receptor superfamily member 9
  • APC antigen-presenting cell
  • CD 137 is a member of the tumor necrosis factor (TNF)-receptor superfamily and activates various immune competent cells, leading to activation, cytokine induction, prevention of activation-induced cell death (AICD), upregulation of cytotoxic T lymphocyte (CTL) activity, and increased survival.
  • TNF tumor necrosis factor
  • AICD activation-induced cell death
  • CTL cytotoxic T lymphocyte
  • CD 137 binds with high affinity to its ligand, CD137L (4-1BBL), which is expressed on antigen-presenting cells, such as dendritic cells, macrophages, and activated B cells.
  • CD 137 is predominantly found on various hematopoietic cells, such as primed T cells, natural killer cells, neutrophils, monocytes, dendritic cells, and mast cells. Its activation has been shown to reverse tolerance to tumor-specific antigens, leading to antigen-specific antitumor immunity.
  • Fc receptors are a family of immune cell surface proteins capable of binding to the Fc portion of antibodies. There are several different types of Fc receptors, including Fey receptors, Fca receptors, Fes receptors, and neonatal Fc receptors (FcRn), which have different binding activities to IgG, IgA, IgE, and IgG antibodies, respectively.
  • the Fey receptor subfamily includes FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIB (CD32c), FcyRIIIA (CDl6a), and FcyRIIIB (CDl6b).
  • FcyRI has high binding affinity to IgGl and IgG3 antibodies, while the other FcyRs have low binding affinity to IgG antibodies.
  • FcyRIII receptors expressed on NK cells and macrophages, bind to antibodies that are attached to infected cells or invading pathogens and trigger antibody-mediated phagocytosis (ADCP) or antibody-dependent cell-mediated cytotoxicity (ADCC) of the immune cells, thereby leading to elimination of the infected cells or invading pathogens.
  • ADCP antibody-mediated phagocytosis
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcyRII receptors expressed on B cells and dendritic cells, can down regulate the activity of the immune cells when binding to IgG antibodies.
  • CD 137-binding molecules e.g CD137 agonists or CD137 antagonists
  • CD137-binding molecules comprising an engineered (variant)
  • Fc region e.g., an engineered Fc region of IgGl, IgG2, or IgG4 molecules
  • desired Fc receptor binding activity and/or selectivity e.g., enhanced binding affinity and/or selected to FcyRIIB or substantially reduced binding affinity to one or more FcyR receptors.
  • CDl37-binding molecules can be used for modulating immune responses (e.g., selectively enhancing or selectively inhibiting an immune response) in subject in need of the treatment.
  • one aspect of the present disclosure provides a CD 137-binding molecule (e.g., a CD137 agonist or a CD137 antagonist) comprising a CD137 binding moiety and an antibody heavy chain constant region comprising an engineered Fc region, which comprises at least one mutation at any of positions 220-331, for example, any of positions 228-329, as compared to the wild-type Fc region counterpart.
  • a CD 137-binding molecule e.g., a CD137 agonist or a CD137 antagonist
  • an antibody heavy chain constant region comprising an engineered Fc region, which comprises at least one mutation at any of positions 220-331, for example, any of positions 228-329, as compared to the wild-type Fc region counterpart.
  • the numbering system is according to the EU index.
  • the CD 137 binding moiety is the extracellular domain of a CD137L, which may comprise the amino acid sequence of SEQ ID NO: 143.
  • a CD137- binding molecule comprising such a CD 137-binding moiety may be a CD 137 agonist.
  • the CD 137 binding moiety can be an anti-CD 137 antibody fragment.
  • the anti-CD 137 antibody fragment comprises a heavy chain that comprises a heavy chain variable region, which is linked to any of the engineered Fc regions described herein, and a light chain that comprises a light chain variable region, and a light chain constant region.
  • the variant Fc region may have an enhanced binding affinity and/or selectivity to FcyRIIB relative to the wild-type counterpart. In other instances, the variant Fc region may have substantially reduced binding affinity to one or more FcyR receptors. In some examples, the variant Fc region has low or no binding activity to all FcyR receptors.
  • the CDl37-binding molecule may comprise a variant Fc region of an IgGl molecule (e.g., a human IgGl molecule), which may comprise a mutation comprising one or more of the following: (a) an amino acid substitution or deletion within positions 233-238 (e.g., 234-238); (b) a substitution at position 265; (c) a substitution at position 267; (d) a substitution at position 297; (e) a substitution at position 328, a substitution at position 329; or a combination thereof.
  • the Fc variant of an IgGl molecule may comprise one or more mutations at positions 220, 226, 229, 238, 273, 327, 330, and/or 331.
  • the mutation comprises substitutions at any of positions 233-236, a deletion at one or more of positions 236-238, or a combination thereof.
  • the substitutions at positions 233-235 may comprise E233P, L234V, L234F, L234A, L235A, and/or L235E.
  • the mutation comprises a deletion at one or more of positions 236-238.
  • the substitution at position 238 can be P238S
  • the substation at position 265 can be D265A, or a combination thereof.
  • the substitution is at position 267 and is S267E.
  • the substitution is at position 329 and is P329G.
  • the mutation may comprise a substitution at position 265, a substitution at position 297, or a combination thereof.
  • the substitution at position 265 may be D265A and the substitution at position 297 may be N297A.
  • the one or more substitutions at positions 220, 226, 229, 327, 330, and 331 can be C220S, C226S, C229S, A327G, A330S, and P331 S.
  • Exemplary variant Fc regions derived from human IgGl may be one of Glml, Glm2, Glml7, Glm27, GlmAA, GlmAAG, G1N297A, Glm240, and Glm40.
  • the CD 137-binding molecule may comprise an Fc region of an
  • IgG2 molecule e.g., a human IgG2 molecule
  • a mutation comprising one or more of the following: (a) a deletion of one or more of positions 237 and 238; (b) a substitution at position 265; (b) a substitution at position 267; (c) a substitution at position 297; (d) a substitution at position 328; or a combination thereof.
  • the deletion is at position 237 or at both positions 237 and 238.
  • the Fc variant of an IgG2 molecule may comprise at least one mutation comprising a substitution at one or more of positions 233-235, 237, 238, 268, 273, 330, and 331.
  • the substitution at one or more of positions 233-235, 237, 238, 268, 273, 330, and 331 can be P233E, V234A, V234L, A235L, A235S, G237A, P238S, H268A, H268Q, V273E, A330S, and P331S.
  • the substitution is at position 267 and is S267E.
  • the substitution is at position 328 and is L328F.
  • the mutation may comprise a substitution at position 265, a substitution as position 297, or a combination thereof.
  • the substitution at position 265 may be D265A and the substitution at position 297 may be N297A.
  • the variant Fc region derived from IgG2 can be one of G2ml, G2ml7, G2ml8, G2ml9, G2m20, G2m27, G2m28, G2m29, G2m2040, G2m43, G2G4, G2mAA, and G2m40.
  • the CDl37-binding molecule may comprise an Fc region of an IgG4 molecule (e.g. , a human IgG4 molecule), which may comprise (a) an amino acid residue substitution at position 228; (b) a substitution or deletion at any one positions 235- 238; (c) a substitution at position 265; (d) a substitution at position 267; (e) a substitution at position 297; (e) a substitution at position 328; or a combination thereof.
  • the substitution at position 228 is S228P.
  • the deletion is at one or more of positions 236, 237, and 238 (e.g. , at both positions 236 and 237).
  • the Fc variant of an IgG4 molecule may comprise a substitution at one or more of positions 233-235, 237, and 273.
  • the substitution at one or more positions 233- 235, 237, and 273 may be E233P, F234V, F234A, F235S, F235E, F235A, G237A, and V273E.
  • the substitution at position 267 is S267E.
  • the substitution at position 328 is F328F.
  • the mutation may comprise a substitution at position 228, a substitution at position 265, a substitution at position 297, or a combination thereof.
  • the substitution at position 228 may be S228P
  • the substitution at position 265 may be D265A
  • the substitution at position 297 may be N297A
  • the variant Fc region is one of G4ml, G4m2, G4m20, G4m28, G4m30, G4m4l, G4m42, G4m46, G4mPE, G4mAA, and G4m40.
  • any of the variant Fc regions described herein may exhibit an enhanced binding activity and/or an enhanced selectivity to FcyRIIB as compared with the wild-type Fc region.
  • the variant Fc regions described herein may have low or no binding activity to any of the FcyR receptors.
  • the variant Fc region may exhibit a decreased binding affinity to FcyRIIB.
  • the variant Fc region binds FcRn.
  • the anti-CD 137 antibody fragment in any of the CD 137-binding molecules described herein may be of a human antibody or a humanized antibody.
  • the anti-CD 137 antibody is an agonist antibody.
  • the anti-CD 137 antibody may comprise the same heavy chain complementary determining regions (HC CDRs) as those in SEQ ID NO: 128 or as those in any one of 20A12D11, 11E10D12, 23D2D6,
  • the antibody fragment may comprise a heavy chain variable region of SEQ ID NO: 128 and/or a light chain variable region of SEQ ID NO: 129. In other examples, the antibody fragment may comprise the same heavy chain variable region and/or the same light chain variable region as one of 20A12D11, 11E10D12, 23D2D6, 22F2C2, 30C11B4, and 26B3D7.
  • a pharmaceutical composition comprising any of the CD 137-binding molecules described herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition may be used to selectively modulate (e.g ., selectively activate or selectively inhibit) an immune response in a subject.
  • the present disclosure provides a method for selectively modulating (e.g. , selectively activating or selectively inhibiting) an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of a CD 137-binding molecule as described herein.
  • the subject can be a human patient having or suspected of having a cancer and the CD 137-binding molecule may be a CD 137 agonist.
  • exemplary cancers include lung cancer, stomach cancer, liver cancer, breast cancer, skin cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, colorectal cancer, sarcoma, bone cancer, lymphoma and a hematological cancer.
  • the subject can be a human patient having or suspected of having an immune -related disorder and the CDl37-binding molecule may be a CD137 antagonist.
  • immune disorders include autoimmune diseases, immune-deficiencies, or allergies.
  • the target disease for treatment is an autoimmune disease.
  • the disclosure provides an isolated anti-CD 137 antibody, which binds to the same epitope of CD 137 as a reference antibody selected from the group consisting of: 20A12D11, 11E10D12, 23D2D6, 22F2C2, 30C11B4, and 26B3D7 or competes against the reference antibody from binding to the epitope.
  • the antibody comprises a heavy chain complementary determining region 1 (CDR1), a heavy chain complementary determining region 2 (CDR2), and a heavy chain complementary determining region 3 (CDR3), which collectively are at least 85% identical to the respective heavy chain CDRs of the reference antibody; and/or wherein the antibody comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, which collectively are at least 85% identical to the respective light chain CDRs of the reference antibody.
  • CDR1 heavy chain complementary determining region 1
  • CDR2 heavy chain complementary determining region 2
  • CDR3 heavy chain complementary determining region 3
  • the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 collectively comprise 10 or fewer amino acid mutations relative to the respective heavy chain CDRs of the reference antibody; and/or wherein the light chain CDR1, light chain CDR2, and light chain CDR3 collectively comprise 10 or fewer amino acid mutations relative to the respective light chain CDRs of the reference antibody.
  • the antibody comprises a heavy chain variable region that is at least 85% identical to the heavy chain variable region of the reference antibody and/or a light chain variable region that is at least 85% identical to the light chain variable region of the reference antibody.
  • the heavy chain variable region comprises 10 or fewer amino acid residue mutations relative to the heavy chain variable region of the reference antibody; and/or and a light chain variable region that comprises 10 or fewer amino acid mutations relative to the light chain variable region of the reference antibody.
  • the anti-CD 137 antibody disclosed herein may comprise the same heavy chain variable region CDRs as the reference antibody and/or the same light chain variable region CDRs as the reference antibody.
  • any of the anti-CD 137 antibodies described herein may be a human antibody or a humanized antibody. Also provided herein is a pharmaceutical composition comprising any of the anti-CD 137 antibodies described herein and a pharmaceutically acceptable carrier.
  • the instant disclosure features an isolated nucleic acid or set of nucleic acids which collectively encode any of the CD 137 binding molecules or any of the anti-CD 137 antibodies described herein.
  • the nucleic acid or set of nucleic acids may be located on one or two vectors, for example, expression vectors. Also provided herein are host cells comprising such vector(s).
  • the instant disclosure also features pharmaceutical compositions comprising one or more of the CDl37-binding molecules described herein and/or one or more of the anti- CD 137 antibodies disclosed herein for use in treating a target disorder as described herein, or uses of such CDl37-binding molecules and/or anti-CDl37 antibodies for manufacturing a medicament for use in treating the target disorder.
  • FIGs. 1A-1W are charts showing binding activity of various IgG variants as indicated to different types of Fey receptors expressed on CHO-K1 cells at the various concentrations as indicated.
  • the concentrations of each IgG variant, from left to right, are 0 pg/ml, 0.3 pg/ml, 1 pg/ml, 3 pg/ml, and 10 pg/ml in FIGs. 1A-1C, 1E-1J, 1F-1M, 1P-1R, and 1T-1V.
  • FIGs. 1D, 1K, 10, 1S, and 1W the bars, from left to right, correspond to the following concentrations of IgG variant: 0.3 pg/ml, 1 pg/ml, 3 pg/ml, and 10 pg/ml.
  • FIGs. 1A-1D binding activity to FcyRI.
  • FIGs. 1E-1G binding activity to FcyRIIA(Hl3 l).
  • FIGs. 1H-1K binding activity to FcyRIIA(Rl 31).
  • FIGs. 1F- 10 binding activity to FcyRIIB.
  • FIGs. 1P-1S binding activity to FcyRIIC.
  • FIGs. 1T-1W binding activity to FcyRIII.
  • FIGs. 2A-2C are charts showing stimulation of human CD8 + T cells in co-culture with parental or FcyR expressing cells by a number of IgG variants as indicated by IFN-y secretion.
  • the groups, from left to right, correspond to: no OKT, 0.01 pg/ml, 0.03 pg/ml, 0.1 pg/ml, and 0.3 pg/ml, in FIG. 2A.
  • the groups, from left to right correspond to 0.01 pg/ml, 0.03 pg/ml, and 0.1 pg/ml.
  • FIG. 2A G2m2.
  • FIG. 2B various Fc variants as indicated.
  • FIG. 2C additional Fc variants as indicated.
  • FIGs. 3A-3D are charts showing binding activity of various IgG variants, as indicated, to different types of Fey receptors expressed on CHO-K1 cells at the various concentrations indicated.
  • concentrations of each IgG variant, from left to right, are 0.1 pg/ml, 0.3 pg/ml, 1 pg/ml, 3 pg/ml, 10 pg/ml, and 30 pg/ml.
  • FIG. 3A binding activity to FcyRI.
  • FIG. 3B binding activity to FcyRIIA.
  • FIG. 3C binding activity to FcyRIIB.
  • FIG. 3D binding activity to FcyRIIIA.
  • each IgG variant is a chart showing stimulation of human CD 137 activation in a reporter assay by the indicated IgG variants.
  • concentrations of each IgG variant from left to right, are 0.01 pg/ml, 0.03 pg/ml, 0.1 pg/ml, 0.3 pg/ml, 1 pg/ml, 3 pg/ml, and 10 pg/ml.
  • CD137-binding molecules e.g., CD137 agonists or CD137 antagonists
  • CD137 binding moiety e.g., an extracellular domain of a CD137 ligand or an anti-CD 137 antibody fragment
  • engineered (variant) Fc regions which may have altered binding affinity and/or specificity to one or more Fc receptors, for example, enhanced binding affinity to FcyRIIB, enhanced binding specificity to FcyRIIB, or substantially reduced binding affinity to one or more FcyR receptors (e.g. , low or no binding activity to all FcyR receptors).
  • CD 137-binding molecules are expected to exhibit unexpected, superior therapeutic activity.
  • CD 137-binding molecules having a variant Fc region that has enhanced binding affinity to FcyRIIB would be expected to exhibit enhanced agonistic activity and CD 137-binding molecules having a variant Fc region that has enhanced binding specificity to FcyRIIB would be expected to exhibit higher tumor selectivity relative to the wild-type counterpart.
  • CD 137-binding molecules having low or no binding affinity to FcyR receptors would help activating CD 137-positive immune cells in tumor
  • an Fc variant which may be of an IgG molecule (e.g., IgGl, IgG2, and IgG4 molecules such as human IgGl, human IgG2, and human IgG4 molecules), and uses thereof for modulating immune responses.
  • an Fc variant may have enhanced binding affinity to FcyRIIB (CD32B) relative to the wild-type counterpart and/or binding selectivity as relative to other Fc receptors such as FcyRIII (CD 16).
  • such an Fc variant may have substantially reduced binding affinity to one or more FcyR receptors (e.g., to all FcyR receptors) relative to the wild-type counterpart.
  • “Substantially reduced” means that the binding affinity of a Fc variant to a FcyR receptor is at least 60% lower (e.g. , 70% lower, 80% lower, 90% lower, 95% lower, 98% lower, or 99% lower) than the binding affinity of the wild-type counterpart to the same FcyR receptor.
  • the Fc variant may have low or no binding affinity to all FcyR receptors, i.e., binding affinity cannot be detected by conventional assays or binding affinity is substantially low such that no significant bioactivity would be triggered.
  • CDl37-binding molecules that comprise a CDl37-binding moiety linked to an engineered Fc region having altered binding affinity and/or specificity to one or more Fc receptors as described herein.
  • Such CD 137-binding molecules may be CD137 agonists, which are capable of triggering the signaling mediated by CD137/CD137L upon binding to cell surface CD137.
  • the CDl37-binding molecules described herein are CD 137 antagonists, which are capable of inhibiting the signaling mediated by CD137/CD137L upon binding to cell surface CD137.
  • the CDl37-binding molecules are useful in modulating (selectively modulating) immune responses when administered to a subject in need of the treatment.
  • a CD 137-binding moiety as described herein may be any peptide or polypeptide that binds CD137, for example human CD137.
  • CD137 is an immune cell receptor well known in the art.
  • NCBI GenBank Accession Nos. NP_00l552.2 and NP_00l070977 provide information for human and mouse CD137, respectively.
  • Provided below is an amino acid sequence of an exemplary human CD 137 polypeptide.
  • CD 137 polypeptides from other species are known in the art and can be obtained from publicly available gene databases, for example, GenBank, using either the human sequence or the mouse sequence as a query.
  • the CD 137-binding moieties can be a polypeptide comprising an extracellular domain of a CD137 ligand (CD137L or 4-1BBL).
  • CD137L is a membrane glycoprotein expressed antigen-presenting cells (APCs), namely, activated B and T cells.
  • CD137L can drive monocyte to dendritic cell differentiation.
  • the sequence of CD137L and its extracellular domain are well known in the art. For example, NCBI GenBank Accession No. NP_003802.1 (extracellular domain, amino acids 50-254) provides information for human CD137L. Provided below is an amino acid sequence of an extracellular domain of an exemplary human CD137L.
  • CD137L polypeptides from other species are known in the art and can be obtained from publicly available gene databases, for example, GenBank, using the human sequence as a query.
  • the CD 137-binding moiety described herein can comprise an anti-CDl37 antibody.
  • anti-CDl37 antibody refers to any antibody capable of binding to a CD137 polypeptide, which can be of a suitable source, for example, human or a non-human mammal (e.g., mouse, rat, rabbit, primate such as monkey, etc.).
  • the anti-CD 137 antibodies described herein comprise a heavy chain that comprises a heavy chain variable domain, which is linked to any of the Fc variants described herein, and optionally a light chain that comprises a light chain variable region and a light chain constant region.
  • the heavy chain variable region (V H ) and optionally the light chain variable region (V L ) are usually involved in antigen (CD 137 in this case) binding.
  • the V H and V L regions can be further subdivided into regions of hypervariability, also known as“complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as“framework regions” (“FR”).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Rabat definition, the Chothia definition, the AbM definition, and/or the contact definition, all of which are well known in the art. See, e.g.
  • the anti-CD 137 antibody as described herein can bind and inhibit the activity of CD137 by at least 50% (e.g. , 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).
  • the apparent inhibition constant (Ki app or K Lapp ) which provides a measure of inhibitor potency, is related to the concentration of inhibitor required to reduce enzyme activity and is not dependent on enzyme concentrations.
  • the inhibitory activity of an anti-CD 137 antibody described herein can be determined by routine methods known in the art.
  • the K L app value of an antibody may be determined by measuring the inhibitory effect of different concentrations of the antibody on the extent of the reaction (e.g. , enzyme activity); fitting the change in pseudo-first order rate constant (v) as a function of inhibitor concentration to the modified Morrison equation (Equation 1) yields an estimate of the apparent Ki value.
  • the Ki app can be obtained from the y-intercept extracted from a linear regression analysis of a plot of K L app versus substrate concentration.
  • the anti-CD 137 antibody described herein may have a Ki app value of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 pM or less for the target antigen or antigen epitope.
  • any of the anti-CD 137 antibodies may be further affinity matured to reduce the Ki app of the antibody to the target antigen or antigenic epitope thereof.
  • the anti-CD 137 antibody may suppress the signaling triggered by CD137/CD137L interaction by at least 50% (e.g. , 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).
  • Such inhibitory activity can be determined by conventional methods or the assays described herein.
  • the antibodies described herein can be murine, rat, human, or any other origin
  • Such antibodies are non-naturally occurring, i.e.. , would not be produced in an animal without human act (e.g. , immunizing such an animal with a desired antigen or fragment thereof or isolated from antibody libraries).
  • the anti-CD 137 antibody is a humanized antibody, which may have one of more of the elements or characteristics described below or elsewhere herein.
  • Humanized antibodies refer to forms of non-human (e.g. , murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.
  • the humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
  • Antibodies may have Fc regions modified as described in WO 99/58572.
  • humanized antibodies have one or more CDRs (one, two, three, four, five, or six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.
  • variable regions of V H and V L of a parent non-human antibody are subjected to three- dimensional molecular modeling analysis following methods known in the art. Next, framework amino acid residues predicted to be important for the formation of the correct
  • CDR structures are identified using the same molecular modeling analysis.
  • human V H and V L chains having amino acid sequences that are homologous to those of the parent non-human antibody are identified from any antibody gene database using the parent V H and V L sequences as search queries. Human V H and V L acceptor genes are then selected.
  • the CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the parent non-human antibody or functional variants thereof.
  • residues within the framework regions of the parent chain that are predicted to be important in interacting with the CDR regions can be used to substitute for the corresponding residues in the human acceptor genes.
  • the anti-CD 137 antibodies described herein specifically bind to the corresponding target antigen or an epitope thereof, e.g., CD 137 antigen or epitope.
  • An antibody that“specifically binds” to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit“specific binding” if it reacts more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen than it does with alternative targets.
  • An antibody“specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.
  • an antigen CD 137
  • an antigenic epitope therein is an antibody that binds this target antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens or other epitopes in the same antigen. It is also understood with this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen.
  • “specific binding” or“preferential binding” does not necessarily require (although it can include) exclusive binding.
  • an antibody that“specifically binds” to a target antigen or an epitope thereof may not bind to other antigens or other epitopes in the same antigen (i.e., only baseline binding activity can be detected in a conventional method).
  • the anti-CD 137 antibodies described herein specifically bind to
  • the anti-CDl37 antibody described herein specifically binds human CD 137 or a fragment thereof as relative to the mouse counterpart, or vice versa (e.g., having a binding affinity at least 10-fold higher to one antigen than the other as determined in the same assay under the same assay conditions).
  • the anti- CD 137 antibody described herein may cross-react to human and a non-human CD 137 (e.g., mouse), e.g., the difference in binding affinity to the human and the non-human CD 137 is less than 5-fold, e.g. , less than 2-fold, or substantially similar.
