[go: up one dir, main page]

WO2016207324A1 - Biomarqueurs pour une résistance et une sensibilité à la nad(+)-diphtamide adp-ribosyl-transférase - Google Patents

Biomarqueurs pour une résistance et une sensibilité à la nad(+)-diphtamide adp-ribosyl-transférase Download PDF

Info

Publication number
WO2016207324A1
WO2016207324A1 PCT/EP2016/064629 EP2016064629W WO2016207324A1 WO 2016207324 A1 WO2016207324 A1 WO 2016207324A1 EP 2016064629 W EP2016064629 W EP 2016064629W WO 2016207324 A1 WO2016207324 A1 WO 2016207324A1
Authority
WO
WIPO (PCT)
Prior art keywords
diphthamide
eef2
seq
residue
patient
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2016/064629
Other languages
English (en)
Inventor
Ulrich Brinkmann
Michael Gerg
Sebastian Stahl
Gerhard Niederfellner
Axel Ducret
Ira H. Pastan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
F Hoffmann La Roche AG
Hoffmann La Roche Inc
US Department of Health and Human Services
Original Assignee
F Hoffmann La Roche AG
Hoffmann La Roche Inc
US Department of Health and Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by F Hoffmann La Roche AG, Hoffmann La Roche Inc, US Department of Health and Human Services filed Critical F Hoffmann La Roche AG
Publication of WO2016207324A1 publication Critical patent/WO2016207324A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6815—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/02036—NAD(+)--diphthamide ADP-ribosyltransferase (2.4.2.36)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91091—Glycosyltransferases (2.4)
    • G01N2333/91142—Pentosyltransferases (2.4.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to methods of assessing resistance to treatment with NAD (+) -diphthamide ADP ribosyltransferases, for example Pseudomonas exotoxin A (PE), and to related methods of treatment and medical uses.
  • NAD (+) -diphthamide ADP ribosyltransferases for example Pseudomonas exotoxin A (PE)
  • PE Pseudomonas exotoxin A
  • Eukaryotic translation elongation factor 2 (eEF2) is a highly conserved protein and essential for protein biosynthesis. eEF2 enables peptide-chain elongation by translocating the peptide-tRNA complex from the A- to the P- site of the ribosome in a GTP-dependent manner [1_, 2] .
  • a modification at His715 is conserved in all eukaryotes [3], as well as in its archaeal counterpart. This diphthamide modification is generated by the concerted action of proteins that are encoded by 7 genes [4] . Proteins encoded by DPH1, DPH2, DPH3 and DPH4 attach a 3-amino-3-carboxypropyl (ACP) group to His715 of eEF2.
  • ACP 3-amino-3-carboxypropyl
  • This intermediate is subsequently converted to diphthine by the methyitransferase DPH5.
  • DPH5 methyitransferase
  • Amidation of diphthine to diphthamide requires DPH6 and DPH7 [5] .
  • the diphthamide biosynthetic pathway is shown in Fig. 5.
  • Diphthamide modified eEF2 is the target of ADP-ribosylating toxins including Pseudomonas exotoxin A (PE), Diphtheria toxin (DT) [15] and cholix toxin from Vibrio cholerae (J0rgensen et al . 2008 J Biol Chem
  • ribosyltransferase enzymes (EC 2.4.2.36) .
  • cholix toxin is distinct from cholera toxin, which ADP-ribosylates an arginine residue of the GTP- binding protein G s .
  • Tumor-targeted truncated PE and DT derivatives are applied in cancer therapies [ 18-24 ] , and their therapeutic efficacy depends to a large degree on sensitivity of target cells to these ADP-ribosylating toxins. Therefore, information about the factors (and their relative contributions) that influences cellular sensitivities towards diphthamide-modi tying toxins may be applicable to predict therapy responses. For example, alterations in OVCA1 (i.e. the human DPH1 gene) have been described for many ovarian cancer samples [1_2, 25] , yet it is still not known if and to what degree such alterations would affect sensi ivities of tumor cells towards PE- derived drugs.
  • OVCA1 i.e. the human DPH1 gene
  • the inventors have surprisingly found that cells containing eEF2 that lacks the diphthamide modification at the His715 residue remain sensitive to killing by PE when the cells also contain eEF2 with the diphthamide modification at the His715 residue. That is, although eEF2 lacking the diphthamide modification is not inactivated by PE, its presence is insufficient to bestow resistance to PE .
  • the inventors propose that (in addition to direct inactivation of eEF2 in translation elongation) ADP-ribosylation of the diphthamide group on the His715 residue of eEF2 may trigger an additional mechanism that interrupts protein synthesis even though unmodified, translation-competent eEF2 remains available.
  • the inventors propose to assess sensitivity or resistance to PE by assaying for the presence or absence of eEF2 protein with the diphthamide modification at the His715 residue.
  • the invention provides a method for assessing sensitivity and/or resistance of diseased cells in a patient to treatment with a NAD (+) -diphthamide ADP-ribosyltransferase, the method comprising assaying for the presence of eEF2 protein having diphthamide modification at the His715 residue in a sample containing the diseased cells, wherein the presence of eEF2 protein having diphthamide modification at the His715 residue is indicative that the diseased cells are sensitive to treatment with a MAD (+) -diphthamide ADP-ribosyltrans ferase and/or wherein the absence of eEF2 protein having diphthamide modification at the His715 residue is indicative that the diseased cells are resistant to treatment with a NAD (+) -diphththamide
  • ribosyltrans ferase coupled to a cell-binding agent targeted to diseased cells of the patient if the diseased cells are assessed to be sensitive to treatment with MAD (+) -diphthamide ADP ribosyltransferase .
  • the method may include a step of deselecting the patient for treatment with a NAD (+) -diphthamide ADP ribosyltransferase if the diseased cells are assessed to be resistant to NAD ( + ⁇ ) -diphthamide ADP ribosyltrans ferase .
  • the invention provides a method for selecting and/or deselecting a patient for treatment with a targeted therapeutic agent comprising a NAD (+) -diphthamide ADP ribosyltransferase coupled to a cell-binding agent targeted to diseased cells of the patient, the method comprising : (i) assaying for the presence of eEF2 protein having diphthamide modification at the His715 residue, in a sample containing diseased cells from the patient; and
  • the patient may be treated with the targeted therapeutic agent .
  • the invention provides a method for treating a patient having a condition that is treatable by cytotoxic activity targeted to diseased cells of the patient, the method comprising: assaying a sample containing diseased cells from a patient for the presence of eEF2 protein having diphthamide modification at the His715 residue; and treating a patient in whose sample the assay is positive for the presence of eEF2 protein having diphthamide modification at the His715 residue with a targeted therapeutic agent comprising a NAD (+) -diphthamide ADP ribosyltransferase coupled to a cell-binding agent targeted to diseased cells of the patient.
  • the invention provides a method for treating a patient having a condition that is treatable by cytotoxic activity targeted to diseased cells of the patient, the method comprising: assaying for the presence of eEF2 protein having diphthamide modification at the His715 residue in a sample containing diseased cells from the patient; assessing sensitivity or resistance of the diseased cells to treatment with a NAD (+) -diphthamide ADP-ribosyltransferase, wherein the presence of eEF2 protein having diphthamide modification at the His715 residue is indicative that the diseased cells are sensitive to treatment with a NAD (+) -diphthamide ADP-ribosyltransferase and/or wherein the absence of eEF2 protein having diphthamide modification at the His715 residue is indicative that the diseased cells are resistant to treatment with a NAD (+) -diphthamide ADP-ribosyltransferase; and treating a patient whose diseased cells are assessed to be sensitive
  • the invention provides a method for treating a patient having a condition that is treatable by cytotoxic activity targeted to diseased cells of the patient, the method comprising: treating the patient with a targeted therapeutic agent comprising a
  • NAD (+) -diphthamide ADP ribosyltransferase coupled to a cell-binding agent targeted to diseased cells of the patient, wherein the patient is selected for treatment with the targeted therapeutic agent on the basis of a positive assay result for the presence of eEF2 protein having diphthamide modification at the His715 residue in a sample containing diseased cells from the patient.
  • the invention provides a NAD (+) -diphthamide ADP ribosyltransferase for use in a method of medical treatment of a patient from whom a sample containing diseased cells has given a positive result in an assay for the presence of eEF2 protein having diphthamide modification at the His715 residue.
  • the invention also provides a NAD ( + ) -diphthamide ADP ribosyltransferase for use in a method of medical treatment of a patient from whom, a sample containing diseased cells has been assayed for the presence of eEF2 protein having diphthamide modification at the His715 residue and assessed as sensitive to NAD (+) -diphthamide ADP ribosyltransferase treatment.
  • the invention also provides a NAD (+) -diphthamide ADP ribosyltrans ferase for use in any of the methods of treatment otherwise described herein.
  • ribosyltransferase is preferably coupled to a cell-binding agent targeted to diseased cells of the patient, as a targeted therapeutic agent.
  • NAD (+) -diphthamide ADP ribosyltransferase-related aspects and embodiments of the invention a NAD (+) -diphthamide ADP ribosyltransferase that is administered to (or that is for administration to) a patient will be coupled to a cell-binding agent targeted against diseased cells of the patient as a targeted therapeutic agent.
  • the NAD (+) -diphthamide ADP ribosyltransferase is preferably coupled to the cell-binding agent as a fusion polypeptide, either directly or indirectly via a linker. In preferred embodiments, the fusion is direct. Coupling may also be by chemical conjugation.
  • a preferred cell-binding agent is an antibody, in particular an antibody directed against a tumour- or cancer-specific antigen.
  • the NAD (+) -diphthamide ADP ribosyltransferase is preferably a PE toxin, diphtheria toxin or cholix toxin, more preferably a PE toxin or diphtheria toxin, still more preferably a PE toxin. Further preferred forms of PE toxin are described below. These preferences apply independently to the NAD (+) -diphthamide ADP ribosyltransferase that is administered (or that is for administration) to the patient and to the methods of assessing sensitivity and/or resistance to treatment with a
  • a method of the invention may involve determining that diseased cells of a patient are sensitive to treatment with NAD (+) -diphthamide ADP ribosyltransferases generally, and administering a preferred NAD (+) -diphthamide ADP
  • ribosyltransferase such as a PE toxin.
  • the patient is preferably a patient having a condition that is treatable by cytotoxic activity targeted to diseased cells of the patient.
  • the condition is preferably a cancer or tumour.
  • the invention is not limited to the treatment of cancer and tumour.
  • Other conditions may also be treatable by cytotoxic activity targeted to diseased cells of the patient, including viral infections such as HIV, rabies, EBV and Kaposi's sarcoma-associated herpesvirus, and autoimmune diseases such as multiple sclerosis and graft-versus-host disease drugs.
  • the assay may exclude any direct assay for the presence or absence of eEF2 that lacks diphthamide modification at the His715 residue, since the inventors propose that the presence of eEF2 lacking the diphthamide modification is insufficient to confer resistance to PE and other NAD (+) -diphthamide ADP ribosyltransferases .
  • the inventors have also surprisingly found that cells in which a
  • eEF2 lacks the diphthamide modification at the His715 residue show increased sensitivity to TNFa-mediated apoptosis compared to comparable cells in which substantially all the eEF2 has the diphthamide modification.
  • the inventors also propose that comparable results will also be obtained with other direct or indirect inducers of NFkappaB-signaling pathways or related signaling pathways (hereafter, "other inducer”) .
  • the invention provides a method for assessing increased sensitivity of diseased cells in a patient to treatment with TNFa or other inducer, the method comprising assaying for the proportion of eEF2 protein that lacks diphthamide modification at the His715 residue in a sample containing the diseased cells, wherein the presence of a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue is indicative that the diseased cells have increased sensitivity to treatment with TNFa or other inducer compared to cells in which eEF2 protein lacking diphthamide modification is substantially absent.
  • the method may include a step of selecting the patient for treatment with TNFa or other inducer if the diseased cells are assessed to have increased sensitivity to treatment with TNFa or other inducer. Additionally or alternatively, the method may include a step of deselecting the patient for treatment with TNFcx or other inducer if the diseased cells are assessed, not to have increased sensitivity to TNFa or other inducer.
  • the invention provides a method for selecting and/or deselecting a patient for treatment with TNFa or other inducer, the method comprising:
  • diphthamide modification at the His715 residue in a sample containing diseased cells from the patient; and (ii) (a) selecting the patient for treatment with TNFa or other inducer if the assay is positive for the presence of a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue; and/or
  • the invention provides a method for treating a patient having a condition that is treatable by TNFa or other inducer, the method comprising: assaying a sample containing diseased cells from a patient for the proportion of eEF2 protein that lacks diphthamide modification at the His715 residue; and treating a patient in whose sample the assay is positive for the presence of a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue with TNFa or other inducer.
  • the invention provides a method for treating a patient having a condition that is treatable by TNFa or other inducer, the method
  • eEF2 protein lacking diphthamide modification at the His715 residue comprising : assaying for the presence of a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue in a sample containing diseased cells from the patient; assessing whether the diseased cells have increased sensitivity to treatment with TNFa compared to cells in which eEF2 protein lacking diphthamide modification is substantially absent, wherein the presence of a significant portion of eEF2 protein lacking diphthamide modification at the His715 residue is indicative that the diseased cells have increased sensitivity to treatment with TNFa or other inducer and/or wherein the absence of a significant portion of eEF2 protein lacking diphthamide modification at the His715 residue is indicative that the diseased cells do not have increased sensitivity to treatment with TNFa or other inducer; and treating a patient whose diseased cells are assessed to have increased sensitivity with TNFa or other inducer.
  • the invention provides a method for treating a patient having a condition that is treatable with TNFa or other inducer, the method comprising : treating the patient with TNFa or other inducer, wherein the patient is selected for treatment with TNFa or other inducer on the basis of a positive assay result for the presence of a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue in a sample containing diseased cells from the patient.
  • the invention provides TNFa or other inducer for use in a method of medical treatment of a patient from whom a sample containing diseased cells has given a positive result in an assay for the presence of a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue.
  • the invention also provides TNFa or other inducer for use in a method of medical treatment of a patient from whom a sample containing diseased cells has been assayed for the presence of a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue and assessed as having increased sensitivity to TNFa or other inducer compared to cells in which eEF2 protein lacking diphthamide modification is substantially absent .
  • the invention also provides TNFa or other inducer for use in any of the methods of treatment otherwise described herein.
  • the steps of selecting and/or deselecting patients for treatment with TNFa or other inducer may be steps of selecting and/or deselecting patients for treatment with TNFa or other inducer in preference to other treatment options. That is, the presence of a significant proportion of eEF2 lacking diphthamide modification at the His715 residue may result in the selection of the patient for treatment with TNFa or other inducer in preference to other treatment options that are available for the disease in question; similarly, the absence of a significant proportion of eEF2 lacking diphthamide modification at the
  • His715 residue may result in the deselection of the patient for treatment with TNFa or other inducer in preference to other treatment options that are available for the disease in question.
  • the presence of a significant proportion of eEF2 lacking diphthamide modification at the His715 residue may result in the selection of the patient for treatment with TNFa or other inducer as a first-line treatment, when TNFa or other inducer would not normally be considered as a first-line treatment for the disease in question.
  • the absence of a significant proportion of eEF2 lacking diphthamide modification at the His715 residue may result in the deselection of the patient for treatment with TNFa or other inducer as a first-line treatment, when TNFa or other inducer would normally be considered as a first-line treatment for the disease in question.
  • eEF2 protein lacking diphthamide modification at the His715 residue encompasses eEF2 protein that has no modification, 3-amino-3- carboxypropyl (ACP) modification, or diphthine modification at the His715 residue, and preferably refers to eEF2 protein that has no modification or ACP-modification at the His715 residue.
  • ACP 3-amino-3- carboxypropyl
  • a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue preferably refers to the situation where at least about 50% of the eEF2 in the sample lacks diphthamide modification, more preferably at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%. Most preferably, it refers to the situation where eEF2 protein having diphthamide modification at the His715 residue is undetectable in the sample .
  • eEF2 protein lacking diphthamide modification is substantially absent preferably refers to the situation where no more than about 10% of the eEF2 in the sample lacks diphthamide modification at the His715 residue, more preferably no more than about 5%, no more than about 2%, no more than about 1%, no more than about 0.5% or no more than about 0.1%. Most preferably, it refers to the situation where eEF2 protein lacking diphthamide modification at the His715 residue is undetectable in the sample .
  • the assaying may be direct, in the sense that the read-out of the assay directly reflects the presence, absence and/or amount of that form of eEF2 in the sample.
  • a step of assaying for the presence of eEF2 having diphthamide modification at the His715 residue may be carried out by mass spectrometry, which is capable of discriminating between the different forms of modification (no
  • the assaying may be indirect, in the sense that the presence, absence and/or amount of that form of eEF2 in the sample is deduced from the amount (s) of one or more other forms of eEF2 in the sample.
  • the absence of eEF2 having diphthamide modification at the His715 residue may be inferred if the amount of eEF2 that is unmodified at the His715 residue matches the total amount of eEF2 in the sample.
  • the assays used in the NAD (+) -diphthamide ADP ribosyltransferase-related aspects of the invention preferably employ antibodies that selectively bind to eEF2 having diphthamide modification at the His715 residue, relative to eEF2 that is unmodified at this residue and preferably also relative to eEF2 that has 3-amino-3-carboxypropyl (AC?) modification and/or diphthine modification at thisresidue.
  • antibodies of the invention as defined below.
  • the assays used in the TNFa-related aspects of the invention preferably employ antibodies that selectively bind to eEF2 that is unmodified at the His715 residue, relative to eEF2 having diphthamide modification at this residue and preferably also relative to eEF2 that has 3-amino-3- carboxypropyl (ACP) modification and/or diphthine modification at this residue.
  • ACP 3-amino-3- carboxypropyl
  • the term preferably refers to cells that are otherwise comparable to the diseased cells in the patient sample. That is, the term preferably refers to cells displaying similar signs of disease to the diseased cells in the patient sample (for example, cells taken from the same kind of cancer, or infected with the same virus) .
  • Anti-eEF2 antibodies In the work underlying the present invention, the inventors have produced a monoclonal antibody (referred to in the examples as MGb) that specifically binds to eEF2 that is unmodified at the His715 residue and does not bind to eEF2 with the diphthamide modification at the His715 residue. That is, the MGb antibody is fully selective for eEF2 that is unmodified at the His715 residue compared to diphthamide-modified eEF2. Two other antibodies ( Ga and MGd) show preferential but not fully selective binding.
  • MGb monoclonal antibody
  • the invention provides a monoclonal anti- eEF2 antibody, wherein the antibody binds to eEF2 that is unmodified at the His715 residue with higher binding affinity than to eEF2 having diphthamide modification at the His715 residue.
  • the higher binding affinity is preferably at least 10-fold higher, more preferably at least 100-fold higher, more preferably at least 1000-fold higher. More preferably, the antibody substantially does not bind to eEF2 having diphthamide
