Method of treating cancer

Abstract

The disclosure relates to a method of treating cancer in a subject, the method comprising administering (a) a wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof; or (b) a Staphylococcus aureus biofilm-conditioned medium (BCM) or a purified fraction thereof comprising a wild-type Staphylococcus aureus Hla polypeptide to the subject, wherein the cancer expresses a disintegrin and metalloprotease 10 (ADAM10). Also disclosed is a method of preparing Staphylococcus aureus BCM comprising culturing Staphylococcus aureus within a wool scaffold, such as aquaria filter wool, in a culture media.

Description

[0001] Method of Treating Cancer

[0002] Field of Invention

[0003] The invention relates generally to the field of oncology. In particular, the specification teaches a method of treating cancer and pharmaceutical compositions thereof.

[0004] Background

[0005] SCC is the cancer of squamous epidermal keratinocytes and adnexal structures and usually develops on sun-exposed skin such as the head and neck. As of 2015, there arc approximately 2.2 million people with SCC at any one time. Surgical excision remains the gold standard treatment strategy for fully developed SCC. Surgical excision relies on the use of large excisional margin to prevent cancer recurrence, which often results in large excisional wounds that are difficult to close. Patients unable to undergo surgical excision have to rely on radiotherapy, which while providing a good cure rate increases the chances of future skin cancer. The efficacy of chemotherapy in SCC therapy has not yet been well established. Other treatment strategics currently available, such as cryotherapy and topical imiquimod creams, are ineffective against fully developed SCC. The use of epidermal growth factor receptor (EGFR) inhibitors such as monoclonal antibodies or small molecule kinase inhibitors in SCC therapy is currently investigated. Although many such compounds are in clinical trials, they have not yet demonstrated their potential as a stand-alone treatment strategy.

[0006] It would be desirable to overcome or ameliorate at least one of the above-described problems, or at least to provide a useful alternative.

[0007] Summary

[0008] Disclosed herein is a method of treating cancer in a subject, the method comprising administering a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof to the subject, wherein the cancer expresses A Disintegrin and Metalloprotease 10 (ADAM 10).

[0009] Disclosed herein is a method of killing a cancer cell, the method comprising contacting the cancer cell with a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof for a sufficient time and under conditions to kill the cancer cell, wherein the cancer expresses ADAM10.

[0010] Disclosed herein is a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof for use in treating cancer in a subject, wherein the cancer expresses ADAM10.

[0011] Disclosed herein is the use of a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof in the manufacture of a medicament for treating cancer in a subject, wherein the cancer expresses ADAM10.

[0012] Disclosed herein is a pharmaceutical composition comprising a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof.

[0013] Disclosed herein is a pharmaceutical composition comprising a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof for use in treating cancer in a subject, wherein the cancer expresses a ADAMIO.

[0014] Disclosed herein is the use of a pharmaceutical composition comprising a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof in the manufacture of a medicament for treating cancer in a subject, wherein the cancer expresses ADAMIO.

[0015] Disclosed herein is a method of treating cancer in a subject, the method comprising administering a pharmaceutical composition comprising a Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof to the subject, wherein the Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof comprises a wild-type Staphylococcus aureus HLA polypeptide, wherein the cancer expresses ADAMIO.

[0016] Disclosed herein is a pharmaceutical composition comprising a Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof, wherein the Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof comprises a wild-type Staphylococcus aureus HLA polypeptide.

[0017] Disclosed herein is a pharmaceutical composition comprising a Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof for use in treating cancer in a subject, wherein the Staphylococcus aureus biofilm-conditioncd medium or a purified fraction thereof comprises a wild-type Staphylococcus aureus HLA polypeptide, wherein the cancer expresses ADAMIO.

[0018] Disclosed herein is the use of a pharmaceutical composition comprising a Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof in the manufacture of a medicament for treating cancer in a subject, wherein the Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof comprises a wild-type Staphylococcus aureus HLA polypeptide, wherein the cancer expresses ADAMIO.

[0019] Disclosed herein is a method of preparing Staphylococcus aureus biofilm-conditioned media (BCM), the method comprising a) culturing Staphylococcus aureus within a wool scaffold in a culture media for a sufficient time and under conditions to allow accumulation of Staphylococcus aureus biofilm, and b) isolating the culture medium to obtain Staphylococcus aureus BCM.

[0020] Brief Description of Drawings

[0021] Embodiments of the present invention are hereafter described, by way of non-limiting example only, with reference to the accompanying drawings in which: Figure 1. Generation of TSB-based static culture SA29213 BCM. (A) Schematic of static BCM generation protocol. BCM was harvested every 24 h for 4 days. Bacteria in suspension was defined as the planktonic population and bacteria adherent on the aquaria filter wool was defined as the biofilm population. (B) CFU / mL of SA29213 planktonic and biofilm population of each BCM culture day. CFU was adjusted to per mL of BCM (40 mL). (C) SA29213 biofilm accumulation on aquaria filter wool. Observable biofilm accumulation on aquaria filter wool is indicated with white arrows. Image acquired using SPE confocal microscope at 20 X magnification. (D) Protein concentration of BCM of each culture day as quantified by Bradford Protein Assay. (E) Effect of BCM of each culture day on SCC-12 metabolic activity. Cells were treated with 10% BCM of each culture day. Metabolic activity was assessed at 24 h post treatment using MTT assay. Data is shown as mean ± SEM of three technical replicates. N=l. Cell viability was normalised to control. Two-way ANOVA analyses with post hoc Bonferroni's multiple comparisons test were performed between the various BCMs and control. (*: p^0.05, ns: p>0.05).

[0022] Figure 2: Low dose BCM, while highly toxic towards SCC-12, caused minimal HaCaT cell death. (A) Co-culture of HaCaT (green) and SCC-12 treated with 2.5% BCM. SCC- 12 was shown to readily accumulate PI while HaCaT remain viable. HaCaT colonies are highlighted with a green dotted line. Image taken at 0, 6, 12, and 18 h post-treatment at 20 X magnification. HaCaT were dyed with Vybrant DiO (green). Dead cells were stained with PI (red). (B) Cell death profile of SCC-12, HaCaT, and BJ-5ta upon BCM treatment was assessed using live / dead fluorescent time-lapse microscopy. Cells were treated with 0.03 to 300 μg / mL concentrated SA29213 BCM. Cell lysis was observed in SCC-12 and HaCaT treated with 100 and 300 pg / mL concentrated SA29213 BCM and w as hence excluded from cell death quantification. Dotted line represents non-linear regression. N=3. (C) SCC-12 cell viability upon 24 h treatment with heat-treated BCM as assessed by MTT assay. Unconcentrated D3 BCM was heated for 1 h at 55°C and applied onto SCC-12 at 10% (BCM-H). (D) SCC-12 cell viability upon 24 h treatment ith undigested BCM (BCM-UD) ProK digested BCM (BCM-ProK), and ProK digested media (C-ProK) as assessed by MTT assay. Data is shown as mean ± SEM of three technical replicates. N=l. Cell viability was normalised to control. Two-way ANOVA analyses with post hoc Bonferroni s multiple comparisons test were performed between the various BCM treatments and control. (****: p≤ 0.0001, *: p≤0.05, ns: p>0.05).

