BIOMARKERS FOR NAD (+) -DIPHTHAMIDE ADP RIBOSYLTRANSFERASE SENSITIVITY AND

RESISTANCE

Statement of Government Support This invention was created in the performance of a Cooperative Research and Development Agreement with the National Institutes of Health, an Agency of the Department of Health and Human Services. The Government of the United States of America has certain rights in this invention.

Field of the invention

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.

Background

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. This intermediate is subsequently converted to diphthine by the methyitransferase DPH5. Amidation of diphthine to diphthamide requires DPH6 and DPH7 [5] . The diphthamide biosynthetic pathway is shown in Fig. 5.

So far, the biological function of diphthamide on eEF2 remained rather elusive. Reports indicate that it contributes to protein translation fidelity [§~9_] . On the other hand, DPH genes or eEF2 can be mutated to prevent diphthamide attachment, yet cells carrying such mutations are still viable [5, 7, lfJ, 1_1] . Animals carrying heterozygous knockouts of DPH genes can be generated, but homozygous knockouts of the genes DPHl, DPH3 and DPH4 are embryonic lethal [9, 12-14 ] . 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

288 (16) : 10671-10678) . These bacterial proteins enter eukaryotic cells and catalyze ADP- ribosylation of the His715-diphthamide using NAD as substrate [1_6, 17] . This inactivates eEF2, arrests protein synthesis and kills cells [10].

These toxins are accordingly classified as NAD (+) -diphthamide ADP

ribosyltransferase enzymes (EC 2.4.2.36) . (Note, 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.

Variations of some of the genes in the diphthamide biosynthesis pathway that affect activity or expression have previously been described to affect sensitivity of cancer cells towards anticancer agents that contain PE- or DT-derivatives as cytotoxic payloads . So far, assessment of the importance of diphthamide synthesis genes for toxin sensitivity is limited to individual reports in various different tumor cells, or to effects observed in yeast. However, a comprehensive assessment of the importance of these individual genes and of their relative contributions to antitumoral activity of PE- derived targeted agents is highly desired for the

development of response biomarkers for tumor therapy.

Technologies and reagents that assess the activity and contributions and cellular 'status' of diphthamide synthesis genes and gene products have not previously been available. The inventors have generated technologies and reagents that assess the activity and contributions and cellular 'status' of diphthamide synthesis genes and gene products by combining a cocktail of seemingly unrelated technologies including (state of the art) gene knockout technologies with (newly developed) toxin-selection cloning, additionally implementing (specially optimized and fine-tuned) mass spec technologies and generation of ancibodies with very specific and unusual binding properties. The integration of all these technologies has enabled the inventors to define biomarkers and develop assays predictive for therapy response of patients upon treatment with targeted toxins.

Summary of the invention

NAD (+) -diphthamide ADP-ribosyltransferase sensitivity / resistance

In the work underlying the invention, 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 . Without wishing to be bound by theory, 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.

Thus, 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 inventors also propose that the findings with PE will apply also to other NAD (+) -diphthamide ADP ribosyltransferase enzymes having the same mechanism of action as PE. Accordingly, in a first aspect, 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 (+) -diphthamide ADP-ribosyltrans ferase . The method may include a step of selecting the patient for treatment with a targeted therapeutic agent comprising a NAD (+) -diphthamide ADP

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 .

Additionally or alternatively, 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 . In a related second aspect, 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

(ii) (a) selecting the patient for treatment with a NAD(+)- ribosyltransferase targeted to diseased cells of the patient if the assay is positive for the presence of eEF2 protein having diphthamide modification at the His715 residue; and/or

(ii) (b) deselecting the patient for treatment with a NAD(+)- ribosyltransferase targeted to diseased cells of the patient if the assay is negative for the presence of eEF2 protein having diphthamide modification at the His715 residue.

Following the selection of a patient for treatment with the targeted therapeutic agent, the patient may be treated with the targeted therapeutic agent .

Accordingly, in a third aspect, 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.

Similarly, 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 with a targeted therapeutic agent comprising a NAD (+) -diphthamide ADP ribosyltransferase coupled to a cell-binding agent targeted to diseased cells of the patient.

Further, 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.

In a fourth aspect, 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.

In this aspect of the invention, the NAD (+) -diphthamide ADP

ribosyltransferase is preferably coupled to a cell-binding agent targeted to diseased cells of the patient, as a targeted therapeutic agent.

In all 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. Exemplary

antibodies and antigens are described below.

In all NAD (+) -diphthamide ADP ribosyltransferase-related aspects and embodiments of the invention, 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

NAD (+) -diphthamide ADP ribosyltransferase . Thus, for example, 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.

In all NAD (+) -diphthamide ADP ribosyltrans ferase-related aspects and embodiments of the invention, 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. However, 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.

In all NAD (+) -diphthamide ADP ribosyltransferase-related aspects and embodiments of the invention, 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 .

TNFa sensitivity

The inventors have also surprisingly found that cells in which a

significant portion of 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") . Accordingly, in a fifth aspect, 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.

In a related sixth aspect, the invention provides a method for selecting and/or deselecting a patient for treatment with TNFa or other inducer, the method comprising:

(i) assaying for the proportion of eEF2 protein that lacks

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

(ii) (b) deselecting the patient for treatment with TNFa or other

inducer if the assay is negative for the presence of a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue.

Following the selection of a patient for treatment with the TNFa or other inducer, the patient may be treated with the TNFa or other inducer. Accordingly, in a seventh aspect, 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.

Similarly, the invention provides a method for treating a patient having a condition that is treatable by TNFa or other inducer, the method

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.

Further, 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. In an eighth aspect, 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.

In the TNFa-related aspects of the invention, 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. For example, 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. 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 as a first-line treatment, when TNFa or other inducer would normally be considered as a first-line treatment for the disease in question.

The term "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.

The term "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 .

The term "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 . In all the aspects and embodiments of the invention that require assaying for specified forms of eEF2, 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. For example, 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

modification, ACP, diphthine, diphthamide) at this residue. On the other hand, 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. For example, in the TNFa-related aspects and embodiments of the invention, 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.

Although a variety of assays are available and suitable for the practice of the invention, the use of antibody-based assays is preferred.

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. Especially preferred are 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. Again, especially preferred are antibodies of the invention as defined below.

In the TNFa-related aspects of the invention, the term "cells in which eEF2 protein lacking diphthamide modification is substantially absent"

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.

Accordingly, in a ninth aspect, 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

modification at the His715 residue. Preferably 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.

In common with the exemplified MGb antibody, 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. As shown in Fig. 5, ACP is an intermediate modification in the biosynthetic pathway for the diphthamide modification. Again, 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.

Likewise, 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. Again, 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.

In particularly preferred embodiments, 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.

Additionally or alternatively, 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) . Preferably 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) . More preferably 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) . Additionally or alternatively, 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) .

Preferably 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) .

However, it is also contemplated that one or more of the CDR sequences of the MGb antibody may be altered to a certain extent without loss of the binding properties described above. Accordingly, 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.

In light of the findings reported herein, 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.

Accordingly, in a tenth aspect, 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. Again, the higher binding affinity is preferably at least 10-fold, more preferably at least 100-fold, more preferably at least 1000-fold. More preferably, 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

electroluminescent label or biotin.

The antibodies of the invention may be used in the methods of the

invention. In particular, 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. Further, 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. The descriptions and definitions of suitable and preferred NAD (+) -diphthamide ADP

ribosyltransferases provided elsewhere herein apply here also. Further, 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. Preferably the apoptosis is TNFa- or other inducer-mediated apoptosis. The description and definitions of suitable and preferred other inducers provided elsewhere herein apply here also .

In all aspects and embodiments of the invention, the patient is preferably a human. In all aspects and embodiments of the invention, the eEF2 protein is preferably human eEF2 protein having the amino acid sequence shown in SEQ ID NO:101.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and

embodiments which are described.

Embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying tables and figures. However various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. Brief description of the figures and tables

Table 1: Generation of MCF-7 derivatives. 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

'markers' for DPH2 and DPH5 inactivation in MCF7 are induction of TGFbeta and TNFSF15.

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.

Figure 3: Diphthamide modification and ADP-ribosylation of MCF-7

derivatives. (A) Western-blot detection of unmodified eEF2 in extracts of parent and mutated MCF-7 cells. Loading controls included detection of actin and total eEF2, eEF2 without diphthamide was detected by a rabbit monoclonal antibody that specifically detects eEF2 without diphthamide (Suppl. Data S4) . (B) MS-based detection and semi-quantitative assessment of eEF2 modifications. (C) ADP-ribosylation of eEF2 was assessed by applying with PE as enzyme and biotinylated NAD as substrate. Bio-ADPR-eEF2 was detected in Western-blots as previously described [28] .

Figure 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. The resulting overlapping network of upstream regulators revealed pre-activation of death receptor signaling regulators as a consequence of dph5 deficiency and loss of diphthamide (oval - activated as consequence of TNF treatment as well as dph5 knockout: TNF, IFNG, IL1B, NFKBIA, STAT3, STAT1, RELA and IRF1; hexagon - observed only in TNF treated MCF7: IKBKB, NFKB1 , JUN; rectangle - observed only in dph5 knockout: EGF, CREB1 , EP300, CREBBP, SMAD3, SP1, TP53) . This is also in full agreement with TNF-alpha hypersensitivity of cells that cannot synthesize diphthamide (all DPH1 and DPH2 and DPH4 and DPH5 deficient cells are TNF

hypersensitive, Tab. 2) .

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.

Detailed description of the invention and preferred embodiments Definitions

"Affinity" refers to the strength of the sum total of noncovalent

interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen) . Unless indicated otherwise, as used herein, "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.

In one embodiment, 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. Solution binding affinity of FABs for antigen is measured by equilibrating Fab with a minimal

concentration of ( ¹²⁵ I ) -labelled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881) . To establish conditions for the assay, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In 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

transferred to the capture plate for incubation at room temperature (e.g., for one hour) . The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150 μΐ/well of scintillant (MICROSCINT-20 TM; Packard) is added, and the plates are counted on a TOPCOUNT TM gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays. According to another embodiment, ¾ 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) . Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N' - ( 3-dimethylaminopropyl) - carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (-0.2 μΜ) before injection at a flow rate of 5 μΐ/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-20™) surfactant (PBST) at 25°C at a flow rate of approximately 25 μΐ/min. 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. If the on-rate exceeds 10 ⁶ M ^"1 s ^"1 by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25°C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing

concentrations of antigen as measured in a spectrometer, such as a stop- flow equipped spectrophotometer (Aviv Instruments) or a 8000-series SLM- AMINCO TM spectrophotometer ( ThermoSpectronic ) with a stirred cuvette.

In all aspects and embodiments of the invention, unless the context requires otherwise, absolute binding affinity values and relative binding affinities are preferably determined by surface plasmon resonance. In the case of antibodies, binding affinity is preferably determined using the Fab (or other monovalent) form of the antibody.

As will be evident from the foregoing, "higher binding affinity" therefore corresponds to a numerically lower K _D value. Conversely, the term "a 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.

Additionally or alternatively, it may refer to a K _D of about 1 μΜ or higher, preferably about 10 μΜ or higher, about 100 μΜ or higher or about 1 mM or higher.

"And/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein .

The term "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.,

Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York) . 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,

IgAl and IgA2) or subclass of immunoglobulin molecule. The 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;

fragments produced by a Fab expression library; 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;

antibody-dependent cell-mediated cytotoxicity (ADCC) ; phagocytosis; and down regulation of cell surface receptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of their heavy chains, 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 α, δ, ε, γ, and μ, 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

homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, 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. ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), 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 pemphigoid, and cutaneous lupus erythematosus), hematologic disorders (such as, for example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, posttransfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases (such as, for example, inner ear disease and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders (such as, for example, diabetic-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison's disease, and autoimmune thyroid disease (e.g. Graves' disease and thyroiditis)) . 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 kidney or ureter, renal cell

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,

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 .

The term "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. In particular, 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

necessary, to achieve the desired therapeutic or prophylactic result. The treatment methods of the invention will use effective amounts of the specified agents. The term "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.

The term "immunotoxin" is 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. In the context of CDRs, 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

preferably up to 4 amino acids, more preferably up to 3 amino acids, more preferably 1 or 2 amino acids, more preferably a single amino acid.

The term "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,

combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

A "patient" is a mammal. Mammals include, but are not limited to,

domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats) . In all aspects and embodiments of the invention, 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

introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % 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. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code.

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:

100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program' s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

The term "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

formulation would be administered.

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.

The term "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

preliminary to malignancy. 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 .

As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") 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. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

The terms "treat" and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the treatment aspects invention can provide any amount of any level of treatment or prevention of disease (such as cancer) in a mammal. Furthermore, the invention can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.

The term "tumour" as used herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumour" are not mutually exclusive as referred to herein.

