[go: up one dir, main page]

WO2018188761A1 - Procédé de réduction de taille de population de treg et/ou de mdsc - Google Patents

Procédé de réduction de taille de population de treg et/ou de mdsc Download PDF

Info

Publication number
WO2018188761A1
WO2018188761A1 PCT/EP2017/059041 EP2017059041W WO2018188761A1 WO 2018188761 A1 WO2018188761 A1 WO 2018188761A1 EP 2017059041 W EP2017059041 W EP 2017059041W WO 2018188761 A1 WO2018188761 A1 WO 2018188761A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acids
peptide
peptidomimetic
cells
lipophilic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2017/059041
Other languages
English (en)
Inventor
Laurence Zitvogel
Takahiro Yamazaki
Baldur SVEINBJØRNSSON
Øystein REKDAL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lytix Biopharma AS
Original Assignee
Lytix Biopharma AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lytix Biopharma AS filed Critical Lytix Biopharma AS
Priority to PCT/EP2017/059041 priority Critical patent/WO2018188761A1/fr
Publication of WO2018188761A1 publication Critical patent/WO2018188761A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • 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.
  • tumour therapy 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.
  • immunotherapies e.g. antibody therapies
  • Tregs Regulatory T cells
  • Tregs 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.
  • CTLs cytotoxic T cells
  • T helper cells T helper cells.
  • MDSCs Myeloid-Derived Suppressor Cells
  • MDSCs are another type of immune suppressor cells. MDSCs are a
  • Cytotoxic T cells are T cells that can kill damaged cells such as cancer cells or infected cells.
  • TME tumour microenvironement
  • 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.
  • 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.
  • Tregs regulatory T cells
  • MDSCs Myeloid-Derived Suppressor Cells
  • 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.
  • 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.
  • CAPs Cationic antimicrobial peptides
  • 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-
  • 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 ⁇ 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.
  • 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
  • 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-
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • the number of cationic residues will likely be proportional to the length of the peptide, e.g. 1 ⁇ 2 to 3 ⁇ 4 of the residues are cationic. Likewise, 1 ⁇ 4 to 3 ⁇ 4 of the residues are lipophilic (preferably with 7 or more non- hydrogen atoms).
  • 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.
  • these amino acids will be referred to in the following sections as "cationic residues”.
  • the peptide 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.
  • the peptide may contain 3, 4, 5, 6, 7, 8, 9, 10, 1 1 ,
  • the peptide 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.
  • the peptide may comprise 3 to 8, e.g. 4 to 7 or 4 or 5, bulky and lipophilic residues.
  • the peptide may comprise 2 to 5, e.g. 3 or 4 bulky and lipophilic residues.
  • amino acid with a cationic side chain 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.
  • 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 may exist as two or more stereoisomers.
  • 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.
  • 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.
  • 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.
  • the positively charged amphipathic amino acid derivative, peptide or peptidomimetic of the present invention is further defined as set out in the sections below.
  • the peptide (or peptidomimetic) consists of 9 amino acids in a linear arrangement.
  • methods of the invention employ a peptide having the following characteristics:
  • 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
  • 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
  • the lipophilic amino acids 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.
  • 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.
  • one of the lipophilic amino acids is a non-genetically coded amino acid.
  • the molecule consists of 3 genetically coded lipophilic amino acids, 5 genetically coded cationic amino acids and 1 non-genetically coded lipophilic amino acid.
  • 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.
  • 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-
  • 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.
  • CLCCLLCCL (SEQ ID NO: 5) ⁇ and ⁇ 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.
  • the compounds of the invention incorporate at least one, and preferably one, non-genetically coded amino acid.
  • this residue is denoted L'
  • preferred compounds are represented by the following formulae: CCL'LCCLLC ( ⁇ ') (SEQ ID NO: 6)
  • peptides of formula I and II are especially preferred.
  • 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
  • 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.
  • 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.
  • peptidomimetic or amino acid derivative may have a net positive charge of at least +2 and incorporate a disubstituted ⁇ amino acid, each of the substituting groups in the ⁇ 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 ⁇ 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 ⁇ amino acid has the amino group attached to the ⁇ 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 ⁇ amino acid the backbone of a peptide incorporating one or more ⁇ amino acids. In this arrangement the a and/or the ⁇ carbon atom can be substituted.
  • the a or ⁇ carbon atom may be disubstituted; where the a carbon atom is disubstituted a ⁇ 2,2 amino acid results and where the ⁇ carbon atom is disubstituted a ⁇ 3,3 amino acid is generated.