  • an anti-CD 137 antibody as described herein has a suitable binding affinity for the target antigen (e.g., CD137) or antigenic epitopes thereof.
  • binding affinity refers to the apparent association constant or K A .
  • the K A is the reciprocal of the dissociation constant (K D ).
  • the anti-CDl37 antibody described herein may have a binding affinity (KD) of at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 M, or lower for the target antigen or antigenic epitope.
  • KD binding affinity
  • An increased binding affinity corresponds to a decreased K D .
  • the antibody has specificity for the first antigen (e.g. , a first protein in a first conformation or mimic thereof) relative to the second antigen (e.g., the same first protein in a second conformation or mimic thereof; or a second protein). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10 5 fold. In some embodiments, any of the anti-CDl37 antibodies may be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.
  • Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay).
  • Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl,
  • anti-CD 137 antibodies are provided below (CDRs are indicated in boldface).
  • the anti-CD 137 antibodies described herein bind to the same epitope as any of the exemplary antibodies described herein or competes against the exemplary antibody from binding to the CD 137 antigen.
  • An“epitope” refers to the site on a target antigen that is recognized and bound by an antibody.
  • the site can be entirely composed of amino acid components, entirely composed of chemical modifications of amino acids of the protein ( ., glycosyl moieties), or composed of combinations thereof.
  • Overlapping epitopes include at least one common amino acid residue.
  • An epitope can be linear, which is typically 6-15 amino acids in length. Alternatively, the epitope can be conformational.
  • the epitope to which an antibody binds can be determined by routine technology, for example, the epitope mapping method (see, e.g. , descriptions below).
  • An antibody that binds the same epitope as an exemplary antibody described herein may bind to exactly the same epitope or a substantially overlapping epitope (e.g., containing less than 3 non-overlapping amino acid residue, less than 2 non-overlapping amino acid residues, or only 1 non-overlapping amino acid residue) as the exemplary antibody. Whether two antibodies compete against each other from binding to the cognate antigen can be determined by a competition assay, which is well known in the art.
  • the anti-CD 137 antibodies disclosed herein comprise the same heavy chain and light chain CDRs as those in (a) SEQ ID NO: 128 and SEQ ID NO: 129, (b) SEQ ID NO: 130 and SEQ ID NO: 131 (20A12D11); (c) SEQ ID NO: 132 and SEQ ID NO: 133 (11E10D12); (d) SEQ ID NO: 134 and SEQ ID NO: 135 (23D2D6); (e) SEQ ID NO: 136 and SEQ ID NO: 137 (22F2C2), (f) SEQ ID NO: 138 and SEQ ID NO: 139 (30C11B4), or SEQ ID NO: 140 and SEQ ID NO: 141 (26B3D7).
  • the anti-CDl37 antibody comprises a heavy chain variable domain of SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: l34, SEQ ID NO:l36, SEQ ID NO: 138, or SEQ ID NO: l40 and/or a light chain variable domain of SEQ ID NO: 129, SEQ ID NO: l3l, SEQ ID NO: l33, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, or SEQ ID NO: 141.
  • a functional variant comprises substantially the same V H and V L CDRS as any one of the exemplary anti-CD 137 antibodies.
  • it may comprise only up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid residue variations in the total CDR regions of the antibody (collectively) and binds the same epitope of CD 137 with substantially similar affinity (e.g., having a K D value in the same order).
  • amino acid residue variations are conservative amino acid residue substitutions.
  • a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
  • Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al, eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al, eds., John Wiley & Sons, Inc., New York.
  • amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
  • the anti-CD 137 antibody described herein may comprise heavy chain CDRs, which in combination (collectively) have at least 80% (e.g. , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and any incremental percent therein) sequence identity with the heavy chain CDRs in SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, or SEQ ID NO: 140.
  • the anti-CD 137 antibody may comprises light chain CDRs, which collectively have at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% and any incremental percent therein) sequence identity with those in SEQ ID NO: 129, SEQ ID NO: l3l, SEQ ID NO: l33, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, or SEQ ID NO: 141.
  • the anti-CD 137 antibody described herein may comprise a heavy chain variable domain that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%,
  • Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 25(l7):3389-3402, 1997.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the CD 137-binding molecules described herein comprise a modified heavy chain constant region, in which the Fc domain is modified to modulate its binding affinity and/or specificity to Fc receptors such as FcyRs.
  • the Fc variants in the CD 137-binding molecules described herein have enhanced selectivity to FcyRIIB relative to its wild-type counterpart.
  • An Fc fragment having selectivity to FcyRIIB, selectively binding to FcyRIIB, or specifically binding to FcyRIIB is a term well understood in the art.
  • a molecule is said to exhibit “selective binding” or“specific binding” if it reacts more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen (e.g., an FcyRIIB receptor) than it does with alternative targets (e.g., FcyRIII receptors).
  • an Fc fragment “specifically binds” to an Fc receptor if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other Fc receptors.
  • an Fc fragment that specifically (or preferentially) binds to FcyRIIB is an Fc fragment that binds this Fc receptor with greater affinity, avidity, more readily, and/or with greater duration than it binds to other Fc receptors. It is also understood with this definition that, for example, an Fc fragment that selectively or specifically binds to a first Fc receptor may or may not specifically or preferentially bind to a second Fc receptor.
  • an Fc fragment that“selectively binds,” or “specifically binds” to a target Fc receptor may not bind to other Fc receptors (i.e., binding not detectable by routine methods).
  • the variant Fc fragment does not bind to any FcyRs.
  • the Fc variants described herein may have enhanced selectivity to FcyRIIB relative to their wild-type counterparts (the wild-type parent Fc region in which mutations are introduced to produce the Fc variants).
  • the relative binding activity to FcyRIIB versus another Fc receptor (e.g. , FcyRIII) of such an Fc variant is higher than the relative binding activity to FcyRIIB versus the other Fc receptor (e.g., FcyRIII) of the wild-type counterpart.
  • the Fc variant may have enhanced binding activity to FcyRIIB and/or decreased binding activity to another Fc receptor, for example, FcyRIII.
  • the Fc variants described herein may have decreased binding activity to both FcyRIIB and another Fc receptor (for example, FcyRIII); however, the level of decreased binding activity to the other Fc receptor (e.g., FcyRIII) is greater than the level of decreased binding activity to FcyRIIB.
  • FcyRIII another Fc receptor
  • an Fc variant as described herein has a suitable binding affinity for FcyRIIB, e.g. , enhanced as compared with the wild-type parent Fc from which the Fc variant is derived.
  • binding affinity refers to the apparent association constant or K A .
  • the K A is the reciprocal of the dissociation constant (KD).
  • the Fc variant described herein may have a binding affinity (KD) of at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 M, or lower for FcyRIIB.
  • An increased binding affinity corresponds to a decreased K D .
  • Fc variants described herein have a higher binding affinity (a higher K A or smaller KD) to FcyRIIB as compared to the binding affinity to FcyRIII (either FcyRIIIA or FcyRIIIB). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10 5 fold.
  • the Fc variants described herein may be designed by mutating one or more amino acid residues in the wild-type of human IgGl, IgG2, or IgG4 Fc fragments in light of the amino acid residues in the corresponding mouse IgG, for example, mouse IgGl.
  • a sequence comparison of human and mouse IgGs is provided below (SEQ ID NOs: 60-64, from top to bottom, each representing a combination of fragments 211-245, 260-278, and 320-332 of the corresponding Fc region):
  • the Fc variants described herein is a human IgGl, G2, or G4 Fc variants comprising one or more mutations (e.g. , amino acid substitutions, deletions, or additions) in the hinge domain of an Fc fragment.
  • Human IgGs contain a core motif of CPPC or CPSC in the hinge domain (positions 226-229 according to the EU index). Positions 216 to 225 are deemed as the upper portion of the hinge domain and positions 230-238 are deemed as the lower portion of the hinge domain.
  • the numbering system used herein, unless explicitly indicated, is according to the EU index.
  • the one or more mutations can be located in the upper portion of the hinge domain.
  • the one or more mutations can be located in the lower portion of the hinge domain.
  • the Fc region of any of the anti-CDl37 molecules disclosed herein may comprise at least one mutation at any of positions 220-331, preferably at any of positions 228-329.
  • Such Fc variants may have altered binding affinity and/or specificity to one or more of Fc receptors (e.g ., FcyllB, FcyRI, FcyRIIA, or FcyRIIIA) as compared with their native counterparts.
  • the mutations to a human IgG Fc can be made according to the corresponding amino acid residues in the hinge domain of mouse IgGl.
  • mouse IgGl does not contain the GGP motif at positions 236-238. Accordingly, one or more of the residues in this GGP motif can be deleted from a human IgGl, IgG2, or IgG4 Fc fragment to produce the Fc variants described herein.
  • the human Fc variants may contain one or more amino acid substitutions in the upper portion, in the lower portion, or both of the hinge domain.
  • the Fc variant may comprise one or more amino acid substitutions at one or more of positions 233, 234, 235, and/or 236.
  • Such an amino acid substitution may be in combination with the deletion of one or more of the GGP motif (236-238) noted herein.
  • These mutations may be introduced into a human IgG2 or IgG4 Fc fragment to produce the Fc variants described herein.
  • the Fc variants described herein contains a deletion at one or more of the positions 236-238 (e.g., 236, 237, 238 or any combination thereof)
  • any of the mutations in the hinge domain described herein may be in combination with a mutation (e.g., amino acid substitutions) at one or more positions that are involved in interaction with an Fc receptor.
  • a mutation e.g., amino acid substitutions
  • positions include, but are not limited to, positions 265, 267, 273, 297, and 327-331, or a combination thereof.
  • Exemplary amino acid substitutions at those positions include D265A, S261E, V271E, N297A, F328F, P329G, A330S, and/or P331S.
  • the Fc variants disclosed herein can be derived from an IgGl molecule (e.g., human IgGl) and contain one or more mutations at positions at one or more of positions 220, 226, 229, 234-238, 265, 267, 297, and 327-331.
  • the Fc variant may comprise a substitution or deletion within positions 234-238, a substitution at position 265 (e.g ., D265A), a substitution at position 267 (e.g., S267E), a substitution at position 297 ( e.g ., N297A), a substitution at position 328 (e.g.
  • the Fc variants derived from an IgGl molecule may comprise a deletion at one or more of positions 236-238.
  • the Fc variants disclosed herein can be derived from an IgG2 molecule (e.g., human IgG2) and contain one or more mutations at one or more of positions 233-235, 237-238, 265-268, 273, 297, 328, 330, and 331.
  • a Fc variant may comprise comprises a deletion within positions 237-238 (e.g. , a deletion at position 237 or a deletion of both positions 237 and 238), a substitution at position 265 (e.g.
  • the Fc variant derived from an IgG2 molecule may comprise a substitution at one or more of positions 233-235, 237, 238, 268, 273, 330, and 331 (e.g., P233E, V234A, V234F, A235F, A235S, G237A, P238S, H268A, H268Q, V273E, A330S, and P331S).
  • the Fc variants disclosed herein can be derived from an IgG4 molecule (e.g., human IgG4) and contain one or more mutations at one or more positions of 228, 233-238, 265, 267, 273, 297, and 328.
  • such a Fc variant may comprise a substitution at position 228 (e.g.
  • positions 235-238 e.g., a deletion at one of positions 236-238, for example, position 236 and position 237
  • a substitution at position 265 e.g., D265A
  • a substitution at position 267 e.g., S267E
  • a substitution at position 273 e.g., V273E
  • a substitution at position 297 e.g.,
  • the Fc variant may comprise a substitution at one or more of positions 233-235 and 237 (e.g., E233P, F234V, F234A, F235S, F235E, F235A, and G237A).
  • Fc variants derived from IgG2 or IgG4 molecules that contain one or more mutations at positions 265, 267, 273, 297, 328, and/or 329 are also within the scope of the present disclosure. Such mutations may include amino acid substitutions at one or more of these positions, for example, D265A, S261E, V271E, N297A, F328F, and/or P329G. In some instances, any of the Fc variants disclosed herein may further comprise a mutation at position 309, for example, a substitution ( e.g ., V309L). Such an Fc variant may be derived from an IgG2 molecule (e.g., human IgG2). In some embodiments, the mutation at position 309 may be in combination with mutations at one or more of positions 234, 268, 330, and 331. Examples of such Fc variants include G2m43.
  • an Fc variant described herein may comprise an amino acid sequence at least 85% identical (e.g., 90%, 95%, 98%, 99%, or above) to that of its wild-type counterpart (e.g. , the Fc fragment of wild-type human IgGl , IgG2, or IgG4 described herein).
  • amino acid residue substitutions in an Fc variant described herein are conservative amino acid residue substitutions.
  • VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 1)
  • VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 2)
  • VDKKVEPKCCVECPPCPAPELLSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 7)
  • Glm-4 VDKKVEPKYGPPCPPCPAPELLGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 8)
  • VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEEKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 9)
  • VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVEHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 10)
  • VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 11)
  • VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVEHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 12)
  • VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 21)
  • VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 22)
  • VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVAVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 148)
  • VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYaSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 68)
  • VDKKVEPKSCDKTHTsPPsPAPpvaGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 177)
  • VDKKVEPKSCDKTHTCPPCPAPEfeGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAsIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 178)
  • VDKTVERKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 26)
  • VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEEQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 27)
  • VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVEHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 28)
  • VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGEPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 29)
  • VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVEHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 30)
  • VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEEQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 32)
  • VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMI SRTPEVTCWVDVEHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 33)
  • VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 34)
  • VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMI SRTPEVTCWVDVEHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 35)
  • VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVAVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 149)
  • VDKTVERKCCVECPPCPAPPaaasSVFLFPPKPKDTLMI SRTPEVTCWVDVSaEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVIHQDWLNGKEYKCKVSNKGLPs SlEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 179)
  • VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSqEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVIHQDWLNGKEYKCKVSNKGLPssIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 180)
  • VDKTVERKCCVECPPCPAPPaaaPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 181)
  • VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK (SEQ ID NO: 183)
  • G4m-2 VDKRVESKCCVEPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK (SEQ ID NO: 42)
  • G4m30 VDKRVESKYGPPCPPCPAPEFLPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 58)
  • the Fc variants described herein may exhibit an enhanced binding activity to FcyRIIB as compared with the wild-type counterpart.
  • Examples include G2m2, G2m5, G2m7, G2m8, G2m9, G2ml9, G2m44, Glm7, Glm9, Glm27, Glm45, Glm47, Glm49, Glm50, G4m7, G4m46 and G4mPE.
  • the Fc variants may have an enhanced selectivity to FcyRIIB as compared with their wild-type counterparts, for example, G2ml, G2m20, G2m43, G2m44, G2G4, G2mAA, Glm2, Glml5, Glml7, Glml8, Glml9,
  • Fc variants may retain the binding activity to FcRn.
  • Fc variants can be used for constructing therapeutic agents described herein capable of cross-linking immune receptors and FcyRIIB receptor.
  • certain Fc variants as described herein may have selectivity to FcyRIIB and/or apparent low or no binding activity to any FcyR.
  • Examples include Glm2, Glm25, Glm40, GlmAAG, Glm240, G2ml, G2m20, G2m40, G2m2040, G4m5, G4ml8, G4ml9, G4m20, G4m30, G4m40, G4m4l, and G4m42.
  • Such Fc variants may retain the binding activity to FcRn.
  • Therapeutic agents e.g . , antibodies
  • containing such Fc variants may be capable of cross-linking immune receptors and FcyRIIB receptor due to avidity effect.
  • binding activity of an Fc variant is “increased” or“decreased” as relative to its wild-type counterpart means that the binding activity of the Fc variant is higher or lower than that of the wild-type counterpart as determined by the same assay under the same experimental conditions and the variation is significant (e.g., biologically significant) as known to those skilled in the art.
  • binding activity of an Fc variant is“slightly increased” or“slightly decreased” as relative to its wild-type counterpart means that the binding activity of the Fc variant is higher or lower than that of the wild-type counterpart as determined by the same assay under the same experimental conditions and the variation is statistically significant but to a limited level (e.g., up to 10%).
  • Glm40 would be expected to have decreased or no apparent binding activity to FcyRI, FcyRI I A (H131), FcyRIIB, FcyRIIC, and FcyRIII.
  • Table 3. FcyR Binding Activity of Human IgG2 Mutants as Relative to Wild-Type Human IgG2
  • G2m40 would be expected to have decreased or no apparent binding activity to FcyRI, FcyRI I A (H131), FcyRI IB, FcyRIIC, and FcyRIII.
  • Table 4. FcyR Binding Activity of Human IgG4 Mutants as Relative to Wild-Type Human IgG4
  • G4m40 would be expected to have decreased or no apparent binding activity to FcyRI, FcyRI I A (H131), FcyRIIB, FcyRIIC, and FcyRIII.
  • Fc variant as described herein can be designed following the guidance provided herein and produced via routine recombinant technology. Its binding affinity and specificity to various Fc receptors can be determined via routine methods. See also Examples below.
  • Fc variants for Fey receptors can be examined in alternative assays.
  • CD137 reporter assays with or without co-culture of FcyRIIB expressing cells can be used to demonstrate effect of receptor cross-linking and activation. Because of avidity effect resulting from simultaneous binding of antibody to two targets (e.g ., CD 137 and FcyRIIB), this assay is more sensitive to detect FcyRIIB binding. Minimal or no apparent binding detectable by FACS may show positive results in the reporter assay.
  • CD 137-binding molecules described herein may be prepared by conventional methodology, for example, recombinant technology. Some examples follow.
  • a coding sequence of the CD137L extracellular domain can be fused in frame with a coding sequence of a suitable Fc variant.
  • the coding sequence for the whole CD 137-binding molecule can be cloned into a suitable expression vector, which can be introduced into a suitable host cell for protein expression.
  • Antibodies binding to CD 137 can be prepared by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.
  • antibodies specific to CD 137 or an extracellular domain thereof can be made by the conventional hybridoma technology.
  • the full-length target receptor or a fragment thereof, optionally coupled to a carrier protein such as KLH, can be used to immunize a host animal for generating antibodies binding to that antigen.
  • the route and schedule of immunization of the host animal are generally in keeping with established and conventional techniques for antibody stimulation and production, as further described herein. General techniques for production of mouse, humanized, and human antibodies are known in the art and are described herein.
  • any mammalian subject including humans or antibody producing cells therefrom can be manipulated to serve as the basis for production of mammalian, including human hybridoma cell lines.
  • the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar, and/or intradermally with an amount of immunogen, including as described herein.
  • Hybridomas can be prepared from the lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D. W., et al, In Vitro, 18:377-381 (1982).
  • myeloma lines including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, may be used in the hybridization.
  • the technique involves fusing myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art.
  • the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized parent cells.
  • HAT hypoxanthine-aminopterin-thymidine
  • EBV immortalized B cells may be used to produce the anti-immune cell receptor monoclonal antibodies described herein.
  • the hybridomas are expanded and subcloned, if desired, and supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (e.g ., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).
  • Hybridomas that may be used as source of antibodies encompass all derivatives, progeny cells of the parent hybridomas that produce monoclonal antibodies capable of modulating the activity of the target immune cell receptor.
  • Hybridomas that produce such antibodies may be grown in vitro or in vivo using known procedures.
  • the monoclonal antibodies may be isolated from the culture media or body fluids, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired.
  • Undesired activity if present, can be removed, for example, by running the preparation over adsorbents made of the immunogen attached to a solid phase and eluting or releasing the desired antibodies off the immunogen.
  • a target antigen or a fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum album
  • an antibody (monoclonal or polyclonal) of interest (e.g., produced by a hybridoma) may be sequenced and the polynucleotide sequence may then be cloned into a vector for further construction of the anti-CD 137 antibodies described herein.
  • the sequence encoding the antibody of interest may be maintained in vector in a host cell and the host cell can then be expanded and frozen for future use.
  • the polynucleotide sequence may be used for genetic manipulation to“humanize” the antibody or to improve the affinity (affinity maturation), or other characteristics of the antibody.
  • the constant region may be engineered to more resemble human constant regions to avoid immune response if the antibody is used in clinical trials and treatments in humans. It may be desirable to genetically manipulate the antibody sequence to obtain greater affinity to the target antigen and greater efficacy in inhibiting or activating the activity of the immune cell receptor. It will be apparent to one of skill in the art that one or more polynucleotide changes can be made to the antibody and still maintain its binding specificity to the target receptor.
  • Fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins.
  • Transgenic animals that are designed to produce a more desirable (e.g. , fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are Xenomouse R TM from Amgen, Inc. (Fremont, Calif.) and HuMAb-Mouse R TM and TC MouseTM from Medarex, Inc. (Princeton, N.J.).
  • antibodies may be made recombinantly by phage display or yeast technology. See, for example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et a , (1994) Annu. Rev. Immunol. 12:433-455.
  • antibody library technology such as the phage display technology
  • yeast display technology or mammalian cell display technology, can be used to isolated antibodies such as human antibodies specific to a target immune receptor.
  • variable regions of V H and V L of a parent non-human antibody are subjected to three- dimensional molecular modeling analysis following methods known in the art.
  • framework amino acid residues predicted to be important for the formation of the correct CDR structures are identified using the same molecular modeling analysis.
  • human VH and VL chains having amino acid sequences that are homologous to those of the parent non-human antibody are identified from any antibody gene database using the parent VH and VL sequences as search queries. Human VH and VL acceptor genes are then selected.
  • the CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the parent non-human antibody or functional variants thereof.
  • residues within the framework regions of the parent chain that are predicted to be important in interacting with the CDR regions can be used to substitute for the corresponding residues in the human acceptor genes.
  • the coding sequence of its heavy chain can be fused in-frame with the coding sequence of a suitable Fc variant, which may selectively bind FcyRIIB or which does not bind any FcyRs, for example, any of the Fc variants described herein via routine recombinant technology.
  • the antibody is first investigated for its agonistic effect to activate the immune cell receptor to which it binds.
  • Such an agonistic antibody can be selected for making the CD 137-binding molecules described herein to enhance the agonistic effects.
  • the antibody is first investigated for its antagonistic effect to inhibit the immune cell receptor to which it binds.
  • an antagonistic antibody can be selected for making the CD 137-binding molecule described herein to down-regulate immune responses.
  • an Fc variant having low or no binding activity to any Fc receptor can be selected for making CD 137 antagonists.
  • the resultant antibody molecules or CD 137-binding molecules described herein can be produced via routine recombinant technology as exemplified below.
  • Nucleic acids encoding the heavy and light chain of an antibody or the polypeptide of a CD 137-binding molecule as described herein can be cloned into one expression vector, each nucleotide sequence being in operable linkage to a suitable promoter.
  • each of the nucleotide sequences encoding the heavy chain and light chain is in operable linkage to a distinct prompter.
  • the nucleotide sequences encoding the heavy chain and the light chain can be in operable linkage with a single promoter, such that both heavy and light chains are expressed from the same promoter.
  • an internal ribosomal entry site IRS
  • the nucleotide sequences encoding the two chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells.
  • the two chains are expressed in different cells, each of them can be isolated from the host cells expressing such and the isolated heavy chains and light chains can be mixed and incubated under suitable conditions allowing for the formation of the antibody.
  • a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art.
  • the nucleotide sequence and vector can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of a gene. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector.
  • expression vectors/promoter would depend on the type of host cells for use in producing the antibodies.
  • promoters can be used for expression of the antibodies described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-l LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and the herpes simplex tk virus promoter.
  • CMV cytomegalovirus
  • a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-l LTR, the simian virus 40 (SV40) early promoter
  • E. coli lac UV5 promoter the herpes simplex tk virus promoter
  • Herpes simplex tk virus promoter Herpes simplex tk virus promoter.
  • Regulatable promoters can also be used.
  • Such regulatable promoters include those using the lac repressor from E. coli as a transcription
  • Regulatable promoters that include a repressor with the operon can be used.
  • the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown et al, Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci.
  • cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells.
  • a tetracycline inducible switch is used.
  • the tetracycline repressor (tetR) alone, rather than the tetR- mammalian cell transcription factor fusion derivatives can function as potent trans-modulator to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy).
  • this tetracycline inducible switch does not require the use of a tetracycline repressor-mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells (Gossen et al, Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al, Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)), to achieve its regulatable effects.
  • the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA.
  • a selectable marker gene such as the neomycin gene for selection of stable or transient transfectants in mammalian cells
  • enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription
  • transcription termination and RNA processing signals from SV40 for mRNA stability
  • SV40 polyoma origins of replication and ColEl for proper episomal replication
  • polyadenylation signals useful to practice the methods described herein include, but are not limited to, human collagen I polyadenylation signal, human collagen II polyadenylation signal, and SV40 polyadenylation signal.
  • One or more vectors comprising nucleic acids encoding any of the antibodies may be introduced into suitable host cells for producing the antibodies.
  • the host cells can be cultured under suitable conditions for expression of the antibody or any polypeptide chain thereof.
  • Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g., from the cells or the culture supernatant) via a conventional method, e.g., affinity purification. If necessary, polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time allowing for production of the antibody.
  • methods for preparing an antibody described herein involve a recombinant expression vector that encodes both the heavy chain and the light chain of an antibody as described herein.
  • the recombinant expression vector can be introduced into a suitable host cell (e.g. , a dhfr- CHO cell) by a conventional method, e.g., calcium phosphate- mediated transfection.
  • a suitable host cell e.g. , a dhfr- CHO cell
  • Positive transformant host cells can be selected and cultured under suitable conditions allowing for the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or from the culture medium.
  • the two chains recovered from the host cells can be incubated under suitable conditions allowing for the formation of the antibody.
  • two recombinant expression vectors are provided, one encoding the heavy chain of the anti -immune cell receptor antibody and the other encoding the light chain of the same antibody.
  • Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr- CHO cell) by a conventional method, e.g., calcium phosphate- mediated transfection.
  • each of the expression vectors can be introduced into a suitable host cells. Positive transformants can be selected and cultured under suitable conditions allowing for the expression of the polypeptide chains of the antibody.
  • the antibody produced therein can be recovered from the host cells or from the culture medium.
  • the polypeptide chains can be recovered from the host cells or from the culture medium and then incubated under suitable conditions allowing for formation of the antibody.
  • the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cells or from the corresponding culture media. The two polypeptide chains can then be incubated under suitable conditions for formation of the antibody.
  • Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recovery of the antibodies from the culture medium.
  • some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.
  • compositions comprising the CD 137- binding molecules or any of the anti-CD 137 antibodies described herein and uses of such for modulating immune responses triggered by CD137/CD137L signaling.
  • Such CDl37-binding molecules as described herein or anti-CD 137 antibodies as also described herein can be used for treating diseases such as cancer or immune-related disorders.
  • CDl37-binding molecules or anti-CDl37 antibodies as described herein can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a target disease.
  • a pharmaceutically acceptable carrier excipient
  • “Acceptable” means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated.
  • compositions including buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.
  • compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
  • the pharmaceutical composition described herein comprises liposomes containing the antibodies (or the encoding nucleic acids) which can be prepared by methods known in the art, such as described in Epstein, et al, Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al, Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat.
  • Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidyl ethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • the CDl37-binding molecules, anti-CDl37 antibodies, or the encoding nucleic acid(s), may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano particles and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano particles and nanocapsules
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl- methacrylate), or poly(v nylalcohol)), polylactides (U.S. Pat. No.
  • microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
  • compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes.
  • Therapeutic antibody compositions are generally 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.
  • compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.
  • the principal active ingredient can be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as com starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof.
  • a pharmaceutical carrier e.g., conventional tableting ingredients such as com starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water
  • preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention.
  • the tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
  • Suitable surface-active agents include, in particular, non-ionic agents, such as polyoxyethylenesorbitans (e.g ., TweenTM 20, 40, 60, 80 or 85) and other sorbitans (e.g., SpanTM 20, 40, 60, 80 or 85).
  • Compositions with a surface-active agent will conveniently comprise between 0.05 and 5% surface-active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.
  • Suitable emulsions may be prepared using commercially available fat emulsions, such as IntralipidTM, LiposynTM, InfonutrolTM, LipofundinTM and LipiphysanTM.
  • the active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g. , soybean oil, safflower oil, cottonseed oil, sesame oil, com oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g., egg phospholipids, soybean phospholipids or soybean lecithin) and water.
  • an oil e.g. , soybean oil, safflower oil, cottonseed oil, sesame oil, com oil or almond oil
  • a phospholipid e.g., egg phospholipids, soybean phospholipids or soybean lecithin
  • Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%.
  • the fat emulsion can comprise fat droplets between 0.1 and 1.0 pm, particularly 0.1 and 0.5 pm, and have a pH in the range of 5.5 to 8.0.
  • the emulsion compositions can be those prepared by mixing an antibody with IntralipidTM or the components thereof (soybean oil, egg phospholipids, glycerol and water).
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above.
  • the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
  • compositions in preferably sterile pharmaceutically acceptable solvents may be nebulized by use of gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner. IV. Therapeutic Applications
  • CD 137-binding molecules or anti-CD 137 antibodies disclosed herein may be used to modulating (e.g ., enhancing or inhibiting) immune responses against invading pathogens and/or diseased cells such as cancer cells.
  • an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal,
  • a suitable route such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal,
  • nebulizers for liquid formulations including jet nebulizers and ultrasonic nebulizers are useful for administration.
  • Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution.
  • the antibodies as described herein can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.
  • the subject to be treated by the methods described herein can be a mammal, more preferably a human.
  • Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.
  • the subject is a human patient having or at risk for a cell-mediated disease or disorder, such as cancer including but not limited to lung cancer, stomach cancer, liver cancer, breast cancer, skin cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, or colorectal cancer.
  • a cell-mediated disease or disorder such as cancer including but not limited to lung cancer, stomach cancer, liver cancer, breast cancer, skin cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, or colorectal cancer.
  • cancers include, but are not limited to, breast cancer; biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia, e.g., B Cell CLL; T-cell acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic myelogenous leukemia, multiple myeloma; AIDS-associated leukemias and adult T-cell leukemia/lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from
  • a subject having a cancer can be identified by routine medical examination, e.g., laboratory tests, organ functional tests, CT scans, or ultrasounds.
  • the subject to be treated by the method described herein may be a human cancer patient who has undergone or is subjecting to an anti-cancer therapy, for example, chemotherapy, radiotherapy, immunotherapy, or surgery.
  • the subject is a human patient having or at risk for an immune- related disorder.
  • Immune-related disorders refer to a dysfunction of the immune system, including autoimmune diseases, immunodeficiencies, and/or allergies.
  • the immune-related disorder is an autoimmune disease.
  • immune-related disorders include, but are not limited to, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Myasthenia Gravis (MG), Graves’ Disease, Idiopathic
  • Thrombocytopenia Purpura ITP
  • Guillain-Barre Syndrome autoimmune myocarditis
  • Membrane Glomerulonephritis diabetes mellitus, Type I or Type II diabetes
  • multiple sclerosis Reynaud's syndrome, autoimmune thyroiditis, gastritis, Celiac Disease, Vitiligo, Hepatitis, primary biliary cirrhosis, inflammatory bowel disease, spondyloarthropathies, experimental autoimmune encephalomyelitis, immune neutropenia, juvenile onset diabetes, and immune responses associated with delayed hypersensitivity mediated by cytokines, T- lymphocytes typically found in tuberculosis, sarcoidosis, and polymyositis, polyarteritis, cutaneous vasculitis, pemphigus, pemphigold, Goodpasture's syndrome, Kawasaki's disease, systemic sclerosis, anti-phospholipid syndrome, Sjogren's syndrome, graft-versus-
  • a subject having an immune-related disorder can be identified by routine medical examination, e.g., with laboratory tests.
  • the subject to be treated by the method described herein may be a human subject with an immune-related disorder who has undergone or is subjecting to an immune- related disorder treatment, for example, immunosuppressive mediation, hormone replacement therapy, blood transfusions, anti-inflammatory medication, and/or pain medication.
  • an effective amount refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents.
  • the therapeutic effect is modulating (e.g. , activating) the target immune receptor, thereby triggering or enhancing immune responses mediated by the receptor. Determination of whether an amount of the antibody achieved the therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.
  • Empirical considerations such as the half-life, generally will contribute to the determination of the dosage.
  • molecules that are compatible with the human immune system such as humanized antibodies or fully human antibodies, may be used to prolong half-life of the antibody and to prevent the antibody being attacked by the host's immune system.
  • Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a target disease/disorder.
  • sustained continuous release formulations of an antibody may be appropriate.
  • formulations and devices for achieving sustained release are known in the art.
  • dosages for a CD 137-binding molecule or an anti-CD 137 antibody as described herein may be determined empirically in individuals who have been given one or more administration(s) of the antibody. Individuals are given incremental dosages of the CDl37-binding molecule or an anti-CDl37 antibody. To assess efficacy of the CD137- binding molecule or an anti-CDl37 antibody, an indicator of the disease/disorder can be followed.
  • an initial candidate dosage can be about 2 mg/kg.
  • a typical daily dosage might range from about any of 0.1 pg/kg to 3 pg/kg to 30 pg/kg to 300 pg/kg to 3 mg/kg, to 30 mg/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate a target disease or disorder, or a symptom thereof.
  • An exemplary dosing regimen comprises administering an initial dose of about 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg of the antibody, or followed by a maintenance dose of about 1 mg/kg every other week.
  • an initial dose of about 2 mg/kg followed by a weekly maintenance dose of about 1 mg/kg of the antibody, or followed by a maintenance dose of about 1 mg/kg every other week.
  • other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve.
  • dosing from one-four times a week is contemplated.
  • dosing ranging from about 3 pg/mg to about 2 mg/kg (such as about 3 pg/mg, about 10 pg/mg, about 30 pg/mg, about 100 pg/mg, about 300 pg/mg, about 1 mg/kg, and about 2 mg/kg) may be used.
  • dosing frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer.
  • the progress of this therapy is easily monitored by conventional techniques and assays.
  • the dosing regimen (including the therapeutic used) can vary over time.
  • doses ranging from about 0.3 to 5.00 mg/kg may be administered.
  • the dosage of the therapeutic agents such as antibodies described herein can be 10 mg/kg.
  • the particular dosage regimen i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history, as well as the properties of the individual agents (such as the half-life of the agent, and other considerations well known in the art).
  • the appropriate dosage of a CD 137-binding molecule or anti-CD 137 antibody as described herein will depend on the specific antibody, antibodies, and/or non-antibody peptide (or compositions thereof) employed, the type and severity of the disease/disorder, whether the CDl37-binding molecule or anti-CDl37 antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the agonist, and the discretion of the attending physician.
  • the clinician will administer a CD 137-binding molecule or anti-CD 137 antibody, until a dosage is reached that achieves the desired result.
  • the desired result is a decrease in thrombosis.
  • Administration of one or more CD 137-binding molecule or anti-CD 137 antibody can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of a CDl37-binding molecule or anti-CDl37 antibody may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing a target disease or disorder.
  • the term“treating” refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease or disorder, a symptom of the disease/disorder, or a predisposition toward the disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder.
  • Alleviating a target disease/disorder includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, "delaying" the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated.
  • a method that“delays” or alleviates the development of a disease, or delays the onset of the disease is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.
  • “Development” or“progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein“onset” or“occurrence” of a target disease or disorder includes initial onset and/or recurrence.
  • the antibodies described herein are administered to a subject in need of the treatment at an amount sufficient to activate the activity of the target receptor by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo.
  • Conventional methods known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease.
  • This composition can also be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous,
  • intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, and intracranial injection or infusion techniques can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.
  • the pharmaceutical composition is administered intraocularly or intravitreally.
  • Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like).
  • water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused.
  • Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer’s solution or other suitable excipients.
  • Intramuscular preparations e.g., a sterile formulation of a suitable soluble salt form of the antibody
  • a pharmaceutical excipient such as Water- for- Injection, 0.9% saline, or 5% glucose solution.
  • the particular dosage regimen i.e., dose, timing and repetition, used in the method described herein will depend on the particular subject and that subject's medical history.
  • more than one antibody, or a combination of an antibody and another suitable therapeutic agent may be administered to a subject in need of the treatment.
  • the CD 137-binding molecule or anti-CD 137 antibody can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the agents.
  • Treatment efficacy for a target disease/disorder can be assessed by methods well- known in the art.
  • the therapeutic agent described herein may be utilized in conjunction with other types of therapy for the target disease such as cancer. Additional anti-cancer therapy includes chemotherapy, surgery, radiation, gene therapy, and so forth. When a second therapeutic agent is used, such an agent can be administered simultaneously or sequentially (in any order) with the CD 137-binding molecule or anti-CD 137 antibody described herein.
  • suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.
  • the treatments of the disclosure can be combined with other immunomodulatory treatments such as, e.g. , therapeutic vaccines (including but not limited to GVAX, DC-based vaccines, etc.), or checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.).
  • therapeutic vaccines including but not limited to GVAX, DC-based vaccines, etc.
  • checkpoint inhibitors including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.
  • the treatment of the present disclosure can be combined with a chemotherapeutic agent, for example, pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine), purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupters such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carb
  • antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L- asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents;
  • antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and
  • methylmelamines hexamethylmelamine and thiotepa
  • alkyl sulfonates-busulfan nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate);
  • platinum coordination complexes cisplatin, carboplatin
  • procarbazine hydroxyurea, mitotane, aminoglutethimide
  • hormones hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole)
  • anticoagulants heparin, synthetic heparin salts and other inhibitors of thrombin
  • fibrinolytic agents such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab
  • antimigratory agents antisecretory agents (breveldin);
  • immunosuppressives cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil
  • anti-angiogenic compounds e.g ., TNP-470, genistein, bevacizumab
  • growth factor inhibitors e.g., fibroblast growth factor (FGF) inhibitors
  • angiotensin receptor blocker e.g., fibroblast growth factor (FGF) inhibitors
  • angiotensin receptor blocker e.g., fibroblast growth factor (FGF) inhibitors
  • angiotensin receptor blocker e.g., fibroblast growth factor (FGF) inhibitors
  • angiotensin receptor blocker e.g., fibroblast growth factor (FGF) inhibitors
  • angiotensin receptor blocker e.g., fibroblast growth factor (FGF) inhibitors
  • angiotensin receptor blocker e.g., fibroblast growth factor
  • mitochondrial dysfunction inducers and caspase activators; and chromatin disrupters.
  • the kit can comprise instructions for use in accordance with any of the methods described herein.
  • the included instructions can comprise a description of administration of the therapeutic agent to treat, delay the onset, or alleviate a target disease as those described herein.
  • the kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease.
  • the instructions comprise a description of administering a therapeutic agent such as an antibody to an individual at risk of the target disease.
  • the instructions relating to the use of the therapeutic agent generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
  • the containers may be unit doses, bulk packages ( e.g ., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g. , a paper sheet included in the kit), but machine- readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
  • the label or package insert indicates that the composition is used for treating, delaying the onset and/or alleviating a target disease or disorder such as cancer. Instructions may be provided for practicing any of the methods described herein.
  • kits of this invention are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g. , sealed Mylar or plastic bags), and the like.
  • packages for use in combination with a specific device such as an inhaler, nasal administration device (e.g. , an atomizer) or an infusion device such as a minipump.
  • a kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • a sterile access port for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle.
  • At least one active agent in the composition is the therapeutic agent as those described herein.
  • Kits may optionally provide additional components such as buffers and interpretive information.
  • the kit comprises a container and a label or package insert(s) on or associated with the container.
  • the invention provides articles of manufacture comprising contents of the kits described above.
  • Example 1 Engineering human IgGl, IgG2 and IgG4 for selective FcyRIIB/CD32B binding
  • IgG4 on binding activity to FcyRIIB/CD32B the human Fc variants Glml, Glm2, G2ml, G2m2, G2ml0, G4ml, G4m2, G4ml0, and G4m20 disclosed herein were linked to the VH fragment of an anti-CDl37 antibody and the corresponding IgGl, G2, and G4 CH1 fragments to produce a full-length IgG heavy chain.
  • These mutant IgG heavy chains were cloned, co- expressed with the light chain of the anti-CD 137 antibody, and purified using standard molecular biology and antibody protocols.
  • the amino acid sequences of the VH-CH1 fragment (VH domain italicized) and the light chain are provided below:
  • VH-CH1 (IgGl): EVQLVQSGAEVKKPGESLRISCKGSGYSFSTYWISWVRQMPGKGLEWMGKIYPGDSYTNYSPSFQGQVTISADKS ISTAYLQWSSLKASDTAMYYCARGYGIFDYWGQGTLVTVSSASTKGRSVFREARS SKSTSGGTAAEGCEVKOYFR E PVTVSWNSGALT SGVHTFPAVLQS SGLY SL S SWTVPS S SLGTQTY ICNVNHKP SNTK (SEQ ID NO: 188)
  • VH-CH1 (IgG2):
  • VH-CH1 (IgG4):
  • FcRs human Fc receptors
  • FcRs human Fc receptors
  • FcRs human Fc receptors
  • Human FcR proteins were purchased commercially (FcRn, MednaBio, E1032; FcRI, MednaBio, E1031; FcRIIA, MednaBio, E1033; FcRIIB/C, MednaBio, E1034; FcRIIIA-Fl58, MednaBio, E1036; FcRIIIA-Vl58, MednaBio, E1035; FcRIIIB, MednaBio, E1037).
  • the human Fc variants Glm5, Glm7, Glm8, Glm9, G2m5, G2m7, G2m8, G2m9, G4m5, G4m7, G4m8, and G4m9 disclosed herein were linked to the VH fragment of an aud CD 137 antibody and the corresponding IgGl, G2, and G4 CH1 fragments to produce a full- length IgG heavy chain. See Example 1 above. These mutant IgG heavy chains were cloned, co-expressed with the light chain of the anti-CD 137 antibody (see Example 1 above), and purified using standard molecular biology and antibody protocols. The results are shown in Tables 7-9 below.
  • the human Fc variants Glml5, Glml7, Glml8, Glml9, G2m25, G2m27, G2m28, G4ml5, G4ml7, G4ml8, and G4ml9, G4m25, G4m27, G4m28, and G4m29 disclosed herein were linked to the VH fragment of an anti-CDl37 antibody and the corresponding IgGl, G2, and G4 CH1 fragments to produce a full-length IgG heavy chain. See Example 1 above.
  • These Fc variants contain a combination of one or mutations in the hinge domain and one or more mutations in the CH2 and/or CH3 domains.
  • These mutant IgG heavy chains were cloned, co-expressed with the light chain of the anti-CD 137 antibody (see Example 1 above), and purified using standard molecular biology and antibody protocols. The results are shown in Tables 10-12 below.
  • CHO cells were genetically engineered to express human FcyRs (FcyRI, FcyRIIA(Hl3 l), FcyRIIA(Rl3 l), FcyRIIB, FcyRIIC, and FcyRIII) using a lentivirus delivery system as known in the art.
  • FcyRs FcyRI, FcyRIIA(Hl3 l), FcyRIIA(Rl3 l), FcyRIIB, FcyRIIC, and FcyRIII
  • IgG Fc mutants including Glm-2, Glm-4, GlmAA, GlmAG, G2m-l G2m-4,
  • G2ml5, G2ml7, G2ml7, G2ml8, G2ml9, G2m20, G2m27, G2m28, G4m-l, G4m-2, G430, and G4PE were designed and constructed following the disclosures herein. These IgG mutants contain mutations in either the upper hinge domain or the lower hinge domain as indicated.
  • FcyR For FACS analysis of the IgG mutants’ binding to different FcyRs, FcyR
  • overexpressing CHO cells were harvested using trypsin-EDTA and were suspended in cold staining buffer (3% BSA in PBS).
  • Test IgG mutants which were diluted in staining buffer, were added into the cells. The mixture was incubated 4°C for 2 hours, and then washed twice with cold staining buffer and re-suspended in PE-labeled anti-human IgG followed by incubation at 4°C for 2 hours. The mixture was washed twice with staining buffer and re suspended in 2% PFA in PBS for FACS.
  • Example 5 Human IgG Mutants Capable of Binding to Cellular FcyRIIB Showed Enhanced Agonist Activity
  • a co-culture assay was developed, which involves CHO cells expressing FcyR2B and human CD8 positive T cells.
  • CHO-FcyRIIB cells were plated at 2xl0 4 /well in 96-well cell culture plate and were incubated overnight.
  • human CD8 positive T cells fresh blood from healthy donors were mixed with equal volume of DPBS gently. The blood sample was then placed on top of Ficoll underlay and centrifuged 30 min at lOOOg at RT without brake. The buffy coat containing PBMC was harvested into a new tube and was washed with DPBS.
  • CD8 positive T cells were isolated from the PBMC using EasySepTM Human CD8+ T Cell Isolation Kit (Stemcell #17953) according to the kit manual.
  • the isolated CD8+ T cells suspended in RPMI media were added to the plates with CHO- FcyRIIB cells.
  • the anti-human CD3 antibody OKT3 was added to a final concentration of 0. lug/ml followed by addition of test antibodies diluted at desired concentrations.
  • the plates were cultured for 3 days, and then the culture supernatants were harvested for measurement of IFNg concentration by ELISA using Human IFN-gamma ELISA Ready-SET-GO kit (EBIOSCIENCE, #88-7316-88).
  • the cDNA sequences encoding the anti-CD 137 antibody variable domain with various heavy chain CH1 and Fc region or human kappa light chain constant region were synthesized and cloned. CHO transient expression was carried out with plasmids containing the corresponding heavy and light chain sequences. These antibodies were purified by protein A affinity chromatography.
  • the amino acid sequences of the heavy chain (HC) and the light chain (LC) are provided below:
  • CHO cells were genetically engineered to express human FcyRs (FcyRI,
  • FcyRIIA Hl3 l
  • FcyRIIB FcyRIIB
  • FcyRIII FcyRIII
  • IgG Fc mutants including l73-msIgGl, l73-msIgGl DANA, l73-msIgG2a, 173- msIgG2a DANA, l73-huIgG2, l73-huIgG2m2, l73-huIgG4m2, l73-huIgGlm27 and 173- huIgG4SP (amino acid sequences provided above) were designed and constructed following the disclosures herein. These IgG mutants contain mutations in either the upper hinge domain or the lower hinge domain or within the CH2 domain. For FACS analysis of the IgG mutants’ binding to different FcyRs, FcyR
  • overexpressing CHO cells were harvested using trypsin-EDTA and were suspended in cold staining buffer (3% BSA in PBS).
  • Test IgG mutants which were diluted in staining buffer, were added into the cells. The mixture was incubated 4°C for 2 hours, and then washed twice with cold staining buffer and re-suspended in PE-labeled anti-human IgG followed by incubation at 4°C for 2 hours. The mixture was washed twice with staining buffer and re suspended in 2% PFA in PBS for FACS.
  • a CD 137 reporter assay was developed, which involves Jakat cells expressing human CD 137-positive T cells.
  • GS-H2- huCDl37 reporter cells were re-suspended in assay buffer and cell density and viability were determined with trypan blue.
  • the cell suspension was diluted to 1 x 10 4 cells/mL with assay buffer (MEM containing 1% FBS).
  • the cells were added at lOOuL/well, such that the final cell number was 1000 cells/well in the assay plate (Nunc, Cat#l 67425). Serial working dilutions of the test samples were prepared in assay buffer at 2x final concentrations.
  • Samples were added at lOOuL/well test sample at 2x final concentrations to the assay plate.
