WO2020002668A1 - Peptides and cancer treatment - Google Patents

Abstract

The present invention provides a peptide which: i) consists of 12 to 15 amino acids; ii) has at least 7 cationic amino acids with either a) a side chain comprising a guanidinium group, or b) a side chain comprising an amino group; and iii) has at least 4 tryptophans which are clustered at the N-terminus or the C-terminus of the peptide or a peptidomimetic of said peptide, the peptide or mimetic thereof optionally in the form of a salt, ester or amide, for use in cancer treatment; and a peptide which i) consists of 12 to 15 amino acids; ii) has at least 7 cationic amino acids with either a) a side chain comprising a guanidinium group, or b) a side chain comprising an amino group; and iii) has at least 4 tryptophans which are clustered at the N-terminus or the C-terminus of the peptide; iv) and wherein said peptide c) comprises at least one spacer residue as defined in claim 7; d) has at least 9 cationic amino acids as defined in part (ii); e) has at least 1 cationic amino acid which is not arginine; f) has a total of 5-6 hydrophobic amino acids; and/or g) wherein said at least 4 tryptophans are clustered at the C-terminus of the peptide, as well as salts, esters, amides and peptidomimetics of said peptide.

Description

Peptides and cancer treatment

The present invention relates to novel peptides and their use in cancer treatment, in particular in the treatment of non-haematological and/or deep seated tumours such as liver and/or colorectal cancer.

During the last decade our group has been developing strategies for the intralesional treatment of solid tumors using cationic amphipathic peptides (CAP's). The rationale underlying this approach is that cancer cell membranes are often enriched with negatively charged macromolecules due to the overexpression of, for example, phosphatidyl serine, and sialic acid. This increase in anionic character makes cancer cells more amenable to ionic interactions with positively charged CAP's than their non-cancerous counterparts. The initial ionic interaction is followed by membrane disruption and, ultimately, the semi-selective lysis of the cancer cells.

A further class of peptides is described in WO2015/1 18028, for example WRWRWGGRRRRRRR (SEQ ID NO:27) (using single letter amino acid code). These peptides are effective anti-lymphoma agents but the clinical problem addressed by the present invention is the treatment of further tumours, particularly deep-seated tumours e.g. liver tumours. The peptides of WO2015/1 18028 are selective for lymphoma.

Cancer is one of the world’s most serious diseases. Hepatocellular carcinoma (HCC) is the sixth most common cancer in the world and the third most common cause of cancer related deaths. Just a minority of the patients can be offered potential curative surgical therapy. The liver is also the main location for metastatic colorectal cancer (CRC), and patients with non-resectable liver lesions have no potential curative therapeutic option. CRC is the third most common type of cancer in the western world. Approximately 25% of patients present with metastases at initial diagnosis and almost 50% of patients with CRC will develop metastases, contributing to the high mortality rates reported for CRC. The median survival time is about 24-28 months with the best chemotherapeutic regimens.

Liver malignancies therefore represent a huge unmet medical need.

Thus there is a need for alternative or improved therapies which utilise an effective agent which can be used to treat cancers, particularly non-haematological and/or deep seated tumours. Preferably the agent will have a direct anti-tumour effect and a protective effect against recurrence and metastasis by inducing specific immunity.

In addressing these needs the present inventors have identified a class of peptides with anti-tumoural activity, especially against carcinomas, particularly deep seated tumour types. Thus, according to a first aspect, the present invention provides a peptide for use in cancer treatment, which peptide:

(i) consists of 12 to 15 amino acids;

(ii) has at least 7 cationic amino acids with either

(a) a side chain comprising a guanidinium group, or

(b) a side chain comprising an amino group; and

(iii) has at least 4 tryptophans which are clustered at the N-terminus or the C-terminus of the peptide

said peptide optionally in the form of a salt, ester or amide

or a peptidomimetic of said peptide, optionally in the form of a salt, ester or amide for use in cancer treatment.

According to another aspect, provided is a peptide which:

(i) consists of 12 to 15 amino acids;

(ii) has at least 7 cationic amino acids with either

(a) a side chain comprising a guanidinium group, or

(b) a side chain comprising an amino group; and

(iii) has at least 4 tryptophans which are clustered at the N-terminus or the C-terminus of the peptide;

(iv) and wherein said peptide

(c) comprises at least one X residue as defined herein;

(d) has at least 9 cationic amino acids as defined above;

(e) has at least 1 cationic amino acid which is not arginine, e.g. which has a side chain comprising an amino group;

(f) has at least 1 further hydrophobic amino acid, which may be tryptophan or a different hydrophobic amino acid; and/or

(g) wherein said at least 4 tryptophans are clustered at the C-terminus of the peptide

said peptide optionally in the form of a salt, ester or amide

or a peptidomimetic of said peptide, optionally in the form of a salt, ester or amide.

Thus the peptide or peptidomimetic may be in salt form, may be esterified or may be amidated. Preferably the molecule has a modified C-terminus, more preferably it has an amidated C-terminus. Reference herein to a molecule ‘consisting of certain residues or sequences does not exclude these (amidation, esterification, salt) modifications.

The discussion provided herein regarding any characteristics or applications (uses) of peptides and peptidomimetics applies to any of the aspects provided herein.

The peptide may consist of 12 to 15 amino acids, e.g. 13-15, or exactly 12, 13, 14, or 15 amino acids. By“consists of” a specified number of amino acids is meant in this regard that the peptide has the specified number of amino acids; no additional amino acids may be present. However, the presence of modifying groups, such as amidation, is explicitly contemplated and therefore not excluded, although the peptide may, of course, be one that does not include a modifying group.

The peptide may comprise non-genetic amino acids, but alternatively the peptide only comprises amino acids selected from the 20 genetically encoded amino acids.

By“cationic amino acid” is meant an amino acid carrying a positive charge at pH 7.0. The cationic amino acids may be the same or different, e.g. the peptide may include a single species of cationic amino acid, such as arginine; or it may include two or more different species of cationic amino acids, such as arginine and lysine. Thus, each cationic amino acid may be selected independently.

The peptide has at least 7 cationic amino acids, e.g. at least or exactly 8 or 9; and preferably no more than 10.

The peptides of the invention preferably contain at least or exactly 4, 5, 6 or 7, more preferably at least or exactly 8 or 9 guanidinium containing residues and preferably all cationic amino acids are of this type. Alternatively, the peptide may have all its cationic amino acids of the type which comprise an amino group in the side chain (e.g. Lys), or it may have a mixture of the two types of cationic amino acid, e.g. 1-3 cationic amino acids with a side chain comprising an amino group, the remaining cationic acids all containing guanidinium.

Amino acid residues with a side chain comprising a guanidinium group may, e.g., be selected from arginine (Arg or R according to the accepted 3 and 1 letter codes used herein) and/or homoarginine. A guanidinium group is the protonated cation of guanidine (it is protonated under physiological conditions) and can be represented by the following formula RNHC(NH₂)2⁺· For convenience herein amino acid residues with a side chain comprising a guanidinium group are referred to as guanidinium containing residues.

'Side chain' refers to the variable group (often called the R group) of an amino acid, typically attached to the ocarbon, but which may be attached to the b-carbon in a b-amino acid and so on.

Amino acids with a side chain comprising an amino group may, e.g., be selected from lysine (Lys or K according to the 3 and 1 letter codes) and/or derivatives thereof, such as aminocaproic acid and ornithine. Thus, the side chain may contain more than one amino group. The amino group (-NH₂) or groups is/are typically attached to a linear (or branched) alkyl group (e.g. C₂-C₈ alkyl) and the side chain typically contains no other heteroatoms.

Preferably, at least or exactly 4, 5, 6, 7, 8, 9, or 10, e.g. all, of the cationic amino acids are arginine. Preferred peptides have at least or exactly 8 or 9 cationic amino acids, which preferably include at least or exactly 6, 7, 8 or 9 arginines.

The peptide comprises at least 4 tryptophans (Trp or W according to the accepted 3 and 1 letter codes) and preferably comprises no more than 6, more preferably no more than 5 tryptophans. For example, the peptide may comprise exactly 4, 5 or 6 tryptophans, more preferably 4-5 tryptophans.

Tryptophan is a hydrophobic amino acid. In addition to the at least 4 tryptophans, the peptide may, e.g., also comprise one or more further hydrophobic amino acids. Alternatively, no“further” hydrophobic amino acids are present, i.e. the only hydrophobic amino acids present in the peptide are tryptophans; or the peptide does not comprise any non-genetic further hydrophobic amino acids. Any such “further” hydrophobic amino acid(s), if present, may contain at least 7 non-hydrogen atoms, e.g. 7-22 non-hydrogen atoms in the side chain. They may be selected from genetically encoded amino acids and/or non-genetically encoded amino acids. The side chains of such hydrophobic amino acid(s) may, e.g. contain 1 -3 cyclic groups. Cyclic groups may be fused, will typically be 5 or 6 membered and may contain heteroatoms. If 2 or more cyclic groups are present, these may be in linear, branched or fused arrangements. Any cyclic groups may, e.g. be aromatic. Cyclic groups may be substituted, generally by non-polar, uncharged moieties, for example by C C₄ alkyl or alkenyl groups, but are preferably unsubstituted.

For example, the further hydrophobic amino acid(s) may independently be selected from Phenylalanine (Phe or F), Tyrosine (Tyr or Y), diphenylalanine (Dip), biphenylalanine (Bip), 2,5,7,-tri-tert-butyl-tryptophan (Tbt), naphthylalanine (Nal), which may be 1 -Nal or 2-Nal, anthracenylalanine (Ath), which may be 2-Ath or 9- Ath, benzothienylalanine (Bal), 2-amino-3-[1 ,T:4',1"-terphenyl-4-yl]-propionic acid, 2-amino-3-(2,5,7-tri-fe/f-butyl-1 H-indol-3-yl)-propanoic acid, 2-amino-3-[1 ,T:3',1"- terphenyl-4-yl]-propionic acid, 2-amino-3-[1 ,T:2',1"-terphenyl-4-yl]-propionic acid, 2- amino-3-(4-naphthalen-2-yl-phenyl)-propionic acid, 2-amino-3-(4'-butylbiphenyl-4- yl)propanoic acid, 2-amino-3-[1 ,T:3',1"-terphenyl-5'-yl]-propionic acid, and/or 2- amino-3-(4-(2,2-diphenylethyl)phenyl)propanoic acid.

Each further hydrophobic amino acid, if present, may be selected independently.

The total number of hydrophobic amino acids (the at least 4 tryptophans plus any further hydrophobic amino acids) should be no more than 6, e.g. it may be no more than or exactly 6, 5 or 4.

The peptides may also contain other amino acids which are neither cationic amino acids as defined herein nor a hydrophobic amino acid. The term“amino acid” is used herein to denote a moiety that may, e.g., be an a, b or g amino acid, or an amino acid derivative such as an N-substituted glycine. Such other amino acids are conveniently referred to as X residues or “spacer” residues.