The term, "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

50% amino acid sequence identity to the reference polypeptide or a fragment thereof as also defined herein, more preferably at least 60%, 70%, 80, 90% 95%, 98% or 99% amino acid sequence identity. Preferred variants retain the specified level of amino acid identity to the full length of the reference polypeptide.

Discriminating between different forms of eEF2

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 .

Such techniques are well known in the field. For example, mass

spectrometry can discriminate between these different forms of eEF2, such as ESI-MS as described in Example 3 herein, aidi-TOF and SELDI-TOF.

Alternatively, 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. In particular, 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. For example, as shown herein, eEF2 that is

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.

Alternatively, to generate antibodies that selectively bind the diphthamide modified form of eEF2, animals (preferentially rabbits, hamsters or mice) are immunized with 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. In these experiments, 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.

Essentially the same technique may be used to generate antibodies that selectively bind the ACP intermediate modified form of eEF2. In this case, 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.

Again, essentially the same technique may be used to generate antibodies that selectively bind the diphthine-modified form of eEF2. In this case, 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

knockouts, generate no signals on extracts of derivatives with complete DPH2 knockouts, generate no signals on extracts of derivatives with complete DPH4 knockouts, and generate no signals on extracts of derivatives with complete DPH5 knockouts. Since all these extracts contain eEF2 (as confirmed by polyclonal detection) , and as the antibody does bind the diphthine-modified eEF2 antigen peptide, such antibodies are specific for diphthine-modified eEF2. As a confirmation for antibody specificity, antibodies that specifically detect diphthine-modified. 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. For example, 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

indicative of sensitivity to treatment with a NAD (+) -diphthamide ADP ribosyltransferase and/or the absence of eEF2 having the diphthamide modification at the His715 residue is indicative of resistance to treatment with a NAD (+) -diphthamide ADP ribosyltrans ferase . That 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.

Alternatively, 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 . Thus, it will be understood that a negative assay result and/or an assessment that the diseased cells are resistant is not necessarily absolute: 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

modification, in particular if the proportion is below a threshold value. Likewise, 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.

Similarly, a positive assay result and/or an assessment that the diseased cells are sensitive is not necessarily absolute: 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. Likewise, 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. It will be understood, however, that the inventors surprisingly did not observe a reduction in potency of PE in MCF-7 cells heterozygous for a dph- 2 knock-out mutation, in which approximately 25% of the eEF2 was unmodified at the His715 residue. Accordingly, the inventors expect any such thresholds to 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.

Alternatively, 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

modification at the His715 residue and substantially does not bind to eEF2 lacking the diphthamide modification. 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) .

Patient samples In the methods of the invention, the sample containing diseased cells is not particularly limited. In preferred embodiments, the sample is a precancer, cancer or tumour sample. In the case of solid pre-cancers, cancers and tumours, the sample may be from a biopsy, or a sample taken following surgical removal of the pre-cancer, cancer or tumour. In the case of haematoiogical pre-cancers and cancers such as leukaemias and lymphomas, the sample may be a blood sample containing pre-cancerous or cancerous blood cells. However, 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. So the applicability of the NAD (+) -diphthamide ADP-ribosyltransferase-related aspects and embodiments of the invention is not limited to pre-cancers, cancers and tumours. The sample may contain virally infected cells or autoimmune effector cells, such as autoimmune T-cells or autoantibody- expressing B-cells.

Similarly, 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. In particular, the patient sample may be treated to separate cells (such as by homogenisation) and/or to disrupt cells to release

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) . Particularly in the case of antibody-based assay techniques, 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. Particularly in the case of mass spectrometry-based assay techniques, the eEF2 protein may be purified and/or digested into peptide fragments prior to detection. Trea tmen ts

Likewise, insofar as 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) . In both cases, the treatment is preferably of a tumour or cancer. However, the applicability of the invention is not limited to tumours and cancers. For example, 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.

In addition, Resimmune© (A-dmDT390-bisFv (UCHTl) , described below)

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. Thus, the applicability of NAD (+) -diphthamide ADP ribosyltransferase treatment is limited only by the availability of suitable targeting moieties. Likewise, 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

(J0rgensen 2008a) . It was characterised as a NAD {+) -diphthamide ADP ribosyltransferase by primary sequence identity to PE (32% identity), the presence of a furin protease site for cellular activation, the presence of a C-terminal KDEL sequence that is presumed to direct the toxin to the endoplasmic reticulum (though this property is not shared by DT) and three signature regions that characterise the catalytic domain of diphthamide- specific toxins according to Yates et al. 2006. In particular, 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. Other candidate NAD (+) -diphthamide ADP

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.

Further NAD (+) -diphthamide 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.

Nevertheless, in all aspects and embodiments of the invention, the NAD(+)- diphthamide ADP ribosyltransferase is preferably a PE, DT or cholix toxin. For the avoidance of doubt, 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. In the case of PE, 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. 267:12420-12423; Pastrana et al . 2005 Biochim Biophys Acta. 1741:234-239) . Cholix toxin may have the same cellular targets. In the case of DT, 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

disulphide linkage to activate and release the cytotoxic A subunit.

Both PE and DT have been modified for targeted therapy by removing the B subunits and replacing them with other cell-targeting agents having desired target cell specificity, such as antibodies. For example, 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. Numerous

antibodies have been coupled to truncated forms of PE for a variety of therapeutic purposes, as detailed below. In the practice of the present invention, forms of NAD ( + ) -diphthamide ADP ribosyltransferase which lack a functional native receptor-binding portion are greatly preferred.

Pseudomonas exotoxins (PEs)

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. In the practice of the present invention, 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. However, 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. These form an endoplasmic reticulum localisation sequence (WO 91/09948, Chaudhary et al 1991, Seetharam et al . 1991). Cytotoxicity can be maintained or enhanced by replacing the native ER localisation sequence with one or more other ER localisation sequences. Accordingly the functional domain III of native PE is considered to consist of residues 395-601.

An extensive body of work has been published disclosing variants and improvements of the native PE molecule for use in targeted cytotoxins . The terms "Pseudomonas exotoxin A", " Pseudomonas exotoxin" and "PE" as used herein are intended to encompass these and other variants and improvements of native PE that retain cytotoxic activity. In particular, the terms "Pseudomonas exotoxin A", "Pseudomonas exotoxin" and "PE" are specifically intended to include the variants and improvements disclosed in

WO88/02401A1, WO90/12592A1 , WO91/09949, WO91/09965, WO93/25690, W097/13529, WO98/20135, WO2005/052006, WO2007/016150, WO2007/031741, WO2009/32954 ,

WO2011/32022, WO2012/154530, WO2012/170617 , WO2013/40141, Mazor R, et al PNAS 111 (2014) 8571-8576, Alewine C, et al, Mol Cancer Ther. (2014) 2653- 61 and WO2015/051199. All these publications are incorporated herein in their entirety for the purpose of exemplifying variants and improvements of Pseudomonas exotoxin A / PE that are suitable for use in the present invention and that are included, without limitation, within the terms "Pseudomonas exotoxin A", "Pseudomonas exotoxin" and "PE", with those variants and improvements disclosed in WO2005/052006, WO2007/016150, WO2007/031741, WO2009/32954 , WO2011/32022, WO2012/154530, WO2012/170617, and WO2013/40141 being preferred and those disclosed in WO2009/32954 ,

WO2011/32022, WO2012/154530, WO2012/170617 , WO2013/40141, Mazor R, et al PNAS 111 (2014) 8571-8576, Alewine C, et al, Mol Cancer Ther. (2014) 2653- 61 and WO2015/051199 being particularly preferred. It is anticipated that further variants and improvements of PE will be developed in future. Since the present invention relates to resistance against the cytotoxic effects of PE, it is anticipated that any such future variants and improvements of PE that retain cytotoxic activity may also be used in the practice of the invention and are therefore included within the terms "Pseudomonas exotoxin A" and "PE".

Generally, 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. 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. The cleavable linker (such as an FCS) will generally be on the N-terminal side of the PE functional domain III.

Other cleavable linkers may be used provided that they permit cleavage of the PE from the cell-binding agent following uptake into the target cell. Furthermore, 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. For example, 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.

Much of the work on PE has focussed on eliminating portions of the native sequence that are unnecessary and/or disadvantageous for use in targeted therapies. For example, replacement of the B (receptor-binding) subunit with another cell-binding agent has reduced the non-specific toxicity of the molecule. Further removal of inessential sequences has reduced immunogenicity . This has led in particular to the development of the following truncated forms of PE : PE40, PE35, PE38, PE38QQR, PE-LR and PE24. 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. 263:9470-9475) . 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. Thus, 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:

D403, D406, R412, R427, E431, R432, R458, D461, R467, R490, R505, R513, E522, R538, E548, R551, R576, K590, Q592 and L597. Preferred 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:

E420, D463, Y481, L516, R563, D581, D589 and K606.

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:

R421, L422, L423, A425, R427, L429, Y439, H440, F443, L444, A446, A447, 1450, 463-519, R551, L552, T554, 1555, L556 and W558. Preferred 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:

L294, L297, Y298, L299 and R302.

Preferred 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 :

E282, E285, P290, R313, N314, P319, D324, E327, E331 and Q332.

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

R427A/R456A/D463A/R467A/R490A/R505A/R538A (that is, with R456A instead of R458A) . Mazor et al. discloses that a combination of 6 point mutations within PE domain III, together with deletion of most of PE domain II, reduced T cell responses by 93%. The mutations are R427A/F 43A/L477H/R494A/R505A/L552E .

Accordingly, the PE functional domain III may comprise mutations at any one or any combination of more than one of the following sites:

D403, D406, R412, E420, R421, L422, L423, A425, R427, L429, E431, R432, Y439, H440, F443, L444, A446, A447, 1450, R456, R458, D461, 463-519 (preferably D463, R467, L477, Y481, R490, R494, R505, R513 and/or L516) , E522, R538, E548, R551, L552, T554, 1555, L556, W558, R563, R576, D581, D589, K590, Q592, L597 and K606.

Preferably the mutation (s) reduce (s) the immunogenicity compared to the unmutated sequence of the amino acids 395-613 of SEQ ID NO:l.

Insofar as 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:

E282, E285, P290, L294, L297, Y298, L299, R302, R313, N314, P319, D324, E327, E331 and Q332.

Preferably the mutation (s) reduce (s) the immunogenicity compared to the unmutated sequence from domain II. In particular, in embodiments in which the FCS is derived from the native furin-cleavable sequence of PE consisting of amino acids 274-284

(RHRQPRGWEQL, SEQ ID NO: 31) may comprise a substitution of the E282 residue, especially if the adjacent sequence from the native PE sequence is included downstream of the FCS. In embodiments where the adjacent sequence from the native PE sequence is not included (such as PE-LR, in which the FCS is fused to domain III either directly or via a non-native linker sequence), the epitope from the native sequence may anyway be disrupted such that a mutation at the E282 residue may not be advantageous.

Mazor et al. 2014, Liu et al . 2012, Kreitman et al. 2012, Pastan et al . 2011, Onda et al. 2011, Hansen et al. 2010, Kreitman et al . 2009c, Onda et al. 2008, Ho et al. 2005, Kreitman et al . 2000 and Roscoe et al. 1994 are all directed towards reducing the immunogenicity of PE .

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

immunogenicity. Alternatively, 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

endoplasmic reticulum or to recycle proteins into the endoplasmic

reticulum. Such sequences are referred to here as "endoplasmic reticulum localisation sequences" or "ER localisation sequences". Preferred 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 ) . One or more

additional ER localisation sequences, preferably independently selected from KDEL, REDL, REDLK, RDEL and KEDLK, may be added to the C-terminal end of the PE polypeptide sequence. The substitution of KDEL, or 2 or 3 tandem repeats of KDEL (KDELKDEL, SEQ ID NO 38 ; KDELKDELKDEL, SEQ ID NO 39 ) for the native REDLK sequence, or the addition of 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.

Furin-cleavable sequence (FCS)

As described in WO2012/154530, 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, is one of several mammalian members of the PCSK family and is involved in processing several endogenous human proteins. See generally, Thomas 2002 Nat Rev Mol Cell Biol 10:753-66. It is a membrane-associated protein found mainly in the trans-Golgi network. The sequence of human furin has been known since the early 1990s. See for example Hatsuzawa et al. 1992 J Biol Chem 267: 16094-16099; and Molloy et al . 1992 J Biol Chem 267:16396-16402.