  • the assay plate was incubated in 37°C, 5% C0 2 incubator for 18-20 hours. After the 18-20 hour incubation, 8 ul of the supernatant from each well of the assay plate was collected and added to HTRF detection assay plate (Nunc).
  • a Human Interleukin 8 (reporter of CD 137 activation) detection assay was performed using a Human IL-8 Assay Kit (Cisbio,
  • CD137 Antibody in IgG Mutants Exhibit differential Anti-tumor Activity in Animal Models in Vivo
  • CD137 antibodies with various Fc regions are tested in mouse syngeneic tumor models in vivo to determine how variant Fc regions affect the efficacy and toxicity of these antibodies.
  • C57BL6 mice with human CD 137 extracellular domains knocked in are used to develop syngeneic mouse tumor models.
  • CD137 antibodies are then administered via intraperitoneal injections and tumor sizes are measured during 4-6 weeks of antibody treatment. The anti-tumor efficacy is evaluated between tumor sizes of the control group and antibody treatment group.
  • Exemplary anti-CD 137 antibodies are provided in Example 6 above.
  • inventive embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.
  • inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein.
  • a reference to“A and/or B”, when used in conjunction with open-ended language such as“comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • “or” should be understood to have the same meaning as“and/or” as defined above.
  • “or” or“and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as“only one of’ or“exactly one of,” or, when used in the claims,“consisting of,” will refer to the inclusion of exactly one element of a number or list of elements.
  • the phrase“at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase“at least one” refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

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Abstract

The disclosure provides CD 137-binding molecules comprising engineered human IgG domains, for example, human IgGl, IgG2, and IgG4 variants having mutations in the hinge domain, which exhibit altered binding activity to one or more Fcγ receptors. Also described herein are methods for selectively activating or inhibiting immune responses in a subject using the CD 137-binding molecules.

Description

Anti-CD137 Binding Molecules Having Engineered Fc Domains and
Therapeutic Uses Thereof
RELATED APPLICATION
This application claims the benefit of International Patent Application No.
PCT/CN2018/0108284, filed on September 28, 2018, which is incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
Cluster of differentiation 137 (CD137), also known as 4-1BB and TNFRSF9 (tumor necrosis factor receptor superfamily member 9) is an antigen-presenting cell (APC) costimulatory protein required for APC activation. CD 137 is a member of the tumor necrosis factor (TNF)-receptor superfamily and activates various immune competent cells, leading to activation, cytokine induction, prevention of activation-induced cell death (AICD), upregulation of cytotoxic T lymphocyte (CTL) activity, and increased survival. CD 137 binds with high affinity to its ligand, CD137L (4-1BBL), which is expressed on antigen-presenting cells, such as dendritic cells, macrophages, and activated B cells. CD 137 is predominantly found on various hematopoietic cells, such as primed T cells, natural killer cells, neutrophils, monocytes, dendritic cells, and mast cells. Its activation has been shown to reverse tolerance to tumor-specific antigens, leading to antigen-specific antitumor immunity.
Fc receptors (FcR) are a family of immune cell surface proteins capable of binding to the Fc portion of antibodies. There are several different types of Fc receptors, including Fey receptors, Fca receptors, Fes receptors, and neonatal Fc receptors (FcRn), which have different binding activities to IgG, IgA, IgE, and IgG antibodies, respectively. The Fey receptor subfamily includes FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIB (CD32c), FcyRIIIA (CDl6a), and FcyRIIIB (CDl6b). FcyRI has high binding affinity to IgGl and IgG3 antibodies, while the other FcyRs have low binding affinity to IgG antibodies.
Different types of Fc receptors play different roles in the immune system. For example, FcyRIII receptors, expressed on NK cells and macrophages, bind to antibodies that are attached to infected cells or invading pathogens and trigger antibody-mediated phagocytosis (ADCP) or antibody-dependent cell-mediated cytotoxicity (ADCC) of the immune cells, thereby leading to elimination of the infected cells or invading pathogens. On the other hand, FcyRII receptors, expressed on B cells and dendritic cells, can down regulate the activity of the immune cells when binding to IgG antibodies.
Therapies involving activated immune cells are promising approaches for eliminating diseased cells such as cancer cells. However, such therapeutic approaches often raise safety concerns. For example, overly activated immune cells would lead to undesired cytotoxicity, causing tissue damage. It is therefore of great interest to develop new immune therapies that are effective and safe.
SUMMARY OF THE INVENTION
The present disclosure is based, at least in part, on the design of CD 137-binding molecules ( e.g CD137 agonists or CD137 antagonists) comprising an engineered (variant)
Fc region (e.g., an engineered Fc region of IgGl, IgG2, or IgG4 molecules) that exhibit desired Fc receptor binding activity and/or selectivity, e.g., enhanced binding affinity and/or selected to FcyRIIB or substantially reduced binding affinity to one or more FcyR receptors. Such CDl37-binding molecules can be used for modulating immune responses (e.g., selectively enhancing or selectively inhibiting an immune response) in subject in need of the treatment.
Accordingly, one aspect of the present disclosure provides a CD 137-binding molecule (e.g., a CD137 agonist or a CD137 antagonist) comprising a CD137 binding moiety and an antibody heavy chain constant region comprising an engineered Fc region, which comprises at least one mutation at any of positions 220-331, for example, any of positions 228-329, as compared to the wild-type Fc region counterpart. The numbering system is according to the EU index.
In some embodiments, the CD 137 binding moiety is the extracellular domain of a CD137L, which may comprise the amino acid sequence of SEQ ID NO: 143. A CD137- binding molecule comprising such a CD 137-binding moiety may be a CD 137 agonist.
In other embodiments, the CD 137 binding moiety can be an anti-CD 137 antibody fragment. In one embodiment, the anti-CD 137 antibody fragment comprises a heavy chain that comprises a heavy chain variable region, which is linked to any of the engineered Fc regions described herein, and a light chain that comprises a light chain variable region, and a light chain constant region.
In some instances, the variant Fc region may have an enhanced binding affinity and/or selectivity to FcyRIIB relative to the wild-type counterpart. In other instances, the variant Fc region may have substantially reduced binding affinity to one or more FcyR receptors. In some examples, the variant Fc region has low or no binding activity to all FcyR receptors.
In some embodiments, the CDl37-binding molecule may comprise a variant Fc region of an IgGl molecule (e.g., a human IgGl molecule), which may comprise a mutation comprising one or more of the following: (a) an amino acid substitution or deletion within positions 233-238 (e.g., 234-238); (b) a substitution at position 265; (c) a substitution at position 267; (d) a substitution at position 297; (e) a substitution at position 328, a substitution at position 329; or a combination thereof. Alternatively or in addition, the Fc variant of an IgGl molecule may comprise one or more mutations at positions 220, 226, 229, 238, 273, 327, 330, and/or 331. In one example, the mutation comprises substitutions at any of positions 233-236, a deletion at one or more of positions 236-238, or a combination thereof. For example, the substitutions at positions 233-235 may comprise E233P, L234V, L234F, L234A, L235A, and/or L235E. In one example, the mutation comprises a deletion at one or more of positions 236-238. In another example, the substitution at position 238 can be P238S, the substation at position 265 can be D265A, or a combination thereof. In yet another example, the substitution is at position 267 and is S267E. In a further example, the substitution is at position 329 and is P329G. In an additional example, the mutation may comprise a substitution at position 265, a substitution at position 297, or a combination thereof. For example, the substitution at position 265 may be D265A and the substitution at position 297 may be N297A. Further, the one or more substitutions at positions 220, 226, 229, 327, 330, and 331 can be C220S, C226S, C229S, A327G, A330S, and P331 S.
Exemplary variant Fc regions derived from human IgGl may be one of Glml, Glm2, Glml7, Glm27, GlmAA, GlmAAG, G1N297A, Glm240, and Glm40.
In other embodiments, the CD 137-binding molecule may comprise an Fc region of an
IgG2 molecule (e.g., a human IgG2 molecule), which may comprise a mutation comprising one or more of the following: (a) a deletion of one or more of positions 237 and 238; (b) a substitution at position 265; (b) a substitution at position 267; (c) a substitution at position 297; (d) a substitution at position 328; or a combination thereof. In some examples, the deletion is at position 237 or at both positions 237 and 238. Alternatively or in addition, the Fc variant of an IgG2 molecule may comprise at least one mutation comprising a substitution at one or more of positions 233-235, 237, 238, 268, 273, 330, and 331. For example, the substitution at one or more of positions 233-235, 237, 238, 268, 273, 330, and 331 can be P233E, V234A, V234L, A235L, A235S, G237A, P238S, H268A, H268Q, V273E, A330S, and P331S. In one example, the substitution is at position 267 and is S267E. In another example, the substitution is at position 328 and is L328F. In an additional example, the mutation may comprise a substitution at position 265, a substitution as position 297, or a combination thereof. For example, the substitution at position 265 may be D265A and the substitution at position 297 may be N297A. In some particular examples, the variant Fc region derived from IgG2 can be one of G2ml, G2ml7, G2ml8, G2ml9, G2m20, G2m27, G2m28, G2m29, G2m2040, G2m43, G2G4, G2mAA, and G2m40.
In yet other embodiments, the CDl37-binding molecule may comprise an Fc region of an IgG4 molecule (e.g. , a human IgG4 molecule), which may comprise (a) an amino acid residue substitution at position 228; (b) a substitution or deletion at any one positions 235- 238; (c) a substitution at position 265; (d) a substitution at position 267; (e) a substitution at position 297; (e) a substitution at position 328; or a combination thereof. In one example, the substitution at position 228 is S228P. In another example, the deletion is at one or more of positions 236, 237, and 238 (e.g. , at both positions 236 and 237). Alternatively or in addition, the Fc variant of an IgG4 molecule may comprise a substitution at one or more of positions 233-235, 237, and 273. For example, the substitution at one or more positions 233- 235, 237, and 273 may be E233P, F234V, F234A, F235S, F235E, F235A, G237A, and V273E. In another example, the substitution at position 267 is S267E. In a further example, the substitution at position 328 is F328F. In an additional example, the mutation may comprise a substitution at position 228, a substitution at position 265, a substitution at position 297, or a combination thereof. For example, the substitution at position 228 may be S228P, the substitution at position 265 may be D265A and the substitution at position 297 may be N297A. In some particular examples, the variant Fc region is one of G4ml, G4m2, G4m20, G4m28, G4m30, G4m4l, G4m42, G4m46, G4mPE, G4mAA, and G4m40.
Any of the variant Fc regions described herein may exhibit an enhanced binding activity and/or an enhanced selectivity to FcyRIIB as compared with the wild-type Fc region. Alternatively, the variant Fc regions described herein may have low or no binding activity to any of the FcyR receptors. In some instances, the variant Fc region may exhibit a decreased binding affinity to FcyRIIB. Alternatively or in addition, the variant Fc region binds FcRn. The anti-CD 137 antibody fragment in any of the CD 137-binding molecules described herein may be of a human antibody or a humanized antibody. In some embodiments, the anti-CD 137 antibody is an agonist antibody. In some examples, the anti-CD 137 antibody may comprise the same heavy chain complementary determining regions (HC CDRs) as those in SEQ ID NO: 128 or as those in any one of 20A12D11, 11E10D12, 23D2D6,
22F2C2, 30C11B4, and 26B3D7, and/or the same light chain complementary determining regions (LC CDRs) as those in SEQ ID NO: 129 or those in any one of 20A12D11,
11E10D12, 23D2D6, 22F2C2, 30C11B4, and 26B3D7. In one example, the antibody fragment may comprise a heavy chain variable region of SEQ ID NO: 128 and/or a light chain variable region of SEQ ID NO: 129. In other examples, the antibody fragment may comprise the same heavy chain variable region and/or the same light chain variable region as one of 20A12D11, 11E10D12, 23D2D6, 22F2C2, 30C11B4, and 26B3D7.
Additionally, provided herein is a pharmaceutical composition, comprising any of the CD 137-binding molecules described herein and a pharmaceutically acceptable carrier. Such a pharmaceutical composition may be used to selectively modulate ( e.g ., selectively activate or selectively inhibit) an immune response in a subject.
In yet another aspect, the present disclosure provides a method for selectively modulating (e.g. , selectively activating or selectively inhibiting) an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of a CD 137-binding molecule as described herein.
In any of the methods described herein, the subject can be a human patient having or suspected of having a cancer and the CD 137-binding molecule may be a CD 137 agonist. Exemplary cancers include lung cancer, stomach cancer, liver cancer, breast cancer, skin cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, colorectal cancer, sarcoma, bone cancer, lymphoma and a hematological cancer.
Alternatively, the subject can be a human patient having or suspected of having an immune -related disorder and the CDl37-binding molecule may be a CD137 antagonist. Exemplary immune disorders include autoimmune diseases, immune-deficiencies, or allergies. In some embodiments, the target disease for treatment is an autoimmune disease.
In yet another aspect, the disclosure provides an isolated anti-CD 137 antibody, which binds to the same epitope of CD 137 as a reference antibody selected from the group consisting of: 20A12D11, 11E10D12, 23D2D6, 22F2C2, 30C11B4, and 26B3D7 or competes against the reference antibody from binding to the epitope.
In some embodiments, the antibody comprises a heavy chain complementary determining region 1 (CDR1), a heavy chain complementary determining region 2 (CDR2), and a heavy chain complementary determining region 3 (CDR3), which collectively are at least 85% identical to the respective heavy chain CDRs of the reference antibody; and/or wherein the antibody comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, which collectively are at least 85% identical to the respective light chain CDRs of the reference antibody.
In another embodiment, the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 collectively comprise 10 or fewer amino acid mutations relative to the respective heavy chain CDRs of the reference antibody; and/or wherein the light chain CDR1, light chain CDR2, and light chain CDR3 collectively comprise 10 or fewer amino acid mutations relative to the respective light chain CDRs of the reference antibody.
In one embodiment, the antibody comprises a heavy chain variable region that is at least 85% identical to the heavy chain variable region of the reference antibody and/or a light chain variable region that is at least 85% identical to the light chain variable region of the reference antibody.
In some embodiments, the heavy chain variable region comprises 10 or fewer amino acid residue mutations relative to the heavy chain variable region of the reference antibody; and/or and a light chain variable region that comprises 10 or fewer amino acid mutations relative to the light chain variable region of the reference antibody.
In some examples, the anti-CD 137 antibody disclosed herein may comprise the same heavy chain variable region CDRs as the reference antibody and/or the same light chain variable region CDRs as the reference antibody.
Any of the anti-CD 137 antibodies described herein may be a human antibody or a humanized antibody. Also provided herein is a pharmaceutical composition comprising any of the anti-CD 137 antibodies described herein and a pharmaceutically acceptable carrier.
Further, the instant disclosure features an isolated nucleic acid or set of nucleic acids which collectively encode any of the CD 137 binding molecules or any of the anti-CD 137 antibodies described herein. The nucleic acid or set of nucleic acids may be located on one or two vectors, for example, expression vectors. Also provided herein are host cells comprising such vector(s). The instant disclosure also features pharmaceutical compositions comprising one or more of the CDl37-binding molecules described herein and/or one or more of the anti- CD 137 antibodies disclosed herein for use in treating a target disorder as described herein, or uses of such CDl37-binding molecules and/or anti-CDl37 antibodies for manufacturing a medicament for use in treating the target disorder.
The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS FIGs. 1A-1W are charts showing binding activity of various IgG variants as indicated to different types of Fey receptors expressed on CHO-K1 cells at the various concentrations as indicated. The concentrations of each IgG variant, from left to right, are 0 pg/ml, 0.3 pg/ml, 1 pg/ml, 3 pg/ml, and 10 pg/ml in FIGs. 1A-1C, 1E-1J, 1F-1M, 1P-1R, and 1T-1V. For FIGs. 1D, 1K, 10, 1S, and 1W the bars, from left to right, correspond to the following concentrations of IgG variant: 0.3 pg/ml, 1 pg/ml, 3 pg/ml, and 10 pg/ml. FIGs. 1A-1D: binding activity to FcyRI. FIGs. 1E-1G: binding activity to FcyRIIA(Hl3 l). FIGs. 1H-1K: binding activity to FcyRIIA(Rl 31). FIGs. 1F- 10: binding activity to FcyRIIB. FIGs. 1P-1S: binding activity to FcyRIIC. FIGs. 1T-1W: binding activity to FcyRIII.
FIGs. 2A-2C are charts showing stimulation of human CD8+ T cells in co-culture with parental or FcyR expressing cells by a number of IgG variants as indicated by IFN-y secretion. The groups, from left to right, correspond to: no OKT, 0.01 pg/ml, 0.03 pg/ml, 0.1 pg/ml, and 0.3 pg/ml, in FIG. 2A. In FIGs. 2B and 2C, the groups, from left to right, correspond to 0.01 pg/ml, 0.03 pg/ml, and 0.1 pg/ml. FIG. 2A: G2m2. FIG. 2B: various Fc variants as indicated. FIG. 2C: additional Fc variants as indicated.
FIGs. 3A-3D are charts showing binding activity of various IgG variants, as indicated, to different types of Fey receptors expressed on CHO-K1 cells at the various concentrations indicated. The concentrations of each IgG variant, from left to right, are 0.1 pg/ml, 0.3 pg/ml, 1 pg/ml, 3 pg/ml, 10 pg/ml, and 30 pg/ml. (FIG. 3A: binding activity to FcyRI. FIG. 3B: binding activity to FcyRIIA. FIG. 3C: binding activity to FcyRIIB. FIG. 3D: binding activity to FcyRIIIA. FIG. 4 is a chart showing stimulation of human CD 137 activation in a reporter assay by the indicated IgG variants. The concentrations of each IgG variant, from left to right, are 0.01 pg/ml, 0.03 pg/ml, 0.1 pg/ml, 0.3 pg/ml, 1 pg/ml, 3 pg/ml, and 10 pg/ml.
DETAILED DESCRIPTION OF THE INVENTION
Provided herein are CD137-binding molecules (e.g., CD137 agonists or CD137 antagonists) comprising a CD137 binding moiety (e.g., an extracellular domain of a CD137 ligand or an anti-CD 137 antibody fragment) and an engineered (variant) Fc regions, which may have altered binding affinity and/or specificity to one or more Fc receptors, for example, enhanced binding affinity to FcyRIIB, enhanced binding specificity to FcyRIIB, or substantially reduced binding affinity to one or more FcyR receptors (e.g. , low or no binding activity to all FcyR receptors).
Such CD 137-binding molecules are expected to exhibit unexpected, superior therapeutic activity. For example, CD 137-binding molecules having a variant Fc region that has enhanced binding affinity to FcyRIIB would be expected to exhibit enhanced agonistic activity and CD 137-binding molecules having a variant Fc region that has enhanced binding specificity to FcyRIIB would be expected to exhibit higher tumor selectivity relative to the wild-type counterpart. Further, CD 137-binding molecules having low or no binding affinity to FcyR receptors would help activating CD 137-positive immune cells in tumor
microenvironment and/or block CD 137-positive immune cells in immune disorders.
Accordingly, described herein are approaches for designing CD 137-binding molecules comprising a CD 137-binding moiety and an engineered Fc region (an Fc variant) which may be of an IgG molecule (e.g., IgGl, IgG2, and IgG4 molecules such as human IgGl, human IgG2, and human IgG4 molecules), and uses thereof for modulating immune responses. Such an Fc variant may have enhanced binding affinity to FcyRIIB (CD32B) relative to the wild-type counterpart and/or binding selectivity as relative to other Fc receptors such as FcyRIII (CD 16). Alternatively, such an Fc variant may have substantially reduced binding affinity to one or more FcyR receptors (e.g., to all FcyR receptors) relative to the wild-type counterpart. “Substantially reduced” means that the binding affinity of a Fc variant to a FcyR receptor is at least 60% lower (e.g. , 70% lower, 80% lower, 90% lower, 95% lower, 98% lower, or 99% lower) than the binding affinity of the wild-type counterpart to the same FcyR receptor. In some examples, the Fc variant may have low or no binding affinity to all FcyR receptors, i.e., binding affinity cannot be detected by conventional assays or binding affinity is substantially low such that no significant bioactivity would be triggered.
I. CD137-Binding Molecules
Described herein are CDl37-binding molecules that comprise a CDl37-binding moiety linked to an engineered Fc region having altered binding affinity and/or specificity to one or more Fc receptors as described herein. Such CD 137-binding molecules may be CD137 agonists, which are capable of triggering the signaling mediated by CD137/CD137L upon binding to cell surface CD137. Alternatively, the CDl37-binding molecules described herein are CD 137 antagonists, which are capable of inhibiting the signaling mediated by CD137/CD137L upon binding to cell surface CD137. The CDl37-binding molecules are useful in modulating (selectively modulating) immune responses when administered to a subject in need of the treatment.
(i) CD 137-Bindim Moieties
A CD 137-binding moiety as described herein may be any peptide or polypeptide that binds CD137, for example human CD137. CD137 is an immune cell receptor well known in the art. For example, NCBI GenBank Accession Nos. NP_00l552.2 and NP_00l070977 provide information for human and mouse CD137, respectively. Provided below is an amino acid sequence of an exemplary human CD 137 polypeptide.
Human CD 137:
MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICS PCPPNSFS SAGGQRTCDICRQC KGVFRTRKECS STSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPW TNCSLDGKSVLVNGTKERDVVCGPS PADLSPGAS SVTPPAPAREPGHS PQI I SFFLALTSTALLFLLF FLTLRFSVVKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL ( SEQ ID NO : 142 )
CD 137 polypeptides from other species are known in the art and can be obtained from publicly available gene databases, for example, GenBank, using either the human sequence or the mouse sequence as a query.
In some embodiments, the CD 137-binding moieties can be a polypeptide comprising an extracellular domain of a CD137 ligand (CD137L or 4-1BBL). CD137L is a membrane glycoprotein expressed antigen-presenting cells (APCs), namely, activated B and T cells.
The molecule has been shown to play a role in T cell proliferation and cytokine production through its interaction with its receptor. In addition, CD137L can drive monocyte to dendritic cell differentiation. The sequence of CD137L and its extracellular domain are well known in the art. For example, NCBI GenBank Accession No. NP_003802.1 (extracellular domain, amino acids 50-254) provides information for human CD137L. Provided below is an amino acid sequence of an extracellular domain of an exemplary human CD137L.
Human CD137L (extracellular domain) :
ACPWAVSGARAS PGSAAS PRLREGPELS PDDPAGLLDLRQGMFAQLVAQNVLL I DGPLSWYSDPGLAG VSLTGGLSYKEDTKELWAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDL PPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPE I PAGLPS PRS
E ( SEQ I D NO : 1 43 )
CD137L polypeptides from other species are known in the art and can be obtained from publicly available gene databases, for example, GenBank, using the human sequence as a query.
In other embodiments, the CD 137-binding moiety described herein can comprise an anti-CDl37 antibody. As used herein, the term“anti-CDl37 antibody” refers to any antibody capable of binding to a CD137 polypeptide, which can be of a suitable source, for example, human or a non-human mammal (e.g., mouse, rat, rabbit, primate such as monkey, etc.).
The anti-CD 137 antibodies described herein comprise a heavy chain that comprises a heavy chain variable domain, which is linked to any of the Fc variants described herein, and optionally a light chain that comprises a light chain variable region and a light chain constant region. The heavy chain variable region (VH) and optionally the light chain variable region (VL) are usually involved in antigen (CD 137 in this case) binding.
The VH and VL regions can be further subdivided into regions of hypervariability, also known as“complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as“framework regions” (“FR”). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Rabat definition, the Chothia definition, the AbM definition, and/or the contact definition, all of which are well known in the art. See, e.g. , Rabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17: 132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk/abs).
