WO2011137245A2 - Lytic-peptide-her2/neu (human epidermal growth factor receptor 2) ligand conjugates and methods of use - Google Patents

Lytic-peptide-her2/neu (human epidermal growth factor receptor 2) ligand conjugates and methods of use Download PDF

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Publication number
WO2011137245A2
WO2011137245A2 PCT/US2011/034338 US2011034338W WO2011137245A2 WO 2011137245 A2 WO2011137245 A2 WO 2011137245A2 US 2011034338 W US2011034338 W US 2011034338W WO 2011137245 A2 WO2011137245 A2 WO 2011137245A2
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Prior art keywords
conjugate
cell
cancer
tumor
her2
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PCT/US2011/034338
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French (fr)
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WO2011137245A3 (en
Inventor
Hector Alila
Carola Leuschner
Martha Juban
Ann Coulter
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Esperance Pharmaceuticals, Inc.
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Priority to US13/695,390 priority Critical patent/US9586996B2/en
Priority to JP2013508251A priority patent/JP2013531474A/en
Publication of WO2011137245A2 publication Critical patent/WO2011137245A2/en
Publication of WO2011137245A3 publication Critical patent/WO2011137245A3/en
Priority to US15/433,887 priority patent/US10729740B2/en
Priority to US16/941,383 priority patent/US20200353032A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • C07K2317/20—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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • 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)

Definitions

  • Lytic-Peptide-Her2/neu Human Epidermal growth factor Receptor 2
  • the invention relates to lytic peptide and Her2/neu (Human Epidermal growth factor Receptor 2, also known as ErbB-2) ligand conjugates, methods of using conjugates, for example, in methods of treating undesirable or aberrant cell proliferation or hyperproliferative disorders, such as non-metastatic and metastatic neoplasias, cancers, tumors and malignancies that express Her2/neu.
  • Her2/neu Human Epidermal growth factor Receptor 2, also known as ErbB-2
  • conjugates for example, in methods of treating undesirable or aberrant cell proliferation or hyperproliferative disorders, such as non-metastatic and metastatic neoplasias, cancers, tumors and malignancies that express Her2/neu.
  • the invention is based, at least in part on lytic domains fused or conjugated to a ligand that binds to Her2/neu (Human Epidermal growth factor Receptor 2, also known as ErbB-2), referred to herein as conjugates or fusion constructs.
  • Her2/neu Human Epidermal growth factor Receptor 2, also known as ErbB-2
  • Contact of a cell with a lytic domain is believed to cause disruption of the cell membrane which results in cell death.
  • the ligand that binds to Her2/neu targets cells that express Her2/neu for destruction by the ligand that binds to Her2/neu and/or lytic domain, including undesirable or aberrant proliferating cells or hyperproliferating cells, such as non-metastatic and metastatic neoplasias, cancers, tumors and malignancies.
  • non-metastatic and metastatic neoplastic, cancer, tumor and malignant cells overexpress receptors or ligands.
  • many non-metastatic and metastatic neoplasias, cancers, tumors and malignancies express Her2/neu that can be used as a target of the conjugate or fusion construct.
  • Conjugates can be designed to target any cell or cell population that expresses Her2/neu.
  • Ligands that bind to Her2/neu which include antibodies and fragments thereof, hormones, growth factors, cytokines, etc., can be linked to a lytic domain to provide targeted killing of cells that express or contain Her2/neu thereby reducing or inhibiting cell proliferation or growth.
  • Conjugates do not require cells to divide in order to kill the target cells. Furthermore, the conjugates are not likely to be immunogenic because they can be made to be relatively small in size. In addition, the conjugates kill multi-drug resistant cells. [0007] In accordance with the invention, there are provided conjugates that include a first and a second domain.
  • a conjugate includes or consists of a ligand that binds to Her2/neu consisting and a second domain consisting of a 10-120 amino acid fragment, such as a L- or D-amino acid sequence that includes a peptide sequence selected from for example, KFAKFAKKFAKFAKK, FA K F A KKFAKFA KKF, KFAKFAKKFAKFAKKFA, KFA FAKKFAKFA KFA ,
  • KFAKFAKKFAKFAKKFAKFAKFAKFAKFAKFAK or a sequence that includes a peptide selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,
  • a conjugate includes or consists of a ligand that binds to Her2/neu and a second domain consisting of an L- or D-amino acid sequence selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKPA,
  • KFAKFAKKFAKFAKKFAK KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKF, KFAKFAKKFAKFAKFA and KFAKFAKKFAKFAKFAKFAKFAKFAKFAK, or a sequence that includes a peptide selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, or a sequence that includes a peptide selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,
  • a conjugate includes a ligand that binds to Her2/neu and a second domain consisting of an L- or D-amino acid sequence selected from,
  • KFAKFAKKFAKFAKK KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,
  • KFAKFAKKFAKFAKKFAK KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKF, KFAKFAKKFAKFAKFA and KFAKFAKKFAKFAKFAKFAKFAKFAKFAK, or a sequence that includes a peptide selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, or a sequence that includes a peptide selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,
  • isolated and purified peptides that include or consist of a ligand that binds to Her2/neu and a second domain.
  • a second domain is KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKF or KFAKFAKKFAKFAKFA.
  • a second domain is KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKF or KFAKFAKKFAKFAKFA having one or more of the K residues substituted with any of an F or
  • Conjugates include a ligand that binds to Her2/neu.
  • Ligands include or consist of a molecule that binds to a Her2/neu (Human Epidermal growth factor Receptor 2, also known as ErbB-2), which can be an agonist or antagonist.
  • a ligand or lytic peptide can include or consist of a linear or cyclic structure.
  • a ligand includes an antibody and antibody fragments.
  • ligands include one or more amino acids (e.g., peptides, polypeptides, proteins), nucleic acids and carbohydrates.
  • Specific non-limiting classes of ligands include hormones, growth factors, hormone and growth factor analogues, and fragments of hormones, hormone analogs, growth factors, growth factor analogues, and fragments of growth factors and analogues, antibodies and fragments of antibodies that bind to a Her2/neu.
  • antibody fragments include Fab, Fab', F(ab')2, Fv, Fd, single-chain Fv (scFv), disulfide- linked Fvs (sdFv), V L , V H , Camel Ig, V-NAR, VHH, trispecific (Fab 3 ), bispecific (Fak), diabody ((V L - V H )2 or (VH-V L )?), triabody (trivalent), tetrabody (tetravalent), minibody ((scF v -C H 3)2), bispecific single-chain Fv (Bis-scFv), lgGdeltaCH2, scFv-Fc, (scFv) 2 -Fc, affibody (e.g., ZHer2-neu:2, ZHer2- neu:4 ZHer2-neu:7 ZHer2-neu:8), affibody, aptamer, avimer or nanobody.
  • Fab 3 bispecific (Fak), dia
  • Her2/neu and/or ligands can be optionally expressed on a cell.
  • Cells that express Her2/neu or that can be targeted in accordance with methods of the invention include hyperproliferative cells.
  • Cells that express Her2/neu or that can be targeted in accordance with methods of the invention for example include breast, ovarian, uterine, cervical, stomach, lung, gastric, colon, bladder, glial, hematologic and endometrial cells.
  • a ligand that binds to Her2/neu and second domain can include or consist of an amino acid, or an amino acid sequence.
  • a first or second domain has about 1 to 10, 10 to 20, 15 to 20 (i.e., 15, 16, 17, 18, 19 or 20 amino acids), 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino acid residues.
  • Her2/neu binding ligands and second domains can be positioned at either (or both) the NH 2 - terminus or the C-terminus relative to each other.
  • the first (binding ligand) domain is positioned at the NH-terminus relative to the second (lytic peptide) domain
  • the second (lytic peptide) domain is positioned at the C-terminus relative to the first (binding ligand) domain
  • a first (binding ligand) domain is positioned at the C-terminus relative to the second (lytic peptide) domain
  • a second (lytic peptide) domain is positioned at the NH-terminus relative to the first (binding ligand) domain.
  • Such second (lytic peptide) domains can be positioned at the C-terminus, the NH-terminus, or both the C-terminus and the NH-terminus. Accordingly, multiple such second (lytic peptide) domains can be included in a conjugate of the invention.
  • Her2/neu binding ligands and second domains can include or consist of one or more D-amino acids.
  • a first domain has a D-amino acid, for example, at any K, F or A residue.
  • Her2/neu binding ligands and second domains can be joined by a covalent (e.g., peptide or nonpeptide) bond.
  • a first and a second domain can be joined by a peptide or a non- peptide linker.
  • Her2/neu binding ligands and second domains are joined by a peptide sequence having from about 1 to 25 amino acid residues, or having a linear carbon chain.
  • Her2/neu binding ligands and second domains are joined by a peptide sequence that includes or consist of one or more A, S or G amino acid residues.
  • Her2/neu binding ligands and second domains are joined by a peptide sequence a first and second domain is joined by peptide sequence including or consisting of GSGGS, ASAAS, or a linear carbon chain such as CCCCCC.
  • Other peptide linkers include but are not limited to GS, AF, FK, VK, FFK,
  • Her2/neu binding ligands and second domains can further include or consist of additional domains.
  • a conjugate includes a third, fourth, fifth, sixth, seventh domain, etc.
  • Conjugates include or consist of isolated and purified forms. Conjugates also include or consist of a mixture. Such mixtures include compositions, such as a mixture of conjugate and a pharmaceutically acceptable carrier or excipient appropriate for administration to or in vivo contact with a subject, or a mixture of conjugate and an anti-cell proliferative or immune stimulating agent.
  • Conjugates include or consist of a unit dosage form.
  • a conjugate is a unit dosage in an amount effective to treat a subject having undesirable cell proliferation or a hyperproliferative disorder.
  • a conjugate is a unit dosage in an amount effective to treat a subject having a neoplasia, tumor or cancer.
  • a conjugate is a unit dosage in an amount effective to reduce fertility of a subject.
  • kits can be included within kits, optionally with instructions for practicing a method.
  • a kit includes a conjugate and instructions for reducing or inhibiting proliferation of a cell, reducing or inhibiting proliferation of a hyperproliferating cell, reducing or inhibiting proliferation of a neoplastic, tumor or cancer cell, treating a subject having a hyperproliferative disorder, treating a subject having a neoplasia, tumor or cancer, or reducing fertility of an animal.
  • nucleic acids that encode conjugates.
  • a nucleic acid encodes a conjugate including or consisting of a ligand that binds to Her2/neu and a second domain and a peptide sequence selected from for example, KFAKFAKKFAKFAKK, KFA FAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKF, KFAKFAKKFAKFAKFA and KF AKF AKXFAKTAKKTAKFAKKFAKFAK, or a sequence that includes a peptide selected from KFAKFAKKFAKFAKK, KFAKFAKK AK AKKF, KFAK AKKFAKFAKKFA,
  • a nucleic acid encodes a conjugate including or consisting of a ligand that binds to Her2/neu and a second domain and a peptide consisting of an L- or D-amino acid sequence selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,
  • KFAKFAKKFAKFAKKFA KFAKFAKKFAKFAKFAK, KFAKFAKKFAKFAK, KFAKFAKKFAKFAKF, KFAKFAKKFAKFAKFA and KFAKFAKKFAKF AKKFAKFAK.
  • Nucleic acids can be included in a vector, such as an expression vector that when expressed in a cell encodes a conjugate.
  • Host cells can be transformed with a nucleic acid in a vector, such that the cell expresses a conjugate encoded by the nucleic acid.
  • Conjugates are useful for, among other things, reducing or inhibiting proliferation of a cell, reducing or inhibiting cell proliferation, reducing or inhibiting proliferation of a hyperproliferating cell, reducing or inhibiting proliferation of a neoplastic, tumor, cancer or malignant cell and treating undesirable or aberrant cell proliferation, such as hyperproliferating cells or hyperproliferative disorders.
  • hyperproliferative disorders include benign hyperplasia, non- metastatic and metastatic neoplasias, cancers tumors and malignancies.
  • methods of reducing or inhibiting proliferation of a cell comprising methods of reducing or inhibiting cell proliferation; methods of reducing or inhibiting proliferation of a hyperproliferating cell; and methods of reducing or inhibiting proliferation of a neoplastic, tumor, cancer or malignant cell.
  • a method includes contacting a cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the cell; contacting a cell with a conjugate in an amount sufficient to reduce or inhibit cell proliferation; contacting a cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the hyperproliferating cell; and contacting a cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the neoplastic, tumor, cancer or malignant cell.
  • a method includes contacting a cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the cell; contacting a cell with the conjugate in an amount sufficient to reduce or inhibit proliferation of the hyperproliferating cell; and contacting a cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the neoplastic, tumor, cancer or malignant cell, wherein the ligand of the conjugate binds to the Her2/neu expressed by the cell.
  • Cells targeted in accordance with the invention methods include cells that express Her2/neu. Cells targeted in accordance with the invention methods therefore include, for example, breast, ovarian, uterine, cervical, stomach, lung, gastric, colon, bladder, glial, hematologic and endometrial cells.
  • Methods performed include, among others, contacting a subject in need of inhibiting, reducing or preventing proliferation, survival, differentiation, death, or activity of a cells, such as a hyperprolifertive cell or an undesirably proliferating cell.
  • exemplary subjects include a subject having or at risk of having undesirable or aberrant cell proliferation; a subject having or at risk of having a benign hyperplasia; or a non-metastatic or metastatic neoplasia, cancer, tumor or malignancy (e.g., a solid or liquid tumor, in any of breast, ovarian, uterine, cervical, stomach, lung, gastric, colon, bladder, glial, hematologic or endometrial cells).
  • a method includes, administering to a subject an amount of the conjugate sufficient to treat the hyperproliferative disorder; and administering to a subject an amount of the conjugate sufficient to reduce or inhibit proliferation of the neoplasia, tumor, cancer or malignancy.
  • Methods include treating a subject having or at risk of having a metastasis. For example, an amount of a conjugate effective to reduce or inhibit spread or dissemination of a tumor, cancer or neoplasia to other sites, locations or regions within the subject.
  • a method reduces or inhibits metastasis of a primary tumor or cancer to one or more other sites, formation or establishment of a metastasis at one or more other sites, locations or regions thereby reducing or inhibiting tumor or cancer relapse or tumor or cancer progression.
  • a method reduces or inhibits growth, proliferation, mobility or invasiveness of tumor or cancer cells that potentially or do develop metastases (e.g., disseminated tumor cells); reduces or inhibits formation or establishment of metastases arising from a primary tumor or cancer to one or more other sites, locations or regions distinct from the primary tumor or cancer; reduces or inhibits growth or proliferation of a metastasis at one or more other sites, locations or regions distinct from the primary tumor or cancer after the metastasis has formed or has been established; or reduces or inhibits formation or
  • metastases e.g., disseminated tumor cells
  • a method reduces or inhibits relapse or progression of the neoplasia, tumor, cancer or malignancy.
  • a method includes administering to a subject an amount of the conjugate sufficient to reduce or inhibit metastasis of the neoplasia, tumor, cancer or malignancy to other sites, or formation or establishment of metastatic neoplasia, tumor, cancer or malignancy at other sites distal from the primary neoplasia, tumor, cancer or malignancy.
  • Neoplasia, tumor, cancer and malignancy treatable in accordance with the invention include solid cellular mass, hematopoietic cells, or a carcinoma, sarcoma (e.g. lymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma or fibrosarcoma), lymphoma, leukemia, adenoma, adenocarcinoma, melanoma, glioma, glioblastoma, meningioma, neuroblastoma, retinoblastoma, astrocytoma, oligodendrocytoma, mesothelioma, reticuloendothelial, lymphatic or haematopoietic (e.g., myeloma, lymphoma or leukemia) neoplasia, tumor, cancer or
  • Neoplasia, tumor, cancer and malignancy treatable in accordance with the invention can be present in or affect a lung (small cell lung or non-small cell lung cancer), breast, ovarian, uterine, cervical, stomach, lung, gastric, colon, bladder, glial, hematologic, endometrial, lymph, blood, muscle, or skin cell neoplasia, tumor, cancer, or malignancy.
  • Methods may be practiced with other treatments or therapies (e.g., surgical resection, radiotherapy, ionizing or chemical radiation therapy, chemotherapy, immunotherapy, local or regional thermal (hyperthermia) therapy, or vaccination).
  • treatments or therapies can be administered prior to, substantially contemporaneously with (separately or in a mixture), or following administration of a conjugate.
  • a method includes administering an anti-cell proliferative, antineoplastic, anti-tumor, anti-cancer or immune-enhancing treatment or therapy.
  • a method includes administering an alkylating agent, anti-metabolite, plant extract, plant alkaloid, nitrosourea, hormone, nucleoside or nucleotide analog; cyclophosphamide, azathioprine, cyclosporin A, prednisolone, melphalan, chlorambucil, mechlorethamine, busulphan, methotrexate, 6-mercaptopurine, thioguanine, 5-fluorouracil, cytosine arabinoside , 5-azacytidine (5-AZC) and 5-azacytidine related compounds, bleomycin, actinomycin D, mithramycin, mitomycin C, carmustine, lomustine, semustine, streptozotocin, hydroxyurea, cisplatin, carboplatin, oxiplatin, mitotane, procarbazine, dacarbazine, taxol, vinblastine, vincris
  • Cell or immunotherapies include lymphocytes, plasma cells, macrophages, dendritic cells, T-cells, NK cells or B-cells; an antibody, a cell growth factor, a cell survival factor, a cell differentiative factor, a cytokine or a chemokine
  • IL-2 interleukins IL-2, IL-la, IL- ⁇ ⁇ , IL-3, IL-6, IL-7, granulocyte-macrophage-colony stimulating factor (GMCSF), IFN- ⁇ , IL-12, TNF-a, TNFp, MIP-la, ⁇ - ⁇ , RANTES, SDF-1 , MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, eotaxin-2, 1-309 TCA3, AT AC, HCC-1, HCC-2, HCC-3, LARC/MIP-3cc, PARC, TARC, CK , CKp6, CKp7, ⁇ 8, CKp9, CKpl 1, CKpl2, CIO, IL-8, GRO , GROp, ENA-78, GCP-2, PBP/CTAPIIIp-TG/NAP-2, Mig, PBSF/SDF-1, or lymphotactin) etc.
  • GMCSF gran
  • trastuzumab Herceptin
  • pertuzumab cetuximab
  • cetuximab Erbitux
  • ipilimumab zalutumumab
  • dalotuzumab figitumumab
  • ramucirumab galiximab
  • farletuzumab ocrelizumab
  • ofatumumab alemtuzumab (Campath)
  • panitumumab Vectibix
  • ibritumomab tiuxetan Zavalin
  • Methods of the invention include providing a subject with a benefit.
  • a method of treatment results in partial or complete destruction of the neoplastic, tumor, cancer or malignant cell mass, volume, size or numbers of cells, stimulating, inducing or increasing neoplastic, tumor, cancer or malignant cell necrosis, lysis or apoptosis, reducing neoplasia, tumor, cancer or malignancy volume size, cell mass, inhibiting or preventing progression or an increase in neoplasia, tumor, cancer or malignancy volume, mass, size or cell numbers, or prolonging lifespan; results in reducing or decreasing severity, duration or frequency of an adverse symptom or
  • complication associated with or caused by the neoplasia, tumor, cancer or malignancy results in reducing or decreasing pain, discomfort, nausea, weakness or lethargy; or results in increased energy, appetite, improved mobility or psychological well being.
  • Subjects treatable in accordance with the methods include mammals.
  • a subject is a human.
  • Figures 1A and IB show cytotoxicity of recombinantly scFv-CH 3 (naked antibody), scFv- C H 3 -GS-Phorl8 and scFv-C H 3 -GS-(KLAKLAK) 2 KLAK to Her2-neu receptor positive A) breast (SKBR-3); and B) ovarian (S OV-3) cancer cell lines determined after 48 hours.
  • Figures 2 A and 2B show median tumor volumes from mice treated with MAb (naked), A) MAb-Phorl 8 and B) recombinant scFv-C H 3 naked antibody and scFv-C H 3 -Phor-18 conjugates during the study period of 64 days in SKOV-3 xenografted mice in comparison with saline injected mice.
  • Figure 3 shows mean tumor weights from mice treated with MAb (naked), MAb-Phor 18 and recombinantly scFv-C H 3 naked antibody and scFv-C H 3 -Phor-18 conjugates on day 64 in SKOV-3 xenografted mice in comparison with saline injected mice.
  • Figure 4 shows mean body weights from mice treated with with with MAb (naked), MAb-Phorl 8 and recombinantly scFv-C H 3 naked antibody and scFv-C H 3 -Phor-18 conjugates during the study period of 64 days in SKOV-3 xenografted mice in comparison with saline injected mice.
  • W
  • the invention is based at least in part on a conjugate that includes a ligand that binds to Her2/neu joined or fused to a second lytic domain.
  • a conjugate first domain includes a ligand that binds to Her2/neu and a second domain that includes lytic portion, which is directly or indirectly toxic to a cell, which can thereby reduce cell proliferation or survival, or stimulate, induce, increase or enhance cell death, killing or apoptosis.
  • conjugates that include or consist of a first ligand that binds to Her2/neu domain and include or consist of a second lytic or toxic domain.
  • a conjugate in another embodiment, includes a first ligand that binds to Her2/neu domain and a second domain consisting of an L- or D-amino acid sequence selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,
  • KFAKFAKKFAKFAKKFA KFAKFAKKFAKFAKKFA, KFAKFAKKFAK, KFAKFAKKFAKFAKF, KFAKFAKKFAKFAKFA and KFAKFAKKFAKFAKFAKFAKFAK.
  • conjugate or “fusion construct” and grammatical variations thereof, means that the construct contains portions or sections that are derived from, obtained or isolated from, or are based upon or modeled after two different molecular entities that are distinct from each other and do not typically exist together in nature. That is, for example, one portion of the conjugate includes or consists of a ligand that binds to Her2/neu and a second portion of the conjugate includes or consists of a lytic portion, each of first and second domains structurally distinct.
  • a conjugate can also be referred to as a "fusion construct,” wherein the conjugate includes or consists of a second domain ligand that binds to Her2/neu and a second domain lytic portion.
  • First domains and or second domains of conjugates include or consist of amino acid sequences (peptides, polypeptides, proteins, lectins), nucleic acids (DNA, RNA) and carbohydrates (saccharides, sialic acid, galactose, mannose, fucose, acetylneuraminic acid, etc.).
  • amino acid sequences peptides, polypeptides, proteins, lectins
  • nucleic acids DNA, RNA
  • carbohydrates saccharide, sialic acid, galactose, mannose, fucose, acetylneuraminic acid, etc.
  • Amino acid sequences can be linked by non-natural and non-amide chemical bonds including, for example, those formed with glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, or N, N'- dicyclohexylcarbodiimide (DCC).
  • Non-amide bonds include, for example, ketomethylene, aminomethylene, olefin, ether, thioether and the like (see, e.g., Spatola in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357 (1983), "Peptide and Backbone
  • First and second domains of a conjugate or fusion construct include L-amino acid sequences, D-amino acid sequences and amino acid sequences with mixtures of L-amino acids and D-amino acids.
  • Amino acid sequences of first and second domains can be a linear or a cyclic structure, conjugated to a distinct moiety (e.g., third, fourth, fifth, sixth, seventh, etc. domains), form intra or intermolecular disulfide bonds, and also form higher order multimers or oligomers with the same or different amino acid sequence, or other molecules.
  • exemplary lengths of conjugates are from about 5 to 15, 20 to 25, 25 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 300 or more amino acid residues in length.
  • a first or second domain includes or consists of an amino acid sequence of about 1 to 10, 10 to 20, 15 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more residues.
  • a first domain consists of a 15, 16, 17, 18, 19, 20, 28 or more residue amino acid sequence.
  • Conjugate with first and a second domain, optionally the ligand that binds to Her2/neu lytic domain or second lytic domain form an amphipathic alpha-helix.
  • An amphipathic alpha-helix contains mostly hydrophilic amino acids on one side of the alpha-helix and the other side contains mostly hydrophobic amino acids. Since the alpha helix makes a complete turn for every 3.6 residues, the amino acid sequence of an amphipathic alpha helix alternates between hydrophilic and hydrophobic residues every 3 to 4 residues.
  • a PNNPNNP repeat pattern or motif is predicted to form an amphipathic alpha- helix where P represents a positively charged amino acid residue and N a neutral amino acid residue.
  • a PNNPNNP repeat pattern provides a cationic binding site for the lytic peptide to a negatively charged cell membrane and a hydrophobic site for membrane interaction/penetration.
  • Conjugates therefore include second domains with one or more uninterrupted PNNPNNP repeat patterns or motifs, or one or more interrupted PNNPNNP repeat patterns or motifs, which can form an amphipathic alpha-helix. For example, a 15 or 18 residue amino acid sequence, such as KF AKFA KFAKFAKK and
  • FAKFAKKFAKFAKKFAK has uninterrupted and interrupted PNNPNNP repeat motifs.
  • a conjugate first domain such as a ligand that binds to Her2/neu, includes or consists of a ligand, antibody, hormone, growth factor, or an analogue or fragment thereof.
  • Her2/neu is typically expressed by or present on (e.g., a membrane receptor) or within a cell. Her2/neu may associate with the cell membrane surface or traverse the cell membrane. For example, Her2/neu can have a transmembrane domain that traverses the cell membrane, optionally with a portion that is cytoplasmic or extracellular, or both. Her2/neu therefore include full length intact native Her2/neu containing an extracellular, transmembrane or cytoplasmic portion, as well as truncated forms or fragments thereof (e.g., an extracellular, transmembrane or cytoplasmic portion or subsequence of Her2/neu alone, or in combination).
  • a soluble Her2/neu typically lacks a transmembrane and may optionally also lack all or a part of the native extracellular or cytoplasmic region (if present in native Her2/neu). Such truncated forms and fragments can retain at least partial binding to a ligand.
  • Ligands that bind to Her2/neu of conjugates include or consist of any entity that binds to Her2/neu, specifically or non-specifically.
  • Non-limiting examples of ligands therefore include antibodies, antibody fragments, hormones, hormone analogues, fragments of a hormone or hormone analogue that binds to Her2/neu, growth factors, growth factor analogues, fragments of a growth factor or growth factor analogue that binds to Her2/neu, etc.
  • Exemplary ligands that bind to Her2/neu include affibodies, such as ZHer2-neu:2, ZHer2- neu:4 ZHer2-neu:7 ZHer2-neu:8 and Fab63.
  • affibody sequences are as follows:
  • Exemplary growth factors useful as ligands that bind to Her2/neu include epidermal growth factor and epidermal growth factor analogues.
  • Her2/neu ligands and binding moieties further include antibodies, and antibody fragments.
  • An ''antibody refers to any monoclonal or polyclonal immunoglobulin molecule, such as IgM, IgG, IgA, IgE, IgD, and any subclass thereof. Exemplary subclasses for IgG are IgG], IgG 2 , IgG 3 and IgG 4 including engineered antibody subclasses and recombinant antibodies with various glycosylation patterns.
  • Antibodies include those produced by or expressed on cells, such as B cells, or synthesized or engineered to be produced by other cells, e.g., CHO cells.
  • Such antibodies include those with improved characteristics, such as increased serum stability and/or half life in vivo, PK, etc. (e.g., as described in Antibody Engineering Vol 1, Konterman R and Duebel S, eds., 2010, Springer, WO 2006/130834 and Horton et al., Cancer Res 68:8049 (2008)).
  • Non-limiting mutations in the Fc include, for example, I253A, H310A, H435 R, H435Q, G236R, L328 R, S239D, I332E.
  • Non-limiting mutations in IgGlcan beat residues 238, 252, 253, 254, 255, 256, 265, 272, 289, 288, 303, 305, 307, 309, 31 1, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 439 and/or 477 of the Fc region.
  • An antibody fragment or subsequence refers to a portion of a full length antibody that retains at least partial antigen binding capability of a comparison full length antibody.
  • Exemplary antibody fragments include Fab, Fab', F(ab') 2 , Fv, Fd, single-chain Fv (scFv), disulfide-linked Fvs (sdFv), V L ,
  • V H Camel Ig, V-NAR, VHH, trispecific (Fab 3 ), bispecific (Fab:), diabody ((V L -V H ) 2 or (VH-V L ) 2 ), triabody (trivalent), tetrabody (tetravalent), minibody ((scFv-C H 3)2), bispecific single-chain Fv (Bis- scFv), IgGdeltaC H 2, scFv-Fc, (scFv) 2 -Fc, affibody, aptamer, avimer, nanobody or other antigen binding fragment.
  • Exemplary antibodies bind to epitopes present on Her2/neu.
  • Exemplary Her2/neu epitopes include HER-2 (p5-13) A2, HER-2 (p8-16) A24, HER-2 (p48-56) A2, HER-2 (p63-71) A24, HER-2 (pi 06-114) A2, HER-2 (p369-377) A2, A3, A26, HER-2 (p435-443) A2, HER-2 (p654-662) A2, HER- 2 (p665-673) A2, HER-2 (p689-697) A2, HER-2 (p754-762) A3, Al 1 , A33, HER-2 (p773-782) A2, HER-2 (p780-788) A24, HER-2 (p785-794) A2, HER-2 (p789-797) A2, HER-2 (p799-807) A2,
  • Exemplary antibodies include humanized anti-ErbB2 antibodies huMA Dl-l, huMAb4D5- 1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (HERCEPTINTM) as described in U.S. Patent No.5,821,337; humanized 520C9 (WO 93/21319) and humanized 2C4 (pertuzumab) as described in U.S. Patent No.7,097,840 and pertuzumab variants as described in US2009/0285837A1.
  • V j _ Light chain
  • huMAb4D5-6 Vi_ (Light chain):
  • Vr Light chain: Claim 1 mutation
  • Pertuzumab Sequences humanized 2C4, 7C2, 7 F3, 7D3, 3 E 8, 4D5, 2H11 , 3 H4 (U.S. Patent
  • Conjugates include those with a first domain at the amino-terminus and a second domain at the carboxyl-terminus. Conjugates also include those with a first domain at the carboxyl -terminus and a second domain at the amino-terminus. Where additional domains are present (e.g., third, fourth, fifth, sixth, seventh, etc. domains), a first domain is positioned at the NH 2 -terminus relative to a second domain, or a second domain is positioned at the NFF-terminus relative to a first domain.
  • a subsequence of a first or second domain has at least 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, or more amino acid residues.
  • the invention therefore includes modifications or variations, such as substitutions, additions or deletions of a first or second domain, or both first and second domains.