  • One substituting group on each of the a or ⁇ carbon atoms results in a ⁇ 2,3 amino acid, ⁇ 2,2 and ⁇ 3,3 disubstituted amino acids are preferred, ⁇ 2,2 disubstituted amino acids being especially preferred.
  • the ⁇ amino acid is substituted by two groups incorporating at least 7 non-hydrogen atoms.
  • 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-
  • 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 ⁇ carbon atom of the ⁇ 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 ⁇ 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.
  • 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.
  • the molecule 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.
  • 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 ⁇ , ⁇ -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 ⁇ 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.
  • 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:
  • 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 ⁇ 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • the two substituting groups are R-i and R 2 or R 3 and R 4 , R 3 and R 4 being most preferred.
  • the cyclic groups of R 1-4 are not attached directly to either the a or ⁇ 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.
  • the N atom may be substituted, for example by an aminoalkyl group, e.g. aminoethyl or aminopropyl or dimethylaminoethyl.
  • 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.
  • 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 ⁇ , 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 ⁇ -amino acid being flanked by two cationic groups.
  • 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.
  • 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.
  • 'stable' is meant more resistant to enzymatic degradation by hydrolytic enzymes.
  • the bond which replaces the amide bond conserves many of the properties of the amide bond, e.g. conformation, steric bulk, electrostatic character, possibility for hydrogen bonding etc.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a reducing agent e.g. borane or a hydride reagent such as lithium aluminium-hydride.
  • 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.
  • the reactive groups present for example amino, thiol and/or carboxyl
  • the final step in the synthesis will thus be the deprotection of a protected derivative of the invention.
  • 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 described in detail below.
  • 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.
  • Boc t-butyloxycarbonyl
  • Fmoc can be used for protection of the alpha-amino groups.
  • peptides containing Arg 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl can be used for protection of the guanidine side chain.
  • 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.
  • the molecules used in accordance with the present invention are able to lyse tumour cell membranes.
  • 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.
  • 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.
  • the present invention provides a method of reducing the size of a population of regulatory T cells (Tregs) and/or Myeloid-Derived
  • MDSCs Suppressor Cells
  • the size of a population of regulatory T cells (Tregs) is reduced.
  • MDSCs Suppressor Cells
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 .
  • the reduction in the size of a Treg and/or 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%, 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%.
  • the reduction in the size of a Treg population (number of such cells) in a subject 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%.
  • 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).
  • 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).
  • 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%.
  • 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).
  • 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).
  • 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.
  • a tumour microenvironment or tumour bed
  • MDSCs Suppressor Cells
  • 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).
  • ECM extracellular matrix
  • 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).
  • a tumour sample or samples
  • tumour microenvironment sample or samples
  • 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.
  • Tregs regulatory T cells
  • MDSCs Myeloid-Derived Suppressor Cells
  • 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.
  • Tregs regulatory T cells
  • MDSCs Myeloid-Derived Suppressor Cells
  • the present invention provides a method of decreasing the local abundance of regulatory T cells (Tregs) and/or Myeloid-Derived
  • MDSCs Suppressor Cells in a tumour microenvironment (or a tumour bed or tumour tissue) in a subject.
  • 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.
  • Tregs regulatory T cells
  • MDSCs Myeloid-Derived Suppressor Cells
  • 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.
  • Tregs regulatory T cells
  • MDSCs Myeloid- Derived Suppressor Cells
  • 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.
  • a reduction is observed at about seven days after administration (e.g. after first administration).
  • cytotoxic T cells cytotoxic T lymphocytes, CTLs
  • Tregs e.g. CD3 + CD4 + CD25 + FoxP3 + Tregs
  • the ratio is the intratumoural ratio.
  • 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.
  • there is an increase in the ratio of CD8 + IFNv + T cells over Tregs such as CD3 + CD4 + CD25 + FoxP3 + Tregs.
  • there is an increase in the ratio of CD8 + TNFa + T cells over Tregs such as CD3 + CD4 + CD25 + FoxP3 + Tregs.
  • 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 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.
  • 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.
  • 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 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).
  • CTL/Treg ratio CTL:Treg ratio
  • 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.
