[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2010059315A1 - Lieurs de sérum-albumine humaine et conjugués de ceux-ci - Google Patents

Lieurs de sérum-albumine humaine et conjugués de ceux-ci Download PDF

Info

Publication number
WO2010059315A1
WO2010059315A1 PCT/US2009/060721 US2009060721W WO2010059315A1 WO 2010059315 A1 WO2010059315 A1 WO 2010059315A1 US 2009060721 W US2009060721 W US 2009060721W WO 2010059315 A1 WO2010059315 A1 WO 2010059315A1
Authority
WO
WIPO (PCT)
Prior art keywords
hsa linker
hsa
seq
amino acid
linker conjugate
Prior art date
Application number
PCT/US2009/060721
Other languages
English (en)
Inventor
Charlotte Mcdonagh
Michael Feldhaus
Alexandra Huhalov
Original Assignee
Merrimack Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2009/040259 external-priority patent/WO2009126920A2/fr
Application filed by Merrimack Pharmaceuticals, Inc. filed Critical Merrimack Pharmaceuticals, Inc.
Priority to JP2011536365A priority Critical patent/JP5677972B2/ja
Priority to CN2009801545099A priority patent/CN102282168A/zh
Priority to BRPI0921586A priority patent/BRPI0921586A2/pt
Priority to US13/130,007 priority patent/US8927694B2/en
Priority to US12/757,801 priority patent/US20110059076A1/en
Publication of WO2010059315A1 publication Critical patent/WO2010059315A1/fr
Priority to US14/582,719 priority patent/US20150191545A1/en
Priority to US15/251,158 priority patent/US20170204192A1/en

Links

Classifications

    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • 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
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