  • the antibody binds to eEF2 that is unmodified at the His715 residue with a D of 100 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, 10 pM or less, or 1 pM or less.
  • the antibody of the invention may bind to eEF2 that is unmodified at the His715 residue also with higher binding affinity than to eEF2 having 3-amino-3-carboxypropyl (ACP) modification at the His715 residue.
  • ACP is an intermediate modification in the biosynthetic pathway for the diphthamide modification.
  • the higher binding affinity is preferably at least 10-fold higher, more preferably at least 100-fold higher, more preferably at least 1000-fold higher. More preferably, the antibody substantially does not bind to eEF2 having ACP modification at the His715 residue.
  • the antibody of the invention may bind to eEF2 that is unmodified at the His715 residue also with higher binding affinity than to eEF2 having diphthine modification at the His715 residue.
  • the higher binding affinity is preferably at least 10-fold higher, more preferably at least 100-fold higher, more preferably at least 1000-fold higher. More preferably, the antibody substantially does not bind to eEF2 having ACP modification at the His715 residue.
  • the anti-eEF2 antibody of the invention substantially does not bind to eEF2 having either diphthamide or ACP modification at the His715 residue.
  • the antibody may have the heavy chain variable domain sequence of SEQ ID NO: 102, or a heavy chain variable domain sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO:102.
  • the antibody may have the light chain variable domain sequence of SEQ ID NO: 103, or a light chain variable domain sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 103.
  • the antibody may have at least the heavy chain complementarity-determining region (CDR) sequence H3 of the heavy chain variable domain sequence shown in Figure 6 (SEQ ID NO: 106) .
  • the antibody has the heavy chain CDRs H2 and H3 of the heavy chain variable domain sequence shown in Figure 6 (SEQ ID NOs:105 and 106) .
  • the antibody has the heavy chain CDRs HI, H2 and H3 of the heavy chain variable domain sequence shown in Figure 6 (SEQ ID NOs:104, 105 and 106) .
  • the antibody may have at least the light chain, complementarity-determining region (CDR) sequence L3 of the light chain variable domain sequence shown in Figure 6 (SEQ ID NO: 109) .
  • the antibody has the light chain CDRs L2 and L3 of the light chain variable domain sequence shown in Figure 6 (SEQ ID N0s:108 and 109) . More preferably the antibody has the light chain CDRs LI, L2 and L3 of the light chain variable domain sequence shown in Figure 6 (SEQ ID NOs:107, 108 and 109) .
  • the antibody may have the CDR-Hl sequence of the heavy chain variable domain sequence shown in Figure 6 (SEQ ID NO: 104), or said CDR-Hl sequence with one or more amino acid insertions, deletions and/or substitutions.
  • the antibody may have the CDR- H2 sequence of the heavy chain variable domain sequence shown in Figure 6 (SEQ ID NO:105), or said CDR-H2 sequence with one or more amino acid insertions, deletions and/or substitutions.
  • the antibody may have the CDR- H3 sequence of the heavy chain variable domain sequence shown in Figure 6 (SEQ ID NO: 106), or said CDR-H3 sequence with one or more amino acid insertions, deletions and/or substitutions.
  • the antibody may have the CDR- Ll sequence of the heavy chain variable domain sequence shown in Figure 6 (SEQ ID NO: 107), or said CDR-L1 sequence with one or more amino acid insertions, deletions and/or substitutions.
  • the antibody may have the CDR- L2 sequence of the heavy chain variable domain sequence shown in Figure 6 (SEQ ID NO:108), or said CDR-L2 sequence with one or more amino acid insertions, deletions and/or substitutions.
  • the antibody may have the CDR- L3 sequence of the heavy chain variable domain sequence shown in Figure 6 (SEQ ID NO: 109), or said CDR-L3 sequence with one or more amino acid insertions, deletions and/or substitutions.
  • the invention further provides a monoclonal antibody comprising the CDR Hl- H3 sequences shown in SEQ ID NOs:104 to 106 and the CDR L1-L3 sequences shown in SEQ ID NOs:107 to 109.
  • the invention further provides a monoclonal antibody comprising the heavy chain variable domain sequence shown in SEQ ID NO: 102 and the light chain variable domain sequence shown in SEQ ID NO: 103.
  • the inventors are also developing a monoclonal anti-eEF2 antibody that binds to eEF2 having the diphthamide modification at the His715 residue in preference to eEF2 that is unmodified at the His715 residue.
  • the invention provides a monoclonal anti- eEF2 antibody that binds to eEF2 having diphthamide modification at the His715 residue with higher binding affinity than to eEF2 that is unmodified at the His715 residue.
  • the higher binding affinity is preferably at least 10-fold higher, more preferably at least 100-fold higher, more preferably at least 1000-fold higher. More preferably, the antibody substantially does not bind to eEF2 that is unmodified at the His715 residue.
  • the antibody may bind to eEF2 having the diphthamide modification at the His715 residue also with higher binding affinity than to eEF2 having 3-amino-3- carboxypropyl (ACP) modification and/or (preferably and) diphthine modification at the His715 residue.
  • ACP 3-amino-3- carboxypropyl
  • the higher binding affinity is preferably at least 10-fold, more preferably at least 100-fold, more preferably at least 1000-fold.
  • the antibody substantially does not bind to eEF2 having ACP modification and/or (preferably and) diphthine modification at the His715 residue.
  • the antibodies of the invention are preferably labelled with a detectable label, such as an enzyme, a fluorescent label, a radiolabel, an enzyme, a fluorescent label, a radiolabel, an enzyme, a fluorescent label, a radiolabel, an enzyme, a fluorescent label, a radiolabel, an enzyme, a fluorescent label, a radiolabel, an enzyme, a fluorescent label,
  • the antibodies of the invention may be used in the methods of the invention.
  • the antibodies of the tenth aspect (which bind to eEF2 having diphthamide modification at the His715 residue with higher binding affinity than to eEF2 that is unmodified at the His715 residue) may be used in the methods of the first to fourth aspects; the antibodies of the ninth aspect (which bind to eEF2 that is unmodified at the His715 residue with higher binding affinity than to eEF2 having diphthamide modification at the His715 residue) may be used in the methods of the fifth to eighth aspects.
  • the invention also provides the use of an antibody of the tenth aspect of the invention in an in vitro method of assessing resistance or non-resistance of a cell population to NAD(+)- diphthamide ADP ribosyltransferases treatment.
  • NAD(+)- diphthamide ADP ribosyltransferases treatment The descriptions and definitions of suitable and preferred NAD (+) -diphthamide ADP
  • the invention provides the use of an antibody of the ninth aspect of the invention in an in vitro method of assessing whether a cell population has increased sensitivity to apoptosis.
  • the apoptosis is TNFa- or other inducer-mediated apoptosis.
  • suitable and preferred other inducers provided elsewhere herein apply here also .
  • the patient is preferably a human.
  • the eEF2 protein is preferably human eEF2 protein having the amino acid sequence shown in SEQ ID NO:101.
  • MCF-7 derivatives were obtained by the toxin-selection or genetic screen procedures described.
  • * t/g indicates number of transfected cells subjected to toxin selection (t) and genetic screen (g) .
  • # of clones x/y lists the number (x) of completely (ko- ko) or heterozygous (wt-ko) mutated individual clones (i.e. with different mutations) that were identified by sequence analyses among the number (y) of all clones that were obtained (toxin selection) or chosen as candidates due to their HRM curve shapes (genetic screens) .
  • Table 2 Influence of heterozygous and complete DPH gene inactivation on sensitivity of MCF-7 cells to ADP-ribosylating toxins, protein synthesis inhibitors and TNFalpha. Sensitivity of MCF-7 clones to PE, DT, CHX or TNFa was determined by BrdU incorporation assays. IC50 values were calculated from dose-response curves, x/y describes values of two independent DPH ko clones.
  • Table 3 Transcriptome (mRNAseq) comparisons of MCF-7 derivatives. mRNAs showing the highest level of induction as a consequence of DPH2 as well as DPH5 inactivation were defined by calling transcripts that are among the 50 genes (rank) with highest induction levels (log2 value) compared to parent MCF7 in DPH2ko cells as well as in DPH5ko cells. The most prominent
  • Figure 1 ZFN target sequences and allele sequences of mutated MCF-7 clones (SEQ ID Os : 5 - 30) .
  • Figure 2 Western blot analyses identify antibodies that specifically detect eEF2 without diphthamide.
  • FIG. 4 Inactivation of DPH5 induces pathways resembling pre-activation of response to TNFalpha.
  • Whole transcriptome RNAseq data were obtained for untreated MCF7 cells, TNF-alpha treated MCF7 cells and dph5 inactivated MCF7 derivatives.
  • Genes with changed transcript levels (MCF7 vs TNFalpha treated MCF7 and MCF7 vs MCF7dph5koko) were subjected to ingenuity upstream pathway analyses.
  • Figure 5 Biosynthetic pathway for diphthamide modification at the His715 residue of eFE2 and schematic showing substrate specificity of PE.
  • Figure 6 Amino acid sequences of the heavy and light chain variable domains of antibody MGb (SEQ ID NOs : 102 & 103) .
  • the CDR sequences defined by analogy to Kabat are underlined.
  • binding affinity refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K D ) . Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.
  • K D is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay.
  • RIA radiolabeled antigen binding assay
  • a non-adsorbent plate (Nunc #269620) 100 pM or 26 pM [ 125 I ] -antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab- 12, in Presta, L.G. et al . , Cancer Res. 57 (1997) 4593-4599) .
  • the Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are
  • 3 ⁇ 4 is measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE ®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at -10 response units (RU) .
  • CM5 carboxymethylated dextran biosensor chips
  • EDC N-ethyl-N' - ( 3-dimethylaminopropyl) - carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 ⁇ g/ml (-0.2 ⁇ ) before injection at a flow rate of 5 ⁇ /minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20TM) surfactant (PBST) at 25°C at a flow rate of approximately 25 ⁇ /min.
  • TWEEN-20TM polysorbate 20
  • association rates (k on ) and dissociation rates (k 0ff ) are calculated using a simple one-to-one Langmuir binding model (BIACORE ® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams.
  • the equilibrium dissociation constant (K D ) is calculated as the ratio k 0ff /k on . See, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881.
  • a spectrometer such as a stop- flow equipped spectrophotometer (Aviv Instruments) or a 8000-series SLM- AMINCO TM spectrophotometer ( ThermoSpectronic ) with a stirred cuvette.
  • a spectrometer such as a stop- flow equipped spectrophotometer (Aviv Instruments) or a 8000-series SLM- AMINCO TM spectrophotometer ( ThermoSpectronic ) with a stirred cuvette.
  • absolute binding affinity values and relative binding affinities are preferably determined by surface plasmon resonance.
  • binding affinity is preferably determined using the Fab (or other monovalent) form of the antibody.
  • K D of [value] or higher refers to antibodies with the specified binding affinity or lower binding affinity (higher K 3 ) .
  • Substantially does not bind may refer to a level of binding that is undetectable and/or indistinguishable from non-specific binding by standard techniques for assessing antibody binding, such as western blot, denaturing or non-denaturing gel electrophoresis, immunostaining or ELISA.
  • K D of about 1 ⁇ or higher, preferably about 10 ⁇ or higher, about 100 ⁇ or higher or about 1 mM or higher.
  • antibody herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour, of Immunology 170:4854-4861) .
  • Antibodies may be murine, human, humanized, chimeric, or derived from other species.
  • An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C, Travers, P., Walport, M.,
  • a target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody.
  • An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cells, virally-infected cells, cells that produce autoimmune antibodies associated with an autoimmune disease, or eEF2 with or without modification at the His715 residue.
  • the immunoglobulin can be of any type (e.g. IgG, IgE, IgM, IgD, and IgA) , class (e.g. IgGl, IgG2, IgG3, IgG4,
  • immunoglobulins can be derived from any species, including human, murine, or rabbit origin.
  • Antibody fragments comprise a portion of a full length antibody, generally the antigen binding or variable region thereof.
  • Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab' )2, Fv and scFv fragments; diabodies; single-domain antibodies; linear antibodies;
  • antibody fragments produced by a Fab expression library include CDR (complementary determining region) fragments, and epitope-binding fragments of any of the above which immunospecifically bind to an antigen of a target of interest; single-chain antibody molecules; and multispecific (such as bispecific) antibodies formed from antibody fragments.
  • Particularly preferred antibody fragments for use in accordance with the invention by coupling to NAD(+)- diphthamide ADP ribosyltransferases include Fab fragments, scFv fragments and disulphide-stabilised Fv fragments, especially Fab fragments.
  • an “intact antibody” herein is one comprising VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CHI, CH2 and CH3.
  • the constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof.
  • the intact antibody may have one or more "effector functions" which refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding;
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • intact 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.
  • 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 polyclonal antibody preparations which 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 may be synthesized uncontaminated by other antibodies.
  • the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially
  • 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 (1975) Nature 256:495, or may be made by recombinant D A methods (see, US 4816567) .
  • the monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581- 597.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies 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 (US 4816567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851- 6855) .
  • Chimeric antibodies include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey or Ape) and human constant region sequences.
  • “Autoimmune disease” includes rheumatologic disorders (such as, for example, rheumatoid arthritis, Sjogren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, anti-phospholipid antibody syndrome, and psoriatic arthritis), osteoarthritis, autoimmune gastrointestinal and liver disorders (such as, for example, inflammatory bowel diseases (e.g.
  • autoimmune gastritis and pernicious anemia such as, for example, ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and polyarteriitis
  • vasculitis such as, for example, ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and polyarteriitis
  • autoimmune neurological disorders such as, for example, multiple sclerosis, opsoclonus myoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathies
  • renal disorders such as, for example, glomerulonephritis, Goodpasture's syndrome, and Berger's disease
  • autoimmune dermatologic disorders such as, for example, psoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigo
  • Graves' disease and thyroiditis are preferred such diseases. More preferred such diseases include, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjogren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.
  • Cancer as used herein include both solid and haematologic cancers, such as lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the
  • carcinoma carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS) , spinal axis tumours, brain stem glioma, glioblastoma multiforme,
  • CNS central nervous system
  • astrocytomas sch anomas, ependymomas, medulloblastomas , meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings sarcoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers .
  • condition that is treatable by TNFa or other inducer refers to a condition that is ameliorated by triggering apoptosis in target cells by activation of TNFR1 and/or by induction of other NFkappaB-signalling pathways or related signaling pathways.
  • the target cells may express a death receptor such as TNFRl, Fas receptor, DR4 and/or DR5, wherein the condition is ameliorated by triggering apoptosis via activation of the death receptor.
  • Preferred diseases are pre-cancers, cancers or tumours or viral infections.
  • An "effective amount" of an agent e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time
  • fragment when used in relation to a reference polypeptide other than an antibody refers to a polypeptide containing N-terminal and/or C- terminal deletions compared to the reference polypeptide, such that its amino acid sequence represents a contiguous portion of the amino acid sequence of the reference polypeptide.
  • Preferred fragments retain at least 10% of the reference amino acid sequence, more preferably 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the reference sequence.
  • the "His715" residue of eEF2 refers to a histidine residue at a position in an eEF2 sequence that corresponds to residue 715 in the human eEF2 sequence represented by NCBI accession number NP-001952 (version 1; GI : 4503483) when the two eEF2 sequences are aligned.
  • immunotoxins are used herein to refer to a composition comprising an antibody or antigen-binding fragment thereof, coupled to a toxic moiety.
  • An alternative term for certain immunotoxins that are generated by genetic fusion of protein components is "cytolytic fusion protein (cFP)". While immunotoxins represent a preferred class of targeted therapeutic agents of the present invention, the targeted therapeutic agents may comprise alternative cell-binding agents as described herein. The applicability of the present invention is therefore not limited to immunotoxins.
  • the term "one or more amino acid substitutions, deletions and/or insertions” preferably refers to the substitution, deletion and/or insertion of up to 5 amino acids in any CDR, more
  • up to 4 amino acids preferably up to 3 amino acids, more preferably 1 or 2 amino acids, more preferably a single amino acid.
  • 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,
  • a "patient” is a mammal. Mammals include, but are not limited to,
  • the patient is preferably a human.
  • Percent (%) amino acid seguence identity with respect to a reference polypeptide seguence is defined as the percentage of amino acid residues in a candidate seguence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program, and do not vary. In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
  • pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • pre-cancer refers to a condition or lesion that typically precedes or develops into a cancer, in particular a condition characterized by the presence of cells that show pathological changes that are
  • Examples include actinic keratosis, Barrett's esophagus, atrophic gastritis, ductal carcinoma in situ, lobular carcinoma in situ, dyskeratosis congenita, sideropenic dysphagia, lichen planus, oral submucous fibrosis, solar elastosis, cervical intra-epithelial neoplasia, leukoplakia and erythroplakia .
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
  • the treatment aspects invention can provide any amount of any level of treatment or prevention of disease (such as cancer) in a mammal.
  • the invention can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented.
  • prevention can encompass delaying the onset of the disease, or a symptom or condition thereof.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and tumor are not mutually exclusive as referred to herein.
  • variant refers to a polypeptide comprising one or more amino acid sequence insertions, deletions, substitutions and/or additions compared to a reference polypeptide. Preferred variants retain at least
  • the invention includes methods that comprise assaying for eEF2 having or lacking the diphthamide modification at the His715 residue. These assays employ techniques that are capable of distinguishing between different eEF2 species, namely eEF2 in which the His715 residue (1) is unmodified, (2) has the intermediate ACP-modification, (3) has the intermediate diphthine-modification and (4) has the diphthamide modification .
  • spectrometry can discriminate between these different forms of eEF2, such as ESI-MS as described in Example 3 herein, aidi-TOF and SELDI-TOF.
  • the assays of the invention may employ antibody-based techniques to discriminate between these different forms of eEF2.
  • An antibody that selectively binds to eEF2 that is unmodified at the His715 residue is exemplified herein and an antibody that selectively binds to eEF2 having the diphthamide modification is being developed.
  • Antibodies capable of selectively binding to other forms of eEF2 can be obtained using routine techniques.
  • anti-eEF2 antibodies are available commercially and may be used to isolate different eEF2 species, which may then be used for positive- and negative-screening of antibody libraries (such as phage display libraries) or to generate monoclonal antibodies by immmunisation and for positive- and negative-screening of the resultant antibodies, to obtain antibodies that are capable of selectively binding to desired eEF2 species.
  • antibody libraries such as phage display libraries
  • monoclonal antibodies by immmunisation and for positive- and negative-screening of the resultant antibodies
