The present invention relates generally to the field of immunology. More specifically, the present invention relates to methods of modulating the immune system via the administration of certain peptides which elicit therapeutically relevant immune responses.

In the field of tumour therapy, it is becoming increasingly clear that immunotherapies (e.g. antibody therapies) generally require a T cell inflamed, or so-called "hot", tumour microenvironment in order for good efficacy to be observed. Put another way, it is becoming clear that immunotherapies of tumours having a non-T cell inflamed, or so-called "cold", tumour microenvironment are sub-optimal. Unfortunately, a significant proportion of cancer (tumour) patients have cancers that are non-T cell inflamed, i.e. a significant proportion of patients have "cold" tumours.

Thus, a challenge in this field is to provide new methods for converting

"cold" (non-T cell inflamed) tumours to "hot" (T cell inflamed) tumours so that such tumours are able to exhibit an improved therapeutic response to immunotherapy.

Regulatory T cells (Tregs), sometimes called suppressor T cells, are cells which modulate the immune system, maintain tolerance to self-antigens and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells such as cytotoxic T cells (CTLs) and T helper cells. Myeloid-Derived Suppressor Cells (MDSCs) are another type of immune suppressor cells. MDSCs are a

heterogenous group of immune cells from the myeloid lineage. Cytotoxic T cells (CTLs) are T cells that can kill damaged cells such as cancer cells or infected cells.

What is needed in the art is a new method of reducing the size of a population of regulatory T cells (Tregs) and/or of Myeloid-Derived Suppressor Cells (MDSCs), for example in a tumour microenvironement (TME). Such a method of reducing the suppressor immune cell population would facilitate accumulation of effector T cells such as CTLs and T helper cells in a tumour microenvironment. Such a method would thus enable medical practitioners to "heat-up" a tumour microenvironment, and thus render it more receptive to immunotherapies.

The present inventors have found that certain cationic amphipathic molecules are able to shape the tumour microenvironment by reducing the size of a population of Tregs and/or MDSCs, and have found that the ratio of cytotoxic T cells over Tregs is also markedly increased.

Thus, in a first aspect, the present invention provides a method of reducing the size of a population of regulatory T cells (Tregs) and/or Myeloid-Derived Suppressor Cells (MDSCs) in a subject, said method comprising administration to said subject of an effective amount of a positively charged amphipathic amino acid derivative, peptide or peptidomimetic.

The molecules of use in the methods of the invention are amphipathic in that they have a hydrophilic, i.e. cationic, part or parts and a hydrophobic part or parts.

The amino acids which may be used are derivatives as they are not naturally occurring amino acids and typically include modifications to the standard amino acid structure, e.g. a modified carboxyl group.

The molecules of use according to the invention include the group of peptides commonly known as Cationic antimicrobial peptides (CAPs). These are positively charged amphipathic peptides and peptides of this type are found in many species and form part of the innate immune system.

Each molecule of use in methods of the present invention preferably contains at least two cyclic groups. The cyclic group is preferably a 5 or 6 membered ring (although larger rings, e.g. rings of 7, 8, 9 or 10 non-hydrogen atoms, can be used) which may be aliphatic or aromatic, preferably aromatic, and may be substituted, substituting groups may include heteroatoms such as oxygen, nitrogen, sulphur or a halogen, in particular fluorine, bromine or chlorine. Preferred substituting groups include C-|-C ₄ alkyl (especially t-butyl), methoxy, fluoro and fluoromethyl groups. The cyclic group may be homo- or heterocyclic, preferably a homocyclic ring of carbon atoms. The cyclic groups may be connected or fused, preferably fused.

Single amino acid derivatives may be employed provided they have the necessary amphipathicity. They will carry at least one, preferably at least 2 positive charges and to exhibit adequate cationicity will typically have a modified C terminus, e.g. amidated or esterified, possibly with addition of a lipophilic group of 6 or more non-hydrogen atoms. A single amino acid derivative will also need to contain sufficient lipophilic group(s), e.g. a single group of 10 or more or 12 or more non-hydrogen atoms such as tri-butyl tryptophan. The amino acid may include 2 or more lipophilic groups, each of at least 6 non-hydrogen atoms. Preferred amino acid derivatives are β amino acids which are disubstituted, as described in further detail below.

Preferred peptides may consist of 2 to 25 (preferably 2 to 20 or 2 to 15, more usually 6 to 10 or 6 to 12, e.g. 8 to 10 or 8 to 1 1 or 8 to 12) amino acids and have a net positive charge at pH 7.2-7.6. More particularly, preferred peptides have a net positive charge at pH 7.4 of at least +3 or +4, preferably at least +5, usually no more than +10, preferably no more than +7 or +8. Preferably (i) 2 or more (e.g. 2 or 3 to 15 or 18, more usually 4 to 10, preferably 4 to 8) of the amino acids have a cationic side chain, and (ii) one or more (e.g. 1 or 2 to 6, preferably 3 to 5) amino acids have a lipophilic side chain, e.g. incorporating at least one cyclic group and at least 7 non-hydrogen atoms.

Peptides typically comprise one or more amino acids having a lipophilic side chain incorporating at least one cyclic group and at least 7 non-hydrogen atoms (including the cyclic group).

The cyclic group is preferably a 5 or 6 membered ring (although larger rings, e.g. rings of 7, 8, 9 or 10 non-hydrogen atoms, can be used) which may be aliphatic or aromatic, preferably aromatic, and may be substituted, substituting groups may include heteroatoms such as oxygen, nitrogen, sulphur or a halogen, in particular fluorine, bromine or chlorine. Preferred substituting groups include C-|-C ₄ alkyl (especially t-butyl), methoxy, fluoro and fluoromethyl groups. The cyclic group may be homo- or heterocyclic, preferably a homocyclic ring of carbon atoms. The cyclic groups may be connected or fused, preferably fused. Particularly preferred side- chains comprise a naphthalene or an indole group. A further preferred group of lipophilic side chains have a single substituted or unsubstituted cyclic group, preferably a phenyl or cyclohexyl group.

Preferably the peptides comprise 1 to 6, more preferably 1 to 5 or 1 to 4, e.g. 2 to 4, amino acids with such lipophilic side chains. All such amino acids and side chains thereof may conveniently be referred to as "bulky and lipophilic" amino acids/side chains. Preferably, the side chain contains at least 8, more preferably at least 10 non-hydrogen atoms. Preferred lipophilic side chains incorporate two or three cyclic groups, preferably two cyclic groups, as defined above.

Of the genetically coded amino acids phenylalanine (7 non hydrogen atoms), tryptophan (10 non hydrogen atoms) and tyrosine (8 non hydrogen atoms) are suitable bulky and lipophilic amino acids. Tryptophan, because of its two fused ring structure and additional bulk is particularly preferred. Non-genetic amino acids, which may be naturally occurring, and tryptophan, phenylalanine and tyrosine analogues and amino acids which have been modified to incorporate a lipophilic group as defined above may also be used, e.g. tryptophan residues which have been substituted at the 1-, 2-, 5- and/or 7-position of the indole ring, positions 1- or 2- being preferred e.g. 5' hydroxy tryptophan. A variety of other amino acid derivatives having a bulky and lipophilic character are known to the man skilled in the art.

Preferred non-genetically coded bulky and lipophilic amino acids include adamantylalanine; 3-benzothienylalanine; biphenylalanine, e.g. 4,4'- biphenylalanine; diphenylalanine, e.g. 3,3-diphenylalanine; a biphenylalanine derivative, e.g. Bip (4-(2-Naphthyl)), Bip (4-(1 -Naphthyl)), Bip (4-n-Bu), Bip (4-Ph) or Bip (4-T-Bu) or Phe (4-(2'-naphthyl)), Phe (4-(1 '-naphthyl)), Phe (4-n-butylphenyl), Phe (4-4'-biphenyl) or Phe (4'-t-butylphenyl); homophenylalanine; 2,6- dichlorobenzyltyrosine, cyclohexyltyrosine; 7-benzyloxytryptophan; tributyl tryptophan (Tbt), e.g. tri-tert.-butyltryptophan; homotryptophan; 3-(-anthracenyl)-L- alanine; L-p-iso-propylphenylalanine; thyroxine; 3,3',5-triiodo-L-thyronine; triiodo- tyrosine; 2-amino-3-(anthracen-9-yl)propanoic acid; 2-amino-3-(naphthalen-2- yl)propanoic acid; 2-amino-3-(naphthalen-1-yl)propanoic acid; 2-amino-3-[1 ,1 ':4',1 "- terphenyl-4-yl]-propionic acid; 2-amino-3-(2,5,7-tri-tert-butyl-1 H-indol-3-yl)propanoic acid; 2-amino-3-[1 ,1 ':3',1 "-terphenyl-4-yl]-propionic acid; 2-amino-3-[1 ,1 ':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 ,1 ':3',1 "-terphenyl-5'- yl]-propionic acid; and 2-amino-3-(4-(2,2-diphenylethyl)phenyl)propanoic acid.

