WO2018229706A1 - Polythérapie pour le traitement du cancer - Google Patents

Polythérapie pour le traitement du cancer Download PDF

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WO2018229706A1
WO2018229706A1 PCT/IB2018/054369 IB2018054369W WO2018229706A1 WO 2018229706 A1 WO2018229706 A1 WO 2018229706A1 IB 2018054369 W IB2018054369 W IB 2018054369W WO 2018229706 A1 WO2018229706 A1 WO 2018229706A1
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Haihui Lu
Matthew John MEYER
Ryan Sullivan
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Novartis Ag
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • 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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • 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

  • the present invention relates to antibody molecules which bind human CD32b in combination with an additional agent that enhances antibody dependent cellular cytotoxicity (ADCC), such as a complex comprising interleukin-15 (“IL-15”) bound to IL-15 receptor alpha (“IL-15Ra”).
  • ADCC antibody dependent cellular cytotoxicity
  • IL-15 interleukin-15
  • IL-15Ra IL-15 receptor alpha
  • the combination is useful in the prevention, treatment, and/or management of disorders in which activating the immune system is beneficial, such as cancer.
  • Fc gamma receptors bind IgG and they are expressed by many immune cells, enabling them to serve as the link between innate and humoral immunity. Activatory FcyRs mediate immune responses including phagocytosis and ADCC (Nimmerjahn & Ravetch (2008) Nature Rev. Immunol. 8(1): 34-47).
  • CD32b (FcyRIIb) is the sole inhibitory FcyR. It is expressed by immune cells including dendritic cells and macrophages and is the only FcyR expressed on B cells (Nimmerjahn & Ravetch, supra;
  • CD32b results in inhibition of activatory FcyR functions, i.e. inhibition of phagocytosis and ADCC (Smith & Clatworthy, (2010) Nat. Rev. Immunol. 10(5): 328-343), or when cross-linked to the B cell receptor, reduced B cell function (Horton et al., (2011) J. Immunol. 186(7): 4223-4233).
  • an antibody molecule that binds to CD32b can reduce the inhibitory effects of this receptor and when combined with an agent that triggers ADCC, will give rise to an enhanced ADCC response.
  • cytokine interleukin-15
  • IL-15 is a member of the four alpha-helix bundle family of lymphokines produced by many cells in the body.
  • IL-15 plays a pivotal role in modulating the activity of both the innate and adaptive immune system, e.g., maintenance of the memory T-cell response to invading pathogens, inhibition of apoptosis, activation of dendritic cells, and induction of Natural Killer (NK) cell proliferation and cytotoxic activity.
  • NK Natural Killer
  • IL-15 signaling has been shown to occur through the heterodimeric complex of the IL-15 receptor, which consists of three polypeptides, the type-specific IL- 15 receptor alpha (“IL-15Ra”), the IL-2/IL-15 receptor beta (or CD122) (" ⁇ "), and the common gamma chain (or CD 132) ("y”) that is shared by multiple cytokine receptors.
  • IL-15Ra type-specific IL- 15 receptor alpha
  • ⁇ IL-2/IL-15 receptor beta
  • CD 132 common gamma chain
  • IL-15 and IL-15/IL-15Ra complexes have been shown to promote to different degrees the expansion of memory CD8 T cells and NK cells and enhance tumor rejection in various preclinical models. Furthermore, tumor targeting of IL-15 or IL-15/IL-15Ra complex containing constructs in mouse models, resulted in improved anti-tumor responses in either
  • Enhanced anti -tumor activity is thought to be dependent on increased half-life of the IL-15 -containing moiety as well as the trans-presentation of IL-15 on the surface of tumor cells leading to enhanced NK and/or CD8 cytotoxic T cell expansion within the tumor.
  • tumor cells engineered to express IL-15 were also reported to promote rejection of established tumors by enhancing T cell and NK cell recruitment, proliferation and function (Zhang et al, (2009) PNAS USA. 106:7513-7518; Munger et al, (1995) Cell Immunol.
  • NK cells are one cellular mediator of ADCC and given the role shown for IL-15 and IL-15/IL- 15Ra complexes in enhancing NK-cell functionality, a role for IL-15 in enhancing ADCC has also been investigated.
  • IL-15 has been shown to enhance NK-cell ADCC in vitro (Carson et al, (1994) J. Exp. Med. 180: 1395-403), including that directed by anti-CD20 mAbs (Moga et al, (2008) Exp. Hematol. 36: 69-77).
  • therapeutic approaches that enhance anti-tumor immunity could work more effectively when the immune response is optimized by targeting multiple components at one or more stages of an immune response.
  • approaches that enhance cellular and humoral immune responses e.g., by stimulating (e.g., NK cells) and e.g., disinhibiting ADCC (e.g., by inhibiting CD32b)
  • combination therapies that enhance ADCC and as such can provide a superior beneficial effect, e.g., in the treatment of a disorder, such as an enhanced anti -cancer effect, reduced toxicity and/or reduced side effects, compared to monotherapy administration of the therapeutic agents of the combination.
  • a disorder such as an enhanced anti -cancer effect, reduced toxicity and/or reduced side effects
  • one or more of the therapeutic agents in the combination can be administered at a lower dosage, or for a shorter period of administration or less frequently, than would be required to achieve the same therapeutic effect compared to the monotherapy administration.
  • one of the therapeutic agents in the combination can be administered to enhance the effect of the other agent.
  • the combination therapy comprises an agent that modulates the inhibition of activatory FcyR functions such as an antibody to CD32b, in combination with immune enhancing agents such as IL-15 complexed with sIL-15Ra, to enhance the immune system.
  • the present invention provides a combination comprising an anti-human CD32b antibody molecule in combination with an IL-15/IL-15Ra complex.
  • the anti- human CD32b antibody molecule of the combination comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 113, a VHCDR2 amino acid sequence of SEQ ID NO: 114 and a VHCDR3 amino acid sequence of SEQ ID NO: 115, as described in Table 1 and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 120, a VLCDR2 amino acid sequence of SEQ ID NO: 121 and a VLCDR3 amino acid sequence of SEQ ID NO: 122 as described in Table 1.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-CD32b antibody molecule of the combination comprises a heavy chain variable domain (VH) comprising an amino acid sequence at least 90% identical to SEQ ID NO: 116 and a light chain variable domain (VL) comprising an amino acid sequence at least 90% identical to SEQ ID NO: 123, as described in Table 1.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the anti-CD32b antibody molecule of the combination comprises a VH comprising an amino acid sequence of SEQ ID NO: 1 16 and a VL comprising an amino acid sequence of SEQ ID NO: 123, as described in Table 1.
  • the anti-CD32b antibody molecule of the combination comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 118 and a light chain comprising the amino acid sequence of SEQ ID NOs: 125, as described in Table 1.
  • the present application discloses an isolated antibody molecule that specifically binds to CD32b within the Fc binding domain of CD32b.
  • the antibody binds within amino acid residues 107-123 (VLRCHSWKDKPLVKVTF; SEQ ID NO: 250) of CD32b, as described in Table 1.
  • the anti-CD32b antibody molecule of the combination comprises the antibody 2B6 as described in PCT Publication WO2004/016750 (Koenig & Maria-Concetta) and Maria-Concetta et al (2007) Immunology, 121 : 392-404.
  • 2B6 comprises a VH having the amino acid sequence of SEQ ID NO: 200 and a VL having the amino acid sequence of SEQ ID NO: 205, as described in Table 1.
  • the anti-CD32b antibody molecule of the combination comprises any one of Antibodies 20, 24, 26 and 28, having a VH and VL amino acid sequence of SEQ ID NO: 210 and 214, 218 and 222, 226 and 230, or 234 and 238, respectively as described in PCT Publication
  • the anti-CD32b antibody of the combination comprises the antibody 6G11 as described in PCT Publication
  • the IL-15/IL-15Ra complex of the combination may comprise wild-type IL- 15 or an IL-15 derivative covalently or noncovalently bound to wild-type IL-15Ra or an IL-15Ra derivative.
  • the IL-15/IL-15Ra complex comprises wild-type IL-15 and IL-15Ra.
  • the IL-15/IL-15Ra complex comprises an IL-15 derivative and wild-type IL-15Ra.
  • the IL-15/IL-15Ra complex is in the wild-type heterodimeric form.
  • the IL-15 is human IL-15 and IL-15Ra is human IL-15Ra.
  • the human IL-15 comprises the amino acid sequence of SEQ ID NO: 251 or amino acid residues 49 to 162 of SEQ ID NO: 251 and the human IL-15Ra comprises the amino acid sequence of SEQ ID NO: 256 or a fragment thereof, as described in Table 1.
  • the IL-15 comprises the amino acid sequence of SEQ ID NO: 251 or amino acid residues 49 to 162 of SEQ ID NO: 251 and the IL-15Ra comprises the amino acid sequence of SEQ ID NO: 257 or 260, as described in Table 1.
  • the human IL-15 comprises amino acid residues 49 to 162 of the amino acid sequence of SEQ ID NO: 251 and human IL-15Ra comprises the amino acid sequence of SEQ ID NO: 260, as described in Table 1.
  • the IL-15Ra is glycosylated such that glycosylation accounts for at least or more than 20%, 30%, 40% or 50% of the mass of the IL-15Ra.
  • the IL-15/IL- 15Ra complex comprises wild-type IL-15 and an IL-15Ra derivative.
  • the IL- 15/IL-15Ra complex comprises an IL-15 derivative and an IL-15Ra derivative.
  • the IL-15Ra derivative is a soluble form of the wild-type IL-15Ra.
  • the IL-15Ra derivative comprises a mutation that inhibits cleavage by an endogenous protease.
  • the extracellular domain cleavage site of IL-15Ra is replaced with a cleavage site that is specifically recognized by a heterologous protease.
  • the extracellular domain cleavage site of IL-15Ra is replaced with a heterologous extracellular domain cleavage site (e.g., heterologous transmembrane domain that is recognized and cleaved by another enzyme unrelated to the endogenous processing enzyme that cleaves the IL-15Ra).
  • the present invention provides a combination comprising an anti- human CD32b antibody molecule in combination with an IL-15/IL-15Ra complex, wherein
  • the anti- human CD32b antibody molecule of the combination comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 113, a VHCDR2 amino acid sequence of SEQ ID NO: 114 and a VHCDR3 amino acid sequence of SEQ ID NO: 115, as described in Table 1 and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 120, a VLCDR2 amino acid sequence of SEQ ID NO: 121 and a VLCDR3 amino acid sequence of SEQ ID NO: 122, as described in Table 1; and
  • the IL-15/IL-15Ra complex is a heterodimeric complex of human IL-15 and human soluble IL-15Ra and wherein the human IL-15 and comprises residues 49 to 162 of the amino acid sequence of SEQ ID NO: 251 and the human soluble IL-15Ra comprises the amino acid sequence of SEQ ID NO: 260, as described in Table 1.
  • the combinations disclosed herein can result in one or more of: an increase in antigen presentation, an increase in effector cell function (e.g., one or more of T cell proliferation, IFN-a secretion or cytolytic function), inhibition of regulatory T cell function, an effect on the activity of multiple cell types, such as regulatory T cell, effector T cells and NK cells), an increase in tumor infiltrating lymphocytes and an increase in T-cell receptor mediated proliferation.
  • an anti-CD32b antibody molecule in the combination inhibits, reduces or neutralizes one or more activities of CD32b, resulting in enhanced effector function/ADCC.
