WO2009000297A1 - Tpp ii inhibitors for use in combination with chemotherapy for the treatment of cancer - Google Patents

Abstract

TPP Il (tripeptidyl peptidase II) inhibitors are useful in enhancing the efficacy of cancer chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells. Suitable compounds comprise tripeptide compounds of general formula RN1RN2-A1-A2-A-CO-RC1 wherein RN1, RN2, A1, A2, A3 and RC1 are as defined herein, and which include for example the tripeptide sequences GLA and GPG. In vivo experiments show that TPPII inhibitors are effective in combination with each of a range of chemotherapeutic agents of varying types.

Description

TPP Il INHIBITORS FOR USE IN COMBINATION WITH CHEMOTHERAPY FOR THE TREATMENT OF CANCER

The present invention relates to compounds for use in combination with chemotherapy for the treatment of cancer. 5

Many detailed studies have been carried out on tripeptidyl-peptidase Il (TPP II). TPPII is built from a unique 138 kDa sub-unit expressed in multi-cellular organisms from Drosophila to Homo Sapiens (Tomkinson B, Lindas AC. Tripeptidyl-peptidase II: a multi-purpose peptidase, lnt J Biochem Cell Biol 2005;37:1933-7) (Renn SC₁ Tomkinson B, Taghert PH.

10 Characterization and cloning of tripeptidyl peptidase Il from the fruit fly, Drosophila melanogaster. J Biol Chem 1998;273:19173-82) (Rockel B, Peters J, Kuhlmorgen B, Glaeser RM, Baumeister W. A giant protease with a twist: the TPPII complex from Drosophila studied by electron microscopy. EMBO J 2002;21 :5979-84). Data from Drosophila suggests that the TPPII complex consists of repeated sub-units forming two

15 twisted strands with a native structure of about 6 MDa (Rockel B, Peters J, Kuhlmorgen B, Glaeser RM, Baumeister W. A giant protease with a twist: the TPPII complex from Drosophila studied by electron microscopy. EMBO J 2002;21 :5979-84). TPPII degrades cytosolic polypeptides (Glas R, Bogyo M, McMaster JS, Gaczynska M, Ploegh HL. A proteolytic system that compensates for loss of proteasome function. Nature

20 1998;392:618-22) (Geier E, Pfeifer G, WiIm M, Lucchiari-Hartz M₁ Baumeister W,

Eichmann K, et. al. A giant protease with potential to substitute for some functions of the proteasome. Science 1999;283:978-81 ) (Gavioli R₁ Frisan T, Vertuani S₁ Bomkamm GW, Masucci MG. c-myc overexpression activates alternative pathways for intracellular proteolysis in lymphoma cells. Nat Cell Biol 2001 ;3:283-8.), generates certain MHC class I

25 ligands (Reits E, Neijssen J, Herberts C₁ Benckhuijsen W, Janssen L₁ Drijfhout JW₁ et. al. A major role for TPPII in trimming proteasomal degradation products for MHC class I antigen presentation. Immunity 2004;20:495-506) (York IA₁ Bhutani N, Zendzian S, Goldberg AL₁ Rock KL. Tripeptidyl Peptidase Il Is the Major Peptidase Needed to Trim Long Antigenic Precursors, but Is Not Required for Most MHC Class I Antigen Presentation. J Immunol

30 2006;177:1434-43.) and complements the proteasome in protein turnover. However, other roles of this complex may also exist, that may be unrelated to protein turnover. TPPII regulates transduction of apoptotic signals as well as centrosome homeostasis, by unclear mechanisms (Hong X, Lei L, Glas R. Tumors acquire inhibitor of apoptosis protein (IAP)- mediated apoptosis resistance through altered specificity of cytosolic proteolysis. J Exp

35 Med 2003;197:1731-43.) (Hilbi H, Puro RJ, Zychlinsky A. Tripeptidyl peptidase Il promotes maturation of caspase-1 in Shigella flexneri-induced macrophage apoptosis. Infect lmmun 2000;68:5502-8) (Stavropoulou V, Xie J, Henriksson M, Tomkinson B, lmreh S₁ Masucci MG. Mitotic infidelity and centrosome duplication errors in cells overexpressing tripeptidyl- peptidase II. Cancer Res 2005;65:1361-8) (Stavropoulou V, Vasquez V, Cereser B, Freda E, Masucci MG. TPPII promotes genetic instability by allowing the escape from apoptosis of cells with activated mitotic checkpoints. Biochem Biophys Res Commun 2006;346:415- 25).

Whilst many scientists have carried out work on many aspects of the above-mentioned pathways, there has been much confusion and uncertainty concerning the actual therapeutic potential of targeting the TPP Il pathway. The present inventors have carried out detailed and extensive studies and show herein that cancer chemotherapy can be enhanced in vivo by the use of TPP Il inhibitors. This new therapeutic use is supported by data presented below for combination with several classes of chemotherapeutic drugs.

From a first aspect the present invention provides a compound for use in enhancing the efficacy of cancer chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells, wherein said compound is a TPP Il inhibitor.

As used herein the term "cancer chemotherapy" covers the treatment of a cancerous condition by a chemical compound which is known to have some therapeutic effect against cancer. The TPP Il inhibitor may be used in combination with one or more drugs of which at least one is known to possess anti-cancer properties.

The therapy herein may also include preventative therapy and the treatment of a precancerous condition.

As used herein the term "tumour cells" includes cancerous or pre-cancerous cells. Such cells may have cancerous or pre-cancerous defects. Thus the cells may have acquired one or several alterations characteristic of malignant progression.

The invention not only allows chemotherapy-resistant tumours to be treated, but is also advantageous even with tumours that can be treated with chemotherapy, in allowing lower doses of chemotherapy to be used. From a further aspect the present invention provides a compound for use in enhancing the efficacy of cancer chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells, wherein said compound is selected from the following formula (i) or is a pharmaceutically acceptable salt thereof:

(i) R^N1R^N2N-A¹-A²-A³-CO-R^C1

wherein A¹, A² and A³ are amino acid residues having the following definitions according to the standard one-letter abbreviations or names:

A¹ is G, A, V, L, I, P, 2-a mi no butyric acid, norvaline or tert-butyl glycine,

A² is G, A, V, L₁ I₁ P, F₁ W, C, S₁ K₁ R₁ 2-aminobutyric acid, norvaline, norleucine, tert-butyl alanine, alpha-methyl leucine, 4,5-dehydro-leucine, allo-isoleucine, alpha- methyl valine, tert-butyl glycine, 2-allylglycine, ornithine or alpha, gamma- diaminobutyric acid,

A³ is G, A, V, L, I₁ P, F, W, D, E, Y, 2-aminobutyric acid, norvaline or tert-butyl glycine,

R^N1 and R^N2are each attached to the N terminus of the peptide, are the same or different, and are each independently

R^N3,

(Iinker1 )-R^N3, CO-(linker1 )-R^N3,

CO-O-(linker1 )-R^N3,

CO-N-((linker1 )-R^N3)R^N4 or

SO₂-(linker1 )-R^N3,

(linker! ) may be absent, i.e. a single bond, or CH₂, CH₂CH₂, CH₂CH₂CH₂,

CH₂CH₂CH₂CH₂ or CH=CH,

R^N3and R^N4 are the same or different and are hydrogen or any of the following optionally substituted groups: saturated or unsaturated, branched or unbranched Ci_-6 alkyl; saturated or unsaturated, branched or unbranched C₃.₁₂ cycloalkyl; benzyl; phenyl; naphthyl; mono- or bicyclic C_MO heteroaryl; or non-aromatic d.-io heterocyclyl;

wherein there may be zero, one or two (same or different) optional substituents on R^N3 and/or R^N4 which may be: hydroxy-; thio-: amino-; carboxylic acid; saturated or unsaturated, branched or unbranched C_1-6 alkyloxy; saturated or unsaturated, branched or unbranched C_3-12 cycloalkyl;

N-, O-, or S- acetyl; carboxylic acid saturated or unsaturated, branched or unbranched Ci_-6 alkyl ester; carboxylic acid saturated or unsaturated, branched or unbranched C_3-I2 cycloalkyl ester phenyl; mono- or bicyclic Ci_-10 heteroaryl; non-aromatic Ci.i₀ heterocyclyl; or halogen;

R^C1 is attached to the C terminus of the tripeptide, and is: O-R^C2,

O-(linker2)-R^C2, N((linker2)R^C2)R^C3, or N(linker2)R^C2-NR^C3R^C4,

(Iinker2) may be absent, i.e. a single bond, or Ci_-6 alkyl or C_2-4 alkenyl, preferably a single bond or CH₂, CH₂CH₂, CH₂CH₂CH₂, CH₂CH₂CH₂CH₂ or CH=CH, R^c2, R^C3 and R^CA are the same or different, and are hydrogen or any of the following optionally substituted groups: saturated or unsaturated, branched or unbranched d.₆ alkyl; saturated or unsaturated, branched or unbranched C₃.₁₂ cycloalkyl; benzyl; phenyl; naphthyl; mono- or bicyclic C_MO heteroaryl; or non-aromatic C_MO heterocyclyl;

wherein there may be zero, one or two (same or different) optional substituents on each of R^c2 and/or R^C3 and/or R^C4 which may be one or more of: hydroxy-; thio-: amino-; carboxylic acid; saturated or unsaturated, branched or unbranched Ci_-6 alkyloxy; saturated or unsaturated, branched or unbranched C₃_i₂ cycloalkyl; N-, O-, or S- acetyl; carboxylic acid saturated or unsaturated, branched or unbranched Ci_-6 alkyl ester; carboxylic acid saturated or unsaturated, branched or unbranched C_3-I2 cycloalkyl ester phenyl; halogen; mono- or bicyclic Ci_io heteroaryl; or non-aromatic C_MO heterocyclyl.

The N and CO indicated in the general formula for formula (i) are the nitrogen atom of amino acid residue A¹ and the carbonyl group of amino acid residue A³ respectively.

From a further aspect the invention provides a method of enhancing the efficacy of cancer chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells comprising administering to a patient in need thereof a therapeutically effective amount of a TPPII inhibitor or a compound selected from formula (i) or a pharmaceutically acceptable salt thereof. The compound may be administered in combination with cancer chemotherapy in order to decrease resistance to said cancer chemotherapy.

The administration is preferably repeated until treatment of the tumour is enhanced.

Similarly, from a further aspect the present invention provides the use of a TPPII inhibitor or a compound selected from formula (i) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for enhancing the efficacy of cancer chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells.

Without wishing to be bound by theory, the invention may be considered to recognize that TPP Il inhibitors are useful in combination with cancer chemotherapy in the treatment of cancer.

From a further aspect the present invention provides a pharmaceutical composition comprising a TPP Il inhibitor and a cancer chemotherapy compound. Said pharmaceutical composition may also comprise a pharmaceutically acceptable diluent or carrier. Said pharmaceutical composition may comprise more than one cancer drug.

The TPP Il inhibitor and cancer chemotherapy compound may be present in the same composition, so that they can be administered together. Alternatively the TPP Il inhibitor and the cancer therapy agent may be present in a kit of parts so that they may be administered separately.

From a further aspect the present invention provides a combination of a TPP Il inhibitor and a cancer chemotherapy compound for use as in therapy. The combination may be such that the TPP Il inhibitor and cancer chemotherapy compound are administrable simultaneously or sequentially.

From a further aspect the invention provides a method for identifying a compound suitable for enhancing the efficacy of cancer chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells comprising contacting TPP Il with a compound to be screened, and identifying whether the compound inhibits the activity of TPP II. The cancer chemotherapy referred to herein may for example be cytostatic therapy or angiogenesis inhibition. Thus two classes of cancer chemotherapeutic agents which may be used in combination with TPP Il inhibitors include cytostatic drugs and angiogenesis inhibitors.

Cytostatic drugs are substances with an inhibitory effect on cancer cell growth, which may be used for the treatment of cancer. Cytostatic drugs comprise, but are not limited to, DNA damaging drugs (e.g. topoisomerase inhibitors, alkylators, or anti-metabolites), tubulin inhibitors, proteasome inhibitors, Hsp90 inhibitors, Corticosteroids, inhibitors of growth factor signaling (e.g. inhibitors of PI3K or MAPK pathways), inhibitors of the anti-apoptotic signals (e.g. antagonists of Bcl-2 or XIAP) or activators of pro-apoptotic signals (e.g. p53 activators). The present invention therefore relates, inter alia, to the use of molecules that inhibit or antagonize the function of TPPII in combination with cytostatic drugs. In vivo data presented below support the use of TPP Il inhibitors in combination with numerous types of cytostatic drugs across the spectrum of cytostatic therapy.

The TPP Il inhibitor may be used in combination with a drug selected from any one or more of the following classes: DNA damaging drugs (e.g. topoisomerase inhibitors, alkylators, or anti-metabolites), tubulin inhibitors, proteasome inhibitors, Hsp90 inhibitors, Corticosteroids, inhibitors of growth factor signaling (e.g. inhibitors of PI3K or MAPK pathways), inhibitors of the anti-apoptotic signals (e.g. antagonists of Bcl-2 or XIAP) or activators of pro-apoptotic signals (e.g. p53 activators). The TPP Il inhibitor may be used with other chemotherapeutic drugs.

One preferred class of cytostatic drugs for use in combination with TPP Il inhibitors is the class of alkylators, for example cyclophosphamide. Another preferred class of cytostatic drugs for use in combination with TPP Il inhibitors is the class of tubulin inhibitors, for example paclitaxel, taxoter or vinorelbine, preferably paclitaxel.

Angiogenesis inhibitors treat cancer by targeting the growth of blood vessels, since these are needed to supply nutrients and oxygen to an expanding tumour mass (Folkman, J. Angiogenesis. Annnu Rev. Med. 2006;57:1-18). This concept has been much studied in pre-clinical studies with inhibitors that block growth factor receptors on endothelial cells (e.g. VEGF-R), which in some cases can cause complete regression of a growing tumour mass in mice. Recent studies in human patients have also shown significant, although less dramatic, effects on tumour growth. This has led to approval of certain inhibitors of angiogenesis for therapy in cancer patients. However, there is a great need for agents which improve the effectiveness of cancer treatment with angiogenesis inhibitors (Gasparini G, Longo R, Toi M, Ferrara N. Angiogenic inhibitors: a new therapeutic strategy in oncology. Nat Clin Pract Oncol 2005; 1 1 :562-77). The present invention therefore relates, inter alia, to the use of molecules that inhibit or antagonize the function of TPPII in combination with angiogenesis inhibitors. In vivo data presented below support the use of TPP Il inhibitors in combination with angiogenesis inhibitors.

One preferred angiogenesis inhibitor for use in combination with TPP Il inhibitors is TNP- 470. Another is Thalidomide.