Thus the peptide may contain 0-4, e.g. 1-3, 1-2 or 0-1 X residues, e.g.

exactly 1 , 2, 3 or 4. Alternatively, no X residues are present, i.e. the only amino acids present in the peptide are the at least 7 cationic amino acids, the at least 4 tryptophans, and any further hydrophobic amino acids (if present).

Each X residue may be selected independently. X residues are preferably non-charged. They may, e.g., be selected from Gly, Ala, Val, Leu, Met, lie, beta- alaninine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5- aminovaleric acid (AVA), 6-aminocaproic acid (Ahx), and 8-Amino-3,6- dioxaoctanoic acid (AEEA, mini-PEG1 ), preferably Gly.

The X residue may, e.g., have no more than 5 or 4 non-hydrogen atoms in the side chain.

The peptide should preferably not contain a hydrophobic amino acid (tryptophan or further hydrophobic amino acid) immediately adjacent to a cationic amino acid as defined herein. In particular, the peptide preferably does not contain a motif of ccW, wherein‘cc’ stands for 2 or more consecutive cationic amino acids as defined herein and W stands for tryptophan. This may be achieved through the presence of one or more X residues. Thus, any X residue(s), if present, may, e.g. act as a spacer between a hydrophobic residue, particularly tryptophan, and a cationic residue.

Examples of peptides incorporating X residues are shown in Table 1.

The peptides may contain“consecutive” cationic amino acids and/or “consecutive” hydrophobic amino acids respectively and in the context of the present invention by that is meant a“block” of at least 2, 3 or 4 consecutive cationic or hydrophobic amino acids respectively. Thus, the peptides may contain one or more blocks of cationic amino acids and/or one or more blocks of hydrophobic amino acids. It should be understood that any amino acids adjacent to such a block cannot be of the same type. For example, a“block” of, e.g., 4 cationic amino acids, should be understood to be 4 consecutive cationic amino acids that may be adjacent to any non-cationic acids, and/or be at the N- or C-terminus of the peptide. Thus, for example, a peptide of the sequence WWWWGGRRRRRRRRR (SEQ ID NO:8) contains a block of 9 arginines and a block of 4 tryptophans (separated by 2 X amino acids).

The T rp residues contained in the peptide are clustered, and if any further hydrophobic amino acids are present, the Trp cluster preferably includes these further hydrophobic amino acids. The cationic residues contained in the peptide are preferably also clustered.

By“clustered” is meant that the residues of one type are in close proximity to one another, with few or no intervening residues of another type. A cluster may, e.g., consist of a single block, or it may consist of 2 or more blocks, in which case the blocks are preferably separated by X residues. Thus the peptides of the invention will typically have at least 4 tryptophan residues within the 6 N-terminal or C-terminal residues (i.e. within the 6 residues at the N-terminus or the 6 residues at the C-terminus). Preferably the 4 N-terminal or C-terminal residues are Trp, more preferably the 4 N-terminal residues are Trp. Peptidomimetics will preferably include the peptidomimetic equivalent of such arrangements of Trp residues.

The cluster of T rp and optionally any further hydrophobic amino acids preferably contains no more than 2, preferably no more than 1 , e.g., 1 or 0 intervening residues. Any intervening residues may be cationic, but are preferably X residues.

Preferably, at least 3 of the hydrophobic residues are consecutive, i.e. in a block of 3 or more consecutive hydrophobic residues. More preferably at least or exactly 4 or 5 of the hydrophobic residues are consecutive. For example, all of the hydrophobic amino acids may be consecutive.

The cluster of Trp is at the N-terminus or at the C-terminus, so the N- terminal or C-terminal residue should preferably be Trp or a further hydrophobic amino acid.

At least 5, 6 or 7 of the cationic amino acids, e.g. at least or exactly 8, 9 or 10, are preferably also clustered and such a cationic cluster preferably contains no more than 3 or 2, preferably no more than 1 , e.g., 1 or 0 intervening residues. Any intervening residues should be X residues.

Thus, at least some of the cationic amino acids will be present in 1 or more blocks. For example, the peptide may contain a single block of cationic amino acids consisting of exactly or at least at least 4, 5, 6, 7, 8, 9 or 10 cationic amino acids. Alternatively it may contain exactly or at least 2, 3 or 4 blocks of cationic amino acids, which may each independently contain exactly or at least 2, 3, 4 or 5 cationic amino acids.

In particular, contemplated in any of the aspects provided herein is a peptide which:

(i) consists of 12 to 15 amino acids, preferably 13-15;

(ii) has at least 7 cationic amino acids, preferably at least or exactly 8 or

9, with either

(a) a side chain comprising a guanidinium group, or

(b) a side chain comprising an amino group,

wherein preferably at least 5, 6, 7, 8 or 9 of said cationic amino acids are consecutive; and

(iii) has 4 to 5 consecutive hydrophobic amino acids, of which at least 4 are tryptophans, which are clustered at the N-terminus or the C- terminus of the peptide; and (iv) has at least 1 X residue, preferably as a spacer between the cluster of hydrophobic amino acids and the cationic amino acids

said peptide optionally in the form of a salt, ester or amide or a peptidomimetic of said peptide, optionally in the form of a salt, ester or amide.

The peptide may, e.g., comprise or consist of a sequence disclosed in Table 1 , i.e. a sequence selected from SEQ ID NOs: 1-13. The peptide may comprise or consist of a sequence of the reversed order of such a sequence, i.e. be an inverted sequence, e.g. RRRRRRRRRGGWWWW (SEQ ID NO: 14) being the reversed order/inverted sequence of WWWWGGRRRRRRRRR (SEQ ID NO:1 ). Thus, the peptide may, e.g., comprise or consist of a sequence disclosed in Table 1 , i.e. a sequence selected from SEQ ID NOs: 14-26. Any of these peptides may be in the form of a salt, ester or amide, e.g., have an amidated C-terminus.

Table 1

X= spacer residue as defined herein

Ahx = 6-aminocaproic acid

h= hydrophobic amino acid

^* at least 4 of the hydrophobic amino acids (h) are W

The peptides and peptidomimetics of the invention include all enantiomeric forms. In particular, they may comprise D and/or L forms of one or more or all of the amino acids.

In a further aspect, the present invention provides a pharmaceutical composition comprising a peptide or peptidomimetic of the invention as defined herein together with a pharmaceutically acceptable diluent, carrier or excipient. ln a further aspect, the present invention provides a peptide or

peptidomimetic of the invention as defined herein or a pharmaceutical composition as defined herein for use in therapy.

In a further aspect, the present invention provides a peptide or

peptidomimetic of the invention as defined herein or a pharmaceutical composition as defined herein for use in the treatment of cancer, particularly tumours.

Treatment includes preventing or reducing the growth, establishment, spread or metastasis of a tumour. Tumours treated according to the present invention will typically be solid and cancerous, usually malignant. Treatment includes prophylactic treatment, e.g. through the induction of specific immunity to an initially treated tumour which can prevent the establishment of a further tumour of the same or a similar type. Such prophylactic treatments may be secondary to treatment of a target tumour or may, in certain circumstances, be the primary purpose of treatment.

Compositions, uses and treatment methods described herein may further comprise a second therapeutic agent, in particular a further anti-cancer agent or an agent which supports the efficacy of the peptides or peptidomimetics of the present invention.

The peptides described herein can be considered antitumoural, anticancer or antineoplastic agents. Cancer cells may exist within a tumour or may lack the morphological characteristics of a tumour, e.g. be circulating within the body, for example to establish metastases.

The tumour is preferably malignant and may, e.g., be a tumour that has a significant risk of becoming metastatic, or be a tumour that is metastatic.

The cancer may be selected from a carcinoma, sarcoma and/or glioma, preferably carcinoma, which may, e.g. be selected from adenocarcinoma and/or squamous cell carcinoma, preferably adenocarcinoma. Preferred cancer targets exclude lymphoma, and may exclude any haematological cancers affecting blood, bone marrow and lymph nodes, including leukaemia, lymphoma and myeloma.

Alternatively, preferred cancer targets are haematological

cancers, preferably leukaemia, lymphoma and myeloma, more preferably lymphoma.

Preferred cancer targets are solid tumours, which are preferably not transdermally accessible. Preferably, the cancer is a deep-seated tumour. The term “deep-seated tumour” is widely understood in the art.

A deep-seated tumour is a solid tumour that is not a surface or superficial tumour. In some embodiments it may be at least 2cm or at least 4cm from the outside of the body.

The tumour preferably affects an internal organ and preferably at least partially penetrates the organ. The tumour may, e.g., be predominantly or fully located inside an internal organ. Some or all of the tumour may, e.g., be at least 0.5, 1 , 1.5 or 2 cm from the outer boundary of the organ.

The internal organ may, e.g., be selected from heart, lung, oesophagus, stomach, kidney, diaphragm, spleen, liver, pancreas, intestine, colon, gallbladder, bladder, ovary, prostate and/or brain, preferably colon and/or liver.

Alternatively, the tumour may, e.g. be located within tissue such as breast tissue, or bone.

The tumour may, e.g., be an unresectable tumour, meaning one that cannot be removed completely through surgery.

The cancer may, e.g. be selected from breast cancer, heart cancer, lung cancer, oesophagus cancer, stomach cancer, kidney cancer, diaphragm cancer, spleen cancer, liver cancer, pancreas cancer, intestine cancer, colon cancer, gallbladder cancer, bladder cancer ovarian cancer, prostate cancer and/or brain cancer, preferably colon cancer and/or liver cancer.

The tumour may be a primary tumour or a secondary tumour that has arisen through metastasis, e.g. colon cancer may give rise to a secondary tumour in the liver.

Thus, the peptides of the invention are especially preferred for use in treating liver cancer or liver tumours, including secondary liver tumours caused, e.g., by colon cancer.

Primary and/or secondary liver cancer, such as hepatocellular carcinoma and/or live metastases of colorectal cancer are of particular interest.

Treatment is preferably intralesional, i.e. intratumoural.

When the peptides of the invention are used to treat tumours intralesionally, a high selectivity of the peptide for cancer cells may not be necessary, especially if the tumour is large. High activity may be the more important criterion. In particular, when the peptides are used to treat liver tumours intralesionally, it is not important that the peptide is selective for tumour cells due to the regenerative capacity of the liver. Thus, the peptides may be used to treat tumours intralesionally, particularly large tumours and/or liver tumours.

In a further aspect, the present invention provides a method of treating a tumour or cancer cells, which method comprises administering to a subject a pharmaceutically effective amount of a peptide or peptidomimetic of the invention as defined herein. Typically the subject has been identified as being in need of the treatment, e.g. diagnosed as having cancer.

The subject or patient treated may be human, but non-human animals are also contemplated, for example livestock (e.g. cows, horses, sheep and pigs) or domestic or companion animals (e.g. dogs and cats). Treatment of dogs is preferred. In a further aspect, the present invention provides the use of a peptide or peptidomimetic of the invention as defined herein in the manufacture of a medicament for the treatment of cancer.

