Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
  • C07K14/43572—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from bees
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/24—Heavy metals; Compounds thereof
  • A61K33/243—Platinum; Compounds thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70546—Integrin superfamily
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/28—Compounds containing heavy metals
  • A61K31/282—Platinum compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins

Definitions

• the present invention relates to the field of compositions and methods for the treatment of cancer.
• the present invention relates to melittin fusion peptides and the use of melittin fusion peptides to treat cancers.
• honeybee venom is melittin, which comprises about half of honeybee venom by dry weight.
• Melittin is a positively charged, amphipathic 26 amino acid peptide of SEQ ID NO: 1 that associates with the phospholipids of the membrane bilayer, causing cell death by forming transmembrane toroidal pores. These pores of approximately 4.4 nm in diameter may also enable the internalisation of additional small molecules with cytotoxic activities.
• honeybee venom and melittin have demonstrated anti-tumour effects in melanoma, non-small-cell lung cancer, glioblastoma, leukemia, hepatocellular carcinoma, ovarian, cervical, and pancreatic cancers, and have demonstrated higher killing potency in cancer cells compared to non-transformed cells.
• Additive and synergistic anticancer effects between honeybee venom and cisplatin have also been reported in cervical and laryngeal malignancies, and with docetaxel in lung cancer cells.
• honeybee venom reduced breast cancer lung metastasis, inhibited tumour growth, and prolonged survival in spontaneous mammary carcinoma murine tumours.
• Honeybee venom and melittin induced apoptosis in luminal MCF7 cells, and reduced cell viability and migration in triple negative MDA-MB-231 cells.
• honeybee venom Most of the anti-neoplastic activity of honeybee venom has been attributed to melittin through inhibition of the PI3K/Akt/mTOR axis in breast cancer, MAPK in hepatocyte and melanoma cells, JAK2/STAT3 in ovarian cancer, and NFKB signaling pathways in lung carcinoma cells.
• TNBC triple negative breast cancer
• TNBCs overexpress epidermal growth factor receptor (EGFR), and HER2-enriched tumours overexpress HER2, another receptor tyrosine kinase (RTK) of the same family.
• RTKs confer oncogenic signaling often dependent on the PI3K/Akt pathway downstream.
• Blocking EGFR signaling in TNBC has demonstrated limited clinical efficacy in early phase clinical trials due to a lack of dependence on the EGFR pathway and the importance of collateral pathways.
• FIER2-targeted therapies have dramatically improved median survival in the metastatic setting, resistance is almost inevitable over the longer term for this subtype.
• Ovarian cancer is a type of gynecologial cancer that originate in the reproductive system. OC is the deadliest gynecological cancer, associated with a very poor 5-year survival of 46%, with 1 ,068 estimated deaths and 1 ,532 new diagnoses in Australia in 2020. Whist survival rates from other solid cancers have improved in last decades, the rates survival from OC have remained unchanged since the 1980s. This is associated to several factors, including the non-specific symptoms of the disease, the lack of effective screening strategies, and consequently, most (75- 80%) of these cancers are diagnosed at advanced stages.
• OCs have an epithelial origin, and among these, high grade serious ovarian cancers (FIGSOCs) are highly proliferative and aggressive, and thought to originate from the fallopian tube.
• FIR homologous recombination
• Ovarian cancer is typically associated with a poor prognosis for patients, so there is a need for improved treatments.
• the effects of melittin have been reported in a limited number of ovarian cancer cell lines, being PA-1 , SKOV3 and OVCAR3.
• the present invention provides a novel fusion peptide, that can reduce cell viability in cancer cells, including in particular, TNBC and HER2-enriched breast cancer cells, and ovarian cancer cells.
• cancer cells including in particular, TNBC and HER2-enriched breast cancer cells, and ovarian cancer cells.
• the present invention provides a novel fusion peptide, that can reduce cell viability in cancer cells, including in particular, TNBC and HER2-enriched breast cancer cells, and ovarian cancer cells.
• references to “fusion peptides” and “melittin fusion peptides” of the invention, throughout this specification, should be read as a reference to a fusion peptide comprising (1 ) SEQ ID NO: 1 , or a variant thereof, and (2) an RGD-motif containing peptide.
• the invention provides a fusion peptide comprising: (1 ) SEQ ID NO: 1 , or a variant thereof, and (2) an RGD-motif containing peptide.
• the fusion peptide comprises SEQ ID NO: 1 , or a variant thereof, bound to an RGD-motif containing peptide, wherein the fusion peptide binds to the integrins anbq and anb3.
• the integrins anbq and/or anb3 are commonly overexpressed in TNBC and HER2-enriched breast cancer cells, as well as ovarian cancer cells.
• the invention also provides variants of the fusion peptides described herein.
• the RGD-motif containing peptide comprises SEQ ID NO: 2 or a variant thereof.
• the fusion peptide comprises SEQ ID NO: 3, or a variant thereof.
• the invention provides a fusion peptide of SEQ ID NO: 3 or a variant thereof.
• the invention provides a pharmaceutical composition comprising a fusion peptide of the invention.
• the pharmaceutical composition additionally comprises a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
• the pharmaceutical composition may additionally comprise a chemotherapeutic agent.
• the chemotherapeutic agent is selected from docetaxel or cisplatin.
• the invention provides a method of treating cancer in a subject comprising administering to the said subject a therapeutically effective amount of a fusion peptide of the invention.
• the cancer is selected from the group of HER2-enriched breast cancer, triple negative breast cancer or ovarian cancer.
• the method of treating cancer additionally comprises the step of administering to said subject a chemotherapeutic agent.
• the chemotherapeutic agent is selected from one or more of pertuzumab, trastuzumab, docetaxel or cisplatin.
• the method of treating cancer additionally comprises administering to said subject an anticancer therapy.
• the anticancer therapy is omomyc or a Cas9-gRNA complex or variant thereof.
• the invention provides a fusion peptide of the invention for use in a method of treating cancer in a subject in need thereof.
• the invention provides the use of a fusion peptide of the invention in the manufacture of a medicament for treating cancer in a subject in need thereof.
• the invention provides a method of treating triple negative breast cancer, HER2 enriched breast cancer or ovarian cancer in a subject comprising the steps of: (1) administering to said subject a therapeutically effective amount of a combination therapy of melittin or fusion peptide; and (2) administering a therapeutically effective amount of a chemotherapeutic agent.
• the chemotherapeutic agent is selected from pertuzumab, trastuzumab, docetaxel, and/or cisplatin.
• the invention provides a method of treating triple negative breast cancer, HER2 enriched breast cancer or ovarian cancer in a subject comprising administering to said subject a therapeutically effective amount of a combination therapy of melittin or melittin fusion peptide and an anticancer therapy.
• the anticancer therapy is omomyc or a Cas9-gRNA complex or variant thereof.
• the invention provides a method of reducing tumour cell viability in a subject, said method comprising the step of administration of a therapeutically effective amount of a melittin fusion peptide of the invention.
• the melittin fusion peptide comprises SEQ ID NO: 3.
• the method further comprises the step of administering to said subject a chemotherapeutic agent.
• the chemotherapeutic agent is selected from one or more of pertuzumab, trastuzumab, docetaxel, or cisplatin.
• the invention provides a kit for treating cancer in a subject comprising a melittin fusion peptide of the invention together with instructions as to how to use that fusion peptide in accordance with this invention.
• the kit additionally comprises a chemotherapeutic agent.
• the chemotherapeutic agent is selected from pertuzumab, trastuzumab, docetaxel, or cisplatin.
• the invention provides a nucleic acid encoding a melittin fusion peptide.
• the nucleic acid encodes a melittin fusion peptide of SEQ ID NO: 3, of SEQ ID NO: 7.
• the invention provides a method of inhibiting PD-L1 expression in a subject suffering from cancer comprising the step of administering melittin or a fusion peptide in a therapeutically effective amount to the subject.
• the method additionally comprises the step of administering a chemotherapeutic agent.
• the chemotherapeutic agent is selected from one or more of pertuzumab, trastuzumab, docetaxel or cisplatin.
• the subject is suffering from triple negative breast cancer, HER2-enriched breast cancer or ovarian cancer.
• the invention provides the use of the fusion peptide of the invention in the manufacture of a medicament for inhibiting PD-L1 expression in a subject suffering from cancer.
• the invention provides an isolated monoclonal antibody that specifically binds to the peptides of SEQ ID NO:1 and SEQ ID NO: 3.
• the invention provides a method of facilitating the cell penetration of an anticancer therapy in a subject suffering from cancer, comprising the step of administering a therapeutically effective amount of a combination therapy comprising administering: (1 ) of melittin or a or a fusion peptide; and (2) an anticancer therapy to the subject.