Without wishing to be bound by theory, an understanding of cellular stress sensing and signal transduction at the molecular level is useful for development of therapies against many pathogenic conditions, including cancer, ischemia and viral infections (Brown JM, Attardi LD. The role of apoptosis in cancer development and treatment response. Nat Rev Cancer 2005;5:231-7) (Yonekura I, Takai K, Asai A, Kawahara N, Kirino T. p53 potentiates hippocampal neuronal death caused by global ischemia. J Cereb Blood Flow Metab 2006;26:1332-40) (Lau A, Swinbank KM, Ahmed PS, Taylor DL, Jackson SP, Smith GC, et. al. Suppression of HIV-1 infection by a small molecule inhibitor of the ATM kinase. Nat Cell Biol 2005;7:493-500). Several types of stress activate Phospho-lnositide-3-OH- Kinase-related kinases (PIKKs); a family that includes several kinases, DNA-PKcs, ATM, and ATR in the nucleus; as well as mTOR that resides in the cytosol and mitochondria (Bakkenist CJ, Kastan MB. Initiating cellular stress responses. Cell 2004;118:9-17) (McKinnon PJ. ATM and ataxia telangiectasia. EMBO Rep 2004;5:772-6). PIKKs play a role as signal transducers from sensor molecules in response to stress, and phosphorylate a network of regulatory factors to initiate DNA repair and cell cycle arrest; pathways often constitutively activated in transformed cells (Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, et. al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 2005;434:907-13) (Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, et. al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 2005;434:864-70). Cells and tissues failing to express PIKKs have deficient p53 stabilization, cell cycle checkpoints and an increased susceptibility to apoptosis induced by gamma-irradiation; as present in genetic diseases with failure to express ATM (i.e. Ataxia Telangiectasia) and related diseases (McKinnon PJ. ATM and ataxia telangiectasia. EMBO Rep 2004;5:772- 6) (Barlow C, Hirotsune S, Paylor R, Liyanage M, Eckhaus M, Collins F, et. al. Atm-deficient mice: a paradigm of ataxia telangiectasia. Cell 1996;86: 159-71)

Cancer therapy frequently depends on the induction of DNA damage, e.g. treatment with gamma-irradiation or DNA topoisomerase inhibitors. Nuclear PIKKs (i.e. ATM, ATR and DNA-PKcs) are therefore also possible targets to increase the efficiency of such therapy. Incubation of tumour cells with inhibitors of these PIKKs block DNA repair responses, which increases susceptibility to gamma-irradiation- induced apoptosis in vitro (Cowell IG, Durkacz BW, Tilby MJ. Sensitization of breast carcinoma cells to ionizing radiation by small molecule inhibitors of DNA- dependent protein kinase and ataxia telangiectsia mutated. Biochem Pharmacol 2005;71 : 13-20). This occurs despite that PIKK inhibitors prevent stabilization of p53, suggesting that apoptosis of such gamma-irradiated cells is p53 independent. However, also normal tissues require PIKKs for protection against DNA damage (Bakkenist CJ, Kastan MB. Initiating cellular stress responses. Cell 2004;118:9- 17) (McKinnon PJ. ATM and ataxia telangiectasia. EMBO Rep 2004;5:772-) (Barlow C, Hirotsune S, Paylor R, Liyanage M, Eckhaus M, Collins F, et. al. Atm-deficient mice: a paradigm of ataxia telangiectasia. Cell 1996 ;86: 159-71 ). Experimental in vivo studies of mice treated with inhibitors of nuclear PIKKs have shown significant effects on tumour growth, but tumour regression responses are usually not observed using such protocols (Choi EK, Ji IM, Lee SR, Kook YH, Griffin RJ, Lim BU, et. al. Radiosensitization of tumor cells by modulation of ATM kinase, lnt J Radiat Biol 2006;82:277-83) (Zhao Y, Thomas HD, Batey MA, Cowell IG, Richardson CJ, Griffin RJ, et. al. Preclinical evaluation of a potent . novel DNA-dependent protein kinase inhibitor NU7441. Cancer Res 2006;66:5354-62). mTOR, a cytosolic PIKK-family member, has a crucial role in the integration of signals from nutritional sensing, regulation of protein translation and control of Akt kinase activation (Faivre S, Kroemer G, Raymond E. Current development of mTOR inhibitors as anticancer agents. Nat Rev Drug Discov 2006;5:671-88). Inhibitors of mTOR sensitize tumors to gamma-irradiation in mice, with the occasional observation of tumor regression, and such inhibitors show promising results in trials against several forms of cancer (Faivre S, Kroemer G, Raymond E. Current development of mTOR inhibitors as anticancer agents. Nat Rev Drug Discov 2006; 5: 671-88) (Eshleman JS, Carlson BL, Mladek AC, Kastner BD, Shide KL, Sarkaria JN. Inhibition of the mammalian target of rapamycin sensitizes U87 xenografts to fractionated radiation therapy. Cancer Res 2002; 62:7291-7) (Weppler SA, Krause M, Zyromska A, Lambin P, Baumann M, Wouters BG. Response of U87 glioma xenografts treated with concurrent rapamycin and fractionated radiotherapy: possible role for thrombosis. Radiother Oncol 2007;82 :96-104) (Tirado OM, Mateo-Lozano S, Sanders S, Dettin LE, Notario V. The PCPH oncoprotein antagonizes the proapoptotic role of the mammalian target of rapamycin in the response of normal fibroblasts to ionizing radiation. Cancer Res 2003;63 :6290-8). In addition, inhibition of Chkl and Chk2 kinases, downstream targets of PIKK signaling pathways, increase the susceptibility to tumor treatment; an effect that may include targeting of cancer stem cells (Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, et. al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006;444:756-60) (Reinhardt HC, Aslanian AS, Lees JA, Yaffe MB. p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell 2007;11 :175-89). It is yet unclear which PIKKs, or PIKK-dependent pathways, represent targets for efficient cancer therapy.

Several types of stress, proteotoxic, oncogenic as well as nutrient stress, cause increased expression of TPPII (18-21). We believe that TPPII has a crucial role in gamma- irradiation-induced DNA damage responses in vitro and in resistance to gamma-irradiation-based cancer therapy in vivo.

Our results show that the expression of TPPII requires PIKK signaling, and that TPPII is rapidly translocated into the nucleus of gamma-irradiated cells. These events are dependent on mTOR, a cytosolic/ mitochondrial PIKK that is activated by gamma-irradiation. Lymphoma cells with inhibited expression of TPPII fail to efficiently stabilize p53, and have reduced ability to arrest proliferation in response to gamma-irradiation. We observe that TPPII contains a BRCA C-terminal (BRCT)-like motif, contained within sequences of several proteins involved in DNA damage signaling pathways, and this motif is important for nuclear translocation of TPPII and stabilization of p53. We have found tri-peptide-based inhibitors of TPPII which cause complete in vivo tumour regression in mice, in response to relatively low doses of gamma-irradiation (3-4 Gy/week). We have observed this with established mouse and human tumours of diverse tissue backgrounds, with no tumour re- growth after cancellation of treatment. We have also found that these TPPII inhibitors do not have adverse cellular toxicity. Our data indicate that TPPII connects signaling by cytosolic/mitochondrial and nuclear PIKK-dependent pathways, and that TPPII can be 5 targeted for inhibition of tumor therapy resistance.

TPP Il accepts a relatively broad range of substrates. All the compounds falling within formula (i) are peptides or peptide analogues. Compounds of formulae (i) are readily synthesizable by methods known in the art (see for example Ganellin et al., J. Med. Chem. 10 2000, 43, 664-674) or are readily commercially available (for example from Bachem AG). In a preferred aspect the compound may be selected from formulae (i). Such tripeptides and derivatives are particularly effective therapeutic agents.

According to the invention the compound for use in enhancing the efficacy of cancer 15 chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells may be a compound which is known to be a TPP Il inhibitor in vivo.

For example, the compound may be selected from compounds identified in Winter et al., Journal of Molecular Graphics and Modelling 2005, 23, 409-418 as TPP Il inhibitors. The 20 compounds may be selected from the following formula (ii) because these compounds are particularly suited to the TPP Il pharmacophore:

(ϋ) R¹ 5 wherein R' is H, CH₃, CH₂CH₃, CH₂CH₂CH₃ or CH(CH₃)₂,

R" is H, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)_2, CH₂CH₂CH₂CH₃, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃ or C(CH₃)₃, and R¹¹¹ Js H₁ CH₃₁ OCH₃₁ F₁ Cl Or Br;

Compounds of formula (ii) are synthesizable by known methods (see for example Winter et al., Journal of Molecular Graphics and Modelling 2005, 23, 409-418 and Breslin et al., Bioorg. Med. Chem. Lett. 2003, 13, 4467-4471 ).

Also by way of example, the compound may be selected from compounds identified in US 6,335,360 of Schwartz et al. as TPP Il inhibitors. Such compounds include those of the following formula (iii).

(iii)

wherein:

each R¹ may be the same or different, and is selected from the group consisting of halogen, OH; Ci -C₆ alkyl optionally substituted by one or more radicals selected from the group consisting of halogen and OH; (Ci -C₆) alkenyl optionally substituted by one or more radicals selected from the group consisting of halogen and OH; (C1 -C₆) alkynyl, optionally substituted by one or more radicals selected from the group consisting of halogen and OH, X(Ci -C₆)alkyl, wherein X is S₁ 0 or OCO, and the alkyl is optionally substituted by one or more radicals selected from the group consisting of halogen and OH; SO₂ (Ci -C₆)alkyl, optionally substituted by at least one halogen, YSO₃ H, YSO₂ (Ci -C₆)alkyl, wherein Y is O or NH and the alkyl is optionally substituted by at least one halogen, a diradical -X1-(Ci -C₂)alkylene-X1- wherein X₁ is O or S; and a benzene ring fused to the indoline ring;

n is from 0 to 4; R² is CH₂R⁴, wherein R⁴ is Ci -C₆ alkyl substituted by one or more radicals selected from the group consisting of halogen and OH; (CH₂)_pZ(CH₂)qCH₃, wherein Z is O or S, p is from 0 to 5 and q is from 0 to 5, provided that p+q is from 0 to 5; (C2 -C₆) unsaturated alkyl; or (C₃ -C₆) cycloalkyl;

or R² is (Ci -C₆)alkyl or O(Ci -C₆)alkyl, each optionally substituted by at least one halogen;

R³ is H; (Ci -C₆)alkyl optionally substituted by at least one halogen; (CH₂)_P ZR⁵ wherein p is from 1 to 3, Z is O or S and R⁵ is H or (Ci -C₃)alkyl; benzyl.

Compounds of formula (iii) are readily synthesizable by known methods (see for example US 6,335,360 of Schwartz et al.).

Compounds of formula (iii) are less preferable than other TPP Il inhibitors. Thus butabindide compounds and UCL1371 are less preferable than other TPP Il inhibitors. For example, it is preferred that the compound be selected from formulae (i) and (ii), more preferably formula (i).

It is also possible for the compound to be a compound of formula (i) wherein R^N1, R^N2 and R^C1 are as defined above or in any of the preferences below and wherein:

A¹ is G, A, V, L, I, P, S, T, C, N, Q, 2-aminobutyric acid, norvaline, norleucine, tert- butyl alanine, alpha-methyl leucine, 4,5-dehydro-leucine, allo-isoleucine, alpha- methyl valine, tert-butyl glycine or 2-allylglycine,

A² is G, A, V, L, I, P, S, T₁ C, N, Q, F, Y, W, K₁ R, histidine, 2-aminobutyric acid, norvaline, norleucine, tert-butyl alanine, alpha-methyl leucine, 4,5-dehydro-leucine, allo-isoleucine, alpha-methyl valine, tert-butyl glycine, 2-allylglycine, ornithine, alpha.gamma-diaminobutyric acid or 4,5-dehydro-lysine, and

A³ is G, A₁ V₁ L, I, P₁ S₁ T, C, N₁ Q, D, E, F, Y₁ W, 2-aminobutyric acid, norvaline, norleucine, tert-butyl alanine, alpha-methyl leucine, 4,5-dehydro-leucine, allo- isoleucine, alpha-methyl valine, tert-butyl glycine or 2-allylglycine. Preferred compounds of formula (\)

Various groups and specific examples of compounds of formula (i) are preferred.

In general, amino acids of natural (L) configuration are preferred, particularly at the A² position.

In general, it is preferred that R^N1 is hydrogen, and that

R^N2 is:

R^N3,

(Iinker1)-R^N3, CO-(linker1 )-R^N3, or CO-O-(linker1 )-R^N3,

wherein

(linkeri ) may be absent, i.e. a single bond, or CH₂₁ CH₂CH₂, CH₂CH₂CH₂, CH₂CH₂CH₂CH₂ or CH=CH, and

R^N3 is hydrogen or any of the following unsubstituted groups: saturated or unsaturated, branched or unbranched Ci_-4 alkyl; benzyl; phenyl; or monocyclic heteroaryl.

In general, it is preferred that R^C1 is: O-R",

O-(linker2)-R^C2, or NH-(linker2)R^C2

wherein

(Iinker2) may be absent, i.e. a single bond, Ci_-6 alkyl or C_2-4 alkenyl, preferably a single bond or CH₂, CH₂CH₂, CH₂CH₂CH₂, CH₂CH₂CH₂CH₂ or CH=CH₁ R^N2 is hydrogen, C(=O)-O-saturated or unsaturated, branched or unbranched, Ci_-4 alkyl, optionally substituted with phenyl or 2-furyl, or C(=O)- saturated or unsaturated, branched or unbranched, Ci_-4 alkyl, optionally substituted with phenyl or 2-furyl, and R^C1 is OH₁ O-Ci-₆ alkyl, 0-Ci_-6 alkyl-phenyl, NH-Ci_-6 alkyl, or NH-Ci_-6 alkyl-phenyl.

Group (i)(b):

A¹ is G, A or 2-aminobutyric acid,

A² is L, I, norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine, 2- allylglycine, P, 2-aminobutyric acid, alpha-methyl leucine, alpha-methyl valine or tert-butyl glycine,

A³ is G, A₁ V, P, 2-aminobutyric acid or norvaline,

R^N1 is H,

R^N2 is hydrogen, C(=O)-O-saturated or unsaturated, branched or unbranched, Ci_-4 alkyl, optionally substituted with phenyl or 2-furyl, or C(=O)- saturated or unsaturated, branched or unbranched, Ci_-4 alkyl, optionally substituted with phenyl or 2-furyl, and

R^C1 is OH, O-Ci.₆ alkyl, 0-Ci_-6 alkyl-phenyl, NH-Ci_-6 alkyl, or NH-Ci_-6 alkyl-phenyl.

Group (i)(c):

A¹ is G, A or 2-aminobutyric acid, A² is L, I₁ norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine or

2-allylglycine,

A³ is G, A₁ V, P, 2-aminobutyric acid or norvaline,

R^N1 is H,

R^N2 is hydrogen, C(=O)-O-satu rated or unsaturated, branched or unbranched, C1.4 alkyl, optionally substituted with phenyl or 2-furyl, or C(=O)- saturated or unsaturated, branched or unbranched, Ci_-4 alkyl, optionally substituted with phenyl or 2-furyl, and

R^C1 is OH, O-Ci.₆ alkyl, O-Ci_-6 alkyl-phenyl, NH-C_1-6 alkyl, or NH-Ci_-6 alkyl-phenyl.

Group (i)(d): A¹ is G or A,

A² is L, I₁ or norleucine, A³ is G or A, R^N1 is H,

16 R^C2 is hydrogen or any of the following unsubstituted groups: saturated or unsaturated, branched or unbranched C1.₅ alkyl; benzyl; phenyl; or monocyclic Ci.i₀ heteroaryl.

In general, with regard to the substituents at the N-terminus, it is further preferred that: R^N1 is hydrogen, and

R^N2 is hydrogen, C(=O)-O-(linker1 )-R^N3 or C(=O)-(linker1 )-R^N3, (linker1 ) is CH₂ or CH=CH, and R^N3 is phenyl or 2-furyl.

It is further preferred that R^m is hydrogen, R^N2 is hydrogen, C(=O)-OCH₂Ph or C(=O)-CH=CH-(2-furyl).

Another preferred grouping for the substituents on the N-terminus is such that: R^N1 is hydrogen, and

R^N2 is a is benzyloxycarbonyl, benzyl, benzoyl, tert-butyloxycarbonyl, 9- fluorenylmethoxycarbonyl or FA, more preferably benzyloxycarbonyl or FA.