  • unmodified at the His715 residue can be isolated from cells (such as MCF-7 cells) that are homozygous for a dphl, dph2 or dph4 mutation; eEF2 having the ACP modification at the His715 residue can be isolated from cells (such as MCF-7 cells) that are homozygous for a dph5 knock-out mutation; and eEF2 having the diphthamide modification at the His715 residue can be obtained from wild-type cells (such as MCF-7 cells) . Furthermore, eEF2 that carries a diphthine at the His715 residue can be isolated from cells (such as MCF-7 cells) that are compromised in DPH6 or DPH7 activity. This can be achieved by applying transient siRNA-mediated inhibition of DPH6 or DPH7 expression, or by other technologies that inactivate DPH6 or DPH7.
  • mice to generate antibodies that selectively bind the diphthamide modified form of eEF2
  • animals preferentially rabbits, hamsters or mice
  • synthetic peptides that span the His715 residue, and that carry a diphthamide-histidine at the appropriate position.
  • the diphthamide-histidine is generated by synthesis of the His-amino acid derivative, which becomes incorporated into the peptide during chemical peptide synthesis.
  • the diphthamide-modified peptide serves as the immunogen for immunizations in combination with an adjuvant, in accordance with well- known techniques, as well as for subsequent boosting of the immune response.
  • B-cell clones and/or hybridomas that produce antibodies that react with these peptides are isolated by standard methods including technologies that are described in Example 2.
  • Antibodies that bind the peptides applied as immunogen are thereafter subjected to Western blot analyses on immobilized cell extracts of wildtype MCF7 cells and MCF7 derivatives with inactivated DPH genes.
  • antibodies that selectively detect diphthamide-modified eEF2 generate signals on extracts of wildtype MCF7, but are negative on complete DPH1 knockout extracts, are negative on complete DPH2 knockout extracts, are negative on complete DPH4 knockout extracts, and are negative on complete DPH5 knockout extracts.
  • the same technique may be used to generate antibodies that selectively bind the ACP intermediate modified form of eEF2.
  • the animals are immunized with synthetic peptides that span the His715 residue, and that carry an ACP-histidine at the appropriate position.
  • the ACP-histidine is generated by synthesis of the His-amino acid derivative, which becomes incorporated into the peptide during chemical peptide synthesis .
  • Antibodies that selectively detect ACP-modified eEF2 generate signals on extracts of MCF7 derivatives with complete DPH5 knockouts, but are negative on MCF7 wildtype extracts, are negative on complete DPH2 knockout extracts.
  • the animals are immunized with synthetic peptides that span the His715 residue and that carry a diphthine-histidine at the appropriate position.
  • the diphthine-histidine is generated by synthesis of the His-amino acid derivative which becomes incorporated into the peptide during chemical peptide synthesis.
  • Antibodies that selectively detect diphthine-modified eEF2 are identified by a ⁇ Exclusion Western' approach: antibodies that selectively detect diphthine-modified eEF2 generate no signals on extracts of wildtype MCF7, generate no signals on extracts of derivatives with complete DPH1
  • eEF2 also generate specific eEF2-detection signals in cells that produce diphthine-modified eEF2 due to a block in or reduced efficacy of conversion of the diphthine to diphthamide.
  • a block in or reduced efficacy of conversion of the diphthine to diphthamide can be achieved by transient siRNA-mediated inactivation of the expression of the DPH6 or DPH7 genes.
  • ACP-, diphthine-, and diphthamide-modified histidine may be synthesized by routine techniques. Diphthamide-modified histidine is also commercially available from various sources (such as Angene, London, UK) under CAS registry number 75645-22-6.
  • Preferred antibody-based techniques include Western blotting as described above, IHC and ELISA.
  • the assays of the invention may be qualitative, semi-quantitative or quantitative.
  • the methods of the invention may involve determining qualitatively the presence and/or absence of eEF2 having the diphthamide modification at the His715 residue, wherein the presence of eEF2 having the diphthamide modification at the His715 residue is
  • the diseased cells may be assessed to be sensitive to treatment with NAD (+) -diphthamide ADP ribosyltransferase when eEF2 having the diphthamide modification at the His715 residue is present and resistant when eEF2 having the diphthamide modification at the His715 residue is absent.
  • the proportion of eEF2 having the diphthamide modification may be compared to a reference value, wherein the diseased cells are assessed to be sensitive to treatment with a NAD (+) -diphthamide ADP ribosyltransferase if the proportion of eEF2 having the diphthamide modification exceeds a threshold value and/or wherein the diseased cells are assessed to be resistant to treatment with a NAD (+) -diphthamide ADP ribosyltransferase if the proportion of eEF2 having the diphthamide modification is below a threshold value.
  • the thresholds may be the same or differen .
  • diseased cells may be assayed as negative for eEF2 having the diphthamide modification at the His715 residue and/or assessed to be resistant if a proportion of the eEF2 protein has the diphthamide
  • a patient may be deselected for treatment with a NAD(+)- diphthamide ADP ribosyltransferase if a proportion of the eEF2 protein has the diphthamide modification, in particular if the proportion is below a threshold value.
  • diseased cells may be assayed as positive for eEF2 having the diphthamide modification at the His715 residue and/or assessed to be sensitive only if the proportion eEF2 protein having the diphthamide modification is above a threshold value.
  • a patient may be selected for treatment with a NAD (+) -diphthamide ADP ribosyltransferase only if the proportion of the eEF2 protein having the diphthamide modification is above a threshold value.
  • any such thresholds be no more than about 50% of total eEF2, preferably no more than about 40%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1%, 0.5%, 0.2% or 0.1% of total eEF2, preferably between 5% and 50% of total eEF2, more preferably between 10% and 25% of total eEF2.
  • antibody-based technologies that selectively detect the diphthamide modification can be applied to detecting and/or quantifying eEF2 having the diphthamide modification, for example using a monoclonal antibody that specifically binds to eEF2 having the diphthamide
  • the proportion of total eEF2 that has the diphthamide modification at the His715 residue may be quantified by comparing the signal obtained using such an antibody to that obtained using an antibody that cross-reacts with the different species of eEF2 (such as an antibody that recognizes an epitope that excludes the His715 residue) .
  • the sample containing diseased cells is not particularly limited.
  • the sample is a precancer, cancer or tumour sample.
  • the sample may be from a biopsy, or a sample taken following surgical removal of the pre-cancer, cancer or tumour.
  • the sample may be a blood sample containing pre-cancerous or cancerous blood cells.
  • NAD (+) -diphthamide ADP-ribosyltrans ferases may also be used for the treatment of other conditions in which the destruction of diseased cells is desired, such as viral infections and autoimmune diseases.
  • the sample may contain virally infected cells or autoimmune effector cells, such as autoimmune T-cells or autoantibody- expressing B-cells.
  • TNFa and other inducers may also be used for the treatment of other conditions for which stimulation of the immune response is desired, such as viral infections. So the applicability of the TNFa -related aspects and embodiments of the invention is not limited to pre-cancers, cancers and tumours.
  • the sample may contain virally infected cells.
  • Sample preparation Various aspects and embodiments of the invention provide or include assays for the presence or absence of eEF2 protein having or lacking diphthamide modification at the His715 residue. In all such assays, a sample
  • containing diseased cells of the patient may be processed prior to or as part of the assay to produce protein-containing extracts of the patient sample.
  • the patient sample may be treated to separate cells (such as by homogenisation) and/or to disrupt cells to release
  • eEF2 intracellular components (such as by incubation in lysis buffer) and/or to remove cellular debris (such as by centrifugation) and/or to isolate eEF2 from the sample (such as by immunoprecipitation with anti-eEF2 antibodies) .
  • protein- containing extracts may then be fractionated by chromatography techniques or subject to electrophoresis prior to detection of the eEF2 protein having or lacking diphthamide modification at the His715 residue.
  • the eEF2 protein may be purified and/or digested into peptide fragments prior to detection.
  • the invention relates to treatment methods and to products for use in methods of treatment, it is applicable to any condition, that is treatable by cytotoxic activity targeted to diseased cells of the patient (in the case of the NAD ( + ) -diphthamide ADP-ribosyltrans ferase- related aspects and embodiments of the invention) or by TNFa or other inducer (in the case of the TNFa-related aspects and embodiments of the invention) .
  • the treatment is preferably of a tumour or cancer.
  • the applicability of the invention is not limited to tumours and cancers.
  • the treatment may also be of viral infection .
  • Immunotoxins directed against viral antigens expressed on the surface of infected cells have been investigated for a variety of viral infections such as HIV, rabies and EBV. Cai and Berger 2011 Antiviral Research
  • 90(3) 143-50 used an immunotoxin containing PE38 for targeted killing of cells infected with Kaposi's sarcoma-associated herpesvirus.
  • TNFa selectively kills human malignant T cells and transiently depletes normal T cell and is considered to have potential for the treatment of T-cell driven autoimmune diseases such as multiple sclerosis and graft-versus-host disease, as well as T cell blood cancers for which it is undergoing clinical trials.
  • T-cell driven autoimmune diseases such as multiple sclerosis and graft-versus-host disease, as well as T cell blood cancers for which it is undergoing clinical trials.
  • NAD (+) -diphthamide ADP ribosyltransferase treatment is limited only by the availability of suitable targeting moieties.
  • TNFa is known for use as an immunostimulant and anti-neoplastic agent, so may find use in the treatment of viral infection, as well as neoplasms .
  • NAD (+) -diphthamide ADP ribosyltransferases As explained above, three NAD (+) -diphthamide ADP ribosyltransferases are known to occur naturally as bacterial virulence factors, namely PE, DT and cholix toxin. Of these, cholix toxin was identified most recently
  • Yates 2006 reports the identification of several putative NAD (+) -diphthamide ADP ribosyltransferases based on a sequence-based search pattern described in Box I of Yates 2006. These putative NAD (+) -diphthamide ADP
  • ribosyltransferases included cholix toxin, which was subsequently verified in J0rgensen 2008a.
  • ribosyltransferases may be identified by similar criteria, followed by verification of diphthamide-specific activity, for example following the techniques of Example 4 herein, in which PE and DT are shown to kill MCF-7 cancer cells, but not MCF-7 cells in which diphthamide modification of eEF2 had been prevented.
  • ADP ribosyltransferase enzymes suitable for use in accordance with the present invention may be identified by subjecting the eEF2 to ADP-ribosylation reactions described herein (e.g. utilizing biotinylated-NAD) or similar assays, and monitoring for the production of ADP-ribosylated eEF2.
  • Protein preparations or fractions, bacterial extracts, plant extracts or cell extracts may be applied as the candidate 'toxin-source' .
  • Positive controls for such reactions include PE or DT or cholix toxin.
  • Negative controls to differentiate from non-specific labelling of eEF2 by the 'toxin sources' include eEF2 variants that do not contain a diphthamide.
  • the NAD(+)- diphthamide ADP ribosyltransferase is preferably a PE, DT or cholix toxin.
  • the terms 'PE' , 'DT' and 'cholix toxin' are not limited to the naturally occurring protein sequences, but also include recombinant toxins derived from the naturally occurring toxins that retain NAD ⁇ +) -diphthamide ADP ribosyltransferase activity. Indeed, the full- length naturally occurring sequences are generally not preferred.
  • the native PE, DT and cholix toxin proteins belong to the A-B class of cytotoxic proteins, which consist of a cell-binding subunit (B subunit) and a subunit with cytotoxic activity (A subunit) .
  • the B subunits of PE and DT in particular have different cell surface targets, but the A subunit of all three proteins has the NAD (+) -diphthamide ADP ribosyltransferase toxic activity.
  • the cell surface target is the low density lipoprotein receptor related protein (LRP1; also known as CD91 or the a 2- macroglobulin receptor) or the closely related variant LRP1B (Kounnas et al. 1992 J Biol Chem.
  • Cholix toxin may have the same cellular targets.
  • the cell surface target is a membrane-anchored form of the heparin-binding EGF-like growth factor (HB-EGF precursor) (Naglich et al. (1992) Cell 69: 1051-61) .
  • PE, DT and cholix toxin are taken up into cells by receptor-mediated endocytosis, and are processed by furin cleavage and reduction of
  • A-dmDT390- bisFv (UCHT1) (Resimmune®) is composed of the first 390 amino acid residues of DT (containing the catalytic domain and translocation domain that translocates the catalytic domain into the cytosol) coupled to two tandem sFv molecules derived from the anti-CD3e antibody UCHT1.
  • DT has also been coupled to IL-2 as denileukin difitox for targeting to cells bearing IL-2 receptors in the treatment of leukaemias and lymphomas.
  • Pseudomonas exotoxins Pseudomonas exotoxins
  • Native, wild-type Pseudomonas exotoxin A is a 66kD bacterial toxin secreted by Pseudomonas aeruginosa , having the 613 amino acid sequence shown in SEQ ID NO:l and also disclosed in US 5,602,095. This sequence is shown without the native signal peptide, which is shown as the first 25 amino acids of OniProt accession number PI1439.2 (gi: 12231043).
  • the native protein has three major structural domains.
  • the N-terminal domain I comprises two subdomains la (amino acids 1-252) and lb (amino acids 365-399) that are structurally adjacent but separated in the primary amino acid sequence.
  • Domain I and in particular domain la is the cell-binding domain.
  • the function of domain lb remains undefined.
  • Domain I forms the major component of the B subunit.
  • forms of PE in which the native domain la sequence is omitted or disrupted, and which consequently are unable to bind to LRP1 or LRP1B, are greatly preferred.
  • Domain II (amino acids 253-364) has been reported to mediate translocation into the cytosol, but this remains controversial ( eldon & Pastan 2011).
  • Domain III (amino acids 400-613) mediates ADP ribosylation of elongation factor 2.
  • the structural boundary between domain lb and domain III is not fully settled. According to WO2013/040141 it lies between residues 399 and 400, but Weldon and Pastan 2011 place it between residues 404 and 405.
  • full catalytic activity requires a portion of domain lb as well as domain III. Accordingly, the functional domain III of the native toxin is defined, to start at residue 395.
  • Amino acids 602-613 have been found to be inessential for NAD (+) -ribosyltransferase activity, but amino acids 609-613 of the native sequence are required for cytotoxic activity.
  • a PE toxin will have a polypeptide sequence comprising a PE functional domain III having at least 50% amino acid sequence identity over the full length of residues 395-601 of SEQ ID NO : 1 , wherein the PE toxin has cytotoxic activity when introduced into a eukaryotic (preferably mammalian) cell.
  • Preferred forms of PE comprise (1) a PE functional domain III having at least 50% amino acid sequence identity over the full length of residues 395-601 of SEQ ID NO : 1 and having NAD ⁇ + ) -diphthamide ADP ribosyltransferase activity, and (2) at least one endoplasmic reticulum localisation sequence.
  • the PE in embodiments in which the PE is coupled to a cell-binding agent as a fusion polypeptide, the PE preferably also comprises (3) a cleavable linker sequence such as a furin-cleavable sequence (FCS) that permits cleavage of the PE functional domain III from the cell-binding agent following uptake into the target cell.
  • a cleavable linker sequence such as a furin-cleavable sequence (FCS) that permits cleavage of the PE functional domain III from the cell-binding agent following uptake into the target cell.
  • FCS furin-cleavable sequence
  • cleavable linkers may be used provided that they permit cleavage of the PE from the cell-binding agent following uptake into the target cell.
  • other means of coupling the PE to the cell-binding agent are contemplated, provided again that they permit separation of the PE from the cell-binding agent following uptake into the target cell.
  • the cell-binding agent may be non-covalently linked to the PE, or linked by disulfide bonds which permit release of the PE moiety under reducing conditions, or linked by other conjugation chemistries that are known in the field of immunoconj ugate production.
  • the PE for use in accordance with the present invention will generally lack a functional cell-binding domain I.
  • PE40 is a truncated derivative of PE (Pai et al 1991 Proc. Natl. Acad. Sci. USA 88:3358-62 and Kondo et al. 1988 J. Biol. Chem.
  • PE35 is a 35 kD carboxyl-terminal fragment of PE in which amino acid residues 1- 279 have been deleted and the molecule commences with a Met at position 280 followed by amino acids 281-364 and 381-613 of PE as defined by reference to SEQ ID NO:l.
  • PE35 and PE40 are disclosed, for example, in US 5,602,095, US 4,892,827, WO93/25690 and WO88/02401, each of which is incorporated herein by reference in its entirety.
  • PE38 contains the translocating and ADP ribosylating domains of PE but not the cell-binding portion (Hwang et al . 1987 Cell 48:129-136) .
  • PE38 (SEQ ID N0:2) is a truncated PE pro-protein composed of amino acids 253-364 and 381-613 of SEQ ID NO : 1 which is activated to its cytotoxic form upon processing within a cell (see US 5,608,039, which is incorporated by reference in its entirety herein, and Pastan et al. 1997 Biochim. Biophys . Acta, 1333:C1-C6) .
  • PE38QR is a variant of PE38 having mutations of the lysines at positions 590, 606 and 613 of domain III, to permit conjugation to antibodies.
  • PE-LR contains a deletion of domain II except for a furin-cleavable sequence (FCS) corresponding to amino acid residues 274-284 of SEQ ID NO:l ( RHRQPRGWEQL (SEQ ID NO: 31) ) and a deletion of amino acid residues 365-394 of domain lb.
  • FCS furin-cleavable sequence
  • PE-LR contains amino acid residues 274-284 and 395-613 of SEQ ID NO:l.
  • PE-LR is described in WO 2009/032954 and Weldon et al 2009, which are each incorporated herein by reference in their entirety.
  • WO2012/154530 describes that the addition of a short, flexible linker of between 3 and 8 amino acids each independently selected from glycine and serine between the FCS and the PE functional domain III improves the cytotoxicity of the PE-LR molecule without disrupting binding by furin.
  • exemplary linkers are GGS and GGSGGS ( SEQ ID NO: 32) .
  • Other work has sought to further reduce the immunogenicity of PE.
  • WO2012/154530 reports that substitutions at the following amino acid residues within PE domain III reduce immunogenicity:
  • substitutions are with a glycine, serine or alanine residue.
  • WO2012/170617 reports that substitutions at these residues may reduce immunogenicity of B cell epitopes, and that substitutions at one or more of residues R427, R458, R467, R490, R505 and F538 are preferred, particularly with alanine.
  • WO2013/040141 reports that substitutions at the following additional amino acid residues may reduce the immunogenicity of B cell epitopes within PE domain III:
  • Preferred substitutions are with a glycine, serine, alanine or glutamine residue.
  • WO2012/170617 reports that substitutions at the following residues can reduce the immunogenicity of T-cell epitopes within PE domain III:
  • substitutions are at one or more of residues D463, Y481 and L516, which may also reduce the immunogenicity of B cell epitopes.
  • Preferred substitutions are with a glycine, serine, alanine or glutamine residue.
  • WO2012/170617 also reports that substitutions at the following amino acid residues can reduce the immunogenicity of T cell epitopes within PE domain II:
  • substitutions are with a glycine, serine, alanine or glutamine residue .
  • O2012/170617 also reports that substitutions at the following amino acid residues can reduce the immunogenicity of B cell epitopes within PE domain II :
  • WO2012/170617 also reports that a particularly preferred combination of substitutions is D463A/R427A/R458A/R467A/R490A/R505A/R538A.
  • Alewine et al discloses a similar combination of 7 point mutations within PE domain III that reduce B-cell immunogenicity, namely
  • the PE functional domain III may comprise mutations at any one or any combination of more than one of the following sites:
  • the mutation (s) reduce (s) the immunogenicity compared to the unmutated sequence of the amino acids 395-613 of SEQ ID NO:l.
  • the PE contains some or all of domain II, it may comprise mutations at any one or any combination of more than one of the following sites:
  • the mutation (s) reduce (s) the immunogenicity compared to the unmutated sequence from domain II.
  • the FCS is derived from the native furin-cleavable sequence of PE consisting of amino acids 274-284
  • RHRQPRGWEQL SEQ ID NO: 31
  • RHRQPRGWEQL SEQ ID NO: 31
  • the epitope from the native sequence may anyway be disrupted such that a mutation at the E282 residue may not be advantageous.
  • Reduced immunogenicity in variant PE toxins may refer to a reduced ability of the variant sequence to induce a T cell response and/or a reduced ability of the variant sequence to induce a B cell (antibody) response, preferably both.