• a combination therapy comprising administering: (1 ) of melittin or a or a fusion peptide; and (2) an anticancer therapy to the subject.
• the anticancer therapy is omomyc or a Cas9-gRNA complex or variant thereof.
• the invention provides a fusion peptide comprising (1) SEQ ID NO: 1 , or a variant thereof, and (2) an RGD-motif containing peptide, wherein the fusion peptide binds to the integrins anbq and anb3.
• Figure 1 presents results of tumour viability assays for cell lines exposed to bee venom and melittin.
• Figure 1 A presents cell viability assays of a panel of human normal and breast cancer cell lines treated with Australian honeybee venom (left) or melittin (right), with the IC50 values presented in Figure 1 B.
• Figure 1 C presents cell viability dose-response curves of a panel of murine non-transformed (NIH/3T3), basal-like breast cancer (BRCA- B.15) and claudin-low breast cancer (p53 T11 ) cell lines treated with Australian honeybee venom (left) or melittin (right).
• Figure 1 D presents the IC50 values for each cell type exposed to honeybee venom and melittin.
• Figure 2 presents cell viability dose-response curves of human dermal fibroblasts (FIDFa) and breast cancer cell lines (SUM159 and SKBR3) treated with venom from populations of honeybees in Ireland (left) and England (right).
• Figure 2B presents cell viability dose-response curves of human dermal fibroblasts (FIDFa) and breast cancer cell lines (SUM159 and SKBR3) treated with venom from populations of England worker (left) and queen (right) bumblebees.
• Figure 3 presents absorbance (405 nm) of aqueous solutions of melittin and bee venom assessed by ELISA with the anti-melittin antibody and IgG control.
• Figure 3B presents cell viability assays in FIDFa and SUM 159 after blocking melittin using the anti-melittin antibody with honeybee venom (top) and melittin (bottom). Data are represented as mean ⁇ SEM.
• Figure 4 presents results of the mechanism and kinetics of cell death for TNBC (SUM159), FIER2-enriched (SKBR3), and normal (FIDFa) cells treated with honeybee venom or melittin.
• Figure 4A presents a Western blot for the detection of cleaved caspase-3 (CL-csp-3) in SUM159 cells treated with vehicle (1 ), honeybee venom (2-3) and melittin (4-5) for 18 and 24 hours.
• Figure 4B presents flow cytometry analysis of SUM 159 cells treated with the IC50 of honeybee venom (5.58 ng/pL) and the IC50 of melittin (4.24 ng/pL) for one hour.
• Figure 4C presents cell viability assays of human dermal fibroblasts (FIDFa) and breast cancer cells (SUM159 and SKBR3) treated with honeybee venom (left) or melittin (right) over one hour.
• Figure 4D presents live cell confocal microscopy of SKBR3 cells treated with the IC50 of honeybee venom (5.77 ng/pL) over one hour, with time in minutes’ post-treatment.
• Figure 4E presents scanning electron microscopy of SUM159 cells treated with vehicle, the IC50 of honeybee venom (5.58 ng/pL) or the IC50 of melittin (4.24 ng/pL) over one hour, with two representative images shown for each treatment group. Scale bars represent 200 nm, 1 pm or 10 pm as indicated. Data are represented as mean ⁇ SEM.
• Figure 5 presents the lack of anticancer activity of DEDE-melittin, and the breast cancer cell selectivity of the melittin fusion peptide RGD1 -melittin.
• Figure 5A presents cell viability assays of TNBC (SUM159) and HER2-enriched (SKBR3) cells treated with DEDE-melittin for 24 hours.
• Figure 5B presents cell viability assays of T11 cells treated with melittin, RGD1 -melittin, SV40- melittin, TAT-melittin and DEDE-melittin.
• Figure 5C presents cell viability assays of human dermal fibroblasts (FIDFa) and SUM159 treated with melittin (top) and RGD1 -melittin (bottom) for 24 hours.
• Figure 6 presents the lack of anticancer activity of DEDE-melittin, and the breast cancer cell selectivity of the melittin fusion peptide RGD1 -melittin.
• Figure 6A presents a Western blot for the detection of cleaved caspase-3 (CL-csp-3) in cell lysates from SUM159 cells treated with either vehicle, melittin, DEDE-melittin or RGD1 -melittin for 24 hours.
• Figure 6B presents absorbance (405 nm) of aqueous solutions of melittin, RGD1 -melittin, DEDE-melittin and SV40- melittin subjected to an ELISA with the anti-melittin antibody.
• Figure 6C presents the amino acid sequence and top predicted 3D model of melittin, RGD1 -melittin, DEDE-melittin and SV40- melittin.
• Figure 7 presents immunofluorescence images of SUM159 treated with vehicle, honeybee venom, melittin, RGD1 -melittin or DEDE-melittin for 30 minutes.
• Figure 8 presents the phosphorylation kinetics of EGFR, FIER2 and downstream MAPK and Akt pathways after treatment with honeybee venom and melittin in (A) SKBR3 and (B) SUM159, and melittin in (C) MDA-MB-231 breast cancer cells, assessed by immunoblotting.
• Figure 9 presents a Western blot for the detection of JAK/STAT pathway inhibitors in SUM159 cells treated with vehicle, honeybee venom or melittin for 60 minutes, with and without 20 ng/mL EGF for 5 minutes. a-Tubulin was used as the loading control.
• Figure 10 presents the kinetic analysis of (A) TAMRA-EGF, (B) FITC-melittin, (C) FITC- DEDE-melittin, and (D) FITC-EN1 -mutant interaction with NanoLuc-EGFR by bioluminescence resonance energy transfer (BRET) in FIEK293FT cells.
• A TAMRA-EGF
• B FITC-melittin
• C FITC- DEDE-melittin
• D FITC-EN1 -mutant interaction with NanoLuc-EGFR by bioluminescence resonance energy transfer (BRET) in FIEK293FT cells.
• Figure 11 presents the saturation binding analysis of increasing concentrations of (A) TAMRA-EGF, (B) FITC-melittin and (C) FITC-DEDE-melittin in HEK293FT cells transfected with NanoLuc-EGFR in the presence or absence of unlabeled EGF (1 mM).
• Figure 12 presents the results of in vitro assays of the combination of honeybee venom or melittin with docetaxel or cisplatin administered to TNBC cells.
• Figure 12A presents cell viability assays of T11 breast cancer cells treated with honeybee venom or melittin alone and in combination with docetaxel or cisplatin for 24 hours. Representative plots of the combination treatments are presented as mean ⁇ SEM.
• Figure 12B presents Combination Index graphs obtained for different fractions of cells affected in each combination treatment in 12A, calculated using CompuSyn software. A synergistic drug combination is indicated by Cld .
• Figure 13 presents the results of in vivo assays of the combination of docetaxel administered to mice.
• Figure 13A presents tumour volumes of mice treated intratumourally with vehicle, 5 mg/Kg melittin, 7 mg/Kg docetaxel, or 5 mg/Kg melittin + 7 mg/Kg docetaxel. Arrows indicate the treatment days. Corresponding scatter plots of relative change in tumour volumes at days 3, 7 and 9 are indicated.
• Figure 13B presents representative bioluminescence imaging (BLI) images of T11 -luciferase tumours in mice at days 4, 10, 12 and 14 post-inoculation of the cells.
• BLI bioluminescence imaging
• Figure 14 presents the quantification of immunohistochemistry and immunofluorescence in tumour biopsies from mice extracted on day 14 post-T11 inoculation stained with anti-melittin, anti-Ki-67, anti-PD-L1 , with TUNEL assay, and with Floechst and FI&E.
• Figure 14B presents the quantification of immunohistochemistry in tumour biopsies from mice extracted on day 14 post-T11 cells inoculation, evaluating the effect of single or combined inhibition with melittin and docetaxel on the expression levels of EGFR and FIER2 and reduction of p-EGFR (Tyr1068) and p-HER2 (Tyr1248).
• Figure 15 presents the results of cell viability assays of T11 breast cancer cells treated with melittin or RGD1 -melittin alone and in combination with docetaxel for 48 hours. Representative plots of the combination treatments are presented as mean ⁇ SEM.
• Figure 16 presents the results of cell viability assays of T11 breast cancer cells treated with melittin or RGD1 -melittin alone and in combination with omomyc for 24 hours. Representative plots of the combination treatments are presented as mean ⁇ SEM.
• Figure 17 presents the results of cell viability assays of OVCAR3 and COV362 ovarian cancer cells treated with active RGD1 -melittin and mutant RGD1 -melittin (RGD1-DEDE melittin).
• Figure 17B presents the results of cell viability assays of OVCAR3 and COV362 ovarian cancer cells with active melittin and mutant melittin (DEDE melittin).
