WO2010117448A2 - Chimeric immunocytokines and methods of use thereof - Google Patents

Chimeric immunocytokines and methods of use thereof Download PDF

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Publication number
WO2010117448A2
WO2010117448A2 PCT/US2010/001034 US2010001034W WO2010117448A2 WO 2010117448 A2 WO2010117448 A2 WO 2010117448A2 US 2010001034 W US2010001034 W US 2010001034W WO 2010117448 A2 WO2010117448 A2 WO 2010117448A2
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immunocytokine
dog
heavy chain
sequence
seq
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PCT/US2010/001034
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French (fr)
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WO2010117448A3 (en
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Stephen D. Gillies
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Provenance Biopharmaceuticals Corp.
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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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3084—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated gangliosides
    • 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
    • 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]
    • C07K14/55—IL-2
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3053—Skin, nerves, brain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • 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
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to therapeutic antibodies and antibody-cytokine fusion proteins, and methods for their preparation and use.
  • Immunocytokines (antibody-cytokine fusion proteins) were first reported in the literature in the early 1990s and consisted of whole antibody fusions with cytokines such as lymphotoxin (TNF- ⁇ ) or interleukin 2 (IL-2). Subsequent studies in GD2-expressing tumor models in mice indicated that the chl4.18 antibody and chl4.18-IL2 immunocytokine both had anti-tumor activity but that the immunocytokine was far more potent than the antibody, even when combined with free IL-2.
  • cytokines such as lymphotoxin (TNF- ⁇ ) or interleukin 2 (IL-2).
  • TNF- ⁇ lymphotoxin
  • IL-2 interleukin 2
  • a related humanized immunocytokine hul4.18-IL2
  • Many publications describe the ability of this molecule to activate several components of the immune system to kill tumor cells and to evoke a long lasting CD8 T cell memory response that resists subsequent tumor challenge.
  • aspects of the invention relate to recombinant antibodies containing one or more non- human and/or non-rodent (e.g. , canine, feline) antibody sequences.
  • Recombinant antibodies of the invention are useful for administering to non-human and/or non-rodent animals (e.g., for targeted cytokine therapy, for targeted radiotherapy, for targeted imaging, or for any combination thereof).
  • recombinant antibodies are provide that have improved ADCC effector function.
  • antibodies are provided that have improved protein A binding.
  • antibodies are provided that have improved Fc-receptor binding.
  • antibodies are provided that bind specifically with non-human and/or non-rodent antigens.
  • antibodies are provided that bind specifically with non-proteinaceous antigens, e.g., DNA, polysaccharide.
  • immunocytokines are provided that comprise a non-human immunoglobulin gamma heavy chain.
  • non-human immunoglobulin gamma heavy chain refers to a gamma heavy chain comprising an amino acid sequence that is unique to a non-human animal compared with the sequence of a gamma heavy chain of a human animal.
  • a non-human immunoglobulin gamma heavy chain is an immunoglobulin gamma heavy chain of a non- human animal, or a functional fragment thereof.
  • a non-human immunoglobulin gamma heavy chain may be a chimeric immunoglobulin gamma heavy chain comprising amino acid sequences corresponding to (derived from) more than one animal, at least one of which is a non-human animal.
  • the immunocytokines comprise a non- human cytokine.
  • a "non-human cytokine” is a cytokine of a non-human animal.
  • a "non-human, non-rodent cytokine” is a cytokine of an animal that is not a human and not a rodent.
  • Non-limiting examples of a non-human, non-rodent cytokine include a cytokine of a dog, a cytokine of a cat, a cytokine of a non-human primate, etc.
  • aspects of the invention are based in part on the discovery of certain motifs in immunoglobulin heavy chain constant region that mediate Fc-Receptor binding and ADCC effector function.
  • a CPX- motif bridges the hinge and CH2 domain of the immunoglobulin.
  • the CPX-motif has a sequence Of CPXiPX 2 X 3 X 4 X 5 LGGPSX 6 X 7 (SEQ ID NO: 36).
  • the CPX-motif mediates Fc-Receptor binding and ADCC effector function.
  • a GKX-motif is in the CH2 domain of the immunoglobulin.
  • the GKX-motif has a sequence of GKX 8 FX 9 CX 10 V (SEQ ID NO: 39).
  • the GKX-motif mediates Fc- Receptor binding and ADCC effector function.
  • an immunoglobulin heavy chain that has a GKX-motif that has a negatively charged amino acid (e.g., glutamate, aspartate) at its X 8 position binds to Fc-Receptor and activates ADCC.
  • an immunoglobulin heavy chain that has a combination of a CPX-motif that does not have proline at its X 5 position and a GKX-motif that has a negatively charged amino acid (e.g., glutamate) at its X 8 position binds to Fc-Receptor and activates ADCC.
  • an immunoglobulin heavy chain (e.g., a non-human and/or non-rodent immunoglobulin heavy chain) does not have proline at the amino acid position corresponding to amino acid position 116 of the human immunoglobulin gamma- 1 heavy chain constant region (amino acid numbering according to GenBank Accession Number: AAC82527).
  • an immunoglobulin heavy chain has a hydrophobic amino acid (e.g., leucine, isoleucine, etc.) at the amino acid position corresponding to amino acid position 116 of the human immunoglobulin gamma- 1 heavy chain constant region (amino acid numbering according to GenBank Accession Number: AAC82527).
  • an immunoglobulin heavy chain has a negatively charged amino acid (e.g., glutamate, aspartate) at the amino acid position corresponding to amino acid position 200 of the human immunoglobulin gamma- 1 heavy chain constant region (amino acid numbering according to GenBank Accession Number: AAC82527).
  • a negatively charged amino acid e.g., glutamate, aspartate
  • an immunoglobulin heavy chain does not have proline at the amino acid position corresponding to amino acid position 116 of the human immunoglobulin gamma- 1 heavy chain constant region and has a negatively charged amino acid (e.g., glutamate, aspartate) at the amino acid position corresponding to amino acid position 200 of the human immunoglobulin gamma- 1 heavy chain constant region (amino acid numbering according to GenBank Accession Number: AAC82527).
  • a negatively charged amino acid e.g., glutamate, aspartate
  • canine IgG-A has a CPX-motif having a proline at position X 5 that is undesirable for inducing ADCC-function.
  • canine IgG-B and IgG-C have a GKX-motif having a glutamine at position X 8 that is undesirable for inducing ADCC-function.
  • recombinant antibodies e.g., recombinant dog IgG antibodies
  • have improved ADCC effector function are provided that have improved ADCC effector function.
  • recombinant antibodies that induce ADCC-function have a CPX-motif of canine IgG-B, IgG-C or IgG-D and a GKX-motif of canine IgG-A or IgG-D.
  • recombinant antibodies that induce ADCC-function have a CPX-motif of canine IgG-B and a GKX-motif of canine IgG-A.
  • recombinant antibodies that induce ADCC-function have a CPX-motif of canine IgG-C and a GKX-motif of canine IgG-A.
  • recombinant antibodies that induce ADCC-function have a CPX-motif of canine IgG-D and a GKX-motif of canine IgG-A. In some embodiments, recombinant antibodies that induce ADCC-function have a CPX-motif of canine IgG-B and a GKX-motif of canine IgG-D. In some embodiments, recombinant antibodies that induce ADCC-function have a CPX-motif of canine IgG-C and a GKX-motif of canine IgG-D.
  • recombinant antibodies that induce ADCC-function have a CPX-motif of canine IgG-D and a GKX-motif of canine IgG-D.
  • recombinant antibodies may be synthesized by fusing regions from different immunoglobulin proteins.
  • fusion may be made in a region of sequence identity to avoid generating new sequences at the fusion junction.
  • recombinant antibodies may be synthesized by mutating one or more residues of a immunoglobulin protein.
  • recombinant IgG may be produced by mutating the proline at position 1 15 of canine IgG-A (See figure 22 A for amino acid numbering) to a hydrophobic amino acid (e.g., leucine).
  • recombinant IgG may be produced by mutating the glutamine at position 204 of canine IgG-B (See figure 22A for amino acid numbering) to a negatively charged amino acid (e.g., glutamate).
  • recombinant IgG may be produced by mutating the glutamine at position 202 of canine IgG-C (See figure 22 A for amino acid numbering) to a negatively charged amino acid (e.g., glutamate).
  • immunocytokines are provided that comprise the foregoing recombinant antibodies.
  • the immunoglobulin gamma heavy chain (e.g., non-human immunoglobulin heavy chain) comprises a CPX-motif that does not have a sequence of CPVPEPLGGPSVL (SEQ ID NO: 32), and a cytokine (e.g., a non-human, non-rodent cytokine).
  • the immunoglobulin gamma heavy chain (e.g., non-human immunoglobulin heavy chain) comprises a CPX-motif that has a sequence Of CPX 1 PX 2 X 3 X 4 X 5 LGGPSX 6 X 7 (SEQ ID NO: 36); wherein X 1 , X 4, X 6 and X 7 are each independently any amino acid, wherein X 2 and X 3 are each independently any amino acid or absent, and wherein X 5 is any amino acid other than proline; and a cytokine (e.g., a non-human, non-rodent cytokine).
  • a cytokine e.g., a non-human, non-rodent cytokine
  • the immunoglobulin gamma heavy chain comprises a GKX-motif that does not have a sequence of GKQFTCKV (SEQ ID NO: 38), and a cytokine (e.g., a non-human, non-rodent cytokine).
  • the immunoglobulin gamma heavy chain comprises a GKX-motif that has a sequence of GKXsFXgCXi 0 V (SEQ ID NO: 39); wherein X 8 , X 9 , and X] 0 are each independently any amino acid; and a cytokine (e.g. , a non-human, non-rodent cytokine).
  • the immunoglobulin gamma heavy chain comprises a CPX-motif that has a sequence Of CPX 1 PX 2 X 3 X 4 X 5 LGGPSX 6 X 7 (SEQ ID NO: 36) and a GKX-motif that has a sequence Of GKX 8 FX 9 CX 10 V (SEQ ID NO: 39); wherein Xi, X 41 X 6, X 7, X 8, X 9 and Xj 0 are each independently any amino acid, wherein X 2 and X 3 are each independently any amino acid or absent, and wherein X 5 is any amino acid other than proline; and a cytokine (e.g., a non-human, non-rodent cytokine).
  • a cytokine e.g., a non-human, non-rodent cytokine
  • Xi is alanine, valine, cysteine or proline.
  • X 2 is glycine or absent.
  • X 3 is cysteine or absent.
  • X 4 is glycine or glutamine.
  • X 5 is leucine, methionine, or serine.
  • X 6 is isoleucine or valine.
  • X 7 is phenylalanine or leucine.
  • the non-human immunoglobulin gamma heavy chain is selected from the group consisting of canine IgB, canine IgC, and canine IgD.
  • the CPX-motif has a sequence of CP APEMLGGPS VF (SEQ ID NO: 33). In some embodiments, the CPX-motif has a sequence of CPCPGCGLLGGPSVF (SEQ ID NO: 34). In some embodiments, the CPX-motif has a sequence of CPVPESLGGPSVF (SEQ ID NO: 35). In some embodiments, the CPX-motif has a sequence of CPPPEMLGGPSIF (SEQ ID NO: 37). In some embodiments, the constant region of the immunoglobulin gamma heavy chain is a canine or a feline immunoglobulin gamma heavy chain constant region.
  • the immunoglobulin gamma heavy chain is chimeric. In some embodiments, the immunoglobulin gamma heavy chain comprises a mouse variable region and a feline or canine constant region. In certain embodiments, X 8 is aspartate or glutamate. In certain embodiments, X 8 is glutamate. In certain embodiments, X 9 is lysine or threonine. In certain embodiments, Xj 0 is lysine or arginine. In some embodiments, the immunoglobulin gamma heavy chain is a non-human immunoglobulin gamma heavy chain.
  • the immunocytokine binds specifically to a tumor antigen.
  • the tumor antigen is not a nucleic acid.
  • the tumor antigen is not a DNA molecule.
  • the tumor antigen is a non- proteinaceous tumor antigen.
  • the tumor antigen is a polysaccharide.
  • the tumor antigen is a polypeptide.
  • the tumor antigen is selected from GD2, CD20, CD 19, CSPG and EpCAM.
  • the cytokine (e.g., non-human, non-rodent cytokine) is not IL- 12.
  • the cytokine is selected from the group consisting of: IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-I l, IL-12, IL-13, IL-14, IL-15, IL-16, IL- 17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, G-CSF, GM-CSF, TNF- ⁇ , TGF- ⁇ , IFN- ⁇ , and IFN- ⁇ / ⁇ .
  • the cytokine (e.g., non-human, non-rodent cytokine) is a cytokine that induces production of natural killer cells. In some embodiments, the cytokine (e.g., non-human, non- rodent cytokine) is a cytokine that induces production of cytotoxic T-CeIIs.
  • immuncytokines are provided that bind specifically to CD20, wherein the immunocytokine comprises a dog or feline C ⁇ region.
  • the immunocytokines bind specifically to an extracellular loop of CD20.
  • the immunocytokines bind specifically to a peptide having a sequence set forth as:
  • the immunocytokines bind specifically to a peptide having a sequence set forth as:
  • the immunocytokines bind specifically to a peptide having a sequence set forth as:
  • DIFNIAICHFFKMENLNLLKSPKP YICIHTCQPESKPSEKNSLSIKYCDSIRS (SEQ ID NO: 26), wherein the peptide forms a loop between the cysteine at position 8 and the cysteine at position 27.
  • immunocytokines are provided that bind specifically to CSPG, wherein the immunocytokine comprises a dog or feline C ⁇ region.
  • the immunocytokines bind specifically to a peptide having a sequence set forth as:
  • immunocytokines are provided that bind specifically to EpCAM, wherein the immunocytokine comprises a dog or feline C ⁇ region.
  • the immunocytokines bind specifically to a peptide having a sequence set forth as:
  • methods for producing an immunoglobulin gamma heavy chain constant region.
  • the methods comprise: (a) identifying a motif (a CPX-motif) in the immunoglobulin gamma heavy chain constant region having between one mismatch and six mismatches with the sequence set forth as: CPAPELLGGPSVF (SEQ ID NO: 31); and (b) producing a mutated version of the immunoglobulin gamma heavy chain constant region having at least one fewer mismatch with the sequence set forth as: CPAPELLGGPSVF (SEQ ID NO: 31).
  • the motif is at the junction of a heavy chain hinge and a CH2 domain of the constant region.
  • the immunoglobulin gamma heavy chain constant region is a canine or a feline immunoglobulin gamma heavy chain constant region. In some embodiments, the immunoglobulin gamma heavy chain constant region is a canine immunoglobulin gamma heavy chain constant region of IgG-A, IgG-B, IgG-C or IgG-D. In some embodiments, the motif identified in (a) has a proline in place of the leucine at position 6 of SEQ ID NO: 31. In certain embodiments, the mutated version of the immunoglobulin gamma heavy chain constant region has a substitution at position 6 that is a conservative substitution of leucine.
  • the mutated version of the immunoglobulin gamma heavy chain constant region has improved Fc-receptor binding compared with the immunoglobulin gamma heavy chain constant region. In some embodiments, the mutated version of the immunoglobulin gamma heavy chain constant region has improved ADCC effector function compared with the immunoglobulin gamma heavy chain constant region. In some embodiments, the mutated version of the immunoglobulin gamma heavy chain constant region has improved protein A binding compared with the immunoglobulin gamma heavy chain constant region.
  • a recombinant antibody is provided that comprises an immunoglobulin gamma heavy chain constant region produced by the foregoing method.
  • immunocytokines are provided that comprise the foregoing recombinant antibody.
  • methods are provided for targeting a cytokine in a non-human animal.
  • the targeting methods comprise administering any one of the immunocytokines disclosed herein to a mammal.
  • methods for promoting ADCC in a non-human animal comprise administering any one of the immunocytokines disclosed herein to a mammal.
  • aspects of the invention relate to antibodies with reduced immunogenicity in a mammal (e.g., dogs, cats).
  • Recombinant antibodies can be immunogenic when administered to a mammal, particularly when they contain antibody sequences from another species.
  • Recombinant antibodies fused to cytokines can create even greater immunogenicity problems due to the presence of the cytokine.
  • aspects of the invention relate to recombinant antibodies that contain one or more dog sequences to avoid or reduce immunogenicity-associated problems when administered to dogs.
  • recombinant antibodies are immunocytokines and include one or more cytokine proteins or portions thereof.
  • Immunocytokines of the invention generally include an antibody, or an antigen binding fragment or derivative thereof, that is capable of binding specifically to a target antigen, linked to a cytokine.
  • Immunocytokines of the invention may include one or more peptide sequences that are suitable for administration to a non-human and/or non-rodent animal, e.g., dog, cat, etc.
  • one or more of the antibody, antigen binding fragment or derivative thereof, and/or cytokine contains a dog amino acid sequence or an amino acid sequence that has been modified for administration to a dog.
  • the cytokine is a dog cytokine.
  • the antibody is a dog antibody.
  • a portion of the antibody contains a dog sequence.
  • an immunocytokine of the invention includes a dog heavy chain and/or light chain constant region, or portion thereof.
  • the invention provides immunocytokines that include a dog C ⁇ region, as well as methods for preparing same and uses thereof.
  • the invention provides immunocytokines that include a dog Cj 1 region, as well as methods for preparing same and uses thereof.
  • a cytokine can be fused to the N-terminus or C-terminus of an antibody heavy chain polypeptide.
  • the C-terminus of the cytokine can be fused to the N-terminus of a heavy chain polypeptide (e.g., to the N-terminus of the heavy chain variable region).
  • the N-terminus of the cytokine can be fused to the C-terminus of a heavy chain polypeptide (e.g., to the C-terminus of the heavy chain constant region).
  • an immunocytokine of the invention can contain a cytokine at both the N-terminus and C-terminus of one or more heavy chain antibody polypeptides.
  • immunocytokines are described herein primarily in the context of antibodies having both heavy and light chains, some embodiments of immunocytokines can include single chain antibodies (e.g., scFy, scFy- Fc, or other single chain antibodies) or other antigen binding peptides instead of a heavy and/or light chain.
  • an immunocytokine may include a cytokine fused to the N- terminus and/or C-terminus of a single chain antibody or other antigen binding peptide.
  • an immunocytokine includes an scFy-Fc-cytokine fusion, wherein the Fc is from a dog or cat constant region. In some embodiments an immunocytokine includes a scF ⁇ -Fc-IL2 protein fusion, wherein the IL2 protein is a dog or cat IL2.
  • a recombinant antibody heavy chain includes a constant region of a dog IgG A, IgG B, IgG C, or IgG D.
  • a recombinant antibody light chain includes a dog kappa light chain constant region.
  • the dog kappa light chain constant region is selected to be fused to a kappa light chain variable region (e.g., from a mouse antibody or from an antibody of another species).
  • a recombinant antibody light chain includes a dog lambda light chain constant region.
  • an immunocytokine includes a dog cytokine. In some embodiments an immunocytokine includes a cytokine fused to an antibody heavy chain, wherein the immunocytokine comprises a light chain having a dog C ⁇ region.
  • an antibody domain of the invention can be fused to an imaging marker (e.g., for use in imaging applications).
  • an antibody domain is labeled or fused to a radiolabeled agent (e.g. , for use in radiotherapy).
  • the antibody domain may be labeled and/or fused to the imaging marker and/or radiolabeled agent without including a cytokine in the recombinant molecule.
  • imaging marker and/or a radiolabeled agent can be a peptide that is used for imaging or labeling.
  • the antibody domain can include one or more points mutations or a CH2 deletion to shorten the half-life of the antibody (e.g., for use in imaging, or radiotherapy).
  • an antibody or an immunocytokine is a recombinant protein that can be expressed from a recombinant gene (e.g., that includes coding sequences for the antigen binding and/or cytokine polypeptides fused in frame in the appropriate configuration and under suitable genetic control). Accordingly, embodiments of the invention relate to recombinant nucleic acids that encode an antibody or an immunocytokine. Other embodiments include host cells.
  • nucleic acids e.g., isolated nucleic acids
  • the nucleic acid coding sequences e.g., of an antibody region and/or a cytokine
  • the nucleic acids are included in vectors (e.g., plasmids) having one or more replication and/or selectable sequences (e.g., origins of replication, antibiotic markers, etc.).
  • vectors e.g., plasmids
  • selectable sequences e.g., origins of replication, antibiotic markers, etc.
  • an isolated nucleic acid molecule comprises a sequence encoding an antibody variable region that binds specifically to a tumor antigen and a sequence encoding a non-human, non-rodent cytokine.
  • the nucleic acid further comprises a sequence encoding a non-human light chain.
  • the nucleic acid further comprises a sequence encoding a non-human heavy chain.
  • a sequence of the nucleic acid is a variant of a naturally occurring sequence, wherein the variant confers reduced ribonuclease mediated degradation of an mRNA encoded by the nucleic acid.
  • a sequence of the nucleic acid is a variant of a naturally occurring sequence, wherein the variant eliminates at least one adenosine-thymidine rich (AT-rich in the DNA, AU-rich in the mRNA) sequence that is a target for a ribonuclease (e.g. ATTTA).
  • AT-rich in the DNA, AU-rich in the mRNA adenosine-thymidine rich sequence that is a target for a ribonuclease
  • at least one AT-rich sequence is in a coding region of the nucleic acid.
  • the at least one AT-rich sequence is in a non-coding region of the nucleic acid.
  • the non- human, non-rodent cytokine is selected from the group consisting of: IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-I l, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL- 32, IL-33, IL-35, G-CSF, GM-CSF, TNF- ⁇ , TGF- ⁇ , IFN- ⁇ , and IFN- ⁇ / ⁇ .
  • the non-human, non-rodent cytokine is not IL- 12.
  • an immunocytokine is a synthetic protein that is produced using synthetic chemistry techniques.
  • the different portions of the protein may be fused via one or more linker peptides.
  • the linkers are encoded by nucleic acid sequences that are located, in frame, in between the coding regions for the different immunocytokine portions.
  • the linker peptides are introduced during synthesis.
  • synthetic immunocytokines may include non-peptide linkers that connect the different portions of the protein.
  • the immunocytokine is an anti-GD2-dog IL2 immunocytokine that binds specifically to GD2, wherein the immunocytokine includes a dog C ⁇ region.
  • the dog C ⁇ region includes an amino acid sequence comprising SEQ ID NO:1.
  • the immunocytokine includes a dog C H region, wherein the dog C H region optionally can include an amino acid sequence including SEQ ID NO:2.
  • the anti-GD2-dog IL2 immunocytokine includes a heavy chain variable region and a light chain variable region from a mouse anti-GD2 antibody.
  • the heavy chain variable region includes an amino acid sequence including SEQ ID NO:3. In one embodiment, the light chain variable region includes an amino acid sequence comprising SEQ ID NO:4. In one embodiment, the heavy chain variable region includes an amino acid sequence including SEQ ID NO:3 and the light chain variable region includes an amino acid sequence including SEQ ID NO:4.
  • the anti-GD2-dog IL-2 immunocytokine includes a heavy chain polypeptide including an amino acid sequence including SEQ ID NO: 5 and a light chain polypeptide including an amino acid sequence including SEQ ID NO:7.
  • the invention in one aspect is an isolated nucleic acid molecule encoding a heavy chain polypeptide of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2, wherein the heavy chain polypeptide includes an amino acid sequence including SEQ ID NO:5.
  • the nucleic acid molecule includes the sequence SEQ ID NO:6.
  • the invention in one aspect is an isolated nucleic acid molecule encoding a light chain polypeptide of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2 and includes a dog C ⁇ region, wherein the light chain polypeptide includes an amino acid sequence including SEQ ID NO:7.
  • the nucleic acid molecule includes the sequence SEQ ID NO: 8.
  • the invention is an isolated vector including any one or more of the foregoing nucleic acid molecules.
  • the invention is a cell including a vector of the invention.
  • the invention in one aspect is a composition including an anti-GD2-dog IL-2 immunocytokine of the invention.
  • compositions are provided.
  • the pharmaceutical compositions comprise (i) a therapeutically effective amount of any of one the immunocytokines disclosed herein and (ii) a pharmaceutically acceptable carrier.
  • the methods comprise administering a pharmaceutical composition comprising a therapeutically effective amount of any one of the immunocytokines disclosed herein and a pharmaceutically acceptable carrier, wherein the immunocytokine binds specifically to an antigen that is expressed on the extracellular surface of a cell in the non-human and/or non-rodent animal.
  • the cell is a tumor cell and the antigen is a tumor antigen.
  • the cell is a B-cell and the antigen is CD20.
  • the methods comprise: administering a pharmaceutical composition comprising a therapeutically effective amount of any one of the immunocytokines disclosed herein and a pharmaceutically acceptable carrier, wherein the immunocytokine binds specifically to a tumor antigen that is expressed on the extracellular surface of a tumor cell of the cancer.
  • the methods comprise determining that a tumor antigen is expressed on the extracellular surface of a tumor cell of the cancer; and administering a pharmaceutical composition comprising a therapeutically effective amount of any one of the immunocytokines disclosed herein and a pharmaceutically acceptable carrier, wherein the immunocytokine binds specifically to the tumor antigen.
  • the methods further comprise administering an anti-cancer compound other than the immunocytokine in combination with the pharmaceutical composition.
  • the methods further comprise subjecting the non-human animal to any one of the following protocols: CHOP therapy, the Wisconsin-Madison protocol, the AMC protocol and the VELCAP protocol, in combination with administering the pharmaceutical composition.
  • the invention is a method of treating a GD2-expressing cancer in a dog.
  • the method includes the step of administering to a dog having a GD2-expressing cancer an effective amount of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2, wherein the immunocytokine comprises a dog C ⁇ region, to treat the cancer.
  • the immunocytokine comprises a dog C H region.
  • the immunocytokine includes a heavy chain variable region and a light chain variable region from a mouse anti-GD2 antibody, wherein optionally the heavy chain variable region includes an amino acid sequence including SEQ ID NO: 3 and the light chain variable region includes an amino acid sequence including SEQ ID NO:4.
  • the immunocytokine includes a heavy chain polypeptide including an amino acid sequence including SEQ ID NO: 5 and a light chain polypeptide including an amino acid sequence including SEQ ID NO:7.
  • the GD2-expressing cancer is selected from the group consisting of melanoma, osteosarcoma, neuroblastoma, and small cell lung cancer.
  • the GD2-expressing cancer is melanoma.
  • the GD2-expressing cancer is osteosarcoma.
  • antibodies or immunocytokines may be used for human administration and/or therapy.
  • Figure 1 depicts a genetically engineered anti-GD2 immunocytokine having mouse antibody variable regions, canine antibody constant regions and canine IL-2.
  • Figure 2A depicts an expression map for an immunocytokine expression vector containing transcription units for both light and heavy chains together with a transcription unit for the selectable marker gene, dihydrofolate reductase (DHFR).
  • DHFR dihydrofolate reductase
  • Figure 2B depicts a strategy for inserting V regions in the light chain.
  • Figure 2C depicts a strategy for inserting V regions in the heavy chain.
  • Figure 3 depicts SDS-PAGE analysis of the human chl4.18 antibody and chl4.18-IL2 immunocytokine. Both non-reduced (left side of the gel) and reduced (right side of the gel) samples of purified protein were boiled and run on the gel together with molecular weight markers. The separated L, H, and H-IL2 fusion protein bands are indicated.
  • Figure 4 depicts a strategy for inserting introns into IgG cDNA sequences to create artificial genes with enhanced expression capacity.
  • Figure 5 A depicts the sequences for splice donor-acceptor sites at the junction between human C ⁇ i and dog CHl. Nucleic acid sequence is provided along with the amino acid sequences in three reading frames. The proper reading frame is underlined.
  • Figure 5 B depicts the sequences for splice donor-acceptor sites at the junction between dog CHl and human C ⁇ i. Nucleic acid sequence is provided along with the amino acid sequences in three reading frames. The proper reading frame is underlined.
  • Figure 5 C depicts the sequences for splice donor-acceptor sites at the junction between human C ⁇ i and the hinge domain (H), and the junction between H and dog CHl. Nucleic acid sequence is provided along with the amino acid sequences in three reading frames. The proper reading frame is underlined.
  • Figure 6 depicts a homology comparison between dog and cat IgGl H chain sequences.
  • Figure 7 depicts sequences for splice donor-acceptor sites at the junctions between dog CH2 and CH3 and the cat intron. Nucleic acid sequence is provided along with the amino acid sequences in three reading frames. The proper reading frame is underlined.
  • Figure 8 depicts the end of the CH3 domain of dog IgG, which includes a stop codon after the Pro-Gly-Lys C-terminus and a unique Fse I restriction site downstream of the stop codon.
  • Figure 9 depicts dog IL-2 protein and mRNA sequences.
  • Figure 10 depicts a Canis familiari genomic sequence with 97% identity to a 32 bp C ⁇ homology probe.
  • Figure 1 IA depicts a homology comparison between dog and cat C ⁇ proteins.
  • Figure 11 B depicts a vector comprising a dog C ⁇ insert.
  • Figure 12 depicts a gel analysis of immunocytokines transiently expressed in human 293 T cells.
  • Construct 1 had introns between CHl and H domains as well as between the CH2 and CH3 exons.
  • Construct 2 had only the latter intron in the H chain gene.
  • the arrows denote the H-IL2 fused protein and the L chain.
  • the positive control was the chimeric mouse /human 14.18 antibody.
  • Figure 13 shows binding of dog 14.18-IL2 to GD2+ melanoma cells and detection with labeled anti-dog IL2 antibody.
  • the dog IC produced by transient expression was incubated with human melanoma cells and then detected with either an anti-dog (middle panel) or anti-human antibody (right panel) followed by flow cytometry.
  • the cells in the left panel were incubated with the secondary anti-dog IL2 antibody but not the dog 14.18-IL2 (negative control).
  • Figure 14 shows bioactivity of dog 14.18-IL2 as measured by 3 H incorporation into mouse CTLL-2 cells. Dilutions of the culture supernatant containing transiently expressed dog 14.18-IL2 were tested (red squares) in the same assay as a known amount of purified human 14.18-IL2 (blue diamonds).
  • Figure 15 shows an alignment of FcR binding sequences at the junction of the hinge and CH2 exons of human and dog IgG H chains.
  • the human IgGl has very high FcR binding and corresponding high ADCC effector function.
  • Figure 16 shows a western blot analysis of 293 cells transiently transfected with pcDNA3.1-dogCD20 expression vector.
  • Cells were collected 72 hours after transfection and cytoplasmic extracts were prepared and analyzed by SDS-PAGE followed by Western Blotting and detection with a rabbit polyclonal anti-CD20 antisera known to be cross-reactive with CD20 of multiple species.
  • the CD20 protein runs at roughly 33 and 37 kD with the larger species believed to represent the phosphorylated form. These bands are only seen in cells receiving the indicated amounts of the plasmid DNA.
  • Figure 17 shows protein sequence for a soluble extracellular loop of canine CD20 (SEQ ID NO: 71) fused to mouse Fc. Only the CD20 sequence is shown and includes the introduction of two cysteine residues (bold C). These are predicted to form a disulfide bond and artificial loop.
  • Figure 18 shows expression of canine CD20 loop as an Fc fusion protein.
  • the left and right panels show SDS-PAGE analysis of Protein A Affinity Enrichment from conditioned media of pdHP-dogCD201oop-mFc (PBC00021).
  • samples were resolved by SDS-PAGE (4-20%) under reducing conditions (100 ⁇ M 2-mercaptoethanol) and stained with Coomassie Brilliant Blue R250.
  • samples were resolved by SDS-PAGE (4-20%) under non-reducing conditions (100 ⁇ M 2- mercaptoethanol) and stained with Coomassie Brilliant Blue R250.