Treatment, as mentioned above, may include a reduction in growth of the tumours, e.g. a reduction in size of the tumour, e.g. by at least 20%, preferably at least 30, 40 or 50%, more preferably at least 70%. Treatment may lead to eradication of the tumour, but treatment can be considered successful even without eradication of the tumour. The primary objective will be to increase life expectancy and/or to improve quality of life. Prognosis after a diagnosis of cancer can be estimated by clinicians and improved survival rates, whether at 1 , 5 or 10 years may be judged against data or expectations.

The peptides of the invention may be amidated, esterified or in salt form and may be administered in these forms. Suitable physiologically acceptable salts are well known in the art and include salts of inorganic or organic acids, preferred salts include trifluoroacetate, acetate and salts formed with HCI. The peptides may incorporate a modified N and/or C terminus. Preferred examples include acetylation at the N terminus to form an alkylated amine or incorporation of a thiol modification, e.g. to form a cysteamine group at the N terminus. The C terminus is conveniently amidated, but may alternatively form an ester, amidation of the C terminus is preferred. Thus‘peptides’ include peptides with a modified N and/or C terminus and peptidomimetics may be similarly modified.

The cationic residues may be non-genetically coded and the peptide may contain other non-genetically coded amino acids, e.g. amino acids other than the standard 20 amino acids of the genetic code may be included within any of the X residues (if present) and/or any of the further hydrophobic residues (if present).

Peptidomimetics of any of the peptides of the invention are also

contemplated, and any discussion of features of the peptides of the invention applies mutatis mutandis to the peptidomimetics.

A peptidomimetic is typically characterised by retaining the polarity, three dimensional size and functionality (bioactivity) of its peptide equivalent but wherein one or more of the peptide bonds have been replaced, often by more stable linkages. By 'stable' is meant more resistant to enzymatic degradation by hydrolytic enzymes. Generally, the bond which replaces the amide bond (amide bond surrogate) conserves many of the properties of the amide bond, e.g. conformation, steric bulk, electrostatic character, possibility for hydrogen bonding etc. Chapter 14 of "Drug Design and Development", Krogsgaard, Larsen, Liljefors and Madsen (Eds) 1996, Horwood Acad. Pub provides a general discussion of techniques for the design and synthesis of peptidomimetics. Suitable amide bond surrogates include the following groups: N-alkylation (Schmidt, R. et al., Int. J. Peptide Protein Res., 1995, 46,47), retro-inverse amide (Chorev, M and Goodman, M., Acc. Chem. Res, 1993, 26, 266), thioamide (Sherman D.B. and Spatola, A.F. J. Am. Chem.

Soc., 1990, 112, 433), thioester, phosphonate, ketomethylene (Hoffman, R.V. and Kim, H.O. J. Org. Chem., 1995, 60, 5107), hydroxymethylene, fluorovinyl

(Allmendinger, T. et al., Tetrahydron Lett., 1990, 31 , 7297), vinyl, methyleneamino (Sasaki, Y and Abe, J. Chem. Pharm. Bull. 1997 45, 13), methylenethio (Spatola, A.F., Methods Neurosci, 1993, 13, 19), alkane (Lavielle, S. et. al., Int. J. Peptide Protein Res., 1993, 42, 270) and sulfonamido (Luisi, G. et al. Tetrahedron Lett.

1993, 34, 2391 ).

The peptidomimetic compounds of the present invention will typically have identifiable sub-units which are approximately equivalent in size and function to amino acids, e.g. to guanidinium containing residues such as Arg, or to hydrophobic residues such as Trp. The term 'amino acid' may thus conveniently be used herein to refer to the equivalent sub-unit of a peptidomimetic compound.

As is discussed in the text book referenced above, as well as replacement of amide bonds, peptidomimetics may involve the replacement of larger structural moieties with di- or tripeptidomimetic structures and in this case, mimetic moieties involving the peptide bond, such as azole-derived mimetics may be used as dipeptide replacements.

Peptidomimetics and thus peptidomimetic backbones wherein just the amide bonds have been replaced as discussed above are, however, preferred.

Suitable peptidomimetics include reduced peptides where the amide bond has been reduced to a methylene amine by treatment with a reducing agent e.g. borane or a hydride reagent such as lithium aluminium-hydride. Such a reduction has the added advantage of increasing the overall cationicity of the molecule.

Other peptidomimetics include peptoids formed, for example, by the stepwise synthesis of amide-functionalised polyglycines. Some peptidomimetic backbones will be readily available from their peptide precursors, such as peptides which have been permethylated, suitable methods are described by Ostresh, J.M. et al. in Proc. Natl. Acad. Sci. USA(1994) 91 , 11 138-1 1142. Strongly basic conditions will favour N-methylation over O-methylation and result in methylation of some or all of the nitrogen atoms in the peptide bonds and the N-terminal nitrogen.

Preferred peptidomimetic backbones include polyesters, polyamines and derivatives thereof as well as substituted alkanes and alkenes. The

peptidomimetics will preferably have N and C termini which may be modified as discussed herein.

b and g amino acids as well as a amino acids are included within the term 'amino acids', as are N-substituted glycines. The compounds of the invention include beta peptides and depsipeptides.

Peptides of the present invention may be cyclic, whether cyclisation involves the amino and/or the carboxy terminus or one or more amino acid side chains, but will preferably be linear. Likewise, peptidomimetics of the present invention may also be cyclic molecules but preferably are linear. As mentioned above, features and preferred embodiments described in relation to the peptides of the invention apply, mutatis mutandis, to peptidomimetics of the invention.

Assays described in the Examples with a peptide of the invention revealed no significant effect on median arterial blood pressure. The peptides of the invention are preferably substantially not hypotensive. By“substantially not hypotensive” is meant that any hypotensive effect produced by administration of (an appropriate dose of) the peptides of the invention to a patient would be sufficiently moderate or low so as to not necessitate administration of an agent to counteract any hypotensive effect produced by the peptides. Preferably, the peptides of the invention do not produce any significant hypotensive effect, e.g. no detectable hypotensive effect, when administered to a patient.

The peptides are preferably substantially not hypertensive. By“substantially not hypertensive” is meant that administration of (an appropriate dose of) the peptides of the invention to a patient would be sufficiently moderate or low so as to not necessitate administration of an agent to counteract any hypertensive effect produced by the peptides. Preferably, the peptides do not produce any significant hypertensive effect, e.g. no detectable hypertensive effect, when administered to a patient.

Any change in arterial blood pressure produced by administration of (an appropriate dose of) the peptides of the invention to a patient would preferably be sufficiently moderate or low so as to not necessitate administration of an agent to counteract any effect on arterial blood pressure produced by the peptides.

Preferably, the peptides of the invention do not induce any significant change in arterial blood pressure, e.g. no detectable change therein, when administered to a patient.

The effect of a given peptide on blood pressure can be determined by any means known in the art, such as the experiment described in the Examples (mean arterial pressure using telemeterized rats). Methods for measuring blood pressure of human subjects are well known.

Preferably, the peptides do not have any substantial haemodynamic effects.

The activity of peptides against cancer cells may be assayed, e.g., via the MTT cell proliferation assay. The MTT Cell Proliferation Assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability.

The reduction of tetrazolium salts is now widely accepted as a reliable way to examine cell proliferation. The yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl- 2)-2, 5-diphenyltetrazolium bromide) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan can be solubilized and quantified by spectrophotometric means.

MTT assays were carried out with the peptide of SEQ ID NO: 1 compared to some comparator peptides (see Examples). The Examples show an activity against a variety of different cancer cells. Preferably, the peptides may therefore have an anti-cancer activity against a variety of different cancers, e.g. against carcinomas, sarcomas and/or gliomas, e.g. carcinomas or adenocarcinomas, preferably at least colon carcinoma and/or liver carcinoma.

When applied in vitro to cancer cell lines (for example, JM1 , BEL-7402, HEPG2, CT-26, HT-29, HepaRG and/or CC531 cells), peptides of the invention preferably have an IC₅₀ value less than 30mM, more preferably less than 25mM and for selected cancer cell lines such as HepaRG preferably less than 20 or 15mM.

IC₅₀ is herein defined as the peptide concentration which kills 50 % of the cells after 4 hours incubation.

As shown in the Examples, treatment of a tumour with a peptide of the invention may result not only in a beneficial effect in relation to that tumour (such as inhibition of growth or even tumour regression); such treatment may also prevent re-growth of such a tumour, including the prevention of the establishment/growth of metastases.

The peptides may have certain characteristics, as illustrated by the

Examples. In particular, the peptides may, e.g., selectively enrich in the lysosomal and/or mitochondrial compartment of cancer cells and; the cytotoxic effects of the peptides may, e.g. be inhibited by serum and/or anti-oxidants, which may be strong inhibition; the cytotoxic effects of the peptides may, e.g. be inhibited by lysosomal inhibition with bafilomycin A1 and/or double knockout of BAX and BAK, which may be partial inhibition; the cytotoxic effects of the peptides may, e.g. be refractory to caspase inhibition; the peptides may, e.g., cause the exposure of calreticulin at the cell surface; the peptides may, e.g., cause the release of HMGB1 from the cells; and/or the peptides may, e.g., stimulate a necrosis-like disruption of cellular morphology.

The stimulation of a necrosis-like disruption of cellular morphology may be determined by transmission electron microscopy. A necrosis-like disruption of cellular morphology is evident when cells have an intact plasma membrane (and hence retain the cytoplasm) and lipid droplet in the cytoplasm are a prominent morphological feature. The formation of lipid droplets in the cytoplasm may be confirmed via staining with the red-fluorescent lipophilic dye Nile red.

Confocal fluorescence microscopy with appropriate fluorescent biosensors may be used to determine whether a peptide selectively enriches in the lysosomal compartment of cancer cells. In particular, co-localisation with a labelled lysosomal marker such as LAMP1-GFP may be analysed. Suitable fluorescent biosensors for various cellular compartments are known, e.g. for the nucleus (histone H2B fused to red fluorescent protein, RFP), the endoplasmic reticulum (calreticulin, CALR fused to green fluorescent protein, GFP), the Golgi apparatus (GALT1 fused to GFP), the mitochondria (DIABLO fused to GFP) and the lysosomes (LAMP1 fused to GFP).

Localisation to the lysosomal compartment may be confirmed by pre- incubation of the cells with the vacuolar ATPase inhibitor bafilomycin A1 (BAFA1 ), which is known to abolish lysosomal acidification and therefore typically abrogates such localisation. The results may be compared to cells that were not treated with BAFA1.

The effect of the peptides on the lysosomal stability may be measured by means of a LysoTracker™.

Apoptotic signaling involves mitochondrial membrane permeabilization that often depends on the expression of pro-apoptotic multidomain members of the BCL2 family, such as BAX and BAK. The induction of apoptotic signaling by a peptide may therefore be assessed by comparing the effect of the peptide on cells lacking BAX and/or BAK (BAX and/or BAK knockout) to wild-type cells.