[0023] Figure 3: SA29213 BCM reduces metabolic activity and initiates regulated cell death in SCC- 12. (A) BCM treatment resulted in DNA damage and PARP- 1 cleavage in SCC- 12. SCC-12 were treated with 2.5 g / mL BCM. (B) Relative protein expression of PH H2A.X of SCC-12 when treated with 2.5 μg / mL BCM. Western blot band intensity was quantified using ImageJ. Relative expression was normalised to untreated control. (C) Caspase 3 / 7 activity upon treatment with 2.5 μg / mL BCM (BCM) or 0.1 pM staurosporine (Stauro) was assessed via luminescence assay 24 h post-treatment. Luminescence was normalised to untreated control. Data is shown as mean ± SEM of three technical replicates. (D) Cleaved Caspase-3 is highly expressed in a small population of SCC-12 4 h post BCM treatment. SCC-12 was treated with 2.5 μg / mL BCM. Cell monolayer was labelled for nucleus (blue), cleaved caspase-3 (green) and actin (red). Image acquired using SP8 confocal microscope at 40 X magnification. Scale bar represents 50 pm. (E) HMGB 1 translocates out of the nucleus into the cytoplasm upon BCM treatment. SCC-12 was treated with 2.5 μg / mL BCM. Cell monolayer was labelled for nucleus (blue), HMGB1 (green) and actin (red). Image acquired using SP8 confocal microscope at 40 X magnification. Scale bar represents 50 pm.

[0024] Figure 4: Caspase inhibition delayed SA29213 BCM-induced SCC-12 cell death. (A) Morphology of SCC-12 treated with 2.5 μg / mL concentrated SA29213 BCM 18 h posttreatment. White arrow heads highlight giant membrane blebs observed in BCM- induced SCC-12 cell death. (B) Cell death profile of SCC-12 upon BCM treatment was assessed using live / dead fluorescent time-lapse microscopy. Cells were treated with 2.5 μg / mL BCM along with 50 pM of either Fer-1, Nec-1, Z-VAD-FMK, or no inhibitor (BCM). Data is shown as mean ± SEM of three technical replicates. (C) Representative images of SCC-12 treated with 2.5 μg / mL BCM along with 100 pM of either Fer-1, Nec-1, Z-VAD-FMK, or no inhibitor (BCM) at 0, 6, 12 h post-treatment. Scale bar represents 150 pm. Figure 5. Staphylococcal Hla is the primary source of SCC-12-specific toxicity in SA29213 BCM. (A &B) Effect of Hla antibody neutralisation on SCC-12 cell viability. Cell were treated with 5 μg / mL BCM with or without 1:100 anti-Staphylococcal Hla rabbit antisera. (A) Representative images of SCC-12 24 h post-treatment taken at 10 X magnification. Dead cells were stained with PI (red). Data is shown as mean ± SEM of three technical replicates. Scale bar represents 250 pm. (B) Cell metabolic activity was assessed by MTT assay 24 h post-treatment. (C) Cell death profile of SCC-12, HaCaT, and BJ-5ta upon Hla treatment was assessed using live / dead fluorescent time-lapse microscopy. Cells were treated with 0.001 to 30 μg / mL purified Hla. Line represents non-linear regression. N=3. (D&E) ADAM 10 knockdown increases SCC-12 tolerance towards Hla. (D) Percentage live cells of ADAMI 0 transfected cells (ADAM10), sham transfected (NEG) and untransfected cells (CTRL) treated with 2.5 pg / mL SA29213 BCM was assessed by livc / dcad fluorescence microscopy 24 h post-treatment. Ordinary one-way ANOVA analyses with post hoc Dunnett’s multiple comparisons test of conditions were performed with respect to ADAM 10 transfected cells (****: p <0.0001; ns: p> 0.05). (E) Cell death profile of DsiRNA ADAM10 transfected and untransfected SCC-12 upon Hla treatment was assessed using live / dead fluorescent time-lapse microscopy. Cells were treated with 0.001 to 30 pg / mL purified Hla. Line represents non-linear regression. (F) Ribbon diagram of Hla monomer. Cap, stem, and rim domain of Hla is annotated. The approximate location of amino acid residue 35 is marked with a black star. (G) Cell death profile of SCC-12 upon WT or H35L Hla treatment was assessed using live / dead fluorescent time-lapse microscopy. Cells were treated with 0.001 to 30 μg / mL purified Hla. Line represents non-linear regression. N=3.

[0025] Figure 6. Staphylococcal Hla is the primary source of SCC-12-specific toxicity in SA29213 BCM. (A) Effect of SA29213 mutant BCM (M5-8) on SCC-12 cell viability. Cells were treated with 3 μg / mL BCM, and the percentage live cells was assessed by live / dead fluorescence microscopy 24 h post-treatment. (B) Hla mRNA fold change of mutant biofilm, normalised to WT SA29213. N=4 (C) Western blot of Hla abundance in mutant (M5-8), SA29213 (WT), and HG001 (HG) D3 BCM. BCMs were loaded at equal volumes. (D) Western blot of Hla abundance in wild type SA29213 Dl-5 BCM. BCMs from various culture days were loaded at equal volumes. (E & F) Effect of Hla antibody neutralisation on SCC-12 cell viability. Cell were treated with 5 pg / mL BCM with or without 1:100 anti-Staphylococcal Hla rabbit antisera. (E) Cell metabolic activity was assessed by MTT assay 24 h post-treatment. (F) Representative images of SCC-12 24 h post-treatment taken at 10 X magnification. Dead cells were stained with Pl (red). Data is shown as mean ± SEM of three technical replicates. Scale bar represents 250 pm.

[0026] Detailed Description

[0027] The present specification teaches a method of treating cancer in a subject. Disclosed herein is a method of treating cancer in a subject, the method comprising administering a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof to the subject, wherein the cancer expresses A Disintcgrin and Mctalloprotcasc 10 (ADAM10).

[0028] In one embodiment, there is provided a method of treating skin cancer in a subject, the method comprising administering a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof to the subject.

[0029] Without being bound by theory, the inventors have found that Staphylococcus aureus (SA) strain SA29213 produces a toxin that possesses highly specific toxicity towards cutaneous squamous cell carcinoma (cSCC) cell line SCC-12. The inventors have optimized a protocol to generate Staphylococcus aureus biofilm-conditioned media (BCM), where soluble bacterial products enriched in the development of biofilms were isolated from bacterial culture and applied onto SCC cell lines. Bioactive components of SA29213 BCM display selective toxicity towards SCC-12 at low doses while non- cancerous human keratinocyte HaCaT are minimally affected. SA29213 BCM treatment causes DNA damage to SCC-12 and initiates Caspase 3-dependent regulated cell death. The use of the novel SA29213 bursa aurealis transposon mutant library led to the identification of S. aureus alpha hemolysin to be the main bioactive compound responsible for the observed SCC-12-specific toxicity. Antibody neutralisation of Hla eradicates cytotoxicity of SA29213 BCM towards SCC-12. Hla displays high SCC-12- specific toxicity which is exerted through primarily Hla- AD AM 10 interaction and Hla oligomerisation and pore formation. The high target- specificity and potential to cause cell death in a controlled manner highlights SA29213 Hla as a good candidate as an alternative SCC therapeutic.

[0030] In one embodiment, the polypeptide is a recombinant polypeptide.