Preferred peptides include at least one, e.g. 1-4, typically 1 or 2 non- genetically coded amino acids, e.g. biphenylalanine or diphenylalanine.

A lipophilic molecule is one which associates with its own kind in an aqueous solution, not necessarily because the interactions between the lipophilic molecules are stronger than between the lipophilic molecule and water but because interactions between a lipophilic molecule and water would destroy the much stronger interactions between the water molecules themselves. It is therefore preferable that the lipophilic side chain should not contain many polar functional groups e.g. no more than 4, preferably 2 or less, e.g. one or none. Such groups would increase the binding interaction with the aqueous surroundings and hence lower the lipophilicity of the molecule. The slight polarity of a side-chain like tryptophan's is tolerated and indeed, tryptophan is a preferred bulky and lipophilic amino acid.

Standard chemical protecting groups when attached to an amino acid side chain can provide suitable bulky and lipophilic side chains. Suitable amino acid protecting groups are well known in the art and include Pmc (2,2,5,7,8- pentamethylchroman-6-sulphonyl), Mtr (4-methoxy-2,3,6-trimethylbenzenesulfonyl) and Pbf (2,2,4,6,7-pentamethyldihydrobenzofuransulfonyl), which may conveniently increase the bulk and lipophilicity of aromatic amino acids, e.g. phenylalanine, tryptophan and tyrosine. Also, the tert.-butyl group is a common protecting group for a wide range of amino acids and is capable of providing a bulky and lipophilic group to amino acid side chains, particularly when modifying aromatic side chains. The Z-group (carboxybenzyl) is a further protecting group which can be used to provide a bulky and lipophilic group.

A further lipophilic group incorporating at least one cyclic group and at least

7 non-hydrogen atoms may be present as an N or C-terminal modification and the above discussion of preferred bulky and lipophilic groups applies, mutatis mutandis, to this group.

N-terminal modifications providing the further bulky and lipophilic group may be attached directly to the N-terminal amine by any convenient means to form a mono-, di- and possibly cationic trialkylated N-terminal amine. Alternatively, the further bulky and lipophilic group ("R" in the following paragraphs) may be attached via a linking moiety e.g. a carbonyl group (RCO) e.g. adamantyl or benzyl, carbamate (ROCO), or a linker which forms urea (RNHCO) or (R ₂ NCO) or by a linker which forms a sulfonamide, boronamide or phosphonamide. Sulfonamide forming linkers may be particularly useful when a more stable peptide is required.

A bulky and lipophilic group as defined above may also be provided by a C- terminal modifying group. Bulky and lipophilic groups may be attached directly to the C-terminal carboxy group to form a ketone. Alternatively, bulky and lipophilic groups may be attached via a linking moiety, e.g. (OR) which forms an ester at the C-terminus, (NH-R) or (NR ₂ , wherein the two R groups needs not be the same) which form primary and secondary amide groups respectively at the C-terminus or groups (B-(OR) ₂ ) which form boronic esters or phosphorous analogues. Dae (diaminoethyl) is a further linking moiety which may be used to attach a bulky and lipophilic group, e.g. carbobenzoxy (Z) to the C-terminus.

It will be appreciated that the number of cationic residues will likely be proportional to the length of the peptide, e.g. ½ to ¾ of the residues are cationic. Likewise, ¼ to ¾ of the residues are lipophilic (preferably with 7 or more non- hydrogen atoms).

In certain embodiments the peptide may contain 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, or 19 amino acids with a cationic side chain. For ease of reference, these amino acids will be referred to in the following sections as "cationic residues".

In further embodiments where the peptide consists of 8, 9, 10 or 1 1 amino acids, it may comprise 3 to 10, e.g. 4 to 9, 5 to 8, 6 to 7, 4 to 6 or 5 cationic residues. In still further embodiments where the peptide consists of 4, 5, 6 or 7 amino acids, it may comprise 2 to 6, e.g. 3 or 4 cationic residues.

In certain embodiments the peptide may contain 3, 4, 5, 6, 7, 8, 9, 10, 1 1 ,

12, 13, 14, 15, 16, 17 or 18 bulky and lipophilic amino acids.

In certain embodiments where the peptide consists of 16, 17, 18, 19 or 20 amino acids, it may comprise 3 to 17, 4 to 16, 5 to 15, 6 to 14, 7 to 13, 8 to 12, 9 to 1 1 or 10 bulky and lipophilic residues. In further embodiments where the peptide consists of 8, 9, 10 or 1 1 amino acids, it may comprise 3 to 8, e.g. 4 to 7 or 4 or 5, bulky and lipophilic residues. In still further embodiments where the peptide consists of 4, 5, 6 or 7 amino acids, it may comprise 2 to 5, e.g. 3 or 4 bulky and lipophilic residues.

The arrangement of the bulky and lipophilic and cationic residues and the further bulky and lipophilic group in the peptide is not of paramount importance to the functioning of the invention.

By "amino acid with a cationic side chain" it is meant an amino acid that has a side chain that has a net positive charge at the intracellular pH of a tumour cell, e.g. around pH 7.4. Of the genetically coded amino acids this would include lysine and arginine but any non-genetically coded or modified amino acid carrying such a net positive charge on its side chain may be used, e.g. those amino acids carrying a side-chain with a guanidino group or an amine group or another cationic moiety, e.g. derivatives of lysine, and arginine in which any hydrogen in the side chain, except the protonating hydrogen, is substituted with a halogen atom, e.g. fluorine, chlorine or bromine, or a linear, branched aliphatic unsaturated or saturated C1-C4 alkyl or alkoxy group, e.g. methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec- butyl, tert-butyl, ethylene, propylene, butylene, hydroxy, methoxy, ethyloxy, propyloxy, iso-propyloxy, butyloxy group, iso-butyloxy, sec-butyloxy, tert-butyloxy or halogen substituted versions thereof.

Suitable non-genetically coded amino acids with cationic side chains include homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid and homoarginine as well as trimethylysine and trimethylornithine, 4- aminopiperidine-4-carboxylic acid, 4-amino-1-carbamimidoylpiperidine-4-carboxylic acid and 4-guanidinophenylalanine.

Amino acids, with the exception of glycine, may exist as two or more stereoisomers. In particular the ocarbon of an amino acid other than glycine is a chiral centre and so gives rise to two enantiomeric forms of each amino acid.

These forms are often referred to as D and L forms, e.g. D-alanine and L-alanine. Amino acids with further chiral centres will exist in four or more possible

stereoisomers, e.g. threonine has two chiral centres and so may exist in one of four stereoisomeric forms. Any stereoisomeric form of an amino acid may be used in the molecules of the invention. For the purposes of describing the present invention, where the term "non-genetically encoded" is applied to amino acids, this does not include the D forms of amino acids that occur in nature in the L form.

Preferably, the positively charged amphipathic amino acid derivative, peptide or peptidomimetic of the present invention is further defined as set out in the sections below.

Preferably, the peptide (or peptidomimetic) consists of 9 amino acids in a linear arrangement.

Preferably, methods of the invention employ a peptide having the following characteristics:

a) consisting of 9 amino acids in a linear arrangement;

b) of those 9 amino acids, 5 are cationic and 4 have a lipophilic R

group;

c) at least one of said 9 amino acids is a non-genetically coded amino acid or a modified derivative of a genetically coded amino acid; and optionally d) the lipophilic and cationic residues are arranged such that there are no more than two of either type of residue adjacent to one another; and further optionally

e) the molecule comprises two pairs of adjacent cationic amino acids and one or two pairs of adjacent lipophilic residues, or a peptidomimetic thereof.

The cationic amino acids, which may be the same or different, are preferably lysine or arginine.

Suitable non-genetically coded cationic amino acids and modified cationic amino acids include analogues of lysine and arginine such as homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid and homoarginine as well as trimethylysine and trimethylornithine.

The lipophilic amino acids (i.e. amino acids with a lipophilic R group), which may be the same or different, all possess an R group with at least 7, preferably at least 8 or 9, more preferably at least 10 non-hydrogen atoms. An amino acid with a lipophilic R group is referred to herein as a lipophilic amino acid. Typically the lipophilic R group has at least one, preferably two cyclic groups, which may be fused or connected.

The lipophilic R group may contain hetero atoms such as O, N or S but typically there is no more than one heteroatom, preferably it is nitrogen. This R group will preferably have no more than 2 polar groups, more preferably none or one, most preferably none.