  • Techniques for assessing the effect of mutations on T cell immunogenicity are well known in the art and described in the examples of WO 2012/170617.
  • Techniques for assessing the effect of mutations on the B cell immunogenicity are likewise well known in the art and described in WO 2013/040141, for example.
  • Human antibodies may be raised against the native PE sequence by phage display using a human antibody library. The ability of mutations in the PE sequence to disrupt binding of such antibodies to the variant PE molecule is indicative of reduced
  • the titre of PE-specific antibodies raised in transgenic mice carrying the human antibody repertoire may be compared for the native and mutated PE sequences.
  • the C-terminal end of the PE functional domain III may contain the native sequence of residues 609-613, namely REDLK (SEQ ID NO: 33 ) . Additionally or alternatively to any other modifications of the native PE sequence, the PE functional domain III may contain a variant of the REDLK sequence, or other sequences, that function to maintain the PR protein in the
  • ER localisation sequences include such as KDEL (SEQ ID NO: 34 ) , REDL ⁇ SEQ ID N0: 35 ) , RDEL (SEQ ID NO: 36 ) or KEDLK (SEQ ID N0: 37 ) .
  • KDEL SEQ ID NO: 34
  • RDEL SEQ ID NO: 36
  • KEDLK SEQ ID N0: 37
  • additional ER localisation sequences may be added to the C-terminal end of the PE polypeptide sequence.
  • KDEL, or 2 or 3 tandem repeats of KDEL KDELKDEL, SEQ ID NO 38 ; KDELKDELKDEL, SEQ ID NO 39
  • KDEL KDELKDEL, SEQ ID NO 38
  • KDELKDELKDEL SEQ ID NO 39
  • KDEL after the native REDLK sequence is preferred. See for example WO91/099949, Chaudhary et al 1991 Seetharam et al 1991.
  • WO91/09949 discloses that the C-terminal end of the PE functional domain III may lack some or all of residues 602-608, which are not essential for the NAD ( + ) -diphthamide ADP ribosyltransferase activity.
  • FCS Furin-cleavable sequence
  • the furin-cleavable sequence can be any polypeptide sequence cleavable by furin.
  • Duckert et al . 2004, Protein Engineering, Design & Selection 17 ( 1 ) : 107-112 (hereafter, "Duckert et al.") is incorporated herein by reference in its entirety and particularly with regard to the furin-cleavable sequences and motifs it discloses.
  • Duckert et al. discloses that furin is an enzyme in a family of evolutionarily conserved dibasic- and monobasic-specific CA2 "-dependent serine proteases called substilisin/kexin-like proprotein convertases. See page 107.
  • Furin also known as "paired basic amino acid cleaving enzyme", "PACE", or PCSK3
  • PACE paired basic amino acid cleaving enzyme
  • PCSK3 paired basic amino acid cleaving enzyme
  • the minimal furin-cleavable sequence typically is, in the single letter code for amino acid residues, R-X-X-R (SEQ ID N0: 40 ) , with cleavage occurring after the second "R".
  • Duckert et al. summarizes the information available on the sequences of 38 proteins reported in the literature to have furin-cleavable sequences, including mammalian proteins, proteins of pathogenic bacteria, and viral proteins.
  • the residues surrounding the furin cleavage site are numbered from the scissile bond (which is typically indicated by a downward arrow) .
  • the substrate residues are designated PI, P2, and so on, while counting towards the C-terminus, the residues are designated Pi', P2 ' , and so on.
  • the following sequence can be used to align and number the residues of the minimal cleavage sequence and the surrounding residues:
  • furin cleavage occurs between arginine 279 and glycine 280 in an arginine-rich loop located in domain II of the toxin.
  • the native furin- cleavable sequence in domain II of PE is set forth below ⁇ with numbers indicating the positions of the residues in the 613-amino acid native PE sequence), and aligned to show its numbering under the convention noted above :
  • This sequence has shown a cleavage rate faster than that of the native sequence, and when used in an exemplary immunotoxin resulted in
  • a furin-cleavable sequence used to attach the targeting molecule to PE domain III can be the minimal furin- cleavable sequence, R-X-X-R (wherein each X is independently any naturally occurring amino acid), preferably R-X-[R/K]-R (wherein X is any naturally occurring amino acid and [R/K] denotes either arginine or lysine), or any of the other furin-cleavable sequences known in the art or permitted by Fig.
  • the sequence can be RKKR ( SEQ ID NO: 44 ) , RRRR (SEQ ID N0: 45 ) , RKAR ( SEQ ID NO: 46 ) , SRVARS (SEQ ID NO: 47 ) , TSSRKRRFW (SEQ ID NO: 48 ) , or ASRRKARSW (SEQ ID NO: 49) .
  • the furin-cleavable sequence is RRVKKRFW (SEQ ID NO: 50 ) , RNVVRRDW (SEQ ID NO: 51 ) , or TRAVRRRSW (SEQ ID NO: 52 ) .
  • the residue at position PI can be the arginine present in the native sequence, or lysine.
  • a lysine can be
  • the furin-cleavable sequence contains the native furin-cleavable sequence of PE: R-H-R-Q-P-R-G-W-E-Q-L (SEQ ID NO: 31 ) or a truncated version of the native sequence, so long as it contains the minimal furin-cleavable sequence and is cleavable by furin.
  • the furin-cleavable sequence can be R-Q-P-R (SEQ ID NO: 53 ) , R-H-R-Q-P-R-G- (SEQ ID NO: 54 ) , R-H-R-Q-P-R-G-W-E (SEQ ID NO: 55 ) , H-R-Q- P-R-G-W-E-Q (SEQ ID NO: 56 ) , or R-Q-P-R-G-W-E (SEQ ID NO: 57 ) .
  • the sequence is R-H-R-S-K-R-G-W-E-Q-L (SEQ ID NO: 43 ) or a truncated version of this sequence, so long as it contains the minimal furin-cleavable sequence and is cleavable by furin.
  • the furin-cleavable sequence can be R-S-K-R (SEQ ID NO: 58), R-H-R-S-K-R-G-W (SEQ ID NO: 59 ) , H-R-S-K-R-G-W-E (SEQ ID NO: 60 ) , R-S-K-R- G-W-E-Q-L (SEQ ID NO: 61 ) , H-R-S-K-R-G-W-E-Q-L (SEQ ID NO: 62 ) , or R-H-R-S- K-R (SEQ ID NO: 63 ) .
  • the E282 residue at the P3 ' position of PCS sequences derived from PE may be replaced by another amino acid to reduce B cell immunogenicity .
  • the sequence lacks native PE residues downstream of this residue, or where the FCS contains other mutations relative to the native PE sequence, such replacement may not be necessary.
  • Whether or not any particular sequence is cleavable by furin can be determined by methods known in the art. For example, whether or not a sequence is cleavable by furin can be tested by incubating the sequence with furin in furin buffer (0.2 M NaOAc (pH 5.5), 5 mM CaC12) at a 1 :10 enzyme : substrate molar ratio at 25°C for 16 hours. These conditions have previously been established as optimal for furin cleavage of PE .
  • the furin used is human furin.
  • Recombinant truncated human furin is commercially available, for example, from New England Biolabs (Beverly, MA) . See also, Bravo et al. 1994 J Biol Chem 269 ( 14 ) : 25830-25837.
  • a furin-cleavable sequence can be tested by making it into an immunotoxin with an antibody against a cell surface protein and testing the resulting immunotoxin on a cell line expressing that cell surface protein.
  • Suitable antibody sequences are disclosed in, for example, WO2012/154530 and WO2009/032954.
  • General formula for preferred PE toxins are disclosed in, for example, WO2012/154530 and WO2009/032954.
  • FCS - Rl m - R 2 n - R 3 p - PE functional domain III - R 4 wherein :
  • 1, m, n, p and q are each, independently, 0 or 1;
  • FCS is a furin-cleavable sequence, preferably (i) R-H-R-Q-P-R-G-W-E- Q-L or a truncated version thereof containing R-Q-P-R, optionally R-Q-P-R, R-H-R-Q-P-R-G-W, R-H-R-Q-P-R-G-W-E, H-R-Q-P-R-G-W-E-Q, or R-Q-P-R-G-W-E; or (ii) R-H-R-S-K-R-G-W-E-Q-L or a truncated version thereof containing R-S-K- R, optionally R-S-K-R, R-H-R-S- -R-G-W, H-R-S-K-R-G-W-E, R-S-K-R-G-W-E-Q-L, H-R-S-K-R-G
  • R is a linker sequence of 1 to 10 amino acids, preferably GGS or GGSGGS;
  • R 2 is one or more consecutive amino acid residues of residues 285-364 of SEQ ID NO:l, in which any one or more of residues E285, P290, L294, L297, Y298, L299, R302, R313, N314, P319, D324, E327, E331 and Q332, where present, is/are optionally independently replaced by another amino acid, preferably glycine, serine, alanine or giutamine;
  • R 3 is one or more consecutive amino acid residues of residues 365-394 of SEQ ID NO: 1;
  • PE functional domain III comprises residues 395-613 of SEQ ID NO : 1 in which :
  • residues 602-608 are optionally deleted.
  • residues 609-613 are optionally replaced by another ER localisation sequence, preferably KDEL, REDL, RDEL or EDLK, and
  • K606 is/are optionally independently replaced by another amino acid, preferably glycine, serine, alanine or glutamine, or histidine in the case of L477;
  • R 4 is one or more (preferably 1 or 2) additional ER localisation sequences, preferably REDLK, KDEL, REDL, RDEL or KEDLK.
  • 1 is preferably 1; that is, an FCS is preferably present; m is preferably 1; that is, a linker is preferably present especially in the case that 1 is 1; n is preferably 0; that is, residues 285-364 of SEQ ID NO : 1 are preferably absent ; p is preferably 0; that is residues 365-394 of SEQ ID NO : 1 are preferably absent;
  • PE functional domain III preferably includes the combination of mutations R427A/F443A/L477H/R494A/R505A/L552E, or the combination of mutations R427A/R456A/D463A/R467A/R490A/R505A/R538A, or the combination of mutations
  • SEQ ID NO: 110 corresponds to amino acid residues 395-613 of SEQ ID NO:l with Ala substitutions at positions 427, 456, 463, 467, 490, 505 and 538 and is disclosed in WO2015/51199 as LO10R-456A and SEQ ID NO: 37.
  • SEQ ID NO: 111 corresponds to amino acid residues 395-613 of SEQ ID NO:l with Ala substitutions at positions 427, 443, 477, 494, 505 and 552 and is disclosed in WO2015/051199 as T18/T20 and SEQ ID NO:289.
  • amino acid sequences of SEQ ID NO: 110 and SEQ ID NO: 111 are each preferably fused to the C-terminal end of the amino acid sequence of SEQ ID NO: 112, which corresponds to SEQ ID NO: 36 of WO2015/051199 and contains an FCS and linker sequences.
  • diphtheriae and has the 535 amino acid sequence shown in SEQ ID NO: 3. This sequence is shown without the native signal peptide, which is shown as the first 25 amino acids of UniProt accession number Q6NK15, version 1.
  • N-terminal domain I amino acids 1-191 of SEQ ID NO : 3
  • C-terminal domain III amino acids 385-535 of SEQ ID NO : 3
  • activation of the native DT protein depends on furin cleavage within domain II.
  • Truncated or modified forms of DT that lack receptor-binding activity have been widely used in the form of immunotoxins , coupled to other targeted therapeutic agents.
  • Exemplary truncated forms of DT include residues 1-384, 1-387, 1-388, 1-389 or 1-485 of SEQ ID NO : 3 ) , optionally with an additional N-terminal methionine residue from.
  • a DT toxin will have a polypeptide sequence comprising a DT functional domain I having at least 50% amino acid sequence identity over the full length of residues 1-191 of SEQ ID NO: 3 and having cytotoxic activity when introduced into a eukaryotic (preferably mammalian) cell.
  • Preferred forms of DT comprise a DT functional domain I having at least 50% amino acid sequence identity over the full length of residues 1-191 of SEQ ID NO : 3 and having NAD (+) -diphthamide ADP ribosyltransferase activity.
  • the DT in embodiments in which the DT is coupled to a cell-binding agent as a fusion polypeptide, the DT preferably also comprises (2) a cleavable linker sequence such as a furin-cleavabie sequence (FCS) that permits cleavage of the DT functional domain I from the cell-binding agent following uptake into the target cell.
  • a cleavable linker sequence such as a furin-cleavabie sequence (FCS) that permits cleavage of the DT functional domain I from the cell-binding agent following uptake into the target cell.
  • FCS furin-cleavabie sequence
  • the cleavable linker (such as an FCS) will generally be on the C-terminal side of the DT functional domain I.
  • FCS cleavable linker
  • the furin-cleavable sequence preferably includes the minimal furin-cleavable sequence motif from the native DT sequence, namely the R-V-R-R (SEQ ID NO 64 ) sequence at residues 190-193 of SEQ ID NO : 3. It may also include N-terminal and/or C-terminal flanking regions from the native sequence.
  • the furin-cleavable sequence includes the sequence GNRVRRSVGSS (SEQ ID NO 65 ) or a fragment thereof comprising RVRR.
  • GNRVRRSVGSS SEQ ID NO 65
  • other cleavable linkers and other means of coupling the DT to cell-binding agents are contemplated.
  • a DT for use in accordance with the invention will generally lack an ER localisation sequence.
  • DT may contain deletions within domain II, particularly downstream of the furin-cleavable sequence (that is, within residues 194- 384 of SEQ ID NO: 3, preferably within residues 200-384 so as to preserve a longer native furin-cleavable sequence) .
  • the DTs for use in accordance with the invention may also be mutated to reduce immunogenicity .
  • the DT for use in accordance with the present invention will generally lack a functional cell-binding domain III.
  • Native, wild-type cholix toxin is secreted by Vibrio cholerae and has the 634 amino acid sequence shown in SEQ ID NO: 4. This sequence is shown without the native signal peptide, which is shown as the first 32 amino acids of UniProt accession number Q5EK40.1 ( gi : 75355041 ) .
  • domain la (amino acids 1-264) is the cell-binding domain
  • domain II (amino acids 265-386) is the translocation domain and contains the furin-cleavable sequence R PR (SEQ ID NO 66) at residues 289-292;
  • domain lb (amino acids 387-423) is of unknown function and domain III (amino acids 424-634) is the catalytic domain, comprising an ER
  • RKDELK (SEQ ID NO: 67 ) at positions 629-634. See Awasthi et al . 2013, which also provides naturally occurring variant cholix toxin sequences.
  • a cholix toxin will have a polypeptide sequence comprising a cholix toxin functional domain III having at least 50% amino acid sequence identity over the full length of residues 424-628 of SEQ ID NO: 4, wherein the cholix toxin has cytotoxic activity when introduced into a eukaryotic (preferably mammalian) cell.
  • Preferred forms of cholix toxin comprise (1) a cholix toxin functional domain III having at least 50% amino acid sequence identity over the full length of residues 424-628 of SEQ ID NO: 4 and having NAD (+) -diphthamide ADP ribosyltransferase activity, and (2) at least one endoplasmic reticulum localisation sequence.
  • the cholix toxin preferably also comprises (3) a cleavable linker sequence such as a furin-cleavable sequence (FCS) that permits cleavage of the cholix toxin functional domain III from the cell- binding agent following uptake into the target cell.
  • FCS furin-cleavable sequence
  • the cleavable linker (such as an FCS) will generally be on the N-terminal side of the cholix toxin functional domain III.
  • the furin-cleavable sequence preferably includes the minimal furin- cleavable sequence motif from the native cholix toxin sequence, namely the R PR (SEQ ID NO 66) sequence of residues 289-292 of SEQ ID NO : 4. It may also include N-terminal and/or C-terminal flanking regions from the native sequence.
  • the furin-cleavable sequence includes the sequence RSRKPRDLTDD (SEQ ID NO 68) of amino acids 287-297 of SEQ ID NO : 4 or a fragment thereof comprising RKPR.
  • it may comprises the sequence RGRKPRDLTDD (SEQ ID NO: 69) of ChxA III of Awasthi et al.
  • the ER localisation sequence may be the native RKDELK sequence of SEQ ID NO: 4 or the HDELK (SEQ ID NO: 71) sequence of ChxA III of Awasthi et al. 2013 or any of the ER localisation sequences disclosed above for PE .
  • the cholix toxin may include one or more additional ER
  • the cholix toxin may include some or all of domain lb (amino acids 387-423), preferably at least about 10 amino acids from the C- terminus of domain lb, that is, at least about amino acids 413-422.
  • cholix toxins for use in accordance with the present invention will generally lack a functional cell-binding domain I.
  • cholix toxin may contain deletions, particularly upstream of the furin-cleavable sequence and/or between the furin-cleavable sequence and the cholix toxin domain III (that is, within residues 1-288 (preferably 1-286) and/or 293-423 (preferably 298-423 or 293-413, more preferably 298- 413) of SEQ ID NO : 4.
  • the cholix toxins for use in accordance with the invention may also be mutated to reduce immunogenicity .
  • cytotoxic activity of the NAD(+)- diphthamide ADP-ribosyitransferases of the present invention may be tested using a cytotoxic activity assay.
  • the NAD (+) -diphthamide ADP- ribosyltransferase is coupled to a cell-binding agent that is targeted to the cells used in the assay.
  • a wide variety of cytotoxicity assays are available, such as the WST assay used in WO 2011/032022, which measures cell proliferation using the tetrazolium salt WST-1. Reagents and kits are commercially available from Roche Applied Sciences.
  • NAD (+) -diphthamide ADP-ribosyltransferase activity may be assayed by the ability to incorporate biotinylated ADP into eEF2 protein, as described in Example 3 herein.
  • Immunotoxins that combine an antibody with a PE toxin and that have progressed to clinical trials are reviewed in Weldon & Pastan (2011) and include the following:
  • RFB4 (dsFv) PE38 (also known as BL22 or CAT-3888) directed against
  • CD22 for the treatment of B-cell malignancies (Kreitman et al. 2005, Kreitman et al. 2009a, Wayne et al. 2010) .
  • ClinicalTrial.gov identifiers NCT00462189, NCT00457860, NCT00515892, NCT01086644, NCT00659425, and NCT00586924) .
  • SSI (dsFv) PE38 also known as SS1P directed against mesothelin, for the treatment of lung cancer and mesothelioma (Hassan et al . ,
  • anti-TAC (scFv) PE38 also known as LMB-22 directed against IL-2R, for the treatment of hematologic malignancies (Kreitman et al. 2000, ClinicalTrial.gov identifiers NCT00924170, NCT00077922, NCT00080535, and NCT00321555) .
  • Wolf et al. 2009 and Shapira et al. 2010 review other targeted therapeutic agents in pre-clinical and clinical development, which combine either an antibody or another cell-binding agent with a PE or DT toxin.
  • Table 1 of Shapira et al. 2010 refers to targeted therapeutic agents incorporating a variety of truncated or modified forms of DT that lack receptor-binding activity (truncated: DABjsg, DAB 8S, DT388, DT390;
  • CRM107 CRM107
  • PE toxins full-length PE, PE38, PE40, modified PE38 and modified PE40
  • cell-binding agents IL-2, transferrin, GM-CSF, EGF, anti-CD3£, variant IL-3, anti- ovarian antigen, anti-HER2, anti-mesothelin, anti-Lewis Y, anti-CD22, anti- CD25, TGFa , circularly permuted IL-4 and IL-13
  • leukaemia and lymphoma including cutaneous T cell lymphoma (CTCL) , non-Hodgkin' s lymphoma (NHL), chronic lymphoblastic lymphoma (CLL) , Hodgkin' s disease (HD) , small lymphatic lymphoma (SLL), prolymphocytic leukaemia (PLL) , acute myelogenous leukaemia (AML) , hairy cell leukaemia (HCL) , acute lymphoblastic leukaemia (ALL) and T-cell lymphoma/leukaemia, ⁇ lung cancer including non-small cell lung cancer (NSCLC) and mesothelioma; other cancers including adenocarcinoma, EGFR-expressing carcinomas, melanoma, ovarian cancer, breast cancer, kidney cancer, Kaposi's sarcoma (KS), brain and CNS tumours, oesophageal cancer, pancreatic cancer, colon cancer, bladder
  • any of these targeted therapeutic agents may be used in accordance with the present invention.
  • the cell-binding agents of these targeted therapeutic agents may be used with other NAD (+) -diphthamide ADP ribosyltransferases, especially for the indications shown.
  • Still further cell-binding agents are disclosed in the context of other (non-NAD(+)- diphthamide ADP ribosyltransferase) toxins and may similarly be used with NAD (+) -diphthamide ADP ribosyltransferases , especially for the indications shown.
  • Shapira et al. is incorporated herein by reference in its entirety and (along with the references cited in Table 1) especially for the purpose of exemplifying both specific targeted therapeutic agents and cell-binding agents and their associated indications, suitable for use in accordance with the present invention.
  • the NAD (+) -diphthamide ADP ribosyltransferases herein are coupled to a cell binding agent to produce a targeted therapeutic agent.
  • targeted therapeutic agent is used in the broadest sense and is not intended to imply that the cell binding agent is necessarily an antibody or immunoglobulin.
  • a wide variety of cell binding agents may be included in targeted therapeutic agents in accordance with the invention.
  • the cell binding agent is a peptide, polypeptide, or protein
  • NAD ( + ) -dipthamide ADP ribosyltransferase is preferably coupled to the ceil binding agent as a fusion polypeptide or protein. Fusion may be direct or via a linker peptide. The fusion polypeptide or protein may be produced recombinantly, avoiding any need for conjugation chemistry.
  • the furin-cleavable sequence will generally be positioned between the cell binding agent and the cytotoxic domain of the NAD ⁇ +) -dipthamide ADP ribosyltransferase, such that cleavage of the fusion polypeptide inside the target cell will separate the cytotoxin domain from the cell binding agent.
  • the NAD ( + ) -diphthamide ADP ribosyltransferase will be positioned on the C-terminal side of the ceil binding agent.
  • NAD (+) -diphthamide ADP ribosyltransferase may be conjugated to the cell binding agent.
  • a cell binding agent may be of any kind, and include peptides and non- peptides. These can include antibodies or a fragment of an antibody that contains at least one binding site, iymphokines, hormones, growth factors, nutrient-transport molecules, or any other cell binding molecule or substance .
  • the cell binding agent may be, or comprise, a polypeptide.
  • the cell binding agent is preferably an antibody.
  • the peptide may comprise 4-20, preferably 6-20, contiguous amino acid residues.
  • Antibodies for use in the targeted therapeutic agents of the invention include those antibodies described in WO 2005/082023 which is incorporated herein. Particularly preferred are those antibodies for tumour-associated antigens. Examples of those antigens known in the art include, but are not limited to, those tumour-associated antigens set out in WO 2005/082023. See, for instance, pages 41-55.
  • the cell-binding agents described herein are designed to target diseased cells such as tumour cells via their cell surface antigens.