In general, with regard to the substituents at the C-terminus, it is preferred that: R^C1 is OH, 0-C_1-6 alkyl, 0-Ci_-6 alkyl-phenyl, NH-Ci_-6 alkyl, or NH-Ci_-6 alkyl-phenyl, more preferably OH.

Several preferred groups are as follows.

Group (i)(a):

A¹ is G, A, V, L, I₁ P, 2-aminobutyric acid, norvaline or tert-butyl glycine, A² is G, A, V, L, I, P, F, W, C, S, K, R, 2-aminobutyric acid, norvaline, norleucine, tert-butyl alanine, alpha-methyl leucine, 4,5-dehydro-leucine, allo-isoleucine, alpha-methyl valine, tert-butyl glycine, 2-allylglycine, ornithine or alpha, gamma-diaminobutyric acid, A³ is G, A, V₁ L, I, P, F, W, D, E, Y, 2-aminobutyric acid, norvaline or tert-butyl glycine, R^N1 is H,

15 R^N2 is hydrogen, C(=O)-O-saturated or unsaturated, branched or unbranched, C_1-4 alkyl, optionally substituted with phenyl or 2-furyl, or C(=O)- saturated or unsaturated, branched or unbranched, Ci_-4 alkyl, optionally substituted with phenyl or 2-furyl, and R^C1 is OH, 0-C_1-6 alkyl, 0-Ci_-6 alkyl-phenyl, NH-C_L6 alkyl, or NH-C_1-6 alkyl-phenyl.

A first set of specific preferred compounds are those in which: A¹ is G, A² is L,

A³ is G, A, V, L, I, P, F, W, D, E, Y, 2-aminobutyric acid, norvaline or tert-butyl glycine, more preferably G, A, V, P₁ 2-aminobutyric acid or norvaline, more preferably G or A, R^N1 is hydrogen, R^N2 is benzyloxycarbonyl, and R^C1 is OH.

A second set of specific preferred compounds are those in which: A¹ is G,

A² is G, A, V, L, I, P, F, W, C, S, 2-aminobutyric acid, norvaline, norleucine, tert-butyl alanine, alpha-methyl leucine, 4,5-dehydro-leucine, allo-isoleucine, alpha-methyl valine, tert-butyl glycine or 2-allylglycine, more preferably L, I, norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine, 2-allylglycine, P, 2-aminobutyric acid, alpha- methyl leucine, alpha-methyl valine or tert-butyl glycine, more preferably L, I, norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine or 2-allylglycine, more preferably L, I, or norleucine, A³ is A, R^N1 is hydrogen,

R^N2 is benzyloxycarbonyl, and R^C1 is OH.

A third set of specific preferred compounds are those in which: A¹ is G, A, V, L, I, P, 2-aminobutyric acid, norvaline or tert-butyl glycine, more preferably G,

A or 2-aminobutyric acid, more preferably G or A,

A² is L,

A³ is A,

R^N1 is hydrogen, R^N2 is benzyloxycarbonyl, and R^C1 is OH.

Preferably the sequence A¹-A²-A³ is GLA₁ GLF₁ GVA₁ GIA, GPA or ALA₁ most preferably GLA₁ and: R^N1 is hydrogen,

R^N2 is benzyloxycarbonyl, and R^C1 is OH.

Where alkyl groups are described as saturated or unsaturated, this encompasses alkyl, alkenyl and alkynyl hydrocarbon moieties.

Ci_-6 alkyl is preferably Ci_-4 alkyl, more preferably methyl, ethyl, n-propyl, isopropyl, or butyl (branched or unbranched), most preferably methyl.

C_3-i₂ cycloalkyl is preferably C_5-I0 cycloalkyl, more preferably C₅.7 cycloalkyl.

"aryl" is an aromatic group, preferably phenyl or naphthyl,

"hetero" as part of a word means containing one or more heteroatom(s) preferably selected from N, O and S.

"heteroaryl" is preferably pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrimidinyl, indolyl, pyrazinyl, indazolyl, pyrimidinyl, thiophenetyl, pyranyl, carbazolyl, acridinyl, quinolinyl, benzimidazolyl, benzthiazolyl, purinyl, cinnolinyl or pteridinyl.

"non-aromatic heterocyclyl" is preferably pyrrolidinyl, piperidyl, piperazinyl, morpholinyl, tetrahydrofuranyl or monosaccharide.

"halogen" is preferably Cl or F, more preferably Cl. Further preferred compounds of formula (i)

In general, A¹ may preferably be selected from G, A or 2-aminobutyric acid; more preferably G or A₁ most preferably G.

In general, A² may preferably be selected from L, I, norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine, 2-allylglycine, P, K, 2-aminobutyric acid, alpha-methyl leucine, alpha-methyl valine or tert-butyl glycine; more preferably L, I, norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine, 2- allylglycine, P or K; more preferably L, I, norleucine, P or K; more preferably L or P.

In general, A³ may preferably be selected from G, A, V, P, 2-aminobutyric acid or norvaline; more preferably G or A. One general preference is that A³ is G. Another general preference is that A³ is A, particularly when R^C1 is OH.

In general, it is preferred that R^N1 is hydrogen.

In general, R^N2 is preferably:

R^N3, (linkeri )-R^N3,

CO-(linker1 )-R^N3, or CO-O-(linker1 )-R^N3, wherein

(linkeri ) may be absent, i.e. a single bond, or CH₂, CH₂CH₂, CH₂CH₂CH₂,

CH₂CH₂CH₂CH₂ or CH=CH, and

R^N3 is hydrogen or any of the following unsubstituted groups: saturated or unsaturated, branched or unbranched Ci.₄ alkyl; benzyl; phenyl; or monocyclic heteroaryl.

In general, R^N2 is more preferably hydrogen, benzyloxycarbonyl, benzyl, benzoyl, tert- butyloxycarbonyl, 9-fluorenylmethoxycarbonyl or FA, more preferably hydrogen, benzyloxycarbonyl or FA. In general, it is preferred that R^C1 is: O-R^C2,

O-(linker2)-R^C2 ₁ or NH-(linker2)R^C2

wherein

(Iinker2) may be absent, i.e. a single bond, Ci_-6 alkyl or C₂-₄ alkenyl, preferably a single bond or CH₂, CH₂CH₂, CH₂CH₂CH₂, CH₂CH₂CH₂CH₂ or CH=CH,

R^C2 is hydrogen or any of the following unsubstituted groups: saturated or unsaturated, branched or unbranched C_1-5 alkyl; benzyl; phenyl; or monocyclic Ci_-10 heteroaryl.

In general, R^C1 is more preferably OH, 0-Ci_-6 alkyl, 0-C_1-6 alkyl-phenyl, NH₂, NH-Ci_-6 alkyl, or NH-C_1-6 alkyl-phenyl, more preferably OH, 0-Ci_-6 alkyl, NH₂, or NH-C_1-6 alkyl, more preferably OH or NH₂.

Compounds of particular interest include those wherein A² is P.

Compounds of particular interest include those wherein R^C1 is NH₂.

In general the following amino acids are less preferred for A³: F, W, D, E and Y. Similarly, in general A³ may be selected not to be P and/or E due to compounds containing these exhibiting lower activity.

Preferred compounds of formula (iθ

Compounds of formula (ii) are preferably such that:

R' is CH₂CH₃ or CH₂CH₂CH₃,

R" is CH₂CH₂CH₃ or CH(CH₃)₂, and

R'" is H or Cl. Preferred compounds of formula (NH

Various preferred groups and specific examples of compounds of formula (iii) are as defined in any of the claims, taken separately, of US 6,335,360 B1 of Schwartz et al..

One example of a therapeutic compound of formula (i) is Z-GLA-OH, i.e. tripeptide GLA which is derivatized at the N-terminus with a Z group and which is not derivatized at the C- terminus. Z denotes benzyloxycarbonyl. This is a compound of formula (i) wherein R^N1 is H, R^N2 is Z, A¹ is G, A² is L, A³ is A and R^C1 is OH. This compound is available commercially from Bachem AG and has been found to inhibit the bacterial homologue of the eukaryotic TPP II, Subtilisin. Z-GLA-OH is of low cost and works well in vivo to induce rejection of tumours that are resistant to cancer chemotherapy. Novel treatments of therapy resistant cancers are of substantial interest to public health.

Whilst preferred compounds include those containing GLA, such as Z-GLA-OH, Bn-GLA- OH, FA-GLA-OH and H-GLA-OH, for example Z-GLA-OH; according to the present invention any disclosures of any compounds or groups of compounds herein may optionally be subject to the proviso that the sequence A¹A²A³ is not GLA, or the proviso that the compound is not selected from the group consisting of Z-GLA-OH, Bn-GLA-OH, FA-GLA-OH or H-GLA-OH, or the proviso that the compound is not Z-GLA-OH.

In the treatment of tumours that fail to respond to standard cancer chemotherapy Z-GLA- OH or other compounds described herein may be administered to improve such treatment in patients with malignant disease, for example increasing the in vivo response to such treatment in solid tumours.

Other preferred compounds include those wherein A¹A²A³ is GPG, such as GPG-NH₂ or Z- GPG-NH₂.

The skilled person will be aware that the compounds described herein may be administered in any suitable manner. For example, the administration may be parenteral, such as intravenous or subcutaneous, oral, transdermal, intranasal, by inhalation, or rectal. In one preferred embodiment the compounds are administered by injection. Examples of pharmaceutically acceptable addition salts for use in the pharmaceutical compositions of the present invention include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids. The pharmaceutically acceptable excipients described herein, for example, vehicles, adjuvants, carriers or diluents, are well-known to those who are skilled in the art and are readily available to the public. The pharmaceutically acceptable carrier may be one that is chemically inert to the active compounds and that has no detrimental side effects or toxicity under the conditions of use. Pharmaceutical formulations are found e.g. in Remington: The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995).

The composition may be prepared for any route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal. The precise nature of the carrier or other material will depend on the route of administration. For a parenteral administration, a parenterally acceptable aqueous solution is employed, which is pyrogen free and has requisite pH, tonicity and stability. Those skilled in the art are well able to prepare suitable solutions and numerous methods are described in the literature. A brief review of methods of drug delivery is also found in e.g. Langer, Science 249:1527-1533 (1990).

The dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable time frame. One skilled in the art will recognize that dosage will depend upon a variety of factors including the age, condition and body weight of the patient, as well as the stage/severity of the disease. The dose will also be determined by the route (administration form) timing and frequency of administration. In the case of oral administration the dosage can vary for example from about 0.01 mg to about 10 g, preferably from about 1 mg to about 8 g, preferably from about 10 mg to about 5 g, more preferably from about 10 mg to about 2 g, more preferably from about 100 mg to about 1 g per day of a compound or the corresponding amount of a pharmaceutically acceptable salt thereof.

The compounds may be administered before, during or after cancer chemotherapy. It is clear to the skilled person how to screen compounds for their inhibition of the activity of TPP II. TPP Il protein may be purified in a first step, and a TPP ll-preferred fluorogenic substrate may be used in a second step. This results in an effective method to measure TPP Il activity.

It is not necessary to achieve a particularly high level of purification, and conventional simple techniques can be used to obtain TPP Il of sufficient quality to use in a screening method. In one non-limiting example of purification of TPP II, 100 x 10⁶ cells (such as EL-4 cells) were sedimented and^'lysed by vortexing in glass beads and homogenisation buffer (50 mM Tris Base pH 7.5, 250 mM Sucrose, 5 mM MgCI₂, 1 mM DTT). Cellular lysates were subjected to differential centrifugation; first the cellular homogenate was centrifuged at 14,000 rpm for 15 min, and then the supernatant was transferred to ultra-centrifugation tubes. Next the sample was ultra-centrifugated at 100,000 x g for 1 hour, and the supernatant (denoted as cytosol in most biochemical literature) was subjected to 100,000 x g centrifugation for 5 hours, which sedimented high molecular weight cytosolic proteins/protein complexes. The resulting pellet dissolved in 50 mM Tris Base pH 7.5, 30%Glycerol, 5 mM MgCb, and 1 mM DTT, and 1 ug of high molecular weight protein was used as enzyme in peptidase assays.

It is possible to test the activity of TPP Il using for example the substrate AAF-AMC

(Sigma, St. Louis, MO). This may for example be used at 100 uM concentration in 100 ul of test buffer composed of 50 mM Tri Base pH 7.5, 5 mM MgCI₂ and 1 mM DTT. It is possible to stop reactions using dilution with 900 ul 1 % SDS solution. Cleavage activity may be measured for example by emission at 460 nm in a LS50B Luminescence Spectrometer (Perkin Elmer, Boston, MA).

The compounds of use in the present invention may be defined as those which result in partial or preferably complete tumour regression compared to control experiments when used in an in vivo model which comprises the steps of: (i) inoculation of tumor cells into mice; (ii) administration of a cancer chemotherapeutic agent to said mice and administration of compound to said mice; and (iii) measuring the tumour size at periodic intervals. The step of administration of a cancer chemotherapeutic agent is omitted in the control experiments. Further details and examples of tumour growth experiments are described below. We found it convenient to inject the compound shortly after application of cancer chemotherapy, but the invention should not be understood as limited to this sequence of administration. The compounds used in the present invention are sufficiently serum-stable, i.e. in vivo they retain their identity long enough to exert the desired therapeutic effect.

Without wishing to be bound by theory, the present invention is described in more detail in 5 the non-limiting Examples below with reference to the accompanying drawings which are now summarized:

Figure 1. TPPII in growth arrest regulation by gamma-irradiation exposure. (A)

Western blotting analysis using anti-TPPII of cellular lysates from EL-4 cells exposed to

10 1000 Rad of gamma-irradiation in the presence or absence of 1 micro-M wortmannin; with subsequent exposure to wortmannin in the presence of 25 micro-M NLVS (right lanes). (B) TPPII activity (enzymatic cleavage of AAF-AMC, top) and expression (by western blotting with anti-TPPU, bottom) as determined by testing high molecular weight cytosolic protein from EL-4 cells stably transfected with either empty pSUPER vector (denoted EL-4.wt,

15 empty bars) or with pSUPER-TPPIIi (anti-TPPII siRNA, denoted EL-4.TPPM', filled bars). AAF-CMK is a Serine peptidase inhibitor. (C) Immuno-cytochemical analysis of TPPII in EL-4. wt (top) versus EL-4. TPPH¹ cells (bottom), either left untreated (left panels) or gamma- irradiated (5 Gy) and analyzed after 1 hour. DAPI was used as controls for nuclear staining. (D) DNA synthesis of gamma-irradiated EL-4.wt (open symbols) and EL-4. TPPII'

20 cells (closed symbols) following exposure to 1000 Rad, as measured by ³H-Thymidin incorporation (bars indicate +/- standard deviation). (E) Cell cycle analysis of EL-4.wt (top) versus EL-4.TPPN' cells (bottom), before or 20 hours after exposure to 10 Gy of gamma- irradiation. (F) Phospho-Ser139-H2AX (gamma-H2AX) expression in EL-4.wt control versus EL-4.TPPII' cells exposed to 2,5 Gy of gamma-irradiation. 5

Figure 2. TPPII expression is required for stabilization of p53. Western blot analysis of cellular lysates after exposure of the indicated cells to gamma-irradiation (10 Gy): (A) p53 expression in EL-4.wt control versus EL-4.TPPN' cells. (B) p21 expression in EL-4.wt control versus EL-4. TPPII' cells. (C) p53 expression in EL-4.pcDNA3control versus EL- 0 4.pcDNA3-TPPII cells. (D) Western blotting analysis of TPPII using p53- immunoprecipitates from lysates of EL-4.wt versus EL-4. TPPII¹ cells (top); or from EL-4.wt cells treated with 1 micro-M wortmannin, versus untreated (bottom). Lanes labeled "+" indicates gamma-irradiated cells, whereas "-" were untreated (incubated for 16 hours at 37⁰C, prior to lysis). (E) p53 expression in ALC.pcDNA3 versus ALC.pSUPER-TPPII¹ (left), YAC-1 versus YAC-1.pSUPER-TPPII' (middle) and LLCpSUPER control versus LLC.TPPir cells (right), exposed to gamma-irradiation.