Tryptophan is a preferred lipophilic amino acid and the molecules preferably comprise 1 to 3, more preferably 2 or 3, most preferably 3 tryptophan residues. Further genetically coded lipophilic amino acids which may be incorporated are phenylalanine and tyrosine.

Preferably one of the lipophilic amino acids is a non-genetically coded amino acid. Most preferably the molecule consists of 3 genetically coded lipophilic amino acids, 5 genetically coded cationic amino acids and 1 non-genetically coded lipophilic amino acid. In this context, a D amino acid, while not strictly genetically coded, is not considered to be a "non-genetically coded amino acid", which should be structurally, not just stereospecifically, different from the 20 genetically coded L amino acids. The molecules of the invention may have some or all of the amino acids present in the D form, preferably however all amino acids are in the L form. When the molecules include a non-genetically coded lipophilic amino acid (or amino acid derivative), the R group of that amino acid preferably contains no more than 35 non-hydrogen atoms, more preferably no more than 30, most preferably no more than 25 non-hydrogen atoms.

Preferred non-genetically coded amino acids include: 2-amino-3-(biphenyl-

4-yl)propanoic acid (biphenylalanine), 2-amino-3,3-diphenylpropanoic acid

(diphenylalanine), 2-amino-3-(anthracen-9-yl)propanoic acid, 2-amino-3- (naphthalen-2-yl)propanoic acid, 2-amino-3-(naphthalen-1-yl)propanoic acid, 2- amino-3-[1 ,1 ':4',1 "-terphenyl-4-yl]-propionic acid, 2-amino-3-(2,5,7-tri-ie f-butyl-1 H- indol-3-yl)propanoic acid, 2-amino-3-[1 ,1 ':3',1 "-terphenyl-4-yl]-propionic acid, 2- amino-3-[1 ,1 ':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 , 1':3',1 "-terphenyl-5'-yl]-propionic acid and 2-amino-3-(4-(2,2- diphenylethyl)phenyl)propanoic acid.

In a preferred embodiment the peptides for use in the methods of the invention have one of formulae I to V listed below, in which C represents a cationic amino acid as defined above and L represents a lipophilic amino acid as defined above. The amino acids being covalently linked, preferably by peptide bonds resulting in a true peptide or by other linkages resulting in a peptidomimetic. The free amino or carboxy terminals of these molecules may be modified, the carboxy terminus is preferably modified to remove the negative charge, most preferably the carboxy terminus is amidated, this amide group may be substituted.

CCLLCCLLC (l) (SEQ ID NO: 1 )

LCCLLCCLC (II) (SEQ ID NO: 2)

CLLCCLLCC (III) (SEQ ID NO: 3)

CCLLCLLCC (IV) (SEQ ID NO: 4)

CLCCLLCCL (V) (SEQ ID NO: 5) β and γ 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.

As discussed above, the compounds of the invention incorporate at least one, and preferably one, non-genetically coded amino acid. When this residue is denoted L', preferred compounds are represented by the following formulae: CCL'LCCLLC (Ι') (SEQ ID NO: 6)

CCLLCCLL'C (I") (SEQ ID NO: 7)

CCLL'CCLLC (I'") (SEQ ID NO: 8)

LCCLL'CCLC (ΙΓ) (SEQ ID NO: 9)

Particularly preferred are peptides of formula I and II, and of these, peptides of formula I" are especially preferred.

The following peptides as presented in Table 1 are most preferred.

Table 1

SEQ ID NO Sequence

10 Dip-K-K-W-W-K-K-W-K-NH ₂

1 1 W-K-K-W-Dip-K-K-W-K-NH ₂

12 W-K-K-W-W-K-K-Dip-K-NH ₂

13 e/p-K-K-W-W-K-K-W-K-NH ₂

14 W-K-K-e/p-W-K-K-W-K-NH ₂

15 w-k-k-w-dip-k-k-w-k-NH ₂

16 K-K-W-Dip-K-K-W-W-K-NH ₂

17 k-k-W-Dip-k-k-W-W-k-NH ₂

18 K-K-W-Dip-K-K-W-Dip-K-NH ₂

19 K-K-W-e/p-K-K-W-W-K-N H ₂

20 Κ-β/ρ-Κ-Κ-W-W-K-K-W-N H ₂

21 Κ-Κ-β/ρ-W-K-K-W-W-K-N H ₂

22 K-K-W-W-K-K-Dip-W-K-NH ₂

23 K-K-W-W-K-K-W-Dip-K-NH ₂

24 K-W-Dip-K-K-W-W-K-K-NH ₂

25 K-K-W-W-K-W-Dip-K-K-NH ₂

26 Orn-Orn-W-Dip-Orn-Orn-W-W-Orn-NH ₂

27 Dap-Dap-W-Dip-Dap-Dap-W-W-Dap-NH ₂

28 R-R-W-Dip-R-R-W-W-R-NH ₂

29 K-W-W-K-K-Dip-W-K-K-NH ₂

30 K-Dip-K-K-W-W-K-K-W-NH ₂

31 K-K-Dip-W-K-K-W-W-K-NH ₂ 32 k-w-w-k-k-dip-w-k-k-NH ₂

33 R-R-e/p-W-R-R-W-W-R-NH ₂

34 R-R-Dip-W-R-R-W-W-R-NH ₂

35 k-k-bip-w-k-k-w-w-k-NH ₂

36 k-k-Bip-w-k-k-w-w-k-NH ₂

37 K-K-bip-W-K-K-W-W-K-NH ₂

38 Dab-Dab-W-Dip-Dab-Dab-W-W-Dab-NH ₂

39 K-K-W-1-Nal-K-K-W-W-K-NH ₂

40 K-K-W-2-Nal-K-K-W-W-K-NH ₂

41 K-K-W-Ath-K-K-W-W-K-NH ₂

42 K-K-W-Phe(4-4'Bip)-K-K-W-W-K-NH ₂

In which:

• the standard single letter code is used for the genetically coded amino acids

• lower case denotes D amino acids

• Dip is diphenylalanine

• Bip is biphenylalanine

• Orn is ornithine

• Dap is 2,3-diaminopropionic acid

• Dab is 2,4-diaminobutyric acid

• 1-Nal is 1 -naphthylalanine

• 2-Nal is 2-naphthylalanine

• Ath is 2-amino-3-(anthracen-9-yl)propanoic acid

• Phe(4,4'Bip) is 2-amino-3-[1 ,1 ':4',1 "-terphenyl-4-yl]propionic acid

All of the molecules described herein may be in salt, ester or amide form.

In some embodiments of methods of the present invention, the compound has a formula selected from the group consisting of: SEQ ID NOs: 10 and 12 to 42, or a salt, ester or amide thereof.

Especially preferred compounds for use in methods of the present invention are the peptide of SEQ ID NO:23 (K-K-W-W-K-K-W-Dip-K-NH ₂ ) or a peptidomimetic thereof.

The molecules are preferably peptides and preferably have a modified, particularly an amidated, C-terminus. Amidated peptides may themselves be in salt form and acetate forms are preferred. Suitable physiologically acceptable salts are well known in the art and include salts of inorganic or organic acids, and include trifluoracetate as well as acetate and salts formed with HCI.

In some embodiments, peptides or peptidomimetics, such as those having preferred peptide sequences described herein, are not modified at the C-terminus or have a C-terminal modification other than amidation.

The molecules described herein are amphipathic in nature, their 2° structure, which may or may not tend towards the formation of an a-helix, provides an amphipathic molecule in physiological conditions.

As described in patent publication WO201 1/051692, the peptide,

peptidomimetic or amino acid derivative may have a net positive charge of at least +2 and incorporate a disubstituted β amino acid, each of the substituting groups in the β amino acid, which may be the same or different, comprises at least 7 non-hydrogen atoms, is lipophilic and has at least one cyclic group, one or more cyclic groups within a substituting group may be linked or fused to one or more cyclic groups within the other substituting group and where cyclic groups are fused in this way the combined total number of non-hydrogen atoms for the two substituting groups is at least 12. The 2 substituting groups on the β amino acid are preferably the same.

Lipophilicity can be measured by a molecule's distribution in a biphasic system, e.g. liquid-liquid such as 1-octanol/water. It is well known in the art that polar substituents such as hydroxy, carboxy, carbonyl, amino and ethers decrease the partition coefficient in a biphasic system such as 1-octanol/water as they reduce lipophilicity; the lipophilic substituting groups will therefore preferably contain no more than two, more preferably one or no such polar groups.