  • a conjugate that includes a peptide sequence first or second domain can incorporate any number of conservative or non- conservative amino acid substitutions, as long as such substitutions do not destroy activity (Her2/neu binding or lytic) of first or second domains.
  • a modified ligand that binds to Her2/neu or a second lytic portion domain can retain at least partial Her2/neu binding or lytic activity, such as cell killing or apoptosis, of an unmodified second domain.
  • a "conservative substitution” is a replacement of one amino acid by a biologically, chemically or structurally similar residue.
  • Biologically similar means that the substitution is compatible with a biological activity, e.g., lytic activity.
  • Structurally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or having similar size, or the structure of a first, second or additional domain is maintained, such as an amphipathic alpha helix.
  • Chemical similarity means that the residues have the same charge or are both hydrophilic and hydrophobic.
  • Particular examples include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, serine for threonine, etc.
  • Routine assays can be used to determine whether a conjugate variant has activity, e.g., binding activity or lytic activity.
  • a modified conjugate can have a peptide sequence with 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more identity to a reference sequence (e.g., a first domain, such as a ligand that binds to Her2/neu or a second domain such as KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,
  • a conjugate includes a peptide first domain that includes or consists of a ligand that binds to Her2/neu and a second lytic domain that includes or consists of an L- or D-amino acid sequence that includes a peptide selected from KPAKFAKKFAKFAKK,
  • KFAKFAKKFAKFAKKF KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKFAK,
  • KFAKFAKKFAKFAKKFAKF KFAKFAKKFAKFAKKF AKFA
  • a conjugate includes a ligand that binds to Her2/neu and a second domain consisting of an L- or D-amino acid sequence selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF.
  • identity and “homology” and grammatical variations thereof mean that two or more referenced entities are the same. Thus, where two amino acid sequences are identical, they have the same amino acid sequence. "Areas, regions or domains of identity” mean that a portion of two or more referenced entities are the same. Thus, where two amino acid sequences are identical or homologous over one or more sequence regions, they share identity in these regions.
  • complementary when used in reference to a nucleic acid sequence means the referenced regions are 100%) complementary, i.e., exhibit 100% base pairing with no mismatches.
  • the amount of sequence identity required to retain a function or activity depends upon the protein, the region and the function or activity of that region.
  • a function or activity e.g., Her2/neu binding or lytic
  • multiple PNNPNNP sequence repeat patterns or motifs can be present, but one or more interrupted or non-interrupted PNNPNNP sequence repeat patterns or motifs need not be present.
  • BLAST e.g., BLAST 2.0
  • Altschul et al., J. Mol. Biol. 215:403 (1990), publicly available through NCB1 has exemplary search parameters as follows: Mismatch -2; gap open 5; gap extension 2.
  • a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAM100, PAM 250, BLOSUM 62 or BLOSUM 50.
  • FASTA e.g., FASTA2 and FASTA3
  • SSEARCH sequence comparison programs are also used to quantitate the extent of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132: 185 (2000); and Smith et al., J. Mol. Biol. 147: 195 (1981)).
  • Programs for quantitating protein structural similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).
  • covalent bonds are amide bonds, non-natural and non-amide chemical bonds, which include, for example, glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N, N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC).
  • aminomethylene CPL-NH
  • ethylene olefin
  • ether CH 2 -0
  • thioether CFL-S
  • First and second domains can be fused or joined immediately adjacent to each other by a covalent or a non-covalent bond.
  • First and second domains can be separated by an intervening region, such as a hinge, spacer or linker positioned between a first and a second domain.
  • a first and second domain are joined by a carbon chain (e.g., CCCCC).
  • Multi-carbon chains include carboxylic acids (e.g., dicarboxylic acids) such as glutaric acid, succinic acid and adipic acid.
  • a first and second domain are joined by an amino acid, peptide or a non-peptide hinge, spacer or linker positioned between the first and second domains.
  • Peptide hinge, spacer or linker sequences can be any length, but typically range from about 1-10, 10-20, 20-30, 30-40, or 40-50 amino acid residues.
  • a peptide hinge, spacer or linker positioned between a first and second domain is from 1 to 5, 1 to 10, 1 to 20, 1 to 25 L- or D-amino acid residues, or 1 to 6 L- or D-amino acid residues.
  • Particular amino acid residues that are included in sequences positioned between the first and second domains include one or more of or C, A, S or G amino acid residues.
  • Specific non-limiting examples of peptides positioned between the first and second domains include a sequence within or set forth as: GSGGS, ASAAS, or a carbon chain such as CCCCCC.
  • Derivatives of amino acids andpeptides can be positioned between the first and second domain.
  • a specific non-limiting example of an amino acid derivative is a lysine derivative, or a 6 carbon linker such as a-amino-caproic acid.
  • Conjugates with or without a hinge, spacer or linker, or a third, fourth, fifth, sixth, seventh, etc. domain can be entirely composed of natural amino acids or synthetic, non-natural amino acids or amino acid analogues, or can include derivatized forms.
  • a conjugate includes in a first or second domain one or more D-amino acids substituted for L-amino acids, mixtures of D- amino acids and L-amino acids, or a sequence composed entirely of D-amino acid residues.
  • Conjugates can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., induce or stabilize a secondary structure, e.g., an alpha helix conformation.
  • Conjugates include cyclic structures such as an end-to-end amide bond between the amino and carboxy-terminus of the molecule or intra- or inter-molecular disulfide bond(s).
  • Conjugates may be modified in vitro or in vivo, e.g., post-translationally modified to include, for example, sugar or carbohydrate residues, phosphate groups, fatty acids, lipids, etc.
  • Specific examples of an addition include a third, fourth, fifth, sixth or seventh domain.
  • Conjugates with a first and second domain therefore include one or more additional domains (third, fourth, fifth, sixth, seventh, etc.) covalently linked thereto to impart a distinct or complementary function or activity.
  • additional domains include domains facilitating isolation, which include, for example, metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals; protein A domains that allow purification on immobilized immunoglobulin; and domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA).
  • a cleavable sequence such as Factor Xa or enterokinase between a purification domain and the conjugate can be used to facilitate purification.
  • an expression vector can include a conjugate-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site.
  • the histidine residues facilitate detection and purification of the conjugate while the enterokinase cleavage site provides a means for purifying the construct from the remainder of the protein (see e.g., roll, DNA Cell. Biol. 12:441 (1993)).
  • Conjugate activity can be affected by various factors and therefore conjugates can be designed or optimized by taking into consideration one or more of these factors. Such factors include, for example, length, which can affect toxicity to cells. Cell killing activity of alpha helix forming lytic peptide domains can also depend on the stability of the helix. Hinge and spacers can affect membrane interaction of a first domain and the helical structure of a peptide lytic domain. The charge of lytic peptide domains, which is determined in part by the particular amino acid residues present in the domain, also affects cell killing potency. The positioning of the ligand that binds to Her2/neu relative to the lytic domain (N- or C-terminus) also can affect cell killing activity of conjugates.
  • Conjugate in vivo half-life can be increased by constructing peptide domains with one or more non-naturally occurring amino acids or derivatives.
  • conjugates with D-amino acids can be increased by constructing peptide domains with one or more non-naturally occurring amino acids or derivatives.
  • conjugates with D-amino acids can be increased by constructing peptide domains with one or more non-naturally occurring amino acids or derivatives.
  • conjugates with D-amino acids for example, conjugates with D-amino acids
  • peptide domains with one or more non-naturally occurring amino acids or derivatives can reduce hemolytic activity.
  • conjugates with D-enantiomers also have a greater tendency to be monomeric in solution- they do not significantly aggregate.
  • Peptides and peptidomimetics can be produced and isolated using methods known in the art.
  • Peptides can be synthesized, whole or in part, using chemical methods known in the art (see, e.g., Caruthers (1980). Nucleic Acids Res. Symp. Ser. 215; Horn (1980); and Banga, A. ., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA).
  • Peptide synthesis can be performed using various solid-phase techniques (see, e.g., Roberge Science 269:202 (1995); Merrifield, Methods Enzymol.
  • the invention further provides nucleic acids encoding the conjugates of the invention and vectors that include nucleic acid that encodes conjugates.
  • Nucleic acid which can also be referred to herein as a gene, polynucleotide, nucleotide sequence, primer, oligonucleotide or probe refers to natural or modified purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides and a-anomeric forms thereof.
  • the two or more purine- and pyrimidine-containing polymers are typically linked by a phosphoester bond or analog thereof.
  • nucleic acid can be single strand, double, or triplex, linear or circular.
  • Nucleic acids include genomic DNA, cDNA, and antisense.
  • RNA nucleic acid can be spliced or unspliced mRNA, rRNA, tRNA or antisense.
  • Nucleic acids include naturally occurring, synthetic, as well as nucleotide analogues and derivatives.
  • Nucleic acid can be produced using any of a variety of known standard cloning and chemical synthesis methods, and can be altered intentionally by site-directed mutagenesis or other recombinant techniques known to one skilled in the art. Purity of polynucleotides can be determined through sequencing, gel electrophoresis, UV spectrometry.
  • Nucleic acids may be inserted into a nucleic acid construct in which expression of the nucleic acid is influenced or regulated by an "expression control element," referred to herein as an "expression cassette.”
  • expression control element refers to one or more nucleic acid sequence elements that regulate or influence expression of a nucleic acid sequence to which it is operatively linked.
  • An expression control element can include, as appropriate, promoters, enhancers, transcription terminators, gene silencers, a start codon (e.g., ATG) in front of a protein-encoding gene, etc.
  • An expression control element operatively linked to a nucleic acid sequence controls transcription and, as appropriate, translation of the nucleic acid sequence.
  • the term "operatively linked” refers to a juxtaposition wherein the referenced components are in a relationship permitting them to function in their intended manner.
  • expression control elements are juxtaposed at the 5' or the 3' ends of the genes but can also be intronic.
  • Expression control elements include elements that activate transcription constitutively, that are inducible (i.e., require an external signal for activation), or derepressible (i.e., require a signal to turn transcription off; when the signal is no longer present, transcription is activated or "derepressed”). Also included in the expression cassettes of the invention are control elements sufficient to render gene expression controllable for specific cell-types or tissues (i.e., tissue-specific control elements).
  • Promoters are generally positioned 5' of the coding sequence. Promoters, produced by recombinant DNA or synthetic techniques, can be used to provide for transcription of the polynucleotides of the invention.
  • a "promoter” is meant a minimal sequence element sufficient to direct transcription.
  • Nucleic acids may be inserted into a plasmid for propagation into a host cell and for subsequent genetic manipulation if desired.
  • a plasmid is a nucleic acid that can be stably propagated in a host cell; plasmids may optionally contain expression control elements in order to drive expression of the nucleic acid.
  • a vector is used herein synonymously with a plasmid and may also include an expression control element for expression in a host cell. Plasmids and vectors generally contain at least an origin of replication for propagation in a cell and a promoter.
  • Plasmids and vectors are therefore useful for genetic manipulation of conjugate encoding nucleic acids, producing conjugates or antisense nucleic acid, and expressing conjugates in host cells and organisms, for example.
  • Bacterial system promoters include T7 and inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter) and tetracycline responsive promoters.
  • Insect cell system promoters include constitutive or inducible promoters (e.g., ecdysone).
  • Mammalian cell constitutive promoters include SV40, RSV, bovine papilloma virus (BPV) and other virus promoters, or inducible promoters derived from the genome of mammalian cells (e.g., metallothionein IIA promoter; heat shock promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the inducible mouse mammary tumor virus long terminal repeat).
  • a retroviral genome can be genetically modified for introducing and directing expression of a conjugate in appropriate host cells.
  • Expression systems further include vectors designed for in vivo use.
  • vectors designed for in vivo use include adenoviral vectors (U.S. Patent Nos. 5,700,470 and 5,731,172), adeno-associated vectors (U.S. Patent No. 5,604,090), herpes simplex virus vectors (U.S. Patent No. 5,501,979), retroviral vectors (U.S. Patent Nos. 5,624,820, 5,693,508 and 5,674,703), BPV vectors (U.S. Patent No.
  • Yeast vectors include constitutive and inducible promoters (see, e.g., Ausubel et al., In:
  • Yeast artificial chromosomes are typically used when the inserted polynucleotides are too large for more conventional vectors (e.g., greater than about 12 Kb).
  • Expression vectors also can contain a selectable marker conferring resistance to a selective pressure or identifiable marker (e.g., beta-galactosidase), thereby allowing cells having the vector to be selected for, grown and expanded.
  • a selectable marker can be on a second vector that is cotransfected into a host cell with a first vector containing a nucleic acid encoding a conjugate.
  • Selection systems include but are not limited to herpes simplex virus thymidine kinase gene (Wigler et al., Cell 1 1 :223 (1977)), hypoxanthine-guanine phosphoribosyltransferase gene (Szybalska et al., Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes which can be employed in tk-, hgprt- or aprt- cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (O'Hare et al., Proc. Natl. Acad. Sci. USA 78: 1527 (1981)); the gpt gene, which confers resistance to mycophenolic acid (Mulligan et al., Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neomycin gene, which confers resistance to aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol. 150: 1 (1981)); puromycin; and hygromycin gene, which confers resistance to hygromycin
  • Additional selectable genes include trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman et al., Proc. Natl. Acad. Sci. USA 85:8047 (1988)); and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue (1987) In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory).
  • a host cell that express conjugates, and host cells transformed with nucleic acids encoding conjugates and vectors including a nucleic acid that encodes the conjugates are also provided.
  • a host cell is a prokaryotic cell.
  • a host cell is a eukaryotic cell.
  • the eukaryotic cell is a yeast or mammalian (e.g., human, primate, etc.) cell.
  • a "host cell” is a cell into which a nucleic acid is introduced that can be propagated, transcribed, or encoded conjugate expressed. The term also includes any progeny or subclones of the host cell. Host cells include cells that express conjugates.
  • Host cells include but are not limited to microorganisms such as bacteria and yeast; and plant, insect and mammalian cells.
  • bacteria transformed with recombinant bacteriophage nucleic acid, plasmid nucleic acid or cosmid nucleic acid expression vectors yeast transformed with recombinant yeast expression vectors
  • plant cell systems infected with recombinant virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
  • recombinant plasmid expression vectors e.g., Ti plasmid
  • insect cell systems infected with recombinant virus expression vectors e.g., baculovirus
  • animal cell systems infected with recombinant virus expression vectors e.g., retroviruses, adenovirus, vaccinia virus, or transformed animal cell systems engineered for transient or stable propagation or expression.
  • Conjugates, nucleic acids encoding conjugates, vectors and host cells expressing conjugates or transformed with nucleic acids encoding conjugates and antisense include isolated and purified forms.
  • isolated when used as a modifier of an invention composition, means that the composition is made by the hand of man or is separated, substantially completely or at least in part, from the naturally occurring in vivo environment. Generally, an isolated composition is substantially free of one or more materials with which it normally associates with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane.
  • isolated does not exclude alternative physical forms of the composition, such as muitimers/oligomers, variants, modifications or derivatized forms, or forms expressed in host cells produced by the hand of man.
  • isolated also does not exclude forms (e.g., pharmaceutical formulations and combination compositions) in which there are combinations therein, any one of which is produced by the hand of man.
  • An “isolated” composition can also be “purified” when free of some, a substantial number of, most or all of the materials with which it typically associates with in nature.
  • an isolated conjugate that also is substantially pure does not include polypeptides or polynucleotides present among millions of other sequences, such as proteins of a protein library or nucleic acids in a genomic or cDNA library, for example.
  • a "purified” composition can be combined with one or more other molecules.
  • a mixture includes one or more conjugates and a pharmaceutically acceptable carrier or excipient.
  • a mixture includes one or more conjugates and an anti-cell proliferative, anti-tumor, anti-cancer, or anti-neoplastic treatment or agent.
  • a mixture includes one or more conjugates and an immune enhancing agent.
  • Combinations such as one or more conjugates in a pharmaceutically acceptable carrier or excipient, with one or more of an anti-cell proliferative, anti-tumor, anti-cancer, or anti-neoplastic treatment or agent, and an immune enhancing treatment or agent, are also provided.
  • Conjugates of the invention such as polypeptides having an amino acid sequence including a first ligand that binds to Her2/neu (Her2/neu binding moiety) domain and a second lytic domain, can be used to target cells for lysis, cell death or apoptosis. Such cells can be selectively targeted. For example a cell that expresses Her2/neu can be targeted by a conjugate and thereby be preferentially killed compared to cells that express none or less of the Her2/neu.
  • a method includes contacting a cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the cell. In another embodiment, a method includes contacting a cell with a conjugate in an amount sufficient to reduce or inhibit cell proliferation.
  • a method includes contacting a hyperproliferative cell or hyperprol iterating cells with a conjugate in an amount sufficient to reduce or inhibit proliferation.
  • a method includes contacting a neoplastic, cancer, tumor or malignant cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the cell.
  • a method includes contacting a dormant or non-dividing neoplastic, cancer, tumor or malignant cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the dormant or non-dividing cell.
  • a method includes contacting the cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the cell (e.g., hyperproliferating cell), wherein the conjugate binds to Her2/neu expressed by the cell.
  • a method includes contacting the cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the neoplastic, tumor, cancer or malignant cell, wherein the conjugate binds to the Her2/neu expressed by the cell.
  • contacting means direct or indirect binding or interaction between two or more entities (e.g., between a conjugate and a cell).
  • Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration or delivery.
  • Cells to target for reducing or inhibiting proliferation include cells that express Her2/neu.
  • Non-limiting exemplary cells include breast, ovarian, uterine, cervical, stomach, lung, gastric, colon, bladder, glial, hematologic and endometrial cells.
  • target cells include cells that express Her2/neu.
  • Conjugates and methods of the invention are also applicable to treating undesirable or aberrant cell proliferation and hyperproliferative disorders, which include cells expressing Her2/neu.
  • methods of treating undesirable or aberrant cell proliferation and hyperproliferative disorders are provided.
  • a method includes administering to a subject (in need of treatment) an amount of a conjugate sufficient to treat the undesirable or aberrant cell proliferation or the hyperproliferative disorder.
  • hyperproliferative disorder refers to any undesirable or aberrant cell survival (e.g., failure to undergo programmed cell death or apoptosis), growth or proliferation.
  • disorders include benign hyperplasias, non-metastatic and metastatic neoplasias, cancers, tumors and
  • Undesirable or aberrant cell proliferation and hyperproliferative disorders can affect any cell, tissue, organ in a subject. Undesirable or aberrant cell proliferation and hyperproliferative disorders can be present in a subject, locally, regionally or systemically.
  • a hyperproliferative disorder can arise from a multitude of tissues and organs, including but not limited to breast, lung (e.g., small cell or non- small cell), thyroid, head and neck, brain, nasopharynx, throat, nose or sinuses, lymphoid, adrenal gland, pituitary gland, thyroid, lymph, gastrointestinal (mouth, esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon, rectum), genito-urinary tract (uterus, ovary, vagina, cervix, endometrium, fallopian tube, bladder, testicle, penis, prostate), kidney, pancreas, liver, bone, bone marrow, lymph, blood (hematologic), brain (glial), muscle, skin, and stem cells, which may or may not metastasize to other secondary sites, regions or locations.
  • breast breast
  • lung e.g., small cell or non- small cell
  • thyroid e.g., head and neck
  • Conjugates and methods of the invention are also applicable to metastatic or non-metastatic tumor, cancer, malignancy or neoplasia of any cell, organ or tissue origin.
  • Such disorders can affect virtually any cell or tissue type, e.g., carcinoma, sarcoma, melanoma, neural, and reticuloendothelial or hematopoietic neoplastic disorders (e.g., myeloma, lymphoma or leukemia).
  • neoplasia and “tumor” refer to a cell or population of cells whose growth, proliferation or survival is greater than growth, proliferation or survival of a normal counterpart cell, e.g. a cell proliferative or differentiative disorder.
  • a tumor is a neoplasia that has formed a distinct mass or growth.
  • cancer or “malignancy” refers to a neoplasia or tumor that can invade adjacent spaces, tissues or organs.
  • a “metastasis” refers to a neoplasia, tumor, cancer or malignancy that has disseminated or spread from its primary site to one or more secondary sites, locations or regions within the subject, in which the sites, locations or regions are distinct from the primary tumor or cancer. All or a portion of such cells can express Her2/neu and can therefore be targeted with conjugates in accordance with the invention.
  • Neoplastic, tumor, cancer and malignant cells include dormant or residual neoplastic, tumor, cancer and malignant cells, all or a portion of which express Her2/neu. Such cells typically consist of remnant tumor cells that are not dividing (G0-G1 arrest). These cells can persist in a primary site or as disseminated neoplastic, tumor, cancer or malignant cells as a minimal residual disease. These dormant neoplastic, tumor, cancer or malignant cells remain asymptomatic, but can develop severe symptoms and death once these dormant cells proliferate. Invention methods can be used to reduce or inhibit proliferation of dormant neoplastic, tumor, cancer or malignant cells, which can in turn inhibit or reduce tumor or cancer relapse, or tumor or cancer metastasis or progression.
  • a method includes administering to a subject (in need of treatment) an amount of a conjugate of sufficient to treat (e.g., reduce or inhibit proliferation) the metastatic or non-metastatic tumor, cancer, malignancy or neoplasia.
  • the metastatic or non-metastatic tumor, cancer, malignancy or neoplasia may be in any stage, e.g., early or advanced, such as a stage I, II, III, IV or V tumor.
  • the metastatic or non-metastatic tumor, cancer, malignancy or neoplasia may have been subject to a prior treatment or be stabilized (non- progressing) or in remission.
  • invention methods can be used to reduce or inhibit metastasis of a primary tumor or cancer to other sites, or the formation or establishment of metastatic tumors or cancers at other sites distal from the primary tumor or cancer thereby inhibiting or reducing tumor or cancer relapse or tumor or cancer progression.
  • methods of the invention include, among other things, 1) reducing or inhibiting growth, proliferation, mobility or invasiveness of tumor or cancer cells that potentially or do develop metastases (e.g., disseminated tumor cells, DTC); 2) reducing or inhibiting formation or establishment of metastases arising from a primary tumor or cancer to one or more other sites, locations or regions distinct from the primary tumor or cancer; 3) reducing or inhibiting growth or proliferation of a metastasis at one or more other sites, locations or regions distinct from the primary tumor or cancer after a metastasis has formed or has been established; and 4) reducing or inhibiting formation or establishment of additional metastasis after the metastasis has been formed or established.
  • metastases e.g., disseminated tumor cells, DTC
  • DTC disseminated tumor cells
  • Cells of a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia may be aggregated in a "solid” cell mass or be dispersed or diffused.
  • a “solid” tumor refers to cancer, neoplasia or metastasis that typically aggregates together and forms a mass. Specific non-limiting examples include breast, ovarian, uterine, cervical, stomach, lung, gastric, colon, bladder, glial, and endometrial tumors/cancers.
  • Carcinomas which refer to malignancies of epithelial or endocrine tissue, include respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
  • Exemplary carcinomas include those forming from the uterus, cervix, lung, prostate, breast, head and neck, colon, pancreas, testes, adrenal, kidney, esophagus, stomach, liver and ovary.
  • the term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • Adenocarcinoma includes a carcinoma of a glandular tissue, or in which the tumor forms a gland like structure.
  • Sarcomas refer to malignant tumors of mesenchymal cell origin.
  • exemplary sarcomas include for example, lymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma and fibrosarcoma.
  • Neural neoplasias include glioma, glioblastoma, meningioma, neuroblastoma,
  • retinoblastoma retinoblastoma, astrocytoma and oligodendrocytoma.
  • Particular examples include neoplasia of the reticuloendothelial or hematopoietic system, such as lymphomas, myelomas and leukemias.
  • leukemias include acute and chronic lymphoblastic, myeolblastic and multiple myeloma.
  • diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia.
  • Lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), which includes B-lineage ALL (B-ALL) and T-lineage ALL (T-ALL), chronic lymphocytic leukemia (CLL), prolymphocyte leukemia (PLL), hairy cell leukemia (HLL) and Waldenstroem's macroglobulinemia (WM).
  • ALL acute lymphoblastic leukemia
  • ALL includes B-lineage ALL (B-ALL) and T-lineage ALL (T-ALL)
  • CLL chronic lymphocytic leukemia
  • PLL prolymphocyte leukemia
  • HLL hairy cell leukemia
  • W Waldenstroem's macroglobulinemia
  • a method includes administering to a subject an amount of a conjugate sufficient to treat a uterus, breast, vagina, cervix, endometrium or fallopian tube hyperproliferative disorder.
  • compositions, treatment, protocol, therapy or regimen having an anti-cell proliferative activity or effect can be combined with a conjugate or used in combination in a method of the invention.
  • Conjugates and methods of the invention therefore include anti-proliferative, anti-tumor, anti-cancer, anti-neoplastic and anti-metastatic treatments, protocols and therapies, which include any other composition, treatment, protocol or therapeutic regimen that inhibits, decreases, retards, slows, reduces or prevents a hyperproliferative disorder, such as tumor, cancer, malignant or neoplastic growth, progression, metastasis, proliferation or survival, or worsening in vitro or in vivo.
  • a hyperproliferative disorder such as tumor, cancer, malignant or neoplastic growth, progression, metastasis, proliferation or survival, or worsening in vitro or in vivo.
  • an anti-proliferative (e.g., tumor) therapy include chemotherapy, immunotherapy, radiotherapy (ionizing or chemical), local thermal (hyperthermia) therapy, surgical resection and vaccination.
  • a conjugate can be administered prior to, substantially contemporaneously with or following administration of the anti-cell proliferative, anti-neoplastic, anti-tumor, anti-cancer, anti-metastatic or immune-enhancing treatment or therapy.
  • a conjugate can be administered as a combination composition with the anti-cell proliferative, anti-neoplastic, anti-tumor, anti-cancer, anti- metastatic or immune-enhancing treatment or therapy, metastatic or non-metastatic tumor, cancer, malignancy or neoplasia.
  • Anti-proliferative, anti-neoplastic, anti-tumor, anti-cancer and anti-metastatic compositions, therapies, protocols or treatments include those that prevent, disrupt, interrupt, inhibit or delay cell cycle progression or cell proliferation; stimulate or enhance apoptosis or cell death, inhibit nucleic acid or protein synthesis or metabolism, inhibit cell division, or decrease, reduce or inhibit cell survival, or production or utilization of a necessary cell survival factor, growth factor or signaling pathway (extracellular or intracellular).
  • Non-limiting examples of chemical agent classes having anti-cell proliferative, anti-neoplastic, anti-tumor, anti-cancer and anti-metastatic activities include alkylating agents, anti-metabolites, plant extracts, plant alkaloids, nitrosoureas, hormones, nucleoside and nucleotide analogues.
  • drugs having anti-cell proliferative, anti-neoplastic, antitumor, anti-cancer and anti-metastatic activities include cyclophosphamide, azathioprine, cyclosporin A, prednisolone, melphalan, chlorambucil, mechlorethamine, busulphan, methotrexate, 6- mercaptopurine, thioguanine, 5-fluorouracil, cytosine arabinoside, AZT, 5-azacytidine (5-AZC) and 5- azacytidine related compounds such as decitabine (5-aza-2'deoxycytidine), cytarabine, 1-beta-D- arabinofuranosyl-5-azacytosine and dihydro-5-azacytidine, bleomycin, actinomycin D, mithramycin, mitomycin C, carmustine, lomustine, semustine, streptozotocin, hydroxyurea, c
  • Additional agents that are applicable with conjugates and methods are known in the art and can be employed.
  • biologicals such as antibodies, cell growth factors, cell survival factors, cell differentiative factors, cytokines and chemokines can be administered.
  • monoclonal antibodies include rituximab (Rituxan®), trastuzumab (Herceptin), pertuzumab
  • Targets that are applicable for use with the conjugates are imatinib (Gleevec), gefitinib (Iressa), bortzomib (Velcade), lapatinib (Tykerb), sunitinib (Sutent), sorafenib (Nevaxar), nilotinib (Tasigna) etc.
  • Non-limiting examples of cell growth factors, cell survival factors, cell differentiative factors, cytokines and chemokines include IL-2, IL-l a, IL- ⁇ ⁇ , IL-3, IL-6, IL-7, granulocyte-macrophage-colony stimulating factor (GMCSF), IFN- ⁇ , IL-12, TNF-a, TNFp, MIP-l , ⁇ - ⁇ ⁇ , RANTES, SDF-1, MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, eotaxin-2, 1-309/TCA3, ATAC, HCC-1 , HCC-2, HCC-3, LARC/MIP-3a, PARC, TARC, CK , CKp6, CK 7, CKp8, CKp9, C pl 1 , CKpl2, CIO, IL-8, GROa, GRO , ENA- 78, GCP-2, PBP/CTAPlIIp-TG/NAP
  • immune-enhancing treatments and therapies include cell based therapies.
  • immune-enhancing treatments and therapies include administering lymphocytes, plasma cells, macrophages, dendritic cells, NK cells and B-cells.
  • Methods of treating a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia methods of treating a subject in need of treatment due to having or at risk of having a metastatic or non- metastatic tumor, cancer, malignancy or neoplasia, and methods of increasing effectiveness or improving an anti-proliferative, anti-tumor, anti-cancer, anti-neoplasia or anti-malignancy, therapy are provided.
  • a method includes administering to a subject with or at risk of a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia, an amount of a conjugate sufficient to treat the metastatic or non-metastatic tumor, cancer, malignancy or neoplasia;
  • administering to the subject an amount of a conjugate sufficient to treat the subject; and administering to a subject that is undergoing or has undergone metastatic or non-metastatic tumor, cancer, malignancy or neoplasia therapy, an amount of a conjugate sufficient to increase effectiveness of the antiproliferative, anti-tumor, anti-cancer, anti-neoplasia or anti-malignancy therapy.
  • Methods of the invention may be practiced prior to (i.e. prophylaxis), concurrently with or after evidence of the presence of undesirable or aberrant cell proliferation or a hyperproliferative disorder, disease or condition begins (e.g., one or more symptoms).
  • Administering a conjugate prior to, concurrently with or immediately following development of a symptom of undesirable or aberrant cell proliferation or a hyperproliferative disorder may decrease the occurrence, frequency, severity, progression, or duration of one or more symptoms of the undesirable or aberrant cell proliferation or a hyperproliferative disorder, disease or condition in the subject.
  • administering a conjugate prior to, concurrently with or immediately following development of one or more symptoms of the undesirable or aberrant cell proliferation or a hyperproliferative disorder, disease or condition may inhibit, decrease or prevent the spread or dissemination of hyperproliferating cells (e.g., metastasis) to other sites, regions, tissues or organs in a subject, or establishment of hyperproliferating cells (e.g., metastasis) at other sites, regions, tissues or organs in a subject.