• Figure 18 presents immunofluorescence images of PD-L1 expression in T11 , SUM159 and SKBR3 breast cancer cells treated with RGD1 -melittin and mutant RGD1 -melittin (DEDE-RGD1 -melittin).
• Figure 18B presents qRT-PCT analysis of PD-L1 expression levels in T11 , SUM159 and SKBR3 treated with active RGD1 -melittin and mutant RGD1 -melittin peptides, or untreated (media only) for 24h.
• Figure 19 presents the results of cell viability assays of T11 , SUM159, OVCAR3, and SKBR3 breast and ovarian cancer cells treated with RGD1 -melittin and docetaxel alone and in combination.
• Figure 20 presents the results of cell viability assays of T11 , OVCAR3, and COV362 breast and ovarian cancer cells treated with RGD1 -melittin and cisplatin alone and in combination.
• the present invention is based on the discovery that certain melittin fusion peptides have enhanced selectivity for certain subtypes of cancer cells.
• the melittin fusion peptides have enhanced selectivity for specific subtypes of breast cancer or ovarian cancer.
• the invention described herein may include one or more range of values (e.g. dosage, concentration etc.).
• a range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Hence “about 80 %” means “about 80 %” and also “80 %”. At the very least, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.
• polypeptide means a macromolecule having the amino acid sequence of a native protein, that is, a protein produced by a naturally-occurring and non recombinant cell; or it is produced by a genetically-engineered or recombinant cell, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence.
• polypeptide specifically encompass melittin and melittin fusion peptides or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of the melittin or melittin fusion peptide without adversely affecting the functionality of the peptide.
• a "variant" of a polypeptide comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence, but which retains the biological activity of the original polypeptide sequence by reference to assays known in the art.
• Variants include fusion proteins.
• a “variant” of a melittin fusion peptide comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to the melittin fusion peptide, which retains melittin fusion peptide like biological activity, which can be measured by assays known in the art, such as cell viability assays.
• therapeutically effective amount refers to the amount of melittin or a melittin fusion peptide determined to produce a therapeutic response in a mammal, including a human. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art.
• patient and “subject” are used interchangeably and include human and non human animal subjects with formally diagnosed disorders, those without formally recognized disorders, those receiving medical attention and those at risk of developing the disorders.
• treat and treatment includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms, underlying risk factors or delays progression of the disorder.
• cancer refers to an abnormal growth of cells that tend to proliferate in an uncontrolled way and, in some cases, to metastasize (spread).
• Types of cancer include, but are not limited to, solid tumours (such as those of the bladder, bowel, brain, breast, endometrium, heart, kidney, lung, liver, uterus, lymphatic tissue [lymphoma], ovary, pancreas or other endocrine organ [thyroid], prostate, skin [melanoma or basal cell cancer]), or haematological tumours (such as the leukemias and lymphomas) at any stage of the disease with or without metastases.
• solid tumours such as those of the bladder, bowel, brain, breast, endometrium, heart, kidney, lung, liver, uterus, lymphatic tissue [lymphoma], ovary, pancreas or other endocrine organ [thyroid], prostate, skin [melanoma or basal cell cancer]
• haematological tumours such as the leukemias and lymphomas
• TNBC triple negative breast cancer
• HER2-enriched breast cancer refers to a type of breast cancer where human epidermal growth factor receptor 2 (HER2) is overexpressed by the tumour cells.
• ovarian cancer refers to cancer that forms in or on an ovary.
• binding in connection with the interaction between a one compound or molecule and another compound or molecule, such as a target and a potential binding compound, indicates that the potential binding compound associates with the target to a statistically significant degree as compared to association with proteins generally.
• specific binding refers to binding between two molecules or compounds that is statistically significantly higher than non-specific binding to another molecule.
• a binding compound interacts with a specified target with a dissociation constant (k.sub.d) of 1 mM or less, for example 0.1 -100 nM.
• a binding compound can bind with “low affinity”, “very low affinity”, “extremely low affinity”, “moderate affinity”, “moderately high affinity”, or “high affinity”.
• the term “greater affinity” indicates that the compound binds more tightly than a reference compound, or than the same compound in a reference condition, i.e., with a lower dissociation constant.
• the greater affinity is at least 2, 3, 4, 5, 8, 10, 50, 100, 200, 400, 500, 1000, or 10,000-fold greater affinity.
• the term “greater specificity” indicates that a compound binds to a specified target to a greater extent than to another biomolecule or biomolecules that may be present under relevant binding conditions, where binding to such other biomolecules produces a different biological activity than binding to the specified target.
• the specificity is with reference to a limited set of other biomolecules.
• the greater specificity is at least 2, 3, 4, 5, 8, 10, 50, 100, 200, 400, 500, or 1000-fold greater specificity.
• Binding can be measured by various assays known in the art, including mobility shift experiments, surface plasmon resonance, fluorescence polarization, fluorescence resonance energy transfer, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) methods.
• melittin refers to the 26 amino acid length peptide of SEQ ID NO: 1 that is derived from honeybee venom.
• melittin fusion peptide refers to melittin of SEQ ID NO: 1 or a variant thereof fused with another peptide, protein or fragment thereof.
• isolated peptide means that a subject protein (1) is free of at least some other proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (6) does not occur in nature.
• an "isolated peptide” constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample.
• Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof can encode such an isolated protein.
• the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use.
• chemotherapeutic agent means a chemical compound useful in the treatment of cancer.
• examples of chemotherapeutic agents include alkylating agents, such as, for example, temozolomide, the imidazotetrazine derivative of the alkylating agent dacarbazine.
• Additional examples of chemotherapeutics agents include, e.g., paclitaxel or topotecan or pegylated liposomal doxorubicin (PLD).
• chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin; bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic ana
• dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins (bleomycin A2), cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin and deoxydox
• an "anticancer therapy” is any compound, composition or treatment that prevents or delays the growth and/or metastasis of cancer cells.
• anticancer therapies include but are not limited to chemotherapeutic agents, radiation, gene therapy, hormone therapy, immunotherapy, polypeptides and antisense oligonucleotide therapy.
• active agent may mean one active agent or may encompass two or more active agents.
• the invention provides a melittin fusion peptide comprising melittin of SEQ ID NO: 1 bound to an RGD-motif containing peptide.
• the invention provides a melittin fusion peptide comprising (1 ) melittin of SEQ ID NO: 1 and (2) an RGD-motif containing peptide.
• the melittin fusion peptide binds to the integrins anbq and anb3.
• the RGD-motif containing peptide comprises SEQ ID NO: 2.
• the invention provides a melittin fusion peptide of SEQ ID NO: 3, or a variant thereof.
• Melittin is a polypeptide component of honeybee venom. Melittin is known to reduce tumour cell viability. Melittin is thought to reduce tumour cell viability by binding to the plasma membrane, inducing subsequent pore formation, which leads to the leaking of cellular ions and molecules, an enhancement of cell permeability and ultimately cell lysis. [0083] Melittin may also have an effect on apoptosis and necrosis of cells. The specific mechanism of action of melittin in reducing tumour cell viability can depend on the type of tumour cell. In respect of triple negative breast cancer expressing EGF receptors and HER2, melittin can supress ligand induced phosphorylation of EGFR and FIER2, dynamically modulating downstream signaling pathways in breast cancer cells.
• Immune checkpoint protein Programmed Death Ligand-1 binds to the immune checkpoint protein PD-1 and can inhibit the activation and immune recognition of T-cells that would otherwise be involved in the natural immune response against tumour cells.
• the inhibition of PD-L1 results in the activation of T cells.
• certain chemotherapeutic agents can increase the expression of PD-L1 , resulting in reduced immune response to the tumour cell, and reduced overall efficacy of the treatment.
• trastuzumab anti-HER2 therapy was shown to result in the upregulation of PD-L1 (B.
• ovarian cancer cells have been shown to upregulate PD-L1 expression in response to chemotherapy exposure (Grabosch, S., Zeng, F., Zhang, L. et al. PD-L1 biology in response to chemotherapy in vitro and in vivo in ovarian cancer j. immunotherapy cancer 3, P302 (2015)).
• melittin may reduce the levels of PD-L1 immune check-point protein. Melittin therefore may decrease the immune-suppressive effects of the tumour microenvironment, which are prevalent in cancers such as triple negative breast cancer in the presence of chemotherapy. In some embodiments, melittin may attenuate the increased expression of PD-L1 caused by chemotherapeutic agents, such as trastuzumab. Melittin has been shown to reduce the tumour promoting M2-like tumour-associated macrophage population in the tumour microenvironment in a lung carcinoma model.
• EGFR and FIER2 signaling in breast cancer cells modulates PD- L1 expression in tumour cells.