In some embodiments, the anti-CD 137 antibody as described herein can bind and inhibit the activity of CD137 by at least 50% (e.g. , 60%, 70%, 80%, 90%, 95% or greater, including any increment therein). The apparent inhibition constant (Kiapp or KLapp), which provides a measure of inhibitor potency, is related to the concentration of inhibitor required to reduce enzyme activity and is not dependent on enzyme concentrations. The inhibitory activity of an anti-CD 137 antibody described herein can be determined by routine methods known in the art.
The KL app value of an antibody may be determined by measuring the inhibitory effect of different concentrations of the antibody on the extent of the reaction (e.g. , enzyme activity); fitting the change in pseudo-first order rate constant (v) as a function of inhibitor concentration to the modified Morrison equation (Equation 1) yields an estimate of the apparent Ki value. For a competitive inhibitor, the Kiapp can be obtained from the y-intercept extracted from a linear regression analysis of a plot of KL app versus substrate concentration.
1 (Equation 1 )
Figure imgf000012_0001
Where A is equivalent to vJE, the initial velocity ( v of the enzymatic reaction in the absence of inhibitor (I) divided by the total enzyme concentration (E).
In some embodiments, the anti-CD 137 antibody described herein may have a Kiapp value of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 pM or less for the target antigen or antigen epitope. In some embodiments, any of the anti-CD 137 antibodies may be further affinity matured to reduce the Kiapp of the antibody to the target antigen or antigenic epitope thereof.
In some instances, the anti-CD 137 antibody may suppress the signaling triggered by CD137/CD137L interaction by at least 50% (e.g. , 60%, 70%, 80%, 90%, 95% or greater, including any increment therein). Such inhibitory activity can be determined by conventional methods or the assays described herein.
The antibodies described herein can be murine, rat, human, or any other origin
(including chimeric or humanized antibodies). Such antibodies are non-naturally occurring, i.e.. , would not be produced in an animal without human act (e.g. , immunizing such an animal with a desired antigen or fragment thereof or isolated from antibody libraries).
In some embodiments, the anti-CD 137 antibody is a humanized antibody, which may have one of more of the elements or characteristics described below or elsewhere herein. Humanized antibodies refer to forms of non-human (e.g. , murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. In general, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human
immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In some instances, the humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have Fc regions modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.
Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989). In one example, variable regions of VH and VL of a parent non-human antibody are subjected to three- dimensional molecular modeling analysis following methods known in the art. Next, framework amino acid residues predicted to be important for the formation of the correct
CDR structures are identified using the same molecular modeling analysis. In parallel, human VH and VL chains having amino acid sequences that are homologous to those of the parent non-human antibody are identified from any antibody gene database using the parent VH and VL sequences as search queries. Human VH and VL acceptor genes are then selected.
The CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the parent non-human antibody or functional variants thereof. When necessary, residues within the framework regions of the parent chain that are predicted to be important in interacting with the CDR regions can be used to substitute for the corresponding residues in the human acceptor genes.
In some embodiments, the anti-CD 137 antibodies described herein specifically bind to the corresponding target antigen or an epitope thereof, e.g., CD 137 antigen or epitope. An antibody that“specifically binds” to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit“specific binding” if it reacts more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen than it does with alternative targets. An antibody“specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically (or
preferentially) binds to an antigen (CD 137) or an antigenic epitope therein is an antibody that binds this target antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens or other epitopes in the same antigen. It is also understood with this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen.
As such,“specific binding” or“preferential binding” does not necessarily require (although it can include) exclusive binding. In some examples, an antibody that“specifically binds” to a target antigen or an epitope thereof may not bind to other antigens or other epitopes in the same antigen (i.e., only baseline binding activity can be detected in a conventional method). In some embodiments, the anti-CD 137 antibodies described herein specifically bind to
CD137. Alternatively, or in addition, the anti-CDl37 antibody described herein specifically binds human CD 137 or a fragment thereof as relative to the mouse counterpart, or vice versa (e.g., having a binding affinity at least 10-fold higher to one antigen than the other as determined in the same assay under the same assay conditions). In other instances, the anti- CD 137 antibody described herein may cross-react to human and a non-human CD 137 (e.g., mouse), e.g., the difference in binding affinity to the human and the non-human CD 137 is less than 5-fold, e.g. , less than 2-fold, or substantially similar. In some embodiments, an anti-CD 137 antibody as described herein has a suitable binding affinity for the target antigen (e.g., CD137) or antigenic epitopes thereof. As used herein,“binding affinity” refers to the apparent association constant or KA. The KA is the reciprocal of the dissociation constant (KD). The anti-CDl37 antibody described herein may have a binding affinity (KD) of at least 10 5, 106, 10 7, 10 8, 109, 10 10 M, or lower for the target antigen or antigenic epitope. An increased binding affinity corresponds to a decreased KD. Higher affinity binding of an antibody for a first antigen relative to a second antigen can be indicated by a higher KA (or a smaller numerical value KD) for binding the first antigen than the KA (or numerical value KD) for binding the second antigen. In such cases, the antibody has specificity for the first antigen (e.g. , a first protein in a first conformation or mimic thereof) relative to the second antigen (e.g., the same first protein in a second conformation or mimic thereof; or a second protein). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 105 fold. In some embodiments, any of the anti-CDl37 antibodies may be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.
Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl,
0.005% (v/v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation: [Bound] = [Free]/(Kd+[Free])
It is not always necessary to make an exact determination of KA, though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to KA, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay. Provided below is an example anti-CDl37 antibody 173 (VH and VL amino acid sequences; CDRs are indicated in boldface):
VH:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRV TMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSS (SEQ ID NO: 128)
VL:
DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSG TDFT LTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIK (SEQ ID NO: 129)
Further exemplary anti-CD 137 antibodies are provided below (CDRs are indicated in boldface).
20A12D11
VH:
QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVKQAPGKGLKWMGWINTYSGVPTYADDFK GRFAFSLETSASTAYLQINNLKNEDMAIYFCARGYGSPDYWGQGTTLTVSS
(SEQ ID NO: 130)
VL:
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIFDTSKLASGVPARFSGSG SGTSYSLTISSMEAEDAATYYCQQWSGNPPISTFGSGTKLEIKR
(SEQ ID NO: 131)
11E10D12
VH:
QVQLQQSGAELVRPGASVTLSCKASGYTFAGFEMHWIKQTPVHGLGWIGAIDPKTGGTDYNQKFK DKALLTADKSSNTAYMELRSLTSEDSAVYYCTRDLGYFDVWGTGTTVTVSS
(SEQ ID NO: 132)
VL:
DIQMTQTTSSLSASLGDRVTISCRASQDIRSNLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGS
GSGTDYSLTISNLEQEDIATYFCQQSEKLPRTFGGGTKLEIRR
(SEQ ID NO: 133)
23D2D6
VH:
QVQLQQSGAELVRPGSSVKISCKASGYAFSIYWMNWVKQRPGQGLEWIGQIYPGDGYTNYNGKFK GKATLTADKSSSTAYMQLSSLTSEDSAVYFCARGQLGLDGYWGQGTTLTVSS
(SEQ ID NO: 134) VL:
QIVLTQSPTIMSASPGEKVTMTCSASSSVSYIYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSG SGTSYSLTISRMEAEDAATYYCQQWNIYPYTFGGGTKLEIKR
(SEQ ID NO: 135)
22F2C2
VH:
EVKLVESGAELVRPGSSVKISCKASGYAFSLYWMNWVKQRPGQGLEWIGQIYPGDGYTNYNGKFK GKATLTADKSSSTAYMQLSSLTSEDSAVYFCARGQLGLDGYWGQGTTLTVSS
(SEQ ID NO: 136)
VL:
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYIYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSG SGTSYSLTISRMEAEDAATYYCQQWNIYPYTFGGGTKLEIKR
(SEQ ID NO: 137)
30C11B4
VH:
QIQLVQSGPELKKPGESVKISCKASGYTFTDYAMHWVKQAPGKALKWMGLINTYTGKPTYVDDLK GRFVFSLEASASTAKLQISNLKNEDTAIYFCARYYHDGTYYGWFANWGQGTLVTVSS
(SEQ ID NO: 138)
VL:
DTVLTQSPALAVSPGERVTVSCGATESVSTALNWYQQKPGQQPRLLIYGASNLESGVPARFSGSG SGTDFTLSIDPVEADDTATYFCQQTWNDPLTFGSGTKLEIK
(SEQ ID NO: 139)
26B3D7
VH:
EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSNPINYTPSLK DKFI ISRDNAKNTLYLQMSKVRSEDTALYYCARDGSSSRYFDVWGAGTTVTVSS
(SEQ ID NO: 140)
VL:
DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYVSQSISGIPSRFSGS
GSGSDFTLSINSVEPEDVGVYYCQNGHSFPPTFGGGTKLEIKR
(SEQ ID NO: 141)
In some embodiments, the anti-CD 137 antibodies described herein bind to the same epitope as any of the exemplary antibodies described herein or competes against the exemplary antibody from binding to the CD 137 antigen.
An“epitope” refers to the site on a target antigen that is recognized and bound by an antibody. The site can be entirely composed of amino acid components, entirely composed of chemical modifications of amino acids of the protein (
Figure imgf000017_0001
., glycosyl moieties), or composed of combinations thereof. Overlapping epitopes include at least one common amino acid residue. An epitope can be linear, which is typically 6-15 amino acids in length. Alternatively, the epitope can be conformational. The epitope to which an antibody binds can be determined by routine technology, for example, the epitope mapping method (see, e.g. , descriptions below). An antibody that binds the same epitope as an exemplary antibody described herein may bind to exactly the same epitope or a substantially overlapping epitope (e.g., containing less than 3 non-overlapping amino acid residue, less than 2 non-overlapping amino acid residues, or only 1 non-overlapping amino acid residue) as the exemplary antibody. Whether two antibodies compete against each other from binding to the cognate antigen can be determined by a competition assay, which is well known in the art.
In some embodiments, the anti-CD 137 antibodies disclosed herein comprise the same heavy chain and light chain CDRs as those in (a) SEQ ID NO: 128 and SEQ ID NO: 129, (b) SEQ ID NO: 130 and SEQ ID NO: 131 (20A12D11); (c) SEQ ID NO: 132 and SEQ ID NO: 133 (11E10D12); (d) SEQ ID NO: 134 and SEQ ID NO: 135 (23D2D6); (e) SEQ ID NO: 136 and SEQ ID NO: 137 (22F2C2), (f) SEQ ID NO: 138 and SEQ ID NO: 139 (30C11B4), or SEQ ID NO: 140 and SEQ ID NO: 141 (26B3D7). In some examples, the anti-CDl37 antibody comprises a heavy chain variable domain of SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: l34, SEQ ID NO:l36, SEQ ID NO: 138, or SEQ ID NO: l40 and/or a light chain variable domain of SEQ ID NO: 129, SEQ ID NO: l3l, SEQ ID NO: l33, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, or SEQ ID NO: 141.
Also within the scope of the present disclosure are functional variants of any one of the exemplary anti-CDl37 antibodies as disclosed herein. Such functional variants are substantially similar to any one of the exemplary anti-CD 137 antibodies, both structurally and functionally. A functional variant comprises substantially the same VH and VL CDRS as any one of the exemplary anti-CD 137 antibodies. For example, it may comprise only up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid residue variations in the total CDR regions of the antibody (collectively) and binds the same epitope of CD 137 with substantially similar affinity (e.g., having a KD value in the same order). Alternatively or in addition, the amino acid residue variations are conservative amino acid residue substitutions. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al, eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al, eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
In some embodiments, the anti-CD 137 antibody described herein may comprise heavy chain CDRs, which in combination (collectively) have at least 80% (e.g. , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and any incremental percent therein) sequence identity with the heavy chain CDRs in SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, or SEQ ID NO: 140. Alternatively or in addition, the anti-CD 137 antibody may comprises light chain CDRs, which collectively have at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% and any incremental percent therein) sequence identity with those in SEQ ID NO: 129, SEQ ID NO: l3l, SEQ ID NO: l33, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, or SEQ ID NO: 141.
In some embodiments, the anti-CD 137 antibody described herein may comprise a heavy chain variable domain that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%,
99% and any incremental percent therein) to SEQ ID NOs: 128, 130, 132, 134, 136, 138, or 140 and/or a light chain variable domain that is at least 80% (e.g., 85%, 90%, 95%, 96%,
97%, 98%, 99% and any incremental percent therein) identical to SEQ ID NOs: 129, 131,
133, 135, 137, 139, or 141.
The“percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 25(l7):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. (ii) Engineered Fc Regions
The CD 137-binding molecules described herein comprise a modified heavy chain constant region, in which the Fc domain is modified to modulate its binding affinity and/or specificity to Fc receptors such as FcyRs.
In some embodiments, the Fc variants in the CD 137-binding molecules described herein have enhanced selectivity to FcyRIIB relative to its wild-type counterpart. An Fc fragment having selectivity to FcyRIIB, selectively binding to FcyRIIB, or specifically binding to FcyRIIB is a term well understood in the art. A molecule is said to exhibit “selective binding” or“specific binding” if it reacts more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen (e.g., an FcyRIIB receptor) than it does with alternative targets (e.g., FcyRIII receptors). An Fc fragment “specifically binds” to an Fc receptor if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other Fc receptors. For example, an Fc fragment that specifically (or preferentially) binds to FcyRIIB is an Fc fragment that binds this Fc receptor with greater affinity, avidity, more readily, and/or with greater duration than it binds to other Fc receptors. It is also understood with this definition that, for example, an Fc fragment that selectively or specifically binds to a first Fc receptor may or may not specifically or preferentially bind to a second Fc receptor. As such,“selective binding,” “specific binding” or“preferential binding” does not necessarily require (although it can include) exclusive binding. In some examples, an Fc fragment that“selectively binds,” or “specifically binds” to a target Fc receptor (e.g. , FcyRIIB) may not bind to other Fc receptors (i.e., binding not detectable by routine methods). In other embodiments, the variant Fc fragment does not bind to any FcyRs.
Relative binding affinities of IgGl, IgG2, and IgG4 to different Fc receptors are given in Table 1 below.
Table 1. Relative Binding Affinities of Human and Mouse Immunoglobulins to Fc Receptors
Figure imgf000020_0001
Figure imgf000021_0001
The Fc variants described herein may have enhanced selectivity to FcyRIIB relative to their wild-type counterparts (the wild-type parent Fc region in which mutations are introduced to produce the Fc variants). The relative binding activity to FcyRIIB versus another Fc receptor (e.g. , FcyRIII) of such an Fc variant is higher than the relative binding activity to FcyRIIB versus the other Fc receptor (e.g., FcyRIII) of the wild-type counterpart. The Fc variant may have enhanced binding activity to FcyRIIB and/or decreased binding activity to another Fc receptor, for example, FcyRIII. In some embodiments, the Fc variants described herein may have decreased binding activity to both FcyRIIB and another Fc receptor (for example, FcyRIII); however, the level of decreased binding activity to the other Fc receptor (e.g., FcyRIII) is greater than the level of decreased binding activity to FcyRIIB.
In some embodiments, an Fc variant as described herein has a suitable binding affinity for FcyRIIB, e.g. , enhanced as compared with the wild-type parent Fc from which the Fc variant is derived. As used herein,“binding affinity” refers to the apparent association constant or KA. The KA is the reciprocal of the dissociation constant (KD). The Fc variant described herein may have a binding affinity (KD) of at least 10 5, 106, 10 7, 10 8, 109, 10 10 M, or lower for FcyRIIB. An increased binding affinity corresponds to a decreased KD. Higher affinity binding of an Fc fragment for a first Fc receptor relative to a second Fc receptor can be indicated by a higher KA (or a smaller numerical value KD) for binding the first Fc receptor than the KA (or numerical value KD) for binding the second Fc receptor. In such cases, the Fc variant has specificity for the first Fc receptor relative to the second Fc receptor. In some embodiments, the Fc variants described herein have a higher binding affinity (a higher KA or smaller KD) to FcyRIIB as compared to the binding affinity to FcyRIII (either FcyRIIIA or FcyRIIIB). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 105 fold.
In some embodiments, the Fc variants described herein may be designed by mutating one or more amino acid residues in the wild-type of human IgGl, IgG2, or IgG4 Fc fragments in light of the amino acid residues in the corresponding mouse IgG, for example, mouse IgGl. A sequence comparison of human and mouse IgGs (hlgG and mlgG, respectively) is provided below (SEQ ID NOs: 60-64, from top to bottom, each representing a combination of fragments 211-245, 260-278, and 320-332 of the corresponding Fc region):
21- 22- 23- 24- ...26- 27- ...32- 33-
12345678 901234567890123456789012345...0123456789012345678...0123456789012
Upper Corel·ower CH2
hlgGl VDKKVEPK-SCDKTHTCPPCPAPE11GGPSVF1FPP...TCVWDVSHEDPEVKFNWY...KCKVSNKA1PAPI hIgG2 VDKTVERK-CC-V-E-CPPCPAPPVA-GPSVF1FPP...TCVWDVSHEDPEVQFNWY...KCKVSNKG1PAPI hIgG4 VDKRVESKYG-PPCPSCPAPEF1GGPSVF1FPP...TCVWDVSQEDPEVQFNWY...KCKVSNKG1PSSI mlgGl VDKKIVPR-DC—GCKPCJCTVPEVS-SVFIFPP...TCVWDISKDDPEVQFSWF...KCRVNSAAFPAPI mIgG2a VDKKIEPRGPTIKP PPCKCPAPNLLGGPSVFIFPP...TCVWDVSEDDPDVQISWF...KCKVNNKDLPAPI
The amino acid sequences of wild-type murine IgGl and IgG2 Fc fragments, as well as exemplary Fc variants with reduced FcyR binding are provided below:
Amino acid sequence of wild-type mouse IgGl Fc fragment:
VDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVWDI SKDDPEVQFSWFVDDVEVHTAQTQ PREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSL TCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKS LSHSPGK (SEQ ID NO: 144)
Amino acid sequence of wild-type mouse IgG2a Fc fragment:
VDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVWDVSEDDPDVQISWFVNNVEV HTAQTQTHREDYNSTLRWSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMT KKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSWHEGLHN HHTTKSFSRTPGK (SEQ ID NO: 145)
Amino acid sequence of mutant mouse IgGlmDANA Fc fragment:
VDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVWAI SKDDPEVQFSWFVDDVEVHTAQTQ PREEQFASTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSL TCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKS LSHSPGK (SEQ ID NO: 146)
Amino acid sequence of mutant mouse IgG2almDANA Fc fragment:
VDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVWAVSEDDPDVQISWFVNNVEV HTAQTQTHREDYASTLRWSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMT KKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSWHEGLHN HHTTKSFSRTPGK (SEQ ID NO: 147)
In some embodiments, the Fc variants described herein is a human IgGl, G2, or G4 Fc variants comprising one or more mutations (e.g. , amino acid substitutions, deletions, or additions) in the hinge domain of an Fc fragment. Human IgGs contain a core motif of CPPC or CPSC in the hinge domain (positions 226-229 according to the EU index). Positions 216 to 225 are deemed as the upper portion of the hinge domain and positions 230-238 are deemed as the lower portion of the hinge domain. The numbering system used herein, unless explicitly indicated, is according to the EU index. In some examples, the one or more mutations can be located in the upper portion of the hinge domain. Alternatively or in addition, the one or more mutations can be located in the lower portion of the hinge domain. In some embodiments, the Fc region of any of the anti-CDl37 molecules disclosed herein may comprise at least one mutation at any of positions 220-331, preferably at any of positions 228-329. Such Fc variants may have altered binding affinity and/or specificity to one or more of Fc receptors ( e.g ., FcyllB, FcyRI, FcyRIIA, or FcyRIIIA) as compared with their native counterparts.
The mutations to a human IgG Fc can be made according to the corresponding amino acid residues in the hinge domain of mouse IgGl. For example, mouse IgGl does not contain the GGP motif at positions 236-238. Accordingly, one or more of the residues in this GGP motif can be deleted from a human IgGl, IgG2, or IgG4 Fc fragment to produce the Fc variants described herein.
Alternatively or in addition, the human Fc variants may contain one or more amino acid substitutions in the upper portion, in the lower portion, or both of the hinge domain. For example, the Fc variant may comprise one or more amino acid substitutions at one or more of positions 233, 234, 235, and/or 236. Such an amino acid substitution may be in combination with the deletion of one or more of the GGP motif (236-238) noted herein. These mutations may be introduced into a human IgG2 or IgG4 Fc fragment to produce the Fc variants described herein. In some examples, the Fc variants described herein contains a deletion at one or more of the positions 236-238 (e.g., 236, 237, 238 or any combination thereof)
Any of the mutations in the hinge domain described herein may be in combination with a mutation (e.g., amino acid substitutions) at one or more positions that are involved in interaction with an Fc receptor. Such positions include, but are not limited to, positions 265, 267, 273, 297, and 327-331, or a combination thereof. Exemplary amino acid substitutions at those positions include D265A, S261E, V271E, N297A, F328F, P329G, A330S, and/or P331S.
In some embodiments, the Fc variants disclosed herein can be derived from an IgGl molecule (e.g., human IgGl) and contain one or more mutations at positions at one or more of positions 220, 226, 229, 234-238, 265, 267, 297, and 327-331. For example, the Fc variant may comprise a substitution or deletion within positions 234-238, a substitution at position 265 ( e.g ., D265A), a substitution at position 267 (e.g., S267E), a substitution at position 297 ( e.g ., N297A), a substitution at position 328 (e.g. , L328F), a substitution at position 329 (e.g., P329G), or a combination thereof. Alternatively or in addition, it may comprise a substitution at one or more of the positions 220 (e.g., C220S), 226 (e.g., C226S), 229 (e.g., C229S), 327 (e.g., A327G), 330 (e.g., A330S), and 331 (e.g., P331S). In some instances, the Fc variants derived from an IgGl molecule may comprise a deletion at one or more of positions 236-238.
In some embodiments, the Fc variants disclosed herein can be derived from an IgG2 molecule (e.g., human IgG2) and contain one or more mutations at one or more of positions 233-235, 237-238, 265-268, 273, 297, 328, 330, and 331. In some examples, such a Fc variant may comprise comprises a deletion within positions 237-238 (e.g. , a deletion at position 237 or a deletion of both positions 237 and 238), a substitution at position 265 (e.g. , D265A), a substitution at position 267 (e.g., S267E), a substitution at position 297 (e.g., N297A), a substitution at position 328 (e.g., F328F), or a combination thereof. Alternatively or in addition, the Fc variant derived from an IgG2 molecule may comprise a substitution at one or more of positions 233-235, 237, 238, 268, 273, 330, and 331 (e.g., P233E, V234A, V234F, A235F, A235S, G237A, P238S, H268A, H268Q, V273E, A330S, and P331S).
In yet other embodiments, the Fc variants disclosed herein can be derived from an IgG4 molecule (e.g., human IgG4) and contain one or more mutations at one or more positions of 228, 233-238, 265, 267, 273, 297, and 328. In some examples, such a Fc variant may comprise a substitution at position 228 (e.g. , S228P), a substitution or deletion at any of positions 235-238 (e.g., a deletion at one of positions 236-238, for example, position 236 and position 237), a substitution at position 265 (e.g., D265A), a substitution at position 267 (e.g., S267E), a substitution at position 273 (e.g., V273E), a substitution at position 297 (e.g.,
N297A), a substitution at position 328 (e.g., F328F), or a combination thereof. Alternatively or in addition, the Fc variant may comprise a substitution at one or more of positions 233-235 and 237 (e.g., E233P, F234V, F234A, F235S, F235E, F235A, and G237A).
Fc variants derived from IgG2 or IgG4 molecules that contain one or more mutations at positions 265, 267, 273, 297, 328, and/or 329 are also within the scope of the present disclosure. Such mutations may include amino acid substitutions at one or more of these positions, for example, D265A, S261E, V271E, N297A, F328F, and/or P329G. In some instances, any of the Fc variants disclosed herein may further comprise a mutation at position 309, for example, a substitution ( e.g ., V309L). Such an Fc variant may be derived from an IgG2 molecule (e.g., human IgG2). In some embodiments, the mutation at position 309 may be in combination with mutations at one or more of positions 234, 268, 330, and 331. Examples of such Fc variants include G2m43.