  • an IL-15/IL-15Ra complex in the combination stimulates the immune response and can enhance ADCC resulting from the use of an anti-CD32b antibody molecule.
  • combinations can be used to treat or prevent disorders where enhancing an immune response in a subject is desired, e.g. cancer.
  • Such combination therapies can be used, e.g., for cancer immunotherapy and treatment of other conditions, such as chronic infection.
  • a disorder e.g., a hyperproliferative condition or disorder (e.g., a cancer) in a subject by administering to the subject an anti-CD32b antibody molecule in combination with an IL-15/IL-15Ra complex.
  • an anti-CD32b antibody molecule in combination with an IL-15/IL-15Ra complex for use in the treatment of (e.g., inhibiting, reducing, ameliorating, or preventing) a disorder, e.g., a hyperproliferative condition or disorder (e.g., a cancer) in a subject.
  • an anti-CD32b antibody molecule in combination with an IL-15/IL-15Ra complex for use in the preparation of a medicament for the treatment of (e.g., inhibiting, reducing, ameliorating, or preventing) a disorder, e.g., a hyperproliferative condition or disorder (e.g., a cancer) in a subject.
  • a disorder e.g., a hyperproliferative condition or disorder (e.g., a cancer) in a subject.
  • an anti-CD32b antibody of the present invention showed dose dependent ADCC activity against effector (Daudi) cells.
  • the addition of an IL-15/IL-15Ra complex enhanced the potency of the ADCC activity.
  • the present invention provides a method of enhancing the ADCC activity of an anti-CD32b antibody comprising administering an effective amount of an IL-15/IL-15Ra complex in combination with an anti-CD32b antibody.
  • the disorder is selected from B cell malignancies, Hodgkins lymphoma, Non-Hodgkins lymphoma, multiple myeloma, diffuse large B cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, small lymphocytic lymphoma, diffuse small cleaved cell lymphoma, MALT lymphoma, mantel cell lymphoma, marginal zone lymphoma, follicular lymphoma, systemic light chain amyloidosis or haematological malignancies.
  • the disorder is selected from B-cell malignancies, non-Hodgkins lymphoma multiple myeloma or chronic lymphocytic leukemia.
  • the combination of anti-CD32b antibody molecule and IL-15/IL-15Ra complex are administered to a subject separately or together. In another embodiment, the combination of anti-CD32b antibody molecule and IL-15/IL-15Ra complex are administered simultaneously or sequentially.
  • the present application also provides nucleic acids encoding the anti-CD32b antibody molecule and/or the IL-15/IL-15Ra complex disclosed herein, as well as a vector comprising the nucleic acid, and a host cell comprising the nucleic acid or the vector. Also provided are methods of producing the anti-CD32b antibody molecule and/or the IL-15/IL-15Ra complex disclosed herein, the method comprising: culturing a host cell expressing a nucleic acid encoding the antibody molecule or complex; and collecting the antibody molecule or complex from the culture.
  • FIG. 1 shows that ADCC mediated by the anti-CD32b antibody NOV2108 against Daudi cells is enhanced by the IL-15/IL-15Ra complex, hetIL-15.
  • ADCC assays were performed using primary NK cells as effector cells in the absence (grey lines) or presence (black lines) of hetIL-15.
  • Luciferized Daudi cells as target cells were incubated with primary NK cells and antibody NOV2108 in different formats ((A) wild type, (B) N297A Fc silent, (C) afucosylated or (D) an IgG control), in serial dilution for 4 hr.
  • Effector: target ratio (E:T) 1. Luciferase signal was then measured to determine % lytic activity. The experiment was repeated with two different donor NK cells with the representative data from one experiment is shown.
  • ADCC is a mechanism of immune defense whereby an effector cell (NK cells, macrophages, neutrophils) actively lyse a target cell whose cell surface antigens are bound by specific antibodies engaged with Fc Receptors on the immune cell.
  • NK cells effector cell
  • Many anti-tumor antibodies have been developed into therapeutics for cancer and an important mechanism of their anti-tumor activity is ADCC.
  • CD32b is expressed on various B cell and plasma cell malignancies, and anti-CD32b antibodies can be used to target the malignant cells.
  • a complex of IL-15 with IL-15Ra IL-15/IL-15Ra
  • potently activates NK cells potently activates NK cells, resulting in proliferation of NK cells as well as enhanced target cell lysis.
  • the present invention provides a combination comprising an antibody molecule that specifically binds to human CD32b protein with an IL-15/IL-15Ra complex, and pharmaceutical compositions, production methods, and methods of use of such a combination.
  • each agent will be administered at a dose and/or on a time schedule determined for that agent. It will further be appreciated that each therapeutic agent utilized in this combination may be administered together in a single composition or administered separately in different compositions.
  • the therapeutic agents of the combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels of the agents utilized in combination will be lower than those utilized individually. In some embodiments, the agents of the combination may also be used as entirely separate pharmaceutical dosage forms or pharmaceutical formulations that are also sold independently of each other and where instructions of the possibility of their combined use is or are provided in the package equipment, e.g. leaflet or the like, or in other information e.g. provided to physicians and medical staff.
  • CD32A or “CD32a”, as used herein, means human CD32a protein, also referred to as human FCy Receptor 2A or FCyR2A or FCGR2a or FCGR2A.
  • FCy Receptor 2A or FCyR2A or FCGR2a or FCGR2A.
  • H131 and R131 when referenced without the signal sequence
  • HI 67 and R167 when referenced with the signal sequence.
  • the amino acid sequence of the H167 variant is deposited under accession number UniProtKB P12318 and is detailed below:
  • CD32B or “CD32b”, as used herein, means human CD32b protein, also referred to as human FCy Receptor 2B or FCyR2B or FCGR2b or FCGR2B.
  • FCy Receptor 2B or FCyR2B or FCGR2b or FCGR2B The amino acid sequence for CD32b variant 1 is deposited under accession number UniProtKB P31994-1 and is detailed below:
  • CD32b variant 2 The amino acid sequence for CD32b variant 2 is deposited under accession number UniProtKB P31994-2 and is detailed below:
  • an antibody molecule which binds to CD32b binds to human CD32b protein or a fragment thereof.
  • huCD32b refers to human CD32b protein or a fragment thereof.
  • antibody molecule and the like, as used herein, include whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof.
  • a naturally occurring "antibody molecule” is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • the precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al., (1991) "Sequences of Proteins of Immunological Interest," 5th Ed.
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDRl), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3).
  • the CDR amino acids in the VH are numbered 26-32 (HCDRl), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).
  • the CDRs consist of amino acid residues 26- 35 (HCDRl), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.
  • LCDR1 amino acid residues 24-34
  • LCDR2 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • antigen binding fragment refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., CD32b).
  • Antigen binding functions of an antibody can be performed by antibody fragments.
  • antigen binding fragments encompassed within the term "antigen binding portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F (ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH and CHI domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and an isolated CDR.
  • Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
  • F (ab)2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • an Fd fragment consisting of the VH and CHI domains an Fv fragment consist
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by an artificial peptide linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) PNAS USA. 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies include one or more "antigen binding fragments" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • Antigen binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger & Hudson (2005), Nature Biotechnology, 23 (9): 1126-1136).
  • Antigen binding fragments of antibodies can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No. 6,703, 199, which describes fibronectin polypeptide monobodies).
  • Fn3 Fibronectin type III
  • Antigen binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al, (1995) Protein Eng. 8 (10): 1057-1062; and U.S. Pat. No. 5,641,870).
  • Affinity refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody “arm” interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity.
  • amino acid refers to wild-type and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the wild-type amino acids.
  • Wild- type amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a wild-type amino acid, i.e., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a wild- type amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a wild-type amino acid.
  • binding specificity refers to the ability of an individual antibody combining site to react with one antigenic determinant and not with a different antigenic determinant.
  • the combining site of the antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains. Binding affinity of an antibody is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody.
  • Specific binding between two entities means a binding with an equilibrium constant (KA or K A ) of at least 1 x 10 7 M “1 , 10 8 M “1 , 10 9 M “1 , 10 10 M “1 , 10 11 M “1 , 10 12 M “1 , 10 13 M “1 , or 10 14 M “1 .
  • the phrase “specifically (or selectively) binds" to an antigen refers to a binding reaction that is determinative of the presence of a cognate antigen (e.g., a human CD32b protein) in a heterogeneous population of proteins and other biologies.
  • a CD32b-binding antibody of the invention binds to CD32b with a greater affinity than it does to a non-specific antigen (e.g., CD32a).
  • a non-specific antigen e.g., CD32a.
  • the phrases "an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen”.
  • conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • conservatively modified variants include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine
  • G Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • the term "conservative sequence modifications" are used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.
  • recognition refers to an antibody antigen-binding fragment thereof that finds and interacts (e.g., binds) with its conformational epitope.
  • blocks refers to reducing an interaction or a process, e.g., stopping ligand-dependent or ligand-independent signaling.
  • the interaction or process can be reduced by up to 50%, up to 60%, up to 70%, up to 80%, up to 90% or up to 100%.
  • cross-block “cross-blocked”, “cross-blocking”, “compete”, “cross compete” and related terms are used
  • an antibody or other binding agent to interfere with the binding of other antibodies or binding agents to CD32b in a standard competitive binding assay.
  • the ability or extent to which an antibody or other binding agent is able to interfere with the binding of another antibody or binding molecule to CD32b, and therefore whether it can be said to cross-block according to the invention, can be determined using standard competition binding assays.
  • One suitable assay involves the use of the BIAcore ® technology (e.g. by using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of interactions using surface plasmon resonance technology.
  • Another assay for measuring cross-blocking uses an ELISA-based approach. Although the techniques are expected to produce substantially similar results, measurement by the Biacore technique is considered definitive.
  • neutralizes means that an antibody, upon binding to its target, reduces the activity, level or stability of the target; e.g., a CD32b antibody, upon binding to CD32b neutralizes CD32b by at least partially reducing an activity, level or stability of CD32b, such as its role in engaging Fc portions of antibodies.
  • epitope means a protein determinant capable of specific binding to an antibody.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • epitopope includes any protein determinant capable of specific binding to an immunoglobulin or otherwise interacting with a molecule.
  • Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • An epitope may be "linear” or “conformational”.
  • linear epitope refers to an epitope with all of the points of interaction between the protein and the interacting molecule (such as an antibody) occurring linearly and/or continuously along the primary amino acid sequence of the protein.
  • high affinity for an IgG antibody refers to an antibody having a KD of 10 "8 M or less, 10 "9 M or less, or 10 "10 M, or 10 "11 M or less for a target antigen, e.g., CD32b.
  • high affinity binding can vary for other antibody isotypes.
  • “high affinity” binding for an IgM isotype refers to an antibody having a KD of 10 "7 M or less, or 10 "8 M or less.
  • human antibody (or antigen-binding fragment thereof), as used herein, is intended to include antibodies (and antigen-binding fragments thereof) having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences.
  • the human antibodies and antigen-binding fragments thereof of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • monoclonal antibody or “monoclonal antibody composition” (or antigen-binding fragment thereof) as used herein refers to polypeptides, including antibodies, antibody fragments, bispecific antibodies, etc. that have substantially identical to amino acid sequence or are derived from the same genetic source. This term also includes preparations of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • human monoclonal antibody refers to antibodies (and antigen-binding fragments thereof) displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human sequences.