  • hyperproliferating cells e.g., metastasis
  • Conjugates and the methods of the invention can provide a detectable or measurable therapeutic benefit or improvement to a subject.
  • a therapeutic benefit or improvement is any measurable or detectable, objective or subjective, transient, temporary, or longer- term benefit to the subject or improvement in the condition, disorder or disease, an adverse symptom, consequence or underlying cause, of any degree, in a tissue, organ, cell or cell population of the subject.
  • Therapeutic benefits and improvements include, but are not limited to, reducing or decreasing occurrence, frequency, severity, progression, or duration of one or more symptoms or complications associated with a disorder, disease or condition, or an underlying cause or consequential effect of the disorder, disease or condition. Conjugates and methods of the invention therefore include providing a therapeutic benefit or improvement to a subject.
  • a conjugate of the invention can be administered in a sufficient or effective amount to a subject in need thereof.
  • An “amount sufficient” or “amount effective” refers to an amount that provides, in single or multiple doses, alone or in combination, with one or more other compositions (therapeutic agents such as a chemotheraputic or immune stimulating drug), treatments, protocols, or therapeutic regimens agents, a detectable response of any duration of time (long or short term), a desired outcome in or a benefit to a subject of any measurable or detectable degree or for any duration of time (e.g., for hours, days, months, years, or cured).
  • the doses or "sufficient amount” or “effective amount” for treatment typically are effective to ameliorate a disorder, disease or condition, or one, multiple or all adverse symptoms, consequences or complications of the disorder, disease or condition, to a measurable extent, although reducing or inhibiting a progression or worsening of the disorder, disease or condition or a symptom, is considered a satisfactory outcome.
  • Ameliorate means a detectable objective or subjective improvement in a subject's condition.
  • a detectable improvement includes a subjective or objective reduction in the occurrence, frequency, severity, progression, or duration of a symptom caused by or associated with a disorder, disease or condition, an improvement in an underlying cause or a consequence of the disorder, disease or condition, or a reversal of the disorder, disease or condition.
  • Treatment can therefore result in inhibiting, reducing or preventing a disorder, disease or condition, or an associated symptom or consequence, or underlying cause; inhibiting, reducing or preventing a progression or worsening of a disorder, disease, condition, symptom or consequence, or underlying cause; or further deterioration or occurrence of one or more additional symptoms of the disorder, disease condition, or symptom.
  • a successful treatment outcome leads to a "therapeutic effect,” or “benefit” or inhibiting, reducing or preventing the occurrence, frequency, severity, progression, or duration of one or more symptoms or underlying causes or consequences of a condition, disorder, disease or symptom in the subject.
  • Treatment methods affecting one or more underlying causes of the condition, disorder, disease or symptom are therefore considered to be beneficial.
  • Stabilizing or inhibiting progression or worsening of a disorder or condition is also a successful treatment outcome.
  • a therapeutic benefit or improvement therefore need not be complete ablation of any one, most or all symptoms, complications, consequences or underlying causes associated with the condition, disorder or disease. Thus, a satisfactory endpoint is achieved when there is an incremental
  • a method of treatment results in partial or complete destruction of a metastatic or non-metastatic tumor, cancer, malignant or neoplastic cell mass, volume, size or numbers of cells; results in stimulating, inducing or increasing metastatic or non-metastatic tumor, cancer, malignant or neoplastic cell necrosis, lysis or apoptosis; results in reducing metastatic or non- metastatic tumor, cancer, malignant or neoplastic volume, size, cell mass; results in inhibiting or preventing progression or an increase in metastatic or non-metastatic tumor, cancer, malignant or neoplastic volume, mass, size or cell numbers; results in inhibiting or decreasing the spread or dissemination of hyperproliferating cells (e.g., metastasis) to other (secondary) sites, regions, tissues or organs in a subject, or establishment of hyperproliferating cells (e.g., metastasis) at other (secondary) sites, regions, tissues or organs in a subject;
  • hyperproliferating cells
  • An amount sufficient or an amount effective can but need not be provided in a single administration and, can but need not be, administered alone or in combination with another composition (e.g., chemotherapeutic or immune enhancing or stimulating agent), treatment, protocol or therapeutic regimen.
  • another composition e.g., chemotherapeutic or immune enhancing or stimulating agent
  • the amount may be proportionally increased as indicated by the need of the subject, status of the disorder, disease or condition treated or the side effects of treatment.
  • an amount sufficient or an amount effective need not be sufficient or effective if given in single or multiple doses without a second composition (e.g., chemotherapeutic or immune stimulating agent), treatment, protocol or therapeutic regimen, since additional doses, amounts or duration above and beyond such doses, or additional compositions (e.g., chemotherapeutic or immune stimulating agents), treatments, protocols or therapeutic regimens may be included in order to be considered effective or sufficient in a given subject.
  • Amounts considered sufficient also include amounts that result in a reduction of the use of another treatment, therapeutic regimen or protocol.
  • An amount sufficient or an amount effective need not be effective in each and every subject treated, prophylactically or therapeutically, nor a majority of treated subjects in a given group or population. As is typical for treatment or therapeutic methods, some subjects will exhibit greater or less response to a given treatment, therapeutic regimen or protocol. An amount sufficient or an amount effective refers to sufficiency or effectiveness in a particular subject, not a group or the general population.
  • Such amounts will depend in part upon the condition treated, such as the type or stage of undesirable or aberrant cell proliferation or hyperproliferative disorder (e.g., a metastatic or non- metastatic tumor, cancer, malignancy or neoplasia), the therapeutic effect desired, as well as the individual subject (e.g., the bioavailability within the subject, gender, age, etc.).
  • a metastatic or non- metastatic tumor e.g., a metastatic or non- metastatic tumor, cancer, malignancy or neoplasia
  • the therapeutic effect desired e.g., the individual subject (e.g., the bioavailability within the subject, gender, age, etc.).
  • a hyperproliferative disorder e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia
  • a hyperproliferative disorder include a reduction in cell size, mass or volume, inhibiting an increase in cell size, mass or volume, a slowing or inhibition of worsening or progression, stimulating cell necrosis, lysis or apoptosis, reducing or inhibiting neoplastic or tumor malignancy or metastasis, reducing mortality, and prolonging lifespan of a subject.
  • inhibiting or delaying an increase in cell size, mass, volume or metastasis can increase lifespan (reduce mortality) even if only for a few days, weeks or months, even though complete ablation of the metastatic or non-metastatic tumor, cancer, malignancy or neoplasia has not occurred.
  • Adverse symptoms and complications associated with a hyperproliferative disorder e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia
  • a hyperproliferative disorder e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia
  • a hyperproliferative disorder e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia
  • a hyperproliferative disorder include, for example, pain, nausea, discomfort, lack of appetite, lethargy and weakness.
  • a reduction in the occurrence, frequency, severity, progression, or duration of a symptom of undesirable or aberrant cell proliferation such as a hyperproliferative disorder (e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia), such as an improvement in subjective feeling (e.g., increased energy, appetite, reduced nausea, improved mobility or psychological well being, etc.), are therefore all examples of therapeutic benefit or improvement.
  • a hyperproliferative disorder e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia
  • an improvement in subjective feeling e.g., increased energy, appetite, reduced nausea, improved mobility or psychological well being, etc.
  • a sufficient or effective amount of a conjugate is considered as having a therapeutic effect if administration results in less chemotherapeutic drug, radiation or immunotherapy being required for treatment of undesirable or aberrant cell proliferation, such as a hyperproliferative disorder (e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia).
  • a hyperproliferative disorder e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia.
  • subject' * refers to animals, typically mammalian animals, such as humans, non human primates (apes, gibbons, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), farm animals (horses, cows, goats, sheep, pigs) and experimental animal (mouse, rat, rabbit, guinea pig).
  • Subjects include animal disease models, for example, animal models of undesirable or aberrant cell proliferation, such as a hyperproliferative disorder (e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia) for analysis of conjugates in vivo.
  • a hyperproliferative disorder e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia
  • Subjects appropriate for treatment include those having or at risk of having a metastatic or non-metastatic tumor, cancer, malignant or neoplastic cell, those undergoing as well as those who are undergoing or have undergone anti-proliferative (e.g., metastatic or non-metastatic tumor, cancer, malignancy or neoplasia) therapy, including subjects where the tumor is in remission.
  • anti-proliferative e.g., metastatic or non-metastatic tumor, cancer, malignancy or neoplasia
  • “At risk” subjects typically have risk factors associated with undesirable or aberrant cell proliferation, development of hyperplasia (e.g., a tumor).
  • At risk or candidate subjects include those with cells that express Her2/neu to which a conjugate can bind, particularly where cells targeted for necrosis, lysis, killing or destruction express greater numbers or amounts of Her2/neu than non-target cells. Such cells can be selectively or preferentially targeted for necrosis, lysis or killing.
  • At risk subjects also include those that are candidates for and those that have undergone surgical resection, chemotherapy, immunotherapy, ionizing or chemical radiotherapy, local or regional thermal (hyperthermia) therapy, or vaccination.
  • the invention is therefore applicable to treating a subject who is at risk of a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia or a complication associated with a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia, for example, due to metastatic or non-metastatic tumor, cancer, malignancy or neoplasia reappearance or regrowth following a period of stability or remission.
  • Risk factors include gender, lifestyle (diet, smoking), occupation (medical and clinical personnel, agricultural and livestock workers), environmental factors (carcinogen exposure), family history (autoimmune disorders, diabetes, etc.), genetic predisposition, etc.
  • subjects at risk for developing melanoma include excess sun exposure (ultraviolet radiation), fair skin, high numbers of naevi (dysplastic nevus), patient phenotype, family history, or a history of a previous melanoma.
  • Subjects at risk for developing cancer can therefore be identified by lifestyle, occupation, and
  • Subjects at risk for developing breast cancer lack Brcal, for example.
  • Subjects at risk for developing colon cancer have early age or high frequency polyp formation, or deleted or mutated tumor suppressor genes, such as adenomatous polyposis coli (APC), for example.
  • APC adenomatous polyposis coli
  • Subjects also include those precluded from other treatments. For example, certain subjects may not be good candidates for surgical resection, chemotherapy, immunotherapy, ionizing or chemical radiotherapy, local or regional thermal (hyperthermia) therapy, or vaccination.
  • candidate subjects for treatment in accordance with the invention include those that are not a candidate for surgical resection, chemotherapy, immunotherapy, ionizing or chemical radiotherapy, local or regional thermal (hyperthermia) therapy, or vaccination.
  • Conjugates may be formulated in a unit dose or unit dosage form.
  • a conjugate is in an amount effective to treat a subject having undesirable or aberrant cell proliferation or a hyperproliferative disorder.
  • a conjugate is in an amount effective to treat a subject having a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia.
  • Exemplary unit doses range from about 25-250, 250-500, 500-1000, 1000-2500 or 2500- 5000, 5000-25,000, 5000-50,000 ng; and from about 25-250, 250-500, 500-1000, 1000-2500 or 2500- 5000, 5000-25,000, 5000-50,000
  • compositions and methods of the invention may be contacted or provided in vitro, ex vivo or in vivo.
  • Compositions can be administered to provide the intended effect as a single or multiple dosages, for example, in an effective or sufficient amount.
  • Exemplary doses range from about 25-250, 250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000, 5000-50,000 pg kg; from about 50-500, 500-5000, 5000-25,000 or 25,000-50,000 ng/kg; and from about 25-250, 250-500, 500-1000, 1000- 2500 or 2500-5000, 5000-25,000, 5000-50,000 ⁇ g/kg, on consecutive days, or alternating days or intermittently.
  • Single or multiple doses can be administered on consecutive days, alternating days or intermittently.
  • compositions can be administered and methods may be practiced via systemic, regional or local administration, by any route.
  • a conjugate can be administered systemically, regionally or locally, intravenously, orally ⁇ e.g., ingestion or inhalation), intramuscularly,
  • compositions and methods of the invention including pharmaceutical formulations can be administered via a (micro)encapsulated delivery system or packaged into an implant for administration.
  • the invention further provides conjugates and methods wherein the conjugates are included in pharmaceutical compositions.
  • a pharmaceutical composition refers to "pharmaceutically acceptable” and “physiologically acceptable” carriers, diluents or excipients.
  • pharmaceutically acceptable and “physiologically acceptable,” when referring to carriers, diluents or excipients includes solvents (aqueous or non-aqueous), detergents, solutions, emulsions, dispersion media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration and with the other components of the formulation.
  • Such formulations can be contained in a tablet (coated or uncoated), capsule (hard or soft), microbead, emulsion, powder, granule, crystal, suspension, syrup or elixir.
  • compositions can be formulated to be compatible with a particular route of administration.
  • Compositions for parenteral, intradermal, or subcutaneous administration can include a sterile diluent, such as water, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents.
  • the preparation may contain one or more preservatives to prevent microorganism growth (e.g., antibacterial agents such as benzyl alcohol or methyl parabens;
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as
  • ethylenediaminetetraacetic acid ethylenediaminetetraacetic acid
  • buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose).
  • compositions for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and polyetheylene glycol), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of a coating such as lecithin, or by the use of surfactants.
  • Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal.
  • Including an agent that delays absorption, for example, aluminum monostearate and gelatin can prolonged absorption of injectable compositions.
  • kits including conjugates of the invention, combination compositions and pharmaceutical formulations thereof, packaged into suitable packaging material.
  • a kit optionally includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein.
  • Exemplary instructions include instructions for reducing or inhibiting proliferation of a cell, reducing or inhibiting proliferation of undesirable or aberrant cells, such as a hyperproliferating cell, reducing or inhibiting proliferation of a metastatic or non-metastatic tumor, cancer, malignant or neoplastic cell, treating a subject having a hyperproliferative disorder, treating a subject having a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia, or reducing fertility of an animal.
  • a kit can contain a collection of such components, e.g., two or more conjugates alone, or in combination with another therapeutically useful composition (e.g., an anti-proliferative or immune- enhancing drug).
  • packaging material refers to a physical structure housing the components of the kit.
  • the packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.).
  • Kits of the invention can include labels or inserts.
  • Labels or inserts include "printed matter," e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component.
  • Labels or inserts can additionally include a computer readable medium, such as a disk (e.g., floppy diskette, hard disk, ZIP disk), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media or memory type cards.
  • Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location and date.
  • Labels or inserts can include information on a condition, disorder, disease or symptom for which a kit component may be used.
  • Labels or inserts can include instructions for the clinician or for a subject for using one or more of the kit components in a method, treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency or duration, and instructions for practicing any of the methods, treatment protocols or therapeutic regimes set forth herein. Exemplary instructions include, instructions for treating an undesirable or aberrant cell proliferation, hyperproliferating cells and disorders (e.g., metastatic or non-metastatic tumor, cancer, malignancy or neoplasia). Kits of the invention therefore can additionally include labels or instructions for practicing any of the methods of the invention described herein including treatment methods.
  • Labels or inserts can include information on any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition. Adverse side effects could also occur when the subject has, will be or is currently taking one or more other medications that may be incompatible with the composition, or the subject has, will be or is currently undergoing another treatment protocol or therapeutic regimen which would be incompatible with the composition and, therefore, instructions could include information regarding such incompatibilities. [0166] Invention kits can additionally include other components. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package.
  • Invention kits can be designed for cold storage. Invention kits can further be designed to contain host cells expressing conjugates of the invention, or that contain nucleic acids encoding conjugates. The cells in the kit can be maintained under appropriate storage conditions until the cells are ready to be used. For example, a kit including one or more cells can contain appropriate cell storage medium so that the cells can be thawed and grown.
  • reference to a range of 90-100% includes 91- 99%, 92-98%, 93-95%, 91-98%, 91-97%, 91 -96%, 91-95%, 91-94%, 91-93%, and so forth.
  • Reference to a range of 90-100% also includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1 %, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth.
  • reference to a range of 1-5,000 fold includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1.1 , 1.2, 1.3, 1.4, 1.5, fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5, fold, etc., and any numerical range within such a ranges, such as 1-2, 5-10, 10-50, 50-100, 100-500, 100-1000, 500-1000, 1000-2000, 1000-5000, etc.
  • reference to a range of KD 10 -5 M to about KD 10 "13 M includes any numerical value or range within or encompassing such values.
  • 50-75, 75-100, 100-150, and 150-171 includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-
  • the invention is generally disclosed herein using affirmative language to describe the numerous embodiments.
  • the invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis.
  • the invention is generally not expressed herein in terms of what the invention does not include aspects that are not expressly included in the invention are nevertheless disclosed herein.
  • Peptides studied were: Phorl 8- scFv-C H 3 -Phor-18 (2 molecules of Phor -18 joined at N- and C-terminal ends of the antibody, scFv- C H 3 -GS-Phor-18 (one molecule of Phor-18 joined to the antibody at the C-terminus by GS linker, scFv- C H 3 -GS-(KLAKLAK) 2 KLAK (one molecule of (KLAKLAK) 2 KLAK linked to the antibody at the C-terminus by GS linker, scFv- C H 3 - -NRVRRS- Phor-18 (one molecule of Phor-18 to the antibody at the C-terminus by NRVRRS linker, and scFv- C H 3 -NRVRRS-(KLAKLAK) 2 KLAK (one molecule of (KLAKLAK) 2 KLAK to the antibody at the C- terminus by NRVRRS linker).
  • Cytotoxicity was compared to a naked antibody (antibody without a lytic peptide) in Her-2 receptor positive cells (SKBR-3 and SKOV-3, human breast and ovarian cancer cells, respectively) and Her-2 receptor negative breast cancer cells (MDA-MB-231).
  • the plasmid was acquired through gene codon optimization.
  • the gene was synthesized with a N-His tag sequence and the plasmid was subcloned into an E. coli bacteria expression vector pUC57. After expression optimization and evaluation the His-tag product was selected and 1 L of the bacteria expression product was purified in a one-step affinity purification.
  • the sequences of the plasmid gene insertion for each construct is described in Table 1.
  • Table 1 Nucleotide sequence of the plasmid insertion for the production of each recombinant Her2/neu antibody and antibody conjugate.
  • the cytotoxic molecule containing cysteine is thoroughly reduced by reaction with a 10-fold excess of reductacryl reagent before mixing in 10-fold excess with the antibody-linker construct.
  • the reaction is allowed to incubate at room temperature for 18 hours, then desalted to remove unreacted cytotoxin molecule.
  • the solution is filter-sterilized before storage.
  • the Her2-antibody-Phor-18 conjugates (Phorl 8- scFv- C H 3 -Phor-18, scFv- C H 3 -GS-Phor-18, scFv- C H 3 -NRVRRS-Phor-18, the Her2-antibody-(KLAKLAK) 2 KLAK (scFv- C H 3 -GS- (KLAKLAK) 2 KLAK, and scFv- C H 3 -NRVRRS-(KLAKLAK) 2 LAK), or scFv- C H 3 - receptor antibody (naked) in Tris HCL-buffer were diluted with saline and added to cells at increasing concentrations of 0, 0.0012, 0.012, 0.12, 1.2, 6.0, 12.0, 120, 360 and 720 nM.
  • the anti-Her2-Phor-l 8 antibody conjugates (Phorl 8- scFv- C H 3 -Phor- 18, scFv- C H 3 -GS-Phor-18, scFv-CH 3 -NRVRRS-Phor-18) killed Her2 positive human breast cancer SKJBR-3 and ovarian cancer SKOV-3 cell lines by 48 hours, whereas the Her2 negative human breast cancer MDA-MB-231cell line was not killed.
  • Evidence of cytotoxicity was observed microscopically at as early as 24 hours of incubation. As expected, unconjugated Phor-18 showed only modest cytotoxicity.
  • the HER2 antibody conjugated to the Phor-18 was significantly more cytotoxic than antibody conjugated to the lytic peptide (KLAKLAK) 2 KLA .
  • Figure 1, Table 2 The Her2 negative MDA-MB-231 cells were not killed by any of the recombinant antibody- lytic peptide conjugates indicating that the cytotoxicity of the antibodies was mediated via Her2/neu receptors.
  • the "naked" (unconjugated) antibody (scFv- CH3) was not cytotoxic in all 3 cell lines indicating that the cell-killing properties of the antibody-lytic peptide conjugates were due to the presence of lytic peptide payload and sequence of the lytic peptide.
  • unconjugated Phor-18 showed very minimal nonspecific cytotoxicity in all cell lines (Table 2).
  • Table 2 In vitro cytotoxicity of anti-Her2-Phor-18 antibody conjugates (scFv- C H 3 -Phor-18 and - scFv- CH3 -(KLAKLAK) 2 KLAK conjugates, Her2/neu scFv- CH3 and unconjugated Phor-18 in Her2 receptor positive S OV-3, SKJBR-3 and Her2 receptor negative MDA-MB-231 cancer cells. Values are IC expressed in nM.
  • This example includes a description of in vitro cytotoxicity studies of recombinantly produced antibody to Her-2 receptor conjugated to lytic peptide, Phor-18 ( FAKFAK KFA FAK KFAK) (scFv- C H 3 -GS- Phor-18), and a chemically conjugated MAb-Phorl 8 conjugate against Her2 positive ovarian cancer cell line SKOV-3.
  • This example describes an in vivo study in a mouse xenograft model of human ovarian cancer with various doses of anti-Her2-Phor-18 antibody conjugates (scFv- CH3 -GS-Phor-18, MAb-Phorl8), naked whole antibody (MAb) and naked recombinant antibody (scFv- C H 3) treatments.
  • Tumor weights from mice that were killed on day 42 served as baseline.
  • treatment started on day 43 after tumor cell injection on tumors of median tumor volume of 130.3 ⁇ 10.25 mm 3 and continued on days 47, 50, 54, 57 and 60 as a single bolus injection into the lateral tail vein.
  • Treatments were: saline control, whole naked monoclonal anti-Her2-antibody, MAb, (3 mg/kg), recombinant naked-Her2-antibody (scFv- C H 3) (3 mg kg), scFv- C H 3 -GS-Phor-18 (0.3 and 3 mg/kg), MAb-Phor-18 (0.3 and 3mg/kg).
  • Tumors from mice sacrificed at treatment start underwent immunohistochemistry evaluation of Her2/neu receptors. Each group consisted of 8-9 mice.
  • Figure 2A and 2B show median tumor volumes during the course of the study and mean tumor weights on day 64 for each individual treatment group for saline controls, and mice treated with MAb (naked)
  • This example describes in vitro cytotoxicity studies of recombinantly produced antibody to Her-2 receptor conjugated to lytic peptide, Phor-18 (KFAKFAK KFAKFAK KFAK) against ovarian cancer cells.
  • scFv-C H 2- C H 3 -GS-Phor-18 one molecule of Phor-18 joined to the antibody at the C- terminus by GS linker, consisting of V L — G linker— VH— CH2- ( 3 ⁇ 43 ⁇ G linker— CH3-C h 2 ⁇ VH— G linker— VL -GS-(Phor- 18). Cytotoxicity was compared to a naked antibody (scFv- CH2- C h 3; antibody without a lytic peptide) in Her-2 receptor positive cells (SKOV-3, human ovarian cancer cells).
  • SKOV-3 Her-2 receptor positive cells
  • Table 3 Amino Acid sequence for the production of each recombinant antibody, (A) scFv- CR2- C h 3; and antibody conjugate (B) scFv- C H 2- CH3 -GS-Phor-18.
  • DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ PG AP LLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
  • Phor-18, scFv-C H 3 GS-Phor-18) or the naked antibodies (scFv- C H 2- C H 3, scFv-C H 3) in Tris/HCL- buffer were diluted with saline and added to cells at increasing concentrations of 0, 0.0012, 0.012, 0.12,

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Abstract

The invention relates to conjugates that bind to Her2/neu, methods of using conjugates that bind to Her2/neu and methods of treating undesirable or aberrant cell proliferation or hyperproliferative disorders, such as tumors, cancers, neoplasia and malignancies that express Her2/neu.

Description

Lytic-Peptide-Her2/neu (Human Epidermal growth factor Receptor 2) Ligand Conjugates and
Methods of Use
Related Applications
[0001] This application claims priority to application serial no. 61/330,005, filed April 30, 2010, and application serial no. 61/362,603, filed July 8, 2010, each of which applications are incorprated herein by reference in their entirety.
Technical Field
[0002] The invention relates to lytic peptide and Her2/neu (Human Epidermal growth factor Receptor 2, also known as ErbB-2) ligand conjugates, methods of using conjugates, for example, in methods of treating undesirable or aberrant cell proliferation or hyperproliferative disorders, such as non-metastatic and metastatic neoplasias, cancers, tumors and malignancies that express Her2/neu.
Introduction
[0003] The need to develop new therapeutics for treatment of primary tumors and metastases is clearly evident when the five year survival rate of cancer patients is considered: Only 10-40 % for patients with lung, colorectal, breast and prostate cancer survive if diagnosed with distant metastatic disease.
Summary
[0004] The invention is based, at least in part on lytic domains fused or conjugated to a ligand that binds to Her2/neu (Human Epidermal growth factor Receptor 2, also known as ErbB-2), referred to herein as conjugates or fusion constructs. Contact of a cell with a lytic domain is believed to cause disruption of the cell membrane which results in cell death. The ligand that binds to Her2/neu targets cells that express Her2/neu for destruction by the ligand that binds to Her2/neu and/or lytic domain, including undesirable or aberrant proliferating cells or hyperproliferating cells, such as non-metastatic and metastatic neoplasias, cancers, tumors and malignancies. A number of non-metastatic and metastatic neoplastic, cancer, tumor and malignant cells overexpress receptors or ligands. For example, many non-metastatic and metastatic neoplasias, cancers, tumors and malignancies, express Her2/neu that can be used as a target of the conjugate or fusion construct.
[0005] Conjugates can be designed to target any cell or cell population that expresses Her2/neu.
Ligands that bind to Her2/neu which include antibodies and fragments thereof, hormones, growth factors, cytokines, etc., can be linked to a lytic domain to provide targeted killing of cells that express or contain Her2/neu thereby reducing or inhibiting cell proliferation or growth.
[0006] Conjugates do not require cells to divide in order to kill the target cells. Furthermore, the conjugates are not likely to be immunogenic because they can be made to be relatively small in size. In addition, the conjugates kill multi-drug resistant cells. [0007] In accordance with the invention, there are provided conjugates that include a first and a second domain. In one embodiment, a conjugate includes or consists of a ligand that binds to Her2/neu consisting and a second domain consisting of a 10-120 amino acid fragment, such as a L- or D-amino acid sequence that includes a peptide sequence selected from for example, KFAKFAKKFAKFAKK, FA K F A KKFAKFA KKF, KFAKFAKKFAKFAKKFA, KFA FAKKFAKFA KFA ,
KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA and
KFAKFAKKFAKFAKKFAKFAKKFAKFAK, or a sequence that includes a peptide selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,
KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA having one or more of the K residues substituted with any of an F or L residue, one or more of the F residues substituted with any of a K, A or L residue, or one or more of the A residues substituted with any of a K, F or L residue. In another embodiment, a conjugate includes or consists of a ligand that binds to Her2/neu and a second domain consisting of an L- or D-amino acid sequence selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKPA,
KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA and KFAKFAKKFAKFAKKFAKFAKKFAKFAK, or a sequence that includes a peptide selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,
KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA having one or more of the K residues substituted with any of an F or L residue, one or more of the F residues substituted with any of a K, A or L residue, or one or more of the A residues substituted with any of a K, F or L residue. In a further emodiment, a conjugate includes a ligand that binds to Her2/neu and a second domain consisting of an L- or D-amino acid sequence selected from,
KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,
KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA and KFAKFAKKFAKFAKKFAKFAKKFAKFAK, or a sequence that includes a peptide selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,
KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA having one or more of the K residues substituted with any of an F or L residue, one or more of the F residues substituted with any of a K, A or L residue, or one or more of the A residues substituted with any of a K, F or L residue.
[0008] In accordance with the invention, there are also provided isolated and purified peptides that include or consist of a ligand that binds to Her2/neu and a second domain. In various embodiments, a second domain is KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF or KFAKFAKKFAKFAKKFAKFA. In additional embodiments, a second domain is KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF or KFAKFAKKFAKFAKKFAKFA having one or more of the K residues substituted with any of an F or
L residue, one or more of the F residues substituted with any of a K, A or L residue, or one or more of the A residues substituted with any of a K, F or L residue.
[0009] Conjugates include a ligand that binds to Her2/neu. Ligands include or consist of a molecule that binds to a Her2/neu (Human Epidermal growth factor Receptor 2, also known as ErbB-2), which can be an agonist or antagonist. A ligand or lytic peptide can include or consist of a linear or cyclic structure. A ligand includes an antibody and antibody fragments.
[0010] Specific non-limiting examples of ligands include one or more amino acids (e.g., peptides, polypeptides, proteins), nucleic acids and carbohydrates. Specific non-limiting classes of ligands include hormones, growth factors, hormone and growth factor analogues, and fragments of hormones, hormone analogs, growth factors, growth factor analogues, and fragments of growth factors and analogues, antibodies and fragments of antibodies that bind to a Her2/neu. Specific non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, Fv, Fd, single-chain Fv (scFv), disulfide- linked Fvs (sdFv), VL, VH, Camel Ig, V-NAR, VHH, trispecific (Fab3), bispecific (Fak), diabody ((VL- VH)2 or (VH-VL)?), triabody (trivalent), tetrabody (tetravalent), minibody ((scFv-CH3)2), bispecific single-chain Fv (Bis-scFv), lgGdeltaCH2, scFv-Fc, (scFv)2-Fc, affibody (e.g., ZHer2-neu:2, ZHer2- neu:4 ZHer2-neu:7 ZHer2-neu:8), affibody, aptamer, avimer or nanobody.
[0011] Her2/neu and/or ligands can be optionally expressed on a cell. Cells that express Her2/neu or that can be targeted in accordance with methods of the invention include hyperproliferative cells. Cells that express Her2/neu or that can be targeted in accordance with methods of the invention for example include breast, ovarian, uterine, cervical, stomach, lung, gastric, colon, bladder, glial, hematologic and endometrial cells.
[0012] A ligand that binds to Her2/neu and second domain can include or consist of an amino acid, or an amino acid sequence. In particular aspects, a first or second domain has about 1 to 10, 10 to 20, 15 to 20 (i.e., 15, 16, 17, 18, 19 or 20 amino acids), 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino acid residues.