  • the antigens are usually normal cell surface antigens which are either over-expressed or expressed at abnormal times. Ideally the target antigen is expressed only on diseased cells (such as tumour cells), however this is rarely observed in practice. As a result, target antigens are usually selected on the basis of differential expression between diseased and healthy tissue.
  • the cell-binding agent may specifically bind to any suitable cell surface marker.
  • the choice of a particular cell-binding agent and/or cell surface marker may be chosen depending on the particular cell population to be targeted.
  • Cell surface markers are known in the art (see, e.g., Mufson et al., Front. Biosci., 11:337-43 (2006); Frankel et al., Clin. Cancer Res., 6:326-334 (2000); and Kreitman et al . , AAPS Journal, 8(3) : E532-E551 (2006)) and may be, for example, a protein or a carbohydrate.
  • the cell-binding agent is a ligand that specifically binds to a receptor on a cell surface.
  • ligands include, but are not limited to, vascular endothelial growth factor (VEGF) , Fas, TNF-related apoptosis-inducing ligand (TRAIL), a cytokine (e.g., IL-2, IL-15, IL-4, IL-13), a lymphokine, a hormone, and a growth factor (e.g., transforming growth factor (TGFa) , neuronal growth factor, epidermal growth factor) .
  • VEGF vascular endothelial growth factor
  • Fas Fas
  • a cytokine e.g., IL-2, IL-15, IL-4, IL-13
  • TGFa transforming growth factor
  • epidermal growth factor epidermal growth factor
  • the cell surface marker can be, for example, a tumour-associated antigen.
  • tumour-associated antigen refers to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) solely or predominantly expressed or over-expressed by tumour cells and/or cancer cells, such that the antigen is associated with the tumour (s) and/or cancer (s) .
  • the tumour- associated antigen can additionally be expressed by normal, non-tumour, or non-cancerous cells. However, in such cases, the expression of the tumour- associated antigen by normal, non-tumour, or non-cancerous cells is not as robust as the expression by tumour or cancer cells.
  • the tumour or cancer cells can over-express the antigen or express the antigen at a significantly higher level, as compared to the expression of the antigen by normal, non-tumour, or non-cancerous cells.
  • the tumour- associated antigen can additionally be expressed by cells of a different state of development or maturation.
  • the tumour-associated antigen can be additionally expressed by cells of the embryonic or fetal stage, which cells are not normally found in an adult host.
  • the tumour-associated antigen can be additionally expressed by stem cells or precursor cells, which cells are not normally found in an adult host.
  • the tumour-associated antigen can be an antigen expressed by any cell of any cancer or tumour, including the cancers and tumours described herein.
  • the tumour-associated antigen may be a tumour-associated antigen of only one type of cancer or tumour, such that the tumour-associated antigen is associated with or characteristic of only one type of cancer or tumour.
  • the tumour-associated antigen may be a tumour-associated antigen (e.g., may be characteristic) of more than one type of cancer or tumour.
  • the tumour-associated antigen may be expressed by both breast and prostate cancer cells and not expressed at all by normal, non- tumour, or non-cancer cells.
  • tumour-associated antigens to which the cell-binding agent may specifically bind include, but are not limited to, mucin 1 ( UCl; tumour- associated epithelial mucin) , melanoma associated antigen (MAGE) ,
  • FRAME carcinoembryonic antigen
  • CEA carcinoembryonic antigen
  • PSA prostate-specific antigen
  • PSMA prostate specific membrane antigen
  • GM-CSFR granulocyte-macrophage colony-stimulating factor receptor
  • CD56 human epidermal growth factor receptor 2 (HER2/neu) (also known as erbB-2), CDS, CD7, tyrosinase tumour antigen, tyrosinase related protein (TRP) I, TRP2, NY-ESO-1, telomerase, and p53.
  • TRP tyrosinase related protein
  • Mesothelin is expressed in, e.g., ovarian cancer, mesothelioma, non- small cell lung cancer, lung adenocarcinoma, fallopian tube cancer, head and neck cancer, cervical cancer, and pancreatic cancer.
  • CD22 is expressed in, e.g., hairy cell leukemia, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL) , non-Hodgkin ' s lymphoma, small lymphocytic lymphoma (SLL), and acute lymphatic leukemia (ALL) .
  • CD25 is expressed in, e.g., leukemias and lymphomas, including hairy cell leukemia and Hodgkin's lymphoma.
  • Lewis Y antigen is expressed in, e.g., bladder cancer, breast cancer, ovarian cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancer, and pancreatic cancer.
  • CD33 is expressed in, e.g., acute myeloid leukemia (AML) , chronic myelomonocytic leukemia (CML) , and myeloproliferative disorders.
  • the cell-binding agent is an antibody that specifically binds to a tumour-associated antigen.
  • antibodies that specifically bind to tumour-associated antigens include, but are not limited to, antibodies against the transferrin receptor (e.g., HB21 and variants thereof), antibodies against CD22 (e.g., RFB4 and variants thereof), antibodies against CD25 (e.g., anti-Tac and variants thereof), antibodies against mesothelin (e.g., SS 1, MORAb-009, SS, HN1, HN2, MN, MB, and variants thereof) and antibodies against Lewis Y antigen (e.g., B3 and variants thereof) .
  • the transferrin receptor e.g., HB21 and variants thereof
  • CD22 e.g., RFB4 and variants thereof
  • CD25 e.g., anti-Tac and variants thereof
  • mesothelin e.g., SS 1, MORAb-009, SS, HN1, HN2, MN, MB, and variants thereof
  • Lewis Y antigen e
  • the cell-binding agent may be an antibody selected from the group consisting ofB3, RFB4, SS, SSI, MN, MB, HN1, HN2 , HB21, and MORAb-009, and antigen binding portions thereof.
  • Further exemplary targeting moieties suitable for use in the inventive chimeric molecules are disclosed e.g., in U.S.
  • Antibodies have been raised to target specific tumour related antigens including: Cripto, CD30, CD19, CD33, Glycoprotein NMB, CanAg, Her2
  • CD56 (ErbB2/Neu), CD56 (NCAM) , CD22 (Siglec2), CD33 (Siglec3), CD79, CD138, PSCA, PSMA (prostate specific membrane antigen), BCMA, CD20, CD70, E- selectin, EphB2, Melanotransferin, Mucl6 and TMEFF2.
  • Native human TNFa is a 157-amino acid polypeptide that binds to and activates TNF receptor 1, leading to an enhancement of apoptosis.
  • TNFa examples include native human TNFa and active fragments and variants thereof that are capable of binding to and activating TNF receptor 1.
  • the applicability of the invention is not limited to TNFa but extends also to other inducers of NFkappaB- and related signaling pathways, such as other death receptor ligands .
  • the term, 'death receptor' refers to members of the TNFR superfamily that contain a death domain, such as TNFR1, Fas receptor, DR4 and DR5.
  • Suitable death receptor ligands include TNF- related apoptosis-inducing ligand (TRAIL), which targets the death receptors DR4 and DR5, and FasL, which targets Fas receptor.
  • TRAIL TNF- related apoptosis-inducing ligand
  • references herein to TRAIL include both native human TRAIL and active fragments and variants thereof that are capable of binding to and
  • FasL includes both native human FasL and active fragments and variants thereof that are capable of binding to and activating Fas receptor.
  • the TNFoi or other inducer of the invention may be directly or indirectly coupled to a cell-binding agent such as an antibody (or antigen-binding fragment thereof) directed against a tumour-related antigen or viral antigen to reduce non-specific activity against non-target cells, as described in Scherf et al . (1996) Clinical Cancer Research 2:1523-31 and Siegemund et al (2012) Cell Death & Disease 3(4) :295 with specific reference to TNFa and. TRAIL.
  • the TNFa or other inducer of the invention (such as a death receptor ligand, such as TNFa, TRAIL or FasL) is fused to the cell-binding agent as a fusion polypeptide.
  • the cell-binding agent is a single chain antibody such as an scFv.
  • the cell- binding agent-coupled inducer (such as a death receptor ligand, such as TNFa, TRAIL or FasL) may be multimeric, such as dimeric as described in
  • TNFa may be fused at its C terminus to a cell-binding agent to reduce binding affinity of the TNFa moiety for TNFR expressed on non-target cells.
  • the TNFa or other inducer is indirectly coupled to a cell-binding agent to reduce non-specific activity
  • the TNFa or other inducer such as a death receptor ligand such as TNFa, TRAIL or FasL
  • a coating such as a lipid coating
  • a cell-binding agent such as an antibody (or antigen-binding fragment thereof) directed against a tumour-related antigen or viral antigen.
  • the TNFa or other inducer (such as a death receptor ligand, such as TNFa, TRAIL or FasL) may be displayed on nanoparticles , which are themselves encapsulated, as described in Messerschmidt et al.
  • TNFa and other inducers of NFkappaB- and related signaling pathways such as death receptor ligands, especially TNFa, TRAIL and FasL.
  • Still other inducers of NFkappaB- and related signaling pathways include agonist anti-death receptors antibodies, such as agonist anti-TNFRl antibodies, agonist anti-Fas receptor antibodies, agonist anti-DR4 antibodies and agonist anti-DR5 antibodies.
  • the antibodies are preferably monoclonal .
  • the TNFa and other inducers of the invention are preferably capable of stimulating apoptosis in cancer cells, in particular in cancer ceils that express death receptors such as TNFR1, Fas receptor, DR4 and/or DR5.
  • the death receptors and ligands are preferably human.
  • the diseased cells of the patient preferably express the corresponding death receptor.
  • TNFa and agonist anti-TNFRl antibodies may be used to treat diseased cells that express TNF receptor 1;
  • TRAIL and agonist anti-DR4 antibodies may be used to treat diseased cells that express DR4 ;
  • TRAIL and agonist anti-DR5 antibodies may be used to treat diseased cells that express DR5;
  • FasL and agonist anti-Fas receptor antibodies may be used to treat diseased cells that express Fas receptor.
  • Labelled antibodies In certain embodiments, the antibodies of the invention, in particular the anti-eEF2 antibodies of the invention, are labelled. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent , and
  • radioactive labels include, but are not limited to, the radioisotopes 32 P, 1 C, 125 I, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases , e.g., firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3- dihydrophthalazinediones , horseradish peroxidase (HRP) , alkaline
  • phosphatase e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate
  • dehydrogenase heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.
  • heterocyclic oxidases such as uricase and xanthine oxidase
  • an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.
  • amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. a . Substitution, Insertion, and Deletion Variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional
  • mutagenesis include the HVRs and FRs .
  • Conservative substitutions are shown in the table below under the heading of "conservative substitutions.” More substantial changes are provided in Table 1 under the heading of "exemplary substitutions,” and as further described below in reference to amino acid side chain classes.
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
  • Amino acids may be grouped according to common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody) .
  • a parent antibody e.g. a humanized or human antibody
  • the resulting variant (s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity) .
  • Alterations may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or SDKs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity.
  • HVR "hotspots” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or SDKs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity.
  • Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom
  • affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis) .
  • a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized.
  • HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling.
  • CDR-H3 and CDR-L3 in particular are often targeted .
  • substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example,
  • each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning
  • mutagenesis as described by Cunningham and Wells (1989) Science, 244:1081- 1085.
  • a residue or group of target residues e.g., charged residues such as arg, asp, his, lys, and glu
  • a neutral or negatively charged amino acid e.g., alanine or
  • polyalanine to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody. b .
  • Glycosylation variants include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed. Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al . TIBTECH 15:26-32 (1997). The
  • oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc) , galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
  • antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream, of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos . US 2003/0157108
  • knockout cell lines such as alpha-1, 6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al . Biotech. Bioeng. 87: 614 (2004); anda, Y. et al . , Biotechnol. Bioeng., 94 ( ) : 680-688 (2006); and WO2003/085107 ) .
  • Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.) . Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided.
  • Such antibody variants may have improved CDC function.
  • Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S . ) ; and WO 1999/22764 (Raju, S.) . c .
  • Fc region variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S . ) ; and WO 1999/22764 (Raju, S.) . c . Fc region variants
  • one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
  • the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96 ® non-radioactive cytotoxicity assay (Promega, Madison, WI ) .
  • 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 in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Nat' 1 Acad. Sci.
  • Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)) .
  • FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al . , Int'l. Immunol. 18 ( 12 ) : 1759-1769 (2006)) .
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056) .
  • Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581) .
  • Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al . , J. Biol. Chem. 9(2) : 6591-6604 (2001) .)
  • an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues) .
  • alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No.
  • Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus are described in US2005/0014934A1 (Hinton et al . ) .
  • Those antibodies comprise an Fc region with one or more
  • Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826) .
  • cysteine engineered antibodies e.g., "thioMAbs”
  • one or more residues of an antibody are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the antibody.
  • reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker- drug moieties, to create an immunoconjugate, as described further herein.
  • any one or more of the following residues may be substituted with cysteine: V205 ( abat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.
  • Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No. 7,521,541. e . Antibody Derivatives
  • an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l, 3-dioxolane, poly-1 , 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyarninoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone ) polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., g
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc .
  • conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided.
  • the nonproteinaceous moiety is a carbon nanotube (Kam et al . , Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)) .
  • the radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.
  • an antibody provided herein is an antibody fragment.
  • Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab' ) 2r Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003) . For a review of scFv fragments, see, e.g.,
  • Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;
  • Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl) .
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein .
  • recombinant host cells e.g. E. coli or phage
  • an antibody provided herein is a chimeric antibody.
  • Certain chimeric antibodies are described, e.g., in. U.S. Patent No. 4,816,567; and Morrison et al . , Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)) .
  • a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region.
  • a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody.
  • Chimeric antibodies include antigen-binding fragments thereof.
  • a chimeric antibody is a humanized antibody.
  • a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non- human antibody.
  • a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
  • a humanized antibody optionally will also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit” method (see, e.g., Sims et al . J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al . Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J.
  • an antibody provided herein is a human antibody.
  • Human antibodies can be produced using various techniques known in the art.
  • Human antibodies are described generally in van Di k and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008) .
  • Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge.
  • Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes.
  • the endogenous immunoglobulin loci which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes.
  • the endogenous immunoglobulin loci which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes
  • Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.
  • Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., ozbor J. Immunol., 133: 3001 (1984); Brodeur et al . , Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991) .) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) . Additional methods include those described, for example, in U.S. Patent No. 7,189,826
  • Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.
  • Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al . in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, e.g., in the McCafferty et al . , Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al . , J. Mol . Biol. 222: 581-597 (1992); Marks and
  • repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455
  • Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.
  • Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas .
  • the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993) .
  • naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381- 388 (1992) .
  • Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.
  • Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.
  • an antibody provided herein is a multispecific antibody, e.g. a bispecific antibody.
  • Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites.
  • one of the binding specificities is for linear linked polyubiquitin and the other is for any other antigen.
  • bispecific antibodies may bind to two different epitopes of linear linked polyubiquitin.
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express linear linked polyubiquitin.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
  • Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al . , EMBO J. 10: 3655 (1991)), and "knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168) .
  • diabody technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g. Gruber et al . , J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991) .
  • Engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies,” are also included herein (see, e.g.
  • the antibody or fragment herein also includes a "Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to linear linked
  • polyubiquitin as well as another, different antigen (see, US 2008/0069820, for example ) .
  • DPH gene sequences cDNA sequences of the DPH1-7 genes are available via the following NCBI accession numbers and may be used for the generation of knockout cells, for example by miRNA, zinc finger nuclease, RNAi or CRISPR/Cas 9-mediated gene knockdown :
  • DPH3 NM_206831 (transcript variant 1); M_001047434 (transcript variant 2)
  • DPH5 NM_001077394 (transcript variant 1); NM_015958 (transcript variant 2); NM_001077395 (transcript variant 3)
  • DPH6 NM_080650 (transcript variant 1); NM_001141972 (transcript variant 2)
  • the zinc finger nuclease sequences used in the examples were chosen to permit knockdown of all known variants.
  • the DPH gene sequences are preferably those of the most recent version of the database entries as at 24 June 2015.
  • Targeted therapeutic agents of the invention that comprise a NAD(+)- diphthamide ADP-ribosyltransferase coupled to a cell-binding agent (in particular those coupled by fusion) , and anti-eEF2 antibodies of the invention, may be obtained, for example, by solid-state peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production.
  • solid-state peptide synthesis e.g. Merrifield solid phase synthesis
  • one or more polynucleotides together encoding the targeted therapeutic agent or antibody are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • Such polynucleotide may be readily isolated and sequenced using conventional procedures . Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequence (s) of the targeted therapeutic agent or antibody along with appropriate
  • transcriptional/translational control signals include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Maniatis et al . , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al . , Current
  • the expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment.
  • the expression vector includes an expression cassette into which the polynucleotide encoding the targeted therapeutic agent or antibody (i.e. the coding region) is cloned in operable association with a promoter and/or other transcription or translation control elements.
  • a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids.
  • a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5' and 3' untranslated regions, and the like, are not part of a coding region.