  • the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • recombinant human antibody includes all human antibodies (and antigen-binding fragments thereof) that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DNA sequences.
  • recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline
  • such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • a “humanized antibody” (or antigen-binding fragment thereof), as used herein, is an antibody molecule that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts (i.e., the constant region as well as the framework portions of the variable region). See, e.g., Morrison et al, (1984) PNAS. USA, 81 :6851-6855; Morrison & Oi, (1988) Adv. Immunol., 44:65-92; Verhoeyen et al, (1988) Science, 239: 1534-1536; Padlan, (1991) Molec. Immun, 28:489-498 and Padlan, (1994) Molec. Immun., 31 : 169-217.
  • Other examples of human engineering technology include, but is not limited to, Xoma technology disclosed in U.S. Pat. No.
  • chimeric antibody (or antigen-binding fragment thereof) is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • a mouse antibody can be modified by replacing its constant region with the constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced antigenicity in human as compared to the original mouse antibody.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same.
  • Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
  • the identity exists over a region that is at least 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman & Wunsch, (1970) J. Mol. Biol.
  • BLAST and BLAST 2.0 algorithms Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al, (1990) J. Mol. Biol. 215:403-410, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for
  • HSPs high scoring sequence pairs
  • neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative -scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, (1993) PNAS USA 90:5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P (N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., (1988) 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman & Wunsch (J. Mol, Biol.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions describes conditions for hybridization and washing.
  • Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used.
  • Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1% SDS at least at 50°C (the temperature of the washes can be increased to 55°C for low stringency conditions); 2) medium stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; 3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.
  • isolated antibody refers to an antibody molecule that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds CD32b is substantially free of antibodies that specifically bind antigens other than CD32b). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • isotype refers to the antibody class (e.g., IgM, IgE, IgG such as IgGl or IgG4) that is provided by the heavy chain constant region genes. Isotype also includes modified versions of one of these classes, where modifications have been made to after the Fc function, for example, to enhance or reduce effector functions or binding to Fc receptors.
  • Kassoc is intended to refer to the association rate of a particular antibody-antigen interaction
  • Kdis is intended to refer to the dissociation rate of a particular antibody-antigen interaction
  • KD is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art.
  • a method for determining the KD of an antibody is by using surface plasmon resonance, or using a biosensor system such as a Biacore ® system. Where the dissociation constant is less than about 10 "9 M, solution equilibrium kinetic exclusion KD measurement (MSD-SET) is a preferred method for determining the KD of an antibody (see, e.g. Friquet et al., (1985) J Immnunol. Meth. 77, 305-319; herein incorporated by reference).
  • IC 50 refers to the concentration of an antibody or an antigen-binding fragment thereof, which induces an inhibitory response, either in an in vitro or an in vivo assay, which is 50% of the maximal response, i.e., halfway between the maximal response and the baseline.
  • effector function refers to an activity of an antibody molecule that is mediated by binding through a domain of the antibody other than the antigen-binding domain, usually mediated by binding of effector molecules.
  • Effector function includes complement-mediated effector function, which is mediated by, for example, binding of the CI component of the complement to the antibody. Activation of complement is important in the opsonisation and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. Effector function also includes Fc receptor (FcR)-mediated effector function, which may be triggered upon binding of the constant domain of an antibody to an Fc receptor (FcR).
  • FcR Fc receptor
  • Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, release of inflammatory mediators, placental transfer, control of immunoglobulin production and lysis of antibody- coated target cells by killer cells. The latter response, whereby an effector cell of the immune system (e.g.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCC is part of the adaptive immune response due to its dependence on a prior antibody response.
  • An effector function of an antibody may be altered by altering, e.g., enhancing or reducing, the affinity of the antibody for an effector molecule such as an Fc receptor or a complement component. Binding affinity will generally be varied by modifying the effector molecule binding site, and in this case it is appropriate to locate the site of interest and modify at least part of the site in a suitable way. It is also envisaged that an alteration in the binding site on the antibody for the effector molecule need not alter significantly the overall binding affinity but may alter the geometry of the interaction rendering the effector mechanism ineffective as in non-productive binding. It is further envisaged that an effector function may also be altered by modifying a site not directly involved in effector molecule binding, but otherwise involved in performance of the effector function.
  • nucleic acid is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, wild-type, and non-wild-type, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, (1991) Nucleic Acid Res. 19:5081; Ohtsuka ei /., (1985) J. Biol. Chem. 260:2605-2608; and Rossolini et al, (1994) Mol. Cell. Probes 8:91-98).
  • operably linked refers to a functional relationship between two or more
  • polynucleotide e.g., DNA
  • a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
  • promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
  • some transcriptional regulatory sequences, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
  • the term, "optimized" means that a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, generally a eukaryotic cell, for example, a cell of Pichia, a Chinese Hamster Ovary cell (CHO) or a human cell.
  • the optimized nucleotide sequence is engineered to retain completely or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence, which is also known as the "parental" sequence.
  • the optimized sequences herein have been engineered to have codons that are preferred in mammalian cells. However, optimized expression of these sequences in other eukaryotic cells or prokaryotic cells is also envisioned herein.
  • the amino acid sequences encoded by optimized nucleotide sequences are also referred to as optimized.
  • polypeptide and "protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding wild-type amino acid, as well as to wild-type amino acid polymers and non-wild-type amino acid polymer. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • recombinant host cell refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • subject includes human and non-human animals.
  • Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or “subject” are used herein
  • treat include the administration of compositions or antibodies to alleviate or delay the onset of the symptoms, complications, or biochemical indicia of a disease, preventing the development of further symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Treatment can be measured by the therapeutic measures described herein.
  • the methods of "treatment” of the present invention include administration of a CD32b antibody molecule to a subject in order to cure, reduce the severity of, or ameliorate one or more symptoms of cancer or condition associated with cancer, in order to prolong the health or survival of a subject beyond that expected in the absence of such treatment.
  • treatment includes the alleviation of a disease symptom in a subject by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.
  • prevent includes administration of compositions or antibodies to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof i.e.
  • vector is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.
  • Anti-human CD32b antibody molecules for use in a combination of the present invention include isolated antibodies or antigen-binding fragments thereof that bind with a higher affinity for human CD32b protein, than to human CD32a protein. Selectivity for CD32b over CD32a is desired to ensure selective binding to CD32b positive B-cell malignancies and B-cells while lacking binding to CD32a positive immune cells, including monocytes and neutrophils.
  • anti-CD32b antibody molecules for use in a combination of the present invention include antibody 2B6 (PCT Publication WO2004/016750), antibodies 20, 24, 26 or 28 (PCT Publication WO2009/083009) and antibody 6G11 (PCT Publications WO2012/022985 and WO2015/173384), whose sequences are listed in Table 1.
  • Additional antibody molecules that specifically bind to CD32b include those comprising (or alternatively, consisting of) a VH amino acid sequence listed in Table 1, wherein no more than about 10 amino acids in a framework sequence (for example, a sequence which is not a CDR) have been mutated (wherein a mutation is, as various non-limiting examples, an addition, substitution or deletion) or wherein no more than about 20 amino acids in a framework sequence (for example, a sequence which is not a CDR) have been mutated (wherein a mutation is, as various non-limiting examples, an addition, substitution or deletion).
  • antibody molecules that specifically bind to CD32b include those comprising (or alternatively, consisting of) a VL amino acid sequence listed in Table 1, wherein no more than about 10 amino acids in a framework sequence (for example, a sequence which is not a CDR) have been mutated (wherein a mutation is, as various non-limiting examples, an addition, substitution or deletion) or wherein no more than about 20 amino acids in a framework sequence (for example, a sequence which is not a CDR) have been mutated (wherein a mutation is, as various non-limiting examples, an addition, substitution or deletion).
  • antibody molecules for use in a combination of the present invention include amino acids that have been mutated, yet have at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity in the CDR regions with the CDR regions depicted in the sequences described in Table 1.
  • other antibody molecules of the invention includes mutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the CDR regions when compared with the CDR regions depicted in the sequence described in Table 1.
  • antibody molecules for use in a combination of the present invention include those wherein the amino acids or nucleic acids encoding the amino acids have been mutated, yet have at least 60, 70, 80, 90 or 95 percent identity to the sequences described in Table 1. In one embodiment, it includes mutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the variable regions when compared with the variable regions depicted in the sequence described in Table 1, while retaining substantially the same therapeutic activity.
  • VH, VL, full length light chain, and full length heavy chain sequences (amino acid sequences and the nucleotide sequences encoding the amino acid sequences) can be "mixed and matched" to create other CD32b-binding antibody molecules of the invention.
  • Such "mixed and matched" CD32b-binding antibodies can be tested using the binding assays known in the art (e.g., ELISAs, and other assays described in the Example section). When these chains are mixed and matched, a VH sequence from a particular VH/VL pairing should be replaced with a structurally similar VH sequence.
  • a full length heavy chain sequence from a particular full length heavy chain/full length light chain pairing should be replaced with a structurally similar full length heavy chain sequence.
  • a VL sequence from a particular VH/VL pairing should be replaced with a structurally similar VL sequence.
  • a full length light chain sequence from a particular full length heavy chain/full length light chain pairing should be replaced with a structurally similar full length light chain sequence.
  • a heavy chain variable region VHCDR3 comprising an amino acid sequence selected from any of SEQ ID NOs: 3, 17, 31, 45, 59, 73, 87, 101, 115, 129, 143, 157, 171, 185, 199, 209, 217, 225, 233 and 241;
  • a light chain variable region VLCDR1 comprising an amino acid sequence selected from any of SEQ ID NOs: 8, 22, 36, 50, 64, 78, 92, 106, 120, 134, 148, 162, 176, 190, 202, 211, 219, 227, 235 and 243;
  • a light chain variable region VLCDR2 comprising an amino acid sequence selected from any of SEQ ID NOs: 9, 23, 37, 51, 65, 79, 93, 107, 121, 135, 149, 163, 177, 191, 203, 212
  • VH heavy chain variable region
  • VL light chain variable region
  • an antibody molecule, for use in a combination of the present invention and that specifically binds to CD32b is an antibody that is described in Table 1.
  • an antibody that specifically binds to CD32b is NOV0281.
  • an antibody that specifically binds to CD32b is NOV0308.
  • an antibody that specifically binds to CD32b is NOV0563.
  • an antibody that specifically binds to CD32b is NOV1216.
  • an antibody that specifically binds to CD32b is NOV1218.
  • an antibody that specifically binds to CD32b is NOV1219.
  • an antibody that specifically binds to CD32b is NOV2106.
  • an antibody that specifically binds to CD32b is NOV2107. In one embodiment, an antibody that specifically binds to CD32b is NOV2108. In one embodiment, an antibody that specifically binds to CD32b is NOV2109. In one embodiment, an antibody that specifically binds to CD32b is NOV2112. In one embodiment, an antibody that specifically binds to CD32b is NOV2113. In one embodiment, an antibody that specifically binds to CD32b is 2B6 (WO2004/016750). In one embodiment, an antibody that specifically binds to CD32b is Ab 20, 24, 26 or 28
  • an antibody that specifically binds to CD32b is 6G11
  • an antibody of the invention optimized for expression in a mammalian cell has a full length heavy chain sequence and a full length light chain sequence, wherein one or more of these sequences have specified amino acid sequences based on the antibody molecules described herein or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the CD32b-binding antibodies and antigen-binding fragments thereof of the invention.