[0013] Her2/neu binding ligands and second domains can be positioned at either (or both) the NH2- terminus or the C-terminus relative to each other. Thus, in one embodiment the first (binding ligand) domain is positioned at the NH-terminus relative to the second (lytic peptide) domain, and in another embodiment, the second (lytic peptide) domain is positioned at the C-terminus relative to the first (binding ligand) domain, and in a further embodiment, a first (binding ligand) domain is positioned at the C-terminus relative to the second (lytic peptide) domain, and in still another embodiment, a second (lytic peptide) domain is positioned at the NH-terminus relative to the first (binding ligand) domain. Such second (lytic peptide) domains can be positioned at the C-terminus, the NH-terminus, or both the C-terminus and the NH-terminus. Accordingly, multiple such second (lytic peptide) domains can be included in a conjugate of the invention. [0014] Her2/neu binding ligands and second domains can include or consist of one or more D-amino acids. In particular aspects, a first domain has a D-amino acid, for example, at any K, F or A residue.
[0015] Her2/neu binding ligands and second domains can be joined by a covalent (e.g., peptide or nonpeptide) bond. For example, a first and a second domain can be joined by a peptide or a non- peptide linker. In particular aspects, Her2/neu binding ligands and second domains are joined by a peptide sequence having from about 1 to 25 amino acid residues, or having a linear carbon chain. In more particular aspects, Her2/neu binding ligands and second domains are joined by a peptide sequence that includes or consist of one or more A, S or G amino acid residues. In further particular aspects,
Her2/neu binding ligands and second domains are joined by a peptide sequence a first and second domain is joined by peptide sequence including or consisting of GSGGS, ASAAS, or a linear carbon chain such as CCCCCC. Other peptide linkers include but are not limited to GS, AF, FK, VK, FFK,
FA, GSGRSA, RVRRSV, SS, Cit-V, F-Cit, at various length. Thioether, N-succinidyl-3-(2- pyridylothio)propionate, thio ether bonds such as SLAB [N-succinimidyl (4-iodoacetyl)
aminobenzoate], SMCC [succinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate], MBS (m-
Maleimidobenzoyl-N-hydroxysuccinimide) and SMPB [succinimidyl-4-(p-maleimidophenyl)butyrate], maleimide and hydrazone linkers.
[0016] Her2/neu binding ligands and second domains can further include or consist of additional domains. Thus, in various aspects, a conjugate includes a third, fourth, fifth, sixth, seventh domain, etc.
[0017] Conjugates include or consist of isolated and purified forms. Conjugates also include or consist of a mixture. Such mixtures include compositions, such as a mixture of conjugate and a pharmaceutically acceptable carrier or excipient appropriate for administration to or in vivo contact with a subject, or a mixture of conjugate and an anti-cell proliferative or immune stimulating agent.
[0018] Conjugates include or consist of a unit dosage form. In one embodiment, a conjugate is a unit dosage in an amount effective to treat a subject having undesirable cell proliferation or a hyperproliferative disorder. In another embodiment, a conjugate is a unit dosage in an amount effective to treat a subject having a neoplasia, tumor or cancer. In an additional embodiment, a conjugate is a unit dosage in an amount effective to reduce fertility of a subject.
[0019] Conjugates can be included within kits, optionally with instructions for practicing a method. In one embodiment, a kit includes a conjugate and instructions for reducing or inhibiting proliferation of a cell, reducing or inhibiting proliferation of a hyperproliferating cell, reducing or inhibiting proliferation of a neoplastic, tumor or cancer cell, treating a subject having a hyperproliferative disorder, treating a subject having a neoplasia, tumor or cancer, or reducing fertility of an animal.
[0020] In accordance with the invention, there are also provided nucleic acids that encode conjugates. In one embodiment, a nucleic acid encodes a conjugate including or consisting of a ligand that binds to Her2/neu and a second domain and a peptide sequence selected from for example, KFAKFAKKFAKFAKK, KFA FAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA and KF AKF AKXFAKTAKKTAKFAKKFAKFAK, or a sequence that includes a peptide selected from KFAKFAKKFAKFAKK, KFAKFAKK AK AKKF, KFAK AKKFAKFAKKFA,
KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA having one or more of the K residues substituted with any of an F or L residue, one or more of the F residues substituted with any of a K, A or L residue, or one or more of the A residues substituted with any of a K, F or L residue. In another embodiment, a nucleic acid encodes a conjugate including or consisting of a ligand that binds to Her2/neu and a second domain and a peptide consisting of an L- or D-amino acid sequence selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,
KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA and KFAKFAKKFAKFAKKFAKF AKKFAKFAK.
[0021] Nucleic acids can be included in a vector, such as an expression vector that when expressed in a cell encodes a conjugate. Host cells can be transformed with a nucleic acid in a vector, such that the cell expresses a conjugate encoded by the nucleic acid.
[0022] Conjugates are useful for, among other things, reducing or inhibiting proliferation of a cell, reducing or inhibiting cell proliferation, reducing or inhibiting proliferation of a hyperproliferating cell, reducing or inhibiting proliferation of a neoplastic, tumor, cancer or malignant cell and treating undesirable or aberrant cell proliferation, such as hyperproliferating cells or hyperproliferative disorders. Non-limiting examples of hyperproliferative disorders include benign hyperplasia, non- metastatic and metastatic neoplasias, cancers tumors and malignancies.
[0023] In accordance with the invention, there are further provided methods of reducing or inhibiting proliferation of a cell; methods of reducing or inhibiting cell proliferation; methods of reducing or inhibiting proliferation of a hyperproliferating cell; and methods of reducing or inhibiting proliferation of a neoplastic, tumor, cancer or malignant cell. In various embodiments, a method includes contacting a cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the cell; contacting a cell with a conjugate in an amount sufficient to reduce or inhibit cell proliferation; contacting a cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the hyperproliferating cell; and contacting a cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the neoplastic, tumor, cancer or malignant cell.
[0024] In accordance with the invention, there are moreover provided methods of selectively reducing or inhibiting proliferation of a cell that expresses Her2/neu; selectively reducing or inhibiting proliferation of a hyperproliferating cell that expresses Her2/neu; and selectively reducing or inhibiting proliferation of a neoplastic, tumor, cancer or malignant cell that expresses Her2/neu. In various embodiments, a method includes contacting a cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the cell; contacting a cell with the conjugate in an amount sufficient to reduce or inhibit proliferation of the hyperproliferating cell; and contacting a cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the neoplastic, tumor, cancer or malignant cell, wherein the ligand of the conjugate binds to the Her2/neu expressed by the cell.
[0025] Cells targeted in accordance with the invention methods include cells that express Her2/neu. Cells targeted in accordance with the invention methods therefore include, for example, breast, ovarian, uterine, cervical, stomach, lung, gastric, colon, bladder, glial, hematologic and endometrial cells.
[0026] Methods performed include, among others, contacting a subject in need of inhibiting, reducing or preventing proliferation, survival, differentiation, death, or activity of a cells, such as a hyperprolifertive cell or an undesirably proliferating cell. Exemplary subjects include a subject having or at risk of having undesirable or aberrant cell proliferation; a subject having or at risk of having a benign hyperplasia; or a non-metastatic or metastatic neoplasia, cancer, tumor or malignancy (e.g., a solid or liquid tumor, in any of breast, ovarian, uterine, cervical, stomach, lung, gastric, colon, bladder, glial, hematologic or endometrial cells).
[0027] In accordance with the invention, there are additionally provided methods of treating a subject having a hyperproliferative disorder and methods of treating a subject having a neoplasia, tumor, cancer or malignancy (metastatic, non-metastatic or benign). In various embodiments, a method includes, administering to a subject an amount of the conjugate sufficient to treat the hyperproliferative disorder; and administering to a subject an amount of the conjugate sufficient to reduce or inhibit proliferation of the neoplasia, tumor, cancer or malignancy.
[0028] Methods include treating a subject having or at risk of having a metastasis. For example, an amount of a conjugate effective to reduce or inhibit spread or dissemination of a tumor, cancer or neoplasia to other sites, locations or regions within the subject. In various embodiments, a method reduces or inhibits metastasis of a primary tumor or cancer to one or more other sites, formation or establishment of a metastasis at one or more other sites, locations or regions thereby reducing or inhibiting tumor or cancer relapse or tumor or cancer progression. In further embodiments, a method reduces or inhibits growth, proliferation, mobility or invasiveness of tumor or cancer cells that potentially or do develop metastases (e.g., disseminated tumor cells); reduces or inhibits formation or establishment of metastases arising from a primary tumor or cancer to one or more other sites, locations or regions distinct from the primary tumor or cancer; reduces or inhibits growth or proliferation of a metastasis at one or more other sites, locations or regions distinct from the primary tumor or cancer after the metastasis has formed or has been established; or reduces or inhibits formation or
establishment of additional metastasis after the metastasis has been formed or established. In yet another embodiment, a method reduces or inhibits relapse or progression of the neoplasia, tumor, cancer or malignancy.
[0029] In accordance with the invention, there are still further provided methods of reducing or inhibiting metastasis of a neoplasia, tumor, cancer or malignancy to other sites, or formation or establishment of metastatic neoplasia, tumor, cancer or malignancy at other sites distal from a primary neoplasia, tumor, cancer or malignancy. In various embodiments, a method includes administering to a subject an amount of the conjugate sufficient to reduce or inhibit metastasis of the neoplasia, tumor, cancer or malignancy to other sites, or formation or establishment of metastatic neoplasia, tumor, cancer or malignancy at other sites distal from the primary neoplasia, tumor, cancer or malignancy.
[0030] Neoplasia, tumor, cancer and malignancy treatable in accordance with the invention include solid cellular mass, hematopoietic cells, or a carcinoma, sarcoma (e.g. lymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma or fibrosarcoma), lymphoma, leukemia, adenoma, adenocarcinoma, melanoma, glioma, glioblastoma, meningioma, neuroblastoma, retinoblastoma, astrocytoma, oligodendrocytoma, mesothelioma, reticuloendothelial, lymphatic or haematopoietic (e.g., myeloma, lymphoma or leukemia) neoplasia, tumor, cancer or malignancy.
[0031] Neoplasia, tumor, cancer and malignancy treatable in accordance with the invention can be present in or affect a lung (small cell lung or non-small cell lung cancer), breast, ovarian, uterine, cervical, stomach, lung, gastric, colon, bladder, glial, hematologic, endometrial, lymph, blood, muscle, or skin cell neoplasia, tumor, cancer, or malignancy.
[0032] Methods may be practiced with other treatments or therapies (e.g., surgical resection, radiotherapy, ionizing or chemical radiation therapy, chemotherapy, immunotherapy, local or regional thermal (hyperthermia) therapy, or vaccination). Such treatments or therapies can be administered prior to, substantially contemporaneously with (separately or in a mixture), or following administration of a conjugate. In one embodiment, a method includes administering an anti-cell proliferative, antineoplastic, anti-tumor, anti-cancer or immune-enhancing treatment or therapy. In further embodiments, a method includes administering an alkylating agent, anti-metabolite, plant extract, plant alkaloid, nitrosourea, hormone, nucleoside or nucleotide analog; cyclophosphamide, azathioprine, cyclosporin A, prednisolone, melphalan, chlorambucil, mechlorethamine, busulphan, methotrexate, 6-mercaptopurine, thioguanine, 5-fluorouracil, cytosine arabinoside , 5-azacytidine (5-AZC) and 5-azacytidine related compounds, bleomycin, actinomycin D, mithramycin, mitomycin C, carmustine, lomustine, semustine, streptozotocin, hydroxyurea, cisplatin, carboplatin, oxiplatin, mitotane, procarbazine, dacarbazine, taxol, vinblastine, vincristine, doxorubicin or dibromomannitol, topoisomerase inhibitors, (irinotecan, topotecan, etoposide, teniposide), gemcitabine, pemetrexed etc. Cell or immunotherapies include lymphocytes, plasma cells, macrophages, dendritic cells, T-cells, NK cells or B-cells; an antibody, a cell growth factor, a cell survival factor, a cell differentiative factor, a cytokine or a chemokine
(examples are interleukins IL-2, IL-la, IL-Ι β, IL-3, IL-6, IL-7, granulocyte-macrophage-colony stimulating factor (GMCSF), IFN-γ, IL-12, TNF-a, TNFp, MIP-la, ΜΙΡ-Ιβ, RANTES, SDF-1 , MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, eotaxin-2, 1-309 TCA3, AT AC, HCC-1, HCC-2, HCC-3, LARC/MIP-3cc, PARC, TARC, CK , CKp6, CKp7, Οίβ8, CKp9, CKpl 1, CKpl2, CIO, IL-8, GRO , GROp, ENA-78, GCP-2, PBP/CTAPIIIp-TG/NAP-2, Mig, PBSF/SDF-1, or lymphotactin) etc. [0033] Additional agents that are applicable with conjugates are targeted drugs or biologicals such as antibodies or small molecules. Non-limiting examples of monoclonal antibodies include rituximab
(Rituxan®), trastuzumab (Herceptin), pertuzumab, cetuximab (Erbitux), ipilimumab, zalutumumab, dalotuzumab, figitumumab, ramucirumab, galiximab, farletuzumab, ocrelizumab, ofatumumab, alemtuzumab (Campath), panitumumab (Vectibix), ibritumomab tiuxetan (Zevalin), tositumomab
(Bexxar) etc. which can be used in combination with, inter alia, a conjugate in accordance with the invention. Other targeted drugs that are applicable for use with conjugates are imatinib (Gleevec), gefitinib (Iressa), bortzomib (Velcade), lapatinib (Tykerb), sunitinib (Sutent), sorafenib (Nevaxar), nilotinib (Tasigna) etc.
[0034] Methods of the invention include providing a subject with a benefit. In particular embodiments, a method of treatment results in partial or complete destruction of the neoplastic, tumor, cancer or malignant cell mass, volume, size or numbers of cells, stimulating, inducing or increasing neoplastic, tumor, cancer or malignant cell necrosis, lysis or apoptosis, reducing neoplasia, tumor, cancer or malignancy volume size, cell mass, inhibiting or preventing progression or an increase in neoplasia, tumor, cancer or malignancy volume, mass, size or cell numbers, or prolonging lifespan; results in reducing or decreasing severity, duration or frequency of an adverse symptom or
complication associated with or caused by the neoplasia, tumor, cancer or malignancy; results in reducing or decreasing pain, discomfort, nausea, weakness or lethargy; or results in increased energy, appetite, improved mobility or psychological well being.
[0035] Subjects treatable in accordance with the methods include mammals. In particular embodiments, a subject is a human.
Description of Drawings
[0036] Figures 1A and IB show cytotoxicity of recombinantly scFv-CH3 (naked antibody), scFv- CH3 -GS-Phorl8 and scFv-CH3 -GS-(KLAKLAK)2KLAK to Her2-neu receptor positive A) breast (SKBR-3); and B) ovarian (S OV-3) cancer cell lines determined after 48 hours.
[0037] Figures 2 A and 2B show median tumor volumes from mice treated with MAb (naked), A) MAb-Phorl 8 and B) recombinant scFv-CH3 naked antibody and scFv-CH3 -Phor-18 conjugates during the study period of 64 days in SKOV-3 xenografted mice in comparison with saline injected mice.
[0038] Figure 3 shows mean tumor weights from mice treated with MAb (naked), MAb-Phor 18 and recombinantly scFv-CH3 naked antibody and scFv-CH3 -Phor-18 conjugates on day 64 in SKOV-3 xenografted mice in comparison with saline injected mice.
[0039] Figure 4 shows mean body weights from mice treated with with MAb (naked), MAb-Phorl 8 and recombinantly scFv-CH3 naked antibody and scFv-CH3 -Phor-18 conjugates during the study period of 64 days in SKOV-3 xenografted mice in comparison with saline injected mice. W
Detailed Description
[0040] The invention is based at least in part on a conjugate that includes a ligand that binds to Her2/neu joined or fused to a second lytic domain. In a typical configuration, a conjugate first domain includes a ligand that binds to Her2/neu and a second domain that includes lytic portion, which is directly or indirectly toxic to a cell, which can thereby reduce cell proliferation or survival, or stimulate, induce, increase or enhance cell death, killing or apoptosis.
[0041] In accordance with the invention, there are provided conjugates that include or consist of a first ligand that binds to Her2/neu domain and include or consist of a second lytic or toxic domain. In one embodiment, a conjugate includes a ligand that binds to Her2/neu and a second domain consisting of a 12-100 residue L- or D-amino acid sequence that includes a peptide sequence (selected from amino acids such as Lysine=K, Phenylalanine = F and Alanine = A), for example, KFAKFA KFAKFAK , FAKFA KFAKFAKKF, FA FAKKFAKFAKKFA, KFAKFAK FAKFAKKFAK,
FAKFAK K PA K FAKKFAK F, KFA FAKKFAKFAKKFAKFA and
KFAKFAKKFAKFAKKFAKFAK FAKFAK. In another embodiment, a conjugate includes a first ligand that binds to Her2/neu domain and a second domain consisting of an L- or D-amino acid sequence selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF,
KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA and KFAKFAKKFAKFAKKFAKFAKKFAKFAK.
[0042] As used herein, the term "conjugate" or "fusion construct" and grammatical variations thereof, means that the construct contains portions or sections that are derived from, obtained or isolated from, or are based upon or modeled after two different molecular entities that are distinct from each other and do not typically exist together in nature. That is, for example, one portion of the conjugate includes or consists of a ligand that binds to Her2/neu and a second portion of the conjugate includes or consists of a lytic portion, each of first and second domains structurally distinct. A conjugate can also be referred to as a "fusion construct," wherein the conjugate includes or consists of a second domain ligand that binds to Her2/neu and a second domain lytic portion.
[0043] First domains and or second domains of conjugates include or consist of amino acid sequences (peptides, polypeptides, proteins, lectins), nucleic acids (DNA, RNA) and carbohydrates (saccharides, sialic acid, galactose, mannose, fucose, acetylneuraminic acid, etc.). The terms "amino acid sequence," "protein," "polypeptide" and "peptide" are used interchangeably herein to refer to two or more amino acids, or "residues," covalently linked by an amide bond or equivalent. Amino acid sequences can be linked by non-natural and non-amide chemical bonds including, for example, those formed with glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, or N, N'- dicyclohexylcarbodiimide (DCC). Non-amide bonds include, for example, ketomethylene, aminomethylene, olefin, ether, thioether and the like (see, e.g., Spatola in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357 (1983), "Peptide and Backbone
Modifications," Marcel Decker, NY).
[0044] First and second domains of a conjugate or fusion construct include L-amino acid sequences, D-amino acid sequences and amino acid sequences with mixtures of L-amino acids and D-amino acids. Amino acid sequences of first and second domains can be a linear or a cyclic structure, conjugated to a distinct moiety (e.g., third, fourth, fifth, sixth, seventh, etc. domains), form intra or intermolecular disulfide bonds, and also form higher order multimers or oligomers with the same or different amino acid sequence, or other molecules.
[0045] Exemplary lengths of conjugates are from about 5 to 15, 20 to 25, 25 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 300 or more amino acid residues in length. In particular embodiments, a first or second domain includes or consists of an amino acid sequence of about 1 to 10, 10 to 20, 15 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more residues. In more particular embodiments, a first domain consists of a 15, 16, 17, 18, 19, 20, 28 or more residue amino acid sequence.
[0046] Conjugate with first and a second domain, optionally the ligand that binds to Her2/neu lytic domain or second lytic domain form an amphipathic alpha-helix. An amphipathic alpha-helix contains mostly hydrophilic amino acids on one side of the alpha-helix and the other side contains mostly hydrophobic amino acids. Since the alpha helix makes a complete turn for every 3.6 residues, the amino acid sequence of an amphipathic alpha helix alternates between hydrophilic and hydrophobic residues every 3 to 4 residues. A PNNPNNP repeat pattern or motif is predicted to form an amphipathic alpha- helix where P represents a positively charged amino acid residue and N a neutral amino acid residue. A PNNPNNP repeat pattern provides a cationic binding site for the lytic peptide to a negatively charged cell membrane and a hydrophobic site for membrane interaction/penetration. Conjugates therefore include second domains with one or more uninterrupted PNNPNNP repeat patterns or motifs, or one or more interrupted PNNPNNP repeat patterns or motifs, which can form an amphipathic alpha-helix. For example, a 15 or 18 residue amino acid sequence, such as KF AKFA KFAKFAKK and
FAKFAKKFAKFAKKFAK, has uninterrupted and interrupted PNNPNNP repeat motifs.
[0047] A conjugate first domain, such as a ligand that binds to Her2/neu, includes or consists of a ligand, antibody, hormone, growth factor, or an analogue or fragment thereof.
[0048] Her2/neu is typically expressed by or present on (e.g., a membrane receptor) or within a cell. Her2/neu may associate with the cell membrane surface or traverse the cell membrane. For example, Her2/neu can have a transmembrane domain that traverses the cell membrane, optionally with a portion that is cytoplasmic or extracellular, or both. Her2/neu therefore include full length intact native Her2/neu containing an extracellular, transmembrane or cytoplasmic portion, as well as truncated forms or fragments thereof (e.g., an extracellular, transmembrane or cytoplasmic portion or subsequence of Her2/neu alone, or in combination). For example, a soluble Her2/neu typically lacks a transmembrane and may optionally also lack all or a part of the native extracellular or cytoplasmic region (if present in native Her2/neu). Such truncated forms and fragments can retain at least partial binding to a ligand.
[0049] Ligands that bind to Her2/neu of conjugates include or consist of any entity that binds to Her2/neu, specifically or non-specifically. Non-limiting examples of ligands therefore include antibodies, antibody fragments, hormones, hormone analogues, fragments of a hormone or hormone analogue that binds to Her2/neu, growth factors, growth factor analogues, fragments of a growth factor or growth factor analogue that binds to Her2/neu, etc.
[0050] Exemplary ligands that bind to Her2/neu include affibodies, such as ZHer2-neu:2, ZHer2- neu:4 ZHer2-neu:7 ZHer2-neu:8 and Fab63. Exemplary affibody sequences are as follows:
VDNKFNK EQQNAFYEILH LPNLNE EQRNAFIQSLKD DPSQ SANLLAEAKKLNDA QAPK
¾er2 :4 VDNKFNK ELRQAYWEIQA LPNLNW TQSRAFIRSLYD DPSQ SANLLAEAKKLNDA QAPK
¾er2 :7 VDNKFNK EPKTAYWEIVK LPNLNP EQRRAFIRSLYD DPSQ SANLLAEAKKLNDA QAPK
¾er2 :24 VDNKFNK EPREAYWEIQR LPNLNN KQKAAFIRSLYD DPSQ SANLLAEAKKLNDA QAPK
¾er2 :79 VDNKFNK EWMTAGKEIYR LPNLNG TQVRAFIQSLSD DPSQ SANLLAEAKKLNDA QAPK
¾er2 :2 VDNKFNK EWVQAGSEIYN LPNLNR AQMRAFIRSLSD DPSQ SANLLAEAKKLNDA QAPK
¾er2 :8 VDNKFNK EIKQAFHEI R LPNLNA DQVRAFIYSLGD DPSQ SANLLAEAKKLNDA QAPK
¾er2 :25 VDNKFNK EMVDAGAEI R LPNLNA KQM*AFIDSLGD DPSQ SANLLAEAKKLNDA QAPK
[0051] Exemplary growth factors useful as ligands that bind to Her2/neu include epidermal growth factor and epidermal growth factor analogues.
[0052] Her2/neu ligands and binding moieties further include antibodies, and antibody fragments. An ''antibody" refers to any monoclonal or polyclonal immunoglobulin molecule, such as IgM, IgG, IgA, IgE, IgD, and any subclass thereof. Exemplary subclasses for IgG are IgG], IgG2, IgG3 and IgG4 including engineered antibody subclasses and recombinant antibodies with various glycosylation patterns. Antibodies include those produced by or expressed on cells, such as B cells, or synthesized or engineered to be produced by other cells, e.g., CHO cells. Such antibodies include those with improved characteristics, such as increased serum stability and/or half life in vivo, PK, etc. (e.g., as described in Antibody Engineering Vol 1, Konterman R and Duebel S, eds., 2010, Springer, WO 2006/130834 and Horton et al., Cancer Res 68:8049 (2008)).
[0053] Non-limiting mutations in the Fc include, for example, I253A, H310A, H435 R, H435Q, G236R, L328 R, S239D, I332E. Non-limiting mutations in IgGlcan beat residues 238, 252, 253, 254, 255, 256, 265, 272, 289, 288, 303, 305, 307, 309, 31 1, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 439 and/or 477 of the Fc region.
[0054] An antibody fragment or subsequence refers to a portion of a full length antibody that retains at least partial antigen binding capability of a comparison full length antibody. Exemplary antibody fragments include Fab, Fab', F(ab')2, Fv, Fd, single-chain Fv (scFv), disulfide-linked Fvs (sdFv), VL,
VH, Camel Ig, V-NAR, VHH, trispecific (Fab3), bispecific (Fab:), diabody ((VL-VH)2 or (VH-VL)2), triabody (trivalent), tetrabody (tetravalent), minibody ((scFv-CH3)2), bispecific single-chain Fv (Bis- scFv), IgGdeltaCH2, scFv-Fc, (scFv)2-Fc, affibody, aptamer, avimer, nanobody or other antigen binding fragment.
[0055] Exemplary antibodies bind to epitopes present on Her2/neu. Exemplary Her2/neu epitopes (the amino acid position numbers of Her2 epitope is refered to by a "p" followed by arabic numbers) include HER-2 (p5-13) A2, HER-2 (p8-16) A24, HER-2 (p48-56) A2, HER-2 (p63-71) A24, HER-2 (pi 06-114) A2, HER-2 (p369-377) A2, A3, A26, HER-2 (p435-443) A2, HER-2 (p654-662) A2, HER- 2 (p665-673) A2, HER-2 (p689-697) A2, HER-2 (p754-762) A3, Al 1 , A33, HER-2 (p773-782) A2, HER-2 (p780-788) A24, HER-2 (p785-794) A2, HER-2 (p789-797) A2, HER-2 (p799-807) A2, HER- 2 (p952-961) A2 and HER-2 (pi 023- 1032) A2.
[0056] Exemplary antibodies include humanized anti-ErbB2 antibodies huMA Dl-l, huMAb4D5- 1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN™) as described in U.S. Patent No.5,821,337; humanized 520C9 (WO 93/21319) and humanized 2C4 (pertuzumab) as described in U.S. Patent No.7,097,840 and pertuzumab variants as described in US2009/0285837A1.