[0031] In one embodiment, the polypeptide is or comprises a wild-type Streptococcal alpha hemolysin (Hla) (such as wild-type Staphylococcus aureus alpha hemolysin (Hla)) polypeptide or a fragment thereof to the subject. In one embodiment, there is provided a polypeptide having at least 70% (or at least 75%, 85%, 90%, 95% or 99%) sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof to the subject. The polypeptide may be modified to have enhanced efficacy in treating cancer.

[0032] In one embodiment, the polypeptide is or comprises a wild-type Staphylococcus aureus HLA polypeptide.

[0033] In one embodiment, the polypeptide consists or comprises an amino acid sequence having at least 70% sequence identity to aa7-319 of wild-type Staphylococcus aureus HLA polypeptide (Accession: HEA2945143):

[0034] ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLL VIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNS IDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESP TDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAAEN FLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTS TNWKGTNTKDKWTDRSSERYKI DWEKEEMTN (SEQ ID NO: 1).

[0035] In one embodiment, the polypeptide consists or comprises an amino acid sequence having at least 70% sequence identity to:

[0036] MADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKK LLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPR NSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILE SPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAA ENFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHW TSTNWKGTNTKDKWTDRSSERYK1DWEKEEMTNAHHHHHH (SEQ ID NO: 2).

[0037] In one embodiment, the polypeptide consists or comprises an amino acid sequence having at least 70% sequence identity to:

[0038] MKTRIVSSVTTTLLLGSILMNPVANAADSDTNIKTGTTDIGSNTTVKTGDLVTY

[0039] DKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLA WPSAFKVQLQLPDNEVAQTSDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKT GGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDR DSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDR KASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWE KEEMTN (SEQ ID NO: 3).

[0040] In one embodiment, the polypeptide is provided in a Staphylococcus aureus biofilm- conditioned media or a purified fraction thereof. The Staphylococcus aureus biofilm- conditioned media may be Staphylococcus aureus biofilm-conditioned media from SA29213.

[0041] The terms "polypeptide", "peptide", or "protein" are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acid residues are usually in the natural "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide

[0042] The term “sequence identity” as used herein refers to the extent that sequences arc identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g.. A, T, C, G and I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

[0043] In one embodiment, there is provided a polynucleotide or polynucleotide construct encoding a polypeptide as defined herein.

[0044] The term “polynucleotide” or “nucleic acid” are used interchangeably herein to refer to a polymer of nucleotides, which can be mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

[0045] The polynucleotide or polynucleotide construct may be, or may be comprised in, a vector. A “vector” as used herein may be a nucleic acid used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the target cell. Such vectors may include a promoter sequence operably linked to the nucleic acid sequence to be expressed. A vector may also include a termination codon and expression enhancers. In this specification the term “operably linked” may include the situation where a selected nucleic acid sequence and regulatory nucleic acid sequence (e.g., promoter and / or enhancer) are covalently linked in such a way as to place the expression of the nucleotide sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. Where appropriate, the resulting transcript may then be translated into a desired polypeptide.

[0046] Any suitable vectors, promoters, enhancers and termination codons known in the art may be used. Suitable vectors include viral vectors, e.g. retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, herpesvirus vectors, transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), e.g., as described in Maus el al., Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines 2016, 4, 9, which are both hereby incorporated by reference in its entirety.

[0047] The term “subject” as used throughout the specification is to be understood to mean a human or may be a domestic or companion animal. While it is particularly contemplated that the methods of the invention are for treatment of humans, they are also applicable to veterinary treatments, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as primates, felids, canids, bovids, and ungulates. The “subject” may include a person, a patient or individual, and may be of any age or gender.

[0048] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized in part by unregulated cell growth. As used herein, the term “cancer” refers to non-merastatic and metastatic cancers, including early stage and late stage cancers. By “non-metastatic” is meant a cancer that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site. The term "metastatic cancer" refers to cancer that has spread or is capable of spreading from one part of the body to another. Generally, a non- metastatic cancer is any cancer that is a Stage 0, 1, or II cancer, and occasionally a Stage III cancer. A metastatic cancer, on the other hand, is usually a stage IV cancer.

[0049] The term "cancer" includes but is not limited to, breast cancer, large intestinal cancer, lung cancer, small cell lung cancer, gastric (stomach) cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and / or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal or colorectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vulval cancer, squamous cell carcinoma, vaginal carcinoma, Hodgkin's disease, nonHodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue tumor, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, glioma, astrocytoma, glioblastoma multiforme, primary CNS lymphoma, bone marrow tumor, brain stem nerve gliomas, pituitary adenoma, uveal melanoma (also known as intraocular melanoma), testicular cancer, oral cancer, pharyngeal cancer or a combination thereof.

[0050] In one embodiment, the cancer is a cancer that expresses ADAM10. In one embodiment, the cancer is a cancer that overexpresses ADAM 10 as compared to a reference. In one embodiment, the cancer is a cancer wherein ADAM10 expression is increased (or elevated) as compared to surrounding normal / non-cancerous cell or tissue. The cancer may be a skin cancer (such as a melanoma or carcinoma), an oral cancer (such as oral squamous cell carcinoma), a hepatocellular cancer, a pancreatic cancer, a lung cancer, a gastric cancer, a bladder cancer, a breast cancer or a prostate cancer.

[0051] In one embodiment, the cancer is a skin cancer. The skin cancer may be a skin carcinoma. The skin cancer may be squamous cell carcinoma (SCC) or basal cell carcinoma (BCC).

[0052] The cancer as referred to in the present application may be determined to be expressing (or overexpressing) ADAM 10 by obtaining a cancer sample from the subject and detecting ADAM 10 expression in the cancer sample. The cancer sample (which may be, for example, a cell or tissue sample) may be determined to be overexpressing ADAM10 when ADAM10 expression is increased (or elevated) as compared to a reference (such as a sample from surrounding normal / non-cancerous cell or tissue).

[0053] The term “sample” herein is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, including both biological and environmental sources. A “biological sample” includes within its scope a collection of similar fluids, cells, or tissues isolated from a biological source, such as a whole organism or in vitro culture. Samples include but are not limited to tissue biopsies, tissue resections, tissue aspirates, swabs (c.g., buccal swabs), whole blood, plasma, scrum, urine, saliva, cerebrospinal fluid, and cell cultures, and may be obtained using any suitable method known in the art. Archival tissues, such as those having treatment or outcome history may also be used for sample extraction. The sample may be pooled from multiple aliquots. Samples include untreated, treated, diluted and concentrated samples.

[0054] Methods of detecting ADAM 10 expression are well known in the art and include methods that quantify the level of an RNA or protein expression product of the ADAM10 gene. Non-limiting examples of gene RNA expression products include messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA) and circular RNA (circRNA). Methods of detecting expression products such as RNA and proteins are well known to persons skilled in the art. Exemplary nucleic acid detection methods include blotting techniques (e.g., Northern blots), probe hybridisation-based methods, nucleic acid amplification-based methods and nucleic acid sequencing. Exemplary protein detection methods include gel electrophoresis (e.g., 2D electrophoresis), immunoassays, protein activity assays and mass spectrometry.

[0055] In some embodiments, methods herein comprise detecting an RNA expression product of ADAM 10. In one embodiment, methods herein comprise detecting the level of ADAM10 mRNA.