  • 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.
  • 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
  • the increase may, for example, be up to about 5000% or up to about 6000%.
  • 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%.
  • 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%.
  • CD8 + IFNy + TNFa + T cells (CTLs) over Tregs (e.g. CD3 + CD4 + CD25 + FoxP3 + Tregs) in a subject 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.
  • 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
  • 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).
  • appropriate antibodies e.g. antibodies which bind to the various markers described herein.
  • Suitable reagents and suitable flow cytometry methodology is described in the Example section herein. Samples are typically processed before analysis and suitable processing methods are described in the Examples herein.
  • 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
  • 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.
  • 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).
  • Tregs regulatory T cells
  • MDSCs Myeloid-Derived Suppressor Cells
  • Other features and properties of other aspects of the invention apply, mutatis mutandis, to this aspect of the invention.
  • 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).
  • Tregs regulatory T cells
  • MDSCs Myeloid-Derived Suppressor Cells
  • Other features and properties of other aspects of the invention apply, mutatis mutandis, to this aspect of the invention.
  • 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
  • MDSCs Suppressor Cells in a subject (e.g. in a tumour microenvironment, tumour bed, tumour or tumour tissue).
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • a tumour e.g. a solid tumour
  • This administration is typically, and preferably, via intratumoural delivery (e.g. by intratumoural injection), but other administration routes may be used.
  • 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.
  • solid tumour targets are preferred.
  • sarcomas are preferred cancer targets.
  • a positively charged amphipathic amino acid derivative, peptide or peptidomimetic targets (or is active in) the tumour microenvironment of preferred cancer targets described herein.
  • 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.
  • a cancer e.g. a solid tumour
  • lymphomas e.g. a solid tumour
  • leukaemias e.g. from the brain
  • carcinomas and adenocarcinomas particularly from the breast, colon, kidney, liver, lung, ovary, pancreas, prostate and skin
  • melanomas and sarcomas e.g. adenocarcinomas.
  • the subject has a sarcoma.
  • 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.
  • cancer e.g. a solid tumour
  • Tregs regulatory T cells
  • MDSCs Myeloid-Derived Suppressor Cells
  • 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.
  • Tregs regulatory T cells
  • MDSCs Myeloid- Derived Suppressor Cells
  • 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.
  • the active molecule is typically present in an amount to achieve a serum level of the active molecule of at least about 5 ⁇ g/ml.
  • the serum level need not exceed 500 ⁇ g/ml.
  • a preferred serum level is about 100 ⁇ g/ml.
  • Such serum levels may be achieved by
  • bioactive molecule in a composition to be administered systemically at a dose of from 1 to about 10 mg/kg.
  • the molecule(s) need not be administered at a dose exceeding 100 mg/kg.
  • the subject may be administered with multiple doses of the molecules used in the invention (e.g. on consecutive days).
  • the subject may receive 2, 3, 4 (or more) doses, preferably on consecutive days.
  • 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.
  • 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).
  • Tregs regulatory T cells
  • MDSCs myeloid-derived suppressor cells
  • FIG. 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
  • IFNv + interferon ⁇ -positive CTLs
  • TNFa + tumour necrosis factor a-positive CTLs
  • c double-positive CD8 + CTLs
  • the Tregs are CD4 + CD25 + CD3 + FoxP3 + .
  • LTX-315 K-K-W-W-K-K-W-Dip-K-NH 2 , SEQ ID NO:23
  • Tregs immunosuppressive regulatory T cells
  • MDSCs myeloid-derived suppressor cells
  • 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.
  • 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
  • mice were subcutaneously injected into the right flank with 1 ⁇ 10 6 MCA205 cells. Tumour cell lines were inoculated into C57BL/6 mice. Tumour surfaces (longest dimension ⁇ perpendicular dimension) were routinely monitored by caliper. When tumours reached a size of 20-40 mm 2 (day 0), mice were administered
  • 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;
  • 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).
  • PMA phorbol 12-myristate 13-acetate
  • ionomycin Sigma, St. Louis, MO, USA
  • BD Golgi STOP BD Biosciences, San Jose, CA, USA.
  • Anti-CD45.2 (104), anti-Foxp3 (FJK-16s), anti-IFN- ⁇ (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
  • Tregs immunosuppressive regulatory T cells
  • MDSCs myeloid-derived suppressor cells
  • LTX-315 has the potential to convert "cold” (or non T-cell inflamed) tumours into “hot” (or T-cell inflamed) tumours.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un procédé de réduction de la taille d'une population de lymphocytes T régulateurs (Treg) et/ou de cellules suppresseurs d'origine myéloïde (MDSC) chez un sujet, ledit procédé comprenant l'administration audit sujet d'une quantité efficace d'un dérivé d'acide aminé amphipathique positivement chargé, d'un peptide ou d'un peptidomimétique.
PCT/EP2017/059041 2017-04-13 2017-04-13 Procédé de réduction de taille de population de treg et/ou de mdsc Ceased WO2018188761A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/EP2017/059041 WO2018188761A1 (fr) 2017-04-13 2017-04-13 Procédé de réduction de taille de population de treg et/ou de mdsc