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. It reports that 31, or 81%, of the cleavage motifs reviewed had the R-X-[R/K]-R (SEQ ID NOs: 41 & 42 ) consensus sequence, of which 11, or 29%, had R-X-R-R ( SEQ ID NO: 41 ) , and 20, or 52%, were R-X-K-R (SEQ ID N0: 42 ) . Three of the cleavage motifs contained only the minimal cleavage sequence. Duckert et al . further aligned the motifs and identified the residues found at each position in each furin both for the cleavage motif itself and in the surrounding residues. Fig. 1A of Duckert et al. shows by relative size the residues most commonly found at each position. By convention, the residues surrounding the furin cleavage site are numbered from the scissile bond (which is typically indicated by a downward arrow) . Counting toward the N terminus, 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. See Rockwell and Thorner 2004 Trends Biochem. Sci. 29:80-87; and Thomas 2002 Nat. Rev. Mol. Cell Biol 3:753-766. Thus, following the convention, the following sequence can be used to align and number the residues of the minimal cleavage sequence and the surrounding residues:

P6-P5-P4-P3-P2-P1-P1 ' -P2 ' -P3 ' -P4 ' -P5 ' , in which the minimal furin-cleavable sequence is numbered as P4-P1. Duckert et al. 's alignment of 38 sequences cleaved by furin identifies the variations permitted depending on the residues present at various

positions. For example, if the residue at P4 is not an R, that can be compensated for by having arginine or lysine residues at P2 and P6. See page 109. In native PE, 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 :

274- R H R Q P R G W E Q L -284 (SEQ ID N0: 31 ) P6— P5—P4—P3—P2—PI—PI ' -P2 ' -P3 ' -P4 ' -P5 '

In studies underlying WO2012/154530, substitutions were made at positions ?3 and P2 to form the following sequence, with the substitutions

underlined : 274- R H R S K R G W E Q L -284 (SEQ ID NO: 43 ) .

This sequence has shown a cleavage rate faster than that of the native sequence, and when used in an exemplary immunotoxin resulted in

cytotoxicity to target cells approximately the same as that of the native sequence.

Based on this and previous studies, 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. 1 A of Duckert et al., with the proviso that, if there is a residue present at the position identified as P2 ' , it should be tryptophan or, if not tryptophan, should not be valine or alanine. For example, in some embodiments, 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) .

As noted in Duckert et al., a less favorable residue than R (primarily valine) can be used position P4 if compensated for by arginine or lysine residues at positions P2 and P6, so that at least two of the three residues at P2, P4 and P6 are basic. Thus, in some embodiments, 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. Thus, a lysine can be

substituted for the arginine at position PI in, for example, any the sequences set forth above. In some embodiments, 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. Thus, in some embodiments, 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 ) . In some embodiments, 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. Thus, in some embodiments, 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 ) .

As mentioned above, 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 . Where 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 .

Preferably, 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.

Alternatively, 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

Preferred PE toxins for use in accordance with the presen

the following structure:

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-W-E-Q-L, or R-H-R-S-K-R, wherein the glutamic acid residue corresponding to position 282 of the native PE sequence (where present) is optionally replaced by another residue, preferably glycine, serine, alanine or giutamine;

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 :

(a) some or all of residues 602-608 are optionally deleted, and

(b) residues 609-613 are optionally replaced by another ER localisation sequence, preferably KDEL, REDL, RDEL or EDLK, and

(c) any one or more of residues D403, D406, R412, E420, R421,

L422, L423, Ά425, R427, L429, E431, R432, Y439, H440, F443, L444,

A446, A447, 1450, R456, R458, D461, 463-519 (preferably D463, R467,

L477, Y481, R490, R494, R505, R513 and/or L516) , E522, R538, E548,

R551, L552, T554, 1555, L556, W558, R563, R576, D581, D589, K590, Q592, L597 and (where present) 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.

Within the formula above:

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

R427A/F443A/R456A/D463A/R467A/L477H/R490A/R494A/R505A/R538A/ L552E.

Particularly preferred PE toxins for use in accordance with the present invention comprise the amino acid sequence of SEQ ID NO: 110 or SEQ ID

NO: 111. 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.

The 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.

Diphtheria toxins (DTs)

Native, wild-type Diphtheria toxin is secreted by Corynebacterium

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.

The order of the structural domains in DT is reversed compared to that of PE . That is, the N-terminal domain I (amino acids 1-191 of SEQ ID NO : 3 ) is NAD (+) -diphthamide ADP ribosyltransferase domain; and the C-terminal domain III (amino acids 385-535 of SEQ ID NO : 3 ) is the cell-binding domain. As with PE, activation of the native DT protein depends on furin cleavage within domain II.

Truncated or modified forms of DT that lack receptor-binding activity (truncated: DAB ₃₈ 9, DAB ₄₃₆ , DT ₃₉ 5, DT ₃₈ 3 , DT390; modified: CRM107; point mutation of S525F) 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.

recombinant expression in bacterial cells.

Generally, 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.

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.

The cleavable linker (such as an FCS) will generally be on the C-terminal side of the DT functional domain I. 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. Preferably the furin-cleavable sequence includes the sequence GNRVRRSVGSS (SEQ ID NO 65 ) or a fragment thereof comprising RVRR. As with PE toxins, however, other cleavable linkers and other means of coupling the DT to cell-binding agents are contemplated. In contrast to PE, it is thought that DT is not processed in target cells via the endoplasmic reticulum, so a DT for use in accordance with the invention will generally lack an ER localisation sequence.

As with PE, 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) . As with PE, 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.

Cholix toxins

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 ) .

The order of the structural domains in cholix toxin is the same as that of PE. That is, 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

localisation sequence RKDELK (SEQ ID NO: 67 ) at positions 629-634. See Awasthi et al . 2013, which also provides naturally occurring variant cholix toxin sequences.

Generally, 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. In embodiments in which the cholix toxin is coupled to a cell-binding agent as a fusion polypeptide, 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. 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. Preferably 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. Alternatively, it may comprises the sequence RGRKPRDLTDD (SEQ ID NO: 69) of ChxA III of Awasthi et al. 2013 or a fragment thereof comprising RKPR. Alternatively, it may comprises the sequence RSRKPRDLPDD (SEQ ID NO:70) of ChxA I and II of Awasthi et al. 2013 or a fragment thereof containing RKPR. As with PE toxins, however, other cleavable linkers and other means of coupling the cholix toxin to cell- binding agents are contemplated.

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 . As with PE, the cholix toxin may include one or more additional ER

localisation sequences.

As with PE, 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.

The cholix toxins for use in accordance with the present invention will generally lack a functional cell-binding domain I.

As with PE, 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.

As with PE, the cholix toxins for use in accordance with the invention may also be mutated to reduce immunogenicity . Cytotoxic activity

Confirmation or comparison of the 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

NAD (+) -diphthamide ADP-ribosyltransferase activity may be assayed by the ability to incorporate biotinylated ADP into eEF2 protein, as described in Example 3 herein.

Exemplary targeted therapeutic agents

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:

1. 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) .

2. A second generation, affinity matured version of (1), moxetumomab pasudotox (also known as RFB4 [GTHW] (dsFv) -PE38 , HA22 or CAT-8015) (Salvatore et al . 2002), for the treatment of hematologic

malignancies (Alderson et al . 2009, Kreitman et al. 2009b,

ClinicalTrial.gov identifiers NCT00462189, NCT00457860, NCT00515892, NCT01086644, NCT00659425, and NCT00586924) .

3. SSI (dsFv) PE38 (also known as SS1P) directed against mesothelin, for the treatment of lung cancer and mesothelioma (Hassan et al . ,

Kreitman et al . 2009c, ClinicalTrial.gov identifiers NCT01041118, NCT00575770, and NCT01051934)

4. anti-TAC (scFv) PE38 (also known as LMB-2) 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. In

particular, 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;

modified: CRM107) or a variety of PE toxins (full-length PE, PE38, PE40, modified PE38 and modified PE40), coupled to a variety of 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) for a variety of

indications (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 cancer, glioblastoma, and glioma; graft-versus host disease (GVHD) ; psoriasis; Rheumatoid arthritis (RA) ; and myelodyplastic syndrome (MDS) . Any of these targeted therapeutic agents may be used in accordance with the present invention. Furthermore, 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.

Targeted therapeutic agents

For therapeutic use, the NAD (+) -diphthamide ADP ribosyltransferases herein are coupled to a cell binding agent to produce a targeted therapeutic agent. The term "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. As discussed below, a wide variety of cell binding agents may be included in targeted therapeutic agents in accordance with the invention. Where the cell binding agent is a peptide, polypeptide, or protein, the

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. When the NAD (+) -dipthamide ADP ribosyltransferase fused to the cell binding agent comprises a furin-cieavable sequence, 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. In preferred embodiments of the invention, the NAD ( + ) -diphthamide ADP ribosyltransferase will be positioned on the C-terminal side of the ceil binding agent.

Alternatively, however, the NAD (+) -diphthamide ADP ribosyltransferase may be conjugated to the cell binding agent.

Cell binding agents

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. Where the cell-binding agent is a peptide, 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.

Thus, 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. In an embodiment of the invention, the cell-binding agent is a ligand that specifically binds to a receptor on a cell surface. Exemplary 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) .

The cell surface marker can be, for example, a tumour-associated antigen. The term "tumour-associated antigen" as used herein 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. In this regard, 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. Also, the tumour- associated antigen can additionally be expressed by cells of a different state of development or maturation. For instance, 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. Alternatively, 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. Alternatively, the tumour-associated antigen may be a tumour-associated antigen (e.g., may be characteristic) of more than one type of cancer or tumour. For example, 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.

Exemplary 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) ,

preferentially expressed antigen of melanoma (FRAME), carcinoembryonic antigen (CEA) , prostate-specific antigen (PSA), prostate specific membrane antigen (PSMA), granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR), 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. In a preferred embodiment, the cell surface marker, to which the cell-binding agent specifically binds, is selected from the group consisting of cluster of differentiation (CD) 19, CD21, CD22, CD25, CD30, CD33 (sialic acid binding Ig-like lectin 3, myeloid cell surface antigen), CD79b, CD123 (interleukin 3 receptor alpha), transferrin receptor, EGF receptor, mesothelin, cadherin, Lewis Y, Glypican-3, FAP (fibroblast activation protein alpha), PSMA (prostate specific membrane antigen) , CA9 = CAIX (carbonic anhydrase IX) , LI CAM (neural cell adhesion molecule L I ), endosialin, HER3

(activated conformation of epidermal growth factor receptor family member 3), Alkl/BMP9 complex (anaplastic lymphoma kinase 1/bone morphogenetic protein 9), TPBG = 5T4 (trophoblast glycoprotein), R0R1 (receptor tyrosine kinase-like surface antigen) , HER1 (activated conformation of epidermal growth factor receptor), and CLLl (C-type lectin domain family 12, member A) . 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. In an embodiment of the invention, the cell-binding agent is an antibody that specifically binds to a tumour-associated antigen. Exemplary

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) . In this regard, 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. Patents 5,242,824 (anti- transferrin receptor); 5,846,535 (anti-CD25) ; 5,889,157 (anti-Lewis Y) ; 5,981,726 (anti-Lewis Y) ; 5,990,296 (anti-Lewis Y) ; 7,081,518 (anti- mesothelin); 7,355,012 (anti-CD22 and anti-CD25); 7,368,110 (anti- mesothelin) ; 7,470,775 (anti-CD30) ; 7,521,054 (anti-CD25); and 7,541,034 (anti-CD22) ; U.S. Patent Application Publication 2007/0189962 (anti-CD22); Frankel et al . , Clin. Cancer Res., 6: 326-334 (2000), and Kreitman et al., AAPS Journal, 8(3) : E532-E551 (2006), each of which is incorporated herein by reference.

Antibodies have been raised to target specific tumour related antigens including: Cripto, CD30, CD19, CD33, Glycoprotein NMB, CanAg, Her2

(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.

TNFa and other inducers of NFkappaB- and related signaling pathways

Native human TNFa is a 157-amino acid polypeptide that binds to and activates TNF receptor 1, leading to an enhancement of apoptosis.

References herein to TNFa include native human TNFa and active fragments and variants thereof that are capable of binding to and activating TNF receptor 1. However, 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. Likewise, references herein to TRAIL include both native human TRAIL and active fragments and variants thereof that are capable of binding to and

activating DR4 and/or DR5; references herein to FasL include 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 (such as a death receptor ligand, such as TNFa, TRAIL or FasL) 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. In certain preferred embodiments, 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. Preferably 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

Siegemund et al. As described in Scherf et al . , 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.

In other preferred embodiments in which 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) is encapsulated with a coating (such as a lipid coating) that bears 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. (2009) Journal of Controlled Release 137: 69-77. The disclosure elsewhere herein of suitable and preferred cell-binding agents applies also in the present context of targeted 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.

In the case where the TNFa or other inducer is a death receptor ligand or agonist anti-death receptor antibody, the diseased cells of the patient preferably express the corresponding death receptor. Thus, for example, 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; and 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), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction. Exemplary 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, β-galactosidase, glucoamylase , lysozyme, saccharide oxidases, 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.

Antibody Variants

In certain embodiments, 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.