  • Two or more coding regions can be present in a single polynucleotide construct, e.g. on a single vector, or in separate polynucleotide constructs, e.g. on separate (different) vectors.
  • any vector may contain a single coding region, or may comprise two or more coding regions, e.g.
  • a vector of the present invention may encode one or more polypeptides, which are post- or co-translationally separated into the final protein via proteolytic cleavage (for example in the case of antibodies of the invention that are composed of multiple chains, or in the case of targeted therapeutic agents in which the NAD(+)- diphthamide ADP ribosyltransferase is coupled to the cell-binding agent via a disulphide bond, rather than as a fusion polypeptide via a peptide bond) .
  • a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a
  • heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.
  • An operable association is when a coding region for a gene product, e.g. a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence (s) .
  • Two DNA fragments are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
  • a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid.
  • the promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells.
  • transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.
  • Suitable promoters and other transcription control regions are disclosed herein.
  • a variety of transcription control regions are known to those skilled in the art. These include, without limitation,
  • transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g. the immediate early promoter, in conjunction with intron-A) , simian virus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Rous sarcoma virus) .
  • Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit a-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as inducible promoters (e.g.
  • promoters inducible tetracycline inducible tetracycline
  • translation control elements include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence) .
  • the expression cassette may also include other features such as an origin of replication, and/or chromosome integration elements such as retroviral long terminal repeats (LTRs), or adeno-associated viral (AAV) inverted terminal repeats ( ITRs ) .
  • LTRs retroviral long terminal repeats
  • AAV adeno-associated viral
  • Polynucleotide and nucleic acid coding regions may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of the targeted therapeutic agent or antibody of the present invention.
  • DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding the targeted therapeutic agent or antibody of the invention.
  • polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or "mature" form of the polypeptide.
  • the native signal peptide e.g. an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it.
  • a heterologous mammalian signal peptide may be used.
  • the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse ⁇ -glucuronidase .
  • TPA tissue plasminogen activator
  • DNA encoding a short protein sequence that could be used to facilitate later purification (e.g. a histidine tag) or assist in labeling the targeted therapeutic agent or antibody may be included within or at the ends of the targeted therapeutic agent or antibody.
  • the term "host cell” refers to any kind of cellular system which can be engineered to generate the targeted therapeutic agent or antibody of the invention.
  • Host cells suitable for replicating and for supporting expression of the targeted therapeutic agents or antibodies of the invention are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the targeted therapeutic agent or antibody for clinical applications.
  • Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO) , insect cells, or the like.
  • polypeptides may be produced in bacteria in particular when glycosylation is not needed. After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of a polypeptide with a partially or fully human glycosylation pattern.
  • fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of a polypeptide with a partially or fully human glycosylation pattern.
  • Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates) . Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of
  • Plant cell cultures can also be utilized as hosts. See e.g. US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTM technology for producing
  • Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham et al .
  • TM4 cells mouse Sertoli cells (TM4 cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK) , buffalo rat liver cells (BRL 3A) , human lung cells (W138), human liver cells (Hep G2 ) , mouse mammary tumour cells (MMT 060562), TRI cells (as described, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68
  • MRC 5 cells MRC 5 cells
  • FS4 cells MRC 5 cells
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr " CHO cells
  • Host cells include cultured cells, e.g., mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
  • the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NSO , Sp20 cell) .
  • CHO Chinese Hamster Ovary
  • HEK human embryonic kidney
  • a lymphoid cell e.g., Y0, NSO , Sp20 cell
  • the NAD ( + ) -diphthamide ADP ribosyltransferases of the invention are produced in prokaryotic cells, such as E. coli. Standard technologies are known in the art to express foreign genes in these systems.
  • Cells expressing a polypeptide comprising either the heavy or the light chain of an antigen binding domain such as an antibody may be engineered so as to also express the other of the antibody chains fused to a NAD (+) -diphthamide ADP ribosyltransferase such that the expressed product is an antibody that has both a heavy and a light chain.
  • a method of producing a targeted therapeutic agent or antibody according to the invention may comprise culturing a host cell comprising a
  • polynucleotide encoding the targeted therapeutic agent or antibody under conditions suitable for expression of the targeted therapeutic agent or antibody, and recovering the targeted therapeutic agent or antibody from the host cell (or host cell culture medium) .
  • the components of the targeted therapeutic agent or antibody may be genetically fused to each other.
  • the targeted therapeutic agent or antibody can be designed such that its components are fused directly to each other or indirectly through a linker sequence.
  • the composition and length of the linker may be determined in accordance with methods well known in the art and may be tested for efficacy.
  • the antibodies of the invention and, in certain embodiments, the antibodies that form part of the targeted therapeutic agents of the invention comprise at least an antibody variable region capable of binding an antigenic determinant.
  • Variable regions can form part of and be derived from naturally or non-naturally occurring antibodies and fragments thereof.
  • Non-naturally occurring antibodies can be constructed using solid phase-peptide synthesis, can be produced recombinantly (e.g. as described in U.S. patent No. 4,186,567) or can be obtained, for example, by screening combinatorial libraries comprising variable heavy chains and variable light chains (see e.g. U.S. Patent. No. 5,969,108 to McCafferty) .
  • Any animal species of antibody, antibody fragment, antigen binding domain or variable region can be used as an antibody of the invention and/or in the targeted therapeutic agents of the invention.
  • Non-limiting antibodies, antibody fragments, antigen binding domains or variable regions useful in the present invention can be of murine, primate, or human origin. If the targeted therapeutic agent or antibody is intended for human use, a chimeric form of antibody may be used wherein the constant regions of the antibody are from a human.
  • a humanized or fully human form of the antibody can also be prepared in accordance with methods well known in the art (see e. g. U.S. Patent No. 5,565,332 to Winter) .
  • Humanization may be achieved by various methods including, but not limited to (a) grafting the non-human (e.g., donor antibody) CDRs onto human (e.g. recipient antibody) framework and constant regions with or without retention of critical framework residues (e.g. those that are important for retaining good antigen binding affinity or antibody functions), (b) grafting only the non-human CDRs onto human (e.g. recipient antibody) framework and constant regions with or without retention of critical framework residues (e.g. those that are important for retaining good antigen binding affinity or antibody functions), (b) grafting only the non-human
  • SDRs or a-CDRs specificity-determining regions
  • a-CDRs the residues critical for the antibody-antigen interaction
  • transplanting the entire non-human variable domains but "cloaking" them with a human-like section by replacement of surface residues.
  • Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332, 323-329 (1988); Queen et al . , Proc Natl Acad Sci USA 86, 10029-10033 (1989); US Patent Nos .
  • Human antibodies and human variable regions can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008) . Human variable regions can form part of and be derived from, human monoclonal antibodies made by the hybridoma method (see e.g. Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)) .
  • Human antibodies and human variable regions may also be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge (see e.g. Lonberg, Nat Biotech 23, 1117- 1125 (2005). Human antibodies and human variable regions may also be generated by isolating Fv clone variable region sequences selected from human-derived phage display libraries (see e.g., Hoogenboom et al . in Methods in Molecular Biology 178, 1-37 (O'Brien et al .
  • Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.
  • the antibodies of the present invention or that, in certain embodiments, form part of the targeted therapeutic agent of the present invention are engineered to have enhanced binding affinity according to, for example, the methods disclosed in U.S. Pat. Appl. Publ. No. 2004/0132066, the entire contents of which are hereby incorporated by reference.
  • the ability of the targeted therapeutic agent or antibody of the invention to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g.
  • ELISA enzyme-linked immunosorbent assay
  • Competition assays may be used to identify an antibody, antibody fragment, antigen binding domain or variable domain that competes with a reference antibody for binding to a particular antigen.
  • a competing antibody binds to the same epitope (e.g. a linear or a conformational epitope) that is bound by the reference antibody.
  • immobilized antigen is incubated in a solution comprising a first labeled antibody that binds to the antigen and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to the antigen.
  • the second antibody may be present in a hybridoma supernatant.
  • immobilized antigen is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody.
  • Targeted therapeutic agents and antibodies prepared as described herein may be purified by art-known techniques such as high pe.rform.ance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
  • the actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art.
  • affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the targeted therapeutic agent or antibody binds.
  • a matrix with protein A or protein G may be used.
  • Sequential Protein A or G affinity chromatography and size exclusion chromatography can be used to isolate the antibody or targeted therapeutic agent.
  • the purity of the targeted therapeutic agent or antibody can be determined by any of a variety of well known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like.
  • compositions of a targeted therapeutic agent as described herein are prepared by mixing such targeted therapeutic agent having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as 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;
  • buffers such as 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 parab
  • resorcinol cyclohexanol ; 3-pentanol; and m-cresol
  • low molecular weight polypeptides include proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides , and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol;
  • salt-forming counter-ions such as sodium
  • metal complexes e.g. Zn-protein complexes
  • non-ionic surfactants such as polyethylene glycol (PEG).
  • exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP) , for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX ® , Baxter
  • sHASEGPs and methods of use including rHuPH20, are described in US Patent Publication Nos . 2005/0260186 and 2006/0104968.
  • a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases .
  • additional glycosaminoglycanases such as chondroitinases .
  • Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958.
  • Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO2006/044908, the latter formulations including a histidine-acetate buffer.
  • the formulation herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-
  • microcapsules respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions , nano-particles and nanocapsules ) or in macroemulsions .
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions , nano-particles and nanocapsules
  • macroemulsions for example, macroemulsions .
  • Sustained-release preparations may be prepared. Suitable examples
  • sustained-release preparations include semipermeable matrices of hydrophobic polymers containing the targeted therapeutic agent, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • TNFa or other inducer A recombinant form of TNFa known as tasonermin is available from Boehringer Ingelheim as BeromunTM, which is approved as an anti-neoplastic and is formulated for injection or infusion.
  • the targeted therapeutic agents may be used in therapeutic methods.
  • a targeted therapeutic agent can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are
  • Targeted therapeutic agents would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the targeted therapeutic agent need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in guestion. The effective amount of such other agents depends on the amount of targeted therapeutic agent present in the formulation, the type of disorder or treatment, and other factors discussed above.
  • a targeted therapeutic agent of the invention will depend on the type of disease to be treated, the type of targeted therapeutic agent, the severity and course of the disease, whether the targeted therapeutic agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the targeted therapeutic agent, and the discretion of the attending physician.
  • the targeted therapeutic agent is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg/kg to 15 mg/kg (e.g. 0.
  • lmg/kg-lOmg/kg) of the targeted therapeutic agent can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • One exemplary dosage of the targeted therapeutic agent would be in the range from about 0.05 mg/kg to about 10 mg/kg.
  • one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg may be administered to the patient.
  • Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g.
  • an article of manufacture containing materials useful for the treatment and/or prevention of the disorders described above is provided.
  • the article of manufacture comprises
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating and/or preventing the condition 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) .
  • At least one active agent in the composition is a targeted therapeutic agent of the invention.
  • the label or package insert indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • a pharmaceutically-acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • Example 1 Generation of MCF-7 cells with heterozygous or completely inactivated DPH genes
  • ZFN Zinc finger nucleases
  • transfected cells were exposed to lethal doses of PE (100 nM) , sufficient to kill all MCF-7 cells whose eEF2 is a substrate for the toxin. After additional 48 hrs, dead cells were removed and the culture further propagated in toxin containing media. This procedure generated colonies in cells that were transfected with ZFN that target DPHl, DPH2, DPH4 and DPH5 ( Figure 1) . These were isolated and re-cloned from single cells. No colonies were obtained under toxin selection in cells that were mock transfected, or with ZFNs that target DPH3 , DPH6, and DPH7.
  • the 'genetic screen' approach flow delivered clones that had one gene copy inactivated and another functional (wildtype) gene for all DPH genes (Table 1) . Also, clones that had both genes inactivated with a different mutation on each allele were obtained for DPH4 and DPH5. For the DPH3, DPH6 and DPH7 genes, clones that had both genes inactivated could not be obtained in repeated attempts, even though the ZFNs were effective (generated heterozygotes ) and the number of colonies screened delivered complete knockouts for other DPH genes.
  • Example 2 Generation and characterization of antibodies that specifically detect unmodified eEF2
  • Antibodies that specifically detect eEF2 without diphthamide, but do not bind eEF2 containing diphthamide are highly desired for the analysis of the diphthamide status of tumor cells. So far, such antibodies validated in extracts of tumor cells or on tumor tissues are not available due to lack of (positive and appropriate negative) control reagents. Applying our set of knockout cell lines, we applied a rabbit immunization and subsequent B- cell-cloning procedure [49] for the generation of antibodies that
  • TLHADAIHRGGGQI I PT SEQ ID NO: 72
  • B-cells expressing peptide-binding antibodies were isolated by FACS (using fluorescence-labeled eEF2 peptide), and subsequently converted to
  • Figure 2 shows Western blots of 8 different antibody candidates on extracts from parent MCF-7 cells (contain only diphthamide-modified eEF2), and MCF-7 DPHlko-ko cells which contain only eEF2 without diphthamide modification as
  • Example 3 Combination of antibody-based, mass-spec based and enzyme based methods to comprehensively address diphthamide modifications at H715 of eEF2
  • Antibody-based assays Extracts of MCF-7 and DPH knockout cells were subjected to Western blot analyses with a rabbit monoclonal antibody that detects specifically only unmodified but not diphthamide-modified eEF2 (see Example 2 & Figure 3 ⁇ ) . Western blot analyses of ceil extracts with this antibody enables the determination of relative amounts of eEF2 without diphthamide. To detect actin and total human eEF2 as controls, monoclonal Anti-p-Actin (AC-74, Sigma) and eEF2 (H-118, Santa Cruz) were applied.
  • cell extracts for analyses of eEF2 modifications were prepared by lysing cells (4xl0 5 cells in 6-well dishes) in RIPA buffer. Then, 100 ⁇ g of proteins were reduced with DTE and alkylated using iodoacetarrtide and further digested with trypsin. Resulting peptides were desalted using solid phase extraction and approximately 1 ⁇ g of the digest was analyzed by nanoLC-ESI-MS using Parallel Reaction Monitoring on a Thermo Q-Exactive Plus mass spectrometer (Thermo Electron, San Jose, USA) . The most abundant precursor ions of the tryptic peptides F702-R716 containing the native or modified His7is at mass-over-charge 437.2234 (native peptide), 462.4854
  • diphtham.ide The diphthine intermediate was not found in any of the cells. If it had been present, it would also have been detected and its relative level determined, with a mass-over-charge of
  • inactivated MCF-7 derivatives DPH1-7 .
  • eEF2 from cells that have completely inactivated DPH1 or DPH2 or DPH4 or DPH5 genes is not amenable to toxin-mediated ADP-ribosylation. This confirms that only eEF2 with diphthamide is a substrate for ADP-ribosylating toxins.
  • EEF2 without modification (DPH1, 2, 4) or with partial modification (ACP in DPH5) shows no evidence for ADP-ribosylation. This indicates that such eEF2 forms are no toxin substrates at all, and that there is no remaining toxin activity towards these eEF2 forms.
  • Example 4 Measuring the influence of heterozygous and complete DPH gene inactivations on toxin sensitivity
  • CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega
  • Absolute resistance correlates with complete absence of diphthamide-modified eEF2 and lack of ADP-ribosylation (Fig. 3) . This indicates that cytotoxicity inferred by PE and DT is solely due to diphthamide-dependent ADP-ribosylation of eEF2. PE or DT does not harbor other complementary or additional cytotoxic
  • MCF-7 derivatives that contained one inactivated and one functional DPH gene copy (DPH1-DPH7) remained fully sensitive to PE and DT .
  • Cytotoxicity assays revealed no significant difference in the IC50 concentrations of these toxins.
  • MCF-7 derivatives contain as the major species diphthamlde-modified eEF2, toxin sensitivity correlates with the presence of eEF2-diphthamide .
  • heterozygous DPH1 or DPH2 clones which contain significant amounts of unmodified toxin-resistant eEF2 remain as sensitive to the toxins as the wildtype cells which contain exclusively toxin-sensitive eEF2.
  • the DPH gene-dose modulates the relative amount of unmodified eEF2 in MCF-7 cells, but does not affect their toxin sensitivity.
  • Example 5 Relevance of DPH genes for diphthamide synthesis and sensitivity towards ADP-ribosylating toxins:
  • diphthamide has previously been described in yeast and other cells [4_, _5, 3_5] . These defined the proteins encoded by 7 genes, DPH1-DPH7 to be necessary for eEF2 modification [ 5 ] . Further reports describe that modulation of expression levels or inactivation of single DPH genes can reduce cellular sensitivity towards toxins that ADP-ribosylate diphthamide-eEF2 [36-38] .
  • the combined evidence clearly supports the existing diphthamide synthesis scheme in eukaryotic cells. However, the 'complete picture' is (with the exception of the yeast pathway) to a large portion composed of observations made in different cell types on single genes.
  • MCF-7 derivatives with defined inactivation of DPH genes enabled a comprehensive assessment of their relative contribution in mammalian cells in a defined cellular background.
  • Our set of MCF-7 derivatives contains heterozygotes still retaining one functional allele to study gene dose effects for all genes, as well as complete knockouts with no functional gene copy left for DPH1, DPH2, DPH4 and DPH5.
  • DPH5 is the sole diphthine synthase in MCF-7 and its absence does not prevent ACP generation.