A β amino acid has the amino group attached to the β carbon atom;

genetically coded amino acids are a amino acids in which the amino group is attached to the a carbon atom. This arrangement lengthens by one atom per β amino acid the backbone of a peptide incorporating one or more β amino acids. In this arrangement the a and/or the β carbon atom can be substituted. The a or β carbon atom may be disubstituted; where the a carbon atom is disubstituted a β ^2,2 amino acid results and where the β carbon atom is disubstituted a β ^3,3 amino acid is generated. One substituting group on each of the a or β carbon atoms results in a β ^2,3 amino acid, β ^2,2 and β ^3,3 disubstituted amino acids are preferred, β ^2,2 disubstituted amino acids being especially preferred.

The β amino acid is substituted by two groups incorporating at least 7 non-hydrogen atoms. Preferably one, more preferably both of the substituting groups contains at least 8, more preferably at least 10 non-hydrogen atoms. These groups are lipophilic in nature and while they may be different, are preferably the same. Each contains at least one cyclic group, typically a 6 membered ring which may be aliphatic or aromatic, preferably aromatic, and may be substituted, substituting groups may include hetero atoms such as oxygen, nitrogen, sulphur or a halogen, in particular fluorine or chlorine. Preferred substituting groups include C-|-C ₄ alkyl (especially t-butyl), methoxy, fluoro and fluoromethyl groups. The cyclic groups may be homo- or heterocyclic, preferably they are homocyclic ring of carbon atoms. Preferred lipophilic substituting groups incorporate two or three cyclic groups, preferably two cyclic groups, which may be connected or fused, preferably fused. Particularly preferred substituting groups comprise a naphthalene group.

A further preferred group of lipophilic substituting groups have a single substituted or unsubstituted cyclic group, preferably a phenyl or cyclohexyl group.

The cyclic group or groups is typically spaced away from the peptide backbone (i.e. from the a or β carbon atom of the β amino acid) by a chain of 1 to 4, preferably 1 to 3 atoms; these linking atoms may include nitrogen and/or oxygen but will typically be carbon atoms, preferably the linking atoms are unsubstituted. These spacers are of course part of the substituting groups as defined herein.

Each substituting moiety of the disubstituted β amino acid will typically comprise 7 to 20 non-hydrogen atoms, preferably 7 to 13, more preferably 8 to 12, most preferably 9-1 1 non-hydrogen atoms.

These molecules will preferably be peptides or peptidomimetics of 1 or 2 to 12 amino acids or equivalent subunits in length. Unless otherwise clear from the context, reference herein to 'amino acids' includes the equivalent subunit in a peptidomimetic. The preferred molecules have either 1 to 3 or 4 amino acids, but alternatively may be 3 to 12, preferably 5 to 12 amino acids in length. Molecules of use according to the invention may only comprise a single amino acid but this will be a 'modified' amino acid in order to fulfil the requirements for charge.

Single amino acids as well as peptides and peptidomimetics will preferably incorporate a modified C terminus, the C terminal modifying group typically resulting in charge reversal, i.e. removing the negative charge of the carboxyl group and adding a positive charge, e.g. through the presence of an amino group. This modification alone, assuming the N terminus is not modified, will give the molecule overall a net charge of +2. Whether the C terminus is modified to give charge reversal or simply to remove the negative charge of the carboxyl group, the molecule preferably also contains one or more cationic amino acids. Thus the overall charge of the molecule may be +3, +4 or higher for larger molecules.

Suitable C-terminal groups, which are preferably cationic in nature, will typically have a maximum size of 15 non-hydrogen atoms. The C-terminus is preferably amidated and the amide group may be further substituted to form an N-alkyl or Ν,Ν-dialkyl amide. Primary and secondary amide groups are preferred. Suitable groups to substitute the amide group include aminoalkyl, e.g. amino ethyl or dimethylaminoethyl; the nitrogen atom of the amide group may form part of a cyclic group e.g. pyrazolidine, piperidine, imidazolidine and piperazine, piperazine being preferred, these cyclic groups may themselves be substituted, for example by alkyl or aminoalkyl groups.

Peptides preferably incorporate one or more cationic amino acids, lysine and arginine are preferred, but non-genetically coded or modified amino acids may also be incorporated (as described elsewhere herein).

Suitable non-genetically coded cationic amino acids and modified cationic amino acids include analogues of lysine, arginine and histidine such as homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid and homoarginine as well as trimethylysine and trimethylornithine, 4-aminopiperidine-4- carboxylic acid, 4-amino-1-carbamimidoylpiperidine-4-carboxylic acid and 4- guanidinophenylalanine.

Dipeptides will typically incorporate one cationic amino acid and longer peptides will usually incorporate additional cationic amino acids, thus a peptide of 4 or 5 amino acids may have 2 or 3 cationic amino acids and peptides of 6 to 9 amino acids may have 3 to 6 cationic amino acids.

A preferred group of molecules comprise a β ^2,2 disubstituted amino acid coupled to a C-terminal L-arginine amide residue and dipeptides having this arrangement are particularly preferred.

Peptides with three or more amino acids will typically have one or more additional lipophilic amino acids, i.e. amino acids with a lipophilic R group. Typically the lipophilic R group has at least one, preferably two cyclic groups, which may be fused or connected. The lipophilic R group may contain hetero atoms such as O, N or S but typically there is no more than one heteroatom, preferably it is nitrogen. This R group will preferably have no more than 2 polar groups, more preferably none or one, most preferably none.

Tryptophan is a preferred lipophilic amino acid and peptides preferably comprise 1 to 3 tryptophan residues. Further genetically coded lipophilic amino acids which may be incorporated are phenylalanine and tyrosine.

The lipophilic amino acids may be non-genetically coded, including genetically coded amino acids with modified R groups.

Especially preferred peptides, peptidomimetics or (modified) amino acids have a net positive charge of at least +2 and incorporate a group of formula I:

wherein any 2 from R-i, R ₂ , R3 and R ₄ are hydrogen atoms and 2 are substituting groups, which may be the same or different, comprise at least 7 non-hydrogen atoms, are lipophilic and include a cyclic group, said cyclic group not being attached directly either to the a or β carbon atom but optionally being linked or fused to a cyclic group in the other substituting group, where cyclic groups are fused the combined total number of non-hydrogen atoms for the two substituting groups is at least 12, and wherein X represents O, C, N or S.

It will be appreciated that the minimum figure of 12 for the combined total of non-hydrogen atoms in the two groups of Ri _-4 when the cyclic groups of each moiety are fused is arrived at by adding the minimum number for the unfused groups (7+7 = 14) and subtracting 2 because two of the non-hydrogen atoms effectively participate in ring formation in each group. Preferably the combined total of non-hydrogen atoms in the two groups of Ri _-4 when the cyclic groups of each moiety are fused is 14. Complex fused and linked groups can be envisaged where the two groups attached to the C ^a or C ^p may contain more than one pair of fused cyclic groups, with or without additional linking bonds between the substituting groups. Nevertheless, the two substituting groups are preferably not fused or linked as molecules in which these groups have greatest flexibility of movement are preferred. The nitrogen atom in the group of formula (I) is preferably not bound to any atom of groups R _1-4 , except, of course, indirectly through C ^p or C ^a . Preferably the 5 atoms in the above backbone (N- C ^p - C ^a -C-X) are connected to each other only in a linear, not cyclic, fashion. It will be appreciated that X and N in formula (I) have their normal valencies and thus will typically be further substituted as they are bound to other parts of the compound, e.g. further amino acids or N- or C- terminal capping groups.

The substituting groups of R _1-4 are generally lipophilic in nature and preferably carry no charge and preferably have no more than two, more preferably no more than one polar group. One or both of the substituting groups of R _1-4 preferably contain at least 8, more preferably at least 9 or 10 non-hydrogen atoms, e.g. 7-13, 7-12, 8-12 or 9-1 1 non-hydrogen atoms. These two substituting groups are preferably the same, if only for ease of synthesis. Preferably the two substituting groups are R-i and R ₂ or R ₃ and R ₄ , R ₃ and R ₄ being most preferred.

As stated above, the cyclic groups of R _1-4 are not attached directly to either the a or β carbon atom because they are spaced therefrom by a chain of 1 to 4, preferably 1 to 3 atoms; these linking atoms may include nitrogen and/or oxygen but will typically be carbon atoms, preferably the linking atoms are unsubstituted. X may be substituted or unsubstituted and is preferably a N atom and preferably substituted. When X is N it may form part of an amide bond with a further amino acid. Alternatively, the N atom may be substituted, for example by an aminoalkyl group, e.g. aminoethyl or aminopropyl or dimethylaminoethyl. In a further alternative the N atom may form part of a cyclic group such as piperazine, which may itself be substituted by alkyl or aminoalkyl groups.

The peptides or peptidomimetics incorporating a group of formula I will preferably have a modified C terminus, which is preferably amidated and is described above.