In some embodiments, an Fc variant described herein may comprise an amino acid sequence at least 85% identical (e.g., 90%, 95%, 98%, 99%, or above) to that of its wild-type counterpart (e.g. , the Fc fragment of wild-type human IgGl , IgG2, or IgG4 described herein).
In one example, the amino acid residue substitutions in an Fc variant described herein are conservative amino acid residue substitutions.
Provided below is a sequence alignment showing exemplary positions where mutations can be introduced into hlgGl, hIgG2, and IgG4 to produce various Fc variants for the present disclosure.
Sequence alignment of human IgGl variants relative to wild-type human IgGl (SEQ ID NOs: 70-88 and 161-166 from top to bottom ):
21 22- 23- 24- 26- 27- ...32- 33-
12345678 901234567890123456789012345. 0123456789012345678...0123456789012
Upper Corelower CH2
hlgGl VDKKVEPK-SCDKTHTCPPCPAPELL FLFPP. .TCVWDVSHEDPEVKFNWY. .KCKVSNKALPAPI hlgGlml KVEPK-SCDKTHTCPPCPAPELL- FLFPP. .TCVWDVSHEDPEVKFNWY. .KCKVSNKALPAPI hIgGlm2 KVEPK-SCDKTHTCPPCPAPELL-C
Figure imgf000025_0001
FLFPP. .TCVWDVSHEDPEVKFNWY. .KCKVSNKALPAPI hIgGlm-2 KKVEPK-C -v-e-CPPCPAPELLGC FLFPP. .TCVWDVSHEDPEVKFNWY. .KCKVSNKALPAPI hIgGlm-4 KKVEPKYG|:|||ppCPPCPAPELLGGPSVFLFPP .TCVWDVSHEDPEVKFNWY. .KCKVSNKALPAPI hIgGlm5 KVEPK-SCDKTHTCPPCPAPELLGGPSVFLFPP .TCVWDVSHEDPEVKFNWY. .KCKVSNKALPAPI hIgGlm7 KVEPK-SCDKTHTCPPCPAPELLGGPSVFLFPP TCVWDV|HEDPEVKFNWY. .KCKVSNKALPAPI hIgGlm8 KVEPK-SCDKTHTCPPCPAPELLGGPSVFLFPP .TCVWDVSHEDPEVKFNWY. .KCKVSNKAfPAPI hIgGlm9 KVEPK-SCDKTHTCPPCPAPELLGGPSVFLFPP .TCVWDViHEDPEVKFNWY. .KCKVSNKAIPAPI hIgGlml5 KVEPK-SCDKTHTCPPCPAPELLiiiSVFLFPP. .TCVWDVSHEDPEVKFNWY. .KCKVSNKALPAPI hIgGlml7 KVEPK-SCDKTHTCPPCPAPELL|1|:SVFLFPP. .TCVWDVVHEDPEVKFNWY. .KCKVSNKALPAPI hIgGlml8 KVEPK-SCDKTHTCPPCPAPELL|1|:SVFLFPP. .TCVWDVSHEDPEVKFNWY. .KCKVSNKAfPAPI hlgGlml 9 KVEPK-SCDKTHTCPPCPAPELLiilSVFLFPP. .TCVWDVVHEDPEVKFNWY. .KCKVSNKAfPAPI hIgGlm25 KVEPK-SCDKTHTCPPCPAPELL|GPSVFLFPP. .TCVWDVSHEDPEVKFNWY. .KCKVSNKALPAPI hIgGlm27 KVEPK-SCDKTHTCPPCPAPELL|GPSVFLFPP. .TCVWDVVHEDPEVKFNWY. .KCKVSNKALPAPI hIgGlm28 KVEPK-SCDKTHTCPPCPAPELL|GPSVFLFPP. .TCVWDVSHEDPEVKFNWY. .KCKVSNKAfPAPI hIgGlm29 KVEPK-SCDKTHTCPPCPAPELL|GPSVFLFPP. .TCVWDVVHEDPEVKFNWY. .KCKVSNKAfPAPI hlgGlmAA KKVEPK-SCDKTHTCPPCPAPE^GGPSVFLFPP. .TCVWDVSHEDPEVKFNWY. .KCKVSNKALPAPI hlgGlmAG KKVEPK-SCDKTHTCPPCPAPEiSGGPSVFLFPP. .TCVWDVSHEDPEVKFNWY. .KCKVSNKAL API hIgGlm40 VDKKVEPK-SCDKTHTCPPCPAPELLGGPSVFLFPP. .TCWvivSHEDPEVKFNWY. .KCKVSNKALPAPI hIgGlm45 VDKKVEPK-SCDKTHTCPPCPAPpva-G-SVFLFPP. .TCVWDVSHEDPEVKFNWY..KCKVSNKALPAPI hIgGlm24 OVDKKVEPK-SCDKTHTCPPCPAPELL-GPSVFLFPP. .TCWvivSHEDPEVKFNWY..KCKVSNKALPAPI hIgGlm47 VDKKVEPK-SCDKTHTCPPCPAPpva-G-SVFLFPP. .TCVWDVSHEDPEVKFNWY..KCKVSNKg -ssl hIgGlm48 VDKKVEPK-slDKTHTiPP|PAPELLGG|SVFLFPP. .TCVWDVSHEDPEVKFNWY..KCKVSNKALPAPI hIgGlm49 VDKKVEPK-SCDKTHT§PP|PAP|i§GGPSVFLFPP. .TCVWDVSHEDPEVKFNWY..KCKVSNKALPAPI h!gGlm50 VDKKVEPK-SCDKTHTCPPCPAPE!IGGPSVFLFPP. .TCVWDVSHEDPEVKFNWY. .KCKVSNKALPA!I
Sequence alignment of human IgG2 variants relative to wild-type human IgG2 (SEQ ID NOs: 89-105 and 167-171, from top to bottom ):
Figure imgf000026_0001
Sequence alignment of human IgG4 variants relative to wild-type human IgG4 (SEQ ID NOs: 65, 106-127, and 172-175, from top to bottom)
21- 22- 23- 24- ...26- 27- ...32- 33-
12345678 901234567890123456789012345. 0123456789012345678...0123456789012 Upper CoreLower CH2
hIgG4 VDKRVESKYG-PPCPSCPAPEFLGGPSVFLFPP. .TCVWDVSQEDPEVQFNWY. .KCKVSNKGLPSSI hIgG4ml KRVESKYG-PPCPPCPAPEFL-SVFLFPP. .TCVWDVSQEDPEVQFNWY. .KCKVSNKGLPSSI hIgG4m-l KRVESKISCDKTHTCPPCPAPEFLGGPSVFLFPP. .TCVWDVSQEDPEVQFNWY. .KCKVSNKGLPSSI hIgG4m2 KRVESKYG-PPCPPCPAPEFL|GPSVFLFPP. .TCVWDVSQEDPEVQFNWY. .KCKVSNKGLPSSI hIgG4m-2 KRVESK-CC-v-efCPPCPAPEFLGGPSVFLFPP. .TCVWDVSQEDPEVQFNWY. .KCKVSNKGLPSSI hIgG4m3 KRVESKYG-PPCPPCPAPEFLGGPSVFLFPP. TCVWDVSQEDPE|QFNWY. .KCKVSNKGLPSSI hIgG4m4 KRVESKYG-PPCPPCPAPEFL-SVFLFPP. TCVWDVSQEDPE|QFNWY. .KCKVSNKGLPSSI hIgG4m5 KRVESKYG-PPCPPCPAPEFL-GPSVFLFPP. TCVWDVSQEDPE|QFNWY. .KCKVSNKGLPSSI hIgG4m7 KRVESKYG-PPCPPCPAPEFLGGPSVFLFPP. TCVWDV|QEDPEVQFNWY. .KCKVSNKGLPSSI hIgG4m8 KRVESKYG-PPCPPCPAPEFLGGPSVFLFPP. .TCVWDVSQEDPEVQFNWY. .KCKVSNKGifPSSI hIgG4m9 KRVESKYG-PPCPPCPAPEFLGGPSVFLFPP. TCVWDV|QEDPEVQFNWY. .KCKVSNKG¾PSSI hIgG4mlO KRVESKYG-PPCPPCPAPEVS-SVFLFPP. .TCVWDVSQEDPEVQFNWY. .KCKVSNKG|PSSI hIgG4ml5 KRVESKYG-PPCPPCPAPEFL!!HSVFLFPP. TCVWDVSQEDPE|QFNWY. .KCKVSNKGLPSSI hIgG4ml7 KRVESKYG-PPCPPCPAPEFLlllisVFLFPP.TCVWDV|QEDPEVQFNWY. .KCKVSNKGLPSSI hIgG4ml8 KRVESKYG-PPCPPCPAPEFLlllisVFLFPP. .TCVWDVSQEDPEVQFNWY. .KCKVSNKGifPSSI hIgG4ml 9 KRVESKYG-PPCPPCPAPEFLlllisVFLFPP. TCVWDV|QEDPEVQFNWY. .KCKVSNKG PSSI hIgG4m20 KRVESKYG-PPCPPCPAPEFLGiiSVFLFPP. .TCVWDVSQEDPEVQFNWY. .KCKVSNKGLPSSI hIgG4m25 KRVESKYG-PPCPPCPAPEFL|GPSVFLFPP. TCVWDVSQEDPE|QFNWY. .KCKVSNKGLPSSI hIgG4m27 KRVESKYG-PPCPPCPAPEFL|GPSVFLFPP. TCVWDV|QEDPEVQFNWY. .KCKVSNKGLPSSI hIgG4m28 KRVESKYG-PPCPPCPAPEFL|GPSVFLFPP. .TCVWDVSQEDPEVQFNWY. .KCKVSNKG PSSI hIgG4m29 KRVESKYG-PPCPPCPAPEFL|GPSVFLFPP. TCVWDV|QEDPEVQFNWY. .KCKVSNKGfPSSI hIgG4m30 KRVESKYG-PPCPPCPAPEFL||PSVFLFPP. .TCVWDVSQEDPEVQFNWY. .KCKVSNKGLPSSI hIgG4mPE KRVESKYG-PPCPPCPAPEFIGGPSVFLFPP. .TCVWDVSQEDPEVQFNWY. .KCKVSNKGLPSSI hIgG4m40 VDKRVESKYG-PPCPPCPAPEFLGGPSVFLFPP. .TCVWiVSQEDPEVQFNWY. .KCKVSNKGLPSSI hIgG4m41 VDKRVESKYG-PPCPPCPAPpvaGGPSVFLFPP. .TCVWiVSQEDPEVQFNWY. .KCKVSNKGLPSSI hIgG4m42 VDKRVESKYG-PPCPPCPAPpvaGGPSVFLFPP. .TCVWiVSQEDPEVQFNWY. .KCKVSNKGLPSSI hIgG4m46 VDKRVESKYG-PPCPPCPAPE§LGiPSVFLFPP. .TCVWDVSQEDPEVQFNWY. .KCKVSNKGLPSSI* hIgG4mAA VDKRVESKYG-PPCPPCPAPEISGGPSVFLFPP. .TCVWDVSQEDPEVQFNWY. .KCKVSNKGLPSSI The amino acid sequences of wild-type human IgGl, IgG2, and IgG4 Fc fragments, and a number of exemplary hlgGl, hIgG2, and hIgG4 Fc variants (positions 221 and onward based on EU numbering) are provided below:
Amino acid sequence of wild-type human IgGl Fc fragment:
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 1)
Amino acid sequence of wild-type human IgG2 Fc fragment:
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 2)
Amino acid sequence of wild-type human IgG4 Fc fragment:
VDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 3)
Amino acid sequence of human IgG4 S228P Fc variant:
VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 4)
Amino acid sequences of exemplary human IgGl Fc variants:
Glml :
VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO: 5)
Glm2 :
VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 6)
Glm-2 :
VDKKVEPKCCVECPPCPAPELLSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 7)
Glm-4 : VDKKVEPKYGPPCPPCPAPELLGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 8)
Glm5 :
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEEKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 9)
Glm7 :
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVEHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 10)
Glm8 :
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 11)
Glm9 :
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVEHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 12)
Glml5 :
VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEEKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO: 13)
Glml7\
VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVWDVEHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO: 14)
Glml8 :
VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO: 15)
Glml9 :
VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVWDVEHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO: 16)
Glm25 :
VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEEKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 17)
Glm27:
VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVWDVEHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 18)
Glm28 :
VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 19)
Glm29 :
VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVWDVEHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 20)
GlmAA :
VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 21)
GlmAG :
VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 22)
Glm40 :
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVAVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 148)
Glm45 VDKKVEPKSCDKTHTCPPCPAPpvaGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 66)
Glm240
VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVWAVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 67)
G1 N297A
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYaSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 68)
Glm47
VDKKVEPKSCDKTHTCPPCPAPpvaGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKgLPss IEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 69)
Glm48
VDKKVEPKSsDKTHTsPPsPAPELLGGsSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 176)
Glm49
VDKKVEPKSCDKTHTsPPsPAPpvaGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 177)
Glm50
VDKKVEPKSCDKTHTCPPCPAPEfeGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAsIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 178)
Amino acid sequences of exemplary human IgG2 Fc variants:
G2ml :
VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 23) G2m-1:
VDKTVERKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 24)
G2m2:
VDKTVERKCCVECPPCPAPPFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO: 25)
G2m-4:
VDKTVERKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 26)
G2m5:
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEEQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 27)
G2m7:
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVEHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 28)
G2m8 :
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGEPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 29)
G2m9 :
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVEHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 30)
G2ml0:
VDKTVERKCCVECPPCPAPEVSSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 31) G2ml5 :
VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEEQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 32)
G2ml7:
VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMI SRTPEVTCWVDVEHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 33)
G2ml8 :
VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 34)
G2ml9 :
VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMI SRTPEVTCWVDVEHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 35)
G2m20:
VDKTVERKCCVECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 36)
G2m27 :
VDKTVERKCCVECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVWDVEHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 37)
G2m28:
VDKTVERKCCVECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 38)
G2m40 :
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVAVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 149)
G2m43
VDKTVERKCCVECPPCPAPPaaasSVFLFPPKPKDTLMI SRTPEVTCWVDVSaEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVIHQDWLNGKEYKCKVSNKGLPs SlEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 179)
G2m44
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSqEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVIHQDWLNGKEYKCKVSNKGLPssIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 180)
G2mAA
VDKTVERKCCVECPPCPAPPaaaPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 181)
G2m2040
VDKTVERKCCVECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVWAVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFASTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 182)
G2G4
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK (SEQ ID NO: 183)
Amino acid sequences of exemplary human IgG4 Fc variants:
G4ml :
VDKRVESKYGPPCPPCPAPEFLSVFLFPPKPKDTLMI SRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK (SEQ ID NO: 39)
G4m-1 :
VDKRVESKSCDKTHTPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK (SEQ ID NO: 40)
G4m2:
VDKRVESKYGPPCPPCPAPEFLGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK (SEQ ID NO: 41)
G4m-2: VDKRVESKCCVEPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK (SEQ ID NO: 42)
G4m3:
VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEEQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 43)
G4m4:
VDKRVESKYGPPCPPCPAPEFLSVFLFPPKPKDTLMI SRTPEVTCWVDVSQEDPEEQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK (SEQ ID NO: 44)
G4m5 :
VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEEQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 45)
G4m7 :
VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVEQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 46)
G4m8:
VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 47)
G4m9 :
VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVEQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 48)
G4ml0 :
VDKRVESKYGPPCPPCPAPEVSSVFLFPPKPKDTLMI SRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK (SEQ ID NO: 49)
G4ml7 :
VDKRVESKYGPPCPPCPAPEFLSVFLFPPKPKDTLMI SRTPEVTCWVDVEQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK (SEQ ID NO: 50)
G4ml8:
VDKRVESKYGPPCPPCPAPEFLSVFLFPPKPKDTLMI SRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK (SEQ ID NO: 51)
G4ml9 :
VDKRVESKYGPPCPPCPAPEFLSVFLFPPKPKDTLMI SRTPEVTCWVDVEQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK (SEQ ID NO: 52)
G4m20:
VDKRVESKYGPPCPPCPAPEFLGSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 53)
G4m25 :
VDKRVESKYGPPCPPCPAPEFLGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSQEDPEEQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK (SEQ ID NO: 54)
G4m27 :
VDKRVESKYGPPCPPCPAPEFLGPSVFLFPPKPKDTLMI SRTPEVTCWVDVEQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK (SEQ ID NO: 55)
G4m28:
VDKRVESKYGPPCPPCPAPEFLGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK (SEQ ID NO: 56)
G4m29:
VDKRVESKYGPPCPPCPAPEFLGPSVFLFPPKPKDTLMI SRTPEVTCWVDVEQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK (SEQ ID NO: 57)
G4m30: VDKRVESKYGPPCPPCPAPEFLPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 58)
G4mPE :
VDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 59)
G4m40\
VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWAVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFASTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 150)
G4m41
VDKRVESKYGPPCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVWAVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTWHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 184)
G4m42
VDKRVESKYGPPCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVWAVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 185)
G4m46
VDKRVESKYGPPCPPCPAPEaLGaPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTVLHQDWLNGKaYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 186)
G4mAA
VDKRVESKYGPPCPPCPAPEaaGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 187)
The Fc variants described herein may exhibit an enhanced binding activity to FcyRIIB as compared with the wild-type counterpart. Examples include G2m2, G2m5, G2m7, G2m8, G2m9, G2ml9, G2m44, Glm7, Glm9, Glm27, Glm45, Glm47, Glm49, Glm50, G4m7, G4m46 and G4mPE. Alternatively or in addition, the Fc variants may have an enhanced selectivity to FcyRIIB as compared with their wild-type counterparts, for example, G2ml, G2m20, G2m43, G2m44, G2G4, G2mAA, Glm2, Glml5, Glml7, Glml8, Glml9,
Glm27, Glm28, Glm29, G4ml, G4m2, G4m7, G4m8, G4m9, G4m20, G4m25, G4m27, G4m28, G4m30, G4m46 and G4mPE. Such Fc variants may retain the binding activity to FcRn. These Fc variants can be used for constructing therapeutic agents described herein capable of cross-linking immune receptors and FcyRIIB receptor.
Alternatively, certain Fc variants as described herein may have selectivity to FcyRIIB and/or apparent low or no binding activity to any FcyR. Examples include Glm2, Glm25, Glm40, GlmAAG, Glm240, G2ml, G2m20, G2m40, G2m2040, G4m5, G4ml8, G4ml9, G4m20, G4m30, G4m40, G4m4l, and G4m42. Such Fc variants may retain the binding activity to FcRn. Therapeutic agents ( e.g . , antibodies) containing such Fc variants may be capable of cross-linking immune receptors and FcyRIIB receptor due to avidity effect.
The changes of binding affmity/specificity of the exemplary Fc variants as compared with their wild-type counterparts are provided in Tables 2-4 below.“N/A” indicates no data available. When the binding activity of an Fc variant is found to be“no change” as compared with the wild-type counterpart, it means that there is no significant variation of the binding activity between the Fc variant and the wild-type counterpart as indicated by the same assay under the same experimental conditions. When the binding activity of an Fc variant is “increased” or“decreased” as relative to its wild-type counterpart means that the binding activity of the Fc variant is higher or lower than that of the wild-type counterpart as determined by the same assay under the same experimental conditions and the variation is significant (e.g., biologically significant) as known to those skilled in the art. When the binding activity of an Fc variant is“slightly increased” or“slightly decreased” as relative to its wild-type counterpart means that the binding activity of the Fc variant is higher or lower than that of the wild-type counterpart as determined by the same assay under the same experimental conditions and the variation is statistically significant but to a limited level (e.g., up to 10%).
Table 2. FcyR Binding Activity of Human IgGl Mutants as Relative to Wild-Type Human IgGl
ity Relative to Wild-Type Counterparts
Figure imgf000037_0001
Figure imgf000037_0002
Figure imgf000038_0001
Further, Glm40 would be expected to have decreased or no apparent binding activity to FcyRI, FcyRI I A (H131), FcyRIIB, FcyRIIC, and FcyRIII. Table 3. FcyR Binding Activity of Human IgG2 Mutants as Relative to Wild-Type Human IgG2
Figure imgf000038_0002
Figure imgf000039_0001
Further, G2m40 would be expected to have decreased or no apparent binding activity to FcyRI, FcyRI I A (H131), FcyRI IB, FcyRIIC, and FcyRIII. Table 4. FcyR Binding Activity of Human IgG4 Mutants as Relative to Wild-Type Human IgG4
Figure imgf000039_0002
Figure imgf000040_0001
Further, G4m40 would be expected to have decreased or no apparent binding activity to FcyRI, FcyRI I A (H131), FcyRIIB, FcyRIIC, and FcyRIII.
An Fc variant as described herein can be designed following the guidance provided herein and produced via routine recombinant technology. Its binding affinity and specificity to various Fc receptors can be determined via routine methods. See also Examples below.
Besides the FACS binding assay described herein, the activity of Fc variants for Fey receptors can be examined in alternative assays. For example, CD137 reporter assays with or without co-culture of FcyRIIB expressing cells can be used to demonstrate effect of receptor cross-linking and activation. Because of avidity effect resulting from simultaneous binding of antibody to two targets ( e.g ., CD 137 and FcyRIIB), this assay is more sensitive to detect FcyRIIB binding. Minimal or no apparent binding detectable by FACS may show positive results in the reporter assay. II. Preparation of CD137-binding Molecules Comprising Fc Variants
The CD 137-binding molecules described herein may be prepared by conventional methodology, for example, recombinant technology. Some examples follow.
For CDl37-binding molecules comprising an extracellular domain of a CD137L, a coding sequence of the CD137L extracellular domain can be fused in frame with a coding sequence of a suitable Fc variant. The coding sequence for the whole CD 137-binding molecule can be cloned into a suitable expression vector, which can be introduced into a suitable host cell for protein expression.
Antibodies binding to CD 137 can be prepared by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, antibodies specific to CD 137 or an extracellular domain thereof can be made by the conventional hybridoma technology. The full-length target receptor or a fragment thereof, optionally coupled to a carrier protein such as KLH, can be used to immunize a host animal for generating antibodies binding to that antigen. The route and schedule of immunization of the host animal are generally in keeping with established and conventional techniques for antibody stimulation and production, as further described herein. General techniques for production of mouse, humanized, and human antibodies are known in the art and are described herein. It is contemplated that any mammalian subject including humans or antibody producing cells therefrom can be manipulated to serve as the basis for production of mammalian, including human hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar, and/or intradermally with an amount of immunogen, including as described herein.
Hybridomas can be prepared from the lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D. W., et al, In Vitro, 18:377-381 (1982).
Available myeloma lines, including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, may be used in the hybridization. Generally, the technique involves fusing myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art. After the fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized parent cells. Any of the media described herein, supplemented with or without serum, can be used for culturing hybridomas that secrete monoclonal antibodies. As another alternative to the cell fusion technique, EBV immortalized B cells may be used to produce the anti-immune cell receptor monoclonal antibodies described herein. The hybridomas are expanded and subcloned, if desired, and supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures ( e.g ., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).
Hybridomas that may be used as source of antibodies encompass all derivatives, progeny cells of the parent hybridomas that produce monoclonal antibodies capable of modulating the activity of the target immune cell receptor. Hybridomas that produce such antibodies may be grown in vitro or in vivo using known procedures. The monoclonal antibodies may be isolated from the culture media or body fluids, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired. Undesired activity if present, can be removed, for example, by running the preparation over adsorbents made of the immunogen attached to a solid phase and eluting or releasing the desired antibodies off the immunogen. Immunization of a host animal with a target antigen or a fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOC1, or RlN=C=NR, where R and Rl are different alkyl groups, can yield a population of antibodies (e.g., monoclonal antibodies).