  • FIG. 19 shows sequence homology (SEQ ID NO: 72) between human (SEQ ID NO: 73), mouse (SEQ ID NO: 74) and a potential canine (SEQ ID NO: 75) D3 domain of CSPG. (human, AAQ62842; mouse, NP_620570)
  • Figure 20 shows sequence homology (SEQ ID NO: 76) between canine (SEQ ID NO: 77) and other mammalian EpCAM proteins, (human, NP_002345 (SEQ ID NO: 78); mouse, NP_032558 (SEQ ID NO: 79); bovine, NP_001030367 (SEQ ID NO: 80)).
  • Figure 21 shows results of Protein A pull down assays using a dog specific molecule, dl4.18-IL2, which contains an IgG-B isotype for the H chain constant region ( Figure 21 A) or which contains an IgG-A isotype for the H chain constant region ( Figure 21B).
  • Figure 22 shows alignments of portions of various IgG heavy chains with regions associated with ADCC effector function denoted by underline or bracket.
  • Figure 22A shows alignments of portions of canine IgG-A, IgG-B, IgG-C, and IgG-D.
  • Figure 22B shows an alignment of portions of canine IgG-A and feline IgGl.
  • the present invention provides antibodies useful for targeting tissues in non-human and/or non-rodent animals (e.g., dogs, cats, etc.).
  • Embodiments of the invention are useful for targeting diseased cells (e.g., cancer cells) in non-human animals.
  • Antibodies of the invention may be fused to cytokines (e.g., to form immunocytokines for therapeutic applications), imaging molecules (e.g., for targeted imaging applications), and/or radiolabeled molecules (e.g., for targeted radiotherapy). It should be appreciated that in some embodiments immunocytokines also may be radiolabeled as aspects of the invention are not limited in this respect.
  • Immunocytokines as a class of drug may have a particular utility for the treatment of veterinary cancer due to their low dosing requirements and therefore their low cost of goods.
  • their anti-tumor activity is greater than that of the antibody from which they are derived.
  • Due to their potential immunogenicity ICs prepared for human therapy cannot be effectively used for the veterinary market. Therefore it is useful to develop non-human animal specific (e.g., dog and cat) ICs, useful for the treatment of cancers, utilizing coding sequences for the species-relevant immunoglobulin and cytokine components.
  • the variable regions from mouse antibodies can be used to confer the antigen binding property of tradition monoclonal antibodies.
  • variable regions are derived from antibody display libraries, including those generated from the repertoire of the animal to be treated.
  • non-human animal refers to any animal that is not a human. Examples of non-human animals include, but are not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Non-human animals can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), zoo animals (e.g., lions, giraffes, etc.), but are not so limited. Often the non-human animals are mammals. As used herein, the term “non-rodent animal” refers to any animal that is not a rodent.
  • V regions derived from an existing antibody that binds to a non- species-specific antigen (such as a carbohydrate or a glycolipid or a ganglioside) that is expressed on the cancer cells of the animal;
  • a second source is to screen available anti-tumor antibodies against protein antigens for cross-reactivity between the original species (typically human), and the animal (e.g., dog or cat);
  • the third source is to obtain the particular antigen and to use it to create new antibodies either through immunization or through selective binding of a synthetic antibody library.
  • immunocytokines for human use include those specific for gangliosides (e.g., GD2), carbohydrates (e.g., LewisY ), nucleic acids (e.g., tumor necrosis targeting) and several proteins expressed in cancer cells of epithelial origin (e.g., EpCAM, EGFR, Her2) as well as hematological origin (e.g.,CD20, CD 19, Lym-1, CD30, etc.).
  • gangliosides e.g., GD2
  • carbohydrates e.g., LewisY
  • nucleic acids e.g., tumor necrosis targeting
  • proteins expressed in cancer cells of epithelial origin e.g., EpCAM, EGFR, Her2
  • hematological origin e.g.,CD20, CD 19, Lym-1, CD30, etc.
  • modular expression vectors have been created to produce veterinary ICs.
  • the modular expression vectors have unique restriction sites flanking the gene fragments encoding the immunoglobulin and cytokine components, allowing the species-specific sequences to be replaced according to the application.
  • a vector containing a set of V regions of a mouse anti-GD2 antibody can be adapted to contain a light chain constant region flanked by BcI I and Not I sites that are unique in the plasmid vector.
  • the originally synthesized vector encoded the dog C kappa region but his can easily be replaced by removing this fragment and replacing it with another BcI I to Not I fragment encoding the cat C kappa sequence.
  • the same process is followed using other unique sites for the heavy chain C region and the cytokine (IL-2) coding sequences.
  • IL-2 cytokine
  • antibodies can include a dog heavy chain and a dog light chain or a portion thereof (e.g. , a kappa or lambda constant region).
  • antibodies include a variable region from a mouse antibody (optionally humanized or canonized) fused to a constant region from a dog antibody.
  • Mouse variable regions have been identified for many different antigens. Since most mouse antibodies have kappa light chains, a mouse light chain variable region should generally be fused to a kappa constant region from a dog antibody for optimal stability and performance of recombinant mouse/dog antibodies. However, in some embodiments, a mouse variable region may be fused to a dog lambda constant region.
  • immunocytokines include a heavy chain having a variable region (e.g., a mouse variable region, or a dog variable region, or any other suitable variable region) fused to a dog heavy chain constant region (e.g., full length, or containing one or more point mutations and/or deletions) fused to a cytokine (e.g., a dog cytokine) or a portion thereof.
  • the recombinant heavy chain can be combined with a light chain (e.g., a recombinant light chain) that contains either a lambda or a kappa light chain constant region (e.g. , from dog).
  • a kappa constant region may be selected (even though a lambda constant region could be used in some embodiments).
  • tumor antigens refers to a substance produced directly or indirectly by a tumor cell that induces a specific immune response in a host to the substance. Typically, the tumor antigen is expressed on the extracellular surface of a tumor cell. In some embodiments, the tumor antigen is a non-human homologue of a human tumor antigen. In some embodiments, the tumor antigen is GD2-ganglioside, CD 19, CD20, EPCAM, or CSPG. Other suitable tumor antigens include, for example, pi 85 HER2/neu (erb-Bl; Pisk et al., J. Exp.
  • EGFR epidermal growth factor receptor
  • CEA carcinoembryonic antigens
  • MUC- 1 gene products Jerome et al.,. J. Immunol., 151 :1654-1662 (1993)
  • E7 and E6 proteins of human papillomavirus Ressing et al., J.
  • the invention is not limited to immunocytokines that bind tumor antigens.
  • autoimmune diseases e.g., Rheumatoid Arthritis, Multiple Sclerosis, Crohn's Disease, Psoriasis, etc.
  • immunocytokines that bind specifically to a cell surface antigen of a cell that mediates an autoimmune response (e.g., a CD20 antigen, alpha-4 ( ⁇ x4) integrin, CDl Ia, etc.).
  • immunocytokines are provided for killing GD2-expressing malignant cells in dogs.
  • Immunocytokines of the invention can be used in dogs in order to characterize their clinical efficacy in vivo. Preclinical data obtained from such studies are useful for the development of therapeutic agents for use in veterinary medicine, as well as further development and use of hul4.18-IL2 in human subjects in some embodiments.
  • immunocytokines of the invention generally include an anti- GD2 antibody that is fused to at least one dog IL-2 polypeptide.
  • the anti-GD2 antibody of the immunocytokine binds specifically to GD2 and is characterized in part by the presence of a dog kappa light chain constant (C ⁇ ) region.
  • at least one heavy chain of the anti-GD2 antibody of the immunocytokine is fused to a dog IL-2 polypeptide.
  • the C-terminal amino acid of at least one heavy chain of the anti-GD2 antibody of the immunocytokine is covalently linked to the N-terminal amino acid of a dog IL-2 polypeptide.
  • the anti-GD2 antibody of the immunocytokine is an IgG antibody.
  • An IgG antibody is a tetramer that includes two heavy chains and two light chains, each heavy chain being linked to the other heavy chain and also to one light chain.
  • Each heavy chain includes an N-terminal variable (V H ) region linked to a C-terminal constant (C H ) region.
  • the two heavy chains are linked to each other through one or more disulfide bonds between the respective C H regions.
  • Each light chain includes an N-terminal variable (V L ) region linked to a C-terminal constant (C L ) region.
  • the light chain can be a kappa chain or a lambda chain, depending on its V L and C L regions.
  • a kappa light chain includes a V ⁇ and a C ⁇ region, while a lambda light chain includes a V ⁇ and C ⁇ region.
  • Each heavy chain is linked to one light chain through one or more disulfide bonds between the C H region and the C L (e.g., CK) region.
  • the V H and VL regions of an antibody determine the antigen specificity and affinity of the antibody. Together, the C H regions, in part, define the Fc portion of the antibody that is capable of directing effector functions antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the V H and V L regions of a first antibody are substituted for the VH and V L regions of a second antibody, resulting in an antibody with the antigen specificity of the first antibody and the effector function characteristics of the second antibody.
  • an immunoglobulin has a CPX-motif that bridges the hinge and CH2 domain of the immunoglobulin.
  • the CPX-motif has a sequence Of CPXiPX 2 X3 X4 X 5 LGGPSX 6 X 7 (SEQ ID NO: 36). Certain non-limiting examples of CPX motifs are shown in Figure 15. In some embodiments, the CPX-motif mediates Fc- Receptor binding and ADCC effector function.
  • an immunoglobulin heavy chain that has a CPX-motif that has an amino acid other than a proline (e.g., a hydrophobic amino acid (e.g., isoleucine, leucine, methionine, etc.)) at its X 5 position binds to Fc-Receptor and activates ADCC.
  • a GKX-motif is in the CH2 domain of an immunoglobulin.
  • the GKX-motif has a sequence Of GKXgFXgCXi 0 V (SEQ ID NO: 39).
  • the GKX-motif mediates Fc-Receptor binding and ADCC effector function.
  • an immunoglobulin heavy chain that has a GKX- motif that has a negatively charged amino acid (e.g., glutamate, aspartate) at its X 8 position binds to Fc-Receptor and activates ADCC.
  • an immunoglobulin heavy chain that has a combination of a CPX-motif that does not have proline at its X 5 position and a GKX-motif that has a negatively charged amino acid (e.g., glutamate) at its X 8 position binds to Fc-Receptor and activates ADCC.
  • antibodies containing light chain variable and constant regions derived from different species are more stable and/or have higher performance characteristics when the variable and constant regions are of the same isotype (e.g., matched to both be either kappa or lambda).
  • Mouse antibody light chains are typically kappa light chains
  • dog antibody light chains are typically lambda light chains.
  • a mouse variable region e.g., a mouse kappa variable region
  • the variable region of the 14.18 antibody is a kappa variable region and in some embodiments is fused to a dog kappa constant region or a portion thereof.
  • the anti-GD2 antibody of the immunocytokine is a chimeric mouse-dog IgG antibody, with anti-GD2 specificity provided by the variable regions of murine monoclonal anti-human GD2 antibody 14.18 and the remainder of the antibody provided by constant regions of dog IgG, including dog C ⁇ .
  • Dog IgG includes at least four subtypes, IgG A, IgG B, IgG C, and IgG D. Of these dog IgG subtypes, dog IgG A is highly similar to human IgGl . Tang L et al. (2001) Vet Immunol Immunopath 80:259-70.
  • the anti-GD2 antibody of the immunocytokine is a chimeric mouse-dog IgG antibody, with anti-GD2 specificity provided by the variable regions of murine monoclonal anti-human GD2 antibody 14.18 and the remainder of the antibody provided by constant regions of dog IgG A, including dog C ⁇ .
  • the C-terminal amino acid of at least one heavy chain of the chimeric mouse-dog anti-GD2 IgG A antibody of the immunocytokine is covalently linked to the N-terminal amino acid of a dog IL-2 polypeptide. In one embodiment, the C-terminal amino acid of at least one heavy chain of the chimeric mouse-dog anti-GD2 IgG A antibody of the immunocytokine is genetically linked to the N-terminal amino acid of a dog IL-2 polypeptide.
  • the C-terminal amino acid of each heavy chain of the chimeric mouse-dog anti-GD2 IgG A antibody of the immunocytokine is covalently linked to the N- terminal amino acid of a dog IL-2 polypeptide. In one embodiment, the C-terminal amino acid of each heavy chain of the chimeric mouse-dog anti-GD2 IgG A antibody of the immunocytokine is genetically linked to the N-terminal amino acid of a dog IL-2 polypeptide.
  • the anti-GD2 antibody and the IL-2 component parts of the immunocytokine are linked such that anti-GD2 antibody of the immunocytokine binds specifically to GD2 via its V regions, and the dog IL-2 of the immunocytokine is capable of signaling through an IL-2 receptor.
  • the term "binds specifically” means that the immunocytokine or recombinant antibody is capable of specific binding to its target antigen in the presence of the antigen under suitable binding conditions known to one of skill in the art.
  • the immunocytokine or recombinant antibody has an affinity constant, K 3 in a range of 10 7 NT 1 to 10 8 M '1 , 10 8 IVT 1 to 10 9 M “1 , 10 9 M “1 to 10 10 M “1 , 10 10 Nf 1 to l ⁇ ” M “1 , or 10 11 M “1 to 10 12 M “1 .
  • the immunocytokine or recombinant antibody has an affinity constant, K 3 of at least 10 7 M '1 , at least 10 8 M "1 , at least 10 9 M "1 , at least 10 10 M- 1 , at least 10 11 M '1 , or at least 10 12 M '1 .
  • "binds specifically” means that at least 90 percent of antibody- antigen immune complexes formed when the antibody is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include a specified antigen.
  • the immunocytokine of the invention is said to bind specifically to GD2 when at least 90 percent of antibody-antigen immune complexes formed when the antibody-containing immunocytokine is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include GD2.
  • binds specifically means that at least 95 percent of antibody- antigen immune complexes formed when the antibody is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include a specified antigen.
  • the immunocytokine of the invention is said to bind specifically to GD2 when at least 95 percent of antibody-antigen immune complexes formed when the antibody-containing immunocytokine is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include GD2.
  • binds specifically means that at least 98 percent of antibody- antigen immune complexes formed when the antibody is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include a specified antigen.
  • the immunocytokine of the invention is said to bind specifically to GD2 when at least 98 percent of antibody-antigen immune complexes formed when the antibody-containing immunocytokine is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include GD2.
  • binds specifically means that at least 99 percent of antibody- antigen immune complexes formed when the antibody is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include a specified antigen.
  • the immunocytokine of the invention is said to bind specifically to GD2 when at least 99 percent of antibody-antigen immune complexes formed when the antibody-containing immunocytokine is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include GD2.
  • immunocytokines of the invention include a dog C ⁇ region.
  • the dog C ⁇ region includes an amino acid sequence provided as SEQ ID NO: 1. Because the sequence for dog C ⁇ region was not in PubMed/NCI database, the sequence was identified as follows. A cat C ⁇ cDNA sequence is available as GenBank accession no. AFl 98257. Based on homology comparison of several mammalian C ⁇ sequences, a common 32 bp sequence was identified and then used to probe the dog genome. A sequence with 97 percent identity was found and the source sequence down loaded. Accession #AAEX02013460 (72,415 nt). The homology was in reverse orientation, so the sequence was reversed and the coding region identified by the open reading frame and by the protein similarity to cat C ⁇ .
  • the immunocytokine of the invention includes a dog C H region.
  • the dog C H region includes an amino acid sequence provided as SEQ ID NO:2.
  • SEQ ID NO:2 is an amino acid sequence from dog IgG A. Tang L et al. (2001) Vet Immunol Immunopath 80:259-70.
  • the immunocytokine of the invention includes a heavy chain variable region that includes an amino acid sequence provided as SEQ ID NO:3.
  • SEQ ID NO: 3 is an amino acid sequence of the VH region of the mouse monoclonal anti-GD2 antibody 14.18.
  • the immunocytokine of the invention includes a light chain variable region that includes an amino acid sequence provided as SEQ ID NO:4.
  • SEQ ID NO:4 is an amino acid sequence of the V L region of the mouse monoclonal anti-GD2 antibody 14.18.
  • the immunocytokine of the invention includes a heavy chain variable region that includes an amino acid sequence provided as SEQ ID NO: 3 and a light chain variable region that includes an amino acid sequence provided as SEQ ID NO:4.
  • the immunocytokine of the invention includes a heavy chain polypeptide that includes an amino acid sequence provided as SEQ ID NO:5.
  • SEQ ID NO: 5 is an amino acid sequence of a polypeptide that includes, from its N-terminus to its C- terminus, the V H region of the mouse monoclonal anti-GD2 antibody 14.18 (SEQ ID NO:3), linked to the C H region of dog IgG A (SEQ ID NO:2), linked to dog IL-2 (SEQ ID NO:9).
  • the immunocytokine of the invention includes a light chain polypeptide that includes an amino acid sequence provided as SEQ ID NO:7.
  • SEQ ID NO: 7 is an amino acid sequence of a polypeptide that includes, from its N-terminus to its C- terminus, the V L region of the mouse monoclonal anti-GD2 antibody 14.18 (SEQ ID NO:4), linked to the C ⁇ region of dog IgG (SEQ ID NO:1).
  • the immunocytokine of the invention includes a heavy chain polypeptide that includes an amino acid sequence provided as SEQ ID NO: 5 and a light chain polypeptide that includes an amino acid sequence provided as SEQ ID NO:7.
  • the light chain, heavy chain, and cytokine portions of the immunocytokines can be connected with or without an intervening linker ⁇ e.g., peptide linker).
  • FIG. 1 A non-limiting schematic representation of an embodiment of an immunocytokine of the invention is shown in FIG. 1.
  • the immunocytokine is a covalently linked homodimer composed of two pairs of polypeptides, each pair of polypeptides including (a) a chimeric mouse-dog anti-GD2 immunoglobulin heavy chain linked at its C-terminus to the N-terminus of dog IL-2, and (b) a chimeric mouse-dog anti-GD2 immunoglobulin light chain that includes a dog C ⁇ region.
  • the invention in one aspect is an isolated nucleic acid molecule encoding a heavy chain polypeptide of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2, wherein the heavy chain polypeptide comprises an amino acid sequence comprising SEQ ID NO: 5.
  • the nucleic acid molecule includes the sequence SEQ ID NO:6.
  • SEQ ID NO: 6 includes, from its 5' end to its 3' end, sequence for a human C ⁇ l intron, dog CHl cDNA, human C ⁇ l intron, dog hinge and CH2 cDNA, cat CH2-CH3 intron, and dog CH3 cDNA fused in correct translational reading frame to an artificial dog IL-2 coding sequence.
  • Dog IL-2 protein and cDNA sequences are available from Knapp et al. (1995) Gene 159:281-2. Due to the instability of cytokine mRNAs, the mature dog IL-2 sequence was reverse transcribed to a coding sequence using mammalian optimized codon usage.
  • the invention in one aspect is an isolated nucleic acid molecule encoding a light chain polypeptide of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2 and comprises a dog C ⁇ region, wherein the light chain polypeptide comprises an amino acid sequence comprising SEQ ID NO:7.
  • the nucleic acid molecule includes the sequence SEQ ID NO: 8.
  • SEQ ID NO:8 includes, from its 5' end to its 3' end, a sequence for a dog C ⁇ gene.
  • an isolated molecule is a molecule that is substantially pure and is free of other substances with which it is ordinarily found in nature or in vivo systems to an extent practical and appropriate for its intended use.
  • the molecular species are sufficiently pure and are sufficiently free from other biological constituents of host cells so as to be useful in, for example, producing pharmaceutical preparations or sequencing if the molecular species is a nucleic acid, peptide, or polypeptide.
  • Mammalian expression vectors can include non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a poly-adenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • suitable promoter and enhancers are derived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence.
  • a nucleic acid molecule of the invention can be inserted into an appropriate expression vector using standard methods of molecular biology which need not be described in further detail here.
  • the expression vector can include a promoter or promoter/enhancer element that is positioned upstream of the coding nucleic acid molecule that is inserted into the vector.
  • Expression vectors can optionally include at least one coding region for a selection marker and/or gene amplification element, e.g., dihydrofolate reductase (DHFR).
  • DHFR dihydrofolate reductase
  • a vector or vectors containing nucleic acid sequences encoding the various polypeptides of the immunocytokine can be introduced into a suitable host cell or population of host cells.
  • the vector includes a nucleic acid molecule that encodes a heavy chain polypeptide of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2, wherein the heavy chain polypeptide comprises an amino acid sequence comprising SEQ ID NO: 5.
  • the vector includes a nucleic acid molecule that includes the sequence SEQ ID NO:6.
  • the vector includes a nucleic acid molecule that encodes a light chain polypeptide of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2 and comprises a dog C ⁇ region, wherein the light chain polypeptide comprises an amino acid sequence comprising SEQ ID NO:7.
  • the vector includes a nucleic acid molecule that includes the sequence SEQ ID NO: 8.
  • the vector includes a nucleic acid molecule that includes the sequence SEQ ID NO: 6 and a nucleic acid molecule that includes the sequence SEQ ID NO:8.
  • the vector or vectors can be introduced into a host cell or cells using any suitable method, including, for example, electroporation, biolistic delivery ⁇ e.g., using a gene gun), lipofection, calcium phosphate precipitation, microinjection, viral transduction, nucleofection, sonoporation, magnetofection, and heat shock. Such methods are well known by persons skilled in the art and need not be described here.
  • the cell or cells are maintained under physiologically suitable conditions suitable for in vitro cell culture, for a period of time sufficient to permit the cell or cells to express the immunocytokine.
  • a host cell is a eukaryotic cell.
  • the host cell is a mammalian cell.
  • the host cell is a mammalian cell line.
  • the mammalian cell line is non-Ig-secreting myeloma such as NS/0 or Sp2/0- Agl4.
  • the mammalian cell line is HEK293.
  • the mammalian cell line is a Chinese hamster ovary (CHO) line.
  • the immunocytokine is secreted into the culture medium by the cells containing the expression vector or vectors.
  • Secreted expressed immunocytokine can be readily isolated from culture by centrifugation (to remove cells) followed by immunoaffinity separation, for example using protein A or protein G chromatography.
  • the immunoaffinity separation can alternatively or in addition involve an anti-cytokine antibody, e.g., and anti-IL-2 antibody, as the immunoaffinity reagent.
  • compositions that include an immunocytokine of the invention.
  • the composition is a pharmaceutical composition that includes an immunocytokine of the invention and a pharmaceutically acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being commingled with other compounds, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
  • Immunocytokines of the invention can be used to treat cancers (e.g, GD2-expressing cancers) in non-human animals ⁇ e.g., dogs, cats, etc.).
  • cancer refers to an abnormal uncontrolled growth of cells in a subject.
  • cancer as used herein can refer to solid tumors, primary as well as metastatic cancers, as well as hematogenous (“liquid”) cancers.
  • GD2-expressing cancers are cancers that have detectable GD2 expressed on their cell surface.
  • GD2-expressing cancers are generally cancers of neuroectodermal origin and specifically can include, without limitation, melanoma, neuroblastoma, osteosarcoma, and small cell lung cancer.
  • to treat means to slow or halt the progression of, or to reduce or eliminate, a disease in a subject having the disease.
  • a subject having a disease is a subject that has at least one objectively identifiable manifestation of the disease.
  • a dog having a GD2-expressing cancer is a dog that has at least one objectively identifiable manifestation of a GD2-expressing cancer.
  • Certain embodiments of the invention also include methods of treating a GD2- expressing cancer in a dog by administering to a dog having a GD2-expressing cancer an effective amount of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2, wherein the immunocytokine comprises a dog C ⁇ region, to treat the cancer.
  • the immunocytokine of the invention can be administered alone to a dog having a GD2-expressing cancer to treat the cancer.
  • an "effective amount" refers to the amount necessary or sufficient to realize a desired biologic effect.
  • an effective therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is effective to treat the particular subject.
  • the effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular active agent being administered, the size of the subject, or the severity of the disease or condition.
  • One of ordinary skill in the art can empirically determine the effective amount of a particular active agent and/or other therapeutic agent without necessitating undue experimentation.
  • a maximum dose that is, the highest safe dose according to some medical judgment, although this is not necessarily the case for immune-stimulating agents.
  • Multiple doses per week may be contemplated to achieve appropriate systemic levels of compounds. Appropriate systemic levels can be determined by, for example, measurement of the subject's peak or sustained plasma level of the drug. "Dose” and “dosage” are used interchangeably herein.
  • daily doses of active compounds will be from about 0.01 milligrams/kg per day to 10 milligrams/kg per day. It is expected that intravenous doses in the range of 0.05 milligrams/kg per day to 5 milligrams/kg per day, in one or several administrations per day, will yield the desired results. Similarly, it is expected that subcutaneous doses in the range of 0.05 milligrams/kg per day to 5 milligrams/kg per day, in one or several administrations per day, will yield the desired results. Dosage may be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration.
  • the therapeutically effective amount can be initially determined from animal models.
  • a therapeutically effective dose can also be determined from human data for immunocytokines which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents.
  • the applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.
  • the immunocytokine may be administered in combination with a therapeutic antibody ⁇ e.g., a CD20 antibody).
  • the immunocytokine of the invention can be administered in conjunction with at least one other anti-cancer treatment agent or anti-cancer treatment modality to treat the cancer.
  • "in conjunction with” or “in combination with” refers to any suitable form of combination therapy, for example simultaneous, overlapping, and/or sequential treatments.
  • Anti-cancer treatment agents ⁇ e.g., anti-cancer compounds) and anti-cancer treatment modalities other than treatment with an immunocytokine of the invention can include chemotherapy (including combination chemotherapy), radiation therapy, surgery, other immunotherapy, and any combination thereof.
  • the anti-cancer treatment is local radiation or radiofrequency ablation.
  • Anti-cancer treatments such as cyclophosphamide, doxorubicin, valinomycin, hormone therapy, and other therapies disclosed herein or otherwise known in the art may be used.
  • an "anti-cancer compound” refers to an agent which is administered to a subject for the purpose of treating a cancer.
  • Anti-cancer compounds include, but are not limited to antiproliferative compounds, anti-neoplastic compounds, anti-cancer supplementary potentiating agents and radioactive agents.
  • One of ordinary skill in the art is familiar with a variety of anti-cancer compounds.
  • anti-cancer compounds include, but are not limited to, the following: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Adriamycin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Buniodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorombucil; Cirole
  • Radioactive agents may also be used.
  • radioactive agents include but are not limited to Fibrinogen I 125; Fludeoxyglucose F18; Fluorodopa F 18; Insulin I 125; Insulin I 131; Iobenguane I 123; Iodipamide Sodium 1 131; Iodoantipyrine 1 131; Iodocholesterol I 131; Iodohippurate Sodium 1 123; Iodohippurate Sodium I 125; Iodohippurate Sodium 1 131 ; Iodopyracet I 125; Iodopyracet 1 131 ; Iofetamine Hydrochloride I 123; Iomethin I 125; Iomethin 1 131; Iothalamate Sodium I 125; Iothalamate Sodium 1 131; Iotyrosine 1 131; Liothyronine I 125; Liothyronine 1 131; Merisoprol Acetate Hg
  • an immunocytokine is administered in combination with (before, during or after) CHOP therapy.
  • CHOP therapy is a common chemotherapy protocol for treating cancer ⁇ e.g., lymphoma (e.g., Non- Hodgkin lymphoma)).
  • the following drugs are typically used in the CHOP protocol: Cyclophosphamide, Adriamycin, Vincristine, and Prednisone.
  • Immunocytokines may also be administered in combination any one of a variety of alternative cancer therapy protocols that may differ from CHOP therapy in terms of scheduling, dosages and use of other chemotherapeutics.
  • Alternative protocols include, but are not limited to, the Wisconsin- Madison protocol, AMC protocol and VELCAP protocol.
  • Non-limiting examples of cancer therapy regimens with which the immunocytokines may be administered in combination are disclosed in: L ⁇ eureux DA, Moore AS et al. Evaluation of a Discontinuous Protocol (VELCAP-S) for Canine Lymphoma. J Vet Intern Med 2001; 15:348-354; Ogilvie GK, Berman PJ. Drug Resistance and Cancer Therapy. Compendium 1995; 17:549-556; Page RL, Lee JJ et al. P-Glycoprotein Expression in Canine Lymphoma. Cancer 1996;77: 1892-1898; Leifer CE, Calvert CA. Doxorubicin for Treatment of Canine Lymphosarcoma After Development of Resistance to Combination Chemotherapy.
  • formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
  • Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).
  • Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).
  • an effective amount of the immunocytokine can be administered to a subject by any mode that delivers the immunocytokine to the desired surface.
  • Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to intravenous and subcutaneous.
  • the compounds when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • GD2 is a disialoganglioside expressed on tumors of neuroectodermal origin, including human neuroblastoma and melanoma, with highly restricted expression on normal tissues, principally to the cerebellum and peripheral nerves in humans.
  • the relatively tumor-specific expression of GD2 makes it an attractive target for immunotherapy, for example with monoclonal antibodies.
  • Melanomas, sarcomas, and neuroblastomas abundantly express GD2 on the cell surface where it is susceptible to immune attack by antibodies. Overexpression of GD2 on these tumors is striking, as is the frequency of clinical responses after treatment of neuroblastoma with monoclonal antibodies against GD2.
  • Chimeric mouse-human antibody, chl4.18, was found to have potent effector activities of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), as well as the ability to target GD2-positive melanoma cell xenografts in mice. Mueller et al. (supra).
  • An anti-GD2 antibody-IL-2 fusion protein that is suitable for therapeutic use in dogs with neuroectodermal cancer is genetically engineered.
  • the immunocytokine contains canine immunoglobulin heavy and light chain constant (C) regions and canine IL-2 sequences and is not immunogenic in dogs.
  • Dog antibody and IL-2 sequence information from database sources is adapted for high-level expression in mammalian cells by insertion in a vector containing the mouse 14.18 variable heavy (V H ) and variable light (V L ) coding sequences, as well as appropriate splicing signals that result in the joining of the V and C regions to create a chimeric mouse-dog molecule after transfection into mammalian cells in culture.
  • the dog IL-2 sequence is fused in-frame to the carboxyl terminus of the chimeric H chain.
  • the result is a whole antibody HL chain dimer containing two molecules of dog IL-2 per antibody.
  • the final vector sequence is assembled using the DNASTAR Lasergene program and the sequence is synthesized.
  • the immunocytokine is produced in vitro from cells transfected with expression constructs encoding the immunocytokine.
  • Expression vector DNA is used for transient expression of the protein in human 293 cells using standard protocols.
  • Conditioned media from the cultures serves as a source of immunocytokine material for biochemical analyses to ensure that correctly sized proteins are secreted and that the L chain and H chain-IL2 fusion protein are assembled into a heterodimeric structure.
  • the immunocytokine is captured on protein A Sepharose beads and subsequently analyzed by SDS-polyacrylamide gel electrophoresis.
  • PBMC dog peripheral blood monocytic cells
  • Transient cell cultures are scaled-up and moderate quantities of the dog immunocytokine are purified for further analyses.
  • Multi-milligram quantities of the immunocytokine are purified from cell culture supernatants and captured using standard protein A Sepharose and ion exchange chromatography methods.
  • This material is used to establish a reference standard for biochemical assays and for further characterization of biochemical and biophysical properties such as solubility, aggregation and in vitro stability.
  • the material is used to establish enzyme-linked immunosorbent assay (ELISA) methods necessary for identity and potency assays, as well as for measurement of the immunocytokine in biological samples such as blood, plasma, or serum.