  • the invention provides an isolated monoclonal antibody optimized for expression in a mammalian cell comprising a full length heavy chain and a full length light chain wherein: the full length heavy chain comprises an amino acid sequence selected from any of SEQ ID NOs: 6, 20, 34, 48, 62, 76, 90, 104, 118, 132, 174 and 188, as described in Table 1, and conservative modifications thereof; and the full length light chain comprises an amino acid sequence selected from any of SEQ ID NOs: 13, 27, 41, 55, 69, 83, 97, 111, 125, 139, 181 and 195, as described in Table 1, and conservative modifications thereof; wherein the antibody specifically binds to CD32b and mediates both macrophage and NK cell killing of antibody bound, CD32b positive target cells.
  • the antibodies comprise a wild type (WT) Fc sequence.
  • antibody molecules of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • an antibody of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
  • the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fc-gamma receptor by modifying one or more amino acids.
  • ADCC antibody dependent cellular cytotoxicity
  • This approach is described further, for example, in PCT Publication WO 00/42072 by Presta and by Lazar et al, (2006) PNAS 103(110): 4005- 4010.
  • the binding sites on human IgGl for Fc-gamma RI, Fc-gamma RII, Fc-gamma RIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al, (2001) J. Biol. Chem. 276:6591-6604).
  • the glycosylation of an antibody is modified.
  • an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for "antigen ' .
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated or afucosylated antibody having reduced amounts of fucosyl residues, or an antibody having increased bisecting GlcNac structures.
  • altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation.
  • EP 1,176,195 by Hang et al describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation.
  • PCT Publication WO 03/035835 by Presta describes a variant
  • GnTIII acetylglucosaminyl-transferase III
  • the CD32b-binding antibody is afucosylated NOV2108, comprising a WT Fc.
  • the CD32b-binding antibody comprises an HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 113, 114, and 115, respectively, and a LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 120, 121, and 122 respectively, and wherein the antibody is afucosylated.
  • the CD32b- binding antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 116 and a VL comprising the amino acid sequence of SEQ ID NO: 123, and wherein the antibody is afucosylated.
  • the CD32b-binding antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 118 and a light chain comprising the amino acid sequence of SEQ ID NO: 125, wherein the antibody is afucosylated.
  • the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased.
  • This approach is described further in U.S. Pat. No. 5,677,425 by Bodmer et al.
  • the number of cysteine residues in the hinge region of CHI is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
  • the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired
  • Staphylococcyl protein A binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745 by Ward et al.
  • the antibody is modified to increase its biological half-life.
  • Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to Ward.
  • the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6, 121,022 by Presta et al.
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody.
  • one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the C 1 component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.
  • one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.
  • the invention also provides substantially purified nucleic acid molecules which encode polypeptides comprising segments or domains of the CD32b-binding antibody chains described above.
  • Some of the nucleic acids of the invention comprise the nucleotide sequence encoding the heavy chain variable region shown in any of SEQ ID NOs: 4, 18, 32, 46, 60, 74, 88, 102, 116, 130, 144, 158, 172, 186, 200, 210, 218, 226, 234 or 242, and/or the nucleotide sequence encoding the light chain variable region shown in any of SEQ ID NOs: 11, 25, 39, 53, 67, 81, 95, 109, 123, 137, 151, 165, 179, 193, 205, 214, 222, 230, 238 or 246.
  • the nucleic acid molecules are those identified in Table 1 and comprise any of SEQ ID Nos: 5, 19, 33, 47, 61, 75, 89, 103, 117, 131, 145, 159, 173, 187 or 201, encoding a heavy chain variable region, and/or the nucleic acid molecules identified in Table 1 that comprise any of SEQ ID Nos: 12, 26, 40, 54, 68, 82, 96, 110, 124, 138, 152, 166, 180, 194 or 206, encoding a light chain variable region.
  • nucleic acid molecules of the invention comprise nucleotide sequences that are substantially identical (e.g., at least 65, 80%, 95%, or 99%) to the nucleotide sequences of those identified in Table 1.
  • polypeptides encoded by these polynucleotides are capable of exhibiting CD32b antigen binding capacity.
  • polynucleotides which encode at least one CDR region and usually all three CDR regions from the heavy or light chain of the CD32b-binding antibody set forth in Table 1. Some other polynucleotides encode all or substantially all of the variable region sequence of the heavy chain and/or the light chain of the CD32b-binding antibody set forth in Table 1. Because of the degeneracy of the code, a variety of nucleic acid sequences will encode each of the immunoglobulin amino acid sequences.
  • nucleic acid molecules of the invention can encode both a variable region and a constant region of the antibody.
  • Some of the nucleic acid sequences of the invention comprise nucleotides encoding a mature heavy chain sequence that is identical or substantially identical (e.g., at least 80%, 90%, or 99%) to the mature heavy chain sequence set forth in any of SEQ ID NOs: 6, 20, 34, 48, 62, 76, 90, 104, 118, 132, 174 or 188.
  • nucleic acid sequences of the invention comprise nucleotide encoding a mature light chain sequence that is identical or substantially identical (e.g., at least 80%, 90%, or 99%) to the mature light chain sequence set forth in any of SEQ ID NOs: 13, 27, 41, 55, 69, 83, 97, 111, 125, 139, 181 or 195.
  • the nucleic acid molecules are those identified in Table 1 and comprise any of SEQ ID Nos: 7, 21, 35, 49, 63, 77, 91, 105, 119, 133, 175 or 189, encoding a mature heavy chain, and/or the nucleic acid molecules identified in Table 1 that comprise any of SEQ ID Nos: 14, 28, 42, 56, 70, 84, 98, 112, 126, 140, 182 or 196, encoding a mature light chain.
  • the polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence (e.g., sequences as described in Table 1) encoding a CD32b-binding antibody or its binding fragment.
  • Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al, (1979), Meth. Enzymol. 68:90; the phosphodiester method of Brown et al, (1979) Meth. Enzymol. 68: 109; the
  • IL-15 and "interleukin-15” refer to a wild-type IL-15 or an IL-15 derivative. In specific embodiments, the IL-15 is isolated and recombinantly produced. As used herein, the terms “wild-type IL-15” and “wild-type interleukin-15” in the context of proteins or polypeptides refer to any mammalian interleukin-15 amino acid sequences, including immature or precursor and mature forms. Non-limiting examples of GeneBank Accession Nos.
  • NP_000576 human, immature form
  • CAA62616 human, immature form
  • NP_001009207 ⁇ Felis catus, immature form
  • AAB94536 Raster norvegicus, immature form
  • AAB41697 ⁇ Rattus norvegicus, immature form
  • NP_032383 ⁇ Mus musculus, immature form
  • AAR19080 canine
  • AAB60398 Macaca mulatta, immature form
  • IL-15 is the immature or precursor form of a mammalian IL-15.
  • IL-15 is the mature form of a mammalian IL-15.
  • IL-15 is the precursor form of human IL-15.
  • IL-15 is the mature form of human IL-15.
  • the IL-15 protein/polypeptide is isolated or purified.
  • IL-15 and "interleukin-15” in the context of nucleic acids refer to any nucleic acid sequences encoding mammalian interleukin-15, including the immature or precursor and mature forms.
  • Non-limiting examples of GeneBank Accession Nos. for the nucleotide sequence of various species of wild-type mammalian IL-15 include NM_000585 (human), NM_008357 ⁇ Mus musculus), and RNU69272 ⁇ Rattus norvegicus).
  • nucleic acid is an isolated or purified nucleic acid.
  • nucleic acids encode the immature or precursor form of a mammalian IL-15.
  • nucleic acids encode the mature form of a mammalian IL-15.
  • nucleic acids encoding IL-15 encode the precursor form of human IL-15.
  • nucleic acids encoding IL-15 encode the mature form of human IL-15.
  • IL-15 derivative and "interleukin-15 derivative” in the context of proteins or polypeptides refer to: (a) a polypeptide that is at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to a wild-type mammalian IL-15 polypeptide; (b) a polypeptide encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical a nucleic acid sequence encoding a wild-type mammalian IL-15 polypeptide; (c) a polypeptide that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions and/or substitutions) relative to a wild-type mammalian IL-15 polypeptide; (d) a polypeptide encoded by nucleic acids can hybridize under high or medium stringency hybridization conditions to nucleic acids
  • IL-15 derivatives also include a polypeptide that comprises the amino acid sequence of a mature form of a mammalian IL-15 polypeptide and a heterologous signal peptide amino acid sequence.
  • an IL-15 derivative is a derivative of a wild-type human IL-15 polypeptide.
  • an IL-15 derivative is a derivative of an immature or precursor form of human IL-15 polypeptide.
  • an IL-15 derivative is a derivative of a mature form of human IL-15 polypeptide.
  • an IL-15 derivative is the IL- 15N72D described in, e.g., Zhu et al., (2009), J. Immunol. 183: 3598 or U.S. Patent No. 8,163,879.
  • an IL-15 derivative is one of the IL-15 variants described in U.S. Patent No.
  • an IL-15 derivative is isolated or purified.
  • IL-15 derivatives retain at least 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of wild-type mammalian IL-15 polypeptide to bind IL-15Ra polypeptide, as measured by assays well known in the art, e.g., ELISA, BIAcore ® , co-immunoprecipitation.
  • IL-15 derivatives retain at least 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of wild-type mammalian IL-15 polypeptide to induce IL-15 -mediated signal transduction, as measured by assays well-known in the art, e.g., electromobility shift assays, ELISAs and other immunoassays.
  • IL-15 derivatives bind to IL-15Ra and/or ⁇ -15 ⁇ as assessed by, e.g., ligand/receptor binding assays well-known in the art. Percent identity can be determined using any method known to one of skill in the art and as described supra.
  • IL-15 derivative and "interleukin-15 derivative” in the context of nucleic acids refer to: (a) a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the nucleic acid sequence encoding a mammalian IL-15 polypeptide; (b) a nucleic acid sequence encoding a polypeptide that is at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical the amino acid sequence of a wild-type mammalian IL-15 polypeptide; (c) a nucleic acid sequence that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acid base mutations (i.e., additions, deletions and/or substitutions) relative to the nucleic acid sequence encoding a mammalian IL-15 polypeptide; (d) a nucleic acid sequence that hybridizes under high or medium stringency hybrid
  • an IL-15 derivative in the context of nucleic acids is a derivative of a nucleic acid sequence encoding a human IL-15 polypeptide.
  • an IL-15 derivative in the context of nucleic acids is a derivative of a nucleic acid sequence encoding an immature or precursor form of a human IL-15 polypeptide.
  • an IL-15 derivative in the context of nucleic acids is a derivative of a nucleic acid sequence encoding a mature form of a human IL-15 polypeptide.
  • an IL-15 derivative in the context of nucleic acids is the nucleic acid sequence encoding the IL-15N72D described in, e.g., Zhu et al, (2009; supra), or U.S. Patent No. 8,163,879.
  • an IL-15 derivative in the context of nucleic acids is the nucleic acid sequence encoding one of the IL-15 variants described in U.S. Patent No. 8, 163,879.
  • IL-15 derivative nucleic acid sequences include codon-optimized nucleic acid sequences that encode mammalian IL-15 polypeptide, including mature and immature forms of IL-15 polypeptide.