• Immunohistochemical studies indicate that PD-L1 has the highest expression in TNBC tumours, followed by HER2-enriched tumours, and PD-L1 expression is associated with poor survival.
• the absence of the protein ALIX may correlate with EGFR activation, impairing exosome biogenesis.
• PD-L1 is secreted via exosomes in an ALIX-dependent manner such that exosome impairment increases PD-L1 on the cell membrane.
• ALIX downregulation promotes tumour survival through enhancement of EGFR activation, and through PD-L1 membrane accumulation, leading to immunosuppression.
• the C-terminus of melittin forms a positively charged a-helix.
• the positively charged alpha helix mediates binding to the negatively charged plasma membrane, then resulting in the formation of pores in the cell plasma membrane.
• the positive K21 RKR24 sequence of the C-terminus of melittin mediates binding to the negatively charged plasma membrane and is therefore essential to its function in disrupting the cell plasma membrane.
• Selectivity of melittin to tumour cell types is important for enhancing the ability of melittin to target tumour cells rather than exerting its effects on healthy cells.
• the selectivity of melittin to specific tumour cells is enhanced by fusing melittin to another peptide.
• the melittin is fused with a peptide containing an RGD peptide motif.
• RGD is a tri-peptide motif consisting of the amino acids arginine, glycine and aspartate.
• RGD motifs are present on a number of different extracellular proteins. Many such proteins are recognised by some members of the integrin protein family via the RGD motif.
• eight integrin dimers i.e. , anb1, anb3, anb5, anbq, anbd, a5b1, a8b1 , and aII b3, recognize the tripeptide RGD-motif within extracellular matrix proteins.
• Certain proteins containing RGD motifs are selective for particular members of the integrin protein family.
• the ligand TGF ⁇ 3 which is known to regulate molecules involved in cellular adhesion and extracellular matrix formation during the process of palate development, contains an RGD-motif and is known to interact with the integrins anbq and anb3 via the RGD- motif.
• RGD-motif contains an RGD-motif and is known to interact with the integrins anbq and anb3 via the RGD- motif.
• RGD-motif an RGD-motif and is known to interact with the integrins anbq and anb3 via the RGD- motif.
• TGF-bI the foot-and-mouth-disease virus (FMDV) and fibronectin are known to bind to anbq.
• RGD-motif containing peptides there are other peptide options: see Dong, X., Fludson, N. E., Lu, C. & Springer, T. A. Structural determinants of integrin b-subunit specificity for latent TGF-b. Nat. Struct. Mol. Biol. 21, 1091-1096 (2014); Sorolla, A. et al. Triple-hit therapeutic approach for triple negative breast cancers using docetaxel nanoparticles, EN1 -iPeps and RGD peptides. Nanomedicine Nanotechnology, Biol. Med. 102003 (2019), which are incorporated in their totality by reference.
• integrin protein family Certain members of the integrin protein family are overexpressed on the plasma membrane of specific tumour cell types and associated vasculature. Therefore, peptides containing an RGD-motif can be useful for targeting tumour cell types that overexpress the integrins to which the RGD-containing peptides bind.
• Integrins anb6 and anb3 are over-expressed on the plasma membrane of certain breast cancer cells and tumour associated vasculature.
• anb6 and anb3 are overexpressed on the surface of triple negative breast cancer and FIER2-enriched breast cancer cells. Therefore, peptides that selectively bind integrins anbq and anb3 can be selective towards triple negative breast cancer and HER2-enriched breast cancer cells.
• Integrin anbq is over-expressed on the plasma membrane of ovarian cancer cells (see N Ahmed et. al. Carcinogenesis vol.23 no.2 pp.237-244, 2002).
• the RGD motif of SEQ ID NO: 2 (RGD1) has the best attraction, that is binding affinity, to anbq integrins making it potent to breast cancer and ovarian cancer cells.
• RGD1 also binds, with lower affinity, to anb3 integrin.
• the invention provides for a melittin fusion peptide wherein the melittin of SEQ ID NO: 1 is fused with a peptide containing an RGD peptide motif, which recognises integrins anbq and anb3.
• the melittin fusion peptide of the invention has enhanced selectivity for HER2-enriched, triple negative breast cancer cell subtypes and/or ovarian cancer.
• the melittin fusion peptide retains the anti-tumoral activity of melittin, and exerts substantially the same, or superior, effects on tumour cell viability as melittin.
• the melittin fusion peptide experts substantially the same, or superior, effects on PD-L1 expression in tumour cells.
• the sequence of the RGD motif containing peptide is derived from TGF ⁇ 3. More preferably, the RGD motif containing peptide has the sequence HGRGDLGRLKK (SEQ ID NO: 2) (RGD1). Preferably, RGD1 interacts with both anbq and anb3 integrins, with particularly high affinity for anbq integrin.
• melittin is fused with the RGD motif of TGF ⁇ 3 of SEQ ID NO: 2 at the N-terminus, and has the following sequence:
• HGRGDLGRLKKGIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 3)
• the fusion peptide of the invention is an isolated peptide.
• the melittin fusion peptides of the invention are bi-functional in that the fusion peptide has an anti-tumour effect derived from the melittin, and also is selective for cells over expressing the integrins anb6 and/or anb3, its selectivity is enhanced from the RGD motif containing peptide.
• the melittin in order to facilitate the bi-functional characteristics of the melittin fusion peptides, is directly fused or bound to the RGD motif containing peptide without a linker.
• the melittin can be fused or bound to the RGD motif containing peptide via a linker.
• Linkers can in some embodiments assist with improving the specificity of delivery to a particular tumour site.
• the linker is cleavable in vivo. Most preferably, the linker is cleaved at the tumour site.
• the melittin fusion peptide comprises one or more amino acid substitutions of the melittin fusion peptides discussed above.
• a skilled person will be able to determine suitable variants of the melittin fusion peptides which retain functionality, using well- known techniques.
• one skilled in the art can identify suitable areas of the peptide that can be modified without destroying activity by, for example, targeting regions not believed to be important for activity. For example, such area can be modified by conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
• the C-terminus of the melittin fusion peptides of this invention and their variants retain a positive charge.
• the retention of the positive charge is advantageous for maintaining the biological activity of the melittin fusion peptide.
• the melittin fusion peptides of the invention and their variants do not comprise a negatively charged C-terminus sequence.
• a variant melittin fusion peptide retains the original positive K21RKR24 (SEQ ID NO: 4) sequence of the C-terminus of melittin of SEQ ID NO: 1.
• melittin fusion peptide variants that retain SEQ ID NO: 4 sequence at the C-terminus exhibit the biological activities characteristic of melittin and melittin fusion peptides of SEQ ID NO: 3.
• the melittin fusion peptide comprises an alternative C-terminus sequence that is positively charged.
• the melittin fusion peptide comprises a C-terminal sequence of ((K21 KKRKV26) present in the Simian Virus 40 (SV40) large T antigen (peptide SV40-melittin)) (SEQ ID NO:5), derived from the Simian Virus 40 (SV40) large T antigen (peptide SV40-melittin).
• the melittin fusion peptides of this invention can be produced by routine and standard techniques known in the art.
• the melittin fusion peptides can be synthesised through the use of peptide synthesisers well known in the art, or through standard recombinant DNA technology known in the art.
• Peptides encompassed by the present invention include peptide variants based on a common scaffold, wherein each peptide variant comprises an amino acid sequence with an amino acid sequence identity compared to the primary peptide sequence selected from the group consisting of: 99 % or greater identity; 98 % or greater identity; 97 % or greater identity; 96 % or greater identity; 95 % or greater identity; 90 % or greater identity; 85 % or greater identity; 80 % or greater identity; 75 % or greater identity; and 70 % or greater identity.
• any changes to the primary peptide sequence to create a variant peptide includes amino acid deletions, amino acid additions and/or amino acid substitutions.
• Amino acid substitutions are preferably conservative amino acid substitutions known to those skilled in the art.
• the person skilled in the art may perform an amino acid substitution by selecting an amino acid from within the same class of amino acid that is shared with the specific amino acid that is identified for substitution.
• the amino acid classes where the amino acids are sorted into six main classes on the basis of their structure and general chemical characteristics of their side chains (R groups), are: Class 1 - Glycine, Alanine, Valine, Leucine, Isoleucine;
• Class 2 Serine, Cysteine, Selenocysteine, Threonine, Methionine;
• Class 5 Histidine, Lysine, Arginine; Class 6 - Aspartate, Glutamate, Asparagine, Glutamine
• Val (V) lie Met, Leu, Phe, Ala, Norleucine
• % identity may for example be calculated as follows.
• the query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al, Nucleic Acids Research, 22: 4673-4680 (1994)).