  • HSA linker conjugates and binding, diagnostic, and therapeutic conjugates thereof.
  • the HSA linker includes two amino acid substitutions.
  • Another embodiment is a conjugate in which the HSA linker is covalently bonded to amino and carboxy terminal binding moieties that are first and second single-chain Fv molecules (scFvs).
  • scFvs first and second single-chain Fv molecules
  • Antibody-like binding moieties are often used for therapeutic applications.
  • Antibody fragments and scFvs generally exhibit shorter serum half lives than intact antibodies, and in some therapeutic applications increased in vivo half lives would be desireable for therapeutic agents possessing the functionality of such fragments and scFvs.
  • Human serum albumin is a protein of about 66,500 kD and is comprised of 585 amino acids including at least 17 disulphide bridges. As with many of the members of the albumin family, human serum albumin plays an important role in human physiology and is located in virtually every human tissue and bodily secretion. HSA has the ability to bind and transport a wide spectrum of ligands throughout the circulatory system, including the long-chain fatty acids, which are otherwise insoluble in circulating plasma.
  • the serum albumins belong to a family of proteins that includes alpha-fetoprotein and human group-specific component, also known as vitamin-D binding protein.
  • the serum albumins are the major soluble proteins of the circulatory system and contribute to many vital physiological processes.
  • Serum albumin generally comprises about 50% of the total blood component by dry weight.
  • the albumins and their related blood proteins also play an important role in the transport, distribution, and metabolism of many endogenous and exogenous ligands in the human body, including a variety of chemically diverse molecules, such as fatty acids, amino acids, steroids, calcium, metals such as copper and zinc, and various pharmaceutical agents.
  • the albumin family of molecules is generally thought to facilitate transfer of many of these ligands across organ-circulatory interfaces, such as the liver, intestines, kidneys, and the brain.
  • the albumins are thus involved in a wide range of circulatory and metabolic functions.
  • the invention provides an HSA linker conjugate that includes a human serum albumin (HSA) linker that comprises an amino acid sequence set forth in any one of SEQ ID NOS:6-15 and first and second binding moieties selected from antibodies, single-chain Fv molecules, bispecific single chain Fv ((scFv') 2 ) molecules, domain antibodies, diabodies, triabodies, hormones, Fab fragments, F(ab') 2 molecules, tandem scFv (taFv) fragments, receptors (e.g., cell surface receptors), ligands, aptamers, and biologically-active fragments thereof, in which the first binding moiety is bonded to the amino terminus of the HSA linker and the second binding moiety is bonded to the carboxy terminus of the HSA linker.
  • the first binding moiety specifically binds ErbB3 and the second binding moiety specifically binds ErbB2.
  • the HSA linker comprises an amino acid sequence set forth in
  • binding moieties e.g., 4, 5, 6, 7, 8, 9, 10, or more
  • these additional binding moieties can be added to the agent, e.g., in tandem (e.g., 2, 3, 4, or 5 or more in tandem) with the first or second binding moiety.
  • the invention provides an HSA linker that comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:1, and a serine residue at position 34 and a glutamine residue at position 503 of the amino acid sequence set forth in SEQ ID NO: 1.
  • the amino acid sequence has at least 95% sequence identity to the sequence set forth in SEQ ID NO: 1.
  • the HSA linker comprises the amino acid sequence set forth in SEQ ID NO:1.
  • the HSA linker has the amino acid sequence set forth in SEQ ID NO: 1.
  • the invention provides an HSA linker conjugate that includes an HSA linker having at least 90% amino acid sequence identity to the sequence set forth in SEQ ID NO:1 and at least a first binding moiety.
  • the HSA linker conjugate includes a first peptide connector that binds the first binding moiety to the HSA linker.
  • the invention features an USA linker conjugate that includes an HSA linker having an amino acid sequence set forth in any one of SEQ ID NOs:l 1-15, or a fragment or variant of any one of these sequences, and at least a first binding moiety.
  • the HSA linker conjugate further includes a first peptide connector (e.g., AAS, AAQ, or AAAL (SEQ ID NO:5)) that binds the first binding moiety to the amino or carboxy terminus of the HSA linker.
  • a first peptide connector e.g., AAS, AAQ, or AAAL (SEQ ID NO:5)
  • the first connector covalently binds the first binding moiety to the HSA linker.
  • the HSA linker conjugate further includes at least a second binding moiety.
  • the HSA linker conjugate further includes a second peptide connector (e.g., AAS, AAQ, or AAAL (SEQ ID NO:5)) that binds the second binding moiety to the HSA linker.
  • the second connector binds the second binding moiety to the amino or carboxy terminus of the HSA linker.
  • the second connector covalently binds the second binding moiety to the HSA linker.
  • the HSA linker conjugate further includes three or more binding moieties which are included in tandem with the first or second binding moiety; the three or more binding moieties can further include a connector sequence that joins the three or more binding moieties to the first or second binding moiety and to each other.
  • the HSA linker conjugate includes a first peptide connector that covalently binds a first binding moiety to the amino terminus of the HSA linker and a second peptide connector that covalently binds a second binding moiety to the carboxy terminus of the HSA linker.
  • the first connector has the amino acid sequence AAS or AAQ and the second connector has the amino acid sequence set forth in SEQ ID NO:5.
  • the first or second binding moiety is an antibody, single-chain Fv molecule, bispecific single chain Fv ((scFv') 2 ) molecule, domain antibody, diabody, triabody, hormone, Fab fragment, F(ab') 2 molecule, tandem scFv (taFv) fragment, receptor (e.g., cell surface receptor), ligand, aptamer, or biologically-active fragment thereof.
  • the HSA linker conjugatse provided herein include combinations of these different types of binding moieties.
  • at least the first or second binding moiety is a human or humanized single-chain Fv molecule.
  • one or more of the first or second binding moiety is or specifically binds to a protein selected from the group consisting of an insulin-like growth factor 1 receptor (IGFlR), IGF2R, insulin-like growth factor (IGF), mesenchymal epithelial transition factor receptor (c-met; also known as hepatocyte growth factor receptor (HGFR)), hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), heregulin, fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), platelet- derived growth factor (PDGF), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor (VEGF), tumor necrosis factor receptor (TNFR), tumor necrosis factor alpha (TNF- ⁇ ), TNF- ⁇ , folate receptor (FOLR),
  • IGFlR insulin-like growth factor 1 receptor
  • IGF2R insulin-like growth factor
  • IGF insulin-like growth factor
  • IGF
  • one or more of the first or second binding moiety is or specifically binds to erythroblastic leukemia viral oncogene homolog (ErbB) receptor (e.g., ErbBl receptor; ErbB2 receptor; ErbB3 receptor; and ErbB4 receptor).
  • ErbB erythroblastic leukemia viral oncogene homolog
  • one or more of the first or second binding moiety is or specifically binds to alpha- fetoprotein (AFP) or an interferon, or a biologically-active fragment thereof.
  • AFP alpha- fetoprotein
  • one or more of the first or second binding moiety is natalizumab, infliximab, adalimumab, rituximab, alemtuzumab, bevacizumab, daclizumab, efalizumab, golimumab, certolizumab, trastuzumab, abatacept, etanercept, pertuzumab, cetuximab, panitumumab, or anakinra.
  • the HSA linker conjugate is conjoined to a diagnostic, a therapeutic agent, or both.
  • the diagnostic agent is a detectable label, such as a radioactive, fluorescent, or heavy metal label.
  • the therapeutic agent is a cytotoxic, cytostatic, or immunomodulatory agent. Cytotoxic agents include alkylating agents, antibiotics, antineoplastic agents, antiproliferative agents, antimetabolites, tubulin inhibitors, topoisomerase I or II inhibitors, hormonal agonists or antagonists, immunomodulators, DNA minor groove binders, and radioactive agents, or any agent capable of binding to and killing a tumor cell or inhibiting tumor cell proliferation.
  • Antineoplastic agents include cyclophosphamide, camptothecin, homocamptothecin, colchicine, combrestatin, combrestatin, rhizoxin, dolistatin, ansamitocin p3, maytansinoid, auristatin, caleachimicin, methotrexate, 5-fluorouracil (5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin, tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab, trastuzumab, and their derivatives.
  • the HSA linker conjugate is admixed with a pharmaceutically acceptable carrier, excipient, or diluent.
  • the agent exhibits an in vivo half-life of between 6 hours and 7 days. In another embodiment, the agent exhibits an in vivo half-life greater than 8 hours.
  • the invention features a method for treating a mammal having a disease or disorder by administering any one of the HSA linker conjugates described herein.
  • the disease or disorder is associated with cellular signaling through a cell surface receptor.
  • the mammal is a human.
  • the disease or disorder is a proliferative or autoimmune disease.
  • Proliferative diseases includesuch cancers as melanoma, clear cell sarcoma, head and neck cancer, bladder cancer, breast cancer, colon cancer, ovarian cancer, endometrial cancer, gastric cancer, pancreatic cancer, renal cancer, prostate cancer, salivary gland cancer, lung cancer, liver cancer, skin cancer, and brain cancer.
  • HSA linker conjugate is administered in combination with one or more therapeutic agents, such as an antineoplastic agent.
  • the invention features a method for making an HSA linker conjugate by bonding at least a first binding moiety to the amino terminus and a second binding moiety to the carboxy terminus of an HSA linker having the amino acid sequence set forth in any one of SEQ ID NOS: 1, 3, or 6-15, or a sequence having at least 90%, 95%, 97%, or 100% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1, 3, or 6-15.
  • the first or second binding moiety is covalently joined to the amino or carboxy terminus of the HSA linker.
  • a third or additional binding moiety e.g., a fourth, fifth, sixth, seventh, eighth, ninth, or tenth binding moiety
  • a third or additional binding moiety is covalently joined in tandem with the first or second binding moiety to the amino or carboxy terminus of the HSA linker.
  • one or more of the first or second binding moiety is an antibody, single-chain Fv molecule, bispecific single chain Fv ((scFv') 2 ) molecule, domain antibody, diabody, triabody, hormone, Fab fragment, F(ab') 2 molecule, tandem scFv (taFv) fragment, receptor (e.g., cell surface receptor), ligand, or aptamer.
  • the first or second binding moiety is a human or humanized single-chain Fv molecule.
  • one or more of the first or second binding moiety is or specifically binds to insulin-like growth factor 1 receptor (IGFlR), IGF2R, insulin-like growth factor (IGF), mesenchymal epithelial transition factor receptor (c-met; also known as hepatocyte growth factor receptor (HGFR)), hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), heregulin, fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), platelet-derived growth factor (PDGF), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor (VEGF), tumor necrosis factor receptor (TNFR), tumor necrosis factor alpha (TNF- ⁇ ), TNF- ⁇ , folate receptor (FOLR), folate, transferrin receptor (TfR), mesothelin, Fc receptor, c-kit receptor,
  • IGFlR insulin-like growth factor 1 receptor
  • IGF insulin-like
  • one or more of the first or second binding moiety is or specifically binds to erythroblastic leukemia viral oncogene homolog (ErbB) receptor (e.g., ErbBl receptor; ErbB2 receptor; ErbB3 receptor; and ErbB4 receptor).
  • ErbB erythroblastic leukemia viral oncogene homolog
  • one or more of the first or second binding moiety is or specifically binds to alpha-fetoprotein (AFP) or an interferon, or a biologically-active fragment thereof.
  • AFP alpha-fetoprotein
  • one or more of the first or second binding moiety is natalizumab, infliximab, adalimumab, rituximab, alemtuzumab, bevacizumab, daclizumab, efalizumab, golimumab, certolizumab, trastuzumab, abatacept, etanercept, pertuzumab, cetuximab, panitumumab, or anakinra.
  • the agent is conjoined to a diagnostic or therapeutic agent.
  • the diagnostic agent is a detectable label, such as a radioactive, bioluminescent, fluorescent, heavy metal, or epitope tag.
  • the therapeutic agent is a cytotoxic agent, cytostatic, or immunomodulatory agent. Cytotoxic agents include alkylating agents, antibiotics, antineoplastic agents, antiproliferative agents, antimetabolites, tubulin inhibitors, topoisomerase I and II inhibitors, hormonal agonists or antagonists, immunomodulators, DNA minor groove binders, and radioactive agents, or any agent capable of binding to and killing a tumor cell or inhibiting tumor cell proliferation.
  • Antineoplastic agents include cyclophosphamide, camptothecin, homocamptothecin, colchicine, combrestatin, combrestatin, rhizoxin, dolistatin, ansamitocin p3, maytansinoid, auristatin, caleachimicin, methotrexate, 5-fluorouracil (5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin, tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab, trastuzumab, and their derivatives.
  • the agent is admixed with a pharmaceutically acceptable carrier, excipient, or diluent.
  • the invention features a method for making an HSA linker by substituting one or more surface-exposed amino acid residues in the amino acid sequences set forth in any one of SEQ ID NOS: 1, 3, and 6-15 with a substitute amino acid capable of chemical modification that allows conjugation of a diagnostic or therapeutic agent.
  • the substitute amino acid is cysteine and the surface exposed amino acid residues are serine or threonine.
  • the chemical modification results in a covalent bond between the substitute amino acid and the diagnostic or therapeutic agent.
  • the surface-exposed amino acid residues is threonine at position 496, serine at position 58, threonine at position 76, threonine at position 79, threonine at position 83, threonine at position 125, threonine at position 236, serine at position 270, serine at position 273, serine at position 304, serine at position 435, threonine at position 478, threonine at position 506, or threonine at position 508.
  • the invention features a method for making an HSA linker by substituting one or more of the residues in the amino acid sequences set forth in any one of SEQ ID NOS: 1, 3, and 6-15 with an asparagine, serine, or threonine, thereby incorporating a glycosylation site within the HSA agent.
  • the invention features a method for making an HSA linker by substituting one or more of the asparagine, serine, or threonine residues in the amino acid sequences set forth in any one of SEQ ID NOS: 1, 3, and 6-15 with any amino acid other than asparagine, serine, or threonine, thereby removing a glycosylation site from the HSA agent.
  • the invention features an HSA linker that comprises a sequence that has at least 90% sequence identity to one of the amino acid sequences set forth in SEQ ID NOS: 16-25.
  • the HSA linker has at least 95% sequence identity to one of the amino acid sequences set forth in SEQ ID NOS: 16-25.
  • the HSA linker comprises one of the amino acid sequences set forth in SEQ ID NOS: 16-25.
  • the HSA linker has one of the amino acid sequences set forth in SEQ ID NOS: 16-25.
  • the HSA linker or HSA linker conjugate is conjoined to a diagnostic or therapeutic agent.
  • Diagnostic agents include detectable labels, such as a radioactive, bioluminesccnt, fluorescent, or heavy metal labels, or epitope tags.
  • Fluorescent molecules that can serve as detectable labels include green fluorescent protein (GFP), enhanced GFP (eGFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), and dsRed.
  • the bioluminescent molecule is luciferase.
  • the epitope tag is c-myc, hemagglutinin, or a histidine tag.
  • the therapeutic agent is a cytotoxic polypeptide such as cytochrome c, caspase 1-10, granzyme A or B, tumor necrosis factor- alpha (TNF- ⁇ ), TNF- ⁇ , Fas, Fas ligand, Fas-associated death doman-like IL-I ⁇ converting enzyme (FLICE), TRAIL/APO2L, TWEAK/APO3L, Bax, Bid, Bik, Bad, Bak, RICK, vascular apoptosis inducing proteins 1 and 2 (VAPl and VAP2), pierisin, apoptosis- inducing protein (AIP), IL- l ⁇ propiece polypeptide, apoptin, apoptin-associated protein 1 (AAP-I), endostatin, angiostatin, and biologically-active fragments thereof.
  • cytotoxic polypeptide such as cytochrome c, caspase 1-10, granzyme A or B, tumor necrosis factor- alpha
  • HSA linker or HSA linker conjugate can be combined with (e.g., conjoined to or mixed with in a pharmaceutical composition) one or more therapeutic agents such as cyclophosphamide, camptothecin, homocamptothecin, colchicine, combrestatin, combrestatin, rhizoxin, dolistatin, ansamitocin p3, maytansinoid, auristatin, caleachimicin, methotrexate, 5- fluorouracil (5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin, tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab, trastuzumab, and derivatives thereof.
  • therapeutic agents such as cyclophosphamide, camptothecin, homocamptothecin, colchicine, combrestatin, comb
  • the first and second binding moieties specifically bind the same target molecule. In another embodiment of any aspect, the first and second binding moieties (and, if present, one or more of the third or further binding moiety) specifically bind different target molecules. In a further embodiment of any aspect, the first and second binding moieties (and, if present, one or more of the third or further binding moiety) specifically bind different epitopes on the same target molecule.
  • the invention features an HSA linker that comprises one or both of amino acid residues 25-44 and 494-513 of the amino acid sequence set forth in SEQ ID NO:1.
  • the HSA linker comprises amino acid residues 25-70 and 450-513 of the amino acid sequence set forth in SEQ ID NO:1.
  • the HSA linker comprises amino acid residues 15-100 and 400-520 of the amino acid sequence set forth in SEQ ID NO: 1.
  • the HSA linker comprises amino acid residues 10-200 and 300-575 of the amino acid sequence set forth in SEQ ID NO:1.
  • the HSA linker comprises amino acid residues 5-250 and 275-580 of the amino acid sequence set forth in SEQ TD NO: 1.
  • the HSA linker is conjoined to at least a first binding moiety, for form an HSA linker conjugate.
  • the HSA linker conjugate includes at least a first peptide connector that binds the first binding moiety to the amino or carboxy terminus of the HSA linker.
  • the first peptide connector covalently binds the first binding moiety to the HSA linker.
  • the HSA linker includes a second binding moiety.
  • the HSA linker includes a second peptide connector that binds the second binding moiety to the HSA linker.
  • the second connector binds the second binding moiety to the amino or carboxy terminus of the HSA linker. In a further embodiment, the second connector covalently binds the second binding moiety to the HSA linker. In other embodiments, the HSA linker includes a third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth binding moiety. In other embodiments, these additional binding moieties are present in tandem with one or both of the first or second binding moiety. In yet other embodiments, a peptide connector (e.g., AAS, AAQ, or AAAL (SEQ ID NO:5)) separates one or more of these additional binding moities from each other, the first or second binding moiety, or the HSA linker.
  • a peptide connector e.g., AAS, AAQ, or AAAL (SEQ ID NO:5)
  • the HSA linker includes a first peptide connector that covalently binds a first binding moiety to the amino terminus of the polypeptide linker and a second peptide connector that covalently binds a second binding moiety to the carboxy terminus of the HSA linker.
  • the first connector has the amino acid sequence AAS or AAQ and the second connector has the amino acid sequence set forth in SEQ ID NO: 5.
  • one or more of the first or second binding moiety is an antibody, single-chain Fv molecule, bispecific single chain Fv ((scFv') 2 ) molecule, domain antibody, diabody, triabody, hormone, Fab fragment, F(ab') 2 molecule, tandem scFv (taFv) fragment, receptor (e.g., cell surface receptor), ligand, aptamer, or biologically-active fragment thereof.
  • one or more of the first or second binding moiety is a human or humanized single-chain Fv molecule.
  • one or more of the first or second binding moiety is or specifically binds to a protein selected from the group consisting of an insulin-like growth factor 1 receptor (IGFlR), IGF2R, insulin-like growth factor (IGF), mesenchymal epithelial transition factor receptor (c-met; also known as hepatocyte growth factor receptor (HGFR)), hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), heregulin, fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), platelet-derived growth factor (PDGF), vascular endothelial growth factor receptor (VEGFR).
  • IGFlR insulin-like growth factor 1 receptor
  • IGF2R insulin-like growth factor
  • IGF insulin-like growth factor
  • IGF insulin-like growth factor
  • c-met mesenchymal epithelial transition factor receptor
  • HGF hepatocyte growth factor
  • EGFR epidermal growth factor receptor
  • EGF epi
  • vascular endothelial growth factor VEGF
  • tumor necrosis factor receptor TNFR
  • tumor necrosis factor alpha TNF- ⁇
  • folate receptor folate
  • transferrin receptor TfR
  • mesothelin Fc receptor
  • c-kil receptor c-kit
  • an integrin e.g., an ⁇ 4 integrin or a ⁇ -1 integrin
  • P-selectin P-selectin
  • sphingosine- 1 -phosphate receptor- 1 SlPR
  • hyaluronate receptor leukocyte function antigen- 1 (LFA- 1)
  • CD 106 vascular cell adhesion molecule 1 (VCAMl), CD 166 (activated leukocyte cell adhesion molecule (ALCAM)), CD 178 (Fas ligand), CD253 (TNF
  • the first or second binding moiety is or specifically binds to erythroblastic leukemia viral oncogene homolog (ErbB) receptor (e.g., ErbBl receptor; ErbB2 receptor; ErbB3 receptor; and ErbB4 receptor).
  • ErbB erythroblastic leukemia viral oncogene homolog
  • one or more of the first or second binding moiety (or, if present, the third or further binding moiety) is or specifically binds to alpha-fetoprotcin (AFP) or an interferon, or a biologically-active fragment thereof.
  • AFP alpha-fetoprotcin
  • one or more of the first or second binding moiety is natalizumab, infliximab, adalimumab, rituximab, alemtuzumab, bevacizumab, daclizumab, efalizumab, golimumab, certolizumab, trastuzumab, abatacept, etanercept, pertuzumab, cetuximab, panitumumab, or anakinra.
  • the HSA linker is conjoined to a diagnostic agent, a therapeutic agent, or both.
  • the diagnostic agent is a detectable label, such as a radioactive, fluorescent, or heavy metal label.
  • the therapeutic agent is a cytotoxic agent, cytostatic, or immunomodulatory agent. Cytotoxic agents include alkylating agents, antibiotics, antineoplastic agents, antiproliferative agents, antimetabolites, tubulin inhibitors, topoisomerase I or II inhibitors, hormonal agonists or antagonists, immunomodulators, DNA minor groove binders, and radioactive agents, or any agent capable of binding to and killing a tumor cell or inhibiting tumor cell proliferation.
  • Antineoplastic agents include cyclophosphamide, camptothecin, homocamptothecin, colchicine, combrestatin, combrestatin, rhizoxin, dolistatin, ansamitocin p3, maytansinoid, auristatin, caleachimicin, methotrexate, 5- fluorouracil (5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin, tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab, trastuzumab, and their derivatives.
  • the conjoined HSA linker is admixed with a pharmaceutically acceptable carrier, excipient, or diluent.
  • the HSA linker exhibits an in vivo half-life of between 6 hours and 7 days. In a further embodiment, the HSA linker exhibits an in vivo half-life greater than 8 hours.
  • a thirteenth aspect of the invention features an agent of any of the prior aspects of the invention (one through twelve), in which the HSA linker is replaced by another polypeptide linker.
  • the polypeptide linker sequence could be a mammalian, non-human serum albumin polypeptide sequence, such as, e.g., a bovine, murine, feline, and canine serum albumin (BSA) polypeptide sequence.
  • this polypeptide linker sequence is between 5 and 1,000 amino acids in length, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 amino acids in length, or any number of amino acids within this range.
  • the polypeptide linker sequence includes a single amino acid (including, but not limited to, e.g., glycine, alanine, serine, glutamine, leucine, and valine), or combinations of amino acids.
  • the HSA linker is replaced by an alpha-fetoprotein (AFP) polypeptide, e.g., mammalian AFP polypeptide, such as a human, murine, bovine, or canine AFP polypeptide.
  • AFP linker corresponds to the full-length human AFP polypeptide sequence (a.a. 1-609; SEQ ID NO:58), the mature human AFP polypeptide sequence lacking amino acids 1-18 of the signal sequence (a.a., 19-609 of SEQ ID NO: 58), or fragments thereof.
  • the AFP polypeptide linker contains at least 5 to 8 contiguous amino acids, preferably at least 10, 20, or 50 contiguous amino acids, more preferably at least 100 contiguous amino acids, and most preferably at least 200, 300, 400, or more contiguous amino acids of SEQ ID NO:58, or has at least 90% sequence identity (e.g., at least 95%, 97%, 99%, or more sequence identity) to a continguous polypeptide sequence of SEQ ID NO:58 having one or more of these lengths.
  • an AFP polypeptide linker sequence having 90% sequence identity to a 34-mer human AFP peptide corresponding to amino acids 446-479 of SEQ ID NO:58 may contain up to 3 amino acids altered from the 446-479 segment of SEQ ID NO:58.
  • sequence deviation in biologically active human AFP fragments is found in, e.g., U.S. Patent No. 5,707,963 (incorporated by reference herein), which discloses a 34-amino acid fragment of human AFP (SEQ ID NO:59) with flexibility at two amino acid residues (amino acid 9 and 22 of SEQ ID NO:59).
  • AFP polypeptide linker sequences include, e.g., amino acids 19-198 of SEQ ID NO:58 (human AFP Domain I), amino acids 217-408 of SEQ ID NO:58 (human AFP Domain 11), amino acids 409-609 of SEQ ID NO:58 (human AFP Domain III), amino acids 19-408 of SEQ ID NO:58 (human AFP Domain I+II), amino acids 217-609 of SEQ ID NO:58 (human AFP Domain II+III), and amino acids 285-609 of SEQ ID NO:58 (human AFP Fragment I).
  • the human AFP polypeptide linker sequence is an 8-amino acid sequence that includes amino acids 489-496 (i.e., EMTPVNPG) of SEQ ID NO:58.
  • kits that include any of the HSA linkers, HSA linker conjugates, or any other agents described in the first, second, third, fourth, fifth, eleventh, twelfth, and thirteenth aspects discussed above.
  • the kits further include instructions to allow a practitioner (e.g., a physician, nurse, or patient) to administer the compositions and agents contained therein.
  • the kits include multiple packages of a single- or multi-dose pharmaceutical composition containing an effective amount of an agent, e.g,.
  • kits may provide one or more pre-filled syringes containing an effective amount of an HSA linker conjugate or HSA linker, or any binding, diagnostic, and/or therapeutic agent conjugated thereto.
  • binding moieties e.g., antibodies or antibody fragments (e.g., scFv)
  • diagnostic agents e.g., radionuclide or chelating agents
  • therapeutic agents e.g., cytotoxic or immunomodulatory agents.
  • instruments or devices necessary for administering the pharmaceutical composition(s) may be included in the kits.
  • a kit may provide one or more pre-filled syringes containing an effective amount of an HSA linker conjugate or HSA linker, or any binding, diagnostic, and/or therapeutic agent conjugated thereto.
  • kits may also include additional components such as instructions or administration schedules for a patient suffering from a disease or condition (e.g., a cancer, autoimmune disease, or cardiovascular disease) to use the pharmaceutical composition(s) containing, e.g., an HSA linker conjugate or HSA linker, or any binding, diagnostic, and/or therapeutic agent conjugated thereto.
  • a disease or condition e.g., a cancer, autoimmune disease, or cardiovascular disease
  • pharmaceutical composition(s) containing, e.g., an HSA linker conjugate or HSA linker, or any binding, diagnostic, and/or therapeutic agent conjugated thereto.
  • antibody as used interchangeably herein, includes whole antibodies or immunoglobulins and any antigen-binding fragment or single chains thereof.
  • Antibodies can be mammalian (e.g., human or mouse), humanized, chimeric, recombinant, synthetically produced, or naturally isolated. In most mammals, including humans, whole antibodies have at least two heavy (H) chains and two light (L) chains connected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, C H 1, C R 2, and C H 3 and a hinge region between C H 1 and C H 2.
  • Each light chain consists of a light chain variable region (abbreviated herein as V L ) and a light chain constant region.
  • the light chain constant region consists of one domain, C L .
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system.
  • Antibodies of the present invention include all known forms of antibodies and other protein scaffolds with antibody-like properties.
  • the antibody can be a human antibody, a humanized antibody, a bispecific antibody, a chimeric antibody, or a protein scaffold with antibody-like properties, such as fibronectin or ankyrin repeats.
  • the antibody also can be a Fab, Fab'2, scFv, SMIP, diabody, nanobody, aptamers, or a domain antibody.
  • the antibody can have any of the following isotypes: IgG (e.g., IgGl, IgG2, IgG3, and IgG4), IgM, IgA (e.g., IgAl , IgA2, and IgAsec), IgD, or IgE.
  • IgG e.g., IgGl, IgG2, IgG3, and IgG4
  • IgM e.g., IgAl , IgA2, and IgAsec
  • IgD e.gAl , IgA2, and IgAsec
  • IgE IgE.
  • Antibodies that can be used as binding moieties, as defined herein, in combination with an HSA linker include, but are not limited to, natalizumab, infliximab, adalimumab, rituximab, alemtuzumab, bevacizumab, daclizumab, efalizumab, golimumab, certolizumab, trastuzumab, abatacept, etanercept, pertuzumab, cetuximab, and panitumumab.
  • antibody fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., ErbB2).
  • an antigen e.g., ErbB2
  • the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L , and C H I domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H 1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb including V H and V L domains; (vi) a dAb fragment (Ward et al, Nature 341 :544-546 (1989)), which consists of a V H domain; (vii) a dAb which consists of a V H or a V L domain; (viii) an isolated complementarity determining region (CDR); and
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al, Science 242:423-426 (1988) and Huston et al, Proc. Natl. Acad. ScL USA 85:5879-5883 (1988)).
  • scFv single chain Fv
  • These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • Antibody fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.
  • autoimmune disease is meant a disease in which an immune system response is generated against self epitopes or antigens.
  • autoimmune diseases include, but are not limited to, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac Sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Grave's disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary
  • binding moiety any molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Binding moieties include but are not limited to antibodies (e.g., monoclonal, polyclonal, recombinant, humanized, and chimeric antibodies), antibody fragments (e.g., Fab fragments, Fab'2, scFv antibodies, SMIP, domain antibodies, diabodies, minibodies, scFv-Fc, affibodies, nanobodies, and domain antibodies), receptors, ligands, aptamers, and other molecules having a known binding partner.
  • antibodies e.g., monoclonal, polyclonal, recombinant, humanized, and chimeric antibodies
  • antibody fragments e.g., Fab fragments, Fab'2, scFv antibodies, SMIP, domain antibodies, diabodies, minibodies, scFv-Fc, affibodies, nanobodies, and
  • biologically-active is meant that a molecule, including biological molecules, such as nucleic acids, peptides, polypeptides, and proteins, exerts a physical or chemical activity on itself or other molecule.
  • a “biologically-active” molecule may possess, e.g., enzymatic activity, protein binding activity (e.g., antibody interactions), or cytotoxic activities are “biologically-active.”
  • chimeric antibody refers to an immunoglobulin or antibody whose variable regions derive from a first species and whose constant regions derive from a second species. Chimeric antibodies can be constructed, for example, by genetic engineering, from immunoglobulin gene segments belonging to different species (e.g., from a mouse and a human).
  • connector or “peptide connector” is meant an amino acid sequence of 2 to 20 residues in length that is covalently attached to one or both of the amino or carboxy termini of an HSA linker, or is covalently attached to one or more residues of an HSA linker (e.g., a residue between the amino and carboxy terminal residues).
  • the peptide connector attached to the amino terminus of an HSA linker has the amino acid sequence AAS or AAQ and the connector attached to the carboxy terminus has the amino acid sequence "AAAL" (SEQ ID NO: 5).
  • an agent e.g., an HSA linker bonded with one or more binding moieties or diagnostic or therapeutic agents with or without a connector sequence
  • an agent e.g., an HSA linker bonded with one or more binding moieties or diagnostic or therapeutic agents with or without a connector sequence
  • sufficient to produce a desired result for example, killing a cancer cell, reducing tumor cell proliferation, reducing inflammation in a diseased tissue or organ, or labeling a specific population of cells in a tissue, organ, or organism (e.g., a human).
  • human antibody is intended to include antibodies, or fragments thereof, having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences as described, for example, by Kabat et al, (Sequences of proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)). Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
  • the human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • human antibody is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., a humanized antibody or antibody fragment).
  • humanized antibody refers to any antibody or antibody fragment that includes at least one immunoglobulin domain having a variable region that includes a variable framework region substantially derived from a human immunoglobulin or antibody and complementarity determining regions (e.g., at least one CDR) substantially derived from a non-human immunoglobulin or antibody.
  • an "inflammatory signaling inhibitor” or “ISI” is an agent that decreases the binding between a pro-inflammatory cytokine (e.g., TNF-alpha, TNF-beta, or IL-I) and its receptor (e.g., TNF receptor 1 or 2, or IL-I receptor, respectively); decreases the binding of activating molecules to pro-inflammatory cell surface signaling molecules (e.g., CD20, CD25, CTLA-4, CD80/CD86, or CD28); or decreases the downstream activation of, or activity of, intracellular signaling molecules that are activated following the binding of pro-inflammatory cytokines to their receptors or the binding of activating molecules to pro-inflammatory cell surface signaling molecules (e.g., an agent that decreases the activation of, or activity of, signaling molecules in the p38 MAPK signaling pathway).
  • a pro-inflammatory cytokine e.g., TNF-alpha, TNF-beta, or IL-I
  • its receptor e.g.,
  • the decrease mediated by an ISI may be a decrease in binding between a pro-inflammatory cytokine and its receptor, a decrease in binding of an activating molecule to a pro-inflammatory cell surface signaling molecule, or a decrease in intracellular signaling which occurs following the binding of pro-inflammatory cytokines to their receptors or activating molecules to pro-inflammatory cell surface signaling molecules.
  • a decrease mediated by an ISI is a decrease of at least about 10%, preferably at least 20%, 30%, 40%, or 50%, more preferably at least 60%, 70%, 80%, or 90% (up to 100%).
  • An ISI may act by reducing the amount of proinflammatory cytokine (e.g., TNF-alpha, TNF -beta, or IL-I) freely available to bind the receptor.
  • cytokine e.g., TNF-alpha, TNF -beta, or IL-I
  • an ISI may be a soluble pro-inflammatory cytokine receptor protein (e.g., a soluble TNF receptor fusion protein such as etanercept (ENBREL®) or lenercept), or a soluble pro-inflammatory cell surface signaling molecule (e.g., a soluble CTLA-4 (abatacept)), or an antibody directed against a pro-inflammatory cytokine or a pro-inflammatory cell surface signaling molecule (e.g., an anti-TNF antibody, such as adalimumab.
  • an anti-TNF antibody such as adalimumab.
  • an ISI may act by disrupting the ability of the endogenous wild- type pro-inflammatory cytokine or the proinflammatory cell surface signaling molecule to bind to its receptor (e.g., TNF receptor 1 or 2, IL-I receptor - e.g., anakinra, or CDl Ia - e.g., efalizumab (RAPTIV A®, Genentech)).
  • its receptor e.g., TNF receptor 1 or 2, IL-I receptor - e.g., anakinra, or CDl Ia - e.g., efalizumab (RAPTIV A®, Genentech
  • dominant-negative TNF-alpha variants examples include XENP345 (a pegylated version of TNF variant A145R/I97T) and XproTM1595, and further variants disclosed in U.S. Patent Application Publication Nos. 20030166559 and 20050265962, herein incorporated by reference.
  • An example of a dominant negative IL-I variant is anakinra (KINERET®), which is a soluble form of IL-I that binds to the IL-I receptor without activating intracellular signaling pathways.
  • Inflammatory signaling inhibitors which can be used in the present invention, are also small molecules which inhibit or reduce the signaling pathways downstream of pro-inflammatory cytokine or proinflammatory cell surface signaling molecules (e.g., DE 096).
  • Examples of ISIs of this kind include inhibitors of p38 MAP kinase, e.g., 5-amino-2-carbonylthiopene derivatives (as described in WO 04/089929, herein incorporated); ARRY-797; BIRB 796 BS, (1-5- tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-2(morpholin-4-yl-ethoxy)-naphtalen-l-yl]- urea); CHR-3620; CNI-1493; FR-167653 (Fujisawa Pharmaceutical, Osaka, Japan); ISIS 101757 (Isis Pharmaceuticals); ML3404; NPC31145; PD 169316; PHZ
  • an ISI may interfere with the processing of a pro-inflammatory cytokine (e.g., TNF-alpha and TNF-beta) from its membrane bound form to its soluble form.
  • Inhibitors of TACE are ISIs of this class. Examples of inhibitors of TACE include BB-1101, BB-3103, BMS-561392, butynyloxyphenyl ⁇ -sulfone piperidine hydroxomates, CH4474, DPC333, DPH-067517, GM6001, GW3333, Ro 32-7315, TAPI-I, TAPI-2, and TMI 005.
  • Additional examples of ISIs include short peptides derived from the E. coli heat shock proteins engineered for disease-specific immunomodulatory activity (e.g.. dnaJPl).
  • integrin antagonist any agent that reduces or inhibits the biological activity of an integrin molecule (e.g., a reduction or inhibition of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more relative to the biological activity in the absence of the integrin antagonist), such as the ⁇ 4 subunit of an integrin molecule.
  • the agent may act directly or indirectly on the ⁇ 4 integrin subunit (NCBI Accession No. P13612; Takada et al, EMBO J.
  • Non-limiting exemplary integrin antagonists suitable for use with the present invention may include proteins, blocking peptides, antibodies, such as natalizumab (TYSAB RI ®), and small molecule inhibitors.
  • ⁇ 4 integrin antagonists include, but are not limited to, natalizumab (Elan/Biogen poutier; see, e.g., U.S. Patent Nos. 5,840,299; 6,033,665; 6,602,503; 5,168,062; 5,385,839; and 5,730,978; incorporated by- reference herein), oMEPUPA-V (Biogen; U.S. Patent No.
  • ⁇ 4 integrin antagonists include the small molecules described in U.S. Patent Nos.
  • ⁇ 4 integrin antagonists include the phenylalanine derivatives described in: U.S. Patent Nos. 6,197,794; 6,229,011 ; 6,329,372; 6,388,084; 6,348,463; 6,362,204; 6,380,387; 6,445,550; 6,806,365; 6,835,738; 6,855,706; 6,872,719; 6,878,718; 6,911,451 ; 6,916,933; 7,105,520; 7,153,963; 7,160,874; 7,193,108; 7,250,516; and 7,291,645 (each herein incorporated by reference).
  • Additional amino acid derivatives that are ⁇ 4 integrin antagonists include those described in, e.g., U.S. Patent Application Publication Nos. 2004/0229859 and 2006/021 1630 (herein incorporated by reference), and PCT Publication Nos. WO 01/36376; WO 01/47868; and WO 01/70670; all of which are incorporated by reference herein.
  • Other examples of ⁇ 4 integrin antagonists include the peptides, and the peptide and semi-peptide compounds described in, e.g., PCT Publication Nos.
  • An additional example of an ⁇ 4 integrin antagonist is the pegylated molecule described in U.S. Patent Application Publication No. 2007/066533 (herein incorporated by reference). Examples of antibodies that are ⁇ 4 integrin antagonists include those described in, e.g., PCT Publication Nos.
  • interferon is meant a mammalian (e.g., a human) interferon -alpha, -beta, - gamma, or -tau polypeptide, or biologically-active fragment thereof, e.g., IFN- ⁇ (e.g., IFN- ⁇ -la; see U.S. Patent Application No. 20070274950, incorporated herein by reference), IFN- ⁇ -lb, IFN- ⁇ -2a (see PCT Application No.
  • IFN- ⁇ -2b IFN- ⁇
  • IFN- ⁇ IFN- ⁇
  • IFN- ⁇ e.g., described in U.S. Patent No. 7,238,344, incorporated by reference; IFN-b-la (AVONEX® and REB1F®), as described in U.S. Patent No. 6,962,978, incorporated by reference
  • IFN- ⁇ - Ib BETASERON®, as described in U.S. Patent Nos. 4,588,585; 4,959,314; 4,737,462; and 4,450,103; incorporated by reference in their entirety
  • IFN-g, and IFN-t as described in U.S. Patent No. 5,738,845 and U.S. Patent Application Publication Nos. 20040247565 and 20070243163; incorporated by reference).
  • HSA linker conjugate is meant a human serum albumin (HSA) linker in combination with (preferably covalently linked to) one or more binding moieties, peptide connectors, diagnostic agents, or therapeutic agents.
  • HSA human serum albumin
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • Monoclonal antibodies can be prepared using any art recognized technique and those described herein such as, for example, a hybridoma method, as described by Kohler et ah, Nature 256:495 (1975), a transgenic animal (e.g., Lonberg et al, Nature 368(6474):856-859 (1994)), recombinant DNA methods (e.g., U.S. Pat. No. 4,816,567), or using phage, yeast, or synthetic scaffold antibody libraries using the techniques described in, for example, Clackson et al., Nature 352:624-628 (1991) and Marks et al, J. MoI. Biol. 222:581-597 (1991).
  • pharmaceutically acceptable carrier a carrier which is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered.
  • physiological saline a carrier which is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered.
  • physiological saline a carrier which is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered.
  • physiological saline is physiological saline.
  • Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (18 th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA.
  • proliferative disease or “cancer” is meant any condition characterized by abnormal or unregulated cell growth.
  • proliferative diseases include, for example, solid tumors such as: sarcomas (e.g., clear cell sarcoma), carcinomas (e.g., renal cell carcinoma), and lymphomas; tumors of the breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, bilecyst, bile duct, small intestine, urinary system (including the kidney, bladder, and epithelium of the urinary tract), female genital system (including the uterine neck, uterus, ovary, chorioma, and gestational trophoblast), male genital system (including the prostate, seminal vesicle, and testicles), endocrine glands (including the thyroid gland, adrenal gland, and pituitary body), skin (including angioma, melanoma, sarcoma originating from bone or soft tissue, and Kaposi's sarcom
  • recombinant antibody refers to an antibody prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for immunoglobulin genes (e.g., human immunoglobulin genes) or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library (e.g., containing human antibody sequences) using phage, yeast, or synthetic scaffold display, and (d) antibodies prepared, expressed, created, or isolated by any other means that involve splicing of immunoglobulin gene sequences (e.g., human immunoglobulin genes) to other DNA sequences.
  • a host cell transformed to express the antibody e.g., from a transfectoma
  • combinatorial antibody library e.g., containing human antibody
  • binding moiety e.g., an antibody, antibody fragment, receptor, ligand, or small molecule portion of an agent as described herein
  • a target molecule e.g., a secreted target molecule, such as a cytokine, chemokine, hormone, receptor, or ligand
  • a cell or tissue bearing the target molecule e.g., a cell surface antigen, such as a receptor or ligand
  • a certain degree of nonspecific interaction may occur between a binding moiety and a non-target molecule (present alone or in combination with a cell or tissue).
  • specific binding may be distinguished as mediated through specific recognition of the target molecule.
  • Specific binding results in a stronger association between the binding moiety (e.g., an antibody) and e.g., cells bearing the target molecule (e.g., an antigen) than between the binding moiety and e.g., cells lacking the target molecule.
  • Specific binding typically results in greater than 2-fold, preferably greater than 5 -fold, more preferably greater than 10-fold and most preferably greater than 100-fold increase in amount of bound binding moiety (per unit time) to e.g., a cell or tissue bearing the target molecule or marker as compared to a cell or tissue lacking that target molecule or marker.