  • IL-15 derivative nucleic acids include nucleic acids that encode mammalian IL-15 RNA transcripts containing mutations that eliminate potential splice sites and instability elements (e.g., A/T or A/U rich elements) without affecting the amino acid sequence to increase the stability of the mammalian IL-15 RNA transcripts.
  • the IL-15 derivative nucleic acid sequences include the codon-optimized nucleic acid sequences described in PCT Publication WO2007/084342.
  • the IL-15 derivative nucleic acid sequence is the codon-optimized sequence in SEQ ID NO: 254 in Table 1 (the amino acid sequence encoded by such a nucleic acid sequence is provided in SEQ ID NO: 255 in Table 1).
  • IL-15 derivative nucleic acid sequences encode proteins or polypeptides that retain at least 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a wild-type mammalian IL-15 polypeptide to bind IL-15Ra, as measured by assays well known in the art, e.g., ELISA, BIAcore ® , co- immunoprecipitation.
  • IL-15 derivative nucleic acid sequences encode proteins or polypeptides that retain at least 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a wild-type mammalian IL-15 polypeptide to induce IL-15 -mediated signal transduction, as measured by assays well-known in the art, e.g., electromobility shift assays, ELISAs and other immunoassays.
  • IL-15 derivative nucleic acid sequences encode proteins or polypeptides that bind to IL-15Ra and/or ⁇ -15 ⁇ as assessed by, e.g., ligand/receptor assays well-known in the art.
  • IL-15Ra and "interleukin-15 receptor alpha” refer to a wild-type IL- 15Ra, an IL-15Ra derivative, or a wild-type IL-15Ra and an IL-15Ra derivative.
  • the IL-15Ra is isolated and recombinantly produced.
  • wild-type IL-15Ra and wild-type interleukin-15 receptor alpha in the context of proteins or polypeptides refer to mammalian interleukin-15 receptor alpha ("IL-15Ra") amino acid sequence, including immature or precursor and mature forms and isoforms.
  • IL-15Ra mammalian interleukin-15 receptor alpha
  • Non-limiting examples of GeneBank Accession Nos. for the amino acid sequence of various wild-type mammalian IL-15Ra include NP 002180 (human), ABK41438 (Macaca mulatto), NP_032384 (Mus musculus), Q60819 (Mus musculus), CAI41082 (human).
  • IL-15Ra is the immature form of a mammalian IL-15Ra polypeptide. In other embodiments, IL-15Ra is the mature form of a mammalian IL-15Ra polypeptide.
  • IL-15Ra is the soluble form of mammalian IL-15Ra polypeptide. In other embodiments, IL-15Ra is the full-length form of a mammalian IL-15Ra polypeptide. In a specific embodiment, IL-15Ra is the immature form of a human IL-15Ra polypeptide. In another embodiment, IL-15Ra is the mature form of a human IL-15Ra polypeptide. In certain embodiments, IL-15Ra is the soluble form of human IL-15Ra polypeptide. In other embodiments, IL-15Ra is the full-length form of a human IL-15Ra polypeptide. In one embodiment, an IL-15Ra protein or polypeptide is isolated or purified.
  • IL-15Ra and "interleukin-15 receptor alpha" in the context of nucleic acids refer to any nucleic acid sequences encoding mammalian interleukin-15 receptor alpha, including the immature or precursor and mature forms.
  • the nucleotide sequence encoding the immature form of wild-type human IL-15Ra which comprises the nucleotide sequence encoding the signal peptide (underlined) and the nucleotide sequence encoding the mature human IL- 15Ra (italicized), as provided in SEQ ID NO: 258 in Table 1.
  • the nucleotide sequence encoding the immature form of soluble human IL-15Ra protein or polypeptide which comprises the nucleotide sequence encoding the signal peptide (underlined) and the nucleotide sequence encoding the mature human soluble IL-15Ra (italicized), as provided in SEQ ID NO in 259 in Table 1).
  • nucleic acid is an isolated or purified nucleic acid.
  • nucleic acids encode the immature form of a mammalian IL-15Ra polypeptide.
  • nucleic acids encode the mature form of a mammalian IL-15Ra polypeptide.
  • nucleic acids encode the soluble form of a mammalian IL-15Ra polypeptide.
  • nucleic acids encode the full-length form of a mammalian IL-15Ra polypeptide.
  • nucleic acids encode the precursor form of human IL-15 polypeptide.
  • nucleic acids encode the mature of human IL-15 polypeptide.
  • nucleic acids encode the soluble form of a human IL-15Ra polypeptide.
  • nucleic acids encode the full-length form of a human IL-15Ra polypeptide.
  • IL-15Ra derivative and "interleukin-15 receptor alpha derivative” in the context of a protein or polypeptide refer to: (a) a polypeptide that is at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to a wild-type mammalian IL-15 polypeptide; (b) a polypeptide encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical a nucleic acid sequence encoding a wild-type mammalian IL-15Ra polypeptide; (c) a polypeptide that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions and/or substitutions) relative to a wild-type mammalian IL-15Ra polypeptide; (d) a polypeptide encoded by a nucleic acid sequence that can hybridize under
  • IL-15Ra derivatives also include a polypeptide that comprises the amino acid sequence of a mature form of mammalian IL-15Ra polypeptide and a heterologous signal peptide amino acid sequence.
  • an IL-15Ra derivative is a derivative of a wild-type human IL-15Ra polypeptide.
  • an IL-15Ra derivative is a derivative of an immature form of human IL-15 polypeptide.
  • an IL-15Ra derivative is a derivative of a mature form of human IL-15 polypeptide.
  • an IL-15Ra derivative is a soluble form of a mammalian IL-15Ra polypeptide.
  • an IL-15Ra derivative includes soluble forms of mammalian IL-15Ra, wherein those soluble forms are not naturally occurring.
  • Other examples of IL-15Ra derivatives include the truncated, soluble forms of human IL-15Ra described herein.
  • an IL-15Ra derivative is purified or isolated.
  • IL-15Ra derivatives retain at least 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a wild-type mammalian IL-15Ra polypeptide to bind an IL-15 polypeptide, as measured by assays well known in the art, e.g., ELISA, BIAcore ® , co-immunoprecipitation.
  • IL-15Ra derivatives retain at least 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a wild-type mammalian IL-15Ra polypeptide to induce IL- 15 -mediated signal transduction, as measured by assays well-known in the art, e.g., electromobility shift assays, ELISAs and other immunoassays.
  • IL-15Ra derivatives bind to IL-15 as assessed by methods well-known in the art, such as, e.g., ELISAs.
  • IL-15Ra derivative and "interleukin-15 receptor alpha derivative” in the context of nucleic acids refer to: (a) a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the nucleic acid sequence encoding a mammalian IL-15Ra polypeptide; (b) a nucleic acid sequence encoding a polypeptide that is at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical the amino acid sequence of a wild-type mammalian IL-15Ra polypeptide; (c) a nucleic acid sequence that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acid mutations (i.e., additions, deletions and/or substitutions) relative to the nucleic acid sequence encoding a mammalian IL-15Ra polypeptide; (d) a nucleic acid sequence that
  • an IL-15Ra derivative in the context of nucleic acids is a derivative of a nucleic acid sequence encoding a human IL-15Ra polypeptide.
  • an IL-15Ra derivative in the context of nucleic acids is a derivative of a nucleic acid sequence encoding an immature form of a human IL-15Ra polypeptide.
  • an IL-15Ra derivative in the context of nucleic acids is a derivative of a nucleic acid sequence encoding a mature form of a human IL-15Ra polypeptide.
  • an IL-15Ra derivative in the context of nucleic acids refers to a nucleic acid sequence encoding a derivative of mammalian IL-15Ra polypeptide that is soluble.
  • an IL-15Ra derivative in context of nucleic acids refers to a nucleic acid sequence encoding a soluble form of mammalian IL-15Ra, wherein the soluble form is not naturally occurring.
  • an IL-15Ra derivative in the context of nucleic acids refers to a nucleic acid sequence encoding a derivative of human IL-15Ra, wherein the derivative of the human IL-15Ra is a soluble form of IL-15Ra that is not naturally occurring.
  • an IL- 15 Ra derivative nucleic acid sequence is isolated or purified.
  • IL-15Ra derivative nucleic acid sequences include codon-optimized nucleic acid sequences that encode an IL-15Ra polypeptide, including mature and immature forms of IL-15Ra polypeptide.
  • IL-15Ra derivative nucleic acids include nucleic acids that encode IL-15Ra RNA transcripts containing mutations that eliminate potential splice sites and instability elements (e.g., A/T or A/U rich elements) without affecting the amino acid sequence to increase the stability of the IL-15Ra RNA transcripts.
  • the IL-15Ra derivative nucleic acid sequence is the codon- optimized sequence in SEQ ID NO: 261 or 263 in Table 1 (the amino acid sequences encoded by such a nucleic acid sequences are provided in SEQ ID NO: 262 and 264 in Table 1, respectively).
  • IL-15Ra derivative nucleic acid sequences encode proteins or polypeptides that retain at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a wild-type mammalian IL-15Ra polypeptide to bind IL-15, as measured by assays well known in the art, e.g., ELISA, BIAcore ® , co-immunoprecipitation.
  • IL- 15Ra derivative nucleic acid sequences encode proteins or polypeptides that retain at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a wild-type mammalian IL- 15Ra to induce IL-15 -mediated signal transduction, as measured by assays well-known in the art, e.g., electromobility shift assays, ELISAs and other immunoassays.
  • IL-15Ra derivative nucleic acid sequences encode proteins or polypeptides that bind to IL-15 as assessed by methods well-known in the art, such as, e.g., ELISAs.
  • IL-15Ra Described herein is the wild type soluble form of human IL-15Ra. Also described herein are specific IL-15Ra derivatives that are truncated, soluble forms of human IL-15Ra. These specific IL-15Ra derivatives and the soluble form of human IL-15Ra are based, in part, on the identification of the proteolytic cleavage site of human IL-15Ra. Further described herein are soluble forms of IL-15Ra that are characterized based upon glycosylation of the IL-15Ra.
  • proteolytic cleavage of human IL-15Ra takes place between the residues (i.e., Glyl70 and His 171) which are in shown in bold and underlined in the provided amino acid sequence of the immature form of the wild-type full length human IL-15Ra:
  • a soluble form of human IL-15Ra e.g., a purified soluble form of human IL-15Ra
  • the amino acid sequence of the soluble form of human IL-15Ra terminates at the site of the proteolytic cleavage of the wild-type membrane -bound human IL-15Ra.
  • a soluble form of human IL-15Ra e.g., a purified soluble form of human IL-15Ra
  • the amino acid sequence of the soluble form of human IL-15Ra terminates with PQG (SEQ ID NO: 270 in Table 1), wherein G is Glyl70.
  • a soluble form of human IL-15Ra e.g., a purified soluble form of human IL-15Ra
  • an IL- 15Ra derivative e.g., a purified and/or soluble form of IL-15Ra derivative
  • PQG amino acid sequence PQG
  • a soluble form of human IL-15Ra e.g., a purified soluble form of human IL-15Ra which has the amino acid sequence of SEQ ID NO: 260 in Table 1).
  • an IL-15Ra derivative e.g., a purified and/or soluble form of an IL- 15Ra derivative
  • PQG amino acid sequence of the soluble form of the IL-15Ra derivative terminates with PQG (SEQ ID NO: 270 in Table 1).