Previous passages defining preferred substituting groups of the β amino acid apply, mutatis mutandis, to the two substituting groups of R _1-4 . The peptides and peptidomimetics incorporating a group of formula I are a preferred sub-set of the molecules described earlier in this application and so all previous passages defining preferred characteristics of the molecules, for example their length and the other amino acids they contain, apply also to these molecules defined by their incorporation of a group of formula I, and vice versa. Particularly preferred molecules are 1 to 7 or 8 (e.g. 1 to 5), more preferably 1 , 2, 3 or 4 amino acids in length. Peptidomimetic molecules will include the same number of subunits but these subunits will typically be linked by amide bond mimics; preferred linkages are discussed above and include esters and aminomethyl and ketomethylene.

The peptides, peptidomimetics and amino acids of the invention may be in salt form, cyclic or esterified, as well as the preferred amidated derivatives discussed above.

A preferred class of molecules are β, preferably p ^2,2 -amino acid derivatives which have a single p ^2,2 -amino acid incorporating two lipophilic side chains as defined above, the di-substituted β-amino acid being flanked by two cationic groups. As described previously, the two substituting groups are preferably the same, include a 6 membered cyclic group and at least 8, preferably at least 10 non-hydrogen atoms.

Preferably, the molecule is LTX-401 . LTX-401 has the following structure:

The molecules for use in the methods of the invention may be in the form of a peptidomimetic. A peptidomimetic is typically characterised by retaining the polarity, three dimensional size and functionality (bioactivity) of its peptide equivalent but wherein 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, 1 12, 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 may have a number sub-units which are approximately equivalent in size and function to the sub-units of an equivalent peptide. The term 'amino acid' may thus conveniently be used herein to refer to the equivalent sub-units of a peptidomimetic compound. Moreover, peptidomimetics may have groups equivalent to the R groups of amino acids and discussion herein of suitable R groups and of N and C terminal modifying groups applies, mutatis mutandis, to peptidomimetic compounds.

As is discussed in "Drug Design and Development", Krogsgaard et al.,

1996, 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 , 1 1 138-1 1 142. 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.

Peptidomimetic equivalents of all peptides described as preferred are also preferred.

The molecules described herein may be synthesised in any convenient way.

Generally the reactive groups present (for example amino, thiol and/or carboxyl) will be protected during overall synthesis. The final step in the synthesis will thus be the deprotection of a protected derivative of the invention.

In building up the peptide, one can in principle start either at the C-terminal or the N-terminal although the C-terminal starting procedure is preferred.

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, such supports being well known in the art.

A wide choice of protecting groups for amino acids are known and suitable amine protecting groups may include carbobenzyloxy (also designated Z) t- butoxycarbonyl (also designated Boc), 4-methoxy-2,3,6-trimethylbenzene sulphonyl (Mtr) and 9-fluorenylmethoxy-carbonyl (also designated Fmoc). It will be

appreciated that when the 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.

Carboxyl protecting groups which may, for example be employed include readily cleaved ester groups such as benzyl (Bzl), p-nitrobenzyl (ONb), or t-butyl (OtBu) groups as well as the coupling groups on solid supports, for example the Rink amide linked to polystyrene.

Thiol protecting groups include p-methoxybenzyl (Mob), trityl (Trt) and acetamidomethyl (Acm).

Preferred peptides of the invention may conveniently be prepared using the t-butyloxycarbonyl (Boc) protecting group for the amine side chains of Lys, Orn, Dab and Dap as well as for protection of the indole nitrogen of the tryptophan residues. Fmoc can be used for protection of the alpha-amino groups. For peptides containing Arg, 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl can be used for protection of the guanidine side chain.

A wide range of procedures exists for removing amine- and carboxyl- 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 a-amino protecting group prior to the next coupling step.

Amine protecting groups such as Boc and carboxyl protecting groups such as tBu may be removed simultaneously by acid treatment, for example with trifluoroacetic acid. Thiol protecting groups such as Trt may be removed selectively using an oxidation agent such as iodine.

References and techniques for synthesising peptidomimetic compounds are provided above and known to the skilled man.

Typically, the molecules used in accordance with the present invention are able to lyse tumour cell membranes. Typically, molecules used in accordance with the present invention exert a cytotoxic effect against tumour cells through a direct membrane-affecting mechanism and thus may be termed tumour membrane acting agents. These compounds may be lytic, destabilising or even perforating the cell membrane. This may offer a distinct therapeutic advantage over agents which act on or interact with proteinaceous components of the target cells, e.g. cell surface receptors. While mutations may result in new forms of the target proteins leading to resistance to agents, it is much less likely that radical changes to the lipid membranes could occur to prevent the cytotoxic effect. Without wishing to be bound by theory it is believed that molecules used in accordance with the invention are attracted to the negatively charged phospholipids of the tumour cell membrane by virtue of the presence of the cationic groups, and their lipophilic groups are able to destabilise the normal three dimensional lipid bi-layer configuration of cell membranes. This interaction may increase permeability and result in a loss of membrane integrity and eventually cell death.

As outlined above, the present invention provides a method of reducing the size of a population of regulatory T cells (Tregs) and/or Myeloid-Derived

Suppressor Cells (MDSCs) in a subject.

In some embodiments, the size of a population of regulatory T cells (Tregs) is reduced.

In some embodiments, the size of a population of Myeloid-Derived

Suppressor Cells (MDSCs) is reduced.

In some embodiments, the size of a population of regulatory T cells (Tregs) and Myeloid-Derived Suppressor Cells (MDSCs) is reduced. Regulatory T cells (Tregs) have a characteristic marker profile and can be readily identified, and their population size (e.g. number of Tregs) can be readily quantified, by a person skilled in the art (e.g. using flow cytometry methods and reagents such as those described in the Example section herein). Tregs typically express the biomarkers CD3, CD4, CD25, and FoxP3. Thus, Tregs in accordance with the present invention are typically CD3 ⁺ CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T cells (or CD3 ⁺ CD4 ⁺ CD25 ⁺ FoxP3 ⁺ tumour infiltrating lymphocytes, TILs).

MDSCs have a characteristic marker profile and can be readily identified, and their population size (e.g. number of MDSCs) can be readily quantified, by a person skilled in the art (e.g. using flow cytometry methods and reagents such as those described in the Example section herein). MDSCs typically express the biomarker CD1 1 b and have low expression of the biomarker Ly6c. Thus, MDSCs in accordance with the present invention are typically CD1 1 b ⁺ , Ly6c ^low cells (i.e. polymorphonuclear MDSCs).

The "reduction" in the size of a Treg and/or a MDSC population (number of such cells) in a subject is typically a reduction as compared to the size of the population of such cells in the subject at an earlier time point, for example prior to the administration of a positively charged amphipathic amino acid derivative, peptide or peptidomimetic as defined elsewhere herein, or as compared to the size of the population of such cells in a subject at an earlier time point (or stage) in the treatment regimen. Thus, alternatively viewed, the "reduction" in the size of a Treg and/or MDSC population may be a reduction in comparison with the size of a control Treg and/or MDSC population, wherein the control Treg and/or MDSC population size is the population size of such cells at an earlier time point, for example prior to administration to the subject of the positively charged amphipathic amino acid derivative, peptide or peptidomimetic, or at an earlier time point (or stage) in the treatment regimen of the subject. Alternatively viewed, the "reduction" in the size of a Treg and/or MDSC population may be a reduction in comparison with a "baseline" population at an earlier time point in that subject.

A reduction in the size of a Treg and/or MDSC population (number of such cells) in a subject can be readily observed and assessed by a person skilled in the art, for example by analysing (for example by flow cytometry) a sample (or samples) that have been obtained from a subject for the presence or absence of Tregs and/or MDSCs (e.g. based on the marker profiles described elsewhere herein) and quantifying the population size of such cells. As described elsewhere herein, the size of a Treg and/or MDSC population can be compared with the size of the population of such cells in the subject at an earlier time point to establish whether or not there has been a reduction in the population size. Suitable methods are described in the Example section herein.

The "reduction" in the size of a Treg and/or of a MDSC population (number of such cells) in a subject as described herein includes any measurable reduction (or decrease). Preferably, the population size is significantly reduced, e.g. as compared to the population size observed in the subject at an earlier time point (as discussed elsewhere herein). More preferably, the significantly decreased population size is statistically significant, preferably with a p-value of <0.05, more preferably <0.01 .

In some embodiments, the reduction in the size of a Treg and/or MDSC population (number of such cells) in a subject (e.g. population in a tumour microenvironment) is a reduction of at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30% or at least 40%, e.g. in comparison to the population size observed in the subject at an earlier time point. The reduction may, for example, be up to about 40% or up to about 50%.