• a comparison is made over the window corresponding to one of the aligned sequences, for example the shortest.
• the window may in some instances be defined by the target sequence. In other instances, the window may be defined by the query sequence.
• the amino acid residues at each position are compared, and the percentage of positions in the query sequence that have identical correspondences in the target sequence is reported as % identity.
• the invention provides a pharmaceutical composition comprising a melittin fusion peptide of the invention.
• the melittin fusion peptide is of SEQ ID NO: 3 or a variant thereof.
• the pharmaceutical composition additionally comprises pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
• the optimal pharmaceutical composition can be determined by a person skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. Such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the melittin fusion peptides of the invention.
• the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature.
• a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
• the saline comprises isotonic phosphate-buffered saline.
• neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
• the pharmaceutical composition components are present in concentrations that are acceptable to the site of administration.
• buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
• the pharmaceutical composition of this invention may be administered by intravenous, intraarterial, or intramuscular injection of a liquid preparation.
• suitable liquid compositions include solutions, suspensions, dispersions, emulsions, oils and the like.
• a therapeutic composition when parenteral administration is contemplated, can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising a desired melittin fusion peptide, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle.
• a vehicle for parenteral injection is sterile distilled water in which a melittin fusion peptide, with or without at least one additional therapeutic agent, is formulated as a sterile, isotonic solution, properly preserved.
• the preparation can involve the formulation of the desired melittin fusion peptide with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, nanoparticles, or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection.
• an agent such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, nanoparticles, or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection.
• hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation.
• implantable drug delivery devices can be used to introduce the desired molecule.
• the pharmaceutical composition once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.
• the pharmaceutical composition comprises preservatives, including water soluble preservatives.
• Water soluble preservatives that may be employed include, but are not limited to, benzalkonium chloride, chlorobutanol, thimerosal, sodium bisulfate, phenylmercuric acetate, phenylmercuric nitrate, ethyl alcohol, methylparaben, polyvinyl alcohol, benzyl alcohol and phenylethyl alcohol.
• the pharmaceutical composition herein may also contain additional therapeutic agents, preferably with a complementary activity that does not adversely affect the functioning of the melittin fusion peptide.
• the pharmaceutical composition further comprises a chemotherapeutic agent or other anticancer therapy.
• Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
• the pharmaceutical composition further comprises one or more chemotherapeutic agents.
• the chemotherapeutic agent is docetaxel, trastuzumab, pertuzumab and/or cisplatin: see for example, Swain, S. M. etal.
• the pharmaceutical composition comprises an anticancer therapy.
• the anticancer therapy is omomyc or a Cas9-gRNA complex or variant thereof.
• the melittin fusion peptides, melittin and chemotherapeutic agents, and other anticancer therapeutics (and composition comprising these agents) herein can be administered for prophylactic and/or therapeutic treatments.
• the compositions are administered to a patient already suffering from a disease or condition, in an amount sufficient to cure or at least partially arrest the symptoms or progression of the disease or condition. Amounts effective for this use will depend on the severity and course of the disease or condition, previous therapy, the patient's health status, weight, and response to the drugs, and the judgment of the treating physician. It is considered well within the skill of the art for one to determine such therapeutically effective amounts by routine experimentation (including, but not limited to, a dose escalation clinical trial).
• the melittin fusion peptides, melittin, chemotherapeutic agents and other anticancer therapeutics (and compositions comprising these agents) described herein are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder, or condition.
• a patient susceptible to or otherwise at risk of a particular disease, disorder, or condition for example, in some embodiments, there is an increase in the expression of anb3, anb5, anbq, anbd, a5b1 , and/or aII b3 integrins during tumour formation which can be targeted by the melittin fusion peptides of the invention.
• Such an amount is defined to be a "prophylactically effective amount or dose”. In this use, the precise amounts also depend on the patient's state of health, weight, and the like.
• a typical dosage can range from about 1 pg/Kg to up to about 10 mg/Kg or more, depending on the factors mentioned above. In certain embodiments, the dosage can range from 1 pg/Kg up to about 10 mg/Kg; or 10 pg/Kg up to about 10 mg/Kg; or 100 pg/Kg up to about 10 mg/Kg. Preferably the human does is in the range of from 0.5 mg/Kg or 500 pg/kg.
• the melittin or melittin fusion peptide is administered at a dosage of about 10 pg/Kg to 10 mg/Kg, more preferably at a dosage between 100 pg/Kg to 1 mg/Kg. In some embodiments, it is administered at a dosage of 500 pg/Kg.
• the chemotherapeutic agent is administered at a dosage of about 10- 200 mg/m 2 IV every three weeks, more preferably at a dosage between 60-100 mg/m 2 IV every three weeks. In some embodiments, it is administered at a dosage of 75 mg/m 2 IV every three weeks.
• the chemotherapeutic agent is docetaxel or cisplatin.
• the chemotherapeutic agent is carboplatin.
• the carboplatin is administered to a subject suffering from triple negative breast cancer in six cycles of carboplatin with target AUC 6, every 3 weeks until disease progression, and the melittin fusion peptide is administered at a dosage of 0.5 mg/Kg to 100 mg/Kg.
• the anticancer therapy additionally comprises treatment with pertuzumab or trastuzumab.
• the pertuzumab is administered at a dose of 840 mg in the first cycle, and then at 420 mg every three weeks.
• trastuzumab is administered at a dose of 8 mg/Kg in the first cycle and at 6 mg/Kg every 3 weeks.
• the anticancer therapy is omomyc.
• omomyc is administered at a dosage of 0.5 mg/Kg to 10mg/Kg. Most preferably, omomyc is administered at a dosage of 2.6 mg/Kg, as set out in Wang et. al. Oncogene (2019) 38:140-150 which is incorporated by reference.
• the anticancer therapy is a Cas9-gRNA complex or variant thereof.
• the Cas9-gRNA complex or variant thereof is administered at a dose of 250pM-1000pM.
• the Cas9-gRNA complex or variant thereof is administered at a dose of 250pM.
• the melittin, melittin fusion peptide or pharmaceutical composition comprising the melittin fusion peptide can be administered to the subject in a range of treatment regimens.
• the melittin, melittin fusion peptide or pharmaceutical composition can be administered hourly, three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once monthly, once every two months, once every six months, and once yearly.
• the appropriate regimen can be determined by the person skilled in the art based on the nature of the condition to be treated.
• the melittin, melittin fusion peptide or pharmaceutical composition comprising the melittin fusion peptide can be administered every two days.
• the invention provides a method of treating cancer in a subject comprising administering to said subject a therapeutically effective amount of a melittin fusion peptide of the invention.
• the cancer to be treated is any tumour cell sub-type wherein the integrins anbq and/or anb3 are overexpressed on the plasma membrane of the tumour cell and/or associated vasculature.
• the cancer is selected from the group of HER2-enriched breast cancer, triple negative breast cancer or ovarian cancer.
• the method of treating cancer additionally comprises the step of administering to said subject an anticancer therapy.
• Anticancer therapies include but are not limited to natural killer cell-based immunotherapy cells (NK-92MI), HER2-targeted agents including monoclonal antibodies, trastuzumab-emtansine, other antibody-drug conjugates, ribonucleoproteins such as Cas9-gRNA and variants, or polypeptides such as omomyc where the membrane disrupting properties of melittin could enhance the internalization kinetics of the cytotoxic payload. It is to be understood that anticancer therapies for use in the present invention also include novel compounds or treatments developed in the future.
• NK-92MI natural killer cell-based immunotherapy cells
• HER2-targeted agents including monoclonal antibodies, trastuzumab-emtansine, other antibody-drug conjugates, ribonucleoproteins such as Cas9-gRNA and variants, or polypeptides such as omomyc where the membrane disrupting properties of melittin could enhance the internalization kinetics of the cyto
• the anticancer therapy is a chemotherapeutic agent and is selected from docetaxel, cisplatin, pertuzumab, and/or trastuzumab.
• the anticancer therapy is omomyc.
• Omomyc is a dominant-negative MYC mutant comprising the b-HLH-LZ domain of Myc, with four amino acid substitutions in the leucine zipper of Myc ( Soucek , L. et al., 1998, Oncogene 17, 2463-2472; Soucek, L, et at. (2002), Cancer Res 62: 3507-3510). These substitutions (E61T, E68I, R74Q, and R75N) confer altered dimerization specificity to Myc, which retains the ability to heterodimerize with its natural partner Max and to form homodimers with wild type c-, N- and L- Myc.
• Omomyc negates the ability of Myc to bind its DNA recognition binding site, the E box, and is able to prevent Myc-dependent gene transactivation functions both in vitro and in vivo.