  • an IL-15Ra derivative of human IL-15Ra wherein the IL-15Ra derivative is soluble and: (a) the last amino acids at the C-terminal end of the IL-15Ra derivative consist of amino acid residues PQGHSDTT (SEQ ID NO: 265 in Table 1); (b) the last amino acids at the C-terminal end of the IL-15Ra derivative consist of amino acid residues PQGHSDT (SEQ ID NO: 266 in Table 1); (c) the last amino acids at the C-terminal end of the IL-15Ra derivative consist of amino acid residues PQGHSD (SEQ ID NO: 267 in Table 1); (d) the last amino acids at the C-terminal end of the IL- 15Ra derivative consist of amino acid residues PQGHS (SEQ ID NO: 268 in Table 1); or (e) the last amino acids at the C-terminal end of the IL-15Ra derivative consist of amino acid residues PQGH (SEQ ID NO:
  • amino acid sequences of these IL-15Ra derivatives are at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 271 in Table 1. In some embodiments, these IL-15Ra derivatives are purified.
  • glycosylated forms of IL-15Ra e.g., purified glycosylated forms of IL-15Ra
  • the glycosylation of the IL-15Ra accounts for at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or 20% to 25%, 20% to 30%, 25% to 30%, 25% to 35%, 30% to 35%, 30% to 40%, 35% to 40%, 35% to 45%, 40% to 50%, 45% to 50%, 20% to 40%, or 25% to 50% of the mass (molecular weight) of the IL-15Ra as assessed by techniques known to one of skill in the art.
  • the percentage of the mass (molecular weight) of IL-15Ra (e.g., purified IL- 15Ra) that glycosylation of IL-15Ra accounts for can be determined using, for example and without limitation, gel electrophoresis and quantitative densitometry of the gels, and comparison of the average mass (molecular weight) of a glycosylated form of IL-15Ra (e.g., a purified glycosylated form of IL- 15Ra) to the non-glycosylated form of IL-15Ra (e.g., a purified non-glycosylated form of IL-15Ra).
  • the average mass (molecular weight) of IL-15Ra (e.g., purified IL-15Ra) can be determined using MALDI-TOF MS spectrum on Voyager De-Pro equipped with CovalX HM-1 high mass detector using sinapic acid as matrix, and the mass of a glycosylated form of IL-15Ra (e.g., purified glycosylated form of IL-15Ra) can be compared to the mass of the non-glycosylated form of IL-15Ra (e.g., purified non-glycosylated form of IL-15Ra) to determine the percentage of the mass that glycosylation accounts for.
  • a glycosylated form of IL-15Ra e.g., purified glycosylated form of IL-15Ra
  • glycosylated forms of IL-15Ra wherein the IL-15Ra is glycosylated (N- or O-glycosylated) at certain amino acid residues.
  • a human IL-15Ra which is glycosylated at one, two, three, four, five, six, seven, or all, of the following glycosylation sites: (i) O-glycosylation on threonine at position 5 of the amino acid sequence NWELTASASHQPPGVYPQG (SEQ ID NO: 272 in Table 1) in the IL-15Ra;
  • ITCPPPMSVEHADIWVKSYSLYSRERYICNS (SEQ ID NO: 274 in Table 1) in the IL-15Ra;
  • ITCPPPMSVEHADIWVKSYSLYSRERYICNS (SEQ ID NO: 274 in Table 1) in the IL-15Ra;
  • ITCPPPMSVEHADIWVKSYSLYSRERYICNS (SEQ ID NO: 274 in Table 1) in the IL-15Ra;
  • ITCPPPMSVEHADIWVKSYSLYSRERYICNS (SEQ ID NO: 274 in Table 1) in the IL-15Ra.
  • the glycosylated IL-15Ra is a wild-type human IL-15Ra. In other specific embodiments, the glycosylated IL-15Ra is an IL-15Ra derivative of human IL-15Ra. In some embodiments, the glycosylated IL-15Ra is a wild-type soluble human IL-15Ra, such as SEQ ID NO:257 or 260 in Table 1. In other embodiments, the glycosylated IL-15Ra is an IL-15Ra derivative that is a soluble form of human IL-15Ra. In certain embodiments, the glycosylated IL-15Ra is purified or isolated.
  • the term "IL-15/IL-15Ra complex” refers to a complex comprising IL-15 and IL- 15Ra covalently or noncovalently bound to each other.
  • the IL-15Ra has a relatively high affinity for IL-15, e.g., KD of 10 to 50 pM as measured by a technique known in the art, e.g., KinEx A assay, plasma surface resonance (e.g., BIAcore ® assay).
  • the IL-15/IL-15Ra complex induces IL-15 -mediated signal transduction, as measured by assays well- known in the art, e.g., electromobility shift assays, ELISAs and other immunoassays.
  • the IL-15/IL-15Ra complex retains the ability to specifically bind to the ⁇ chain.
  • the IL-15/IL-15Ra complex is isolated from a cell.
  • complexes that bind to the ⁇ subunits of the IL-15 receptor, induce IL-15 signal transduction (e.g., Jak/Stat signal transduction) and enhance IL-15 -mediated immune function, wherein the complexes comprise IL-15 covalently or noncovalently bound to interleukin-15 receptor alpha ("IL-15Ra") (a "IL-15/IL-15Ra complex").
  • IL-15Ra interleukin-15 receptor alpha
  • the IL-15/IL-15Ra complex is able to bind to the ⁇ receptor complex.
  • the IL-15/IL-15Ra complexes may be composed of wild-type IL-15 or an IL-15 derivative and wild-type IL-15Ra or an IL-15Ra derivative.
  • an IL-15/IL-15Ra complex comprises IL-15 or an IL-15 derivative and an IL-15Ra described above.
  • an IL-15/IL-15Ra complex comprises IL-15 or an IL-15 derivative and IL-15Ra with the amino acid sequence of SEQ ID NO: 260 in Table 1.
  • an IL-15/IL-15Ra complex comprises IL-15 or an IL-15 derivative and a glycosylated form of IL-15Ra described supra.
  • an IL-15/IL-15Ra complex comprises wild-type IL-15 or an IL-15Ra derivative and soluble IL-15Ra (e.g., wild-type soluble human IL-15Ra).
  • an IL-15/IL-15Ra complex is composed of an IL-15 derivative and an IL-15Ra derivative.
  • an IL-15/IL-15Ra complex is composed of wild-type IL-15 and an IL-15Ra derivative.
  • the IL-15Ra derivative is a soluble form of IL-15Ra. Specific examples of soluble forms of IL-15Ra are described above.
  • the soluble form of IL-15Ra lacks the transmembrane domain of wild-type IL-15Ra, and optionally, the intracellular domain of wild- type IL-15Ra.
  • the IL-15Ra derivative is the extracellular domain of wild-type IL-15Ra or a fragment thereof. In certain embodiments, the IL-15Ra derivative is a fragment of the extracellular domain comprising the sushi domain or exon 2 of wild-type IL-15Ra.
  • the IL-15Ra derivative comprises a fragment of the extracellular domain comprising the sushi domain or exon 2 of wild-type IL-15Ra and at least one amino acid that is encoded by exon 3.
  • the IL-15Ra derivative comprises a fragment of the extracellular domain comprising the sushi domain or exon 2 of wild-type IL-15Ra and an IL-15Ra hinge region or a fragment thereof.
  • the IL-15Ra comprises the amino acid sequence of SEQ ID NO: 260 in Table 1.
  • the IL-15Ra derivative comprises a mutation in the extracellular domain cleavage site that inhibits cleavage by an endogenous protease that cleaves wild-type IL-15Ra.
  • the extracellular domain cleavage site of IL-15Ra is replaced with a cleavage site that is recognized and cleaved by a heterologous known protease.
  • Non-limiting examples of such heterologous protease cleavage sites include Arg-X-X-Arg (SEQ ID NO: 275 in Table 1), which is recognized and cleaved by furin protease; and A-B-Pro-Arg-X-Y (SEQ ID NO: 276 in Table 1) (A and B are hydrophobic amino acids and X and Y are non-acidic amino acids) and Gly-Arg-Gly, which are recognized and cleaved by thrombin protease.
  • the IL-15 is encoded by a nucleic acid sequence optimized to enhance expression of IL-15, e.g., using methods as described in PCT Publications WO 2007/084342 and WO 2010/020047; and U.S. Patent Nos. 5,965,726; 6, 174,666; 6,291,664; 6,414,132; and 6,794,498.
  • an IL-15/IL-15Ra complex comprising human IL- 15Ra which is glycosylated at one, two, three, four, five, six, seven, or all, of the glycosylation sites as described supra and with reference to SEQ ID NOs: 272, 273 and 274 in Table 1.
  • the glycosylated IL-15Ra is a wild-type human IL-15Ra.
  • the glycosylated IL-15Ra is an IL-15Ra derivative of human IL-15Ra.
  • the glycosylated IL-15Ra is a wild-type soluble human IL-15Ra, such as SEQ ID NO: 257 or 260 in Table 1.
  • the glycosylated IL-15Ra is an IL-15Ra derivative that is a soluble form of human IL-15Ra.
  • the IL-15/IL-15Ra complex is purified or isolated.
  • the IL-15/IL-15Ra complexes may comprise a heterologous molecule.
  • the heterologous molecule increases protein stability.
  • Non-limiting examples of such molecules include polyethylene glycol (PEG), Fc domain of an IgG immunoglobulin or a fragment thereof, or albumin that increase the half-life of IL-15 or IL-15Ra in vivo.
  • IL-15Ra is conjugated/fused to the Fc domain of an immunoglobulin (e.g., an IgGl) or a fragment thereof.
  • the IL-15RaFc fusion protein comprises the amino acid sequence of SEQ ID NO: 277 or 278 in Table 1.
  • the IL-15RaFc fusion protein is the IL-15Ra/Fc fusion protein described in Han et al, (2011), Cytokine 56: 804-810, U.S. Patent No. 8,507,222 or U.S. Patent No. 8, 124,084.
  • the heterologous molecule may be conjugated to IL-15 and/or IL-15Ra.
  • the heterologous molecule is conjugated to IL-15Ra.
  • the heterologous molecule is conjugated to IL-15.
  • the components of an IL-15/IL-15Ra complex may be directly fused, using either non-covalent bonds or covalent bonds (e.g., by combining amino acid sequences via peptide bonds), and/or may be combined using one or more linkers.
  • Linkers suitable for preparing the IL-15/IL-15Ra complexes comprise peptides, alkyl groups, chemically substituted alkyl groups, polymers, or any other covalently- bonded or non-covalently bonded chemical substance capable of binding together two or more components.
  • Polymer linkers comprise any polymers known in the art, including polyethylene glycol (PEG).
  • the linker is a peptide that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids long. In a specific embodiment, the linker is long enough to preserve the ability of IL-15 to bind to the IL-15Ra. In other embodiments, the linker is long enough to preserve the ability of the IL-15/IL-15Ra complex to bind to the ⁇ receptor complex and to act as an agonist to mediate IL-15 signal transduction.
  • IL-15/IL-15Ra complexes are pre-coupled prior to use in the methods described herein (e.g., prior to contacting cells with the IL-15/IL-15Ra complexes or prior to
  • the IL-15/IL-15Ra complexes are not pre-coupled prior to use in the methods described herein.