[0056] RNA may be analysed directly without an amplification step, using nucleic acids that specifically hybridise with a target RNA. Alternatively, the RNA may be reverse- transcribed into complementary' DNA (cDNA) before further amplification for analysis. Such reverse transcription may be performed alone or in combination with an amplification step. One example of a method combining reverse transcription and amplification steps is reverse transcription polymerase chain reaction (RT-PCR), which may be further modified to be quantitative, e.g., quantitative RT-PCR (qRT-PCR). Methods for reverse transcription with and without amplification axe generally known in the art.

[0057] Nucleic acid amplification methods include, without limitation, polymerase chain reaction (PCR) and its variants such as in situ PCR and quantitative PCR, self-sustained sequence replication and its variants, and transcriptional amplification system and its variants (Kwoh et al., 1989), followed by the detection of the amplified molecules using techniques well known to those of skill in the art. Especially useful are those detection schemes designed for the detection of nucleic acid molecules if such molecules are present in very low numbers.

[0058] Complementary DNA may also be amplified using isothermal amplification, i.e., amplification of DNA that occurs at substantially the same temperature. A number of isothermal amplification methods are known in the art, including but not limited to transcription mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), signal mediated amplification of RNA technology (SMART), strand displacement amplification (SDA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), isothermal multiple displacement amplification (MDA), helicase-dependent amplification (HDA), single primer isothermal amplification (SPIA), and cross-primed amplification (CPA).

[0059] The DNA that is amplified can be detected with generic double- stranded DNA detection reagents or with sequence-specific probes. Any method of detecting amplified DNA is suitable for use in the present methods.

[0060] Methods for assessing RNA levels that do not require conversion of the RNA to cDNA are also known in the art and are suitable for use in the methods herein. For example, the nCounter™ Analysis system from NanoString Technologies uses a digital molecular barcoding technology for multiplex measurement of RNA levels. The RNA sample is mixed with pairs of capture and reporter probes, tailored to each RNA sequence of interest. After hybridisation and washing, probe-bound target nucleic acids are immobilised to a surface to detect the fluorescent barcodes of the reporter probes. This allows for up to 1000-plex measurement with high sensitivity and without amplification bias.

[0061] RNA can also be sequenced to determine gene expression. Sequencing methods can include but are not limited to RNA-seq. In some embodiments, RNA-seq comprises reverse transcribing at least one RNA molecule to produce at least one double-stranded complementary' DNA molecule (dscDNA). Methods known in the art for creating a dscDNA library' may be used. RNA-seq can further comprise appending sequencing adaptors to the at least one dscDNA molecule, followed by amplification, and finally sequencing. Methods of sequencing known in the art, including sequencing by synthesis, can be used. The various RNA-seq methods known in the art may be used, e.g., those described in Kukurba et al. (Kukurba K.R., Montgomery S.B. RNA sequencing and analysis. Cold Spring Harb Protoc. 2015; 2015(11):951-69). Base abundances obtained using RNA-seq methods can be measured as read counts and normalised using methods known in the art. Gene abundances can also be reported in Reads Per Million (RPM) or Transcripts Per Million (TPM).

[0062] “Next-generation” sequencing (NGS) or high-throughput sequencing may also performed for DNA or RNA detection. These sequencing techniques allow for the identification of nucleic acids present in low or high abundance in a sample, or which arc otherwise not detected by more conventional hybridisation methods or a quantitative PCR method. NGS typically incorporates the addition of nucleotides followed by washing steps.

[0063] In some embodiments, methods herein comprise detecting a polypeptide expression product of ADAM10. In one embodiment, methods herein comprise detecting the level of ADAM 10 protein.

[0064] The level of ADAM 10 may be detected using antibody-based techniques such as enzyme-linked immunosorbent assay (ELISA), Luminex® assay or Western blotting. An antibody or antigen-binding fragment thereof that binds to ADAM10 may be used. The antibody may be further conjugated to a detectable label (such as a fluorescent, luminescent or enzyme label) to allow detection. Alternatively, the antibody may be detected using a secondary antibody that is conjugated to a label (such as a fluorescent, luminescent or enzyme label). In other embodiments, surface plasmon resonance (SPR) may be used to detect the interaction between ADAM 10 and an antibody or antigenbinding fragment that recognises it, and used to quantify the amount of the polypeptide in a sample.

[0065] Non-immunological methods may also be used to detect a polypeptide product of ADAM10. For instance, labelled aptamers (e.g., a radiolabeled, chromophore-labeled. fluorophore-labeled, or enzyme-labeled aptamer) may be used for polypeptide binding and detection. An aptamer refers to a nucleic acid that has a specific binding affinity for a target molecule. A suitable aptamer can be identified using any known method, including but not limited to the SELEX process, and prepared or synthesised in accordance with any known method, including chemical synthesis methods and enzymatic synthesis methods. Other binding agents for detecting a polypeptide may include, e.g., small molecules, lectins, ligand-binding receptors, affybodies, ankyrins, alternative antibody scaffolds (e.g., diabodies), imprinted polymers, avimers, peptidomimetics, peptoids, peptide nucleic acids, threose nucleic acids, synthetic receptors, and modifications and fragments of these.

[0066] ADAM10 protein may also be quantified, via its proteotypic peptides, by known mass spectrometry techniques, non-limiting examples of which include: clcctrospray ionization mass spectrometry (ESI-MS), ES1-MS / MS, ESI-MS / (MS)n, matrix-assisted laser desorption ionization timc-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI- TOF-MS), desorption / ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), tandem time-of-flight (TOF / TOF) technology, atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS / MS, APCI-(MS)n, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS, and APPI-(MS)n, quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), quantitative mass spectrometry, and ion trap mass spectrometry. Any of these techniques may be combined with selected reaction monitoring (SRM) to produce more targeted quantitative measurements for a polypeptide product.

[0067] In one embodiment, the cancer is determined to overexpress ADAMIO if it has increased expression of ADAMIO (e.g. polypeptide or polynucleotide expression product) as compared to a reference.

[0068] As used herein, the term “increase” or “increased” with reference to a biomarker such as ADAM 10 refers to a statistically significant and measurable increase in the biomarker as compared to a reference. The increase may be an increase of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%.

[0069] In one embodiment, an increase in the level of ADAM 10 as compared to a reference is an increase of 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, 24 fold, 25 fold, 26 fold, 27 fold, 28 fold, 29 fold, 30 fold, 31 fold, 32 fold, 33 fold, 34 fold, 35 fold, 36 fold, 37 fold, 38 fold, 39 fold, 40 fold, 41 fold, 42 fold, 43 fold, 44 fold, 45 fold, 46 fold, 47 fold, 48 fold, 49 fold, 50 fold, 51 fold, 52 fold, 53 fold, 54 fold, 55 fold, 56 fold, 57 fold, 58 fold, 59 fold, 60 fold, 61 fold, 62 fold, 63 fold, 64 fold, 65 fold, 66 fold, 67 fold, 68 fold, 69 fold, 70 fold, 71 fold, 72 fold, 73 fold, 74 fold, 75 fold, 76 fold, 77 fold, 78 fold, 79 fold, 80 fold, 81 fold, 82 fold, 83 fold, 84 fold, 85 fold, 86 fold, 87 fold, 88 fold, 89 fold, 90 fold, 91 fold, 92 fold, 93 fold, 94 fold, 95 fold, 96 fold, 97 fold, 98 fold, 99 fold or 100 fold increase, or anywhere in between.

