WO2009140469A2 - Procédés d'utilisation d'apo2l/trail pour traiter le cancer - Google Patents

Procédés d'utilisation d'apo2l/trail pour traiter le cancer Download PDF

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WO2009140469A2
WO2009140469A2 PCT/US2009/043908 US2009043908W WO2009140469A2 WO 2009140469 A2 WO2009140469 A2 WO 2009140469A2 US 2009043908 W US2009043908 W US 2009043908W WO 2009140469 A2 WO2009140469 A2 WO 2009140469A2
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apo2l
day
subject
treatment
antibody
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PCT/US2009/043908
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WO2009140469A3 (fr
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William Novotny
David Michael Reese
Peter James Wyld
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Genentech, Inc.
Amgen, Inc.
Immunex Corporation
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Publication of WO2009140469A2 publication Critical patent/WO2009140469A2/fr
Publication of WO2009140469A3 publication Critical patent/WO2009140469A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/282—Platinum compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • TNF tumor necrosis factor
  • TNF-alpha tumor necrosis factor-alpha
  • TNF-beta tumor necrosis factor-beta
  • LT-beta lymphotoxin-beta
  • CD30 ligand CD27 ligand
  • CD40 ligand OX-40 ligand
  • 4-lBB ligand LIGHT
  • Apo-1 ligand also referred to as Fas ligand or CD95 ligand
  • Apo-2 ligand also referred to as Apo2L or TRAIL
  • Apo-3 ligand also referred to as TWEAK
  • APRIL OPG ligand
  • OPG ligand also referred to as RANK ligand, ODF, or TRANCE
  • TALL-I also referred to as BIyS, BAFF
  • TNF family ligands bind to, and induce various biological activity through, cell surface "death receptors" to activate caspases, or enzymes that carry out the cell death or apoptosis pathway (Salvesen et al . , Cell, 91:443-446 (1997) .
  • TNFRl TNFR2
  • TACI GITR
  • CD27 CD27
  • OX-40 CD30
  • CD40 HVEM
  • Fas also referred to as Apo-1 or CD95
  • DR4 also referred to as TRAIL-Rl
  • DR5 also referred to as Apo-2 or TRAIL- R2
  • DcRl DcR2
  • osteoprotegerin OPG
  • RANK RANK
  • a ⁇ o-3 also referred to as DR3 or TRAMP
  • TNF receptor family members share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular regions, while others are found naturally as soluble proteins lacking a transmembrane and intracellular domain.
  • the extracellular portion of typical TNFRs contains a repetitive amino acid sequence pattern of multiple cysteine-rich domains (CRDs), starting from the NH 2 -terminus .
  • CCDs cysteine-rich domains
  • the ligand referred to as Apo-2L or TRAIL was identified several years ago as a member of the TNF family of cytokines, (see, e.g., Wiley et al., Immunity, 3:673-682 (1995); Pitti et al . , J. Biol.
  • the full-length native sequence human Apo2L/TRAIL polypeptide is a 281 amino acid long, Type II transmembrane protein. Some cells can produce a natural soluble form of the polypeptide, through enzymatic cleavage of the polypeptide's extracellular region (Ma ⁇ ani et al., J. Cell. Biol., 137:221-229 (1997)) .
  • Apo2L/TRAIL unlike other TNF family members however, was found to have a unique structural feature in that three cysteine residues (at position 230 of each subunit in the homot ⁇ mer) together coordinate a zinc atom, and that the zinc binding is important for trimer stability and biological activity. (Hymowitz et al . , supra; Bodmer et al., J. Biol. Chem., 275:20632-20637 (2000)) .
  • Apo2L/TRAIL may play a role in immune system modulation, including autoimmune diseases such as rheumatoid arthritis [see, e.g., Thomas et al . , J. Immunol., 161:2195-2200 (1998); Johnsen et al., Cytokine, 11:664-672 (1999); Griffith et al . , J. Exp. Med., 189:1343-1353 (1999); Song et al., J. Exp. Med., 191:1095-1103 (2000) ] .
  • autoimmune diseases such as rheumatoid arthritis
  • Soluble forms of Apo2L/TRAIL have also been reported to induce apoptosis in a variety of cancer cells, including colon, lung, breast, prostate, bladder, kidney, ovarian and brain tumors, as well as melanoma, leukemia, and multiple myeloma (see, e.g., Wiley et al., supra; Pitti et al . , supra; US Patent 6,030,945 issued February 29, 2000; US Patent 6,746,668 issued June 8, 2004; Rieger et al., FEBS Letters, 427:124-128 (1998) ; Ashkenazi et al., J. Clin. Invest., 104:155-162 (1999); Walczak et al .
  • Apo2L/TRAIL preparations may vary in terms of biochemical properties and biological activities on diseased versus normal cells, depending, for example, on the presence or absence of a tag molecule, zinc content, and % trimer content (See, Lawrence et al . , Nature Med., Letter to the Editor, 7:383-385 (2001) ; Qin et al . , Nature Med., Letter to the Editor, 7:385-386 (2001)) .
  • Apo2L/TRAIL has been found to bind at least five different receptors. At least two of the receptors which bind Apo2L/TRAIL contain a functional, cytoplasmic death domain.
  • One such receptor has been referred to as "DR4" (and alternatively as TR4 or TRAIL-Rl) (Pan et al . , Science, 226:111-113 (1997) ; see also WO98/32856 published July 30, 1998; WO99/37684 published July 29, 1999; WO 00/73349 published December 7, 2000; US 6,433,147 issued August 13, 2002; US 6,461,823 issued October 8, 2002, and US 6,342,383 issued January 29, 2002) .
  • DR5 Another such receptor for Apo2L/TRAIL has been referred to as Apo-2; TRAIL-R or TRAIL-R2, TR6, Tango-63, hAPO8, TRICK2 or KILLER
  • Apo-2 TRAIL-R or TRAIL-R2, TR6, Tango-63, hAPO8, TRICK2 or KILLER
  • Sheridan et al. Science, 222:818-821 (1997); Pan et al., Science, 222:815-818 (1997); WO98/51793 published November 19, 1998; WO98/41629 published September 24, 1998; Screaton et al . , Curr.
  • DR5 is reported to contain a cytoplasmic death domain and be capable of signaling apoptosis upon ligand binding (or upon binding a molecule, such as an agonist antibody, rfhich mimics the activity of the ligand) .
  • the crystal structure of the complex formed between Apo-2L/TRAIL and DR5 is described in Hymowitz et al . , Molecular Cell, 4_:563-571 (1999) .
  • both DR4 and DR5 can trigger apoptosis independently by recruiting and activating the apoptosis initiator, caspase-8, through the death-domain-containing adaptor molecule referred to as FADD/Mortl [Kischkel et al . , Immunity, 12:611-620 (2000); Sprick et al . , Immunity, L2:599-609 (2000); Bodmer et al., Nature Cell Biol. , ⁇ :241-243 (2000) ] .
  • DcRl receptors referred to as DcRl, DcR2 and OPG
  • DCRl also referred to as TRID, LIT or TRAIL-R3
  • TRID TRID, LIT or TRAIL-R3
  • Embodiments of the invention include methods of treating cancer, comprising exposing cancer cells to an effective amount of Apo2L/TRAIL and selected chemotherapy decisionsines and/or VEGF antibodies.
  • the cancer cells are non- small cell lung cancer (NSCLC) cells.
  • NSCLC non- small cell lung cancer
  • Articles of manufacture and kits comprising Apo2L/TRAIL and package inserts that provide, eg, administration information for treating cancers such as NSCLC are also included.
  • Embodiments of the invention include methods of treating cancer, comprising exposing cancer cells to an effective amount of Apo2L/TRAIL and CD20 antibodies.
  • the cancer cells are non-hodgkin' s lymphoma (NHL) cells.
  • NDL non-hodgkin' s lymphoma
  • Articles of manufacture and kits comprising Apo2L/TRAIL and package inserts that provide, eg, administration information for treating cancers such as NHL are also included.
  • a method of treating non-small cell lung cancer (NSCLC) in a subject comprising administering on Day 1 of treatment (a) an effective amount of Paclitaxel, Carboplatin and anti-VEGF antibody and (b) Apo2L/TRAIL polypeptide comprising amino acids 114-281 of Figure 1 (SEQ ID N0:l) in an amount ranging from about 4 mg/kg to 20 mg/kg, wherein following administration of said Apo2L/TRAIL polypeptide on Day 1 of treatment, further doses of Apo2L/TRAIL polypeptide are administered to said subject in an amount ranging from about 4 mg/kg/day to about 20 mg/kg/day for up to 5 consecutive days .
  • NSCLC non-small cell lung cancer
  • a method of treating non-Hodgkin' s lymphoma (NHL) in a subject comprising administering on Day 1 of treatment (a) an effective amount of anti-CD20 antibody and (b) Apo2L/TRAIL polypeptide comprising amino acids 114-281 of Figure 1 (SEQ ID NO:1) in an amount of about 4 mg/kg to about 8 mg/kg, wherein following administration of said Apo2L/TRAIL polypeptide on Day 1 of treatment, further doses of Apo2L/TRAIL polypeptide are administered to said subject in an amount ranging from about 4 mg/kg/day to about 8 mg/kg/day for up to 5 consecutive days.
  • said anti-CD20 antibody is Rituximab administered in an amount of about 375 mg/m 2 .
  • an Apo2L/TRAIL polypeptide in the manufacture of a medicament for the treatment of NSCLC in a subject, wherein the medicament comprises administering on Day 1 of treatment (a) an effective amount of Paclitaxel, Carboplatin and anti-VEGF antibody and (b) Apo2L/TRAIL polypeptide comprising amino acids 114-281 of
  • Figure 1 in an amount ranging from about 4 mg/kg to 20 mg/kg, and wherein following administration of said Apo2L/TRAIL polypeptide on Day 1 of treatment, further doses of Apo2L/TRAIL polypeptide are administered to said subject in an amount ranging from about 4 mg/kg/day to about 20 mg/kg/day for up to 5 consecutive days .
  • An Apo2L/TRAIL polypeptide for use in a method of medical treatment of NSCLC in a subject wherein said use includes administering on Day 1 of treatment (a) an effective amount of Paclitaxel, Carboplatin and anti-VEGF antibody and (b) Apo2L/TRAIL polypeptide comprising amino acids 114-281 of Figure 1 (SEQ ID NO:1) in an amount ranging from about 4 mg/kg to 20 mg/kg, and wherein following administration of said Apo2L/TRAIL polypeptide on Day 1 of treatment, further doses of A ⁇ o2L/TRAIL polypeptide are administered to said subject in an amount ranging from about 4 mg/kg/day to about 20 mg/kg/day for up to 5 consecutive days.
  • an Apo2L/TRAIL polypeptide in the manufacture of a medicament for the treatment of NHL in a subject, wherein the medicament comprises administering on Day 1 of treatment (a) an effective amount of anti-CD20 antibody and (b) Apo2L/TRAIL polypeptide comprising amino acids 114-281 of Figure 1 (SEQ ID NO:1) in an amount of about 4 mg/kg to about 8 mg/kg, and wherein following administration of said Apo2L/TRAIL polypeptide on Day 1 of treatment, further doses of Apo2L/TRAIL polypeptide are administered to said subject in an amount ranging from about 4 mg/kg/day to about 8 mg/kg/day for up to 5 consecutive days.
