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CA-3060667-C - TUMOUR-TARGETING PEPTIDE VARIANTS

CA3060667CCA 3060667 CCA3060667 CCA 3060667CCA-3060667-C

Abstract

The present invention provides a peptide that selectively binds αvβ6 integrin, the peptide having an amino acid sequence comprising the motif X1BnAGDLX2X3X4ZmX5, wherein X1 is any D-amino acid, B n is a sequence of any n amino acids, which may be natural or unnatural, D- or L-, and may be the same or different, wherein n is a number between 1 and 10, X2 and X3 are independently selected from any amino acid, X4 is Leu or Ile, Z m is a sequence of any m amino acids, which may be natural or unnatural, D- or L-, and may be the same or different, wherein m is a number between 1 and 10, X5 is any L- or D-amino acid. Also provided are conjugates comprising said peptide, pharmaceutical compositions comprising said peptide or said conjugates, and uses of said peptide, conjugate or composition, for example, in the treatment, imaging and/or diagnosis of an αvβ6- expressing tumour in a mammalian subject.

Inventors

  • John Marshall
  • Margaret Brimble

Assignees

  • CANCER RESEARCH TECHNOLOGY LIMITED

Dates

Publication Date
20260505
Application Date
20180424
Priority Date
20170424

Claims (20)