Immunogenic cell death (ICD) is characterized by the cellular

release/exposure of DAMPs (ATP, CALR, HMGB1 , type-1 interferons) that make dead-cell antigens recognizable to the immune system. The induction of ICD may be assayed by measuring ATP release, CALR exposure on the cell surface, nuclear HMGB1 release, and/or the transcription of genes coding for type-1 interferons.

The stimulation of an anticancer immune response may be assayed by treatment of a tumour in an immunocompetent animal, e.g. via intratumoral injection of the peptide. T reatment of animals that were depleted of T cells by injection of specific antibodies blocking CD4 and CD8 may be compared to treatment of animals that were not depleted of T cells.

Analysis of the peptides of the invention suggests they may be able to penetrate the cell membrane of cancer cells. Without wishing to be bound by theory, it is believed that the peptides may induce immunogenic cell death via a process that may be BAX/BAK dependent and/or that may involve localisation of the peptide to the lysosomal compartment.

Without wishing to be bound by theory, it is believed that the peptides of the invention may induce immunogenic cell death (ICD), which may result in a systemic immune response, providing a protective effect against future tumours of the same or a similar type.

The molecules of the invention (by which is meant herein the peptides and/or peptidomimetics of the invention) may be administered by any convenient means, in particular by enteral, parenteral or topical routes. Parenteral routes are preferred and intratumoural (intralesional) administration is particularly preferred. Topical administration may be appropriate in certain cases, in particular when treating a cutaneous tumour. Thus administration may be local, e.g. topical or intratumoural, or systemic.

The skilled man will be able to formulate the molecules of the invention into pharmaceutical compositions that are adapted for these routes of administration according to any of the conventional methods known in the art and widely described in the literature.

The active ingredient may be incorporated, optionally together with other active agents, with one or more conventional carriers, diluents and/or excipients, to produce conventional galenic preparations such as tablets, pills, powders (e.g. inhalable powders), lozenges, sachets, cachets, elixirs, suspensions, emulsions, creams, foams, gels, solutions, syrups, aerosols (as a solid or in a liquid medium), lotions, ointments, soft and hard gelatine capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like.

Examples of suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, inert alginates, tragacanth, gelatine, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, water, water/ethanol, water/ glycol, water/polyethylene, hypertonic salt water, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof.

Preferred excipients and diluents are mannitol and/or hypertonic salt water (saline).

The compositions may additionally include lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, and the like.

Parenterally administrable forms should be sterile and free from

physiologically unacceptable agents, and should have low osmolarity to minimize irritation or other adverse effects upon administration and thus solutions should preferably be isotonic or slightly hypertonic, e.g. hypertonic salt water (saline). Suitable vehicles include aqueous vehicles customarily used for administering parenteral solutions such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection and other solutions such as are described in Remington's Pharmaceutical Sciences,

15th ed., Easton: Mack Publishing Co., pp. 1405-1412 and 1461-1487 (1975) and The National Formulary XIV, 14th ed. Washington: American Pharmaceutical Association (1975). The solutions can contain preservatives, antimicrobial agents, buffers and antioxidants conventionally used for parenteral solutions, excipients and other additives which are compatible with the peptides/peptidomimetic and which will not interfere with the manufacture, storage or use of products. A more sophisticated delivery vehicle, e.g. liposomes, may be used, in particular when delivery is systemic, the liposome encasing molecules of the invention in aqueous solution.

In medical applications, the peptide or mimetic may conveniently be administered at concentrations of 5-500 mg/I, preferably 20-100 mg/I, e.g. 40-60 mg/I.

The molecules described herein may be synthesised in any convenient way. Generally the reactive groups present (amino and/or carboxyl terminus and some side chains) will be protected during overall synthesis.

Methods of peptide synthesis are well known in the art but for the present invention it may be particularly convenient to carry out the synthesis on a solid phase support (SPPS), such supports and associated techniques being well known in the art. In building up the peptides, one can in principle start either at the C- terminus or the N-terminus, although step-wise addition to a free N-terminus is preferred.

Methods for the synthesis of the molecules of the invention constitute a further aspect of the present invention. For example, in one embodiment there is provided a method of synthesising a molecule of the invention as defined herein, which method includes the generation of a molecule of the invention having one or more protecting groups attached thereto and then the removal of said protecting group(s). Preferably the molecule is formed on a solid support by stepwise addition of amino acids or equivalent sub-units and then, in either order, (i) any remaining protecting groups are removed and (ii) the peptide is removed from the solid support.

A wide choice of protecting groups for amino acids are known (see for example Isidro-Llobet et al. Chem. Rev. [2009] 109, 2455-2504). It will be appreciated that when a peptide is built up from the C-terminal end, an amine- protecting group will be present on the a-amino group of each new residue added and will need to be removed selectively prior to the next coupling step. Suitable amine protecting groups include t-butoxycarbonyl (also designated Boc) and 9- fluorenylmethoxy-carbonyl (also designated Fmoc). Fmoc based SPPS

procedures are preferred. These procedures may be automated and amino acids with Fmoc protected N-termini and any necessary side-chain protection are readily available. When Fmoc is used as the N-terminal protecting group, conveniently mild acid-labile protecting groups that are stable under basic conditions, such as Boc or benzyl groups may be used to protect reactive amino acid side chains.

A wide range of procedures exists for removing protecting groups. These must, however, be consistent with the synthetic strategy employed. The side chain protecting groups must be stable to the conditions used to remove the temporary o amino protecting group prior to the next coupling step. Dosage units containing the active molecules preferably contain 0.1-1 Omg, for example 1.5mg of the antitumour molecule of the invention. The pharmaceutical compositions may additionally comprise further active ingredients, including other cytotoxic or antitumour agents such as other antitumour peptides. Other active ingredients may include different types of cytokines e.g. IFN-g, TNF, CSF and growth factors, immunomodulators, chemotherapeutics e.g. cisplatin or antibodies or cancer vaccines.

In employing such compositions systemically, the active molecule is present in an amount to achieve a serum level of the active molecule of at least about 5 pg/mL. In general, the serum level need not exceed 500 pg/mL. A preferred serum level is about 100 pg/mL. Such serum levels may be achieved by incorporating the bioactive molecule in a composition to be administered systemically at a dose of from 1 to about 10 mg/kg. In general, the molecule(s) need not be administered at a dose exceeding 100 mg/kg.

In a further aspect the present invention provides a molecule as defined herein and (b) a further antitumoural agent as a combined preparation for separate, simultaneous or sequential use in the treatment of a tumour or cancer cells or preventing or reducing the growth, establishment spread, or metastasis of a tumour.

The above description describes numerous features of the present invention and in most cases preferred embodiments of each feature are described. It will be appreciated that each preferred embodiment of a given feature may provide a molecule, use, method etc. of the invention which is preferred, both when combined with the other features of the invention in their most general form and when combined with preferred embodiments of other features. The effect of selecting multiple preferred embodiments may be additive or synergistic. Thus all such combinations are contemplated unless the technical context obviously makes them mutually exclusive or contradictory. In general each feature and preferred embodiments of it are independent of the other features and hence combinations of preferred embodiments may be presented to describe sub-sets of the most general definitions without providing the skilled reader with any new concepts or information as such.

The invention will now be described with reference to the following non-limiting Examples and with reference to the following Figures:

Fig. 1 shows results of Examples 1A and 1 B. Figs. 1A-1 C are graphs showing the change in volume of MC38 subcutaneous tumour in C57BL/6 mice from the initiation of treatment with vehicle (Fig. 1A), 10 mg/ml peptide of SEQ ID NO: 1 (peptide 1 ) (Fig. 1 B), or 20 mg/ml peptide 1 (Fig. 1 C).

Fig. 1 D shows results of Example 1 B. Fig. 1 D is a Kaplan-Meier curve showing the survival of C57BL/6 mice after rechallenge with MC38 cells.

Rechallenged mice were treated with 10 mg/ml or 20 mg/ml of peptide 1. As a control, naive mice were challenged with MC38 cells and treated with vehicle control. The lines for 10 mg/ml peptide 1 and 20 mg/ml peptide 1 overlap, i.e. the survival rate for both groups was 100%.

Fig. 2 shows results of Example 2. Fig. 2A is a Kaplan-Meier curve showing the survival of BALB/c mice after rechallenge with CT26 cells. Rechallenged mice were treated with 10 mg/ml or 20 mg/ml of peptide 1. As a control, naive mice were challenged with MC38 cells and treated with vehicle control. Figs. 2B-C are representative magnetic resonance images from control (Fig. 2B) and peptide 1- treated (Fig. 2C) BALB/c mice established with liver tumour prior to imaging. The liver in the control mouse is indicated by the white outline, and the white arrow points to a CT26 liver tumour in that animal.

Fig. 3 shows results of Example 3. Fig. 3A is a graph of quantified transmission electron microscopy data showing the mean number of lipid droplets per electron micrograph (mean ± SD of a minimum of 5 viewfields). Fig. 3B is a graph showing the area stained by the lipophilic dye Nile Red following treatment (mean ± SD of triplicate assessments, Student’s t-test, ^*p<0.5, ^**p<0.01 ). Fig. 3C is a graph showing the area stained by the lipophilic dye Nile Red following treatment and at different temperatures. Fig. 3D is a graph showing the viability of cells after peptide 1 treatment In the presence of different serum levels.

Fig. 4 shows results of Example 4. Fig. 4A is a graph of quantified fluorescence microscopy data showing the relative co-occurrence of organellar markers with Pacific Blue-labelled peptide 1 in the presence or absence of BAFA1. Fig. 4B is a graph of quantified fluorescence microscopy data showing the relative LysoTracker area in U20S cells after treatment with vehicle (Ctr (control)) or 0.65 or 1.25 mM peptide 1. Fig. 4C is a graph of quantified fluorescence microscopy data showing the viability of cells after treatment with vehicle (Ctr) or 0.65 - 10 mM peptide 1 , using the exclusion dye propidium iodide (mean ± SD of triplicate assessments, Student’s t-test, ^*p<0.5, ^**p<0.01 , ^***p<0.001 ).

Fig. 5 shows results of Example 5. Figure 5A is a graph of quantified fluorescence microscopy data showing the level of activation of caspase 3 after treatment with different concentrations of peptide 1 or STS (positive control). Fig. 5B is a graph of quantified fluorescence microscopy data showing degree of nuclear shrinkage caused to cells following treatment with different concentrations of peptide 1 or STS (positive control). Figs. 5C-E are graphs showing the viability of cells administered with various compounds prior to treatment with peptide 1. Fig. 5F is a graph showing the viability of cells lacking Bax and/or Bak expression after treatment with different concentrations of peptide 1 (mean ± SD of triplicate assessments, Student’s t-test, ^*p<0.5, ^**p<0.01 , ^***p<0.001 ).