[0070] The reference may be ADAM10 expression level in a sample from a subject of the same species without cancer, or an average expression level in samples from a population of subjects of the same species (e.g., of varying ages, ethnic backgrounds and genders) without cancer. The reference may be ADAM10 expression level in a non-cancerous tissue sample from the same subject. The reference may also be the expression level in a sample from the same subject before the suspected onset of a cancer. Reference values may be ADAM10 mRNA or protein levels. The reference values can be a predetermined value and can be stored in a database and used as a reference in subsequent analyses. In one embodiment, methods herein comprise the step of comparing the level of ADAM10 expression in the sample to the reference.

[0071] The measured expression level of a gene may first be normalised before comparison with a reference. Normalisation is typically used to control for unwanted biological variation. In a non-limiting example, biological variation can result from some feature of the subject or the sample collection that is not relevant to the methods of the present disclosure, such as variations created by collecting samples at different times of the day and variations due to the age or gender of the subject.

[0072] Normalisation can be performed using methods known in the art. In a non-limiting example, normalisation is performed by dividing the measured expression level of ADAM10 by the expression level of a reference or housekeeping gene. Useful reference or housekeeping genes are genes that show a low variation in their expression level across a variety of different samples and subjects. For example, a useful reference gene will show the same expression level in samples derived from subjects who have cancer and in samples derived from subjects who do not have cancer. Such reference or housekeeping genes are typically genes which are crucial for fundamental cellular processes such as metabolism and cell structure maintenance.

[0073] In one embodiment, there is provided a method of treating cancer in a subject, the method comprising: a) detecting an increased level of ADAM10 in a cancer sample obtained from the subject, and b) administering a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof to the subject, wherein the subject is found to have increased level of ADAM 10 in the cancer sample as compared to a reference.

[0074] In one embodiment, an increased level of ADAM 10 as compared to a reference indicates that the subject is likely to responsive to treatment with a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof.

[0075] The methods as defined herein may comprise topically or parenterally administering the polypeptide to the subject.

[0076] In one embodiment, the polypeptide is provided at a dose of about O.Olpg / ml to Ipg / ml (including O.Olpg / ml to O.lpg / ml or O.lpg / ml to Ipg / ml). For example, the polypeptide may be provided at a dose of about O.Olpg / ml, 0.02pg / ml, 0.03pg / ml, 0.04pg / ml, 0.05pg / ml, 0.06pg / ml, 0.07pg / ml, 0.08pg / ml, 0.09pg / ml, O.lpg / ml, 0.2pg / ml, 0.3pg / ml, 0.4pg / ml, 0.5pg / ml, 0.6pg / ml, 0.7pg / ml, 0.8pg / mI, 0.9pg / ml or Ipg / ml. As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition.

[0077] Disclosed herein is a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof for use in treating cancer in a subject, wherein the cancer expresses ADAM10.

[0078] Disclosed herein is the use of a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof in the manufacture of a medicament for treating cancer in a subject, wherein the cancer expresses ADAM10.

[0079] Disclosed herein is a method of killing a cancer cell, the method comprising contacting the cancer cell with a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof for a sufficient time and under conditions to kill the cancer cell, wherein the cancer cell expresses ADAM10.

[0080] The cancer cell may be a skin carcinoma cell. The cancer cell may be a skin carcinoma stem cell. The cancer cell may be a keratinocyte.

[0081] Provided herein is a pharmaceutical composition comprising a Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof, wherein the Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof comprises a wild-type Staphylococcus aureus HLA polypeptide.

[0082] The pharmaceutical composition may comprise other virulence factors such as exotoxins (superantigens, cytotoxins and / or cytotoxic enzymes), cofactors for host zymogen activation and / or exoenzymes. Disclosed herein is a pharmaceutical composition comprising a Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof for use in treating cancer in a subject, wherein the Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof comprises a wild-type Staphylococcus aureus HLA polypeptide, wherein the cancer expresses ADAM10.

[0083] Disclosed herein is the use of a pharmaceutical composition comprising a Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof in the manufacture of a medicament for treating cancer in a subject, wherein the Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof comprises a wild-type Staphylococcus aureus HLA polypeptide, wherein the cancer expresses ADAMIO.

[0084] Disclosed herein is a method of treating cancer in a subject, the method comprising administering a pharmaceutical composition comprising a Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof to the subject, wherein the Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof comprises a wild-type Staphylococcus aureus HLA polypeptide, wherein the cancer expresses ADAMIO.

[0085] The methods as defined herein may comprise administering an effective amount of the polypeptide, BCM or composition as defined herein to the subject.

[0086] The term “administering” refers to contacting, applying, or providing a polypeptide, BCM or composition as defined herein to the subject.

[0087] As used herein, the term "effective amount" relates to an amount of polypeptide, BCM or composition which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. Tn one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.

[0088] Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the severity of the condition as well as the general age, health and weight of the patient to be treated.

[0089] Disclosed herein is a pharmaceutical composition comprising a polypeptide as defined herein and a pharmaceutically acceptable carrier.

[0090] The polypeptide, BCM or composition of the invention may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition. The formulation of such compositions is well known to those skilled in the art. The composition may contain any suitable carriers, diluents or excipients. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.

[0091] The carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

[0092] The polypeptide, BCM or composition of the invention may be suitable for topical administration to the skin may comprise the compounds dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gel, creams, pastes, ointments and the like. Suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Transdermal patches may also be used to administer the compounds of the invention. In one embodiment, the polypeptide, BCM or composition is provided as a topical hydrogel to the subject.

[0093] The polypeptide, BCM or composition of the invention may be suitable for topical administration in the mouth including lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatine and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0094] The polypeptide, BCM or composition of the invention may be suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes which render the compound, composition or combination isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compound, composition or combination may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0095] Preferred unit dosage composition are those containing a daily dose or unit, daily subdose, as herein above described, or an appropriate fraction thereof, of the active ingredient.

[0096] It should be understood that in addition to the active ingredients particularly mentioned above, the composition of this invention may include other agents conventional in the art having regard to the type of composition or combination in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents. preservatives, lubricants and / or time delay agents. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include cornstarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of Wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alphatocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl di stearate.

[0097] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0098] As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

[0099] Throughout this specification and the statements which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0100] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0101] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0102] Certain embodiments of the invention will now be described with reference to the following examples which arc intended for the purpose of illustration only and arc not intended to limit the scope of the generality hereinbefore described.

[0103] EXAMPLES

[0104] Methods

[0105] Cell Culture

[0106] Human skin SCC-12 (RRID: CVCL_4026), the spontaneously immortalised human keratinocytes HaCaT (RRID: CVCL_0038), and h-TERT-immortalised human fibroblast BJ-5ta (RRID: CVCL_6573) were used. Cell lines were maintained in SCC- 12 cell culture media, which were comprised of 1:1 Gibco™ Dulbccco’s Modified Eagle Medium, high glucose, sodium pyruvate (DMEM) to Gibco™ Ham’s F12 Nutrient Mixture (F12) (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS) (HyClone Laboratories, South Logan, UT, USA) and 1 % Gibco™ penicillin / streptomycin (P / S) (Thermo Fisher Scientific, Waltham, MA, USA). All cells were grown as monolayers in a 5% CO2humidified chamber at 37 °C.