Figure 3. TPPII controls pathways that respond to PIKK signaling. (A) Western blotting analysis of Akt kinase expression, total Akt and Ser473-phosphorylated (p-Akt), in EL-4.wt control versus EL-4.TPPII¹ cells (top), or in EL-4.pcDNA3 versus EL-4.pcDNA3- TPPII cells (bottom). (B) Growth in vitro of EL-4.wt and EL-4.TPPII¹ cells in cell culture medium with either high (5%, left) or low (1%, right) serum content. Both live (empty circles) and dead (filled circles) cells were counted. (C) In vitro growth of EL-4.pcDNA3 and EL-4.PCDNA3-TPPII cells in cell culture medium with either high (5%, empty circles) or low (0,5%, filled circles) serum content. (D) XIAP expression by Western blotting analysis in EL-4.wt control cells versus EL-4.TPPN' cells exposed to 25 micro-M etoposide. (E) Cell surface Rae-1 expression of EL-4 (left) and YAC-1 (right) lymphoma cells with (right panels) and without (left panels) expression of pSUPER-TPPN' plasmid, as analyzed by flow cytometry. Filled curves represent cells stained with conjugate only.

Figure 4. TPPII controls interactions that mediate p53 stabilization.

(A) Sequence alignment of mouse versus human TPPII (a. a. 715-813) with BRCA C- terminal repeat domains previously described in BRCA1 (mouse), 53BP1 (human), MDC1 (human), C19G10.7 (S. pombe) and Rev1 (S. cerevisiae). U denotes hydrophobic amino acid (Bork, P, Hofmann, K, Bucher, P, Neuwald, AF₁ Altschul, SF, Koonin, EV. A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins. FASEB J. 1997; 11 :68-76). (B) Western blotting analysis of TPPII using cellular lysates from EL-4.TPPII"¹ or EL-4.TPPII^wt/G725E cells, exposed to 1000 Rad gamma- irradiation or left untreated. (C) p53 expression in EL-4.TPPir versus EL-4.TPPII^wt/G725E cells exposed to 1000 Rads of gamma-irradiation. (D) Western blotting analysis of TPPII using p53-immuno-precipitates from EL^.TPPir" or EL-4.TPPII^wt/G725E cells exposed to 1000 Rad gamma-irradiation, or left untreated. (E) Western blotting analysis of p53- immunoprecipitates from lysates of EL-4.wt versus EL-4.TPPII¹ cells, treated with NLVS versus untreated, using anti-sera specific for (left) ATM, (middle) Mre1 1 , (right) 53BP 1. Lanes labeled "+" indicates gamma-irradiated cells, whereas "-" were untreated.

Figure 5. TPPII is required for in vivo tumor resistance to gamma-irradiation. (A, B)

Tumor growth of 10⁶ EL-4.wt (A) or EL-4.TPPII' cells (B) in syngeneic C57BI/6 mice, gamma-irradiated with 4 Gy at time-points indicated with arrows. (C) Tumor growth of 5 x 10⁶ EL-4.ATM¹ cells in syngeneic C57BI/6 mice, left untreated (top) or gamma-irradiated with 4 Gy at time-points indicated with arrows (bottom). (D) Tumor growth of 5 x 10⁶ EL- 4.TPPirVG725E cells in syngeneic C57BI/6 mice, left untreated (top) or gamma-irradiated (bottom).

Figure 6. The Subtilisin inhibitor Z-Gly-Leu-Ala-OH inhibits TPPII and allows efficient radio-sensitization of tumors in vivo. (A) Cleavage of AAF-AMC by partially purified TPPII enzyme, as measured by fluorimetry, in the presence of Z-GLA-OH or butabindide. (B) Tumor growth of 10⁶ EL-4 lymphoma cells in syngeneic C57BI/6 mice, left untreated (8 mice, empty circles), treated with gamma-irradiation (7 mice, closed circles, 4 Gy doses indicated by arrow) or treated with Z-GLA-OH injection (13,8 mg/kg, indicated with +) as well as gamma-irradiation (8 mice, crossed circles). The data represent the mean tumor size. (C) Tumor growth of 10⁶ EL-4 lymphoma cells in syngeneic C57BI/6 mice, treated with gamma-irradiation doses of 3 Gy, 2 Gy or 1 Gy in combination with Z-GLA-OH injection (left panel); versus gamma-irradiation doses of 4 Gy or Z-GLA-OH alone and untreated (middle panel). Tumor growth of 10⁶ EL-4 lymphoma cells inoculated into C57BI/6 mice, treated with gamma-irradiation doses of 3 Gy and Z-GLA-OH at the indicated doses (right panel). In C linear scale was used, to better visualize differences at larger tumor sizes. (D) Tumor growth of 10⁶ Lewis Lung Carcinoma (LLC) cells in syngeneic C57BI/6 mice, left untreated (open squares) or treated with gamma-irradiation (4 Gy doses indicated by arrow), in the presence (bottom) or absence (top) of Z-GLA-OH. All data points in C and D represent data from at least 4 mice. (F) Tumor growth of 5 x 10⁶ HeLa cells (human cervical carcinoma) in CB.17 SCID mice, left untreated (open circles), injected with Z-GLA-OH (open squares) or injected with Z-GLA-OH and treated with gamma-irradiation (closed circles, 1 ,5 Gy per dose, indicated with arrows, closed circles).

Figure 7. Radio-sensitization of freshly transformed leukemic cells in vivo.

(A) Flow cytometric analysis of DBA/2 spleen cells 13 days post-transplantation of stem cells transduced with pMSCV-Bcl-XL-IRES-E-GFP and pMSCV-c-Myc-IRES-E-YFP. (B) In vivo tumor growth of DBA/2-c-myc/Bcl-xL cells in the presence or absence of gamma- irradiation treatment and Z-GLA-OH. (C-G) Flow cytometric detection of vector encoded YFP (c-Myc+) and GFP (Bcl-xL+) from DBA-c-Myc/Bcl-xL cells in tissues derived from tumour-carrying mice from untreated (C-E) versus treated (F, G) mice (gamma-irradiation and Z-GLA-OH), tissues used were from subcutaneous tumor (C)₁ lung (D, F), and spleen (E, G). Gates indicated in top panels correspond to cells analyzed for GFP/YFP- fluorescence in bottom panels. (H-J) Histological sections of livers from mice inoculated with DBA/2-c-Myc/Bcl-xL cells, receiving no treatment (H), gamma-irradiation (I) or both gamma-irradiation and Z-GLA-OH (J). Arrows indicate sinusoids filled with tumor cells.

Figure 8. Strong response to in vivo treatment with GPG-NH₂ or Z-GPG-NH₂ in combination with gamma-irradiation.

Tumour size (vertical axis, mm³) against time (horizontal axis, days) in mice carrying EL-4 tumours treated with gamma-irradiation alone, and treated with gamma-irradiation in combination with each of Z-GLA-OH, GPG-NH₂ and Z-GPG-NH₂.

Figure 9. Inhibition of TPP Il affects Mre11 foci formation The results of further immunocytochemical experiments are shown. Lewis Lung Carcinoma (LLC, A)₁ ALC (B) and YAC-1 (C) cells were stably transfected with pSUPER- TPPIIi, or with empty pSUPER vector, and were exposed to 5 Gy of gamma-irradiation. Immunocytochemical expression of TPPII and Mre11 was measured, as indicated in figure, and DAPI was used for nuclear control staining.

Figures 10 and 11. Treatment with Z-GLA-OH in combination with a range of cytostatic drugs enhances therapeutic effects in vivo. Tumour size (vertical axis, mm³) against time (horizontal axis, days) in mice carrying EL-4 tumours with conventional treatment alone, and in combination with Z-GLA-OH.

Figure 12. Treatment with Z-GLA-OH in combination with angiogenesis inhibitors enhances therapeutic effects in vivo. Tumour size (vertical axis, mm³) against time (horizontal axis, days) in mice carrying EL-4 tumours wwhen treated with angiogenesis inhibitors alone, and in combination with Z-GLA- OH.

Figure 13 shows growth of EL-4 T-lymphoma cells in vivo, in syngeneic mice, treated with Dexamethasone (5 mg/kg) and/or Z-GLA-OH (13.8 mg/kg), or left untreated.

Examples

The materials and methods used were as follows. Cells and Culture Conditions. EL-4 is a Benzpyrene-induced lymphoma cell line derived from the C57BI/6 mouse strain. EL-4.wt and EL-4.TPPII¹ are EL-4 cells transfected with the pSUPER vector (Brummelkamp, TR, Bernards, R, Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550-3), empty 5 versus containing the siRNA directed against TPPII. HeLa cells are human cervical carcinoma cells. YAC-1 is a Moloney Leukemia Virus-induced lymphoma cell line derived from the A/Sn mouse strain. ALC is a T cell lymphoma induced by radiation leukemia virus D-RadLV, derived from the C57BI/6 mouse strain. For measurement of DNA synthesis cells were seeded into 96-well plates and ³H-Thymidin was added after 16 or 36 hours and 10 incubated for 6 hours before wash. For induction of stress, cells were gamma-irradiated 500 - 1000 Rad's, or starved by growth in 50%-75% Phosphate Buffered Saline (PBS); and incubated at 37⁰C and 5.3%CO₂.

Enzyme Inhibitors. NLVS is an inhibitor of the proteasome that preferentially targets the 15 chymotryptic peptidase activity, and efficiently inhibits proteasomal degradation in live cells. Butabindide is described in the literature (Rose, C, Vargas, F, Facchinetti, P, Bourgeat, P, Bambal, RB, Bishop, PB, et. al. Characterization and inhibition of a cholecystokinin- inactivating serine peptidase. Nature 1996;380:403-9). Z-Gly-Leu-Ala-OH (Z-GLA-OH) is an inhibitor of Subtilisin (Bachem, Weil am Rhein, Germany), a bacterial enzyme with an 20 active site that is homologous to that of TPPII. Wortmannin is an inhibitor of PIKK (PI3- kinase-related) -family kinases (Sigma, St. Louis, MO). All inhibitors were dissolved in DMSO and stored at -2O⁰C until use.

Protein Purification, Peptidase Assays and Analysis of DNA Fragmentation. 100 x

25 10⁶ cells were sedimented and lysed by vortexing in glass beads and homogenisation buffer (50 mM Tris Base pH 7.5, 250 mM Sucrose, 5 mM MgCI2, 1 mM DTT). Cellular lysates were submitted to differential centrifugation where a supernatant from a 1 hour centrifugation at 100,000 x g (cytosol) was submitted to 100,000 x g centrifugation for 3-5 hours, which sedimented high molecular weight cytosolic proteins/protein complexes. The

30 resulting pellet dissolved in 50 mM Tris Base pH 7.5, 30%Glycerol, 5 mM MgCI2, and 1 mM DTT₁ and 1 micro-g of high molecular weight protein was used as enzyme in peptidase assays or in Western blotting for TPP Il expression. To test the activity of TPP Il we used the substrate AAF-AMC (Sigma, St. Louis, MO), at 100 micro-M concentration in 100 micro-l of test buffer composed of 50 mM Tri Base pH 7.5, 5 mM MgCI2 and 1 mM DTT.

35 Cleavage activity was measured by emission at 460 nm in a LS50B Luminescence Spectrometer (Perkin Elmer, Boston, MA). For analysis of DNA fragmentation cells were seeded in 12-well plates at 10⁶ cells /ml and exposed to 25 micro-M etoposide, a DNA topoisomerase Il inhibitor commonly used as an apoptosis-inducing agent, to starvation (50% PBS). Cells were seeded at 10⁶ cells/ml in 12-well plates and incubated for the indicated times, usually 18-24 hours. DNA from EL-4 control and adapted cells was purified by standard chloroform extraction, and 2.5 micro-g of DNA was loaded on 1.8% agarose gel for detection of DNA from apoptotic cells.

Plasmids and Gene Transfection. TPPII siRNA-expressing pSUPER (Brummelkamp, TR, Bernards, R, Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550-3.) plasmids were constructed as follows. Non- phosphorylated DNA oligomers (Thermo Hybaid, UIm, Germany) were resuspended to a concentration of 3 micro-g/micro-l. 1 micro-l of each oligo pair was mixed with 48 micro-l of annealing buffer (100 mM KAc; 30 mM HEPES-KOH pH 7.4; 2 mM MgAc) and heated to 95° C for 4 min, 70° C for 10 min, then slowly cooled to room temperature. 2 micro-l of annealed oligomers were mixed with 100 ng of pSUPER plasmid (digested with BgIII and Hindlll), ligated, transformed, and plated on Amp/LP plates, as previously described (Brummelkamp, TR, Bernards, R, Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550-3.). Colonies were screened for the presence of inserts by EcoRI-Hindlll digestion and DNA sequencing. Annealed oligomer pairs were as follows, for pSUPER-TPPN', forward primer:

5'GATCCCCGATGTATGGGAGAGGCCTTTCAAGAGAAGGCCTCTCCCATACATCTTTTT GGAAA-3'; reverse primer: 5'AGCTTTTCCAAAAAGATGTATGGGAGAGGCCTTCTCTTGAAAGGCCTCTCCCATACA TCGGG-3'.

For generation of stable transfectants, 5 x 10⁶ cells were washed in PBS, then resuspended into 500 micro-l of PBS in a Bio-Rad gene-pulser and pulsed with 10 micro-g DNA and 250 V at 960 micro-F; and selected by resistance to G418.

Antibodies and Antisera. The following molecules were detected by the antibodies specified: Akt by rabbit anti-Akt serum (Cell Signaling Technology, Beverly, MA); Phospho- Akt (Ser 473) by 193H2 rabbit anti-phospho-Akt serum (Cell Signaling Technology, Beverly, MA); gamma-H2AX by rabbit anti-gamma-H2AX (Cell Signalling Technology, Beverly, MA); Mre11 by polyclonal rabbit anti-human Mre1 1 (Cell Signalling Technology, Beverly, MA); p21 by SX118 (R & D Systems, Minneapolis, MN); p53 (R & D Systems, Minneapolis, MN); Rae-1 by monoclonal Rat anti-mouse Rae-1 , 199215 (R &D Systems, Minneapolis, MN); XIAP by monoclonal mouse anti-human XIAP, 117320 (R&D Systems, Minneapolis, MN). For detection of TPPII we used chicken anti-TPPII serum (Immunsystem, Uppsala, Sweden). Western blotting was performed by standard techniques. Protein concentration was measured by BCA Protein Assay Reagent (Pierce Chemical Co.). 5 micro-g of protein was loaded per lane for separation by SDS/PAGE unless stated otherwise.

Immunohistochemistry. Cells were attached to glass cover slips through cytospin and fixed in acetone: methanol (1 :1 ) for 1 hour; then the slides were rehydrated in BSS buffer for 1 hour. The first antibody was added and remained for 1 hour until a brief wash in BSS, after which a secondary conjugate (anti-rabbit-FITC) was added and incubated for 1 hour. Then the slides were washed and stained with Hoescht 333258 for 30 min. Finally, the slides were mounted with DABCO mounting buffer and kept at 4⁰C until analysis.