Fig. 6 shows results of Example 6. Fig. 6A is a graph of quantified fluorescence microscopy data showing the aggregation of RIP3-GFP after treatment with different concentrations of peptide 1 or TSZ (positive control) . Fig. 6B is Western blotting data showing the relative levels of MLKL and p-MLKL after treatment with different concentrations of peptide 1 or TSZ (positive control). Fig. 6C is Western blotting data demonstrating the lack of expression of RIP3 or MLKL in the corresponding RIP3 or MLKL knockout cell line used in Fig. 6D. Figure 6D is a graph showing the viability of RIP3-deficient or MLKL-deficient cells after treatment with different concentrations of peptide 1 or TSZ (positive control) (mean ± SD of triplicate assessments, Student’s t-test, ^*p<0.5, ^**p<0.01 , ^***p<0.001 ).

Fig. 7 shows results of Example 7. Fig. 7A is a graph showing the level of induction of autophagy in U20S cells treated with vehicle (Ctr), 0.65 - 10 mM peptide 1 , or fatty acid oleate (OL). Fig. 7B is a graph showing the level of ATP release from U20S cells stably expressing LC3 coupled to GFP treated with vehicle (Ctr), 0.65 - 10 mM peptide 1 , or MTX. Fig. 7C is a graph showing exposure of CALR on the cell surface of live (PL) U20S cells. Fig. 7D is a graph showing release of HMGB1 from U20S cells after treatment. Fig. 7E is a graph showing the level of transcription of genes coding for type I interferons in U20S cells after treatment (mean ± SD of triplicate assessments; Student’s t-test, ^*p<0.5, ^**p<0.01 ,

^***p<0.001 ).

Fig. 8 shows results of Example 7. Fig. 8A gives an overview of the experimental procedure followed in Fig. 8B-E. Fig. 8B is a graph showing the change in MCA205 tumour size after treatment with vehicle (Ctr) or peptide 1. Fig. 8C is a graph showing the change in MCA205 tumour size after treatment with vehicle (Ctr), or with peptide 1 and anti-CD4 and anti-CD8 antibodies (Peptide 1 + antibodies). Fig. 8D is a graph showing the results of Figs. 8B and 8C as mean values(Chi-squared test, ^***p<0.001 ). Fig. 8E is a Kaplan-Meier curve showing the survival of MCA205 injected mice after treatment with vehicle or peptide 1 in mice with or without depleted CD4 and CD8 T-cell populations (Chi-squared test, ^**p<0.01 ). Fig. 8F is a cartoon depicting the location of administration of MCA205 cell rechallenge and TC-1 cell challenge on mice cured from TC-1 (and control mice with no previous cancers or treatments). Fig. 8G is a graph showing the change in TC-1 and MCA205 tumour size in the mice treated as shown in Fig. 8F.

Fig. 9 shows results of Example 7. Fig. 9A gives an overview of the experimental procedure. Fig. 9B is a graph showing the change in TC-1 tumour size after treatment with vehicle (Ctr) (n=6 mice) or peptide 1 (n=6 mice). Figure 9C is a graph showing the results of Fig. 9B as mean values (Chi-squared test, ^***p<0.001 ). Fig. 9D is a Kaplan-Meier curve showing the survival of the TC-1 injected mice of Fig. 9B-C after treatment with vehicle or peptide 1 (Chi-squared test, ^***p<0.001 ). Fig. 9E is a cartoon depicting the locations of administration of TC-1 cell rechallenge and MCA205 cell challenge on mice cured from TC-1 (and control mice i.e. no previous cancers or treatments). Fig. 9F is a graph showing the change in TC-1 and MCA205 tumour size in 4 mice treated as shown in Fig. 9E.

Examples

Materials and methods

Chemicals and cell cultures.

Cell culture media and media supplements were purchased from Thermo Fisher Scientific (Carlsbad, CA, US) and chemicals came from Sigma-Aldrich (St. Louis, MO, US) with the exception of the peptides, which were provided by Lytix Biopharma (Oslo, Norway). Plastic ware was obtained from Greiner Bio-One (Monroe, CA, US), primary antibody (cleaved caspase-3; #9661 ) came from Cell Signaling (Danvers; MA; US) and AlexaFluor-coupled secondary antibody from Thermo Fisher Scientific. Mouse embryonic fibroblast (MEF) murine lung cancer TC-1 , human osteosarcoma U20S, wild type or stably expressing GALT1-GFP, H2B-RFP, CALR-GFP, DIABLO-GFP, LAMP1-GFP or GFP-LC3 and HT29 stably expressing RIP3-GFP (Cho et al., Cell, 2009, 137(6): 1 112-1123) cells were cultured in Glutamax^®-containing DMEM medium supplemented with 10 % fetal calf serum (FCS), and 10 mM HEPES. Cells were grown in a humidified incubator at 37 °C under a 5 % C0₂ atmosphere.

High-throughput assessment of cell death

Five x 10³ U20S cells were seeded into black 96-well pclear imaging plates (Greiner Bio-One) and allowed to adapt for 24 h. Thereafter the cells were treated with peptide and respective controls and incubated for additional 6 or 24 h before either 1 mM of DAPI or a mixture 1 mM Hoechst and 1 pM propidium iodide were added upon treatment immediately before monitoring the uptake of the exclusion dye in a minimum of four view fields per well by means of an ImageXpress micro XL automated bioimager (Molecular Devices) equipped with a PlanApo 20X/0.75 NA objective (Nikon, Tokyo, Japan).

High-content screening microscopy

U20S cells stably expressing GFP-LC3, GALT1-GFP, CALR-KDEL-GFP, H2B-RFP, LAMP1-GFP and DIABLO-GFP (Example 4) or HT-29 cells stably expressing RIP3-GFP (Example 6) were seeded in 96-well black microplates for 24 h. After treatment, cells were fixed with 3.7 % PFA for 20 min at room temperature and stained with 10 pg/ml Hoechst 33342 in PBS. Image acquisition was performed using an ImageXpress Micro XL automated microscope (Molecular Devices). A minimum of 4 view fields were captured per well. Upon acquisition, images were analyzed using the Custom Module Editor of the MetaXpress software (Molecular Devices). Briefly, cells were segmented and divided into nuclear and cytoplasmic regions based on the nuclear Hoechst staining and GFP or RFP cytoplasmic signals. GFP-LC3 and RIP3-GFP dots were detected using automated

thresholding, and their number and surface were measured in the cytoplasmic compartment. Cooccurrence of GALT1 -GFP, CALR-KDEL-GFP, H2B-RFP, LAMP1-GFP and DIABLO-GFP fluorescence signals with Pacific Blue-labeled peptides were systematically analyzed to assess subcellular targeting. Data processing and statistical analyses were performed using the R software

(http://www.r-proiect.org/)·

Data processing and statistical analyses

Unless otherwise specified, experiments were performed in triplicate parallel instances and repeated at least once, and data were analyzed with the R software (http://www.r-proiect.org/^')· Microscopy images were segmented and analyzed by means of the MetaXpress (Molecular Devices) software and numerical data was further processed with R. Unless otherwise specified, data are presented as means ± SD. Thresholds for the minimum number of events in each analysis necessary to apply further statistics were calculated based on a medium effect size (according to Cohen's conventional criteria) using the pwr package for R with a targeted value of 0.95. Samples that did not match the requirements were marked ND and were excluded from the analysis.

Peptide synthesis

Peptides were synthesized on solid-phase with a Prelude instrument (Protein Technologies Inc. Tucson, AZ, US) using standard Fmoc protocols and amino acid derivatives. Each peptide was prepared as a C-terminal amide by using a 100-200 mesh Rink amide resin (Novabiochem, Merck Millipore, Billerica, MA, US) as solid support. The Fmoc-amino acids used were standard derivatives from Novabiochem. Double couplings (2 x 30 min, 5 eq to the resin) were performed.

The incoming Fmoc-amino acids were activated with 5 eq (2-(6-Chloro-1 H- benzotriazole-1-yl)-1 ,1 ,3,3-tetramethylaminium hexafluorophosphate) (HCTU) and 10 eq diisopropylethylamine (DIPEA) with dimethylformamide (DMF) as solvent. Coupling reactions were concluded with a washing (DMF, 3 x 30 s) and Fmoc- removal step (20% piperidine in DMF, 5 + 10 min). Completed peptides were cleaved from the resin using a cocktail containing 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% triisopropylsilane for 3 h. The TFA was removed using a rotavapor (Hei-VAP Advantage rotavapor, Heidolph Instruments, Schwabach, Germany) and the fully deprotected peptides precipitated with diethyl ether as C- terminal amides. The ether was decanted and the precipitated crude peptide allowed to air dry before analysis and purification. All the peptides tested have an amidated C-terminus.

Peptide purification and characterization

The solvents used in the analytical and preparative systems were MilliQ water (Solvent A) and acetonitrile (Solvent B), both modified with 0.1% TFA. The crude and purified peptides were analyzed on an ACQUITY UPLC H-class system with a photodiode array (PDA) detector (Waters, Milford, MA, US) equipped with an ACQUITY CEH C18 UPLC column (Waters, 2.1 x 50 mm, 1.7 pm). A gradient of 0- 50% Solvent B over 30 min with a flow rate of 1 mL/min was used, and detection was set at 200-500 nm. The crude peptides were purified to >95% on a XSelect CSH C18 OBD prep column (Waters, 19 x 250 mm, 5 pm) installed in an

AutoPurificayion System (Waters). A default gradient of 10-40% Solvent B over 30 min with a flow rate of 20 mL/min was used, but it was adjusted as required. The detection was set at 200-500 nm.The molecular weight of the peptides was confirmed on the Xevo G2 Q-TOF with ACQUITY UPLC l-Class system (Waters). The purified peptides were then freeze dried (Labconco FreeZone 4.5 Plus, Kansas City, MO, US) as TFA-salts and stored at -20°C until further use.

Examples - General remarks

The results of these studies suggest that the oncolytic peptide‘peptide 1’ targets lysosomes, as indicated by its selective accumulation in this organelle. In this way, peptide 1 is rather distinct from other oncolytic peptides, which often target mitochondria. Peptide 1 triggered the release of pro-immunogenic DAMPs in vitro and activated anticancer immune responses in vivo. In this study, animals that were cured from primary tumors generated long-term immunological memory that mediated the rejection of isogenic tumors, which underlines the immunotherapeutic potential of the peptides of the invention.

Example 1 A - Peptide-mediated inhibition of subcutaneous tumour growth in a MC38-C57BL/6 mouse model

Methods

MC38 cells are a cell line derived from C57BL/6 murine colon

adenocarcinoma cells. MC38 cells were obtained from Kerafast, Inc., 27 Drydock Avenue, 2nd Floor Boston, MA 02210, catalogue No. ENH204. The cells were injected subcutaneously in C57BL/6 mice. Each tumour was allowed to grow for 7 days until it reached a size of approximately 75 mm³.

The Peptide of SEQ ID NO: 1 (peptide 1 ) was dissolved in saline to a concentration of 10 or 20 mg/ml. Tumours in the test mice were treated

intratumourally with 50 m I of 10 mg/ml (n=6 mice) or 20 mg/ml (n=9) peptide 1 solution. Tumours in the control mice (n=7) were treated intratumourally with 50 pi vehicle. The mice were treated for 3 consecutive days. Subcutaneous tumour growth was measured using a calliper.