  • An Apo2L/TRAIL polypeptide for use in a method of medical treatment of NHL in a subject wherein said use includes administering on Day 1 of treatment (a) an effective amount of anti- CD20 antibody and (b) Apo2L/TRAIL polypeptide comprising amino acids 114-281 of Figure 1 (SEQ ID N0:l) in an amount of about 4 mg/kg to about 8 mg/kg, and wherein following administration of said Apo2L/TRAIL polypeptide on Day 1 of treatment, further doses of Apo2L/TRAIL polypeptide are administered to said subject in an amount ranging from about 4 mg/kg/day to about 8 mg/kg/day for up to 5 consecutive days.
  • An article of manufacture comprising: a container including Apo2L/TRAIL polypeptide comprising amino acids 114-281 Figure 1 (SEQ ID N0:l); and a package insert with instructions for treating NSCLC in a subject, wherein the instructions indicate dosing and amounts of (a) and (b) administered to the subject according to claim 1.
  • An article of manufacture comprising: a container including Apo2L/TRAIL polypeptide comprising amino acids 114-281 Figure 1 (SEQ ID N0:l); and a package insert with instructions for treating NHL in a subject, wherein the instructions indicate dosing and amounts of (a) and (b) administered to the subject according to claim 11.
  • Figure 1 shows the encoding DNA (SEQ ID NO: 2) and amino acid sequence (SEQ ID N0:l) for human Apo-2 ligand or TRAIL ("Apo2L/TRAIL”) polypeptide.
  • Figure 2 illustrates the study schema and the dosing mitine for Cohorts 1, 2, 3 and 4.
  • Figure 3 identifies the baseline demographics and disease characteristics of the human patients (24) who received >; 1 dose of rhApo2L/TRAIL plus PCB in the trial.
  • Figure 4 describes the disposition of the patients treated in Cohorts 1, 2, 3 and 4.
  • Figure 5 provides the incidence of treatment-emergent adverse events in the study.
  • Figure 6 provides the incidence of rhApo2L/TRAIL-related adverse events.
  • Figure 7 provides pharmacokinetic data on serum concentration-time profiles of rhApo2L/TRAIL on Day 1 of cycles 1 and 3 in the study.
  • Figure 8 provides the mean pharmacokinetic parameters of rhApo2L/TRAIL following a 1-hour infusion in human patients.
  • Figure 9 provides the tumor response assessment in the 24 human patients in the study.
  • Figure 10 shows the reduction of tumor burden in a male patient with Stage IV NSCLC following 6 cycles of rhApo2L/TRAIL 4 mg/kg for 5 days plus PCB treatment.
  • Figure 11 shows the reduction of tumor burden in a male patient with Stage IV NSCLC following 4 cycles of rhApo2L/TRAIL 20mg/kg for 2 days plus PCB treatment.
  • Figure 12 is a graph illustrating representative alanine amino transferase (ALT) variations for Cohort 1 during treatment .
  • Figure 13 is a graph illustrating representative aspartate amino transferase (AST) variations for Cohort 4 during treatment.
  • Figure 14 is a graph illustrating representative total bilirubin variations for Cohort 1 during treatment.
  • Figures 15A-B show the clinical study design, dose selection and schedule of dosing, respectively, for administering Rituximab and rhApo2L/TRAIL (study referred to in the figure as "APO3585”) .
  • Figure 16 is a table showing patient characteristics for the patients enrolled in the clinical trial.
  • Figures 17A-B are tables providing the incidence of adverse events.
  • Figure 18 provides the efficacy assessements for patients receiving Rituximab and Apo2L/TRAIL at the specified doses .
  • Figure 19 shows the mean pharmacokinetic parameters.
  • Apo-2 ligand refers to a polypeptide seguence which includes amino acid residues 114-281, inclusive, 95-281, inclusive, residues 92-281, inclusive, residues 91-281, inclusive, residues 41-281, inclusive, residues 39- 281, inclusive, residues 15-281, inclusive, or residues 1-281, inclusive, of the amino acid seguence shown in Figure 1, as well as biologically active fragments, deletional, insertional, or substitutional variants of the above seguences.
  • the polypeptide seguence comprises residues 114-281 of Figure 1.
  • the polypeptide sequence comprises residues 92-281 or residues 91-281 of Figure 1.
  • the Apo-2L polypeptides may be encoded by the native nucleotide sequence shown in Figure 1.
  • the codon which encodes residue Proll9 ( Figure 1) may be "CCT” or "CCG”.
  • the fragments or variants are biologically active and have at least about 80% amino acid sequence identity, more preferably at least about 90% sequence identity, and even more preferably, at least 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the above sequences.
  • the definition encompasses substitutional variants of Apo-2 ligand in which at least one of its native amino acids are substituted by another amino acid such as an alanine residue.
  • Optional substitutional variants include one or more of the residue substitutions.
  • Optional variants may comprise an amino acid sequence which differs from the native sequence Apo-2 ligand polypeptide sequence of Figure 1 and has one or more of the following amino acid substitutions at the residue position (s) in Figure 1: S96C; SlOlC; SlIlC; R170C; K179C.
  • the definition also encompasses a native sequence Apo-2 ligand isolated from an Apo-2 ligand source or prepared by recombinant or synthetic methods.
  • the Apo-2 ligand of the invention includes the polypeptides referred to as Apo-2 ligand or TRAIL disclosed in WO97/01633 published January 16, 1997, WO97/25428 published July 17, 1997, WO99/36535 published July 22, 1999, WO 01/00832 published January 4, 2001, WO02/09755 published February 7, 2002, and WO 00/75191 published December 14, 2000.
  • the terms are used to refer generally to forms of the Apo-2 ligand which include monomer, dimer, trimer, hexamer or hight oligomer forms of the polypeptide. All numbering of amino acid residues referred to in the Apo-2L sequence use the numbering according to Figure 1, unless specifically stated otherwise. For instance, "D203" or "Asp203” refers to the aspartic acid residue at position 203 in the sequence provided in Figure 1.
  • Apo2L/TRAIL polypeptide comprising amino acids 114-281 of Figure 1 has been assigned the USAN name "Dulanermin” and references to “Dulanermin” refer to this form of Apo2L/TRAIL polypeptide.
  • Apo-2 ligand selective variant refers to an Apo-2 ligand polypeptide which includes one or more amino acid mutations in a native Apo-2 ligand sequence and has selective binding affinity for either the DR4 receptor or the DR5 receptor.
  • the Apo-2 ligand variant has a selective binding affinity for the DR4 receptor and includes one or more amino acid substitutions in any one of positions 189, 191, 193, 199, 201 or 209 of a native Apo-2 ligand sequence.
  • the Apo-2 ligand variant has a selective binding affinity for the DR5 receptor and includes one or more amino acid substitutions in any one of positions 189, 191, 193, 264, 266, 267 or 269 of a native Apo-2 ligand sequence.
  • Preferred Apo-2 ligand selective variants include one or more amino acid mutations and exhibit binding affinity to the DR4 receptor which is equal to or greater (>) than the binding affinity of native sequence Apo-2 ligand to the DR4 receptor, and even more preferably, the A ⁇ o-2 ligand variants exhibit less binding affinity ( ⁇ ) to the DR5 receptor than the binding affinity exhibited by native sequence Apo- 2 ligand to DR5.
  • binding affinity of such Apo-2 ligand variant to the DR4 receptor is approximately equal (unchanged) or greater than (increased) as compared to native sequence Apo-2 ligand, and the binding affinity of the Apo-2 ligand variant to the DR5 receptor is less than or nearly eliminated as compared to native sequence Apo-2 ligand, the binding affinity of the Apo-2 ligand variant, for purposes herein, is considered "selective" for the DR4 receptor.
  • Preferred DR4 selective Apo-2 ligand variants of the invention will have at least 10-fold less binding affinity to DR5 receptor (as compared to native sequence Apo-2 ligand) , and even more preferably, will have at least 100-fold less binding affinity to DR5 receptor (as compared to native sequence Apo-2 ligand) .
  • the respective binding affinity of the Apo-2 ligand variant may be determined and compared to the binding properties of native Apo-2L (such as the 114-281 form) by ELISA, RIA, and/or BIAcore assays, known in the art.
  • Preferred DR4 selective Apo-2 ligand variants of the invention will induce apoptosis in at least one type of mammalian cell (preferably a cancer cell) , and such apoptotic activity can be determined by known art methods such as the alamar blue or crystal violet assay.
  • the DR4 selective Apo-2 ligand variants may or may not have altered binding affinities to any of the decoy receptors for Apo-2L, those decoy receptors being referred to in the art as DcRl, DcR2 and OPG.
  • Apo-2 ligand selective variants include one or more amino acid mutations and exhibit binding affinity to the DR5 receptor which is equal to or greater (>) than the binding affinity of native sequence Apo-2 ligand to the DR5 receptor, and even more preferably, such Apo-2 ligand variants exhibit less binding affinity ( ⁇ ) to the DR4 receptor than the binding affinity exhibited by native sequence Apo-2 ligand to DR4.
  • binding affinity of such Apo-2 ligand variant to the DR5 receptor is approximately equal (unchanged) or greater than (increased) as compared to native sequence Apo-2 ligand, and the binding affinity of the Apo-2 ligand variant to the DR4 receptor is less than or nearly eliminated as compared to native sequence Apo-2 ligand, the binding affinity of the Apo-2 ligand variant, for purposes herein, is considered "selective" for the DR5 receptor.
  • Preferred DR5 selective Apo-2 ligand variants of the invention will have at least 10-fold less binding affinity to DR4 receptor (as compared to native sequence Apo-2 ligand) , and even more preferably, will have at least 100-fold less binding affinity to DR4 receptor (as compared to native sequence Apo-2 ligand) .
  • the respective binding affinity of the Apo- 2 ligand variant may be determined and compared to the binding properties of native Apo2L (such as the 114-281 form) by ELISA, RIA, and/or BIAcore assays, known in the art.
  • Preferred DR5 selective Apo-2 ligand variants of the invention will induce apoptosis in at least one type of mammalian cell (preferably a cancer cell) , and such apoptotic activity can be determined by known art methods such as the alamar blue or crystal violet assay.
  • the DR5 selective Apo-2 ligand variants may or may not have altered binding affinities to any of the decoy receptors for Apo-2L, those decoy receptors being referred to in the art as DcRl, DcR2 and OPG.
  • Amino acid identification may use the single-letter alphabet or three-letter alphabet of amino acids, i.e.,
  • Apo2L/TRAIL extracellular domain or "Apo2L/TRAIL ECD” refers to a form of Apo2L/TRAIL which is essentially free of transmembrane and cytoplasmic domains .
  • the ECD will have less than 1% of such transmembrane and cytoplasmic domains, and preferably, will have less than 0.5% of such domains. It will be understood that any transmembrane domain (s) identified for the polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain.
  • transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified.
  • the ECD will consist of a soluble, extracellular domain sequence of the polypeptide which is free of the transmembrane and cytoplasmic or intracellular domains (and is not membrane bound) .
  • Particular extracellular domain sequences of Apo-2L/TRAIL are described in PCT Publication Nos . WO97/01633 and WO97/25428.
  • Apo2L/TRAIL monomer or "Apo2L monomer” refers to a covalent chain of an extracellular domain sequence of Apo2L.
  • Apo2L/TRAIL dimer or "Apo2L dimer” refers to two Apo-2L monomers joined in a covalent linkage via a disulfide bond.