In some embodiments, the reduction in the size of a Treg population (number of such cells) in a subject (e.g. population in a tumour microenvironment) is a reduction of at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30% or at least 40%, e.g. in comparison to the population size observed in the subject at an earlier time point. The reduction may, for example, be up to about 40% or up to about 50%.

In some embodiments, the method of the present invention results in a population of Tregs that represents less than 8%, less than 7.5%, less that 7% or less than 6.5% of the total CD3 ⁺ CD4 ⁺ cell population (e.g. population in a tumour microenvironment). For example, in some embodiments, the method of the present invention results in a population of Tregs that represents 5-8%, 5-7%, 6-8% or 6-7% of the total CD3 ⁺ CD4 ⁺ cell population (e.g. population in a tumour

microenvironment).

In some embodiments, the reduction in the size of a MDSC population (number of such cells) in a subject is a reduction of at least 2%, at least 5%, at least 10%, at least 15% or at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 65%, e.g. in comparison to the population size observed in the subject at an earlier time point. The reduction may, for example, be up to about 60% or up to about 65% or up to about 70%.

In some embodiments, the method of the present invention results in a population of MDSCs that represents less than 20%, less than 18%, less that 15%, or less than 12% of the total CD1 1 b ⁺ cell population (e.g. population in a tumour microenvironment). For example, in some embodiments, the method of the present invention results in a population of MDSCs that represents 10-20%, 10-15%, 10- 18%, 12-20%, 12-15%, 12-18%, 15-20% or 15-18% of the total CD1 1 b ⁺ cell population (e.g. population in a tumour microenvironment).

Typically, the positively charged amphipathic amino acid derivatives, peptides or peptidomimetics in accordance with the present invention act in (or have an effect on) a tumour microenvironment (or tumour bed) in a subject. Thus, typically the population of regulatory T cells (Tregs) and/or Myeloid-Derived

Suppressor Cells (MDSCs) is the population in a tumour microenvironment (or tumour bed) in said subject.

Thus, the present invention provides a method of reducing the size of a population of regulatory T cells (Tregs) and/or Myeloid-Derived Suppressor Cells (MDSCs) in a tumour microenvironment (or tumour bed) in a subject. A tumour microenvironment may be defined as the cellular environment in which the tumor exists, typically including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix (ECM). A person skilled in the art is readily able to assess whether or not there is a reduction in the size of a population of regulatory T cells (Tregs) and/or Myeloid-Derived Suppressor Cells (MDSCs) in a tumour

microenvironment (or tumour bed) in a subject, for example by analysing and quantifying the population size of such cells (e.g. using flow cytometry) in a tumour sample (or samples) or tumour microenvironment sample (or samples) that has been obtained from the subject (e.g. via biopsy such as tumour biopsy).

Alternatively viewed, the present invention provides a method of reducing the size of a population of regulatory T cells (Tregs) and/or Myeloid-Derived Suppressor Cells (MDSCs) in a tumour (or tumour tissue) in a subject.

Alternatively viewed, the present invention provides a method of reducing the size of a population of regulatory T cells (Tregs) and/or Myeloid-Derived Suppressor Cells (MDSCs) that have infiltrated a tumour or have accumulated in a tumour microenvironment (or a tumour bed or tumour tissue) in a subject.

Alternatively viewed, the present invention provides a method of decreasing the local abundance of regulatory T cells (Tregs) and/or Myeloid-Derived

Suppressor Cells (MDSCs) in a tumour microenvironment (or a tumour bed or tumour tissue) in a subject.

Alternatively viewed, the present invention provides a method of depleting regulatory T cells (Tregs) and/or Myeloid-Derived Suppressor Cells (MDSCs) in a tumour microenvironment (or a tumour bed or tumour tissue) in a subject.

Alternatively viewed, the present invention provides a method of reducing the size of an intratumoural population of regulatory T cells (Tregs) and/or Myeloid- Derived Suppressor Cells (MDSCs) in a subject.

A reduction in the size of a Treg and/or a MDSC population in a subject, typically in a tumour microenvironment, may be observed at any time after the administration of a positively charged amphipathic amino acid derivative, peptide or peptidomimetic in accordance with the present invention. For example, in some embodiments, a reduction is observed at about seven days after administration (e.g. after first administration).

Typically, in methods of the present invention, there is an increase in the ratio of cytotoxic T cells (cytotoxic T lymphocytes, CTLs) over Tregs (e.g. CD3 ⁺ CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Tregs). Preferably, there is an increase in the ratio of CTLs over Tregs in a tumour microenvironment (or tumour bed or tumour or tumour tissue). Thus, preferably, the ratio is the intratumoural ratio.

Alternatively viewed, the population size of CTLs is typically increased relative to the population size of Tregs.

Cytotoxic T cells have a characteristic marker profile and can be readily identified, and their population size (e.g. number of CTLs) can be readily quantified, by a person skilled in the art (e.g. using flow cytometry methods and reagents such as those described in the Example section herein). Cytotoxic T cells are sometimes referred to as CD8 ⁺ T cells, T _c , or cytotoxic T lymphocytes (CTLs). CTLs typically express the biomarkers CD3 and CD8 and may express the cytokines IFNy and/or TNFa. Thus, CTLs in accordance with the present invention are typically CD3 ⁺ CD8 ⁺ CTLs (or CD3 ⁺ CD8 ⁺ tumour infiltrating lymphocytes, TILs). CTLs in accordance with the present invention include CD3 ⁺ CD8 ⁺ IFNv ⁺ T cells (or CD3 ⁺ CD8 ⁺ IFNy ⁺ tumour infiltrating lymphocytes, TILs), and CD3 ⁺ CD8 ⁺ TNFa ⁺ T cells (or CD3 ⁺ CD8 ⁺ TNFa ⁺ tumour infiltrating lymphocytes, TILs), and CD3 ⁺ CD8 ⁺ IFNv ⁺ TNFa ⁺ T cells (or CD3 ⁺ CD8 ⁺ IFNv ⁺ TNFa ⁺ tumour infiltrating lymphocytes, TILs). CD3 ⁺ CD8 ⁺ IFNv ⁺ TNFa ⁺ T cells may be referred to as polyfunctional CTLs. CTLs also typically express CD45.

In some embodiments, there is an increase in the ratio of CD8 ⁺ IFNv ⁺ T cells over Tregs (such as CD3 ⁺ CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Tregs). In some embodiments, there is an increase in the ratio of CD8 ⁺ TNFa ⁺ T cells over Tregs (such as CD3 ⁺ CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Tregs). In some embodiments, there is an increase in the ratio of CD8 ⁺ IFNy ⁺ TNFa ⁺ T cells over Tregs (such as CD3 ⁺ CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Tregs).

The "increase" in the ratio of CTLs over Tregs in a subject (e.g. in a tumour microenvironment) is typically an increase as compared to the ratio in the subject at an earlier time point, for example prior to the administration of a positively charged amphipathic amino acid derivative, peptide or peptidomimetic as defined elsewhere herein, or as compared to the ratio in a subject at an earlier time point (or stage) in the treatment regimen. Thus, alternatively viewed, the "increase" in the ratio may be an increase in comparison with a control ratio of CTLs over Tregs, wherein the control ratio is the ratio of CTLs over Tregs at an earlier time point, for example prior to administration to the subject of the positively charged amphipathic amino acid derivative, peptide or peptidomimetic, or at an earlier time point (or stage) in the treatment regimen of the subject. Alternatively viewed, the "increase" in the ratio of CTLs over Tregs may be an increase in comparison with a "baseline" ratio in that subject (e.g. in a tumour microenvironment) at an earlier time point.

An increase in the ratio of CTLs over Tregs in a subject (e.g. in a tumour microenvironment) can be readily determined (or assessed) by a person skilled in the art, for example by analysing (for example by flow cytometry) a sample (or samples) that have been obtained from a subject for the presence or absence of a CTL population and a Treg population (e.g. based on the marker profiles described elsewhere herein), quantifying the population sizes of such cells and deriving a ratio of the two different population sizes (CTL/Treg ratio; CTL:Treg ratio). As described elsewhere herein, the ratio can be compared with the ratio in the subject at an earlier time point to establish whether or not there has been an increase in the ratio. Suitable methods are described in the Example section herein.

The "increase" in the ratio of CTLs over Tregs (e.g. CD3 ⁺ CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Tregs) in a subject (e.g. in a tumour microenvironment) as described herein includes any measurable increase. Preferably, the ratio is significantly increased, e.g. as compared to the ratio in the subject at an earlier time point (as discussed elsewhere herein). More preferably, the significantly increased ratio is statistically significant, preferably with a p-value of <0.05, more preferably <0.01 or <0.001.