Original Exemplary Preferred Residue Slabstitutions Substitutions

Asp (D) Glu; Asn Glu

Cys (C) Ser; Ala Ser

Gin (Q) Asn; Glu Asn

Glu (E) Asp; Gin Asp

Gly (G) Ala Ala

His (H) Asn; Gin; Lys; Arg Arg

He (I) Leu; Val; Met; Ala; Phe; Leu

Norleucine

Leu (L) Norleucine; He; Val; Met; Ala; He

Phe

Lys (K) Arg; Gin; Asn Arg

Met (M) Leu; Phe; He Leu

Phe (F) Trp; Leu; Val; He; Ala; Tyr Tyr

Pro (P) Ala Ala

Ser (S) Thr Thr

Thr (T) Val; Ser Ser

Trp (W) Tyr; Phe Tyr

Tyr (Y) Trp; Phe; Thr; Ser Phe

Val (V) He; Leu; Met; Phe; Ala; Leu

Norleucine

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, lie;

(2) neutral hydrophiiic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe .

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody) . Generally, 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 (e.g., substitutions) 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. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al . in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some embodiments of 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 . In certain embodiments, 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,

conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR "hotspots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, 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. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by 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. Examples of 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

In certain embodiments, 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. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, 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

(Presta, L . ) ; US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd) . Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US

2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US

2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO

2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Hoi. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) . Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al . Arch. Biochem. Biophys. 249:533-545

(1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11), and 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

In certain embodiments, 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.

In certain embodiments, 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. For example, 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. The primary cells for mediating ADCC, NK ceils, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII and Fc(RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu . Rev. Immunol. 9:457-492 (1991) . Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No.

5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA

83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.

166:1351-1361 (1987)) . Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ 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. Alternatively, or additionally, 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. USA 95:652-656 (1998) . 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. To assess complement activation, 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) .)

In certain embodiments, 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) .

In some embodiments, 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.

6,194,551, WO 99/51642, and Idusogie et al . J. Immunol. 164: 4178-4184 (2000) .

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 (Guyer et al . , J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton et al . ) . Those antibodies comprise an Fc region with one or more

substitutions therein which improve binding of the Fc region to FcRn. Such 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) .

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No.

5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants. d . Cysteine engineered antibody variants

In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, 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. In certain embodiments, 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

In certain embodiments, 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. Non- limiting examples of 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., glycerol), polyvinyl alcohol, and mixtures thereof. 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 . In another embodiment, conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, 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.

Antibody Fragments In certain embodiments, 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.,

Pluckthtin, in The Pharmacology of Monoclonal Antibodies, vol. 113,

Rosenburg and Moore eds . , ( Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab' ) 2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;

Hudson et al . , Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) . Triabodies and tetrabodies are also described in Hudson et al., Wat. Med. 9:129-134 (2003) .

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. In certain embodiments, 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 .

Chimeric and Humanized Antibodies In certain embodiments, an antibody provided herein (in particular, an antibody provided as a cell-binding agent) 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)) . In one example, 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. In a further example, 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. In certain embodiments, a chimeric antibody is a humanized antibody.

Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non- human antibody. Generally, 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. In some embodiments, 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.

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. Nat' 1 Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos . 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000)

(describing the "guided selection" approach to FR shuffling) . 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. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Blosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al . , J. Biol. Chem. 271:22611-22618 (1996)) .

Human Antibodies

In certain embodiments, an antibody provided herein (in particular, an antibody provided as a cell-binding agent) 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. In such transgenic mice, the endogenous

immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Wat. Biotech. 23:1117-1125 (2005) . See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HuMab© technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US

2007/0061900, describing VelociMouse© technology) . 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

(describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26 ( 4 ) : 265-268 (2006) (describing human-human hybridomas) . Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20 ( 3 ) : 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3) :185-91 (2005) .

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.

Library -derived antibodies 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

Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2) : 299-310

(2004); Lee et al., J. Mol. Biol. 340(5) : 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al . , J.

Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, 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

(1994) . 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 . Alternatively, 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) . Finally, 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.

Multispecific Antibodies

In certain embodiments, 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. In certain embodiments, one of the binding specificities is for linear linked polyubiquitin and the other is for any other antigen. In certain embodiments, 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) .

Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules

(WO 2009/089004A1 ) ; cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229: 81

(1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al . , J. Immunol., 148 ( 5 ): 154 -1553 (1992)); using

"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.

US 2006/0025576Ά1) . 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 :

DPH1: NM_001383

DPH2 : NM_001384 (transcript variant 1); NM_001039589 (transcript variant 2)

DPH3: NM_206831 (transcript variant 1); M_001047434 (transcript variant 2)

DPH4: NM_181706

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)

DPH7: NM_138778

In the case of genes having multiple transcript variants, the zinc finger nuclease sequences used in the examples were chosen to permit knockdown of all known variants. For the avoidance of doubt, the DPH gene sequences are preferably those of the most recent version of the database entries as at 24 June 2015.

Recombinant Methods and Compositions 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. For recombinant production, 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. These methods 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

Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience , N.Y (1989) . 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. As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids. Although 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. Furthermore, 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) . In addition, a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a

polynucleotide encoding the targeted therapeutic agent or antibody of the invention, or variant or derivative thereof. 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 (such as a polypeptide coding region and a promoter associated therewith) 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. Thus, 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. Other 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 ) . Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These 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 ) .

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. For example, if secretion of the targeted therapeutic agent or antibody is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding the targeted therapeutic agent or antibody of the invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that 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. In certain embodiments, 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.

Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase . 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. As used herein, 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. For example, 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. In addition to prokaryotes, 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. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al . , Nat Biotech 24, 210-215 (2006) . 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

Spodoptera frugiperda cells. 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 PLANTIBODIES™ technology for producing

antibodies in transgenic plants) . Vertebrate cells may also be used as hosts. For example, 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 . , J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK) , 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

(1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr ^" CHO cells

(Urlaub et al . , Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NSO, P3X63 and Sp2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed. , Humana Press,

Totowa, NJ) , pp. 255-268 (2003) . 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. In one embodiment, 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) . Typically, 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.

Methods to produce polyclonal antibodies and monoclonal antibodies are well known in the art (see e.g. Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988) . 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

specificity-determining regions (SDRs or a-CDRs; the residues critical for the antibody-antigen interaction) onto human framework and constant regions, or (c) 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 . 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et al . , Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36, 43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36, 61-68 (2005) and Klimka et al . , Br J Cancer 83, 252-260 (2000) (describing the "guided selection" approach to FR shuffling) . 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 . , ed., Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)) . Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.

In certain embodiments, 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. surface plasmon resonance technique (analyzed on a BIACORE T100 system) (Liljeblad, et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)) . 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. In certain embodiments, such a competing antibody binds to the same epitope (e.g. a linear or a conformational epitope) that is bound by the reference antibody. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ) . In an exemplary competition assay, 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. As a control, immobilized antigen is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to the antigen, excess unbound antibody is removed, and the amount of label associated with immobilized antigen is measured. If the amount of label associated with immobilized antigen is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) .

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. For affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the targeted therapeutic agent or antibody binds. For example, for affinity chromatography purification of antibodies, or of targeted therapeutic agents that include antibody sequences as cell-binding agents, 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.

Pharmaceutical Formulations Pharmaceutical formulations 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;

resorcinol; cyclohexanol ; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as 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); and/or 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

International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos . 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more 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. Such 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-

(methylmethacylate ) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions , nano-particles and nanocapsules ) or in macroemulsions . Such techniques are disclosed in Remington 's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) .

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.

The foregoing disclosure also applies mutatis mutandis to the formulation of TNFa or other inducer. A recombinant form of TNFa known as tasonermin is available from Boehringer Ingelheim as Beromun™, which is approved as an anti-neoplastic and is formulated for injection or infusion.

Therapeutic Methods and Compositions

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

contemplated herein.

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. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate. For the prevention or treatment of disease, the appropriate dosage of 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. For repeated administrations over several days or longer, depending on the condition, 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. Thus, 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.

about six doses) . An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

The foregoing disclosure also applies mutatis mutandis to therapeutic methods and compositions involving TNFa or other inducer.

Articles of Manufacture

In another aspect of the invention, 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

a container and a label or package insert on or associated with the container. 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.

The foregoing disclosure also applies mutatis mutandis to articles of manufacture comprising TNFa or other inducer.

The invention is further illustrated in the following examples, which are not intended to limit the invention in any way.

Example 1: Generation of MCF-7 cells with heterozygous or completely inactivated DPH genes Gene-specific zinc finger nucleases (ZFN) were applied to generate MCF-7 cells with inactivated DPH genes [29] . Therefore, Zinc finger nucleases (ZFN) were obtained from Sigma and transfected into MCF-7 cells in 6-well dishes. The target sequences of the ZFNs are listed in Figure 1. Two days later (to enable ZFN binding to target genes, induction and mis-repair of double strand breaks), cells with gene mutations were identified by either ^phenotype selection' or by genetic analyses.

For phenotype selection, 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. Genetic analyses of single-cell derived clones revealed that all resistant isolates contained only defective ( out-of-frame ) copies of DPHl, DPH2, DPH4, or DPH5 genes. None of these toxin-resistant cells contained an additional unaltered functional gene copy.

For the alternative 'genetic screen' method, single transfected cells were seeded, expanded without toxin selection and subjected to High Resolution Melting (HRM) analyses [26] of gene specific PCR fragments to define clones containing variant DPH genes (genetic screen) . This technique allows the rapid identification of cells that contain two different alleles of the gene to be analyzed, as those generate biphasic or odd-shaped melting curves and can hence be differentiated from parent nonmutated cells (which displayed mono-phasic melting curves) . The sequences of the alleles in clones generated by both procedures were determined by characterizing plasmids containing PCR-fragments derived from genomic DNA encompassing the ZFN-target regions (Figure 1) . 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

specifically bind eEF2 without diphthamide. Therefore, rabbits were immunized with a peptide spanning amino acids 708-724 of human eEF2

( TLHADAIHRGGGQI I PT (SEQ ID NO: 72), without diphthamide modification) coupled to KLH. After 10 rounds of immunization with adjuvant, B-cells expressing peptide-binding antibodies were isolated by FACS (using fluorescence-labeled eEF2 peptide), and subsequently converted to

recombinant antibody clones via B-cell cloning and PCR-mediated V-region extraction [49] . ELISA and Biacore analyses with eEF2 peptide as antigen and scrambled peptides as controls were used to select antibody candidates. Western blot analyses (peptide & purified eEF2 & eEF2 detection in MCF-7 cell extracts) were performed, to evaluate eEF2 specificity and to identify those that selectively recognize eEF2 without diphthamide. 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

determined by MS analyses (see Example 3) : Of the 8 analyzed antibodies, 5 detected eEF2 with low background in extracts of both cell lines. These bind eEF2 independent of presence or absence of the diphthamide and can be applied as eEF2 control reagents. Two antibodies (MGa and MGd) generate stronger eEF2 signals with extracts of DPH-inactivated cells than wildtype cells. These antibodies appear to bind better to non-diphthamide eEF2 than to diphthamide eEF2, but it is not specific for diphthamide-deficient eEF2. Finally, 1 of the 8 antibodies did not generate signals in Western blocs of wildtype MCF-7, but revealed good signals without background in diphthamide deficient MCF-7. This antibody (MGb) can therefore specifically detect eEF2 without diphthamide modification.

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 3Ά) . 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.

Secondary antibodies coupled to horseradish peroxidase were purchased from Dako. The results of the Western biot analyses applying the newly generated antibodies revealed that unmodified eEF2 is virtually absent (below detection levels) in MCF-7 cells. In contrast, cells carrying complete inactivation of DPH1, DPH2, or DPH4 genes contained eEF2 without H715 modification as indicated by strong antibody signals. Signals indicative for unmodified eEF2 were also observed in extracts of cells carrying heterozygous DPH2 mutations, albeit to a much lower degree. All other heterozygous cell lysates generated only background signals. Thus, inactivation of DPHl, DPH2 and DPH4 interferes with H715 modification of eEF2.