  • the term as used herein includes free standing Apo2L dimers and Apo2L dimers that are within tumeric forms of Apo2L (i.e., associated with another, third Apo2L monomer) .
  • Apo2L/TRAIL trimer or "Apo2L trimer” refers to three Apo2L monomers that are non-covalently associated.
  • Apo2L/TRAIL aggregate is used to refer to self- associated higher oligomeric forms of Apo2L/TRAIL, such as Apo2L/TRAIL trimers, which form, for instance, hexameric and nanomeric forms of Apo2L/TRAIL. Determination of the presence and quantity of Apo2L/TRAIL monomer, dimer, or trimer (or other aggregates) may be made using methods and assays known in the art (and using commercially available materials) , such as native size exclusion HPLC (“SEC”) , denaturing size exclusion using sodium dodecyl sulphate (“SDS-SEC”), reverse phase HPLC and capillary electrophoresis.
  • SEC native size exclusion HPLC
  • SDS-SEC denaturing size exclusion using sodium dodecyl sulphate
  • reverse phase HPLC capillary electrophoresis.
  • Apo-2 ligand receptor includes the receptors referred to in the art as "DR4" and "DR5".
  • Pan et al. have described the TNF receptor family member referred to as "DR4" (Pan et al . , Science, 27 ⁇ :111-113 (1997) ; see also WO98/32856 published July 30, 1998; WO 99/37684 published July 29, 1999; WO 00/73349 published December 7, 2000; US 6,433,147 issued August 13, 2002; US 6,461,823 issued October 8, 2002, and US 6,342,383 issued January 29, 2002) .
  • DR5 the receptor has also been alternatively referred to as Apo-2; TRAIL-R, TR6, Tango-63, hAPO8, TRICK2 or KILLER; Screaton et al., Curr. Biol., 2 ⁇ 93 " 696 (1997); Walczak et al., EMBO J., 16:5386-5387 (1997); Wu et al .
  • a ⁇ o-2L receptor when used herein encompasses native sequence receptor and receptor variants. These terms encompass Apo-2L receptor expressed in a variety of mammals, including humans. Apo-2L receptor may be endogenously expressed as occurs naturally in a variety of human tissue lineages, or may be expressed by recombinant or synthetic methods.
  • a "native sequence Apo-2L receptor” comprises a polypeptide having the same amino acid sequence as an Apo-2L receptor derived from nature.
  • a native sequence Apo-2L receptor can have the amino acid sequence of naturally-occurring Apo-2L receptor from any mammal.
  • Such native sequence A ⁇ o-2L receptor can be isolated from nature or can be produced by recombinant or synthetic means.
  • the term "native sequence Apo-2L receptor" specifically encompasses naturally- occurring truncated or secreted forms of the receptor (e.g., a soluble form containing, for instance, an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants.
  • Receptor variants may include fragments or deletion mutants of the native seguence Apo-2L receptor.
  • a transcriptional splice variant of human DR5 is known in the art. This DR5 splice variant encodes the 440 amino acid sequence of human DR5.
  • polyol when used herein refers broadly to polyhydric alcohol compounds.
  • Polyols can be any water-soluble poly (alkylene oxide) polymer for example, and can have a linear or branched chain.
  • Preferred polyols include those substituted at one or more hydroxyl positions with a chemical group, such as an alkyl group having between one and four carbons.
  • the polyol is a poly (alkylene glycol), preferably poly (ethylene glycol) (PEG) .
  • PEG poly (ethylene glycol)
  • PEG poly (ethylene glycol)
  • polyols of the invention include those well known in the art and those publicly available, such as from commercially available sources.
  • conjugate is used herein according to its broadest definition to mean joined or linked together. Molecules are “conjugated” when they act or operate as if joined.
  • extracellular domain refers to a form of ligand or receptor which is essentially free of transmembrane and cytoplasmic domains. Ordinarily, the soluble ECD will have less than 1% of such transmembrane and cytoplasmic domains, and preferably, will have less than 0.5% of such domains.
  • divalent metal ion refers to a metal ion having two positive charges.
  • divalent metal ions for use in the present invention include but are not limited to zinc, cobalt, nickel, cadmium, magnesium, and manganese.
  • Particular forms of such metals that may be employed include salt forms (e.g., pharmaceutically acceptable salt forms) , such as chloride, acetate, carbonate, citrate and sulfate forms of the above mentioned divalent metal ions.
  • Divalent metal ions as described herein, are preferably employed in concentrations or amounts (e.g., effective amounts) which are sufficient to, for example, (1) enhance storage stability of Apo-2L trimers over a desired period of time, (2) enhance production or yield of Apo-2L trimers in a recombinant cell culture or purification method, (3) enhance solubility (or reduce aggregation) of Apo-2L trimers, or (4) enhance Apo-2L trimer formation.
  • Isolated when used to describe the various proteins disclosed herein, means protein that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the protein will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated protein includes protein in situ within recombinant cells, since at least one component of the protein's natural environment will not be present. Ordinarily, however, isolated protein will be prepared by at least one purification step.
  • An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid.
  • An isolated Apo-2 ligand nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated Apo-2 ligand nucleic acid molecules therefore are distinguished from the Apo-2 ligand nucleic acid molecule as it exists in natural cells.
  • an isolated Apo-2 ligand nucleic acid molecule includes Apo-2 ligand nucleic acid molecules contained in cells that ordinarily express Apo-2 ligand where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • Percent (%) amino acid sequence identity with respect to the sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the Apo-2 ligand sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art can determine appropriate parameters for measuring alignment, including assigning algorithms needed to achieve maximal alignment over the full-length sequences being compared. For purposes herein, percent amino acid identity values can be obtained using the sequence comparison computer program, ALIGN-2, which was authored by Genentech, Inc.
  • ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, CA. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers .
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous . Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • VEGF vascular endothelial cell growth factor
  • VEGF-A 165-amino acid human vascular endothelial cell growth factor and related 121-, 189-, and 206- amino acid human vascular endothelial cell growth factors, as described by Leung et al. Science, 246:1306 (1989), and Houck et al. MoI. Endocrm . , 5:1806 (1991), together with the naturally occurring allelic and processed forms thereof.
  • VEGF-A is part of a gene family including VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PlGF.
  • VEGF-A primarily binds to two high affinity receptor tyrosine kinases, VEGFR-I (Flt-1) and VEGFR-2 (Flk-1/KDR) , the latter being the major transmitter of vascular endothelial cell mitogenic signals of VEGF-A. Additionally, neuropilm-1 has been identified as a receptor for heparin-binding VEGF-A isoforms, and may play a role in vascular development.
  • the term "VEGF” or "VEGF- A” also refers to VEGFs from non-human species such as mouse, rat, or primate.
  • VEGF vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • Reference to any such forms of VEGF may be identified in the present application, e.g., by "VEGF (8-109),” “VEGF (1-109)” or “VEGF 165 .”
  • the amino acid positions for a "truncated" native VEGF are numbered as indicated in the native VEGF sequence.
  • amino acid position 17 (methionine) in truncated native VEGF is also position 17 (methionine) in native VEGF.
  • the truncated native VEGF has binding affinity for the KDR and Flt-1 receptors comparable to native VEGF.
  • VEGF variant refers to a VEGF polypeptide which includes one or more amino acid mutations in the native VEGF sequence.
  • the one or more amino acid mutations include amino acid substitution (s) .
  • numbers refer to the amino acid residue position along the amino acid sequence of the putative native VEGF (provided in Leung et al., supra and Houck et al., supra.) .
  • antibody herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • Antibody fragments comprise a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes . Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
  • V H variable domain
  • Each light chain has a variable domain at one end (VJ and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains .
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs) .
  • the variable domains of native heavy and light chains each comprise four FRs, largely adopting a ⁇ -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Rabat et al. r Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) .
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC) .
  • Papain digestion of antibodies produces two identical antigen- binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily.
  • Pepsin treatment yields an F(ab') 2 fragment that has two antigen-bmdmg sites and is still capable of cross-linking antigen.
  • Fv is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hyperva ⁇ able regions of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer. Collectively, the six hyperva ⁇ able regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hyperva ⁇ able regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region.
  • Fab '-SH is the designation herein for Fab' in rfhich the cysteine residue (s) of the constant domains bear at least one free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • the "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ) , based on the amino acid sequences of their constant domains.
  • antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes) , e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • Single-chain Fv or “scFv” antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V n and V L domains which enables the scFv to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V n ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V n - V L ) .
  • V n heavy-chain variable domain
  • V L light-chain variable domain
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al . , Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) .
  • the term "monoclonal antibody” as used herein 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. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567) .
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the technigues described in Clackson et al . , Nature, 352:624-628 (1991) and Marks et al., J. MoI. Biol., 222:581-597 (1991), for example.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)) .
  • chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequence
  • Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey, such as baboon, rhesus or cynomolgus monkey) and human constant region sequences (US Pat No. 5,693,780) .
  • a non-human primate e.g. Old World Monkey, such as baboon, rhesus or cynomolgus monkey
  • human constant region sequences US Pat No. 5,693,780
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non- human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g. residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a "hypervariable loop" (e.g.
  • an antibody "which binds" an antigen of interest e.g. VEGF
  • an antigen of interest is one capable of binding that antigen with sufficient affinity and/or avidity, optionally such that the antibody is useful as a therapeutic agent for targeting a cell expressing the antigen.
  • An "anti-VEGF antibody” is an antibody that binds to VEGF with sufficient affinity and specificity.
  • the antibody selected will normally have a sufficiently strong binding affinity for VEGF, for example, the antibody may bind hVEGF with a K d value of between 100 nM-1 pM.
  • Antibody affinities may be determined by a surface plasmon resonance based assay (such as the BIAcore assay as described in PCT Application Publication No.
  • the anti-VEGF antibody of the invention can be used as a therapeutic agent in targeting and interfering with diseases or conditions wherein the VEGF activity is involved.
  • the antibody may be subjected to other biological activity assays, e.g., in order to evaluate its effectiveness as a therapeutic.
  • Such assays are known in the art and depend on the target antigen and intended use for the antibody.
  • HUVEC inhibition assay examples include the HUVEC inhibition assay ; tumor cell growth inhibition assays (as described in WO 89/06692, for example) ; antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (US Patent 5,500,362) ; and agonistic activity or hematopoiesis assays (see WO 95/27062) .
  • An anti-VEGF antibody will usually not bind to other VEGF homologues such as VEGF-B or VEGF-C, nor other growth factors such as PlGF, PDGF or bFGF.
  • Preferred anti-VEGF antibodies include a monoclonal antibody that binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB 10709; a recombinant humanized anti-VEGF monoclonal antibody generated according to Presta et al. (1997) Cancer Res. 57:4593-4599, including but not limited to the antibody known as bevacizumab (BV; Avastin®) .
  • Bevacizumab includes mutated human IgGl framework regions and antigen-binding complementarity- determining regions from the murine anti-hVEGF monoclonal antibody A.4.6.1 that blocks binding of human VEGF to its receptors.
  • Bevacizumab Approximately 93% of the amino acid sequence of bevacizumab, including most of the framework regions, is derived from human IgGl, and about 7% of the sequence is derived from the murine antibody A4.6.1. Bevacizumab has a molecular mass of about 149,000 daltons and is glycosylated. Bevacizumab and other humanized anti-VEGF antibodies are further described in U.S. Pat. No. 6,884,879 issued Feb. 26, 2005. Additional preferred antibodies include the G6 or B20 series antibodies (e.g., G6-31, B20-4.1), as described in PCT Application Publication No. WO2005/012359. For additional preferred antibodies see U.S. Pat. Nos .