  • Binding moieties bind to the target molecule or marker with a dissociation constant of e.g., less than 10 "6 M, more preferably less than 10 "7 M, 10 "8 M, 10 "9 M, 10 "10 M, 10 "1 ' M, or 10 "12 M, and most preferably less than 10 "13 M, 10 "14 M, or 10 "15 M.
  • Specific binding to a protein under such conditions requires a binding moiety that is selected for its specificity for that particular protein.
  • a variety of assay formats are appropriate for selecting binding moieties (e.g., antibodies) capable of specifically binding to a particular target molecule.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • sequence identity is meant (in the context of comparing a polynucleotide or polypeptide sequence to a reference sequence) that the polynucleotide or polypeptide sequence is the same as the reference sequence or has a specified percentage of nucleotides or amino acid residues that are the same at the corresponding locations within the reference sequence when the two sequences are optimally aligned.
  • surface-exposed amino acid residue or “surface-exposed” is meant an amino acid residue that is present on the exterior face of the folded and conformationally-correct tertiary structure of a HSA polypeptide. Such residues can be substituted with e.g., other, chemically-reactive, amino acids (e.g., cysteine) to allow for site-specific conjugation of diagnostic or therapeutic agents.
  • amino acids e.g., cysteine
  • surface-exposed amino acid residues can be substituted to allow (e.g., by addition of serine, threonine, or asparagine residues, or glycosylation motifs) or prevent (e.g., by removal of serine, threonine, or asparagine residues, or glycosylation motifs) glycosylation.
  • amino acid residues include, but are not limited to, threonine at position 496, serine at position 58, threonine at position 76, threonine at position 79, threonine at position 83, threonine at position 125, threonine at position 236, serine at position 270, serine at position 273, serine at position 304, serine at position 435, threonine at position 478, threonine at position 506, and threonine at position 508 (amino acid numbering is relative to e.g., the sequence of the HSA linker set forth in SEQ ID NO:1).
  • a “subject” refers to a human patient or a nude mouse xenograft model comprising human tumor cells.
  • target molecule or “target cell” is meant a molecule (e.g., a protein, epitope, antigen, receptor, or ligand) or cell to which a binding moiety (e.g., an antibody), or an HSA conjugate that contains one or more binding moieties (e.g., an HSA linker bonded to one or more antibodies or antibody fragments) can specifically bind.
  • a binding moiety e.g., an antibody
  • HSA conjugate that contains one or more binding moieties (e.g., an HSA linker bonded to one or more antibodies or antibody fragments) can specifically bind.
  • Preferred target molecules are exposed on the exterior of a target cell (e.g., a cell-surface or secreted protein) but target molecules may alternately or also be present in the interior of a target cell.
  • Treating preferably provides a reduction (e.g., by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or even 100%) in the progression or severity of a human disease or disorder (e.g., an autoimmune or proliferative disease), or in the progression, severity, or frequency of one or more symptoms of the human disease or disorder in a subject.
  • a human disease or disorder e.g., an autoimmune or proliferative disease
  • Figure 1 is an illustration showing the schematic representation of an exemplary HSA linker conjugate.
  • the connector between the amino terminal binding moiety and the HSA linker has a sequence of alanine, alanine and serine.
  • the connector between the HSA linker and the carboxy terminal binding moiety has a sequence of alanine, alanine, alanine, leucine (SEQ ID NO:5).
  • Figure 2 is a graph showing that B2B3 variants inhibit HRG-induced pErbB3 in ZR75-1 breast cancer cells.
  • Figures 3A-D are graphs showing the inhibition of phosphorylated ErbB3 in BT474 breast cancer cells following 24 hour pre-treatment with the B2B3 HSA linker conjugates B1D2-2 (A5-HSA-B1D2, Figure 3A), B1D2-1 (H3-HSA-B1D2, Figure 3B), B2B3-10 (H3-HSA-F5B6H2, Figure 3C), and B2B3-8 (F4-HSA-F5B6H2, Figure 3D). Further details regarding these HSA linker conjugates is set forth below, e.g., in Table 6.
  • FIGS 4A-D are graphs showing the inhibition of phosphorylated AKT in BT474 breast cancer cells following 24 hour pre-treatment with the B2B3 HSA linker conjugates B1D2-2 (A5-HSA-B1D2, Figure 4A), B1D2-1 (H3-HSA-B1D2, Figure 4B), B2B3-10 (H3-HSA-F5B6H2, Figure 4C), and B2B3-8 (F4-HSA-F5B6H2, Figure 4D). Further details regarding these HSA linker conjugates is set forth below, e.g., in Table 6.
  • FIGS 5A-D are graphs showing that inhibition of phosphorylated ERK in BT474 breast cancer cells following 24 hour pre-treatment with the B2B3 HSA linker conjugates B1D2-2 (A5-HSA-B1D2, Figure 5A), B1D2-1 (H3-HSA-B1D2, Figure 5B), B2B3-10 (H3-HSA-F5B6H2, Figure 5C), and B2B3-8 (F4-HSA-F5B6H2, Figure 5D). Further details regarding these HSA linker conjugates is set forth below, e.g., in Table 6
  • Figure 6 is a graph showing that treatment of BT474 breast cancer cells with B2B3-1 variants causes Gl arrest and a decrease in the number of cells in S phase.
  • Figure 7 is a flow cytometry histogram showing that pre-incubation of BT-474- M3 cells with 1 ⁇ M B2B3-1 substantially blocks binding of HRG.
  • Figures 8A-D are graphs showing that B2B3-1 inhibits ErbB3 and AKT phosphorylation in B-T474-M3 and ZR75-30 cell lines.
  • Breast cancer cell lines BT-474- M3 ( Figures 8 ⁇ and 8C) and ZR75-3 0 ( Figures 8B and 8D) were pre-treatcd with a dose titration of B2B3-1 for 24 hours and then stimulated for 10 minutes with 5 nM of HRG l ⁇ EGF domain. The phosphorylation status of ErbB3 and AKT was then examined using an ELISA assay.
  • Figure 9 is a photograph of a Western blot that shows the effect of treatment with increasing concentrations of B2B3-1 on signaling proteins in BT474 breast cancer cells. "p-" indicates the tyrosine-posphorylated form of the signaling protein. Beta tubulin (not a signaling protein in this context) provides a loading control. Beta tubulin (not a signaling protein in this context) provides a loading control.
  • Figure 10 is a photograph of a Western blot that shows the immunoprecipitation of B2B3-1 treated BT474 breast cancer cells.
  • Beta tubulin provides a control for levels of cellular proteins input into the immunoprecipitation reactions.
  • Figures HA-C are graphs showing B2B3-1 treatment of BT-474 cell line causes Gl arrest and a decrease in the population of cells in S phase ( Figure 1 IA), and inhibits colony formation in both BT-474 and SKBr3 cells compared to untreated cells (Figure 1 IB).
  • B2B3-1 inhibits proliferation of BT-474-M3 cells in a cell impedance assay ( Figure HC).
  • Figure 12 is a graph showing that B2B3-1 does not stimulate ErbB3 phosphorylation in ZR75-1 cells.
  • Figures 13A-B are graphs showing that B2B3-1 binds specifically to ErbB3 ( Figure 13A) and ErbB2 ( Figure 13B).
  • Figure 14 is a graph showing that avidity binding of B2B3-1 to MALME-3 cells results in a significant increase in apparent binding affinity compared to ErbB2-only binding variant, SKO-3, and ErbB3-only binding variant, SKO-2.
  • Figures 16 is a graph showing B2B3-1 dose response in a BT-474-M3 breast cancer xenograft model. The relationship of B2B3-1 dose on tumor response was assessed in the BT-474-M3 breast tumor line at the doses indicated. HSA was given at 52.5 mg/kg, which is an equimolar dose to the 90 mg/kg B2B3-1 dose.
  • FIGS 17A-E are graphs showing that B2B3-1 is effective in multiple xenograft models in an ErbB2 dependent manner.
  • Calu-3 human lung adenocarcinoma; Figure 17A
  • SKOV-3 human ovarian adenocarcinoma; Figure 17B
  • NCI-N87 human gastric carcinoma
  • Figure 17C human kidney adenocarcinoma
  • Figure 17D human kidney adenocarcinoma
  • MDA-MB-361 human breast adenocarcinoma; Figure 17E
  • Figures 18A-B are graphs showing that the over-expression of ErbB2 converts B2B3-1 non-responder ADRr breast cancer xenograft model into a responder.
  • ErbB2 was over-expressed in wild type ADRr xenografts ( Figure 18A) and ADRr-E2 xenografts ( Figure 18B) using a retroviral expression system.
  • Figures 19A-B are graphs showing that B2B3-1 activity correlates positively with ErbB2 expression levels in vitro ( Figure 19A) and in vivo (Figure 19B).
  • Figure 20A-B show that B2B3-1 treatment modifies tumor cell cycling.
  • Figure 2OA includes fluorescent micrographs showing that B2B3-1 treatment of BT474-M3 breast tumor cells for 6 hours results in translocation of cell cycle inhibitor p27 k ⁇ pl to the nucleus. Hoechst stain was used to identify the nucleus.
  • Figure 2OB is a Western blot of BT-474-M3 cells treated with B2B3-1 for 72 hours, which resulted in a decrease in the levels of the cell cycle regulator Cyclin Dl. The cytoskeleton protein vinculin was probed as a protein loading control in this experiment.
  • Figures 21A-B are micrographs showing that B2B3-1 treatment of BT474 breast tumor xenografts results in translocation of p27 kipl to the nucleus.
  • BT474 breast tumor xenografts were treated with B2B3-1 ( Figure 21 A) at a dose of 30 mg/kg or an equimolar dose of HSA ( Figure 21B) every 3 days for a total of 4 doses and stained for p27 k ⁇ l .
  • Figure 22A-B are fluorescent micrographs showing that B2B3-1 treatment results in a reduction of the proliferation marker Ki67 in BT474-M3 breast cancer xenograft.
  • BT474-M3 breast tumor xenografts were treated with B2B3-1 (Figure 22A) at a dose of 30 mg/kg or an equimolar dose of HSA ( Figure 22B) every 3 days for a total of 4 doses.
  • Figures 23A-B are fluorescent micrographs showing that B2B3-1 treatment results in a reduction of vessel density in BT474-M3 breast cancer xenograft tumors (as determined by a reduction in CD31 staining).
  • BT474-M3 breast tumor xenografts were treated with B2B3-1 ( Figure 23A) at a dose of 30 mg/kg or an equimolar dose of HSA ( Figure 23B) every 3 days for a total of 4 doses.
  • Figures 24 A-B are graphs showing that B2B3-1 inhibits phosphorylation of ErbB3 in vivo. Lysates from individual BT-474-M3 xenograft tumors treated with B2B3-1 (M1-M5) or control HSA (H1-H2) were subjected to SDS-PAGE and probed for pErbB3 and beta tubulin using Western blot analysis (Figure 24A). Normalization of the mean pErbB3 signal to the mean beta tubulin signal demonstrated that B2B3-1 treated tumors contained significantly less pErbB3 than HSA tumors ( Figure 24B).
  • Figures 25A and B are graphs showing the in vivo activity of B2B3-1 in BT- 474-M3 shPTEN and shControl xenografts.
  • Cultured BT-474-M3 tumor cells were transfected with a control vector ( Figure 25A) or with a retroviral vector expressing shPTEN ( Figure 25B), which knocks out PTEN activity.
  • BT-474-M3 breast cancer cells thus engineered to knock out PTEN activity were injected into the right flank of mice, while cells transfected with control vector were injected into the left flank of the same mouse.
  • mice were treated with 30mg/kg B2B3-1 every 3 days or 10mg/kg Herceptin every week and HSA was injected as a control at an equimolar dose to B2B3-1.
  • B2B3-1 and Herceptin promoted a reduction in the size of tumors formed by control BT-474-M3 breast cancer cells ( Figure 25 ⁇ ), whereas only B2B3-1 (and not Herceptin) promoted a reduction in the size of tumors formed by BT-474-M3 breast cancer cells lacking expression of PTEN ( Figure 25B).
  • Figures 26A-B show that B2B3-1 inhibits phosphorylation of AKT in BT- 474-M3 xenografts that have reduced PTEN activity. Tumors were lysed following the completion of treatment (q3dxl 1) and tested for PTEN, pErbB3, and pAKT expression levels by Western blot analysis (Figure 26A). Densitometry on the band intensity for pAKT normalized to total AKT and total protein demonstrate that B2B3- 1 was able to inhibit phosphorylation of this protein, when Herceptin did not (Figure 26B).
  • Figures 27A-D are graphs showing the single dose pharmacokinetic properties of 5 (Figure 27A), 15 (Figure 27B), 30 (Figure 27C), and 45 (Figure 27D) mg/kg bolus dose of B2B3-1 in nu/nu mice.
  • B2B3-1 serum concentrations are comparable measured by the HSA assay or ErbB2/ErbB3 assay, indicating that the antigen binding activity of B2B3-1 is retained in circulation.
  • Figure 28 is a graph showing the dose-exposure relationship for 5, 15, 30, and 45 mg/kg bolus doses of B2B3-1 in nude mice. Increases in dose result in a linear increase in overall exposure to B2B3-1.
  • Figure 30 is an illustration of the B2B3-1 expression plasmid pMP10k4H3- mHSA-BlD2.
  • Figure 31 is an illustration of the neomycin resistance plasmid pSV2-neo.
  • Figure 32 is an illustration of the hygromycin resistance plasmid pTK-Hyg.
  • Figure 33 shows data demonstrating that B2B3-1 dosed q7d shows equivalent efficacy to q3d dosing.
  • Figure 34 shows western blot data demonstrating that B2B3-1 and trastuzumab exhibit different mechanisms of ErbB3 inhibition.
  • Figure 35 A-C shows the results of the experiments detailed in Example 43 in which B2B3-1 combination treatment with trastuzumab was studied in spheroids of various human breast cancer cell lines, which serve as a model for human breast tumors.
  • Figure 35A shows data obtained using BT-474-M3 cells
  • Figure 35B shows data obtained using SKBR3 cells
  • Figure 35C shows data obtained using MDA-MB-361 cells.
  • the molar concentration of B2B3-1, alone or in combination is given along the X-axis.
  • the molar concentration of trastuzumab, alone or in combination is one third that of each indicated concentration of B2B3-1.
  • Figure 36 show the results of the in vivo tumor xenograft experiments detailed in Example 44. "Days" on the X-axis indicates days post tumor implant. Error bars for each data point represent the response for at least two independent xenografts.
  • Figure 37 shows data obtained from a xenograft model essentially as described in Example 44, except that the tumor cells used are N-87 gastric tumor cells which may be obtained from the US National Cancer Institute.
  • Figure 38 shows a subset of the data presented in Figure 36.
  • HSA linker conjugates e.g., binding, diagnostic, or therapeutic agents
  • HSA linker conjugates e.g., binding, diagnostic, or therapeutic agents
  • Such HSA linker conjugates have desirable properties such as, for example, an increased in vivo half-life of between 6 hours and 7 days, and do not induce significant humoral or cell-mediated immune responses when administered in vivo to a mammal (e.g., a human).
  • the invention provides a mutated HSA linker that has two defined amino acid substitutions (i.e., the "C34S" and "N503Q" substitutions, as set forth in SEQ ID NO:1).
  • the invention provides an HSA linker bonded to one or more binding moieties (e.g., antibodies, antibody fragments, receptor/ligands, or small molecules) for diagnostic or therapeutic applications in a mammal (e.g., a human) in vivo or for use in vitro in connection with mammalian cells, tissues, or organs.
  • a mammal e.g., a human
  • the HSA linker may be coupled to one or more immunomodulatory agents, cytotoxic or cytostatic agents, detectable labels, or radioactive agents for diagnostic or therapeutic applications in a mammal (or in connection with a mammalian cell, tissue, or organ).
  • An HSA linker conjugate which includes the HSA linker, can be optionally combined with one or more pharmaceutically acceptable carriers or excipients and can be formulated to be administered intravenously, intramuscularly, orally, by inhalation, parenterally, intraperitoneally, intraarterially, transdermally, sublingually, nasally, through use of suppositories, transbuccally, liposomally, adiposally, opthalmically, intraocularly, subcutaneously, intrathecally, topically, or locally.
  • HSA linker conjugate can, but need not, be combined or coadministered with one or more biologically-active agents (e.g., biological or chemical agents, such as chemotherapeutics and antineoplastic agents).
  • biologically-active agents e.g., biological or chemical agents, such as chemotherapeutics and antineoplastic agents.
  • the invention provides a kit, with instructions, for the conjugation of binding moieties (e.g., antibodies, antibody fragments, receptors or ligands), immunomodulatory agents, cytotoxic or cytostatic agents, detectable labels, or radioactive agents to the HSA linker to prepare HSA linker conjugates that can be used for diagnostic or therapeutic applications.
  • binding moieties e.g., antibodies, antibody fragments, receptors or ligands
  • immunomodulatory agents e.g., cytotoxic or cytostatic agents, detectable labels, or radioactive agents
  • An HSA linker may comprise a wild-type HSA amino acid sequence, as set forth in SEQ ID NO:3.
  • the HSA linker may comprise an altered, or mutated, sequence.
  • One mutated HSA linker contains two amino acid mutations, at positions 34 and 503, relative to the wild-type HS ⁇ amino acid sequence set forth in SEQ ID NO:3.
  • the cysteine residue at position 34 i.e., C34
  • the asparagine residue at position 503 can be mutated to any amino acid residue other than asparagine (e.g., glutamine, serine, histidine, or alanine).
  • the HSA linker has the the amino acid and corresponding nucleotide sequence set forth in SEQ ID NOS: 1 and 2, respectively. This mutated HSA linker contains two amino acid substitutions (i.e., serine for cysteine at amino acid residue 34 ("C34S”) and glutamine for asparagine at amino acid residue 503 ("N503Q”)).
  • the HSA linker when bonded to one or more binding moieties (e.g., antibodies, antibody fragments (e.g., single chain antibodies), or other targeting or biologically active agents (e.g., receptors and ligands)), confers several beneficial pharmacologic properties to those conjugates and to additional diagnostic or therapeutic agents also conjoined (e.g., immunomodulatory agents, cytotoxic or cytostatic agents, detectable labels, or radioactive agents)) relative to the pharmacologic properties of these agents in the absence of the HSA linker.
  • binding moieties e.g., antibodies, antibody fragments (e.g., single chain antibodies), or other targeting or biologically active agents (e.g., receptors and ligands)
  • additional diagnostic or therapeutic agents also conjoined (e.g., immunomodulatory agents, cytotoxic or cytostatic agents, detectable labels, or radioactive agents)) relative to the pharmacologic properties of these agents in the absence of the HSA linker.
  • These benefits can include decreased immunogenicity (e.g., decreased host antibody neutralization of linker-antibody conjugates), increased detection of HSA linker conjugates (e.g., by mass spectroscopy) and increased pharmacologic half-life (e.g., a half-life greater than 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) when administered to a mammal (e.g., a human).
  • pharmacologic half-life e.g., a half-life greater than 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days
  • the asparagine at amino acid residue 503 is sensitive to deamination, likely resulting in reduced pharmacologic half-life.
  • the substitution of glutamine for asparagine at amino acid residue 503 can result in increased pharmacologic half-life of the HSA linker, and correspondingly, of conjugate agents that include the HSA linker when administered to a mammal (e.g., a human) or cells, tissues, or organs thereof.
  • the mutated HSA linker includes domain I of HSA (SEQ ID NO:53; residues 1-197 of SEQ ID NO:1), domain III of HSA (SEQ ID NO:55; residues 381-585 of SEQ ID NO:1), combination of domains I and III of HSA, or a combination of domain I or III of HSA with domain II of HSA (SEQ ID N ⁇ :54; residues 189-385 of SEQ ID NO:1).
  • an HSA linker can include domains I and II, I and III, or II and III.
  • cysteine residue at position 34 i.e., C34 of domain I (SEQ ID NO:53) can be mutated to any amino acid residue other than cysteine (e.g., serine, threonine, or alanine).
  • asparagine residue at position 503 i.e., N503 of domain III (SEQ ID NO:55) can be mutated to any amino acid residue other than asparagine (e.g., glutamine, serine, histidine, or alanine).
  • HSA linkers can be incorporated into an HSA linker conjugate, which includes one or more of a peptide connector, a binding moiety, and therapeutic or diagnostic agents, each of which is described in detail below.
  • peptide connectors that can be bonded (e.g, covalently (e.g., a peptidic bond), ionically, or hydrophobically bonded, or via a high- affinity protein-protein binding interaction (e.g., biotin and avidin)) to the amino or carboxy termini of an HSA linker.
  • peptide connectors provide flexible tethers to which any of the binding moieties described herein can be attached.
  • a peptide connector may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids in length.
  • the connector is a sequence of, e.g., glycine, alanine, serine, glutamine, leucine, or valine residues.
  • the connector can be solely glycine, alanine, serine, glutamine, leucine, or valine residues, or it may be any combination of these residues up to about 20 amino acids in length.
  • the connector attached to the amino terminus of an HSA linker has the amino acid sequence AAS or AAQ and the connector attached to the carboxy terminus has the amino acid sequence "AAAL" (SEQ ID NO:5).
  • the connector can be covalently bound to the amino or carboxy terminal residue of the HSA linker, to an amino acid residue within the HSA linker, or can be included between one or more binding moieties, if present.
  • the HSA linker with or without one or more peptide connectors described above, one or more of the binding moieties described below, polypeptide-based detectable labels, and other polypeptide-based therapeutic agents, can be produced recombinantly.
  • a nucleotide sequence encoding the HSA linker (and one or more of the optional elements) may be expressed (e.g., in a plasmid, viral vector, or transgenically) in a bacterial (e.g., E.
  • HSA linker in the host cell, tissue, or organ, the skilled artisan may isolate and purify the HSA linker using standard protein purification methods (e.g., FPLC or affinity chromatography).
  • FPLC protein purification methods
  • affinity chromatography A recombinant expression system for the production of an HSA linker in combination with two binding moieties is illustrated in Figure 1.
  • the HSA linker with or without one or more of the optional elements described above, can be synthetically produced.
  • the HSA linker or HSA linker conjugate can be prepared by techniques generally established in the art of peptide synthesis, such as the solid-phase approach. Solid-phase synthesis involves the stepwise addition of amino acid residues to a growing peptide chain that is linked to an insoluble support or matrix, such as polystyrene. The C-terminus residue of the peptide is first anchored to a commercially available support with its amino group protected with an N-protecting agent such as a t-butyloxycarbonyl group (tBoc) or a fluorenylmethoxycarbonyl (FMOC) group.
  • tBoc t-butyloxycarbonyl group
  • FMOC fluorenylmethoxycarbonyl
  • the amino-protecting group is removed with suitable deprotecting agents such as TFA in the case of tBOC or piperidine for FMOC and the next amino acid residue (in N-protected form) is added with a coupling agent such as dicyclocarbodiimide (DCC).
  • a coupling agent such as dicyclocarbodiimide (DCC).
  • DCC dicyclocarbodiimide
  • the agent is cleaved from the support with a suitable reagent, such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF).
  • TFA trifluoroacetic acid
  • HF hydrogen fluoride
  • HSA linker conjugates may include one or more binding moieties, such as antibodies, antibody fragments (as defined herein, e.g., a single chain Fv (scFv)) or receptor/ligands (i.e., protein or glycoprotein ligands or receptors)) that allow selective and specific binding of the HSA linker conjugate to a target cell, tissue, or organ.
  • the binding moieties can be bonded to the HSA linker (e.g., via a covalent (e.g., a peptide bond), ionic, or hydrophobic bond, or via a high-affinity protein-protein binding interaction (e.g., biotin and avidin)).
  • binding moieties can be bonded to an HSA linker.
  • two or more identical binding moieties i.e., moieties having the same structure and binding affinities
  • two or more different binding moieties e.g., an antibody, such as a scFv, with binding affinities for two or more different target molecules, or scFv with binding affinities for two or more different epitopes on the same target molecule
  • an HSA linker e.g., a bispecific HSA linker conjugate
  • different species of binding moieties can also be bonded to an HSA linker to bestow, for example, two or more different binding specificities or agonistic/antagonistic biological properties on the linker conjugate.
  • binding moiety pairs for use in the preparation of bispecific HSA linker conjugates are disclosed in, e.g., International Patent Application Publications WO 2006/091209 and WO 2005/1 17973, herein incorporated by reference.
  • more than two binding moieties e.g., the same or different binding moieties
  • the invention features an HSA linker conjugate having at least first and second binding moieties, each of which can be bound at either the amino or carboxy terminus of the HSA linker, or to peptide connectors, as defined herein, present at either or both termini.
  • Figure 1 illustrates an exemplary mutated HSA linker in which Iwo binding moieties ("arm 1" and "arm 2") are bonded to the mutated HSA linker by the amino terminal peptide connector AAS and carboxy terminal peptide connector AAAL (SEQ ID NO: 5).
  • Binding moieties can also be bound to other loci (e.g., internal amino acid residues of the HSA linker), for example, covalently or ionically, e.g., using biotin-avidin interactions.
  • Biotinylation of amine (e.g., lysine residues) and sulfhydryl (e.g., cysteine residues) amino acid side chains is known in the art and can be used to attach binding moieties to the HSA linker.
  • Binding moieties that can be included in an HSA linker conjugate include antibodies, antibody fragments, receptors, and ligands. Binding moieties bound to an HSA linker may be recombinant (e.g., human, murine, chimeric, or humanized), synthetic, or natural.
  • binding moieties include, for example, complete antibodies, domain antibodies, diabodies, triabodies, bi-specific antibodies, antibody fragments, Fab fragments, F(ab')2 molecules, single chain Fv (scFv) molecules, bispecific single chain Fv ((scFv') 2 ) molecules, tandem scFv fragments, antibody fusion proteins, hormones, receptors, ligands, and aptamers, and biologically-active fragments thereof.
  • Antibodies include, for example, complete antibodies, domain antibodies, diabodies, triabodies, bi-specific antibodies, antibody fragments, Fab fragments, F(ab')2 molecules, single chain Fv (scFv) molecules, bispecific single chain Fv ((scFv') 2 ) molecules, tandem scFv fragments, antibody fusion proteins, hormones, receptors, ligands, and aptamers, and biologically-active fragments thereof.
  • Antibodies include, for example, complete antibodies, domain antibodies, diabodies
  • Antibodies include the IgG, IgA, IgM, IgD, and IgE isotypes. Antibodies or antibody fragments thereof, as used herein, contain one or more complementarity determining regions (CDR) or binding peptides that bind to target proteins, glycoproteins, or epitopes present on the exterior or in the interior of target cells.
  • CDR complementarity determining regions
  • the HSA linker can also be bonded to one or more fragments of an antibody that retain the ability to bind with specificity to a target antigen.
  • Antibody fragments include separate variable heavy chains, variable light chains, Fab, Fab', F(ab') 2 , Fabc, and scFv. Fragments can be produced by enzymatic or chemical separation of intact immunoglobulins. For example, a F(ab') 2 fragment can be obtained from an IgG molecule by proteolytic digestion with pepsin at pH 3.0-3.5 using standard methods such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Pubs., N.Y. (1988).
  • Fab fragments may be obtained from F(ab') 2 fragments by limited reduction, or from whole antibody by digestion with papain in the presence of reducing agents. Fragments can also be produced by recombinant DNA techniques. Segments of nucleic acids encoding selected fragments are produced by digestion of full- length coding sequences with restriction enzymes, or by de novo synthesis. Often fragments are expressed in the form of phage-coat fusion proteins. This manner of expression is advantageous for affinity-sharpening of antibodies.
  • Humanized antibodies may also be used in combination with the HSA linker, in which one or more of the antibody CDRs are derived from a non-human antibody sequence, and one or more, but preferably all, of the CDRs bind specifically to an antigen (e.g., a protein, glycoprotein, or other suitable epitope).
  • an antigen e.g., a protein, glycoprotein, or other suitable epitope
  • a humanized antibody contains constant framework regions derived substantially from a human antibody (termed an acceptor antibody), as well as, in some instances, a majority of the variable region derived from a human antibody.
  • One or more of the CDRs are provided from a non-human antibody, such as a mouse antibody.
  • the constant region(s) of the antibody may or may not be present.
  • the substitution of one or more mouse CDRs into a human variable domain framework is most likely to result in retention of their correct spatial orientation if the human variable domain framework adopts the same or a similar conformation as the mouse variable framework from which the CDRs originated. This is achieved by obtaining the human variable domains from human antibodies whose framework sequences exhibit a high degree of sequence and structural identity with the murine variable framework domains from which the CDRs were derived.
  • the heavy and light chain variable framework regions can be derived from the same or different human antibody sequences.
  • the human antibody sequences can be the sequences of naturally occurring human antibodies, consensus sequences of several human antibodies, or can be human germline variable domain sequences.
  • Suitable human antibody sequences are identified by alignments of the amino acid sequences of the mouse variable regions with the sequences of known human antibodies. The comparison is performed separately for heavy and light chains but the principles are similar for each.
  • HSA linker conjugates protein or glycoprotein receptors or ligands are bound to an HSA linker.
  • HSA linkers bonded with a receptor or ligand can be used, for example, to specifically target a secreted protein, a cell (e.g., a cancer cell), tissue, or organ.
  • the specific binding of the HSA linker-receptor or -ligand conjugate to cognate target receptors or ligands can cause agonistic or antagonistic biological activity in intracellular or intercellular signaling pathways.
  • receptors and ligands, or fragments thereof can be conjoined to the amino and/or carboxy termini of an HSA linker, to a peptide connector linked to the HSA linker or to an amino acid residue within the HSA linker.
  • Exemplary receptors and ligands that can be joined to an HSA linker include, but are not limited to, insulin-like growth factor 1 receptor (IGFlR), IGF2R, insulin- like growth factor (IGF), mesenchymal epithelial transition factor receptor (c-met; also known as hepatocyte growth factor receptor (HGFR)), hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), heregulin, fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), platelet-derived growth factor (PDGF), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor (VEGF), tumor necrosis factor receptor (TNFR), tumor necrosis factor alpha (TNF- ⁇ ), TNF- ⁇ , folate receptor (FOLR), folate, transferrin receptor (TfR), mesothelin, Fc receptor, c-kit receptor, c-kit, ⁇ 4 integrin
  • Receptors and ligands can be expressed, isolated, or joined to an HSA linker using any of the methods described supra. Diagnostic Agents
  • the HSA linker or any binding moiety conjugated thereto (e.g., antibody, antibody fragment, receptor, or ligand), can be coupled to a chelating agent or to a detectable label to form a diagnostic agent. Also contemplated are HSA linker conjugates that include a detectable label, as described herein, as well as one or more of the therapeutic agents or binding moieties described herein.
  • the HSA linker (or HSA linker conjugate) and chelator components can be coupled by reacting the free amino group of a threonine residue of the HSA linker (or HSA linker conjugate) with an appropriate functional group of the chelator, such as a carboxyl group or activated ester.
  • an appropriate functional group of the chelator such as a carboxyl group or activated ester.
  • a such coupling may be achieved by incorporating the chelator ethylenediaminetetraacetic acid (EDTA), which is common in the art of coordination chemistry, when functionalized with a carboxyl substituent on the ethylene chain. Synthesis of EDTA derivatives of this type are reported in Arya et al.
  • An HSA linker or an HSA linker conjugate may incorporate a metal chelator component that is peptidic, i.e., compatible with solid-phase peptide synthesis.
  • the chelator may be coupled in the same manner as EDTA described above or, more conveniently, the chelator and HSA linker or HSA linker conjugate are synthesized in toto starting from the C-terminal residue of the HSA linker or HSA linker conjugate and ending with the N-terminal residue of the chelator.
  • An HSA linker or an HSA linker conjugate may further incorporate a linking group component that serves to couple the HSA linker to the chelator while not adversely affecting the biological properties of the HSA linker, the targeting function of the binding moiety portion(s) of the HSA linker conjugate, or the metal binding function of the chelator.
  • Suitable linking groups include amino acid chains and alkyl chains functionalized with reactive groups for coupling to the HSA linker or the HSA linker conjugate and to the chelator.
  • An amino acid chain is the preferred linking group when the chelator is peptidic so that the HSA linker or HSA linker conjugate can be synthesized in toto by solid-phase techniques.
  • An alkyl chain-linking group may be incorporated in the HSA linker or HSA linker conjugate by reacting the amino group of a threonine residue of a peptide portion of an HSA linker with a first functional group on the alkyl chain, such as a carboxyl group or an activated ester. Subsequently the chelator is attached to the alkyl chain to complete the formation of the HSA linker or HSA linker conjugate by reacting a second functional group on the alkyl chain with an appropriate group on the chelator.
  • the second functional group on the alkyl chain is selected from substituents that are reactive with a functional group on the chelator while not being reactive with a threonine residue of the mutated HSA linker.
  • the second functional group of the alkyl chain-linking group can be an amino group.
  • the HSA linker or HSA linker conjugate may require protection and deprotection of the functional groups present in order to avoid formation of undesired products. Protection and deprotection are accomplished using protecting groups, reagents, and protocols common in the art of organic synthesis. Particularly, protection and deprotection techniques employed in solid phase peptide synthesis described above may be used.
  • linking group to an alkyl chain is polyethylene glycol (PEG), which is functionalized in the same manner as the alky] chain described above for incorporation in the HSA linker or HSA linker conjugate. It will be appreciated that linking groups may alternatively be coupled first to the chelator and then to the HSA linker or HSA linker conjugate.
  • PEG polyethylene glycol
  • an HSA linker or HSA linker conjugate is coupled to a diagnostically useful metal capable of forming a complex.
  • Suitable metals include, e.g., radionuclides, such as technetium and rhenium in their various forms (e.g., ""'TcO 3+ , " ""TcO 2 + , ReO 3+ , and ReO 2 + ).
  • Incorporation of the metal within the HSA linker or HSA linker conjugate can be achieved by various methods common in the art of coordination chemistry. When the metal is technetium-99 m, the following general procedure may be used to form a technetium complex.
  • HSA linker-chelator conjugate solution is formed initially by dissolving the HSA linker or HSA linker conjugate in aqueous alcohol such as ethanol. The solution is then degassed to remove oxygen then thiol protecting groups are removed with a suitable reagent, for example, with sodium hydroxide, and then neutralized with an organic acid, such as acetic acid (pH 6.0-6.5).
  • a suitable reagent for example, with sodium hydroxide
  • an organic acid such as acetic acid (pH 6.0-6.5).
  • a stoichiometric excess of sodium pertechnetate, obtained from a molybdenum generator is added to a solution of the conjugate with an amount of a reducing agent such as stannous chloride sufficient to reduce technetium and heated.
  • a reducing agent such as stannous chloride
  • labeling of an HSA linker can be accomplished by a transchelation reaction.
  • the technetium source is a solution of technetium complexed with labile ligands facilitating ligand exchange with the selected chelator.
  • Suitable ligands for transchelation include tartarate, citrate, and heptagluconate.
  • the preferred reducing reagent is sodium dithionite.
  • the HSA linker or HSA linker conjugate may be labeled using the techniques described above, or alternatively the chelator itself may be labeled and subsequently coupled to an HSA linker to form an HSA linker-chelator conjugate; a process referred to as the "prelabeled ligand" method.
  • HSA linker-chelator conjugate Another approach for labeling an HSA linker, or any agent conjugated thereto, involves immobilizing the HSA linker-chelator conjugate on a solid-phase support through a linkage that is cleaved upon metal chelation. This is achieved when the chelator is coupled to a functional group of the support by one of the complexing atoms.
  • a complexing sulfur atom is coupled to the support which is functionalized with a sulfur protecting group such as maleimide.
  • an agent that includes an HSA linker-chelator conjugate can be used to detect tissue at risk of developing cancer (e.g., lung cancer, breast cancer, colon cancer, and prostate cancer), age-related diseases (e.g., cardiovascular disease, cerebrovascular disease, or Alzheimer's disease), tobacco-related diseases (e.g., emphysema, aortic aneurysms, esophageal cancer, or squamous cell cancer of the head and neck) by procedures established in the art of diagnostic imaging.
  • cancer e.g., lung cancer, breast cancer, colon cancer, and prostate cancer
  • age-related diseases e.g., cardiovascular disease, cerebrovascular disease, or Alzheimer's disease
  • tobacco-related diseases e.g., emphysema, aortic aneurysms, esophageal cancer, or squamous cell cancer of the head and neck
  • An agent that incorporates an HSA linker labeled with a radionuclide metal, such as technetium-99 m, may be administered to a mammal (e.g., a human) by intravenous injection in a pharmaceutically acceptable solution, such as isotonic saline, or by other methods described herein.
  • a pharmaceutically acceptable solution such as isotonic saline, or by other methods described herein.
  • the amount of a labeled agent appropriate for administration is dependent upon the distribution profile of the chosen HSA linker or HSA linker conjugate in the sense that an agent that incorporates a rapidly cleared HSA linker or HSA linker conjugate may be administered at higher doses than an agent that incorporates an HSA linker or HSA linker conjugate that clears less rapidly.
  • Unit doses acceptable for imaging tissues are in the range of about 5-40 mCi for a 70 kg individual.
  • the in vivo distribution and localization of an agent that incorporates a labeled HSA linker or HSA linker conjugate can be tracked by standard techniques described herein at an appropriate time subsequent to administration, typically between 30 minutes and 180 minutes and up to about 5 days depending upon the rate of accumulation at the target site with respect to the rate of clearance at non-target tissue.
  • HSA linker or any molecule or moiety conjugated thereto, can also be modified or labeled to facilitate diagnostic or therapeutic uses.
  • Detectable labels such as a radioactive, fluorescent, heavy metal, or other molecules may be bound to any of the agents.
  • Single, dual, or multiple labeling of an agent may be advantageous. For example, dual labeling with radioactive iodination of one or more residues combined with the additional coupling of, for example, 90 Y via a chelating group to amine-containing side or reactive groups, would allow combination labeling. This may be useful for specialized diagnostic needs such as identification of widely dispersed small neoplastic cell masses.
  • HSA linker or any molecule or moiety conjugated thereto, can also be modified, for example, by halogenation of the tyrosine residues of the peptide component.
  • Halogens include fluorine, chlorine, bromine, iodine, and astatine.
  • Such halogenated agents may be detectably labeled, e.g., if the halogen is a radioisotope, such as, for example, 18 F, 75 Br, 77 Br, 122 1, 123 I, 124 1, 125 I, 129 1, 131 I, or 211 At.
  • Halogenated agents contain a halogen covalently bound to at least one amino acid, and preferably to D-Tyr residues in each agent molecule.
  • Radioisotopes for radiolabeling an HSA linker, or any molecule or moiety conjugated thereto include any radioisotope that can be covalently bound to a residue of the peptide component of the agent or analog thereof.
  • the radioisotopes can also be selected from radioisotopes that emit either beta or gamma radiation, or alternatively, any of the agents can be modified to contain chelating groups that, for example, can be covalently bonded to lysine residue(s) of the HSA linker or any peptidic agent conjugated thereto.
  • the chelating groups can then be modified to contain any of a variety of radioisotopes, such as gallium, indium, technetium, ytterbium, rhenium, or thallium (e.g., 125 I, 67 Ga, 111 In, "mTc, 169 Yb, 186 Re).
  • an HSA linker or any molecule or moiety conjugated thereto, can be modified by attachment of a radioisotope.
  • a radioisotope Preferable radioisotopes are those having a radioactive half- life corresponding to, or longer than, the biological half-life of the HSA conjugate used. More preferably, the radioisotope is a radioisotope of a halogen atom (e.g. a radioisotope of fluorine, chlorine, bromine, iodine, and astatine), even more preferably 75 Br, 77 Br, 76 Br,
  • a halogen atom e.g. a radioisotope of fluorine, chlorine, bromine, iodine, and astatine
  • radioactive metals can be coupled to radioactive metals and used in radiographic imaging or radiotherapy.
  • Preferred radioisotopes also include 99m Tc, 51 Cr, 67 Ga, 68 Ga, 11 1 In, 168 Yb, 140 La, 90 Y 5 88 Y, 153 Sm, 156 Ho, 165 Dy, 64 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, and 214 Bi.
  • the choice of metal is determined based on the desired therapeutic or diagnostic application.
  • HSA linker or any molecule or moiety conjugated thereto, can be coupled to a metal component, to produce a diagnostic or therapeutic agent.
  • a detectable label may be a metal ion from heavy elements or rare earth ions, such as Gd 3+ , Fe 3+ , Mn 3+ , or Cr 2+ .
  • Agents that incorporate an HSA linker having paramagnetic or superparamagnetic metals conjoined thereto are useful as diagnostic agents in MRI imaging applications.
  • Paramagnetic metals include, but are not limited to, chromium (III), manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), and ytterbium (III).
  • Chelating groups may be used to indirectly couple detectable labels or other molecules to an HSA linker or to an agent conjugated thereto. Chelating groups can link agents with radiolabels, such as a bifunctional stable chelator, or can be linked to one or more terminal or internal amino acid reactive groups.
  • An HSA linker, or any molecule or moeity conjugated thereto, can be linked via an isothiocyanate ⁇ -Ala or appropriate non- ⁇ -amino acid linker which prevents Edman degradation.
  • chelators known in the art include, for example, the ininocarboxylic and polyaminopolycarboxylic reactive groups, ininocarboxylic and polyaminopolycarboxylic reactive groups, diethylenetriaminepentaacetic acid (DTPA), and 1, 4,7,10-tetraazacyclododecane- 1,4,7, 10- tctraacctic acid (DOTA).
  • DTPA diethylenetriaminepentaacetic acid
  • DOTA 1, 4,7,10-tetraazacyclododecane- 1,4,7, 10- tctraacctic acid
  • HSA linker when expressed recombinantly, can be joined to a peptidic detectable label or diagnostic agent.
  • Peptides and proteins that can be used as a detectable label with an HSA linker include, but are not limited to, fluorescent proteins, bioluminescent proteins, and epitope tags, each of which is discussed in detail below.
  • One or more of these detectable labels can also be incorporated into an HSA linker conjugate that also includes a therapeutic, cytotoxic, or cytostatic agent.
  • Fluorescent proteins or fluorochromes such as green fluorescent protein (GFP; SEQ ID NO:47), enhanced GFP (eGFP), yellow fluorescent protein (SEQ IO NO:48; YFP), cyan fluorescent protein (SEQ ID NO:49; CFP), and red fluorescent protein (SEQ ID NO:50; RFP or DsRed), can be used as detectable label joined to an HSA linker.
  • Fluorescent proteins can be recombinantly expressed in a cell (e.g., a blood cell, such as a lymphocyte) following transfection or transduction of the cell with an expression vector that encodes the nucleotide sequence of the fluorescent protein.
  • the fluorescent protein Upon exposure of the fluorescent protein to a stimulating frequency of light, the fluorescent protein will emit light at a low, medium, or high intensity that can be observed by eye under a microscope or by an optical imaging device.
  • Exemplary fluorescent proteins suitable for use as the diagnostic sequence in agents are described in, e.g., U.S. Patent Nos. 7,417,131 and 7,413,874, each of which is herein incorporated by reference.
  • Bioluminescent proteins can also be used as a detectable label incorporated into an HSA linker.