Flow Cytometry. For staining of cell surface Rae-1 antigens we incubated 0,5-1 ,0 x 10⁶ cells with 50 micro-l of Rae-1 monoclonal antibody 199215 (R &D Systems, Minneapolis, MN) at 20 micro-g/ml, and incubated on ice for 30 min. After washing in PBS, we sequentially incubated with Biotinylated Polyclonal Rabbit anti-Rat Ig (Dako Cytomation, Glostrup, Denmark) and Streptavidin-FITC (Pharmingen, San Diego, CA), with washing in PBS after each step. Fluorescence was quantified by a FACScalibur. Flow cytometric cell sorting of live cells was performed by incubation of cells for 5 minutes with 2 micro-g/ml of Propidium Iodide (Pl) and subsequent sorting into Pl⁺ and Pl^" populations with a FACSvantage.

Tumor Growth Experiments. Tumor cells were washed in PBS and resuspended in a volume of 200 micro-l per inoculate. The cells were then inoculated into the right flank at 10⁶ per mouse and growth of the tumor was monitored by measurement two times per week. The initiation of anti-tumor treatment of the mice was to some extent individualized according to when tumor growth started in each mouse. The mice were irradiated with 4 Gy prior to tumor inoculation in order to inhibit anti-tumor immune responses. The tumor volume was calculated as the mean volume in mice with tumors growth, according to (ai x a₂ x a₃)/2 (the numbers a, denote tumor diameter, width and depth). The time of first palpation varied between different mice, although the general pattern of growth was similar in virtually all of the mice. In most diagrams a log-scale is used to better visualize the therapeutic effects against small tumors, i.e. the presence of complete rejections. For inhibition of TPPII in vivo we made intraperitoneal injections with 13.8 mg per kg of body weight (14 micro-l of a 50 mM solution/mouse) of the Subtilisin inhibitor Z-Gly-Leu-Ala-OH (Z-GLA-OH₁ Bachem, Weil am Rhein, Germany) twice per week, diluted into 200 micro-l PBS. All gamma-irradiations were full body exposures.

Retroviral transduction and transplantation. With reference to Example 7, the sequence for c-Myc was amplified from human cDNA (brain) by PCR using the following primers: 5ΑCGTGAATTCCACCATGCCCCTCAACGTTAGCTTC and 3TACGTCTCGAGCTTACGCACAAGAGTTCCGTAG and inserted in the EcoRI site of the retroviral expression vector pMSCV-IRES-EYFP. hBcl-x_L was excised from the pLXIN-hBcl- x_L (Djerbi, M., Darreh-Shori, T., Zhivotovsky, B. & Grandien, A. Characterization of the human FLICE-inhibitory protein locus and comparison of the anti-apoptotic activity of four different flip isoforms. Scand J Immunol. 54, 180-9, 2001) and inserted into the EcoRI site of the pMSCV-IRES-EGFP. Production of retroviral particles, enrichment and transduction of hematopoietic stem cells and transplantation was performed as described previously (Nyakeriga, A.M., Djerbi, M., Malinowski, M. M. & Grandien, A. Simultaneous expression and detection of multiple retroviral constructs in haematopoietic cells after bone marrow transplantation. Scand J Immunol. 61, 545-50, 2005). Briefly, retroviral vectors were transiently transfected into Phoenix-Eco packaging cells using the LipofectAMINE 2000 Reagent (Invitrogen, Life Technologies Inc., Paisley, UK) and viral supernatants containing viral particles were harvested and used to transduce lineage negative cells obtained from bone marrow of 5-fluorouracil treated mice. These cells were thereafter injected into lethally irradiated recipient mice. Between 7 and 14 days after transplantation, the mice developed an acute myeloid leukaemia-like disease. Cells from spleen of such mice could be grown in vitro in regular RPMI medium supplemented with, glutamin and fetal calf serum.

Detection of GFP and YFP expression was performed using a Cyan™ ADP cytometer (Dako, Glostrup, Denmark) where after excitation at 488 nm, a 525-nm long-pass dichroic mirror was used to initially separate the signals followed by a 510/21-nm bandpass filter for detection of EGFP and a 550/30-nm band pass filter for EYFP. Data were analyzed using FlowJo software (Tree Star, Inc., San Carlos, CA). Abbreviations list: ATM, Ataxia Telangiectasia Mutated; BRCT, BRCA C-terminal repeat; NLVS, ^hydroxy-δ-iodo-S-nitrophenylacetyl-Leu-Leu-Leu-vinyl sulphone; Pl, Propidium Iodide; PIKKs, Phosphoinositide-3-OH-kinase-related kinases; TPPII, Tripeptidyl-peptidase Il ; FA, 3-(2-furyl)acryloyl; YFP, Yellow Fluorescent Protein, GFP, Green Fluorescent Protein;

Standard abbreviations are used for chemicals and amino acids herein.

Abbreviation Alternative abbreviation

A Alanine Ala

R Arginine Arg

N Asparagine Asn

D Aspartic acid Asp

C Cysteine Cys

E Glutamic Acid GIu

Q Glutamine GIn

G Glycine GIy

H Histidine His

I lsoleucine He

L Leucine Leu

K Lysine Lys

M Methionine Met

F Phenylalanine Phe

P Proline Pro

S Serine Ser

T Threonine Thr

W Tryptophan Trp

Y Tyrosine Tyr

V Valine VaI

The invention also makes use of several unnatural alpha-amino acids. Abbreviation SIDE CHAIN

Abu 2-aminobutyric acid CH₂CH₃

Nva norvaline CH₂CH₂CH₃

NIe norleucine CH₂CH₂CH₂CH₃ tert-butyl alanine CH₂C(CH₃)₃ alpha-methyl leucine (CH₃)(CH₂C(CH₃)CH₃)

4,5-dehydro-leucine CH₂C(=CH₂)CH₃ allo-iso leucine CH(CH₃)CH₂CH₃ alpha-methyl valine (CH₃)CH(CH₃)(CH₃) tert-butyl glycine C(CH₃)₃

2-allylglycine CH₂CH=CH₂

Om Ornithine CH₂CH₂CH₂NH₂

Dab ' alpha.gamma-diaminobutyric acid CH₂CH₂NH₂

4,5-dehydro-lysine CH₂CH=CHCH₂NH₂

Example 1 and Figure 1

Gamma-irradiation-induced cell cycle arrest depends on TPPII expression.

Since TPPII expression is increased by several types of stress we tested whether this was controlled by PIKKs. By Western blotting analysis of the T cell lymphoma line EL-4 with TPPII anti-serum we found that TPPII expression was increased by gamma-irradiation. Further, this increase was not present in gamma-irradiated EL-4 cells treated with 1 micro- M wortmannin, a PIKK inhibitor, which instead reduced TPPII expression (Fig. 1A). Treatment with NLVS, a proteasomal inhibitor, inhibited down regulation of TPPII in wortmannin treated gamma-irradiated EL-4 cells, suggesting that TPPII is degraded by the proteasome in the absence of PIKK signaling (Fig. 1A). To further study whether TPPII had any role in cellular responses mediated by PIKKs, we generated stable EL-4 transfectants expressing siRNA against TPPII, encoded by the pSUPER vector (denoted EL-4.TPPM', [Brummelkamp, TR, Bernards, R, Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550-3.]). EL-4.TPPII¹ cells had both inhibited expression and activity of TPPII, in comparison to EL-4.wt cells (transfected with empty pSUPER vector, Fig. 1 B). To trigger a cellular stress response where members of the PIKK family members control signal transduction, we used gamma- irradiation (5 Gy). TPPII was previously reported as a soluble cytosolic peptidase (Reits, E, Neijssen, J, Herberts, C, Benckhuijsen, W₁ Janssen, L₁ Drijfhout, JW, et. al. A major role for TPPII in trimming proteasomal degradation products for MHC class I antigen presentation. Immunity 2004;20:495-506), but we here found rapid translocation of TPPII into the nucleus of gamma-irradiated EL-4 cells (Fig. 1C). This was evident already 1 hour following gamma-irradiation exposure of EL-4 cells, as detected by immunohistochemical analysis of TPPII. A similar response was observed in ALC and YAC-1 lymphoma as well as Lewis Lung Carcinoma (LLC) cells.

Activation of PIKKs is required to halt DNA synthesis in response to DNA damage (Bakkenist, CJ₁ Kastan MB. Initiating cellular stress responses. Cell 2004;118:9-17)

(McKinnon, PJ. ATM and ataxia telangiectasia. EMBO Rep. 2004;5:772-6). We observed that DNA synthesis was inhibited in gamma-irradiated EL-4.wt control, but we found high levels of gamma-irradiation-resistant DNA synthesis in EL-4. TPPII¹ cells up to 36 hours after exposure (as measured by ³H-Thymidin incorporation, Fig. 1 D). These data suggested that TPPII was important to halt DNA synthesis of EL-4 cells in response to gamma-irradiation. EL-4. TPPII' cells arrested almost uniformly in G2/M after exposure to gamma-irradiation, whereas EL-4.wt control cells showed both G1 and G2/M arrest, suggesting an absence of a G1/S checkpoint in EL-4.TPPN' cells (Fig. 1 E). However, initial detection of DNA damage was still present in gamma-irradiated EL-4. TPPII¹ cells, as measured by western blotting of gamma-H2AX (Ser139-phosphorylated H2AX, Fig. 1 F). H2AX is phosphorylated in response to ATM activation, which triggers the formation of DNA repair foci (Bakkenist, CJ, Kastan MB. Initiating cellular stress responses. Cell 2004,118:9-17). Thus, TPPII is rapidly translocated into the nucleus following gamma- irradiation-exposure, and required to efficiently halt DNA synthesis in EL-4 cells, but not for phosphorylation of H2AX.

Example 2 and Figure 2

Failure to stabilize p53 in cells with inhibited TPPII expression.

The transcription factor p53 initiates cell cycle arrest in response to many types of stress, and its expression is controlled by direct phosphorylation by PIKKs. By Western blotting analysis in cellular lysates of gamma-irradiated EL-4.wt cells, we found increased levels of p53, whereas those of EL-4.TPPII' cells showed low levels (Fig. 2A). However, treatment with NLVS increased p53 expression of gamma-irradiated EL-4. TPPH' cells, suggesting that p53 was still synthesized but degraded by the proteasome in EL-4. TPPII' cells. p21 , a transcriptional target of p53, was weakly expressed in EL-4.TPPH' cells following exposure to gamma-irradiation, compared to EL-4.wt control cells (Fig. 2B). Further, EL-4.pcDNA- TPPII cells that stably over-express TPPII, showed increased levels of p53 following exposure to gamma-irradiation in comparison to EL-4.pcDNA3 cells (Wang, EW, Kessler, BM, Borodovsky, A, Cravatt, BF, Bogyo, M, Ploegh, HL, et. al. Integration of the ubiquitin- proteasome pathway with a cytosolic oligopeptidase activity. Proc Natl Acad Sci U S A. 2000;97:9990-5.) (Fig. 2C). To test if p53 and TPPII were physically linked we next performed co-immuno-precipitation experiments using an anti-serum directed against the N-terminus of p53, followed by western blot analysis for TPPII. In p53 immuno- precipitates from lysates of EL-4-pSUPER cells we detected TPPII; levels that were increased by gamma-irradiation (Fig. 2D, top). This was not observed in lysates from EL- 4.TPPII¹ cells or from lysates of EL-4.wt cells treated with 1 micro-M wortmannin (Fig. 2D). These data supported a gamma-irradiation-induced physical link between TPPII and p53. We found that p53 expression was also TPPII-dependent in gamma-irradiated YAC-1 and ALC lymphoma cells, where virtually no p53 was detectable following stable expression of pSUPER-TPPir (Fig. 2E). We failed to find expression of p53 in Lewis Lung Carcinoma (LLC) cells (Fig. 2E). We noted substantial levels of p53 in some of our control tumor cell lines also prior to exposure to gamma-irradiation, a phenomenon in line with the frequently up-regulated DNA damage response in transformed cells (Bartkova, J, Horejsi, Z, Koed, K, Kramer, A, Tort, F, Zieger, K, et. al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 2005;434:907-13) (Bartkova, J, Horejsi, Z, Koed, K, Kramer, A, Tort, F, Zieger, K, et. al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 2005;434:864-70). We concluded that TPPII expression was required for efficient stabilization of p53.

Example 3 and Figure 3

TPPII controls activation of several pathways that depend on PIKK signaling.

Since TPPII expression was a requirement for stabilization of p53 we tested also other stress-induced pathways that depend on PIKK signaling (Gasser, S, Orsulic, S₁ Brown, EJ, Raulet, DH. The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor. Nature 2005;436: 1186-90) (Viniegra, JG, Martinez, N, Modirassari, P, Losa, JH, Parada Cobo, C, Lobo, VJ, et. al. Full activation of PKB/Akt in response to insulin or ionizing radiation is mediated through ATM. J Biol Chem. 2005;280:4029-36) (Feng, J, Park, J, Cron, P, Hess, D, Hemmings, BA. Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase. J Biol Chem 2004;279:41 189-96) (Sarbassov, DD, Guertin, DA, AIi, SM, Sabatini, DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005;307:1098-101 ), by comparing their status in EL-4.wt versus EL-4. TPPII' cells. Ser473 phosphorylation of Akt kinase requires PIKK signaling by ATM, DNA-PK or mTOR, the mechanistic details are debated (Viniegra, JG, Martinez, N, Modirassari, P, Losa, JH, Parada Cobo, C, Lobo, VJ, et. al. Full activation 5 of PKB/Akt in response to insulin or ionizing radiation is mediated through ATM. J Biol Chem. 2005;280:4029-36) (Feng, J, Park, J₁ Cron, P, Hess, D, Hemmings, BA. Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase. J Biol Chem 2004;279:41189-96) (Sarbassov, DD, Guertin, DA, AIi, SM, Sabatini, DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science

10 2005:307:1098-101 ). We detected substantial levels of phospho-Ser473-Akt in lysates of EL-4.wt cells. However, EL-4.TPPII¹ cells displayed very low levels of phospho-Ser473- Akt, whereas total expression of Akt was similar (Fig. 3A). In addition, we find increased Ser473-phosphorylation of Akt in EL-4.pcDNA3-TPPII, in comparison to EL-4.pcDNA3 control cells further supporting that TPPII expression controls Akt-Ser473-phosphorylation

15 (Fig. 3B): Akt kinase is important for transduction of cell survival signals, and is over- activated in many tumors. In normal medium (5% serum) EL-4. TPPII¹ cells showed an increased rate of proliferation, compared to EL-4.wt, but also an increased accumulation of dead cells (Fig. 3C). Further, by lowering serum concentrations to 1% this accumulation was accelerated, compared to EL-4.wt cells, suggesting that cell survival mechanisms

20 were impaired in the absence of TPPII (Fig. 3C). In addition, EL-4.pcDNA3-TPPII cells were able perform limited growth in 0,5% serum, which EL-4.pcDNA3 cells did not (Fig. 3D). These phenotypes indicate that TPPII expression is important for Akt Ser473 phosphorylation and cell survival during in vitro culture. XIAP, a direct substrate of Akt kinase (Dan, HC, Sun, M, Kaneko, S, Feldman, Rl, Nicosia, SV, Wang, HG, et. al. Akt

25 phosphorylation and stabilization of X-linked inhibitor of apoptosis protein (XIAP). J Biol Chem. 2004;279:5405-12), is a member of the IAP family of molecules; endogenous caspase inhibitors commonly over-expressed in tumor cells. Up-regulation of TPPII causes increased expression of c-IAP-1 and XIAP molecules in EL-4.pcDNA3-TPPII cells. By treatment with etoposide we found that expression of XIAP was substantially higher in EL-

30 4.wt cells, compared to EL-4. TPPII¹ cells, with a slower rate of degradation (Fig. 3E). Further, activation of ATM and ATR kinases mediate expression of NKG2D lig^'ands, thereby allowing the immune system to detect cells with an ongoing DNA damage response (Gasser, S, Orsulic, S, Brown, EJ, Raulet, DH. The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor. Nature 2005;436:1 186-

35 90). By flow cytometric measurements we detected expression of Rae-1 on EL-4.wt cells, whereas minor amounts of Rae-1 expression were detected on EL-4. TPPII' cells (Fig. 3F). We failed to detect expression of Rae-1 ligands on ALC lymphoma cells, but analysis of mouse YAC-1 lymphomas also showed that Rae-1 expression was dependent on TPPII expression, since stable pSUPER-TPPII' transfectants (YAC-1. TPPII') express minor levels 5 of Rae-1 ligands at the cell surface (Fig. 3G). These data show that that several stress- induced pathways activated by PIKKs require TPPII expression.