[0107] Bacterial Strains Wound isolate Staphylococcus aureus subsp. Aureus Rosenbach (ATCC® 29213™) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Prior to conditioned media generation, an overnight starter culture was prepared (200 rpm, 16 h, 37 °C) in Tryptic soy broth (TSB) (Sigma-Aldrich, Burlington, MA, USA).

[0108] Biofilm-Conditioned Media Preparation

[0109] Static Culture Biofilm Conditioned Media (Figure 1A) SA29213 was inoculated at OD6000.01 in T75 Nunc™ EasYFlask™ Cell Culture Flasks (Thermo Fisher Scientific, Waltham, MA, USA), with a thin layer of aquaria filter wool (0.20 g, 75 cm2) containing 40 mLTSB. Cultures were incubated statically at 37 °C, with spent media harvested and replaced with fresh TSB every 24 h for up to 96 h. Spent TSB was clarified via centrifugation (4000 rpm, 40 min, 4 °C), filter- sterilized (0.2 pm pore size), and pH- adjusted to -7.4. The resultant conditioned media (hereon referred to as BCM) were stored at -80 °C until use. Colony-forming units (CFUs) of biofilm and planktonic population per volume of BCM were enumerated daily via CFU counting. Adherent bacteria on aquaria filter wool were dissociated using sonication in a chilled Elmasonic S 30 (H) water bath sonicator (Elma Schmidbauer GmbH, Singen, Germany) (37 kHz, 10 min per cycle, 3 cycles, 1 min vortex after each cycle). Biofilm accumulation on aquaria filter wool was visualised using brightfield microscopy on Leica SP8 (Leica, Wetzlar, Germany) at 20x magnification.

[0110] High-throughput Static Culture Biofilm-Conditioned Media: SA29213 mutants were inoculated using sterile disposable plastic 96-pin replicators into 150 pL of TSB in 96- well flat bottom TC-treated microplate (Corning, Corning, NY, USA). Cultures were incubated shaking for 5 h (150 rpm, 37 °C) or until average turbidity reached ODeoo 0.05 and static for 48 h at 37 °C in a highly humidified environment without media change. Spent media were filter- sterilised using MultiScreen GV Filter plates (0.22 pm pore size, Merck, Rahway, NJ, USA) and collected onto fresh 96-wcll microplatc. The resultant conditioned media (hereon referred to as BCM) were stored at -80 °C until use.

[0111] Six-well Biofilm-Conditioned Media: S A29213 was inoculated at ODeoo 0.01 in Nunc™ Cell-Culture-Treated 6-well plates (Thermo Fisher Scientific, Waltham, MA, USA) with a thin layer of aquaria filter wool (0.02 g, 9.5 cm2) containing 5 mLTSB. Cultures were incubated statically at 37 °C, with spent media harvested and replaced with fresh TSB every 24 h for 72 h. Spent TSB was clarified via centrifugation (4000 rpm, 40 min, 4 °C) and filter-sterilized (0.2 pm pore size). The resultant conditioned media (hereon referred to as BCM) were stored at -80 °C until use.

[0112] Sample Concentration: SA29213 BCM was concentrated via centrifugal ultrafiltration (4 °C, 3000x g. 7 min cycles) using Vivaspin® 20 ultracentrifuge unit (Cytiva, Chicago, IL, USA) with 10,000 molecular weight cut-off (MWCO) membrane (Vivaspin 20). BCM was concentrated to the dead-stop volume and washed with 1 sample volume of cold sterile PBS, reconstituted to 2 mL with cold sterile PBS, filter-sterilised, and snap- frozen with liquid nitrogen. Protein concentration of the concentrated BCM was quantified using Quick Start™ Bradford Protein Assay (Bio-Rad, Hercules, CA, USA).

[0113] BCM Inactivation

[0114] Heat Treatment: Unconcentrated day 3 (D3) BCM was heated at 55 °C for 1 h using a water bath. Heat-treated BCM was cooled to room temperature and applied at 10% onto an SCC-12 monolayer.

[0115] Proteinase K (ProK) Treatment: D3 BCM was concentrated and buffer-exchanged using Vivaspin 20. Concentrated BCM was adjusted to protein concentration of 200 μg / mL and digested with 20 μg / mL ProK (Qiagen, Hilden, Germany) overnight at 4 °C. ProK- digested BCM was applied at 10% (protein concentration of 20 μg / mL) along with 0.1 mM PMSF onto SCC-12 monolayer.

[0116] BCM Fractionation by Fast Protein Liquid Chromatography (FLPC)

[0117] BCM was purified by acetone precipitation. Briefly, 4 volumes of cold acetone (-20 °C) were added to 1 volume of BCM and incubated at -20 °C for 1 h. Protein precipitate w'as pelleted down (4000x g, 30 min, 4 °C), and the acetone was allowed to dry at room temperature for up to 30 min. The protein pellet was resuspended in cold sterile PBS and allowed to dissolve overnight with gentle rolling at 4 °C. Solution was clarified (4000x g, 30 min, 4 °C) and concentrated using Vivaspin 20. Sample was buffer- exchanged to PBS (pH 7.4) or IEX-Q Start Buffer (20 mM Tris, 50 mM NaCI, pH 8.5).

[0118] Anion-exchange chromatography :

[0119] Purified and concentrated sample was loaded on a HiTrap Q-XL 5 mL (Cytiva, Marlborough, MA, USA) attached to AKTA Pure purification system (Cytiva, Marlborough, MA, USA), which was previously equilibrated with 10 column volumes (CV) of IEX-Q Start Buffer. The column was washed with 10 CV of TEX-Q Start Buffer. BCM was fractionated by step-elution with increasing NaCI concentrations (100, 200, 300, 400, 500 mM, and 1 M) at 5 CV intervals. Fractions containing the peaks of interest were pooled, concentrated using Vivaspin 20, and buffer-exchanged to PBS.

[0120] SCC-12 / HaCaT Co-Culture

[0121] HaCaT cells were stained with Vybrant DiO cell-labelling solution (Life Technologies, Waltham, MA, USA) at 1:200, as per manufacturer’s instructions. Stained HaCaTs were then seeded at 160,000 cells / mL together with 220,000 cells / mL SCC-12 into a 24-well plate 24 h prior to start of experiment.

[0122] Live / Dead Fluorescence Microscopy

[0123] Time-Lapse Microscopy: Cells were grown overnight on a 24-well plate (200,000 cells / mL) to 90% confluence. Cells were stained with 1 μg / mL Propidium iodide (PI) (Life Technologies, Waltham, MA, USA), 0.5 μg / mL Hoechst 33342 (Life Technologies, Waltham, MA, USA), and 0.5 pg / mL Hoechst 33258 (Life Technologies, Waltham, MA, USA) prior to treatment and imaging. Unless otherwise specified, cells were treated with 2.5 pg / mL BCM. Cell death inhibitors Fer-1 (SMLO583, Sigma Aldrich, Burlington, MA, USA), Ncc-1 (N9037, Sigma Aldrich, Burlington, MA, USA), and Z-VAD-FMK (G7231, Promega, Madison, WI, USA), and Hla-neutralising anti- staphylococcal α -toxin rabbit whole anti-serum (S7531, Sigma Aldrich, Burlington, MA, USA) were introduced along with BCM treatment. Time-lapse imaging of treated cells was performed for 18 h with Integrated Modulation Contrast (IMC) and fluorescence microscopy at 20x magnification on a Leica DM16000B inverted microscope (Leica, Wetzlar, Germany) with an attached humidified chamber (37 °C, 5% CO2). Images of random duplicate fields per well were captured with Leica DFC3000G every hour. Hoechst fluorescence of nuclei was visualised with A4 filter cube (excitation: 340-380 nm, emission: 450-490 nm). PI fluorescence of dead cells was visualised with Rho filter cube (excitation: 541-551 nm, emission: 565-605 nm).