• Omomyc also strongly enhances Myc-induced apoptosis dependent on Myc expression level to strengthen Myc transrepression activity. Therefore, while retaining Miz-1 dependent binding to promoters and transrepression, omomyc prevents Myc binding to promoter E-boxes and transactivation of target genes. In the presence of omomyc, the activity of the Myc interactome changes from pro- oncogenic to tumor-suppressive.
• the anticancer therapy is a Cas9-gRNA complex or variant such as a dCas9-gRNA complex.
• Cas9-gRNA complexes consist of two parts: a guide RNA (gRNA) and a Cas9 binding endonuclease.
• the guide RNA is a short synthetic RNA consisting of a scaffold sequence necessary for binding to Cas9, and a specific targeting sequence (target site) that is specific for the target location.
• Cas9-gRNA complexes can be used to edit genes involved in tumour proliferation.
• Variants of Cas9-gRNA complexes such as catalytically inactive dCas9-gRNA can also be used for the purpose of inducing epigenetic modifications, in which dCas9 or variants are linked to domains that modifies chromatin.
• the invention provides a melittin fusion peptide of the invention for use in a method of treating cancer in a subject in need thereof.
• the cancer to be treated is any tumour cell sub-type wherein the integrins anb6 and/or anb3 are overexpressed on the plasma membrane of the tumour cell and/or associated vasculature.
• the cancer is selected from the group of HER2-enriched breast cancer, triple negative breast cancer or ovarian cancer.
• the invention provides the use of a melittin fusion peptide of the invention in the manufacture of a medicament for treating cancer in a subject in need thereof.
• the cancer to be treated is any tumour cell sub-type wherein the integrins anb6 and/or anb3 are overexpressed on the plasma membrane of the tumour cell and/or associated vasculature.
• the cancer is selected from the group of HER2-enriched breast cancer, triple negative breast cancer or ovarian cancer.
• the invention provides a method of treating triple negative breast cancer, HER2 enriched breast cancer or ovarian cancer in a subject comprising administering to said subject a therapeutically effective amount of a combination therapy of a melittin fusion peptide and docetaxel, cisplatin, pertuzumab, and/or trastuzumab.
• the invention also provides a method of reducing tumour cell viability in a subject, said method comprising the step of administration of a therapeutically effective amount of a melittin fusion peptide of the invention.
• the melittin fusion peptide comprises SEQ ID NO: 3 or a variant thereof.
• the method further comprises the step of administering to said subject a chemotherapeutic agent or other anticancer therapy.
• the chemotherapeutic agent is selected from docetaxel, cisplatin, pertuzumab, omomyc and/or trastuzumab.
• the anticancer therapy is selected from omomyc or Cas9-gRNA complexes or a variant thereof.
• the invention provides a method of inhibiting PD-L1 expression in a subject suffering from cancer comprising the step of administering melittin or a melittin fusion peptide in a therapeutically effective amount to the subject.
• the method additionally comprises the step of administering a chemotherapeutic agent.
• the chemotherapeutic agent is docetaxel, cisplatin, pertuzumab, omomyc and/or trastuzumab.
• the subject is suffering from triple negative breast cancer, HER2-enriched breast cancer or ovarian cancer.
• Immune checkpoint protein PD-L1 binds to the immune checkpoint protein PD-1 , and can inhibit the activation and immune recognition of T-cells that would otherwise be involved in the natural immune response against tumour cells.
• the inhibition of PD-L1 results in the activation of T cells. It has also been shown that in certain cancers, the administration of certain chemotherapeutic agents can increase the expression of PD-L1 , resulting in reduced immune response to the tumour cell, and reduced overall efficacy of the treatment.
• the administration of a therapeutically effective amount of melittin or melittin fusion peptide to a subject suffering from cancer reduces the expression of PD-L1 in tumour cells. This can result in improved anti-tumoral immune responses by the subject.
• the cancer to be treated is any tumour cell sub-type wherein the integrins anb6 and/or anb3 are overexpressed on the plasma membrane of the tumour cell and/or associated vasculature.
• the cancer is selected from the group of HER2-enriched breast cancer, triple negative breast cancer or ovarian cancer.
• the invention provides a melittin fusion peptide of the invention for use in a inhibiting PD-L1 expression in a subject suffering from cancer.
• the cancer is any tumour cell sub-type wherein the integrins anb6 and/or anb3 are overexpressed on the plasma membrane of the tumour cell and/or associated vasculature.
• the cancer is selected from the group of HER2-enriched breast cancer, triple negative breast cancer or ovarian cancer.
• the method additionally comprises the administration of an anticancer therapy.
• the invention provides the use of a melittin fusion peptide of the invention in the manufacture of a medicament for inhibiting PD-L1 expression in a subject suffering from cancer.
• the cancer is any tumour cell sub-type wherein the integrins anb6 and/or anb3 are overexpressed on the plasma membrane of the tumour cell and/or associated vasculature.
• the cancer is selected from the group of HER2-enriched breast cancer, triple negative breast cancer or ovarian cancer.
• the invention provides a method of facilitating the cell penetration of an anticancer therapy in a subject suffering from cancer, comprising the step of administering a therapeutically effective amount of a combination therapy of a melittin fusion peptide and an anticancer therapy to the subject.
• the anticancer therapy is omomyc or a Cas9-gRNA complex or variant thereof.
• the cancer to be treated is any cancer cell sub-type wherein the integrins anbq and/or anb3 are overexpressed.
• the cancer is selected from the group of HER2-enriched, triple negative breast cancer or ovarian cancer.
• Another aspect of the present invention provides a method for treating cancer in a subject comprising the step of administering to the said subject a therapeutically effective amount of a melittin fusion peptide of the invention (or pharmaceutical composition comprising a melittin fusion peptide) and additionally the step of administering to said subject an anticancer therapy.
• the melittin fusion peptide can be administered before, during or after treatment with the anticancer therapy in order to treat the subject in need thereof.
• a method for reducing the expression of PD-L1 in a subject suffering from cancer and additionally the step of administering to said subject an anticancer therapy.
• the anticancer therapy is a chemotherapeutic agent.
• the chemotherapeutic agent is an agent that is used in the standard of care for the type of cancer suffered by the subject.
• the chemotherapeutic agent is selected from docetaxel, trastuzumab, pertuzumab and/or cisplatin, used to treat a patient with triple negative breast cancer, HER2 enriched breast cancer or ovarian cancer.
• the chemotherapeutic agent is carboplatin.
• the anticancer therapy is a polypeptide.
• the polypeptide is omomyc.
• the anticancer therapy is a Cas9-gRNA complex or a variant thereof.
• the melittin fusion peptide is administered together with docetaxel.
• docetaxel is administered to a subject suffering from triple negative breast cancer or HER2 enriched breast cancer at a dosage of 60 to 100 mg/m 2 IV every three weeks until disease progression is arrested, and the melittin fusion peptide is administered at a dosage of 0.5 to 100 mg/Kg, in order to treat triple negative breast cancer or HER2 enriched breast cancer.
• docetaxel is administered at a dose of 75 mg/m 2 on day 2, and then at 75 mg/m 2 every 3 weeks, with dose adjustments allowed up to 100 mg/m 2 , or a 25% dose reduction to improve tolerability.
• docetaxel is administered together with one or more additional chemotherapeutic agents.
• the additional chemotherapeutic agent is trastuzumab and/or pertuzumab.
• trastuzumab is administered in combination with pertuzumab or another like antibody in accordance with the methodology set out in Swain, S. M. et al. Pertuzumab, Trastuzumab, and Docetaxel in HER2-Positive Metastatic Breast Cancer. N. Engl. J. Med. 372, 724-734 (2015), to a subject suffering from HER2 positive metastatic breast cancer.
• the trastuzumab is administered at a dosage of 8 mg/Kg on day 1 , then at 6 mg/Kg every three weeks until disease progression is arrested, and the melittin fusion peptide is administered at a dosage of 0.5 to 100 mg/Kg, in order to treat the breast cancer.
• the anticancer therapy additionally comprises treatment with pertuzumab or trastuzumab.
• the pertuzumab is administered at a dose of 840 mg on day 1 , and then at 420 mg every three weeks.
• trastuzumab is administered at a dose of 8 mg/Kg on day 1 , and at 6 mg/Kg every 3 weeks.
• the melittin fusion peptide of the invention is administered together with cisplatin.
• cisplatin or combinations of cisplatin with other agents is (are) administered to a subject suffering from triple negative breast cancer, HER2 enriched breast cancer or ovarian cancer at a cisplatin dosage of 60 to 100 mg/m 2 IV every three weeks until disease progression is arrested, and the melittin fusion peptide is preferably administered at a dosage of 0.5 to 100 mg/Kg.
• the melittin fusion peptide is administered together with carboplatin.