Mass-spec based assays: Optimized mass spec (MS) analyses were

subsequently applied to extracts of DPH-mutated cell lines to determine the composition and modification of eEF2 at the His715 residue in more detail. Therefore, cell extracts for analyses of eEF2 modifications were prepared by lysing cells (4xl0 ⁵ cells in 6-well dishes) in RIPA buffer. Then, 100 μg 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 μg 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

(ACP-derivative ) and 472.7511 (diphthamide-derivative ) were selected and fragmented. The EF2 peptides T237-K298 (mass-over-charge 747.9047), ₄₁₅ -K ₄ 25 (mass-over-charge 554.3241), G667-K675 (mass-over-charge 532.2928) and A785- Rsoo (mass-over-charge 900.4518) were co-analyzed to normalize EF2 amounts across preparations. All PRM chromatograms were processed using Skyline 2.6 [27] . These optimized MS procedures enabled the detection and determination of relative levels of eEF2 in unmodified form, eEF2 with the ACP

intermediate and with 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

472.9971 for the F702-R716 peptide.) In wildtype cells, only diphthamide- modified eEF2 is detectable without evidence for the presence of unmodified eEF2 or diphthine or ACP modifications (Figure 3B) . In contrast, MCF-7 cells with complete inactivation of the DPHl, DPH2, DPH4, as well as DPH5 genes contained no diphthamide modified eEF2. Thus, these genes are essential for diphthamide synthesis and their inactivation cannot be compensated by other genes in MCF-7 cells. Complete inactivation of DPHl, DPH2 or DPH4 generated cells in which only unmodified eEF2 and no other modified form was detectable. Complete inactivation of DPH5 generated cells that harbor the ACP intermediate (eEF2 with this intermediate is not recognized by the antibody applied in the preceding Western blots, Fig. 3A) . The major eEF2 species in MCF-7 cells which carry one inactivated and one functional copy of DPH1-7 is diphthamide-modified eEF2. In contrast to the parent MCF-7 cells, however, unmodified eEF2 was detectable in different amounts (up to 25% of the total eEF2 species, Fig.3B) upon heterozygous inactivation of DPH1 and. DPH2. This demonstrates that gene- dose reduction by inactivation of one allele of DPH1 or DPH2 is

insufficient to prevent diphthamide synthesis, but sufficient to modulate the amount of unmodified eEF2. Enzyme based assays: In-vitro ADP-ribosylation assays were applied to determine the impact of partial or complete DPH inactivation: cell extracts were incubated with PE as toxin/enzyme and biotinylated NAD as substrate, followed by detection of Bio-ADPR-eEF2 in Western blot. This method permits reliable detection of ADP-ribosylation, however cannot be used for quantification of slight differences [28] . The results of these analyses (Figure 3C) demonstrate that the toxin ADP-ribosylates eEF2 of parental (wildtype) MCF-7 cells, as well as the eEF2 of all 7 heterozygote

inactivated MCF-7 derivatives (DPH1-7) . In contrast, 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

So far, it was unkown whether or to which degree gene dose effects of diphthamide synthesis genes might affect sensitivity of tumor cells towards targeted tumor therapies. This question could previously not be addressed due to the lack of specific assays and technologies. To address this question, we exposed MCF-7 derivatives with heterozygous or complete inactivation of individual DPH genes to PE and DT and evaluated their susceptibility towards ADP-ribosylating toxins. Therefore, growth and cytotoxicity assays were performed in flat bottom 96 well plates containing 10.000 cells at 37°C in humidified 5% C02 conditions. 24 hrs after seeding, cells were exposed to toxins. To determine cell growth a cell proliferation assay was performed according to the manufacturer's specifications

(CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega) .

Cell proliferation (DNA replication) was addressed by BrdU incorporation assays (Roche Diagnostics, Mannheim FRG) 72 hrs after toxin exposure. Both assay types delivered growth inhibition curves from which IC50 values (inhibition of growth, ATP content or BrdU incorporation to 50% of non- treated cells) could be derived. The results of these cell viability analyses are listed in Table 2 and showed that complete inactivation of DPH1, DPH2, DPH4 or DPH5 genes confers absolute resistance of CF-7 cells towards PE and DT . Even toxin concentrations exceeding the doses that kill parent MCF-7 cells by more than 10.000 fold (Tab.2) did not influence cell growth or proliferation of the mutant cell derivatives. 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

modalities . In contrast to MCF-7 cells without any functional DPH gene copy, MCF-7 derivatives that contained one inactivated and one functional DPH gene copy (DPH1-DPH7) remained fully sensitive to PE and DT .

Cytotoxicity assays (Tab. 2) revealed no significant difference in the IC50 concentrations of these toxins. As all these MCF-7 derivatives contain as the major species diphthamlde-modified eEF2, toxin sensitivity correlates with the presence of eEF2-diphthamide . It is however interesting that 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. Thus, 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:

The synthesis of 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. Also, in most reports related to diphthamide synthesis of mammalian cells 'partial knockouts' such as reduced expression levels were analyzed, which generated 'partial phenotypes' i.e. reduced levels but not complete loss of diphthamide modification and/or toxin sensitivities [36-38] .

Mammalian genomes are more complex than that of yeast and carry extended gene families and duplications. Therefore, mammalian ceils may compensate at least to some degree functional loss of genes that may be unique and therefore essential in yeast. The generation of 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. One advantage of MCF-7 as line to address

functionality and relative impact of different DPHs on diphthamide syntheses is that all of its (MS- and antibody-) detectable eEF2 contains diphthamide. Thus, lack of unmodified eEF2 in wt-MCF-7 provides a clean background which facilitates the detection of alterations or loss of diphthamide. This also enables to define the relative contribution of each individual DPH gene to eEF2 modification at His715.

Complete inactivation of DPH1, DPH2, DPH4 or DPH5 genes resulted in a complete loss of diphthamide modification at eEF2, with unmodified eEF2 present as major species in these cells. Loss of function of these genes cannot be compensated by other proteins, not even to very small degree. In cells that have DPH1, DPH2 or DPH4 inactivated, no other additional eEF2 species was detectable. This confirms previous reports that these proteins are necessary for the initial step of diphthamide modification. Cells with inactivated DPH5 contained some unmodified eEF2, the ACP (diphthine precursor) intermediate as major eEF2 derivative, but no diphthine or diphthamide. Thus 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. In these cell lines, 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. For example, 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. Interestingly 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. This suggests that in addition to direct inactivation of the functionality of eEF2 in translation elongation, ADP-ribosylation may trigger (signaling?) events that interrupt protein synthesis even though unmodified translation-competent eEF2 is still available.

These observations may be important for cancer therapy with toxin fusion proteins, in particular for defining response biomarkers. Bottlenecks for toxin action represent relevant factors that predict sensitivity. Toxin resistance in MCF-7 occurs only upon complete inactivation of DPH

functionality and partial inactivated gene dose reduced cells retained sensitivity. Thus, tumors with alterations in DPH genes that do not completely block diphthamide synthesis, such as 0VCA1 variants [41], may still respond to therapy with targeted toxins.

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. Thus, 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. To eliminate variations between individual clones, we have isolated and analyzed at least two independent clones for each knockout. 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.

Under normal growth conditions without applying stress, growth assays and cell counts revealed no general or impact of DPH gene inactivation on cell growth for all heterozygous clones. Also, complete inactivation of DPH1, DPH2, or DPH4 did not cause significant reductions in cell growth or viability. We observed only minor growth or shape alterations for some clones, but these effects were not attributable to the gene itself because some clones showed differences, but others with the same gene affected did not. Cells with complete inactivation of DPH1, DPH2 and DPH4 harbor only unmodified eEF2. Thus, the exclusive presence of unmodified eEF2 by itself does not inhibit the growth of MCF-7 under normal conditions. Reduced growth rates were observed for all MCF-7 clones that had DPH5 completely inactivated. These cells contain unmodified eEF2 and in addition the ACP- modified eEF2 derivative. ACP-modified eEF2 occurs only in DPH5 deficient cells and not in any other MCF-7 variant. Therefore, our data cannot differentiate between growth reduction related to the presence of the eEF2- ACP intermediate, or being a consequence of lost DPH5 function in other cellular processes that affect cell growth.

To address the question if the action of different protein synthesis (translation) inhibitors is affected in cells containing eEF2 without diphthamide, parent MCF-7 and DPH-inactivated variants were exposed to saporin and cycloheximide (CHX) . Results revealed no effect of DPH gene inactivation and loss of diphthamide on cellular sensitivity. Saporin and cycloheximide inhibited the growth and killed DPH-inactivated cells to the same degree as wildtype MCF-7 (Table 2). Also, stress posed upon cells by protein synthesis inhibition in general appears to be not aggravated by lack of diphthamide as the (saporin and CHX) IC50 values did not differ between wildtype and DPH-inactivated MCF-7 cells (Table 2) .

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] . Also, 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] . Thus, both processes (diphthamide-dependent ADP-ribosylation and TNFa. triggered apoptosis) may be somehow linked. We therefore analyzed if loss of diphthamide influences the sensitivity of MCF-7 cells towards TNFa mediated apoptosis. The 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. In contrast, 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. Thus, 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.

To address the surprising phenomenon of TNFalpha hypersensitivity in more detail, mRNA 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) . All libraries were pooled and sequenced in four lanes on a HiSeq2500 sequencer (Illumina) for 2 50 cycles using version 3 cluster generation kits and version 3 sequencing reagents (Illumina) . As a sequencing control, 10% of PhiX control library (Illumina) was spiked into each lane. Reads were aligned to the human genome (hgl9) using GSNAP

(http : //bioinformatics . oxfordj ournals . org/content/26/7 /873.abstract ) with splice junctions and exons being defined based on Ensembl v73. Expression was then profiled using in-house tools and RPKM values were computed as proposed by Mortazavi et al. [48J . We achieved more than 20M aligned reads per sample of which more than 50% aligned to exons. Differential expression was computed using DESeq (http://genomebiology.com/2010/ll/10/rl06) and differentially expressed genes ( | Log2 ratio | > 1 and corrected p-Value < 0.01) were analyzed using Ingenuity Pathway Analysis. Expression patterns that are associated with diphthamide deficiency and cause TNF

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. Of 50 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) . The most prominent 'markers' for DPH2 and DPH5 inactivation in MCF7 are strongly induced expression of TGFbeta and TNFSF15. Furthermore, comparing the complete transcriptome of parent and diphthamide deficient (DPH2ko and DPH5ko) MCF-7 cells, we observed pathway inductions that resemble NFkappaB associated events and 'pre-activation' of death receptor signaling pathways (Figure 4 & Table 3) . Thus, cells that lack diphthamide become pre-sensitized to death receptor signaling, which explains their hypersensitivity to TNFalpha.

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. In consequence, we obtained cell lines with inactivated functionalities of different DPHs, all of which cannot produce diphthamide. We therefore can address with this cell line set not only the relative impact of each DPH gene on toxin sensitivity or different steps of diphthamide synthesis, but also the biological function of the highly conserved diphthamide modification itself. Loss of diphthamide was achieved by inactivating different genes, thus common biological effects observed in these cell lines should be attributable to the loss of diphthamide or presence of unmodified eEF2.

Inactivation of DPH1, DPH2 or DPH4 genes has no or only a minor impact on cell growth. This indicates that loss of diphthamide or presence of unmodified eEF2 per se does not severely impact cell growth under normal conditions. Evidence for effects on cell growth was however observed upon complete inactivation of DPH5. DPH5 ko cells lack diphthamide and

accumulate unmodified eEF2, both of which by itself however do not pose a problem for cells (DPHl DPH2, DPH4 knockouts grow fine) . Thus, growth reduction upon DPH5 inactivation is a gene specific phenotype and not related to loss of diphthamide. The gene specific effect could be explained by either intermediate (ACP-eEF2) accumulation, or by a function of DPH5 in other biological processes. EEF2 mediates the translation elongation of nascent polypeptide chains and is essential for protein synthesis.

'Normal' eEF2 carries a diphthamide and all eEF2 molecules within MCF-7 cells carry this modification. Thus, 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 .

Because the 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.

Apoptotic pathways including those that trigger signaling via TNF-receptor families are essential for development [4_3] . We observed that all

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

elongation of selected (non-essential) proteins. Translation of a subset of proteins relevant for differentiation or development may be different between diphthamide modified and unmodified eEF2. The expression of such proteins would then be affected by loss of diphthamide, explaining pathway embryonic lethality due to compromised development of DPH ko mice as well as signaling pathway modulation in diphthamide deficient cells.

Additional statements of invention

The following numbered paragraphs define additional aspects and embodiments of the invention:

101. 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.

102. 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.

103. The NAD (+) -diphthamide ADP ribosyltrans ferase for use of paragraph 101 or paragraph 102, wherein the assay for serine phosphorylation of eEF2 protein was performed with an 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.

104. The NAD (+) -diphthamide ADP ribosyltransferase for use of paragraph 103, wherein the monoclonal antibody substantially does not bind to eEF2 that is unmodified at the His715 residue.

105. The NAD (+) -diphthamide ADP ribosyltrans ferase for use of paragraph 103 or paragraph 104, wherein the monoclonal antibody binds to eEF2 having diphthamide modification 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.

106. The NAD {+) -diphthamide ADP ribosyltransferase for use of paragraph 105, wherein the monoclonal antibody substantially does not bind to eEF2 having 3-amino-3-carboxypropyl (ACP) modification and/or diphthine modification at the His715 residue. 107. The NAD {+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 103 to 106, wherein the assay comprised subjecting an extract of the sample to chromatography and contacting one or more chromatography fractions with said monoclonal antibody.

108. The NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 103 to 106, wherein the assay comprised subjecting an extract of the sample to electrophoresis and contacting the electrophoresis gel or a blot thereof with said monoclonal antibody.

109. The NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 103 to 106, wherein the assay comprised subjecting an extract of the sample to a sandwich assay comprising said monoclonal antibody as a capture antibody or detection antibody.

110. The NAD (+) -diphthamide ADP ribosyltransferase for use of paragraph 109, wherein the assay was an ELISA assay.

111. The NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 103 to 106, wherein the assay comprised subjecting an extract of the sample to a dipstick test comprising said monoclonal antibody.