  • G6 series antibody is an anti-VEGF antibody that is derived from a sequence of a G6 antibody or G6-derived antibody according to any one of Figures 7, 24-26, and 34-35 of PCT Application Publication No. WO 2005/012359.
  • the G6 series antibody binds to a functional epitope on human VEGF comprising residues F17, Y21, Q22, Y25, D63, 183 and Q89.
  • a "B20 series antibody” is an anti-VEGF antibody that is derived from a sequence of the B20 antibody or a B20-derived antibody according to any one of Figures 27-29 of PCT Application Publication No. WO2005/012359.
  • the B20 series antibody binds to a functional epitope on human VEGF comprising residues F17, M18, D19, Y21, Y25, Q89, 191, KlOl, E103, and C104.
  • immunotherapy will refer to a method of treating a mammal (preferably a human patient) with an antibody, wherein the antibody may be an unconjugated or “naked” antibody, or the antibody may be conjugated or fused with heterologous molecule (s) or agent (s) , such as one or more cytotoxic agent(s), thereby generating an "immunoconjugate” .
  • heterologous molecule s
  • agent s
  • an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antagonist or antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody m situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody «rill be prepared by at least one purification step.
  • a "B cell” is a lymphocyte that matures within the bone marrow, and includes a naive B cell, memory B cell, or effector B cells (plasma cells) .
  • the B cell herein may be a normal or non- malignant B cell.
  • the "CD20” antigen is a " 35 kDa, non-glycosylated phosphoprotein found on the surface of greater than 90% of B cells from peripheral blood or lymphoid organs. CD20 is present on both normal B cells as well as malignant B cells, but is not expressed on stem cells. Other names for CD20 in the literature include "B- lymphocyte-restricted antigen” and "Bp35". The CD20 antigen is described in Clark et al. PNAS (USA) 82:1766 (1985), for example. Examples of antibodies which bind the CD20 antigen include: “C2B8" rfhich is now called “Rituximab” ("RITUXANS)”) (US Patent No.
  • the preferred CD20 antibodies herein are chimeric, humanized, or human CD20 antibodies, more preferably rituximab, humanized 2H7, chimeric or humanized A20 antibody (Immunomedics) , and HUMAX-CD20TM human CD20 antibody (Genmab) .
  • rituximab or “RITUXAN®” herein refer to the genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen and designated “C2B8" in US Patent No. 5,736,137, including fragments thereof which retain the ability to bind CD20.
  • humanized 2H7 refers to a humanized CD20 antibody, or an antigen- binding fragment thereof, wherein the antibody is effective to deplete primate B cells in vivo, the antibody comprising in the H chain variable region (V H ) thereof at least a CDR H3 sequence from an anti-human CD20 antibody and substantially the human consensus framework (FR) residues of the human heavy- chain subgroup III (V H III) .
  • a preferred humanized 2H7 is an intact antibody or antibody fragment comprising the variable light chain seguence:
  • DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKR (SEQ ID N0:3); and the variable heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRF TISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSS (SEQ ID NO: 4) .
  • the humanized 2H7 antibody is an intact antibody, preferably it comprises the light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC (SEQ ID NO: 5); and the heavy chain amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRF TISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPS SKS
  • Antibody-dependent cell-mediated cytotoxicity and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • FcRs Fc receptors
  • FcR expression on hematopoietic cells in summarized is Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991) .
  • an m vitro ADCC assay such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed m vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998) .
  • Human effector cells are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least FcyRIII and carry out ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • Fc receptor or “FcR” are used to describe a receptor that binds to the Fc region of an antibody.
  • the preferred FcR is a native sequence human FcR.
  • a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor") and Fc ⁇ RIIB (an “inhibiting receptor”) , which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine- based activation motif (ITAM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine- based activation motif
  • Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • FcRn neonatal receptor
  • FcRn neonatal receptor
  • FcRs herein include polymorphisms such as the genetic dimorphism in the gene that encodes Fc ⁇ RIIIa resulting in either a phenylalanine (F) or a valine (V) at amino acid position 158, located in the region of the receptor that binds to IgGl.
  • the homozygous valine Fc ⁇ RIIIa (FcyRIIIa-158V) has been shown to have a higher affinity for human IgGl and mediate increased ADCC m vitro relative to homozygous phenylalanine Fc ⁇ RIIIa (Fc ⁇ RIIIa-158F) or heterozygous (Fc ⁇ RIIIa- 158F/V) receptors.
  • "Complement dependent cytotoxicity” or “CDC” refer to the ability of a molecule to lyse a target in the presence of complement.
  • the complement activation pathway is initiated by the binding of the first component of the complement system (CIq) to a molecule (e.g. an antibody) complexed with a cognate antigen.
  • a CDC assay e.g. as described in
  • Gazzano-Santoro et al . J. Immunol. Methods 202:163 (1996), may be performed.
  • the term "therapeutically effective amount” refers to an amount of a therapeutic agent to treat or prevent a disease or disorder in a mammal.
  • the therapeutically effective amount of the therapeutic agent may reduce the number of cancer cells; reduce the primary tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder.
  • the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
  • efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression
  • immunosuppressive agent refers to substances that act to suppress or mask the immune system of the mammal being treated herein. This would include substances that suppress cytokine production, downregulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino- ⁇ -aryl-5-substituted py ⁇ midines (see U.S. Pat. No.
  • NSAIDs nonsteroidal antiinflammatory drugs
  • azathioprine cyclophosphamide
  • bromocryptine danazol
  • dapsone glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat. No.
  • anti-idiotypic antibodies for MHC antigens and MHC fragments include cyclosporin A; steroids such as glucocorticosteroids, e.g., prednisone, methylprednisolone, dexamethasone, and hydrocortisone; methotrexate (oral or subcutaneous); hydroxycloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antagonists including anti-interferon- Y, - ⁇ , or - ⁇ antibodies, anti-tumor necrosis factor- ⁇ antibodies (infliximab or adalimumab) , anti-TNF ⁇ immunoahesin (etanercept) , anti-tumor necrosis factor- ⁇ antibodies, anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies; anti-LFA-I antibodies, including anti-CDlla and anti-CD18 antibodies; anti-L3T4 antibodies; heterolog
  • T-cell receptor fragments (Offner et al., Science, 251: 430-432 (1991); WO 90/11294; Ianeway, Nature, 341: 482 (1989) ; and WO 91/01133) ; and T cell receptor antibodies (EP 340,109) such as T10B9.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g. At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 1"3 , Bi 212 , P 32 and radioactive isotopes of Lu) , chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof .
  • radioactive isotopes e.g. At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 1"3 , Bi 212 , P 32 and radioactive isotopes of Lu
  • chemotherapeutic agents e.g. At 211 , I 131 , I 125 , Y 90 , Re 186
  • Synergistic activity or “synergy” or “synergistic effect” or “synergistic effective amount” for the purposes herein means that the effect observed when employing a combination of Apo2L/TRAIL, chemotherapy and/or VEGF antibody (1) greater than the effect achieved when that Apo2L/TRAIL, chemotherapy or VEGF antibody is employed alone (or individually) and (2) greater than the sum added (additive) effect for that Apo2L/TRAIL, chemotherapy or VEGF antibody.
  • Such synergy or synergistic effect can be determined by way of a variety of means known to those in the art.
  • the synergistic effect of Apo2L/TRAIL, chemotherapy and/or VEGF antibody can be observed in in vitro or in vivo assay formats examining reduction of tumor cell number or tumor mass.
  • "Synergistic activity” or “synergy” or “synergistic effect” or “synergistic effective amount” for the purposes herein also refers to the effect observed when employing a combination of Apo2L/TRAIL and CD20 antibody (1) greater than the effect achieved when that Apo2L/TRAIL or CD20 antibody is employed alone (or individually) and (2) greater than the sum added (additive) effect for that Apo2L/TRAIL and CD20 antibody.
  • synergy or synergistic effect can be determined by way of a variety of means known to those in the art.
  • the synergistic effect of Apo2L/TRAIL and CD20 antibody can be observed in in vitro or in vivo assay formats examining reduction of tumor cell number or tumor mass.
  • apoptosis and apoptotic activity are used in a broad sense and refer to the orderly or controlled form of cell death in mammals that is typically accompanied by one or more characteristic cell changes, including condensation of cytoplasm, loss of plasma membrane microvilli, segmentation of the nucleus, degradation of chromosomal DNA or loss of mitochondrial function. This activity can be determined and measured using well known art methods, for instance, by cell viability assays, FACS analysis or DNA electrophoresis, binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies) .
  • cancer Assays which determine the ability of an antibody (e.g. Rituximab) to induce apoptosis have been described in Shan et al. Cancer Immunol Immunther 48:673-83 (2000) ; Pedersen et al. Blood 99:1314-9 (2002); Demidem et al. Cancer Chemotherapy & Radiopharmaceuticals 12 (3) : 177-186 (1997), for example.
  • the terms "cancer”, “cancerous”, and “malignant” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin' s lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, myeloma (such as multiple myeloma) , salivary gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, and various types of head and neck cancer.
  • squamous cell cancer small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin' s lymphoma, pancreatic cancer, glioblastom
  • pre-cancerous refers to a condition or a growth that typically precedes or develops into a cancer.
  • a "precancerous" growth will have cells that are characterized by abnormal cell cycle regulation, proliferation, or differentiation, rfhich can be determined by markers of cell cycle regulation, cellular proliferation, or differentiation.
  • dysplasia is meant any abnormal growth or development of tissue, organ, or cells.
  • the dysplasia is high grade or precancerous.
  • Metastasis is meant the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass.
  • non-metastatic is meant a cancer that is benign or that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site.
  • a non-metastatic cancer is any cancer that is a Stage 0, I, or II cancer, and occasionally a Stage III cancer.
  • primary tumor or “primary cancer” is meant the original cancer and not a metastatic lesion located in another tissue, organ, or location in the subject's body.
  • cancer benign tumor
  • a tumor that remains localized at the site of origin and does not have the capacity to infiltrate, invade, or metastasize to a distant site.
  • tumor burden is meant the number of cancer cells, the size of a tumor, or the amount of cancer in the body. Tumor burden is also referred to as tumor load. By “tumor number” is meant the number of tumors.
  • B cell neoplasms include Hodgkin's disease including lymphocyte predominant Hodgkin's disease (LPHD); non-Hodgkin' s lymphoma (NHL) ; follicular center cell (FCC) lymphomas; acute lymphocytic leukemia (ALL) ; chronic lymphocytic leukemia (CLL) ; and Hairy cell leukemia.
  • LPHD lymphocyte predominant Hodgkin's disease
  • NHL non-Hodgkin' s lymphoma
  • FCC follicular center cell lymphomas
  • ALL acute lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • Hairy cell leukemia hairy cell leukemia
  • the non-Hodgkins lymphoma include lew grade/follicular non-Hodgkin ' s lymphoma (NHL), small lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, plasmacytoid lymphocytic lymphoma, mantle cell lymphoma, AIDS- related lymphoma and Waldenstrom's macroglobulinemia. Treatment of relapses of these cancers are also contemplated. LPHD is a type of Hodgkin's disease that tends to relapse frequently despite radiation or chemotherapy treatment.