[0057] huMAb4D5-l:
Vj_ (Light chain):
1 10 20 30 40
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ PG
50 60 70 80
KAPKLLIYSASFLESGVPSRFS GSGSGTDFTLTISS LQPEDF
90 100
ATYYCQQHYTTPPTFGQGTKVEIK VH (heavy chain):
1 10 20 30 40
EVQLVESGGGLVQPGGSLRLSCAASGFNI DTYIHWVRQAP
50 60 70 80
GKGLEWVARIYPTNGYTRYADSVKGRFTISRDDSKNTLYLQ
90 100 110 120
MNSLRAEDTAVYYCARWGGDGFYAMDVWGQGTLVTVSS
[0058] huMAb4D5-2:
Vr (Light chain):
1 10 20 30 40
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ PG
50 60 70 80
KAPKLLIYSASFLESGVPSRFS GSGSGTDFTLTISS LQPEDF
90 100
ATYYCQQHYTTPPTFGQGTKVEIK VH (heavy chain):
1 10 20 30 40
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
50 60 70 80 GKGLEWVARIYPTNGYTRYADSV GRFTISADDSKNTLYLQ
90 100 110 120
MNSLRAEDTAVYYCARWGGDGFYAMDVWGQGTLVTVSS
[0059] huMA D5-3:
Vr (Light chain):
1 10 20 30 40
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ PG
50 60 70 80
KAPKLLIYSASFLESGVPSRFS GSGSGTDFTLTISS LQPEDF
90 100
ATYYCQQHYTTPPTFGQGTKVEIK VH (heavy chain):
1 10 20 30 40
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
50 60 70 80
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQ
90 100 110 120
MNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS
[0060] huMAb4D5-4:
Vr (Light chain):
1 10 20 30 40
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ PG
50 60 70 80
KAPKLLIYSASFLESGVPSRFS GSRSGTDFTLTISS LQPEDF
90 100
ATYYCQQHYTTPPTFGQGTKVEIK VH (heavy chain):
1 10 20 30 40
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
50 60 70 80
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTLYLQ
90 100 110 120
MNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS
[0061] huMAb4D5-5:
Vr (Light chain):
1 10 20 30 40
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ PG
50 60 70 80
KAPKLLIYSASFLESGVPSRFS GSRSGTDFTLTISS LQPEDF
90 100
ATYYCQQHYTTPPTFGQGTKVEIK VH (heavy chain):
1 10 20 30 40
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
50 60 70 80
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQ
90 100 110 120
MNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS
[0062] huMAb4D5-6: Vi_ (Light chain):
1 10 20 30 40
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPG
50 60 70 80
KAP LLIYSASFLYSGVPSRFS GSRSGTDFTLTISS LQPEDF
90 100
ATYYCQQHYTTPPTFGQGT VEIK VH (heavy chain):
1 10 20 30 40
EVQLVESGGGLVQPGGSLRLSCAASGFNI DTYIHWVRQAP
50 60 70 80
GKGLEWVARIYPT GYTRYADSVKGRFTISADTS NTAYLQ
90 100 110 120
MNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS
[0063] huMA D5-7:
Vr (Light chain):
1 10 20 30 40
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ PG
50 60 70 80
KAPKLLIYSASFLESGVPSRFS GSRSGTDFTLTISS LQPEDF
90 100
ATYYCQQHYTTPPTFGQGTKVEIK VH (heavy chain):
1 10 20 30 40
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
50 60 70 80
GKGLEWVAR1YPTNGYTRYADSVKGRFTISADTSKNTAYLQ
90 100 110 120
MNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
[0064] huMAb4D5-8:
Vr (Light chain):
1 10 20 30 40
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPG
50 60 70 80
KAPKLLIYSASFLYSGVPSRFS GSRSGTDFTLTISS LQPEDF
90 100
ATYYCQQHYTTPPTFGQGTKVEIK VH (heavy chain):
1 10 20 30 40
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
50 60 70 80
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQ
90 100 110 120
MNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
[0065] huMAb4D5:
Vi_ (Li ht chain):
1 10 20 30 40
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPG
50 60 70 80
KAPKLLIYSASFLYSGVPSRFS GSRSGTDFTLTISS LQPEDF 90 100
ATYYCQQHYTTPPTFGQGTKVEIK VH (heavy chain):
1 10 20 30 40
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVPvQAP
50 60 70 80
GKGLEWVARIYPTNGYTRYADSV GRFTISADTS NTAYLQ
90 100 110 120
MNSLRAEDTA VYYCSRWGGDGF YAMD Y WGQGTL VTVSS
[0066] Antibody sequence permutations(U.S. Patent No.7,435,797 SQ 1 and 2))
huMAb4D5:
Vi_ (Light chain):
1 10 20 30 40
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPG
50 60 70 80
KAPKLLIYSASFLYSGVPSRFS GSRSGTDFTLTISS LQPEDF
90 100
ATYYCQQHYTTPPTFGQGTKVEIK VH (heavy chain):
1 10 20 30 40
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
50 60 70 80
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQ
90 100 110 120
MNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
[0067] huMAb4D5-8
(for VL: Q27, D28, N30, T31, A32, Y49, F53, Y55, R66 H91, Y92, T94; for VH: W95, D98. FlOO, Y100, Y102):
Vr (Light chain): Claim 1 mutation
1 10 20 30 40
DIQMTQSPSSLSASVGDRVTITCRASQDVSSAVAWYQQKPG
50 60 70 80
K A P K L L I D/W SASFLYSGVPSRFS GSRSGTDFTLTISS LQPEDF
90 100
ATYYCQQHYTTPPTFGQGTKVEIK
VH (heavy chain):
1 10 20 30 40
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
50 60 70 80
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT^YLQ
90 100 110 120
MNSLRAEDTAVYYCSRWGGWGP K/L AMDYWGQGTLVTVSS
[0068] Pertuzumab Sequences (US 2010/0015157A 1 )
Vi_ (Li ht chain):
1 10 20 30 40
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPG
50 60 70 80
KAPKLLIYSASYRYTGVPSRFS GSGSGTDFTLTISS LQPED
90 100 110 120 FATYYCQQYYIYPYTFGQGT VEI KR TVAAPSVFIFPPSDEQ
130 140 150 160
LKSGTASVVC L LNNN F YPREA VQWKVDNALQSGENSQES
170 180 190 200
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
210
PVTKSFNRGEC
VH (heavy chain):
1 10 20 30 40
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQA
50 60 70 80
PGKGLEWVADVNPNSGGSIYNQRF GRFTLSVDRSKNTZYL
90 100 110 120
QMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSAST
130 140 150 160
KGPSVFPLAPSS STSGGTAALGCLVKDYFPEPVTVSWNSG
170 180 190 200
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
210 220 230 240
H PSNT VD KVEP SCDKTHTCPPCPAPELLGGPSVFLFP
250 260 270 280
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
290 300 310 320
NAKT PREEQYNSTYRVVSVLTVLHQDWLNG EY CKVSN
330 340 350 360
KALPAPIEKTISKA GQPREPQVYTLPPSREEMTKNQVSLTC
370 380 390 400 410
LVKGFYPSDIAVEWESNGQPENNY TTPPVLDSDGSFFLYS
420 430 440
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
[0069] Pertuzumab variants (US2009/0285837 Al)
Vr (Light chain):
1 10 20 30 40
VHSDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQ
50 60 70 80 PGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQP
90 100 110 120
EDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSD
130 140 150 160
EQL SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
170 180 190 200
VTEQDS DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
210
PVTKSFNRGEC
VH (Heavy chain):
1 10 20 30 40
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQA
50 60 70 80
PGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYL
90 100 110 120
QMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSAS 130 140 150 160
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
170 180 190 200
G ALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
210 220 230 240
NHKPSNTKVD KVEPKSCD THTCPPCPAPELLGGPSVFLV
250 260 270 280
PPKP DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
290 300 310 320
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG EY CKVS
330 340 350 360
NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
370 380 390 400
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
410 420 430 440
S LTVDKSRWQQGNVFSCSVMHEALN HY TQKSLSLSPGK
[0070] Pertuzumab Sequences: humanized 2C4, 7C2, 7 F3, 7D3, 3 E 8, 4D5, 2H11 , 3 H4 (U.S. Patent
No.7,097,840)
Vr (Light chain):
1 10 20 30 40
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPG
50 60 70 80
KAPKLLIYSASYRYTGVPSRFS GSGSGTDFTLTISS LQPED
90 100 110 120
FATYYCQQYYIYPYTFGQGTKVEI KR TVAAPSVFIFPPSDEQ
130 140 150 160
LKSGTASVVC L LNNNF Y PRE A V Q WKVDNALQSGENSQES
170 180 190 200
VTEQDSKDSTYSLSSTLTLSKADYE H VYACEVTHQGLSS
210
PVTKSFNRGEC
VH (heavy chain):
1 10 20 30 40
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQA
50 60 70 80
PGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTZYL
90 100 110 120
QMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSAST
130 140 150 160
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG
170 180 190 200
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
210 220 230 240
HKPSNTKVDK VEPKSCD THTCPPCPAPELLGGPSVFLFP
250 260 270 280
PKPKDTLMISRTPEVTCVVVDVSHEDPEV FNWYVDGVEVH
290 300 310 320
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
330 340 350 360
KALPAPIE TIS A GQPREPQVYTLPPSREEMT NQVSLTC
370 380 390 400 410 LVKGFYPSDIAVEWESNGQPENNY TTPPVLDSDGSFFLYS
420 430 440
KLTVD SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
[0071] Additional anti-ErbB2 antibodies with various properties have been described in Tagliabue et al. Int. J. Cancer 47:933-937 (1991); McKenzie et al. Oncogene 4:543 (1989); Maier et al. Cancer Res. 51:5361 (1991); Bacus et al. Molecular Carcinogenesis 3:350 (1990); Stancovski et al. PNAS (USA) 88:8691 (1991); Bacus et al. Cancer Research 52:2580 (1992); Xu et al. Int. J Cancer 53:401-408 (1993); WO 94/00136; Kasprzyk et al. Cancer Research 52:2771 (1992); Hancock et al. Cancer Res.51:4575 (1991); Shawver et al. Cancer Res.54:1367 (1994); Arteaga et al. Cancer Res.
54:3758 (1994); Harwerth et al. J Biol Chem.267:15160 (1992); U.S. Patent No.5,783,186; and Klapper et al. Oncogene 14:2099 (1997).
[0072] Conjugates include those with a first domain at the amino-terminus and a second domain at the carboxyl-terminus. Conjugates also include those with a first domain at the carboxyl -terminus and a second domain at the amino-terminus. Where additional domains are present (e.g., third, fourth, fifth, sixth, seventh, etc. domains), a first domain is positioned at the NH2-terminus relative to a second domain, or a second domain is positioned at the NFF-terminus relative to a first domain.
[0073] Subsequences and amino acid substitutions of the various sequences set forth herein, such as, KFAKFA KFAKFA K, KFAKFAKKFAKFAKKF, KFA FAKKFAKFAKKFA,
KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, K F A FA KFAKFAKK FA K FA and KJAXFAXKPAKTA KFAKFAKKFAKFAK, or a ligand that binds to Fter2/neu, are also included. In particular embodiments, a subsequence of a first or second domain has at least 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, or more amino acid residues.
[0074] The invention therefore includes modifications or variations, such as substitutions, additions or deletions of a first or second domain, or both first and second domains. Thus, a conjugate that includes a peptide sequence first or second domain can incorporate any number of conservative or non- conservative amino acid substitutions, as long as such substitutions do not destroy activity (Her2/neu binding or lytic) of first or second domains. Thus, for example, a modified ligand that binds to Her2/neu or a second lytic portion domain can retain at least partial Her2/neu binding or lytic activity, such as cell killing or apoptosis, of an unmodified second domain.
[0075] A "conservative substitution" is a replacement of one amino acid by a biologically, chemically or structurally similar residue. Biologically similar means that the substitution is compatible with a biological activity, e.g., lytic activity. Structurally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or having similar size, or the structure of a first, second or additional domain is maintained, such as an amphipathic alpha helix. Chemical similarity means that the residues have the same charge or are both hydrophilic and hydrophobic. Particular examples include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, serine for threonine, etc. Routine assays can be used to determine whether a conjugate variant has activity, e.g., binding activity or lytic activity.
[0076] Specific examples include a substitution or deletion of one or more amino acid (e.g., 1 -3, 3-5, 5-10, 10-20, or more) residues of a peptide first or second domain. A modified conjugate can have a peptide sequence with 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more identity to a reference sequence (e.g., a first domain, such as a ligand that binds to Her2/neu or a second domain such as KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,
FAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKK FAKF, KFAKFAKKFAKFAKKF- AKFA or KFAKFAKKFAKFAKKFAKFAKKFAKFAK).
[0077] In a particular embodiment, a conjugate includes a peptide first domain that includes or consists of a ligand that binds to Her2/neu and a second lytic domain that includes or consists of an L- or D-amino acid sequence that includes a peptide selected from KPAKFAKKFAKFAKK,
KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK,
KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKF AKFA and
KFAKFAKKFAKFAKKFAKFAKKFAKFAK having one or more of the K residues substituted with an F or L residue, one or more of the F residues substituted with a K, A or L residue, or one or more of the A residues substituted with a K, F or L residue. In another particular embodiment, a conjugate includes a ligand that binds to Her2/neu and a second domain consisting of an L- or D-amino acid sequence selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF.
KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF, KFAKFAKKFAKFAKKFAKFA and KFAKFAKKFAKFAKKFAKFAKKFAKFAK having one or more of the K residues substituted with an F or L residue, one or more of the F residues substituted with a K, A or L residue, or one or more of the A residues substituted with a K, F or L residue (e.g., 12, 13, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27 or 28 residues in length).
[0078] The term "identity" and "homology" and grammatical variations thereof mean that two or more referenced entities are the same. Thus, where two amino acid sequences are identical, they have the same amino acid sequence. "Areas, regions or domains of identity" mean that a portion of two or more referenced entities are the same. Thus, where two amino acid sequences are identical or homologous over one or more sequence regions, they share identity in these regions. The term
"complementary," when used in reference to a nucleic acid sequence means the referenced regions are 100%) complementary, i.e., exhibit 100% base pairing with no mismatches.
[0079] Due to variation in the amount of sequence conservation between structurally and functionally related proteins, the amount of sequence identity required to retain a function or activity (e.g., Her2/neu binding or lytic) depends upon the protein, the region and the function or activity of that region. For example, for a lytic peptide sequence multiple PNNPNNP sequence repeat patterns or motifs can be present, but one or more interrupted or non-interrupted PNNPNNP sequence repeat patterns or motifs need not be present.
[0080] The extent of identity between two sequences can be ascertained using a computer program and mathematical algorithm known in the art. Such algorithms that calculate percent sequence identity (homology) generally account for sequence gaps and mismatches over the comparison region. For example, a BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschul et al., J. Mol. Biol. 215:403 (1990), publicly available through NCB1) has exemplary search parameters as follows: Mismatch -2; gap open 5; gap extension 2. For polypeptide sequence comparisons, a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAM100, PAM 250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantitate the extent of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132: 185 (2000); and Smith et al., J. Mol. Biol. 147: 195 (1981)). Programs for quantitating protein structural similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).
[0081] Individual residues and first, second and additional domains can be joined by a covalent or a non-covalent bond. Non-limiting examples of covalent bonds are amide bonds, non-natural and non- amide chemical bonds, which include, for example, glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N, N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC). Linking groups alternative to amide bonds include, for example, ketomethylene (e.g., -C(=0)- CH2- for -C(=0)-NH-), aminomethylene (CPL-NH), ethylene, olefin (CH=CH), ether (CH2-0), thioether (CFL-S), tetrazole (CN4-), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide and Backbone Modifications," Marcel Decker, NY).
[0082] First and second domains can be fused or joined immediately adjacent to each other by a covalent or a non-covalent bond. First and second domains can be separated by an intervening region, such as a hinge, spacer or linker positioned between a first and a second domain. In one embodiment, a first and second domain are joined by a carbon chain (e.g., CCCCC). Multi-carbon chains include carboxylic acids (e.g., dicarboxylic acids) such as glutaric acid, succinic acid and adipic acid.
[0083] In another embodiment, a first and second domain are joined by an amino acid, peptide or a non-peptide hinge, spacer or linker positioned between the first and second domains. Peptide hinge, spacer or linker sequences can be any length, but typically range from about 1-10, 10-20, 20-30, 30-40, or 40-50 amino acid residues. In particular embodiments, a peptide hinge, spacer or linker positioned between a first and second domain is from 1 to 5, 1 to 10, 1 to 20, 1 to 25 L- or D-amino acid residues, or 1 to 6 L- or D-amino acid residues. Particular amino acid residues that are included in sequences positioned between the first and second domains include one or more of or C, A, S or G amino acid residues. Specific non-limiting examples of peptides positioned between the first and second domains include a sequence within or set forth as: GSGGS, ASAAS, or a carbon chain such as CCCCCC.
Derivatives of amino acids andpeptides can be positioned between the first and second domain. A specific non-limiting example of an amino acid derivative is a lysine derivative, or a 6 carbon linker such as a-amino-caproic acid.
[0084] Conjugates with or without a hinge, spacer or linker, or a third, fourth, fifth, sixth, seventh, etc. domain can be entirely composed of natural amino acids or synthetic, non-natural amino acids or amino acid analogues, or can include derivatized forms. In various embodiments, a conjugate includes in a first or second domain one or more D-amino acids substituted for L-amino acids, mixtures of D- amino acids and L-amino acids, or a sequence composed entirely of D-amino acid residues.
[0085] Conjugates can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., induce or stabilize a secondary structure, e.g., an alpha helix conformation. Conjugates include cyclic structures such as an end-to-end amide bond between the amino and carboxy-terminus of the molecule or intra- or inter-molecular disulfide bond(s). Conjugates may be modified in vitro or in vivo, e.g., post-translationally modified to include, for example, sugar or carbohydrate residues, phosphate groups, fatty acids, lipids, etc.
[0086] Specific examples of an addition include a third, fourth, fifth, sixth or seventh domain.
Conjugates with a first and second domain therefore include one or more additional domains (third, fourth, fifth, sixth, seventh, etc.) covalently linked thereto to impart a distinct or complementary function or activity. Exemplary additional domains include domains facilitating isolation, which include, for example, metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals; protein A domains that allow purification on immobilized immunoglobulin; and domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA). Optional inclusion of a cleavable sequence such as Factor Xa or enterokinase between a purification domain and the conjugate can be used to facilitate purification. For example, an expression vector can include a conjugate-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site. The histidine residues facilitate detection and purification of the conjugate while the enterokinase cleavage site provides a means for purifying the construct from the remainder of the protein (see e.g., roll, DNA Cell. Biol. 12:441 (1993)).
[0087] Conjugate activity can be affected by various factors and therefore conjugates can be designed or optimized by taking into consideration one or more of these factors. Such factors include, for example, length, which can affect toxicity to cells. Cell killing activity of alpha helix forming lytic peptide domains can also depend on the stability of the helix. Hinge and spacers can affect membrane interaction of a first domain and the helical structure of a peptide lytic domain. The charge of lytic peptide domains, which is determined in part by the particular amino acid residues present in the domain, also affects cell killing potency. The positioning of the ligand that binds to Her2/neu relative to the lytic domain (N- or C-terminus) also can affect cell killing activity of conjugates.
[0088] Conjugate in vivo half-life can be increased by constructing peptide domains with one or more non-naturally occurring amino acids or derivatives. For example, conjugates with D-amino acids
(e.g., up to 30%, 40%, 50%, 60%, or more of all residues are D-enantiomers) are resistant to serum proteolysis and therefore can be active for longer times thereby increasing in vivo potency.
Furthermore, constructing peptide domains with one or more non-naturally occurring amino acids or derivatives can reduce hemolytic activity. Such conjugates with D-enantiomers also have a greater tendency to be monomeric in solution- they do not significantly aggregate.
[0089] Peptides and peptidomimetics can be produced and isolated using methods known in the art. Peptides can be synthesized, whole or in part, using chemical methods known in the art (see, e.g., Caruthers (1980). Nucleic Acids Res. Symp. Ser. 215; Horn (1980); and Banga, A. ., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA). Peptide synthesis can be performed using various solid-phase techniques (see, e.g., Roberge Science 269:202 (1995); Merrifield, Methods Enzymol. 289:3(1997)) and automated synthesis may be achieved, e.g., using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the manufacturer's instructions. Peptides and peptide mimetics can also be synthesized using combinatorial methodologies. Synthetic residues and polypeptides incorporating mimetics can be synthesized using a variety of procedures and methodologies known in the art (see, e.g., Organic Syntheses Collective Volumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY). Modified peptides can be produced by chemical modification methods (see, for example, Belousov, Nucleic Acids Res. 25:3440 (1997); Frenkel, Free Radic. Biol. Med. 19:373 (1995); and Blommers, Biochemistry 33:7886 (1994).
[0090] The invention further provides nucleic acids encoding the conjugates of the invention and vectors that include nucleic acid that encodes conjugates. Nucleic acid, which can also be referred to herein as a gene, polynucleotide, nucleotide sequence, primer, oligonucleotide or probe refers to natural or modified purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides and a-anomeric forms thereof. The two or more purine- and pyrimidine-containing polymers are typically linked by a phosphoester bond or analog thereof. The terms can be used interchangeably to refer to all forms of nucleic acid, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleic acids can be single strand, double, or triplex, linear or circular. Nucleic acids include genomic DNA, cDNA, and antisense. RNA nucleic acid can be spliced or unspliced mRNA, rRNA, tRNA or antisense. Nucleic acids include naturally occurring, synthetic, as well as nucleotide analogues and derivatives. [0091] As a result of the degeneracy of the genetic code, nucleic acids include sequences degenerate with respect to sequences encoding conjugates of the invention. Thus, degenerate nucleic acid sequences encoding conjugates are provided.
[0092] Nucleic acid can be produced using any of a variety of known standard cloning and chemical synthesis methods, and can be altered intentionally by site-directed mutagenesis or other recombinant techniques known to one skilled in the art. Purity of polynucleotides can be determined through sequencing, gel electrophoresis, UV spectrometry.
[0093] Nucleic acids may be inserted into a nucleic acid construct in which expression of the nucleic acid is influenced or regulated by an "expression control element," referred to herein as an "expression cassette." The term "expression control element" refers to one or more nucleic acid sequence elements that regulate or influence expression of a nucleic acid sequence to which it is operatively linked. An expression control element can include, as appropriate, promoters, enhancers, transcription terminators, gene silencers, a start codon (e.g., ATG) in front of a protein-encoding gene, etc.
[0094] An expression control element operatively linked to a nucleic acid sequence controls transcription and, as appropriate, translation of the nucleic acid sequence. The term "operatively linked" refers to a juxtaposition wherein the referenced components are in a relationship permitting them to function in their intended manner. Typically expression control elements are juxtaposed at the 5' or the 3' ends of the genes but can also be intronic.
[0095] Expression control elements include elements that activate transcription constitutively, that are inducible (i.e., require an external signal for activation), or derepressible (i.e., require a signal to turn transcription off; when the signal is no longer present, transcription is activated or "derepressed"). Also included in the expression cassettes of the invention are control elements sufficient to render gene expression controllable for specific cell-types or tissues (i.e., tissue-specific control elements).
Typically, such elements are located upstream or downstream (i.e., 5' and 3') of the coding sequence. Promoters are generally positioned 5' of the coding sequence. Promoters, produced by recombinant DNA or synthetic techniques, can be used to provide for transcription of the polynucleotides of the invention. A "promoter" is meant a minimal sequence element sufficient to direct transcription.
[0096] Nucleic acids may be inserted into a plasmid for propagation into a host cell and for subsequent genetic manipulation if desired. A plasmid is a nucleic acid that can be stably propagated in a host cell; plasmids may optionally contain expression control elements in order to drive expression of the nucleic acid. A vector is used herein synonymously with a plasmid and may also include an expression control element for expression in a host cell. Plasmids and vectors generally contain at least an origin of replication for propagation in a cell and a promoter. Plasmids and vectors are therefore useful for genetic manipulation of conjugate encoding nucleic acids, producing conjugates or antisense nucleic acid, and expressing conjugates in host cells and organisms, for example. [0097] Bacterial system promoters include T7 and inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and tetracycline responsive promoters. Insect cell system promoters include constitutive or inducible promoters (e.g., ecdysone). Mammalian cell constitutive promoters include SV40, RSV, bovine papilloma virus (BPV) and other virus promoters, or inducible promoters derived from the genome of mammalian cells (e.g., metallothionein IIA promoter; heat shock promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the inducible mouse mammary tumor virus long terminal repeat). Alternatively, a retroviral genome can be genetically modified for introducing and directing expression of a conjugate in appropriate host cells.
[0098] Expression systems further include vectors designed for in vivo use. Particular non-limiting examples include adenoviral vectors (U.S. Patent Nos. 5,700,470 and 5,731,172), adeno-associated vectors (U.S. Patent No. 5,604,090), herpes simplex virus vectors (U.S. Patent No. 5,501,979), retroviral vectors (U.S. Patent Nos. 5,624,820, 5,693,508 and 5,674,703), BPV vectors (U.S. Patent No.
5,719,054) and CMV vectors (U.S. Patent No. 5,561,063).
[0099] Yeast vectors include constitutive and inducible promoters (see, e.g., Ausubel et al., In:
Current Protocols in Molecular Biology, Vol. 2, Ch. 13, ed., Greene Publish. Assoc. & Wiley
Interscience, 1988; Grant et al. Methods in Enzymology, 153:516 (1987), eds. Wu & Grossman; Bitter Methods in Enzymology, 152:673 (1987), eds. Berger & Kimmel, Acad. Press, N.Y.; and, Strathern et al., The Molecular Biology of the Yeast Saccharomyces (1982) eds. Cold Spring Harbor Press, Vols. I and II). A constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (R. Rothstein In: DNA Cloning, A Practical Approach. Vol.1 1 , Ch. 3, ed. D.M. Glover, IRL Press, Wash., D.C., 1986). Vectors that facilitate integration of foreign nucleic acid sequences into a yeast chromosome, via homologous recombination for example, are known in the art. Yeast artificial chromosomes (YAC) are typically used when the inserted polynucleotides are too large for more conventional vectors (e.g., greater than about 12 Kb).
[00100] Expression vectors also can contain a selectable marker conferring resistance to a selective pressure or identifiable marker (e.g., beta-galactosidase), thereby allowing cells having the vector to be selected for, grown and expanded. Alternatively, a selectable marker can be on a second vector that is cotransfected into a host cell with a first vector containing a nucleic acid encoding a conjugate.
[0100] Selection systems include but are not limited to herpes simplex virus thymidine kinase gene (Wigler et al., Cell 1 1 :223 (1977)), hypoxanthine-guanine phosphoribosyltransferase gene (Szybalska et al., Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes which can be employed in tk-, hgprt- or aprt- cells, respectively.
Additionally, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (O'Hare et al., Proc. Natl. Acad. Sci. USA 78: 1527 (1981)); the gpt gene, which confers resistance to mycophenolic acid (Mulligan et al., Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neomycin gene, which confers resistance to aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol. 150: 1 (1981)); puromycin; and hygromycin gene, which confers resistance to hygromycin
(Santerre et al., Gene 30: 147 (1984)). Additional selectable genes include trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman et al., Proc. Natl. Acad. Sci. USA 85:8047 (1988)); and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue (1987) In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory).
[0101] Host cells that express conjugates, and host cells transformed with nucleic acids encoding conjugates and vectors including a nucleic acid that encodes the conjugates are also provided. In one embodiment, a host cell is a prokaryotic cell. In another embodiment, a host cell is a eukaryotic cell. In various aspects, the eukaryotic cell is a yeast or mammalian (e.g., human, primate, etc.) cell.
[0102] As used herein, a "host cell" is a cell into which a nucleic acid is introduced that can be propagated, transcribed, or encoded conjugate expressed. The term also includes any progeny or subclones of the host cell. Host cells include cells that express conjugates.
[0103] Host cells include but are not limited to microorganisms such as bacteria and yeast; and plant, insect and mammalian cells. For example, bacteria transformed with recombinant bacteriophage nucleic acid, plasmid nucleic acid or cosmid nucleic acid expression vectors; yeast transformed with recombinant yeast expression vectors; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid); insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); and animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccinia virus), or transformed animal cell systems engineered for transient or stable propagation or expression.
[0104] Conjugates, nucleic acids encoding conjugates, vectors and host cells expressing conjugates or transformed with nucleic acids encoding conjugates and antisense include isolated and purified forms. The term "isolated," when used as a modifier of an invention composition, means that the composition is made by the hand of man or is separated, substantially completely or at least in part, from the naturally occurring in vivo environment. Generally, an isolated composition is substantially free of one or more materials with which it normally associates with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane. The term "isolated" does not exclude alternative physical forms of the composition, such as muitimers/oligomers, variants, modifications or derivatized forms, or forms expressed in host cells produced by the hand of man. The term "isolated" also does not exclude forms (e.g., pharmaceutical formulations and combination compositions) in which there are combinations therein, any one of which is produced by the hand of man.
[0105] An "isolated" composition can also be "purified" when free of some, a substantial number of, most or all of the materials with which it typically associates with in nature. Thus, an isolated conjugate that also is substantially pure does not include polypeptides or polynucleotides present among millions of other sequences, such as proteins of a protein library or nucleic acids in a genomic or cDNA library, for example. A "purified" composition can be combined with one or more other molecules.
[0106] In accordance with the invention, there are provided mixtures of conjugates and combination compositions. In one embodiment, a mixture includes one or more conjugates and a pharmaceutically acceptable carrier or excipient. In another embodiment, a mixture includes one or more conjugates and an anti-cell proliferative, anti-tumor, anti-cancer, or anti-neoplastic treatment or agent. In a further embodiment, a mixture includes one or more conjugates and an immune enhancing agent.
Combinations, such as one or more conjugates in a pharmaceutically acceptable carrier or excipient, with one or more of an anti-cell proliferative, anti-tumor, anti-cancer, or anti-neoplastic treatment or agent, and an immune enhancing treatment or agent, are also provided.
[0107] Conjugates of the invention, such as polypeptides having an amino acid sequence including a first ligand that binds to Her2/neu (Her2/neu binding moiety) domain and a second lytic domain, can be used to target cells for lysis, cell death or apoptosis. Such cells can be selectively targeted. For example a cell that expresses Her2/neu can be targeted by a conjugate and thereby be preferentially killed compared to cells that express none or less of the Her2/neu.
[0108] In accordance with the invention, there are provided methods of reducing or inhibiting proliferation of a cell that expresses Her2/neu and methods of reducing or inhibiting cell proliferation. In one embodiment, a method includes contacting a cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the cell. In another embodiment, a method includes contacting a cell with a conjugate in an amount sufficient to reduce or inhibit cell proliferation.
[0109] Also provided are methods of reducing or inhibiting proliferation of a hyperproliferative cell that expresses Her2/neu, and methods of reducing or inhibiting proliferation of hyperproliferating cells that express Her2/neu. In one embodiment, a method includes contacting a hyperproliferative cell or hyperprol iterating cells with a conjugate in an amount sufficient to reduce or inhibit proliferation.
[0110] Further provided are methods of reducing or inhibiting proliferation of a non-metastatic or metastatic neoplastic, cancer, tumor and malignant cells that express Her2/neu. In one embodiment, a method includes contacting a neoplastic, cancer, tumor or malignant cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the cell.
[0111] Still further provided are methods of reducing or inhibiting proliferation of a dormant or non- dividing non-metastatic or metastatic neoplastic, cancer, tumor and malignant cells that express Her2/neu. In one embodiment, a method includes contacting a dormant or non-dividing neoplastic, cancer, tumor or malignant cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the dormant or non-dividing cell.
[0112] Additionally provided are methods of selectively reducing or inhibiting proliferation of a cell (e.g., a hyperproliferating cell) that expresses Her2/neu. In one embodiment, a method includes contacting the cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the cell (e.g., hyperproliferating cell), wherein the conjugate binds to Her2/neu expressed by the cell.
[0113] Yet additionally provided are methods of selectively reducing or inhibiting proliferation of a neoplastic, tumor, cancer or malignant cell that expresses that expresses Her2/neu. In one embodiment, a method includes contacting the cell with a conjugate in an amount sufficient to reduce or inhibit proliferation of the neoplastic, tumor, cancer or malignant cell, wherein the conjugate binds to the Her2/neu expressed by the cell.
[0114] The term "contacting" means direct or indirect binding or interaction between two or more entities (e.g., between a conjugate and a cell). Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration or delivery.
[0115] Cells to target for reducing or inhibiting proliferation, non-selectively or selectively, include cells that express Her2/neu. Non-limiting exemplary cells include breast, ovarian, uterine, cervical, stomach, lung, gastric, colon, bladder, glial, hematologic and endometrial cells. Thus, target cells include cells that express Her2/neu.
[0116] Conjugates and methods of the invention are also applicable to treating undesirable or aberrant cell proliferation and hyperproliferative disorders, which include cells expressing Her2/neu. Thus, in accordance with the invention, methods of treating undesirable or aberrant cell proliferation and hyperproliferative disorders are provided. In one embodiment, a method includes administering to a subject (in need of treatment) an amount of a conjugate sufficient to treat the undesirable or aberrant cell proliferation or the hyperproliferative disorder.
[0117] The term "hyperproliferative disorder" refers to any undesirable or aberrant cell survival (e.g., failure to undergo programmed cell death or apoptosis), growth or proliferation. Such disorders include benign hyperplasias, non-metastatic and metastatic neoplasias, cancers, tumors and
malignancies. Undesirable or aberrant cell proliferation and hyperproliferative disorders can affect any cell, tissue, organ in a subject. Undesirable or aberrant cell proliferation and hyperproliferative disorders can be present in a subject, locally, regionally or systemically. A hyperproliferative disorder can arise from a multitude of tissues and organs, including but not limited to breast, lung (e.g., small cell or non- small cell), thyroid, head and neck, brain, nasopharynx, throat, nose or sinuses, lymphoid, adrenal gland, pituitary gland, thyroid, lymph, gastrointestinal (mouth, esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon, rectum), genito-urinary tract (uterus, ovary, vagina, cervix, endometrium, fallopian tube, bladder, testicle, penis, prostate), kidney, pancreas, liver, bone, bone marrow, lymph, blood (hematologic), brain (glial), muscle, skin, and stem cells, which may or may not metastasize to other secondary sites, regions or locations.
[0118] Conjugates and methods of the invention are also applicable to metastatic or non-metastatic tumor, cancer, malignancy or neoplasia of any cell, organ or tissue origin. Such disorders can affect virtually any cell or tissue type, e.g., carcinoma, sarcoma, melanoma, neural, and reticuloendothelial or hematopoietic neoplastic disorders (e.g., myeloma, lymphoma or leukemia).