112. The NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 103 to 111, wherein said monoclonal antibody is labelled with a detectable label. 113. The NAD (+) -diphthamide ADP ribosyltransferase for use of paragraph 101 or paragraph 102, wherein the assay for the presence of eEF2 protein having diphthamide modification at the His715 residue was performed by mass spectrometry .

114. The NAD (+) -diphthamide ADP ribosyltransferase for use of paragraph 113, wherein the mass spectrometry was ESI-TOF, Maldi-TOF or SELDI-TOF.

115. The NAD (+) -diphthamide ADP ribosyltransferase for use of paragraph 113 or paragraph 114, wherein the sample was pretreated prior to mass spectrometry

116. The NAD (+) -diphthamide ADP ribosyltransferase for use of paragraph 115, wherein the pre-treatment comprised digestion of eEF2 into peptide fragments .

117. The NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 101 to 116, wherein the NAD (+) -diphthamide ADP

ribosyltransferase is a PE toxin, a DT toxin or a cholix toxin. 118. The NAD (+) -diphthamide ADP ribosyltransferase for use of paragraph 117, wherein the PE toxin has 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 N0:1, wherein the PE toxin has cytotoxic activity when introduced into a mammalian cell. 119. The 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.

120. The NAD (+) -diphthamide ADP ribosyltrans ferase for use of paragraph 117, wherein the cholix toxin has 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 mammalian cell .

121. The NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 117 to 119, wherein the NAD (+) -diphthamide ADP

ribosyltrans ferase is a PE toxin or a DT toxin. 122. The NAD (+) -diphthamide ADP ribosyltransferase for use of paragraph

117 or paragraph 118, wherein the NAD (+) -diphthamide ADP ribosyltransferase is a PE toxin.

123. The NAD (+) -diphthamide ADP ribosyltrans ferase for use of paragraph

122. wherein the PE toxin has the following structure: FCSi - Ρ η - 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- 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), wherein the glutamic acid residue corresponding to position 282 of the native PE sequence (where present) is optionally replaced by another residue, preferably glycine, serine, alanine or glutamine; R ¹ is a linker sequence of 1 to 10 amino acids, preferably GGS or

GGSGGS (SEQ ID NO: 32) ; 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,

(b) 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,

L422, L423, Ά425, R427, L429, E431, R432, Y439, H440, F443, L444, A446, A447, 1450, R456, R458, D461, 463-519

(preferably D463, R467, L477, Y481, R490, R494, R505, R513 and/or L516), E522, R538, E548, R551, L552, T554, 1555, L556, W558, R563, R576, D581, D589, K590, Q592, L597 and

(where present) 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) .

124. The NAD (+) -diphthamide ADP ribosyltransferase for use of paragraph 123, wherein 1 is 1.

125. The NAD (+) -diphthamide ADP ribosyltrans ferase for use of paragraph 123 or paragraph 124, wherein m is 1.

126. The NAD (+) -diphthamide ADP ribosyltrans ferase for use of any one of paragraphs 123 to 125, wherein n is 0.

127. The 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.

129. The NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 123 to 128, wherein the PE functional domain III includes the combination of mutations R427A/F443A/L477H/R494A/R505A/L552E, or the combination of mutations R427A/R456A/D 63A/R467A/R490A/R505A/R538A, or the combination of mutations

R427A/F443A/R456A/D463A/R467A/L477H/R490A/R494A/R505A/R538A/ L552E.

130. The 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.

131. The 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. 132. 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.

133. The NAD (+) -diphthamide ADP ribosyltrans ferase for use of any one of paragraphs 101 to 132, wherein the NAD (+) -diphthamide ADP

ribosyltrans ferase is coupled to the cell-binding agent as a fusion polypeptide .

134. The NAD (+) -diphthamide ADP ribosyltrans ferase for use of paragraph 133, wherein the NAD (+) -diphthamide ADP ribosyltrans ferase is directly coupled to the cell-binding agent as a fusion polypeptide.

135. The NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 132 to 134, wherein the cell-binding agent is an antibody.

136. The NAD (+) -diphthamide ADP ribosyltransferase for use of paragraph 135, wherein the antibody is an antigen-binding antibody fragment. 137. The NAD (+) -diphthamide ADP ribosyltrans ferase for use of paragraph 135 or paragraph 136, wherein the antibody is directed against a tumour- associated antigen.

138. The NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 101 to 137, wherein the diseased cells are pre-cancer, cancer or tumour cells, virally-infected cells or autoimmune effector cells. 139. The NAD (+) -diphthamide ADP ribosyltransferase for use of paragraph 138, wherein the diseased cells are pre-cancer, cancer or tumour cells.

140. The 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.

141. The NAD (+) -diphthamide ADP ribosyltrans ferase for use of paragraph 140, which is for use in the treatment of a pre-cancer, cancer or tumour.

142. The NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 101-141, which is for use in the treatment of a human patient.

143. The NAD (+) -diphthamide ADP ribosyltransferase for use of any one of paragraphs 101-141, wherein the assay excluded any direct assay for the presence or absence of eEF2 that lacks diphthamide modification at the His715 residue.

144. 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.

201. 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.

202. The monoclonal antibody according to paragraph 201, wherein the binding affinity for eEF2 that is unmodified at the His715 residue is at least 10-fold higher than the binding affinity for eEF2 having diphthamide modification at the His715 residue.

203. The monoclonal antibody according to paragraph 201 or paragraph 202, which substantially does not bind to eEF2 having diphthamide modification at the His715 residue.

204. The monoclonal antibody according to any one of paragraphs 201 to 203, which binds to eEF2 that is unmodified at the His715 residue with a K _D of 100 nM or less.

205. The monoclonal antibody according to any one of paragraphs 201 to 204, which binds to eEF2 that is unmodified at the His715 residue with higher binding affinity than to eEF2 having 3-amino-3-carboxypropyl (ACP) modification at the His715 residue.

206. The monoclonal antibody according to paragraph 205, wherein the binding affinity for eEF2 that is unmodified at the His715 residue is at least 10-fold higher than the binding affinity for eEF2 having ACP modification at the His715 residue.

207. The monoclonal antibody according to paragraph 206, wherein the antibody substantially does not bind to eEF2 having ACP modification at the His715 residue. 208. The monoclonal antibody according to any one of paragraphs 201 to 207, which binds to eEF2 that is unmodified at the His715 residue with higher binding affinity than to eEF2 having diphthine modification at the His715 residue.

209. The monoclonal antibody according to paragraph 208, wherein the binding affinity for eEF2 that is unmodified at the His715 residue is at least 10-fold higher than the binding affinity for eEF2 having diphthine modification at the His715 residue.

210. The monoclonal antibody according to paragraph 209, wherein the antibody substantially does not bind to eEF2 having diphthine modification at the His715 residue.

211. The monoclonal antibody according to any one of paragraphs 201-210, having 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. 212. The monoclonal antibody according to any one of paragraphs 201-211, having 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.

213. The monoclonal antibody according to any one of paragraphs 201-212, having the CDR-H1 sequence of the heavy chain variable domain sequence shown in Figure 6 (SEQ ID NO:104), or said CDR-H1 sequence with one or more amino acid insertions, deletions and/or substitutions.

214. The monoclonal antibody according to any one of paragraphs 201-213 having 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. 215. The monoclonal antibody according to any one of paragraphs 201-214 having 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. 216. The monoclonal antibody according to any one of paragraphs 201-215 having the CDR-L1 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.

217. The monoclonal antibody according to any one of paragraphs 201-216 having the CDR-L2 sequence of the heavy chain variable domain sequence shown in Figure 6 (SEQ ID NO: 108), or said CDR-H2 sequence with one or more amino acid insertions, deletions and/or substitutions.

218. The monoclonal antibody according to any one of paragraphs 201-217 having 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.

219. The monoclonal antibody according to any one of paragraphs 201-218, having the heavy chain complementarity-determining region (CDR) sequence H3 of the heavy chain variable domain sequence shown in Figure 6 (SEQ ID NO: 106) .

220. The monoclonal antibody according to paragraph 219, having the heavy chain CDRs H2 and H3 of the heavy chain variable domain sequence shown in Figure 6 (SEQ ID NOs:105 and 106) .

221. 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).

222. The monoclonal antibody according to any one of paragraphs 201-221 having the light chain complementarity-determining region (CDR) sequence L3 of the light chain variable domain sequence shown in Figure 6 (SEQ ID NO: 109) .

223. 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) .

224. 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) . 225. A monoclonal antibody comprising the CDR H1-H3 sequences of the heavy chain variable domain sequence shown in Fig. 6 (SEQ ID NOs:104 to 106 and the CDR L1-L3 sequences of the light chain variable domain sequence shown in Fig. 6 (SEQ ID NOs:107 to 109) . 226. 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.

227. The monoclonal antibody according to any one of paragraphs 201-226, which is labelled with a detectable label. 228. The monoclonal antibody according to paragraph 227, wherein the detectable label is an enzyme, a fluorescent label, a radiolabel, an electroluminescent label or biotin.

229. Dse of the monoclonal antibody according to any one of paragraphs 201-228 in an in vitro method of assessing whether a cell population has increased sensitivity to apoptosis.

230. Use according to paragraph 229, wherein the apoptosis is mediated by induction of NFkappaB signalling.

231. Use according to paragraph 229 or paragraph 230, wherein the apoptosis is mediated by death receptor activation-mediated apoptosis. 232. Use according to paragraph 231, wherein the death receptor is TNFR1, Fas receptor, DR4 or DR5.

233. Use according to any one of paragraphs 229 to 232, wherein the apoptosis is TNF -mediated apoptosis, FasL-mediated apoptosis or TRAIL- mediated apoptosis. 234. Use according to paragraph 233, wherein the apoptosis is TNFa- mediated apoptosis.

235. Use according to any one of paragraphs 229 to 234, wherein the 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.

236. Use according to any one of paragraphs 229 to 235, wherein the monoclonal antibody substantially does not bind to: (i) eEF2 having ACP modification at the His715 residue, (ii) eEF2 having diphthine modification the His715 residue, and (iii) eEF2 having diphthamide modifica

His715 residue.

301. 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.

302. The monoclonal antibody according to paragraph 301, wherein the binding affinity for eEF2 having diphthamide modification at the His715 residue is at least 10-fold higher than the binding affinity for eEF2 that is unmodified at the His715 residue.

303. The monoclonal antibody according to paragraph 301 or paragraph 302, which substantially does not bind to eEF2 that is unmodified at the His715 residue .

304. The monoclonal antibody according to any one of paragraphs 301 to 303, which binds to eEF2 having diphthamide modification at the His715 residue with a K _D of 100 nM or less.

305. The monoclonal antibody according to any one of paragraphs 301 to 304, which binds to eEF2 having diphthamide modification at the His715 residue with higher binding affinity than to eEF2 having 3-amino-3- carboxypropyl (ACP) modification at the His715 residue.

306. The monoclonal antibody according to paragraph 305, wherein the binding affinity for eEF2 having diphthamide modification at the His715 residue is at least 10-fold higher than the binding affinity for eEF2 having ACP modification at the His715 residue. 307. The monoclonal antibody according to paragraph 306, wherein the antibody substantially does not bind to eEF2 having ACP modification at the His715 residue.

308. The monoclonal antibody according to any one of paragraphs 301 to

307. which binds to eEF2 having diphthamide modification at the His715 residue with higher binding affinity than to eEF2 having diphthine modification at the His715 residue.

309. The monoclonal antibody according to paragraph 308, wherein the binding affinity for eEF2 that is unmodified at the His715 residue is at least 10-fold higher than the binding affinity for eEF2 having diphthine modification at the His715 residue. 310. The monoclonal antibody according to paragraph 309, wherein the antibody substantially does not bind to eEF2 having diphthine modification at the His715 residue.

311. The monoclonal antibody according to any one of paragraphs 301-310, which is labelled with a detectable label.

312. The monoclonal antibody according to paragraph 311, wherein the detectable label is an enzyme, a fluorescent label, a radiolabel, an electroluminescent label or biotin.

313. Use of the monoclonal antibody according to any one of paragraphs 301-312 in an in vitro method of assessing resistance or non-resistance of a cell population to NAD ( +) -diphthamide ADP ribosyltransferases treatment.

314. Use of the monoclonal antibody according to any one of paragraphs 301 to 313 in the method of any one of claims 1 to 50.

315. Use according to paragraph 313 or paragraph 314, wherein the monoclonal antibody substantially does not bind to: (i) eEF2 that is unmodified at the His715 residue, (ii) eEF2 having ACP modification at the His715 residue, and (iii) eEF2 having diphthine modification at the His715 residue .

401. 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"), 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.

402. The method according to paragraph 401, further comprising 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.

403. The method according to paragraph 401 or paragraph 402, further comprising a step of: deselecting the patient for treatment with TNFa or other inducer if the diseased cells are assessed not to have increased sensitivity to TNFa or other inducer.