  • Bioluminescent proteins such as luciferase (e.g., firefly (SEQ ID NO:51), Renilla (SEQ ID NO:52), and Omphalotus luciferase) and aequorin, emit light as part of a chemical reaction with a substrate (e.g., luciferin and coelenterazine).
  • luciferase e.g., firefly (SEQ ID NO:51), Renilla (SEQ ID NO:52), and Omphalotus luciferase
  • aequorin emit light as part of a chemical reaction with a substrate (e.g., luciferin and coelenterazine).
  • a vector encoding a luciferase gene provides for the in vivo, in vitro, or ex vivo detection of cells (e.g., blood cells, such as lymphocytes) that have been transduced or transfected according to standard methods, such as those described herein.
  • cells e.g., blood cells, such as lymphocytes
  • bioluminescent proteins suitable for use as a diagnostic sequence and methods for their use are described in, e.g., U.S. Patent Nos. 5,292,658, 5,670,356, 6,171,809, and 7,183,092, each of which is herein incorporated by reference.
  • Epitope tags are short amino acid sequences, e.g., 5-20 amino acid residues in length, that can be incorporated into an HS ⁇ linker conjugate as a detectable label to facilitate detection once expressed in a cell, secreted from the cell, or bound to a target cell.
  • An agent that incorporates an epitope tag as a diagnostic sequence can be detected by virtue of its interaction with an antibody, antibody fragment, or other binding molecule specific for the epitope tag.
  • Nucleotide sequences encoding the epitope tag are produced either by cloning appropriate portions of natural genes or by synthesizing a polynucleotide that encodes the epitope tag.
  • An antibody, antibody fragment, or other binding molecule that binds an epitope tag can directly incorporate a detectable label (e.g., a fluorochrome, radiolabel, heavy metal, or enzyme such as horseradish peroxidase) or serve itself as a target for a secondary antibody, antibody fragment, or other binding molecule that incorporates such a label.
  • a detectable label e.g., a fluorochrome, radiolabel, heavy metal, or enzyme such as horseradish peroxidase
  • Exemplary epitope tags that can be used as a diagnostic sequence include c-myc (SEQ ID NO:33), hemagglutinin (HA; SEQ ID NO:34), and histidine tag (His 6 ; SEQ ID NO:35).
  • fluorescent (e.g., GFP) and bioluminescent proteins can also serve as epitope tags, as antibodies, antibody fragments, and other binding molecules are commercially available for the detection of these proteins.
  • an HSA linker conjugate that incorporates a diagnostic sequence (e.g., a fluorescent protein, bioluminescent protein, or epitope tag) or any cell expressing or bound thereto can be accomplished using a microscope, flow cytometer, luminometer, or other state of the art optical imaging device, such as an IVIS ® Imaging System (Caliper LifeSciences, Hopkinton, MA).
  • a diagnostic sequence e.g., a fluorescent protein, bioluminescent protein, or epitope tag
  • IVIS ® Imaging System Caliper LifeSciences, Hopkinton, MA.
  • An HSA linker or any molecule or moiety conjugated thereto, can be coupled to any known cytotoxic or therapeutic moiety to form an agent (an HSA linker conjugate) that can be administered to treat, inhibit, reduce, or ameliorate disease (e.g., a cancer, autoimmune disease, or cardiovascular disease) or one or more symptoms of disease.
  • diseases e.g., a cancer, autoimmune disease, or cardiovascular disease
  • antineoplastic agents such as: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; adriamycin; aldesleukin; altretamine; ambomycin; a.
  • metantrone acetate aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; camptothecin; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; combretestatin a-4; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine;
  • therapeutic compounds include, but are not limited to, 20-pi- 1 ,25 dihydroxy vitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti- dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argin
  • HSA linker conjugates can also include site-specifically conjugated molecules and moieties. Site-specific conjugation allows for the controlled stoichiometric attachment to specific residues in the HSA linker of cytotoxic, immunomodulatory, or cytostatic agents including, e.g., anti tubulin agents, DNA minor groove binders, DNA replication inhibitors, alkylating agents, anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy or radiation sensitizer, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins. steroids, taxanes, topoisomerase inhibitors, and vinca alkaloids or any other molecules or moieties described herein.
  • site-specific conjugation allows for the controlled stoichiometric attachment to specific residues in the HSA linker of cytotoxic, immunomodulatory, or
  • An HSA linker or any molecule or moiety conjugated thereto, can also be coupled to a lytic peptide.
  • lytic peptides induce cell death and include, but are not limited to, streptolysin O; stoichactis toxin; phallolysin; staphylococcus alpha toxin; holothurin A; digitonin; melittin; lysolecithin; cardiotoxin; and cerebratulus A toxin (Kem el al, J. Biol. Chem. 253(16):5752-5757, 1978).
  • An HSA linker or any molecule or moiety conjugated thereto (e.g., antibody or antibody fragment conjugates), may be coupled to a synthetic peptide that shares some sequence homology or chemical characteristics with any of the naturally occurring peptide lysins; such characteristics include, but are not limited to, linearity, positive charge, amphipathicity, and formation of alpha-helical structures in a hydrophobic environment (Leuschner et al, Biology of Reproduction 73:860-865, 2005).
  • An HSA linker, or any molecule or moiety conjugated thereto can also be coupled to an agent that induces complement-mediated cell lysis such as, for example, the immunoglobulin F c subunit.
  • HSA linker or any molecule or moiety conjugated thereto, can also be coupled to any member of the phospholipase family of enzymes (including phospholipase A, phospholipase B, phospholipase C, or phospholipase D) or to a catalytically-active subunit thereof.
  • An HSA linker or any molecule or moiety conjugated thereto, can also be coupled to a radioactive agent to form an agent that can be used for detection or therapeutic applications.
  • Radioactive agents that can be used include but are not limited to
  • Fibrinogen I Fludeoxyglucose F; Fluorodopa F; Insulin I; Insulin I; lobenguane 123 I; Iodipamide Sodium 131 I; Iodoantipyrine 131 I; Iodocholesterol 131 I ; lodohippurate Sodium 123 I; Iodohippurate Sodium 125 I; Iodohippurate Sodium 131 I; Iodopyracet 125 I; Iodopyracet 131 I; lofetamine Hydrochloride 123 I; Iomethin 125 I; lomethin 131 I; Iothalamate Sodium 125 I; Iothalamate Sodium 131 I; tyrosine 131 I; Liothyronine 125 I; Liothyronine 131 I; Merisoprol Acetate 197 Hg; Merisoprol Acetate 203 Hg; Merisoprol 197 Hg; Selenomethionine 75 Se;
  • a radioisotope can be site-specifically conjoined to an HSA linker or HSA linker conjugate.
  • the available reactive groups could be used to conjugate site- specific bifunctional chelating agents for labeling of radioisotopes, including 123 1, 124 1, 125 I, 131 1, 99m Tc, 111 In, 64 Cu, 67 Cu, 186 Re, 188 Re, 177 Lu, 90 Y, 77 As, 72 As, 86 Y, 89 Zr, 211 At, 212 Bi, 213 Bi, or 225 Ac.
  • Therapeutic or cytotoxic agents incorporated into or coupled with an HSA linker or an HSA linker conjugate may further include, for example, anti-cancer Supplementary Potentiating Agents, including, but not limited to: tricyclic anti-depressant drugs (e.g., imipramine, desipramine, amitryptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine, and maprotiline); non-tricyclic anti-depressant drugs (e.g., sertraline, trazodone, and citalopram); Ca 2+ antagonists (e.g., verapamil, nifedipine, nitrendipine, and caroverine); Calmodulin inhibitors (e.g., prenylamine, trifluoroperazine, and clomipramine); Amphotericin B; Triparanol analogs (e.g., tamoxifen); anti
  • An agent that includes an HSA linker, or any molecule or moiety conjugated thereto can also be coupled to or administered with one or more cytokines (e.g., granulocyte colony stimulating factor, interferon-alpha, and tumor necrosis factor-alpha).
  • cytokines e.g., granulocyte colony stimulating factor, interferon-alpha, and tumor necrosis factor-alpha.
  • An HSA linker, or any molecule or moiety conjugated thereto can also be coupled to an antimetabolic agent.
  • Antimetabolic agents include, but are not limited to, the following compounds and their derivatives: azathioprine, cladribine, cytarabine, dacarbazine, fludarabine phosphate, fluorouracil, gencitabine chlorhydrate, mercaptopurine, methotrexate, mitobronitol, mitotane, proguanil chlorohydrate, pyrimethamine, raltitrexed, trimetrexate glucuronate, urethane, vinblastine sulfate, vincristine sulfate, etc.
  • an HSA linker or conjugate can be coupled to a folic acid-type antimetabolite, a class of agents that includes, for example, methotrexate, proguanil chlorhydrate, pyrimethanime, trimethoprime, or trimetrexate glucuronate, or derivatives of these compounds.
  • An HSA linker or any molecule or moiety conjugated thereto, can also be coupled to a member of the anthracycline family of neoplastic agents, including but not limited to aclarubicine chlorhydrate, daunorubicine chlorhydrate, doxorubicine chlorhydrate, epirubicine chlorhydrate, idarubicine chlorhydrate, pirarubicine, or zorubicine chlorhydrate; a camptothecin, or its derivatives or related compounds, such as 10, 11 methyl enedioxycamptothecin; or a member of the maytansinoid family of compounds, which includes a variety of structurally-related compounds, e.g., ansamitocin P3, maytansine, 2'-N-demethylmaytanbutine, and maytanbicyclinol.
  • aclarubicine chlorhydrate daunorubicine chlorhydrate, doxorubicine chlorhydrate, epirubicine chlorhydrate, idarubicine chlorhydrate, pirarubicine, or z
  • HSA linker or any molecule or moiety conjugated thereto, can be coupled directly to a cytotoxic or therapeutic agent using known chemical methods, or coupled indirectly to a cytotoxic or therapeutic agent via an indirect linkage.
  • an HSA linker can be attached to a chelating group that is attached to the cytotoxic or therapeutic agent.
  • Chelating groups include, but are not limited to, ininocarboxylic and polyaminopolycarboxylic reactive groups, diethylenetriaminepentaacetic acid (DTPA), and l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA).
  • An HSA linker conjugate that includes, e.g., an HSA linker, one or more binding moieties (with or without intervening peptide connectors, as defined herein), and a therapeutic or cytotoxic agent, can be specifically targeted by the binding moiety (e.g., antibody, antibody fragment, or receptor/ligand) to a cell or tissue, thereby allowing selective destruction of the target cell or tissue to which the binding moiety is directed.
  • an HSA linker conjugate can be used to target and destroy cancer cells of the lung, breast, prostate, and colon in order to prevent, stabilize, inhibit the progression of, or treat cancers originating in these organs when the HSA linker conjugate includes a binding moiety that specifically binds to the cancer cells in these organs.
  • an HSA linker conjugate can be used to target and destroy cells of the vasculature, brain, liver, kidney, heart, lung, prostate, colon, nasopharynx, oropharynx, larynx, bronchus, and skin in order to prevent, stabilize, inhibit the progression of, or treat age-related, tobacco-related, or autoimmune diseases or conditions relating to these organs by targeting, in the case of autoimmune disease for example, autoreactive T cells (e.g., by binding to and agonizing tumor necrosis factor receptor 2 (TNFR2) present on the autoreactive T cells).
  • TNFR2 tumor necrosis factor receptor 2
  • An HSA linker when expressed recombinantly, can be joined to a cytotoxic polypeptide.
  • Cytotoxic polypeptides when brought into contact with a target cell (e.g., a cancer cell), exerts cytotoxic or cytostatic effects on the cell.
  • a cytotoxic polypeptide when joined with an HSA linker, can induce events in a target cell upon binding of the target cell that leads to cell death through, for example, apoptosis, necrosis, or senescence.
  • a cytotoxic polypeptide joined with an HSA linker can interfere or inhibit normal cellular biological activities, such as division, metabolism, and growth, or abnormal cellular biological activities, such as metastasis.
  • an HSA linker joined to caspase 3 will bind a target cell (e.g., a cancer cell) and undergoe endocytosis. Once internalized by the target cell, the caspase portion of the HSA linker conjugate can initiate the pro-apoptotic caspase cascade, ultimately resulting in the apoptosis of the target cell.
  • a target cell e.g., a cancer cell
  • the caspase portion of the HSA linker conjugate can initiate the pro-apoptotic caspase cascade, ultimately resulting in the apoptosis of the target cell.
  • an HSA linker conjugate includes a cytotoxic polypeptide capable of killing a cancer cell.
  • the cytotoxic polypeptide inhibits the growth or metastasis of a cancer cell.
  • the cytotoxic polypeptide joined with an HSA linker can also be used to kill or inhibit the growth of cells associated with, necessary for, or beneficial to cancer growth, such as endothelial cells that form blood vessels that perfuse solid tumors.
  • an HSA linker conjugate can include two or more cytotoxic polypeptides so as to modulate (e.g., increase) the specificity, intensity, or duration of the cytotoxic or cytostatic effect on a target cell (e.g., a cancer cell).
  • the HSA linker is joined to an activatable form of cytotoxic polypeptide (e.g., a biologically-inactive pro-agent that is capable of activation upon cleavage by an enzyme or drug).
  • cytotoxic polypeptide e.g., a biologically-inactive pro-agent that is capable of activation upon cleavage by an enzyme or drug.
  • texposure e.g., in vivo
  • an enzyme or drug capable of cleaving the cytotoxic polypeptide renders the cytotoxic polypeptide biologically- active (e.g., cytotoxic or cytostatic).
  • a biologically-inactive cytotoxic polypeptide that can be converted to a biologically-active form for use with an HSA linker is a procaspase (e.g., procaspase 8 or 3).
  • the procaspase 8 domain of an HSA linker can be cleaved by TRAIL or FasL upon internalization by a target cell (e.g., a cancer cell). Once cleaved, the biologically active caspase 8 can promote apoptosis of the target cell.
  • a target cell e.g., a cancer cell
  • the cytotoxic polypeptide joined to an HSA linker can include a full-length peptide, polypeptide, or protein, or biologically-active fragment thereof (e.g., a "death domain " '), known to have cytotoxic or cytostatic properties.
  • Peptides, polypeptides, or proteins with cytotoxic or cytostatic properties can be altered (e.g., by making amino acid substitutions, mutations, truncations, or additions) to facilitate incorporation of the cytotoxic sequence into an agent as described herein. Desirable alterations include, for example, changes to the amino acid sequence that facilitate protein expression, longevity, cell secretion, and target cell toxicity.
  • the present invention also provides a nucleic acid molecule encoding a cytotoxic polypeptide as a fusion protein with an HSA linker, optionally including binding moieities and peptide connectors.
  • the nucleic acid molecule can be incorporated into a vector (e.g., an expression vector), such that, upon expression of the HSA linker in a cell transfected or transduced with the vector, the cytotoxic polypeptide, HSA linker, and binding moieties, if present, are operably linked (e.g., fused, contiguously-joined, or tethered together).
  • cytotoxic polypeptide of the present invention examples include, but are not limited to, apoptosis-inducing proteins such as cytochrome c (SEQ ID NO:39); caspases (e.g., caspase 3 (SEQ ID NO:36) and caspase 8 (SEQ ID NO:37)); procaspases, granzymes (e.g., granzymes A and B (SEQ ID NO:38)); tumor necrosis factor (TNF) and TNF receptor family members, including TNF-alpha (TNF ⁇ ; SEQ ID NO:40)), TNF-beta, Fas (SEQ ID NO:41) and Fas ligand; Fas-associated death domain-like IL-I ⁇ converting enzyme (FLlCE); TRAIL/APO2L (SEQ ID NO:45) and TWEAK/APO3L (see, e.g., U.S.
  • apoptosis-inducing proteins such as cytochrome c
  • Patent Application Publication No. 2005/0187177 herein incorporated by reference
  • pro-apoptotic members of the Bcl-2 family including Bax (SEQ ID NO:46), Bid, Bik, Bad (SEQ ID NO:42), Bak, and RICK (see, e.g., U.S. Patent Application Publication No. 2004/0224389, herein incorporate by reference); vascular apoptosis inducing proteins 1 and 2 (VAPl and VAP2; Masuda et al., Biochem. Biophys. Res. Commun.
  • pierisin SEQ ID NO:44; Watanabe et al., Biochemistry 96:10608-10613 (1999)
  • apoptosis-inducing protein SEQ ID NO:43; AIP; Murawaka et al., Nature 8:298-307 (2001)
  • IL-l ⁇ propiece polypeptide see, e.g., U.S. Patent 6,191,269, herein incorporated by reference
  • apoptin and apoptin-associated proteins such as AAP-I (see, e.g., European Patent Application Publication No.
  • EP 1083224 herein incorporated by reference
  • anti-angiogenic factors such as endostatin and angiostatin
  • other apoptosis-inducing proteins including those described in the following International and U.S. Patent Application Publications, each herein incorporated by reference: U.S. 2003/0054994, U.S. 2003/0086919, U.S. 2007/0031423, WO 2004/078112, WO 2007/012430, and WO 2006/0125001 (intracellular domain of delta 1 and jagged 1). Wild-type USA Linker Conjugates
  • the present invention also encompasses a naturally-occurring wild-type HSA linker, the amino acid and nucleotide sequences of which are provided in SEQ ID NOS :3 and 4, respectively, in the formation of binding, diagnostic, or therapeutic agents.
  • a naturally-occurring wild-type HSA linker the amino acid and nucleotide sequences of which are provided in SEQ ID NOS :3 and 4, respectively, in the formation of binding, diagnostic, or therapeutic agents.
  • one or more peptide connectors are covalently attached to the amino and/or carboxy termini of the HSA linker, or to an amino acid residue within the HSA linker sequence, to facilitate conjugation of one or more binding moieties.
  • the invention further provides an HSA linker conjugate that is formed using a truncated wild-type HSA polypeptide, optionally combined with one or more peptide connectors or binding moieties.
  • a wild-type HSA polypeptide lacking 1, 2, 3, 4, 5, 10, 15, 20, 50, 100, 200 or more amino acids of the full-length wild-type HSA amino acid sequence i.e., SEQ ID NO:3
  • Truncations can occur at one or both ends of the HSA linker, or can include a deletion of internal residues. Truncation of more than one amino acid residue need not be linear (i.e., consecutive).
  • wild-type HSA linkers include those having, in combination with one or more peptide connectors or binding moieties, one or more of domain I (SEQ ID NO:56; residues 1-197 of SEQ ID NO:3), domain II (SEQ ID NO:54; residues 189-385 of SEQ ID NO:3), or domain 111 (SEQ ID NO:57; residues 381-585 of SEQ ID NO:3), or combinations thereof, e.g., domains I and II, I and III, and II and III.
  • Serum clearance rates of a conjugate e.g., a bispecific HSA-drug or radioisotope- containing agent
  • HSA linkers may, but need not, be modified by site-specific chemical modification of amino acid residues in the HSA linker.
  • the correctly-folded tertiary structure of HSA displays certain amino acid residues on the external face of the protein.
  • Chemically- reactive amino acid residues e.g., cysteine
  • HSA linkers may optionally be modified by the addition or removal of asparagine, serine, or threonine residues from an HSA linker sequence to alter glycosylation of these amino acid residues.
  • Glycosylation sites added to an HSA linker are preferably surface-exposed, as discussed herein.
  • Glycosyl or other carbohydrate moieties introduced to an HSA linker can be directly conjugated to diagnostic, therapeutic, or cytotoic agents. Cysteine (Thiol) Conjugation
  • Surface-exposed amino acid residues of the HSA linker may be substituted with cysteine residues to allow for chemical conjugation of diagnostic, therapeutic, or cytotoxic agents.
  • Cysteine residues exposed on the surface of the HSA linker (when folded into its native tertiary structure) allow the specific conjugation of a diagnostic, therapeutic, or cytotoxic agent to a thiol reactive group such as maleimide or haloacetyl.
  • the nucleophilic reactivity of the thiol functionality of a cysteine residue to a maleimide group is about 1000 times higher compared to any other amino acid functionality in a protein, such as the amino group of a lysine residue or the N-terminal amino group.
  • Thiol specific functionality in iodoacctyl and maleimide reagents may react with amine groups, but higher pH (>9.0) and longer reaction times are required (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London).
  • the amount of free thiol in a protein may be estimated using the standard Ellman's assay. In some instances, reduction of the disulfide bonds with a reagent such as dithiothreitol (DTT) or selenol (Singh et al, Anal. Biochem. 304:147-156 (2002)) is required to generate the reactive free thiol.
  • DTT dithiothreitol
  • selenol Singh et al, Anal. Biochem. 304:147-156 (2002)
  • Sites for cysteine substitution can be identified by analysis of surface accessibility of the HSA linker (e.g., the identification of serine and threonine residues as suitable for substitution are described in Example 1 below).
  • the surface accessibility can be expressed as the surface area (e.g., square angstroms) that can be contacted by a solvent molecule, e.g., water.
  • the occupied space of water is approximated as a sphere with a 1.4 angstrom radius.
  • Software for calculating the surface accessibility of each amino acid of a protein is freely available or licensable.
  • an HSA linker is glycosylated.
  • Glycosylation of polypeptides is typically either N-linked or O-linked.
  • N- linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X represents any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • X represents any amino acid except proline
  • O-linked glycosylation refers to the attachment of one of the sugars N- aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • HSA linker conjugates described herein may be administered with one or more of the therapeutic, cytotoxic, or cytostatic agents described herein.
  • a patient suffering from breast cancer can be administered an HSA linker containing ErbB2 and ErbB3 scFvs (e.g., B2B3-1) can be co-administered with, e.g., doxorubicin, cyclophosphamide, and paclitaxel, a common chemotherapeutic regimen for the treatment of breast cancer.
  • HSA linker containing ErbB2 and ErbB3 scFvs e.g., B2B3-1
  • doxorubicin cyclophosphamide
  • paclitaxel a common chemotherapeutic regimen for the treatment of breast cancer.
  • trastuzumab data regarding this combination are set forth in Examples 42-44 below. Additional biological and chemical agents useful for the treatment of cancer are set forth herein, e.g., in Appendix 2.
  • HSA linker conjugates may be administered prior to, concurrent with, or following radiotherapy or surgery.
  • a patient suffering from a proliferative disorder e.g., breast cancer
  • an HSA linker conjugate alone or in combination with other therapeutic, cytotoxic, or cytotoxic agents as described herein concurrent with targeted radiotherapy or surgical intervention (e.g., lumpectomy or mastectomy) at the site of the cancerous tissue.
  • Radiotherapies suitable for use in combination with HSA linker conjugates include brachy therapy and targeted intraoperative radiotherapy (TARGIT).
  • Pharmaceutical Compositions include brachy therapy and targeted intraoperative radiotherapy (TARGIT).
  • compositions provided herein contain a therapeutically or diagnostically effective amount of an HSA linker conjugate that includes one or more of a binding moiety (e.g., antibodies or antibody fragments), a diagnostic agent (e.g., radionuclide or chelating agents), or a therapeutic agent (e.g., cytotoxic or immunomodulatory agents) agent.
  • a binding moiety e.g., antibodies or antibody fragments
  • a diagnostic agent e.g., radionuclide or chelating agents
  • a therapeutic agent e.g., cytotoxic or immunomodulatory agents
  • the active ingredients, an HSA linker conjugate can be formulated for use in a variety of drug delivery systems.
  • One or more physiologically acceptable excipients or carriers can also be included in the compositions for proper formulation. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). For a brief review of methods for drug delivery, see, Lang
  • compositions are intended for parenteral, intranasal, topical, oral, or local administration, such as by a transdermal means, for prophylactic and/or therapeutic treatment.
  • the pharmaceutical compositions are administered parenterally (e.g., by intravenous, intramuscular, or subcutaneous injection), or by oral ingestion, or by topical application.
  • compositions for parenteral administration may include an HSA linker, with or without one or more binding, diagnostic, and/or therapeutic agent conjugated thereto, dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g., water, buffered water, saline, PBS, and the like.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like.
  • the invention also provides compositions for oral delivery, which may contain inert ingredients such as binders or fillers for the formulation of a tablet, a capsule, and the like.
  • compositions for local administration which may contain inert ingredients such as solvents or emulsifiers for the formulation of a cream, an ointment, and the like.
  • compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
  • the pH of the preparations typically will be between 3 and 11 , more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5.
  • the resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) in a sealed package of tablets or capsules, for example.
  • the composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.
  • compositions containing an effective amount of an HSA linker conjugate can be administered to a mammal (e.g., a human) for prophylactic and/or therapeutic treatments.
  • a mammal e.g., a human
  • compositions containing an HSA linker conjugate are administered to a patient susceptible to or otherwise at risk of developing a disease or condition (e.g., a cancer, autoimmune disease, or cardiovascular disease).
  • Such an amount is defined to be a "prophylactically effective dose.”
  • the precise amounts again depend on the patient's state of health, but generally range from about 0.5 mg to about 400 mg of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) per dose (e.g., 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, or 400 mg or more per dose) and from about 0.1 ⁇ g to about 300 mg of one or more immunomodulatory agents per dose (e.g., 10 ⁇ g, 30 ⁇ g, 50 ⁇ g, 0.1 mg, 10 mg, 50 mg, 100 mg, or 200 mg per dose).
  • a dose of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) can be administered prophylactically to a patient one or more times per hour, day, week, month, or year (e.g., 2, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per hour, day, week, month, or year). More commonly, a single dose per week of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) is administered.
  • a dose of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) is administered to a mammal (e.g., a human) already suffering from a disease or condition (e.g., a cancer, autoimmune disease, or cardiovascular disease) in an amount sufficient to cure or at least partially arrest or alleviate one or more of the symptoms of the disease or condition and its complications.
  • a mammal e.g., a human
  • a disease or condition e.g., a cancer, autoimmune disease, or cardiovascular disease
  • Amounts effective for this use may depend on the severity of the disease or condition and general state of the patient, but generally range from about 0.5 mg to about 400 mg of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) per dose (e.g., 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, or 400 mg or more per dose).
  • a dose of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) can be administered therapeutically to a patient one or more times per hour, day, week, month, or year (e.g., 2, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per hour, day, week, month, or year). More commonly, a single dose per week of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) is administered.
  • the patient may receive an HSA linker conjugate (prepared wilh one or more of a binding, diagnostic, and/or therapeutic agent) in the range of about 0.5 to about 400 mg per dose one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 or more per week), preferably about 5 mg to about 300 mg per dose one or more times per week, and even more preferably about 5 mg to about 200 mg per dose one or more times per week.
  • an HSA linker conjugate prepared wilh one or more of a binding, diagnostic, and/or therapeutic agent in the range of about 0.5 to about 400 mg per dose one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 or more per week), preferably about 5 mg to about 300 mg per dose one or more times per week, and even more preferably about 5 mg to about 200 mg per dose one or more times per week.
  • the patient may also receive a biweekly dose of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) in the range of about 50 mg to about 800 mg or a monthly dose of an HSA linker, or any binding, diagnostic, and/or therapeutic agent conjugated thereto, in the range of about 50 mg to about 1,200 mg.
  • an HSA linker conjugate prepared with one or more of a binding, diagnostic, and/or therapeutic agent
  • a biweekly dose of an HSA linker conjugate prepared with one or more of a binding, diagnostic, and/or therapeutic agent in the range of about 50 mg to about 800 mg or a monthly dose of an HSA linker, or any binding, diagnostic, and/or therapeutic agent conjugated thereto, in the range of about 50 mg to about 1,200 mg.
  • an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) may be administered to a patient in a typical dosage range of about 0.5 mg per week to about 2000 mg per week, about 1.0 mg per week to about 1000 mg per week, about 5 mg per week to about 500 mg per week, about 10 mg per week to about 100 mg per week, about 20 mg per week to about 80 mg per week, about 100 mg per week to about 300 mg per week, or about 100 mg per week to about 200 mg per week.
  • the dosages for administration to a 70 kg patient can range from, for example, about 1 ⁇ g to about 5000 mg, about 2 ⁇ g to about 4500 mg, about 3 ⁇ g to about 4000 mg, about 4 ⁇ g to about 3,500 mg, about 5 ⁇ g to about 3000 mg, about 6 ⁇ g to about 2500 mg, about 7 ⁇ g to about 2000 mg, about ⁇ g to about 1900 mg, about 9 ⁇ g to about 1,800 mg, about 10 ⁇ g to about 1,700 mg, about 15 ⁇ g to about 1,600 mg, about 20 ⁇ g to about 1,575 mg, about 30 ⁇ g to about 1,550 mg, about 40 ⁇ g to about 1,500 mg, about 50 ⁇ g to about 1,475 mg, about 100 ⁇ g to about 1,450 mg, about 200 ⁇ g to about 1,425 mg, about 300 ⁇ g to about 1,000 mg, about 400 ⁇ g to about 975 mg, about 500 ⁇ g to about 650 mg, about 0.5 mg to about 625 mg, about 1 mg to about 600
  • an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) may be administered in the range of about 0.5 mg every other day to about 500 mg every other day, preferably about 5 mg every other day to about 75 mg every other day, more preferably about 10 mg every other day to about 50 mg every other day, and even more preferably 20 mg every other day to about 40 mg every other day.
  • An HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) may also be administered in the range of about 0.5 mg three times per week to about 100 mg three times per week, preferably about 5 mg three times per week to about 75 mg three times per week, more preferably about 10 mg three times per week to about 50 mg three times per week, and even more preferably about 20 mg three times per week to about 40 mg three times per week.
  • an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) is administered to a mammal (e.g., a human) continuously for 1, 2, 3, or 4 hours; 1,
  • An HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) may be administered at different frequencies during a therapeutic regime. In additional embodiments, an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) may be administered to a patient at the same frequency or at a different frequency.
  • the amount of one or more diagnostic or therapeutic agents and an HSA linker, or any agent conjugated thereto, required to achieve the desired therapeutic effect depends on a number of factors, such as the specific diagnostic or therapeutic agent(s) chosen, the mode of administration, and clinical condition of the recipient. A skilled artisan will be able to determine the appropriate dosages of one or more diagnostic or therapeutic agents and an HSA linker, or any agent conjugated thereto, to achieve the desired results.
  • Single or multiple administrations of the compositions comprising an effective amount of an HSA linker conjugate can be carried out with dose levels and pattern being selected by the treating physician.
  • the dose and administration schedule can be determined and adjusted based on the severity of the disease or condition in a mammal (e.g., a human), which may be monitored throughout the course of treatment according to the methods commonly practiced by clinicians or those described herein.
  • An HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) can be administered to a mammalian subject, such as a human, directly or in combination with any pharmaceutically acceptable carrier or salt known in the art.
  • Pharmaceutically acceptable salts may include non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry.
  • acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like.
  • Metal complexes include zinc, iron, and the like.
  • One exemplary pharmaceutically acceptable carrier is physiological saline.
  • Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (18 th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA. Diagnostic and Therapeutic Applications
  • HSA linker conjugates can be used for diagnostic and therapeutic applications in a human, including, for example, the diagnosis or treatment of proliferative diseases (e.g., cancers, such as melanoma, clear cell sarcoma, and renal cancer) and autoimmune diseases (e.g., multiple sclerosis, rheumatoid arthritis, and uveitis).
  • proliferative diseases e.g., cancers, such as melanoma, clear cell sarcoma, and renal cancer
  • autoimmune diseases e.g., multiple sclerosis, rheumatoid arthritis, and uveitis.
  • the following discussion of human proliferative and autoimmune diseases is meant to provide the skilled practitioner with a general understanding of how HSA linker conjugates can be applied in diagnostic and therapeutic applications and is not meant to limit the scope of the present invention.
  • Proliferative Diseases Cancer
  • An HSA linker conjugate can be used to diagnose, treat, prevent, or eliminate proliferative diseases such as, but not limited to, breast cancer, melanoma, clear cell sarcoma, renal cancer (e.g., renal cell carcinoma), prostate cancer, lung cancer, gastric cancer, and ovarian cancer.
  • Binding moieties to be conjoined with an HSA linker for diagnostic or therapeutic application in a patient suspected of having or suffering from a proliferative disease may be chosen based on their ability to specifically bind, agonize, activate, antagonize, or inhibit target molecules (e.g., cell surface receptors such as tyrosine kinase receptors) associated with a proliferative disease.
  • target molecules e.g., cell surface receptors such as tyrosine kinase receptors
  • IGFR insulin-like growth factor receptor
  • FGFR fibroblast growth factor receptor
  • PDGFR platelet-derived growth factor receptor
  • VEGFR vascular endothelial growth factor receptor
  • TNFR tumor necrosis factor receptor
  • EGFR epidermal growth factor receptor
  • Fc receptor, c-kit receptor, or mesenchymal epithelial transition factor receptor (c-met; also known as hepatocyte growth factor receptor (HGFR)) can be conjoined to an HSA linker to diagnose or treat a proliferative disease.
  • HSA linker conjugates for the treatment of breast and renal cancer is described below.
  • breast cancer Common forms of breast cancer include invasive ductal carcinoma, a malignant cancer in the breast's ducts, and invasive lobular carcinoma, a malignant cancer in the breast's lobules.
  • Some types of breast cancer cells are known to express high levels of epidermal growth factor receptors, especially ErbB2 (i.e., HER2/neu).
  • ErbB2 i.e., HER2/neu
  • Aberrant signaling or unregulated activation of EGFRs has been linked to the development and progression of many cancers, including breast cancer.
  • Uncontrolled cellular proliferation mediated via dysfunctional EGFR pathways can be found in a wide variety of solid tumors of epithelial origin and data have linked tumor EGFR expression, overexpression, and dysregulation to advanced disease, metastatic phenotype, resistance to chemotherapy, and an overall poorer prognosis.
  • An HSA linker conjoined to one or more binding moieties specific for an EGFR can be used with a diagnostic (e.g., a detectable label) or cytotoxic, cytostatic, or therapeutic agent, as described herein, to diagnose or treat breast cancer.
  • a bispecific HSA linker conjugate that comprises binding moieties specific for ErbB2 and ErbB3. such as "B2B3-1," described further herein, can be employed to diagnose or treat cancers, e.g., breast, kidney, ovarian, and lung cancers.
  • an HSA linker conjugate used to treat breast cancer can be administered prior to (e.g., neoadjuvant chemotherapy), concurrent with, or following (e.g., adjuvant chemotherapy) radiotherapy or surgical intervention.
  • An HSA linker conjugate can also be co-administered with other compounds (e.g., antineoplastic agents, such as biological or chemical therapeutics) useful for the treatment of breast cancer.
  • antineoplastic agents listed in Table 1 including mitotic inhibitors (e.g., taxanes), topoisomerase inhibitors, alkylating agents (including, e.g., platinum-based agents), selective estrogen modulators (SERM), aromatase inhibitors, antimetabolites, antitumor antibiotics (e.g., anthracycline antibiotics), anti-VEGF agents, anti-ErbB2 (HER2/neu) agents, and anti-ErbB3 agents, are known to be particularly useful for the treatment of breast cancer.
  • An HSA linker conjugate can be administered by a clinician in combination with any compound, including those listed in Appendix 2, known or thought to be beneficial for the treatment of breast cancer.
  • Table 1 Exemplary antineoplastic agents for treatment of breast cancer in combination with HSA linker conjugates.
  • Kidney cancers such as renal cell carcinoma, are particularly resistant to traditional radiological and chemical therapies.
  • the application of biological therapeutics, conjoined with an HSA linker represents an attractive option for patients suffering from these cancers.
  • an HSA linker conjoined with binding moieties that agonize type I interferon or interleukin 2 receptors can be used to treat a renal cancer.
  • binding moieties that target and inhibit tumor vascularization e.g., anti-vascular endothelial growth factor (VEGF) antibodies such as bevacizumab
  • VEGF anti-vascular endothelial growth factor
  • An HSA linker conjugate can be used to diagnose, treat, prevent, or stabilize autoimmune diseases and disorders in e.g., a human patient, such as, e.g., multiple sclerosis (MS), insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA), uveitis, Sjogren's syndrome, Grave's disease, psoriasis, and myasthenia gravis.
  • autoimmune diseases and disorders are caused by self-reactive elements of the immune system (e.g., T cells, B cells, and self-reactive antibodies).
  • binding moieties that inhibit, block, antagonize, or deplete e.g., anti-lymphocyte or anti-thymocyte globulins; basiliximab, daclizumab, or muromonab-CD3 monoclonal antibodies
  • self-reactive immune cells and antibodies can be conjoined with an HSA linker for therapeutic use.
  • Binding moieties that function as inflammatory signaling inhibitors (ISI), as defined herein can be conjoined to an HSA linker for the treatment of autoimmunity.
  • binding moieties that inhibit or antagonize integrin function e.g., an integrin antagonist, as defined herein
  • the binding moiety is a soluble TNF receptor, such as etanercept or lenercept; an antibody directed against a pro-inflammatory cytokine or a pro-inflammatory cell surface signaling molecule, such as adalimumab, certolizumab, inflixamab. golimumab, and rituxan; a dominant-negative pro-inflammatory cytokine variant, such as XENP345, XPROTMl 595, anakinra, and variants disclosed in U.S. Patent Application Publication Nos.
  • binding moiety is an interferon, as described herein.
  • Binding moieties that can be conjoined to an HSA linker include, e.g., interferon-beta (REBIF® (IFN- ⁇ -la), AVONEX® (IFN- ⁇ -la), and BETASERON® (IFN- ⁇ -lb)), interferon-t (TAUFERONTM), interferon-alpha (e.g., ROFERON-A® (IFN- ⁇ -2a), INTRON-A® (IFN- ⁇ -2b), REBETRON® (IFN- ⁇ -2b), ALFERON-N® (IFN- ⁇ -n3), PEG- INTRON® (IFN- ⁇ -2b covalently conjugated with monomethoxy polyethylene glycol), INFERGEN® (a non-naturally occurring type 1 interferon with 88% homology to IFN- ⁇ - 2b), or PEGASYS® (pegylated IFN- ⁇ -la
  • the present invention further provides HSA linker conjugates with binding moieties that antagonize these pro-inflammatory molecules or their specific receptors to treat autoimmunity. Specific application of HSA linker conjugates for the diagnosis and treatment of MS and RA are described below.
  • MS Multiple sclerosis
  • CNS central nervous system
  • T H I cells The infiltrating CD4 T cells (T H I cells) produce the pro-inflammatory cytokines IL-2, IFN- ⁇ , and TNF- ⁇ that activate antigen-presenting cells (APCs) such as macrophages to produce additional proinflammatory cytokines (e.g., IL-l ⁇ , IL-6, IL-8, and IL-12.
  • IL-12 induces further IFN- ⁇ synthesis.
  • the result is progressive demyelination of neuronal sheaths, leading to human disease.
  • HSA linker conjugates can be used to aid in the diagnosis of MS. Diagnostic HSA linker conjugates that include binding moieties that specifically target one or more (e.g., a bispecific HSA linker conjugate) immune cell activation markers (e.g., CD69, CD28, HTA-DR, and CD45). An imbalance of one or more of these pro-inflammatory or immune cell activation mediators relative to other factors or cells may be measured using an HSA linker conjugate conjoined with a diagnostic agent (e.g., a radioisotope or fluorochrome).
  • a diagnostic agent e.g., a radioisotope or fluorochrome
  • An HSA linker conjugate can be used to treat a person at risk of developing or suffering from MS or to prevent, ameliorate, or cure the symptoms of the disease.
  • binding moieties that specifically target and antagonize ⁇ 4 integrin e.g., natalizumab
  • CD52 e.g., alemtuzumab
  • CD80 P-selectin
  • SlPRl sphingosine-1 -phosphate receptor- 1
  • hyaluronate receptor hyaluronate receptor
  • LFA-I leukocyte function antigen- 1
  • CDl 1 e.g., efalizumab
  • CDl 8 e.g., rituximab
  • CD85 ICOS ligand
  • CCR2, CXCR3, or CCR5 can be useful when conjoined to an HSA linker for therapeutic use in a patient suffering from MS.
  • binding moieties that neutralize type I interferons e.g., interferons -alpha and -beta
  • antagonize type I interferon receptors e.g., IFNaRl
  • HSA linker for therapeutic application.
  • RA Rheumatoid arthritis
  • HSA linker conjugates specific for one or both of these cell surface molecules are useful for the diagnosis of RA.
  • An HSA linker conjugate can be used to treat a person at risk of developing of suffering from RA to prevent, ameliorate, or cure the symptoms of the disease.
  • binding moieties as defined herein, that specifically target and antagonize TNF- ⁇ (e.g., etanercept, infliximab, and adalimumab), IL-I (e.g., anakinra), or CTLA-4 (e.g., abatacept).
  • Binding moieties that specifically target and deplete B cells e.g., an anti- CD20 antibody, such as rituximab
  • Uveitis e.g., an anti- CD20 antibody, such as rituximab
  • Uveitis specifically refers to inflammation of the middle layer of the eye, but may refer to any inflammatory process involving the interior of the eye. Uveitis may be autoimmune or idiopathic in origin
  • An HSA linker conjugate can be used to treat a person at risk of developing of suffering from autoimmune uveitis to prevent, ameliorate, or cure the symptoms of the disease.
  • alpha-fetoprotein e.g., human AFP; NCBI Accession No. NM_001134
  • biologically-active fragments thereof can be conjoined to an HSA linker to reduce or eliminate inflammation associated with autoimmune or idiopathic uveitis. Kits
  • kits that include a pharmaceutical composition containing an HSA linker, and one or more of a binding moiety (e.g., antibodies or antibody fragments), a diagnostic agent (e.g., radionuclide or chelating agents), and a therapeutic agent (e.g., cytotoxic or immunomodulatory agents) with reagents that can be used to conjugate them to the HSA linker, if necessary, and including a pharmaceutically-acceptable carrier, in a therapeutically effective amount for treating a disease or condition (e.g., a cancer, autoimmune disease, or cardiovascular disease).
  • kits include instructions to allow a practitioner (e.g., a physician, nurse, or patient) to administer the composition contained therein.
  • kits include multiple packages of the single-dose pharmaceutical composition(s) containing an effective amount of an HSA linker, or any binding (e.g., antibodies or antibody fragments (e.g., scFv)), diagnostic (e.g., radionuclide or chelating agents), and/or therapeutic (e.g., cytotoxic or immunomodulatory agents) conjugate thereof.
  • any binding e.g., antibodies or antibody fragments (e.g., scFv)
  • diagnostic e.g., radionuclide or chelating agents
  • therapeutic e.g., cytotoxic or immunomodulatory agents conjugate thereof.
  • instruments or devices necessary for administering the pharmaceutical composition(s) may be included in the kits.
  • a kit may provide one or more pre-filled syringes containing an effective amount of an HSA linker, or any binding, diagnostic, and/or therapeutic agent conjugated thereto.