[0124] Percentage of live cells was calculated with the following formula.

[0125] Dead cells PI positive)

[0126] Percentage (%) live cell = 1 —

[0127] Total cells (Hoechst positive)

[0128] Cell Viability and Caspase Activity Assay

[0129] Cells were grown overnight on 96-well plate (200,000 cells / mL) to 90% confluence. MTT assay was conducted using Cell Proliferation Kit 1 (MTT) (Roche, Basel, Switzerland), and absorbance was measured after incubation with solubilisation buffer for 24 h at OD570. Caspase 3 / 7 activity was assessed using Caspase-Gio 3 / 7 Assay (Promega, Madison, WI, USA), as per manufacturer’s instructions, and luminescence was read after 2 h incubation with substrate. Cytation™ 3 (BioTek, Singapore) was used to measure absorbance and luminescence.

[0130] Immunocytochemistry

[0131] SCC-12 monolayer was grown on glass coverslips (14 mm diameter round) in 24-well plates 24 h prior to the start of experiment. After BCM treatment, cells were fixed using 4% paraformaldehyde (PFA) pH 7.4 for 15 min. Coverslips were washed with cold PBS to remove residual PFA. Cells were permeabilised with 0.1% Triton-X in PBS and blocked with 1% Bovine Serum Albumin (BSA) in PBS overnight. HMGB1 and Cleaved Caspase-3 were probed with rabbit anti-HMGB l antibody (ab79823, Abeam, Cambridge, UK) (1:400) and rabbit Cleaved Caspase-3 (Asp 175) antibody (#9661, Cell Signalling Technology, Danvers, MA, USA) (1:400) for 1 h at room temperature and goat anti-rabbit Alexa 488 (#7074S, Cell Signalling Technology, Danvers, MA, USA) (1:10,000) for 1 h at room temperature. Cells were counterstained with Rhodamine Phalloidin (1:300) for 30 min and DAPI (1:10,000) for 15 min. Coverslips were mounted onto Menzel Glaser polysine glass slides with Citifluor mountant media and sealed with nail varnish. Mounted slides were stored at 4 °C. Images of random triplicate fields per coverslip were captured with Leica SP8 (Leica, Wetzlar, Germany) at 40x magnification.

[0132] Western Blot

[0133] Mammalian cells were lysed with cold RIPA lysis and extraction buffer (Life technologies, Waltham, MA, USA, Cat No. 89900), complete with 0.2 mM EDTA, 10 LI M DTT, and 1 X Protease Inhibitor cocktail (Nacalai Tesque, Kyoto, lapan). Protein concentration was quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The, 20 pg of cell lysate protein or whole BCM was clcctrophorcscd and transferred to nitrocellulose membrane using iBolt 2 Dry Blotting System (Thermo Fisher Scientific, Waltham, MA, USA) (20 V, 7 min). Western blots were blocked with 5% non-fat milk for 1 h at room temperature. Phospho-Histone H2A.X and Hla were probed with rabbit anti-Phospho-H2A.X (#2577, Cell Signalling Technology, Danvers, MA, USA) (1:2000) and anti-staphylococcal a-toxin rabbit whole anti-serum (S7531, Sigma Aldrich, Burlington, MA, USA) (1:1000), respectively, in 5% non-fat milk overnight at 4 °C. Membranes were washed with PBS with 0.1% Tween- 20 and probed with goat anti-rabbit (#7074S, Cell Signalling Technology, Danvers, MA, USA) (1:10,000) secondary' antibody conjugated with horseradish peroxidase (HRP) (NA931V, GE Healthcare Life Science, Marlborough, MA, USA) (1:10,000) in 5% nonfat milk for 1 h at room temperature. Membranes were washed, and the secondary antibody was visualized with ECL Ultra (TMA-6) chemiluminescent reagent (Lumigen, Southfield, MI, USA).

[0134] Transposon Mutagenesis

[0135] Dh5a / pBursa and Dh5ot / pFA545 were kind gifts from Professor Missiakas (University of Chicago). SA29213 bursa aurealis transposon library was prepared using a previously published protocol with modification. Plasmids pFA545 and pBursa were individually passaged through the E. coli IM01B strain before transformation sequentially into SA29213 electrocompetent cells. Successfully transformed colonies were selected using tryptic soy agar (TSA; Sigma Aldrich) containing 10 pg / mL chloramphenicol and 2.5 iig / mL tetracycline (TSAchl10Tet2.5) and incubated at 30 °C. Single colonies of SA29213 / pFA545 / pBursa were diluted 4 times using sterile Milli-Q water pre-heated to 43 °C, spread onto TSA plates containing 10 μg / mL erythromycin (TS AErmio), and incubated at 43 °C for 5-7 days until individual colonies emerge. Single colonies post-mutagenesis were individually streaked onto fresh TSA and incubated at 37 °C overnight or until individual colonies emerged. Single colonies of each mutagenesis sample were inoculated into TSB in 96-well 2 mL deep well plate (Axygen, Union City, CA, USA), sealed with Breathe-easy® sealing membrane (Sigma Aldrich), and incubated overnight at 37 °C in a shaking incubator at 220 rpm. Successful mutagenesis was confirmed using antibiotic patching, where individuals resistant to 10 μg / mL erythromycin and susceptible to 10 μg / mL chloramphenicol and 2.5 μg / mL tetracycline were included in the library collection. Glycerol stocks of each individual of the library' were prepared using 25% glycerol and stored at -80 °C.

[0136] Reverse-Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)

[0137] Biofilm RNA was extracted from bacteria adhering to aquaria filter wool during the generation of static -culture 6-well BCM 3 days post-inoculation. Bacteria were digested with 0.05 mg / mL lysostaphin in TE buffer for 1 h and lysed with Trizol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s recommendations. Reverse transcription and gDNA removal were conducted using ReverTra Ace™ qPCR RT Master Mix with gDNA Remover (Toyobo, Scottsboro, AL, USA). Quantitative PCR was conducted using Luna® Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA) using the following primers: Hla-F: 5'-AAG GCC GCC AAT TTT TCC TG-3'; Hla-R: 5'-AGT GGT TTA GCC TGG CCT TC-3'. RT- qPCR data were analysed according to the double-delta Ct analysis method.