[0119] As used herein, the terms "neoplasia" and "tumor" refer to a cell or population of cells whose growth, proliferation or survival is greater than growth, proliferation or survival of a normal counterpart cell, e.g. a cell proliferative or differentiative disorder. A tumor is a neoplasia that has formed a distinct mass or growth. A "cancer" or "malignancy" refers to a neoplasia or tumor that can invade adjacent spaces, tissues or organs. A "metastasis" refers to a neoplasia, tumor, cancer or malignancy that has disseminated or spread from its primary site to one or more secondary sites, locations or regions within the subject, in which the sites, locations or regions are distinct from the primary tumor or cancer. All or a portion of such cells can express Her2/neu and can therefore be targeted with conjugates in accordance with the invention.
[0120] Neoplastic, tumor, cancer and malignant cells (metastatic or non-metastatic) include dormant or residual neoplastic, tumor, cancer and malignant cells, all or a portion of which express Her2/neu. Such cells typically consist of remnant tumor cells that are not dividing (G0-G1 arrest). These cells can persist in a primary site or as disseminated neoplastic, tumor, cancer or malignant cells as a minimal residual disease. These dormant neoplastic, tumor, cancer or malignant cells remain asymptomatic, but can develop severe symptoms and death once these dormant cells proliferate. Invention methods can be used to reduce or inhibit proliferation of dormant neoplastic, tumor, cancer or malignant cells, which can in turn inhibit or reduce tumor or cancer relapse, or tumor or cancer metastasis or progression.
[0121] In accordance with the invention, methods of treating a subject having a metastatic or non- metastatic tumor, cancer, malignancy or neoplasia are provided. In one embodiment, a method includes administering to a subject (in need of treatment) an amount of a conjugate of sufficient to treat (e.g., reduce or inhibit proliferation) the metastatic or non-metastatic tumor, cancer, malignancy or neoplasia.
[0122] The metastatic or non-metastatic tumor, cancer, malignancy or neoplasia may be in any stage, e.g., early or advanced, such as a stage I, II, III, IV or V tumor. The metastatic or non-metastatic tumor, cancer, malignancy or neoplasia may have been subject to a prior treatment or be stabilized (non- progressing) or in remission.
[0123] In terms of metastasis, invention methods can be used to reduce or inhibit metastasis of a primary tumor or cancer to other sites, or the formation or establishment of metastatic tumors or cancers at other sites distal from the primary tumor or cancer thereby inhibiting or reducing tumor or cancer relapse or tumor or cancer progression. Thus, methods of the invention include, among other things, 1) reducing or inhibiting growth, proliferation, mobility or invasiveness of tumor or cancer cells that potentially or do develop metastases (e.g., disseminated tumor cells, DTC); 2) reducing or inhibiting formation or establishment of metastases arising from a primary tumor or cancer to one or more other sites, locations or regions distinct from the primary tumor or cancer; 3) reducing or inhibiting growth or proliferation of a metastasis at one or more other sites, locations or regions distinct from the primary tumor or cancer after a metastasis has formed or has been established; and 4) reducing or inhibiting formation or establishment of additional metastasis after the metastasis has been formed or established.
[0124] Cells of a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia (all or a portion of which express Her2/neu) may be aggregated in a "solid" cell mass or be dispersed or diffused. A "solid" tumor refers to cancer, neoplasia or metastasis that typically aggregates together and forms a mass. Specific non-limiting examples include breast, ovarian, uterine, cervical, stomach, lung, gastric, colon, bladder, glial, and endometrial tumors/cancers.
[0125] Carcinomas, which refer to malignancies of epithelial or endocrine tissue, include respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from the uterus, cervix, lung, prostate, breast, head and neck, colon, pancreas, testes, adrenal, kidney, esophagus, stomach, liver and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. Adenocarcinoma includes a carcinoma of a glandular tissue, or in which the tumor forms a gland like structure.
[0126] Sarcomas refer to malignant tumors of mesenchymal cell origin. Exemplary sarcomas include for example, lymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma and fibrosarcoma.
[0127] Neural neoplasias include glioma, glioblastoma, meningioma, neuroblastoma,
retinoblastoma, astrocytoma and oligodendrocytoma.
[0128] A "liquid tumor," which refers to neoplasia that is dispersed or is diffuse in nature, as they do not typically form a solid mass. Particular examples include neoplasia of the reticuloendothelial or hematopoietic system, such as lymphomas, myelomas and leukemias. Non-limiting examples of leukemias include acute and chronic lymphoblastic, myeolblastic and multiple myeloma. Typically, such diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Specific myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML). Lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), which includes B-lineage ALL (B-ALL) and T-lineage ALL (T-ALL), chronic lymphocytic leukemia (CLL), prolymphocyte leukemia (PLL), hairy cell leukemia (HLL) and Waldenstroem's macroglobulinemia (WM). Specific malignant lymphomas include, non-Hodgkin lymphoma and variants, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease. [0129] As disclosed herein, undesirable or aberrant cell proliferation or hyperproliferative disorders can occur in uterus, breast, vagina, cervix, endometrium and fallopian tube. Thus, in accordance with the invention, there are provided methods of treating uterus, breast, vagina, cervix, endometrium and fallopian tube hyperproliferative disorders. In one embodiment, a method includes administering to a subject an amount of a conjugate sufficient to treat a uterus, breast, vagina, cervix, endometrium or fallopian tube hyperproliferative disorder.
[0130] Any composition, treatment, protocol, therapy or regimen having an anti-cell proliferative activity or effect can be combined with a conjugate or used in combination in a method of the invention. Conjugates and methods of the invention therefore include anti-proliferative, anti-tumor, anti-cancer, anti-neoplastic and anti-metastatic treatments, protocols and therapies, which include any other composition, treatment, protocol or therapeutic regimen that inhibits, decreases, retards, slows, reduces or prevents a hyperproliferative disorder, such as tumor, cancer, malignant or neoplastic growth, progression, metastasis, proliferation or survival, or worsening in vitro or in vivo. Particular non-limiting examples of an anti-proliferative (e.g., tumor) therapy include chemotherapy, immunotherapy, radiotherapy (ionizing or chemical), local thermal (hyperthermia) therapy, surgical resection and vaccination. A conjugate can be administered prior to, substantially contemporaneously with or following administration of the anti-cell proliferative, anti-neoplastic, anti-tumor, anti-cancer, anti-metastatic or immune-enhancing treatment or therapy. A conjugate can be administered as a combination composition with the anti-cell proliferative, anti-neoplastic, anti-tumor, anti-cancer, anti- metastatic or immune-enhancing treatment or therapy, metastatic or non-metastatic tumor, cancer, malignancy or neoplasia.
[0131] Anti-proliferative, anti-neoplastic, anti-tumor, anti-cancer and anti-metastatic compositions, therapies, protocols or treatments include those that prevent, disrupt, interrupt, inhibit or delay cell cycle progression or cell proliferation; stimulate or enhance apoptosis or cell death, inhibit nucleic acid or protein synthesis or metabolism, inhibit cell division, or decrease, reduce or inhibit cell survival, or production or utilization of a necessary cell survival factor, growth factor or signaling pathway (extracellular or intracellular). Non-limiting examples of chemical agent classes having anti-cell proliferative, anti-neoplastic, anti-tumor, anti-cancer and anti-metastatic activities include alkylating agents, anti-metabolites, plant extracts, plant alkaloids, nitrosoureas, hormones, nucleoside and nucleotide analogues. Specific examples of drugs having anti-cell proliferative, anti-neoplastic, antitumor, anti-cancer and anti-metastatic activities include cyclophosphamide, azathioprine, cyclosporin A, prednisolone, melphalan, chlorambucil, mechlorethamine, busulphan, methotrexate, 6- mercaptopurine, thioguanine, 5-fluorouracil, cytosine arabinoside, AZT, 5-azacytidine (5-AZC) and 5- azacytidine related compounds such as decitabine (5-aza-2'deoxycytidine), cytarabine, 1-beta-D- arabinofuranosyl-5-azacytosine and dihydro-5-azacytidine, bleomycin, actinomycin D, mithramycin, mitomycin C, carmustine, lomustine, semustine, streptozotocin, hydroxyurea, cisplatin, mitotane, procarbazine, dacarbazine, taxol, vinblastine, vincristine, doxorubicin and dibromomannitol etc.
[0132] Additional agents that are applicable with conjugates and methods are known in the art and can be employed. For example, biologicals such as antibodies, cell growth factors, cell survival factors, cell differentiative factors, cytokines and chemokines can be administered. Non-limiting examples of monoclonal antibodies include rituximab (Rituxan®), trastuzumab (Herceptin), pertuzumab
(Omnitarg), bevacizumab (Avastin), cetuximab (Erbitux), alemtuzumab (Campath), panitumumab (Vectibix), ibritumomab tiuxetan (Zevalin), tositumomab (Bexxar) etc. which can be used in combination with, inter alia, a conjugate in accordance with the invention. Other targeted drugs that are applicable for use with the conjugates are imatinib (Gleevec), gefitinib (Iressa), bortzomib (Velcade), lapatinib (Tykerb), sunitinib (Sutent), sorafenib (Nevaxar), nilotinib (Tasigna) etc. Non-limiting examples of cell growth factors, cell survival factors, cell differentiative factors, cytokines and chemokines include IL-2, IL-l a, IL-Ι β, IL-3, IL-6, IL-7, granulocyte-macrophage-colony stimulating factor (GMCSF), IFN-γ, IL-12, TNF-a, TNFp, MIP-l , ΜΙΡ-Ι β, RANTES, SDF-1, MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, eotaxin-2, 1-309/TCA3, ATAC, HCC-1 , HCC-2, HCC-3, LARC/MIP-3a, PARC, TARC, CK , CKp6, CK 7, CKp8, CKp9, C pl 1 , CKpl2, CIO, IL-8, GROa, GRO , ENA- 78, GCP-2, PBP/CTAPlIIp-TG/NAP-2, Mig, PBSF/SDF-1 and lymphotactin.
[0133] Additional non-limiting examples include immune-enhancing treatments and therapies, which include cell based therapies. In particular, immune-enhancing treatments and therapies include administering lymphocytes, plasma cells, macrophages, dendritic cells, NK cells and B-cells.
[0134] Methods of treating a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia, methods of treating a subject in need of treatment due to having or at risk of having a metastatic or non- metastatic tumor, cancer, malignancy or neoplasia, and methods of increasing effectiveness or improving an anti-proliferative, anti-tumor, anti-cancer, anti-neoplasia or anti-malignancy, therapy are provided. In respective embodiments, a method includes administering to a subject with or at risk of a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia, an amount of a conjugate sufficient to treat the metastatic or non-metastatic tumor, cancer, malignancy or neoplasia;
administering to the subject an amount of a conjugate sufficient to treat the subject; and administering to a subject that is undergoing or has undergone metastatic or non-metastatic tumor, cancer, malignancy or neoplasia therapy, an amount of a conjugate sufficient to increase effectiveness of the antiproliferative, anti-tumor, anti-cancer, anti-neoplasia or anti-malignancy therapy.
[0135] Methods of the invention may be practiced prior to (i.e. prophylaxis), concurrently with or after evidence of the presence of undesirable or aberrant cell proliferation or a hyperproliferative disorder, disease or condition begins (e.g., one or more symptoms). Administering a conjugate prior to, concurrently with or immediately following development of a symptom of undesirable or aberrant cell proliferation or a hyperproliferative disorder may decrease the occurrence, frequency, severity, progression, or duration of one or more symptoms of the undesirable or aberrant cell proliferation or a hyperproliferative disorder, disease or condition in the subject. In addition, administering a conjugate prior to, concurrently with or immediately following development of one or more symptoms of the undesirable or aberrant cell proliferation or a hyperproliferative disorder, disease or condition may inhibit, decrease or prevent the spread or dissemination of hyperproliferating cells (e.g., metastasis) to other sites, regions, tissues or organs in a subject, or establishment of hyperproliferating cells (e.g., metastasis) at other sites, regions, tissues or organs in a subject.
[0136] Conjugates and the methods of the invention, such as treatment methods, can provide a detectable or measurable therapeutic benefit or improvement to a subject. A therapeutic benefit or improvement is any measurable or detectable, objective or subjective, transient, temporary, or longer- term benefit to the subject or improvement in the condition, disorder or disease, an adverse symptom, consequence or underlying cause, of any degree, in a tissue, organ, cell or cell population of the subject. Therapeutic benefits and improvements include, but are not limited to, reducing or decreasing occurrence, frequency, severity, progression, or duration of one or more symptoms or complications associated with a disorder, disease or condition, or an underlying cause or consequential effect of the disorder, disease or condition. Conjugates and methods of the invention therefore include providing a therapeutic benefit or improvement to a subject.
[0137] In a method of the invention in which a therapeutic benefit or improvement is a desired outcome, a conjugate of the invention can be administered in a sufficient or effective amount to a subject in need thereof. An "amount sufficient" or "amount effective" refers to an amount that provides, in single or multiple doses, alone or in combination, with one or more other compositions (therapeutic agents such as a chemotheraputic or immune stimulating drug), treatments, protocols, or therapeutic regimens agents, a detectable response of any duration of time (long or short term), a desired outcome in or a benefit to a subject of any measurable or detectable degree or for any duration of time (e.g., for hours, days, months, years, or cured). The doses or "sufficient amount" or "effective amount" for treatment (e.g., to provide a therapeutic benefit or improvement) typically are effective to ameliorate a disorder, disease or condition, or one, multiple or all adverse symptoms, consequences or complications of the disorder, disease or condition, to a measurable extent, although reducing or inhibiting a progression or worsening of the disorder, disease or condition or a symptom, is considered a satisfactory outcome.
[0138] The term "ameliorate" means a detectable objective or subjective improvement in a subject's condition. A detectable improvement includes a subjective or objective reduction in the occurrence, frequency, severity, progression, or duration of a symptom caused by or associated with a disorder, disease or condition, an improvement in an underlying cause or a consequence of the disorder, disease or condition, or a reversal of the disorder, disease or condition. [0139] Treatment can therefore result in inhibiting, reducing or preventing a disorder, disease or condition, or an associated symptom or consequence, or underlying cause; inhibiting, reducing or preventing a progression or worsening of a disorder, disease, condition, symptom or consequence, or underlying cause; or further deterioration or occurrence of one or more additional symptoms of the disorder, disease condition, or symptom. Thus, a successful treatment outcome leads to a "therapeutic effect," or "benefit" or inhibiting, reducing or preventing the occurrence, frequency, severity, progression, or duration of one or more symptoms or underlying causes or consequences of a condition, disorder, disease or symptom in the subject. Treatment methods affecting one or more underlying causes of the condition, disorder, disease or symptom are therefore considered to be beneficial.
Stabilizing or inhibiting progression or worsening of a disorder or condition is also a successful treatment outcome.
[0140] A therapeutic benefit or improvement therefore need not be complete ablation of any one, most or all symptoms, complications, consequences or underlying causes associated with the condition, disorder or disease. Thus, a satisfactory endpoint is achieved when there is an incremental
improvement in a subject's condition, or a partial reduction in the occurrence, frequency, severity, progression, or duration, or inhibition or reversal, of one or more associated adverse symptoms or complications or consequences or underlying causes, worsening or progression (e.g., stabilizing one or more symptoms or complications of the condition, disorder or disease), of one or more of the physiological, biochemical or cellular manifestations or characteristics of the disorder or disease, over a short or long duration of time (hours, days, weeks, months, etc.).
[0141] In particular embodiments, a method of treatment results in partial or complete destruction of a metastatic or non-metastatic tumor, cancer, malignant or neoplastic cell mass, volume, size or numbers of cells; results in stimulating, inducing or increasing metastatic or non-metastatic tumor, cancer, malignant or neoplastic cell necrosis, lysis or apoptosis; results in reducing metastatic or non- metastatic tumor, cancer, malignant or neoplastic volume, size, cell mass; results in inhibiting or preventing progression or an increase in metastatic or non-metastatic tumor, cancer, malignant or neoplastic volume, mass, size or cell numbers; results in inhibiting or decreasing the spread or dissemination of hyperproliferating cells (e.g., metastasis) to other (secondary) sites, regions, tissues or organs in a subject, or establishment of hyperproliferating cells (e.g., metastasis) at other (secondary) sites, regions, tissues or organs in a subject; or results in prolonging lifespan of the subject. In additional particular embodiments, a method of treatment results in reducing or decreasing severity, duration or frequency of an adverse symptom or complication associated with or caused by the metastatic or non-metastatic tumor, cancer, malignancy or neoplasia.
[0142] An amount sufficient or an amount effective can but need not be provided in a single administration and, can but need not be, administered alone or in combination with another composition (e.g., chemotherapeutic or immune enhancing or stimulating agent), treatment, protocol or therapeutic regimen. For example, the amount may be proportionally increased as indicated by the need of the subject, status of the disorder, disease or condition treated or the side effects of treatment. In addition, an amount sufficient or an amount effective need not be sufficient or effective if given in single or multiple doses without a second composition (e.g., chemotherapeutic or immune stimulating agent), treatment, protocol or therapeutic regimen, since additional doses, amounts or duration above and beyond such doses, or additional compositions (e.g., chemotherapeutic or immune stimulating agents), treatments, protocols or therapeutic regimens may be included in order to be considered effective or sufficient in a given subject. Amounts considered sufficient also include amounts that result in a reduction of the use of another treatment, therapeutic regimen or protocol.
[0143] An amount sufficient or an amount effective need not be effective in each and every subject treated, prophylactically or therapeutically, nor a majority of treated subjects in a given group or population. As is typical for treatment or therapeutic methods, some subjects will exhibit greater or less response to a given treatment, therapeutic regimen or protocol. An amount sufficient or an amount effective refers to sufficiency or effectiveness in a particular subject, not a group or the general population. Such amounts will depend in part upon the condition treated, such as the type or stage of undesirable or aberrant cell proliferation or hyperproliferative disorder (e.g., a metastatic or non- metastatic tumor, cancer, malignancy or neoplasia), the therapeutic effect desired, as well as the individual subject (e.g., the bioavailability within the subject, gender, age, etc.).
[0144] Particular non-limiting examples of therapeutic benefit or improvement for undesirable or aberrant cell proliferation, such as a hyperproliferative disorder (e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia) include a reduction in cell size, mass or volume, inhibiting an increase in cell size, mass or volume, a slowing or inhibition of worsening or progression, stimulating cell necrosis, lysis or apoptosis, reducing or inhibiting neoplastic or tumor malignancy or metastasis, reducing mortality, and prolonging lifespan of a subject. Thus, inhibiting or delaying an increase in cell size, mass, volume or metastasis (stabilization) can increase lifespan (reduce mortality) even if only for a few days, weeks or months, even though complete ablation of the metastatic or non-metastatic tumor, cancer, malignancy or neoplasia has not occurred. Adverse symptoms and complications associated with a hyperproliferative disorder (e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia) that can be reduced or decreased include, for example, pain, nausea, discomfort, lack of appetite, lethargy and weakness. A reduction in the occurrence, frequency, severity, progression, or duration of a symptom of undesirable or aberrant cell proliferation, such as a hyperproliferative disorder (e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia), such as an improvement in subjective feeling (e.g., increased energy, appetite, reduced nausea, improved mobility or psychological well being, etc.), are therefore all examples of therapeutic benefit or improvement.
[0145] For example, a sufficient or effective amount of a conjugate is considered as having a therapeutic effect if administration results in less chemotherapeutic drug, radiation or immunotherapy being required for treatment of undesirable or aberrant cell proliferation, such as a hyperproliferative disorder (e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia).
[0146] The term "subject'* refers to animals, typically mammalian animals, such as humans, non human primates (apes, gibbons, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), farm animals (horses, cows, goats, sheep, pigs) and experimental animal (mouse, rat, rabbit, guinea pig). Subjects include animal disease models, for example, animal models of undesirable or aberrant cell proliferation, such as a hyperproliferative disorder (e.g., a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia) for analysis of conjugates in vivo.
[0147] Subjects appropriate for treatment include those having or at risk of having a metastatic or non-metastatic tumor, cancer, malignant or neoplastic cell, those undergoing as well as those who are undergoing or have undergone anti-proliferative (e.g., metastatic or non-metastatic tumor, cancer, malignancy or neoplasia) therapy, including subjects where the tumor is in remission. "At risk" subjects typically have risk factors associated with undesirable or aberrant cell proliferation, development of hyperplasia (e.g., a tumor).
[0148] Particular examples of at risk or candidate subjects include those with cells that express Her2/neu to which a conjugate can bind, particularly where cells targeted for necrosis, lysis, killing or destruction express greater numbers or amounts of Her2/neu than non-target cells. Such cells can be selectively or preferentially targeted for necrosis, lysis or killing.
[0149] At risk subjects also include those that are candidates for and those that have undergone surgical resection, chemotherapy, immunotherapy, ionizing or chemical radiotherapy, local or regional thermal (hyperthermia) therapy, or vaccination. The invention is therefore applicable to treating a subject who is at risk of a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia or a complication associated with a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia, for example, due to metastatic or non-metastatic tumor, cancer, malignancy or neoplasia reappearance or regrowth following a period of stability or remission.
[0150] Risk factors include gender, lifestyle (diet, smoking), occupation (medical and clinical personnel, agricultural and livestock workers), environmental factors (carcinogen exposure), family history (autoimmune disorders, diabetes, etc.), genetic predisposition, etc. For example, subjects at risk for developing melanoma include excess sun exposure (ultraviolet radiation), fair skin, high numbers of naevi (dysplastic nevus), patient phenotype, family history, or a history of a previous melanoma.
Subjects at risk for developing cancer can therefore be identified by lifestyle, occupation,
environmental factors, family history, and genetic screens for tumor associated genes, gene deletions or gene mutations. Subjects at risk for developing breast cancer lack Brcal, for example. Subjects at risk for developing colon cancer have early age or high frequency polyp formation, or deleted or mutated tumor suppressor genes, such as adenomatous polyposis coli (APC), for example. [0151] Subjects also include those precluded from other treatments. For example, certain subjects may not be good candidates for surgical resection, chemotherapy, immunotherapy, ionizing or chemical radiotherapy, local or regional thermal (hyperthermia) therapy, or vaccination. Thus, candidate subjects for treatment in accordance with the invention include those that are not a candidate for surgical resection, chemotherapy, immunotherapy, ionizing or chemical radiotherapy, local or regional thermal (hyperthermia) therapy, or vaccination.
[0152] Conjugates may be formulated in a unit dose or unit dosage form. In a particular embodiment, a conjugate is in an amount effective to treat a subject having undesirable or aberrant cell proliferation or a hyperproliferative disorder. In an additional particular embodiment, a conjugate is in an amount effective to treat a subject having a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia. Exemplary unit doses range from about 25-250, 250-500, 500-1000, 1000-2500 or 2500- 5000, 5000-25,000, 5000-50,000 ng; and from about 25-250, 250-500, 500-1000, 1000-2500 or 2500- 5000, 5000-25,000, 5000-50,000
[0153] Compositions and methods of the invention may be contacted or provided in vitro, ex vivo or in vivo. Compositions can be administered to provide the intended effect as a single or multiple dosages, for example, in an effective or sufficient amount. Exemplary doses range from about 25-250, 250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000, 5000-50,000 pg kg; from about 50-500, 500-5000, 5000-25,000 or 25,000-50,000 ng/kg; and from about 25-250, 250-500, 500-1000, 1000- 2500 or 2500-5000, 5000-25,000, 5000-50,000 μg/kg, on consecutive days, or alternating days or intermittently. Single or multiple doses can be administered on consecutive days, alternating days or intermittently.
[0154] Compositions can be administered and methods may be practiced via systemic, regional or local administration, by any route. For example, a conjugate can be administered systemically, regionally or locally, intravenously, orally {e.g., ingestion or inhalation), intramuscularly,
intraperitoneally, intradermally, subcutaneously, intracavity, intracranially, transdermally (topical), parenterally, e.g. transmucosally or rectally. Compositions and methods of the invention including pharmaceutical formulations can be administered via a (micro)encapsulated delivery system or packaged into an implant for administration.
[0155] The invention further provides conjugates and methods wherein the conjugates are included in pharmaceutical compositions. A pharmaceutical composition refers to "pharmaceutically acceptable" and "physiologically acceptable" carriers, diluents or excipients. As used herein, the term "pharmaceutically acceptable" and "physiologically acceptable," when referring to carriers, diluents or excipients includes solvents (aqueous or non-aqueous), detergents, solutions, emulsions, dispersion media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration and with the other components of the formulation. Such formulations can be contained in a tablet (coated or uncoated), capsule (hard or soft), microbead, emulsion, powder, granule, crystal, suspension, syrup or elixir.
[0156] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration. Compositions for parenteral, intradermal, or subcutaneous administration can include a sterile diluent, such as water, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. The preparation may contain one or more preservatives to prevent microorganism growth (e.g., antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose).
[0157] Pharmaceutical compositions for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and polyetheylene glycol), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of a coating such as lecithin, or by the use of surfactants.
Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal. Including an agent that delays absorption, for example, aluminum monostearate and gelatin can prolonged absorption of injectable compositions.
[0158] Additional pharmaceutical formulations and delivery systems are known to the skilled artisan and are applicable in the methods of the invention (see, e.g., Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12th ed., Merck
Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa., (1993); and Poznansky, et al., Drug Delivery Systems, R. L. Juliano, ed., Oxford, N.Y. (1980), pp. 253-315).
[0159] The invention provides kits including conjugates of the invention, combination compositions and pharmaceutical formulations thereof, packaged into suitable packaging material. A kit optionally includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein. Exemplary instructions include instructions for reducing or inhibiting proliferation of a cell, reducing or inhibiting proliferation of undesirable or aberrant cells, such as a hyperproliferating cell, reducing or inhibiting proliferation of a metastatic or non-metastatic tumor, cancer, malignant or neoplastic cell, treating a subject having a hyperproliferative disorder, treating a subject having a metastatic or non-metastatic tumor, cancer, malignancy or neoplasia, or reducing fertility of an animal. [0160] A kit can contain a collection of such components, e.g., two or more conjugates alone, or in combination with another therapeutically useful composition (e.g., an anti-proliferative or immune- enhancing drug).
[0161] The term "packaging material" refers to a physical structure housing the components of the kit. The packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.).
[0162] Kits of the invention can include labels or inserts. Labels or inserts include "printed matter," e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component. Labels or inserts can additionally include a computer readable medium, such as a disk (e.g., floppy diskette, hard disk, ZIP disk), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media or memory type cards.
[0163] Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location and date.
[0164] Labels or inserts can include information on a condition, disorder, disease or symptom for which a kit component may be used. Labels or inserts can include instructions for the clinician or for a subject for using one or more of the kit components in a method, treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency or duration, and instructions for practicing any of the methods, treatment protocols or therapeutic regimes set forth herein. Exemplary instructions include, instructions for treating an undesirable or aberrant cell proliferation, hyperproliferating cells and disorders (e.g., metastatic or non-metastatic tumor, cancer, malignancy or neoplasia). Kits of the invention therefore can additionally include labels or instructions for practicing any of the methods of the invention described herein including treatment methods.
[0165] Labels or inserts can include information on any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition. Adverse side effects could also occur when the subject has, will be or is currently taking one or more other medications that may be incompatible with the composition, or the subject has, will be or is currently undergoing another treatment protocol or therapeutic regimen which would be incompatible with the composition and, therefore, instructions could include information regarding such incompatibilities. [0166] Invention kits can additionally include other components. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package. Invention kits can be designed for cold storage. Invention kits can further be designed to contain host cells expressing conjugates of the invention, or that contain nucleic acids encoding conjugates. The cells in the kit can be maintained under appropriate storage conditions until the cells are ready to be used. For example, a kit including one or more cells can contain appropriate cell storage medium so that the cells can be thawed and grown.
[0167] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.
[0168] All applications, publications, patents and other references, GenBank citations and ATCC citations cited herein are incorporated by reference in their entirety. In case of conflict, the
specification, including definitions, will control.
[0169] As used herein, the singular forms "a", ''and," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a conjugate" or a "ligand that binds to Her2/neu," or a "lytic domain" includes a plurality of such conjugates, ligands, or lytic domains, and so forth.
[0170] As used herein, numerical values are often presented in a range format throughout this document. The use of a range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention unless the context clearly indicates otherwise. Accordingly, the use of a range expressly includes all possible subranges, all individual numerical values within that range, and all numerical values or numerical ranges including integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, for example, reference to a range of 90-100% includes 91- 99%, 92-98%, 93-95%, 91-98%, 91-97%, 91 -96%, 91-95%, 91-94%, 91-93%, and so forth. Reference to a range of 90-100% also includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1 %, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth.
[0171] In addition, reference to a range of 1-5,000 fold includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1.1 , 1.2, 1.3, 1.4, 1.5, fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5, fold, etc., and any numerical range within such a ranges, such as 1-2, 5-10, 10-50, 50-100, 100-500, 100-1000, 500-1000, 1000-2000, 1000-5000, etc. In a further example, reference to a range of KD 10-5 M to about KD 10"13 M includes any numerical value or range within or encompassing such values.
[0172] As also used herein a series of ranges are disclosed throughout this document. The use of a series of ranges include combinations of the upper and lower ranges to provide another range. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, for example, reference to a series of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50,
50-75, 75-100, 100-150, and 150-171 , includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-
150, 5-171 , and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, 10-171, and 20-40, 20-50, 20-75, 20-100,
20-150, 20-171, and so forth.
[0173] The invention is generally disclosed herein using affirmative language to describe the numerous embodiments. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include aspects that are not expressly included in the invention are nevertheless disclosed herein.
[0174] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate but not limit the scope of invention described in the claims.
Examples
Example 1
[0175] To determine, in in vitro studies, cytotoxicity of recombinantly produced antibody (as a antibody) scFv-CH3 to Her-2 receptor conjugated to the lytic peptide, Phor-18 (KFAKFAK KFAKFAK KFAK) or (KLAKLAK)2KLAK. Various linkers (GS and NRVRRS) and 1 or 2 molecules of lytic peptides per antibody molecule were studied.