  • ELISA enzyme-linked immunosorbent assay
  • Pharmacokinetic properties are determined in mice. Purified immunocytokine is used to measure concentration vs. time kinetics following intravenous dosing in mice. Blood samples are taken over a 24 hour period and the concentration of immunocytokine is measured by ELISA measuring both the antibody and enzyme-;lL-2 portion of the molecule. This defines the amount of intact immunocytokine present in the samples.
  • Example 1 Genetically engineered anti-GD2 immunocytokine having mouse antibody variable regions, canine antibody constant regions and canine IL-2.
  • a mammalian expression vector capable of generating high level production of the anti-GD2 immunocytokine is produced.
  • the chimeric protein is composed of two protein chains: the chimeric mouse-dog light chain; and the chimeric mouse-dog heavy chain fused to dog IL-2 (FIG. 1).
  • the humanized form of this molecule has been expressed using a vector containing the transcription units for both chains in a single vector, together with a transcription unit for the selectable marker gene, dihydrofolate reductase (DHFR) (FIG. 2A).
  • the two immunoglobulin chain transcription units are each driven by a cytomegalovirus (CMV) promoter and enhancer and utilize a leader sequence derived from the 14.18 light (L) chain.
  • CMV cytomegalovirus
  • L 14.18 light
  • a splice site is used at the end of the intron of the leader sequence so that other V region coding sequences can be inserted into this vector and joined at the RNA level to produce any desired antibody (FIG. 2B).
  • V region coding sequences include an AfI II restriction site at their 5' end and a splice donor site at their 3' end. This ensures joining to the leader sequence in the correct reading frame and then correct splicing to the next exon downstream in the vector.
  • splice donors, introns and splice acceptor sequences adapted from human gene sequences are added at the appropriate positions in the sequence. (See Example 5). After transfection and expression in the cell, these human sequences are removed by splicing and leave only the sequences encoding the dog proteins.
  • cDNA molecules also can be used to assemble recombinant genes that are useful to express recombinant proteins of the invention as aspects of the invention are not limited in this respect.
  • Example 2 Testing the biological properties of the immunocytokine such as cytokine activity and antibody effector functions using dog immune effector cells.
  • the ability of the vector to express the desired protein is tested using transient expression and analysis of small amounts of the protein secreted from transfected cells. This is accomplished by producing milligram quantities of the plasmid DNA from the bacterial host and purifying the DNA using high resolution chromatography. Endotoxin-free DNA is used to transfect HEK293 cells in suspension culture and after several days of culture, the conditioned culture media is harvested. A small amount is incubated with protein A Sepharose beads by gentle mixing and then the captured protein is eluted in gel electrophoresis buffer. Half of the sample is treated further with reducing agent ( ⁇ - mercaptoethanol) while the other half is not.
  • reducing agent ⁇ - mercaptoethanol
  • Both samples are heated and analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) together with an immunoglobulin control protein.
  • SDS-PAGE SDS polyacrylamide gel electrophoresis
  • a correctly assembled IL-2 based immunocytokine migrates as a single high molecular weight band on the gel (-200 kD) when it is not reduced, but dissociates into two bands after chemical reduction.
  • An example of such a result is shown in FIG. 3.
  • These bands include an L chain of normal size, and an H-IL2 chain that is larger than the normal IgG H chain by about 16 kD.
  • the remaining conditioned medium containing the chl4.18-dog IL2 molecule is characterized by performing GD2 binding assays and IL-2 proliferation assays using the standard CTLL-2 mouse T cell assay.
  • GD2 binding is performed using 96-well plates coated with GD2 (Calbiochem) and blocked with 5% bovine serum albumin (BSA) and 5% goat serum.
  • Test antibody or antibody-containing culture supernatants are diluted in PBS containing 1% BSA and 1% goat serum and incubated in wells for 1 hr at room temperature. Unbound proteins are washed three times with dilution buffer and bound immunocytokine is detected with a horseradish peroxidase (HRP)-conjugated secondary antisera against canine IgG or canine IL-2. Bound HRP is quantitated by standard protocols.
  • HRP horseradish peroxidase
  • An alternative method for testing GD2 binding is to incubate the test protein with a GD2-expressing cancer cell (e.g. melanoma) and to the detect its binding using a secondary labeled antibody directed against the dog immunoglobulin or cytokine portion (see example 6 below).
  • a GD2-expressing cancer cell e.g. melanoma
  • a secondary labeled antibody directed against the dog immunoglobulin or cytokine portion see example 6 below.
  • IL-2 bioactivity is performed in 96 well plates containing CTLL-2 mouse T cells that have been deprived of IL-2 for 48 hr prior to the assay. Dilution of purified proteins and culture media containing immunocytokines are plated and then mixed with CTLL-2 cells in culture medium and incubated for two days at 37°C. Additional medium containing H- thymidine is added and incubation continued for an additional 16 hr and incorporation measured using standard protocols. The extent to which dog IL-2 induces proliferation via the mouse IL-2 receptor is tested. Dog IL-2 has roughly the same degree of homology to mouse IL-2 as the human cytokine.
  • Human IL-2 is routinely assayed using this mouse cell line (Gillis S, et al., T cell growth factor: parameters of production and a quantitative microassay for activity. J Immunol. 120(6):2027-32, 1978). Dog peripheral blood mononuclear cells (PBMC) are obtained and cultured with recombinant IL-2 to activate them. Concanavalin A is added, if necessary (Kato M, et al., A novel culture method of canine peripheral blood lymphocytes with concanavalin a and recombinant human interleukin-2 for adoptive immunotherapy. J Vet Med Sci.
  • Example 3 Production and purification of sufficient amounts of this protein for further characterization studies.
  • transient expression in HEK293 cells is scaled to either 1 or 10 L of culture using disposable wave bags. At least 10 mg of purified chl4.18-dog IL2 protein are prepared for further characterization and assay development.
  • stable cell line generation is performed in NS/0 mouse myeloma cells using methotrexate as the selection marker. This is performed using linearized plasmid DNA restriction enzyme cut within the bacterial ampicillin resistance (amp R ) gene. DNA is introduced into the myeloma cells (or CHO cells) using well established electroporation methods, and the cells are cultured in section medium containing 0.1 ⁇ M methotrexate.
  • Drug-resistant myeloma clones are tested for secretion of dog IgG using commercially available anti-dog IgG antisera (Table 1). Expressing clones are tested for productivity, stability and growth rate. Subcloning is used to select for the optimal cell line properties.
  • the expressed protein preferably secreted from cells growing in serum free media, is purified using established protocols for producing clinical grade protein. Great care is used to prevent endotoxin contamination.
  • the steps may include a concentration step (e.g. tangential flow filtration), followed by binding to and elution from protein A Sepharose. Dog IgG binds to this reagent under certain conditions of pH and ionic strength. After elution with acidic pH and neutralization, ion exchange chromatography is used as a polishing step. Additional purification methods for dog IgG and related fusion proteins are available from commercial sources [e.g. Dog IgG purification kit (Code : DIKG-FF KIT), Affiland, Ans- vide, Belgium].
  • Purified protein is analyzed by SDS-PAGE and potential aggregation is examined by size-exclusion chromatography (SEC). Immunocytokine stability issues associated with aggregation are monitored closely. Current formulations, including lyophilization, that minimize stability issues are applied if necessary.
  • Example 4 Obtaining pharmacokinetic properties in mice.
  • Example 5 Artificial Gene Design for Dog IgG C regions Introns were designed for insertion into IgG cDNA sequences to create artificial genes with enhanced expression capacity.
  • FIG. 4 provides a strategy for intron insertions to create artificial genes.
  • Human C ⁇ i intron is inserted between V H and CHl domains, and between CHl and hinge (H) domains.
  • No intron is inserted between H and CH2 domains.
  • a cat C ⁇ i intron analog is inserted between CH2 and CH3.
  • FIG. 5A-C Details of sequences at splice donor and acceptor sites in artificial C2 dog gene have been determined (FIG. 5A-C).
  • the splice acceptor of the 5' end of dog C ⁇ domain is similar to human and cat, both of which use a consensus 3' end of the intron. Therefore the corresponding sequence from the human gene was used and fused to the second residue of the Ala codon as shown (FIG. 5A).
  • the splice donor at the end of the CHl exon is also typical and similar to human, so the corresponding intron between CHl and hinge of the human C ⁇ gene was used (FIG. 5B-C).
  • the junction between H and CH2 in the dog cDNA looks much less typical and hard to predict in terms of an intron insertion. Therefore, the fused sequence with no intron was used to avoid potential problems.
  • An expression vector is constructed to express both the IL2 immunocytokine with dog IL2 fused to the end of the CH3 domain of dog IgG A, as well as the same molecule lacking IL2, i.e., the antibody alone. This is done by constructing the antibody sequence (including a stop codon after the Pro-Gly-Lys C-terminus) and adding a unique Fse I restriction site downstream. This site can be used to insert a replacement fragment encoding a cytokine (or other molecule) between the unique Sma I site and the Not I site (FIG. 8).
  • Dog IL-2 protein and mRNA sequences obtained through PubMed Literature Ref. Knapp et al. (1995) Gene 159:281-2 were identified (FIG. 9). Homology analysis indicates an 84% sequence homology to human IL-2. Due to the instability of cytokine mRNAs, the mature protein sequence was reverse translated to a coding sequence using DNA* mammalian optimized codon usage.
  • the homology was in a reverse orientation, so sequence the sequence was reversed and the coding region was identified by the open reading frame and by protein similarity to cat C ⁇ .
  • the dog C ⁇ coding region, identified from the database was translated and the protein compared to cat C K (FIG. 11).
  • the splice acceptor site appeared normal.
  • Example 6 Expression of a dog anti-GD2 — IL2 immunocytokine
  • Construct 1 had introns between CHl and H domains as well as between the CH2 and CH3 exons.
  • Construct 2 had only the latter intron in the H chain gene. This result indicates that the correct protein was made but that it did not bind well to protein A. This was true whether or not the expression construct contained the intron between the CHl domain and the hinge sequence or not.
  • the positive control 14.18 antibody was expressed at about the same level as dog proteins from the two different constructs (with and without the additional intron) but was highly enriched using protein A.
  • the functionality of the dog IC was tested by measuring antigen binding to a GD2 expressing cell line. This was done by incubating the culture supernatant containing transiently-expressed dl4.18-IL2 with M21 melanoma cells and then with a biotinylated anti- dog IL-2 antibody. Binding was visualized by incubation with streptavadin-FITC followed by flow cytometry. Control incubations indicated that the fluorescence was dependent on the IC as well as the anti-dog antibody but is not detected using an anti-human IL2 antibody. As shown in Figure 13, the dog IC produced by transient expression was incubated with human melanoma cells and then detected with either an anti-dog or anti-human antibody followed by flow cytometry. The extent of binding was compared to the human 14.18-IL2 using what was estimated to be the same concentration of IC and the mean fluorescence was virtually the same, despite the fact that different detecting antibodies were used.
  • IL-2 cytokine component
  • Past studies have shown that human IL-2 is active in dogs and companies that sell dog IL-2 (e.g. R&D Systems) test bioactivity of their preparations using the standard mouse T cell line CTLL-2. Thus, standard cell lines from other species were used for testing bioactivity of IL-2.
  • IL-2 that is fused to the carboxyl terminus of a dog IgG H chain is biologically active
  • the dog 14.18-IL2 that was expressed transiently from human 293T cells was serially diluted and tested for the ability to stimulate the proliferation of CTLL-2 cells that were previously starved for IL-2. Purified human 14.18-IL2 IC was used as a positive control.
  • both proteins stimulated the uptake of 3 H- thymidine (a measure of cell proliferation) far above the baseline of un-stimulated cells and at all concentrations.
  • the unpurified cell culture media containing the dog 14.18-IL2
  • the interference stopped and activity increased and then decreased further upon dilution.
  • the activity of the dog IC was somewhat greater than that of the human protein but this could be due to a slight error in estimating the protein concentration or different activities of the different proteins with respect to the stimulation of the mouse IL-2 receptor.
  • ADCC antibody-dependent cellular cytotoxicity
  • human or dog effector cells peripheral blood mononuclear cells
  • human GD2 expressing melanoma cell line a human GD2 expressing melanoma cell line.
  • Target cells were incubated with 51 Cr and then washed to remove free isotope.
  • Isolated peripheral blood lymphocytes from a healthy human volunteer were incubated with labeled target cells for 4 hours in the absence or presence of increasing amounts of the human or dog 14.18-IL2 IC and the amount of release chromium was taken as a measure of specific lysis.
  • Example 7 Switching the isotype of dog H chain to improve FcR binding and ADCC effector function Although it has been reported that some immunocytokines lacking ADCC effector function still exhibit potent anti-tumor activity in pre-clinical models, it may still be desirable to include this function in some cases.
  • sequences of the other dog H chain isotypes were compared to each other and to those of human H chains known to have this effector function.
  • the motif (a CPX-motif) shown to effect binding of human IgG to Fc receptors is located at the junction of the heavy chain hinge and CH2 domain.
  • the corresponding sequences of the human IgGl (strong FcR binding) and the four dog isotypes are shown in Figure 15.
  • the IgG-A isotype is unique in this group for having a proline (P) residue in this middle of this motif and this residue is known to have a dramatic effect on the secondary structure of polypeptides.
  • P proline
  • the dog IgG-B motif has the closest similarity to the human IgGl sequence (a single substitution of an M in place of L) and the change is far more conservative than a P residue.
  • the second most abundant class of human IgG is IgG2 and the sequence in the critical FcR binding motif includes the residues PVA in place of ELL found in IgGl. This change has a dramatic effect on ADCC due to a loss of FcR binding (Isaacs et al. 1998. J. Immunol. 161 :3862-3869. Thus it seemed that the dog IgG-A most resembles human IgG2 with respect to FcR binding and that one of the other isotypes may be more similar to human IgGl . To test this hypothesis, the dog IgG-B H chain gene was synthesized in a form compatible with the expression vector construct as a MIu I to Xma I fragment.
  • the sequence of the gene begins with intron sequences and a functional splice acceptor site, followed by the CHl, hinge and CH2 exons as a continuous sequence (no introns). There is an intron between the CH2 and CH3 exons and the fragment joins the vector sequence encoding the last residues of the antibody sequence. The fragment was inserted in place of the corresponding fragment in the vector encoding dog 14.18-IL2 (construct 2 of Figure 12) and the resulting vector was tested by transient expression in cultures of human 293T cells. The protein contained in the cell culture media was examined directly by SDS-PAGE analysis and after binding to and elution from protein A.
  • the modified IC was produced as a fully assembled IC that broke down after treatment with reducing agent to L chain and a fusion H-IL2 chains of the correct size. Binding to protein A had an improved concentration effect (compared to IgG-A) using the ionic strength and pH conditions of the culture media, but not to the extent normally seen with human IgGl .
  • Figure 21 shows that an IC comprising an IgG-B isotype for the H chain constant region binds to Protein A. This IgG-B containing IC does not exhibit substantial ADCC effector function (similar to IgG-A), as determined using the standard ADCC assay disclosed in Hank JA, et al.
  • Sequences of the Dog IgG-B replacement fragment and the Dog-IgG-B H chain C region protein sequence are provided as SEQ ID NO: 16 and 17, respectively.
  • Other dog H chain isotypes may be constructed in the same manner as the IgG-B fragment.
  • the constant regions of the expression vector of Example 7 is modified using the corresponding protein encoding sequences of the feline light and heavy chains, the flanking sequences and restriction sites described for the dog protein expression vectors.
  • a nucleic acid sequence encoding a feline C kappa fragment is provided in SEQ ID NO: 18.
  • a protein sequence of a feline C kappa fragment is provided in SEQ ID NO: 19.
  • a nucleic acid sequence encoding a feline H chain C gamma 1 region sequence is provided as SEQ ID NO: 20.
  • the expression vector used to express the dog 14.18-IL2 IC also contains a Xma I to Fse I fragment encoding the dog IL-2 molecule.
  • a feline IC having feline cytokine sequences it may be desirable to reduce immunogenicity.
  • a fragment encoding IL-2 flanked by Xma I and Fse I is provided that maintains the protein reading frame with the heavy chain constant region and thus results in the fusion of the antibody and IL-2 sequences.
  • a nucleic acid sequence encoding a feline IL-2, e.g., for fusion to the feline IgG H chain, is provided as SEQ ID NO: 22.
  • a protein sequence encoding a feline IL-2 is provided as SEQ ID NO: 23.
  • Example 9 Additional sources of antibodies for veterinary use
  • CSPG chondroitin sulfate proteoglycan
  • VF20-VT20 had the strongest binding to the melanoma cells and presumably to the canine version of the human CSPG.
  • the V region sequences of this mouse antibody have been obtained and have been adapted for the expression vector used to express the dog 14.18-IL2 IC.
  • Example 10 Specific immunization with animal versions of a useful antigen targets - CD20
  • mouse anti-human can be restricted due to unique characteristics of the antigen or limited differences between the mouse and human versions of a particular protein. Both of these issues could be addressed by immunization of mice with the target protein (or peptide) of the desired species.
  • DNA encoding such proteins can be used for immunization.
  • a potentially useful target is CD20, the molecule recognized by the highly successful antibody, Rituxan, useful for the treatment of B cell tumors and B cell mediated immune disorders.
  • a first approach was used to express the canine CD20 molecule in a cell line useful for the immunization of Balb/c mice.
  • the NS/0 cell line is syngeneic with Balb/c mice and is not itself immunogenic upon cell immunization.
  • an NS/0 transfectant expressing the dog (or other foreign CD20) would be useful in the generation of mouse antibodies against the dog CD20.
  • the cell line can also be used to test for antibodies that recognize this protein as it is expressed on the surface of live cells.
  • the sequence encoding canine CD20 was obtained using PCR cloning from dog peripheral blood cDNA and adapted to have unique restriction sites just before the initiation codon and just after the stop codon.
  • This Xba to Xho I fragment was sequenced to confirm its authenticity and then subcloned in the pcDNA3.1 expression vector (Invitrogen).
  • Transient expression in human 293T cells was performed in order to verify the sequence and test whether a commercially available polyclonal antibody could be used to detect expression in transfected cells (for selecting stable transfectants).
  • a commercially available polyclonal antibody a rabbit antisera raised against a peptide in the cytoplasmic domain (Fisher Scientific), was found to be reactive by Western blot analysis of transiently transfected human 293 cells, as depicted in Figure 16.
  • mice NS/0 myeloma cells which do not express CD20
  • the preferred method for transfecting this cell line is to linearize the plasmid DNA with a single-site restriction enzyme that cuts in the ampicillin resistance gene (used for bacterial expression), mixing the DNA with NS/0 cells and electroporating the expression vector into the cell nucleus (See, e.g., Gillies et al 1998, J. Immunol. 160:6195-6203).
  • Transfectants were selected that are resistant to the marker gene in the pcDNA3.1 vector, Neo, by their growth as colonies in the presence of the antibiotic G418.
  • cells expressing the canine CD20 can be identified in at least two ways. The first is to make cytoplasmic extracts of individual resistant clones and analyze them for expression of CD20 as shown above ( Figure 16). A second way is to make a fusion protein of the major extracellular loop of canine CD20 fused to a carrier protein such as the mouse IgG2a Fc region. This allows for high level expression, easy purification using protein A Sepharose and immunization under conditions where the carrier portion of the fusion protein (mouse Fc) is a self molecule. Expression is aimed at producing an immune response to the desired portion of the fusion protein - the extracellular loop of canine CD20.
  • a carrier protein such as the mouse IgG2a Fc region
  • the protein may be expressed by adding two cysteine residues that should form a disulphide bond between portions of the loop and help recreate the structure that it has when it is protruding from the cell membrane (Figure 17).
  • Figure 17 shows a protein sequence for a soluble extracellular loop of canine CD20 fused to mouse Fc, which includes the introduction of two cysteine residues (bold Cs), that are capable of forming a disulfide bond and artificial loop.
  • An analogous soluble CD20 loop fusion protein can be made using the sequences of other species for use as an immunogen for the generation of antibodies that react with CD20 on the surface of target cells.
  • polyclonal antisera from mice, rabbits or other species are useful for screening transfectants expressing CD20 of the same species.
  • This method of immunization is useful on its own or combined with cell immunization to identify monoclonal antibodies useful for treatment of disease caused by cells expressing CD20.
  • Non-limiting examples of loop sequences are provided in SEQ ID NO: 24-26.
  • Transient expression of the canine CD20 loop fused to mouse Fc was achieved by sub-coning the DNA sequence into an expression vector containing a leader sequence from a mouse Ig light chain driven by the CMV promoter and also containing the hinge, CH2 and CH3 exons of the mouse IgG2a H chain.
  • Purified vector DNA was combined with a lipid transfection reagent and used to treat cultures of human 293T cells. After 96 hr of cell culture incubation, the conditioned media was tested for the presence of the fusion protein directly and after binding to and elution from protein A Sepharose. Samples were heated in the presence or absence of a reducing agent to test for dimerization of the fused H chains of the Fc region (Fig. 18). As depicted in Figure 18, the reduced protein ran with apparent molecular weight that was half the size of the non-reduced sample demonstrating the dimeric structure of the fusion protein.
  • Example 11 Immunization with a veterinary melanoma antigen
  • the CSPG expressed on melanoma, sarcoma and other cancer cells is a good target for immune therapies such as immunocytokines. While it is not essential that the targeting antibody for an IC also have ADCC activity, it may be preferred to include this activity. Also, as described above for the canine IgG isotypes, some mediate ADCC and some do not. This is based on the ability of the Fc portion to bind to Fc receptors. Another requirement for successful ADCC is based on the structure of the target antigen and the proximity of the specific binding epitope to the cell membrane. In the case of CSPG, most if not all mouse antibodies bind to regions of the protein distal to the membrane and do not mediate ADCC.
  • Figure 19 shows sequence homology between human, mouse and a potential canine D3 domain of CSPG. Examples of a fusion protein sub-fragment of canine CSPG D3 sequence and a sequence of a nucleic acid encoding a fusion protein sub-fragment of canine CSPG D3 are provided in SEQ ID NO: 27 and 28.
  • the protein sequences disclosed herein can be modified for use as an Fc fusion protein by adding a functional leader sequence to the amino terminus of each sequence and the coding sequence of the hinge region of a mouse Fc fragment up to the point where there is a unique restriction site for joining to an Fc encoding expression vector.
  • the mouse gamma 2a hinge sequence has a unique Apa I site that is useful for joining the antigen encoding DNA fragment.
  • gag,ccc,aga,ggg,ccc SEQ ID NO: 29
  • the commas denote the correct reading frame of this hinge region.
  • Antibodies that have been obtained from an immunized animal are screened using standard techniques. Typically, an antibody is screened for binding to the antigen against which the antibody was originally raised, e.g., using an ELISA assay. An antibody is typically also evaluated for its ability to bind to the antigen (cell surface antigen) in situ. Binding of antibodies to antigens in situ may be evaluated using standard immunocytochemistry methods, e.g., microscopy, FACS analysis, etc. The antibodies are often evaluated for their ability to specifically bind cells of a tumor, e.g., dog melanoma cells or human melanoma cells, either in vivo or ex vivo, using standard methods known in the art.
  • a tumor e.g., dog melanoma cells or human melanoma cells
  • the antibodies may also be evaluated to determine whether or not binding to an antigen is species specific. In some embodiments, antibodies are selected that recognize an antigen across multiple species. In some embodiments, antibodies are selected that recognize an antigen of a single or limited number of species (e.g., dog and human; cat and human; dog, cat and human, etc.).
  • a useful target antigen for human epithelial cancer treatment is the epithelial cell adhesion molecule (EpCAM) that is widely expressed in most epithelial human cancers and recently shown to be expressed on the cancer stem cells of these tumor types.
  • EpCAM epithelial cell adhesion molecule
  • Antibodies that are capable of mediating effector functions such as ADCC and immunocytokines derived from such antibodies would be useful for the treatment of animals with these diseases.
  • the coding sequence of canine EpCAM has not been reported and was not available from database searches. However a genomic fragment from shotgun sequencing of the canine genome (EMBL-EBI accession # AAEX02022975) was obtained using a homology search with the second exon of the human EpCAM gene, and exons 2 through 9 could be identified by homology to the human exons.
  • exons 2-9 of the canine gene were assembled into a continuous sequence representing the majority of the predicted mRNA and this was translated into the predicted canine EpCAM molecule.
  • the homology between this sequence and the sequences of other mammalian counterparts is shown in Figure 20.
  • the extensive pattern of cyteine residues that is highly conserved between species is evident in the canine sequence and further supports the authenticity of this gene as encoding EpCAM.
  • exon 1 was not contained in this sequence due to the limited 5' sequence (2 Kb) upstream of exon 2 and a homology search of the available genome sequence with exon 1 did not result in any significant homology.
  • exons 1 and 2 of the human gene are approximately 4 kB, and this is consistent with the lack of intron 1 in the dog genomic fragment.
  • Exons 2 through 9 encode all but the first two amino acids (QE in the human form- residues 24 and 25 in Figure 20) of the mature form of the EpCAM protein.
  • the first two amino acid residues of the human molecule could be added to the construct for expression of canine EpCAM on a syngeneic mouse cell line (e.g. NS/0 myeloma cells), together with a functional leader sequence.
  • the transfected cells can be used to immunize mice and then to screen hybridoma clones supernatants for reactivity with canine EpCAM, as described above for CD20.
  • the extracellular domain of canine EpCAM could be expressed as a soluble protein and used for immunization and screening.
  • Such a soluble form could be produced as a fusion protein with an Fc fragment for high level expression and ease of purification using protein A sepharose, preferably using an Fc region derived from mouse IgG so that the Fc fragment itself does not elicit a strong immune response, thus steering the immune system to react predominantly to the canine EpCAM. It is also possible synthesize peptides or to express smaller portions of the EpCAM molecule, alone or as a fusion protein to generate antibodies reactive with that portion (e.g. N terminal or C-terminal part of the EpCAM ectodomain). Sequences are provided for a predicted protein encoding sequence derived from exons 2 through 9 of the proposed canine EpCAM in SEQ ID NO: 29 and 30.
  • SEQ ID NO:6 Nucleotide sequence encoding the dog IgG A H chain fusion to dog IL-2 portion of SEQ ID NO: 5 (the nucleotide sequence encoding the mouse 14.18 H chain variable region is known in the art and can be found in published documents, including US Patent 7,169,904, the sequence-related contents of which are incorporated herein by reference in their entirety).
  • VPDRFSGSGSGTDFTLKiSR VEAEDLGVYFCSQSTHVPPL TFGAGTKLELKKN ⁇ AQ PAVYLF
  • SEQ ID NO:8 Nucleotide sequence encoding Dog c kappa portion of SEQ ID NO: 7 (the nucleotide sequence encoding the mouse 14.18 L chain variable region is known in the art and can be found in published documents, including US Patent 7,169,904, the sequence- related contents of which are incorporated herein by reference in their entirety).
  • CAAAAAGTCCTTTAAATGGCTGCAAAGATTGAAACAAAAACTTTGTTAAGACGTGGGAACTC AAGAGAAACTCAAGATTGTGGAGATTATAAATCTGTTTCTTGGCCTCCCTCATTGCCACACA GAATAAGCTGCTCTATCTGTCCTTTCCGGGCCCTGGGGTTGCCCACAAACAGTACACCCAAG TGGAGAACTTCCCTGTTACTTAACGACCATTCTGTGTGCTTCCTTCTGCAGGGAATGATGCC CAGCCAGCCGTCTATTTGTTCCAACCATCTCCAGACCAGTTACACACAGGAAGTGCCTCTGT TGTGCTTGCTGAATAGCTTCTACCCCAAAGACATCAATGTCAAGTGGAAAGTGGATGGTG TCATCCAAGACACAGGCATCCAGGAAAGTGTCACAGAGCAGGACAAGGACAGTACCTACAGC CTCAGCAGCACCCTGACGATGTCCAGTACCGAGTACCTAAGTCATGAGTTGTACTCCTGTGA GATCACTCACAAGCCTGCCCAGTGG
  • Dog IgG-B replacement fragment acgcgtgtcacatggcaccacctctcttgcagCCTCCACCACGGCCCCCTCGGTTTTCCCAC TGGCCCCCAGCTGCGGGTCCACTTCCGGCTCCACGGTGGCCCTGGCCTGCCTGGTGTCAGGC TACTTCCCCGAGCCTGTAACTGTGTCCTGGAACTCCGGCTCCTTGACCAGCGGTGTGCACAC CTTCCCGTCCGTCCTGCAGTCCTCAGGGCTCTACTCCCTCAGCAGCATGGTGACAGTGCCCT CCAGCAGGTGACAGTGCCCT CCAGCAGGTGACAGTGCCCT CCAGCAGGTGGCCCAGCGAGACCTTCACCTGCAACGTGGCCCACCCGGCCAGCAAAACTAAA GTAGACAAGCCAGTGCCCAAAAGAGAAAATGGAAGAGTTCCTCGCCCACCTGATTGTCCCAA ATGCCCAGCCCCTGAAATGCTGGGAGGGCCCTCGGTCTTCATCTTTCCCCCGAAACCCAAGG
  • feline H chain C gamma 1 region sequence acgcgtgtcacatggcaccacctctcttgcagCCTCCACCACGGCCCCATCGGTGTTCCCAC
  • Flanking Xma I and Fse I sites are underlined. The first residue of the mature IL-2 molecule is in bold type. Protein encoding sequences are in upper case and untranslated sequences are in small case.
  • SEQ ID NO: 24 Canine CD20 loop DNA sequence: gacatatttaatattaccattTGTcatttcttcaagatggagaatttgaatctgattaaggc tcccatgccatatgttTGTatacacaactgtgacccagctaacccctctgagaaaaactctt tgtctatacaatattgtggcagcatacgatct
  • the bold, underlined C residues are replacements to the natural sequence in order to create an artificial loop.
  • SEQ ID NO: 27 A sub-fragment of canine CSPG D3 which is fused to mouse Fc:
  • SEQ ID NO: 28 DNA sequence encoding a fusion protein sub-fragment of canine CSPG D3 gagcagttcacgcagcgggacctggagggcgggaggctggggctgcagctgggccgcgcccc cggccccacgggcgacagcctcacgctggagctgtgggcgcccggcgtggccccccggccgtgg cccctggacttccacaccgagccctacgacgcggcgcgccctacggcgtggccctgctc agcctcccgagcctccccgagcctccccgaggaagcggcacccgacagcggcgcccccggccacgggccagccgggcgcgccagggccagccggg
  • SEQ ID NO: 30 Predicted protein sequence of canine EpCAM lacking residues 1 and 2 of the mature sequence:
  • Xi, X 4, X 6 and X 7 are each any amino acid
  • X 2 and X 3 are each any amino acid or absent, and
  • Xs is any amino acid other than proline.
  • SEQ ID NO: 37. Cat IgG CPPPEMLGGPSIF
  • X 8 , X9 , and Xio are each independently any amino acid

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Abstract

Provided are recombinant antibodies comprising one or more non-human and/or non- rodent sequences, and methods for their preparation and use. In some embodiments, recombinant antibodies, for example immunocytokines, are provided that include a Cκ region, and methods for their preparation and use. In some embodiments, recombinant antibodies, for example immunocytokines, are provided that have improved Fc-Receptor binding and/or improved ADCC effector function, and methods for their preparation and use. Specifically provided in some embodiments are chimeric mouse-dog anti-GD2-dog-IL2 immunocytokines useful in the treatment of GD2 -expressing cancers in dogs.

Description

CHIMERIC IMMUNOCYTOKINES AND METHODS OF USE THEREOF
RELATED APPLICATIONS
This application claims the benefit under 35 U. S. C. §119(e) of U.S. provisional application serial number 61/211,980, filed April 5, 2009 the entire contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
The invention relates to therapeutic antibodies and antibody-cytokine fusion proteins, and methods for their preparation and use.