If desired, an antibody (monoclonal or polyclonal) of interest (e.g., produced by a hybridoma) may be sequenced and the polynucleotide sequence may then be cloned into a vector for further construction of the anti-CD 137 antibodies described herein. The sequence encoding the antibody of interest may be maintained in vector in a host cell and the host cell can then be expanded and frozen for future use.
In an alternative, the polynucleotide sequence may be used for genetic manipulation to“humanize” the antibody or to improve the affinity (affinity maturation), or other characteristics of the antibody. For example, the constant region may be engineered to more resemble human constant regions to avoid immune response if the antibody is used in clinical trials and treatments in humans. It may be desirable to genetically manipulate the antibody sequence to obtain greater affinity to the target antigen and greater efficacy in inhibiting or activating the activity of the immune cell receptor. It will be apparent to one of skill in the art that one or more polynucleotide changes can be made to the antibody and still maintain its binding specificity to the target receptor.
In other embodiments, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals that are designed to produce a more desirable (e.g. , fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are XenomouseR™ from Amgen, Inc. (Fremont, Calif.) and HuMAb-MouseR™ and TC MouseTM from Medarex, Inc. (Princeton, N.J.). In another alternative, antibodies may be made recombinantly by phage display or yeast technology. See, for example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et a , (1994) Annu. Rev. Immunol. 12:433-455.
Alternatively, antibody library technology, such as the phage display technology
(McCafferty et a , (1990) Nature 348:552-553), yeast display technology, or mammalian cell display technology, can be used to isolated antibodies such as human antibodies specific to a target immune receptor.
Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et af, Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989). In one example, variable regions of VH and VL of a parent non-human antibody are subjected to three- dimensional molecular modeling analysis following methods known in the art. Next, framework amino acid residues predicted to be important for the formation of the correct CDR structures are identified using the same molecular modeling analysis. In parallel, human VH and VL chains having amino acid sequences that are homologous to those of the parent non-human antibody are identified from any antibody gene database using the parent VH and VL sequences as search queries. Human VH and VL acceptor genes are then selected.
The CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the parent non-human antibody or functional variants thereof. When necessary, residues within the framework regions of the parent chain that are predicted to be important in interacting with the CDR regions (see above description) can be used to substitute for the corresponding residues in the human acceptor genes.
Once an antibody capable of binding to a target immune cell receptor is obtained, the coding sequence of its heavy chain can be fused in-frame with the coding sequence of a suitable Fc variant, which may selectively bind FcyRIIB or which does not bind any FcyRs, for example, any of the Fc variants described herein via routine recombinant technology. In some instances, the antibody is first investigated for its agonistic effect to activate the immune cell receptor to which it binds. Such an agonistic antibody can be selected for making the CD 137-binding molecules described herein to enhance the agonistic effects.
In other instances, the antibody is first investigated for its antagonistic effect to inhibit the immune cell receptor to which it binds. Such an antagonistic antibody can be selected for making the CD 137-binding molecule described herein to down-regulate immune responses. Alternatively, an Fc variant having low or no binding activity to any Fc receptor can be selected for making CD 137 antagonists.
The resultant antibody molecules or CD 137-binding molecules described herein can be produced via routine recombinant technology as exemplified below. Nucleic acids encoding the heavy and light chain of an antibody or the polypeptide of a CD 137-binding molecule as described herein can be cloned into one expression vector, each nucleotide sequence being in operable linkage to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy chain and light chain is in operable linkage to a distinct prompter. Alternatively, the nucleotide sequences encoding the heavy chain and the light chain can be in operable linkage with a single promoter, such that both heavy and light chains are expressed from the same promoter. When necessary, an internal ribosomal entry site (IRES) can be inserted between the heavy chain and light chain encoding sequences.
In some examples, the nucleotide sequences encoding the two chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each of them can be isolated from the host cells expressing such and the isolated heavy chains and light chains can be mixed and incubated under suitable conditions allowing for the formation of the antibody.
Generally, a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art. For example, the nucleotide sequence and vector can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of a gene. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector.
The selection of expression vectors/promoter would depend on the type of host cells for use in producing the antibodies.
A variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-l LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and the herpes simplex tk virus promoter. Regulatable promoters can also be used. Such regulatable promoters include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al, Cell, 49:603-612 (1987)], those using the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al, Human Gene Therapy, 9: 1939-1950 (1998); Shockelt, P., et al, Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP 16 or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad.
Regulatable promoters that include a repressor with the operon can be used. In one embodiment, the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown et al, Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci.
USA, 89:5547-5551 (1992)] combined the tetracycline repressor (tetR) with the transcription activator (VP 16) to create a tetR-mammalian cell transcription activator fusion protein, tTa (tetR- VP 16), with the tetO-bearing minimal promoter derived from the human
cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline inducible switch is used. The tetracycline repressor (tetR) alone, rather than the tetR- mammalian cell transcription factor fusion derivatives can function as potent trans-modulator to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy). One particular advantage of this tetracycline inducible switch is that it does not require the use of a tetracycline repressor-mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells (Gossen et al, Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al, Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)), to achieve its regulatable effects.
Additionally, the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.
Examples of polyadenylation signals useful to practice the methods described herein include, but are not limited to, human collagen I polyadenylation signal, human collagen II polyadenylation signal, and SV40 polyadenylation signal.
One or more vectors ( e.g ., expression vectors) comprising nucleic acids encoding any of the antibodies may be introduced into suitable host cells for producing the antibodies. The host cells can be cultured under suitable conditions for expression of the antibody or any polypeptide chain thereof. Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g., from the cells or the culture supernatant) via a conventional method, e.g., affinity purification. If necessary, polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time allowing for production of the antibody.
In some embodiments, methods for preparing an antibody described herein involve a recombinant expression vector that encodes both the heavy chain and the light chain of an antibody as described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g. , a dhfr- CHO cell) by a conventional method, e.g., calcium phosphate- mediated transfection. Positive transformant host cells can be selected and cultured under suitable conditions allowing for the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or from the culture medium. When necessary, the two chains recovered from the host cells can be incubated under suitable conditions allowing for the formation of the antibody.
In one example, two recombinant expression vectors are provided, one encoding the heavy chain of the anti -immune cell receptor antibody and the other encoding the light chain of the same antibody. Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr- CHO cell) by a conventional method, e.g., calcium phosphate- mediated transfection. Alternatively, each of the expression vectors can be introduced into a suitable host cells. Positive transformants can be selected and cultured under suitable conditions allowing for the expression of the polypeptide chains of the antibody. When the two expression vectors are introduced into the same host cells, the antibody produced therein can be recovered from the host cells or from the culture medium. If necessary, the polypeptide chains can be recovered from the host cells or from the culture medium and then incubated under suitable conditions allowing for formation of the antibody. When the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cells or from the corresponding culture media. The two polypeptide chains can then be incubated under suitable conditions for formation of the antibody.
Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recovery of the antibodies from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.
The bioactivity of the antibodies described herein can be verified using assays known in the art or described herein.
III. Pharmaceutical Compositions
The present disclosure provides pharmaceutical compositions comprising the CD 137- binding molecules or any of the anti-CD 137 antibodies described herein and uses of such for modulating immune responses triggered by CD137/CD137L signaling. Such CDl37-binding molecules as described herein or anti-CD 137 antibodies as also described herein can be used for treating diseases such as cancer or immune-related disorders.
The CDl37-binding molecules or anti-CDl37 antibodies as described herein can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a target disease. “Acceptable” means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated.
Pharmaceutically acceptable excipients (carriers) including buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.
The pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th
Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt- forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
In some examples, the pharmaceutical composition described herein comprises liposomes containing the antibodies (or the encoding nucleic acids) which can be prepared by methods known in the art, such as described in Epstein, et al, Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al, Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat.
Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidyl ethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
The CDl37-binding molecules, anti-CDl37 antibodies, or the encoding nucleic acid(s), may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano particles and nanocapsules) or in macroemulsions. Such techniques are known in the art, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).
In other examples, the pharmaceutical composition described herein can be formulated in sustained-release format. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl- methacrylate), or poly(v nylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable
microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
The pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic antibody compositions are generally 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 compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.
For preparing solid compositions such as tablets, the principal active ingredient can be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as com starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
Suitable surface-active agents include, in particular, non-ionic agents, such as polyoxyethylenesorbitans ( e.g ., Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions with a surface-active agent will conveniently comprise between 0.05 and 5% surface-active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.
Suitable emulsions may be prepared using commercially available fat emulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g. , soybean oil, safflower oil, cottonseed oil, sesame oil, com oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g., egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%. The fat emulsion can comprise fat droplets between 0.1 and 1.0 pm, particularly 0.1 and 0.5 pm, and have a pH in the range of 5.5 to 8.0.
The emulsion compositions can be those prepared by mixing an antibody with Intralipid™ or the components thereof (soybean oil, egg phospholipids, glycerol and water).
Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulized by use of gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner. IV. Therapeutic Applications
Any of the CD 137-binding molecules or anti-CD 137 antibodies disclosed herein may be used to modulating ( e.g ., enhancing or inhibiting) immune responses against invading pathogens and/or diseased cells such as cancer cells.
To practice the method disclosed herein, an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal,
intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution.
Alternatively, the antibodies as described herein can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.
The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.
In some instances, the subject is a human patient having or at risk for a cell-mediated disease or disorder, such as cancer including but not limited to lung cancer, stomach cancer, liver cancer, breast cancer, skin cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, or colorectal cancer. Further exemplary cancers include, but are not limited to, breast cancer; biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia, e.g., B Cell CLL; T-cell acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic myelogenous leukemia, multiple myeloma; AIDS-associated leukemias and adult T-cell leukemia/lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer including melanoma, Merkel cell carcinoma, Kaposi’s sarcoma, basal cell carcinoma, and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms tumor. A subject having a cancer can be identified by routine medical examination, e.g., laboratory tests, organ functional tests, CT scans, or ultrasounds. In some embodiments, the subject to be treated by the method described herein may be a human cancer patient who has undergone or is subjecting to an anti-cancer therapy, for example, chemotherapy, radiotherapy, immunotherapy, or surgery.
In other instances, the subject is a human patient having or at risk for an immune- related disorder. Immune-related disorders refer to a dysfunction of the immune system, including autoimmune diseases, immunodeficiencies, and/or allergies. In one embodiment, the immune-related disorder is an autoimmune disease. Examples of immune-related disorders include, but are not limited to, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Myasthenia Gravis (MG), Graves’ Disease, Idiopathic
Thrombocytopenia Purpura (ITP), Guillain-Barre Syndrome, autoimmune myocarditis, Membrane Glomerulonephritis, diabetes mellitus, Type I or Type II diabetes, multiple sclerosis, Reynaud's syndrome, autoimmune thyroiditis, gastritis, Celiac Disease, Vitiligo, Hepatitis, primary biliary cirrhosis, inflammatory bowel disease, spondyloarthropathies, experimental autoimmune encephalomyelitis, immune neutropenia, juvenile onset diabetes, and immune responses associated with delayed hypersensitivity mediated by cytokines, T- lymphocytes typically found in tuberculosis, sarcoidosis, and polymyositis, polyarteritis, cutaneous vasculitis, pemphigus, pemphigold, Goodpasture's syndrome, Kawasaki's disease, systemic sclerosis, anti-phospholipid syndrome, Sjogren's syndrome, graft-versus-host (GVH) disease, and immune thrombocytopenia. A subject having an immune-related disorder can be identified by routine medical examination, e.g., with laboratory tests. In some embodiments, the subject to be treated by the method described herein may be a human subject with an immune-related disorder who has undergone or is subjecting to an immune- related disorder treatment, for example, immunosuppressive mediation, hormone replacement therapy, blood transfusions, anti-inflammatory medication, and/or pain medication.
As used herein,“an effective amount” refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is modulating (e.g. , activating) the target immune receptor, thereby triggering or enhancing immune responses mediated by the receptor. Determination of whether an amount of the antibody achieved the therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.
Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. For example, molecules that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, may be used to prolong half-life of the antibody and to prevent the antibody being attacked by the host's immune system. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a target disease/disorder. Alternatively, sustained continuous release formulations of an antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.
In one example, dosages for a CD 137-binding molecule or an anti-CD 137 antibody as described herein may be determined empirically in individuals who have been given one or more administration(s) of the antibody. Individuals are given incremental dosages of the CDl37-binding molecule or an anti-CDl37 antibody. To assess efficacy of the CD137- binding molecule or an anti-CDl37 antibody, an indicator of the disease/disorder can be followed.
Generally, for administration of any of the therapeutic agents such as the CD 137- binding molecule or an anti-CDl37 antibody described herein, an initial candidate dosage can be about 2 mg/kg. For the purpose of the present disclosure, a typical daily dosage might range from about any of 0.1 pg/kg to 3 pg/kg to 30 pg/kg to 300 pg/kg to 3 mg/kg, to 30 mg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate a target disease or disorder, or a symptom thereof. An exemplary dosing regimen comprises administering an initial dose of about 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg of the antibody, or followed by a maintenance dose of about 1 mg/kg every other week. However, other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve.
For example, dosing from one-four times a week is contemplated. In some embodiments, dosing ranging from about 3 pg/mg to about 2 mg/kg (such as about 3 pg/mg, about 10 pg/mg, about 30 pg/mg, about 100 pg/mg, about 300 pg/mg, about 1 mg/kg, and about 2 mg/kg) may be used. In some embodiments, dosing frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the therapeutic used) can vary over time.
In some embodiments, for an adult patient of normal weight, doses ranging from about 0.3 to 5.00 mg/kg may be administered. In some examples, the dosage of the therapeutic agents such as antibodies described herein can be 10 mg/kg. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history, as well as the properties of the individual agents (such as the half-life of the agent, and other considerations well known in the art).
For the purpose of the present disclosure, the appropriate dosage of a CD 137-binding molecule or anti-CD 137 antibody as described herein will depend on the specific antibody, antibodies, and/or non-antibody peptide (or compositions thereof) employed, the type and severity of the disease/disorder, whether the CDl37-binding molecule or anti-CDl37 antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the agonist, and the discretion of the attending physician. Typically the clinician will administer a CD 137-binding molecule or anti-CD 137 antibody, until a dosage is reached that achieves the desired result. In some embodiments, the desired result is a decrease in thrombosis. Methods of determining whether a dosage resulted in the desired result would be evident to one of skill in the art. Administration of one or more CD 137-binding molecule or anti-CD 137 antibody can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of a CDl37-binding molecule or anti-CDl37 antibody may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing a target disease or disorder.
As used herein, the term“treating” refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease or disorder, a symptom of the disease/disorder, or a predisposition toward the disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder.
Alleviating a target disease/disorder includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, "delaying" the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that“delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.
“Development” or“progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein“onset” or“occurrence” of a target disease or disorder includes initial onset and/or recurrence.
In some embodiments, the antibodies described herein are administered to a subject in need of the treatment at an amount sufficient to activate the activity of the target receptor by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo. Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous,
intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods. In some examples, the pharmaceutical composition is administered intraocularly or intravitreally.
Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer’s solution or other suitable excipients.
Intramuscular preparations, e.g., a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as Water- for- Injection, 0.9% saline, or 5% glucose solution.
The particular dosage regimen, i.e., dose, timing and repetition, used in the method described herein will depend on the particular subject and that subject's medical history.
In some embodiments, more than one antibody, or a combination of an antibody and another suitable therapeutic agent, may be administered to a subject in need of the treatment. The CD 137-binding molecule or anti-CD 137 antibody can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the agents.
Treatment efficacy for a target disease/disorder can be assessed by methods well- known in the art.
The therapeutic agent described herein may be utilized in conjunction with other types of therapy for the target disease such as cancer. Additional anti-cancer therapy includes chemotherapy, surgery, radiation, gene therapy, and so forth. When a second therapeutic agent is used, such an agent can be administered simultaneously or sequentially (in any order) with the CD 137-binding molecule or anti-CD 137 antibody described herein.
When co-administered with an additional therapeutic agent, suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.
The treatments of the disclosure can be combined with other immunomodulatory treatments such as, e.g. , therapeutic vaccines (including but not limited to GVAX, DC-based vaccines, etc.), or checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.). Alternatively, the treatment of the present disclosure can be combined with a chemotherapeutic agent, for example, pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine), purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupters such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyhnelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide,
triethylenethiophosphoramide and etoposide (VP 16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L- asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents;
antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and
methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate);
platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin);
immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds ( e.g ., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors;
mitochondrial dysfunction inducers and caspase activators; and chromatin disrupters.
For examples of additional useful agents see also Physician's Desk Reference, 59.sup.th edition, (2005), Thomson P D R, Montvale N.J.; Gennaro et al, Eds. Remington's The Science and Practice of Pharmacy 20.sup.th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, l5.sup.th edition, (2001), McGraw Hill, NY; Berkow et al, Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.
V. Kits
The present disclosure also provides kits for use in enhancing the desired immune responses using any of the CD 137-binding molecule or anti-CD 137 antibody described herein.
In some embodiments, the kit can comprise instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of administration of the therapeutic agent to treat, delay the onset, or alleviate a target disease as those described herein. The kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease. In still other embodiments, the instructions comprise a description of administering a therapeutic agent such as an antibody to an individual at risk of the target disease. The instructions relating to the use of the therapeutic agent generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages ( e.g ., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g. , a paper sheet included in the kit), but machine- readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
The label or package insert indicates that the composition is used for treating, delaying the onset and/or alleviating a target disease or disorder such as cancer. Instructions may be provided for practicing any of the methods described herein.
The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g. , sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g. , an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the therapeutic agent as those described herein.
Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture comprising contents of the kits described above.
General Techniques
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A
Laboratory Manual, second edition (Sambrook, et al, 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al, eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford
University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995).
Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.
Example 1. Engineering human IgGl, IgG2 and IgG4 for selective FcyRIIB/CD32B binding
To verify the impact of mutations in the hinge domain of human IgGl, IgG2, and
IgG4 on binding activity to FcyRIIB/CD32B, the human Fc variants Glml, Glm2, G2ml, G2m2, G2ml0, G4ml, G4m2, G4ml0, and G4m20 disclosed herein were linked to the VH fragment of an anti-CDl37 antibody and the corresponding IgGl, G2, and G4 CH1 fragments to produce a full-length IgG heavy chain. These mutant IgG heavy chains were cloned, co- expressed with the light chain of the anti-CD 137 antibody, and purified using standard molecular biology and antibody protocols. The amino acid sequences of the VH-CH1 fragment (VH domain italicized) and the light chain are provided below:
VH-CH1 (IgGl): EVQLVQSGAEVKKPGESLRISCKGSGYSFSTYWISWVRQMPGKGLEWMGKIYPGDSYTNYSPSFQGQVTISADKS ISTAYLQWSSLKASDTAMYYCARGYGIFDYWGQGTLVTVSSASTKGRSVFREARS SKSTSGGTAAEGCEVKOYFR E PVTVSWNSGALT SGVHTFPAVLQS SGLY SL S SWTVPS S SLGTQTY ICNVNHKP SNTK (SEQ ID NO: 188)
VH-CH1 (IgG2):
EVQLVQSGAEVKKPGESLRISCKGSGYSFSTYWISWVRQMPGKGLEWMGKIYPGDSYTNYSPSFQGQVTISADKS ISTAYLQWSSLKASDTAMYYCARGYGIFDYWGQGTLVTVSSASTKGRSVFREARCSRSTSE STAAEGCEVKOYFR E PVTVSWNSGALT SGVHTFPAVLQS SGLY SL S SWTVPS SNFGTQTYTCNVDHKP SNTK (SEQ ID NO: 189)
VH-CH1 (IgG4):
EVQLVQSGAEVKKPGESLRISCKGSGYSFSTYWISWVRQMPGKGLEWMGKIYPGDSYTNYSPSFQGQVTISADKS ISTAYLQWSSLKASDTAMYYCARGYGIFDYWGQGTLVTVSSASTKGRSVFREARCSRSTSE STAAEGCEVKOYFR E PVTVSWNSGALT SGVHTFPAVLQS SGLY SL S SWTVPS S SLGTKTYTCNVDHKP SNTK (SEQ ID NO: 190)
Light Chain :
SYELTQPPSVSVS PGQTAS I TCSGDNI GDQYAHWYQQKPGQ SPVLVI YQDKNRPSGI PERFSGSNSGNTATLT I S GTQAMDEADYYCATYTGFGSLAVFGGGTKLTVLGQPKAAPSVTLFPP S SEELQANKATLVCLI SDFYPGAVTVAW KADS S PVKAGVETTTPSKQ SNNKYAAS SYLSLTPEQWKSHRSY SCQVTHEGSTVEKTVAPTEC S (SEQ ID
NO: 191)
These IgG mutants were tested for binding to a panel of human Fc receptors (FcRs), including FcRn, using the Octet Red96 System (ForteBio, Model# Red96). Human FcR proteins were purchased commercially (FcRn, MednaBio, E1032; FcRI, MednaBio, E1031; FcRIIA, MednaBio, E1033; FcRIIB/C, MednaBio, E1034; FcRIIIA-Fl58, MednaBio, E1036; FcRIIIA-Vl58, MednaBio, E1035; FcRIIIB, MednaBio, E1037). All Fey receptor assays were performed at pH 7.2; all FcRn assays were performed at pH 6. The Fey receptors were loaded onto anti-penta-HISlK (ForteBio, Cat#l8-5l22) biosensors at a concentration of 20 pg/mL. The loaded biosensors was then be dipped into an 8-point 1:3 dilution series of the test IgG molecules (including controls and the mutants described herein) in PBS with 0.1% BSA, 0.02% Tween-20 (pH 7.2) at a starting concentration of 300 nM. Kinetic analysis was performed using a 1: 1 binding model and global fitting. The results are shown in Tables 5 and 6 below.
Table 5. FcyR Binding Activity of IgGl and IgG2 Mutants to Various Fc Receptors
Figure imgf000061_0001
Figure imgf000062_0001
Table 6. FcyR Binding Activity of IgG4 Mutants
Figure imgf000062_0002
Certain IgG mutants tested in this study, for example, G4m2, showed selective binding activity to FcyRIIB (CD32B), and also maintained binding activity to FcRn, which is important for the half-life of the IgG molecule in vivo. Others, such as Glm2, G2ml or G4m20 lost binding to all FcyRs. Example 2. Additional IgGl, IgG2, and IgG4 Mutants and Binding Activities to Various
Fc Receptors
The human Fc variants Glm5, Glm7, Glm8, Glm9, G2m5, G2m7, G2m8, G2m9, G4m5, G4m7, G4m8, and G4m9 disclosed herein were linked to the VH fragment of an aud CD 137 antibody and the corresponding IgGl, G2, and G4 CH1 fragments to produce a full- length IgG heavy chain. See Example 1 above. These mutant IgG heavy chains were cloned, co-expressed with the light chain of the anti-CD 137 antibody (see Example 1 above), and purified using standard molecular biology and antibody protocols. The results are shown in Tables 7-9 below.
Example 3. Exemplary IgG Mutants Having Combined Mutations to Enhance Selective
Binding to FcyRIIB (CD32B)
The human Fc variants Glml5, Glml7, Glml8, Glml9, G2m25, G2m27, G2m28, G4ml5, G4ml7, G4ml8, and G4ml9, G4m25, G4m27, G4m28, and G4m29 disclosed herein were linked to the VH fragment of an anti-CDl37 antibody and the corresponding IgGl, G2, and G4 CH1 fragments to produce a full-length IgG heavy chain. See Example 1 above. These Fc variants contain a combination of one or mutations in the hinge domain and one or more mutations in the CH2 and/or CH3 domains. These mutant IgG heavy chains were cloned, co-expressed with the light chain of the anti-CD 137 antibody (see Example 1 above), and purified using standard molecular biology and antibody protocols. The results are shown in Tables 10-12 below.