  • MCF-7 cells with complete DPH3, DPH6 or DPH7 deficiency were not obtained. Because of that, effects of their complete inactivation in MCF-7 could not be determined. Cells with partial inactivation (gene dose reduction) with one functional allele still present, however, were obtained. These contained as major species
  • diphthamide-modified eEF2 This proves that loss of function of one allele of DPH3, DPH6 and DPH7 has no major impact on diphthamide synthesis.
  • Gene dose effects i.e. reduced diphthamide generation in cells with one inactivated allele, were observed for DPHI and DPH2.
  • unmodified eEF2 was detectable (up to 25% of total eEF2) in addition to diphthamide-eEF2. Because only diphthamide-eEF2 serves as target for toxin- mediated ADP-ribosylation, gene dose effects that influence
  • diphthamidylation could be relevant for tumor therapy with targeted toxins.
  • alterations of the human DPHI (0VCA1) gene are described for various cancers [12, 25, 3_9, 40], although their impact on diphthamide modification of eEF2 and toxin sensitivity has not been quantified so far.
  • our analyses in MCF-7 cells revealed a gene dose dependent modulation of the diphthamide content of the cellular eEF2 pool, but in the same cells no significant impact towards sensitivity of cells towards ADP- ribosylating toxins PE and DT .
  • a striking example for that are heterozygous deficient DPH2 clones which contain 25% unmodified eEF2 yet are sensitive as parent MCF-7.
  • ADP-ribosylation may trigger (signaling?) events that interrupt protein synthesis even though unmodified translation-competent eEF2 is still available.
  • Example 6 Comprehensive analyses of the effect of diphthamide on cell growth and cellular sensitivity towards protein synthesis inhibitors which do not target eEF2
  • the common denominator of ail MCF-7 derivatives with different DPH gene defects is loss of diphthamide.
  • the generated set of cell lines with each having a different dph gene inactivated can not only specifically address the relative impact of each of these genes on toxin sensitivity, but also generally address the biological function of the diphthamide modification itself.
  • Cytology indicated that morphology and chromosome composition of the individual clones did not diverge from that of the MCF-7 cells from which they were derived. Since loss of diphthamide was achieved by inactivating different genes, any common biological effect observed in these cell lines should be attributable to the loss of diphthamide, and not to loss of individual gene function or potential compensatory effects.
  • Example 7 Application of the 'diphthamide tool box provides evidence for diphthamide dependent NFkappaB and death receptor pathway activation
  • ADP-ribosylation of eEF2 stalls protein synthesis and subsequently triggers induction of apoptosis in MCF-7 cells [30, 31] .
  • the CSE1L protein (identified in a screen for toxin modulators [32—34] ) influences not only PE and DT cytotoxicity in MCF-7 cells, but also their sensitivity towards TNFa induced apoptosis [34] .
  • both processes diphthamide-dependent ADP-ribosylation and TNFa. triggered apoptosis
  • IC50 values listed in table 2 show that MCF-7 cells which have at least one functional copy of each DPH gene and therefore possess diphthamide are as sensitive to TNFa as parent cells.
  • cells with complete inactivation of DPH1, DPH2, DPH4 or DPH5 have an increased sensitivity towards TNFa.
  • Hypersensitivity was observed for all clones carrying complete inactivation of D?H1 or DPH2 or DPH4 or DPH5.
  • the common denominator of all MCF-7 derivatives with these DPH gene defects is loss of diphthamide.
  • TNFa-hypersensitivity is attributable to loss of diphthamide.
  • TNFa hypersensitivity cannot be explained by altered surface expression of the TNF receptor as FACS analyses of parent and DPH ko cells revealed no differences in TNFR cell surface signals.
  • RNA sequencing was applied to analyze the complete transcriptome of parent and mutated MCF7 derivatives.
  • Total RNA was extracted and purified using the High Pure RNA Isolation Kit (Roche) according to the manufacturer's instructions. For all samples high-quality RNA was obtained (RIN >9.5) .
  • RNA-seq libraries were prepared from 250 ng of total RNA using the TruSeq Stranded Total RNA preparation Kit (Illumina) following the manufacturer's instructions. Sequencing libraries were quantified and quality controlled on Bioanalyzer using High Sensitivity chips (Agilent Technologies) and on Qubit using dsDNA HS Assay Kit (Life Technologies) .
  • hypersensitivity should be similar in DPH5 ko and DPH2ko, yet be different in parent MCF-7. Therefore, we identified genes that become differently expressed (> log+1, ⁇ log-l, p ⁇ 0.01) between parent and DPH2ko cells, and between parent and DPH5ko cells.
  • a comparison of both gene sets revealed an overlap of gene expression/pathway patterns that become induced upon inactivation of DPH2 and DPH5.
  • genes with highest induction levels upon inactivation of dph2 or dph5 11 were identical, i.e. highly induced upon inactivation of diphthamide synthesis irrespective of which gene has been compromised (Table 3) .
  • Example 8 Application of the 'diphthamide tool box f J ⁇ o_ address the biological function of diphthamide in mammalian cells
  • the set of MCF-7 derivatives that we generated are based on the same genetic background, retain cell shape and (with exception of complete DPH5 deficiencies) good growth properties, yet still have different genes inactivated.
  • DPH5 ko cells lack diphthamide and
  • diphthamide-eEF2 carries a diphthamide and all eEF2 molecules within MCF-7 cells carry this modification.
  • diphthamide-eEF2 enables translation of all essential proteins.
  • Diphthamide deficient cells complete knockout of DPHl, DPH2, DPH4 , and DPH5 are also viable, indicating that eEF2 without diphthamide supports also the translation of all essential proteins .
  • diphthamide on eEF2 is highly conserved in all eukaryotes as well as in archea [4_2] , it is surprising that lack of diphthamide synthesis has little impact on growth of cultured MCF-7. In contrast, animals with homozygous DPH knockouts do not survive beyond embryonic stages [9, 12-14 ] . This suggests that the diphthamide may be necessary for development.
  • diphthamide synthesis deficient cells (independent from target gene knockout) were hypersensitive to TNFa induced apoptosis, correlating with 'pre-induction' of interferon/NFkappaB and death receptor signaling pathways in those cells. This suggests that absence of the diphthamide- modification affects these signaling pathways. Signaling pathway pre- activation and TNFalpha hypersensitivity upon loss of diphthamide could be due to secondary effects on eEF2 phosphorylation or modulation of eEF2- dependent stress responses [[2, 44-46] 47] . One direct effect, however, could be that eEF2 without diphthamide generates some defective or altered proteins e.g. by allowing translational slippage [6, 9], whose presence subsequently causes pathway induction (although we did not observe induction of genes associated with the unfolded protein response (UPR) ) . It is also possible that loss of diphthamide impacts the translation
  • a NAD (+) -diphthamide ADP ribosyltrans ferase for use in a method of medical treatment of a patient from whom a sample containing diseased cells has given a positive result in an assay for the presence of eEF2 protein having diphthamide modification at the His715 residue.
  • a NAD (+) -diphthamide ADP ribosyltransferase for use in a method of medical treatment of a patient from whom a sample containing diseased cells has been assayed for the presence of eEF2 protein having diphthamide modification at the His715 residue and assessed as sensitive to NAD(+)- diphthamide ADP ribosyltransferase treatment.
  • NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 103 to 111, wherein said monoclonal antibody is labelled with a detectable label.
  • ribosyltransferase is a PE toxin, a DT toxin or a cholix toxin.
  • NAD (+) -diphthamide ADP ribosyltransferase for use of paragraph 117, wherein the DT toxin has a polypeptide sequence comprising a DT functional domain I having at least 50% amino acid sequence identity over the full length of residues 1-191 of SEQ ID NO : 3 , wherein the DT toxin has cytotoxic activity when introduced into a mammalian cell.
  • ribosyltrans ferase is a PE toxin or a DT toxin. 122.
  • NAD (+) -diphthamide ADP ribosyltransferase is a PE toxin.
  • PE toxin has the following structure: FCSi - ⁇ ⁇ - R 2 n - R 3 p - PE functional domain III - R 4 q wherein :
  • 1, m, n, p and q are each, independently, 0 or 1;
  • FCS is a furin-cleavable sequence, preferably (i) R-H-R-Q-P-R-G-W-E- Q-L (SEQ ID NO: 31) or a truncated version thereof containing R-Q-P-R(SEQ ID NO: 53), optionally R-Q-P-R(SEQ ID NO: 53), R-H-R-Q-P-R-G-W ( SEQ ID NO: 54), R-H-R-Q-P-R-G-W-E (SEQ ID NO: 55), H-R-Q-P-R-G-W-E-Q ( SEQ ID NO: 56), or R-Q- P-R-G-W-E (SEQ ID NO: 57); or (ii) R-H-R-S-K-R-G-W-E-Q-L (SEQ ID NO: 43) or a truncated version thereof containing R-S-K-R(SEQ ID NO: 58), optionally R-
  • R 2 is one or more consecutive amino acid residues of residues 285-364 of SEQ ID N0:1, in which any one or more of residues E285, P290, L294, L297, Y298, L299, R302, R313, N314, P319, D324, E327, E331 and Q332, where present, is/are optionally independently replaced by another amino acid, preferably glycine, serine, alanine or glutamine;
  • R 3 is one or more consecutive amino acid residues of residues 365-394 of SEQ ID NO:l;
  • PE functional domain III comprises residues 395-613 of SEQ ID NO:l in which : (a) some or all of residues 602-608 are optionally deleted,
  • residues 609-613 are optionally replaced by another ER localisation sequence, preferably KDEL(SEQ ID NO: 34), REDL ( SEQ ID NO: 35), RDEL (SEQ ID NO: 36) or KEDLK (SEQ ID NO: 37) , (c) any one or more of residues D403, D406, R412, E420, R421,
  • K606 is/are optionally independently replaced by another amino acid, preferably glycine, serine, alanine or glutamine, or histidine in the case of L477;
  • R 4 is one or more (preferably 1 or 2) additional ER localisation sequences, preferably REDLK ( SEQ ID NO: 33), KDEL ( SEQ ID NO: 34), REDL ( SEQ ID NO: 35), RDEL ( SEQ ID NO: 36) or KEDLK ( SEQ ID NO: 37) .
  • NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 123 to 126, wherein p is 0. 128.
  • the NAD (+) -diphthamide ADP ribosyltrans ferase for use of any one of paragraphs 123 to 127, wherein q is 0.
  • NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 123 to 129, wherein the PE toxin comprises the amino acid sequence of SEQ ID NO:110 or SEQ ID NO:lll.
  • NAD (+) -diphthamide ADP ribosyltransferase for use of paragraph 130, wherein the amino acid sequence of SEQ ID NO: 110 or SEQ ID NO: 111 is fused to the C-terminal end of the amino acid sequence of SEQ ID NO: 112.
  • the NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 101 to 131, wherein the NAD (+) -diphthamide ADP
  • ribosyltransferase is coupled to a cell-binding agent targeted to diseased cells of the patient.
  • ribosyltrans ferase is coupled to the cell-binding agent as a fusion polypeptide .
  • NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 132 to 134, wherein the cell-binding agent is an antibody.
  • NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 101-139, which is for use in the treatment of a pre-cancer, cancer, tumour, viral infection or autoimmune disease.
  • a NAD (+) -diphthamide ADP ribosyltrans ferase for use in a method of medical treatment of a patient having a condition that is treatable by cytotoxic activity targeted to diseased cells of the patient, wherein the method is as defined in any one of claims 4 and 6-50.
  • a monoclonal anti-eEF2 antibody wherein the antibody binds to eEF2 that is unmodified at the His715 residue with higher binding affinity than to eEF2 having diphthamide modification at the His715 residue.
  • CDR heavy chain complementarity-determining region
  • the monoclonal antibody according to paragraph 220 having the heavy chain CDRs HI, H2 and H3 of the heavy chain variable domain sequence shown in Figure 6 (SEQ ID NOs:104 to 106).
  • the monoclonal antibody according to paragraph 222 having the light chain CDRs L2 and L3 of the light chain variable domain sequence shown in Figure 6 (SEQ ID NOs:108 and 109) .
  • the monoclonal antibody according to paragraph 223 having the light chain CDRs LI, L2 and L3 of the light chain variable domain sequence shown in Figure 6 (SEQ ID NOs:107 to 109) .
  • apoptosis is mediated by death receptor activation-mediated apoptosis.
  • the death receptor is TNFR1, Fas receptor, DR4 or DR5.
  • apoptosis is agonist anti-TNFRl antibody-mediated apoptosis, agonist anti- Fas receptor antibody-mediated apoptosis, agonist anti-DR4 antibody- mediated apoptosis, or agonist anti-DR5 antibody-mediated apoptosis.
  • a monoclonal anti-eEF2 antibody that binds to eEF2 having diphthamide modification at the His715 residue with higher binding affinity than to eEF2 that is unmodified at the His715 residue.
  • the detectable label is an enzyme, a fluorescent label, a radiolabel, an electroluminescent label or biotin.
  • a method for assessing increased sensitivity of diseased cells in a patient to treatment with TNFa or other inducer of NFkappaB-signalling pathways or related signalling pathways (“other inducer”) comprising : assaying for the proportion of eEF2 protein that lacks diphthamide modification at the His715 residue in a sample containing the diseased cells , wherein the presence of a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue is indicative that the diseased cells have increased sensitivity to treatment with TNFa or other inducer compared to cells in which eEF2 protein lacking diphthamide modification is substantially absent.
  • paragraph 402 or paragraph 403 which further includes a step, following selection of the patient for treatment, of treating the patient with the TNFa or other inducer.
  • a method for selecting and/or deselecting a patient for treatment with TNFa or other inducer comprising:
  • a method for treating a patient having a condition that is treatable by TNFa or other inducer comprising: assaying a sample containing diseased cells from a patient for the proportion of eEF2 protein that lacks diphthamide modification at the His715 residue; and treating a patient in whose sample the assay is positive for the presence of a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue with TNFa or other inducer. 408.
  • a method for treating a patient having a condition that is treatable by TNFa or other inducer comprising: assaying for the presence of a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue in a sample containing diseased cells from the patient; assessing whether the diseased cells have increased sensitivity to treatment with TNFa compared to cells in which eEF2 protein lacking diphthamide modification is substantially absent, wherein the presence of a significant portion of eEF2 protein lacking diphthamide modification at the His715 residue is indicative that the diseased cells have increased sensitivity to treatment with TNFa or other inducer and/or wherein the absence of a significant portion of eEF2 protein lacking diphthamide modification at the His715 residue is indicative that the diseased cells do not have increased sensitivity to treatment with TNFa or other inducer; and treating a patient whose diseased cells are assessed to have increased sensitivity with TNFa or other inducer.
  • a method for treating a patient having a condition that is treatable with TNFa or other inducer comprising: treating the patient with TNFa or other inducer, wherein the patient is selected for treatment with TNFa or other inducer on the basis of a positive assay result for the presence of a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue in a sample containing diseased cells from the patient.
  • modification at the His715 residue is performed using a monoclonal antibody that binds to eEF2 that is unmodified at the His715 residue with higher binding affinity than to eEF2 having diphthamide modification at the His715 residue . 411. The method according to paragraph 410, wherein the monoclonal antibody substantially does not bind to eEF2 having diphthamide
  • the antibody is an agonist monoclonal anti-TNFRl antibody, an agonist monoclonal anti-Fas receptor antibody, an agonist monoclonal anti-DR4 antibody or an agonist monoclonal anti-DR5 antibody.
  • TNFa or other inducer for use in a method of medical treatment of a patient from whom a sample containing diseased cells has given a positive result in an assay for the presence of a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue.
  • TNFa or other inducer for use in a method of medical treatment of a patient from whom a sample containing diseased cells has been assayed for the presence of a significant proportion of eEF2 protein lacking
  • diphthamide modification at the His715 residue assessed as having increased sensitivity to TNFa or other inducer compared to cells in which eEF2 protein lacking diphthamide modification is substantially absent.
  • paragraph 505 The TNFa or other inducer for use of paragraph 503 or paragraph 504, wherein the monoclonal antibody binds to eEF2 that is unmodified at the His715 residue with higher binding affinity than to eEF2 having 3-amino-3- carboxypropyl (ACP) modification and/or diphthine modification at the His715 residue.
  • ACP 3-amino-3- carboxypropyl
  • ACP 3-amino-3- carboxypropyl
  • the assay comprises subjecting an extract of the sample to a sandwich assay comprising said monoclonal antibody as a capture antibody or detection antibody.
  • TNFa or other inducer for use of any one of paragraphs 501 to 517, wherein the TNFa or other inducer is a death receptor ligand.
  • TNFa or other inducer for use of paragraph 520 wherein the ligand is human TNFa or an active fragment or variant thereof that binds to and activates human TNF receptor 1.
  • TNFa or other inducer for use of paragraph 520 wherein the ligand is human TRAIL or an active fragment or variant thereof that binds to and activates human DR4 and/or DR5.
  • TNFa or other inducer for use of paragraph 524 wherein the other inducer is an agonist monoclonal anti-TNFRl antibody, an agonist monoclonal anti-Fas receptor antibody, an agonist monoclonal anti-DR4 antibody or an agonist monoclonal anti-DR5 antibody.
  • TNFa or other inducer for use of any one of paragraphs 501 to 525, wherein the TNFa or other inducer is directly or indirectly coupled to a cell-binding agent.
  • TNFa or other inducer for use of paragraph 526, wherein the TNFa or other inducer is directly coupled to the cell-binding agent as a fusion polypeptide .
  • TNFa or other inducer for use of paragraph 526, wherein the TNFa or other inducer is encapsulated with a coating that bears a cell-binding agent .
  • TNFa or other inducer for use of any one of paragraphs 501 to 531, wherein the diseased cells are pre-cancer, cancer or tumour cells or virally-infected cells.
  • TNFa or other inducer for use of any one of paragraphs 501-533 which is for use in the treatment of a pre-cancer, cancer, tumour or viral infectio .
  • TNFa or other inducer for use of paragraph 534 which is for use in the treatment of a pre-cancer, cancer or tumour.
  • TNFa or other inducer for use in a method of medical treatment of a patient, wherein the method is as defined in any one of paragraphs 404 and 406 to 444. While there are shown and described presently preferred embodiments of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims.
  • diphthamide biosynthesis is essential in mouse development .
  • Molecular and cellular biology 2006. 26(10) : p. 3835-41.
  • Pseudomonas exotoxin A-mediated apoptosis is Bak dependent and preceded by the degradation of Mcl-1. Molecular and cellular biology, 2010. 30(14) : p. 3444-52.
  • chromosome-segregation gene CSE1 in proliferation, apoptosis , and cancer.
  • mutagenesis-based forward genetic approach reveals that the tumor suppressor OVCAl is a component of the biosynthetic pathway of diphthamide on elongation factor 2.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Urology & Nephrology (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biochemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Oncology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention se base sur la découverte que des cellules contenant le facteur 2 d'élongation eucaryote (eEF2) qui ne présente pas la modification du diphtamide au niveau du résidu His715 restent sensibles à la destruction par PE lorsque les cellules contiennent également le facteur eEF2 présentant la modification du diphtamide au niveau du résidu His715. Par conséquent, l'invention concerne des procédés permettant d'évaluer la sensibilité et/ou la résistance de cellules malades chez un patient à un traitement avec une NAD(+)-diphtamide ADP-ribosyl-transférase, le procédé consistant à analyser la présence de la protéine eEF2 présentant la modification du diphtamide au niveau du résidu His715 dans un échantillon contenant les cellules malades, la présence de la protéine eEF2 présentant la modification du diphtamide au niveau du résidu His715 indiquant que les cellules malades sont sensibles à un traitement avec une NAD(+)-diphtamide ADP-ribosyl-transférase et/ou l'absence de la protéine eEF2 présentant la modification du diphtamide au niveau du résidu His715 indiquant que les cellules malades sont résistantes à un traitement avec une NAD(+)-diphtamide ADP-ribosyl-transférase. L'invention porte également sur la NAD(+)-diphtamide ADP-ribosyl-transférase destinée à être utilisée dans des procédés de traitement médical, sur des anticorps monoclonaux anti-eEF2, sur des procédés permettant d'évaluer une sensibilité accrue de cellules malades chez un patient à un traitement avec le TNFα ou un autre inducteur de voies de signalisation NFkappaB ou de voies de signalisation associées ; et sur le TNFα ou d'autres inducteurs destinés à être utilisés dans des procédés de traitement médical.
PCT/EP2016/064629 2015-06-24 2016-06-23 Biomarqueurs pour une résistance et une sensibilité à la nad(+)-diphtamide adp-ribosyl-transférase Ceased WO2016207324A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201562183900P 2015-06-24 2015-06-24
US62/183,900 2015-06-24