  1. 48 8373813 Claims: 1. A peptide that selectively binds αvβ6 integrin, the peptide having an amino acid sequence comprising the motif X1BnRGDLX2X3X4ZmX5 (SEQ ID NO: 3), wherein X1 is D-Asn, Bn is a sequence of any n amino acids, which may be natural or unnatural, D- or L-, and may be the same or different, wherein n is a number between 1 and 10, X2 and X3 are independently selected from any amino acid, X4 is Leu or Ile, Zm is a sequence of any m amino acids, which may be natural or unnatural, D- or L-, and may be the same or different, wherein m is a number between 1 and 10, and wherein X5 is any L- or D-amino acid.
  2. 2. The peptide according to claim 1, wherein the length of the peptide is between 17 and 25 amino acids and/or wherein X5 is L-Thr or D-Thr and/or wherein X4 is Leu.
  3. 3. The peptide of claim 2, wherein the length of the peptide is 20 amino acids.
  4. 4. The peptide according to any one of claims 1 to 3, wherein n is 5 and/or m is 6 and/or wherein Bn is AVPNL (SEQ ID NO: 4), KVPNL (SEQ ID NO: 5) or K(biotin)VPNL (SEQ ID NO: 5) and/or wherein Zm is AQKVAR (SEQ ID NO: 6).
  5. 5. The peptide according to any one of claims 1 to 4, wherein the amino acid sequence of the peptide is selected from the group consisting of: H2N-[D-Asn]-AVPNLRGDLQVLAQKVART-COOH (SEQ ID NO: 7); H2N-[D-Asn]-KVPNLRGDLQVLAQKVART-COOH (SEQ ID NO: 8); H2N-[D-Asn]-K(biotin)VPNLRGDLQVLAQKVART-COOH (SEQ ID NO: 8); H2N-[D-Asn]-KVPNLRGDLQVLAQKVAR[D-Thr]-COOH (SEQ ID NO: 9); H2N-[D-Asn]-K(biotin)VPNLRGDLQVLAQKVAR[D-Thr]-COOH (SEQ ID NO: 9); and H2N-[D-Asn]-AVPNLRGDLQVLAQKVAR[D-Thr]-COOH (SEQ ID NO: 10). 49 8373813
  6. 6. The peptide according to any one of claims 1 to 5, wherein the peptide is N-acetylated and/or C-amidated.
  7. 7. A conjugate comprising the peptide as defined in any one of claims 1 to 6 conjugated directly or via a linker to a therapeutic moiety, a polymer, a polypeptide and/or a detectable moiety.
  8. 8. The conjugate according to claim 7, wherein the therapeutic moiety comprises an anti-cancer agent.
  9. 9. The conjugate according to claim 8, wherein the anti-cancer agent is selected from the group consisting of: an auristatin, a maytansinoids, a tubulysin, duocarmycin, calicheamicin, alpha-amanitin, a pyrrolobenzodiazepine, irinotecan, and an indolecarboxamide, or a prodrug or active metabolite thereof.
  10. 10. The conjugate according to claim 8 or 9, wherein the anti-cancer agent comprises: Monomethyl Auristatin E (MMAE), mertansine (DM1), ravtansine (DM4) or Centanamycin.
  11. 11. The conjugate according to any one of claims 7 and 8, wherein the therapeutic moiety comprises a radioactive isotope selected from the group consisting of: 177Lu, 90Y, 211At, 213Bi, 212Pb, 225Ac, 223Ra, 44Sc, 67Ga, 131I, 188Re, 186Re and 67Cu.
  12. 12. The conjugate according to any one of claims 7 to 11, wherein the detectable moiety is detectable by Magnetic Resonance Imaging (MRI), Magnetic Resonance Spectroscopy (MRS), Single Photon Emission Computed Tomography (SPECT), Positron Emission Tomograph (PET) or optical imaging.
  13. 13. The conjugate according to claim 12, wherein the detectable moiety comprises a fluorophore, a radionuclide or a spin label.
  14. 14. The conjugate according to claim 13, wherein the detectable moiety comprises: 68Ga, IRDye® 700, IRDye® 800, Fluorescein 50 8373813 isothiocyanate (FITC), 89Zr, 124I, 64Cu, 62Cu, 18F, 86Y, 111In, 131I, 123I, 67Ga or 99mTc.
  15. 15. The conjugate according to any one of claims 7 to 14, wherein the detectable moiety comprises 111In coupled to the peptide via a linker that comprises a chelator.
  16. 16. The conjugate according to any one of claims 7 to 15, wherein the polymer comprises one or more ethylene glycol groups or one or more polyethylene glycol (PEG) chains.
  17. 17. A pharmaceutical composition comprising: the peptide as defined in any one of claims 1 to 6 or the conjugate as defined in any one of claims 7 to 16; and a pharmaceutically acceptable carrier.
  18. 18. Use of the peptide as defined in any one of claims 1 to 6, the conjugate as defined in any one of claims 7 to 16 or the pharmaceutical composition as defined in claim 17 for treating or for the manufacture of a medicament for treating an αvβ6-expressing tumour in a mammalian subject.
  19. 19. The use according to claim 18, wherein the tumour is a cervical tumour, a head or neck tumour, a breast tumour, a lung tumour, a skin tumour, a colon tumour, an ovarian tumour or a pancreatic tumour.
  20. 20. The conjugate as defined in any one of claims 12 to 16 for use in an in vivo diagnosis of cancer in a mammalian subject, wherein detection of said detectable moiety diagnoses the presence of an αvβ6-expressing tumour in the subject.