  • CLL is one of four major types of leukemia.
  • a cancer of mature B-cells called lymphocytes, CLL is manifested by progressive accumulation of cells in blood, bone marrow and lymphatic tissues.
  • Indolent lymphoma is a slow-growing, incurable disease in which the average patient survives between six and 10 years following numerous periods of remission and relapse.
  • Hodgkin's lymphomas can generally be distinguished from non-Hodgkin' s lymphomas by the presence of Reed-Sternberg cells in Hodgkin's lymphomas and the absence of said cells in non-Hodgkin' s lymphomas.
  • non-Hodgkin' s lymphomas encompassed by the term as used herein include any that would be identified as such by one skilled in the art (e.g., an oncologist or pathologist) in accordance with classification schemes known in the art, such as the Revised European-American Lymphoma (REAL) scheme as described in Color Atlas of Clinical Hematology, Third Edition; A. Victor Hoffbrand and John E. Pettit (eds .
  • relapsed or refractory NHL front line low grade NHL, Stage III/IV NHL, chemotherapy resistant NHL, precursor B lymphoblastic leukemia and/or lymphoma, small lymphocytic lymphoma, B cell chronic lymphacytic leukemia and/or prolymphocytic leukemia and/or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and/or lymphoplasmacytic lymphoma, marginal zone B cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone - MALT lymphoma, nodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasma cell myeloma, low grade/follicular lymphoma, intermediate grade/follicular NHL, mantle cell
  • autoimmune disease herein is a disease or disorder arising from and directed against an individual's own tissues or a co-segregate or manifestation thereof or resulting condition therefrom.
  • autoimmune diseases or disorders include, but are not limited to arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and ankylosing spondylitis) , psoriasis, dermatitis including atopic dermatitis; chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis/dermatomyositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, responses associated with inflammatory bowel disease (IBD) (Crohn's disease, ulcerative colitis) , and IBD with co-segregate of pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis,
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to cancer cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Harbor (1986) and Stella et al . , “Prodrugs: A Chemical Approach to Targeted Drug Delivery, " Directed Drug Delivery, Borchardt et al . , (ed.), pp. 247-267, Humana Press (1985) .
  • the prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, beta-lactam- containing prodrugs, optionally substituted phenoxyacetamide- containing prodrugs or optionally substituted phenylacetamide- containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.
  • cytotoxic drugs that can be de ⁇ vatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described below.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells .
  • the term is intended to include radioactive isotopes (e.g. At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 1"3 , Bi 212 , P 32 and radioactive isotopes of Lu) , chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • radioactive isotopes e.g. At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 1"3 , Bi 212 , P 32 and radioactive isotopes of Lu
  • chemotherapeutic agents e.g. At 211 , I 131 , I 125 , Y 90
  • chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; azi ⁇ dines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, t ⁇ ethylenemelamine, t ⁇ etylenephosphoramide, t ⁇ ethiylenethiophosphoramide and trimethylolomelamine; acetogenms (especially bullatacin and bullatacinone) ; a camptothecin (including the synthetic analogue topotecan) ; bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizele
  • calicheamicin especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnerf, Chem Intl. Ed. Engl . , 33 : 183-186 (1994))
  • dynemicin including dynemicin A
  • bisphosphonates such as clodronate
  • an esperamicin as well as neocarzinostatm chromophore and related chromoprotein enediyne antiobiotic chromophores
  • aclacinomysins actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxor
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • SERMs selective estrogen receptor modulators
  • tamoxifen including NOLVADEX® tamoxifen
  • raloxifene including NOLVADEX® tamoxifen
  • droloxifene 4-hydroxytamoxifen
  • trioxifene keoxifene
  • LY117018 onapristone
  • aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4 (5) -imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole
  • anti-androgens such as flutamide
  • a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, either in vitro or m vivo.
  • the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressing such genes in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine) , taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicm, etoposide, and bleomycin.
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
  • cytokines lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH) , thyroid stimulating hormone (TSH) , and luteinizing hormone (LH) ; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor- ⁇ and - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO) ; nerve growth factors; platelet-growth factor; transforming growth factors (TGFs) such as TGF- ⁇ and TGF- ⁇ ; insulin-like growth factor-I
  • cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
  • a "package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and/or warnings concerning the use of such therapeutic products, etc.
  • treating refers to curative therapy, prophylactic therapy, and preventative therapy.
  • mammal refers to any mammal classified as a mammal, including humans, cows, horses, dogs and cats. In a preferred embodiment of the invention, the mammal is a human .
  • a "subject” herein is a human subject. Generally, such subject is eligible for treatment, such as per the criteria set out in the Examples and Figures.
  • a cytokine related to the TNF ligand family the cytokine identified herein as "Apo-2 ligand” or "TRAIL” has been described.
  • the predicted mature amino acid sequence of native human Apo-2 ligand contains 281 amino acids, and has a calculated molecular weight of approximately 32.5 kDa.
  • the absence of a signal sequence and the presence of an internal hydrophobic region suggest that Apo- 2 ligand is a type II transmembrane protein.
  • Soluble extracellular domain Apo-2 ligand polypeptides have also been described. See, e.g., WO97/25428 published July 17, 1997.
  • Apo-2L substitutional variants have further been described. Alanine scanning techniques have been utilized to identify various substitutional variant molecules having biological activity.
  • substitutional variants of the Apo-2 ligand include those in which at least one amino acid is substituted by another amino acid such as an alanine residue. These substitutional variants are identified, for example, as “D203A”; “D218A” and “D269A.” This nomenclature is used to identify Apo-2 ligand variants wherein the aspartic acid residues at positions 203, 218, and/or 269 (using the numbering shown in Figure 1) are substituted by alanine residues.
  • the Apo-2L variants of the present invention may comprise one or more of the amino acid substitutions.
  • such Apo-2L variants will be DR4 or DR5 receptor selective variants.
  • the description below relates to methods of producing A ⁇ o-2 ligand, including Apo-2 ligand variants, by culturing host cells transformed or transfected with a vector containing Apo-2 ligand encoding nucleic acid and recovering the polypeptide from the cell culture.
  • the DNA encoding Apo-2 ligand may be obtained from any cDNA library prepared from tissue believed to possess the Apo-2 ligand mRNA and to express it at a detectable level. Accordingly, human Apo-2 ligand DNA can be conveniently obtained from a cDNA library prepared from human tissues, such as the bacteriophage library of human placental cDNA as described in WO97/25428.
  • the Apo-2 ligand- encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis.
  • Probes such as antibodies to the Apo-2 ligand or oligonucleotides of at least about 20-80 bases
  • Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) .
  • An alternative means to isolate the gene encoding Apo-2 ligand is to use PCR methodology [Sambrook et al . , supra; Dieffenbach et al., PCR Primer :A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995) ] .
  • Amino acid sequence fragments or variants of Apo-2 ligand can be prepared by introducing appropriate nucleotide changes into the Apo-2 ligand DNA, or by synthesis of the desired Apo-2 ligand polypeptide.
  • Such fragments or variants represent insertions, substitutions, and/or deletions of residues within or at one or both of the ends of the intracellular region, the transmembrane region, or the extracellular region, or of the amino acid sequence shown for the full-length Apo-2 ligand in Figure 1. Any combination of insertion, substitution, and/or deletion can be made to arrive at the final construct, provided that the final construct possesses, for instance, a desired biological activity, such as apoptotic activity, as defined herein.
  • the fragments or variants have at least about 80% amino acid sequence identity, more preferably, at least about 90% sequence identity, and even more preferably, at least 95%, 96%, 97%, 98% or 99% sequence identity with the sequences identified herein for the intracellular, transmembrane, or extracellular domains of A ⁇ o-2 ligand, or the full-length sequence for Apo-2 ligand.
  • the amino acid changes also may alter post-translational processes of the Apo-2 ligand, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
  • Variations in the Apo-2 ligand sequence as described above can be made using any of the techniques and guidelines for conservative and non-conservative mutations set forth in U.S. Pat. No. 5,364,934. These include oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.
  • Scanning amino acid analysis can be employed to identify one or more amino acids along a contiguous sequence.
  • preferred scanning amino acids are relatively small, neutral amino acids.
  • amino acids include alanine, glycine, serine and cysteine.
  • Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. [Cunningham et al . , Science, 244:1081 (1989)] .
  • Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W. H. Freeman & Co., NY); Chothia, J. MoI. Biol., 150:1 (1976)] .
  • Amino acids may be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)) :
  • non-polar Ala (A), VaI (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W) , Met (M)
  • uncharged polar GIy (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N) , GIn (Q)
  • Naturally occurring residues may be divided into groups based on common side-chain properties:
  • Apo-2 ligand sequences include any of the Apo- 2 ligand polypeptides described herein having a methionine or modified methionine (such as formyl methionyl or other blocked methionyl species) at the N-terminus of the polypeptide sequence.
  • the nucleic acid encoding native or variant Apo-2 ligand may be inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
  • a replicable vector for further cloning (amplification of the DNA) or for expression.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, each of which is described below.
  • Optional signal sequences, origins of replication, marker genes, enhancer elements and transcription terminator sequences that may be employed are known in the art and described in further detail in WO97/25428.
  • Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the Apo-2 ligand nucleic acid sequence. Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of a particular nucleic acid sequence, such as the Apo-2 ligand nucleic acid sequence, to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature.
  • promoters recognized by a variety of potential host cells are well known. These promoters are operably linked to Apo-2 ligand encoding DNA by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Both the native Apo-2 ligand promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the Apo-2 ligand DNA. Promoters suitable for use with prokaryotic and eukaryotic hosts are known in the art, and are described in further detail in WO97/25428.
  • a preferred method for the production of soluble Apo-2L in E. coli employs an inducible promoter for the regulation of product expression.
  • the use of a controllable, inducible promoter allows for culture growth to the desirable cell density before induction of product expression and accumulation of significant amounts of product which may not be well tolerated by the host.
  • Several inducible promoter systems (T7 polymerase, trp and alkaline phosphatase (AP) ) have been evaluated by Applicants for the expression of Apo-2L (form 114-281) .
  • the use of each of these three promoters resulted in significant amounts of soluble, biologically active Apo-2L trimer being recovered from the harvested cell paste.
  • the AP promoter is preferred among these three inducible promoter systems tested because of tighter promoter control and the higher cell density and titers reached in harvested cell paste.
  • Plasmids containing one or more of the above-listed components employs standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and re- ligated in the form desired to generate the plasmids required.
  • the ligation mixtures can be used to transform E. coli K12 strain 294 (ATCC 31,446) and successful transformants selected by ampicillin or tetracycline resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion, and/or sequenced using standard techniques known in the art. [See, e.g., Messing et al., Nucleic Acids Res . , _9:309 (1981); Maxam et al., Methods in Enzymology, 6_5 : 499 (1980) ] .
  • transient expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector [Sambrook et al., supra] .
  • Transient expression systems comprising a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNAs, as well as for the rapid screening of such polypeptides for desired biological or physiological properties.
  • transient expression systems are particularly useful in the invention for purposes of identifying analogs and variants of Apo-2 ligand that are biologically active Apo-2 ligand.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells.
  • Suitable prokaryotes for this purpose include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimunum, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 April 1989) , Pseudomonas such as P. aeruginosa, and Streptomyces .