  • an IL-15/IL-15Ra complex enhances or induces immune function in a subject by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to the immune function in a subject not administered the IL- 15/IL-15Ra complex using assays well known in the art, e.g., ELISPOT, ELISA, and cell proliferation assays.
  • assays well known in the art, e.g., ELISPOT, ELISA, and cell proliferation assays.
  • the immune function is cytokine release (e.g., interferon-gamma, IL-2, IL-5, IL-10, IL-12, or transforming growth factor (TGF) -beta).
  • the IL-15 mediated immune function is NK cell proliferation, which can be assayed, e.g., by flow cytometry to detect the number of cells expressing markers of NK cells (e.g., CD56).
  • the IL-15 mediated immune function is antibody production, which can be assayed, e.g., by ELISA.
  • the IL-15 mediated immune function is effector function, which can be assayed, e.g., by a cytotoxicity assay or other assays well known in the art.
  • Non-viral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., (1997) Nat Genet. 15:345).
  • nonviral vectors useful for expression of polypeptides in mammalian (e.g., human) cells include pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C, (Invitrogen, San Diego, Calif), MPSV vectors, and numerous other vectors known in the art for expressing other proteins.
  • Useful viral vectors include vectors based on retroviruses, adenoviruses, adenoassociated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al, supra; Smith, (1995) Annu. Rev. Microbiol. 49:807; and Rosenfeld et al., (1992) Cell 68: 143.
  • the choice of expression vector depends on the intended host cells in which the vector is to be expressed.
  • the expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) that are operably linked to the polynucleotides.
  • an inducible promoter is employed to prevent expression of inserted sequences except under inducing conditions.
  • Inducible promoters include, e.g., arabinose, lacZ, metallothionein promoter or a heat shock promoter. Cultures of transformed organisms can be expanded under noninducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells.
  • promoters other regulatory elements may also be required or desired for efficient expression.
  • These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences.
  • the efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf et al., (1994) Results Probl. Cell Differ. 20: 125; and Bittner et al., (1987) Meth. Enzymol., 153:516).
  • the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.
  • the expression vectors may also provide a secretion signal sequence position to form a fusion protein with polypeptides encoded by inserted CD32b-binding antibody sequences or IL-15 or IL-15Ra sequences. More often, the inserted sequences are linked to a signal sequences before inclusion in the vector.
  • Vectors to be used to receive sequences encoding CD32b-binding antibody light and heavy chain variable domains sometimes also encode constant regions or parts thereof. Such vectors allow expression of the variable regions as fusion proteins with the constant regions thereby leading to production of intact antibodies and antigen-binding fragments thereof. Typically, such constant regions are human.
  • the host cells for harboring and expressing the CD32b-binding antibody chains and the IL-15 and IL-15Ra proteins can be either prokaryotic or eukaryotic.
  • E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present invention.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • bacilli such as Bacillus subtilis
  • enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species.
  • expression vectors which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.
  • Other microbes, such as yeast can also be employed to express polypeptides of the invention. Insect cells in combination with baculovirus vectors can also be used.
  • mammalian host cells are used to express and produce the polypeptides of the present invention.
  • they can be either a hybridoma cell line expressing endogenous immunoglobulin genes or a mammalian cell line harboring an exogenous expression vector.
  • These include any normal mortal or normal or abnormal immortal animal or human cell.
  • a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed including the CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B-cells and hybridomas.
  • mammalian cell lines include, but are not limited to, COS, CHO, HeLa, NIH3T3, HepG2, MCF7, HEK 293, HEK 293T, RD, PC 12, hybridomas, pre-B cells, 293, 293H, K562, SkBr3, BT474, A204, M07Sb, ⁇ , Raji, Jurkat, MOLT-4, CTLL-2, MC-IXC, SK-N-MC, SK-N-MC, SK-N-DZ, SH-SY5Y, C127, NO, and BE(2)-C cells.
  • Other mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). The use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, FROM GENES TO CLONES, VCH Publishers, N.Y., N.Y., 1987.
  • Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen, et al, (1986) Immunol. Rev. 89:49-68,), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • expression control sequences such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen, et al, (1986) Immunol. Rev. 89:49-68,), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type -specific, stage -specific, and/or modulatable or regulatable
  • Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP poIIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate -early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.
  • Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts. (See generally Sambrook et al., supra).
  • Other methods include, e.g., electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion to the herpes virus structural protein VP22 (Elliot & O'Hare, (1997) Cell 88:223), agent- enhanced uptake of DNA, and ex vivo transduction.
  • stable cell lines can be generated.
  • cell lines can be transformed using the nucleic acid constructs described herein which may contain a selectable marker gene on the same or on a separate nucleic acid construct.
  • the selectable marker gene can be introduced into the same cell by co-transfection. Following the introduction of the vector, cells are allowed to grow for 1-2 days in an enriched media before they are switched to selective media to allow growth and recovery of cells that successfully express the introduced nucleic acids. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques well known in the art that are appropriate to the cell type.
  • the cell line has been adapted to grow in serum- free medium. In one embodiment, the cell line has been adapted to grow in serum-free medium in shaker flasks. In one embodiment, the cell line has been adapted to grow in stir or rotating flasks. In certain embodiments, the cell line is cultured in suspension. In particular embodiments, the cell line is not adherent or has been adapted to grow as nonadherent cells. In certain embodiments, the cell line has been adapted to grow in low calcium conditions. In some embodiments, the cell line is cultured or adapted to grow in low serum medium.
  • a particularly preferred method of high-yield production of a recombinant polypeptide of the present invention is through the use of dihydro folate reductase (DHFR) amplification in DHFR-deficient CHO cells, by the use of successively increasing levels of methotrexate as described in U.S. Patent No. 4,889,803.
  • DHFR dihydro folate reductase
  • the polypeptide obtained from such cells may be in a glycosylated form.
  • cell lines are engineered to express the stable heterodimer of wild-type human IL-15 and wild-type soluble human IL-15Ra, which can then be purified, and administered to a human.
  • the stability of the IL-15/IL-15Ra heterodimer is increased when produced from cell lines recombinantly expressing both IL-15 and IL-15Ra.
  • the host cell recombinant expresses IL-15 and the full length IL- 15Ra. In another specific embodiment, the host cell recombinantly expresses IL-15 and the soluble form of IL-15Ra. In another specific embodiment, the host cell recombinantly expresses IL-15 and a membrane -bound form of IL-15Ra which is not cleaved from the surface of the cell and remains cell associated. In some embodiments, the host cell recombinantly expressing IL-15 and/or IL-15Ra (full- length or soluble form) also recombinantly expresses another polypeptide (e.g., a cytokine or fragment thereof).
  • another polypeptide e.g., a cytokine or fragment thereof.
  • such a host cell recombinantly expresses an IL-15 polypeptide in addition to an IL-15Ra polypeptide.
  • the nucleic acids encoding IL-15 and/or IL-15Ra can be used to generate mammalian cells that recombinantly express IL-15 and IL-15Ra in high amounts for the isolation and purification of IL-15 and IL-15Ra, preferably the IL-15 and the IL-15Ra are associated as complexes.
  • high amounts of IL-15/IL-15Ra complexes refer to amounts of IL-15/IL- 15Ra complexes expressed by cells that are at least 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 20 fold, or more than 20 fold higher than amounts of IL-15/IL-15Ra complexes expressed endogenously by control cells (e.g., cells that have not been genetically engineered to recombinantly express IL-15, IL-15Ra, or both IL-15 and IL-15Ra, or cells comprising an empty vector).
  • control cells e.g., cells that have not been genetically engineered to recombinantly express IL-15, IL-15Ra, or both IL-15 and IL-15Ra, or cells comprising an empty vector.
  • a host cell described herein expresses approximately 0.1 pg to 25 pg, 0.1 pg to 20 pg, 0.1 pg to 15 pg, 0.1 pg to 10 pg, 0.1 pg to 5 pg, 0.1 pg to 2 pg, 2 pg to 10 pg, or 5 to 20 pg of IL-15 as measured by a technique known to one of skill in the art (e.g., an ELISA).
  • a technique known to one of skill in the art e.g., an ELISA
  • a host cell described herein expresses approximately 0.1 to 0.25 pg per day, 0.25 to 0.5 pg per day, 0.5 to 1 pg per day, 1 to 2 pg per day, 2 to 5 pg per day, or 5 to 10 pg per day of IL-15 as measured by a technique known to one of skill in the art (e.g., an ELISA).
  • the IL-15Ra is the soluble form of IL-15Ra.
  • the IL-15Ra is the soluble form of IL-15Ra associated with IL-15 in a stable heterodimer, which increases yields and simplifies production and purification of bioactive heterodimer IL-15/soluble IL-15Ra cytokine.
  • Recombinant IL-15 and IL-15Ra and an anti-CD32b antibody molecule can be purified using methods of recombinant protein production and purification are well known in the art, e.g., see PCT Publication WO 2007/070488. Briefly, the polypeptide can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. Cell lysate or supernatant comprising the polypeptide can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography.
  • IL-15 and IL-15Ra are synthesized or recombinantly expressed by different cells and subsequently isolated and combined to form an IL-15/IL-15Ra complex, in vitro, prior to administration to a subject. In other embodiments, IL-15 and IL-15Ra are synthesized or
  • IL-15 and IL-15Ra are synthesized or expressed together by the same cell, and the IL-15/IL-15Ra complex formed is isolated.
  • the present invention provides methods of treating a disease or disorder associated with increased CD32b activity or expression by administering to a subject in need thereof an effective amount of an anti-CD32b antibody molecule in combination with an IL-15/IL-15Ra complex.
  • the present invention provides a method of treating indications including, but not limited to, B cell malignancies including Hodgkins lymphoma, Non-Hodgkins lymphoma, multiple myeloma, diffuse large B cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, small lymphocytic lymphoma, diffuse small cleaved cell lymphoma, MALT lymphoma, mantel cell lymphoma, marginal zone lymphoma and follicular lymphoma as well as other diseases and conditions including systemic light chain amyloidosis, haematological malignancies and other solid tumors.
  • the present invention provides methods of treating a CD32b-related disease or disorder by administering to a subject in need thereof an effective amount of an anti-CD32b antibody molecule in combination with an IL-15/IL-15Ra complex.
  • CD32b related diseases or disorders for which the disclosed combination may be useful include but are not limited to: B cell malignancies including Hodgkins lymphoma, Non-Hodgkins lymphoma, multiple myeloma, diffuse large B cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, small lymphocytic lymphoma, diffuse small cleaved cell lymphoma, MALT lymphoma, mantel cell lymphoma, marginal zone lymphoma and follicular lymphoma as well as other diseases and conditions including systemic light chain amyloidosis, haematological malignancies and other solid tumors.
  • an anti-CD32b antibody molecule with an IL-15/IL-15Ra complex can be used, inter alia, to treat, prevent, delay or reverse disease progression of patients who have become resistant or refractory to antibody treatment.
  • an anti-CD32b antibody molecule with an IL-15/IL-15Ra complex can be used, inter alia, to treat, prevent, delay or reverse disease progression of patients who have become resistant or refractory to antibody treatment.
  • the efficacy of the ADCC effect of the antibody may be enhanced, in full or in part.
  • the combination of an anti-CD32b antibody molecule with an IL-15/IL- 15Ra complex described herein can be administered to a patient in need thereof in conjunction with another therapeutic agent as discussed below.