Publications (1)

Publication Number Publication Date
WO2016207324A1 true WO2016207324A1 (fr) 2016-12-29

Family

ID=56289484

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2016/064629 Ceased WO2016207324A1 (fr) 2015-06-24 2016-06-23 Biomarqueurs pour une résistance et une sensibilité à la nad(+)-diphtamide adp-ribosyl-transférase

Country Status (1)

Country Link
WO (1) WO2016207324A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019089603A1 (fr) * 2017-10-31 2019-05-09 Dana-Farber Cancer Institute, Inc. Procédés de détermination et de traitement de la résistance cellulaire à la toxine adp-ribosylante
CN114410680A (zh) * 2022-01-25 2022-04-29 乾元康安(苏州)生物科技有限公司 Dph6基因在制备具有毒素抗性细胞系中的应用
CN115475163A (zh) * 2022-06-28 2022-12-16 重庆医科大学附属儿童医院 白喉酰胺在制备治疗或诊断dedssh的药物或试剂盒中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003087348A1 (fr) * 2002-04-08 2003-10-23 The Johns Hopkins University Lignee d'encapsidation pour adenovirus non replicants exprimant la toxine de la diphterie
US20090010966A1 (en) * 2007-06-21 2009-01-08 Angelica Therapeutics, Inc. Modified diphtheria toxins
WO2009014650A2 (fr) * 2007-07-20 2009-01-29 The General Hospital Corporation Exotoxines recombinantes de vibrio cholerae
WO2013040141A1 (fr) * 2011-09-16 2013-03-21 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Exotoxine a de pseudomonas des épitopes de cellules b moins immunogènes
WO2015051199A2 (fr) * 2013-10-06 2015-04-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Exotoxine a de pseudomonas modifiée

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003087348A1 (fr) * 2002-04-08 2003-10-23 The Johns Hopkins University Lignee d'encapsidation pour adenovirus non replicants exprimant la toxine de la diphterie
US20090010966A1 (en) * 2007-06-21 2009-01-08 Angelica Therapeutics, Inc. Modified diphtheria toxins
WO2009014650A2 (fr) * 2007-07-20 2009-01-29 The General Hospital Corporation Exotoxines recombinantes de vibrio cholerae
WO2013040141A1 (fr) * 2011-09-16 2013-03-21 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Exotoxine a de pseudomonas des épitopes de cellules b moins immunogènes
WO2015051199A2 (fr) * 2013-10-06 2015-04-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Exotoxine a de pseudomonas modifiée

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FOLEY B T ET AL: "A mutation in codon 717 of the CHO-K1 elongation factor 2 gene prevents the first step in the biosynthesis of diphthamide.", SOMATIC CELL AND MOLECULAR GENETICS MAY 1992, vol. 18, no. 3, May 1992 (1992-05-01), pages 227 - 231, XP055299036, ISSN: 0740-7750 *
H. WEI ET AL: "A Modified Form of Diphthamide Causes Immunotoxin Resistance in a Lymphoma Cell Line with a Deletion of the WDR85 Gene", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 288, no. 17, 26 April 2013 (2013-04-26), pages 12305 - 12312, XP055212219, ISSN: 0021-9258, DOI: 10.1074/jbc.M113.461343 *
IVANKOVIC M ET AL: "Site-specific mutagenesis of the histidine precursor of diphthamide in the human elongation factor-2 gene confers resistance to diphtheria toxin", MUTATION RESEARCH. GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS, ELSEVIER, AMSTERDAM, NL, vol. 609, no. 1, 10 October 2006 (2006-10-10), pages 34 - 42, XP028035092, ISSN: 1383-5718, [retrieved on 20061010], DOI: 10.1016/J.MRGENTOX.2006.06.027 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019089603A1 (fr) * 2017-10-31 2019-05-09 Dana-Farber Cancer Institute, Inc. Procédés de détermination et de traitement de la résistance cellulaire à la toxine adp-ribosylante
US11674950B2 (en) 2017-10-31 2023-06-13 Dana-Farber Cancer Institute, Inc. Methods determining and treating cellular resistance to ADP-rtbosylating toxin
CN114410680A (zh) * 2022-01-25 2022-04-29 乾元康安(苏州)生物科技有限公司 Dph6基因在制备具有毒素抗性细胞系中的应用
CN115475163A (zh) * 2022-06-28 2022-12-16 重庆医科大学附属儿童医院 白喉酰胺在制备治疗或诊断dedssh的药物或试剂盒中的应用

Similar Documents

Publication Publication Date Title
TWI825431B (zh) FcRH5之人源化及親和力成熟抗體及使用方法
JP2025109867A (ja) 抗ｔｒｅｍ２抗体及び関連する方法
TWI725931B (zh) 抗ｆｃｒｈ５抗體
CN113543808A (zh) 多特异性蛋白质分子及其用途
CN105658237B (zh) 抗Ly6E抗体及使用方法
JP7688579B2 (ja) 抗ｔｒｅｍ２抗体の使用方法
TW201809008A (zh) 抗ｃ５抗體及使用方法
WO2016146833A1 (fr) Biomarqueurs de résistance à la nad(+)-diphtamide adp-ribosyltransférase
CN104039340B (zh) 治疗黑素瘤的方法及治疗剂组合
TWI884911B (zh) 具有改良之免疫療效但減弱之副作用之突變抗ctla-4抗體
TW202043291A (zh) 抗mertk抗體及使用方法
WO2021155307A1 (fr) Produits de recombinaison anti-b7-h4 et leurs utilisations
EP3184547A1 (fr) Anticorps anti-tpbg et procédés d'utilisation
US20200023071A1 (en) Immunomodulatory antibody drug conjugates binding to a human mica polypeptide
WO2016207324A1 (fr) Biomarqueurs pour une résistance et une sensibilité à la nad(+)-diphtamide adp-ribosyl-transférase
CN112204048A (zh) 抗cct5结合分子及其使用方法
EP2788024A1 (fr) Formulation d'anticorps
CN118139894A (zh) 抗pla2g2d构建体及其用途
WO2023118608A1 (fr) Variants de protéine de surface cellulaire discernable de cd45 destinés à être utilisés en thérapie cellulaire
US20140341916A1 (en) Therapeutic combinations and methods of treating melanoma
TWI905235B (zh) 抗cd93之構築體及其用途
RU2800779C2 (ru) Биспецифичные антигенсвязывающие молекулы и способы их применения
CN117715934A (zh) 抗cd3构建体及其用途
WO2025031965A1 (fr) Variants de protéine de surface cellulaire discernable de cd52 destinés à être utilisés en thérapie cellulaire
CN118510900A (zh) 抗体药物缀合物及其制备方法

Legal Events

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

Ref document number: 16733049

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16733049

Country of ref document: EP

Kind code of ref document: A1