[0176] Peptides studied were: Phorl 8- scFv-CH3 -Phor-18 (2 molecules of Phor -18 joined at N- and C-terminal ends of the antibody, scFv- CH3 -GS-Phor-18 (one molecule of Phor-18 joined to the antibody at the C-terminus by GS linker, scFv- CH3 -GS-(KLAKLAK)2KLAK (one molecule of (KLAKLAK)2KLAK linked to the antibody at the C-terminus by GS linker, scFv- CH3 - -NRVRRS- Phor-18 (one molecule of Phor-18 to the antibody at the C-terminus by NRVRRS linker, and scFv- CH3 -NRVRRS-(KLAKLAK)2KLAK (one molecule of (KLAKLAK)2KLAK to the antibody at the C- terminus by NRVRRS linker). Cytotoxicity was compared to a naked antibody (antibody without a lytic peptide) in Her-2 receptor positive cells (SKBR-3 and SKOV-3, human breast and ovarian cancer cells, respectively) and Her-2 receptor negative breast cancer cells (MDA-MB-231).
Example 2
[0177] This example describes various materials and methods used in the studies described herein.
[0178] Materials: Recombinant DNA technique was used to synthesize anti-Her2 antibody as a recombinant antibody in Escherichia coli. The scFv- CH3 antibody (Olafsen T. et al Protein
Engineering, Design & Selection 17, 315-323, 2004) was conjugated via a peptide linker or without a linker as described in Table 1 to either Phor-18 or an amphipathic, alpha-helical lytic peptide,
(KLAKLAK)2KLAK and analyzed for cytotoxicity in vitro. The plasmid was acquired through gene codon optimization. The gene was synthesized with a N-His tag sequence and the plasmid was subcloned into an E. coli bacteria expression vector pUC57. After expression optimization and evaluation the His-tag product was selected and 1 L of the bacteria expression product was purified in a one-step affinity purification. The sequences of the plasmid gene insertion for each construct is described in Table 1.
Table 1: Nucleotide sequence of the plasmid insertion for the production of each recombinant Her2/neu antibody and antibody conjugate.
Figure imgf000043_0001
1 CATATGCATC ACCACCACCA CCACGACGAC GACGACAAAG ATATTCAAAT GACCCAGTCC
61 CCGAGCAGCC TGAGTGCCTC CGTTGGCGAC CGCGTGACCA TTACGTGCCG TGCGAGCCAG
121 GATGTCAACA CCGCGGTGGC CTGGTATCAG CAAAAACCGG GCAAAGCGCC GAAACTGCTG 181 ATCTATTCAG CCTCGTTTCT GTACAGCGGT GTTCCGTCTC GTTTCAGCGG CTCTCGCAGT 241 GGTACCGATT TTACCCTGAC GATTAGCTCT CTGCAGCCGG AAGACTTTGC GACGTATTAC 301 TGCCAGCAAC ATTACACCAC GCCGCCGACC TTCGGCCAGG GTACGAAAGT GGAAATCAAA 361 GGTTCCACCT CAGGCGGTGG CAGTGGTGGC GGTTCCGGCG GTGGCGGTAG TTCCGAAGTT 421 CAGCTGGTCG AAAGTGGCGG TGGCCTGGTT CAACCGGGTG GCTCACTGCG TCTGTCGTGT 481 GCAGCAAGCG GTTTCAACAT CAAAGATACC TACATCCACT GGGTTCGTCA GGCGCCGGGC 541 AAAGGTCTGG AATGGGTCGC CCGCATTTAC CCGACCAATG GCTATACGCG TTACGCAGAT 601 AGCGTGAAAG GTCGCTTTAC CATCTCTGCG GACACCAGTA AAAACACGGC CTATCTGCAG 661 ATGAATAGCC TGCGTGCGGA AGATACGGCC GTTTATTACT GCTCTCGCTG GGGTGGCGAT 721 GGCTTCTATG CTATGGACTA CTGGGGCCAG GGTACCCTGG TGACGGTTTC ATCGGGTCAG 781 CCGCGTGAAC CGCAAGTGTA TACCCTGCCG CCGTCACGCG ATGAACTGAC GAAAAACCAG 841 GTGTCGCTGA CGTGTCTGGT TAAAGGCTTT TACCCGAGCG ACATCGCGGT TGAATGGGAA 901 TCTAATGGTC AACCGGAAAA CAATTATAAA ACCACGCCGC CGGTCCTGGA TAGTGACGGC 961 TCCTTTTTCC TGTACAGTAA ACTGACCGTG GATAAATCCC GTTGGCAGCA GGGTAACGTC 1021 TTCTCGTGTA GCGTGATGCA TGAAGCCCTG CATAATCACT ATACCCAGAA ATCTCTGAGT 1081 CTGTCCCCGG GCAAAGGTTC AACGTCGGGT GGCGGTTCCG GCGGTGGCTC AGGTGGCGGT 1141 GGCAGCTCTG GCCAACCGCG CGAACCGCAG GTTTACACCC TGCCGCCGAG CCGTGACGAA 1201 CTGACCAAAA ACCAAGTCAG CCTGACGTGC CTGGTGAAAG GCTTTTACCC GAGTGACATT 1261 GCAGTTGAAT GGGAATCCAA TGGTCAGCCG GAAAATAACT ACAAAACGAC GCCGCCGGTT 1321 CTGGATTCAG ACGGCTCGTT TTTCCTGTAC TCAAAACTGA CCGTCGATAA ATCGCGCTGG 1381 CAACAGGGTA ACGTTTTCAG CTGCTCTGTC ATGCACGAAG CCCTGCACAA CCATTATACC 1441 CAGAAAAGTC TGTCCCTGTC ACCGGGCAAA GAAGTGCAGC TGGTTGAATC TGGTGGCGGT 1501 CTGGTGCAAC CGGGCGGTTC GCTGCGTCTG AGCTGTGCAG CTTCTGGCTT TAATATTAAA 1561 GACACGTACA TCCACTGGGT GCGTCAGGCA CCGGGTAAAG GCCTGGAATG GGTTGCTCGT 1621 ATCTATCCGA CGAACGGTTA TACGCGTTAC GCCGATAGCG TCAAAGGCCG TTTTACCATC 1681 AGTGCAGACA CCTCCAAAAA CACGGCTTAT CTGCAGATGA ATAGTCTGCG TGCAGAAGAT 1741 ACCGCTGTTT ATTACTGCAG CCGCTGGGGC GGTGATGGCT TCTATGCAAT GGATTATTGG 1801 GGTCAAGGTA CCCTGGTCAC CGTGAGTTCC GGTTCGACCA GCGGCGGTGG CTCAGGTGGC 1861 GGTTCGGGCG GTGGCGGTTC ATCGGACATT CAGATGACGC AAAGCCCGAG CTCTCTGTCT 1921 GCGAGTGTTG GCGATCGTGT CACCATCACG TGTCGCGCCT CTCAGGACGT GAATACCGCA 1981 GTTGCTTGGT ACCAACAAAA ACCGGGCAAA GCACCGAAAC TGCTGATTTA CTCCGCTTCA 2041 TTCCTGTACA GCGGTGTGCC GTCTCGTTTT TCGGGCAGCC GCTCTGGTAC CGATTTCACC 2101 CTGACGATTA GTTCCCTGCA ACCGGAAGAT TTCGCCACCT ACTACTGCCA GCAACACTAT 2161 ACGACCCCGC CGACGTTTGG TCAGGGCACG AAAGTGGAAA TTAAATAATG AAAGCTT
2. SCFV- CH3 -GS -Phor-18:
1 CATATGCATC ACCACCACCA CCACGACGAC GACGACAAAG ATATTCAAAT GACCCAGTCC
61 CCGAGCAGCC TGAGTGCCTC CGTTGGCGAC CGCGTGACCA TTACGTGCCG TGCGAGCCAG
121 GATGTCAACA CCGCGGTGGC CTGGTATCAG CAAAAACCGG GCAAAGCGCC GAAACTGCTG
181 ATCTATTCAG CCTCGTTTCT GTACAGCGGT GTTCCGTCTC GTTTCAGCGG CTCTCGCAGT
241 GGTACCGATT TTACCCTGAC GATTAGCTCT CTGCAGCCGG AAGACTTTGC GACGTATTAC
301 TGCCAGCAAC ATTACACCAC GCCGCCGACC TTCGGCCAGG GTACGAAAGT GGAAATCAAA
361 GGTTCCACCT CAGGCGGTGG CAGTGGTGGC GGTTCCGGCG GTGGCGGTAG TTCCGAAGTT 421 CAGCTGGTCG AAAGTGGCGG TGGCCTGGTT CAACCGGGTG GCTCACTGCG TCTGTCGTGT
481 GCAGCAAGCG GTTTCAACAT CAAAGATACC TACATCCACT GGGTTCGTCA GGCGCCGGGC
541 AAAGGTCTGG AATGGGTCGC CCGCATTTAC CCGACCAATG GCTATACGCG TTACGCAGAT
601 AGCGTGAAAG GTCGCTTTAC CATCTCTGCG GACACCAGTA AAAACACGGC CTATCTGCAG
661 ATGAATAGCC TGCGTGCGGA AGATACGGCC GTTTATTACT GCTCTCGCTG GGGTGGCGAT
721 GGCTTCTATG CTATGGACTA CTGGGGCCAG GGTACCCTGG TGACGGTTTC ATCGGGTCAG
781 CCGCGTGAAC CGCAAGTGTA TACCCTGCCG CCGTCACGCG ATGAACTGAC GAAAAACCAG
841 GTGTCGCTGA CGTGTCTGGT TAAAGGCTTT TACCCGAGCG ACATCGCGGT TGAATGGGAA
901 TCTAATGGTC AACCGGAAAA CAATTATAAA ACCACGCCGC CGGTCCTGGA TAGTGACGGC
961 TCCTTTTTCC TGTACAGTAA ACTGACCGTG GATAAATCCC GTTGGCAGCA GGGTAACGTC
1021 TTCTCGTGTA GCGTGATGCA TGAAGCCCTG CATAATCACT ATACCCAGAA ATCTCTGAGT
1081 CTGTCCCCGG GCAAAGGTTC AACGTCGGGT GGCGGTTCCG GCGGTGGCTC AGGTGGCGGT
1141 GGCAGCTCTG GCCAACCGCG CGAACCGCAG GTTTACACCC TGCCGCCGAG CCGTGACGAA
1201 CTGACCAAAA ACCAAGTCAG CCTGACGTGC CTGGTGAAAG GCTTTTACCC GAGTGACATT
1261 GCAGTTGAAT GGGAATCCAA TGGTCAGCCG GAAAATAACT ACAAAACGAC GCCGCCGGTT
1321 CTGGATTCAG ACGGCTCGTT TTTCCTGTAC TCAAAACTGA CCGTCGATAA ATCGCGCTGG
1381 CAACAGGGTA ACGTTTTCAG CTGCTCTGTC ATGCACGAAG CCCTGCACAA CCATTATACC
14 1 CAGAAAAGTC TGTCCCTGTC ACCGGGCAAA GAAGTGCAGC TGGTTGAATC TGGTGGCGGT
1501 CTGGTGCAAC CGGGCGGTTC GCTGCGTCTG AGCTGTGCAG CTTCTGGCTT TAATATTAAA
1561 GACACGTAC TCCACTGGGT GCGTCAGGCA CCGGGTAAAG GCCTGGAATG GGTTGCTCGT
1621 ATCTATCCGA CGAACGGTTA TACGCGTTAC GCCGATAGCG TCAAAGGCCG TTTTACCATC
1681 AGTGCAGACA CCTCCAAAAA CACGGCTTAT CTGCAGATGA ATAGTCTGCG TGCAGAAGAT
1741 ACCGCTGTTT ATTACTGCAG CCGCTGGGGC GGTGATGGCT TCTATGCAAT GGATTATTGG
1801 GGTCAAGGTA CCCTGGTCAC CGTGAGTTCC GGTTCGACCA GCGGCGGTGG CTCAGGTGGC
1861 GGTTCGGGCG GTGGCGGTTC ATCGGACATT CAGATGACGC AAAGCCCGAG CTCTCTGTCT
1921 GCGAGTGTTG GCGATCGTGT CACCATCACG TGTCGCGCCT CTCAGGACGT GAATACCGCA
1981 GTTGCTTGGT ACCAACAAAA ACCGGGCAAA GCACCGAAAC TGCTGATTTA CTCCGCTTCA
2041 TTCCTGTACA GCGGTGTGCC GTCTCGTTTT TCGGGCAGCC GCTCTGGTAC CGATTTCACC
2101 CTGACGATTA GTTCCCTGCA ACCGGAAGAT TTCGCCACCT ACTACTGCCA GCAACACTAT
2161 ACGACCCCGC CGACGTTTGG TCAGGGCACG AAAGTGGAAA TTAAAGGCAG CAAATTTGCG
2221 AAATTCGCCA AAAAATTCGC AAAATTCGCG AAAAAATTCG CGAAATAATG AAAGCTT
3. scFv- CH3 -GS-fKLAKLAI bKLAK:
1 CATATGGAAA ATCTGTATTT CCAAGGTGAT ATTCAAATGA CCCAGTCCCC GAGCAGCCTG
61 AGTGCCTCCG TTGGCGACCG CGTGACCATT ACGTGCCGTG CGAGCCAGGA TGTCAACACC
121 GCGGTGGCCT GGTATCAGCA AAAACCGGGC AAAGCGCCGA AACTGCTGAT CTATTCAGCC
181 TCGTTTCTGT ACAGCGGTGT TCCGTCTCGT TTCAGCGGCT CTCGCAGTGG TACCGATTTT
241 ACCCTGACGA TTAGCTCTCT GCAGCCGGAA GACTTTGCGA CGTATTACTG CCAGCAACAT
301 TACACCACGC CGCCGACCTT CGGCCAGGGT ACGAAAGTGG AAATCAAAGG TTCCACCTCA
361 GGCGGTGGCA GTGGTGGCGG TTCCGGCGGT GGCGGTAGTT CCGAAGTTCA GCTGGTCGAA
421 AGTGGCGGTG GCCTGGTTCA ACCGGGTGGC TCACTGCGTC TGTCGTGTGC AGCAAGCGGT
481 TTCAACATCA AAGATACCTA CATCCACTGG GTTCGTCAGG CGCCGGGCAA AGGTCTGGAA
541 TGGGTCGCCC GCATTTACCC GACCAATGGC TATACGCGTT ACGCAGATAG CGTGAAAGGT
601 CGCTTTACCA TCTCTGCGGA CACCAGTAAA AACACGGCCT ATCTGCAGAT GAATAGCCTG
661 CGTGCGGAAG ATACGGCCGT TTATTACTGC TCTCGCTGGG GTGGCGATGG CTTCTATGCT
721 ATGGACTACT GGGGCCAGGG TACCCTGGTG ACGGTTTCAT CGGGTCAGCC GCGTGAACCG
781 CAAGTGTATA CCCTGCCGCC GTCACGCGAT GAACTGACGA AAAACCAGGT GTCGCTGACG
841 TGTCTGGTTA AAGGCTTTTA CCCGAGCGAC ATCGCGGTTG AATGGGAATC TAATGGTCAA
901 CCGGAAAACA ATTATAAAAC CACGCCGCCG GTCCTGGATA GTGACGGCTC CTTTTTCCTG
961 TACAGTAAAC TGACCGTGGA TAAATCCCGT TGGCAGCAGG GTAACGTCTT CTCGTGTAGC
1021 GTGATGCATG AAGCCCTGCA TAATCACTAT ACCCAGAAAT CTCTGAGTCT GTCCCCGGGC
1081 AAAGGTTCAA CGTCGGGTGG CGGTTCCGGC GGTGGCTCAG GTGGCGGTGG CAGCTCTGGC
1141 CAACCGCGCG AACCGCAGGT TTACACCCTG CCGCCGAGCC GTGACGAACT GACCAAAAAC
1201 CAAGTCAGCC TGACGTGCCT GGTGAAAGGC TTTTACCCGA GTGACATTGC AGTTGAATGG
1261 GAATCCAATG GTCAGCCGGA AAATAACTAC AAAACGACGC CGCCGGTTCT GGATTCAGAC
1321 GGCTCGTTTT TCCTGTACTC AAAACTGACC GTCGATAAAT CGCGCTGGCA ACAGGGTAAC
1381 GTTTTCAGCT GCTCTGTCAT GCACGAAGCC CTGCACAACC ATTATACCCA GAAAAGTCTG
1441 TCCCTGTCAC CGGGCAAAGA AGTGCAGCTG GTTGAATCTG GTGGCGGTCT GGTGCAACCG
1501 GGCGGTTCGC TGCGTCTGAG CTGTGCAGCT TCTGGCTTTA ATATTAAAGA CACGTACATC
1561 CACTGGGTGC GTCAGGCACC GGGTAAAGGC CTGGAATGGG TTGCTCGTAT CTATCCGACG
1621 AACGGTTATA CGCGTTACGC CGATAGCGTC AAAGGCCGTT TTACCATCAG TGCAGACACC
1681 TCCAAAAACA CGGCTTATCT GCAGATGAAT AGTCTGCGTG CAGAAGATAC CGCTGTTTAT
1741 TACTGCAGCC GCTGGGGCGG TGATGGCTTC TATGCAATGG ATTATTGGGG TCAAGGTACC 1801 CTGGTCACCG TGAGTTCCGG TTCGACCAGC GGCGGTGGCT CAGGTGGCGG TTCGGGCGGT
1861 GGCGGTTCAT CGGACATTCA GATGACGCAA AGCCCGAGCT CTCTGTCTGC GAGTGTTGGC
1921 GATCGTGTCA CCATCACGTG TCGCGCCTCT CAGGACGTGA ATACCGCAGT TGCTTGGTAC
1981 CAACAAAAAC CGGGCAAAGC ACCGAAACTG CTGATTTACT CCGCTTCATT CCTGTACAGC
2041 GGTGTGCCGT CTCGTTTTTC GGGCAGCCGC TCTGGTACCG ATTTCACCCT GACGATTAGT
2101 TCCCTGCAAC CGGAAGATTT CGCCACCTAC TACTGCCAGC AACACTATAC GACCCCGCCG
2161 ACGTTTGGTC AGGGCACGAA AGTGGAAATT AAAGGCAGCA AACTGGCGAA ACTGGCCAAA
2221 AAACTGGCAA AACTGGCGAA AAAACTGGCG AAATAATGAA AGCTT
4. scFv- CH3 -NRVRRS-Phor-18:
1 CATATGGAAA ATCTGTATTT CCAAGGTGAT ATTCAAATGA CCCAGTCCCC GAGCAGCCTG
61 AGTGCCTCCG TTGGCGACCG CGTGACCATT ACGTGCCGTG CGAGCCAGGA TGTCAACACC
121 GCGGTGGCCT GGTATCAGCA AAAACCGGGC AAAGCGCCGA AACTGCTGAT CTATTCAGCC
181 TCGTTTCTGT ACAGCGGTGT TCCGTCTCGT TTCAGCGGCT CTCGCAGTGG TACCGATTTT
241 ACCCTGACGA TTAGCTCTCT GCAGCCGGAA GACTTTGCGA CGTATTACTG CCAGCAACAT
301 TACACCACGC CGCCGACCTT CGGCCAGGGT ACGAAAGTGG AAATCAAAGG TTCCACCTCA
361 GGCGGTGGCA GTGGTGGCGG TTCCGGCGGT GGCGGTAGTT CCGAAGTTCA GCTGGTCGAA
421 AGTGGCGGTG GCCTGGTTCA ACCGGGTGGC TCACTGCGTC TGTCGTGTGC AGCAAGCGGT
481 TTCAACATCA AAGATACCTA CATCCACTGG GTTCGTCAGG CGCCGGGCAA AGGTCTGGAA
541 TGGGTCGCCC GCATTTACCC GACCAATGGC TATACGCGTT ACGCAGATAG CGTGAAAGGT
601 CGCTTTACCA TCTCTGCGGA CACCAGTAAA AACACGGCCT ATCTGCAGAT GAATAGCCTG
661 CGTGCGGAAG ATACGGCCGT TTATTACTGC TCTCGCTGGG GTGGCGATGG CTTCTATGCT
721 ATGGACTACT GGGGCCAGGG TACCCTGGTG ACGGTTTCAT CGGGTCAGCC GCGTGAACCG
781 CAAGTGTATA CCCTGCCGCC GTCACGCGAT GAACTGACGA AAAACCAGGT GTCGCTGACG
841 TGTCTGGTTA AAGGCTTTTA CCCGAGCGAC ATCGCGGTTG AATGGGAATC TAATGGTCAA
901 CCGGAAAACA ATTATAAAAC CACGCCGCCG GTCCTGGATA GTGACGGCTC CTTTTTCCTG
961 TACAGTAAAC TGACCGTGGA TAAATCCCGT TGGCAGCAGG GTAACGTCTT CTCGTGTAGC
1021 GTGATGCATG AAGCCCTGCA TAATCACTAT ACCCAGAAAT CTCTGAGTCT GTCCCCGGGC
1081 AAAGGTTCAA CGTCGGGTGG CGGTTCCGGC GGTGGCTCAG GTGGCGGTGG CAGCTCTGGC
1141 CAACCGCGCG AACCGCAGGT TTACACCCTG CCGCCGAGCC GTGACGAACT GACCAAAAAC
1201 CAAGTCAGCC TGACGTGCCT GGTGAAAGGC TTTTACCCGA GTGACATTGC AGTTGAATGG
1261 GAATCCAATG GTCAGCCGGA AAATAACTAC AAAACGACGC CGCCGGTTCT GGATTCAGAC
1321 GGCTCGTTTT TCCTGTACTC AAAACTGACC GTCGATAAAT CGCGCTGGCA ACAGGGTAAC
1381 GTTTTCAGCT GCTCTGTCAT GCACGAAGCC CTGCACAACC ATTATACCCA GAAAAGTCTG
1441 TCCCTGTCAC CGGGCAAAGA AGTGCAGCTG GTTGAATCTG GTGGCGGTCT GGTGCAACCG
1501 GGCGGTTCGC TGCGTCTGAG CTGTGCAGCT TCTGGCTTTA ATATTAAAGA CACGTACATC
1561 CACTGGGTGC GTCAGGCACC GGGTAAAGGC CTGGAATGGG TTGCTCGTAT CTATCCGACG
1621 AACGGTTATA CGCGTTACGC CGATAGCGTC AAAGGCCGTT TTACCATCAG TGCAGACACC
1681 TCCAAAAACA CGGCTTATCT GCAGATGAAT AGTCTGCGTG CAGAAGATAC CGCTGTTTAT
1741 TACTGCAGCC GCTGGGGCGG TGATGGCTTC TATGCAATGG ATTATTGGGG TCAAGGTACC
1801 CTGGTCACCG TGAGTTCCGG TTCGACCAGC GGCGGTGGCT CAGGTGGCGG TTCGGGCGGT
1861 GGCGGTTCAT CGGACATTCA GATGACGCAA AGCCCGAGCT CTCTGTCTGC GAGTGTTGGC
1921 GATCGTGTCA CCATCACGTG TCGCGCCTCT CAGGACGTGA ATACCGCAGT TGCTTGGTAC
1981 CAACAAAAAC CGGGCAAAGC ACCGAAACTG CTGATTTACT CCGCTTCATT CCTGTACAGC
2041 GGTGTGCCGT CTCGTTTTTC GGGCAGCCGC TCTGGTACCG ATTTCACCCT GACGATTAGT
2101 TCCCTGCAAC CGGAAGATTT CGCCACCTAC TACTGCCAGC AACACTATAC GACCCCGCCG
2161 ACGTTTGGTC AGGGCACGAA AGTGGAAATT AAAAACCGTG TGCGTCGCAG CAAATTTGCG
2221 AAATTCGCCA AAAAATTTGC AAAATTCGCT AAAAAATTTG CGAAATAATG AAAGCTT
5. scFv- CH3 -N R VR R S-( K L A K LAK h L A K :
1 CATATGCATC ACCACCACCA CCACGACGAC GACGACAAAG ATATTCAAAT GACCCAGTCC
61 CCGAGCAGCC TGAGTGCCTC CGTTGGCGAC CGCGTGACCA TTACGTGCCG TGCGAGCCAG
121 GATGTCAACA CCGCGGTGGC CTGGTATCAG CAAAAACCGG GCAAAGCGCC GAAACTGCTG
181 ATCTATTCAG CCTCGTTTCT GTACAGCGGT GTTCCGTCTC GTTTCAGCGG CTCTCGCAGT
241 GGTACCGATT TTACCCTGAC GATTAGCTCT CTGCAGCCGG AAGACTTTGC GACGTATTAC
301 TGCCAGCAAC ATTACACCAC GCCGCCGACC TTCGGCCAGG GTACGAAAGT GGAAATCAAA 361 GGTTCCACCT CAGGCGGTGG CAGTGGTGGC GGTTCCGGCG GTGGCGGTAG TTCCGAAGTT 421 CAGCTGGTCG AAAGTGGCGG TGGCCTGGTT CAACCGGGTG GCTCACTGCG TCTGTCGTGT 481 GCAGCAAGCG GTTTCAACAT CAAAGATACC TACATCCACT GGGTTCGTCA GGCGCCGGGC 541 AAAGGTCTGG AATGGGTCGC CCGCATTTAC CCGACCAATG GCTATACGCG TTACGCAGAT 601 AGCGTGAAAG GTCGCTTTAC CATCTCTGCG GACACCAGTA AAAACACGGC CTATCTGCAG 661 ATGAATAGCC TGCGTGCGGA AGATACGGCC GTTTATTACT GCTCTCGCTG GGGTGGCGAT 721 GGCTTCTATG CTATGGACTA CTGGGGCCAG GGTACCCTGG TGACGGTTTC ATCGGGTCAG 781 CCGCGTGAAC CGCAAGTGTA TACCCTGCCG CCGTCACGCG ATGAACTGAC GAAAAACCAG
841 GTGTCGCTGA CGTGTCTGGT TAAAGGCTTT TACCCGAGCG ACATCGCGGT TGAATGGGAA
901 TCTAATGGTC AACCGGAAAA CAATTATAAA ACCACGCCGC CGGTCCTGGA TAGTGACGGC
961 TCCTTTTTCC TGTACAGTAA ACTGACCGTG GATAAATCCC GTTGGCAGCA GGGTAACGTC
1021 TTCTCGTGTA GCGTGATGCA TGAAGCCCTG CATAATCACT ATACCCAGAA ATCTCTGAGT
1081 CTGTCCCCGG GCAAAGGTTC AACGTCGGGT GGCGGTTCCG GCGGTGGCTC AGGTGGCGGT
1141 GGCAGCTCTG GCCAACCGCG CGAACCGCAG GTTTACACCC TGCCGCCGAG CCGTGACGAA
1201 CTGACCAAAA ACCAAGTCAG CCTGACGTGC CTGGTGAAAG GCTTTTACCC GAGTGACATT
1261 GCAGTTGAAT GGGAATC CAA TGGTCAGCCG GAAAATAACT ACAAAACGAC GCCGCCGGTT
1321 CTGGATTCAG ACGGCTCGTT TTTCCTGTAC TCAAAACTGA CCGTCGATAA ATCGCGCTGG
1381 CAACAGGGTA ACGTTTTCAG CTGCTCTGTC ATGCACGAAG CCCTGCACAA CCATTATACC
1441 CAGAAAAGTC TGTCCCTGTC ACCGGGCAAA GAAGTGCAGC TGGTTGAATC TGGTGGCGGT
1501 CTGGTGCAAC CGGGCGGTTC GCTGCGTCTG AGCTGTGCAG CTTCTGGCTT TAATATTAAA
1561 GACACGTACA TCCACTGGGT GCGTCAGGCA CCGGGTAAAG GCCTGGAATG GGTTGCTCGT
1621 ATCTATCCGA CGAACGGTTA TACGCGTTAC GCCGATAGCG TCAAAGGCCG TTTTACCATC
1681 AGTGCAGACA CCTCCAAAAA CACGGCTTAT CTGCAGATGA ATAGTCTGCG TGCAGAAGAT
1741 ACCGCTGTTT ATTACTGCAG CCGCTGGGGC GGTGATGGCT TCTATGCAAT GGATTATTGG
1801 GGTCAAGGTA CCCTGGTCAC CGTGAGTTCC GGTTCGACCA GCGGCGGTGG CTCAGGTGGC
1861 GGTTCGGGCG GTGGCGGTTC ATCGGACATT CAGATGACGC AAAGCCCGAG CTCTCTGTCT
1921 GCGAGTGTTG GCGATCGTGT CACCATCACG TGTCGCGCCT CTCAGGACGT GAATACCGCA
1981 GTTGCTTGGT ACCAACAAAA ACCGGGCAAA GCACCGAAAC TGCTGATTTA CTCCGCTTCA
2041 TTCCTGTACA GCGGTGTGCC GTCTCGTTTT TCGGGCAGCC GCTCTGGTAC CGATTTCACC
2101 CTGACGATTA GTTCCCTGCA ACCGGAAGAT TTCGCCACCT ACTACTGCCA GCAACACTAT
2161 ACGACCCCGC CGACGTTTGG TCAGGGCACG AAAGTGGAAA TTAAAAACCG TGTGCGTCGC
2221 AGCAAACTGG CGAAACTGGC CAAAAAACTG GCAAAACTGG CTAAAAAACT GGCGAAATAA
2281 TGAAAGCTT
6. Phorl 8- scFv- CH3 - Phor-18:
1 CATATGGAAA ATCTGTATTT CCAAGGTAAA TTTGCGAAAT TCGCCAAAAA ATTCGCAAAA
61 TTCGCGAAAA AATTCGCGAA |GATATTCAA ATGACCCAGT CCCCGAGCAG CCTGAGTGCC
121 TCCGTTGGCG ACCGCGTGAC CATTACGTGC CGTGCGAGCC AGGATGTCAA CACCGCGGTG
181 GCCTGGTATC AGCAAAAACC GGGCAAAGCG CCGAAACTGC TGATCTATTC AGCCTCGTTT
241 CTGTACAGCG GTGTTCCGTC TCGTTTCAGC GGCTCTCGCA GTGGTACCGA TTTTACCCTG
3 01 ACGATTAGCT CTCTGCAGCC GGAAGACTTT GCGACGTATT ACTGCCAGCA ACATTACACC
361 ACGCCGCCGA CCTTCGGCCA GGGTACGAAA GTGGAAATCA AAGGTTCCAC CTCAGGCGGT
421 GGCAGTGGTG GCGGTTCCGG CGGTGGCGGT AGTTCCGAAG TTCAGCTGGT CGAAAGTGGC
481 GGTGGCCTGG TTCAACCGGG TGGCTCACTG CGTCTGTCGT GTGCAGCAAG CGGTTTCAAC
541 ATCAAAGATA CCTACATCCA CTGGGTTCGT CAGGCGCCGG GCAAAGGTCT GGAATGGGTC
601 GCCCGCATTT ACCCGACCAA TGGCTATACG CGTTACGCAG ATAGCGTGAA AGGTCGCTTT
661 ACCATCTCTG CGGACACCAG TAAAAACACG GCCTATCTGC AGATGAATAG CCTGCGTGCG
721 GAAGATACGG CCGTTTATTA CTGCTCTCGC TGGGGTGGCG ATGGCTTCTA TGCTATGGAC
781 TACTGGGGCC AGGGTACCCT GGTGACGGTT TCATCGGGTC AGCCGCGTGA ACCGCAAGTG
841 TATACCCTGC CGCCGTCACG CGATGAACTG ACGAAAAACC AGGTGTCGCT GACGTGTCTG
901 GTTAAAGGCT TTTACCCGAG CGACATCGCG GTTGAATGGG AATCTAATGG TCAACCGGAA
961 AACAATTATA AAACCACGCC GCCGGTCCTG GATAGTGACG GCTCCTTTTT CCTGTACAGT
1021 AAACTGACCG TGGATAAATC CCGTTGGCAG CAGGGTAACG TCTTCTCGTG TAGCGTGATG
1081 CATGAAGCCC TGCATAATCA CTATACCCAG AAATCTCTGA GTCTGTCCCC GGGCAAAGGT
1141 TCAACGTCGG GTGGCGGTTC CGGCGGTGGC TCAGGTGGCG GTGGCAGCTC TGGCCAACCG
1201 CGCGAACCGC AGGTTTACAC CCTGCCGCCG AGCCGTGACG AACTGACCAA AAACCAAGTC
1261 AGCCTGACGT GCCTGGTGAA AGGCTTTTAC CCGAGTGACA TTGCAGTTGA ATGGGAATCC
1321 AATGGTCAGC CGGAAAATAA CTACAAAACG ACGCCGCCGG TTCTGGATTC AGACGGCTCG
1381 TTTTTCCTGT ACTCAAAACT GACCGTCGAT AAATCGCGCT GGCAACAGGG TAACGTTTTC
1441 AGCTGCTCTG TCATGCACGA AGCCCTGCAC AACCATTATA CCCAGAAAAG TCTGTCCCTG
1501 TCACCGGGCA AAGAAGTGCA GCTGGTTGAA TCTGGTGGCG GTCTGGTGCA ACCGGGCGGT
1561 TCGCTGCGTC TGAGCTGTGC AGCTTCTGGC TTTAATATTA AAGACACGTA CATCCACTGG
1621 GTGCGTCAGG CACCGGGTAA AGGCCTGGAA TGGGTTGCTC GTATCTATCC GACGAACGGT
1681 TATACGCGTT ACGCCGATAG CGTCAAAGGC CGTTTTACCA TCAGTGCAGA CACCTCCAAA
1741 AACACGGCTT ATCTGCAGAT GAATAGTCTG CGTGCAGAAG ATACCGCTGT TTATTACTGC
1801 AGCCGCTGGG GCGGTGATGG CTTCTATGCA ATGGATTATT GGGGTCAAGG TACCCTGGTC
1861 ACCGTGAGTT CCGGTTCGAC CAGCGGCGGT GGCTCAGGTG GCGGTTCGGG CGGTGGCGGT
1921 TCATCGGACA TTCAGATGAC GCAAAGCCCG AGCTCTCTGT CTGCGAGTGT TGGCGATCGT
1981 GTCACCATCA CGTGTCGCGC CTCTCAGGAC GTGAATACCG CAGTTGCTTG GTACCAACAA
2041 AAACCGGGCA AAGCACCGAA ACTGCTGATT TACTCCGCTT CATTCCTGTA CAGCGGTGTG 2101 CCGTCTCGTT TTTCGGGCAG CCGCTCTGGT ACCGATTTCA CCCTGACGAT TAGTTCCCTG 2161 CAACCGGAAG ATTTCGCCAC CTACTACTGC CAGCAACACT ATACGACCCC GCCGACGTTT 2221 GGTCAGGGCA CGAAAGTGGA AATTAAAAAA TTTGCGAAAT TCGCCAAAAA ATTCGCAAAA 2281 TTCGCGAAAA AATTCGCGAA ATAATGAAAG CTT
[0179] Chemical conjugation of Phor-18 to a monoclonal anti-Her2 antibody IgGl (MAb): Purified antibody in phosphate buffered saline (PBS) is concentrated to a concentration of approximately 2 mg/mL. A 20 mM solution of N-succinidyl-3-(2-pyridylothio)propionate (SPDP) is freshly prepared in dimethylsulfoxide (DMSO), and added to the antibody solution in 20-fold excess. The mixture is incubated at room temperature for about 30 minutes to produce the antibody-linker intermediate. Excess unreacted SPDP is removed by size exclusion. The cytotoxic molecule containing cysteine is thoroughly reduced by reaction with a 10-fold excess of reductacryl reagent before mixing in 10-fold excess with the antibody-linker construct. The reaction is allowed to incubate at room temperature for 18 hours, then desalted to remove unreacted cytotoxin molecule. The solution is filter-sterilized before storage.