  • kits may also include additional components such as instructions or administration schedules for a patient suffering from a disease or condition (e.g., a cancer, autoimmune disease, or cardiovascular disease) to use the pharmaceutical composition(s) containing an HSA linker, or any binding, diagnostic, and/or therapeutic agent conjugated thereto.
  • a disease or condition e.g., a cancer, autoimmune disease, or cardiovascular disease
  • Example 1 Methods to identify residues in the HSA linker for site-specific conjugation of cytotoxic or cytostatic drugs.
  • the crystal structure is studied and surface exposed serine and threonine residues are identified. These particular surface-exposed amino acids can then be mutated to cysteine allowing drug conjugation to the substituted cysteine using a thiol-specific conjugating agent such as maleimide. Mild reduction may be required prior to drug conjugation.
  • the number of drugs conjugated is controlled by the number of surface exposed cysteine residues introduced into HSA. Serine and threonine are selected as the most suitable residues for mutation as they share the most structural identity with cysteine, however, other surface exposed residues may also be mutated to cysteine and successfully conjugated to cytostatic or cytotoxic drugs.
  • the crystal structure of HSA is deposited in the RSCB Protein Data Bank (lbmO - Sugio et al, "Crystal structure of human serum albumin at 2.5 A resolution," Protein Eng. 12:439-446 (1999)). This structure is analyzed using the Deep View Swiss PDB Viewer downloaded from the Swiss Institute of Bioinformatics. Serine and threonine residues with 50%, 40%, and 30% surface exposure were identified as the most suitable for mutation to cysteine (Table 2). Mutations can be introduced using standard molecular biology procedures. Conjugation of a thiol reactive drug or chelating agent to introduced cysteines can be tested using standard protein chemistry techniques.
  • Example 2 Methods to identify residues in the HSA linker for introduction of asparagine-linked glycosylation sites.
  • B2B3-1 is a bispecific scFv antibody fusion molecule comprising B1D2, a human anti-ErbB2 scFv antibody (SEQ ID NO:27) and H3, a human anti-ErbB3 scFv (SEQ ID NO:26).
  • the two scFvs are joined by a modified human serum albumin (HSA) linker.
  • HSA human serum albumin
  • the anti-ErbB3 scFv, H3 is recombinantly fused to the amino terminus of the HSA linker incorporating a short connector polypeptide and the anti-ErbB2 scFv, B1D2, is recombinantly fused to the carboxy terminus of the modified HSA linker incorporating an additional short connector polypeptide.
  • Each connector polypeptide is selected based on protease resistance properties.
  • the modified HSA linker contains two amino acid substitutions. A cysteine residue at position 34 of native HSA is mutated to serine in order to reduce potential protein heterogeneity due to oxidation at this site.
  • B2B3-1 selectively binds ErbB2 over-expressing tumors by virtue of its high affinity anti-ErbB2 scFv binding moiety, which has a kD in the range of 10.0 nM to 0.01 nM and more preferably a kD of about 0.3 nM.
  • B2B3-1 has a molecular weight of 119.6 kDa and is preferably not glycosylated. B2B3-1 inhibits ligand-induced phosphorylation of ErbB3 with sub- nanomolar potency; this activity is believed to be due, at least in part, to the abundant expression of its dimerization partner, ErbB2, which facilitates specific targeting of cancer cells that express both receptors.
  • Example 4
  • B2B3-1 variants inhibit HRG-induced pErbB3 in ZR75-1 breast cancer cells.
  • ZR75-1 breast cancer cells are treated with a dose range of B2B3-1 variants for 24 hours followed by HRG stimulation.
  • pErbB3 levels are measured in cell lysates by ELISA and IC 50 values are calculated together with the percent of inhibition. Shown are the mean IC 50 values (Y axis) with error bars representing replicate experiments. Percent inhibition values are shown above the corresponding bar.
  • ELISA reagents for total and phospho-ErbB3 ELISAs are purchased from R&D Systems as DUOSET kits.
  • 96-well NUNC MAXISORB plates are coated with 50 ⁇ l of an antibody and incubated overnight at room temperature. Next morning, plates are washed 3 times with 1000 ⁇ l/well in a BIOTEK plate washer with Dulbecco's phosphate buffered saline without calcium or magnesium (PBS) with added Tween detergent (PBST) (0.05% Tween-20). Plates are subsequently blocked for about 1 hr at room temperature with 2 % BSA in PBS. The plates are washed 3 times with 1000 ⁇ l/well in the BIOTEK plate washer with PBST.
  • Cells are grown at 37 0 C and 5% carbon dioxide, washed with cold PBS, then harvested with mammalian protein extract (MPER) lysis buffer (Pierce, 78505) to which 15OmM NaCl, 5mM sodium pyrophosphate, lOuM bpV (phen), 5OuM phenylarsine, ImM sodium orthovanadate, and protease inhibitor cocktail (Sigma, P2714) is added. 50 ⁇ L of cell lysates and standards diluted in 50% Lysis buffer and 1%BSA are used in duplicates for further processing. Samples are incubated for 2hrs at 4°C on a plate shaker and washed as before.
  • MPER mammalian protein extract
  • a detection antibody diluted in 2% BSA, PBST is added and incubated for about 1-2 hrs at room temperature.
  • HRP horseradish peroxidase
  • the detection antibody is directly conjugated to horseradish peroxidase (HRP), while for total ErbB3 and biotinylated mouse anti-human ErbB3 secondary detection antibody is used.
  • HRP horseradish peroxidase
  • the plate is washed as before.
  • For total ErbB3 about 50 ⁇ l of Streptavidin-HRP is added and incubation is for 30 min. and the plates washed as before.
  • About 50 ⁇ L of SUPERSIGNAL ELISA Pico (Thermo Scientific) substrate is added and the plate is read using a FUSION plate reader. Duplicate samples are averaged and, where shown, error bars represent the standard deviation between the two replicates.
  • BT474 breast tumor cells As shown in Figure 6, treatment of BT474 breast tumor cells with B2B3-1 variants causes Gl cell cycle arrest and a decrease in the population of cells in S phase.
  • BT474 cells are treated with 1 ⁇ M of B2B3-1 variants and controls for 72 hours. After the end of treatment, cells are trypsinized, gently resuspended in hypotonic solution containing propidium iodide and single cells are analyzed by flow cytometry. Cell cycle distribution in Gl and S phases is measured using curve-fitting algorithms designed for cell cycle analysis (FlowJo software cell cycle platform).
  • B2B3-1 inhibits ErbB3 activation, utilizing the abundant expression of its dimerization partner, ErbB2, to target tumor cells.
  • a high affinity anti- ErbB2 scFv antibody, B1D2 facilitates targeting of B2B3-1 to tumor cells over- expressing ErbB2.
  • B1D2 is connected by a modified HSA linker to a lower affinity anti- ErbB3 scFv antibody, H3, which blocks binding of ErbB3's ligand, HRG. It is believed that the inhibition of ErbB3 phosphorylation and downstream AKT signaling mediated by B2B3-1 is due to this blockade.
  • the ErbB2 binding scFv, Bl D2 is derived from parent scFv C6.5, which possesses neither agonistic nor antagonistic activity at ErbB2. B1D2, therefore, likely functions solely as a targeting agent.
  • the lower affinity binding of the ErbB3 binding scFv is believed to prevent strong binding of B2B3-1 to normal, noncancerous tissues which express ErbB3 but little or no ErbB2, thereby reducing the potential for non-specific toxicity.
  • B2B3-1 The ability of B2B3-1 to inhibit HRG binding to ErbB3 is investigated using flow cytometry (FACS).
  • FACS flow cytometry
  • Cells of the breast cancer cell line human a variant of BT-474 that over-express ErbB2
  • B2B3-1 are pretreated with 1 ⁇ M B2B3-1 then incubated with 10 nM biotinylated HRG 1 ⁇ EGF domain. After extensive washing, binding is assessed using streptavidin-AlexaFluor 647 conjugate. All incubations are performed at 4°C.
  • Figure 7 shows that B2B3-1 is capable of blocking the binding of HRG to ErbB3. and appears to provide 100% blockade at a concentration of l ⁇ M.
  • Figure 9 shows the effect of B2B3-1 treatment on signaling proteins in BT474 breast cancer cells.
  • Cells are treated with a dose range of B2B3-1 for 24 hours, followed by heregulin stimulation.
  • Levels of pErbB2, pErbB3, pErk and pAKT and their corresponding total protein levels are determined on cell lysates by Western blot analysis. Results indicate that levels of at least pErbB2 and pErbB3 are reduced in a dose-dependent manner by B2B3-1 treatment.
  • Example 10 shows the effect of B2B3-1 treatment on signaling proteins in BT474 breast cancer cells.
  • Figure 10 shows the immunoprecipitation- Western blot analysis of B2B3-1 treated BT474 breast cancer cells.
  • Cells are treated with a dose range of B2B3-1 for 24 hours, followed by heregulin stimulation.
  • ErbB2-associated complexes are isolated from cell lysates using an anti-ErbB2 antibody followed by Western blot analysis to detect pErbB2 and pErbB3 and the corresponding total protein levels.
  • the results show that B2B3-1 crosslinks ErbB2 to ErbB3, so that substantially more ErbB3 and phospho-ErbB3 is precipitated by the anti-ErbB2 antibody.
  • Example 11 shows that B2B3-1 crosslinks ErbB2 to ErbB3, so that substantially more ErbB3 and phospho-ErbB3 is precipitated by the anti-ErbB2 antibody.
  • the anti-tumor activity of B2B3-1 is investigated in vitro using a number of assays.
  • the effect of B2B3-1 on the accumulation of BT-474 or SKBR3 cells in Gl phase and the concomitant decrease in S phase of the cell cycle is examined. Briefly, cells are treated with 1 ⁇ M B2B3-1 or PBS vehicle for 72 hours. After the end of treatment, cells are trypsinized, gently resuspended in hypotonic solution containing propidium iodide and single cells are analyzed by flow cytometry. Cell cycle distribution in Gl and S phases is measured using curve -fitting algorithms designed for cell cycle analysis (FlowJo software cell cycle platform, Tree Star, Inc.).
  • B2B3-1 was found to modestly decrease the percentage of cells in S phase and increase the population of cells in Gl phase ( Figure 11 ⁇ ).
  • the number of cell colonies formed following treatment with B2B3-1 is studied.
  • BT-474 and SKBR3 breast cancer cells are plated in the presence of 1 ⁇ M B2B3-1 and compared to cells plated in media only. Media only or media including treatment is replenished every 3 days. After 14 days the number of colonies is counted and compared to untreated cells.
  • Figure 1 IB illustrates the 40-45% decrease in the number of colonies formed when cells are treated with B2B3-1 compared to control cells.
  • BT- 474 cells are seeded on plates integrated with microelectronic sensor arrays and treated with a dose titration of B2B3-1 or media only for 72 hours. Data reflecting the generation of cell-electrode impedance response are collected every hour for 72 hours and IC 50 values are calculated 68 hours after treatment.
  • Figure 11C illustrates that B2B3-1 was able to inhibit impedance of BT-474 cells with an IC 50 of 33 nM.
  • Example 12 We also investigated whether B2B3-1 exhibits agonistic activity based on its ability to simultaneously bind and cross-link ErbB2 and ErbB3 receptors.
  • Serum starved ZR75-1 breast cancer cells are incubated with 1 ⁇ M B2B3-1 or PBS vehicle for 24 hours. Cells are also treated with B2B3-1 or PBS vehicle for 24 hours followed by a 10-minute stimulation with 5 nM HRG 1 ⁇ EGF domain. Cells are lysed and the pErbB3 content of the lysates is assessed by ELISA generally as described above.
  • Figure 12 shows that cells treated with B2B3-1 alone contained levels of phosphorylated ErbB3 that were comparable to the levels in untreated cells, indicating that B2B3-1 does not act as an agonist promonting ErbB3 phosphorylation.
  • Example 13 shows that cells treated with B2B3-1 alone contained levels of phosphorylated ErbB3 that were comparable to the levels in untreated cells, indicating that B2B3-1 does not act as an agonist promonting ErbB3 phosphorylation.
  • B2B3-1 The ability of B2B3-1 to bind with specificity to ErbB2 and ErbB3 and not to related ErbB family members, EGFR and ErbB4, is investigated by ELISA. Plates are coated with the recombinant extracellular domain of either ErbB2 or ErbB3. Plates are blocked and incubated with a half-maximal binding concentration of B2B3-1 in the presence of a dilution series of competing recombinant extracellular domains of EGFR, ErbB2, ErbB3 or ErbB4. The results show only soluble ErbB2 extracellular domain blocked B2B3-1 binding to ErbB2-coated plates ( Figure 13A).
  • SKO-2 SEQ ID NO:67
  • SKO-3 SEQ ID NO:68
  • SKO-2 and SKO-3 are constructed using the QUIKCHANGE Site Directed Mutagenesis kit (STRATAGENE) which uses oligonucleotide primer pairs, each complementary to opposite strands of the template vector. These are extended during temperature cycling generating a mutated plasmid containing staggered nicks. Following temperature cycling, the product is treated with Dpn I, which is used to digest the parental DNA template. The nicked vector DNA containing the desired mutations is then transformed into XLl-BLUE supercompetent cells STRATA GENE) to propogate plasmid DNA.
  • STRATAGENE QUIKCHANGE Site Directed Mutagenesis kit
  • mutations c295g and a296c are generated using primers 5'- GTA CTT TTG TGC CCG GGC CGA TGT GGG CTA CTG C -3 '(SEQ ID NO:61) and 5'- GCA GTA GCC CAC ATC GGC CCG GGC ACA AAA GTA C -3' (SEQ ID NO:62) and temperature cycling of 95°C for 1 minute followed by 30 cycles of 95°C for 1 minute. 55°C for minute, and 65°C for 17.2 minutes. Mutations are confirmed by DNA sequencing of plasmid DNA.
  • a second round of site-directed mutagenesis is performed on the plasmid with sequence-confirmed mutations at c295g and a296c using primers 5'- GAC ATG TGC CAA GGC CCC CGC GTG GCT GGG AGT G -3' (SEQ ID NO:63) and 5'- CAC TCC CAG CCA CGC GGG GGC CTT GGC ACA TGT C -3' (SEQ ID NO:64) and temperature cycling of 95°C for 30 seconds and 18 cycles of 95°C for 30 seconds, 55°C for 1 minute and 68°C for 17.2 minutes. Mutations are confirmed by DNA sequencing of resulting plasmid DNA.
  • the mutated B1D2 scFv is subcloned into the original B2B3-1 plasmid to replace the anti-ErbB3 scFv, H3.
  • Primers annealing to SKO-2 5 'ACAGTGGCGGCCGCCACCATGGGCTGGTCTCTGATCCTGCTGTTCCTGGTGG CCGTGGCCACGCGTGTGCTGTCCCAGGTGCAGCTCGTCCAGAGCGGCGC (SEQ ID NO:65) and 5'GGAGGCGGCGCCCAGGACTGTCAGCTTGGTGCCACCGCCG (SEQ ID NO:66) are used to isolate the mutated Bl D2 scFv from SKO-2 and to introduce Kas I and Not I restriction sites for subcloning into Kas I/ Not T restriction digested B2B3- 1 plasmid.
  • PCR is performed as follows: 94 0 C for 1 minute followed by 30 cycles of 94°C for 30 seconds, 58°C for 1 minute, 72°C for 1 minute, followed by one cycle of 72°C for 5 minutes to amplify the mutant B1D2.
  • Successful cloning is monitored by DNA sequencing.
  • SKO-2 and SKO-3 plasmids are stably expressed from CHO-Kl cells in shake flasks or 1OL WAVE bags and purified from conditioned media using Blue SEPHAROSE and cation exchange chromatography.
  • MALME-3M melanoma cells which express approximately equal numbers of ErbB2 and ErbB3 receptors, are incubated with a dilution series of B2B3-1, SKO-2, or SKO-3 in the presence of saturating concentrations of a goat anti-HS A Alexafluor-647 conjugated antibody. Cell binding is assessed by flow cytometry and apparent binding affinities are determined for each molecule. Control cells are incubated with secondary antibody alone. No measurable cell binding is observed for SKO-2, which retains only the low affinity binding to ErbB3 mediated by H3 and lacks ErbB2 binding activity.
  • B2B3-1 The stability of B2B3-1 under physiological conditions is assessed by incubating 100 nM B2B3-1 in human, Cynomolgus monkey, or mouse serum at 37°C for a period of 120 hours. Samples are removed at 0, 24, 48, 72, 96 and 120 hours and the ability of B2B3-1 to bind both ErbB2 and ErbB3 is measured by ELISA.
  • This ELISA involves coating a 96-well plate with recombinant ErbB2 extracellular domain overnight followed by a blocking step and then incubation with a dilution series of B2B3-1. Plates are then incubated with an Fc-ErbB3 extracellular domain fusion protein followed by a goat antihuman-Fc-HRP conjugate.
  • Plates are developed by addition of supersignal chemiluminescense substrate.
  • Figures 15A-C show that B2B3-1 remains stable in serum from all three species under physiological conditions, retaining comparable ability to bind both ErbB2 and ErbB3 at all time points measured.
  • Example 16 B2B3-1 dose response in BT-474-M3 human breast cancer in vivo xenograft model.
  • mice bearing human BT- 474-M3 xenografts The efficacy of B2B3-1 in vivo is assessed using nude mice bearing human BT- 474-M3 xenografts. Ten mice per group are treated with 12 doses of 0.3, 1 , 3, 10, 30 or 90 mg/kg of B2B3-1 every 3 days. Control groups are administered PBS vehicle or HSA at an equimolar dose to the 90 mg/kg B2B3-1 dose. All doses are administered interperitoneally (i.p.). Tumor size is measured twice a week and the corresponding tumor volume is calculated. The results show that B2B3-1 treatment leads to significant reduction in BT-474-M3 tumor size as compared to the control group (Figure 16). Complete regressions were observed in each of the B2B3-1 treatment groups except mice treated with the lowest dose of B2B3-1 (0.1 mg/kg).
  • B2B3-1 reduces tumor size in multiple xenograft models in an ErbB2 dependent manner.
  • B2B3-1 was efficacious in the Calu-3 ( Figure 17A), SKOV-3 ( Figure 17B), NCI-N87 ( Figure 17C), and MDA-MB-361 (Figure 17E) xenograft models expressing ErbB2 at > 1 x 10 5 receptors/cell but worked less well in the ACHN (Figure 17D) xenograft model which expresses 4.5 x 10 4 ErbB2 receptors/cell. Mice were treated with 30 mg/kg of B2B3-1 every 3 days.
  • Example 18 Example 18
  • ErbB2 converts B2B3-1 non-responder ADRr breast cancer xenograft model into a responder to B2B3-1 ( Figures 18A and 18B).
  • ErbB2 is over- expressed in ADRr breast cancer cells using a retroviral expression system.
  • Transfected cells expressing high levels of ErbB2 (ADRr -E2) are selected using FACS and subsequently injected subcutaneously into nude mice to establish xenograft tumors. Mice are treated with 30 mg/kg of B2B3-1 every 3 days. While no response to B2B3-1 was observed in wild type ADRr xenografts ( Figure 18A), ADRr-E2 xenografts ( Figure 18B) responded to B2B3-1.
  • Example 19 Example 19
  • B2B3-1 activity correlates positively with ErbB2 expression levels in vitro ( Figure 19A) and in vivo (Figure 19B).
  • B2B3-1 inhibition of ErbB3 phosphorylation is determined in 9 tumor cell lines with expression levels of ErbB2 ranging from 5 x 10 4 receptors/cell to 2.4 x 10 6 receptors/cell using an ELISA assay. The extent of B2B3-1 's ability to inhibit ErbB3 phosphorylation to basal levels (% pErbB3 inhibition) was found to correlate positively with ErbB2 expression levels.
  • B2B3-1 activity is assessed in 10 tumor xenograft models expressing low to high levels of ErbB2. Xenograft response also correlated positively with ErbB2 expression levels.
  • B2B3-1 treatment of BT474-M3 breast tumor cells results in translocation of p27 klp1 to the nucleus ( Figure 20A).
  • BT474-M3 cells are treated with 1 ⁇ M of B2B3-1 for 6 hours.
  • the sub-cellular location of p27 k ⁇ pl is assessed using immunofluorescence techniques.
  • p27 k ⁇ I translocated to the nucleus which has been shown to result in inhibition of cell proliferation.
  • p27 k ⁇ pl remained in the cytoplasm of untreated cells.
  • BT-474-M3 cells treated with B2B3-1 for 72 hours are probed for the cell cycle regulator Cyclin Dl using Western blot analysis ( Figure 20B).
  • the cytoskeleton protein vinculin is used as a protein loading control in this experiment.
  • B2B3-1 treatment resulted in a decrease in the levels of Cyclin Dl compared to untreated cells.
  • B2B3-1 treatment of BT474-M3 breast tumor xenografts results in translocation of p27 kipl to the nucleus.
  • BT474 breast tumor xenografts are treated with B2B3-1 ( Figures 21A) at a dose of 30 mg/kg or an equimolar dose of HSA ( Figures 21B) every 3 days for a total of 4 doses.
  • Increased nuclear staining for p27 klpl was observed in B2B3-1 treated tumors compared to HSA control tumors indicating an antiproliferative effect of B2B3-1 in vivo.
  • B2B3-1 treatment results in a reduction of the proliferation marker Ki67 in BT474 breast cancer xenograft tumors.
  • BT474-M3 breast tumor xenografts are treated with B2B3-1 (Figure 22A) at a dose of 30 mg/kg or an equimolar dose of HSA ( Figure 22B) every 3 days for a total of 4 doses.
  • Subsequent staining of tumor sections for Ki67 demonstrated a reduced expression pattern for B2B3-1 treated tumors compared to HSA treated tumors.
  • B2B3-1 treatment results in a reduction of vessel density in BT474-M3 breast cancer xenograft tumors, as determined by assaying for CD31 expression ( Figures 23 A- B).
  • BT474 breast tumor xenografts are treated with B2B3-1 ( Figure 23A) at a dose of 30 mg/kg or an equimolar dose of HSA ( Figure 23B) every 3 days for a total of 4 doses. Tumors are stained for the presence of vascular marker CD31.
  • Tumors treated with B2B3-1 show a marked decrease in vessel density compared to control tumors treated with HSA.
  • Example 24 B2B3-1 inhibits phosphorylation ofErbB3 in vivo.
  • BT-474-M3 xenograft tumors are treated with 30 mg/kg B2B3-1 or 17.5 mg/kg HSA every 3 days for a total of 4 doses and tumors are harvested 24 hours after the final dose.
  • Tumors are lysed and subjected to SDS-PAGE followed by Western analysis to assess relative levels of phosphorylation of B2B3-1 's target ErbB3.
  • Equal quantities of protein are loaded in each lane and total protein levels are controlled by probing for beta tubulin.
  • Western blot analysis using antibodies specific for phosphorylated ErbB3 show that B2B3-1 treated tumors contain less pErbB3 than HSA treated tumors ( Figure 24A).
  • Example 25 In vivo activity ofB2B3-l in BT-474-M3 xenografts which have reduced PTEN activity.
  • ShRNA technology is applied to knock out the activity of the tumor suppressor gene phosphatase and tensin homolog (PTEN) in BT-474-M3 breast cancer cells. Briefly, cultured BT-474-M3 cells are transfected with shPTEN or shControl RNA by retroviral transfection. Transfected cells with reduced PTEN are selected using FACS and subsequently injected subcutaneously into the right flank of nude mice to establish xenograft tumors. Cells transfected with a control vector are injected into the left flank of the same mouse. Mice are treated with 30mg/kg B2B3-1 every 3 days or 10mg/kg trastuzumab every week. HSA is injected as a control at an equimolar dose to B2B3-1. All injections are done i.p.
  • B2B3-1 and trastuzumab promoted a reduction in the size of tumors formed by control BT-474-M3 breast cancer cells ( Figure 25A), whereas only B2B3-1 (and not trastuzumab) promoted a reduction in the size of tumors formed by BT-474-M3 human breast cancer cells lacking expression of PTEN ( Figure 25B).
  • Example 26 B2B3-1 inhibits ErbB3 signaling in BT-474-M3 breast cancer cells having reduced PTEN activity.
  • B2B3-1 The ability of B2B3-1 to inhibit phosphorylation of ErbB3 signaling in tumor xenografts is studied using the PTEN deficient BT-474-M3 model described above.
  • Xenograft tumors of the engineered cell line or control cell line are treated with 30 mg/kg B2B3-1, 17.5 mg/kg HSA every 3 days or 10 mg/kg trastuzumab weekly and tumors are harvested 24 hours after the final dose. Tumors are lysed and subjected to SDS-PAGE followed by Western analysis to assess relative levels of phosphorylation of B2B3-l 's target ErbB3, AKT and total PTEN levels. Equal quantities of protein are loaded in each lane and total protein levels are controlled by probing for PCNA.
  • the pharmacokinetic parameters for B2B3-1 are investigated in nu/nu mice. Animals are randomized into groups and administered intravenous (IV) with a single dose of 5, 15, 30, or 45 mg/kg B2B3-1 ( Figures 27A-D, respectively). Blood is collected pre- dose and at 0.5, 4, 8, 24, 48, 72, and 120 hours post dose. Three mice are used for each time point. Serum levels of B2B3-1 are measured using two ELISA methods. The first method requires functional binding of B2B3-1 to both ErbB2 and ErbB3 while the second method measures only the HSA component of B2B3-1 in the serum.
  • the HSA ELISA utilizes a polyclonal-anti HSA capture antibody and a HRP-conjugated polyclonal anti- HSA detection antibody.
  • a reduction in B2B3-1 serum concentration measured using the ErbB2/ErbB3 binding method versus the HSA method would indicate a loss in functional B2B3-1.
  • Figures 27A-D show that the pharmacokinetic properties of B2B3-1 are comparable when assessed using either ELISA method, indicating that B2B3-1 is stable in circulation in mice.
  • B2B3-1 serum concentrations are fit using a two-compartment, biexponential model and show biphasic disposition. Terminal half-lives were calculated to be 17, 16, 23, and 18 hrs for the 5, 15, 30, or 45 mg/kg doses, respectively, and are shown in Table 4. Increases in B2B3-1 dose resulted in a linear increase in exposure (Figure 28).
  • Table 4 Pharmacokinetic properties of B2B3-1 in mice and Cynomolgus monkeys.
  • Blood samples for pharmacokinetic analysis are also obtained from a dose range- finding toxicology study in female Cynomolgus monkeys.
  • animals are infused with 4, 20 or 200 mg/kg of B2B3-1 administered every 3 days for 4 doses. Sampling occurred prior to and 5 minutes after dosing on each dosing day (study days 1, 4, 7 and 10) to provide pre-dose and peak/trough concentrations, and at 1, 2, 4, 8, 24 and 48 hours after the end of the first infusion on day 1 and at 1 , 2, 4, 8, 24, 48, 72 and 120 hours after the last infusion on day 10.
  • serum samples are also collected at 168, 336 and 456 hours after the last infusion.
  • the plasmid encoding the B2B3-1 bispecific scFv antibody fusion protein is created combining gene sequences of a unique human anti-ErbB3 scFv (designated "H3"), a human anti-ErbB2 scFv (designated "B1D2”), and a modified human serum albumin (HSA) linker.
  • H3 human anti-ErbB3 scFv
  • B1D2 human anti-ErbB2 scFv
  • HSA human serum albumin
  • the anti-ErbB3 scFv, H3 is recombinantly linked to the amino terminus of the HSA linker via a connecting peptide (Ala-Ala-Ser) and the anti-ErbB2 scFv, B1D2, is genetically linked the carboxy terminus of the HSA linker via a connecting peptide (Ala- Ala- Ala-Leu - SEQ ID NO:5).
  • the peptide connectors are formed through the introduction of restriction sites during construction of the mammalian expression vector and are synthesized with optimized codon usage for mammalian expression together with the single chain antibody fragments and HSA linker.
  • the B1D2 scFv is selected from a combinatorial phage display library created by mutagenesis of the ErbB2-binding scFv C6.5, which is selected from a non-immune phage display library.
  • the H3 scFv is selected from a non-immune phage display library originally made by Sheets et al.
  • the gene sequences encoding the B1D2 and H3 single chain antibody fragments are optimized for CHO cell codon preferences and synthesized for subsequent construction of the B2B3-1 encoding plasmid.
  • the modified HSA linker contains two amino acid substitutions.
  • a cysteine residue at position 34 is mutated to serine in order to reduce potential protein heterogeneity due to oxidation at this site.
  • An asparagine residue at amino acid 503 is mutated to glutamine, which in wild type HSA is sensitive to deamination and can result in decreased pharmacologic half-life.
  • the gene sequence encoding the modified HSA linker is synthesized with optimized codon usage for mammalian expression for subsequent construction of the B2B3-1 encoding plasmid.
  • the B2B3-1 coding sequence is cloned into pMPlOk base vector using standard molecular biology techniques to create plasmid pMP10k4H3-mHSA-BlD2, shown in Figure 30.
  • this construct employs commonly used genetic elements.
  • B2B3-1 expression is driven by the human GAPD promoter.
  • This vector utilizes genetic elements referred to as Matrix Attachment Regions or MAR elements.
  • the MAR genetic elements control the dynamic organization of chromatin, and insulate nearby genes from the effect of surrounding chromatin thereby increasing copy number dependent, position- independent, expression of genes.
  • MAR elements have been shown to improve the probability of isolating a clone exhibiting the desired level of expression for the production of a recombinant protein and to increase the stability of production.
  • the MAR elements used in the B2B3-1 constructs are non-coding human MAR elements.
  • a neomycin antibiotic resistance plasmid Figure 31
  • a hygromycin resistance plasmid Figure 32
  • Chinese Hamster Ovary CHO-Kl cells are purchased from ATCC (ATCC # CCL- 61).
  • the CHO-Kl cell line is a serum and proline dependent adherent sub-clone of the parental CHO cell line created by T.T. Puck.
  • the CHO-Kl cells used for B2B3-1 transfection are pre-adapted for suspension growth in serum free media prior to transfection. An iterative transfection procedure is used to develop the B2B3-1 cell line.
  • CHO-Kl cells are sub passaged to l.Oxl O 6 cells/mL in SFM4CHO (Serum Free) medium (HyClone, Logan, UT) supplemented with 8 mM L-glutamine, 0.1 mM sodium hypoxanthine, and 0.016 mM thymidine.
  • SFM4CHO (Serum Free) medium HyClone, Logan, UT
  • OptiMEM medium Invitrogen Corp, Carlsbad, CA
  • the B2B3-1 expression plasmid (pMP10k4H3-mHSA-BlD2) and the neomycin resistance plasmid (Figure 30; pSV2-neo (Selexis, Inc., Marlborough, MA) are mixed together using a molar plasmid ratio of 75: 1 (B2B3-1 :neomycin resistance).
  • the plasmid mixture is subsequently mixed with a cationic lipid transfection reagent (Lipofectamine LTX, Invitrogen Corp, Carlsbad, CA) and lipid/DNA complexes are allowed to form for thirty minutes.
  • the DNA/Lipid complex is then added to the CHO- Kl cells and the 24-well plates are placed in a 37°C, 5% CO 2 incubator.
  • Example 33 Selection and Screening for High Producers
  • the contents of each transfection well are washed with PBS, trypsinized and distributed across two, ninety-six well plates.
  • the growth media used consists of DMEM/F12 (Invitrogen Corp, Carlsbad, CA) with 10% FBS (Invitrogen Corp, Carlsbad, CA) and 500 mg/L of geneticin (G418; Invitrogen Corp, Carlsbad, CA).
  • Media in the 96- well plates is changed on day 4 to SFM4CHO medium supplemented with 8 mM L- glutamine, 0.1 mM sodium hypoxanthine, 0.016 mM thymidine, and 500mg/L geneticin.
  • SFM4CHO medium Supplemented with 8 mM L- glutamine, 0.1 mM sodium hypoxanthine, 0.016 mM thymidine, and 500mg/L geneticin.
  • surviving cells have formed well-defined colonies.
  • the clones are evaluated using quantitative spot blot techniques.
  • a seven day productivity assay is used to screen for high B2B3-1 producing colonies.
  • the cells in 24-well plates are allowed to proliferate for seven days in SFM4CHO medium supplemented with 8 mM L-glutamine, 0.1 mM sodium hypoxanthine, and 0.016 mM thymidine.
  • the B2B3-1 concentration in the spent media is determined.
  • Top clones from the 24-well scale are expanded into 125 mL baffled shake flasks.
  • the highest producing cell pool determined from the first round of transfection is transfected a second time to increase production. Twenty-four hours before transfection, the cell pool is sub passaged to 1.OxIO 6 cells/mL in SFM4CHO (Serum Free) medium supplemented with 8mM L-glutamine, 0.1 mM sodium hypoxanthine, and 0.016 mM thymidine. On the day of transfection, cells are resuspended in OptiMEM medium (Invitrogen Corp, Carlsbad, CA) and 40,000 cells re placed in each well of a twenty- four well plate.
  • SFM4CHO Serum Free
  • the B2B3-1 and hygromycin resistance plasmid ( Figure 32; pTK-IIyg (Clontech, Mountain View, CA)) are mixed together using a molar plasmid ratio of 50: 1 (B2B3-1 :hygromycin resistance).
  • the plasmid mixture is subsequently mixed with a cationic lipid transfection reagent (Lipofectamine LTX, Invitrogen Corp) and lipid/DNA complexes are allowed to form for thirty minutes.
  • the DNA/Lipid complex is then added to the cell pool and the 24-wcll plates are placed in a 37°C, 5% CO 2 incubator.
  • Example 35 Selection and Screening for High Producers From Second Transfection
  • the contents of each transfection well are washed with PBS, trypsinized and distributed across two, 96-well plates.
  • the growth media used consists of DMEM/F12 supplemented with 10% FBS and 400 mg/L of hygromycin B (Invitrogen Corp).
  • Media in the 96-well plates is changed on day 4 to Hyclone SFM4CHO medium supplemented with 8 mM L-glutamine, 0.1 mM sodium hypoxanthine, 0.016 mM thymidine, and 400 mg/L of hygromycin B.
  • Hyclone SFM4CHO medium supplemented with 8 mM L-glutamine, 0.1 mM sodium hypoxanthine, 0.016 mM thymidine, and 400 mg/L of hygromycin B.
  • surviving cells have formed well-defined colonies.
  • the clones are evaluated using quantitative spot blot techniques.
  • the top producing colonies are trypsinized, and expanded to
  • a seven day productivity assay is used to screen for high B2B3-1 producing colonies.
  • the cells Upon expansion the cells are allowed to proliferate for seven days, and the B2B3-1 concentration in the spent media is determined.
  • Top clones from the 24-well plates are expanded into 125 mL baffled shaker flasks in the Hyclone SFM4CHO medium supplemented with 8mM L-glutamine, 0.1 mM sodium hypoxanthine, and 0.016 mM thymidine.
  • a seven day study in shake flask is used to screen the cell pools for growth and B2B3-1 production.
  • the spent media is quantitated using Protein Aresin and an HPLC instrument.
  • Example 36 Limiting Dilution Cloning
  • the best growing and highest B2B3-1 -producing colony identified by the productivity assay is transferred from the 125 mL shaker flask and plated in five 96-well plates at a cell concentration calculated to give one cell/well.
  • the 96-well plates are placed in an incubator at 37°C and 5% CO 2 .
  • the wells are examined bi-weekly to track formation of colonies. Colonies arising from a single cell are identified based on the symmetrical shape of the colony. Wells containing such colonies are marked for further screening by 24-well 7-day assessment, and 125mL shaker flask 7-day assessment.
  • B2B3-1 is administered once a week via intravenous infusion over a period of 60 or 90 minutes, depending on patient tolerability.
  • B2B3-1 is formulated in a sterile 20 mM L-histidine hydrochloride, 150 mM sodium chloride, pH 6.5 solution at a concentration of 25mg/mL for administration to a patient (e.g., a human).
  • Example 38 Treatment of Breast Cancer
  • a patient's cancer is believed to be expressing high levels of epidermal growth factor receptors, including ErbB2 (HER2/neu)
  • an HSA linker conjoined to an ErbB2 biding moiety, such as B2B3-1, B2B3-2, v-3, B2B3-4, B2B3-5, B2B3-6, B2B3-7, B2B3-8, B2B3-9, or B3B3-10 (see Table 6, below) would be indicated.
  • an HSA linker conjoined to an ErbB2 biding moiety, such as B2B3-1, B2B3-2, v-3, B2B3-4, B2B3-5, B2B3-6, B2B3-7, B2B3-8, B2B3-9, or B3B3-10 (see Table 6, below) would be indicated.
  • genotypic or histologic screens of cancer biopsies reveals increased expression of ErbB2 in the patient's tumor.
  • a B2B3 HSA linker conjugate (e.g., B2B3-1, SEQ ID NO: 16) is administered to a patient diagnosed with breast cancer once a week or twice a week via intravenous infusion over a period of, e.g., 60 or 90 minutes, depending on patient tolerability, at a dose no higher than 30mg/kg.
  • the B2B3 HSA linker conjugate is formulated in a sterile 20 mM L-histidine hydrochloride, 150 mM sodium chloride, pH 6.5 solution at a concentration of 25mg/mL for administration to the patient.
  • a clinician supervising the administration of the B2B3 HSA linker conjugate follows common formulation and dosing practices to determine the proper course of therapy for the patient.
  • the clinician may further co-administer one or more therapies with the B2B3 HSA linker conjugate.
  • one or more therapeutic drugs or compounds may be administered in combination with the B2B3 HSA linker conjugate, such as the common chemotherapeutic regimen for the treatment of breast cancer, which includes doxorubicin, cyclophosphamide, and paclitaxel.
  • a clinician can administer the B2B3 HSA linker conjugate in combination with surgical or radiation therapy to treat breast cancer the patient.
  • Example 39 Treatment of Ovarian Cancer
  • a patient's cancer is believed to be expressing high levels of epidermal growth factor receptors, including ErbB2 (HER2/neu), then treatment with an HSA linker conjoined to an ErbB2 biding moiety, such as B2B3-1, B2B3-2, v-3, B2B3-4, B2B3-5, B2B3-6, B2B3-7, B2B3-8, B2B3-9, or B3B3-10 (see Table 6, below) would be indicated..
  • an HSA linker conjoined to an ErbB2 biding moiety, such as B2B3-1, B2B3-2, v-3, B2B3-4, B2B3-5, B2B3-6, B2B3-7, B2B3-8, B2B3-9, or B3B3-10 (see Table 6, below) would be indicated..
  • an HSA linker conjoined to an ErbB2 biding moiety, such as B2B3-1, B2B3-2, v-3, B
  • a B2B3 HSA linker conjugate (e.g., B2B3-1, SEQ ID NO: 16) is administered to the patient diagnosed with ovarian cancer alone or in combination with one or more other therapies essentially as described in the preceding Example.
  • HSA linker conjugates are constructed using one or more of the elements (groups A-E) listed in Table 5 below.
  • an HSA linker conjugate which is shown as Group C in Table 5 below, incorporates one or more binding moieties selected from groups A and E shown in Table 5.
  • the HSA linker conjugates can also include one or more peptide connectors, which are selected from groups B and D in Table 5, at each of the amino and carboxy terminal ends of the HSA linker. Peptide connectors can be repeated or truncated to increase or decrease the length of the connector sequence.
  • Example 41 in vivo, B2B3-1 dosed q7d shows equivalent efficacy as B2B3-1 dosed q3d
  • B2B3-1 efficacy using a q7d (once every 7 days) dosing regimen is determined in in female athymic nude mice (nu/ nu) from Charles River Labs, 5-6 weeks of age bearing xenograft tumors of the human breast cancer cell line BT-474- M3 ( Figure 33).
  • Mice receive a subcutaneous estrogen-releasing implant in the opposite flank (0.72 mg pellet, 60 day, slow-release, innovative Research of America, Sarasota, FL) 24 h prior to the injection of 2OxIO 6 human BT-474-M3 cells in PBS.
  • Dosing is initiated when tumor growth is established (tumor volumes of approximately 400 mm3) and B2B3-1 is administered to 10 mice per group either once every 3 days (q3d) at 30 mg/kg for the course of the study or once every 7 days at 22 mg/kg, 66 mg/kg, 132 mg/kg or 198 mg/kg by intraperitoneal injection. Tumors are measured twice a week using digital calipers. Tumor volume is calculated using the formula: ⁇ /6 x (W 2 x L), where W is the short diameter and L is the long diameter. Pharmacokinetic calculations suggest that a 66 mg/kg dose q7d should give a similar exposure of B2B3-1 to the xenograft tumor as a 30 mg/kg dose q3d.
  • B2B3-1 efficacy was equivalent for the 30 mg/kg, q3d dose and the 3 highest doses of B2B3-1 administered q7d, indicating a q7d dosing schedule for B2B3-1 is suitable in this model.
  • B2B3-1 to inhibit heregulin induced ErbB3 activity is tested using Western blot analysis.
  • Monolayers of serum-starved BT-474-M3 cells are treated for 24 hours with 100 nM B2B3-1 or trastuzumab and then stimulated with 5 nM HRG l ⁇ EGF for 10 minutes.
  • Cells are also treated with 1OnM and 100 nM B2B3-1 or 1OnM and 100 nM trastuzumab and left unstimulated. Lysates are subjected to immunoblot analysis for ErbB3, pErbB3, AKT, and pAKT.
  • B2B3-1, trastuzumab and the combination of both drugs on the growth of cancer cell spheroids was examined using four differnt breast cancer cell lines.
  • 2,000 cells of BT-474-M3, SKBR3 (ATCC), or MDA-MB-361 (ATCC) human breast cancer cells were seeded in round-bottom low adherence 96-well plates (Corning® 96 Well Clear Round Bottom Ultra Low Attachment Microplate - Product #7007) and the following day the spheroids were measured and treated with a dose range of B2B3-1, trastuzumab or a combination of both at a ratio of 3 fold molar excess B2B3-1 to trastuzumab.
  • Example 44 B2B3-1 has an additive effect when administered with trastuzumab in vivo
  • Tumor volume is calculated using the formula: ⁇ /6 x (W x L), where W is the short diameter and L is the long diameter.
  • Ten mice per group are administered B2B3-1 at 3 mg/kg or 10 mg/kg q3d, trastuzumab at 1 mg/kg or 0.1 mg/kg q7d or the combination of both drugs for the course of the study by intraperitoneal injection.
  • B2B3-1 and trastuzumab (10 mg/kg B2B3-1 + 1 mg/kg trastuzumab, 10 mg/kg B2B3-1 + 0.1 mg/kg trastuzumab, 3 mg/kg B2B3-1 + 1 mg/kg trastuzumab, 3 mg/kg B2B3-1 + 0.1 mg/kg trastuzumab) are dosed as for the corresponding single agent.
  • the combination of at least 3 mg/kg of B2B3-1 and 1 mg/kg trastuzumab induces essentially complete tumor regression in at least about 50% of nude mice carrying human breast tumor cell xenografts, while the same concentrations of either B2B3-1 or trastuzumab alone do not provide complete regression in even 10% of such mice.
  • HSA linkers Assays for HSA linkers, peptide connectors, and binding moieties discussed above. Table 6, below, lists ten HSA linker conjugates with varying ErbB2-specific or ErbB3-specific binding moieties, as well as peptide connectors, at the amino and carboxy termini of an HSA linker.
  • H3 underlined 1-248 with bold italicized CDRs
  • Bl D2 underlined 841-1095 with bold italicized CDRs
  • Connectors to modified HSA are dotted- underlined. Sequence alignments for various HSA Linker Conjugates Comprising B2B3 and mHSA
  • H3-mHSA-BlD2 D QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGL
  • H3-mHSA-BlD2 (46) EWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAED
  • A5-mHSA-BlD2 (91) TAVYYCARDG VATTPFDYWGQGTLVTVS SGGGGSGGGGS
  • A5-mHSA-F5B6H2 (91) TAVYYCARDG VATTPFDYWGQGTLVTVS SGGGGSGGGGS
  • F4-mHSA-F5B6H2 (91) TAVYYCAKGYSSSWSEVASGYWGQGTLVTVSS ⁇ STGGGGSGGGCS
  • H3-mHSA-BlD2 91) TAVYYCARDR GVGYFDLWGRGTLVTVSSASTGGGGSGGGGS
  • H3-mHSA-F5B6H2 (91) TAVYYCARDR GVGYFDLWGRGTLVTVSSASTGGGGSGGGGS
  • A5-rnHSA-BlD2 (130) GGGGSQSVLTQPPS-VSGAPGQRVTISCTGSSSNIGAGYDVHWYQ
  • A5-mHSA-F5B6H2 (130) GGGGSQSVLTQPPS-VSGAPGQRVTISCTGSSSNIGAGYDVHWYQ
  • H3-mHSA-BlD2 (132) GGGGSQSALTQPAS-VSGSPGQSITISCTGTSSDVGGYNFVSWYQ
  • H3-mHSA-F5B6H2 (132) GGGGSQSALTQPAS-VSGSPGQSITISCTGTSSDVGGYNFVSWYQ
  • H3-mHSA-BlD2 (221) EADYYCSSYGSSSTHVIFGGGTKVTVLG-AASDAHKSEVAHRFKD
  • H3-mHSA-F5B ⁇ H2 (221) EADYYCSSYGSSSTHVIFGGGTKVTVLG-AASDAHKSEVAHRFKD
  • A5-mHSA-BlD2 (262) LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES
  • A5-mHSA-F5B6H2 (262) LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADE ⁇
  • H3-mHSA-BlD2 (265) LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES
  • H3-mHSA-F5B6H2 (265) LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES
  • A5-mHSA-ML3.9 (307) AENCDKSLHTLFGDKLCTVATLRETYGEM ⁇ DCCAKQEPERNECFL
  • A5-mHSA-BlD2 (307) AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL
  • A5-mHSA-F5B6H2 (307) AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL
  • F4-mHSA-BlD2 (311) AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL
  • F4-mHSA-F5B6H2 (311) AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL
  • H3-mHSA-BlD2 (310) AENCDKSLIITLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL
  • H3-mHSA-F5B6H2 (310) AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL
  • A5-mHSA-BlD2 (352) OHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYElARRHPY
  • H3-mHSA-BlD2 (355) QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY
  • H3-mHSA-BlD2 (400) FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS
  • H3-mHSA-F5B6H2 (400) FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS
  • H3-mHSA-BlD2 (535) LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG
  • F4-mHSA-BlD2 (581) MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV
  • H3-mHSA-BlD2 (580) MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV
  • H3-mHSA-BlD2 (625) FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV
  • H3-mHSA-F5B6H2 (625) FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV
  • H3-mHSA-BlD2 (670) STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL
  • H3-mHSA-F5B6H2 (670) STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL
  • H3-mHSA-BlD2 (715) HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT
  • H3-mHSA-F5B6H2 (715) HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT
  • F4-mHSA-BlD2 (761) FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA
  • F4-mHSA-F5B6H2 (761) FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA
  • H3-mHSA-BlD2 (760) FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA
  • A5-mHSA-BlD2 (802) FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGA
  • F4-mHSA-BlD2 (806) FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGA
  • F4-mHSA-F5B6H2 (806) FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVESGG
  • H3-mHSA-BlD2 (805) FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGA
  • A5-mHSA-ML3.9 (847) EVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPG
  • A5-mHSA-BlD2 (847) EVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPG
  • A5-mHSA-F5B6H2 (847) GLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGR
  • F4-mHSA-BlD2 (851) EVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPG
  • F4-mHSA-F5B6H2 (851) GLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGR
  • H3-mHSA-BlD2 (850) EVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPG
  • H3-mHSA-F5B6H2 (850) GLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGR
  • H3-mHSA-R] D2 (895) DSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARH
  • H3-mHSA-F5B6H2 (895) GDNTYYADSVKGRFTISRDNSKNTT,YLQMNSLRAEDTAVYYCAKM
  • A5-mHSA-BlD2 ( 937 ) DVGYCTDRTCAKWPEWLGVWGQGTLVTVSSGGGGSSGGGSGGGGS
  • F4-mHSA-F5B6H2 (941) TS NAVGFDYWGQGTLVTVSSGGGGSGGGSGGGGSG
  • H3-mHSA-BlD2 (940) DVGYCTDRTCAKWPEWLGVWGQGTLVTVSSGGGGSSGGG ⁇ GGGGS
  • A5-mHSA-BlD2 (982) QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNY-VSWYQQLPGTA
  • A5-mHSA-F5B6H2 (972) QSVLTQPPSVSGAPGQRVTISCTGRHSNIGLGYGVHWYQQLPGTA
  • H3-mHSA-BlD2 (985) QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNY-VSWYQQLPGTA
  • H3-mHSA-BlD2 (1029) PKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDE ⁇ DYYC
  • H3-mHSA-F5B6H2 (1020) PKLLIYGNTNRPSGVPDRFSGFKSGTSASLAITGLQAEDEADYYC