• the carboplatin is administered to a subject suffering from triple negative breast cancer, HER2 enriched breast cancer or ovarian cancer.
• Carboplatin is administered over six cycles with target AUC 6, every 3 weeks or until disease progression has been arrested.
• the melittin fusion peptide is preferably administered at a dosage of 0.5 to 100 mg/Kg.
• the melittin fusion peptide is administered together with omomyc.
• the omomyc is administered at a dosage of about 10 pg/Kg to 10 mg/Kg, more preferably at a dosage between 100 pg/Kg to 10 mg/Kg. In some embodiments, it is administered at a dosage of 2.6 mg/Kg.
• omomyc is administered at a dosage of 0.5 mg/Kg to 10mg/Kg. Most preferably, omomyc is administered at a dosage of 2.6 mg/Kg.
• the melittin fusion peptide is administered together with a Cas9-gRNA complex or a variant thereof.
• a Cas9-gRNA complex or a variant thereof is administered at a dosage of about 10 pg/Kg to 10 mg/Kg, more preferably at a dosage between 100 pg/Kg to 1 mg/Kg.
• the Cas9-gRNA complex or variant thereof is administered at a dose of 250pM-1000pM.
• the Cas9-gRNA complex or variant thereof is administered at a dose of 250pM.
• the invention provides a method of facilitating the cell penetration of an anticancer therapy in a subject suffering from cancer, comprising the step of administering a therapeutically effective amount of a combination therapy of a melittin fusion peptide and an anticancer therapy to the subject.
• the anticancer therapy is any compound, composition or treatment that prevents or delays the growth and/or metastasis of cancer cells, and can include small molecules, peptides, polypeptides and protein-nucleic acid complexes, including those that are larger than 100 kDa.
• the melittin fusion peptide can generate transmembrane pores with an internal diameter of approximately 4.4 nm. These pores may facilitate the cell penetration of other therapeutics, including anticancer therapies (including chemotherapeutic agents). This can be of particular benefit in respect of anticancer therapies that otherwise do not easily penetrate into tumour cells, for example, due to their size.
• the anticancer therapy is a chemotherapeutic agent, specifically docetaxel, trastuzumab, pertuzumab and/or cisplatin.
• the anticancer therapy is omomyc or Cas9-gRNA or a variant thereof.
• the melittin fusion peptide of the above methods comprises SEQ ID NO: 3 or a variant thereof.
• the present invention provides a method for treating cancer in a subject comprising the step of administering to the said subject a therapeutically effective amount of melittin (or pharmaceutical composition comprising melittin) and additionally the step of administering to said subject an anticancer therapy.
• the melittin can be administered before, during or after treatment with the anticancer therapy in order to treat the subject in need thereof.
• the therapeutically effective amount of melittin is administered together with a therapeutically effective amount of cisplatin, pertuzumab, trastuzumab, and/or docetaxel to treat a patient suffering from cancer.
• the therapeutically effective amount of melittin is administered together with the therapeutically effective amount of cisplatin, pertuzumab, trastuzumab, and/or or docetaxel to treat a patient suffering from triple negative breast cancer, HER2 enriched breast cancer or ovarian cancer.
• the therapeutically effective amount of melittin is administered together with a therapeutically effective amount of omomyc, or a Cas9-gRNA complex or variant thereof. More preferably, the therapeutically effective amount of melittin is administered together with the therapeutically effective amount of omomyc, or a Cas9-gRNA complex or variant thereof to treat a patient suffering from triple negative breast cancer, HER2 enriched breast cancer or ovarian cancer.
• the invention provides a method of facilitating the cell penetration of an anticancer therapy in a subject suffering from cancer, comprising the step of administering a therapeutically effective amount of a combination therapy of melittin and an anticancer therapy to the subject.
• the anticancer therapy is any compound, composition or treatment that prevents or delays the growth and/or metastasis of cancer cells, and can include small molecules, peptides, polypeptides and protein-nucleic acid complexes more, including those that are larger than 100 kDa.
• melittin can generate transmembrane pores with an internal diameter of approximately 4.4 nm. These pores may facilitate the cell penetration of other therapeutics, including anticancer therapies (including chemotherapeutic agents). This can be of particular benefit in respect of anticancer therapies that otherwise do not easily penetrate into tumour cells, for example, due to their size.
• the anticancer therapy is a chemotherapeutic agent, specifically docetaxel, trastuzumab, pertuzumab and/or cisplatin.
• the anticancer therapy is omomyc or Cas9-gRNA or a variant thereof.
• the invention provides a method of inhibiting PD-L1 expression in a subject suffering from cancer comprising the step of administering melittin or a melittin fusion peptide in a therapeutically effective amount to the subject, and the step of administering a chemotherapeutic agent.
• the chemotherapeutic agent is docetaxel, trastuzumab, pertuzumab, and/or cisplatin.
• the administration of a therapeutically effective amount of melittin or melittin fusion peptide to a subject suffering from cancer reduces the expression of PD-L1 in tumour cells, which can otherwise be upregulated by the administration of some chemotherapeutic agents, including trastuzumab. This can result in improved anti-tumoral immune responses by the subject.
• the administration of melittin or a melittin fusion peptide in a therapeutically effective amount can result in improved anti-tumoral immune responses by the subject, and reduce any upregulation of PD-L1 expression by the chemotherapeutic agent in the subject.
• the chemotherapeutic agent is docetaxel, trastuzumab, pertuzumab, and/or cisplatin and the subject is suffering from triple negative breast cancer, HER2-enriched breast cancer or ovarian cancer.
• the invention provides a kit for treating cancer comprising a melittin fusion peptide together with instructions as to how to use that fusion peptide in accordance with this invention.
• the melittin fusion peptide is of SEQ ID NO: 3 or a variant thereof.
• the kit can additionally comprise a chemotherapeutic agent. More preferably, the chemotherapeutic agent is docetaxel, trastuzumab, pertuzumab, and/or cisplatin.
• the kit can additionally comprise another anticancer therapy. More preferably, the anticancer therapy is omomyc or Cas9-gRNA.
• kits can be packaged in separate containers and, associated with such containers, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
• the liquid solution can be an aqueous solution, for example a sterile aqueous solution.
• the expression construct may be formulated into a pharmaceutically acceptable syringeable composition.
• the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an affected area of the animal, such as the breast, injected into an animal, or even applied to and mixed with the other components of the kit.
• the components of the kit may also be provided in dried or lyophilized forms.
• kits of the invention also may comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal or patient.
• an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
• the invention comprises a nucleic acid molecule comprising the melittin fusion peptides of this invention.
• the nucleic acid molecule comprises SEQ ID NO: 7 or 8, which are nucleic acid sequences encoding the melittin fusion peptide of SEQ ID NO: 3:
• the invention comprises a vector comprising a nucleic acid molecule as described herein.
• the invention comprises a host cell comprising a nucleic acid molecule as described herein.
• selective binding agent refers to a molecule that has specificity for peptides of the invention described herein.
• Suitable selective binding agents include, but are not limited to, antibodies and derivatives thereof, polypeptides and small molecules. Suitable selective binding agents may be prepared using methods known in the art. An exemplary selective binding agent is capable of binding a portion of a peptide of the invention. Such binding agents can be utilised to determine the presence of peptides of the invention in tissue or individual cells and determine binding activity and/or localisation with other molecules.
• Selective binding agents such as antibodies and antibody fragments that bind peptides of the invention include monospecific polyclonal, monoclonal (MAbs), recombinant, chimeric, humanized such as CDR-grafted, human, single chain, and/or bispecific, as well as fragments, variants or derivatives thereof.
• Antibody fragments include those portions of the antibody that bind to an epitope on the peptide. Examples of such fragments include Fab and F(ab’) fragments generated by enzymatic cleavage of full-length antibodies.
• Other binding fragments include those generated by recombinant DNA techniques, such as the expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions.
• the selective binding agents are also useful for in vivo imaging of administered peptides of the invention.
• An antibody labelled with a detectable moiety may be administered to an animal, preferably into the bloodstream, and the presence and location of the labelled antibody in the host is assayed.
• the antibody may be labelled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.
• the invention also relates to a kit comprising selective binding agents (such as antibodies) and other reagents useful for detecting the levels and localisation of the peptides described herein in biological samples.
• selective binding agents such as antibodies
• Such reagents may include, a detectable label, blocking serum, positive and negative control samples, and detection reagents.
• antibodies may be used to detect peptides of the invention present in biological samples. Suitable samples are preferably from breast or ovarian tissue but may also include extracts of tissues such as brain, skin, heart, lung, colon, pancreas, testes, liver, muscle, prostate and bone tissues. Such antibodies of the invention may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like. [00193] In an embodiment, the invention comprises an isolated monoclonal antibody that specifically binds to melittin of SEQ ID NO: 1 and/or SEQ ID NO: 3.