  • the combination of the present invention can be co-formulated with, and/or co-administered with, one or more additional therapeutic agents, e.g., one or more anti-cancer agents, cytotoxic or cytostatic agents, hormone treatment, vaccines, and/or other immunotherapies.
  • the combination can be administered with other therapeutic treatment modalities, including surgery, radiation, cryosurgery, and/or thermotherapy.
  • Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.
  • therapies utilizing the combination of the present invention may be administered in conjunction with multiple classes of the agents described above.
  • the combination of the present invention is administered together with another agent or agents, the two (or more) can be administered sequentially in any order, or simultaneously.
  • the combination of the present invention is administered to a subject who is also receiving therapy with one or more other agents or methods.
  • the combination is administered in conjunction with surgical treatments.
  • the therapy regimen may be additive, or it may produce synergistic results.
  • the invention provides pharmaceutical compositions comprising the combination of an anti- CD32b antibody molecule with an IL-15/IL-15Ra complex formulated together or separately with a pharmaceutically acceptable carrier.
  • the anti-CD32b antibody molecule and the IL-15/IL-15Ra complex can be administered to a patient as a "non-fixed combination" meaning that the anti-CD32b antibody molecule and IL-15/IL-15Ra complex are administered as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two agents in the body of the patient.
  • the term "non-fixed combination” thus defines especially a "kit of parts" in the sense that the combination agents (i) an anti- CD32b antibody molecule and (ii) an IL-15/IL-15Ra complex as defined herein can be dosed
  • the combination agents may also be used as entirely separate pharmaceutical dosage forms or pharmaceutical formulations that are also sold independently of each other and where instructions for the possibility of their combined use is or are provided in the package equipment, e.g. leaflet or the like, or in other information e.g. provided to physicians and medical staff.
  • the independent formulations or the parts of the kit of parts can then, e.g. be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts.
  • the time intervals are chosen such that the effect on the treated disease in the combined use of the parts is larger than the effect which would be obtained by use of only any one of the combination agents (i) and (ii), thus being jointly active.
  • the ratio of the total amounts of the combination agent (i) to the combination agent (ii) to be administered in the combined preparation can be varied, e.g. in order to cope with the needs of a patient sub-population to be treated or the needs of the single patient which different needs can be due to age, sex, body weight, etc. of the patients.
  • a pharmaceutical composition of the present invention can additionally contain one or more other therapeutic agents that are suitable for treating or preventing a CD32b-associated disease (e.g., B cell malignancies including Hodgkins lymphoma, Non-Hodgkins lymphoma, multiple myeloma, diffuse large
  • a CD32b-associated disease e.g., B cell malignancies including Hodgkins lymphoma, Non-Hodgkins lymphoma, multiple myeloma, diffuse large
  • B cell lymphoma acute lymphocytic leukemia, chronic lymphocytic leukemia, small lymphocytic lymphoma, diffuse small cleaved cell lymphoma, MALT lymphoma, mantel cell lymphoma, marginal zone lymphoma and follicular lymphoma as well as other diseases and conditions including systemic light chain amyloidosis, haematological malignancies and other solid tumors).
  • a pharmaceutical composition of the present invention can be administered with a
  • compositions to enhance or stabilize the composition, or facilitate preparation of the composition.
  • Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • a pharmaceutical composition of the present invention can be administered by a variety of methods known in the art.
  • the route and/or mode of administration may vary depending upon the desired results. Administration can be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target.
  • the pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound i.e., anti-CD32b antibody molecule and/or IL-15/IL-15Ra complex, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
  • the composition should be sterile and fluid. Fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • compositions of the invention can be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., (2000); and Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, (1978). Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or efficacious dose of the CD32b-binding antibody molecule and the IL-15/IL-15Ra complex, of the combination, is employed in pharmaceutical compositions of the invention.
  • the anti-CD32b antibody molecules and IL-15/IL-15Ra complex are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, for each component of the combination, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the requirements of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular components of the combination of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.
  • a physician can start doses of the combination of the invention employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • effective doses of the compositions of the present invention for the treatment of an disorder described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages need to be titrated to optimize safety and efficacy.
  • the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 15 mg/kg, of the host body weight.
  • An exemplary treatment regime entails systemic administration once every two weeks or once a month or once every 3 to 6 months.
  • the dose ranges from about 0.25 to 8 ⁇ g/kg/day.
  • An exemplary treatment regime entails subcutaneous administration in a treatment cycle of three times a week for two weeks, followed by a two week break before a repeat of the treatment cycle.
  • the antibody and/or IL-15/IL-15Ra complex may be administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of CD32b-binding antibody and/or IL-15 in the patient.
  • dosage is adjusted to achieve a plasma antibody concentration of 1-1000 ⁇ g/ml and in some methods 25-500 ⁇ g/ml.
  • dosage is escalated until an y adverse events or dose limiting toxicity are observed.
  • the components of the combination can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody and complex in the patient. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
  • EXAMPLE 1 Target cell lysis mediated by an anti-CD32b antibody in combination with an IL- 15/IL-15Ra complex
  • NK cells are among the most potent effector cells for the ADCC activity of anti-CD32b antibodies. It is believed that the IL-15/IL-15Ra complex "hetIL-15" can augment their ADCC activity by: 1) increasing effectortarget cell ratio as a result of NK cell proliferation; and 2) enhancing effector cell function, such as release of perforin and granzyme, as well as secretion of IFNy.
  • an ADCC assay to measure target cell lysis mediated by the anti- CD32b human IgGl (NOV2108) in combination with hetIL-15 was performed as detailed below.
  • PBMCs were isolated from a Leukopak (HemaCare catalog# PB001F-3) via a ficoll gradient. Primary NK cells were then negatively selected from PBMCs using Miltenyi beads (catalog# 130-050- 101). Isolated NK cells were incubated with lOOpg/ml of rhIL-2 overnight (Peprotech, #200-02) and then used in the ADCC assay at an appropriate E:T ratio. Luciferized Daudi cells were used as target cells and primary NK cells were used as effector cells; and cells were co-cultured for 4 hr either in the absence or presence of hetIL-15. Following the co-incubation, Bright Glo (Promega, catalog# E2620; 60 ⁇ ) was added to all wells, with the exception of the appropriate control wells and the luminescence signal was subsequently measured on an Envision (Perkin Elmer).
  • Bright Glo Promega, catalog# E2620; 60 ⁇
  • NOV2108 demonstrated Fc- and dose- dependent ADCC activity against Daudi cells.
  • Addition of hetIL-15 enhanced the potency of both the Fc wild type and Fc -enhanced NOV2108 (afucosylated) in the ADCC assay ( Figure 1).
  • the ADCC enhancement by hetIL-15 was evident at concentrations as low as 0.01 and 0.1 ng/ml, and at lng/ml similar ADCC activity as with 0.1 ng/ml hetIL15 was observed.
  • No effect on ADCC activity against Daudi cells was observed with an Fc silent version of the NOV2108 antibody having the modification N297A, with or without the addition of hetIL-15.
  • the EC50 of each antibody in two experiments with different donor NK cells is shown in Table 2, where the increase in potency was reflected by the lower EC50 values.
  • Table 2 EC50 values of anti-CD32b antibody NOV2108-mediated ADCC activity against Daudi in combination with hetIL-15
  • EXAMPLE 2 Assay for IFNy expression mediated by an anti-CD32b antibody in combination with an IL-15/IL-15Ra complex
  • an IL-15/IL-15Ra complex (hetIL-15) can be included in an NK cell killing assay.
  • NK cells can be pre-treated with hetIL-15, and cultured with Daudi cells and anti- CD32b antibody, and the resulting level of IFNy measured.
  • NK cells can be cultured with Daudi cells, hetIL15 and anti-CD32b, and the resulting level of IFNy measured.
  • Microplates containing the capture antibody can be first prepared according to the following method.
  • the Capture Antibody (R&D Systems, Cat. no: 840101) is diluted to a working concentration in PBS without carrier protein and used immediately to coat a 96-well microplate (R&D Systems, Cat. No: DY990) with ⁇ per well of the diluted Capture Antibody.
  • the plate is then sealed and incubated overnight at room temperature. After incubation, each well is aspirated and washed with wash buffer twice (0.05% Tween ® 20 in PBS, pH 7.2-7.4), for a total of three washes.
  • the plates are then blocked by adding 300 ⁇ of block buffer (1% BSA in PBS, pH 7.2-7.4, 0.2 um filtered) to each well and incubated at room temperature for a minimum of 1 hour.
  • block buffer 1% BSA in PBS, pH 7.2-7.4, 0.2 um filtered
  • sample or standard (R&D Systems, Cat. No: 840103) in Reagent Diluent (0.1% BSA, 0.05% Tween 20 in Tris-buffered Saline ) or another appropriate diluent, is added to each well, incubated for 2 hours and then aspirated/washed as described above.
  • Reagent Diluent 0.1% BSA, 0.05% Tween 20 in Tris-buffered Saline
  • Detection Antibody (R&D Systems, Cat. No: 840102), diluted in Reagent Diluent with NGS, is then added to each well, incubated for 2 hours and then aspirated/washed as described above.
  • ⁇ of a working dilution of Streptavidin-HRP (R&D Systems, Cat. No: 893975) is then added to each well, incubated for 20 min at room temperature and then aspirated/washed as described above.
  • Substrate Solution (1 : 1 mixture of Color Reagent A (H202) and Color Reagent B (Tetramethylbenzidine) (R&D Systems, Cat. No: DY999) is then added to each well and the plate incubated for 20 min at room temperature. 50ul of Stop Solution (2 N H 2 SO 4 ) is then added to each well, tapping the plate to mix thoroughly.
  • the optical density of each well should be determined immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate as readings made directly at 450 nm without correction may be higher and less accurate.
  • GQPKAAPSVTLFPPSSEELQANKATLVCLI SDFYPGAVTVAWKADSSPVKAGVE TTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
  • GQPKAAPSVTLFPPSSEELQANKATLVCLI SDFYPGAVTVAWKADSSPVKAGV ETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
  • PSGIPERFSGSNSGNTATLTISRAQAGDEADYYCSSWDSSMDSWFGGGTKLT VL 180 DNA VL AGCTACGAGCTGACTCAGCCCCTGAGCGTCAGCGTGGCCCTGGGTCAGACCGC
  • IL-15 (with MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEAJV!VV signal ISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDAS peptide) aa IHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFI human NTS
  • GMCSF ctcgctcgag tcgggggacg cgtcgatcca cgacacggtg
  • I IL-15 codon cctggccatt gcatacgttg tatccatatc ataatatgta
  • gacgctagca agaaatggcc ccgaggcggg cgcgaggctg

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Abstract

La présente invention concerne une composition pharmaceutique comprenant une molécule d'anticorps anti-CD32b en combinaison avec un complexe IL-15/IL-15Ra. Ladite combinaison peut être administrée indépendamment ou séparément, dans une quantité qui est efficace sur le plan thérapeutique pour le traitement du cancer. L'invention concerne également l'utilisation d'une telle combinaison pour la fabrication d'un médicament ; l'utilisation d'une telle combinaison en tant que médicament ; un kit d'éléments comprenant une telle combinaison ; et un procédé de traitement d'une telle combinaison.
PCT/IB2018/054369 2017-06-16 2018-06-14 Polythérapie pour le traitement du cancer WO2018229706A1 (fr)

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