Results

Results are shown in Fig. 1. T umour size is displayed as relative tumour volume, i.e. relative tumour volume = current tumour volume/tumour volume at initiation of treatment.

All control mice (n=7) exhibited exponential tumour growth in the weeks following treatment (Fig. 1A).

All mice treated with 10 mg/ml peptide solution (n=6) went into complete remission following treatment, i.e. tumour volume was reduced to zero (Fig. 1 B).

Of the mice treated with 20 mg/ml peptide solution (n=9), 7 went into complete remission following treatment. The tumours of the 2 remaining mice had significantly reduced growth compared with the control (treatment with vehicle)(Fig. 1 C).

Example 1B - Peptide-mediated inhibition of liver tumour growth in a MC38- C57BL/6 mouse model

Methods

Selected C57BL/6 mice from Example 1 A which showed complete remission following 10 mg/ml peptide 1 treatment (n=3) and 20 mg/ml peptide 1 treatment (n=6) were then rechallenged, i.e. injected intrasplenically with MC38 cells 5 weeks after treatment to establish tumour in the liver. Naive mice were injected intrasplenically with MC38 cells as a control. Survival was recorded for around 40 days post injection with MC38 cells.

Results

Results are shown in Fig. 1 D. All test mice previously treated with peptide 1 survived, in contrast to 4 out of 8 of the control mice, which had to be sacrificed. None of the mice treated with peptide 1 exhibited tumour growth in the liver.

Example 2 - Peptide-mediated inhibition of liver tumour growth in a CT26-

BALB/c mouse model

Methods

CT26 cells (a colon carcinoma cell line, ATCC number 2638) were injected subcutaneously in BALB/c mice. Each tumour was allowed to grow for 9 days until it reached a size of approximately 80 mm³.

A sample of peptide 1 was dissolved in saline to a concentration of 20 mg/ml. Tumours in the test mice were treated intratumourally with 50 mI of 20 mg/ml peptide 1. Tumours in the control mice were treated intratumourally with 50 pi vehicle. The mice were treated for 3 consecutive days. Subcutaneous tumour growth was measured using a calliper for several weeks from the first day of treatment.

Two BALB/c mice which showed complete remission following 20 mg/ml peptide 1 treatment were then injected intrasplenically with CT26 cells 5 weeks after treatment to establish tumour in the liver. Six naive BALB/c mice were injected intrasplenically with CT26 cells as a control. Survival was recorded for approximately 40 days after injection with CT26 cells.

Results

Results are shown in Fig. 2. Both mice previously treated with 20 mg/ml peptide 1 survived (indicating that an immunological memory against CT26 cancer had been established), in contrast to the 6 control mice, which all had to be sacrificed before 20 days post-injection (Fig. 2A). Magnetic resonance images of control (Fig. 2B) and peptide 1 -treated (Fig. 2C) mice show that liver tumour growth is inhibited through treatment with peptide 1. No tumour is seen in the peptide 1 - treated mouse.

Example 3 - Morphological effects of peptide 1

Human osteosarcoma U20S cells were treated with various concerntations of peptide 1 for 6 h. The cells were fixed in 1.6 % glutaraldehyde (v/v in 0.1 M phosphate buffer) for 1 h, collected by scraping, centrifuged and the pellet was post-fixed 1 % osmium tetroxide (w/v in 0.1 M phosphate buffer). Following dehydration through a graded ethanol series, cells were embedded in Epon™ 812 and ultrathin sections were stained with standard uranyl acetate and lead citrate. Transmission electron microscopy images were taken using a Tecnai 12 electron microscope (FEI, Eindhoven, the Netherlands). The quantified data (mean number of lipid droplets) is shown in Fig. 3A.

The formation of lipid droplets in response to increasing doses from 0.65 to 10 mM peptide 1 was assessed by means of the lipophilic dye Nile Red at 6 h and 24 h post treatment by epifluorescence microscopy. The quantified data is shown in Fig. 3B.

The temperature dependency of the lipid droplet formation was assessed by keeping the cell cultures at the indicated temperature for 6 h after treatment with 0.65 to 10 mM peptide 1 before staining with Nile Red. Increased number of lipid droplets at physiological temperature is indicative for an underlying active biochemical reaction. The quantified data is shown in Fig. 3C.

Discussion When added to human cancer cells, peptide 1 stimulated a necrosis-like disruption of cellular morphology. In cells in which the plasma membrane was still intact and hence retaining the cytoplasm, lipid droplets in the cytoplasm were a prominent morphological feature that was induced in a dose-dependent fashion, as determined by transmission electron microscopy and quantified in Fig. 3A. Staining with the red-fluorescent lipophilic dye Nile red confirmed the formation of lipid droplets in the cytoplasm of cells treated with peptide 1 that occurred in a time and dose-dependent fashion (Fig. 3B). Of note, these effects were only obtained when cellular metabolism and membrane trafficking were active at 37°C, yet not at lower temperatures such as 14 or 22°C (Fig. 3C). As observed with some other oncolytic peptides, serum had an inhibitory effect on the cytotoxic potential of peptide 1 (Fig. 3D).

Example 4 - Lysosomal tropism of peptide 1

Intrigued by the peculiar morphology of peptide 1-treated cells, we investigated the subcellular distribution of the peptide, using peptide 1 with a blue- fluorescent moiety (Pacific Blue) attached to it. Cells that express fluorescent biosensors in the nucleus (histone H2B fused to red fluorescent protein, RFP), endoplasmic reticulum (calreticulin (CALR) fused to green fluorescent protein (GFP)), Golgi apparatus (GALT1 fused to GFP), mitochondria (DIABLO fused to GFP) or lysosomes (LAMP1 fused to GFP) were treated with 1.25 mM Pacific Blue- labelled peptide 1 in the presence or absence of lysosomal acidification (where desired, lysosomal acidification was blocked using bafilomycin A1 (BAFA1 )).

The relative co-occurrence of Pacific Blue label with organellar markers was imaged by confocal microscopy. The quantified data is shown in Fig. 4A.

Wild type U20S cells were stained with LysoTracker green and the decrease in lysosomal content was assessed upon treatment with 0.65 or 1.25 pM peptide 1 for 6 h by epifluorescence microscopy. The quantified data is shown in Fig. 4B.

Viability was measured in living cells by means of the exclusion dye propidium iodide 6 h post treatment with 0.65 to 10 pM peptide 1 in the presence or absence of BAFA1 by live cell microscopy. The quantified data is shown in Fig. 4C.

Discussion

Confocal fluorescence microscopy revealed that peptide 1 co-localized with the lysosomal marker LAMP1-GFP but not with any other organellar probe (Fig. 4A). Of note, this colocalization was abrogated upon pre-incubation of the cells with the vacuolar ATPase inhibitor bafilomycin A1 (BAFA1 ), which is known to abolish lysosomal acidification (Fig. 4A). As is characteristic of lysomotropic agents, peptide 1 affected lysosomal stability and let to a reduction in measurable organellar surface area, as measured by means of LysoTracker (quantified in Fig. 4B). Next, we determined whether the lysosomo-tropism of peptide 1 might explain its cytotoxic activity. For this, we used BAFA1 to prevent lysosomal accumulation of peptide 1. We found that BAFA1 partially reduced cell killing by peptide 1 (Fig. 4C).

Example 5 - Apoptosis-related signalling induced by peptide 1

Immunostaining

Five x 10³ U20S cells were seeded into black 96-well pclear imaging plates (Greiner Bio-One) and allowed to adapt for 24 h. Thereafter the cells were treated with peptide 1 or control (i.e. vehicle alone) and incubated for additional 6 or 24 h before fixation in 3.7 % (w/v) paraformaldehyde (PFA) in PBS supplemented with 1 mM Hoechst 33342 for 20 min. Upon fixation cells were permeabilized with 0.1 % Triton in PBS for 10 min at RT. Unspecific binding was blocked with 2 % BSA in PBS for 10 min at RT followed by primary antibody diluted in BSA 2 % following the manufactures recommendations overnight on an orbital shaker at 4°C. The cells were rinsed twice and stained with AlexaFluor-coupled secondary antibodies for 1 h at RT, rinsed twice and subjected to imaging using an ImageXpress micro XL automated bioimager (Molecular Devices) equipped with a PlanApo 20X/0.75 NA objective (Nikon).

Human osteosarcoma U20S cells were treated with 0.65 to 10 mM peptide 1 for the indicated time and then assessed for the activation of caspase-3 (CASP3). The pan-kinase inhibitor staurosporine (STS) was used as positive control.

Quantifications of CASP3 activation are depicted in Fig. 5A, while pyknosis was assessed by measuring the decrease in Hoechst 33342-stained nuclear area by conventional microscopy (the quantified data is shown in Fig. 5B).

In order to assess cell death modality, U20S cells were pretreated with the ferroptosis inhibitor ferrostatin 1 (FER-1 ), the pan-caspase inhibitor zVAD-fmk (Z- VAD), the antioxidant N-acetylcysteine (NAC), or the antioxidant reduced glutathione (GSH) before the addition of 2.5 pM of peptide 1 (Fig. 5C-E).

Mouse embryonic fibroblasts (MEFs) that were either wildtype, single- or double-knockout for the pro-apoptotic proteins Bax or Bak were treated with 0.65 to 10 pM of peptide 1 and viability was assessed by means of an exclusion dye using epifluorescence microscopy. The quantified data is shown in Fig. 5F.

Discussion

Caspase-3 activation is one of biochemical hallmarks of apoptosis. While a significant fraction of cells treated with the positive control (pan-kinase inhibitor staurosporine (STS)) stained positively with an antibody recognizing the proteolytically mature fragment of caspase-3, only a minor fraction of cells exhibited caspase activation after treatment with peptide 1 (Fig. 5A).

Peptide 1 caused nuclear shrinkage (Fig. 5B), though without the formation of apoptotic bodies (as indicated by the confocal microscopy images). Neither the pan-caspase inhibitor Z-VAD-fmk (z-VAD) nor the ferroptosis inhibitor ferrostatin-1 (FER-1 ) was able to prevent cell killing by peptide 1 (Fig. 5C). In contrast, the two anti-oxidants, namely N-acetylcysteine (NAC) and glutathione, strongly reduced the cytotoxic activity of peptide 1 (Figs. 5C-E).

Apoptotic signalling also involves mitochondrial membrane permeabilization that often depends on the expression of pro-apoptotic multi-domain members of the BCL2 family such as BAX and BAK. Indeed, knockout of BAX or BAX, alone or together, reduced killing by peptide 1 to a variable extent (Fig. 5F). In summary, it appears that peptide 1 kill cells via pro-oxidant and BAX/BAK-dependent mechanisms, but not via caspase activation.