Example 4 and Figure 4

A BRCT-like motif of TPPII required for p53 stabilization in response to gamma-

10 irradiation.

BRCA C-terminal repeat (BRCT)-domains are often contained within proteins controlling DNA damage signaling pathway, where they control interactions with ATM substrates (Bork, P, Hofmann, K, Bucher, P, Neuwald, AF, Altschul, SF, Koonin, EV. A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins. FASEB J.

15 1997;11 :68-76) (Manke, IA, Lowery, DM, Nguyen, A, Yaffe, MB. BRCT repeats as phosphopeptide-binding modules involved in protein targeting. Science 2003;302:636-9) (Yu, X, Chini, CC, He, M, Mer, G, Chen, J. The BRCT domain is a phospho-protein binding domain. Science 2003;302:639-42). We found one region of TPPII centered around the GG-doublet at position 725 which matched most, but not all, requirements of a BRCT motif

20 (Fig. 4A). We performed site-directed mutagenesis of the characteristic Gly-Gly-doublet present in many BRCT sequences (labeled *, Fig. 4A), mutating it into GIy-GIu in our pcDNA3-TPPII vector. To allow expression of this plasmid in EL-4.TPPH' cells, we inserted 3 silent mutations in the 3' region of TPPII among the nucleotides that interact with the pSUPER-TPPM'-encoded siRNA (this plasmid was denoted TPPII"¹), in addition to the

25 mutation in position 725 (denoted TPPir7G725E). We found that both TPPIf¹ as well as TPPII^wt/G725E mutant molecules were stably expressed in EL-4 cells co-transfected with pSUPER-TPPII' (Fig. 4B). Further, the expression of p53 was analyzed in EL-4.TPPir and EL-4.TPPirVG725E transfectant cells exposed to gamma-irradiation. We found that EL-4.TPPII^wt/G725E cells showed much reduced expression of p53, compared to EL-

30 4.TPPIr control cells (Fig. 4C). In addition, we failed to detect TPPII in p53- immunoprecipitates from lysates of EL-4.TPPII^wl/G725E cells, both in the presence and absence of gamma-irradiation, whereas TPPII was detected using EL^.TPPII"¹ control cells (Fig. 4D). We concluded that TPPII possesses a BRCT-like domain important for DNA damage signaling. 5 Regulatory factors are co-localized at sites of DNA damage, to allow the activation of downstream responses (Al Rashid, ST, Dellaire, G, Cuddihy, A, JaIaIi, F, Vaid, M₁ Coackley, C, et. al. Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo. Cancer Res. 2005;65: 10810-21 ) (Lisby, M, Barlow, JH, Burgess, RC, Rothstein, R. Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell. 2004;1 18:699-713). A possible reason behind the failure of p53 stabilization in cells with inhibited TPPII expression is that p53 fails to be recruited to such sites. We examined the presence of DNA repair foci components in p53 immuno-precipitates from EL-4.wt versus EL-4.TPPII' cells. We detected ATM in p53- immuno-precipitates from EL-4.wt, but not from EL-4.TPPH' cells, as measured by Western blotting (Fig. 4 E). We also detected DNA repair foci proteins 53BP1 and Mre11 among p53-linked proteins upon gamma-irradiation, in EL.-4.wt, but not in EL-4. TPPII¹ cells (Fig. 4F, G). Further, NLVS-treated EL-4.TPPII¹ cells also failed to show ATM, 53BP1 and Mre1 1 in p53-immunoprecipitates (Fig. 4E-G). The fact that p53 and ATM are found in proximity to DNA repair foci components is in line with that certain p53 isoforms accumulate at these foci, where they may interact with ATM kinase (Al Rashid, ST, Dellaire, G, Cuddihy, A, JaIaIi, F, Vaid, M, Coackley, C, et. al. Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo. Cancer Res. 2005;65: 10810- 21 ). Our data support that a physical link between p53 and ATM, as well as DNA repair foci components 53BP1 and Mre11 , requires TPPII.

Example 5 and Figure 5

TPPII expression controls gamma-irradiation resistance of EL-4 tumors in vivo.

PIKKs are possible target molecules for the development of novel cancer therapies (Choudhury, A, Cuddihy, A, Bristow, RG. Radiation and new molecular agents part I: targeting ATM-ATR checkpoints, DNA repair, and the proteasome. Semin Radiat Oncol 2006; 16:51 -8). To address whether TPPII-mediated growth regulation was important for in vivo tumor growth we inoculated 10⁶ EL-4.wt control or EL-4.TPPII' cells into syngeneic C57BI/6 mice. We found that both EL-4.wt and EL-4.TPPH' cells established tumors and grew at an approximately equal rate, suggesting when considered in isolation that TPPII was not important for growth of EL-4 tumors in vivo (Fig. 5A, B, panels labeled control). However, we also treated mice carrying either tumors of EL-4.wt or EL-4.TPPII' cells with 2- 4 doses of 4 Gy (400 Rad's) gamma-irradiation. We found that this had minor effects on tumor size after inoculation with 10⁶ EL-4.wt cells that continued to grow despite gamma- irradiation (Fig. 5 A, gamma-irradiation indicated with arrow). In contrast, mice carrying tumors of EL-4. TPPII¹ cells responded to gamma-irradiation treatment with complete regression of established tumors (Fig. 5B). These data resembled those obtained with tumors of EL-4.ATM¹ or EL-4.TPPII^wt/G725E cells, since these also failed to resist gamma- irradiation in vivo (Fig. 5C, D). The data support TPPII as a target to increase in vivo gamma-irradiation susceptibility of tumor cells.

Example 6 and Figure 6

Tri-peptide-based TPPII inhibitors radio-sensitize tumors in vivo.

TPPII is a Subtilisin-type Serine peptidase, with a catalytic domain that is homologous to bacterial Subtilisins (Tomkinson, B, Wemstedt, C, Hellman, U, Zetterqvist, O. Active site of tripeptidyl peptidase Il from human erythrocytes is of the subtilisin type. Proc Natl Acad Sci U S A. 1987;84:7508-12). We found that the tri-peptide Subtilisin inhibitor Z-G Iy-Le u-Ala- OH (Z-GLA-OH) efficiently inhibited TPPII, with a Kj50 of about 10 nM, slightly less efficient than observed for butabindide (which has a Kj50 of 7 nM), as observed by inhibited TPPII cleavage of the substrate AAF-AMC (Fig. 6A). Moreover, Z-GLA-OH was relatively stable in serum.

To test the effects of catalytic TPPII inhibition during tumor gamma-irradiation in vivo, we exposed C57BI/6 mice with established EL-4 tumors to gamma-irradiation doses of 4 Gy (one dose/week) and injections with Z-GLA-OH twice weekly (13.8 mg/kg body weight). Weekly gamma-irradiation doses of 4 Gy had minor effects on growth of established EL-4 tumors in C57BI/6 control mice. In contrast, following injection with Z-GLA-OH we observed complete tumor regression after 3-4 doses of 400 Rad gamma-irradiation in all mice tested (Fig. 6B). When these tumors were no longer palpable the treatment was cancelled, and no re-growth of tumors was observed for the entire period of observation (over 3 months). Titrations of the gamma-irradiation dose, in the presence of Z-GLA-OH injection, also showed complete regression of EL-4 tumors in mice exposed to doses of 3 Gy, whereas lower doses of gamma-irradiation reduced tumor growth also with some complete rejections (2 Gy, 2 out of 5 mice; 1 Gy, 1 out of 4, Fig. 6C). Titrations of the Z-GLA-OH compound showed complete tumor rejection in response to gamma-irradiation in most mice following inoculations with 6.9 mg/kg of Z-GLA-OH (3 out of 4), whereas 3.5 mg/kg and lower doses gave partial effects in terms of tumor regression (2 out of 4; using 3 Gy gamma-irradiation doses; Fig. 6C, right panel). One common reason behind tumor therapy resistance, including in vivo resistance to gamma-irradiation, is p53 mutations (El-Deiry, WS. The role of p53 in chemosensitivity and radiosensitivity. Oncogene 2003;22:7486-95). To test whether also p53-mutated tumors responded to gamma-irradiation in the presence of TPPII inhibitors we similarly inoculated 10⁶ Lewis Lung Carcinoma (LLC) cells in syngeneic C57BI/6 mice. We found that LLC tumors were virtually insensitive to repeated gamma-irradiation doses of 4 Gy, and Z-GLA- OH only (in the absence of gamma-irradiation) gave no effect (Fig. 6D). In contrast, we observed complete regression of established LLC tumors to gamma-irradiation in mice injected with Z-GLA-OH (Fig. 6D). We found that a protected di-peptide Z-GL-OH, was ineffective both in terms of TPPII inhibition and radio-sensitization of LLC tumors, whereas the N-terminal protective Z-group was not strictly required for anti-tumor effects in vivo. TPPII is an evolutionary conserved enzyme with an identity of 96% at the amino acid level between human and mouse, and we observed strong tumor regression also of human HeLa cervical carcinoma cells in Z-GLA-OH-treated SCID mice in response to gamma- irradiation (Fig. 6E). A reduced dose of gamma-irradiation (1 ,5 Gy/dose) was used, since SCID mice have substantially reduced radio-resistance.

Toxicity studies show that Z-GLA-OH had minor effects in vivo as single agent in doses up to 100 mg/kg, in a preliminary study. Furthermore, our mice survived for an extended period of time after the study. Since the gamma-irradiation protocols used here were exclusively whole body exposures, all tissues where Z-GLA-OH was distributed were exposed to gamma-irradiation and Z-GLA-OH in combination. This suggests manageable toxicity for the combined treatment.

Example 7 and Figure 7

Radio-sensitization of freshly transformed leukemic cells in vivo.

To establish tumor cells that more resemble primary tumors we used a retroviral expression system with two separate vectors encoding c-Myc and BCI-X_L (PMSCV-BCI-X_L - IRES-EGFP and pMSCV-c-Myc-IRES-EYFP). DBA/2 bone-marrow cells were retrovirally infected with these Bcl-x_L- and c-Myc-expressing vectors and transplanted into gamma- irradiated syngeneic mice. Vector-encoded Green Fluorescence Protein (GFP) versus Yellow Fluorescence Protein (YFP) allowed monitoring of retroviral gene expression (Nyakeriga, A.M., Djerbi, M., Malinowski, M. M. & Grandien, A. Simultaneous expression and detection of multiple retroviral constructs in haematopoietic cells after bone marrow transplantation. Scand J Immunol. 61, 545-50, 2005). 7-14 days post-transplantation we observed a massive accumulation of YFPVGFP⁺ myeloid (CDH b⁺GrI⁺) blasts in the spleen and bone-marrow (shown for spleen, Fig. 7 A). We inoculated these DBA/2-c- Myc/Bcl-X_L cells subcutaneously into syngeneic DBA/2 mice, and we observed palpable tumors after about 3 weeks that grew to sizes exceeding 1000 mm³ within an additional 2-3 weeks (Fig. 7 B). In all mice inoculated with DBA/2-c-Myc/Bcl-x_L cells we found tumor dissemination into the liver, as observed by histological analysis of fixed organs (Fig. 7 H). These malignant cells were also detected by flow cytometry showing YFP⁺/GFP⁺ cells in the spleen, lung and liver, using the cells from the primary tumor as control (Fig. 7 C-G). By treatment with gamma-irradiation (4 Gy/dose, 1 dose/week), we observed slightly reduced growth but the DBA/2-c-Myc/Bcl-xi. tumors still reached sizes exceeding 1000 mm³ with a delay of less than one week, also with the presence of liver metastasis (Fig. 7 B). In contrast, mice with established DBA/2-c-Myc/Bcl-x_L tumors receiving Z-GLA-OH (13.8 mg/kg body weight) had complete tumor regression in response to 4 Gy-doses of gamma- irradiation (Fig. 7 B). Further, we failed to find tumor cells in either lung, spleen or liver in these Z-GLA-OH-treated mice (Fig. 7 F, G, J). Gamma-irradiation was required for this treatment response, since no reduction of tumor size was observed in mice receiving Z- GLA-OH only (Fig. 7 B). These data support that the radio-sensitizing effect observed from Z-GLA-OH is unlikely to depend on specific tumor defects, but can be observed in cells freshly transformed by a simple two-hit strategy, deregulating proliferation and apoptosis.

Example 8

In vitro testing of di- and tri-peptides and derivatives.

Table 1 contains in vitro data, in fluorometric units which are arbitrary but relative, for the inhibition of cleavage of AAF-AMC (H-Ala-Ala-7-amido-4-methylcoumarin) by compounds at several concentrations. Some beneficial effect is seen for most of the compounds tested.

TPP Il protein was enriched, and then a TPP ll-preferred fluorogenic substrate AAF-AMC was used. 100 x 10⁶ cells were sedimented and lysed by vortexing in glass beads and homogenisation buffer (50 mM Tris Base pH 7.5, 250 mM Sucrose, 5 mM MgCI₂, 1 mM DTT). Cellular lysates were subjected to differential centrifugation; first the cellular homogenate was centrifuged at 14,000 rpm for 15 min, and then the supernatant was transferred to ultra-centrifugation tubes. Next the sample was ultra-centrifugated at 100,000 x g for 1 hour, and the supernatant (denoted as cytosol in most biochemical literature) was subjected to 100,000 x g centrifugation for 5 hours, which sedimented high molecular weight cytosolic proteins/protein complexes. The resulting pellet dissolved in 50 mM Tris Base pH 7.5, 30%Glycerol, 5 mM MgCI₂, and 1 mM DTT, and 1 ug of high molecular weight protein was used as enzyme in peptidase assays.

To test the activity of TPP Il we used the substrate and AAF-AMC (Sigma, St. Louis, MO), at 100 uM concentration in 100 ul of test buffer composed of 50 mM Tri Base pH 7.5, 5 mM MgCI₂ and 1 mM DTT. To stop reactions we used dilution with 900 ul 1% SDS solution. Cleavage activity was measured by emission at 460 nm in a LS50B Luminescence Spectrometer (Perkin Elmer, Boston, MA).

FA = 3-(2-furyl)acryloyl; PBS = phosphate-buffered saline. The text (Z, FA, H, etc.) at the start of each compound name is the substituent at the N-terminus; H indicates that the N- terminus is free NH₂. The text (OH, NBu, etc.) at the end of each compound name is the substituent at the C-terminus; OH indicates that the C-terminus is free CO₂H.