  • the host cell should secrete minimal amounts of proteolytic enzymes.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for Apo-2 ligand-encoding vectors.
  • Suitable host cells for the expression of glycosylated Apo-2 ligand are derived from multicellular organisms. Examples of all such host cells, including CHO cells, are described further in WO97/25428.
  • Host cells are transfected and preferably transformed with the above-described expression or cloning vectors for Apo-2 ligand production and cultured in nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaPO 4 and electroporation . Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell. Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes or other cells that contain substantial cell- wall barriers.
  • Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23_:315 (1983) and WO 89/05859 published 29 June 1989.
  • plants may be transfected using ultrasound treatment as described in WO 91/00358 published 10 January 1991.
  • DNA into cells such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used.
  • polycations e.g., polybrene, polyornithine.
  • Prokaryotic cells used to produce Apo-2 ligand may be cultured in suitable culture media as described generally in Sambrook et al., supra . Particular forms of culture media that may be employed for culturing E. coli are described further in the Examples below. Mammalian host cells used to produce Apo-2 ligand may be cultured in a variety of culture media.
  • Examples of commercially available culture media include Ham's FlO (Sigma), Minimal Essential Medium (“MEM”, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (“DMEM”, Sigma) . Any such media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor) , salts (such as sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleosides (such as adenosine and thymidine) , antibiotics (such as GentamycinTM drug) , trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) , and glucose or an equivalent energy source.
  • hormones and/or other growth factors such as insulin, transferrin, or epidermal growth factor
  • salts such as sodium chloride, calcium, magnesium, and phosphate
  • buffers such as HEPES
  • nucleosides such as
  • any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. In general, principles, protocols, and practical techniques for maximizing the productivity of mammalian cell cultures can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRL Press, 1991) .
  • one or more divalent metal ions will typically be added to or included in the culture media for culturing or fermenting the host cells .
  • the divalent metal ions are preferably present in or added to the culture media at a concentration level sufficient to enhance storage stability, enhance solubility, or assist in forming stable Apo-2L trimers coordinated by one or more zinc ions.
  • the amount of divalent metal ions which may be added will be dependent, in part, on the host cell density in the culture or potential host cell sensitivity to such divalent metal ions. At higher host cell densities in the culture, it may be beneficial to increase the concentration of divalent metal ions.
  • divalent metal ions are added during or after product expression by the host cells, it may be desirable to adjust or increase the divalent metal ion concentration as product expression by the host cells increases. It is generally believed that trace levels of divalent metal ions which may be present in typical commonly available cell culture media may not be sufficient for stable trimer formation. Thus, addition of further quantities of divalent metal ions, as described herein, is preferred.
  • the divalent metal ions are preferably added to the culture media at a concentration which does not adversely or negatively affect host cell growth, if the divalent metal ions are being added during the growth phase of the host cells in the culture.
  • ZnSO 4 added at concentrations of greater than 1 mM can result in lower host cell density.
  • bacterial cells can sequester metal ions effectively by forming metal ion complexes with cellular matrices.
  • divalent metal ions may be added or fed to the cell culture media during the product expression phase.
  • the divalent metal ion concentration in the culture media should not exceed the concentration which may be detrimental or toxic to the host cells.
  • the concentration of the divalent metal ion concentration in the culture media does not exceed about ImM (preferably, ⁇ ImM) .
  • the divalent metal ion concentration in the culture media is about 50 micromolar to about 250 micromolar.
  • the divalent metal ion used in such methods is zinc sulfate. It is desirable to add the divalent metal ions to the cell culture in an amount wherein the metal ions and Apo-2 ligand trimer can be present at a one to one molar ratio.
  • the divalent metal ions can be added to the cell culture in any acceptable form.
  • a solution of the metal ion can be made using water, and the divalent metal ion solution can then be added or fed to the culture media.
  • Expression of the Apo-2L may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 72:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.
  • Various labels may be employed, most commonly radioisotopes, and particularly 32 P.
  • other techniques may also be employed, such as using biotin-modified nucleotides for introduction into a polynucleotide.
  • the biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionucleotides, fluorescers or enzymes.
  • labels such as radionucleotides, fluorescers or enzymes.
  • antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
  • the antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • Gene expression may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product.
  • immunohistochemical staining techniques a cell sample is prepared, typically by dehydration and fixation, followed by reaction with labeled antibodies specific for the gene product coupled, where the labels are usually visually detectable, such as enzymatic labels, fluorescent labels, luminescent labels, and the like.
  • Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native Apo-2 ligand polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to Apo-2 ligand DNA and encoding a specific antibody epitope.
  • Apo-2 ligand preferably is recovered from the culture medium as a secreted polypeptide, although it also may be recovered from host cell lysates when directly produced without a secretory signal. If the Apo-2 ligand is membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or its extracellular region may be released by enzymatic cleavage.
  • a suitable detergent solution e.g. Triton-X 100
  • the Apo-2 ligand When Apo-2 ligand is produced in a recombinant cell other than one of human origin, the Apo-2 ligand is free of proteins or polypeptides of human origin. However, it is usually necessary to recover or purify Apo-2 ligand from recombinant cell proteins or polypeptides to obtain preparations that are substantially homogeneous as to Apo-2 ligand.
  • the culture medium or lysate may be centrifuged to remove particulate cell debris.
  • Apo-2 ligand thereafter is purified from contaminant soluble proteins and polypeptides, with the following procedures being exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE or CM; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; diafiltration and protein A Sepharose columns to remove contaminants such as IgG.
  • the Apo-2 ligand can be isolated by affinity chromatography.
  • Apo-2 ligand fragments or variants in which residues have been deleted, inserted, or substituted are recovered in the same fashion as native Apo-2 ligand, taking account of any substantial changes in properties occasioned by the variation.
  • preparation of an Apo-2 ligand fusion with another protein or polypeptide e.g., a bacterial or viral antigen, facilitates purification; an immunoaffinity column containing antibody to the antigen can be used to adsorb the fusion polypeptide .
  • a protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) also may be useful to inhibit proteolytic degradation during purification, and antibiotics may be included to prevent the growth of adventitious contaminants.
  • PMSF phenyl methyl sulfonyl fluoride
  • purification methods suitable for native Apo-2 ligand may require modification to account for changes in the character of Apo-2 ligand or its variants upon expression in recombinant cell culture.
  • the recovered Apo-2L may be desirable to expose the recovered Apo-2L to a divalent metal ion-containing solution or to purification material (such as a chromatography medium or support) containing one or more divalent metal ions.
  • the divalent metal ions and/or reducing agent is used during recovery or purification of the Apo-2L.
  • both divalent metal ions and reducing agent such as DTT or BME, may be used during recovery or purification of the Apo-2L. It is believed that use of divalent metal ions during recovery or purification will provide for stability of Apo-2L trimer or preserve Apo-2L trimer formed during the cell culturing step.
  • the description below also relates to methods of producing Apo-2 ligand covalently attached (hereinafter "conjugated”) to one or more chemical groups.
  • Chemical groups suitable for use in an Apo-2L conjugate of the present invention are preferably not significantly toxic or immunogenic.
  • the chemical group is optionally selected to produce an Apo-2L conjugate that can be stored and used under conditions suitable for storage.
  • a variety of exemplary chemical groups that can be conjugated to polypeptides are known in the art and include for example carbohydrates, such as those carbohydrates that occur naturally on glycoproteins, polyglutamate, and non-proteinaceous polymers, such as polyols (see, e.g., U.S. Patent No. 6,245,901) .
  • a polyol for example, can be conjugated to polypeptides such as an Apo-2L at one or more amino acid residues, including lysine residues, as is disclosed in WO 93/00109, supra .
  • the polyol employed can be any water-soluble poly (alkylene oxide) polymer and can have a linear or branched chain. Suitable polyols include those substituted at one or more hydroxyl positions with a chemical group, such as an alkyl group having between one and four carbons .
  • the polyol is a poly (alkylene glycol), such as poly (ethylene glycol) (PEG), and thus, for ease of description, the remainder of the discussion relates to an exemplary embodiment wherein the polyol employed is PEG and the process of conjugating the polyol to a polypeptide is termed "pegylation . "
  • PEG poly (ethylene glycol)
  • pegylation the process of conjugating the polyol to a polypeptide
  • other polyols such as, for example, poly (propylene glycol) and polyethylene-polypropylene glycol copolymers, can be employed using the techniques for conjugation described herein for PEG.
  • the average molecular weight of the PEG employed in the pegylation of the Apo-2L can vary, and typically may range from about 500 to about 30,000 daltons (D) .
  • the average molecular weight of the PEG is from about 1,000 to about 25,000 D, and more preferably from about 1,000 to about 5,000 D.
  • pegylation is carried out with PEG having an average molecular weight of about 1,000 D.
  • the PEG homopolymer is unsubstituted, but it may also be substituted at one end with an alkyl group.
  • the alkyl group is a C1-C4 alkyl group, and most preferably a methyl group.
  • PEG preparations are commercially available, and typically, those PEG preparations suitable for use in the present invention are nonhomogeneous preparations sold according to average molecular weight.
  • commercially available PEG (5000) preparations typically contain molecules that vary slightly in molecular weight, usually ⁇ 500 D.
  • the Apo-2 ligand of the invention may be in various forms, such as in monomer form or trimer form (comprising three monomers) .
  • an Apo-2L trimer will be pegylated in a manner such that a PEG molecule is linked or conjugated to one, two or each of the three monomers that make up the trimeric Apo-2L.
  • the PEG employed have an average molecular weight of about 1,000 to about 5,000 D.
  • the Apo-2L trimers may be "partially" pegylated, i.e., wherein only one or two of the three monomers that make up the trimer are linked or conjugated to PEG.
  • proteins conjugated to PEG include the methods described in U.S. Pat. No. 4,179,337, U.S. Pat. No. 4,935,465 and U.S. Patent No. 5,849,535.
  • the protein is covalently bonded via one or more of the amino acid residues of the protein to a terminal reactive group on the polymer, depending mainly on the reaction conditions, the molecular weight of the polymer, etc.
  • the polymer with the reactive group (s) is designated herein as activated polymer.
  • the reactive group selectively reacts with free amino or other reactive groups on the protein.
  • the PEG polymer can be coupled to the amino or other reactive group on the protein in either a random or a site specific manner. It will be understood, however, that the type and amount of the reactive group chosen, as well as the type of polymer employed, to obtain optimum results, will depend on the particular protein or protein variant employed to avoid having the reactive group react with too many particularly active groups on the protein. As this may not be possible to avoid completely, it is recommended that generally from about 0.1 to 1000 moles, preferably 2 to 200 moles, of activated polymer per mole of protein, depending on protein concentration, is employed. The final amount of activated polymer per mole of protein is a balance to maintain optimum activity, while at the same time optimizing, if possible, the circulatory half-life of the protein.
  • Apo2L described herein may be also be linked or fused to leucine zipper sequences using techniques known in the art.
  • VEGF antibodies including humanized and/or affinity matured anti-VEGF antibodies such as the humanized anti-VEGF antibody huA4.6.1 AVASTIN (Kim et al., Growth Factors, 7:53-64 (1992), International Publication No. WO 96/30046, and WO 98/45331, published October 15, 1998) .
  • the VEGF antibody employed according to the present inventions is "Bevacizumab” or Avastin®, commercially available from Genentech, Inc.