BACKGROUND OF THE INVENTION
Immunocytokines (antibody-cytokine fusion proteins) were first reported in the literature in the early 1990s and consisted of whole antibody fusions with cytokines such as lymphotoxin (TNF-β) or interleukin 2 (IL-2). Subsequent studies in GD2-expressing tumor models in mice indicated that the chl4.18 antibody and chl4.18-IL2 immunocytokine both had anti-tumor activity but that the immunocytokine was far more potent than the antibody, even when combined with free IL-2. Sabzevari H et al.. (1994) Proc Natl Acad Sci USA 91:9626-30; Pancook JD et al. (1996) Cancer Immunol Immunother 42:88-92; Becker JC et al. (1996) Proc Natl Acad Sci USA 93:2702-7. In addition, immune-competent mice treated with the immunocytokine, but not the antibody plus IL-2, developed an adaptive immune response dependent on CD8+ T cells that prevented subsequent tumor challenge. Becker JC et al. (1996) J Exp Med 183:2361-6; Becker JC et al. (1996) Proc Natl Acad Sci USA 93:7826-31. Thus, the targeting of IL-2 to the tumor microenvironment induces an antitumor vaccine effect that is not possible with the antibody, either alone or together with the free cytokine. A related humanized immunocytokine, hul4.18-IL2, has recently achieved clinical proof of concept in relapsed neuroblastoma as monotherapy where it induced a significant number of complete responses in patients with no other treatment options. Many publications describe the ability of this molecule to activate several components of the immune system to kill tumor cells and to evoke a long lasting CD8 T cell memory response that resists subsequent tumor challenge. SUMMARY OF THE INVENTION
Aspects of the invention relate to recombinant antibodies containing one or more non- human and/or non-rodent (e.g. , canine, feline) antibody sequences. Recombinant antibodies of the invention are useful for administering to non-human and/or non-rodent animals (e.g., for targeted cytokine therapy, for targeted radiotherapy, for targeted imaging, or for any combination thereof).
Aspects of the invention relate to methods for preparing recombinant antibodies or recombinant antibody fragments (e.g., heavy chain, light chain) having one or more improved properties. In some embodiments, recombinant antibodies are provide that have improved ADCC effector function. In certain embodiments, antibodies are provided that have improved protein A binding. In some embodiments, antibodies are provided that have improved Fc-receptor binding. In some aspects of the invention antibodies are provided that bind specifically with non-human and/or non-rodent antigens. In some embodiments, antibodies are provided that bind specifically with non-proteinaceous antigens, e.g., DNA, polysaccharide.
According to some aspects of the invention, immunocytokines are provided that comprise a non-human immunoglobulin gamma heavy chain. As used herein, the term, "non-human immunoglobulin gamma heavy chain" refers to a gamma heavy chain comprising an amino acid sequence that is unique to a non-human animal compared with the sequence of a gamma heavy chain of a human animal. Typically, a non-human immunoglobulin gamma heavy chain is an immunoglobulin gamma heavy chain of a non- human animal, or a functional fragment thereof. However, a non-human immunoglobulin gamma heavy chain may be a chimeric immunoglobulin gamma heavy chain comprising amino acid sequences corresponding to (derived from) more than one animal, at least one of which is a non-human animal. In some embodiments, the immunocytokines comprise a non- human cytokine. As used herein, a "non-human cytokine" is a cytokine of a non-human animal. As used herein, a "non-human, non-rodent cytokine" is a cytokine of an animal that is not a human and not a rodent. Non-limiting examples of a non-human, non-rodent cytokine include a cytokine of a dog, a cytokine of a cat, a cytokine of a non-human primate, etc.
Aspects of the invention are based in part on the discovery of certain motifs in immunoglobulin heavy chain constant region that mediate Fc-Receptor binding and ADCC effector function. A CPX- motif bridges the hinge and CH2 domain of the immunoglobulin. The CPX-motif has a sequence Of CPXiPX2 X3 X4 X5LGGPSX6X7 (SEQ ID NO: 36). In some embodiments, the CPX-motif mediates Fc-Receptor binding and ADCC effector function. In certain embodiments, an immunoglobulin heavy chain that has a CPX-motif that has an amino acid other than a proline (e.g., a hydrophobic amino acid (e.g., isoleucine, leucine, methionine, etc.)) at its X5 position binds to Fc-Receptor and activates ADCC. A GKX-motif is in the CH2 domain of the immunoglobulin. The GKX-motif has a sequence of GKX8FX9CX10V (SEQ ID NO: 39). In some embodiments, the GKX-motif mediates Fc- Receptor binding and ADCC effector function. In some embodiments, an immunoglobulin heavy chain that has a GKX-motif that has a negatively charged amino acid (e.g., glutamate, aspartate) at its X8 position binds to Fc-Receptor and activates ADCC. In some embodiments, an immunoglobulin heavy chain that has a combination of a CPX-motif that does not have proline at its X5 position and a GKX-motif that has a negatively charged amino acid (e.g., glutamate) at its X8 position binds to Fc-Receptor and activates ADCC.
In some embodiments, an immunoglobulin heavy chain (e.g., a non-human and/or non-rodent immunoglobulin heavy chain) does not have proline at the amino acid position corresponding to amino acid position 116 of the human immunoglobulin gamma- 1 heavy chain constant region (amino acid numbering according to GenBank Accession Number: AAC82527). In some embodiments, an immunoglobulin heavy chain has a hydrophobic amino acid (e.g., leucine, isoleucine, etc.) at the amino acid position corresponding to amino acid position 116 of the human immunoglobulin gamma- 1 heavy chain constant region (amino acid numbering according to GenBank Accession Number: AAC82527). In some embodiments, an immunoglobulin heavy chain has a negatively charged amino acid (e.g., glutamate, aspartate) at the amino acid position corresponding to amino acid position 200 of the human immunoglobulin gamma- 1 heavy chain constant region (amino acid numbering according to GenBank Accession Number: AAC82527). In some embodiments, an immunoglobulin heavy chain does not have proline at the amino acid position corresponding to amino acid position 116 of the human immunoglobulin gamma- 1 heavy chain constant region and has a negatively charged amino acid (e.g., glutamate, aspartate) at the amino acid position corresponding to amino acid position 200 of the human immunoglobulin gamma- 1 heavy chain constant region (amino acid numbering according to GenBank Accession Number: AAC82527).
According to some aspects of the invention it has been discovered that prevalent dog immunoglobulins surprisingly do not stimulate ADCC effector function efficiently. For example, it has been discovered that canine IgG-A has a CPX-motif having a proline at position X5 that is undesirable for inducing ADCC-function. It has also been discovered that canine IgG-B and IgG-C have a GKX-motif having a glutamine at position X8 that is undesirable for inducing ADCC-function. Accordingly, in some embodiments recombinant antibodies (e.g., recombinant dog IgG antibodies) are provided that have improved ADCC effector function. In some embodiments recombinant antibodies that induce ADCC-function have a CPX-motif of canine IgG-B, IgG-C or IgG-D and a GKX-motif of canine IgG-A or IgG-D. In some embodiments, recombinant antibodies that induce ADCC-function have a CPX-motif of canine IgG-B and a GKX-motif of canine IgG-A. In some embodiments, recombinant antibodies that induce ADCC-function have a CPX-motif of canine IgG-C and a GKX-motif of canine IgG-A. In some embodiments, recombinant antibodies that induce ADCC-function have a CPX-motif of canine IgG-D and a GKX-motif of canine IgG-A. In some embodiments, recombinant antibodies that induce ADCC-function have a CPX-motif of canine IgG-B and a GKX-motif of canine IgG-D. In some embodiments, recombinant antibodies that induce ADCC-function have a CPX-motif of canine IgG-C and a GKX-motif of canine IgG-D. In some embodiments, recombinant antibodies that induce ADCC-function have a CPX-motif of canine IgG-D and a GKX-motif of canine IgG-D. In some embodiments, recombinant antibodies may be synthesized by fusing regions from different immunoglobulin proteins. In some embodiments, fusion may be made in a region of sequence identity to avoid generating new sequences at the fusion junction. In some embodiments, recombinant antibodies may be synthesized by mutating one or more residues of a immunoglobulin protein. In some embodiments, recombinant IgG may be produced by mutating the proline at position 1 15 of canine IgG-A (See figure 22 A for amino acid numbering) to a hydrophobic amino acid (e.g., leucine). In some embodiments, recombinant IgG may be produced by mutating the glutamine at position 204 of canine IgG-B (See figure 22A for amino acid numbering) to a negatively charged amino acid (e.g., glutamate). In some embodiments, recombinant IgG may be produced by mutating the glutamine at position 202 of canine IgG-C (See figure 22 A for amino acid numbering) to a negatively charged amino acid (e.g., glutamate). In some embodiments, immunocytokines are provided that comprise the foregoing recombinant antibodies.
In some embodiments, the immunoglobulin gamma heavy chain (e.g., non-human immunoglobulin heavy chain) comprises a CPX-motif that does not have a sequence of CPVPEPLGGPSVL (SEQ ID NO: 32), and a cytokine (e.g., a non-human, non-rodent cytokine). In some embodiments, the immunoglobulin gamma heavy chain (e.g., non-human immunoglobulin heavy chain) comprises a CPX-motif that has a sequence Of CPX1PX2 X3 X4 X5LGGPSX6X7 (SEQ ID NO: 36); wherein X1, X4, X6 and X7 are each independently any amino acid, wherein X2 and X3 are each independently any amino acid or absent, and wherein X5 is any amino acid other than proline; and a cytokine (e.g., a non-human, non-rodent cytokine). In some embodiments, the immunoglobulin gamma heavy chain comprises a GKX-motif that does not have a sequence of GKQFTCKV (SEQ ID NO: 38), and a cytokine (e.g., a non-human, non-rodent cytokine). In some embodiments, the immunoglobulin gamma heavy chain comprises a GKX-motif that has a sequence of GKXsFXgCXi0V (SEQ ID NO: 39); wherein X8, X9, and X]0 are each independently any amino acid; and a cytokine (e.g. , a non-human, non-rodent cytokine). In some embodiments, the immunoglobulin gamma heavy chain comprises a CPX-motif that has a sequence Of CPX1PX2 X3 X4 X5LGGPSX6X7 (SEQ ID NO: 36) and a GKX-motif that has a sequence Of GKX8FX9CX10V (SEQ ID NO: 39); wherein Xi, X41 X6, X7, X8, X9 and Xj0 are each independently any amino acid, wherein X2 and X3 are each independently any amino acid or absent, and wherein X5 is any amino acid other than proline; and a cytokine (e.g., a non-human, non-rodent cytokine). In certain embodiments, Xi is alanine, valine, cysteine or proline. In certain embodiments, X2 is glycine or absent. In one embodiments, X3 is cysteine or absent. In certain embodiments, X4 is glycine or glutamine. In certain embodiments, X5 is leucine, methionine, or serine. In certain embodiments, X6 is isoleucine or valine. In certain embodiments, X7 is phenylalanine or leucine. In some embodiments, the non-human immunoglobulin gamma heavy chain is selected from the group consisting of canine IgB, canine IgC, and canine IgD. In some embodiments, the CPX-motif has a sequence of CP APEMLGGPS VF (SEQ ID NO: 33). In some embodiments, the CPX-motif has a sequence of CPCPGCGLLGGPSVF (SEQ ID NO: 34). In some embodiments, the CPX-motif has a sequence of CPVPESLGGPSVF (SEQ ID NO: 35). In some embodiments, the CPX-motif has a sequence of CPPPEMLGGPSIF (SEQ ID NO: 37). In some embodiments, the constant region of the immunoglobulin gamma heavy chain is a canine or a feline immunoglobulin gamma heavy chain constant region. In some embodiments, the immunoglobulin gamma heavy chain is chimeric. In some embodiments, the immunoglobulin gamma heavy chain comprises a mouse variable region and a feline or canine constant region. In certain embodiments, X8 is aspartate or glutamate. In certain embodiments, X8 is glutamate. In certain embodiments, X9 is lysine or threonine. In certain embodiments, Xj0 is lysine or arginine. In some embodiments, the immunoglobulin gamma heavy chain is a non-human immunoglobulin gamma heavy chain.
In some embodiments, the immunocytokine binds specifically to a tumor antigen. In certain embodiments, the tumor antigen is not a nucleic acid. In certain embodiments, the tumor antigen is not a DNA molecule. In certain embodiments, the tumor antigen is a non- proteinaceous tumor antigen. In certain embodiments, the tumor antigen is a polysaccharide. In certain embodiments, the tumor antigen is a polypeptide. In certain embodiments, the tumor antigen is selected from GD2, CD20, CD 19, CSPG and EpCAM.
In some embodiments, the cytokine (e.g., non-human, non-rodent cytokine) is not IL- 12. In some embodiments, the cytokine is selected from the group consisting of: IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-I l, IL-12, IL-13, IL-14, IL-15, IL-16, IL- 17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, G-CSF, GM-CSF, TNF-β, TGF-β, IFN-γ, and IFN-α/β. In some embodiments, the cytokine (e.g., non-human, non-rodent cytokine) is a cytokine that induces production of natural killer cells. In some embodiments, the cytokine (e.g., non-human, non- rodent cytokine) is a cytokine that induces production of cytotoxic T-CeIIs.
According to some aspects of the invention, immuncytokines are provided that bind specifically to CD20, wherein the immunocytokine comprises a dog or feline Cκ region. In some embodiments, the immunocytokines bind specifically to an extracellular loop of CD20. In certain embodiments, the immunocytokines bind specifically to a peptide having a sequence set forth as:
DIFNITISHFFKMENLNLIKAPMPYVDIHNCDP ANPSEKNSLSIQ YCGSIRS (SEQ ID NO: 89). In certain embodiments, the immunocytokines bind specifically to a peptide having a sequence set forth as:
DIFNITICHFFKMENLNLIKAPMPYVCIHNCDPANPSEKNSLSIQYCGSIRS (SEQ ID NO: 71), wherein the peptide forms a loop between the cysteine at position 8 and the cysteine at position 27. In certain embodiments, the immunocytokines bind specifically to a peptide having a sequence set forth as:
DIFNIAICHFFKMENLNLLKSPKP YICIHTCQPESKPSEKNSLSIKYCDSIRS (SEQ ID NO: 26), wherein the peptide forms a loop between the cysteine at position 8 and the cysteine at position 27.
According to some aspects of the invention, immunocytokines are provided that bind specifically to CSPG, wherein the immunocytokine comprises a dog or feline Cκ region. In certain embodiments, the immunocytokines bind specifically to a peptide having a sequence set forth as:
EQFTQRDLEGGRLGLQLGRAPGPTGDSLTLELWAPGVPPAVASLDFHTEPYDAARPY GVALLSLPEEAGAPDSGAPATGQPGAPGPSPGPTAASGGFLGLLEAN (SEQ ID NO: 27).
According to some aspects of the invention, immunocytokines are provided that bind specifically to EpCAM, wherein the immunocytokine comprises a dog or feline Cκ region. In certain embodiments, the immunocytokines bind specifically to a peptide having a sequence set forth as:
ACICENYKLTTNCSLNINNQCECTSIGAQNSVICSKLATKCLVMKAEMTGTKSGRRA
RPEGAFQNNDGLYDPDCDEKGLFKAKQCNGTTTCWCVNTAGVRRTDKDTEISCTER
VRTYWIIIELKHKTRETPYDTQSLQNALKETLKNRYQLDPKYITNILYENDLITIDLMQ
NSSQKAQNDVDIADVAYYFEKDVKDESLFHSSKMDLRVNGEQLDLDPGRTAIYYVD
EKPPEFSMQGLQAGIIAVIVVVTLAVIAGIVVLVISRKNRMAKYEKAEIKEMGEMHRE
LNA
(SEQ ID NO: 29).
According to some aspects of the invention, methods are provided for producing an immunoglobulin gamma heavy chain constant region. In some embodiments, the methods comprise: (a) identifying a motif (a CPX-motif) in the immunoglobulin gamma heavy chain constant region having between one mismatch and six mismatches with the sequence set forth as: CPAPELLGGPSVF (SEQ ID NO: 31); and (b) producing a mutated version of the immunoglobulin gamma heavy chain constant region having at least one fewer mismatch with the sequence set forth as: CPAPELLGGPSVF (SEQ ID NO: 31). In some embodiments, the motif is at the junction of a heavy chain hinge and a CH2 domain of the constant region. In some embodiments, the immunoglobulin gamma heavy chain constant region is a canine or a feline immunoglobulin gamma heavy chain constant region. In some embodiments, the immunoglobulin gamma heavy chain constant region is a canine immunoglobulin gamma heavy chain constant region of IgG-A, IgG-B, IgG-C or IgG-D. In some embodiments, the motif identified in (a) has a proline in place of the leucine at position 6 of SEQ ID NO: 31. In certain embodiments, the mutated version of the immunoglobulin gamma heavy chain constant region has a substitution at position 6 that is a conservative substitution of leucine. In some embodiments, the mutated version of the immunoglobulin gamma heavy chain constant region has improved Fc-receptor binding compared with the immunoglobulin gamma heavy chain constant region. In some embodiments, the mutated version of the immunoglobulin gamma heavy chain constant region has improved ADCC effector function compared with the immunoglobulin gamma heavy chain constant region. In some embodiments, the mutated version of the immunoglobulin gamma heavy chain constant region has improved protein A binding compared with the immunoglobulin gamma heavy chain constant region. According to some aspects of the invention, a recombinant antibody is provided that comprises an immunoglobulin gamma heavy chain constant region produced by the foregoing method. According to some aspects of the invention, immunocytokines are provided that comprise the foregoing recombinant antibody.
According to other aspects of the invention, methods are provided for targeting a cytokine in a non-human animal. In some embodiments, the targeting methods comprise administering any one of the immunocytokines disclosed herein to a mammal.
According to other aspects of the invention, methods are provided for promoting ADCC in a non-human animal. In some embodiments, the methods for promoting ADCC comprise administering any one of the immunocytokines disclosed herein to a mammal.
In some embodiments, aspects of the invention relate to antibodies with reduced immunogenicity in a mammal (e.g., dogs, cats). Recombinant antibodies can be immunogenic when administered to a mammal, particularly when they contain antibody sequences from another species. Recombinant antibodies fused to cytokines (immunocytokines) can create even greater immunogenicity problems due to the presence of the cytokine. Aspects of the invention relate to recombinant antibodies that contain one or more dog sequences to avoid or reduce immunogenicity-associated problems when administered to dogs.
In some embodiments, recombinant antibodies are immunocytokines and include one or more cytokine proteins or portions thereof. Immunocytokines of the invention generally include an antibody, or an antigen binding fragment or derivative thereof, that is capable of binding specifically to a target antigen, linked to a cytokine. Immunocytokines of the invention may include one or more peptide sequences that are suitable for administration to a non-human and/or non-rodent animal, e.g., dog, cat, etc. In some embodiments, one or more of the antibody, antigen binding fragment or derivative thereof, and/or cytokine contains a dog amino acid sequence or an amino acid sequence that has been modified for administration to a dog. In some embodiments the cytokine is a dog cytokine. In some embodiments the antibody is a dog antibody. In some embodiments a portion of the antibody contains a dog sequence. In some embodiments an immunocytokine of the invention includes a dog heavy chain and/or light chain constant region, or portion thereof. In some embodiments, the invention provides immunocytokines that include a dog Cκ region, as well as methods for preparing same and uses thereof. In some embodiments, the invention provides immunocytokines that include a dog Cj1 region, as well as methods for preparing same and uses thereof.
It should be appreciated that any suitable non-human and/or non-rodent cytokine and antibody combination may be used. It also should be appreciated that a cytokine can be fused to the N-terminus or C-terminus of an antibody heavy chain polypeptide. For example, the C-terminus of the cytokine can be fused to the N-terminus of a heavy chain polypeptide (e.g., to the N-terminus of the heavy chain variable region). Similarly, the N-terminus of the cytokine can be fused to the C-terminus of a heavy chain polypeptide (e.g., to the C-terminus of the heavy chain constant region). In some embodiments, an immunocytokine of the invention can contain a cytokine at both the N-terminus and C-terminus of one or more heavy chain antibody polypeptides. It should be appreciated that although immunocytokines are described herein primarily in the context of antibodies having both heavy and light chains, some embodiments of immunocytokines can include single chain antibodies (e.g., scFy, scFy- Fc, or other single chain antibodies) or other antigen binding peptides instead of a heavy and/or light chain. Accordingly, an immunocytokine may include a cytokine fused to the N- terminus and/or C-terminus of a single chain antibody or other antigen binding peptide. In some embodiments an immunocytokine includes an scFy-Fc-cytokine fusion, wherein the Fc is from a dog or cat constant region. In some embodiments an immunocytokine includes a scFγ-Fc-IL2 protein fusion, wherein the IL2 protein is a dog or cat IL2.
In some embodiments a recombinant antibody heavy chain includes a constant region of a dog IgG A, IgG B, IgG C, or IgG D. In some embodiments a recombinant antibody light chain includes a dog kappa light chain constant region. In some embodiments, the dog kappa light chain constant region is selected to be fused to a kappa light chain variable region (e.g., from a mouse antibody or from an antibody of another species). In some embodiments a recombinant antibody light chain includes a dog lambda light chain constant region.
In some embodiments an immunocytokine includes a dog cytokine. In some embodiments an immunocytokine includes a cytokine fused to an antibody heavy chain, wherein the immunocytokine comprises a light chain having a dog Cκ region.
In some embodiments, an antibody domain of the invention (e.g., a dog antibody domain, a cat antibody domain, etc.) can be fused to an imaging marker (e.g., for use in imaging applications). In some embodiments an antibody domain is labeled or fused to a radiolabeled agent (e.g. , for use in radiotherapy). It should be appreciated that in some embodiments the antibody domain may be labeled and/or fused to the imaging marker and/or radiolabeled agent without including a cytokine in the recombinant molecule. It also should be appreciated that in some embodiments that imaging marker and/or a radiolabeled agent can be a peptide that is used for imaging or labeling. In some embodiments, the antibody domain can include one or more points mutations or a CH2 deletion to shorten the half-life of the antibody (e.g., for use in imaging, or radiotherapy).
In some embodiments an antibody or an immunocytokine is a recombinant protein that can be expressed from a recombinant gene (e.g., that includes coding sequences for the antigen binding and/or cytokine polypeptides fused in frame in the appropriate configuration and under suitable genetic control). Accordingly, embodiments of the invention relate to recombinant nucleic acids that encode an antibody or an immunocytokine. Other embodiments include host cells.
Some embodiments of the invention provide nucleic acids (e.g., isolated nucleic acids) that encode all or a portion of antibodies, antigen-binding domains, cytokines, and/or immunocytokines described herein. In some embodiments, the nucleic acid coding sequences (e.g., of an antibody region and/or a cytokine) are optimized for cloning and/or expression (e.g., in mammalian cells). In some embodiments the nucleic acids are included in vectors (e.g., plasmids) having one or more replication and/or selectable sequences (e.g., origins of replication, antibiotic markers, etc.). Some embodiments of the invention provide host cells transformed with one or more nucleic acids of the invention.
In some aspects of the invention an isolated nucleic acid molecule is provided that comprises a sequence encoding an antibody variable region that binds specifically to a tumor antigen and a sequence encoding a non-human, non-rodent cytokine. In some embodiments, the nucleic acid further comprises a sequence encoding a non-human light chain. In some embodiments, the nucleic acid further comprises a sequence encoding a non-human heavy chain. In some embodiments, a sequence of the nucleic acid is a variant of a naturally occurring sequence, wherein the variant confers reduced ribonuclease mediated degradation of an mRNA encoded by the nucleic acid. In some embodiments, a sequence of the nucleic acid is a variant of a naturally occurring sequence, wherein the variant eliminates at least one adenosine-thymidine rich (AT-rich in the DNA, AU-rich in the mRNA) sequence that is a target for a ribonuclease (e.g. ATTTA). In certain embodiments, at least one AT-rich sequence is in a coding region of the nucleic acid. In certain embodiments, the at least one AT-rich sequence is in a non-coding region of the nucleic acid. In one embodiment, the non- human, non-rodent cytokine is selected from the group consisting of: IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-I l, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL- 32, IL-33, IL-35, G-CSF, GM-CSF, TNF-β, TGF-β, IFN-γ, and IFN-α/β. In one embodiment, the non-human, non-rodent cytokine is not IL- 12.
In some embodiments an immunocytokine is a synthetic protein that is produced using synthetic chemistry techniques.
It should be appreciated that regardless of how an immunocytokine is produced, the different portions of the protein (e.g., the cytokine, the antibody or antigen binding portion, and/or regions thereof) may be fused via one or more linker peptides. In the case of recombinant fusion proteins, the linkers are encoded by nucleic acid sequences that are located, in frame, in between the coding regions for the different immunocytokine portions. In the case of synthetic proteins, the linker peptides are introduced during synthesis. In some embodiments synthetic immunocytokines may include non-peptide linkers that connect the different portions of the protein.
In some embodiments, the immunocytokine is an anti-GD2-dog IL2 immunocytokine that binds specifically to GD2, wherein the immunocytokine includes a dog Cκ region. In one embodiment, the dog Cκ region includes an amino acid sequence comprising SEQ ID NO:1. In one embodiment, the immunocytokine includes a dog CH region, wherein the dog CH region optionally can include an amino acid sequence including SEQ ID NO:2.
In one embodiment, the anti-GD2-dog IL2 immunocytokine includes a heavy chain variable region and a light chain variable region from a mouse anti-GD2 antibody.
In one embodiment, the heavy chain variable region includes an amino acid sequence including SEQ ID NO:3. In one embodiment, the light chain variable region includes an amino acid sequence comprising SEQ ID NO:4. In one embodiment, the heavy chain variable region includes an amino acid sequence including SEQ ID NO:3 and the light chain variable region includes an amino acid sequence including SEQ ID NO:4.
In one embodiment, the anti-GD2-dog IL-2 immunocytokine includes a heavy chain polypeptide including an amino acid sequence including SEQ ID NO: 5 and a light chain polypeptide including an amino acid sequence including SEQ ID NO:7.
The invention in one aspect is an isolated nucleic acid molecule encoding a heavy chain polypeptide of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2, wherein the heavy chain polypeptide includes an amino acid sequence including SEQ ID NO:5. In one embodiment, the nucleic acid molecule includes the sequence SEQ ID NO:6. The invention in one aspect is an isolated nucleic acid molecule encoding a light chain polypeptide of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2 and includes a dog Cκ region, wherein the light chain polypeptide includes an amino acid sequence including SEQ ID NO:7. In one embodiment, the nucleic acid molecule includes the sequence SEQ ID NO: 8.
In one aspect the invention is an isolated vector including any one or more of the foregoing nucleic acid molecules.
In one aspect the invention is a cell including a vector of the invention.
The invention in one aspect is a composition including an anti-GD2-dog IL-2 immunocytokine of the invention.
In some aspects of the invention pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise (i) a therapeutically effective amount of any of one the immunocytokines disclosed herein and (ii) a pharmaceutically acceptable carrier.
In some aspects of the invention methods for potentiating a cell-directed immune response in a non-human and/or non-rodent animal are provided. In some embodiments, the methods comprise administering a pharmaceutical composition comprising a therapeutically effective amount of any one of the immunocytokines disclosed herein and a pharmaceutically acceptable carrier, wherein the immunocytokine binds specifically to an antigen that is expressed on the extracellular surface of a cell in the non-human and/or non-rodent animal. In some embodiments, the cell is a tumor cell and the antigen is a tumor antigen. In some embodiments, the cell is a B-cell and the antigen is CD20. In some aspects of the invention methods for treating cancer in a non-human animal are provided. In some embodiments, the methods comprise: administering a pharmaceutical composition comprising a therapeutically effective amount of any one of the immunocytokines disclosed herein and a pharmaceutically acceptable carrier, wherein the immunocytokine binds specifically to a tumor antigen that is expressed on the extracellular surface of a tumor cell of the cancer. In some embodiments, the methods comprise determining that a tumor antigen is expressed on the extracellular surface of a tumor cell of the cancer; and administering a pharmaceutical composition comprising a therapeutically effective amount of any one of the immunocytokines disclosed herein and a pharmaceutically acceptable carrier, wherein the immunocytokine binds specifically to the tumor antigen. In some embodiments, the methods further comprise administering an anti-cancer compound other than the immunocytokine in combination with the pharmaceutical composition. In some embodiments, the methods further comprise subjecting the non-human animal to any one of the following protocols: CHOP therapy, the Wisconsin-Madison protocol, the AMC protocol and the VELCAP protocol, in combination with administering the pharmaceutical composition.
In one aspect the invention is a method of treating a GD2-expressing cancer in a dog. The method includes the step of administering to a dog having a GD2-expressing cancer an effective amount of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2, wherein the immunocytokine comprises a dog Cκ region, to treat the cancer. In one embodiment, the immunocytokine comprises a dog CH region. In one embodiment, the immunocytokine includes a heavy chain variable region and a light chain variable region from a mouse anti-GD2 antibody, wherein optionally the heavy chain variable region includes an amino acid sequence including SEQ ID NO: 3 and the light chain variable region includes an amino acid sequence including SEQ ID NO:4. In one embodiment, the immunocytokine includes a heavy chain polypeptide including an amino acid sequence including SEQ ID NO: 5 and a light chain polypeptide including an amino acid sequence including SEQ ID NO:7.
In one embodiment the GD2-expressing cancer is selected from the group consisting of melanoma, osteosarcoma, neuroblastoma, and small cell lung cancer.
In one embodiment the GD2-expressing cancer is melanoma.
In one embodiment the GD2-expressing cancer is osteosarcoma.
In some embodiments, antibodies or immunocytokines may be used for human administration and/or therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts a genetically engineered anti-GD2 immunocytokine having mouse antibody variable regions, canine antibody constant regions and canine IL-2.
Figure 2A depicts an expression map for an immunocytokine expression vector containing transcription units for both light and heavy chains together with a transcription unit for the selectable marker gene, dihydrofolate reductase (DHFR).
Figure 2B depicts a strategy for inserting V regions in the light chain.
Figure 2C depicts a strategy for inserting V regions in the heavy chain.
Figure 3 depicts SDS-PAGE analysis of the human chl4.18 antibody and chl4.18-IL2 immunocytokine. Both non-reduced (left side of the gel) and reduced (right side of the gel) samples of purified protein were boiled and run on the gel together with molecular weight markers. The separated L, H, and H-IL2 fusion protein bands are indicated.
Figure 4 depicts a strategy for inserting introns into IgG cDNA sequences to create artificial genes with enhanced expression capacity.
Figure 5 A depicts the sequences for splice donor-acceptor sites at the junction between human Cγi and dog CHl. Nucleic acid sequence is provided along with the amino acid sequences in three reading frames. The proper reading frame is underlined.
Figure 5 B depicts the sequences for splice donor-acceptor sites at the junction between dog CHl and human Cγi. Nucleic acid sequence is provided along with the amino acid sequences in three reading frames. The proper reading frame is underlined.
Figure 5 C depicts the sequences for splice donor-acceptor sites at the junction between human Cγi and the hinge domain (H), and the junction between H and dog CHl. Nucleic acid sequence is provided along with the amino acid sequences in three reading frames. The proper reading frame is underlined.
Figure 6 depicts a homology comparison between dog and cat IgGl H chain sequences.
Figure 7 depicts sequences for splice donor-acceptor sites at the junctions between dog CH2 and CH3 and the cat intron. Nucleic acid sequence is provided along with the amino acid sequences in three reading frames. The proper reading frame is underlined.