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Oncology (AREA)
  • Inorganic Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne un lieur de sérum-albumine humaine (HSA) et un lieur HSA avec des agents de liaison, diagnostiques et thérapeutiques conjugués à celui-ci. La présente invention concerne en outre un conjugué dans lequel le lieur HSA est lié de manière covalente à des fragments de liaison amino- et carboxy-terminaux qui sont des première et deuxième molécules Fv monocaténaires (scFv). Les conjugués exemplifiés sont utiles, par exemple, dans la réduction de la prolifération de cellules tumorales, par exemple, pour des applications thérapeutiques. La présente invention concerne en outre des procédés et des trousses pour l'application diagnostique et thérapeutique d'un conjugué de lieur HSA.
PCT/US2009/060721 2008-11-18 2009-10-14 Lieurs de sérum-albumine humaine et conjugués de ceux-ci WO2010059315A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
JP2011536365A JP5677972B2 (ja) 2008-11-18 2009-10-14 ヒト血清アルブミンリンカーおよびそのコンジュゲート
CN2009801545099A CN102282168A (zh) 2008-11-18 2009-10-14 人血清白蛋白接头以及其结合物
BRPI0921586A BRPI0921586A2 (pt) 2008-11-18 2009-10-14 articuladores de albumina de soro humana e conjugados destes
US13/130,007 US8927694B2 (en) 2008-11-18 2009-10-14 Human serum albumin linkers and conjugates thereof
US12/757,801 US20110059076A1 (en) 2008-11-18 2010-04-09 Human serum albumin linkers and conjugates thereof
US14/582,719 US20150191545A1 (en) 2008-11-18 2014-12-24 Human serum albumin linkers and conjugates thereof
US15/251,158 US20170204192A1 (en) 2008-11-18 2016-08-30 Human serum albumin linkers and conjugates thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US11579708P 2008-11-18 2008-11-18
US61/115,797 2008-11-18
USPCT/US2009/040259 2009-04-10
PCT/US2009/040259 WO2009126920A2 (fr) 2008-04-11 2009-04-10 Lieurs d'albumine de sérum humain, et ses conjugués