  • VEGF Vascular endothelial growth factor
  • Anti-VEGF neutralizing antibodies suppress the growth of a variety of human tumor cell lines in nude mice (Kim et al . , Nature 362:841-844 (1993) ; barren et al . , J. Clin. Invest. 95:1789-1797 (1995); Borgstrom et al . , Cancer Res. 56:4032-4039 (1996); Melnyk et al., Cancer Res. 56:921-924 (1996)) and also inhibit intraocular angiogenesis in models of ischemic retinal disorders. Adamis et al., Arch. Ophthalmol. 114:66-71 (1996) .
  • anti-VEGF monoclonal antibodies or other inhibitors of VEGF action are promising candidates for the treatment of tumors and various intraocular neovascular disorders.
  • Such antibodies are described, for example, in EP 817,648 published January 14, 1998; and in WO98/45331 and WO98/45332, both published October 15, 1998.
  • Bevacizumab also known as “rhuMAb VEGF” or “Avastin ® ", is a recombinant humanized anti-VEGF monoclonal antibody generated according to Presta et al. Cancer Res. 57:4593- 4599 (1997) . It comprises mutated human IgGl framework regions and antigen-binding complementarity-determining regions from the murine anti-hVEGF monoclonal antibody A.4.6.1 that blocks binding of human VEGF to its receptors. Approximately 93% of the amino acid sequence of Bevacizumab, including most of the framework regions, is derived from human IgGl, and about 7% of the sequence is derived from the murine antibody A4.6.1.
  • Bevacizumab has a molecular mass of about 149,000 daltons and is glycosylated. Bevacizumab has been approved by the FDA for use in combination with chemotherapy regimens to treat metastatic colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) . Hurwitz et al . , N. Engl. J. Med. 350:2335-42 (2004); Sandler et al., N. Engl. J. Med. 355:2542-50 (2006) .
  • CRC metastatic colorectal cancer
  • NSCLC non-small cell lung cancer
  • bevacizumab is being investigated in ongoing clinical trials for treating various cancer indications. Kerbel, J. CIm. Oncol. 19:45S- 51S (2001) ; De Vore et al, Proc. Am. Soc.
  • CD20 antibody employed according to the present inventions is "Rituximab” or Rituxan®, commercially available from Genentech, Inc.
  • the CD20 antigen also called human B-lymphocyte-restricted differentiation antigen, Bp35
  • Bp35 human B-lymphocyte-restricted differentiation antigen
  • the antigen is also expressed on greater than 90% of B cell non-Hodgkin' s lymphomas (NHL) (Anderson et al.
  • CD20 regulates an early step(s) in the activation process for cell cycle initiation and differentiation (Tedder et al., supra) and possibly functions as a calcium ion channel (Tedder et al. J. Cell. Biochem. 14D:195 (1990)) .
  • this antigen may serve as a candidate for "targeting" of such lymphomas.
  • the ⁇ tuximab (RITUXAN®) antibody is a genetically engineered chimeric mu ⁇ ne/human monoclonal antibody directed against the CD20 antigen.
  • Rituximab is the antibody called "C2B8" in US Patent No. 5,736,137 issued April 7, 1998 (Anderson et al.) .
  • RITUXAN® is indicated for the treatment of patients with relapsed or refractory low-grade or follicular, CD20 positive, B cell non-Hodgkin' s lymphoma. In vitro mechanism of action studies have demonstrated that RITUXAN® binds human complement and lyses lymphoid B cell lines through complement-dependent cytotoxicity (CDC) (Reff et al .
  • CDC complement-dependent cytotoxicity
  • RITUXAN® has been sho «m to have anti-proliferative effects in t ⁇ tiated thymidine incorporation assays and to induce apoptosis directly, while other anti-CDl9 and CD20 antibodies do not (Maloney et al. Blood 88(10) :637a (1996)) . Synergy between RITUXAN® and certain chemotherapies and toxins has also been observed experimentally.
  • RITUXAN ⁇ sensitizes drug-resistant human B cell lymphoma cell lines to the cytotoxic effects of doxorubicin, CDDP, VP-16, diphtheria toxin and ricin (Demidem et al . Cancer Chemotherapy & Radiopharmaceuticals 12 (3) : 177-186 (1997)) .
  • RITUXAN® depletes B cells from the peripheral blood, lymph nodes, and bone marrow of cynomolgus monkeys, presumably through complement and cell-mediated processes (Reff et al . Blood 83 (2) : 435-445 (1994)) .
  • formulations comprising Apo2L/TRAIL, chemotherapy, and/or VEGF antibodies are also provided by the present invention. It is believed that such formulations will be particularly suitable for storage as well as for therapeutic administration.
  • the formulations may be prepared by known techniques. For instance, the formulations may be prepared by buffer exchange on a gel filtration column. Formulations comprising Apo2L/TRAIL and/or CD20 antibodies are also provided by the present invention. It is believed that such formulations will be particularly suitable for storage as well as for therapeutic administration.
  • the formulations may be prepared by known techniques. Typically, an appropriate amount of an acceptable salt or carrier is used in the formulation to render the formulation isotonic.
  • compositions can be prepared by mixing the desired molecules having the appropriate degree of purity with optional carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), in the form of lyophilized formulations, aqueous solutions or aqueous suspensions.
  • Acceptable carriers, excipients, or stabilizers are preferably nontoxic to recipients at the dosages and concentrations employed, and include buffers such as Tris, HEPES, PIPES, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as gly
  • Such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, colloidal silica, magnesium t ⁇ silicate, polyvinyl pyrrolidone, and cellulose-based substances.
  • Carriers for topical or gel-based forms include polysaccharides such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wood wax alcohols.
  • conventional depot forms are suitably used. Such forms include, for example, microcapsules, nano-capsules, liposomes, plasters, inhalation forms, nose sprays, sublingual tablets, and sustained-release preparations.
  • Formulations to be used for in vivo administration should be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution .
  • the formulation may be stored in lyophilized form or in solution if administered systemically . If in lyophilized form, it is typically formulated in combination with other ingredients for reconstitution with an appropriate diluent at the time for use.
  • An example of a liquid formulation is a sterile, clear, colorless unpreserved solution filled in a single-dose vial for subcutaneous injection.
  • Therapeutic formulations generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the formulations are preferably administered as repeated intravenous (i.v.), subcutaneous (s.c), intramuscular (i.m.) injections or infusions, or as aerosol formulations suitable for intranasal or intrapulmonary delivery (for mtrapulmonary delivery see, e.g., EP 257, 956) .
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl- methacrylate) as described by Langer et al . , J. Biomed. Mater. Res., L5: 167-277 (1981) and Langer, Chem. Tech., L2: 98-105 (1982) or poly(vinylalcohol) ) , polylactides (U.S. Patent No.
  • Diagnosis in mammals of the various pathological conditions described herein can be made by the skilled practitioner. Diagnostic techniques are available in the art which allow, e.g., for the diagnosis or detection of cancer in a mammal. For instance, cancers may be identified through techniques, including but not limited to, palpation, blood analysis, x-ray, NMR and the like. Cancer staging systems describe how far the cancer has spread anatomically and attempt to put patients with similar prognosis and treatment in the same staging group. Several tests may be performed to help stage cancer including biopsy and certain imaging tests such as a chest x-ray, mammogram, bone scan, CT scan, and MRI scan. Blood tests and a clinical evaluation are also used to evaluate a patient's overall health and detect whether the cancer has spread to certain organs .
  • TNM classification system To stage cancer, the American Joint Committee on Cancer first places the cancer, particularly solid tumors, in a letter category using the TNM classification system. Cancers are designated the letter T (tumor size) , N (palpable nodes) , and/or M (metastases) . Tl, T2, T3, and T4 describe the increasing size of the primary lesion; NO, Nl, N2, N3 indicates progressively advancing node involvement; and MO and Ml reflect the absence or presence of distant metastases.
  • stage 0 is referred to as "in situ” or “Tis,” such as ductal carcinoma in situ or lobular carcinoma in situ for breast cancers.
  • stage I cancers are small localized cancers that are usually curable, while stage IV usually represents inoperable or metastatic cancer.
  • Stage II and III cancers are usually locally advanced and/or exhibit involvement of local lymph nodes. In general, the higher stage numbers indicate more extensive disease, including greater tumor size and/or spread of the cancer to nearby lymph nodes and/or organs adjacent to the primary tumor. These stages are defined precisely, but the definition is different for each kind of cancer and is known to the skilled artisan. Many cancer registries, such as the NCI's Surveillance, Epidemiology, and End Results Program (SEER), use summary staging. This system is used for all types of cancer. It groups cancer cases into five main categories: In situ is early cancer that is present only in the layer of cells in which it began.
  • SEER End Results Program
  • Regional is cancer that has spread beyond the original (primary) site to nearby lymph nodes or organs and tissues.
  • Distant is cancer that has spread from the primary site to distant organs or distant lymph nodes.
  • recurrent disease Cancer that recurs after all visible tumor has been eradicated, is called recurrent disease. Disease that recurs in the area of the primary tumor is locally recurrent, and disease that recurs as metastases is referred to as a distant recurrence.
  • the tumor can be a solid tumor or a non-solid or soft tissue tumor.
  • soft tissue tumors include leukemia (e.g., chronic myelogenous leukemia, acute myelogenous leukemia, adult acute lymphoblastic leukemia, acute myelogenous leukemia, mature B- cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, polymphocytic leukemia, or hairy cell leukemia) or lymphoma (e.g., non-Hodgkin' s lymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease) .
  • a solid tumor includes any cancer of body tissues other than blood, bone marrow, or the lymphatic system.
  • Solid tumors can be further divided into those of epithelial cell origin and those of non-epithelial cell origin.
  • epithelial cell solid tumors include tumors of the gastrointestinal tract, colon, breast, prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gall bladder, labium, nasopharynx, skin, uterus, male genital organ, urinary organs, bladder, and skin.
  • Solid tumors of non-epithelial origin include sarcomas, brain tumors, and bone tumors.
  • the Apo2L/TRAIL, chemotherapy, and/or VEGF antibodies or CD20 antibodies can be administered in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • administration may be performed through mini-pump infusion using various commercially available devices .
  • the one or more other therapies may include but are not limited to, administration of radiation therapy, cytokine (s) , growth inhibitory agent (s), chemotherapeutic agent (s) , cytotoxic agent (s), tyrosine kinase inhibitors, ras farnesyl transferase inhibitors, angiogenesis inhibitors, and cyclin-dependent kinase inhibitors which are known in the art and defined further with particularity in Section I above.
  • Exemplary therapeutic antibodies include anti-HER2 antibodies including rhuMAb 4D5 (HERCEPTIN ) (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285-4289 (1992), U.S. Patent No. 5,725,856) ; anti-IL-8 (St John et al . , Chest, 103:932 (1993), and International Publication No. WO 95/23865); anti-PSCA antibodies (WO01/40309) ; anti-CD40 antibodies, including S2C6 and humanized variants thereof (WO00/75348) ; anti-CDlla antibodies including RaptivaTM (US Patent No.
  • anti-human ⁇ 4 - ⁇ 7 integrin antibodies WO 98/06248 published February 19, 1998
  • anti-EGFR antibodies chimerized or humanized 225 antibody as in WO 96/40210 published December 19, 1996
  • anti-CD3 antibodies such as OKT3 (US Patent No. 4,515,893 issued May 7, 1985)
  • anti-CD25 or anti-Tac antibodies such as CHI-621 (SIMULECT ) and ZENAPAX (See US Patent No. 5,693,762 issued December 2, 1997)
  • anti-CD4 antibodies such as the cM-7412 antibody (Choy et al .