Figure 8 depicts the end of the CH3 domain of dog IgG, which includes a stop codon after the Pro-Gly-Lys C-terminus and a unique Fse I restriction site downstream of the stop codon.
Figure 9 depicts dog IL-2 protein and mRNA sequences.
Figure 10 depicts a Canis familiari genomic sequence with 97% identity to a 32 bp Cκ homology probe.
Figure 1 IA depicts a homology comparison between dog and cat Cκ proteins.
Figure 11 B depicts a vector comprising a dog Cκ insert.
Figure 12 depicts a gel analysis of immunocytokines transiently expressed in human 293 T cells. Construct 1 had introns between CHl and H domains as well as between the CH2 and CH3 exons. Construct 2 had only the latter intron in the H chain gene. The arrows denote the H-IL2 fused protein and the L chain. The positive control was the chimeric mouse /human 14.18 antibody. Figure 13 shows binding of dog 14.18-IL2 to GD2+ melanoma cells and detection with labeled anti-dog IL2 antibody. The dog IC produced by transient expression was incubated with human melanoma cells and then detected with either an anti-dog (middle panel) or anti-human antibody (right panel) followed by flow cytometry. The cells in the left panel were incubated with the secondary anti-dog IL2 antibody but not the dog 14.18-IL2 (negative control).
Figure 14 shows bioactivity of dog 14.18-IL2 as measured by 3H incorporation into mouse CTLL-2 cells. Dilutions of the culture supernatant containing transiently expressed dog 14.18-IL2 were tested (red squares) in the same assay as a known amount of purified human 14.18-IL2 (blue diamonds).
Figure 15 shows an alignment of FcR binding sequences at the junction of the hinge and CH2 exons of human and dog IgG H chains. The human IgGl has very high FcR binding and corresponding high ADCC effector function.
Figure 16 shows a western blot analysis of 293 cells transiently transfected with pcDNA3.1-dogCD20 expression vector. Cells were collected 72 hours after transfection and cytoplasmic extracts were prepared and analyzed by SDS-PAGE followed by Western Blotting and detection with a rabbit polyclonal anti-CD20 antisera known to be cross-reactive with CD20 of multiple species. The CD20 protein runs at roughly 33 and 37 kD with the larger species believed to represent the phosphorylated form. These bands are only seen in cells receiving the indicated amounts of the plasmid DNA.
Figure 17 shows protein sequence for a soluble extracellular loop of canine CD20 (SEQ ID NO: 71) fused to mouse Fc. Only the CD20 sequence is shown and includes the introduction of two cysteine residues (bold C). These are predicted to form a disulfide bond and artificial loop.
Figure 18 shows expression of canine CD20 loop as an Fc fusion protein. The left and right panels show SDS-PAGE analysis of Protein A Affinity Enrichment from conditioned media of pdHP-dogCD201oop-mFc (PBC00021). In the left panel, samples were resolved by SDS-PAGE (4-20%) under reducing conditions (100 μM 2-mercaptoethanol) and stained with Coomassie Brilliant Blue R250. Whereas, in the right panel, samples were resolved by SDS-PAGE (4-20%) under non-reducing conditions (100 μM 2- mercaptoethanol) and stained with Coomassie Brilliant Blue R250. The reduced protein (left panel) ran with apparent molecular weight that was half the size of the non-reduced sample (right) demonstrating the dimeric structure of the fusion protein. Figure 19 shows sequence homology (SEQ ID NO: 72) between human (SEQ ID NO: 73), mouse (SEQ ID NO: 74) and a potential canine (SEQ ID NO: 75) D3 domain of CSPG. (human, AAQ62842; mouse, NP_620570)
Figure 20 shows sequence homology (SEQ ID NO: 76) between canine (SEQ ID NO: 77) and other mammalian EpCAM proteins, (human, NP_002345 (SEQ ID NO: 78); mouse, NP_032558 (SEQ ID NO: 79); bovine, NP_001030367 (SEQ ID NO: 80)).
Figure 21 shows results of Protein A pull down assays using a dog specific molecule, dl4.18-IL2, which contains an IgG-B isotype for the H chain constant region (Figure 21 A) or which contains an IgG-A isotype for the H chain constant region (Figure 21B).
Figure 22 shows alignments of portions of various IgG heavy chains with regions associated with ADCC effector function denoted by underline or bracket. Figure 22A shows alignments of portions of canine IgG-A, IgG-B, IgG-C, and IgG-D. Figure 22B shows an alignment of portions of canine IgG-A and feline IgGl.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides antibodies useful for targeting tissues in non-human and/or non-rodent animals (e.g., dogs, cats, etc.). Embodiments of the invention are useful for targeting diseased cells (e.g., cancer cells) in non-human animals. Antibodies of the invention may be fused to cytokines (e.g., to form immunocytokines for therapeutic applications), imaging molecules (e.g., for targeted imaging applications), and/or radiolabeled molecules (e.g., for targeted radiotherapy). It should be appreciated that in some embodiments immunocytokines also may be radiolabeled as aspects of the invention are not limited in this respect.
Immunocytokines (ICs) as a class of drug may have a particular utility for the treatment of veterinary cancer due to their low dosing requirements and therefore their low cost of goods. In many cases with human ICs, their anti-tumor activity is greater than that of the antibody from which they are derived. Due to their potential immunogenicity, ICs prepared for human therapy cannot be effectively used for the veterinary market. Therefore it is useful to develop non-human animal specific (e.g., dog and cat) ICs, useful for the treatment of cancers, utilizing coding sequences for the species-relevant immunoglobulin and cytokine components. In one embodiment, the variable regions from mouse antibodies can be used to confer the antigen binding property of tradition monoclonal antibodies. In another embodiment, the variable regions are derived from antibody display libraries, including those generated from the repertoire of the animal to be treated. As used herein, the term "non-human animal" refers to any animal that is not a human. Examples of non-human animals include, but are not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Non-human animals can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), zoo animals (e.g., lions, giraffes, etc.), but are not so limited. Often the non-human animals are mammals. As used herein, the term "non-rodent animal" refers to any animal that is not a rodent.
According to some aspects of the invention, three basic sources of V regions are possible: the first is to use V regions derived from an existing antibody that binds to a non- species-specific antigen (such as a carbohydrate or a glycolipid or a ganglioside) that is expressed on the cancer cells of the animal; a second source is to screen available anti-tumor antibodies against protein antigens for cross-reactivity between the original species (typically human), and the animal (e.g., dog or cat); the third source is to obtain the particular antigen and to use it to create new antibodies either through immunization or through selective binding of a synthetic antibody library. To date, immunocytokines for human use include those specific for gangliosides (e.g., GD2), carbohydrates (e.g., LewisY ), nucleic acids (e.g., tumor necrosis targeting) and several proteins expressed in cancer cells of epithelial origin (e.g., EpCAM, EGFR, Her2) as well as hematological origin (e.g.,CD20, CD 19, Lym-1, CD30, etc.).
In some aspects of the invention, modular expression vectors have been created to produce veterinary ICs. In some embodiments, the modular expression vectors have unique restriction sites flanking the gene fragments encoding the immunoglobulin and cytokine components, allowing the species-specific sequences to be replaced according to the application. For example, a vector containing a set of V regions of a mouse anti-GD2 antibody can be adapted to contain a light chain constant region flanked by BcI I and Not I sites that are unique in the plasmid vector. The originally synthesized vector encoded the dog C kappa region but his can easily be replaced by removing this fragment and replacing it with another BcI I to Not I fragment encoding the cat C kappa sequence. The same process is followed using other unique sites for the heavy chain C region and the cytokine (IL-2) coding sequences.
In some embodiments antibodies can include a dog heavy chain and a dog light chain or a portion thereof (e.g. , a kappa or lambda constant region). In some embodiments antibodies include a variable region from a mouse antibody (optionally humanized or canonized) fused to a constant region from a dog antibody. Mouse variable regions have been identified for many different antigens. Since most mouse antibodies have kappa light chains, a mouse light chain variable region should generally be fused to a kappa constant region from a dog antibody for optimal stability and performance of recombinant mouse/dog antibodies. However, in some embodiments, a mouse variable region may be fused to a dog lambda constant region.
In some embodiments immunocytokines include a heavy chain having a variable region (e.g., a mouse variable region, or a dog variable region, or any other suitable variable region) fused to a dog heavy chain constant region (e.g., full length, or containing one or more point mutations and/or deletions) fused to a cytokine (e.g., a dog cytokine) or a portion thereof. The recombinant heavy chain can be combined with a light chain (e.g., a recombinant light chain) that contains either a lambda or a kappa light chain constant region (e.g. , from dog). However, when the variable region is a kappa variable region, a kappa constant region may be selected (even though a lambda constant region could be used in some embodiments).
In some embodiments, immunocytokines are provided that bind specifically to tumor antigens. As used herein, the term "tumor antigen" refers to a substance produced directly or indirectly by a tumor cell that induces a specific immune response in a host to the substance. Typically, the tumor antigen is expressed on the extracellular surface of a tumor cell. In some embodiments, the tumor antigen is a non-human homologue of a human tumor antigen. In some embodiments, the tumor antigen is GD2-ganglioside, CD 19, CD20, EPCAM, or CSPG. Other suitable tumor antigens include, for example, pi 85 HER2/neu (erb-Bl; Pisk et al., J. Exp. Med., 181 :2109-2117 (1995)); epidermal growth factor receptor (EGFR) (Harris et al., Breast Cancer Res. Treat, 29 : 1-2 (1994)); carcinoembryonic antigens (CEA) (Kwong et al., J. Natl. Cancer Inst., 85:982-990 (1995); carcinoma-associated mutated mucins (MUC- 1 gene products; Jerome et al.,. J. Immunol., 151 :1654-1662 (1993)); E7 and E6 proteins of human papillomavirus (Ressing et al., J. Immunol, 154:5934-5943 (1995)); prostate specific membrane antigen (PSMA Israeh, et al., Cancer Res., 54:1807-1811 (1994)); and idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes (Chen et al., J. Immunol., 153 : 4775-4787 (1994)).
Still, the invention is not limited to immunocytokines that bind tumor antigens. For example, autoimmune diseases (e.g., Rheumatoid Arthritis, Multiple Sclerosis, Crohn's Disease, Psoriasis, etc.) may be treated using immunocytokines that bind specifically to a cell surface antigen of a cell that mediates an autoimmune response (e.g., a CD20 antigen, alpha-4 (<x4) integrin, CDl Ia, etc.). In some embodiments immunocytokines are provided for killing GD2-expressing malignant cells in dogs. Immunocytokines of the invention can be used in dogs in order to characterize their clinical efficacy in vivo. Preclinical data obtained from such studies are useful for the development of therapeutic agents for use in veterinary medicine, as well as further development and use of hul4.18-IL2 in human subjects in some embodiments.
In some embodiments, immunocytokines of the invention generally include an anti- GD2 antibody that is fused to at least one dog IL-2 polypeptide. The anti-GD2 antibody of the immunocytokine binds specifically to GD2 and is characterized in part by the presence of a dog kappa light chain constant (Cκ) region. In one embodiment, at least one heavy chain of the anti-GD2 antibody of the immunocytokine is fused to a dog IL-2 polypeptide. In one embodiment, the C-terminal amino acid of at least one heavy chain of the anti-GD2 antibody of the immunocytokine is covalently linked to the N-terminal amino acid of a dog IL-2 polypeptide.
In one embodiment the anti-GD2 antibody of the immunocytokine is an IgG antibody. An IgG antibody is a tetramer that includes two heavy chains and two light chains, each heavy chain being linked to the other heavy chain and also to one light chain. Each heavy chain includes an N-terminal variable (VH) region linked to a C-terminal constant (CH) region. The two heavy chains are linked to each other through one or more disulfide bonds between the respective CH regions. Each light chain includes an N-terminal variable (VL) region linked to a C-terminal constant (CL) region. The light chain can be a kappa chain or a lambda chain, depending on its VL and CL regions. A kappa light chain includes a Vκ and a Cκ region, while a lambda light chain includes a Vχ and Cλ region. Each heavy chain is linked to one light chain through one or more disulfide bonds between the CH region and the CL (e.g., CK) region.
The VH and VL regions of an antibody determine the antigen specificity and affinity of the antibody. Together, the CH regions, in part, define the Fc portion of the antibody that is capable of directing effector functions antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). In a chimeric antibody, the VH and VL regions of a first antibody are substituted for the VH and VL regions of a second antibody, resulting in an antibody with the antigen specificity of the first antibody and the effector function characteristics of the second antibody.
In some embodiments, an immunoglobulin is provided that has a CPX-motif that bridges the hinge and CH2 domain of the immunoglobulin. The CPX-motif has a sequence Of CPXiPX2 X3 X4 X5LGGPSX6X7 (SEQ ID NO: 36). Certain non-limiting examples of CPX motifs are shown in Figure 15. In some embodiments, the CPX-motif mediates Fc- Receptor binding and ADCC effector function. In certain embodiments, an immunoglobulin heavy chain that has a CPX-motif that has an amino acid other than a proline (e.g., a hydrophobic amino acid (e.g., isoleucine, leucine, methionine, etc.)) at its X5 position binds to Fc-Receptor and activates ADCC. In some embodiments, a GKX-motif is in the CH2 domain of an immunoglobulin. The GKX-motif has a sequence Of GKXgFXgCXi0V (SEQ ID NO: 39). In some embodiments, the GKX-motif mediates Fc-Receptor binding and ADCC effector function. In some embodiments, an immunoglobulin heavy chain that has a GKX- motif that has a negatively charged amino acid (e.g., glutamate, aspartate) at its X8 position binds to Fc-Receptor and activates ADCC. In some embodiments, an immunoglobulin heavy chain that has a combination of a CPX-motif that does not have proline at its X5 position and a GKX-motif that has a negatively charged amino acid (e.g., glutamate) at its X8 position binds to Fc-Receptor and activates ADCC.
In some embodiments, antibodies containing light chain variable and constant regions derived from different species (e.g., a variable region from mouse, whether humanized or not, and a constant region from dog) are more stable and/or have higher performance characteristics when the variable and constant regions are of the same isotype (e.g., matched to both be either kappa or lambda). Mouse antibody light chains are typically kappa light chains, whereas dog antibody light chains are typically lambda light chains. Accordingly, in some embodiments a mouse variable region (e.g., a mouse kappa variable region) is fused to a dog kappa constant region. The variable region of the 14.18 antibody is a kappa variable region and in some embodiments is fused to a dog kappa constant region or a portion thereof.
In one embodiment the anti-GD2 antibody of the immunocytokine is a chimeric mouse-dog IgG antibody, with anti-GD2 specificity provided by the variable regions of murine monoclonal anti-human GD2 antibody 14.18 and the remainder of the antibody provided by constant regions of dog IgG, including dog Cκ.
Dog IgG includes at least four subtypes, IgG A, IgG B, IgG C, and IgG D. Of these dog IgG subtypes, dog IgG A is highly similar to human IgGl . Tang L et al. (2001) Vet Immunol Immunopath 80:259-70. In one embodiment the anti-GD2 antibody of the immunocytokine is a chimeric mouse-dog IgG antibody, with anti-GD2 specificity provided by the variable regions of murine monoclonal anti-human GD2 antibody 14.18 and the remainder of the antibody provided by constant regions of dog IgG A, including dog Cκ. In one embodiment, the C-terminal amino acid of at least one heavy chain of the chimeric mouse-dog anti-GD2 IgG A antibody of the immunocytokine is covalently linked to the N-terminal amino acid of a dog IL-2 polypeptide. In one embodiment, the C-terminal amino acid of at least one heavy chain of the chimeric mouse-dog anti-GD2 IgG A antibody of the immunocytokine is genetically linked to the N-terminal amino acid of a dog IL-2 polypeptide.
In one embodiment, the C-terminal amino acid of each heavy chain of the chimeric mouse-dog anti-GD2 IgG A antibody of the immunocytokine is covalently linked to the N- terminal amino acid of a dog IL-2 polypeptide. In one embodiment, the C-terminal amino acid of each heavy chain of the chimeric mouse-dog anti-GD2 IgG A antibody of the immunocytokine is genetically linked to the N-terminal amino acid of a dog IL-2 polypeptide.
In certain embodiments, the anti-GD2 antibody and the IL-2 component parts of the immunocytokine are linked such that anti-GD2 antibody of the immunocytokine binds specifically to GD2 via its V regions, and the dog IL-2 of the immunocytokine is capable of signaling through an IL-2 receptor.
As used herein, the term "binds specifically" means that the immunocytokine or recombinant antibody is capable of specific binding to its target antigen in the presence of the antigen under suitable binding conditions known to one of skill in the art. In some embodiments, the immunocytokine or recombinant antibody has an affinity constant, K3 in a range of 107 NT1 to 108 M'1, 108 IVT1 to 109 M"1, 109 M"1 to 1010 M"1, 1010 Nf1 to lθ" M"1, or 1011 M"1 to 1012 M"1. In some embodiments, the immunocytokine or recombinant antibody has an affinity constant, K3 of at least 107 M'1, at least 108 M"1, at least 109 M"1, at least 1010 M-1, at least 1011 M'1, or at least 1012 M'1.
In some embodiments, "binds specifically" means that at least 90 percent of antibody- antigen immune complexes formed when the antibody is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include a specified antigen. For example, the immunocytokine of the invention is said to bind specifically to GD2 when at least 90 percent of antibody-antigen immune complexes formed when the antibody-containing immunocytokine is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include GD2.
In one embodiment "binds specifically" means that at least 95 percent of antibody- antigen immune complexes formed when the antibody is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include a specified antigen. For example, in one embodiment the immunocytokine of the invention is said to bind specifically to GD2 when at least 95 percent of antibody-antigen immune complexes formed when the antibody-containing immunocytokine is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include GD2.
In one embodiment "binds specifically" means that at least 98 percent of antibody- antigen immune complexes formed when the antibody is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include a specified antigen. For example, in one embodiment the immunocytokine of the invention is said to bind specifically to GD2 when at least 98 percent of antibody-antigen immune complexes formed when the antibody-containing immunocytokine is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include GD2.
In one embodiment "binds specifically" means that at least 99 percent of antibody- antigen immune complexes formed when the antibody is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include a specified antigen. For example, in one embodiment the immunocytokine of the invention is said to bind specifically to GD2 when at least 99 percent of antibody-antigen immune complexes formed when the antibody-containing immunocytokine is contacted with a source of antigens, under conditions suitable for the formation of immune complexes, include GD2.
In some embodiments, immunocytokines of the invention include a dog Cκ region. In one embodiment, the dog Cκ region includes an amino acid sequence provided as SEQ ID NO: 1. Because the sequence for dog Cκ region was not in PubMed/NCI database, the sequence was identified as follows. A cat Cκ cDNA sequence is available as GenBank accession no. AFl 98257. Based on homology comparison of several mammalian Cκ sequences, a common 32 bp sequence was identified and then used to probe the dog genome. A sequence with 97 percent identity was found and the source sequence down loaded. Accession #AAEX02013460 (72,415 nt). The homology was in reverse orientation, so the sequence was reversed and the coding region identified by the open reading frame and by the protein similarity to cat Cκ.
The dog Cκ coding region, identified from the database as above, was translated and the protein compared to cat and human Cκ. Unlike human Cκ, the C-terminal amino acid of dog Cκ is not cysteine. Four additional residues, mostly charged, are added to dog Cκ, and three to the cat protein. In one embodiment, the immunocytokine of the invention includes a dog CH region. In one embodiment, the dog CH region includes an amino acid sequence provided as SEQ ID NO:2. SEQ ID NO:2 is an amino acid sequence from dog IgG A. Tang L et al. (2001) Vet Immunol Immunopath 80:259-70.
In one embodiment, the immunocytokine of the invention includes a heavy chain variable region that includes an amino acid sequence provided as SEQ ID NO:3. SEQ ID NO: 3 is an amino acid sequence of the VH region of the mouse monoclonal anti-GD2 antibody 14.18.
In one embodiment, the immunocytokine of the invention includes a light chain variable region that includes an amino acid sequence provided as SEQ ID NO:4. SEQ ID NO:4 is an amino acid sequence of the VL region of the mouse monoclonal anti-GD2 antibody 14.18.
In one embodiment, the immunocytokine of the invention includes a heavy chain variable region that includes an amino acid sequence provided as SEQ ID NO: 3 and a light chain variable region that includes an amino acid sequence provided as SEQ ID NO:4.
In one embodiment, the immunocytokine of the invention includes a heavy chain polypeptide that includes an amino acid sequence provided as SEQ ID NO:5. SEQ ID NO: 5 is an amino acid sequence of a polypeptide that includes, from its N-terminus to its C- terminus, the VH region of the mouse monoclonal anti-GD2 antibody 14.18 (SEQ ID NO:3), linked to the CH region of dog IgG A (SEQ ID NO:2), linked to dog IL-2 (SEQ ID NO:9).
In one embodiment, the immunocytokine of the invention includes a light chain polypeptide that includes an amino acid sequence provided as SEQ ID NO:7. SEQ ID NO: 7 is an amino acid sequence of a polypeptide that includes, from its N-terminus to its C- terminus, the VL region of the mouse monoclonal anti-GD2 antibody 14.18 (SEQ ID NO:4), linked to the Cκ region of dog IgG (SEQ ID NO:1).
In one embodiment, the immunocytokine of the invention includes a heavy chain polypeptide that includes an amino acid sequence provided as SEQ ID NO: 5 and a light chain polypeptide that includes an amino acid sequence provided as SEQ ID NO:7.
It should be appreciated that the light chain, heavy chain, and cytokine portions of the immunocytokines can be connected with or without an intervening linker {e.g., peptide linker).
A non-limiting schematic representation of an embodiment of an immunocytokine of the invention is shown in FIG. 1. As shown in FIG. 1, in one embodiment the immunocytokine is a covalently linked homodimer composed of two pairs of polypeptides, each pair of polypeptides including (a) a chimeric mouse-dog anti-GD2 immunoglobulin heavy chain linked at its C-terminus to the N-terminus of dog IL-2, and (b) a chimeric mouse-dog anti-GD2 immunoglobulin light chain that includes a dog Cκ region.
The invention in one aspect is an isolated nucleic acid molecule encoding a heavy chain polypeptide of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2, wherein the heavy chain polypeptide comprises an amino acid sequence comprising SEQ ID NO: 5. In one embodiment the nucleic acid molecule includes the sequence SEQ ID NO:6. As described in greater detail in Examples 1 and 5 below, SEQ ID NO: 6 includes, from its 5' end to its 3' end, sequence for a human Cγl intron, dog CHl cDNA, human Cγl intron, dog hinge and CH2 cDNA, cat CH2-CH3 intron, and dog CH3 cDNA fused in correct translational reading frame to an artificial dog IL-2 coding sequence. Dog IL-2 protein and cDNA sequences are available from Knapp et al. (1995) Gene 159:281-2. Due to the instability of cytokine mRNAs, the mature dog IL-2 sequence was reverse transcribed to a coding sequence using mammalian optimized codon usage.
The invention in one aspect is an isolated nucleic acid molecule encoding a light chain polypeptide of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2 and comprises a dog Cκ region, wherein the light chain polypeptide comprises an amino acid sequence comprising SEQ ID NO:7. In one embodiment the nucleic acid molecule includes the sequence SEQ ID NO: 8. As described in greater detail in Examples 1 and 5 below, SEQ ID NO:8 includes, from its 5' end to its 3' end, a sequence for a dog Cκ gene.
As used herein, an isolated molecule is a molecule that is substantially pure and is free of other substances with which it is ordinarily found in nature or in vivo systems to an extent practical and appropriate for its intended use. In particular, the molecular species are sufficiently pure and are sufficiently free from other biological constituents of host cells so as to be useful in, for example, producing pharmaceutical preparations or sequencing if the molecular species is a nucleic acid, peptide, or polypeptide.
Also provided are vectors useful for expression of an immunocytokine of the invention. In one embodiment the expression vector is suitable for use in mammalian host cells. Mammalian expression vectors can include non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a poly-adenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Commonly used promoters and enhancers are derived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence. A nucleic acid molecule of the invention can be inserted into an appropriate expression vector using standard methods of molecular biology which need not be described in further detail here. The expression vector can include a promoter or promoter/enhancer element that is positioned upstream of the coding nucleic acid molecule that is inserted into the vector. Expression vectors can optionally include at least one coding region for a selection marker and/or gene amplification element, e.g., dihydrofolate reductase (DHFR).
For expression of an immunocytokine of the invention, a vector or vectors containing nucleic acid sequences encoding the various polypeptides of the immunocytokine can be introduced into a suitable host cell or population of host cells. In one embodiment the vector includes a nucleic acid molecule that encodes a heavy chain polypeptide of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2, wherein the heavy chain polypeptide comprises an amino acid sequence comprising SEQ ID NO: 5. In one embodiment the vector includes a nucleic acid molecule that includes the sequence SEQ ID NO:6. In one embodiment the vector includes a nucleic acid molecule that encodes a light chain polypeptide of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2 and comprises a dog Cκ region, wherein the light chain polypeptide comprises an amino acid sequence comprising SEQ ID NO:7. In one embodiment the vector includes a nucleic acid molecule that includes the sequence SEQ ID NO: 8.
In one embodiment the vector includes a nucleic acid molecule that includes the sequence SEQ ID NO: 6 and a nucleic acid molecule that includes the sequence SEQ ID NO:8.
The vector or vectors can be introduced into a host cell or cells using any suitable method, including, for example, electroporation, biolistic delivery {e.g., using a gene gun), lipofection, calcium phosphate precipitation, microinjection, viral transduction, nucleofection, sonoporation, magnetofection, and heat shock. Such methods are well known by persons skilled in the art and need not be described here. Following introduction of the vector or vectors into the host cell or cells, the cell or cells are maintained under physiologically suitable conditions suitable for in vitro cell culture, for a period of time sufficient to permit the cell or cells to express the immunocytokine.
As used herein, a host cell is a eukaryotic cell. In one embodiment the host cell is a mammalian cell. In one embodiment the host cell is a mammalian cell line. In one embodiment the mammalian cell line is non-Ig-secreting myeloma such as NS/0 or Sp2/0- Agl4. In one embodiment the mammalian cell line is HEK293. In another embodiment the mammalian cell line is a Chinese hamster ovary (CHO) line. These and other suitable host cells are available from American Type Culture Collection (ATCC) (Manassas, VA).
In one embodiment the immunocytokine is secreted into the culture medium by the cells containing the expression vector or vectors. Secreted expressed immunocytokine can be readily isolated from culture by centrifugation (to remove cells) followed by immunoaffinity separation, for example using protein A or protein G chromatography. In one embodiment the immunoaffinity separation can alternatively or in addition involve an anti-cytokine antibody, e.g., and anti-IL-2 antibody, as the immunoaffinity reagent.
Also provided are compositions that include an immunocytokine of the invention. In one embodiment, the composition is a pharmaceutical composition that includes an immunocytokine of the invention and a pharmaceutically acceptable carrier.
The term "pharmaceutically-acceptable carrier" means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with other compounds, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
Immunocytokines of the invention can be used to treat cancers (e.g, GD2-expressing cancers) in non-human animals {e.g., dogs, cats, etc.). As used herein, "cancer" refers to an abnormal uncontrolled growth of cells in a subject. The term "cancer" as used herein can refer to solid tumors, primary as well as metastatic cancers, as well as hematogenous ("liquid") cancers.
GD2-expressing cancers are cancers that have detectable GD2 expressed on their cell surface. GD2-expressing cancers are generally cancers of neuroectodermal origin and specifically can include, without limitation, melanoma, neuroblastoma, osteosarcoma, and small cell lung cancer. As used herein, "to treat" means to slow or halt the progression of, or to reduce or eliminate, a disease in a subject having the disease. A subject having a disease is a subject that has at least one objectively identifiable manifestation of the disease. For example, a dog having a GD2-expressing cancer is a dog that has at least one objectively identifiable manifestation of a GD2-expressing cancer.
Certain embodiments of the invention also include methods of treating a GD2- expressing cancer in a dog by administering to a dog having a GD2-expressing cancer an effective amount of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2, wherein the immunocytokine comprises a dog Cκ region, to treat the cancer. In one embodiment the immunocytokine of the invention can be administered alone to a dog having a GD2-expressing cancer to treat the cancer. As used herein, an "effective amount" refers to the amount necessary or sufficient to realize a desired biologic effect. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side- effects and preferred mode of administration, an effective therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular active agent being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular active agent and/or other therapeutic agent without necessitating undue experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to some medical judgment, although this is not necessarily the case for immune-stimulating agents. Multiple doses per week may be contemplated to achieve appropriate systemic levels of compounds. Appropriate systemic levels can be determined by, for example, measurement of the subject's peak or sustained plasma level of the drug. "Dose" and "dosage" are used interchangeably herein.
Generally, daily doses of active compounds will be from about 0.01 milligrams/kg per day to 10 milligrams/kg per day. It is expected that intravenous doses in the range of 0.05 milligrams/kg per day to 5 milligrams/kg per day, in one or several administrations per day, will yield the desired results. Similarly, it is expected that subcutaneous doses in the range of 0.05 milligrams/kg per day to 5 milligrams/kg per day, in one or several administrations per day, will yield the desired results. Dosage may be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
For any immunocytokine described herein, the therapeutically effective amount can be initially determined from animal models. A therapeutically effective dose can also be determined from human data for immunocytokines which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.
In some embodiments, the immunocytokine may be administered in combination with a therapeutic antibody {e.g., a CD20 antibody). In one embodiment the immunocytokine of the invention can be administered in conjunction with at least one other anti-cancer treatment agent or anti-cancer treatment modality to treat the cancer. As used herein, "in conjunction with" or "in combination with" refers to any suitable form of combination therapy, for example simultaneous, overlapping, and/or sequential treatments. Anti-cancer treatment agents {e.g., anti-cancer compounds) and anti-cancer treatment modalities other than treatment with an immunocytokine of the invention can include chemotherapy (including combination chemotherapy), radiation therapy, surgery, other immunotherapy, and any combination thereof. In some embodiments, the anti-cancer treatment is local radiation or radiofrequency ablation. Anti-cancer treatments such as cyclophosphamide, doxorubicin, valinomycin, hormone therapy, and other therapies disclosed herein or otherwise known in the art may be used.