Table 1

100 10 1 100 10 1

Compound uM uM uM nM nM nM 0

Z-GL-OH 23,14 23,60 24,18 34,6 34,07 44,53 49,55 (comparative) 24,99 24,72 24,4 33,02 33,85 44,21 49,82 23,69 24,59 24,29 34,6 34,38 43,62 49,51 mean 23,94 24,30 24,29 34,07 34,1 44,12 49,63

Z-GLG-OH 14,44 17,49 23,79 31 ,49 34,4 43,42 48,58 15,02 17,58 24,85 28,64 34,16 44,02 49,03 15,8 17,44 24,63 26,13 34,27 43,73 49,2 mean 15,09 17,50 24,42 28,75 34,28 43,72 48,94

Z-GGA-OH 15,5 16,65 21 ,37 24,27 36,01 43,42 51 ,19 15,27 17,27 22,14 31 ,54 36,59 43,87 48,44 15,78 17,18 22,62 31 ,61 36,73 44,14 48,48 mean 15,52 17,03 22,04 29,14 36,44 43,81 49,37

FA-GLA-OH 6,34 14,35 19,99 23,33 31 ,19 43,18 49,96 4,05 8,14 16,21 23,87 33,88 43,49 48,4 4,69 9,44 14,78 24,09 33,9 43,68 49,43 mean 5,03 10,64 16,99 23,76 32,99 43,45 49,26 Table 1

100 10 1 100 10 1

Compound uM uM uM nM nM nM 0

H-APA-OH 13,55 14,35 23,94 24,26 28,85 44,05 48,84

8,46 14,64 24,49 24,48 29,39 41 ,76 49,32

7,65 14,91 25,04 28,44 29,44 43,84 49,16 mean 9,89 14,63 24,49 25,73 29,23 43,22 49,11

H-GLA-OH 8,37 12,4 15,53 17,58 22,67 36,63 48,16

7,42 12,53 19,03 17,94 23,33 38,42 49,91

7,12 14,66 18,34 17,53 ^' 22,93 39,4 48,18 mean 7,64 13,20 17,63 17,68 22,98 38,15 48,75

Bn-GLA-OH 12,92 17,74 21 ,14 23,01 33,30 43,67 48,53

1 1 ,17 14,86 21 ,54 22,71 33,45 42,91 47,02

9,65 13,38 22,01 22,90 33,40 41 ,17 49,55 mean 11,25 15,33 21,56 22,87 33,38 42,58 48,37

Z-GKA-OH 8,17 12,48 14,49 21 ,62 23,57 42,13 49,82

9,44 14,52 . 16,43 21 ,98 23,95 42,02 49

9,44 14,82 15,03 21 ,52 24,36 42,51 47,7 mean 9,02 13,94 15,32 21,71 23,96 42,22 48,84

Z-GLA-Nbu 1 1 ,16 13,06 23,89 32,24 34,06 38,14 47,34

13,86 14,73 23,71 32,41 33,89 38,31 47

14,05 14,34 24,13 32,63 34,85 36,63 48 mean 13,02 14,04 23,91 32,43 34,27 37,69 47,45

Z-GLA-OH 1 ,14 6,47 11 ,43 14,43 21 ,74 32,54 49

1 ,44 7,66 11 ,9 14,26 21 ,93 32,61 49,4

1 ,55 7,49 11 ,46 14,37 24,44 33,41 49,5 mean 1,38 7,21 11,60 14,35 22,70 32,85 49,30

Example 9 In vivo testing of di- and tri-peptides and derivatives.

Table 2 contains in vivo data, showing tumor volume in mm³, in groups of 4 mice with LLC (Lewis Lung Carcinoma). Mice were sacrificed if the tumor volume exceeded 1000 mm³. Some mice were administered with the compounds alone; others were additionally administered with irradiation. Mice were given the compounds, and in some cases also gamma irradition (400 Rad), at days 7, 10, 14, 18 and 21. In combination with irradiation some compounds showed excellent results. The fact that the dipeptide derivative Z-GL- OH performs poorly in vitro as well as in vivo supports the theory that the in vitro results can be extrapolated to in vivo effects.

Table 2 days after tumor inoculation

7 10 14 18 21

Z-GL-OH 4,0 147,0 720,0 1687,5 1792,0

(comparative) 10,0 171 ,5 660,0 1372 1352,0

8,0 192,0 936,0 840

4,0 144,0 500,0 1 176 mean 6,5 163,63 704,0 1268,88 1572,0

Z-GL-OH 0,5 108,0 320,0 600 1575,0 irradiated 6,0 144,0 400,0 864 1372,0

(comparative) 6,0 90,0 1 12,5 840 1 176,0

8,0 144,0 450,0 864 1008 mean 5,13 121,5 320,63 792,0 1282,75

PBS (control) 6,0 192,0 720,0 1575

4,0 240,0 600,0 1568

6,0 192,0 500,0 1274

6,0 256,0 720,0 1008

5,50 220,00 635,0 1356.25

PBS (control) 13,5 192,0 500,0 936 irradiated 0,5 144,0 480,0 1014

4,0 192,0 400,0 650

6,0 144,0 600,0 600 mean 6,00 168,00 495,00 800,00

FA-GLA-OH 4,0 144,0 720,0 1 176

13,0 144,0 600,0 ^• 1687,5

4,0 400,0 864,0 1456

13,0 256,0 600,0 1267,5 mean 8,50 236,00 696,00 1396,75

FA-GLA-OH 4,0 100,0 90,0 48 18,0 irradiated 0,0 90,0 120,0 48 48,0

0,5 108,0 126,0 32 18,0

4,0 96,0 72,0 32 12,0 mean 2,13 98,50 102,00 40,00 24,00

H-GLA-OH 9,0 256,0 480,0 750 1792,0

0,5 126,0 864,0 1 176 1280,0

0,5 126,0 480,0 1008 1890,0

18,0 320,0 864,0 1372 mean 7,00 207,00 672,00 1076,50 1654,00 Table 2 days after tumor inoculation

7 10 14 18 21

H-GLA-OH 13,5 62,5 256,0 108 72,0 irradiated 4,0 4,0 320,0 192 108,0

0,0 60,0 320,0 192 108,0

4,0 108,0 480,0 256 72,0 mean 5,38 58,63 344,00 187,00 90,00

Bn-GLA-OH 0,5 192,0 500,0 1575 1792,0

4,0 240,0 400,0 1372 1764,0

4,0 224,0 594,0 1008

0,5 256,0 720,0 840 mean 2,25 228,00 553,50 1198,75 1778,0(

Bn-GLA-OH 4,0 144,0 144,0 48 24,0 irradiated 3,0 144,0 144,0 32 4,0

8,0 171 ,0 171 ,0 4 0,5

0,5 144,0 144,0 12 0,5 mean 3,88 150,75 150,75 24,00 7.25

Z-GLA-OH 4,0 256,0 660,0 864 2048,0

0,0 192,0 864,0 1470

6,0 9,0 720,0 1568

13,5 144,0 mean 5,88 150,25 748 1300,67

Z-GLA-OH 4,0 48,0 72,0 32 13,5 irradiated 6,0 128,0 144,0 24 0,5

0,0 40,0 72,0 13,5 13,5

6,0 40,0 48,0 32 0,5 mean 4,00 64,00 84,00 25,38 7,00

Example 10

Further in vivo testing of Z-GLA-OH

Table 3 contains further in vivo data, showing tumor volume in mm³, in groups of 7-8 mice, according to the EL-4 tumor model described above. 1.000.000 EL-4 lymphoma cells were inoculated subcutaneously at day 0. No palpable tumors were observed until day 22. At each treatment (twice weekly) mice with palpable tumors were given 400 Rads irradiation alone, or in combination with 14 micro-l 5OmM solution of Z-GLA-OH. Mice with no palpable tumors were not treated, i.e. in mice with rejected tumors, treatment was terminated and the mice were kept under observation. Table 3 shows excellent results, namely complete rejection of established tumors, not just arrest of tumor growth, decreased volume, or a delay of tumor growth.

All mice were 400 Rad (1 Gy = 100 Rad) gamma-irradiated at day 0, a standard procedure to improve tumor acceptance. The compound was inoculated intraperitoneally, whereas tumors were always inoculated subcutaneously.

Table 3 irradiation (*) no add irradiation irradiation

Z-GLA-OH (#) no add no add Z-GLA-OH

Day 22 0,50 4,00 0,50

# 0,50 0,50 0,50

13,50 0,50 4,00

108,00 4,00 6,00

4,00 13,50 0,50

4,00 0,50 0,50

0,50 4,00 4,00

0,50 0,50 mean 16,44 3,86 2,06

26 72,00 75,00 108,00

# and * 256,00 108,00 126,00

75,00 13,50 108,00

108,00 24,00 75,00

108,00 13,50 50,00

48,00 90,00 40,00

60,00 62,50 62,50

90,00 108,00 mean 102,13 55,22 84,69

30 500,00 192,00 108,00

# and * 192,00 64,00 13,50

192,00 192,00 18,00

256,00 144,00 24,00

500,00 192,00 32,00

400,00 256,00 48,00

256,00 432,00 108,00

320,00 48,00 mean 327,00 210,28 49,94

34 864,00 90,00 10,00

# and * • 256,00 144,00 0,50

400,00 400,00 62,50

500,00 256,00 24,00

480,00 320,00 13,50 Table 3 irradiation (*) no add irradiation irradiation

Z-GLA-OH (#) no add no add Z-GLA-OH

400,00 400,00 18,00

600,00 240,00 24,00

400,00 13,50 mean 487,50 264,28 20,75

37 720,00 500,00 8,00

# and * 320,00 400,00 18,00

320,00 550,00 12,00

600,00 600,00 6,00

600,00 320,00 27,00

320,00 320,00 6,00

576,00 396,00 24,00

720,00 0,50 mean 522,00 440,85 12,69

41 1170,00 480,00 0,50

# and * 840,00 480,00 4,00

1092,00 480,00 4,00

900,00 600,00 13,50

720,00 780,00 4,00

1 176,00 800,00 0,50

1008,00 480,00 4,00

910,00 0,50 mean 977,00 585.72 3,88

44 1800,00 1008,00 0,50

# and * 1920,00 720,00 0,10

2304,00 726,00 0,50

2304,00 720,00 2,00

2160,00 1008,00 0,50

1764,00 1792,00 1 ,00

1920,00 1008,00 0,50

1792,00 4,00 mean 1995,50 997,43 1,14

48 1344,00 0,10

# and * sacrificed 1920,00 0,50

1575,00 0,50

2048,00 0,10

2048,00 0,10

2304,00 0,10

0,00

0,00 mean 1873,16 0,18

55 0,00

# and * sacrificed 0,00

0,00 Table 3 irradiation (*) no add irradiation irradiation Z-GLA-OH (#) no add no add Z-GLA-OH

O₁OO 0,10 0,00 0,00 0,50 mean 0,07

62 0,10

# and 0,00 0,00 0,00 0,00 0,00 0,00 0,00 mean 0,01

65 0,10

# and 0,00 0,00 0,00 0,00 0,00 0,00 0,00 mean 0,01

72 0,10

# and * 0,00 0,00 0,00 0,00 0,00 0,00 0,00 mean 0,01

78 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 mean 0,00 Example 11 and Figure 8

We tested GPG-NH₂ and Z-GPG-NH₂ in the same manner as Z-GLA-OH. These were injected twice weekly at 13.8 mg/kg in tumor bearing mice, and compared to Z-GLA-OH for their ability to mediate sensitization to gamma-irradiation in vivo. We found that both GPG- NH₂ and Z-GPG-NH₂ mediated complete regression of established EL-4 tumors following gamma-irradiation.

Example 12 and Figure 9 TPP Il is required for M re 11 foci formation

As shown in Figure 1 C and discussed under Example 1 , TPPII is rapidly translocated into the nucleus of gamma-irradiated cells. The results of further immunocytochemical experiments are shown in Figure 9. TPPII does not appear to form foci, which would have instead shown a dotted appearance (Fig. 9, shown for cells with inhibited TPPII expression, LLC, ALC and YAC-1 ). This failure of cells with inhibited TPP Il expression to assemble Mre1 1 foci upon gamma-irradiation exposure provides further support for the use of TPP Il inhibitors in the present invention.

Example 13 and Figures 10 and 11

TPPII inhibitors allow increased efficiency of in vivo chemotherapy

We tested whether TPPII inhibition complemented cytostatic drugs, of eight different classes, in experimental cancer therapy in mice. We tested whether Z-GLA-OH could increase the efficiency of common cytostatic drugs clinically used, including compounds that belong to the groups of DNA Topoisomerase inhibitors, DNA inter-calators, Alkylators, Anti-metabolites, Tubulin inhibitors, Proteasome inhibitors and Hsp90 inhibitors (Table 4).

All cytostatic drugs were injected twice weekly in mice with established tumors at doses previously used in therapeutic cancer models in mice, and we observed a reduced tumor growth in most treated mice, causing a substantial delay in tumor growth (Fig. 10). The addition of Z-GLA-OH (13,8 mg/kg) in a bi-weekly treatment schedule with DNA Topoisomerase inhibitors (Etoposide, 12,5 mg/kg; Doxorubicin, 16 mg/kg or Irinotecan, 60 mg/kg) as well as a DNA inter-calating drug (Cisplatinum, 12mg/kg) further reduced tumour growth. Whereas these cytostatic drugs given as mono-therapy prolonged survival of the mice about 10 days, the addition of Z-GLA-OH increased this survival by a further 7-10 days (until the mice had to be sacrificed due to large tumours).

Table 4

* References:

• Sato Y, Kashimoto S, MacDonald JR, Nakano K. In vivo antitumour efficacy of MGI- 114 (6-hydroxymethylacylfulvene, HMAF) in various human tumour xenograft models including several lung and gastric tumours. Eur J Cancer. 2001 ;37:1419-28.

• Te C, et. al. In vivo effects of chlorophyllin on the antitumour agent cyclophosphamide, lnt J Cancer. 1997;70:84-9.

• Wu SL. Et. al. Effect of resveratrol and in combination with 5-FU on murine liver cancer. World J Gastroenterol. 2004; 10:3048-52.

• Adams J, et. al. Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res. 1999;59:2615-22. • Lin CC, Inhibitor of Heat-shock Protein 90 Enhances the Antitumor Effect of DNA Vaccine Targeting Clients of Heat-shock Protein. MoI Ther. 2007;15:404-10.

Other DNA damaging drugs, 5-Fluoro-Uracil (anti-metabolite, 20 mg/kg) and Cyclophosphamide (alkylator, 220 mg/kg) also showed therapeutic effect on tumor growth in combination with Z-GLA-OH (Fig. 11 ). In particular the Cyclophosphamide was very effective in combination with Z-GLA-OH, with complete regression of established EL-4 tumors in most tested mice. Many of the tested cytostatic drugs caused occasional complete regressions, e.g. using Velcade or Geldanamycin, although these treatment responses were mostly partial (Fig. 1 , exp. 2). Morever, Paclitaxel (22 mg/kg), Taxoter and Vinorelbine (Tubulin inhibitors) resulted in tumour regressions in most mice injected with Z- GLA-OH, as observed in Cyclophosphamide-treated mice.

Thus, particularly synergistic effects were observed in combinations with for example Paclitaxel or Cyclophosphamide.

We conclude that TPPII inhibition allows greatly improved therapy with several common cytostatic drugs in clinical use.

Example 14 and Figure 12

Tri-peptide TPPII inhibitors allow increased efficiency of treatment with angiogenesis inhibitors in vivo.

We examined tumour treatment by injection with the angiogenesis inhibitors TNP-470 and Thalidomide. C57BI/6 mice receiving TNP-470 or Thalidomide were treated three times per week from the time point of tumor detection (i.e. at least 1 mm³ size) until the tumors reached 1000 mm³ or regressed. We found that both TNP-470 or Thalidomide treatments had partial effects on the growth of EL-4.wt control tumors, which reached the size of 1000 mm³ with a delay of 1-2 weeks, compared to untreated mice (Figure 12). However, the addition of Z-GLA-OH to these injections with angiogenesis inhibitors caused a substantial improvement of the anti-tumour effects, with frequent complete regressions occurring; in about 50% of the mice (Figure 12).

We conclude that inhibitors of TPPII enhance the anti-angiogenesis therapy of cancer.