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/040259 Continuation-In-Part WO2009126920A2 (fr) 2008-04-11 2009-04-10 Lieurs d'albumine de sérum humain, et ses conjugués

Related Child Applications (3)

Application Number Title Priority Date Filing Date
US13/130,007 A-371-Of-International US8927694B2 (en) 2008-11-18 2009-10-14 Human serum albumin linkers and conjugates thereof
US12/757,801 Continuation US20110059076A1 (en) 2008-11-18 2010-04-09 Human serum albumin linkers and conjugates thereof
US14/582,719 Continuation US20150191545A1 (en) 2008-11-18 2014-12-24 Human serum albumin linkers and conjugates thereof

Publications (1)

Publication Number Publication Date
WO2010059315A1 true WO2010059315A1 (fr) 2010-05-27

Family

ID=42198443

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/060721 WO2010059315A1 (fr) 2008-11-18 2009-10-14 Lieurs de sérum-albumine humaine et conjugués de ceux-ci

Country Status (6)

Country Link
US (4) US8927694B2 (fr)
JP (1) JP5677972B2 (fr)
KR (1) KR20110112301A (fr)
CN (1) CN102282168A (fr)
BR (1) BRPI0921586A2 (fr)
WO (1) WO2010059315A1 (fr)

Cited By (72)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120220530A1 (en) * 2009-10-30 2012-08-30 Novozymes Biopharma Dk A/S Albumin variants
WO2012125573A3 (fr) * 2011-03-11 2012-12-27 Merrimack Pharmaceuticals, Inc. Utilisation d'inhibiteurs des récepteurs de la famille egfr dans le traitement de cancers du sein réfractaires aux hormones
US20130053322A1 (en) * 2010-02-16 2013-02-28 Medlmmune, Llc Hsa-related compositions and methods of use
WO2013067355A1 (fr) * 2011-11-04 2013-05-10 Novartis Ag Constructions de protéine 6 liée à la lipoprotéine de basse densité (lrp6) prolongeant leur demi-vie
WO2012116317A3 (fr) * 2011-02-24 2013-11-14 Merrimack Pharmaceuticals, Inc. Polythérapies comprenant des agents anti-erbb3
CN103429262A (zh) * 2011-03-15 2013-12-04 梅里麦克制药股份有限公司 克服对erbb途径抑制剂的抗性
US8691771B2 (en) 2010-05-21 2014-04-08 Merrimack Pharmaceuticals, Inc. Bi-specific fusion proteins for tissue repair
US8822417B2 (en) 2011-05-05 2014-09-02 Novozymes Biopharma DIC A/S Albumin variants
WO2014182970A1 (fr) 2013-05-08 2014-11-13 Zymeworks Inc. Constructions de liaison aux antigènes her2 et her3 bispécifiques
US8927694B2 (en) 2008-11-18 2015-01-06 Merrimack Pharmaceuticals, Inc. Human serum albumin linkers and conjugates thereof
US8945571B2 (en) 2009-08-17 2015-02-03 Roche GlyeArt AG Targeted immunoconjugates
US9045564B2 (en) 2011-02-15 2015-06-02 Medimmune, Llc HSA-related compositions and methods of use
WO2015173248A1 (fr) * 2014-05-14 2015-11-19 F. Hoffmann-La Roche Ag Anticorps bispécifiques anti her3/her2 se liant à l'épingle à cheveux bêta de her3 et du domaine ii de her2
US9226966B2 (en) 2010-12-06 2016-01-05 Merrimack Pharmaceuticals, Inc. Dosage and administration for preventing cardiotoxicity in treatment with ERBB2-targeted immunoliposomes comprising anthracycline chemotherapeutic agents
US9290573B2 (en) 2010-05-06 2016-03-22 Novartis Ag Therapeutic low density lipoprotein-related protein 6 (LRP6) multivalent antibodies
US9345766B2 (en) 2012-08-30 2016-05-24 Merrimack Pharmaceuticals, Inc. Combination therapies comprising anti-ERBB3 agents
US9428583B2 (en) 2010-05-06 2016-08-30 Novartis Ag Compositions and methods of use for therapeutic low density lipoprotein-related protein 6 (LRP6) multivalent antibodies
US9493545B2 (en) 2009-02-11 2016-11-15 Albumedix A/S Albumin variants and conjugates
US9518130B2 (en) 2010-03-11 2016-12-13 Merrimack Pharmaceuticals, Inc. Use of ERBB3 inhibitors in the treatment of triple negative and basal-like breast cancers
US9688761B2 (en) 2013-12-27 2017-06-27 Merrimack Pharmaceuticals, Inc. Biomarker profiles for predicting outcomes of cancer therapy with ERBB3 inhibitors and/or chemotherapies
WO2017112847A1 (fr) 2015-12-22 2017-06-29 Albumedix A/S Souches améliorées pour l'expression de protéines
WO2018011421A1 (fr) 2016-07-14 2018-01-18 Genmab A/S Anticorps multispécifiques dirigés contre cd40 et cd137
US9913901B2 (en) 2012-12-03 2018-03-13 Merrimack Pharmaceuticals, Inc. Combination therapy for treating HER2-positive cancers
US9944691B2 (en) 2012-03-16 2018-04-17 Albumedix A/S Albumin variants
WO2018082758A1 (fr) 2016-11-04 2018-05-11 Aarhus Universitet Identification et traitement de tumeurs caractérisées par une surexpression du récepteur fc néonatal
US10040840B2 (en) 2015-10-02 2018-08-07 Silver Creek Pharmaceuticals, Inc. Bi-specific annexin A5/IGF-1 proteins and methods of use thereof to promote regeneration and survival of tissue
WO2018162749A1 (fr) 2017-03-09 2018-09-13 Genmab A/S Anticorps dirigés contre pd-l1
WO2018234349A1 (fr) 2017-06-20 2018-12-27 Albumedix Ltd Souches améliorées pour l'expression de protéines
US10184006B2 (en) 2015-06-04 2019-01-22 Merrimack Pharmaceuticals, Inc. Biomarkers for predicting outcomes of cancer therapy with ErbB3 inhibitors
WO2019025545A1 (fr) 2017-08-04 2019-02-07 Genmab A/S Agents de liaison se liant à pd-l1 et cd137 et leur utilisation
US10233228B2 (en) 2010-04-09 2019-03-19 Albumedix Ltd Albumin derivatives and variants
WO2019096226A1 (fr) * 2017-11-16 2019-05-23 Chengdu Easton Biopharmaceuticals Co., Ltd. Protéines de fusion vegfr/pdgfr pasylées et leur utilisation en thérapie
US10501524B2 (en) 2012-11-08 2019-12-10 Albumedix Ltd Albumin variants
WO2020060406A1 (fr) 2018-09-19 2020-03-26 Lava Therapeutics B.V. Nouveaux anticorps bispécifiques destinés à être utilisés dans le traitement d'hémopathies malignes
US10633428B2 (en) 2015-08-20 2020-04-28 Albumedix Ltd Albumin variants and conjugates
WO2020094744A1 (fr) 2018-11-06 2020-05-14 Genmab A/S Formulation d'anticorps
US10711050B2 (en) 2011-11-18 2020-07-14 Albumedix Ltd Variant serum albumin with improved half-life and other properties
WO2020159368A1 (fr) 2019-02-01 2020-08-06 Lava Therapeutics B.V. Nouveaux anticorps se liant au cd40
NL2022494B1 (en) 2019-02-01 2020-08-19 Lava Therapeutics B V Novel CD40-binding antibodies
EP3792283A1 (fr) 2019-09-16 2021-03-17 Lava Therapeutics B.V. Traitement du cancer comprenant l'administration d'anticorps de liaison du récepteur des lymphocytes t vgamma9vdelta2
WO2021111143A1 (fr) 2019-12-04 2021-06-10 Albumedix Limited Procédés et compositions produites par ceux-ci
WO2021155916A1 (fr) 2020-02-04 2021-08-12 BioNTech SE Traitement impliquant une vaccination d'antigène et des agents de liaison se liant à pd-l1 et cd137
WO2021185934A1 (fr) 2020-03-18 2021-09-23 Genmab A/S Anticorps se liant à b7h4
WO2021205184A1 (fr) 2020-04-09 2021-10-14 Autolus Limited Polypeptide
WO2021234383A1 (fr) 2020-05-19 2021-11-25 Autolus Limited Anticorps à domaine unique contre hace2 et leur utilisation pour prévenir une infection par sars-cov-2
WO2022008646A1 (fr) 2020-07-08 2022-01-13 LAVA Therapeutics N.V. Anticorps se liant au psma et aux récepteurs des lymphocytes t gamma-delta
WO2022029011A1 (fr) 2020-08-06 2022-02-10 BioNTech SE Agents de liaison pour la protéine s du coronavirus
US11279770B2 (en) 2014-02-28 2022-03-22 Merus N.V. Antibody that binds ErbB-2 and ErbB-3
US11359015B2 (en) 2015-07-15 2022-06-14 Genmab A/S Humanized or chimeric CD3 antibodies
WO2022122973A1 (fr) 2020-12-10 2022-06-16 LAVA Therapeutics N.V. Anticorps qui se lient aux récepteurs des lymphocytes t gamma-delta
WO2022180271A1 (fr) 2021-02-26 2022-09-01 LAVA Therapeutics N.V. Anticorps se liant à des récepteurs des lymphocytes t cd123 et gamma-delta
WO2022234146A1 (fr) 2021-05-07 2022-11-10 Genmab A/S Compositions pharmaceutiques comprenant des anticorps bispécifiques se liant à b7h4 et cd3
WO2022268740A1 (fr) 2021-06-21 2022-12-29 Genmab A/S Régime posologique combiné d'agents de liaison cd137 et pd-l1
WO2023037333A1 (fr) 2021-09-13 2023-03-16 Janssen Biotech, Inc Anticorps multispécifiques cd33 x vδ2 pour traiter le cancer
WO2023057571A1 (fr) 2021-10-08 2023-04-13 Genmab A/S Anticorps se liant à cd30 et cd3
WO2023067138A1 (fr) 2021-10-21 2023-04-27 LAVA Therapeutics N.V. Utilisations d'anticorps d'activation de lymphocytes t gamma delta
WO2023174521A1 (fr) 2022-03-15 2023-09-21 Genmab A/S Agents de liaison se liant à epcam et cd137
US11773170B2 (en) 2017-08-09 2023-10-03 Merus N.V. Antibodies that bind EGFR and cMET
US11780925B2 (en) 2017-03-31 2023-10-10 Merus N.V. ErbB-2 and ErbB3 binding bispecific antibodies for use in the treatment of cells that have an NRG1 fusion gene
US11820825B2 (en) 2014-02-28 2023-11-21 Merus N.V. Methods of treating a subject having an EGFR-positive and/or ErbB-3-positive tumor
EP4285926A1 (fr) 2022-05-30 2023-12-06 LAVA Therapeutics N.V. Traitement combiné de la leucémie lymphocytaire chronique
US11845805B2 (en) 2020-09-10 2023-12-19 Genmab A/S Bispecific antibody against CD3 and CD20 in combination therapy for treating diffuse large B-cell lymphoma
EP4292609A1 (fr) 2022-06-15 2023-12-20 LAVA Therapeutics N.V. Compositions comprenant des anticorps se liant aux récepteurs de lymphocytes t gamma-delta
EP4292610A1 (fr) 2022-06-15 2023-12-20 LAVA Therapeutics N.V. Anticorps variants se liant aux récepteurs de lymphocytes t gamma-delta
US11858995B2 (en) 2020-09-10 2024-01-02 Genmab A/S Bispecific antibodies against CD3 and CD20 for treating chronic lymphocytic leukemia
WO2024010445A1 (fr) 2022-07-06 2024-01-11 ACADEMISCH ZIEKENHUIS LEIDEN (h.o.d.n. LUMC) Anticorps bispécifique et ses utilisations
US11939394B2 (en) 2015-10-23 2024-03-26 Merus N.V. Binding molecules that inhibit cancer growth
EP4438624A1 (fr) 2023-03-27 2024-10-02 LAVA Therapeutics N.V. Anticorps se liant aux récepteurs des lymphocytes t nectin-4 et gamma-delta
WO2024200573A1 (fr) 2023-03-27 2024-10-03 LAVA Therapeutics N.V. Agents de liaison à la nectine-4 et méthodes d'utilisation
EP4442251A1 (fr) 2023-04-05 2024-10-09 Albumedix Ltd Formulations et leurs utilisations
WO2024208898A1 (fr) 2023-04-05 2024-10-10 Genmab A/S Compositions pharmaceutiques comprenant des anticorps se liant à cd30 et cd3
US12139548B2 (en) 2014-02-28 2024-11-12 Merus N.V. Antibody that binds ErbB-2 and ErbB-3

Families Citing this family (68)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7332585B2 (en) 2002-04-05 2008-02-19 The Regents Of The California University Bispecific single chain Fv antibody molecules and methods of use thereof
US20040067532A1 (en) 2002-08-12 2004-04-08 Genetastix Corporation High throughput generation and affinity maturation of humanized antibody
AU2007353412A1 (en) * 2006-11-21 2008-11-20 Fox Chase Cancer Center Anti-EGFR family antibodies, bispecific anti-EGFR family antibodies and methods of use thereof
WO2008100624A2 (fr) 2007-02-16 2008-08-21 Merrimack Pharmaceuticals, Inc. Anticorps contre erbb3 et leur utilisation
US8877688B2 (en) 2007-09-14 2014-11-04 Adimab, Llc Rationally designed, synthetic antibody libraries and uses therefor
DK3124497T3 (da) 2007-09-14 2020-05-11 Adimab Llc Rationelt designede syntetiske antistofbiblioteker og anvendelser deraf
EP2564188A4 (fr) 2010-04-26 2013-09-25 Merrimack Pharmaceuticals Inc Essais pour des anticorps anti-médicaments en présence d'homologues protéique endogènes abondants du médicament
TW201302793A (zh) 2010-09-03 2013-01-16 Glaxo Group Ltd 新穎之抗原結合蛋白
CA2815266C (fr) 2010-11-05 2023-09-05 Zymeworks Inc. Conception d'anticorps heterodimerique stable ayant des mutations dans le domaine fc
US9821073B2 (en) * 2010-12-27 2017-11-21 Apo-T B.V. Polypeptide that binds aberrant cells and induces apoptosis
US9499605B2 (en) 2011-03-03 2016-11-22 Zymeworks Inc. Multivalent heteromultimer scaffold design and constructs
WO2013048243A1 (fr) 2011-09-29 2013-04-04 Apo-T B.V. Molécules de liaison multispécifiques ciblant des cellules aberrantes
BR112014010580B1 (pt) 2011-11-04 2021-01-12 Zymeworks, Inc. constructo de fc heteromultimérico isolado, composição, uso de um constructo de fc heteromultimérico isolado, composição de ácido nucléico e método para expressar o constructo de fc heteromultimérico isolado
US20130165628A1 (en) * 2011-12-23 2013-06-27 Sri International Double Binding Constructs
WO2013096939A1 (fr) * 2011-12-23 2013-06-27 Sri International Composés de liaison sélective
JP2015504895A (ja) 2012-01-13 2015-02-16 エーピーオー‐ティー ビー．ヴイ． 毒性部分を備える異常細胞拘束性免疫グロブリン
US20150011736A1 (en) * 2012-01-20 2015-01-08 Sea Lane Biotechnologies, Llc Binding molecule conjugates
CN104755497A (zh) * 2012-05-11 2015-07-01 梅里麦克制药股份有限公司 结合抗癌治疗剂的双特异性scfv轭合物的剂量和给药
US9717724B2 (en) 2012-06-13 2017-08-01 Ipsen Biopharm Ltd. Methods for treating pancreatic cancer using combination therapies
AU2013202947B2 (en) 2012-06-13 2016-06-02 Ipsen Biopharm Ltd. Methods for treating pancreatic cancer using combination therapies comprising liposomal irinotecan
WO2014004586A1 (fr) 2012-06-25 2014-01-03 Zymeworks Inc. Processus et procédés pour la fabrication efficace d'anticorps asymétriques hautement purs dans des cellules de mammifère
CN104768571B (zh) 2012-07-13 2018-11-09 酵活有限公司 多价异多聚体支架设计和构建体
US9914785B2 (en) 2012-11-28 2018-03-13 Zymeworks Inc. Engineered immunoglobulin heavy chain-light chain pairs and uses thereof
WO2014098464A1 (fr) * 2012-12-18 2014-06-26 이화여자대학교 산학협력단 Composition de stabilisation de la chaleur d'albumine de sérum humain et procédé de production d'albumine de sérum humain à chaleur stabilisée l'utilisant
WO2014121033A1 (fr) * 2013-02-04 2014-08-07 Fl Therapeutics Llc Complexes solubles d'analogues de médicament et d'albumine
EP2986306A4 (fr) 2013-04-18 2016-12-07 Armo Biosciences Inc Méthodes d'utilisation de l'interleukine-10 pour le traitement de maladies et de troubles
MX362001B (es) 2013-05-13 2019-01-03 Vision Global Holdings Ltd Composición farmacéutica que comprende un agente terapéutico a base de hemoglobina modificada para tratamiento dirigido al cáncer e imagenología diagnóstica.
CA2913363A1 (fr) * 2013-05-24 2014-11-27 Zymeworks Inc. Conjugue therapeutique modulaire proteine-medicament
EP3010527B1 (fr) 2013-06-17 2018-08-08 Armo Biosciences, Inc. Méthode d'évaluation de l'identité et de la stabilité d'une protéine
CA2920679A1 (fr) 2013-08-30 2015-03-05 Armo Biosciences, Inc. Methodes d'utilisation d'interleukine 10 dans le traitement de maladies et de troubles
WO2015048802A2 (fr) * 2013-09-30 2015-04-02 Daiichi Sankyo Co., Ltd. Biomarqueurs protéiques et leurs utilisations
KR20160079114A (ko) 2013-11-11 2016-07-05 아르모 바이오사이언시스 인코포레이티드 질환 및 장애를 치료하기 위한 인터류킨-10을 사용하는 방법
RS58173B1 (sr) 2013-12-25 2019-03-29 Daiichi Sankyo Co Ltd Konjugat anti-trop2 antitelo-lek
US10487140B2 (en) 2014-01-14 2019-11-26 Integrated Biotherapeutics, Inc. Targeting immunological functions to the site of bacterial infections using cell wall targeting domains of bacteriolysins
KR20230162159A (ko) 2014-01-31 2023-11-28 다이이찌 산쿄 가부시키가이샤 항-her2 항체-약물 접합체
WO2015156268A1 (fr) * 2014-04-07 2015-10-15 中外製薬株式会社 Molécule d'immunoactivation de liaison à un antigène
JP6612738B2 (ja) * 2014-04-10 2019-11-27 第一三共株式会社 抗ｈｅｒ２抗体−薬物コンジュゲート
US11473080B2 (en) * 2014-04-30 2022-10-18 The Board Of Trustees Of The University Of Illinois Method for generating high affinity, bivalent binding agents for sandwich assays
US11505605B2 (en) 2014-05-13 2022-11-22 Chugai Seiyaku Kabushiki Kaisha T cell-redirected antigen-binding molecule for cells having immunosuppression function
WO2015200837A1 (fr) 2014-06-27 2015-12-30 Fl Therapeutics Llc Dérivés d'abiratérone et complexes non covalents avec l'albumine
US20160045596A1 (en) 2014-08-05 2016-02-18 Merrimack Pharmaceuticals, Inc. Combination therapies for treating her2-positive cancers that are resistant to her2-targeted therapies
GB201417067D0 (en) * 2014-09-26 2014-11-12 South African Nuclear Energy Radiopharmaceutical conjugate
EP3223858A4 (fr) * 2014-10-31 2018-07-11 H. Lee Moffitt Cancer Center And Research Institute, Inc. Agents cytotoxiques ciblés vers tlr9
US11318131B2 (en) 2015-05-18 2022-05-03 Ipsen Biopharm Ltd. Nanoliposomal irinotecan for use in treating small cell lung cancer
US20160361415A1 (en) 2015-05-28 2016-12-15 Armo Biosciences, Inc. Methods of Using Interleukin-10 for Treating Diseases and Disorders
CA2990572C (fr) 2015-06-29 2022-07-26 Daiichi Sankyo Company, Limited Procede pour la preparation selective d'un conjugue anticorps-medicament
KR102464778B1 (ko) * 2015-08-11 2022-11-07 코히런트 바이오파마 I, 리미티드 다중-리간드 약물 접합체 및 그의 용도
SI3337467T1 (sl) 2015-08-20 2021-03-31 Ipsen Biopharm Ltd. Ash Road Kombinacijska terapija, ki vsebuje liposomski irinotekan in zaviralec PARP za zdravljenje raka
CN108495629A (zh) 2015-08-21 2018-09-04 益普生生物制药有限公司 使用包含脂质体伊立替康和奥沙利铂的组合疗法治疗转移性胰腺癌的方法
JP7053453B2 (ja) 2015-08-25 2022-04-12 アルモ・バイオサイエンシーズ・インコーポレイテッド 疾患及び障害を治療するためのインターロイキン１０の使用方法
ES2907486T3 (es) * 2015-09-24 2022-04-25 Univ North Carolina Chapel Hill Métodos y composiciones para reducir metástasis
CN108366965B (zh) 2015-10-16 2021-10-01 易普森生物制药有限公司 稳定喜树碱药物组合物
US11649293B2 (en) 2015-11-18 2023-05-16 Chugai Seiyaku Kabushiki Kaisha Method for enhancing humoral immune response
WO2017086367A1 (fr) 2015-11-18 2017-05-26 中外製薬株式会社 Polythérapie utilisant une molécule de liaison à l'antigène à rôle de redirection des cellules t, ciblant des cellules immunosupressives
KR101746152B1 (ko) 2015-12-07 2017-06-13 주식회사 이수앱지스 ＥｒｂＢ3에 특이적으로 결합하는 항체 및 그의 용도
CA3017622A1 (fr) 2016-03-16 2017-09-21 Merrimack Pharmaceuticals, Inc. Molecule trail modifiee pour la therapie du cancer
CN110402163A (zh) 2016-11-02 2019-11-01 易普森生物制药有限公司 使用包括脂质体伊立替康、奥沙利铂、5-氟尿嘧啶(和甲酰四氢叶酸)的组合疗法治疗胃癌
CN110049779A (zh) 2016-12-12 2019-07-23 第一三共株式会社 抗体-药物缀合物和免疫检查点抑制剂的组合
TWI780104B (zh) 2017-01-17 2022-10-11 日商第一三共股份有限公司 抗ｇｐｒ２０抗體及抗ｇｐｒ２０抗體-藥物結合物、以及其製造方法及用途
TWI794230B (zh) 2017-05-15 2023-03-01 日商第一三共股份有限公司 抗ｃｄｈ６抗體及抗ｃｄｈ６抗體-藥物結合物、以及其製造方法
CN117838881A (zh) 2017-08-31 2024-04-09 第一三共株式会社 制备抗体-药物缀合物的新方法
EP3677589A4 (fr) 2017-08-31 2021-04-21 Daiichi Sankyo Company, Limited Procédé amélioré de production d'un conjugué anticorps-médicament
RS64379B1 (sr) 2018-05-18 2023-08-31 Glycotope Gmbh Anti-muc1 antitelo
CN109627337B (zh) * 2018-12-29 2023-07-21 上海复宏汉霖生物技术股份有限公司 一种抗prlr的单克隆抗体及其制备方法和应用
JP2022525791A (ja) * 2019-03-26 2022-05-19 株式会社島津製作所 光免疫療法およびそれに用いる薬剤
PL3865155T3 (pl) * 2020-02-13 2023-01-30 Orano Med Sposób miejscowo-specyficznego modyfikowania przeciwciała
AU2021377232A1 (en) * 2020-11-10 2023-06-15 New York University Macropinocytosis selective monobody-drug conjugates
AR124414A1 (es) 2020-12-18 2023-03-22 Century Therapeutics Inc Sistema de receptor de antígeno quimérico con especificidad de receptor adaptable

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050186664A1 (en) * 2001-12-21 2005-08-25 Rosen Craig A. Albumin fusion proteins
US20080124334A1 (en) * 1996-03-27 2008-05-29 Robert Akita Isolated Nucleic Acids, Vectors and Host Cells Encoding ErbB3 Antibodies
US20080166358A1 (en) * 2006-12-15 2008-07-10 Concert Pharmaceuticals Inc. Quinazoline derivatives and methods of treatment

Family Cites Families (145)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6009A (en) * 1849-01-09 Improvement in machines for gathering pea-vines
US5800815A (en) 1903-05-05 1998-09-01 Cytel Corporation Antibodies to P-selectin and their uses
US4450103A (en) 1982-03-01 1984-05-22 Cetus Corporation Process for recovering human IFN-β from a transformed microorganism
US4588585A (en) 1982-10-19 1986-05-13 Cetus Corporation Human recombinant cysteine depleted interferon-β muteins
US4737462A (en) 1982-10-19 1988-04-12 Cetus Corporation Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of interferon-β
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4959314A (en) 1984-11-09 1990-09-25 Cetus Corporation Cysteine-depleted muteins of biologically active proteins
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
US4704692A (en) 1986-09-02 1987-11-03 Ladner Robert C Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
US6121424A (en) 1991-11-25 2000-09-19 Enzon, Inc. Multivalent antigen-binding proteins
ATE87659T1 (de) 1986-09-02 1993-04-15 Enzon Lab Inc Bindungsmolekuele mit einzelpolypeptidkette.
US5869620A (en) 1986-09-02 1999-02-09 Enzon, Inc. Multivalent antigen-binding proteins
US5260203A (en) 1986-09-02 1993-11-09 Enzon, Inc. Single polypeptide chain binding molecules
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5091513A (en) 1987-05-21 1992-02-25 Creative Biomolecules, Inc. Biosynthetic antibody binding sites
US5258498A (en) 1987-05-21 1993-11-02 Creative Biomolecules, Inc. Polypeptide linkers for production of biosynthetic proteins
US5132405A (en) 1987-05-21 1992-07-21 Creative Biomolecules, Inc. Biosynthetic antibody binding sites
AU612370B2 (en) 1987-05-21 1991-07-11 Micromet Ag Targeted multifunctional proteins
US5753627A (en) 1988-12-05 1998-05-19 Novartis Ag Use of certain complexed somatostatin peptides for the invivo imaging of somatostatin receptor-positive tumors and metastasis
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5705363A (en) 1989-03-02 1998-01-06 The Women's Research Institute Recombinant production of human interferon τ polypeptides and nucleic acids
ATE92107T1 (de) 1989-04-29 1993-08-15 Delta Biotechnology Ltd N-terminale fragmente von menschliches serumalbumin enthaltenden fusionsproteinen.
US5766883A (en) 1989-04-29 1998-06-16 Delta Biotechnology Limited Polypeptides
FR2650598B1 (fr) 1989-08-03 1994-06-03 Rhone Poulenc Sante Derives de l'albumine a fonction therapeutique
US5730978A (en) 1989-09-01 1998-03-24 Fred Hutchinson Cancer Research Center Inhibition of lymphocyte adherence with α4β1-specific antibodies
US6033665A (en) 1989-09-27 2000-03-07 Elan Pharmaceuticals, Inc. Compositions and methods for modulating leukocyte adhesion to brain endothelial cells
US5292658A (en) 1989-12-29 1994-03-08 University Of Georgia Research Foundation, Inc. Boyd Graduate Studies Research Center Cloning and expressions of Renilla luciferase
WO1992015682A1 (fr) 1991-02-27 1992-09-17 Creative Biomolecules, Inc. Segments de liaison peptidique riches en serine
US5525491A (en) 1991-02-27 1996-06-11 Creative Biomolecules, Inc. Serine-rich peptide linkers
EP0617706B1 (fr) 1991-11-25 2001-10-17 Enzon, Inc. Proteines multivalentes de fixation aux antigenes
FR2686620B1 (fr) 1992-01-27 1995-06-23 Rhone Poulenc Rorer Sa Serum-albumine humaine, preparation et utilisation.
FR2686899B1 (fr) 1992-01-31 1995-09-01 Rhone Poulenc Rorer Sa Nouveaux polypeptides biologiquement actifs, leur preparation et compositions pharmaceutiques les contenant.
EP0625200B1 (fr) 1992-02-06 2005-05-11 Chiron Corporation Proteine de liaison biosynthetique pour marqueur de cancer
WO1994012520A1 (fr) 1992-11-20 1994-06-09 Enzon, Inc. Segment de liaison pour polypeptides fusionnes lies
ATE182625T1 (de) 1993-01-12 1999-08-15 Biogen Inc Rekombinante anti-vla4 antikörpermoleküle
CN1074243A (zh) 1993-02-06 1993-07-14 北京中化生物技术研究所 白介素6-白介素2融合蛋白及其制法和用途
US5780594A (en) 1993-03-01 1998-07-14 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration Biologically active protein fragments containing specific binding regions of serum albumin or related proteins
US5914110A (en) 1993-09-07 1999-06-22 Smithkline Beecham Corporation Recombinant IL4 antibodies useful in treatment of IL4 mediated disorders
US5928904A (en) 1993-09-07 1999-07-27 Smithkline Beecham Corporation DNA encoding recombinant IL4 antibodies useful in treatment of IL4 mediated disorders
US5658763A (en) 1993-10-25 1997-08-19 Creative Biomolecules, Inc. Methods and compositions for high protein production from non-native DNA
US5821231A (en) 1993-12-06 1998-10-13 Cytel Corporation CS-1 peptidomimetics, compositions and methods of using same
US5936065A (en) 1993-12-06 1999-08-10 Cytel Corporation CS-1 peptidomimetics, compositions and methods of using the same
US5840299A (en) 1994-01-25 1998-11-24 Athena Neurosciences, Inc. Humanized antibodies against leukocyte adhesion molecule VLA-4
US8715645B2 (en) 1994-05-27 2014-05-06 The Regents Of The University Of Colorado Viral vectors encoding apoptosis-inducing proteins and methods for making and using the same
US5622701A (en) 1994-06-14 1997-04-22 Protein Design Labs, Inc. Cross-reacting monoclonal antibodies specific for E- and P-selectin
US5811391A (en) 1994-08-25 1998-09-22 Cytel Corporation Cyclic CS-1 peptidomimetics, compositions and methods of using same
US5874540A (en) 1994-10-05 1999-02-23 Immunomedics, Inc. CDR-grafted type III anti-CEA humanized mouse monoclonal antibodies
US5763733A (en) 1994-10-13 1998-06-09 Enzon, Inc. Antigen-binding fusion proteins
US5670356A (en) 1994-12-12 1997-09-23 Promega Corporation Modified luciferase
US6306840B1 (en) 1995-01-23 2001-10-23 Biogen, Inc. Cell adhesion inhibitors
US5656730A (en) 1995-04-07 1997-08-12 Enzon, Inc. Stabilized monomeric protein compositions
EP0873363B1 (fr) 1995-06-14 2010-10-06 The Regents of The University of California Anticorps humains a forte affinite diriges contre des antigenes tumoraux
GB9526733D0 (en) 1995-12-30 1996-02-28 Delta Biotechnology Ltd Fusion proteins
US6686350B1 (en) 1996-07-25 2004-02-03 Biogen, Inc. Cell adhesion inhibitors
US6423512B1 (en) 1996-07-26 2002-07-23 Novartis Ag Fusion polypeptides
PE99498A1 (es) 1996-07-26 1999-01-21 Novartis Ag Polipeptidos de fusion
AU7266698A (en) 1997-04-30 1998-11-24 Enzon, Inc. Polyalkylene oxide-modified single chain polypeptides
US6191269B1 (en) 1997-05-30 2001-02-20 The Regents Of The University Of California Selective induction of cell death by delivery of amino-terminal interleukin-1-α pro-piece polypeptide
WO1998058902A1 (fr) 1997-06-23 1998-12-30 Tanabe Seiyaku Co., Ltd. INHIBITEURS D'ADHERENCE CELLULAIRE INDUITE PAR α4β¿1?
US6229011B1 (en) 1997-08-22 2001-05-08 Hoffman-La Roche Inc. N-aroylphenylalanine derivative VCAM-1 inhibitors
US6455550B1 (en) 1997-08-22 2002-09-24 Hoffmann-La Roche Inc. N-alkanoylphenylalanine derivatives
US6197794B1 (en) 1998-01-08 2001-03-06 Celltech Therapeutics Limited Phenylalanine derivatives
US6329372B1 (en) 1998-01-27 2001-12-11 Celltech Therapeutics Limited Phenylalanine derivatives
US6171809B1 (en) 1998-01-29 2001-01-09 Packard Instrument Company Method and compositions for detecting luciferase biological samples
TR200101541T2 (tr) 1998-04-03 2002-06-21 Chugai Seiyaku Kabushiki Kaisha İnsan doku faktörüne (TF) karşı insanlaştırılmış antikor.
GB9811159D0 (en) 1998-05-22 1998-07-22 Celltech Therapeutics Ltd Chemical compounds
WO1999061421A1 (fr) 1998-05-28 1999-12-02 Biogen, Inc. L'oMePUPA-V, NOUVEL INHIBITEUR DU VLA-4
GB9812088D0 (en) 1998-06-05 1998-08-05 Celltech Therapeutics Ltd Chemical compounds
US6685617B1 (en) 1998-06-23 2004-02-03 Pharmacia & Upjohn Company Inhibitors of α4β1 mediated cell adhesion
US6355678B1 (en) 1998-06-29 2002-03-12 Parker Hughes Institute Inhibitors of the EGF-receptor tyrosine kinase and methods for their use
GB9821061D0 (en) 1998-09-28 1998-11-18 Celltech Therapeutics Ltd Chemical compounds
ES2447772T3 (es) 1998-10-16 2014-03-12 Biogen Idec Ma Inc. Conjugados de polialquilenglicol de interferón beta-1a y sus usos
US6333396B1 (en) 1998-10-20 2001-12-25 Enzon, Inc. Method for targeted delivery of nucleic acids
JP3180906B2 (ja) 1998-11-12 2001-07-03 日本電気株式会社 磁気抵抗効果型複合ヘッドの製造方法
DK1154993T3 (da) 1999-02-18 2005-02-14 Hoffmann La Roche Thioamid-derivater
US8236159B2 (en) * 1999-04-13 2012-08-07 Applied Materials Inc. Electrolytic process using cation permeable barrier
US6265572B1 (en) 1999-04-20 2001-07-24 Hoffmann-La Roche Inc. Pyrrolidincarbonylamino cyclic disulfide anti-inflammatory agents
US7534866B2 (en) 2005-10-19 2009-05-19 Ibc Pharmaceuticals, Inc. Methods and compositions for generating bioactive assemblies of increased complexity and uses
US6579857B1 (en) * 1999-06-11 2003-06-17 Evanston Northwestern Healthcare Research Institute Combination cancer therapy comprising adenosine and deaminase enzyme inhibitors
US6756378B2 (en) 1999-06-30 2004-06-29 Pharmacopeia Drug Discovery, Inc. VLA-4 inhibitor compounds
US7067313B1 (en) 1999-07-14 2006-06-27 D. Collen Research Foundation Ligands for use in therapeutic compositions for the treatment of hemostasis disorders
US6365619B1 (en) 1999-07-22 2002-04-02 Novartis Ag Treatment of arteriosclerosis
NZ517011A (en) 1999-08-13 2004-02-27 Biogen Inc Cell adhesion inhibitors
US6531122B1 (en) 1999-08-27 2003-03-11 Maxygen Aps Interferon-β variants and conjugates
EP1094110A1 (fr) 1999-10-21 2001-04-25 Leadd B.V. Substance protéinique induisant l'apoptose
DE60033684T2 (de) 1999-11-18 2007-12-06 Ajinomoto Co., Inc. Phenylalaninderivate
US6380387B1 (en) 1999-12-06 2002-04-30 Hoffmann-La Roche Inc. 4-Pyrimidinyl-n-acyl-l phenylalanines
US6388084B1 (en) 1999-12-06 2002-05-14 Hoffmann-La Roche Inc. 4-pyridinyl-n-acyl-l-phenylalanines
DE19962936A1 (de) 1999-12-24 2001-06-28 Bayer Ag Neue beta-Aminosäureverbindungen als Integrinantagonisten
WO2001051487A1 (fr) 1999-12-28 2001-07-19 Pfizer Products Inc. Inhibiteurs non peptidyle de liaison cellulaire dependant de vla-4 utiles pour le traitement de maladies inflammatoires, auto-immunes et respiratoires
US7446174B2 (en) 2001-03-02 2008-11-04 Xencor, Inc. Protein based TNF-α variants for the treatment of TNF-α related disorders
EP1270547A4 (fr) 2000-03-23 2005-07-13 Ajinomoto Kk Nouveau derive de phenylalanine
US6946134B1 (en) 2000-04-12 2005-09-20 Human Genome Sciences, Inc. Albumin fusion proteins
EP2295456A1 (fr) 2000-04-12 2011-03-16 Human Genome Sciences, Inc. Protéines de fusion d'albumine
US20050100991A1 (en) 2001-04-12 2005-05-12 Human Genome Sciences, Inc. Albumin fusion proteins
CA2408883A1 (fr) 2000-05-19 2001-11-29 The Center For Blood Research, Inc. Procedes pour diagnostiquer et traiter des troubles hemostatiques par modulation de l'activite de la p-selectine
GB0027328D0 (en) 2000-06-23 2000-12-27 Aventis Pharma Inc Bioengineered vehicles for targeted nucleic acid delivery
US6787636B1 (en) 2000-07-14 2004-09-07 New Century Pharmaceuticals, Inc. Modified serum albumin with reduced affinity for nickel and copper
US20040247565A1 (en) 2000-07-19 2004-12-09 Chih-Ping Liu Method of treatment using interferon-tau
US7015216B2 (en) 2000-07-21 2006-03-21 Elan Pharmaceuticals, Inc. Heteroaryl-β-alanine derivatives as alpha 4 integrin inhibitors
US6689781B2 (en) 2000-07-21 2004-02-10 Elan Pharmaceuticals, Inc. Phenylalanine derivatives as alpha 4 integrin inhibitors
IL154350A0 (en) 2000-08-18 2003-09-17 Ajinomoto Kk New phenylalanine derivatives
EP1323709A1 (fr) 2000-09-14 2003-07-02 Toray Industries, Inc. Derive d'uree et inhibiteur de molecule adhesive contenant celui-ci en tant que substance active
CN1247577C (zh) 2000-09-25 2006-03-29 东丽株式会社 螺环衍生物和以其作为有效成分的粘附分子抑制剂
US6818216B2 (en) 2000-11-28 2004-11-16 Medimmune, Inc. Anti-RSV antibodies
ATE355276T1 (de) 2001-02-22 2006-03-15 Celltech R&D Ltd Phenylalaninenamidderivate mit einer cyclobutengruppe zur verwendung als integrininhibitoren
ES2180416B1 (es) 2001-03-12 2004-06-01 BIOTOOLS BIOTECHNOLOGICAL & MEDICAL LABORATORIES, S.A. Procedimiento para la preparacion de mezclas de reaccion estabilizadas, total o parcialmente desecadas, que comprenden, al menos, una enzima, mezclas de reaccion y kits que las contienen.
JP2004535202A (ja) 2001-07-17 2004-11-25 リサーチ　ディベロップメント　ファンデーション アポトーシス促進性蛋白質を含む治療剤
JP4164871B2 (ja) 2001-07-26 2008-10-15 味の素株式会社 新規フェニルプロピオン酸誘導体
US6900292B2 (en) 2001-08-17 2005-05-31 Lee-Hwei K. Sun Fc fusion proteins of human erythropoietin with increased biological activities
JP4452899B2 (ja) 2001-12-13 2010-04-21 味の素株式会社 新規フェニルアラニン誘導体
EP1463752A4 (fr) 2001-12-21 2005-07-13 Human Genome Sciences Inc Proteines de fusion d'albumine
KR101271635B1 (ko) 2001-12-21 2013-06-12 휴먼 게놈 사이언시즈, 인코포레이티드 알부민 융합 단백질
US7399829B2 (en) 2002-01-04 2008-07-15 Xencor, Inc. Variants of RANKL protein
WO2003080611A1 (fr) 2002-03-22 2003-10-02 Toray Industries, Inc. Derives spiro et inhibiteurs de molecules d'adhesion les contenant en tant que principe actif
US7332580B2 (en) 2002-04-05 2008-02-19 The Regents Of The University Of California Bispecific single chain Fv antibody molecules and methods of use thereof
US7332585B2 (en) 2002-04-05 2008-02-19 The Regents Of The California University Bispecific single chain Fv antibody molecules and methods of use thereof
GB0216571D0 (en) 2002-07-17 2002-08-28 Celltech R&D Ltd Chemical compounds
GB0216568D0 (en) 2002-07-17 2002-08-28 Celltech R&D Ltd Chemical compounds
US7314613B2 (en) 2002-11-18 2008-01-01 Maxygen, Inc. Interferon-alpha polypeptides and conjugates
US20040115204A1 (en) 2002-12-11 2004-06-17 Fanger Gary R. Antibodies to treat cancer
WO2004082640A2 (fr) 2003-03-19 2004-09-30 New Century Pharmaceuticals, Inc. Fragments ou polymeres d'albumine a temps de residence vasculaire modulable utilisables pour la mise au point de therapies d'administration et de vaccins
US7446125B2 (en) 2003-04-09 2008-11-04 Universidad Del Pais Vasco Derivatives of nitroproline compounds, processes of manufacture and uses in the treatment of integrin mediated disorders
EP1687399A4 (fr) 2003-10-03 2007-09-05 Univ Michigan Tech Luciferase modifiee
US7638331B2 (en) 2004-01-02 2009-12-29 The Administration of the Tulane Rducation Fund Directed apoptosis in COX-2 overexpressing cancer cells through expression targeted gene delivery
EP2357201B1 (fr) 2004-04-13 2017-08-30 F. Hoffmann-La Roche AG Anticorps dirigés contre la sélectine P
JP2007536254A (ja) 2004-05-05 2007-12-13 メリマック ファーマシューティカルズ インコーポレーティッド 生物活性を調節するための二重特異性結合剤
KR20070039911A (ko) 2004-06-01 2007-04-13 도만티스 리미티드 증강된 혈청 반감기를 가지는 이특이성 융합 항체
TW200610754A (en) 2004-06-14 2006-04-01 Daiichi Seiyaku Co Vla-4 inhibitor
US7196112B2 (en) 2004-07-16 2007-03-27 Biogen Idec Ma Inc. Cell adhesion inhibitors
US20060018859A1 (en) 2004-07-16 2006-01-26 Carter Daniel C Modified human serum albumin with reduced or eliminated affinity to chemical or biological contaminants at Cys 34
ZA200703478B (en) 2004-10-05 2008-07-30 Genentech Inc Therapeutic agents with decreased toxicity
JP2008526205A (ja) 2004-12-31 2008-07-24 ジェネンテック・インコーポレーテッド Ｂｒ３に結合するポリペプチド及びその使用
RU2007135216A (ru) 2005-02-23 2009-03-27 Мерримак Фармасьютикалз, Инк. (Us) Биспецифические связывающие агенты для модулирования биологической активности
WO2007098106A2 (fr) 2006-02-17 2007-08-30 Pepgen Coporation Délivrance d'interféron-tau dans les voies respiratoires
US8618257B2 (en) 2006-09-08 2013-12-31 Ambrx, Inc. Modified human plasma polypeptide or Fc scaffolds and their uses
KR20090064453A (ko) * 2006-09-14 2009-06-18 휴먼 게놈 사이언시즈, 인코포레이티드 알부민 융합 단백질
AU2007353412A1 (en) 2006-11-21 2008-11-20 Fox Chase Cancer Center Anti-EGFR family antibodies, bispecific anti-EGFR family antibodies and methods of use thereof
CN102057054B (zh) 2008-04-11 2015-06-10 梅里麦克制药股份有限公司 人血清白蛋白接头以及其结合物
JP2012500008A (ja) * 2008-08-15 2012-01-05 メリマック ファーマシューティカルズ インコーポレーティッド 治療薬に対する細胞の応答を予測するための方法およびシステム
WO2010059315A1 (fr) 2008-11-18 2010-05-27 Merrimack Pharmaceuticals, Inc. Lieurs de sérum-albumine humaine et conjugués de ceux-ci
WO2011133668A2 (fr) 2010-04-20 2011-10-27 President And Fellows Of Harvard College Méthodes et compositions utilisées pour le traitement du cancer
AU2011255238B2 (en) * 2010-05-21 2015-06-04 Silver Creek Pharmaceuticals, Inc. Bi-specific fusion proteins
ES2666856T3 (es) * 2011-11-04 2018-05-08 Novartis Ag Proteína 6 relacionada con lipoproteínas de baja densidad (LRP6) - constructos extensores de la vida media
US9345766B2 (en) * 2012-08-30 2016-05-24 Merrimack Pharmaceuticals, Inc. Combination therapies comprising anti-ERBB3 agents