404. The method of 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.

405. A method for selecting and/or deselecting a patient for treatment with TNFa or other inducer, the method comprising:

(i) assaying for the proportion of eEF2 protein that lacks 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 (ii) (b) deselecting the patient for treatment with TNFa or other inducer if the assay is negative for the presence of a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue .

406. The method of paragraph 405, further comprising a step, following the selection of a patient for treatment with TNFa or other inducer, of treating the patient with TNFa or other inducer.

407. 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. 408. A method for treating a patient having a condition that is treatable by TNFa or other inducer, the method 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.

409. 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.

410. The method according to any one of paragraphs 401 to 409, wherein the assay for the proportion of eEF2 protein that lacks diphthamide

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

modification at the His715 residue.

412. The method according to paragraph 410 or paragraph 411, 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. 413. The method according to paragraph 412, wherein the monoclonal antibody substantially does not bind to eEF2 having 3-amino-3-carboxypropyl (ACP) modification and/or diphthine modification at the His715 residue.

414. The method according to any one of paragraphs 410 to 413, wherein the assay comprises subjecting an extract of the sample to chromatography and contacting one or more chromatography fractions with said monoclonal antibody .

415. The method according to any one of paragraphs 410 to 413, wherein the assay comprises subjecting an extract of the sample to electrophoresis and contacting the electrophoresis gel or a blot thereof with said monoclonal antibody.

416. The method according to any one of paragraphs 410 to 413, wherein the assay comprises subjecting an extract of the sample to a sandwich assay comprising said monoclonal antibody as a capture antibody or detection antibody.

417. The method according to paragraph 416, wherein the assay is an ELISA assay .

418. The method according to any one of paragraphs 410 to 413, wherein the assay comprises subjecting an extract of the sample to a dipstick test comprising said monoclonal antibody.

419. The method according to any one of paragraphs 410 to 418, wherein said monoclonal antibody is labelled with a detectable label.

420. The method according to any one of paragraphs 410 to 419, wherein the monoclonal antibody is the monoclonal antibody of any one of paragraphs 201 to 228.

421. -The method according to any one of paragraphs 401 to 409, wherein the assay for the proportion of eEF2 protein that lacks diphthamide

modification at the His715 residue is performed by mass spectrometry.

422. The method according to paragraph 421, wherein the mass spectrometry is ESI-TOF, Maldi-TOF or SELDI-TOF.

423. The method according to paragraph 421 or paragraph 422, wherein the sample is pretreated prior to mass spectrometry.

424. The method according to paragraph 423, wherein the pre-treatment comprises digestion of eEF2 into peptide fragments. 425. The method of any one of paragraphs 401 to 424, wherein the TNF or other inducer is a death receptor ligand.

426. The method of paragraph 425, wherein the death receptor is TNFR1, Fas receptor, DR4 or DR5. 427. The method of paragraph 425 or paragraph 426, wherein the ligand is selected from the group consisting of human TNFa, human TRAIL, human FasL, and active fragments and variants thereof.

428. The method of paragraph 427, wherein the ligand is human TNFa or an active fragment or variant thereof that binds to and activates human TNF receptor 1.

429. The method of paragraph 427, wherein the ligand is human TRAIL or an active fragment or variant thereof that binds to and activates human DR4 and/or DR5.

430. The method of paragraph 427, wherein the ligand is human FasL or an active fragment or variant thereof that binds to and activates human Fas receptor .

431. The method of any one of paragraphs 401 to 430, wherein the other inducer is an agonist anti-death receptor antibody.

432. The method of paragraph 431, wherein 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.

433. The method of any one of paragraphs 401 to 432, wherein the TNFa or other inducer is directly or indirectly coupled to a cell-binding agent. 434. The method of paragraph 433, wherein the TNFa or other inducer is directly coupled to the cell-binding agent as a fusion polypeptide.

435. The method of paragraph 433, wherein the TNFa or other inducer is encapsulated with a coating that bears a cell-binding agent.

436. The method of any one of paragraphs 433 to 435, wherein the cell- binding agent is an antibody.

437. The method of paragraph 436, wherein the cell-binding agent is an antigen-binding antibody fragment. 438. The method of paragraph 436 or paragraph 437, wherein the antibody or antigen-binding antibody fragment is directed against a tumour-associated antigen .

439. The method of any one of paragraphs 401 to 438, wherein the diseased cells are pre-cancer, cancer or tumour cells or virally-infected cells.

440. The method of paragraph 439, wherein the diseased cells are precancer, cancer or tumour cells.

441. The method of any one of paragraphs 407-440, wherein the condition is a pre-cancer, cancer, tumour or viral infection. 442. The method of paragraph 441, wherein the condition is a pre-cancer, cancer or tumour.

443. The method of any one of paragraphs 401-442, wherein the patient is human .

444. The method of any one of paragraphs 402 to 406, or of any one of paragraphs 410 to 443 as dependent from paragraphs 402 to 406, wherein the steps of selecting and/or deselecting patients for treatment with TNFa or other inducer are steps of selecting and/or deselecting patients for treatment with TNFa or other inducer in preference to other treatment options . 445. Use of the antibody according to any one of paragraphs 201-228 in a method according to any one of paragraphs 401-444.

501. 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.

502. 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.

503. The TNFa or other inducer for use of paragraph 501 or paragraph 502, wherein the assay for the presence of a significant proportion of eEF2 protein lacking diphthamide modification at the His715 residue was performed with an 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. 504. The TNFa or other inducer for use of paragraph 503, wherein the monoclonal antibody substantially does not bind to eEF2 having diphthamide modification at the His715 residue.

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.

506. The TNFa or other inducer for use of paragraph 505, wherein the monoclonal antibody substantially does not bind to eEF2 having 3-amino-3- carboxypropyl (ACP) modification and/or diphthine modification at the His715 residue.

507. The TNFa or other inducer for use of any one of paragraphs 503 to 506, wherein the assay comprises subjecting an extract of the sample to chromatography and contacting one or more chromatography fractions with said monoclonal antibody.

508. The TNFa or other inducer for use of any one of paragraphs 503 to 506, wherein the assay comprises subjecting an extract of the sample to electrophoresis and contacting the electrophoresis gel or a blot thereof with said monoclonal antibody. 509. The TNFa or other inducer for use of any one of paragraphs 503 to

506, wherein the assay comprises subjecting an extract of the sample to a sandwich assay comprising said monoclonal antibody as a capture antibody or detection antibody.

510. The TNFa or other inducer for use of paragraph 509, wherein the assay is an ELISA assay.

511. The TNFa or other inducer for use of any one of paragraphs 503 to 506, wherein the assay comprises subjecting an extract of the sample to a dipstick test comprising said monoclonal antibody.

512. The TNFa or other inducer for use of any one of paragraphs 503 to 511, wherein said monoclonal antibody is labelled with a detectable label. 513. The TNFa or other inducer for use of any one of paragraphs 503 to 512, wherein the monoclonal antibody is as the monoclonal antibody of any one of paragraphs 201 to 228.

514. The TNFa or other inducer for use of any one of paragraphs 501 to 502, wherein the assay for the proportion of eEF2 protein that lacks diphthamide modification at the His715 residue was performed by mass spectrometry .

515. The TNFa or other inducer for use of paragraph 514, wherein the mass spectrometry was ESI-TOF, Maldi-TOF or SELDI-TOF. 516. The TNFa or other inducer for use of paragraph 514 or paragraph 515, wherein the sample was pretreated prior to mass spectrometry.

517. The TNFa or other inducer for use of paragraph 516, wherein the pre- treatment comprised digestion of eEF2 into peptide fragments.

518. The 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.

519. The TNFa or other inducer for use of paragraph 518, wherein the death receptor is TNFRl, Fas receptor, DR4 or DR5.

520. The TNFa or other inducer for use of paragraph 518 or paragraph 519, wherein the ligand is selected from the group consisting of human TNFa, human TRAIL, human Fash, and active fragments and variants thereof.

521. The 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.

522. The 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.

523. The TNFa or other inducer for use of paragraph 520, wherein the ligand is human FasL or an active fragment or variant thereof that binds to and activates human Fas receptor. 524. The TNFa or other inducer for use of any one of paragraphs 501 to

523, wherein the other inducer is an agonist anti-death receptor antibody.

525. The 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.

526. The 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.

527. The 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 .

528. The 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 .

529. The TNFa or other inducer for use of any one of paragraphs 526 to 528, wherein the cell-binding agent is an antibody.

530. The TNFa or other inducer for use of paragraph 529, wherein the cell- binding agent is an antigen-binding antibody fragment.

531. The TNFa or other inducer for use of paragraph 529 or paragraph 530, wherein the antibody or antigen-binding antibody fragment is directed against a tumour-associated antigen.

532. The 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.

533. The TNFa or other inducer for use of paragraph 532, wherein the diseased cells are pre-cancer, cancer or tumour cells.

534. The 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 .

535. The TNFa or other inducer for use of paragraph 534, which is for use in the treatment of a pre-cancer, cancer or tumour.

536. The TNFa or other inducer for use of any one of paragraphs 501-535, which is for use in the treatment of a human patient.

537. 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.

Numbered References

Merrick, W.C., Mechanism and regulation of eukaryotic protein synthesis. Microbiological reviews, 1992. 56(2) : p. 291-315. Kaul, G., G. Pattan, and T. Rafeequi, Eukaryotic elongation factor-2 (eEF2) : its regulation and peptide chain elongation. Cell biochemistry and function, 2011. 29(3) : p. 227-34.

Van Ness, B.G., J.B. Howard, and J.W. Bodley, Isolation and properties of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2. The Journal of biological chemistry, 1978. 253(24) : p. 8687-90. Su, X., Z. Lin, and H. Lin, The biosynthesis and biological function of diphthamide. Critical reviews in biochemistry and molecular biology, 2013. 48(6) : p. 515-21.

Liu, S., G.T. Milne, J.G. Kuremsky, G.R. Fink, and S.H.

Leppla, Identification of the proteins required for

biosynthesis of diphthamide, the target of bacterial ADP- ribosylating toxins on translation elongation factor 2.

Molecular and cellular biology, 2004. 24(21) : p. 9487-97.

Arguelles, S., S. Camandola, R.G. Cutler, A. Ayala, and M.P. Mattson, Elongation factor 2 diphthamide is critical for translation of two IRES-dependent protein targets, XIAP and FGF2, under oxidative stress conditions. Free radical biology & medicine, 2014. 67: p. 131-8.

Kimata, Y. and K. ohno, Elongation factor 2 mutants deficient in diphthamide formation show temperature-sensitive cell growth. The Journal of biological chemistry, 1994. 269(18) : p. 13497-501.

Ortiz, P. A., R. Ulloque, G.K. Kihara, H . Zheng, and T.G.

Kinzy, Translation elongation factor 2 anticodon mimicry domain mutants affect fidelity and diphtheria toxin

resistance . The Journal of biological chemistry, 2006.

281(43) : p. 32639-48.

Liu, S., C. Bachran, P. Gupta, S. Miller-Randolph, H. Wang, D. Crown, Y. Zhang, A.N. Wein, R. Singh, R. Fattah, and S.H.

Leppla, Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development . Proceedings of the National Academy of Sciences of the United States of America, 2012. 109(34) : p. 13817-22.

Zhang, Y., S. Liu, G. Lajoie, and A.R. Merrill, The role of the diphthamide-containing loop within eukaryotic elongation factor 2 in ADP-ribosylation by Pseudomonas aeruginosa exotoxin A. The Biochemical journal, 2008. 413(1) : p. 163-74. Roy, V., K. Ghani, and M. Caruso, A dominant-negative approach that prevents diphthamide formation confers resistance to Pseudomonas exotoxin A and diphtheria toxin. PloS one, 2010. 5 (12) : p. el5753.

Chen, CM. and R.R. Behringer, Ovcal regulates cell

proliferation, embryonic development, and tumorigenesis . Genes & development, 2004. 18(3) : p. 320-32.

Liu, S., J.F. Wiggins, T. Sreenath, A.B. Kulkarni, J.M. Ward, and S.H. Leppla, Dph3, a small protein required for

diphthamide biosynthesis , is essential in mouse development . Molecular and cellular biology, 2006. 26(10) : p. 3835-41. Webb, T.R., S.H. Cross, L. McKie, R. Edgar, L. Vizor, J.

Harrison, J. Peters, and I.J. Jackson, Diphthamide

modification of eEF2 requires a J-domain protein and is essential for normal development. Journal of cell science, 2008. 121 (Pt 19) : p. 3140-5.

Van Ness, B.G., J.B. Howard, and J.W. Bodley, ADP-ribosylation of elongation factor 2 by diphtheria toxin. Isolation and properties of the novel ribosyl-amino acid and its hydrolysis products. The Journal of biological chemistry, 1980. 255(22) : p. 10717-20.