Example 15 and Figure 13

We examined growth of the T cell-derived lymphoma line EL-4 in vivo, in mice subjected to Cortisone-treatment. We used the derivate Dexamethasone at 5 mg/kg, a dose previously reported to cause thymocyte apoptosis, and a block of T cell responses in vivo (Brewer, J.A., Kanagawa, O., Sleckman, B. P., Muglia, L.J., Thymocyte apoptosis induced by T cell activation is mediated by glucocorticoids in vivo. J Immunol. 2002, 169:1837-43.). Inhibited activation and proliferation of T cells in vivo by Dexamethasone-treatment is a standard method to treat patients with auto-immune, inflammatory as well as transplantation rejection diseases. It is however clear that disease symptoms, as well as immune activation and proliferation, are sometimes not controlled by Dexamethasone, or other Cortisone derivatives. Certain cytostatic drugs, e.g. Sendoxan or Cyclophosphamide, are treatment options when others have failed.

We inoculated 5 x 10⁶ EL-4 T-lymphoma cells into syngeneic C57BI/6 mice. These cells proliferate in vivo to form large tumors, and we observed some treatment effect of 5 mg/kg Dexamethasone twice weekly, i.e. reduced growth of EL-4 cells in vivo (Fig. 9). However, the addition of the TPPII inhibitor Z-GLA-OH increased the antiproliferative effects of Dexamethasone in some of the mice. This illustrates that a TPPII inhibitor potentiates in vivo cell death of activated cells.

Claims

1. A compound for use in enhancing the efficacy of cancer chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells, wherein said compound is a TPP Il inhibitor.

2. A compound for use as claimed in claim 1 , wherein said compound is selected from formula (i) or is a pharmaceutically acceptable salt thereof:

(i) R^N1R^N2N-A¹-A²-A³-CO-R^C1

wherein A¹, A² and A³ are amino acid residues having the following definitions according to the standard one-letter abbreviations or names:

A¹ is G, A, V, L, I, P, 2-aminobutyric acid, norvaline or tert-butyl glycine,

A² is G₁ A, V, L, I, P, F, W, C, S, K, R, 2-aminobutyric acid, norvaline, norleucine, tert-butyl alanine, alpha-methyl leucine, 4,5-dehydro-leucine, allo-isoleucine, alpha-methyl valine, tert-butyl glycine, 2-allylglycine, ornithine or alpha, gamma- diaminobutyric acid,

A³ is G₁ A, V, L, I, P, F, W, D, E, Y, 2-aminobutyric acid, norvaline or tert-butyl glycine,

R^N1 and R^N2are each attached to the N terminus of the peptide, are the same or different, and are each independently

R^N3,

(linkeri )-R^N3,

CO-(linker1 )-R^N3,

CO-O-(linker1 )-R^N3,

CO-N-((linker1 )-R^N3)R^N4 or

SO₂-(linker1 )-R^N3,

(linker! ) may be absent, i.e. a single bond, Or CH₂₁ CH₂CH₂, CH₂CH₂CH₂, CH₂CH₂CH₂CH₂ or CH=CH, R^N3and R^N4 are the same or different and are hydrogen or any of the following optionally substituted groups: saturated or unsaturated, branched or unbranched C_1-6 alkyl; saturated or unsaturated, branched or unbranched C_3-I₂ cycloalkyl; benzyl; phenyl; naphthyl; mono- or bicyclic Ci_-10 heteroaryl; or non-aromatic C_1-I0 heterocyclyl;

wherein there may be zero, one or two (same or different) optional substituents on R^N3 and/or R^N4 which may be: hydroxy-; thio-: amino-; carboxylic acid; saturated or unsaturated, branched or unbranched C_1-6 alkyloxy; saturated or unsaturated, branched or unbranched C₃-1₂ cycloalkyl;

N-, O-, or S- acetyl; carboxylic acid saturated or unsaturated, branched or unbranched C_1-6 alkyl ester; carboxylic acid saturated or unsaturated, branched or unbranched C_3-I₂ cycloalkyl ester phenyl; mono- or bicyclic C_1-I0 heteroaryl; non-aromatic C_1-10 heterocyclyl; or halogen;

R^C1 is attached to the C terminus of the tripeptide, and is: O-R^C2,

O-(linker2)-R^C2, N((linker2)R^C2)R^C3, or N(linker2)R^C2-NR^C3R^C4, (Iinker2) may be absent, i.e. a single bond, or Ci_-6 alkyl or C_2-4 alkenyl, preferably a single bond or CH₂, CH₂CH₂, CH₂CH₂CH₂, CH₂CH₂CH₂CH₂ or CH=CH,

_Rc² _Rc³ _{and R}c⁴ _{are the same or} different _{ancj are} hydrogen or any of the following optionally substituted groups: saturated or unsaturated, branched or unbranched Ci_-6 alkyl; saturated or unsaturated, branched or unbranched C₃-i₂ cycloalkyl; benzyl; phenyl; naphthyl; mono- or bicyclic Ci_-10 heteroaryl; or non-aromatic Ci_io heterocyclyl;

wherein there may be zero, one or two (same or different) optional substituents on each of R^C2 and/or R^C3 and/or R^C4 which may be one or more of: hydroxy-; thio-: amino-; carboxylic acid; saturated or unsaturated, branched or unbranched Ci_-6 alkyloxy; saturated or unsaturated, branched or unbranched C₃-i₂ cycloalkyl;

N-, O-, or S- acetyl; carboxylic acid saturated or unsaturated, branched or unbranched Ci.₆ alkyl ester; carboxylic acid saturated or unsaturated, branched or unbranched C_3-I2 cycloalkyl ester phenyl; halogen; mono- or bicyclic Ci_io heteroaryl; or non-aromatic C_M0 heterocyclyl;

3. A compound for use as claimed in claim 2 wherein said compound of formula (i) is such that: R^N1 is hydrogen,

R^N2 is hydrogen, C(=O)-O-saturated or unsaturated, branched or unbranched, Ci_-4 alkyl, optionally substituted with phenyl or 2-furyl, or C(=O)- saturated or unsaturated, branched or unbranched, Ci_-4 alkyl, optionally substituted with phenyl or 2-furyl, and

R^c1 is OH, O-Ci-₆ alkyl, 0-C_1-6 alkyl-phenyl, NH-Ci_-6 alkyl, or NH-Ci_-6 alkyl-phenyl.

4. A compound for use as claimed in claim 3, wherein said compound of formula (i) is such that:

A¹ is G, A or 2-aminobutyric acid,

A² is L, I, norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine, 2-allylglycine, P, 2-aminobutyric acid, alpha-methyl leucine, alpha-methyl valine or tert- butyl glycine,

A³ is G, A, V, P, 2-aminobutyric acid or norvaline, R^N1 is H,

R^N2 is hydrogen, C(=O)-O-saturated or unsaturated, branched or unbranched, C_1-4 alkyl, optionally substituted with phenyl or 2-furyl, or C(=O)- saturated or unsaturated, branched or unbranched, Ci_-4 alkyl, optionally substituted with phenyl or 2-furyl, and R^C1 is OH, 0-C_1-6 alkyl, O-C_1-6 alkyl-phenyl, NH-C_1-6 alkyl, or NH-Ci_-6 alkyl-phenyl.

5. A compound for use as claimed in claim 4, wherein said compound of formula (i) is such that:

A¹ is G, A or 2-aminobutyric acid,

A² is L, I, norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine or 2-allylglycine,

A³ is G₁ A, V, P, 2-aminobutyric acid or norvaline,

R^N1 is H,

R^N2 is hydrogen, C(=O)-O-saturated or unsaturated, branched or unbranched, C_1-4 alkyl, optionally substituted with phenyl or 2-furyl, or C(=O)- saturated or unsaturated, branched or unbranched, C_1-4 alkyl, optionally substituted with phenyl or 2-furyl, and

R^C1 is OH, 0-C_1-6 alkyl, 0-Cv₆ alkyl-phenyl, NH-C_1-6 alkyl, or NH-C_1-6 alkyl-phenyl.

6. A compound for use as claimed in claim 5 wherein said compound of formula (i) is such that:

A¹ is G or A,

A² is L, I, or norleucine, A³ is G or A,

R^N1 is hydrogen,

R^N2 is hydrogen, C(=O)-O-saturated or unsaturated, branched or unbranched, Ci_-4 alkyl, optionally substituted with phenyl or 2-furyl, or C(=O)- saturated or unsaturated, branched or unbranched, Ci_-4 alkyl, optionally substituted with phenyl or 2-furyl, and

R^C1 is OH, 0-Ci_-6 alkyl, O-Ci_-6 alkyl-phenyl, NH-C_1-6 alkyl, or NH-Ci_-6 alkyl-phenyl.

7. A compound for use as claimed in any of claims 2 to 6 wherein R^N1 is hydrogen,

R^N2 is hydrogen, C(=O)-OCH₂Ph or C(=O)-CH=CH-(2-furyl), and R^C1 is OH, O-Ci_-6 alkyl, or NH-C_1-6 alkyl.

8. A compound for use as claimed in claim 7 wherein said compound of formula (i) is Z-GLA-OH, Bn-GLA-OH, FA-GLA-OH or H-GLA-OH.

9. A compound for use as claimed in claim 8 wherein said compound of formula (i) is Z-GLA-OH

10. A compound for use as claimed in claim 2 wherein A¹ is G₁ A or 2-aminobutyric acid.

1 1. A compound for use as claimed in claim 10 wherein A¹ is G or A.

12. A compound for use as claimed in any of claims 2, 10 or 1 1 wherein A² is L, I₁ norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine, 2- allylglycine, P, K, 2-aminobutyric acid, alpha-methyl leucine, alpha-methyl valine or tert- butyl glycine.

13. A compound for use as claimed in claim 12 wherein A² is L, I, norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine, 2-allylglycine, P or K.

14. A compound for use as claimed in claim 13 wherein A² is L, I, norleucine, P or K.

15. A compound for use as claimed in claim 14 wherein A² is L or P.

16. A compound for use as claimed in claim 15 wherein A² is P.

17. A compound for use as claimed in any of claims 2 or 10 to 16 wherein A³ is G, A, V₁ P₁ 2-aminobutyric acid or norvaline.

18. A compound for use as claimed in claim 17 wherein A³ is G or A.

19. A compound for use as claimed in any of claims 2 or 10 to 18 wherein R^N1 is hydrogen.

20. A compound for use as claimed in any of claims 2 or 10 to 19 wherein R^N2 is

R^N3,

(linkeri )-R^N3, CO-(linker1 )-R^N3, or CO-O-(linker1 )-R^N3,

wherein

(linkeri ) may be absent, i.e. a single bond, or CH₂, CH₂CH₂, CH₂CH₂CH₂, CH₂CH₂CH₂CH₂ or CH=CH₁ and

R^N3 is hydrogen or any of the following unsubstituted groups: saturated or unsaturated, branched or unbranched Ci_-4 alkyl; benzyl; phenyl; or monocyclic heteroaryl.

21. A compound for use as claimed in claim 20 wherein R^N2 is hydrogen, benzyloxycarbonyl, benzyl, benzoyl, tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl or FA.

22. A compound for use as claimed in claim 21 wherein R^N2 is hydrogen, benzyloxycarbonyl or FA.

23. A compound for use as claimed in any of claims 2 or 10 to 22 wherein R^C1 is: O-R^C2,

O-(linker2)-R^C2, or NH-(linker2)R^C2

wherein

(Iinker2) may be absent, i.e. a single bond, C₁.₆ alkyl or C_2-4 alkenyl, preferably a single bond or CH₂, CH₂CH₂, CH₂CH₂CH₂, CH₂CH₂CH₂CH₂ or CH=CH, and

R^C2 is hydrogen or any of the following unsubstituted groups: saturated or unsaturated, branched or unbranched Ci_-5 alkyl; benzyl; phenyl; or monocyclic Ci_-10 heteroaryl.

24. A compound for use as claimed in claim 23 wherein R^C1 is OH, 0-C_1-6 alkyl, 0-Ci_-6 alkyl-phenyl, NH₂, NH-C_1-6 alkyl, or NH-C_1-6 alkyl-phenyl.

25. A compound for use as claimed in claim 24 wherein R^C1 is OH, 0-C_1-6 alkyl, NH₂, or NH-C_1-6 alkyl.

26. A compound for use as claimed in claim 25 wherein R^C1 is OH or NH₂.

27. A compound for use as claimed in claim 26 wherein R^C1 is NH₂.

28. A compound for use as claimed in claim 2 wherein said compound is GPG-NH₂, Z- GPG-NH₂, Bn-GPG-NH₂, FA-GPG-NH₂, GPG-OH, Z-GPG-OH, Bn-GPG-OH, or FA-GPG- OH.

29. A compound for use as claimed in claim 28 wherein said compound is GPG-NH₂.

30. A compound for use as claimed in claim 2 wherein said compound is ALG-NH₂, Z- ALG-NH₂, Bn-ALG-NH₂, FA-ALG-NH₂, ALG-OH, Z-ALG-OH, Bn-ALG-OH, or FA-ALG-OH.

31. A compound for use as claimed in claim 30 wherein said compound is ALG-NH₂.

32. A compound for use as claimed in any of claims 2 to 31 wherein A³ is not F, W, D, E or Y.

33. A compound for use as claimed in any of claims 2 to 32 wherein A³ is not P.

34. A compound for use as claimed in any of claims 2 to 33 wherein A³ is not E.

35. A method of enhancing the efficacy of cancer chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells comprising administering to a patient in need thereof a therapeutically effective amount of a compound defined in any of claims 1 to 34.

36. Use of a compound in the manufacture of a medicament for enhancing the efficacy of cancer chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells, wherein the compound is as defined in any of claims 1 to 34.

37. A method for identifying a compound suitable for enhancing the efficacy of cancer chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells comprising contacting TPP Il with a compound to be screened, and identifying whether the compound inhibits the activity of TPP II.

38. Pharmaceutical composition comprising a compound as defined in any of claims 1 to 34 and a chemotherapeutic agent, and optionally a pharmaceutically acceptable diluent or carrier.

39. Pharmaceutical composition as claimed in claim 38 wherein said compound is not cinnamoyl-IFP-ethylamide, GPE-OH, GGF-OH, GVF-OH, AAA-OH or IPI-OH.

40. Pharmaceutical composition as claimed in claim 38 or 39 with the proviso that A³ is not proline.

41 Pharmaceutical composition as claimed in any of claims 38 to 40 with the proviso that the compound is not GPE-OH.

42. Pharmaceutical composition as claimed in any of claims 38 to 41 with the proviso that R^C1 is not NH₂.

43. A combination of a compound as defined in any of claims 1 to 34 or 38 to 42, with a chemotherapeutic agent, for use as a medicament.

44. Any of a compound as claimed in any of claims 1 to 34, a method as claimed in claim 35, a use as claimed in claim 36, a method as claimed in claim 37, a pharmaceutical composition as claimed in any of claims 38 to 42, or a combination as claimed in claim 43, wherein the form of chemotherapy is treatment with a cytostatic drug.

45. Any of a compound as claimed in any of claims 1 to 34, a method as claimed in claim 35, a use as claimed in claim 36, a method as claimed in claim 37, a pharmaceutical composition as claimed in any of claims 38 to 42, or a combination as claimed in claim 43, wherein the form of chemotherapy is treatment with a cytostatic drug selected from an alkylator and a tubulin inhibitor.

46. Any of a compound as claimed in any of claims 1 to 34, a method as claimed in claim 35, a use as claimed in claim 36, a method as claimed in claim 37, a pharmaceutical composition as claimed in any of claims 38 to 42, or a combination as claimed in claim 43, wherein the form of chemotherapy is treatment with an angiogenesis inhibitor.