Description

l Tumour-Targeting Peptide Variants Field of the invention The present invention relates to peptide variants, ugates and ical composi ions thereof and to including for the treatment of cancer and Background to the invention I ns a cxf3 rodimeric mol cul that 11 surfa rs in°1ol ved in ir use in medicine, ng of tumours. compass a large family of ral including In humans substrates 11 adhesion, invasion, prolif ration and processes sis [23]. 24 a a V present on ext llul r li f which 8 cl d tr ch i s re ze if Gl [ 7 l . I grin avp6 is express din hi in and vitronectin on numerous cancers [4-9] such as ora, ck, pancreatic, ovarian and breast and increa sio 0 i i s been rrelated with tumour pro gri avp6 e fibrosis [ l l and s ra in s i ng mouth disease [ 28 l L~s such, i grin p, 6 is or arge 9-30 l for sties and therapy in the field of oncology and A2OFMDV2 (H2N- 1NAVPNLRGDLQVLAQKVAR20T-OH) (SEQ ID NO: 1) is a 2Oresidue linear peptide derived from the viral protein of foot-andmouth disease virus [1-3, 10]. This peptide has been shown to 30 exhibit high selectively and affinity for avp6, an integrin transmembrane receptor that is highly expressed in cancer cells [4- 9]. A2OFMDV2 binds to avp6 through the RGD tripeptide of the 7RGDLQV13L (SEQ ID NO: 2) fragment, and the two leucine residues present in the RGDLQVL fragment enhance the binding of the peptide 35 to the receptor via hydrophobic interactions [10] and it is also WO 2018/197490 PCT /EP2018/0604 7 4 2 stabilised by an a-helix generated from the C-terminal peptide motif 10Leu-20Thr [l]. In addition, A2OFMDV2 is an effective imaging agent for av~6 in pulmonary fibrosis when coupled with radioactive Indium ( 111 In) [24]. The [18F] fluorobenzoyl-labelled peptide, [18F FBA- 5 A2OFMDV2, has found use as a PET radiotracer in human clinical trials [25]. A2OFMDV2 has been used in positron emission tomography (PET) for diagnostic imaging applications [31] by conjugating to 4- 10 [18F] fluorobenzoic acid (FBA) (or derivatives) [32-34], and 64Cu labelling using A2OFMDV2 peptide that incorporates a metal chelator such as DOTA [35-37]. Recently, [18F]-FBA-A2OFMDV2 has progressed to a clinical setting [25] in the treatment regime of idiopathic pulmonary fibrosis [24] while an 111In labelled A2OFMDV2 derivative 15 has been shown to be highly specific for imaging of breast cancer expressing the av~6 integrin using single-photon emission computed tomography (SPECT) [9]. These studies outline the importance of A2OFMDV2 to ongoing diagnostic tools targeting the av~6 integrin and underpins av~6 as a valid therapeutic target. W02OO7/O39723 describes av~6 peptide ligands, functional variants thereof and nucleic acids encoding them, as well as their use in the treatment and imaging of av~6 mediated diseases, including cancer. 25 Unfortunately, the therapeutic value of A2OFMDV2 is limited by its short half-life in blood caused, in part, by it high susceptibility to serum proteases, such as endo- and exo-peptidases, as determined in mouse plasma studies [13]. 30 One reported method for improving half-lives of susceptible peptides (and proteins) is the introduction of polyethyleneglycol (PEG) [38] Hausner et al. prepared PEGylated versions of 4- [18F] -FBA-A2OFMDV2, by placement of one or two PEG20 moieties at the N terminus [13] or a single PEG2s at both the N and C termini [39] and concluded that a 35 bi-terminally PEGylated A2OFMDV2 derivative, [:8F]-FBA-PEG2sA2OFMDV2- PEG2s exhibited the most favorable pharmacokinetics. WO 2018/197490 PCT /EP2018/0604 7 4 3 Head-to-tail cyclisation of linear peptides is also a wellestablished method to minimise degradation by exopeptidases [40]; however, careful experimentation is required to prevent undesired side reactions such as polymerisation or racemisation. However, there remains an unmet need for tumour-targeting peptides with potent binding activity, cellular uptake and extended plasma half-lives. The present invention addresses these and other needs. 10 Brief description of the invention Broadly, the present invention relates to A20FMDV2 peptide variants that have unexpectedly been found to exhibit enhanced av~6 binding activity in cell studies, and in some cases, enhanced cellular uptake as compared with A20FMDV2 peptide. In particular, as 15 described in further detail herein, such peptide variants have been found to exhibit enhanced bio-distribution into av~6-expressing xenograft tumours (relative to control xenograft tumours lacking av~6 expression) as compared with a control A20FMDV2 peptide, as assessed by 111In-labelled versions of the respective peptides. Such 20 peptide variants therefore have great potential as tumour-targeted chemotherapeutic delivery agents and tumour imaging agents. Accordingly, in a first aspect the present invention provides a peptide that selectively binds av~6 integrin, the peptide having an 25 amino acid sequence comprising the motif X1B:1RGDLX2X3X4ZmXs (SEQ ID NO: 3), wherein X1 is any D-amino acid,