  • anti-CD52 antibodies such as CAMPATH-IH (Riechmann et al . Nature 332:323-337 (1988); anti-Fc receptor antibodies such as the M22 antibody directed against Fc RI as in Graziano et al. J. Immunol. 155 (10) : 4996-5002 (1995) ; anti-carcinoembryonic antigen (CEA) antibodies such as hMN-14 (Sharkey et al . Cancer Res.
  • CEA anti-carcinoembryonic antigen
  • anti-CD33 antibodies such as Hu M195 (Jurcic et al. Cancer Res 55(23 Suppl) : 5908s-5910s (1995) and CMA-676 or CDP771; anti-CD22 antibodies such as LL2 or LymphoCide (Juweid et al.
  • anti- EpCAM antibodies such as 17-1A (PANOREX ) ; anti-GpIIb/IIIa antibodies such as abciximab or c7E3 Fab (REOPRO ) ; anti-RSV antibodies such as MEDI-493 (SYNAGIS.); anti-CMV antibodies such as PROTOVIR ; anti-HIV antibodies such as PRO542; anti-hepatitis antibodies such as the anti-Hep B antibody OSTAVIR ; anti-CA 125 antibody OvaRex; anti-idiotypic GD3 epitope antibody BEC2; anti-.v.3 antibody VITAXIN ; anti-human renal cell carcinoma antibody such as ch-G250; ING-I; anti-human 17-1A antibody (3622W94) ; anti-human colorectal tumor antibody (A33) ; anti-human melanoma antibody R24 directed against GD3 ganglioside; anti
  • chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation for such chemotherapy are also described in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, MD (1992) .
  • the article of manufacture comprises (a) a container comprising VEGF antibody (preferably the container comprises the antibody and a pharmaceutically acceptable carrier or diluent within the container) ; (b) a container comprising Apo2L/TRAIL (preferably the container comprises the Apo2L/TRAIL and a pharmaceutically acceptable carrier or diluent within the container) ; and (c) a package insert with instructions for treating cancer, wherein the instructions provide information such as that recited in the attached drawing sheets.
  • the package insert comprises information concerning administration, side effects, and/or advisory warnings, etc. set forth by the applicable regulatory agency, such as the FDA.
  • the article of manufacture comprises (a) a container comprising CD20 antibody (preferably the container comprises the antibody and a pharmaceutically acceptable carrier or diluent within the container) ; (b) a container comprising Apo2L/TRAIL (preferably the container comprises the Apo2L/TRAIL and a pharmaceutically acceptable carrier or diluent within the container) ; and (c) a package insert with instructions for treating cancer, wherein the instructions provide information such as that recited in the attached Examples and Figures.
  • the package insert comprises information concerning administration, side effects, and/or advisory warnings, etc. set forth by the applicable regulatory agency, such as the FDA.
  • the package insert is on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds or contains a composition that is effective for treating the cancer and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) .
  • the article of manufacture may further comprise an additional container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and/or dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and/or dextrose solution.
  • NSCLC Cancer
  • Mixed tumors will be categorized by the predominant cell type unless small cell elements are present in which case the patient is ineligible. Cytologic or histologic elements can be established on metastatic tumor aspirates or biopsy.
  • stage IHb with malignant pleural effusion or Stage IV or recurrent disease.
  • Subjects with unmeasurable but evaluable disease can be included in the phase Ib study, but disease must be measurable to be included in the phase 2 study.
  • CNS metastases -Untreated or unstable central nervous system (CNS) metastases.
  • Subjects with treated and stably controlled CNS metastases are eligible for cohorts A and B of the phase 2 part of the study if definitive therapy has been administered (surgery and/or radiation therapy) , there is no planned treatment for brain metastases, and the subject is clinically stable and is off corticosteroids or on a stable dose of corticosteroids for at least 2 weeks prior to enrollment.
  • -Myocardial infarction, or unstable or uncontrolled disease or condition related to or impacting cardiac function e.g., unstable angina, congestive heart failure [New York Heart Association > class II]
  • systolic blood pressure > 150 mm Hg
  • diastolic blood pressure > 100 mm Hg (antihypertensive therapy to achieve these parameters is allowable) .
  • HIV infection -Known (documented in medical notes) HIV infection. -Active infection on day of enrollment.
  • rhApo2L/TRAIL recombinant human Apo2L/TRAIL
  • rhApo2L/TRAIL recombinant human Apo2L/TRAIL
  • SEQ ID NO:1 amino acids 114-281 of Figure 1 (SEQ ID NO:1)
  • the selected chemotherapies Paclitaxel (Taxol®) and Carboplatin (Paraplatin®) were purchased from publicly available, commercial sources.
  • Anti- VEGF antibody, Bevacizumab (Avastin®) was purchased from Genentech, Inc.
  • Paclitaxel is referred to as "P”
  • Carboplatin Carboplatin
  • Bevacizumab Bevacizumab
  • PCB the combination use of these three agents
  • the clinical trial design was as follows:
  • -Primary Endpoints Incidence of dose-limiting toxicities (DLT); incidence and severity of adverse events.
  • PK parameters including area under the drug concentration-time curve (AUC) , maximum concentration (Cmax) , half life (tl/2), clearance, volume of distribution (Vd) .
  • stage IHb with pleural effusion stage IV or recurrent
  • rhA ⁇ o2L/TRAIL treatment appeared to have no effect on the pharmacokinetics of P, C, or B.
  • rhApo2L/TRAIL (at the target doses) plus PCB appeared to be well tolerated and have anti-tumor activity in patients with NSCLC.
  • rhA ⁇ o2L/TRAIL PK values (Cmax, AUC, tl/2) were similar to the FIH study.
  • follicular NHL are eligible for the study if they received this therapy at least 1 year prior to Day 1, they have adequate bone marrow function, and they have no evidence of myelodysplastic syndrome on bone marrow aspirate/biopsy -Prior treatment with Apo2L/TRAIL or an agonist antibody to DR4 or DR5
  • hepatitis B surface antigen sAg
  • hepatitis B IgG or IgM core antibody hepatitis B IgG or IgM core antibody
  • hepatitis C antibody hepatitis C antibody
  • rhApo2L/TRAIL recombinant human Apo2L/TRAIL
  • Figure 1 ammo acids 114-281 of Figure 1 (SEQ ID N0:l)
  • Anti-CD20 antibody, Rituximab was purchased from Genentech, Inc.
  • Figure 15A and Figure 15B A schematic of the clinical trial design and schedule of study drug dosing and assessment are presented in Figure 15A and Figure 15B, respectively, and study design details are listed below:

Abstract

La présente invention concerne des procédés permettant de traiter le carcinome pulmonaire non à petites cellules (NSCLC) et le lymphome non-hodgkinien (NHL). Les procédés consistent à traiter les cellules cancéreuses avec une quantité efficace d’Apo2L/TRAIL ainsi que des régimes chimiothérapeutiques et des antibiothérapies choisis. L’invention concerne également des articles manufacturés et des kits comprenant Apo2L/TRAIL et des notices de boîte qui donnent par exemple des informations sur l’administration et la posologie pour le traitement des cancers tels que le NSCLS et le NHL.
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WO2011080314A3 (fr) * 2009-12-31 2011-12-15 Deutsches Krebsforschungszentrum Nouveaux modulateurs de signalisation par trail
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WO2011161260A1 (fr) 2010-06-25 2011-12-29 Adamed Sp. Z O.O. Protéine de fusion anticancéreuse
WO2012072815A1 (fr) 2010-12-03 2012-06-07 Adamed Sp. Z O.O. Protéine de fusion anticancéreuse
US9175059B2 (en) 2011-01-05 2015-11-03 Adamed Sp. Z O.O. Anticancer fusion protein comprising trail and a growth factor receptor inhibitor
WO2012093158A1 (fr) 2011-01-05 2012-07-12 Adamed Sp. Z O.O. Protéine de fusion anticancéreuse
EA025830B1 (ru) * 2011-01-05 2017-02-28 Адамед Сп. З О.О. Слитый белок против злокачественной опухоли
WO2012143477A2 (fr) 2011-04-19 2012-10-26 Adamed Sp. Z O.O. Protéine hybride anticancéreuse
WO2012151317A1 (fr) * 2011-05-03 2012-11-08 Genentech, Inc. Agents d'interruption vasculaire et utilisations associées
JP2014513128A (ja) * 2011-05-03 2014-05-29 ジェネンテック， インコーポレイテッド 血管破壊剤とその使用
CN102329387A (zh) * 2011-09-23 2012-01-25 昆明理工大学 一种抗病毒蛋白trail114-281及其制备方法
WO2013080147A2 (fr) 2011-11-28 2013-06-06 Adamed Sp. Z O.O. Protéine de fusion anticancer
WO2013098755A2 (fr) 2011-12-28 2013-07-04 Adamed Sp. Z O.O. Protéine hybride anticancéreuse
US11299528B2 (en) 2014-03-11 2022-04-12 D&D Pharmatech Inc. Long acting TRAIL receptor agonists for treatment of autoimmune diseases
US10328056B2 (en) * 2014-07-29 2019-06-25 Alliance of Cardiovascular Researches Priming of pancreatic tumor cells and cancer stem cells to TRAIL-induced apoptosis
WO2016019062A1 (fr) * 2014-07-29 2016-02-04 Alliance Of Cardiovascular Researchers Amorçage de cellules tumorales pancréatiques et de cellules souches cancéreuses pour apoptose induite par trail
WO2016029043A1 (fr) 2014-08-21 2016-02-25 The General Hospital Corporation Mutéines de ligands de la superfamille du facteur de nécrose tumorale (tnfsf) et de ligands de type tnf et leurs procédés de préparation et d'utilisation
US11007251B2 (en) 2015-12-17 2021-05-18 The Johns Hopkins University Ameliorating systemic sclerosis with death receptor agonists
US11084879B2 (en) 2016-04-07 2021-08-10 The Johns Hopkins University Compositions and methods for treating pancreatitis and pain with death receptor agonists
EP3323428A1 (fr) 2016-11-17 2018-05-23 CNRS Centre National de la Recherche Scientifique Inhibiteurs sélectifs de c-flip en tant qu'agents anticancéreux
WO2018091647A1 (fr) 2016-11-17 2018-05-24 Centre National De La Recherche Scientifique - Cnrs - Inhibiteurs sélectifs de c-flip en tant qu'agents anticancéreux
CN109069584A (zh) * 2017-06-29 2018-12-21 成都华创生物技术有限公司 一种trail类蛋白持续抑制肿瘤细胞生长的给药方法
WO2019000327A1 (fr) * 2017-06-29 2019-01-03 成都华创生物技术有限公司 Procédé d'administration de protéine trail de façon à inhiber en continu la croissance de cellules tumorales
EP4257132A1 (fr) 2022-04-08 2023-10-11 iOmx Therapeutics AG Inhibiteurs de sik3 pour le traitement de maladies résistantes à la signalisation du récepteur de la mort
WO2023194544A1 (fr) 2022-04-08 2023-10-12 Iomx Therapeutics Ag Inhibiteurs de sik3 pour le traitement de maladies résistantes à la signalisation du récepteur de mort

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