Example 6 - Peptide 1 does not appear to induce necroptotic signalling

Western blot

Half a million cells were harvested from 6 well plates resuspended in lysis buffer containing 150 mM sodium chloride, 1.0 % NP-40, 0.5% sodium

deoxycholate, 0.1% SDS and protease inhibitor cocktails (Complete protease inhibitor cocktail, Roche, Basel, Switzerland) and incubated on ice for 30 min. To obtain supernatant, cell lysate was centrifuged at 12000xg for 20 min at 4 °C to remove insoluble materials. The lysate was mixed with 4*NuPAGE® LDS Sample Buffer and l OxSample Reducing Agent and proteins were denaturated at 100°C for 10 min. NuPAGE® Novex® 4-12% Bis-Tris Protein Gels (Thermo Fisher Scientific) were used for protein electrophoresis under a 100V constant voltage mode.

Separated proteins were transferred from gel to PVDF membrane (Merck-Millipore, Darmstadt, Germany). After blocking with 5% BSA in 1 *TBS containing 0.1 % Tween®-20 (1 *TBST) for 1 h at room temperature, the membranes were probed with corresponding primary antibodies at 4 °C overnight: anti-mouse RIP1 antibody (clone: 38/RIP, BD Bioscience, San Jose, CA, US), anti-mouse RIP3 polyclonal antibody (r4277; Sigma-Aldrich and AHP1797; Bio-Rad laboratories, Hercules, CA, US), anti-mouse MLKL polyclonal antibody (AP14272b; Abgent, San Diego, CA, US). To visualize phosphorylated MLKL (pMLKL), anti-mouse MLKL (phospho S345) monoclonal antibody (ab196436, Abeam) was used. The membranes were then washed and incubated with HRP-conjugated secondary antibodies

(SouthernBiotech, Birmingham, AL, US) at room temperature for 1 h. The peroxidase activity was detected with ECL Western Blotting Detection Reagent (GE healthcare, Chicago, IL, US) and images were acquired by ImageQuant LAS 4000 (GE healthcare). In vitro cytotoxicity

Murine lung cancer TC-1 cells were CRISPR gene edited in RIP3 and MLKL and reduced gene expression is depicted in Fig. 6C. Those cells were treated with 0.65 to 10 mM of peptide 1 for 6 h and viability was assessed by means of an exclusion dye (quantified in Fig. 6D).

To investigate the potential role of necroptotic signaling in cell death induction by peptide 1 , we took advantage of a biosensor cell line that lacks MLKL (and hence cannot undergo necroptosis) yet expresses a RIP3-GFP fusion protein that can be monitored for its aggregation in the cytoplasm within so-called ‘necroptosomes’. As a positive control, a combination of tumor necrosis factor-a (TNFa), DIABLO mimetic and Z-VAD-fmk (collectively abbreviated as TSZ’) induced full necroptosome activation. Peptide 1 was unable to induce this phenomenon (Fig. 6A). Accordingly, treatment of cells with peptide 1 did not result in phosphorylation of the RIP3 substrate MLKL (Fig. 6B). Cells rendered deficient for RIP3 or MLKL (protein expression profiles shown by Western Blot in Fig. 6C) did not have increased resistance to peptide 1 (Fig. 6D), supporting the notion that peptide 1 does not induce cell death via a signaling pathway that involves the necroptotic cascade.

Example 7 - Immunogenic cell death triggered by peptide 1

Measurement of DAMP release

Human osteosarcoma U20S cells stably expressing LC3 coupled to green fluorescent protein (GFP) were treated with 0.65 to 10 mM peptide 1 for the indicated time and subsequently assessed for the formation of LC3 marked vesicles by epifluorescence microscopy. Treatment with 500 pM of the fatty acid oleate (OL) was used as a positive control for the induction of autophagy (Fig. 7A). The release of ATP was assessed by means of ATP-dependent oxygenation of luciferin in cell culture supernatants. Resulting bioluminescence was measured and the concentration of ATP was calculated based on standards (Fig. 7B).

A TP release assay

Cells were seeded in 24-well plate and let adhere and adapt overnight, before they were treated with peptide 1 or the positive control mitoxantrone (MTX). Supernatants were collected and centrifuged at 500xg for 5 min in order to remove cellular debris and immediately subjected to the assessment of extracellular ATP by means of the ENLITEN® ATP Assay System (Promega, Madison, Wl, US) following the recommendations of the provider. ATP-driven chemiluminescence signal was assessed by means of a Paradigm E3 multilabel reader (Molecular Devices, San Jose, CA, US) (Fig. 7B). Determination of surface-exposed CALR by immunofluorescence

Cells were collected and rinsed twice with cold PBS. The cells were then incubated with an anti-CALR antibody (ab2907; Abeam, Cambridge, UK) diluted in cold blocking buffer (1 % BSA in PBS) for 60 min on ice, followed by washing and incubation with AlexaFluor 488-conjugates (Invitrogen) in blocking buffer (for 30 min). Thereafter cells were washed in cold PBS, the vital dye propidium iodide (PI) was added to a final concentration of 1 pg/mL, and samples were analyzed by flow cytometry (CyAn ADP, Beckman Coulter, Brea, CA, US) coupled to a Hypercite autosampler (IntelliCyte; Albuquerque, NM, US). The analysis was limited to living (PI ) cells, i.e. those cells which retained cytoplasmic membrane integrity and thus resisted the uptake of propidium iodide (PI). Data were statistically evaluated using R (https://www.r-proiect.org) (Fig. 7C).

HMGB1 release assay

The release of high mobility group box 1 protein (HMGB1 ) from the cells into cell culture supernatants was quantified by HMGB1-specific ELISA (#ST51011 ; IBL, Hamburg, Germany) according to the manufacturer’s recommendations and samples were measured with a Paradigm E3 multilabel reader (Molecular Devices) (Fig. 7D).

RNA extraction, reverse transcription and qRT-PCR

Cells were collected for total RNA extraction using the RNeasy Mini kit (Quiagen, Hilden, Germany). Three microgram of RNA was reverse-transcribed to cDNA using Superscript® III First-Strand System (Thermo Fisher Scientific). Type I IFN-related gene expression was quantified with TaqMan® Gene Expression Assays using Universal Master Mix II (with UNG) on a StepOnePlus™ Real-Time PCR system (all from Thermo Fisher Scientific). GAPDH was used as house- keeping gene for normalization. Relative gene expression was quantified using the comparative Ct method and was calculated as fold change. All experiments were conducted in triplicate assessment (Fig. 7E).

Mouse experiments

Female wild-type C57BL/6 mice at the age of 6-8 weeks were obtained from Harlan France (Gannat, France) and maintained in the animal facility at Gustave Roussy Campus Cancer in specific pathogen-free conditions in a temperature- controlled environment with 12 h light, 12 h dark cycles and received food and water ad libitum.

MCA205 or TC-1 tumors were established in C57BL/6 hosts by

subcutaneously inoculating 500,000 cells. When tumors became palpable, 1.5 mg of peptide 1 was injected intratumorally. Another 1.5 mg dose of peptide 1 was injected four days later and mice well-being and tumor growth were monitored. Anti-CD8 and anti-CD4 intraperitoneal injections were repeated every 7 days starting on day 0 (i.e. antibody injections were made on days 0, 7, 14 and 21 )in order to ensure the complete depletion of both T cell populations during the whole experiment. Animals were sacrificed when tumor size reached an end-point, or when signs of obvious discomfort associated to the treatment were observed.

Surviving, tumor-free mice previously injected with MCA205 cells were kept for more than 30 days before re-challenge with 5 x 10⁵ live MCA205 cells on the contralateral side and 5 x 10⁵ live TC-1 cells on the original (ipsilateral) side (Fig. 8F).

Surviving, tumor-free mice previously injected with TC-1 cells were kept for more than 30 days before rechallenge with 5 x 10⁵ live TC-1 cells on the contralateral side and 5 x 10⁵ live MCA205 cells on the original (ipsilateral) side (Fig. 9E). Animals were monitored and tumor growth documented regularly until end-points were reached or signs of obvious discomfort were observed.

Statistical analysis was performed employing 2-way ANOVA analysis followed by Bonferroni’s test comparing to Ctr conditions (^* p < 0.05, ^** p < 0.01 and

^***p < 0.001 ).

Results

Immunogenic cell death (ICD) is characterized by the cellular

release/exposure of DAMPs (ATP, CALR, HMGB1 , type-1 interferons) that make dead-cell antigens recognizable to the immune system. Although peptide 1 did not induce autophagy (Fig. 7A), peptide 1 did induce significant ATP release (Fig. 7B), CALR exposure on the cell surface (Fig. 7C), nuclear HMGB1 release (Fig. 7D), and the transcription of genes coding for type-1 interferons (Fig. 7E).

Based on these in vitro characteristics of peptide 1 -induced cell death, we next investigated whether peptide 1 might mediate anticancer effects through the stimulation of an anticancer immune response. For this, established MCA205 fibrosarcomas growing on 5 immunocompetent haploidentical C57BL/6 mice were injected with peptide 1 , which caused complete oncolysis in 4 out of 5 mice (Fig. 8B and D). Of note, prior depletion of T cells by injection of specific antibodies blocking CD4 and CD8 abolished these anticancer effects (n=4 mice) (Fig. 8C-D). These immune-dependent anticancer effects of peptide 1 were evident both when tumor growth kinetics and overall survival of mice were monitored (Fig. 8E).

Rechallenge of 6 mice cured from MCA205 fibrosarcoma with MCA205 and TC-1 cells resulted in efficient rejection of MCA205 (i.e. complete remission), although antigenically distinct TC-1 lung adenocarcinoma grew in most cases (Fig. 8G). Rechallenge of mice cured from TC-1 carcinoma with MCA205 and TC-1 cells yielded very similar results; none of the mice that were rechallenged with TC-1 developed TC-1 tumours, although MCA205 tumours readily developed in 3 out of 4 mice (Fig. 9F). These results indicate that immune-dependent cancer cure mediated by peptide 1 is coupled to the establishment of a long-term immune memory with at least some specificity for the eradicated cancer type.

Example 8 - IC50 values of peptide 1 against various cell lines

Methods

In vitro cytotoxicity

The MTT assay was adopted to determine cell viability in a panel of cancerous and non-transformed cells after 4 h incubation with peptide. Pre-cultured cells were seeded in 96-well plates at a density of 1 x 10⁴ - 1.5 x 10⁴ cells/well and applied for experiment. In short, cells were washed once with serum-free RPMI 1640 and incubated with increasing concentrations of peptide before adding 10 mI MTT solution to each well. Lastly, acidified isopropanol was added to facilitate formazan crystal solubilization. Absorbance was measured at 570 nm on a spectrophotometric microtiter plate reader (Thermomax Molecular Devices, NJ,

US). Cell survival was calculated as the A₅₇₀ nm of peptide-treated cells relative to the negative control (100% viable cells) using the mean of two or three independent experiments and expressed as a 50% inhibitory concentration (IC₅₀).