• IC 5 o values for each of these dose-response assays are presented in Figure 1 D.
• the table shows the half-maximal inhibitory concentrations (IC50) of honeybee venom and melittin for the panel of breast normal and cancer cell lines tested in human and murine cell lines. Data are presented as mean ⁇ SEM in ng/pL or pM (to two decimal places). Experiments were performed in biological triplicate.
• a mouse monoclonal antibody recognizing melittin was then developed to assess the relative abundance of melittin in all honeybee and bumblebee venom samples by ELISA.
• the relative abundance of melittin was not significantly different across all of the honeybee venom samples from different locations (two-way ANOVA, p>0.999).
• melittin concentrations were significantly higher in honeybee samples compared to bumblebee venom and isotype IgG control ( Figure 3A; two-way ANOVA, p ⁇ 0.001 ).
• TNBC cells were treated with the IC50 of either honeybee venom or melittin for 18 and 24 hours, and examined with a cleaved caspase-3 assay to quantify apoptotic cell death.
• Immunoblotting confirmed the induction of cleaved caspase-3 in SUM159 cells, with melittin alone inducing a higher level of apoptosis than honeybee venom at both 18- and 24-hours post-treatment (Figure 4A).
• a bi-functional melittin peptide was generated by engineering an N-terminal alpha helical RGD peptide motif (RGD1 -melittin, derived from TGF- b3; SEQ ID NO 2: HGRGDLGRLKK).
• Melittin was found to predominantly localize in the plasma membrane of cells overexpressing EGFR with honeybee venom, melittin, and RGD1 -melittin treatments, with a degree of intracellular staining observed for honeybee venom and melittin -treated cells, potentially due to membrane disruption and the formation of endosomes.
• honeybee venom and melittin disrupt RTK- associated signaling pathways by blocking the ligand-dependent activation of EGFR and FIER2 in breast carcinoma cells.
• immunoblotting analysis was conducted on SKBR3 (HER2 + , EGFR + ) and SUM159 (EGFR + ) extracts of cells exposed to EGF and treated with the IC 5 o of honeybee venom or melittin from 2.5-20 minutes ( Figure 8A-B). Both honeybee venom and melittin down- regulated the phosphorylation of the RTKs and modulated associated PI3K/Akt and MAPK signaling pathways in a time dependent manner.
• the MAPK and Akt pathways may have been upregulated in SUM159 cells due to the release of a negative regulatory feedback loop that triggers ERK signaling to protect the cells from apoptotic cell death.
• the anti-melittin antibody indicated an increasing amount of melittin present in the lysates of both cell lines over time, with a stronger signal for the melittin treatment compared to honeybee venom in both cell lines.
• BRET bioluminescence resonance energy transfer
• a range of concentrations of each peptide were selected including the IC 5 o of FITC- melittin, with the corresponding molar concentrations of FITC-DEDE-melittin. It was found that the BRET signal increased in a dose-dependent manner for TAMRA-EGF and FITC-DEDE- melittin, and to a lesser extent FITC-melittin (Figure 10A-C).
• the murine p53 TNBC cell line T11 was treated with docetaxel or cisplatin in combination with either honeybee venom or melittin, and cell viability assays were conducted to determine the combination index (Cl) between the treatments ( Figure 12A).
• a Combination Index less than 1 was observed for all the concentrations tested indicating strong synergistic interactions (Figure 12B).
• mice were transplanted in BALB/c mice. This allograft model recapitulates highly aggressive, TNBC claudin-low disease in mice with an intact immune system.
• tumour control for the combination treatment was superior compared to either treatment alone or vehicle, particularly on days 7 and 9 post-inoculation of T11 cells, with the combination achieving a significant reduction in tumour volume (Figure 13A, one-way ANOVAs, p ⁇ 0.001). This suggests that tumours resistant to docetaxel could be rendered sensitive by the addition of melittin.
• tumour cell proliferation assessed by Ki-67 expression
• melittin and docetaxel 5.7 ⁇ 0.8 % relative to vehicle (59.8 ⁇ 1.7 %), compared to either melittin (31.7 ⁇ 1.3 %), or docetaxel alone (21.0 ⁇ 1.3 %; one-way ANOVA, p ⁇ 0.01 , mean ⁇ SEM).
• TUNEL staining confirmed a significantly higher DNA fragmentation and apoptosis induction in the combination group (81.0 ⁇ 3.1 %) compared to vehicle (1.0 ⁇ 0.4 %; one-way ANOVA, p ⁇ 0.01 , mean ⁇ SEM).
• PD-L1 immune checkpoint protein programmed death ligand-1
• melittin significantly reduced PD-L1 expression in tumours when used alone (52.9 ⁇ 2.4 %) or with the combination (44.3 ⁇ 4.2 %) compared to vehicle (84.9 ⁇ 1 .6 %; one-way ANOVA, p ⁇ 0.01 , mean ⁇ SEM).
• p-EGFR For p-EGFR (Tyr1068), the phosphorylation was reduced to a significantly lower level by melittin and docetaxel combination treatment (9.0 ⁇ 2.4 %) compared to vehicle (100.0 ⁇ 8.1 %; one-way ANOVA, p ⁇ 0.0001 , mean ⁇ SEM).
• the murine p53 TNBC cell line T11 was treated with docetaxel in combination with either melittin or RGD1 -melittin, and cell viability assays were conducted to determine the combination effect between the treatments (Figure 15). Both melittin and RGD1 -melittin had a beneficial effect when combined with docetaxel in vitro. The combination of RGD1 -melttin and docetaxel reduced tumour cell viability more effectively than either RGD-1 melittin or docetaxel alone.
• the murine p53 TNBC cell line T11 was treated with omomyc in combination with either melittin or RGD1 -melittin, and cell viability assays were conducted to determine the combination effect between the treatments (Figure 16). Both melittin and RGD1 -melittin had a beneficial effect when combined with omomyc in vitro. The combination of RGD1-melttin and omomyc reduced tumour cell viability more effectively than either RGD-1 melittin or omomyc alone.
• the OVCAR3 and COV362 ovarian cancer cell lines were treated with active RGD1 - melittin and mutant RGD1 -melittin (DEDE-RGD1 -melittin).
• the COV362 cells were cultured in DMEM low glucose medium supplemented with 10% FBS.
• the OVCAR3 cells were grown in RPMI medium supplemented with 20% FBS. Cells were seeded overnight at a density of 5000 cells/p96-well plate and treated with active RGD1 -melittin and mutant RGD1 -melittin (RGD1 - DEDE melittin), for24h at concentrations up to 20 mM.
• the IC50 for active RGD1 -melittin for the OVCAR3 cell line was 3.358mM, compared with 18.15mM for active melittin (approximately 6 fold lower), whereas IC50 for active RGD1 - melittin for the COV362 cell line was 2.095mM, compared with 7.81 OmM for active melittin (approximately 3.7 fold lower).
• the results of the cell viability assays are provided in Figure 17.
• cells were fixed with 4% paraformaldehyde for 20 min, washed twice with PBS, blocked with 5% normal goat serum for 1 h at room temperature and separately incubated with a- Ki-67 (Cell Signaling Technology #9449, 1 :500) and a-PD-L1 (Abeam #238697; 1 :500) in antibody diluent (1% BSA and 0.3% Triton X-100 in PBS) overnight at 4 oC.
• a- Ki-67 Cell Signaling Technology #9449, 1 :500
• a-PD-L1 Abeam #238697; 1 :500
• the cells were washed three times with PBS and then incubated with a goat a-mouse secondary Alexa Fluor 488-conjugated antibody (Thermo Fisher Scientific #A11001 , 1 :500) and Hoechst-33258 (1 :5000) in PBS for 1 h at room temperature. After three more washes of PBS, the coverslip containing cells were mounted onto glass coverslips with SlowFade Diamond Antifade Mountant (Thermo Fisher Scientific). Slides were imaged using the confocal fluorescence Nikon Ti inverted microscope. Images were taken at 40x magnification and processed using ImageJ.
• the level of PD-L1 expression was also measured in T11 , SUM159 and SKBR3 treated with active RGD1 -melittin and mutant RGD1-melittin peptides, or vehicle treated only, using qRT-PCR.
• Relative quantification of PD-L1 transcript expression was obtained by quantitative real-time PCR (qRT-PCR) using fluorescent TaqMan probes (Applied Biosystems) and run on the ViiA 7TM Real-Time PCR machine (Applied Biosystems). Data were analysed by QuantStudio Real-Time PCR Software (Applied Biosystems) and results were expressed as fold change compared to untreated cells, respectively, after normalization against the GAPDH mRNA levels.

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