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080124334A1 (en) * 1996-03-27 2008-05-29 Robert Akita Isolated Nucleic Acids, Vectors and Host Cells Encoding ErbB3 Antibodies
US20050186664A1 (en) * 2001-12-21 2005-08-25 Rosen Craig A. Albumin fusion proteins
US20080166358A1 (en) * 2006-12-15 2008-07-10 Concert Pharmaceuticals Inc. Quinazoline derivatives and methods of treatment

Cited By (122)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8927694B2 (en) 2008-11-18 2015-01-06 Merrimack Pharmaceuticals, Inc. Human serum albumin linkers and conjugates thereof
US9493545B2 (en) 2009-02-11 2016-11-15 Albumedix A/S Albumin variants and conjugates
US11555061B2 (en) 2009-02-11 2023-01-17 Albumedix, Ltd Albumin variants and conjugates
US8945571B2 (en) 2009-08-17 2015-02-03 Roche GlyeArt AG Targeted immunoconjugates
US10696732B2 (en) 2009-10-30 2020-06-30 Albumedix, Ltd Albumin variants
US20120220530A1 (en) * 2009-10-30 2012-08-30 Novozymes Biopharma Dk A/S Albumin variants
US8748380B2 (en) * 2009-10-30 2014-06-10 Novozymes Biopharma Dk A/S Albumin variants
US8697650B2 (en) 2010-02-16 2014-04-15 Medimmune, Llc HSA-related compositions and methods of use
US20130053322A1 (en) * 2010-02-16 2013-02-28 Medlmmune, Llc Hsa-related compositions and methods of use
US9120875B2 (en) 2010-02-16 2015-09-01 Medimmune, Llc HSA-related compositions and methods of use
US9518130B2 (en) 2010-03-11 2016-12-13 Merrimack Pharmaceuticals, Inc. Use of ERBB3 inhibitors in the treatment of triple negative and basal-like breast cancers
US10233228B2 (en) 2010-04-09 2019-03-19 Albumedix Ltd Albumin derivatives and variants
US9290573B2 (en) 2010-05-06 2016-03-22 Novartis Ag Therapeutic low density lipoprotein-related protein 6 (LRP6) multivalent antibodies
US9428583B2 (en) 2010-05-06 2016-08-30 Novartis Ag Compositions and methods of use for therapeutic low density lipoprotein-related protein 6 (LRP6) multivalent antibodies
US10988547B2 (en) 2010-05-21 2021-04-27 Silver Creek Pharmaceuticals, Inc. Bi-specific fusion proteins
US11814443B2 (en) 2010-05-21 2023-11-14 Silver Creek Pharmaceuticals, Inc. Bi-specific fusion proteins
US8691771B2 (en) 2010-05-21 2014-04-08 Merrimack Pharmaceuticals, Inc. Bi-specific fusion proteins for tissue repair
US9982060B2 (en) 2010-05-21 2018-05-29 Merrimack Pharmaceuticals, Inc. Bi-specific fusion proteins
US10407512B2 (en) 2010-05-21 2019-09-10 Silver Creek Pharmaceuticals, Inc. Bi-specific fusion proteins
US9718892B2 (en) 2010-05-21 2017-08-01 Merrimack Pharmaceuticals, Inc. Method of treating myocardial infarction by administering a bi-specific fusion protein
US10858450B2 (en) 2010-05-21 2020-12-08 Silver Creek Pharmaceuticals, Inc. Bi-specific fusion proteins
US11673970B2 (en) 2010-05-21 2023-06-13 Silver Creek Pharmaceuticals, Inc. Bi-specific fusion proteins
US9238080B2 (en) 2010-05-21 2016-01-19 Merrimack Pharmaceuticals, Inc. Bi-specific fusion proteins
US9610249B2 (en) 2010-12-06 2017-04-04 Merrimack Pharmaceuticals. Inc. Dosage and administration for preventing cardiotoxicity in treatment with ERbB2-targeted immunoliposomes comprising anthracycline chemotherapeutic agents
US9226966B2 (en) 2010-12-06 2016-01-05 Merrimack Pharmaceuticals, Inc. Dosage and administration for preventing cardiotoxicity in treatment with ERBB2-targeted immunoliposomes comprising anthracycline chemotherapeutic agents
US9045564B2 (en) 2011-02-15 2015-06-02 Medimmune, Llc HSA-related compositions and methods of use
EP2678359A2 (fr) * 2011-02-24 2014-01-01 Merrimack Pharmaceuticals, Inc. Polythérapies comprenant des agents anti-erbb3
WO2012116317A3 (fr) * 2011-02-24 2013-11-14 Merrimack Pharmaceuticals, Inc. Polythérapies comprenant des agents anti-erbb3
EP2678359A4 (fr) * 2011-02-24 2015-01-28 Merrimack Pharmaceuticals Inc Polythérapies comprenant des agents anti-erbb3
JP2014511383A (ja) * 2011-02-24 2014-05-15 メリマック ファーマシューティカルズ インコーポレーティッド 抗ＥｒｂＢ３剤を含む併用療法
CN103547598A (zh) * 2011-02-24 2014-01-29 梅里麦克制药股份有限公司 包含抗-erbb3药剂的联合治疗
WO2012125573A3 (fr) * 2011-03-11 2012-12-27 Merrimack Pharmaceuticals, Inc. Utilisation d'inhibiteurs des récepteurs de la famille egfr dans le traitement de cancers du sein réfractaires aux hormones
JP2014508782A (ja) * 2011-03-11 2014-04-10 メリマック ファーマシューティカルズ インコーポレーティッド ホルモン不応性乳癌の治療におけるｅｇｆｒファミリー受容体の阻害剤の使用
CN103429262A (zh) * 2011-03-15 2013-12-04 梅里麦克制药股份有限公司 克服对erbb途径抑制剂的抗性
US8822417B2 (en) 2011-05-05 2014-09-02 Novozymes Biopharma DIC A/S Albumin variants
US9173960B2 (en) 2011-11-04 2015-11-03 Novartis Ag Methods of treating cancer with low density lipoprotein-related protein 6 (LRP6)—half life extender constructs
WO2013067355A1 (fr) * 2011-11-04 2013-05-10 Novartis Ag Constructions de protéine 6 liée à la lipoprotéine de basse densité (lrp6) prolongeant leur demi-vie
EP3290442A1 (fr) * 2011-11-04 2018-03-07 Novartis AG Constructions de protéine 6 liée à la lipoprotéine de basse densité (lrp6) prolongeant leur demi-vie
EP3252075A1 (fr) * 2011-11-04 2017-12-06 Novartis AG Constructions de protéine 6 liée à la lipoprotéine de basse densité (lrp6) prolongeant leur demi-vie
USRE47860E1 (en) 2011-11-04 2020-02-18 Novartis Ag Methods of treating cancer with low density lipoprotein-related protein 6 (LRP6)—half life extender constructs
US8883735B2 (en) 2011-11-04 2014-11-11 Novartis Ag Low density lipoprotein-related protein 6 (LRP6)-half life extender constructs
CN104039830A (zh) * 2011-11-04 2014-09-10 诺华股份有限公司 低密度脂蛋白相关蛋白6(lrp6)-半寿期延长物构建体
US10711050B2 (en) 2011-11-18 2020-07-14 Albumedix Ltd Variant serum albumin with improved half-life and other properties
US10329340B2 (en) 2012-03-16 2019-06-25 Albumedix Ltd Albumin variants
US9944691B2 (en) 2012-03-16 2018-04-17 Albumedix A/S Albumin variants
US9345766B2 (en) 2012-08-30 2016-05-24 Merrimack Pharmaceuticals, Inc. Combination therapies comprising anti-ERBB3 agents
US10934341B2 (en) 2012-11-08 2021-03-02 Albumedix, Ltd. Albumin variants
US10501524B2 (en) 2012-11-08 2019-12-10 Albumedix Ltd Albumin variants
US9913901B2 (en) 2012-12-03 2018-03-13 Merrimack Pharmaceuticals, Inc. Combination therapy for treating HER2-positive cancers
WO2014182970A1 (fr) 2013-05-08 2014-11-13 Zymeworks Inc. Constructions de liaison aux antigènes her2 et her3 bispécifiques
US10273304B2 (en) 2013-12-27 2019-04-30 Merrimack Pharmaceuticals, Inc. Biomarker profiles for predicting outcomes of cancer therapy with ERBB3 inhibitors and/or chemotherapies
US9688761B2 (en) 2013-12-27 2017-06-27 Merrimack Pharmaceuticals, Inc. Biomarker profiles for predicting outcomes of cancer therapy with ERBB3 inhibitors and/or chemotherapies
US11820825B2 (en) 2014-02-28 2023-11-21 Merus N.V. Methods of treating a subject having an EGFR-positive and/or ErbB-3-positive tumor
US11279770B2 (en) 2014-02-28 2022-03-22 Merus N.V. Antibody that binds ErbB-2 and ErbB-3
US12139548B2 (en) 2014-02-28 2024-11-12 Merus N.V. Antibody that binds ErbB-2 and ErbB-3
WO2015173248A1 (fr) * 2014-05-14 2015-11-19 F. Hoffmann-La Roche Ag Anticorps bispécifiques anti her3/her2 se liant à l'épingle à cheveux bêta de her3 et du domaine ii de her2
US10808038B2 (en) 2014-05-14 2020-10-20 Hoffmann-La Roche Inc. HER3/HER2 bispecific antibodies binding to the beta-hairpin of HER3 and domain II of HER2
US10184006B2 (en) 2015-06-04 2019-01-22 Merrimack Pharmaceuticals, Inc. Biomarkers for predicting outcomes of cancer therapy with ErbB3 inhibitors
US11359015B2 (en) 2015-07-15 2022-06-14 Genmab A/S Humanized or chimeric CD3 antibodies
US10633428B2 (en) 2015-08-20 2020-04-28 Albumedix Ltd Albumin variants and conjugates
US12116400B2 (en) 2015-08-20 2024-10-15 Sartorius Albumedix Limited Albumin variants and conjugates
US12122819B2 (en) 2015-10-02 2024-10-22 Silver Creek Pharmaceuticals, Inc. Method of treating skin tissue damage by topically administering a bi-specific protein comprising a human insulin-like growth factor variant and a human annexin A5 variant
US10040840B2 (en) 2015-10-02 2018-08-07 Silver Creek Pharmaceuticals, Inc. Bi-specific annexin A5/IGF-1 proteins and methods of use thereof to promote regeneration and survival of tissue
US11155593B2 (en) 2015-10-02 2021-10-26 Silver Creek Pharmaceuticals, Inc. Method of inhibiting apoptosis or promoting cell survival by providing a bi-specific protein comprising insulin-like growth factor IGF-1 and Annexin A5
US10633425B2 (en) 2015-10-02 2020-04-28 Silver Creek Pharmaceuticals, Inc. Method of protecting tissue from damage by administering a bi-specific therapeutic protein comprising insulin-like growth factor 1 (IGF-1) and Annexin A5
US11879002B2 (en) 2015-10-02 2024-01-23 Silver Creek Pharmaceuticals, Inc. Bi-specific therapeutic proteins, in vivo methods of use thereof and encoding nucleic acids thereof
US11939394B2 (en) 2015-10-23 2024-03-26 Merus N.V. Binding molecules that inhibit cancer growth
WO2017112847A1 (fr) 2015-12-22 2017-06-29 Albumedix A/S Souches améliorées pour l'expression de protéines
US10023618B2 (en) 2015-12-22 2018-07-17 Albumedix A/S Protein expression strains
WO2018011421A1 (fr) 2016-07-14 2018-01-18 Genmab A/S Anticorps multispécifiques dirigés contre cd40 et cd137
WO2018082758A1 (fr) 2016-11-04 2018-05-11 Aarhus Universitet Identification et traitement de tumeurs caractérisées par une surexpression du récepteur fc néonatal
WO2018162749A1 (fr) 2017-03-09 2018-09-13 Genmab A/S Anticorps dirigés contre pd-l1
US11780925B2 (en) 2017-03-31 2023-10-10 Merus N.V. ErbB-2 and ErbB3 binding bispecific antibodies for use in the treatment of cells that have an NRG1 fusion gene
WO2018234349A1 (fr) 2017-06-20 2018-12-27 Albumedix Ltd Souches améliorées pour l'expression de protéines
US11130979B2 (en) 2017-06-20 2021-09-28 Albumedix Ltd Protein expression strains
EP4026908A1 (fr) 2017-06-20 2022-07-13 Albumedix Ltd Souches d'expression de protéines améliorées
US11396670B2 (en) 2017-06-20 2022-07-26 Albumedix Limited Protein expression strains
WO2019025545A1 (fr) 2017-08-04 2019-02-07 Genmab A/S Agents de liaison se liant à pd-l1 et cd137 et leur utilisation
US11773170B2 (en) 2017-08-09 2023-10-03 Merus N.V. Antibodies that bind EGFR and cMET
WO2019096226A1 (fr) * 2017-11-16 2019-05-23 Chengdu Easton Biopharmaceuticals Co., Ltd. Protéines de fusion vegfr/pdgfr pasylées et leur utilisation en thérapie
US11548931B2 (en) 2017-11-16 2023-01-10 Xl-Protein Gmbh PASylated VEGFR/PDGFR fusion proteins and their use in therapy
WO2020060405A1 (fr) 2018-09-19 2020-03-26 Lava Therapeutics B.V. Immunoglobuline cd1d à double action
WO2020060406A1 (fr) 2018-09-19 2020-03-26 Lava Therapeutics B.V. Nouveaux anticorps bispécifiques destinés à être utilisés dans le traitement d'hémopathies malignes
WO2020094744A1 (fr) 2018-11-06 2020-05-14 Genmab A/S Formulation d'anticorps
WO2020159368A1 (fr) 2019-02-01 2020-08-06 Lava Therapeutics B.V. Nouveaux anticorps se liant au cd40
NL2022494B1 (en) 2019-02-01 2020-08-19 Lava Therapeutics B V Novel CD40-binding antibodies
WO2021052995A1 (fr) 2019-09-16 2021-03-25 Lava Therapeutics B.V. TRAITEMENT DU CANCER COMPRENANT L'ADMINISTRATION D'ANTICORPS DE LIAISON AU RÉCEPTEUR DES LYMPHOCYTES T VY9Vẟ2
EP3792283A1 (fr) 2019-09-16 2021-03-17 Lava Therapeutics B.V. Traitement du cancer comprenant l'administration d'anticorps de liaison du récepteur des lymphocytes t vgamma9vdelta2
WO2021111143A1 (fr) 2019-12-04 2021-06-10 Albumedix Limited Procédés et compositions produites par ceux-ci
WO2021155916A1 (fr) 2020-02-04 2021-08-12 BioNTech SE Traitement impliquant une vaccination d'antigène et des agents de liaison se liant à pd-l1 et cd137
WO2021156258A1 (fr) 2020-02-04 2021-08-12 BioNTech SE Traitement impliquant une vaccination d'antigène et des agents de liaison se liant à pd-l1 et cd137
US11261254B1 (en) 2020-03-18 2022-03-01 Genmab A/S Antibodies
WO2021185934A1 (fr) 2020-03-18 2021-09-23 Genmab A/S Anticorps se liant à b7h4
WO2021205184A1 (fr) 2020-04-09 2021-10-14 Autolus Limited Polypeptide
WO2021205183A1 (fr) 2020-04-09 2021-10-14 Autolus Limited Polypeptide
WO2021234383A1 (fr) 2020-05-19 2021-11-25 Autolus Limited Anticorps à domaine unique contre hace2 et leur utilisation pour prévenir une infection par sars-cov-2
WO2022008646A1 (fr) 2020-07-08 2022-01-13 LAVA Therapeutics N.V. Anticorps se liant au psma et aux récepteurs des lymphocytes t gamma-delta
WO2022029011A1 (fr) 2020-08-06 2022-02-10 BioNTech SE Agents de liaison pour la protéine s du coronavirus
US11858995B2 (en) 2020-09-10 2024-01-02 Genmab A/S Bispecific antibodies against CD3 and CD20 for treating chronic lymphocytic leukemia
US11845805B2 (en) 2020-09-10 2023-12-19 Genmab A/S Bispecific antibody against CD3 and CD20 in combination therapy for treating diffuse large B-cell lymphoma
WO2022122973A1 (fr) 2020-12-10 2022-06-16 LAVA Therapeutics N.V. Anticorps qui se lient aux récepteurs des lymphocytes t gamma-delta
WO2022180271A1 (fr) 2021-02-26 2022-09-01 LAVA Therapeutics N.V. Anticorps se liant à des récepteurs des lymphocytes t cd123 et gamma-delta
WO2022234146A1 (fr) 2021-05-07 2022-11-10 Genmab A/S Compositions pharmaceutiques comprenant des anticorps bispécifiques se liant à b7h4 et cd3
WO2022268740A1 (fr) 2021-06-21 2022-12-29 Genmab A/S Régime posologique combiné d'agents de liaison cd137 et pd-l1
WO2023037333A1 (fr) 2021-09-13 2023-03-16 Janssen Biotech, Inc Anticorps multispécifiques cd33 x vδ2 pour traiter le cancer
WO2023057571A1 (fr) 2021-10-08 2023-04-13 Genmab A/S Anticorps se liant à cd30 et cd3
WO2023067138A1 (fr) 2021-10-21 2023-04-27 LAVA Therapeutics N.V. Utilisations d'anticorps d'activation de lymphocytes t gamma delta
WO2023174521A1 (fr) 2022-03-15 2023-09-21 Genmab A/S Agents de liaison se liant à epcam et cd137
WO2023174952A1 (fr) 2022-03-15 2023-09-21 Genmab A/S Agents de liaison se liant à l'epcam et/ou au cd137
EP4285926A1 (fr) 2022-05-30 2023-12-06 LAVA Therapeutics N.V. Traitement combiné de la leucémie lymphocytaire chronique
WO2023232805A1 (fr) 2022-05-30 2023-12-07 LAVA Therapeutics N.V. Polythérapie contre la leucémie lymphoïde chronique
WO2023242319A1 (fr) 2022-06-15 2023-12-21 LAVA Therapeutics N.V. Anticorps variants qui se lient aux récepteurs des lymphocytes t gamma-delta
EP4292610A1 (fr) 2022-06-15 2023-12-20 LAVA Therapeutics N.V. Anticorps variants se liant aux récepteurs de lymphocytes t gamma-delta
WO2023242320A1 (fr) 2022-06-15 2023-12-21 LAVA Therapeutics N.V. Compositions comprenant des anticorps qui se lient aux récepteurs des lymphocytes t gamma-delta
EP4292609A1 (fr) 2022-06-15 2023-12-20 LAVA Therapeutics N.V. Compositions comprenant des anticorps se liant aux récepteurs de lymphocytes t gamma-delta
WO2024010445A1 (fr) 2022-07-06 2024-01-11 ACADEMISCH ZIEKENHUIS LEIDEN (h.o.d.n. LUMC) Anticorps bispécifique et ses utilisations
NL2032398B1 (en) 2022-07-06 2024-01-23 Academisch Ziekenhuis Leiden Bispecific antibody and uses thereof
WO2024200573A1 (fr) 2023-03-27 2024-10-03 LAVA Therapeutics N.V. Agents de liaison à la nectine-4 et méthodes d'utilisation
EP4438624A1 (fr) 2023-03-27 2024-10-02 LAVA Therapeutics N.V. Anticorps se liant aux récepteurs des lymphocytes t nectin-4 et gamma-delta
EP4442251A1 (fr) 2023-04-05 2024-10-09 Albumedix Ltd Formulations et leurs utilisations
WO2024208898A1 (fr) 2023-04-05 2024-10-10 Genmab A/S Compositions pharmaceutiques comprenant des anticorps se liant à cd30 et cd3
WO2024209085A1 (fr) 2023-04-05 2024-10-10 Albumedix Ltd Formulations et leurs utilisations

Also Published As

Publication number Publication date
US20170204192A1 (en) 2017-07-20
KR20110112301A (ko) 2011-10-12
US20150191545A1 (en) 2015-07-09
JP2012509259A (ja) 2012-04-19
JP5677972B2 (ja) 2015-02-25
US20110059076A1 (en) 2011-03-10
US20120003221A1 (en) 2012-01-05
CN102282168A (zh) 2011-12-14
US8927694B2 (en) 2015-01-06
BRPI0921586A2 (pt) 2019-09-24

Similar Documents

Publication Publication Date Title
US8927694B2 (en) Human serum albumin linkers and conjugates thereof
AU2009234253C1 (en) Human serum albumin linkers and conjugates thereof
US20220112297A1 (en) Trispecific proteins and methods of use
AU2018346955B2 (en) B cell maturation antigen binding proteins
US20230383003A1 (en) Antagonistic anti-tumor necrosis factor receptor superfamily polypeptides
CA3109954A1 (fr) Polypeptides antagonistes de la superfamille des recepteurs au facteur de necrose tumorale
CA3220878A1 (fr) Bcma ciblant des proteines trispecifiques et methodes d'utilisation
US20240101693A1 (en) Antagonistic anti-tumor necrosis factor receptor superfamily antibodies

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200980154509.9

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09827943

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2011536365

Country of ref document: JP

ENP Entry into the national phase

Ref document number: 20117014075

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 13130007

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09827943

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: PI0921586

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20110518