[0180] In vitro: In vitro cytotoxicity studies were performed to determine the cytotoxicity of the recombinant antibody preparations (conjugated and unconjugated) and lytic peptide, Phor-18, was used in control incubations. Cells were prepared in 96 well plates using 2,000 cells/well and were allowed to attach for 48 hours. Phor-18 in lyophilized form was freshly dissolved in saline and added into the multi-well plates at increasing concentrations of 0, 0.00001, 0.0001 , 0.001, 0.01, 0.1 , 1 , 10, 25, and 100 μΜ. The Her2-antibody-Phor-18 conjugates (Phorl 8- scFv- CH3 -Phor-18, scFv- CH3 -GS-Phor-18, scFv- CH3 -NRVRRS-Phor-18, the Her2-antibody-(KLAKLAK)2KLAK (scFv- CH3 -GS- (KLAKLAK)2KLAK, and scFv- CH3 -NRVRRS-(KLAKLAK)2 LAK), or scFv- CH3 - receptor antibody (naked) in Tris HCL-buffer were diluted with saline and added to cells at increasing concentrations of 0, 0.0012, 0.012, 0.12, 1.2, 6.0, 12.0, 120, 360 and 720 nM. Incubations were conducted for 48 h at 37°C. Cell viability was determined using formazan conversion assays (MTT assays). Controls contained USP saline or 0.1 % TritonX-100™ as reference for 0 and 100 % cell death, respectively. All data were processed and analyzed using Graph Pad Prizm 4™ software (Graph Pad Prizm, Inc).
Example 3
[0181] This example describes studies indicating that anti-Her2-Phor-l 8 antibody conjugate killed her2 expressing breast cancer cells.
[0182] As shown in Table 2, the anti-Her2-Phor-l 8 antibody conjugates (Phorl 8- scFv- CH3 -Phor- 18, scFv- CH3 -GS-Phor-18, scFv-CH3 -NRVRRS-Phor-18) killed Her2 positive human breast cancer SKJBR-3 and ovarian cancer SKOV-3 cell lines by 48 hours, whereas the Her2 negative human breast cancer MDA-MB-231cell line was not killed. Evidence of cytotoxicity was observed microscopically at as early as 24 hours of incubation. As expected, unconjugated Phor-18 showed only modest cytotoxicity. [0183] The HER2 antibody conjugated to the Phor-18 was significantly more cytotoxic than antibody conjugated to the lytic peptide (KLAKLAK)2KLA . (Figure 1, Table 2). The Her2 negative MDA-MB-231 cells were not killed by any of the recombinant antibody- lytic peptide conjugates indicating that the cytotoxicity of the antibodies was mediated via Her2/neu receptors. The "naked" (unconjugated) antibody (scFv- CH3) was not cytotoxic in all 3 cell lines indicating that the cell-killing properties of the antibody-lytic peptide conjugates were due to the presence of lytic peptide payload and sequence of the lytic peptide. Again, as expected, unconjugated Phor-18 showed very minimal nonspecific cytotoxicity in all cell lines (Table 2).
Table 2: In vitro cytotoxicity of anti-Her2-Phor-18 antibody conjugates (scFv- CH3 -Phor-18 and - scFv- CH3 -(KLAKLAK)2KLAK conjugates, Her2/neu scFv- CH3 and unconjugated Phor-18 in Her2 receptor positive S OV-3, SKJBR-3 and Her2 receptor negative MDA-MB-231 cancer cells. Values are IC expressed in nM.
Figure imgf000048_0001
[0184] The results indicate that recombinantly produced Her2 antibody scFv- ¾3 -Phor-18 and Her2 antibody scFv- CH3 -(KLAKLAK)2KLAK conjugates are active in the nanomolar range against Her2/neu receptor expressing cell lines. The unconjugated antibody or free lytic peptide (Phor-18) were without effect indicating that conjugation of lytic peptides to ligands (e.g., antibodies) that bind to Her2/neu receptor to enhances cell cytotoxic potency. Example 4
[0185] This example includes a description of in vitro cytotoxicity studies of recombinantly produced antibody to Her-2 receptor conjugated to lytic peptide, Phor-18 ( FAKFAK KFA FAK KFAK) (scFv- CH3 -GS- Phor-18), and a chemically conjugated MAb-Phorl 8 conjugate against Her2 positive ovarian cancer cell line SKOV-3.
[0186] Cells were prepared in 96 well plates using 5,000 cells/well and were allowed to attach for 48 hours. MAb-Phorl 8, scFv- CH3 -GS- Phor-18, scFv- CH3 were diluted in saline and added at increasing concentrations of 0, 0.0012, 0.012, 0.12, 1.2, 6.0, 12, 120, 360 and 720 nM, N=8 data points per concentration. Incubations were conducted for 24 h at 37°C. Cell viability was determined using formazan conversion assays (MTT assays). Controls contained USP saline or 0.1 % TritonX-100™ as reference for 0 and 100 % cell death, respectively.
[0187] Data were processed and analyzed using Graph Pad Prizm 4™ software (Graph Pad Prizm, Inc). Statistical analysis for significance was determined by a two-tailed Student's T-test. The whole MAb-Phor-18 resulted in IC50 values of 60.53±3.8 nM and scFv- CH3 -GS- Phor-18 was 59.8±3.8 nM. The "naked" (unconjugated) antibodies (MAb and scFv- CH3) were not cytotoxic. In vitro chemically linked HER2 antibody (MAb-Phorl 8) and recombinant Phorl8 conjugate (scFv- CH3 -GS- Phor-18) showed similar toxicity to SKOV-3 cells, whereas the naked recombinant antibody (scFv- CH3) was not toxic.
Example 5
[0188] This example describes an in vivo study in a mouse xenograft model of human ovarian cancer with various doses of anti-Her2-Phor-18 antibody conjugates (scFv- CH3 -GS-Phor-18, MAb-Phorl8), naked whole antibody (MAb) and naked recombinant antibody (scFv- CH3) treatments.
[0189] Female Nu/Nu mice were injected subcutaneously with a SKOV-3/Matrigel suspension
6
(4x10 cells). Tumor weights from mice that were killed on day 42 served as baseline. In brief, treatment started on day 43 after tumor cell injection on tumors of median tumor volume of 130.3± 10.25 mm3 and continued on days 47, 50, 54, 57 and 60 as a single bolus injection into the lateral tail vein.
[0190] During the entire study tumor volumes were measured twice per week and body weights were determined. Final necropsy was conducted on day 64 after tumor cell injection where tumors were excised, weighed and fixed in formalin for histological evaluation.
[0191] Treatments were: saline control, whole naked monoclonal anti-Her2-antibody, MAb, (3 mg/kg), recombinant naked-Her2-antibody (scFv- CH3) (3 mg kg), scFv- CH3 -GS-Phor-18 (0.3 and 3 mg/kg), MAb-Phor-18 (0.3 and 3mg/kg). Tumors from mice sacrificed at treatment start underwent immunohistochemistry evaluation of Her2/neu receptors. Each group consisted of 8-9 mice.
[0192] All groups of mice tolerated the injections well. No mice died as a consequence of injection. [0193] The effect of antibody conjugated Phor- 18 injections and naked antibodies on the primary tumors (volume and tumor weights, Figures 2A, 2B, and 3) and body weight is illustrated in Figure 4.
Figure 2A and 2B show median tumor volumes during the course of the study and mean tumor weights on day 64 for each individual treatment group for saline controls, and mice treated with MAb (naked)
(3 mg/kg), scFv- CH3 (3 mg/kg), scFv- CH3 -GS-Phor-18 (0.3 and 3 mg kg), MAb-Phor-18 (0.3 and
3mg/kg).
[0194] Tumor volumes and weights decreased significantly in all animals treated with 3 mg/kg MAb-Phor-18 chemically linked (p<0.04) or recombinantly produced scFv-CH3 -Phor-18 conjugates (p<0.02) . Naked MAb or scFv- CH3 were not decreasing tumor volumes or tumor weights compared to saline controls at doses of 3 mg/kg (Figure 2A, 2B, and 3). Statistical analysis was conducted in Graphpad prizm 4 using the Wilcoxon signed rank test. Body weights were stable in all treatment groups and control animals (Figure 4).
Example 6
[0195] This example describes in vitro cytotoxicity studies of recombinantly produced antibody to Her-2 receptor conjugated to lytic peptide, Phor-18 (KFAKFAK KFAKFAK KFAK) against ovarian cancer cells.
[0196] scFv-CH2- CH3 -GS-Phor-18 (one molecule of Phor-18 joined to the antibody at the C- terminus by GS linker, consisting of VL— G linker— VH— CH2- (¾3 ~G linker— CH3-Ch2~VH— G linker— VL -GS-(Phor- 18). Cytotoxicity was compared to a naked antibody (scFv- CH2- Ch3; antibody without a lytic peptide) in Her-2 receptor positive cells (SKOV-3, human ovarian cancer cells).
[0197] Materials: Recombinant DNA technique was used to synthesize anti-Her2 antibody as an scFv- CH2- Ch3 antibody in Escherichia coli. The antibody (Olafsen T. et al Protein Engineering, Design & Selection 17, 315-323, 2004) was conjugated via a peptide linker to either Phor-18 and analyzed for cytotoxicity in vitro. The plasmid was acquired through gene codon optimization. The gene was synthesized with a N-His tag sequence and the plasmid was subcloned into an E. coli bacteria expression vector pUC57. After expression optimization and evaluation the His-tag product was selected and 1 L of the bacteria expression product was purified in a one-step affinity purification. The amino acid sequence of for each construct is described in Table 3.
Table 3: Amino Acid sequence for the production of each recombinant antibody, (A) scFv- CR2- Ch3; and antibody conjugate (B) scFv- CH2- CH3 -GS-Phor-18.
A)
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPG AP LLIYSASFLYSGVPSRFS GSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT VEIK
GSTSGGGSGGGSGGGGSS EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS PCPAPELLGGPSVFLFPP PKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGV
E\¾NAKT1<-PREEQY STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP1EKTISKAK
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN YKTTPPVLD SDGSFFLYSKLTVD SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GSTSGGGSGGGSGGGGSS
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN Y TTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV FNWYVDGV EVld A T PREEQYNSTYRVVSVLWLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG GLEWVARIYPTNGYTRYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS GSTSGGGSGGGSGGGGSS
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ PG AP LLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
B)
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ PGKAPKLLIYSASFLYSGVPSRFS GSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK GSTSGGGSGGGSGGGGSS
EVQLVESGGGLVQPGGSLRLSCAASGFNI DTYIHWVRQAPGKGLEWVARJYPTNGYTRYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS PCPAPELLGGPSVFLFPPKP DTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV EVIT AKTKPREEQYNSTYRVVSVLTVLHQDWLNG EY CKVSNKALPAPIEKTIS AK
GQPREPQVYTLPPSPvDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GSTSGGGSGGGSGGGGSS
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVD SRWQQGNVFSCSVMHEALFINHYTQKSLSLSPGK PCPAPELLGGPSVFLFPP PKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV EVHNAKTK REEQYNSTYRWSVLTVLHQDWLNGKEY CKVSNKALPAPIEKTIS AK
EVQLVESGGGLVQPGGSLRLSCAASGFNI DTYIHWVRQAPG GLEWVARIYPTNGYTRYA DSVKGRFT1SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
GSTSGGGSGGGSGGGGSS
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP LLIYSASFLYSGVPSRFS GSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
GS KFAKFAKKFAKFAKKFAK
[0198] In vitro cytotoxicity studies were performed to determine cytotoxicity of the recombinant antibody preparations (scFv- CH2- CH3, scFv-CH3, and scFv- CH2- CH3 -GS-Phor-18, scFv-CH3 -GS- Phor-18). Her-2 receptor positive SKOV-3 cells were prepared in 96 well plates using 2,000 cells/well and were allowed to attach for 48 hours. The Her2-antibody-Phor-18 conjugates (scFv- CH2- Ch3 -GS-
Phor-18, scFv-CH3 GS-Phor-18) or the naked antibodies (scFv- CH2- CH3, scFv-CH3) in Tris/HCL- buffer were diluted with saline and added to cells at increasing concentrations of 0, 0.0012, 0.012, 0.12,
1.2, 6.0, 12.0, 120, 360 and 720 nM. Incubations were conducted for 48 h at 37°C. Cell viability was determined using Cell Titer Glo luminescent cell viability assay (Promega). Controls contained USP saline or 0.1 % TritonX-100™ as reference for 0 and 100 % cell death, respectively. All data were processed and analyzed using Graph Pad Prizm 4™ software (Graph Pad Prizm, Inc).
[0199] The Her2 antibody (scFv- CH2- CH3, scFv-CH3) conjugated to the Phor- 18 resulted in 1C50 values of 53.7±0.63 nM for scFv-CH3 -Phor 18 and 56.7±0.92 nM for scFv-CH2-CH3 -Fv-Phor-18 .
The "naked" (unconjugated) antibodies, scFv- CH2- CH3, SCFV-CH3, were not cytotoxic. In vitro recombinantly Phor- 18 conjugates show similar toxicity to SKOV-3 cells.

Claims

What is Claimed:
1. A conjugate comprising a ligand that binds to Her2/neu and a second domain, wherein said second domain consists of a 12 to 100 amino acid sequence that includes a peptide selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA,
KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA, or a 12 to 100 amino acid sequence that includes a peptide selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK,
KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA having one or more of the K residues substituted with any of an F or L residue, one or more of the F residues substituted with any of a K, A or L residue, or one or more of the A residues substituted with any of a K, F or L residue.
2. A conjugate comprising a ligand that binds to Her2/neu and a second domain, wherein said second domain consists of an amino acid sequence selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK,
KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA; or an amino acid sequence selected from KFAKFAKKFAKFAKK, KFAKFAKKFAKFAKKF, KFAKFAKKFAKFAKKFA, KFAKFAKKFAKFAKKFAK, KFAKFAKKFAKFAKKFAKF and KFAKFAKKFAKFAKKFAKFA having one or more of the K residues substituted with any of an F or L residue, one or more of the F residues substituted with any of a K, A or L residue, or one or more of the A residues substituted with any of a K, F or L residue.
3. The conjugate of claims 1 or 2, wherein said ligand that binds to Her2/neu comprises a peptide, polypeptide, protein, nucleic acid or carbohydrate.
4. The conjugate of claims 1 or 2, wherein said ligand that binds to Her2/neu has a linear or cyclic structure.
5. The conjugate of claims 1 or 2, wherein said ligand that binds to Her2/neu comprises an antibody or an antibody fragment.
6. The conjugate of claim 5, wherein said antibody fragment comprises an Fab, Fab', F(ab')2, Fv, Fd, single-chain Fv (scFv), disulfide-1 inked Fvs (sdFv), VL, VH, Camel Ig, V-NAR, VFIH, trispecific (Fab3), bispecific (Fabb), diabody ((VL-VH)2 or (VH-VL^), triabody (trivalent), tetrabody (tetravalent), minibody ((scFv-CH3)2), bispecific single-chain Fv (Bis-scFv), IgGdeltaCH2, scFv-Fc, (scFv)2-Fc, affibody, aptamer, avimer or nanobody.
7. The conjugate of claims 1 or 2, wherein said ligand that binds to Her2/neu comprises a hormone or a growth factor.
8. The conjugate of claim 7, wherein said hormone or growth factor comprises a hormone or growth factor analogue, a fragment of a hormone or hormone analogue or growth factor or growth factor analogue that binds to Her2/neu.
9. The conjugate of claims 1 or 2, wherein said ligand comprises an agonist or antagonist.
10. The conjugate of claim 5, wherein said antibody comprises a monoclonal antibody.
1 1. The conjugate of claim 5, wherein said antibody comprises a mammalian antibody.
12. The conjugate of claim 5, wherein said antibody comprises a human, humanized, primatized, or chimeric antibody.
13. The conjugate of claim 5, wherein said antibody comprises trastuzumab or pertuzumab.
14. The conjugate of claims 1 or 2, wherein said Her2/neu is expressed on a cell.
15. The conjugate of claim 14, wherein said cell is a hyperproliferative cell.
16. The conjugate of claim 14, wherein said cell is a breast, ovarian, uterine, cervical, stomach, lung, gastric, colon, bladder, glial, hematologic or endometrial cell.
17. The conjugate of claims 1 or 2, wherein said ligand that binds to Her2/neu or said second domain consists of or comprises an amino acid sequence of about 1 to 10, 10 to 20, 15 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino acids.
18. The conjugate of claims 1 or 2, wherein said second domain consists of a sequence from about 10 to about 50 amino acids.
19. The conjugate of claims 1 or 2, wherein said second domain consists of a sequence from about 10 to about 28 amino acids.
20. The conjugate of claims 1 or 2, wherein said second domain consists of a sequence with 15, 16, 17, 18, 19 or 20 amino acids.
21. The conjugate of claims 1 or 2, wherein said ligand that binds to Her2/neu is positioned at the NHo-terminus relative to said second domain.
22. The conjugate of claims 1 or 2, wherein said second domain is positioned at the N¾- terminus relative to said ligand that binds to Her2/neu, or wherein said second domain is positioned at the C-terminus relative to said ligand that binds to Her2/neu, or wherein said ligand that binds to Her2/neu is positioned at the NH2-terminus relative to said second domain, or wherein said ligand that binds to Her2/neu is positioned at the C-terminus relative to said second domain.
23. The conjugate of claims 1 or 2, wherein said ligand that binds to Her2/neu or said second domain has one or more D-amino acids.
24. The conjugate of claims 1 or 2, wherein said second domain forms an amphipathic alpha-helix.
25. The conjugate of claims 1 or 2, wherein said ligand that binds to Her2/neu and said second domain are joined by a covalent bond.
26. The conjugate of claims 1 or 2, wherein said ligand that binds to Her2/neu and said second domain are joined by a peptide or a non-peptide linker.
27. The conjugate of claims 1 or 2, wherein said ligand that binds to Her2/neu and said second domain are joined by peptide sequence having from 1 to 25 amino acid residues, or a linear carbon chain.
28. The conjugate of claims 1 or 2, further comprising a third, fourth, fifth, sixth or seventh domain.
29. The conjugate of claims 1 or 2, wherein said conjugate is isolated or purified.
30. The conjugate of claims 1 or 2, wherein said conjugate comprises a mixture.
31. A composition comprising the conjugate of claims 1 or 2.
32. A pharmaceutical composition comprising the conjugate of claims 1 or 2.
33. A composition comprising the conjugate of claims 1 or 2, and an anti-cell proliferative or immune stimulating agent.
34. A unit dosage, comprising the conjugate of claims 1 or 2 in an amount effective to treat a subject having undesirable cell proliferation or a hyperproliferative disorder.
35. A unit dosage, comprising the conjugate of claims 1 or 2 in an amount effective to treat a subject having a neoplasia, tumor or cancer.
36. A kit comprising the conjugate of claims 1 or 2 and instructions for reducing or inhibiting proliferation of a cell, reducing or inhibiting proliferation of a hyperprol iterating cell, reducing or inhibiting proliferation of a neoplastic, tumor or cancer cell, treating a subject having a
hyperproliferative disorder, or treating a subject having a neoplasia, tumor or cancer.
37. A nucleic acid molecule that encodes the conjugate of claims 1 or 2.
38. A vector comprising the nucleic acid molecule of claim 37.
39. A host cell transformed with the nucleic acid of claim 37, or the vector of claim 38.
40. A cell that expresses the conjugate of claims 1 or 2.
41. A method of reducing or inhibiting proliferation of a cell, comprising contacting a cell with the conjugate of claims 1 or 2 in an amount sufficient to reduce or inhibit proliferation of the cell.
42. A method of reducing or inhibiting cell proliferation, comprising contacting a cell with the conjugate of claims 1 or 2 in an amount sufficient to reduce or inhibit cell proliferation.
43. A method of reducing or inhibiting proliferation of a hyperproliferating cell, comprising contacting a cell with the conjugate of claims 1 or 2 in an amount sufficient to reduce or inhibit proliferation of the hyperproliferating cell.
44. A method of reducing or inhibiting proliferation of a neoplastic, tumor, cancer or malignant cell, comprising contacting the cell with the conjugate of claims 1 or 2 in an amount sufficient to reduce or inhibit proliferation of the neoplastic, tumor, cancer or malignant cell.
45. The method of any of claims 41 to 44, wherein the cell expresses Her2/neu.
46. A method of selectively reducing or inhibiting proliferation of a cell that expresses Her2/neu, comprising contacting the cell with the conjugate of claims 1 or 2 in an amount sufficient to reduce or inhibit proliferation of the cell that expresses Her2/neu.
47. A method of selectively reducing or inhibiting proliferation of a hyperproliferating cell that expresses Her2/neu, comprising contacting the cell with the conjugate of claims 1 or 2 in an amount sufficient to reduce or inhibit proliferation of the hyperproliferating cell that expresses Her2/neu.
48. A method of selectively reducing or inhibiting proliferation of a neoplastic, tumor, cancer or malignant cell that expresses a receptor or antigen, comprising contacting the cell with the conjugate of claims 1 or 2 in an amount sufficient to reduce or inhibit proliferation of the neoplastic, tumor, cancer or malignant cell that expresses Her2/neu.
49. A method of treating a subject having a hyperproliferative disorder, comprising administering to the subject an amount of the conjugate of claims 1 or 2 sufficient to treat the
hyperproliferative disorder.
50. A method of treating a subject having a neoplasia, tumor, cancer or malignancy, comprising administering to the subject an amount of the conjugate of claims 1 or 2 sufficient to reduce or inhibit proliferation of the neoplasia, tumor, cancer or malignancy.
51. The method of claims 48 or 50, wherein the neoplasia, tumor, cancer or malignancy is metastatic, non-metastatic or benign.
52. The method of claims 48 or 50, wherein the neoplasia, tumor, cancer or malignancy comprises a solid cellular mass.
53. The method of claims 48 or 50, wherein the neoplasia, tumor, cancer or malignancy comprises hematopoietic cells.
54. The method of claims 48 or 50, wherein the neoplasia, tumor, cancer or malignancy comprises a carcinoma, sarcoma, lymphoma, leukemia, adenoma, adenocarcinoma, melanoma, glioma, glioblastoma, meningioma, neuroblastoma, retinoblastoma, astrocytoma, oligodendrocytoma, mesothelioma, reticuloendothelial, lymphatic or haematopoietic neoplasia, tumor, cancer or malignancy.
55. The method of claim 54, wherein the sarcoma comprises a lymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma or fibrosarcoma.
56. The method of claim 54, wherein the haematopoietic neoplasia, tumor, cancer or malignancy comprises a myeloma, lymphoma or leukemia.
57. The method of claims 48 or 50, wherein the neoplasia, tumor, cancer or malignancy comprises a lung, thyroid, head or neck, nasopharynx, throat, nose or sinuses, brain, spine, breast, adrenal gland, pituitary gland, thyroid, lymph, gastrointestinal (mouth, esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon, rectum), genito-urinary tract (uterus, ovary, cervix, endometrial, bladder,
54
t testicle, penis, prostate), kidney, pancreas, liver, bone, bone marrow, lymph, blood, muscle, or skin, wilm's, biliary tract, B-ALL, or hematologic neoplasia, tumor, or cancer.
58. The method of claim 57, wherein the lung neoplasia, tumor, cancer or malignancy comprises small cell lung or non-small cell lung cancer.
59. The method of claims 48 or 50, wherein the neoplasia, tumor, cancer or malignancy comprises a stem cell neoplasia, tumor, cancer or malignancy.
60. The method of claims 48 or 50, wherein the method inhibits or reduces relapse or progression of the neoplasia, tumor, cancer or malignancy.
61. The method of claims 48 or 50, further comprising administering an anti-cell proliferative, anti-neoplastic, anti-tumor, anti-cancer or immune-enhancing treatment or therapy.
62. The method of claim 61 , wherein the treatment or therapy comprises surgical resection, radiotherapy, ionizing or chemical radiation therapy, chemotherapy, immunotherapy, local or regional thermal (hyperthermia) therapy, or vaccination.
63. The method of claim 61 , wherein the conjugate is administered prior to, substantially contemporaneously with or following administration of the anti-cell proliferative, anti-neoplastic, antitumor, anti-cancer or immune-enhancing treatment or therapy.
64. The method of claims 48 or 50, wherein the subject has undergone surgical resection, chemotherapy, immunotherapy, ionizing or chemical radiotherapy, local or regional thermal
(hyperthermia) therapy, or vaccination.
65. The method of claims 48 or 50, wherein the subject is a candidate for surgical resection, chemotherapy, immunotherapy, ionizing or chemical radiotherapy, local or regional thermal
(hyperthermia) therapy, or vaccination.
66. The method of claims 48 or 50, wherein the subject is not a candidate for surgical resection, chemotherapy, immunotherapy, ionizing or chemical radiotherapy, local or regional thermal (hyperthermia) therapy, or vaccination.
67. The method of claims 48 or 50, wherein the treatment results in partial or complete destruction of the neoplastic, tumor, cancer or malignant cell mass, volume, size or numbers of cells, stimulating, inducing or increasing neoplastic, tumor, cancer or malignant cell necrosis, lysis or apoptosis, reducing neoplasia, tumor, cancer or malignancy volume size, cell mass, inhibiting or preventing progression or an increase in neoplasia, tumor, cancer or malignancy volume, mass, size or cell numbers, or prolonging lifespan.
68. The method of claims 48 or 50, wherein the treatment results in reducing or decreasing severity, duration or frequency of an adverse symptom or complication associated with or caused by the neoplasia, tumor, cancer or malignancy.
69. The method of claims 48 or 50, wherein the treatment results in reducing or decreasing pain, discomfort, nausea, weakness or lethargy, or results in increased energy, appetite, improved mobility or psychological well being.
70. The method of claims 48 or 50, wherein the subject is a mammal.
71. The method of claims 48 or 50, wherein the subject is a human.
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