As used herein, an "anti-cancer compound" refers to an agent which is administered to a subject for the purpose of treating a cancer. Anti-cancer compounds include, but are not limited to antiproliferative compounds, anti-neoplastic compounds, anti-cancer supplementary potentiating agents and radioactive agents. One of ordinary skill in the art is familiar with a variety of anti-cancer compounds. Examples of anti-cancer compounds include, but are not limited to, the following: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Adriamycin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Buniodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorombucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; DACA (N-[2- (Dimethyl-amino)ethyl]acridine-4-carboxamide); Dactinomycin; Daunorubicin Hydrochloride; Daunomycin; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Ifesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; 5-FdUMP; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta-la; Interferon Gamma-lb; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin,. Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safmgol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Thymitaq; Tiazofurin; Tirapazamine; Tomudex; TOP-53; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine; Vinblastine Sulfate; Vincristine; Vincristine Sulfate, Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride; 2-Chlorodeoxyadenosine; 2'-Deoxyformycin; 9-aminocamptothecin; raltitrexed; N-propargyl-5, 8-dideazafolic acid, 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine; 2-chloro-2'-deoxyadenosine; anisomycin; trichostatin A; hPRL-G129R; CEP-751 ; linomide; Piritrexim Isethionate; Sitogluside; Tamsulosin Hydrochloride and Pentomone. Radioactive agents may also be used. Examples of radioactive agents include but are not limited to Fibrinogen I 125; Fludeoxyglucose F18; Fluorodopa F 18; Insulin I 125; Insulin I 131; Iobenguane I 123; Iodipamide Sodium 1 131; Iodoantipyrine 1 131; Iodocholesterol I 131; Iodohippurate Sodium 1 123; Iodohippurate Sodium I 125; Iodohippurate Sodium 1 131 ; Iodopyracet I 125; Iodopyracet 1 131 ; Iofetamine Hydrochloride I 123; Iomethin I 125; Iomethin 1 131; Iothalamate Sodium I 125; Iothalamate Sodium 1 131; Iotyrosine 1 131; Liothyronine I 125; Liothyronine 1 131; Merisoprol Acetate Hg 197; Merisoprol Acetate- Hg 203; Merisoprol Hg 197; Selenomethionine Se 75; Technetium Tc 99m Atimony Trisulfide Colloid; Technetium Tc 99m Bicisate; Technetium Tc 99m Disofenin; Technetium Tc 99m Etidronate; Technetium Tc 99m Exametazime; Technetium Tc 99m Furifosmin; Technetium Tc 99m Gluceptate; Technetium 99m Lidofenin; Technetium Tc 99mm Mebrofenin; Technetium Tc 99m Medronate; Technetium Tc 99m Medronate Disodium; Technetium Tc 99m Mertiatide; Technetium Tc 99m Oxidronate; Technetium Tc 99m Pentetate; Technetium Ic 99m Pentetate Calcium Trisodium; Technetium Tc 99m Sestamibi; Technetium Tc 99m Siboroxime; Technetium Tc 99m Succimer; Technetium Tc 99m Sulfur Colloid; Technetium Tc 99m Teboroxime; Technetium Tc 99m Tetrofosmin; Technetium Tc 99m Tiatide; Thyroxine I 125: Thyroxine I 131 ; Tolpovidone 1 131; Triolein I 125; Triolein 1 131.
In some embodiments, an immunocytokine is administered in combination with (before, during or after) CHOP therapy. As will be appreciated by the skilled artisan, CHOP therapy is a common chemotherapy protocol for treating cancer {e.g., lymphoma (e.g., Non- Hodgkin lymphoma)). The following drugs are typically used in the CHOP protocol: Cyclophosphamide, Adriamycin, Vincristine, and Prednisone. Immunocytokines may also be administered in combination any one of a variety of alternative cancer therapy protocols that may differ from CHOP therapy in terms of scheduling, dosages and use of other chemotherapeutics. Alternative protocols include, but are not limited to, the Wisconsin- Madison protocol, AMC protocol and VELCAP protocol. Non-limiting examples of cancer therapy regimens with which the immunocytokines may be administered in combination are disclosed in: LΗeureux DA, Moore AS et al. Evaluation of a Discontinuous Protocol (VELCAP-S) for Canine Lymphoma. J Vet Intern Med 2001; 15:348-354; Ogilvie GK, Berman PJ. Drug Resistance and Cancer Therapy. Compendium 1995; 17:549-556; Page RL, Lee JJ et al. P-Glycoprotein Expression in Canine Lymphoma. Cancer 1996;77: 1892-1898; Leifer CE, Calvert CA. Doxorubicin for Treatment of Canine Lymphosarcoma After Development of Resistance to Combination Chemotherapy. JAVMA 1981;179:1011-1012; Madewell BR, Lucroy MD et al. Evaluation of Single- Agent Mitoxantrone as Chemotherapy for Relapsing Canine Lymphoma. J Vet Inter Med 1998;12:325-329; Mooney SC, Rassnick KM. MOPP chemotherapy for the treatment of resistant lymphoma in dogs: a retrospective study of 177 cases (1989-2000). J Vet Intern Med 2002; 16:576-580; Cuoto GC, Alvarez FJ et al. Dexamethasone, Melphalan, Actinomycin D, Cytosine Arabinoside (DMAC) Protocol for Dogs with Relapsed Lymphoma. J Vet Intern Med 2006;20:l 178-1183; Van Vechten M, Hefland SC. Treatment of Relapsed Canine Lymphoma with Doxorubicin and Dacarbazine. JVIM 1990; 4:187-191; Laing EJ, Fitzpatrick PJ. Half-Body Radiation Therapy in the Treatment of Canine Lymphoma. J Vet Intern Med 1889: 3:102-108, the contents of each of which are incorporated herein by reference in their entireties.
For use in therapy, the formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).
For use in therapy, an effective amount of the immunocytokine can be administered to a subject by any mode that delivers the immunocytokine to the desired surface. Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to intravenous and subcutaneous.
The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.
EXAMPLES
Introduction to and Overview of Examples 1-3:
GD2 is a disialoganglioside expressed on tumors of neuroectodermal origin, including human neuroblastoma and melanoma, with highly restricted expression on normal tissues, principally to the cerebellum and peripheral nerves in humans. The relatively tumor-specific expression of GD2 makes it an attractive target for immunotherapy, for example with monoclonal antibodies. Melanomas, sarcomas, and neuroblastomas abundantly express GD2 on the cell surface where it is susceptible to immune attack by antibodies. Overexpression of GD2 on these tumors is striking, as is the frequency of clinical responses after treatment of neuroblastoma with monoclonal antibodies against GD2. Similar to other types of cancer, conventional approaches to treatment of various GD2-positive cancers include surgery, radiotherapy, and chemotherapy. Antibodies, including monoclonal antibodies, have been developed for use in treating GD2-positive cancers. A murine monoclonal anti-human GD2 antibody, designated 14.18, was reported by Mujoo and colleagues in 1987. Mujoo K et al. (1987) Cancer Res 47:1098- 104. With the advent of antibody engineering, chimeric and humanized forms of 14.18 were subsequently developed. Gillies S et al. (1989) J Immunol Methods 125:191-202; Mueller BM et al. (1990) J Immunol 144:1382-6. Chimeric mouse-human antibody, chl4.18, was found to have potent effector activities of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), as well as the ability to target GD2-positive melanoma cell xenografts in mice. Mueller et al. (supra).
An anti-GD2 antibody-IL-2 fusion protein (immunocytokine) that is suitable for therapeutic use in dogs with neuroectodermal cancer is genetically engineered. The immunocytokine contains canine immunoglobulin heavy and light chain constant (C) regions and canine IL-2 sequences and is not immunogenic in dogs. Dog antibody and IL-2 sequence information from database sources is adapted for high-level expression in mammalian cells by insertion in a vector containing the mouse 14.18 variable heavy (VH) and variable light (VL) coding sequences, as well as appropriate splicing signals that result in the joining of the V and C regions to create a chimeric mouse-dog molecule after transfection into mammalian cells in culture. The dog IL-2 sequence is fused in-frame to the carboxyl terminus of the chimeric H chain. The result is a whole antibody HL chain dimer containing two molecules of dog IL-2 per antibody. The final vector sequence is assembled using the DNASTAR Lasergene program and the sequence is synthesized.
Biochemical properties of the immunocytokine are tested. These properties include antibody assembly, antigen binding activity and IL-2 bioactivity. The immunocytokine is produced in vitro from cells transfected with expression constructs encoding the immunocytokine. Expression vector DNA is used for transient expression of the protein in human 293 cells using standard protocols. Conditioned media from the cultures serves as a source of immunocytokine material for biochemical analyses to ensure that correctly sized proteins are secreted and that the L chain and H chain-IL2 fusion protein are assembled into a heterodimeric structure. The immunocytokine is captured on protein A Sepharose beads and subsequently analyzed by SDS-polyacrylamide gel electrophoresis. Media samples are used to test for antigen binding activity as well as IL-2 bioactivity, using a standard mouse cell line, CTLL-2, in a proliferation assay. Results are also confirmed using dog peripheral blood monocytic cells (PBMC) in culture. Transient cell cultures are scaled-up and moderate quantities of the dog immunocytokine are purified for further analyses. Multi-milligram quantities of the immunocytokine are purified from cell culture supernatants and captured using standard protein A Sepharose and ion exchange chromatography methods. This material is used to establish a reference standard for biochemical assays and for further characterization of biochemical and biophysical properties such as solubility, aggregation and in vitro stability. The material is used to establish enzyme-linked immunosorbent assay (ELISA) methods necessary for identity and potency assays, as well as for measurement of the immunocytokine in biological samples such as blood, plasma, or serum.
Pharmacokinetic properties are determined in mice. Purified immunocytokine is used to measure concentration vs. time kinetics following intravenous dosing in mice. Blood samples are taken over a 24 hour period and the concentration of immunocytokine is measured by ELISA measuring both the antibody and enzyme-;lL-2 portion of the molecule. This defines the amount of intact immunocytokine present in the samples.
Example 1: Genetically engineered anti-GD2 immunocytokine having mouse antibody variable regions, canine antibody constant regions and canine IL-2.
A mammalian expression vector capable of generating high level production of the anti-GD2 immunocytokine is produced. The chimeric protein is composed of two protein chains: the chimeric mouse-dog light chain; and the chimeric mouse-dog heavy chain fused to dog IL-2 (FIG. 1).
The humanized form of this molecule has been expressed using a vector containing the transcription units for both chains in a single vector, together with a transcription unit for the selectable marker gene, dihydrofolate reductase (DHFR) (FIG. 2A). The two immunoglobulin chain transcription units are each driven by a cytomegalovirus (CMV) promoter and enhancer and utilize a leader sequence derived from the 14.18 light (L) chain. A splice site is used at the end of the intron of the leader sequence so that other V region coding sequences can be inserted into this vector and joined at the RNA level to produce any desired antibody (FIG. 2B). The vector is designed so that V region coding sequences include an AfI II restriction site at their 5' end and a splice donor site at their 3' end. This ensures joining to the leader sequence in the correct reading frame and then correct splicing to the next exon downstream in the vector. To increase expression efficiency, splice donors, introns and splice acceptor sequences adapted from human gene sequences are added at the appropriate positions in the sequence. (See Example 5). After transfection and expression in the cell, these human sequences are removed by splicing and leave only the sequences encoding the dog proteins. It should be appreciated that cDNA molecules also can be used to assemble recombinant genes that are useful to express recombinant proteins of the invention as aspects of the invention are not limited in this respect.
Once all DNA sequences are assembled using the DNASTAR Lasergene 8 program, all coding sequences are checked to ensure there are no errors in coding the correct protein sequences during merging of input sequences. The final sequences are submitted to a contract supply organization with experience in gene synthesis and assembly (e.g., Blue Sky Biotech, Worcester, MA). After assembly, the sequence of the entire plasmid is verified and corrected, if necessary.
Example 2: Testing the biological properties of the immunocytokine such as cytokine activity and antibody effector functions using dog immune effector cells.
Before generating stable cell lines for long-term production of the anti-GD2 immunocytokine, the ability of the vector to express the desired protein is tested using transient expression and analysis of small amounts of the protein secreted from transfected cells. This is accomplished by producing milligram quantities of the plasmid DNA from the bacterial host and purifying the DNA using high resolution chromatography. Endotoxin-free DNA is used to transfect HEK293 cells in suspension culture and after several days of culture, the conditioned culture media is harvested. A small amount is incubated with protein A Sepharose beads by gentle mixing and then the captured protein is eluted in gel electrophoresis buffer. Half of the sample is treated further with reducing agent (β- mercaptoethanol) while the other half is not. Both samples are heated and analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) together with an immunoglobulin control protein. A correctly assembled IL-2 based immunocytokine migrates as a single high molecular weight band on the gel (-200 kD) when it is not reduced, but dissociates into two bands after chemical reduction. An example of such a result is shown in FIG. 3. These bands include an L chain of normal size, and an H-IL2 chain that is larger than the normal IgG H chain by about 16 kD. Once the structural integrity of the immunocytokine is confirmed, the remaining conditioned medium containing the chl4.18-dog IL2 molecule is characterized by performing GD2 binding assays and IL-2 proliferation assays using the standard CTLL-2 mouse T cell assay.
GD2 binding is performed using 96-well plates coated with GD2 (Calbiochem) and blocked with 5% bovine serum albumin (BSA) and 5% goat serum. Test antibody or antibody-containing culture supernatants are diluted in PBS containing 1% BSA and 1% goat serum and incubated in wells for 1 hr at room temperature. Unbound proteins are washed three times with dilution buffer and bound immunocytokine is detected with a horseradish peroxidase (HRP)-conjugated secondary antisera against canine IgG or canine IL-2. Bound HRP is quantitated by standard protocols.
An alternative method for testing GD2 binding is to incubate the test protein with a GD2-expressing cancer cell (e.g. melanoma) and to the detect its binding using a secondary labeled antibody directed against the dog immunoglobulin or cytokine portion (see example 6 below).
IL-2 bioactivity is performed in 96 well plates containing CTLL-2 mouse T cells that have been deprived of IL-2 for 48 hr prior to the assay. Dilution of purified proteins and culture media containing immunocytokines are plated and then mixed with CTLL-2 cells in culture medium and incubated for two days at 37°C. Additional medium containing H- thymidine is added and incubation continued for an additional 16 hr and incorporation measured using standard protocols. The extent to which dog IL-2 induces proliferation via the mouse IL-2 receptor is tested. Dog IL-2 has roughly the same degree of homology to mouse IL-2 as the human cytokine. Human IL-2 is routinely assayed using this mouse cell line (Gillis S, et al., T cell growth factor: parameters of production and a quantitative microassay for activity. J Immunol. 120(6):2027-32, 1978). Dog peripheral blood mononuclear cells (PBMC) are obtained and cultured with recombinant IL-2 to activate them. Concanavalin A is added, if necessary (Kato M, et al., A novel culture method of canine peripheral blood lymphocytes with concanavalin a and recombinant human interleukin-2 for adoptive immunotherapy. J Vet Med Sci. 69(5):481-6, 2007), and the activated cells are washed and added to wells as just described above for the CTLL-2 assay. Thymidine incorporation is used as an indicator of dog cell proliferation and compared to results with recombinant human IL-2. These ELISA and bioactivity assays are summarized in Table 1, below. Table 1 : Assays for ch!4.18-dog IL2
Figure imgf000038_0001
Example 3: Production and purification of sufficient amounts of this protein for further characterization studies.
Based on the estimated amount of immunocytokine produced by cells in Example 2, transient expression in HEK293 cells is scaled to either 1 or 10 L of culture using disposable wave bags. At least 10 mg of purified chl4.18-dog IL2 protein are prepared for further characterization and assay development. In parallel, stable cell line generation is performed in NS/0 mouse myeloma cells using methotrexate as the selection marker. This is performed using linearized plasmid DNA restriction enzyme cut within the bacterial ampicillin resistance (ampR) gene. DNA is introduced into the myeloma cells (or CHO cells) using well established electroporation methods, and the cells are cultured in section medium containing 0.1 μM methotrexate. Drug-resistant myeloma clones are tested for secretion of dog IgG using commercially available anti-dog IgG antisera (Table 1). Expressing clones are tested for productivity, stability and growth rate. Subcloning is used to select for the optimal cell line properties.
The expressed protein, preferably secreted from cells growing in serum free media, is purified using established protocols for producing clinical grade protein. Great care is used to prevent endotoxin contamination. The steps may include a concentration step (e.g. tangential flow filtration), followed by binding to and elution from protein A Sepharose. Dog IgG binds to this reagent under certain conditions of pH and ionic strength. After elution with acidic pH and neutralization, ion exchange chromatography is used as a polishing step. Additional purification methods for dog IgG and related fusion proteins are available from commercial sources [e.g. Dog IgG purification kit (Code : DIKG-FF KIT), Affiland, Ans- Liege, Belgium]. Purified protein is analyzed by SDS-PAGE and potential aggregation is examined by size-exclusion chromatography (SEC). Immunocytokine stability issues associated with aggregation are monitored closely. Current formulations, including lyophilization, that minimize stability issues are applied if necessary.
Example 4: Obtaining pharmacokinetic properties in mice.
Pharmacokinetic properties of the intact immunocytokine molecule are determined. Mice are injected in the tail vein with approximately 10 μg of the chl4.18-dog IL2 molecule and sampled immediately thereafter by retro-orbital bleeding to establish the to point. Additional samples are taken at 15, 30, 60 min, 2, 4, 8, 24 and 48 hr. Serum is prepared from blood samples by standard protocols and stored cold until assayed. A specific ELISA for measuring intact immunocytokine is modified from an existing protocol (Gan J, et al., Specific enzyme-linked immunosorbent assays for quantitation of antibody-cytokine fusion proteins. Clin Diagn Lab Immunol. 6(2):23642, 1999) and is based on capture in a 96-well plate coated with an anti-idiotype antibody (1A7) specific for the 14.18 antibody, followed by detection of any captured protein with an anti-IL2 specific antisera (Table 1). A standard curve is generated using the purified chl4.18-dog IL2 as a control. A time vs. concentration curve is generated and the data are compared to results with a control hul4.18-IL2 molecule. The results help predict the serum half-life in dogs and help determine what doses are useful and safe to administer to dogs. This is most important for doses given intravenously, since the toxicity of both IL-2 and IL-2 based immunocytokines is related to its maximum concentration in the vascular compartment, which in turn is based on the activation of cells bearing the intermediate affinity IL-2 receptor- NK cells and neutrophils (Assier E, et al., NK cells and polymorphonuclear neutrophils are both critical for IL-2-induced pulmonary vascular leak syndrome. J Immunol. 172(12):7661-8, 2004.). If the canine immunocytokine is cleared too slowly, lower doses for pharmacokinetic studies are used. Alternatively, the molecule is administered by infusion, rather than bolus injection, so that lower vascular concentrations are reached and toxicity is minimized.
Example 5: Artificial Gene Design for Dog IgG C regions Introns were designed for insertion into IgG cDNA sequences to create artificial genes with enhanced expression capacity.
- Sources for dog IgG Constant region cDNA were identified. Tang et al. (2001) Vet. Immunol. Immunopath. 80: 259-270 describe 4 γ-chains and argue that one designated "A" is the analogous to human IgG, based on hinge structure and relative abundance. The reported sequences are from cloned cDNAs so the intron sequences are unknown.
FIG. 4 provides a strategy for intron insertions to create artificial genes. Human Cγi intron is inserted between VH and CHl domains, and between CHl and hinge (H) domains. No intron is inserted between H and CH2 domains. A cat Cγi intron analog is inserted between CH2 and CH3.
Details of sequences at splice donor and acceptor sites in artificial C2 dog gene have been determined (FIG. 5A-C). The splice acceptor of the 5' end of dog Cγ domain is similar to human and cat, both of which use a consensus 3' end of the intron. Therefore the corresponding sequence from the human gene was used and fused to the second residue of the Ala codon as shown (FIG. 5A). The splice donor at the end of the CHl exon is also typical and similar to human, so the corresponding intron between CHl and hinge of the human Cγ gene was used (FIG. 5B-C). The junction between H and CH2 in the dog cDNA looks much less typical and hard to predict in terms of an intron insertion. Therefore, the fused sequence with no intron was used to avoid potential problems.
For the CH2-CH3 domain intron, a corresponding sequence has been reported for the Cat IgG gamma chain. Kanai et al. (2000) Vet. Immunol. Immunpath. 73:53-60. A homology analysis of dog and cat domain sequences was performed (FIG. 6). The amino acid and nucleotide sequences are quite similar between dog and cat, so the cat intron for the γja isotype, which is most common, was used for this artificial dog gene. The final sequence representing the dog exon fused with the cat intron was determined (FIG. 7). The proper reading frame is in underlined.
An expression vector is constructed to express both the IL2 immunocytokine with dog IL2 fused to the end of the CH3 domain of dog IgG A, as well as the same molecule lacking IL2, i.e., the antibody alone. This is done by constructing the antibody sequence (including a stop codon after the Pro-Gly-Lys C-terminus) and adding a unique Fse I restriction site downstream. This site can be used to insert a replacement fragment encoding a cytokine (or other molecule) between the unique Sma I site and the Not I site (FIG. 8).
Dog IL-2 protein and mRNA sequences obtained through PubMed Literature Ref. Knapp et al. (1995) Gene 159:281-2 were identified (FIG. 9). Homology analysis indicates an 84% sequence homology to human IL-2. Due to the instability of cytokine mRNAs, the mature protein sequence was reverse translated to a coding sequence using DNA* mammalian optimized codon usage.
Sequences for dog Cκ light chain were investigated. These sequences were not available in PubMed/NCI database. Cat Cκ mRNA was found at accession #AF 198257. A homology comparison of several mammalian CK sequence, including human, mouse, cow, sheep, was performed. This comparison identified a common 32 bp sequence as a probe. A FASTA - genomes similarity search was performed at www.ebi.ac.uk using "canis lupus familiaris" database. A sequence with 97% identity to the 32 bp probe was found and the source sequence was downloaded (Accession #AAEX02013460 - 72,415 nt) (FIG. 10). The homology was in a reverse orientation, so sequence the sequence was reversed and the coding region was identified by the open reading frame and by protein similarity to cat Cκ. The dog Cκ coding region, identified from the database was translated and the protein compared to cat CK (FIG. 11). The splice acceptor site appeared normal.
Example 6: Expression of a dog anti-GD2 — IL2 immunocytokine
First attempts at transient expression of the dog specific molecule (dl4.18-IL2 containing the IgG-A isotype for the H chain constant region) included the transient transfection of human 293T cells and testing the supernatant of these cells after 72 hours for proteins capable of binding to protein A, followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). No proteins were detected in these cultures so the experiment was repeated using a positive control vector encoding the human form of this antibody. Again no dog proteins were detected despite the high-level expression if the human version (confirming the methods were appropriate). One potential source of the problem, based on the use of human intron sequences in constructing the dog heavy chain gene, was examined by removing the intron between the CHl and hinge exons, but this did not resolve the problem.
Another potential source of the problem was the 14.18 L chain V region (Gillies et al 1989, J. Immunol. Methods 125:191-202). It was thought that a hybrid leader sequence- V region cDNA would be useful for expressing the chimeric light chain. To test this, the V region of the modified cDNA was inserted into the dog IC expression vector and used to transfect 293T cells. In this case, protein was detected that corresponded to the correct molecular weight of an intact IC both in the culture supernatant, but it was only slightly enriched in the material first bound and eluted from protein A Sepharose. Upon incubation with a reducing agent, the large molecular weight protein broke down into the appropriately sized H and L chains (Fig. 12).
As shown in Figure 12, which depicts a gel analysis of immunocytokines transiently expressed in human 293T cells, Construct 1 had introns between CHl and H domains as well as between the CH2 and CH3 exons. Construct 2 had only the latter intron in the H chain gene. This result indicates that the correct protein was made but that it did not bind well to protein A. This was true whether or not the expression construct contained the intron between the CHl domain and the hinge sequence or not. The positive control 14.18 antibody was expressed at about the same level as dog proteins from the two different constructs (with and without the additional intron) but was highly enriched using protein A. Additional studies indicated that the dog protein could be enriched using ion exchange chromatography, specifically using SP Sepharose Fastflow and binding in 20 mM Bis Tris pH5.5. It was noted that polyclonal dog IgG has been reported to bind to protein A and protein G but it is not known which H chain isotypes bind most effectively or if some have little or no binding. As mentioned above, the H chain C region used in these first experiments were of the IgG-A isotype).
The functionality of the dog IC was tested by measuring antigen binding to a GD2 expressing cell line. This was done by incubating the culture supernatant containing transiently-expressed dl4.18-IL2 with M21 melanoma cells and then with a biotinylated anti- dog IL-2 antibody. Binding was visualized by incubation with streptavadin-FITC followed by flow cytometry. Control incubations indicated that the fluorescence was dependent on the IC as well as the anti-dog antibody but is not detected using an anti-human IL2 antibody. As shown in Figure 13, the dog IC produced by transient expression was incubated with human melanoma cells and then detected with either an anti-dog or anti-human antibody followed by flow cytometry. The extent of binding was compared to the human 14.18-IL2 using what was estimated to be the same concentration of IC and the mean fluorescence was virtually the same, despite the fact that different detecting antibodies were used.
Another function of the IC molecule is bioactivity of the cytokine component, in this case IL-2. Past studies have shown that human IL-2 is active in dogs and companies that sell dog IL-2 (e.g. R&D Systems) test bioactivity of their preparations using the standard mouse T cell line CTLL-2. Thus, standard cell lines from other species were used for testing bioactivity of IL-2. To confirm that IL-2 that is fused to the carboxyl terminus of a dog IgG H chain is biologically active, the dog 14.18-IL2 that was expressed transiently from human 293T cells was serially diluted and tested for the ability to stimulate the proliferation of CTLL-2 cells that were previously starved for IL-2. Purified human 14.18-IL2 IC was used as a positive control. As shown in Figure 14, both proteins stimulated the uptake of 3H- thymidine (a measure of cell proliferation) far above the baseline of un-stimulated cells and at all concentrations. At the higher concentrations of the unpurified cell culture media (containing the dog 14.18-IL2) there appeared to be an inhibitory effect of the culture media, but upon dilution the interference stopped and activity increased and then decreased further upon dilution. In this non-inhibited portion of the curve, the activity of the dog IC was somewhat greater than that of the human protein but this could be due to a slight error in estimating the protein concentration or different activities of the different proteins with respect to the stimulation of the mouse IL-2 receptor.
Finally, the ability of the dog 14.18-IL2 to mediate the effector function of ADCC (antibody-dependent cellular cytotoxicity) was tested using either human or dog effector cells (peripheral blood mononuclear cells) and a human GD2 expressing melanoma cell line. Target cells were incubated with 51Cr and then washed to remove free isotope. Isolated peripheral blood lymphocytes from a healthy human volunteer were incubated with labeled target cells for 4 hours in the absence or presence of increasing amounts of the human or dog 14.18-IL2 IC and the amount of release chromium was taken as a measure of specific lysis. While the human IC mediated the typical amount of specific lysis in this assay, the dog IC did not cause the lysis of cells above the background level. The experiment was repeated a second time but in this case the effector cells were from dog peripheral blood. The same results were obtained as in the first experiment. Therefore, it is likely that this molecule, as currently constructed, does not mediate this effector function. A likely explanation is that it has poor binding to both human and dog Fc receptors on the effector cells such as FcRIII on natural killer cells. It is know from other species that even abundant isotypes do not mediate ADCC and this is mediated by a lack of binding to Fc receptors. These include the IgGl mouse and the human IgG2 isotypes, both of which lack this effector function despite being abundant in the blood. It is also interesting to note that the mouse IgGl isotype also binds poorly to protein A.
Example 7 - Switching the isotype of dog H chain to improve FcR binding and ADCC effector function Although it has been reported that some immunocytokines lacking ADCC effector function still exhibit potent anti-tumor activity in pre-clinical models, it may still be desirable to include this function in some cases. To this end the sequences of the other dog H chain isotypes were compared to each other and to those of human H chains known to have this effector function. The motif (a CPX-motif) shown to effect binding of human IgG to Fc receptors is located at the junction of the heavy chain hinge and CH2 domain. The corresponding sequences of the human IgGl (strong FcR binding) and the four dog isotypes are shown in Figure 15. The IgG-A isotype is unique in this group for having a proline (P) residue in this middle of this motif and this residue is known to have a dramatic effect on the secondary structure of polypeptides. Of the four isotypes, the dog IgG-B motif has the closest similarity to the human IgGl sequence (a single substitution of an M in place of L) and the change is far more conservative than a P residue.
The second most abundant class of human IgG is IgG2 and the sequence in the critical FcR binding motif includes the residues PVA in place of ELL found in IgGl. This change has a dramatic effect on ADCC due to a loss of FcR binding (Isaacs et al. 1998. J. Immunol. 161 :3862-3869. Thus it seemed that the dog IgG-A most resembles human IgG2 with respect to FcR binding and that one of the other isotypes may be more similar to human IgGl . To test this hypothesis, the dog IgG-B H chain gene was synthesized in a form compatible with the expression vector construct as a MIu I to Xma I fragment. The sequence of the gene begins with intron sequences and a functional splice acceptor site, followed by the CHl, hinge and CH2 exons as a continuous sequence (no introns). There is an intron between the CH2 and CH3 exons and the fragment joins the vector sequence encoding the last residues of the antibody sequence. The fragment was inserted in place of the corresponding fragment in the vector encoding dog 14.18-IL2 (construct 2 of Figure 12) and the resulting vector was tested by transient expression in cultures of human 293T cells. The protein contained in the cell culture media was examined directly by SDS-PAGE analysis and after binding to and elution from protein A. The results indicate that the modified IC was produced as a fully assembled IC that broke down after treatment with reducing agent to L chain and a fusion H-IL2 chains of the correct size. Binding to protein A had an improved concentration effect (compared to IgG-A) using the ionic strength and pH conditions of the culture media, but not to the extent normally seen with human IgGl . Figure 21 shows that an IC comprising an IgG-B isotype for the H chain constant region binds to Protein A. This IgG-B containing IC does not exhibit substantial ADCC effector function (similar to IgG-A), as determined using the standard ADCC assay disclosed in Hank JA, et al. Activation of Human Effector Cells by a Tumor Reactive Recombinant Anti-Ganglioside GD2 Interleukin- 2 Fusion Proteion (chl4.18-IL2). Vol. 2 1951-59. (1996) Clinical Cancer Research, the contents of which relating to ADCC assays are incorporated herein by reference. These results indicate that the prevalent dog immunoglobulins do not stimulate ADCC efficiently.
Sequences of the Dog IgG-B replacement fragment and the Dog-IgG-B H chain C region protein sequence are provided as SEQ ID NO: 16 and 17, respectively. Other dog H chain isotypes may be constructed in the same manner as the IgG-B fragment.
Example 8 - Feline Immunocytokines
For applications for the treatment of feline cancer, the constant regions of the expression vector of Example 7 is modified using the corresponding protein encoding sequences of the feline light and heavy chains, the flanking sequences and restriction sites described for the dog protein expression vectors. A nucleic acid sequence encoding a feline C kappa fragment is provided in SEQ ID NO: 18. A protein sequence of a feline C kappa fragment is provided in SEQ ID NO: 19. A nucleic acid sequence encoding a feline H chain C gamma 1 region sequence is provided as SEQ ID NO: 20.
The expression vector used to express the dog 14.18-IL2 IC also contains a Xma I to Fse I fragment encoding the dog IL-2 molecule. For the expression of a feline IC having feline cytokine sequences it may be desirable to reduce immunogenicity. In the current example a fragment encoding IL-2 flanked by Xma I and Fse I is provided that maintains the protein reading frame with the heavy chain constant region and thus results in the fusion of the antibody and IL-2 sequences. A nucleic acid sequence encoding a feline IL-2, e.g., for fusion to the feline IgG H chain, is provided as SEQ ID NO: 22. A protein sequence encoding a feline IL-2 is provided as SEQ ID NO: 23.
Example 9 — Additional sources of antibodies for veterinary use
Laboratories in academia and industry have generated thousands of antibodies against human cancer antigens. Very few of these have been screened against dog and cat cancer antigens although some work has been done to identify anti-dog CD20 cross reactivity, but with no success against epitopes exposed on the surface of the cell. This may be due in great part to restricted reactivity of mouse antibodies made against human CD20. Approaches are disclosed herein (e.g. , See Examples) that are useful to overcome these reactivity issues.
Other targeting antigens have been considered for the use in preparing ICs, including many antigens already described in the literature. Additional targets that have been used for naked antibody therapy have been considered as well. For example, there is a high molecule weight proteoglycan expressed on melanoma, sarcoma and certain other human cancers that has been under intense investigation as a target for immune therapy. This is called melanoma-specific chondroitin sulfate proteoglycan (CSPG) and antibodies against non- overlapping epitopes have been described (Campoli et al. 2004. Critical Reviews in Immunology 24:267-296).