Example 4. Determination of Human IgG Mutants Binding Activity to Cellular FcyR To determine the binding activity of human IgG mutants to cellular Fc receptors,
CHO cells were genetically engineered to express human FcyRs (FcyRI, FcyRIIA(Hl3 l), FcyRIIA(Rl3 l), FcyRIIB, FcyRIIC, and FcyRIII) using a lentivirus delivery system as known in the art. IgG Fc mutants, including Glm-2, Glm-4, GlmAA, GlmAG, G2m-l G2m-4,
G2ml5, G2ml7, G2ml7, G2ml8, G2ml9, G2m20, G2m27, G2m28, G4m-l, G4m-2, G430, and G4PE (amino acid sequences provided above) were designed and constructed following the disclosures herein. These IgG mutants contain mutations in either the upper hinge domain or the lower hinge domain as indicated.
For FACS analysis of the IgG mutants’ binding to different FcyRs, FcyR
overexpressing CHO cells were harvested using trypsin-EDTA and were suspended in cold staining buffer (3% BSA in PBS). Test IgG mutants, which were diluted in staining buffer, were added into the cells. The mixture was incubated 4°C for 2 hours, and then washed twice with cold staining buffer and re-suspended in PE-labeled anti-human IgG followed by incubation at 4°C for 2 hours. The mixture was washed twice with staining buffer and re suspended in 2% PFA in PBS for FACS.
As shown in FIGs. 1A-1W, a number of human IgGl, IgG2, and IgG4 mutants showed binding activity to FcyRs expressed on the cell surface. Qualitative summaries of the changes of the mutants’ Fey binding activity relative to the wild-type counterpart are provided in Tables 2-4 above.
Table 7. FcR Binding Activity of IgGl Mutants
Figure imgf000065_0001
Table 8. FcR Binding Activity of IgG2 Mutants
Figure imgf000066_0001
Table 9. FcR Binding Activity of IgG4 Mutants
Figure imgf000067_0001
Table 10. FcR Binding Activity of Human IgGl Mutants
Figure imgf000068_0001
Table 11. FcR Binding Activity of Human IgG2 Mutants
Figure imgf000069_0001
Table 12. FcR Binding Activity of Human IgG4 Mutants
Figure imgf000070_0001
Example 5. Human IgG Mutants Capable of Binding to Cellular FcyRIIB Showed Enhanced Agonist Activity
To verify that binding to cellular FcyRIIB would enhance agonist activity of the IgG mutants, a co-culture assay was developed, which involves CHO cells expressing FcyR2B and human CD8 positive T cells. CHO-FcyRIIB cells were plated at 2xl04/well in 96-well cell culture plate and were incubated overnight. To isolate human CD8 positive T cells, fresh blood from healthy donors were mixed with equal volume of DPBS gently. The blood sample was then placed on top of Ficoll underlay and centrifuged 30 min at lOOOg at RT without brake. The buffy coat containing PBMC was harvested into a new tube and was washed with DPBS. CD8 positive T cells were isolated from the PBMC using EasySep™ Human CD8+ T Cell Isolation Kit (Stemcell #17953) according to the kit manual. The isolated CD8+ T cells suspended in RPMI media were added to the plates with CHO- FcyRIIB cells. The anti-human CD3 antibody OKT3 was added to a final concentration of 0. lug/ml followed by addition of test antibodies diluted at desired concentrations. The plates were cultured for 3 days, and then the culture supernatants were harvested for measurement of IFNg concentration by ELISA using Human IFN-gamma ELISA Ready-SET-GO kit (EBIOSCIENCE, #88-7316-88).
As shown in FIG. 2A-2C, a number of tested IgG mutants stimulated human CD8+ cells in the presence of CHO-FcyRIIB cells as evidenced by the secretion of IFN-y.
Example 6. Engineering Anti-CD137 Antibodies with Mutant Fc
The cDNA sequences encoding the anti-CD 137 antibody variable domain with various heavy chain CH1 and Fc region or human kappa light chain constant region were synthesized and cloned. CHO transient expression was carried out with plasmids containing the corresponding heavy and light chain sequences. These antibodies were purified by protein A affinity chromatography. The amino acid sequences of the heavy chain (HC) and the light chain (LC) are provided below:
l73-hulg LC
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID
NO: 151) l73-huIgGlm27
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSK NQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGPSVFLFPPKPKDTLMI SRTPEVTCWVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 152)
l73-msIgG LC
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKW KIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID
NO: 153)
l73-msIgGl DANA
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSK NQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSASTTPPSVYPLAPGSAAQTNSMVTLGCLV KGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGC KPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVWAI SKDDPEVQFSWFVDDVEVHTAQTQPREEQFASTFRS VSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDI TVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
(SEQ ID NO: 154)
l73-msIgGl HC
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSK NQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSASTTPPSVYPLAPGSAAQTNSMVTLGCLV KGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGC KPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVWDI SKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRS VSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDI TVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
(SEQ ID NO: 155)
l73-msIgG2a DANA HC
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSK
NQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSASTTAPSVYPLAPVCGDTTGSSVTLGCLV
KGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTI
KPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVWAVSEDDPDVQISWFVNNVEVHTAQTQTHREDY
ASTLRWSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTD
FMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSWHEGLHNHHTTKSFSRTPK
(SEQ ID NO: 156)
l73-msIgG2a HC
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSK
NQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSASTTAPSVYPLAPVCGDTTGSSVTLGCLV
KGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTI
KPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVWDVSEDDPDVQISWFVNNVEVHTAQTQTHREDY
NSTLRWSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTD
FMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSWHEGLHNHHTTKSFSRTPK (SEQ ID NO: 157) l73-huIgGlm40 HC
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSK
NQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWaVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY aSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
K (SEQ ID NO: 158)
l73-huIgG2m40 HC
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSK NQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCV ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWaVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFaSTF RWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 159)
l73-huIgG4m40 HC
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSK NQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCWVaVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFaST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 160)
Example 7. Determination of CD137 Antibody with Fc Mutants Binding Activity to
Cellular FcyR
To determine the binding activity of CD 137 antibody in IgG mutants to cellular Fc receptors, CHO cells were genetically engineered to express human FcyRs (FcyRI,
FcyRIIA(Hl3 l), FcyRIIB, and FcyRIII) using a lentivirus delivery system as known in the art.
IgG Fc mutants, including l73-msIgGl, l73-msIgGl DANA, l73-msIgG2a, 173- msIgG2a DANA, l73-huIgG2, l73-huIgG2m2, l73-huIgG4m2, l73-huIgGlm27 and 173- huIgG4SP (amino acid sequences provided above) were designed and constructed following the disclosures herein. These IgG mutants contain mutations in either the upper hinge domain or the lower hinge domain or within the CH2 domain. For FACS analysis of the IgG mutants’ binding to different FcyRs, FcyR
overexpressing CHO cells were harvested using trypsin-EDTA and were suspended in cold staining buffer (3% BSA in PBS). Test IgG mutants, which were diluted in staining buffer, were added into the cells. The mixture was incubated 4°C for 2 hours, and then washed twice with cold staining buffer and re-suspended in PE-labeled anti-human IgG followed by incubation at 4°C for 2 hours. The mixture was washed twice with staining buffer and re suspended in 2% PFA in PBS for FACS.
As shown in FIGs. 3A-3D, a number of human IgGl, IgG2, and IgG4 mutants showed binding activity to FcyRs expressed on the cell surface, whereas others exhibited no binding to the FcyRs examined.
Example 8. CD137 Antibody in IgG Mutants Showed Differential CD137 Agonist
Activity
To determine agonist activity of the IgG mutants, a CD 137 reporter assay was developed, which involves Jakat cells expressing human CD 137-positive T cells. GS-H2- huCDl37 reporter cells were re-suspended in assay buffer and cell density and viability were determined with trypan blue. The cell suspension was diluted to 1 x 104 cells/mL with assay buffer (MEM containing 1% FBS). The cells were added at lOOuL/well, such that the final cell number was 1000 cells/well in the assay plate (Nunc, Cat#l 67425). Serial working dilutions of the test samples were prepared in assay buffer at 2x final concentrations.
Samples were added at lOOuL/well test sample at 2x final concentrations to the assay plate. The assay plate was incubated in 37°C, 5% C02 incubator for 18-20 hours. After the 18-20 hour incubation, 8 ul of the supernatant from each well of the assay plate was collected and added to HTRF detection assay plate (Nunc). A Human Interleukin 8 (reporter of CD 137 activation) detection assay was performed using a Human IL-8 Assay Kit (Cisbio,
Cat#62IL8PEB). In particular, l6ul assay volume was used. The results were read using Time Resolved Fluorescence by Tecan F200pro and the relative light unit data was recorded.
As shown in FIG. 4, all the tested CD 137 antibody IgG mutants stimulated human CD137 activation as evidenced by the secretion of IL8. The magnitude of CD137 activation was influenced by Fc. Example 9. CD137 Antibody in IgG Mutants Exhibit differential Anti-tumor Activity in Animal Models in Vivo
Selected CD137 antibodies with various Fc regions ( e.g ., those disclosed in Example 6 above) are tested in mouse syngeneic tumor models in vivo to determine how variant Fc regions affect the efficacy and toxicity of these antibodies. C57BL6 mice with human CD 137 extracellular domains knocked in are used to develop syngeneic mouse tumor models. Murine colon cancer MC38 cells are subcutaneously implanted into the homozygous human CD 137 knock-in mice. The mice are grouped when the tumor size is approximately l50±50mm3 (n=6). CD137 antibodies are then administered via intraperitoneal injections and tumor sizes are measured during 4-6 weeks of antibody treatment. The anti-tumor efficacy is evaluated between tumor sizes of the control group and antibody treatment group.
Example 10. Preparation and Characterization of Additional CD137 Antibodies in IgG
Mutants
A panel of human CD 137 antibodies or fusion proteins with wild-type or variant Fc regions, is cloned and expressed using standard protocols. Exemplary anti-CD 137 antibodies are provided in Example 6 above.
These antibodies and fusion proteins are tested in vitro for CD 137 binding and agonist activity, as well as in vivo for antitumor efficacy and toxicity, as described in the Examples above. Human T cell activation assays are carried out using standard protocols in the literature. OTHER EMBODIMENTS
All of the features disclosed in this specification may be combined in any
combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
EQUIVALENTS
While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles“a” and“an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean“at least one.” The phrase“and/or,” as used herein in the specification and in the claims, should be understood to mean“either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e.,“one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to“A and/or B”, when used in conjunction with open-ended language such as“comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims,“or” should be understood to have the same meaning as“and/or” as defined above. For example, when separating items in a list,“or” or“and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as“only one of’ or“exactly one of,” or, when used in the claims,“consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term“or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e.“one or the other but not both”) when preceded by terms of exclusivity, such as“either,”“one of,”“only one of,” or “exactly one of.”“Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase“at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase“at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example,“at least one of A and B” (or, equivalently,“at least one of A or B,” or, equivalently“at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

What Is Claimed Is:
1. A CDl37-binding molecule, comprising (a) a CD137 binding moiety, and (b) an engineered Fc region, which comprises at least one mutation at any of positions 220-331, preferably at any of positions 228-329 as compared to a wild-type Fc region counterpart, wherein the numbering is according to the EU index.
2. The CDl37-binding molecule of claim 1, which is a CD137 agonist or a CD 137 antagonist.
3. The CDl37-binding molecule of claim 1 or claim 2, wherein the CD137 binding moiety is a CD 137 ligand (CD137L).
4. The CDl37-binding molecule of claim 3, wherein the CD137L comprises the amino acid sequence of SEQ ID NO: 142.
5. The CDl37-binding molecule of claim 1 or claim 2, wherein the CD137 binding moiety is an anti-CD 137 antibody fragment, which comprising (a) a heavy chain that comprises a heavy chain variable region, and (b) a light chain that comprises a light chain variable region and a light chain constant region, wherein the heavy chain variable domain is linked to the engineered Fc region.
6. The CDl37-binding molecule of any one of claims 1-5, wherein the variant Fc region has an enhanced binding affinity or selectivity to FcyRIIB relative to its wild-type counterpart.
7. The CDl37-binding molecule of any one of claims 1-5, wherein the variant Fc region has a reduced binding affinity to one or more FcyR receptors as relative to its wild- type counterpart.
8. The CDl37-binding molecule of claim 7, wherein the variant Fc region has substantially reduced binding affinity to all FcyR receptors as relative to its wild-type counterpart.
9. The CD 137-binding molecule of any one of claims 1-8, wherein the molecule is an IgGl, and wherein the at least one mutation comprises a substitution or deletion within positions 233-238, a substitution at position 265, a substitution at position 267, a substitution at position 297, a substitution at position 328, a substitution at position 329, or a combination thereof.
10. The CD 137-binding molecule of any one of claims 1-9, wherein the molecule is an IgGl, and wherein the at least one mutation comprises a substitution at one or more of the positions 220, 226, 229, 238, 273, 327, 330, and 331.
11. The CD 137-binding molecule of claim 9 or claim 10, wherein the at least one mutation comprises substitutions at any positions of 233-236, a deletion at one or more of positions 236-238, or a combination thereof.
12. The CDl37-binding molecule of claim 11, wherein the substitutions at positions 233-235 are E233P, L234V, L234F, L235A, and/or L235E.
13. The CDl37-binding molecule of any one of claims 9-12, wherein the deletion is at position 236 or at one or more positions 236-238.
14. The CDl37-binding molecule of any one of claims 9-13, wherein the substitution at position 238 is P238S, wherein the substitution at position 273 is V273E, and/or wherein the substitution at position 265 is D265A.
15. The CDl37-binding molecule of any one of claims 9-14, wherein the substitution at position 267 is S267E.
16. The CDl37-binding molecule of any one of claims 9-13, wherein the substitution at position 329 is P329G.
17. The CDl37-binding molecule of claim 9, wherein the at least one mutation comprises a substitution at position 265, a substitution at position 297, or a combination thereof.
18. The CDl37-binding molecule of claim 15, wherein the substitution at position 265 is D265A and the substitution at position 297 is N297A.
19. The CDl37-binding molecule of claim 9 or claim 10, wherein the one or more substitutions at positions 220, 226, 229, 327, 330, and 331 are C220S, C226S, C229S, A327G, A330S, and P331S.
20. The CDl37-binding molecule of claim 9, wherein the variant Fc region is selected from the group consisting of Glm2, Glml7, Glm27, GlmAA, GlmAAG, G1N279A, Glm240, and Glm40.
21. The CD 137-binding molecule of any one of claims 1-8, wherein the molecule is an IgG2, and wherein the at least one mutation comprises a deletion within positions 237-
238, a substitution at position 265, a substitution at position 267, a substitution at position 297, a substitution at position 328, or a combination thereof.
22. The CDl37-binding molecule of any one of claims 1-8 and 21, wherein the molecule of an IgG2, and wherein the at least one mutation comprises a substitution at one or more of positions 233-235, 237, 238, 268, 273, 330, and 331.
23. The CD 137-binding molecule of claim 21 or claim 22, wherein the deletion is at position 237 or at both positions 237 and 238.
24. The CDl37-binding molecule of any one of claims 21-23, wherein the substitution at position 267 is S267E.
25. The CD 137-binding molecule of any one of claims 21-24, wherein the substitution at position 328 is L328F.
26. The CDl37-binding molecule of any one of claims 22-25, wherein the substitution at one or more of positions 233-235, 237, 238, 268, 273, 330, and 331 are P233E, V234A, V234L, A235L, A235S, G237A, P238S, H268A, H268Q, V273E, A330S, and P331S.
27. The CDl37-binding molecule of any one of claims 21-26, wherein the at least one mutation comprises a substitution at position 265, a substitution at position 297, or a combination thereof.
28. The CDl37-binding molecule of claim 27, wherein the substitution at position 265 is D265A and the substitution at position 297 is N297A.
29. The CDl37-binding molecule of claim 21, wherein the variant Fc region is selected from the group consisting of G2ml, G2ml7, G2ml8, G2ml9, G2m20, G2m27,
G2m28, G2m29, G2m2040, G2m43, G2G4, G2mAA, and G2m40.
30. The CD 137-binding molecule of any one of claims 1-8, wherein the molecule is an IgG4, and wherein the at least one mutation comprises a substitution at position 228, a substitution or deletion at any of positions 235-238, a substitution at position 265, a substitution at position 267, a substitution at position 297, a substitution at position 273, a substitution at position 328, or a combination thereof.
31. The CDl37-binding molecule of any one of claims 1-8 and 30, wherein the molecule is an IgG4, and wherein the at least one mutation comprises a substitution at one or more of positions 233-235 or 237.
32. The CDl37-binding molecule of claim 30 or claim 31, wherein the substitution at position 228 is S228P.
33. The CD 137-binding molecule of any one of claims 30-32, wherein the deletion is at least one of positions 236-238.
34. The CDl37-binding molecule of any one of claims 30-33, wherein the substitution at one or more positions 233-235 and 237 comprises E233P, F234V, F234A, L235S, L235E, L235A, and G237A.
35. The CD 137-binding molecule of any one of claims 30-34, wherein the deletion is at both positions 236 and 237.
36. The CDl37-binding molecule of any one of claims 30-35, wherein the substitution at position 267 is S267E, and/or wherein the substitution at position 273 is
V273E.
37. The CDl37-binding molecule of any one of claims 30-36, wherein the substitution at position 238 is L238F.
38. The CDl37-binding molecule of claim 30, wherein the at least one mutation comprises a substitution at position 228, a substitution at position 265, a substitution at position 297, or a combination thereof.
39. The CDl37-binding molecule of claim 31, wherein the substitution at position
228 is S228P, the substitution at position 265 is D265A and the substitution at position 297 is N297A.
40. The CDl37-binding molecule of claim 30, wherein the variant Fc region is selected from the group consisting of G4m2, G4m20, G4m28, G4m30, G4m4l, G4m42, G4m46, G4mPE, G4mAA, and G4m40.
41. The CD 137-binding molecule of any one of claims 1-40, wherein the variant Fc region exhibits an enhanced binding affinity to FcyRIIB as compared with the parent Fc region.
42. The CDl37-binding molecule of any one of claims 1-41, wherein the variant
Fc region exhibits a decreased binding affinity and/or an enhanced selectivity to FcyRIIB as compared with the parent Fc region.
43. The CD 137-binding molecule of any one of claims 1-42, wherein the variant Fc region exhibits low or no binding activity to any FcyR.
44. The CDl37-binding molecule of any one of claims 1-43, wherein the variant Fc region binds FcRn.
45. The CD 137-binding molecule of any one of claims 5-44, wherein the heavy chain variable region comprises the same heavy chain complementary determining regions (HC CDRs) as those in SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, or SEQ ID NO: 140, and/or the light chain variable region comprises the same light chain complementary determining regions (LC CDRs) as those in SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, or SEQ ID NO: 141.
46. The CDl37-binding molecule of claim 45, wherein the heavy chain variable region comprises SEQ ID NO: 128 SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, or SEQ ID NO: 140, and/or the light chain variable region comprises SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, or SEQ ID NO: 141.
47. The CDl37-binding molecule of any one of claims 5-46, wherein the antibody fragment is of a human antibody or a humanized antibody.
48. An isolated nucleic acid or set of nucleic acids which collectively encode a CD 137-binding molecule of any one of claims 1-47.
49. A vector or set of vectors, which collectively comprises the nucleic acid or set of nucleic acids of claim 48.
50. The vector or set of vectors of claim 49, which is an expression vector(s).
51. A host cell comprising the vector or set of vectors of claim 49 or claim 50.
52. A pharmaceutical composition, comprising a CDl37-binding molecule of any one of claims 1-51 and a pharmaceutically acceptable carrier.
53. A method for selectively modulating an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of a
CD 137-binding molecule set forth in any one of claims 1-52.
54. The method of claim 53, wherein the subject is a human patient having or suspected of having a cancer and the CDl37-binding molecule is a CD137 agonist.
55. The method of claim 54, wherein the cancer is selected from the group consisting of lung cancer, stomach cancer, liver cancer, breast cancer, skin cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, colorectal cancer, sarcoma, bone cancer, lymphoma and a hematological cancer.
56. The method of claim 53, wherein the subject is a human patient having or suspected of having an immune-related disorder, and wherein the CD 137-binding molecule is a CD 137 antagonist.
57. The method of claim 56, wherein the immune-related disorder is selected from the group consisting of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Myasthenia Gravis (MG), Graves’ Disease, Idiopathic Thrombocytopenia Purpura (ITP), Guillain-Barre Syndrome, autoimmune myocarditis, Membrane Glomerulonephritis, diabetes mellitus, Type I or Type II diabetes, multiple sclerosis, Reynaud's syndrome, autoimmune thyroiditis, gastritis, Celiac Disease, Vitiligo, Hepatitis, primary biliary cirrhosis, inflammatory bowel disease, spondyloarthropathies, experimental autoimmune
encephalomyelitis, immune neutropenia, juvenile onset diabetes, and immune responses associated with delayed hypersensitivity mediated by cytokines, T-lymphocytes typically found in tuberculosis, sarcoidosis, and polymyositis, polyarteritis, cutaneous vasculitis, pemphigus, pemphigold, Goodpasture's syndrome, Kawasaki's disease, systemic sclerosis, anti-phospholipid syndrome, Sjogren's syndrome, graft-versus-host (GVH) disease, and immune thrombocytopenia.
58. An isolated anti-CD 137 antibody, which binds to the same epitope of CD 137 as a reference antibody selected from the group consisting of 20A12D11, 11E10D12, 23D2D6, 22F2C2, 30C11B4, 26B3D7, or competes against the reference antibody from binding to the epitope.
59. The isolated anti-CDl37 antibody of claim 58, wherein the antibody comprises a heavy chain complementary determining region 1 (CDR1), a heavy chain complementary determining region 2 (CDR2), and a heavy chain complementary determining region 3 (CDR3), which collectively are at least 85% identical to the heavy chain CDRs of the reference antibody; and/or wherein the antibody comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, which collectively are at least 85% identical to the light chain CDRs of the reference antibody.
60. The isolated anti-CDl37 antibody of claim 58, wherein the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 collectively comprise up to 10 amino acid variations relative to the heavy chain CDRs of the reference antibody; and/or wherein the light chain CDR1, light chain CDR2, and light chain CDR3 collectively comprise up to 10 amino acid variations relative to the light chain CDRs of the reference antibody.
61. The isolated anti-CD 137 antibody of any one of claims 58-60, wherein the antibody comprises a heavy chain variable region that is at least 85% identical to the heavy chain variable region of the reference antibody; and/or a light chain variable region that is at least 85% identical to the light chain variable region of the reference antibody.
62. The isolated anti-CDl37 antibody of claim 58, wherein the antibody comprises the same heavy chain variable region CDRs as the reference antibody and/or the same light chain variable region CDRs as the reference antibody.
63. The isolated anti-CDl37 antibody of any one of claims 58-62, wherein the antibody is a human antibody or a humanized antibody.
64. An isolated nucleic acid or set of nucleic acids which collectively encode an anti-CDl37 antibody of any one of claims 58-63.
65. A vector or set of vectors, which collectively comprises the nucleic acid or set of nucleic acids of claim 64.
66. The vector or set of vectors of claim 65, which is an expression vector(s).
67. A host cell comprising the vector or set of vectors of claim 65 or claim 66.
68. A pharmaceutical composition, comprising the anti-CD 137 antibody of any one of claims 58-63, the encoding nucleic acid of any one of claims 64-66, or the host cell of claim 67, and a pharmaceutically acceptable carrier.
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