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2017/059041 WO2018188761A1 (fr) 2017-04-13 2017-04-13 Procédé de réduction de taille de population de treg et/ou de mdsc

Publications (1)

Publication Number Publication Date
WO2018188761A1 true WO2018188761A1 (fr) 2018-10-18

Family

ID=58664649

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2017/059041 Ceased WO2018188761A1 (fr) 2017-04-13 2017-04-13 Procédé de réduction de taille de population de treg et/ou de mdsc

Country Status (1)

Country Link
WO (1) WO2018188761A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011051692A1 (fr) 2009-11-02 2011-05-05 Lytix Biopharma As Peptides thérapeutiques
WO2015118028A1 (fr) * 2014-02-04 2015-08-13 University of Tromsø Peptides dirigés contre les lymphomes
WO2016091490A1 (fr) * 2014-12-11 2016-06-16 Lytix Biopharma As Associations chimiothérapeutiques de peptides cationiques antimicrobiens et d'agents chimiothérapeutiques
WO2016091487A1 (fr) * 2014-12-11 2016-06-16 Lytix Biopharma As Combinaisons d'inhibiteurs de points de contrôle immunitaires

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011051692A1 (fr) 2009-11-02 2011-05-05 Lytix Biopharma As Peptides thérapeutiques
WO2015118028A1 (fr) * 2014-02-04 2015-08-13 University of Tromsø Peptides dirigés contre les lymphomes
WO2016091490A1 (fr) * 2014-12-11 2016-06-16 Lytix Biopharma As Associations chimiothérapeutiques de peptides cationiques antimicrobiens et d'agents chimiothérapeutiques
WO2016091487A1 (fr) * 2014-12-11 2016-06-16 Lytix Biopharma As Combinaisons d'inhibiteurs de points de contrôle immunitaires

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
ALLMENDINGER, T. ET AL., TETRAHYDRON LETT., vol. 31, 1990, pages 7297
CHOREV, M; GOODMAN, M., ACC. CHEM. RES, vol. 26, 1993, pages 266
GALON ET AL., J. TRANSL. MED., vol. 14, 2016, pages 273
H ZHOU ET AL: "The oncolytic peptide LTX-315 triggers immunogenic cell death", CELL DEATH AND DISEASE, vol. 7, no. 3, 10 March 2016 (2016-03-10), pages e2134, XP055426753, DOI: 10.1038/cddis.2016.47 *
HOFFMAN, R.V.; KIM, H.O., J. ORG. CHEM., vol. 60, 1995, pages 5107
KROGSGAARD ET AL., DRUG DESIGN AND DEVELOPMENT, 1996
LAVIELLE, S., INT. J. PEPTIDE PROTEIN RES., vol. 42, 1993, pages 270
LUISI, G. ET AL., TETRAHEDRON LETT., vol. 34, 1993, pages 2391
OSTRESH, J.M. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 91, 1994, pages 11138 - 11142
SASAKI, Y; ABE, J., CHEM. PHARM. BULL., vol. 45, 1997, pages 13
SCHMIDT, R. ET AL., INT. J. PEPTIDE PROTEIN RES., vol. 46, 1995, pages 47
SHERMAN D.B.; SPATOLA, A.F, J. AM. CHEM. SOC., vol. 112, 1990, pages 433
SPATOLA, A.F., METHODS NEUROSCI, vol. 13, 1993, pages 19
T YAMAZAKI ET AL: "The oncolytic peptide LTX-315 overcomes resistance of cancers to immunotherapy with CTLA4 checkpoint blockade", CELL DEATH AND DIFFERENTIATION., vol. 23, no. 6, 15 April 2016 (2016-04-15), GB, pages 1004 - 1015, XP055427532, ISSN: 1350-9047, DOI: 10.1038/cdd.2016.35 *

Similar Documents

Publication Publication Date Title
KR101445910B1 (ko) 항종양 활성을 가지는 나노펩티드
US12239686B2 (en) Immune checkpoint inhibitor combinations
US20250002854A1 (en) Methods of generating populations of tumour-infiltrating t cells
US20040071691A1 (en) Pharmaceutical formulation
WO2018136779A2 (fr) Facteurs dérivés de oxalobacter formigenes (of) pour le traitement ou le traitement/prévention de taux d'oxalate en excès
JP2007500693A (ja) 移植片拒絶反応を予防するための合成ペプチドコポリマーを含む併用療法
WO2018188761A1 (fr) Procédé de réduction de taille de population de treg et/ou de mdsc
WO2020002668A1 (fr) Peptides et traitement du cancer
US20250049930A1 (en) Protac molecules targeting foxp3 and uses thereof
WO2015089316A1 (fr) Méthodes permettant de prévenir et de traiter les cancers de la bouche
CN110139659B (zh) 用于治疗干燥综合征的肽
JP6813258B2 (ja) 化学療法の組み合わせ
WO2013048226A1 (fr) Métallopeptides immunomodulateurs (immp) et compositions les contenant
EP3836949A1 (fr) Compositions comprenant des peptides dérivés de sel1 et méthodes de traitement/prévention de taux d'oxalate en excès et pathologies/maladies associés à ceux-ci
HK40012327A (en) Peptides for treating sjogren's syndrome

Legal Events

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

Ref document number: 17720699

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17720699

Country of ref document: EP

Kind code of ref document: A1