Hemolytic activity

The cytotoxic activity of peptides against human red blood cells (hRBCs) was determined by a hemolytic assay using freshly isolated blood from healthy individuals who gave their signed informed consent. RBCs were resuspended to a 10% hematocrit solution before being incubated for 1 h at 37°C with peptides dissolved in PBS at concentrations ranging from 438-928 mM (1500-3000 pg/ml). RBCs with PBS and 1 % Triton solution alone served as a negative and positive control, respectively. After centrifuging the samples at 4,000 rpm for 5 minutes, the absorbance of the supernatant was measured at 405 nm on a spectrophotometric microliter plate reader (Thermomax, Molecular Devices). The protocol used for blood sampling and handling has been reviewed and approved by the Regional Ethic Committee (REK)-approved protocol (2016/376).

Results

Table 2A - Peptides tested.

^*Denotes comparators

Table 2B shows IC₅₀ values of peptide 1 against various cell lines. The data represents two or more independent experiments conducted in triplicates (IC50 pM±SD). Standard concentration gradient 1 - 100 pg/ml, equalling -0.3 - 31 mM. hRBCs: human red blood cells, ND: not determined.

Table 2B - IC₅o values (mM)

Example 9 - Effect of peptides on mean arterial pressure

Test System: Male Sprague-Dawley (Charles River, Portage, Ml) rats (320- 400g) were instrumented with DSI (St. Paul, MN) telemetry transmitters by

CorDynamics, Inc.

The peptide of SEQ ID NO: 1 was stored in a freezer set to maintain approximately -20°C and protected from light. Formulations comprising SEQ ID NO:1 were prepared at dose volumes of around 0.5 mL/kg/dose in 0.9% sodium chloride for injection, U.S.P., for IV dosing. Doses were filtered through a 0.2 mM PP filter prior to injection. Test Compound Administration: Each rat was manually restrained and vehicle or SEQ ID NO: 1 dosed intravenously over approximately 1-2 minutes. Two doses were given on the same day, separated by approximately 4 hours.

The initial dosage was 0,3 mg/kg, but this was increased to 1 ,0 mg/kg for the second dosage.

Hemodynamic Assessment: Mean arterial pressure was recorded for -1 to 24 hours following the first daily dose via telemetry and reported in 10 minute averages from 1 to 24 hours following the first daily dose.

No significant difference in mean arterial pressure was seen after administration of SEQ ID NO: 1 when compared to control, indicating that no hypotensive effect is associated with this peptide. Equally, no hypertensive effect is associated with this peptide.

Example 10 - IC50 values of peptide 1 against various cell lines

Methods

Cell culture method

Adherent tumour cells were grown as a monolayer at 37°C in a humidified atmosphere (5% C0₂, 95% air) while non-adherent tumour cells were grown in suspension at 37°C in a humidified atmosphere (5% C0₂, 95% air). Cells were grown in their respective culture media. For experimental use, tumour cells were detached from the culture flasks through 5 minute treatment with trypsin (or equivalent ) in Hanks’ medium without calcium or magnesium and neutralised by addition of complete culture medium.

Cells were counted and viability was assessed using a 0.25% trypan blue exclusion assay.

Cell lines amplification

For the MTS assay the optimal density determined from historical data performed in 96-well flat-bottom microtitration plates was used. The cells were incubated at 37°C overnight before treatment in drug-free medium supplemented with FBS. Volumes for seeding were 90 pi. Before treatment, cells were washed in serum-free RPMI 1640, and fresh medium (90 mI) without serum was added.

Compounds were tested in technical triplicates in one independent experiment.

Determination of activities

At the start of treatment, 10 mI of the peptide or positive control was added to the well to reach the following final concentrations (final volume per well = 100 mΐ)^: • For peptide 106 and peptide 122 (pg/ml): 250, 83.33, 27.78, 9.26, 3.09,

1.03, 0.34, 0.11 , 0.04

• For peptide 1 (pg/ml): 100, 33.33, 1 1.1 1 , 3.70, 1.23, 0.41 , 0.14, 0.05, 0.02

• For positive control: 2% T riton X-100 final concentration

Cells were incubated in triplicate for 4 hours in a 10Opl final volume of culture medium containing peptide at 37°C under 5% C0₂.

MTS assay

The effect of the compounds on the viability of cancer cells was revealed by MTS assay using tetrazolium compound (MTS, 3-(4,5-dimethylthiazol-2-yl)-5-)3- carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and an electron coupling reagent named PMS (phenazine methosulfate). Like MTT, MTS is bioreduced by cells into a formazan product, this is directly soluble in culture medium without processing, unlike MTT.

After treatment of the cells, 20pl of a 0.22pm-filtered, freshly-combined solution of MTS (20 ml at 2mg/ml) and PMS (1 ml at 0.92 mg/ml) in Dulbecco’s Phosphate Buffered Saline (DPBS) was added to each well.

Culture plates were incubated for 1 to 4hr depending on the cell line at 37°C. Optical Density (OD) was measured at 490nm in each well using Envision microplate reader (PerkinElmer, Courtaboeuf, France).

IC₅₀ determination

The dose response inhibition of survival (IC) was calculated as follows:

IC ^_ (O D(j_rUg_exposed weiis/O D_vehicie-exposed weiis) 100. The OD values are the mean of 3 experimental measurements.

Results

Table 3 - IC50 values (pM) of peptides against various cell lines

Example 11 - IC50 values of peptides against JM1 cell line

Methods

In vitro cytotoxicity

The MTT assay was adopted to determine cell viability after 4 h incubation with peptide. Pre-cultured cells were seeded in 96-well plates at a density of 1 x 10⁴ - 1.5 x 10⁴ cells/well and applied for experiment. In short, cells were washed once with serum-free RPMI 1640 and incubated with increasing concentrations of peptide before adding 10 mI MTT solution to each well. Lastly, acidified isopropanol was added to facilitate formazan crystal solubilization. Absorbance was measured at 570 nm on a spectrophotometric microtiter plate reader (Thermomax Molecular Devices, NJ, US). Cell survival was calculated as the A₅₇₀ nm of peptide-treated cells relative to the negative control (100% viable cells) using the mean of two independent experiments and expressed as a 50% inhibitory concentration (IC₅₀).

Results

Table 4 shows IC₅₀ values of peptides against the JM1 cell line. Each peptide was tested in duplicate.

Table 4 - IC50 values of peptides against JM1 cell line

Claims

Claims

1. A peptide which:

(i) consists of 12 to 15 amino acids;

(ii) has at least 7 cationic amino acids with either

(a) a side chain comprising a guanidinium group, or

(b) a side chain comprising an amino group; and

(iii) has at least 4 tryptophans which are clustered at the N-terminus or the C-terminus of the peptide

or a peptidomimetic of said peptide, the peptide or mimetic thereof optionally in the form of a salt, ester or amide, for use in cancer treatment.

2. The peptide or peptidomimetic for use according to claim 1 , wherein the peptide has 4-6 hydrophobic amino acids, of which at least or exactly 4, 5 or 6 are tryptophan.

3. The peptide or peptidomimetic for use according to claim 2, wherein at least or exactly 3, 4, 5 or 6 of the hydrophobic residues are consecutive.

4. The peptide or peptidomimetic for use according to any one of claims 1-3, wherein the N-terminal or C-terminal residue is a hydrophobic amino acid.

5. The peptide or peptidomimetic for use according to any one of claims 1-4, wherein the peptide has 8, 9 or 10 cationic amino acids.

6. The peptide or peptidomimetic for use according to any preceding claim,

wherein 4, 5, 6, 7, 8, 9 or 10 of said cationic amino acids have a side chain comprising a guanidinium group.

7. The peptide or peptidomimetic for use according to any preceding claim,

wherein the peptide further comprises 1-4 spacer residues that are neither a cationic amino acid nor a hydrophobic amino acid.

8. The peptide or peptidomimetic for use according to any preceding claim,

wherein the peptide comprises or consists of a sequence selected from SEQ ID NOs: 1-26, wherein X is a spacer amino acid as defined in claim 7 and each h residue in SEQ ID NOs: 12 or 25 is a hydrophobic amino acid and at least 4 of said hydrophobic amino acids are W.

9. The peptide or peptidomimetic for use according to any preceding claim which consists of the amino acid sequence of SEQ ID NO: 1.

10. A peptide which:

(i) consists of 12 to 15 amino acids;

(ii) has at least 7 cationic amino acids with either

(a) a side chain comprising a guanidinium group, or

(b) a side chain comprising an amino group; and

(iii) has at least 4 tryptophans which are clustered at the N-terminus or the C-terminus of the peptide;

(iv) and wherein said peptide

(c) comprises at least one spacer residue as defined in claim 7;

(d) has at least 9 cationic amino acids as defined in part (ii);

(e) has at least 1 cationic amino acid which is not arginine;

(f) has a total of 5-6 hydrophobic amino acids; and/or

(g) wherein said at least 4 tryptophans are clustered at the C-terminus of the peptide,

said peptide optionally in the form of a salt, ester or amide

or a peptidomimetic of said peptide optionally in the form of a salt, ester or amide.

11. The peptide or peptidomimetic according to claim 10, wherein said peptide or mimetic thereof is further as defined in any one of claims 2-9.

12. A pharmaceutical composition comprising a peptide or peptidomimetic as

defined in any one of claims 10 or 1 1 and a pharmaceutically acceptable diluent, carrier or excipient.

13. The peptide or peptidomimetic according to claim 10 or 11 or the

pharmaceutical composition of claim 12 for use in therapy, preferably for use in treating cancer.

14. The peptide or peptidomimetic for use according to any one of claims 1 - 9 or 13, wherein said cancer is selected from a carcinoma, sarcoma and/or glioma.

15. The peptide or peptidomimetic for use according to any one of claims 1 to 9, 13 or 14, wherein the cancer is a deep seated tumour.

16. The peptide or peptidomimetic for use according to any one of claims 1 to 9 or 13 to 15, wherein the cancer is selected from liver cancer, colon cancer, breast cancer, bone cancer, heart cancer, lung cancer, oesophagus cancer, stomach cancer, kidney cancer, diaphragm cancer, spleen cancer, pancreas cancer, intestine cancer, gallbladder cancer, bladder cancer, ovarian cancer, prostate cancer and/or brain cancer, preferably colon cancer and/or liver cancer.

17. A method of treating cancer, which method comprises administering to a subject in need thereof a pharmaceutically effective amount of a peptide or

peptidomimetic as defined in any one of claims 1 to 1 1 or a pharmaceutical composition as defined in claim 12.

18. Use of a peptide or peptidomimetic as defined in any one of claims 1 to 11 in the manufacture of a medicament for the treatment of cancer.

19. In vitro use of a peptide or peptidomimetic as defined in any one of claims 1 to 11 for killing cancer cells.

20. The peptide or peptidomimetic as defined in any one of claims 1 to 11 and a further antitumoural agent as a combined preparation for separate,

simultaneous or sequential use in the treatment of cancer.

21. The peptide or peptidomimetic for use according to any one of claims 1 to 9 or the peptide or peptidomimetic according to claim 10 or 11 wherein the peptide has at least 4 tryptophan residues within the 6 N-terminal or 6 C-terminal residues, or the peptidomimetic equivalent thereof.