In some embodiments, the increase in the ratio (e.g. intratumoural ratio) of CTLs over Tregs (e.g. CD3 ⁺ CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Tregs) in a subject (e.g. in a tumour microenvironment) is an increase of at least 2%, at least 5%, at least 10%, at least 25%, at least 50%, at least 100%, at least 200%, at least 300% or at least 400%, at least 500%, at least 1000%, at least 2000%, at least 3000%, at least

4000% or at least 5000% e.g. in comparison to the ratio observed in the subject at an earlier time point. The increase may, for example, be up to about 5000% or up to about 6000%.

In some embodiments, the increase in the ratio (e.g. intratumoural ratio) of CD8 ⁺ IFNy ⁺ T cells (CTLs) over Tregs (e.g. CD3 ⁺ CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Tregs) in a subject (e.g. in a tumour microenvironment) is an increase of at least 2%, at least 5%, at least 10%, at least 25%, at least 50%, at least 100%, at least 200%, at least 300% or at least 400%, at least 500%, at least 1000%, at least 1250% or at least 1800%, e.g. in comparison to the ratio observed in the subject at an earlier time point. The increase may, for example, be up to about 1250%, up to about 1800% or up to about 2000%.

In some embodiments, the increase in the ratio (e.g. intratumoural ratio) of CD8 ⁺ TNFa ⁺ T cells (CTLs) over Tregs (e.g. CD3 ⁺ CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Tregs) in a subject (e.g. in a tumour microenvironment) is an increase of at least 2%, at least 5%, at least 10%, at least 25%, at least 50%, at least 100%, at least 200%, at least 300% or at least 400%, at least 500%, at least 1000%, at least 2000%, at least 3000%, at least 4000% or at least 5000% e.g. in comparison to the ratio observed in the subject at an earlier time point. The increase may, for example, be up to about 5000% or up to about 6000%.

In some embodiments, the increase in the ratio (e.g. intratumoural ratio) of

CD8 ⁺ IFNy ⁺ TNFa ⁺ T cells (CTLs) over Tregs (e.g. CD3 ⁺ CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Tregs) in a subject (e.g. in a tumour microenvironment) is an increase of at least 2%, at least 5%, at least 10%, at least 25%, at least 50%, at least 100%, at least 200%, at least 300% or at least 400%, at least 500%, at least 1000%, at least 2000%, at least 3000%, at least 4000%, e.g. in comparison to the ratio observed in the subject at an earlier time point. The increase may, for example, be up to about 4000% or up to about 5000%.

The increase in the ratio of CTLs over Tregs in a subject is typically achieved (or observed) in a tumour microenvironment (or tumour bed or tumour tissue or tumour) in a subject. A person skilled in the art is readily able to assess whether or not there is an increase in the ratio of CTLs over Tregs in a tumour microenvironment (or tumour bed) in a subject, for example by analysing and quantifying the population sizes CTLs and Tregs (e.g. using flow cytometry) in a tumour sample (or samples) or tumour microenvironment sample (or samples) that has been obtained from the subject (e.g. via biopsy such as tumour biopsy).

An increase in the ratio of CTLs over Tregs in a subject, typically in a tumour microenvironment, may be observed at any time after the administration of a positively charged amphipathic amino acid derivative, peptide or peptidomimetic in accordance with the present invention. For example, in some embodiments, an increase is observed at about seven days after administration (e.g. after first administration).

The presence or absence, and quantity, of cells (e.g. in a sample, such as an intratumoural sample, obtained from a subject) having marker profiles of the various cell types described herein can be readily determined by a person skilled in the art using routine techniques and reagents, for example via flow cytometry of cells labelled with appropriate antibodies (e.g. antibodies which bind to the various markers described herein). Suitable reagents and suitable flow cytometry methodology (including suitable apparatus and software) is described in the Example section herein. Samples are typically processed before analysis and suitable processing methods are described in the Examples herein.

In some embodiments, methods of the present invention may further comprise a step (or steps) of determining the size of the population (or relative size of a population) of one or more of the cell types described herein, for example Tregs, MDSCs and/or cytotoxic T cells. Preferably, this is done by flow cytometry. This step may be done one or more times, for example prior to administration of a positively charged amphipathic amino acid derivative, peptide or peptidomimetic as defined herein and/or during a treatment regimen which includes administration of a positively charged amphipathic amino acid derivative, peptide or peptidomimetic as defined herein and/or after a treatment regimen which includes administration of a positively charged amphipathic amino acid derivative, peptide or peptidomimetic as defined herein. This determination will typically be performed on a sample taken from the subject, e.g. a tumour sample or a sample of the tumour

microenvironment.

An effective amount will be one able to generate a reduction in the size of a population of Tregs or MDSCs as defined above, which can be readily determined as described herein. An effective amount will typically be an amount sufficient to cause loss of membrane integrity in the tumour cells in the subject.

Alternatively viewed, the present invention provides a positively charged amphipathic amino acid derivative, peptide or peptidomimetic for use in reducing the size of a population of regulatory T cells (Tregs) and/or Myeloid-Derived Suppressor Cells (MDSCs) in a subject (e.g. in a tumour microenvironment, tumour bed, tumour or tumour tissue). Other features and properties of other aspects of the invention apply, mutatis mutandis, to this aspect of the invention.

Alternatively viewed, the present invention provides the use of a positively charged amphipathic amino acid derivative, peptide or peptidomimetic in the manufacture of a medicament for reducing the size of a population of regulatory T cells (Tregs) and/or Myeloid-Derived Suppressor Cells (MDSCs) in a subject (e.g. in a tumour microenvironment, tumour bed, tumour or tumour tissue). Other features and properties of other aspects of the invention apply, mutatis mutandis, to this aspect of the invention.

Alternatively viewed, the present invention provides the use of a positively charged amphipathic amino acid derivative, peptide or peptidomimetic for reducing the size of a population of regulatory T cells (Tregs) and/or Myeloid-Derived

Suppressor Cells (MDSCs) in a subject (e.g. in a tumour microenvironment, tumour bed, tumour or tumour tissue). Other features and properties of other aspects of the invention apply, mutatis mutandis, to this aspect of the invention.

The subject will typically be a human patient but non-human animals, such as domestic or livestock animals may also be treated and laboratory or test animals (e.g. mice) may be treated.

The subject will typically have been identified as likely to benefit from administration of the compounds defined herein. In particular, the subject has preferably been identified as suffering from cancer, typically possessing one or more solid tumours, said solid tumours usually being classified as "cold" on the basis of low levels of infiltration of the tumours by CTLs, e.g. as assessed by the "Immunoscore" approach (Galon et al., 2016, J. Transl. Med., 14: 273). In this way the immune status of the tumour is determined. Thus, the methods of the present invention may include a step prior to the administration described herein wherein the immune status of a solid tumour within said subject is determined, e.g. by taking a sample of the tumour or tumour microenvironment and analysing T cell populations therein. It being understood that "cold" tumours are most likely to benefit from the methods and treatments described herein.

The administered molecule may be presented, for example, in a form suitable for oral, topical, nasal, parenteral, intravenal, intratumoural, rectal or regional (e.g. isolated limb perfusion) administration. Administration is typically by a parenteral route, preferably by injection subcutaneously, intramuscularly, intracapsularly, intraspinaly, intratumouraly or intravenously. Intratumoural administration is preferred (e.g. by injection).

In preferred embodiments of methods of the present invention, molecules are administered to (or delivered to or targeted to or locally administered to) a tumour (e.g. a solid tumour), preferably an established tumour. This administration is typically, and preferably, via intratumoural delivery (e.g. by intratumoural injection), but other administration routes may be used.

Preferred cancer (or tumour) targets are lymphomas, leukaemias, neuroblastomas and glioblastomas (e.g. from the brain), carcinomas and adenocarcinomas (particularly from the breast, colon, kidney, liver, lung, ovary, pancreas, prostate and skin), melanomas and sarcomas. Typically, solid tumour targets are preferred. In some embodiments, sarcomas are preferred cancer targets.

Preferably, a positively charged amphipathic amino acid derivative, peptide or peptidomimetic (as defined elsewhere herein) targets (or is active in) the tumour microenvironment of preferred cancer targets described herein.

Thus, in some embodiments, the subject is a subject having a cancer (e.g. a solid tumour) selected from the group consisting of lymphomas, leukaemias, neuroblastomas and glioblastomas (e.g. from the brain), carcinomas and adenocarcinomas (particularly from the breast, colon, kidney, liver, lung, ovary, pancreas, prostate and skin), melanomas and sarcomas. In some embodiments, the subject has a sarcoma.