Although the dog CSPG sequence is not known, the species conservation of this protein and its importance in tumorigenicity makes it likely to be expressed on canine melanoma cell lines. To test the possibility that this molecule is indeed expressed on dog melanoma cells and to see if any of the mouse monoclonal antibodies generated against the human protein are reactive with the dog counterpart, a binding assay was conducted against two canine melanoma cell lines - CLM-I and 17CM98 - using a concentration of 5 μg of antibody/ml of culture media and then detecting binding with an anti-mouse antibody conjugate. Out of 24 antibodies tested, four of these anti-human CSPG antibodies reacted with both canine melanoma cell lines. One in particular, VF20-VT20, had the strongest binding to the melanoma cells and presumably to the canine version of the human CSPG. The V region sequences of this mouse antibody have been obtained and have been adapted for the expression vector used to express the dog 14.18-IL2 IC.
Many other useful anti-tumor antibodies, originally screened against human cancer cells can be tested in the manner described herein and cross-reactive antibodies identified that could be useful for the treatment of veterinary cancers.
Example 10 - Specific immunization with animal versions of a useful antigen targets - CD20
As disclosed herein, the reactivity of mouse anti-human can be restricted due to unique characteristics of the antigen or limited differences between the mouse and human versions of a particular protein. Both of these issues could be addressed by immunization of mice with the target protein (or peptide) of the desired species. Alternatively, DNA encoding such proteins can be used for immunization. One example of a potentially useful target is CD20, the molecule recognized by the highly successful antibody, Rituxan, useful for the treatment of B cell tumors and B cell mediated immune disorders. There is also extensive pre-clinical data demonstrating the potency of a human anti-CD20 - IL2 IC, DI-Leul6-IL2, in multiple mouse tumor models of disseminated human lymphoma (Gillies et al. 2005. Blood 105: 3972-3978; Singh et al. 2007. Cancer Res. 67:2872-2880). The sequences for the canine and feline CD20 molecules are known.
A first approach was used to express the canine CD20 molecule in a cell line useful for the immunization of Balb/c mice. The NS/0 cell line is syngeneic with Balb/c mice and is not itself immunogenic upon cell immunization. In contrast, an NS/0 transfectant expressing the dog (or other foreign CD20) would be useful in the generation of mouse antibodies against the dog CD20. The cell line can also be used to test for antibodies that recognize this protein as it is expressed on the surface of live cells. The sequence encoding canine CD20 was obtained using PCR cloning from dog peripheral blood cDNA and adapted to have unique restriction sites just before the initiation codon and just after the stop codon. This Xba to Xho I fragment was sequenced to confirm its authenticity and then subcloned in the pcDNA3.1 expression vector (Invitrogen). Transient expression in human 293T cells was performed in order to verify the sequence and test whether a commercially available polyclonal antibody could be used to detect expression in transfected cells (for selecting stable transfectants). One such polyclonal antibody, a rabbit antisera raised against a peptide in the cytoplasmic domain (Fisher Scientific), was found to be reactive by Western blot analysis of transiently transfected human 293 cells, as depicted in Figure 16. Cells were collected 72 hours after transfection and cytoplasmic extracts were prepared and analyzed by SDS-PAGE followed by Western Blotting and detection with a rabbit polyclonal anti-CD20 antisera known to be cross-reactive with CD20 of multiple species. The CD20 protein runs at roughly 33 and 37 kD with the larger species believed to represent the phosphorylated form. These bands are only seen in cells receiving the indicated amounts of the plasmid DNA.
Next, stable transfection of mouse NS/0 myeloma cells (which do not express CD20) were performed using the same expression vector encoding canine CD20. The preferred method for transfecting this cell line is to linearize the plasmid DNA with a single-site restriction enzyme that cuts in the ampicillin resistance gene (used for bacterial expression), mixing the DNA with NS/0 cells and electroporating the expression vector into the cell nucleus (See, e.g., Gillies et al 1998, J. Immunol. 160:6195-6203). Transfectants were selected that are resistant to the marker gene in the pcDNA3.1 vector, Neo, by their growth as colonies in the presence of the antibiotic G418. After picking and expanding the drug- resistant transfectants, cells expressing the canine CD20 can be identified in at least two ways. The first is to make cytoplasmic extracts of individual resistant clones and analyze them for expression of CD20 as shown above (Figure 16). A second way is to make a fusion protein of the major extracellular loop of canine CD20 fused to a carrier protein such as the mouse IgG2a Fc region. This allows for high level expression, easy purification using protein A Sepharose and immunization under conditions where the carrier portion of the fusion protein (mouse Fc) is a self molecule. Expression is aimed at producing an immune response to the desired portion of the fusion protein - the extracellular loop of canine CD20. The protein may be expressed by adding two cysteine residues that should form a disulphide bond between portions of the loop and help recreate the structure that it has when it is protruding from the cell membrane (Figure 17). Figure 17 shows a protein sequence for a soluble extracellular loop of canine CD20 fused to mouse Fc, which includes the introduction of two cysteine residues (bold Cs), that are capable of forming a disulfide bond and artificial loop.
An analogous soluble CD20 loop fusion protein can be made using the sequences of other species for use as an immunogen for the generation of antibodies that react with CD20 on the surface of target cells. After the initial immunization, polyclonal antisera from mice, rabbits or other species are useful for screening transfectants expressing CD20 of the same species. This method of immunization is useful on its own or combined with cell immunization to identify monoclonal antibodies useful for treatment of disease caused by cells expressing CD20. Non-limiting examples of loop sequences are provided in SEQ ID NO: 24-26.
Transient expression of the canine CD20 loop fused to mouse Fc was achieved by sub-coning the DNA sequence into an expression vector containing a leader sequence from a mouse Ig light chain driven by the CMV promoter and also containing the hinge, CH2 and CH3 exons of the mouse IgG2a H chain. Purified vector DNA was combined with a lipid transfection reagent and used to treat cultures of human 293T cells. After 96 hr of cell culture incubation, the conditioned media was tested for the presence of the fusion protein directly and after binding to and elution from protein A Sepharose. Samples were heated in the presence or absence of a reducing agent to test for dimerization of the fused H chains of the Fc region (Fig. 18). As depicted in Figure 18, the reduced protein ran with apparent molecular weight that was half the size of the non-reduced sample demonstrating the dimeric structure of the fusion protein.
Example 11 — Immunization with a veterinary melanoma antigen
The CSPG expressed on melanoma, sarcoma and other cancer cells is a good target for immune therapies such as immunocytokines. While it is not essential that the targeting antibody for an IC also have ADCC activity, it may be preferred to include this activity. Also, as described above for the canine IgG isotypes, some mediate ADCC and some do not. This is based on the ability of the Fc portion to bind to Fc receptors. Another requirement for successful ADCC is based on the structure of the target antigen and the proximity of the specific binding epitope to the cell membrane. In the case of CSPG, most if not all mouse antibodies bind to regions of the protein distal to the membrane and do not mediate ADCC. Many things determine what portion of a protein antigen induce an antibody immune response including the protein structure, the dominance of adjacent epitopes and the similarity of the sequence to the species being immunized. One way around this problem is to immunize animals with the isolated region of interest that is then not affected by other dominant epitopes. If the sequence is also very similar to the immunizing species, it may be difficult to break immune tolerance. This latter problem might be overcome by immunizing with the same antigen isolated from another species (e.g., canine) that is more foreign and therefore able to break tolerance. Antibodies induced in this way may then bind to the original species but also cross-react with protein of the other species, including human, as well.
To test this hypothesis a database search was conducted to see if any sequences in the dog genomic database were homologous to a portion of the membrane proximal D3 region of CSPG common to human, mouse and rat sequences. A sequence with significant homology was identified in a whole genome shotgun sequence fragment (EMBL-EBI accession # AAEX02018596). This membrane-proximal region of the protein can be expressed in isolation or as a fusion protein as the entire D3 region or as sub-fragments that upon immunization, would induce antibodies to this selected portion of this very large protein and increase the likelihood that such antibodies mediate ADCC. It should be noted that sequence differences (Fig. 19) between the canine CSPG homolog and the mouse are significant and, in many cases, represent epitopes that are shared between the mouse and human sequence, and thus, unlikely to have been induced by immunization with the human protein. Figure 19 shows sequence homology between human, mouse and a potential canine D3 domain of CSPG. Examples of a fusion protein sub-fragment of canine CSPG D3 sequence and a sequence of a nucleic acid encoding a fusion protein sub-fragment of canine CSPG D3 are provided in SEQ ID NO: 27 and 28.
It will be appreciated that the protein sequences disclosed herein can be modified for use as an Fc fusion protein by adding a functional leader sequence to the amino terminus of each sequence and the coding sequence of the hinge region of a mouse Fc fragment up to the point where there is a unique restriction site for joining to an Fc encoding expression vector. For example, the mouse gamma 2a hinge sequence has a unique Apa I site that is useful for joining the antigen encoding DNA fragment. To use this site and keep the protein sequence in the correct reading frame the DNA sequence, gag,ccc,aga,ggg,ccc (SEQ ID NO: 29) is added to the sequences provided above for canine and feline CD20 and canine CSPG D3 domain. The commas denote the correct reading frame of this hinge region.
Antibodies that have been obtained from an immunized animal are screened using standard techniques. Typically, an antibody is screened for binding to the antigen against which the antibody was originally raised, e.g., using an ELISA assay. An antibody is typically also evaluated for its ability to bind to the antigen (cell surface antigen) in situ. Binding of antibodies to antigens in situ may be evaluated using standard immunocytochemistry methods, e.g., microscopy, FACS analysis, etc. The antibodies are often evaluated for their ability to specifically bind cells of a tumor, e.g., dog melanoma cells or human melanoma cells, either in vivo or ex vivo, using standard methods known in the art. The antibodies may also be evaluated to determine whether or not binding to an antigen is species specific. In some embodiments, antibodies are selected that recognize an antigen across multiple species. In some embodiments, antibodies are selected that recognize an antigen of a single or limited number of species (e.g., dog and human; cat and human; dog, cat and human, etc.).
Example 12 - Immunization with canine EpCAM
A useful target antigen for human epithelial cancer treatment is the epithelial cell adhesion molecule (EpCAM) that is widely expressed in most epithelial human cancers and recently shown to be expressed on the cancer stem cells of these tumor types. Antibodies that are capable of mediating effector functions such as ADCC and immunocytokines derived from such antibodies would be useful for the treatment of animals with these diseases. The coding sequence of canine EpCAM has not been reported and was not available from database searches. However a genomic fragment from shotgun sequencing of the canine genome (EMBL-EBI accession # AAEX02022975) was obtained using a homology search with the second exon of the human EpCAM gene, and exons 2 through 9 could be identified by homology to the human exons. The sequences encoded in exons 2-9 of the canine gene were assembled into a continuous sequence representing the majority of the predicted mRNA and this was translated into the predicted canine EpCAM molecule. The homology between this sequence and the sequences of other mammalian counterparts is shown in Figure 20. The extensive pattern of cyteine residues that is highly conserved between species is evident in the canine sequence and further supports the authenticity of this gene as encoding EpCAM. Unfortunately, exon 1 was not contained in this sequence due to the limited 5' sequence (2 Kb) upstream of exon 2 and a homology search of the available genome sequence with exon 1 did not result in any significant homology. The intron between exons 1 and 2 of the human gene is approximately 4 kB, and this is consistent with the lack of intron 1 in the dog genomic fragment. Exons 2 through 9 encode all but the first two amino acids (QE in the human form- residues 24 and 25 in Figure 20) of the mature form of the EpCAM protein.
Therefore, for the purpose of immunization, the first two amino acid residues of the human molecule could be added to the construct for expression of canine EpCAM on a syngeneic mouse cell line (e.g. NS/0 myeloma cells), together with a functional leader sequence. The transfected cells can be used to immunize mice and then to screen hybridoma clones supernatants for reactivity with canine EpCAM, as described above for CD20. Alternatively, the extracellular domain of canine EpCAM could be expressed as a soluble protein and used for immunization and screening. Such a soluble form could be produced as a fusion protein with an Fc fragment for high level expression and ease of purification using protein A sepharose, preferably using an Fc region derived from mouse IgG so that the Fc fragment itself does not elicit a strong immune response, thus steering the immune system to react predominantly to the canine EpCAM. It is also possible synthesize peptides or to express smaller portions of the EpCAM molecule, alone or as a fusion protein to generate antibodies reactive with that portion (e.g. N terminal or C-terminal part of the EpCAM ectodomain). Sequences are provided for a predicted protein encoding sequence derived from exons 2 through 9 of the proposed canine EpCAM in SEQ ID NO: 29 and 30.
NUCLEOTIDE AND AMINO ACID SEQUENCES SEQ ID NO: 1. Dog C kappa protein
RNDAQPAVYLFQPSPDQLHTGSASWCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKD STYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD
SEQ ID NO:2. Dog IgG A H chain constant region
ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGL HSLSSMVTVPSSRWPSETFTCNWHPASNTKVDKPVFNECRCTDTPPCPVPEPLGGPSVLIF PPKPKDI LRI TRTPEVTCWLDLGREDPEVQI SWFVDGKEVHTAKTQSREQQFNGTYRWSV LPIEHQDWLTGKEFKCRVNHIDLPSPIERTI SKARGRAHKPSVYVLPPSPKELSSSDTVSIT CLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCA VMHETLQNHYTDLSLSHSPGK
SEQ ID NO:3. Mouse 14.18 H chain variable region
EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQ KFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS
SEQ ID NO:4. Mouse 14.18 L chain variable region
DWMTQTPLSLPVSLGDQAS I SCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSG VPDRFSGSGSGTDFTLKI SRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK
SEQ ID NO:5. Heavy chain of a non-limiting example of a chimeric mouse-dog anti-GD2- dog-IL2 immunocytokine
EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQ KFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSASΤΤAPSVFPL APSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLHSLSSMVTVPS SRWPSETFTCNWHPASNTKVDKPVFNECRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRIT RTPEVTCWLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRWSVLPIEHQDWLTG KEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDI DVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYT ΌLSLSHSPGAPITSSSTKETEQQMEQLLLDLQLLLNGVNNYENPQLSRMLTFKFYTPKKATE FTHLQCLAEELKNLEEVLGLPQSKNVHLTDTKELISNMNVTLLKLKGSETSYNCEYDDETAT ITEFLNKWITFCQSIFSTLT
SEQ ID NO:6. Nucleotide sequence encoding the dog IgG A H chain fusion to dog IL-2 portion of SEQ ID NO: 5 (the nucleotide sequence encoding the mouse 14.18 H chain variable region is known in the art and can be found in published documents, including US Patent 7,169,904, the sequence-related contents of which are incorporated herein by reference in their entirety).
GCCTCCACCACGGCCCCCTCGGTTTTCCCACTGGCCCCCAGCTGCGGGTCCACTTCCGGCTC CACGGTGGCCCTGGCCTGCCTGGTGTCAGGCTACTTCCCCGAGCCTGTAACTGTGTCCTGGA ATTCCGGCTCCTTGACCAGCGGTGTGCACACCTTCCCGTCCGTCCTGCAGTCCTCAGGGCTT CACTCCCTCAGCAGCATGGTGACAGTGCCCTCCAGCAGGTGGCCCAGCGAGACCTTCACCTG CAACGTGGTCCACCCAGCCAGCAACACTAAAGTAGACAAGCCAGTGTTCAATGAATGCAGAT GCACTGATACACCCCCATGCCCAGTCCCTGAACCTCTGGGAGGGCCTTCGGTCCTCATCTTT CCCCCGAAACCCAAGGACATCCTCAGGATTACCCGAACACCCGAGGTCACCTGTGTGGTGTT AGATCTGGGCCGTGAGGACCCTGAGGTGCAGATCAGCTGGTTCGTGGATGGTAAGGAGGTGC ACACAGCCAAGACCCAGTCTCGTGAGCAGCAGTTCAACGGCACCTACCGTGTGGTCAGCGTC CTCCCCATTGAGCACCAGGACTGGCTCACAGGGAAGGAGTTCAAGTGCAGAGTCAACCACAT AGACCTCCCGTCTCCCATCGAGAGGACCATCTCTAAGGCCAGAGGGAGGGCCCATAAGCCCA GTGTGTATGTCCTGCCGCCATCCCCAAAGGAGTTGTCATCCAGTGACACAGTCAGCATCACC TGCCTGATAAAAGACTTCTACCCACCTGACATTGATGTGGAGTGGCAGAGCAATGGACAGCA GGAGCCCGAGAGGAAGCACCGCATGACCCCGCCCCAGCTGGACGAGGACGGGTCCTACTTCC TGTACAGCAAGCTCTCTGTGGACAAGAGCCGCTGGCAGCAGGGAGACCCCTTCACATGTGCG GTGATGCATGAAACTCTACAGAACCACTACACAGATCTATCCCTCTCCCATTCTCCGGGTAA ATGAGCAACACGCCCGGCACCCAGCGCCCCCATCACCTCCTCCTCCACCAAGGAGACCGAGC AGCAGATGGAGCAGCTCCTGCTGGACCTGCAGCTCCTGCTGAACGGCGTGAACAACTACGAG AACCCCCAGCTcTCCCGCATGCTGACCTTCAAGTTCTACACCCCCAAGAAGGCCACCGAGTT CACCCACCTGCAGTGCCTGGCCGAGGAGCTGAAGAACCTGGAGGAGGTGCTGGGCCTGCCCC AGTCCAAGAACGTGCACCTGACCGACACCAAGGAGCTGATCTCCAACATGAACGTGACCCTG CTGAAGCTGAAGGGCTCCGAGACCTCCTACAACTGCGAGTACGACGACGAGACCGCCACCAT CACCGAGTTCCTGAACAAGTGGATCACCTTCTGCCAGTCCATCTTCTCCACCCTGACC
SEQ ID NO:7. Light chain of a non-limiting example of a chimeric mouse-dog anti-GD2- dog-IL2 immunocytokine
DWMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSG
VPDRFSGSGSGTDFTLKiSR VEAEDLGVYFCSQSTHVPPL TFGAGTKLELKKNΌAQ PAVYLF
QPSPDQLHTGSASWCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTM SSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD
SEQ ID NO:8. Nucleotide sequence encoding Dog c kappa portion of SEQ ID NO: 7 (the nucleotide sequence encoding the mouse 14.18 L chain variable region is known in the art and can be found in published documents, including US Patent 7,169,904, the sequence- related contents of which are incorporated herein by reference in their entirety).
TGATCAACTTCCCTGTTACTTAACGACCATTCTGTGTGCTTCCTTCTGCAGGGAATGATGCC CAGCCAGCCGTCTATTTGTTCCAACCATCTCCAGACCAGTTACACACAGGAAGTGCCTCTGT TGTGTGCTTGCTGAATAGCTTCTACCCCAAAGACATCAATGTCAAGTGGAAAGTGGATGGTG TCATCCAAGACACAGGCATCCAGGAAAGTGTCACAGAGCAGGACAAGGACAGTACCTACAGC CTCAGCAGCACCCTGACGATGTCCAGTACCGAGTACCTAAGTCATGAGTTGTACTCCTGTGA GATCACTCACAAGAGCCTGCCCAGTACTCTCATCAAGAGCTTCCAAAGATCTGAGTGTCAGA GAGTGGACTAACAGCGGCCGC
SEQ ID NO:9. Amino acid sequence for dog IL-2
APITSSSTKETEQQMEQLLLDLQLLLNGVNNYENPQLSRMLTFKFYTPKKATEFTHLQCLAE ELKNLEEVLGLPQSKNVHLTDTKELISNMNVTLLKLKGSETSYNCEYDDETATITEFLNKWI TFCQSIFSTLT
SEQ ID NO: 10. Dog C kappa coding sequence
CGGAATGATGCCCAGCCAGCCGTCTATTTGTTCCAACCATCTCCAGACCAGTTACACACAGG AAGTGCCTCTGTTGTGTGCTTGCTGAATAGCTTCTACCCCAAAGACATCAATGTCAAGTGGA AAGTGGATGGTGTCATCCAAGACACAGGCATCCAGGAAAGTGTCACAGAGCAGGACAAGGAC AGTACCTACAGCCTCAGCAGCACCCTGACGATGTCCAGTACCGAGTACCTAAGTCATGAGTT GTACTCCTGTGAGATCACTCACAAGAGCCTGCCCAGTACTCTCATCAAGAGCTTCCAAAGGA GCGAGTGTCAGAGAGTGGAC
SEQ ID NO: 11. Dog C kappa gene from EMBL shotgun library
CAAAAAGTCCTTTAAATGGCTGCAAAGATTGAAACAAAAACTTTGTTAAGACGTGGGAACTC AAGAGAAACTCAAGATTGTGGAGATTATAAATCTGTTTCTTGGCCTCCCTCATTGCCACACA GAATAAGCTGCTCTATCTGTCCTTTCCGGGCCCTGGGGTTGCCCACAAACAGTACACCCAAG TGGAGAACTTCCCTGTTACTTAACGACCATTCTGTGTGCTTCCTTCTGCAGGGAATGATGCC CAGCCAGCCGTCTATTTGTTCCAACCATCTCCAGACCAGTTACACACAGGAAGTGCCTCTGT TGTGTGCTTGCTGAATAGCTTCTACCCCAAAGACATCAATGTCAAGTGGAAAGTGGATGGTG TCATCCAAGACACAGGCATCCAGGAAAGTGTCACAGAGCAGGACAAGGACAGTACCTACAGC CTCAGCAGCACCCTGACGATGTCCAGTACCGAGTACCTAAGTCATGAGTTGTACTCCTGTGA GATCACTCACAAGAGCCTGCCCAGTACTCTCATCAAGAGCTTCCAAAGGAGCGAGTGTCAGA GAGTGGACTAACAGGCCCCACCACCTGCCCCTCAGGCCTAGCCTTCCTGTCTCCTAGCTCAG GCCTTGGGCCCTTTCCCCATCAGAGACCCACCTCTGTTGCAGGCACCACCCTTCTCCCCACC TCCCCCTCTTGGGCTTTAACAATGCTAATGGTATCTGGGGGAAAAGGAATAAATAAAGTGAA TTGTTGCACCTGTGCTATCCCTCTTCTTCCTGATTTAATGATTGTTATACATTGGGGTTTTT TTTTTCCAATTACTCAATTTATCTTCTACAGAACTAAATGTTGAATTTATCCAGTTTATCCA GTTGAACTTACCAGGAGAATTTACCAAAAGCA SEQ ID NO: 12. Modified C kappa gene sequence
TGATCAACTTCCCTGTTACTTAACGACCATTCTGTGTGCTTCCTTCTGCAGGGAATGATGCC CAGCCAGCCGTCTATTTGTTCCAACCATCTCCAGACCAGTTACACACAGGAAGTGCCTCTGT TGTGTGCTTGCTGAATAGCTTCTACCCCAAAGACATCAATGTCAAGTGGAAAGTGGATGGTG TCATCCAAGACACAGGCATCCAGGAAAGTGTCACAGAGCAGGACAAGGACAGTACCTACAGC CTCAGCAGCACCCTGACGATGTCCAGTACCGAGTACCTAAGTCATGAGTTGTACTCCTGTGA GATCACTCACAAGAGCCTGCCCAGTACTCTCATCAAGAGCTTCCAAAGATCTGAGTGTCAGA GAGTGGACTAACAGCGGCCGC
SEQ ID NO: 13. Dog IgG A H chain coding sequence cDNA
GCCTCCACCACGGCCCCCTCGGTTTTCCCACTGGCCCCCAGCTGCGGGTCCACTTCCGGCTC CACGGTGGCCCTGGCCTGCCTGGTGTCAGGCTACTTCCCCGAGCCTGTAACTGTGTCCTGGA ATTCCGGCTCCTTGACCAGCGGTGTGCACACCTTCCCGTCCGTCCTGCAGTCCTCAGGGCTT CACTCCCTCAGCAGCATGGTGACAGTGCCCTCCAGCAGGTGGCCCAGCGAGACCTTCACCTG CAACGTGGTCCACCCAGCCAGCAACACTAAAGTAGACAAGCCAGTGTTCAATGAATGCAGAT GCACTGATACACCCCCATGCCCAGTCCCTGAACCTCTGGGAGGGCCTTCGGTCCTCATCTTT CCCCCGAAACCCAAGGACATCCTCAGGATTACCCGAACACCCGAGGTCACCTGTGTGGTGTT AGATCTGGGCCGTGAGGACCCTGAGGTGCAGATCAGCTGGTTCGTGGATGGTAAGGAGGTGC ACACAGCCAAGACCCAGTCTCGTGAGCAGCAGTTCAACGGCACCTACCGTGTGGTCAGCGTC CTCCCCATTGAGCACCAGGACTGGCTCACAGGGAAGGAGTTCAAGTGCAGAGTCAACCACAT AGACCTCCCGTCTCCCATCGAGAGGACCATCTCTAAGGCCAGAGGGAGGGCCCATAAGCCCA GTGTGTATGTCCTGCCGCCATCCCCAAAGGAGTTGTCATCCAGTGACACAGTCAGCATCACC TGCCTGATAAAAGACTTCTACCCACCTGACATTGATGTGGAGTGGCAGAGCAATGGACAGCA GGAGCCCGAGAGGAAGCACCGCATGACCCCGCCCCAGCTGGACGAGGACGGGTCCTACTTCC TGTACAGCAAGCTCTCTGTGGACAAGAGCCGCTGGCAGCAGGGAGACCCCTTCACATGTGCG GTGATGCATGAAACTCTACAGAACCACTACACAGATCTATCCCTCTCCCATTCTCCGGGTAA ATGAGCAACACGCCCGGCACCCAGCAAG
SEQ ID NO: 14. Artificial dog IgG A H chain gene sequence
ACGCGTGTCACATGGCACCACCTCTCTTGCAGCCTCCACCACGGCCCCCTCGGTTTTCCCAC TGGCCCCCTCATGCGGGTCCACTTCCGGCTCCACGGTGGCCCTGGCCTGCCTGGTGTCAGGC TACTTCCCCGAGCCTGTAACTGTGTCCTGGAACTCCGGCTCCTTGACCAGCGGTGTGCACAC CTTCCCGTCCGTCCTGCAGTCCTCAGGGCTTCACTCCCTCAGCAGCATGGTGACAGTGCCCT CCAGCAGGTGGCCCAGCGAGACCTTCACCTGCAACGTGGTCCACCCAGCCAGCAACACTAAA GTAGACAAGCCAGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCTCA GCGCTCCTGCCGCGGACGCATCCCGGCTATGCAGTCCCAGTCCAGGGCAGCAAGGCAGGCCC CGTCTGCCTCTTCACCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGAGAGGGTCTTCT GGCTTTTTCCCCAGGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAACCCAGGCCCTGCACAC AAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATATCCGGGAGGACCCTGCCCCTGA CCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCGGACACCTTCTCTCCTCCC AGATTCCAGTAACTCCCAATCTTCTCTCTGCAGTGTTCAATGAATGCAGATGCACTGATACA CCCCCATGCCCAGTCCCTGAACCTCTGGGAGGGCCTTCGGTCCTCATCTTTCCCCCGAAACC CAAGGACATCCTCAGGATTACCCGAACACCCGAGGTCACCTGTGTGGTGTTAGACCTGGGCC GTGAGGACCCTGAGGTGCAGATCAGTTGGTTCGTGGATGGTAAGGAGGTGCACACAGCCAAG ACCCAGTCTCGTGAGCAGCAGTTCAACGGCACCTACCGTGTGGTCAGCGTCCTCCCCATTGA GCACCAGGACTGGCTCACAGGGAAGGAGTTCAAGTGCAGAGTCAACCACATAGACCTCCCGT CTCCCATCGAGAGGACCATCTCTAAGGCCAGAGGTGGGCAACAGGACAGATGGGGCACAGGG AGGTCGAGTGGGGCCTGGTGGACCCAGGCCAGCCCTCCACTCTGGGAGTGACCATCTGTGCT GACCTCTGACCCCACAGGGAGGGCCCATAAGCCCAGTGTGTATGTCCTGCCGCCATCCCCAA AGGAGTTGTCATCCAGTGACACAGTCAGCATCACCTGCCTGATAAAAGACTTCTACCCACCT GACATTGATGTGGAGTGGCAGAGCAATGGACAGCAGGAGCCCGAGAGGAAGCACCGCATGAC CCCGCCCCAGCTCGACGAGGACGGGTCCTACTTCCTGTACAGCAAGCTCTCTGTGGACAAGA GCCGCTGGCAGCAGGGAGACCCCTTCACATGTGCGGTGATGCATGAAACTCTACAGAACCAC TACACAGACCTATCCCTCTCCCATTCCCCGGGTAAA
SEQ ID NO: 15. Sequence encoding dog IL-2 modified for manipulation and expression
GCCCCCATCACCTCCTCCTCCACCAAGGAGACCGAGCAGCAGATGGAGCAGCTCCTGCTGGA
CCTGCAGCTcCTGCTGAACGGCGTGAACAACTACGAGAACCCCCAGCTcTCCCGCATGCTGA
CCTTCAAGTTCTACACCCCCAAGAAGGCCACCGAGTTCACCCACCTGCAGTGCCTGGCCGAG
GAGCTGAAGAACCTGGAGGAGGTGCTGGGCCTGCCCCAGTCCAAGAACGTGCACCTGACCGA
CACCAAGGAGCTGATCTCCAACATGAACGTGACCCTGCTGAAGCTGAAGGGCTCCGAGACCT
CCTACAACTGCGAGTACGACGACGAGACCGCCACCATCACCGAGTTCCTGAACAAGTGGATC
ACCTTCTGCCAGTCCATCTTCTCCACCCTGACCtagtggccggcc
Small letters include Stop codon and restriction site.
SEQ ID NO: 16. Dog IgG-B replacement fragment: acgcgtgtcacatggcaccacctctcttgcagCCTCCACCACGGCCCCCTCGGTTTTCCCAC TGGCCCCCAGCTGCGGGTCCACTTCCGGCTCCACGGTGGCCCTGGCCTGCCTGGTGTCAGGC TACTTCCCCGAGCCTGTAACTGTGTCCTGGAACTCCGGCTCCTTGACCAGCGGTGTGCACAC CTTCCCGTCCGTCCTGCAGTCCTCAGGGCTCTACTCCCTCAGCAGCATGGTGACAGTGCCCT CCAGCAGGTGGCCCAGCGAGACCTTCACCTGCAACGTGGCCCACCCGGCCAGCAAAACTAAA GTAGACAAGCCAGTGCCCAAAAGAGAAAATGGAAGAGTTCCTCGCCCACCTGATTGTCCCAA ATGCCCAGCCCCTGAAATGCTGGGAGGGCCCTCGGTCTTCATCTTTCCCCCGAAACCCAAGG ACACCCTCTTGATTGCCCGAACACCCGAGGTCACCTGTGTGGTGGTGGATCTGGACCCAGAA GACCCTGAGGTGCAGATCAGCTGGTTCGTGGACGGTAAGCAGATGCAAACAGCCAAGACTCA GCCTCGTGAGGAGCAGTTCAATGGCACCTACCGTGTGGTCAGTGTCCTCCCCATTGGGCACC AGGACTGGCTCAAGGGGAAGCAGTTCACGTGCAAAGTCAACAACAAAGCCCTCCCATCCCCG ATCGAGAGGACCATCTCCAAGGCCAGAGgtgggcaacaggacagatggggcacagggaggtc gagtggggcctggtggacccaggccagccctccactctgggagtgaccatctgtgctgacct ctgaccccacagGGCAAGCCCATCAGCCCAGTGTGTATGTCCTGCCGCCATCGCGGGAGGAG TTGAGCAAGAACACAGTCAGCTTGACATGCCTGATCAAAGACTTCTTCCCACCTGACATTGA TGTGGAGTGGCAGAGCAATGGACAGCAGGAGCCTGAGAGCAAGTACCGCACGACCCCGCCCC AGCTGGACGAGGACGGGTCCTACTTCCTGTACAGCAAGCTCTCTGTGGACAAGAGCCGCTGG CAGCGGGGAGACACCTTCATATGTGCGGTGATGCATGAAGCTCTACACAACCACTACACACA GGAATCCCTCTCCCATTCCCCGGG
Flanking unique MIu I and Xma I sites are underlined. Intron sequences are in small caps while exon sequences are in large caps.