Honjo, T . , Y. Nishizuka, and 0. Hayaishi, Diphtheria toxin- dependent adenosine diphosphate ribosylation of aminoacyl transferase II and inhibition of protein synthesis . The

Journal of biological chemistry, 1968. 243(12) : p. 3553-5. Mateyak, M.K. and T.G. Kinzy, ADP-ribosylation of translation elongation factor 2 by diphtheria toxin in yeast inhibits translation and cell separation. The Journal of biological chemistry, 2013. 288(34) : p. 24647-55.

Hassan, R., S. Bullock, A. Premkumar, R.J. Kreitman, H.

Kindler, M.C. Willingham, and I. Pastan, Phase I study of SS1P, a recombinant anti-mesothelin immunotoxin given as a bolus I.V. infusion to patients with mesothelin-expressing mesothelioma , ovarian, and pancreatic cancers. Clinical cancer research : an official journal of the American Association for Cancer Research, 2007. 13(17) : p. 5144-9.

Kelly, R.J., E. Sharon, I. Pastan, and R. Hassan, Mesothelin- targeted agents in clinical trials and in preclinical

development. Molecular cancer therapeutics, 2012. 11(3) : p. 517-25.

Kreitman, R.J., R. Hassan, D.J. Fitzgerald, and I. Pastan,

Phase I trial of continuous infusion anti-mesothelin

recombinant immunotoxin SS1P. Clinical cancer research : an official journal of the American Association for Cancer

Research, 2009. 15(16) : p. 5274-9.

Kreitman, R.J., D.R. Squires, M. Stetler-Stevenson, P. Noel,

D. J. FitzGerald, W.H. Wilson, and I. Pastan, Phase I trial of recombinant immunotoxin RFB4 (dsFv) -PE38 (BL22) in patients with B-cell malignancies. Journal of clinical oncology :

official journal of the American Society of Clinical Oncology, 2005. 23 (27) : p. 6719-29.

Kreitman, R.J., M.S. Tallman, T. Robak, S. Coutre, W.H.

Wilson, M. Stetler-Stevenson, D.J. Fitzgerald, R. Lechleider, and I. Pastan, Phase I trial of anti-CD22 recombinant

immunotoxin moxetumomab pasudotox (CAT-8015 or HA22) in patients with hairy cell leukemia. Journal of clinical oncology : official journal of the American Society of

Clinical Oncology, 2012. 30(15) : p. 1822-8.

Kreitman, R.J., W.H. Wilson, J.D. White, M. Stetler-Stevenson,

E. S. Jaffe, S. Giardina, T.A. Waldmann, and I. Pastan, Phase I trial of recombinant immunotoxin anti-Tac (Fv) -PE38 (1MB-2) in patients with hematologic malignancies . Journal of clinical oncology : official journal of the American Society of

Clinical Oncology, 2000. 18(8) : p. 1622-36.

Wayne, A.S., R.J. Kreitman, H.W. Findley, G. Lew, C. Delbrook, S.M. Steinberg, M. Stetler-Stevenson, D.J. Fitzgerald, and I. Pastan, Anti-CD22 immunotoxin RFB4 (dsFv) -PE38 (BL22) for CD22- positive hematologic malignancies of childhood: preclinical studies and phase I clinical trial. Clinical cancer research : an official journal of the American Association for Cancer Research, 2010. 16(6) : p. 1894-903.

Chen, CM. and R.R. Behringer, OVCA1 : tumor suppressor gene. Current opinion in genetics & development, 2005. 15(1) : p. 49- 54.

Liew, M., R. Pryor, R. Palais, C. Meadows, M. Erali, E. Lyon, and C. Wittwer, Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons . Clinical chemistry, 2004. 50(7) : p. 1156-64.

Maclean, B., D.M. Tomazela, N. Shulman, M. Chambers, G.L.

Finney, B. Frewen, R. Kern, D.L. Tabb, D.C. Liebler, and M.J.

MacCoss, Skyline: an open source document editor for creating and analyzing targeted proteomics experiments . Bioinformatics , 2010. 26(7) : p. 966-8.

Gupta, P.K., S. Liu, and S.H. Leppla, Characterization of a Chinese hamster ovary cell mutant having a mutation in elongation factor-2. PloS one, 2010. 5(2) : p. e9078.

Milstone, D.S., T. Flisikowska, I.S. Thorey, S. Offner, F. Ros, V. Lifke, B. Zeitler, 0. Rottmann, A. Vincent, L. Zhang, S. Jenkins, H. Niersbach, A.J. Kind, P.D. Gregory, A.E.

Schnieke, and J. Platzer, Efficient Immunoglobulin Gene

Disruption and Targeted Replacement in Rabbit Using Zinc Finger Nucleases. PloS one, 2011. 6(6) : p. e21045.

Du, X., R.J. Youle, D.J. FitzGerald, and I. Pastan,

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.

Morimoto, H. and B. Bonavida, Diphtheria toxin- and

Pseudomonas A toxin-mediated apoptosis . ADP ribosylation of elongation factor-2 is required for DNA fragmentation and cell lysis and synergy with tumor necrosis factor-alpha . Journal of immunology, 1992. 149(6) : p. 2089-94.

Brinkmann, U., CAS, the human homologue of the yeast

chromosome-segregation gene CSE1, in proliferation, apoptosis , and cancer. American journal of human genetics, 1998. 62(3) : p. 509-13.

Brinkmann, U., E. Brinkmann, M. Gallo, and I. Pastan, Cloning and characterization of a cellular apoptosis susceptibility gene, the human homologue to the yeast chromosome segregation gene CSE1. Proceedings of the National Academy of Sciences of the United States of America, 1995. 92(22): p. 10427-31.

Brinkmann, U., E. Brinkmann, M. Gallo, U. Scherf, and I.

Pastan, Role of CAS, a human homologue to the yeast chromosome segregation gene CSE1, in toxin and tumor necrosis factor mediated apoptosis. Biochemistry, 1996. 35(21) : p. 6891-9. Schaffrath, R. , W. Abdel-Fattah, R. Klassen, and M.J. Stark, The diphthamide modification pathway from Saccharomyces cerevisiae - revisited. Molecular microbiology, 2014.

Wei, H., L. Xiang, A.S. Wayne, 0. Chertov, D.J. FitzGerald, T.K. Bera, and I. Pastan, Immunotoxin resistance via

reversible methylation of the DPH4 promoter is a unique survival strategy. Proceedings of the National Academy of Sciences of the United States of America, 2012. 109(18) : p. 6898-903. Hu, X., H. Wei, L. Xiang, 0. Chertov, A.S. Wayne, T.K. Bera, and I. Pastan, Methylation of the DPHl promoter causes immunotoxin resistance in acute lymphoblastic leukemia cell line KOPN-8. Leukemia research, 2013. 37(11) : p. 1551-6.

Wei, H., T.K. Bera, A.S. Wayne, L. Xiang, S. Colantonio, 0. Chertov, and I. Pastan, A Modified Form of Diphthamide Causes Immunotoxin Resistance in a Lymphoma Cell Line with Deletion of WDR85. The Journal of biological chemistry, 2013.

L'Allemain, G., [Ovcal gene, deleted in ovarian cancer is a special tumor suppressor] . Bulletin du cancer, 2004. 91(4) : p. 301-2.

Li, A.J. and B.Y. Karlan, Genetic factors in ovarian

carcinoma. Current oncology reports, 2001. 3(1) : p. 27-32. Nobukuni, Y., K. Kohno, and K. iyagawa, Gene trap

mutagenesis-based forward genetic approach reveals that the tumor suppressor OVCAl is a component of the biosynthetic pathway of diphthamide on elongation factor 2. The Journal of biological chemistry, 2005. 280(11) : p. 10572-7.

Greganova, E., M. Altmann, and P. Butikofer, Unique

modifications of translation elongation factors. The FEBS journal, 2011. 278(15) : p. 2613-24.

Yeh, W.C., J.L. de la Pompa, M.E. McCurrach, H.B. Shu, A.J. Elia, A. Shahinian, M. Ng, A. Wakeham, W. Khoo, K. Mitchell, W.S. El-Deiry, S.W. Lowe, D.V. Goeddel, and T.W. Mak, FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis . Science, 1998. 279(5358) : p. 1954-8.

Hizli, A. A., Y. Chi, J. Swanger, J.H. Carter, Y. Liao, M.

Welcker, A.G. Ryazanov, and B.E. Clurman, Phosphorylation of eukaryotic elongation factor 2 (eEF2) by cyclin A-cyclin- dependent kinase 2 regulates its inhibition by eEF2 kinase. Molecular and cellular biology, 2013. 33(3) : p. 596-604.

Ovchinnikov, L.P., L.P. Motuz, P.G. Natapov, L.J. Averbuch, R.E. Wettenhall, R. Szyszka, G. Kramer, and B. Hardesty, Three phosphorylation sites in elongation factor 2. FEBS letters, 1990. 275 (1-2) : p. 209-12.

Sans, M.D., Q. Xie, and J.A. Williams, Regulation of

translation elongation and phosphorylation of eEF2 in rat pancreatic acini. Biochemical and biophysical research communications, 2004. 319(1) : p. 144-51.

Kruiswijk, F., L. Yuniati, R. Magliozzi, T.Y. Low, R. Lim, R. Bolder, S. Mohammed, C.G. Proud, A.J. Heck, M. Pagano, and D. Guardavaccaro, Coupled activation and degradation of eEF2K regulates protein synthesis in response to genotoxic stress. Science signaling, 2012. 5(227) : p. ra40.

Mortazavi, A., B.A. Williams, K. McCue, L. Schaeffer, and B. Wold, Mapping and quantifying mammalian transcriptomes by RNA- Seq. Nature methods, 2008. 5(7) : p. 621-8.

Seeber, S., F. Ros, I. Thorey, G. Tiefenthaler , K. Kaluza, V. Lifke, J. A. Fischer, S. Klostermann, J. Endl, E. Kopetzki, A. Pashine, B. Siewe, B. Kaluza, J. Platzer, and S. Offner, A robust high throughput platform to generate functional recombinant monoclonal antibodies using rabbit B cells from peripheral blood. PloS one, 2014. 9(2) : p. e86184. Other Selected References

Alderson RF et al . 2009 Clin Cancer Res. 15:832-839. [PubMed: 19188153] Alewine C et al . 2014 Mol. Cancer Ther. 13(11): 2653-61

Awasthi et al. 2013 Infect Immun 81 ( 2 ) : 531-541

Chaudhary et al 1991 Proc. Natl. Acad. Sci. USA 87: 308-312

Hansen et al. 2010 Journal of Immunotherapy 33(3): 297-304

Hassan R et al. Clin Cancer Res. 13:5144-5149. [PubMed: 17785569]

Ho et al. 2005 Clin. Cancer Res. 11(10) : 3814-20

J0rgensen et al . 2008a J Bio Chem 283(16) : 10671-10678

Kreitman RJ et al . 2000 J Clin Oncol. 18:1622-1636. [PubMed: 10764422] Kreitman RJ et al . 2005 J Clin Oncol. 23:6719-6729. [PubMed: 16061911] Kreitman RJ et al . 2009a J Clin Oncol. 27:2983-2990. [PubMed: 19414673] Kreitman RJ et al. 2009b Curr Pharm Des. 15:2652-2664. [PubMed: 19689336] Kreitman RJ et al . 2009c Clin Cancer Res. 15:5274-5279. [PubMed:

19671873]

Kreitman RJ et al . 2012 J. Clin. Oncol. 30(15) : 1822-8

Liu et al. 2012 Proc. Natl. Acad. Sci. USA 109(29): 11782-7

Mazor R et al. 2014 Proc. Natl. Acad. Sci. USA 111(23) : 8571-8576

Onda et al. 2008 Proc. Natl. Acad. Sci. USA 105(32) : 11311-6

Onda et al. 2011 Proc. Natl. Acad. Sci. USA 108(14)_: 5742-7

Pastan et al . 2011 Leukemia & Lymphoma 52 Suppl 2:87-90

Roscoe et al. 1994 Infection and Immunity 62(11) : 5055-65

Salvatore G et al. 2002 Clin Cancer Res. 8:995-1002. [PubMed: 11948105] Seetharam et al 1991 J. Biol. Chem. 266: 17376-17381

Shapira A and Benhar I 2010 Toxins 2:2519-2583. [PubMed: 22069564]

Wayne AS et al. 2010 Clin Cancer Res. 16:1894-1903. [PubMed: 20215554] Weldon JE et al . 2009 Blood 113:3792-3800. [PubMed: 18988862] Weldon and Pastan 2011 FEBS J 278 (23 ) : 4683-4700

Wolf P and Elsasser-Beile U 2009 J Med Microbiol. 299:161-176. [PubMed: 18948059]

Yates et al. 2006 Trends Biochem Sci 31(2) :123-33

All documents cited in the text (whether or not included in these lists numbered and other selected references) are incorporated by reference herein in their entirety and for all purposes.

Table 1

Table 3

Table 2