In another aspect, the present invention provides a method for treating cancer (e.g. a solid tumour) by reducing the size of a population of regulatory T cells (Tregs) and/or Myeloid-Derived Suppressor Cells (MDSCs) in a subject, said method comprising administration to said subject of an effective amount of a positively charged amphipathic amino acid derivative, peptide or peptidomimetic as defined elsewhere herein. The reduction of the size of a population of regulatory T cells (Tregs) and/or Myeloid-Derived Suppressor Cells (MDSCs) is typically a reduction of the size of a population of regulatory T cells (Tregs) and/or Myeloid- Derived Suppressor Cells (MDSCs) in a tumour microenvironment (or tumour bed or tumour tissue) in a subject. Preferred cancers are as described elsewhere herein. Other features and properties of other aspects of the invention apply, mutatis mutandis, to this aspect of the invention.

The molecules defined herein may be presented in the conventional pharmacological forms of administration, such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, powders, capsules or sustained release forms. Conventional pharmaceutical excipients as well as the usual methods of production may be employed for the preparation of these forms. Organ specific carrier systems may also be used.

Injection solutions may, for example, be produced in the conventional manner, such as by the addition of preservation agents, such as p

hydroxybenzoates, or stabilizers, such as EDTA. The solutions are then filled into injection vials or ampoules.

Preferred formulations are in saline. Such formulations being suitable for local administration, e.g. intratumoural, e.g. by injection or by perfusion/infusion.

Dosage units containing the active molecules preferably contain 0.1-1 Omg, for example 1 .5mg of the molecule of the invention.

In employing such compositions systemically, the active molecule is typically present in an amount to achieve a serum level of the active molecule of at least about 5 μg/ml. In general, the serum level need not exceed 500 μg/ml. A preferred serum level is about 100 μg/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 some embodiments, the subject may be administered with multiple doses of the molecules used in the invention (e.g. on consecutive days). For example, the subject may receive 2, 3, 4 (or more) doses, preferably on consecutive days. In some embodiments, three consecutive daily doses, preferably three consecutive daily intratumoural injections, are administered.

The molecules used in the methods of the invention include salt forms. Appropriate pharmaceutically acceptable salts for peptides and similar molecules are well known to those skilled in the art.

The invention is further described in the following Examples and with reference to the figures in which:

Figure 1 shows, through flow cytometry determination, that LTX-315 markedly decreased the regulatory T cells (Tregs) (b), and markedly decreased the myeloid-derived suppressor cells (MDSCs) (a), in tumour beds after dissociation of fresh MCA205 sarcoma 7 days post LTX-315 (versus PBS). Each dot represents data of one mouse; at least two experiments were gathered in each graph.

Student's i-test: ^** = P<0.01.

Figure 2 shows, through flow cytometry determination, that LTX-315 markedly increased the CD8 ⁺ cytotoxic T lymphocyte (CTL) to Treg ratio (CTL/Treg) in tumour beds after dissociation of fresh MCA205 sarcoma 7 days post LTX-315 (versus PBS). The ratio between CD8 ⁺ CTLs over Tregs was calculated

considering either interferon γ-positive (IFNv ⁺ ) CTLs (a), or tumour necrosis factor a-positive (TNFa ⁺ ) CTLs (b), or double-positive CD8 ⁺ CTLs (c). The Tregs are CD4 ⁺ CD25 ⁺ CD3 ⁺ FoxP3 ⁺ . Each dot represents data of one mouse; at least two experiments were gathered in each graph. Student's i-test: ^** = P<0.01 and ^*** = P<0.001. Example

In the present study, LTX-315 (K-K-W-W-K-K-W-Dip-K-NH ₂ , SEQ ID NO:23) was assessed for its effect on immunosuppressive regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs).

1 MATERIALS AND METHODS

1.1 Chemicals and cell cultures Media and supplements for cell culture were obtained from Gibco-Life Technologies (Carlsbad, CA, USA), chemicals from Sigma-Aldrich (St. Louis, MO, USA) with the exception of LTX-315 that was provided by Lytix Biopharma (Troms0, Norway) and plasticware from Corning BV Life Sciences (Amsterdam, The Netherlands).

MCA205 was cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, and 2 mM l-glutamine, 100 lU/ml penicillin G sodium salt, 100 /jg/ml streptomycin sulfate, 1 mM sodium pyruvate and 1 mM non-essential amino acids. Cells were grown at 37 °C in a humidified incubator under a 5% C0 ₂ atmosphere.

1.2 Mice

Mice were maintained 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. Animal experiments followed the Federation of European

Laboratory Animal Science Association (FELASA) guidelines, were in compliance with EU Directive 63/2010 and were approved by the Ethical Committee of the Gustave Roussy Cancer Campus (Villejuif, France). Mice were used between 7 and 14 weeks of age. WT-specific pathogen-free (SPF) C57BL/6 J were obtained from Envigo (Gannat, France) and were kept in SPF conditions at Gustave Roussy, Villejuif, France.

1.3 Tumour models

Mice were subcutaneously injected into the right flank with 1 ^χ 10 ⁶ MCA205 cells. Tumour cell lines were inoculated into C57BL/6 mice. Tumour surfaces (longest dimension ^χ perpendicular dimension) were routinely monitored by caliper. When tumours reached a size of 20-40 mm ² (day 0), mice were administered

intratumourally with three consecutive daily injections of 300 /jg LTX-315. 1.4 Flow cytometry

Tumours and spleens were harvested 7 days after the first injection of LTX-315. Excised tumours were cut into small pieces and digested in RPMI-1640 medium containing Liberase at 25 /jg/ml (Roche, Boulogne-Billancourt, France) and DNasel at 150 Ul/ml (Roche) for 30 min at 37°C. The mixture was subsequently passaged through a 100 /jm cell strainer. 2 10 ⁶ splenocytes (after red blood cells lysis) or tumour cells were preincubated with purified anti-mouse CD16/CD32 (93;

eBioscience, San Diego, CA, USA) for 15 min at 4 °C, before membrane staining. For intracellular staining, the FoxP3 staining kit (eBioscience) was used. Dead cells were excluded using the Live/Dead Fixable Yellow dead cell stain kit (Life

Technologies, Carlsbad, CA, USA). For cytokine staining, cells were stimulated for 4 h at 37 °C with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA; Calbiochem, San Diego, CA, USA), 1 /vg/ml of ionomycin (Sigma, St. Louis, MO, USA), and BD Golgi STOP (BD Biosciences, San Jose, CA, USA). Anti-CD45.2 (104), anti-Foxp3 (FJK-16s), anti-IFN-γ (XMG1.2), anti-TNF-a (MP6- XT22), and isotype controls rat lgG1 (eBRG1 ), lgG2a (eBRG2a), lgG2b (eBRG2b), Armenian Hamster IgG (eBio299Arm) were purchased from eBioscience. Anti-CD3 (145-2C1 1 ), anti-CD25 (PC61 .5.3), anti-Ly-6C (AL-21 ), KI67 (FITC mouse anti- human KI67 set), rat IgGl K were obtained from BD Bioscience. Anti-CD4 (GK1 .5), anti-CD83 (YTS1567.7), anti-CD1 1 b (M1/70), Rat lgG2a (RTK2758), Armenian Hamster IgG (HTK888), Rat lgG2b (RTK4530) were purchased from Biolegend (San Diego, CA, USA). Eight-colour flow cytometry analysis was performed with antibodies conjugated to fluorescein isothiocyanate, phycoerythrin, phycoerythrin cyanin 7, peridinin chlorophyll protein cyanin 5.5, allophycocyanin cyanin 7, Pacific blue or allophycocyanin. All cells were analysed on a CyAn ADP (Beckman

Coulter, Marseille, France) flow cytometer with FlowJo (Tree Star, Ashland, OR) software. 1.5 Statistical analysis

Data were analysed with Microsoft Excel (Microsoft Co., Redmont, WA, USA) and Prism 5 (GraphPad, San Diego, CA, USA). Data are presented as meansiS.E.M. and P-values computed by unpaired Student's i-tests or one-way AN OVA followed by Tukey's test where applicable. All reported tests are two-tailed and were considered significant at P-values <0.05.

2 RESULTS In the present study it has been demonstrated that LTX-315 rapidly reprograms the tumour microenvironment by decreasing the local abundance of

immunosuppressive regulatory T cells (Tregs) and of myeloid-derived suppressor cells (MDSCs) (Figure 1 ). It has also been shown that LTX-315 administration results in an increase in the ratio of CD8 ⁺ T effector cells over Tregs (Figure 2).

3 DISCUSSION

In this study it has been found that LTX-315 has the potential to effect

therapeutically relevant immune responses such as the reduction in number of Tregs and MDSCs. More specifically, LTX-315 has the potential to convert "cold" (or non T-cell inflamed) tumours into "hot" (or T-cell inflamed) tumours.