SEQ ID NO: 17. Dog-IgG-B H chain C region protein sequence:
ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGL YSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGP SVFIFPPKPKDTLLIARTPEVTCVWDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTY RWSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNT VSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDT FICAVMHEALHNHYTQESLSHSPGK
SEQ ID NO: 18. Feline C kappa fragment: tgatcaacttccctgttacttaacgaccattctgtgtgcttccttctgcagGGAGTGATGCT CAGCCATCTGTCTTTCTCTTCCAACCATCTCTGGACGAGTTACATACAGGAAGTGCCTCTAT CGTGTGCATATTGAATGACTTCTACCCCAAAGAGGTCAATGTCAAGTGGAAAGTGGATGGCG TAGTCCAAAACAAAGGCATCCAGGAGAGCACCACAGAGCAGAACAGCAAGGACAGCACCTAC AGCCTCAGCAGCACCCTGACGATGTCCAGTACGGAGTACCAAAGTCATGAAAAGTTCTCCTG CGAGGTCACTCACAAGAGCCTGGCCTCCACCCTCGTCAAGAGCTTCAACAGATCTGAGTGTC AGAGAGAGtagcagcggccgc
The unique BcI I and Not I flanking restriction sites are underlined. Intron sequences are in small caps while exon sequences are in large caps.
SEQ ID NO: 19. Feline C kappa protein sequence:
RSDAQPSVFLFQPSLDELHTGSASIVCILNDFYPKEVNVKWKVDGWQNKGIQESTTEQNSK DSTYSLSSTLTMSSTEYQSHEKFSCEVTHKSLASTLVKSFNRSECQRE
SEQ ID NO: 20. feline H chain C gamma 1 region sequence: acgcgtgtcacatggcaccacctctcttgcagCCTCCACCACGGCCCCATCGGTGTTCCCAC
TGGCCCCCAGTTGCGGGACCACATCTGGCGCCACCGTGGCCCTGGCCTGCCTGGTGTTAGGC
TACTTCCCTGAGCCGGTGACCGTGTCCTGGAACTCCGGCGCCCTGACCAGCGGTGTGCACAC
CTTCCCGGCCGTCCTGCAGGCCTCGGGGCTGTACTCTCTCAGCAGCATGGTGACAGTGCCCT
CCAGCAGGTGGCTCAGTGACACCTTCACCTGCAACGTGGCCCACCCGCCCAGCAACACCAAG
GTGGACAAGACCGTTCGCAAAACAGACCACCCACCGGGACCCAAACCCTGCGACTGTCCCAA
ATGCCCACCCCCTGAGATGCTTGGAGGACCGTCCATCTTCATCTTCCCCCCAAAACCCAAGG
ACACCCTCTCGATTTCCCGGACGCCCGAGGTCACATGCTTGGTGGTGGACTTGGGCCCAGAT
GACTCCGATGTCCAGATCACATGGTTTGTGGATAACACCCAGGTGTACACAGCCAAGACGAG
TCCGCGTGAGGAGCAGTTCAACAGCACCTACCGTGTGGTCAGTGTCCTCCCCATCCTACACC
AGGACTGGCTCAAGGGGAAGGAGTTCAAGTGCAAGGTCAACAGCAAATCCCTCCCCTCCCCC
ATCGAGAGGACCATCTCCAAGGCCAAAGgtgggcaacaggacagatggggcacagggaggtc gagtggggcctggtggacccaggccagccctccactctgggagtgaccatctgtgctgacct ctgaccccacagGACAGCCCCACGAGCCCCAGGTGTACGTCCTGCCTCCAGCCCAGGAGGAG
CTGAGCAGGAACAAAGTCAGTGTGACCTGCCTCATCAAGAGCTTCCACCCGCCTGACATTGC
CGTCGAGTGGGAGATCACCGGACAGCCGGAGCCAGAGAACAACTACCGGACGACCCCGCCCC
AGCTCGACAGCGACGGGACCTACTTCGTGTACAGCAAGCTCTCGGTGGACAGGTCCCACTGG
CAGAGGGGAAACACCTACACCTGCTCGGTGTCACACGAAGCTCTGCACAGCCACCACACACA
GAAATCCCTCACCCAGTCCCCGGG
Flanking unique MIu I and Xma I sites are underlined. Intron sequences are in small caps while exon sequences are in large caps. SEQ ID NO: 21 Feline C gamma 1 protein sequence:
ASTTAPSVFPLAPSCGTTSGATVALACLVLGYFPEPVTVSWNSGALTSGVHTFPAVLQASGL YSLSSMVTVPSSRWLSDTFTCNVAHPPSNTKVDKTVRKTDHPPGPKPCDCPKCPPPEMLGGP SIFIFPPKPKDTLSISRTPEVTCLWDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTY RWSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNK VSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNT YTCSVSHEALHSHHTQKSLTQSPGK
SEQ ID NO: 22 Feline IL-2 encoding fragment for fusion to the feline IgG H chain:
CCCGGGTGCCCCCGCCTCCTCCTCCACCAAGGAGACCCAGCAGCGCCTGGAGCAGCTCCTGC TGGACCTGCGCCTGCTGCTGAACGGCGTGAACAACCCCGAGAACCCCAAGCTGTCCCGCATG CTGACCTTCAAGTTCTACGTGCCCAAGAAGGCCACCGAGCTGACCCACCTGCAGTGCCTGGT GGAGGAGCTGAAGCCCCTGGAGGAGGTGCTGTACCTGGCCCAGTCCAAGAACTTCCACCTGA ACCACATCAAGGAGCTGATGTCCAACATCAACGTGACCGTGCTGAAGCTGAAGGGCTCCGAG ACCCGCTTCACCTGCAACTACGACGACGAGACCGCCACCATCGTGGAGTTCCTGAACAAGTG GATCACCTTCTGCCAGTCCATCTTCTCCACCCTGACCtagtggccggcc
Flanking Xma I and Fse I sites are underlined. The first residue of the mature IL-2 molecule is in bold type. Protein encoding sequences are in upper case and untranslated sequences are in small case.
SEQ ID NO: 23. Feline IL-2 protein sequence:
APASSSTKETQQRLEQLLLDLRLLLNGVNNPENPKLSRMLTFKFYVPKKATELTHLQCLVEE LKPLEEVLYLAQSKNFHLNHIKELMSNINVTVLKLKGSETRFTCNYDDETATIVEFLNKWIT FCQSIFSTLT
SEQ ID NO: 24. Canine CD20 loop DNA sequence: gacatatttaatattaccattTGTcatttcttcaagatggagaatttgaatctgattaaggc tcccatgccatatgttTGTatacacaactgtgacccagctaacccctctgagaaaaactctt tgtctatacaatattgtggcagcatacgatct
The TGT codon in large caps indicates where the normal amino residue in that position was replaced with a C residue. SEQ ID NO: 25. Feline CD20 loop DNA sequence: gacatatttaatattgcaattTGTcatttttttaaaatggagaatctgaatcttcttaaaag tcccaagccatatattTGTatccacacctgtcaaccagagagtaagccctctgagaaaaact ctctgtctataaaatattgtgacagcatacgatct
SEQ ID NO: 26. Feline CD20 loop protein sequence:
DIFNIAICHFFKMENLNLLKSPKPYICIHTCQPESKPSEKNSLSIKYCDSIRS
The bold, underlined C residues are replacements to the natural sequence in order to create an artificial loop.
SEQ ID NO: 27. A sub-fragment of canine CSPG D3 which is fused to mouse Fc:
EQFTQRDLEGGRLGLQLGRAPGPTGDSLTLELWAPGVPPAVASLDFHTEPYDAARPYGVALL SLPEEAGAPDSGAPATGQPGAPGPSPGPTAASGGFLGLLEAN
SEQ ID NO: 28. DNA sequence encoding a fusion protein sub-fragment of canine CSPG D3 gagcagttcacgcagcgggacctggagggcgggaggctggggctgcagctgggccgcgcccc cggccccacgggcgacagcctcacgctggagctgtgggcgcccggcgtccccccggccgtgg cctccctggacttccacaccgagccctacgacgcggcgcgcccctacggcgtggccctgctc agcctccccgaggaagccggggcacccgacagcggcgccccggccacgggccagccgggcgc gccaggccccagccccgggcccaccgcggccagcggcggcttcctgggcctcctggaggcca ac
SEQ ID NO: 29. Predicted protein encoding sequence derived from exons 2 through 9 of the proposed canine EpCAM: gcatgtatctgtgaaaactacaaactgaccacaaactgctctttgaacataaataatcagtg cgaatgtacttcaattggtgcacaaaattctgtcatttgctcaaaactggcaaccaaatgtt tggttatgaaggcagaaatgaccggcacaaagtctgggagaagagcgagacctgagggagct ttccagaataacgacgggctctatgatcccgactgtgatgagaaggggctctttaaagccaa gcagtgcaatggcaccaccacgtgctggtgtgtgaacactgctggggtccgaagaactgata aggacactgaaatatcctgcactgaacgagtgaggacctactggatcatcattgaattaaaa cacaaaacaagagaaacaccttatgatacacaaagtttgcaaaatgcacttaaggagacact caaaaaccgttatcaactggatccaaaatacatcacaaatattctgtatgagaatgatctta tcactattgatctgatgcaaaattcatctcagaaagctcagaatgatgtggacatagctgat gtggcttattattttgaaaaagatgttaaagacgaatccttgttccattccagtaaaatgga cctaagagtaaacggggaacaattggatctggatcctggtcgaactgcaatttactatgttg atgaaaaaccacctgaattttcaatgcagggtctacaagctggtattatcgctgtcattgtg gttgtgacactagcagttattgctggaatcgttgtgctggttatttccagaaagaacagaat ggcaaagtatgagaaggctgagataaaggagatgggtgagatgcatagggaactcaatgca
SEQ ID NO: 30. Predicted protein sequence of canine EpCAM lacking residues 1 and 2 of the mature sequence:
ACICENYKLTTNCSLNINNQCECTS IGAQNSVICSKLATKCLVMKAEMTGTKSGRRARPEGA FQNNDGLYDPDCDEKGLFKAKQCNGTTTCWCVNTAGVRRTDKDTEISCTERVRTYWI I IELK HKTRETPYDTQSLQNALKETLKNRYQLDPKYITNILYENDLITIDLMQNSSQKAQNDVDIAD VAYYFEKDVKDESLFHSSKMDLRVNGEQLDLDPGRTAIYYVDEKPPEFSMQGLQAGI IAVIV WTLAVIAGIWLVISRKNRMAKYEKAEIKEMGEMHRELNA
SEQ ID NO: 31. Human Cγl CPAPELLGGPSVF
SEQ ID NO: 32. Dog IgG-A CPVPEPLGGPSVL
SEQ ID NO: 33. Dog IgG-B CPAPEMLGGPSVF
SEQ ID NO: 34. Dog IgG-C CPCPGCGLLGGPSVF
SEQ ID NO: 35. Dog IgG-D CPVPESLGGPSVF
SEQ ID NO: 36. CPX-Motif
CPXiPX2 X3 X4 X5LGGPSX6X7, where:
Xi, X4, X6 and X7 are each any amino acid,
X2 and X3 are each any amino acid or absent, and
Xs is any amino acid other than proline. SEQ ID NO: 37. Cat IgG CPPPEMLGGPSIF
SEQ ID NO: 38. GKX-Motif GKQFTCKV
SEQ ID NO: 39. GKX-Motif
GKX8FX9CXioV, where:
X8, X9, and Xio are each independently any amino acid
SEQ ID NO: 40. Example of a scFV for 14.18 VH-linker-VL
EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGG TSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS GGGGSGGGGSGGGGDVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYL QKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPP LTFGAGTKLELK
SEQ ID NO: 90. gb|AAC82527.1 | immunoglobulin gamma- 1 heavy chain constant region
[Homo sapiens]
STKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
EQUIVALENTS
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

Claims

CLAIMS What is claimed is:
1. An immunocytokine comprising: an immunoglobulin gamma heavy chain comprising a CPX-motif that does not have a sequence of CPVPEPLGGPSVL (SEQ ID NO: 32), and a non-human, non-rodent cytokine.
2 An immunocytokine comprising: an immunoglobulin gamma heavy chain comprising a CPX-motif that has a sequence of CPX,PX2 X3 X4 X5LGGPSX6X7 (SEQ ID NO: 36); wherein Xi, X4, X6 and X7 are each independently any amino acid, wherein X2 and X3 are each independently any amino acid or absent, and wherein X5 is any amino acid other than proline; and a non-human, non-rodent cytokine.
3. An immunocytokine comprising: an immunoglobulin gamma heavy chain comprising a GKX-motif that does not have a sequence of GKQFTCKV (SEQ ID NO: 38), and a non-human, non-rodent cytokine.
4. An immunocytokine comprising: an immunoglobulin gamma heavy chain comprising a GKX-motif that has a sequence Of GKX8FX9CX10V (SEQ ID NO: 39); wherein Xg, Xg1 and Xi0 are each independently any amino acid; and a non-human, non-rodent cytokine.
5. An immunocytokine comprising: an immunoglobulin gamma heavy chain comprising a CPX-motif that has a sequence Of CPXiPX2 X3 X4 X5LGGPSX6X7 (SEQ ID NO: 36) and a GKX-motif that has a sequence Of GKX8FX9CXi0V (SEQ ID NO: 39); wherein Xi, X4 X6 X7 X8 X9 and Xio are each independently any amino acid, wherein X2 and X3 are each independently any amino acid or absent, and wherein X5 is any amino acid other than proline; and a non-human, non-rodent cytokine.
6. An immunocytokine comprising a non-human, non-rodent cytokine other than IL-12.
7. An immunocytokine comprising a dog Cκ region.
8. The immunocytokine of claim 2 or 5, wherein Xi is alanine, valine, cysteine or proline.
9. The immunocytokine of any one of claims 2, 5 and 8, wherein X2 is glycine or absent.
10. The immunocytokine of any one of claims 2, 5, 8 and 9, wherein X3 is cysteine or absent.
1 1. The immunocytokine of any one of claims 2, 5, and 8 to 10, wherein X4 is glycine or glutamine.
12. The immunocytokine of any one of claims 2, 5, and 8 to 1 1, wherein X5 is leucine, methionine, or serine.
13. The immunocytokine of any one of claims 2, 5, and 8 to 12, wherein X6 is isoleucine or valine.
14. The immunocytokine of any one of claims 2, 5, and 8 to 13, wherein X7 is phenylalanine or leucine.
15. The immunocytokine of any one of claims 2, 5, and 8 to 14, wherein the non- human immunoglobulin gamma heavy chain is selected from the group consisting of canine IgB, canine IgC, and canine IgD.
16. The immunocytokine of claim 2 or 5, wherein the CPX-motif has a sequence of CPAPEMLGGPSVF (SEQ ID NO: 33).
17. The immunocytokine of claim 2 or 5, wherein the CPX-motif has a sequence of CPCPGCGLLGGPSVF (SEQ ID NO: 34).
18. The immunocytokine of claim 2 or 5, wherein the CPX-motif has a sequence of CPVPESLGGPSVF (SEQ ID NO: 35).
19. The immunocytokine of claim 2 or 5, wherein the CPX-motif has a sequence of CPPPEMLGGPSIF (SEQ ID NO: 37).
20. The immunocytokine of any one of claims 4 to 19, wherein X8 is aspartate or glutamate.
21. The immunocytokine of any one of claims 4 to 20, wherein X8 is glutamate.
22. The immunocytokine of any one of claims 4 to 21, wherein Xg is lysine or threonine.
23. The immunocytokine of any one of claims 4 to 22, wherein X|0 is lysine or arginine.
24. The immunocytokine of any one of claims 1 to 23, wherein the constant region of the immunoglobulin gamma heavy chain is a canine or a feline immunoglobulin gamma heavy chain constant region.
25. The immunocytokine of any one of claims 1 to 24, wherein the immunoglobulin gamma heavy chain is chimeric.
26. The immunocytokine of claim 25, wherein the immunoglobulin gamma heavy chain comprises a mouse variable region and a feline or canine constant region.
27. The immunocytokine of any one of claims 1 to 26, wherein the immunocytokine binds specifically to a tumor antigen.
28. The immunocytokine of claim 27, wherein the tumor antigen is not a nucleic acid.
29. The immunocytokine of claim 27 or 28, wherein the tumor antigen is not a DNA molecule.
30. The immunocytokine of claim 27 or 28, wherein the tumor antigen is a non- proteinaceous tumor antigen.
31. The immunocytokine of any one of claims 27 to 30, wherein the tumor antigen is a polysaccharide.
32. The immunocytokine of claim 27 or 28, wherein the tumor antigen is a polypeptide.
33. The immunocytokine of claim 27 or 28, wherein the tumor antigen is selected from GD2, CD20, CD 19, CSPG, and EpCAM.
34. The immunocytokine of any one of claims 1 to 5 and 8 to 33, wherein the non- human, non-rodent cytokine is not IL- 12.
35. The immunocytokine of any one of claims 1 to 5 and 8 to 33, wherein the non- human, non-rodent cytokine is selected from the group consisting of: IL-I , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-I l , IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL- 19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL- 32, IL-33, IL-35, G-CSF, GM-CSF, TNF-β, TGF-β, IFN-γ, and IFN-α/β.
36. The immunocytokine of any one of claims 1 to 5 and 8 to 35, wherein the non-human, non-rodent cytokine is a cytokine that induces production of natural killer cells.
37. The immunocytokine of any one of claims 1 to 5 and 8 to 36, wherein the non- human, non-rodent cytokine is a cytokine that induces production of Cytotoxic T-CeIIs.
38. The immunocytokine of any one of claims 1 to 5 and 8 to 37, wherein the immunoglobulin gamma heavy chain is a non-human immunoglobulin gamma heavy chain.
39. An immunocytokine that binds specifically to CD20, wherein the immunocytokine comprises a dog or feline Cκ region.
40. An immunocytokine of claim 39 that binds specifically to an extracellular loop of CD20.
41. The immunocytokine of claim 39 or 40, that binds specifically to a peptide having a sequence set forth as: DIFNITICHFFKMENLNLIKAPMPYVCIHNCDPANPSEKNSLSIQYCGSIRS
(SEQ ID NO: 26), wherein the peptide forms a loop between the cysteine at position 8 and the cysteine at position 27.
42. An immunocytokine that binds specifically to CSPG, wherein the immunocytokine comprises a dog or feline Cκ region.
43. The immunocytokine of claim 42, that binds specifically to a peptide having a sequence set forth as:
EQFTQRDLEGGRLGLQLGRAPGPTGDSLTLELWAPGVPPAVASLDFHTEPYDAARPY GVALLSLPEEAGAPDSGAPATGQPGAPGPSPGPTAASGGFLGLLEAN
(SEQ ID NO: 27).
44. An immunocytokine that binds specifically to EpCAM, wherein the immunocytokine comprises a dog or feline Cκ region.
45. The immunocytokine of claim 44, that binds specifically to a peptide having a sequence set forth as:
ACICENYKLTTNCSLNΓNNQCECTSIGAQNSVICSKLATKCLVMKAEMTGTKSGRRA RPEGAFQNNDGLYDPDCDEKGLFKAKQCNGTTTCWCVNTAGVRRTDKDTEISCTER
VRTYWIIIELKHKTRETPYDTQSLQNALKETLKNRYQLDPKYITNILYENDLITIDLMQ
NSSQKAQNDVDIADVAYYFEKDVKDESLFHSSKMDLRVNGEQLDLDPGRTAIYYVD
EKPPEFSMQGLQAGIIAVIVVVTLAVIAGIVVLVISRKNRMAKYEKAEIKEMGEMHRE
LNA
(SEQ ID NO: 29).
46. A method for producing an immunoglobulin gamma heavy chain constant region, the method comprising:
(a) identifying a motif in the immunoglobulin gamma heavy chain constant region having between one mismatch and six mismatches with the sequence set forth as: CPAPELLGGPSVF (SEQ ID NO: 31); and
(b) producing a mutated version of the immunoglobulin gamma heavy chain constant region having at least one fewer mismatch with the sequence set forth as: CPAPELLGGPSVF (SEQ ID NO: 31).
47. The method of claim 46, wherein the motif is at the junction of a heavy chain hinge and a CH2 domain of the constant region.
48. The method of claim 46, wherein the immunoglobulin gamma heavy chain constant region is a canine or a feline immunoglobulin gamma heavy chain constant region.
49. The method of claim 46, wherein the immunoglobulin gamma heavy chain constant region is a canine immunoglobulin gamma heavy chain constant region of IgG-A, IgG-B, IgG-C or IgG-D.
50. The method of claim 46, wherein the motif identified in (a) has a proline in place of the leucine at position 6 of SEQ ID NO: 31.
51. The method of claim 50, wherein the mutated version of the immunoglobulin gamma heavy chain constant region has a substitution at position 6 that is a conservative substitution of leucine.
52. The method of claim 46, wherein the mutated version of the immunoglobulin gamma heavy chain constant region has improved Fc-receptor binding compared with the immunoglobulin gamma heavy chain constant region.
53. The method of claim 46, wherein the mutated version of the immunoglobulin gamma heavy chain constant region has improved ADCC effector function compared with the immunoglobulin gamma heavy chain constant region.
54. The method of claim 46, wherein the mutated version of the immunoglobulin gamma heavy chain constant region has improved protein A binding compared with the immunoglobulin gamma heavy chain constant region.
55. A recombinant antibody comprising an immunoglobulin gamma heavy chain constant region produced by the method of any one of claims 46 to 54.
56. An immunocytokine comprising the recombinant antibody of claim 55.
57. A method of targeting a cytokine in a non-human animal, the method comprising administering the immunocytokine of any one of claims 1 to 45 and 56 to a mammal.
58. A method of promoting ADCC in a non-human animal, the method comprising administering the immunocytokine of any one of claims 1 to 45 and 56 to a mammal.
59. An immunocytokine comprising a cytokine fused to an antibody heavy chain, wherein the immunocytokine comprises a light chain having a dog Cκ region.
60. The immunocytokine of claim 59, wherein the antibody heavy chain comprises a constant region of a dog IgG A, IgG B, IgG C, or IgG D.
61. The immunocytokine of any one of claims 1 to 45, 56 and 59, wherein the cytokine is a dog cytokine.
62. The immunocytokine of any one of claims 1 to 45, 56 and 59, wherein the cytokine is fused to the C-terminus of the heavy chain constant region.
63. A recombinant antibody comprising a mouse variable region and a dog constant region.
64. A recombinant antibody comprising a dog Cκ region.
65. The recombinant antibody of claim 63, wherein the dog constant region is a dog Cκ region.
66. A recombinant antibody of any one of claims 63 to 65, wherein the antibody is fused to an imaging agent and/or a radiolabeled agent.
67. The recombinant antibody of claim 66, wherein the heavy chain lacks a CH2 domain.
68. The recombinant antibody of claim 66, wherein the heavy chain contains one or more point mutations that shorten the half-life of the antibody.
69. An anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2, wherein the immunocytokine comprises a dog Cκ region.
70. The anti-GD2-dog IL-2 immunocytokine of claim 69, wherein the dog Cκ region comprises an amino acid sequence comprising SEQ ID NO: 1.
71. The anti-GD2-dog IL-2 immunocytokine of claim 69, wherein the immunocytokine comprises a dog CH region.
72. The anti-GD2-dog IL-2 immunocytokine of claim 71, wherein the dog CH region comprises an amino acid sequence comprising SEQ ID NO:2.
73. The anti-GD2-dog IL-2 immunocytokine of any one of claims 69 to 72, wherein the immunocytokine comprises a heavy chain variable region and a light chain variable region from a mouse anti-GD2 antibody.
74. The anti-GD2-dog IL-2 immunocytokine of claim 73, wherein the heavy chain variable region comprises an amino acid sequence comprising SEQ ID NO:3.
75. The anti-GD2-dog IL-2 immunocytokine of claim 73, wherein the light chain variable region comprises an amino acid sequence comprising SEQ ID NO:4.
76. The anti-GD2-dog IL-2 immunocytokine of claim 73, wherein the heavy chain variable region comprises an amino acid sequence comprising SEQ ID NO:3 and the light chain variable region comprises an amino acid sequence comprising SEQ ID NO:4.
77. The anti-GD2-dog IL-2 immunocytokine of claim 69, comprising a heavy chain polypeptide comprising an amino acid sequence comprising SEQ ID NO:5 and a light chain polypeptide comprising an amino acid sequence comprising SEQ ID NO:7.
78. An isolated nucleic acid molecule encoding a heavy chain polypeptide of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2, wherein the heavy chain polypeptide comprises an amino acid sequence comprising SEQ ID NO:5.
79. The nucleic acid molecule of claim 78, comprising the sequence SEQ ID NO:6.
80. An isolated nucleic acid molecule encoding a light chain polypeptide of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2 and comprises a dog Cκ region, wherein the light chain polypeptide comprises an amino acid sequence comprising SEQ ID NO:7.
81. The nucleic acid molecule of claim 80, comprising the sequence SEQ ID NO:8.
82. An isolated nucleic acid molecule comprising a sequence encoding an antibody variable region that binds specifically to a tumor antigen and a sequence encoding a non-human, non-rodent cytokine.
83. The isolated nucleic acid of claim 82, wherein the nucleic acid further comprises a sequence encoding a non-human light chain.
84. The isolated nucleic acid of claim 83, wherein the nucleic acid further comprises a sequence encoding a non-human heavy chain.
85. The isolated nucleic acid of any one of claims 82 to 84, wherein a sequence of the nucleic acid is a variant of a naturally occurring sequence, wherein the variant confers reduced ribonuclease mediated degradation of an mRNA encoded by the nucleic acid.
86. The isolated nucleic acid of any one of claims 82 to 85, wherein a sequence of the nucleic acid is a variant of a naturally occurring sequence, wherein the variant eliminates at least one AT-rich sequence that is a target for a ribonuclease.
87. The isolated nucleic acid of claim 86 wherein the at least one AT-rich sequence is in a coding region of the nucleic acid.
88. The isolated nucleic acid of claim 86 wherein the at least one AT-rich sequence is in a non-coding region of the nucleic acid.
89. The isolated nucleic acid of any one of claims 82 to 86, wherein the non- human, non-rodent cytokine is selected from the group consisting of: IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-1 1 , IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL- 32, IL-33, IL-35, G-CSF, GM-CSF, TNF-β, TGF-β, IFN-γ, and IFN-α/β.
90. The isolated nucleic acid of any one of claims 82 to 86, wherein the non- human, non-rodent cytokine is not IL-12.
91. An isolated vector comprising the nucleic acid molecule of claim 78 or claim 79.
92. An isolated vector comprising the nucleic acid molecule of claim 80 or claim 81.
93. An isolated vector comprising (a) the nucleic acid molecule of claim 78 or claim 79 and (b) the nucleic acid molecule of claim 80 or claim 81.
94. An isolated vector comprising (a) the nucleic acid molecule of claim 79 and (b) the nucleic acid molecule of claim 81.
95. An isolated vector comprising the nucleic acid molecule of any one of claims 82 to 90.
96. A cell comprising the vector of claim 93.
97. A cell comprising the vector of claim 94.
98 A cell comprising the vector of claim 95.
99. A composition comprising the anti-GD2-dog 1L-2 immunocytokine of any one ofclaims 69 to 72.
100. A composition comprising the anti-GD2-dog IL-2 immunocytokine of claim
73.
101. A composition comprising the anti-GD2-dog IL-2 immunocytokine of claim
74.
102. A composition comprising the anti-GD2-dog IL-2 immunocytokine of claim 75.
103. A composition comprising the anti-GD2-dog IL-2 immunocytokine of claim 76.
104. A composition comprising the anti-GD2-dog IL-2 immunocytokine of claim
77.
105. A composition comprising the immunocytokine of any one of claims 1 to 45 and 56 to 62.
106. A pharmaceutical composition comprising (i) a therapeutically effective amount of the immunocytokine of any one of claims 1 to 45, 56 to 62, 69 to 77, 122 and 123 and (ii) a pharmaceutically acceptable carrier.
107. A method for potentiating a cell-directed immune response in a non-human animal, the method comprising: administering the pharmaceutical composition of claim 106, wherein the immunocytokine binds specifically to an antigen that is expressed on the extracellular surface of a cell in the non-human animal.
108. The method of claim 107, wherein the cell is a tumor cell and the antigen is tumor antigen.
109. The method of claim 107, wherein the cell is a B-cell and the antigen is CD20.
1 10. A method for treating cancer in a non-human animal, the method comprising: administering the pharmaceutical composition of claim 106, wherein the immunocytokine binds specifically to a tumor antigen that is expressed on the extracellular surface of a tumor cell of the cancer.
1 1 1. A method for treating cancer in a non-human animal, the method comprising: determining that a tumor antigen is expressed on the extracellular surface of a tumor cell of the cancer; and administering the pharmaceutical composition of claim 106, wherein the immunocytokine binds specifically to the tumor antigen.
1 12. The method of any one of claims 107 to 1 1 1 further comprising administering an anti-cancer compound other than the immunocytokine in combination with the pharmaceutical composition.
1 13. The method of any one of claims 107 to 1 1 1 further comprising subjecting the non-human animal to any one of the following protocols: CHOP therapy, the Wisconsin- Madison protocol, the AMC protocol and the VELCAP protocol, in combination with administering the pharmaceutical composition.
1 14. A method of treating a GD2-expressing cancer in a dog, comprising administering to a dog having a GD2-expressing cancer an effective amount of an anti-GD2-dog IL-2 immunocytokine that binds specifically to GD2, wherein the immunocytokine comprises a dog Cκ region, to treat the cancer.
1 15. The method of claim 1 14, wherein the immunocytokine comprises a dog CH region.
1 16. The method of claim 1 14 or claim 1 15, wherein the immunocytokine comprises a heavy chain variable region and a light chain variable region from a mouse anti- GD2 antibody.
117. The method of claim 1 16, wherein the heavy chain variable region comprises an amino acid sequence comprising SEQ ID NO:3 and the light chain variable region comprises an amino acid sequence comprising SEQ ID NO:4.
1 18. The method of claim 1 14 or claim 1 15, wherein the immunocytokine comprises a heavy chain polypeptide comprising an amino acid sequence comprising SEQ ID NO:5 and a light chain polypeptide comprising an amino acid sequence comprising SEQ ID NO:7.
1 19. The method of claim 1 14, wherein the GD2 -expressing cancer is selected from the group consisting of melanoma, osteosarcoma, neuroblastoma, and small cell lung cancer.
120. The method of claim 1 14, wherein the GD2-expressing cancer is melanoma.
121. The method of claim 1 14, wherein the GD2-expressing cancer is osteosarcoma.
122. An immunocytokine comprising an scFv-Fc-cytokine protein fusion, wherein one or more of the scFv, Fc, and/or cytokine is a dog or cat protein.
123. An immunocytokine comprising an scFγ-Fc-IL2 protein fusion, wherein the IL2 protein is a dog IL2 or cat IL2.
PCT/US2010/001034 2009-04-05 2010-04-05 Chimeric immunocytokines and methods of use thereof WO2010117448A2 (en)

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