[0138] Liquid Chromatography Tandem Mass Spectrometry (LC-MS / MS) and Protein Identification (ID)

[0139] LC-MS / MS and protein ID were conducted in collaboration with NTU School of Biological Sciences (SBS) Mass Spectrometry facility. Samples were subjected to ingel tryspin digestion. Peptides were separated and analysed using a Dionex Ultimate 3000 RSLCnano system coupled to a Q Exactive instrument (Thermo Fisher Scientific, Waltham, MA, USA). Separation was performed on a Dionex EASY-Spray 75 pm x 10 cm column packed with PepMap C18 3 pm, 100 A (Thermo Fisher Scientific) using solvent A (0.1% formic acid) and solvent B (0.1% formic acid in 100% ACN) at flow rate of 300 nL / min with a 60 min gradient. Peptides were then analysed on a Q Exactive apparatus with an EASY nanospray source (Thermo Fisher Scientific, Waltham, MA, USA) at an electrospray potential of 1.5 kV. Raw data files were processed and searched using Proteome Discoverer 2.1 (Thermo Fisher Scientific) against database constructed for SA29213 (NCBI Accession: PRJNA292059). The raw LC-MS / MS data files were loaded into Spectrum Files (default parameters set in Spectrum Selector). The Mascot algorithm was then used for data searching to identify proteins using the following parameters: missed cleavage of two; dynamic modifications were oxidation (+15.995 Da) (M) and phosphorylation (+79.966 Da) (S, T, Y). The static modification was Carbamidomethyl (+57 Da) (C). Percolator was applied to filter out the false MS2 assignments at a strict falsc-discovcry rate of 1% and relaxed falsc-discovcry rate of 5%.

[0140] Example 1

[0141] SA29213 Biofilm-Conditioned Media (BCM) displays selective toxicity towards cancerous keratinocytes

[0142] To enrich S. aureus secreted factors upon biofilm accumulation, a BCM generation protocol that allows for both high output of conditioned media and a favourable biofilm- forming environment was designed and optimized. Biofilm was cultured in a T75 tissue culture flask lined with a thin layer of aquaria filter wool. This allows for biofilm accumulation in a controlled sterile environment with a large air-liquid interface for sufficient aeration and surface area for bacterial adhesion. Biofilm was cultured for 4 days in tryptic soy broth (TSB) with media change every 24 h (Figure 1 A) to encourage a higher degree of biofilm maturity without the risk of sloughing resulting from nutrient deprivation. Day 3 and 4 BCM were considered to be representative of secreted products of mature S. aureus biofilm and were pooled and concentrated for use. The selective toxicity of SA29213 BCM towards SCC-12 was first demonstrated with a co-culture of SCC- 12 / HaCaT. The use of a cancerous and non-cancerous keratinocyte co-culture allows for the modelling of tumour-edge environment. To differentiate the two cell populations, HaCaT cells were stained with Vybrant DiO and seeded alongside unstained SCC-12 cells. Low dose BCM treatment of SCC-12 / HaCaT co-culture resulted in SCC-12 cell death while HaCaT cells remained viable (Figure 2A). At 18h post-treatment with 2.5% BCM, a large population of SCC-12 accumulated PI dye (red), indicating cell death. The lack of PI staining in Vybrant DiO-stained HaCaT (green) suggested that the cells remained viable despite being in close proximity to dead SCC- 12. Furthermore, mitosis was observed in HaCaT colonies despite the presence of BCM, suggesting that the critical S A29213 BCM dosage for SCC-12 minimally affect the cell viability of HaCaT and that BCM-induced cell death does not cause secondary toxicity towards non-cancerous kcratinocytcs.

[0143] The effect of SA29213 BCM on cell viability of cancerous kcratinocytcs SCC-12, non- cancerous keratinocytes HaCaT, and non-cancerous fibroblasts BI-5ta was assessed. The 50% cytotoxic concentration (CC50) of each cell line was determined by quantitating the degree of cell death upon treatment with concentrated SA29213 for 24 h. SCC-12 displayed a much higher susceptibility (CC50=2.033 μg / mL) to BCM treatments than non-cancerous HaCaT (CC50=48.66 μg / mL) and BJ-5ta (CC50=259.2 μg / mL) (Figure 2B). The selectivity index (SI) of SA29213 BCM on SCC-12 was 23.72 and 127.5 for HaCaT and BJ-5ta, respectively.

[0144] Heat sensitivity of the bioactive compound was investigated by heating unconcentrated BCM at 55 °C for 1 h. Heat-treated BCM possessed significantly reduced toxicity towards SCC-12, whereby treatment at 10% led to approximately 60% reduction in cell metabolic activity 24 h post-treatment (p< 0.0001) (Figure 2C). This suggests that the bioactive compound rendering toxicity towards SCC-12 is heat labile. To investigate if the bioactivc compound is proteinaceous, concentrated BCM was digested with broadspectrum serine protease Proteinase K (ProK) at a low digestion temperature to degrade all proteins. ProK activity as terminated with the use of serine protease inhibitor phenylmethane sulfonyl fluoride (PMSF) prior to application on cells. Treatment ith digested BCM led to no decrease in SCC- 12 cell viability, indicating a complete removal of toxic bioactive components in BCM (p=0.5659) (Figure 2D).

[0145] Example 2

[0146] Characterisation of SA29213 BCM toxicity on cancerous and non-cancerous keratinocytes

[0147] The involvement of DNA damage, caspase activation and regulated cell death pathway in SA29213 BCM-treated SCC- 12 was investigated. Treatment with 2.5 μg / mL S A29213 BCM led to the upregulation of DNA damage marker Phospho-Histone (PH) H2A.X, indicating the presence of double- stranded DNA breaks in treated SCC- 12 (Figure 3A & B). At 24 h post-treatment of SCC-12 with 2.5 μg / mL SA29213 BCM showed a 4-fold increase in Caspase 3 / 7 activity. This is higher compared to the approximately 2.6-fold increase in apoptotic SCC-12 induced through 0.1 pM staurosporine treatment (Figure 3C). Treatment with 2.5 μg / mL SA29213 BCM led to increased expression in cleaved Caspase-3 (Figure 3D) and the global translocation of HMBG1 out of the nucleus into the cytoplasm by 4 h post-treatment (Figure 3E).

[0148] To distinguish the cell death pathway involved in BCM-induced SCC-12 cell death, a panel of cell death inhibitors was employed. Necrostatin-1 (Nec-1), Ferroststain-1 (Peril, and carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD- FMK) were used to inhibit cell death via necroptosis, ferroptosis and apoptosis respectively. Cell death inhibitors were introduced to SCC-12 along with 2.5 pg / mL BCM at the point of treatment. The addition of Z-VAD-FMK at 50 pM caused a delay in BCM-induced SCC-12 cell death, with percentage live cell 12 h post-treatment reduced to 60% as compared to approximately 30% without inhibitors (Figure 4A). The addition of Nec-1 at 50 pM did not influence the cell death profile of SCC-12 upon BCM treatment. Interestingly, the addition of Fcr-1 at 50 pM resulted in the formation of small membrane blebs and the lack of PI staining in blebbing cells, both of which are characteristic of classical apoptosis (Figure 4B).

[0149] Example 3 Characterisation of SA29213 Alpha Hemolysin (Hla) as an SCC-12-specific protein toxin

[0150] Staphylococcal alpha hemolysin (Hla) was identified to be the major bioactive component of SA29213 BCM responsible for the observed SCC-12-specific toxicity. The addition of anti- staphylococcal Hla antibody attenuates BCM-induced cellular toxicity, with a rescue of metabolic activity to 90.21% of untreated control (Figure 5B) and near-complete eradication of cell death (Figure 5 A). The effect of Hla in isolation on cell viability of cancerous keratinocyte (SCC-12) and non-cancerous keratinocyte (HaCaT) cell lines was assessed. Purified SA29213 Hla was produced in collaboration with NTU Protein Purification Platform (NTU-PPP). The CC50 of each cell line was determined by quantitating the degree of cell death upon treatment with purified Hla for 24 h. SCC-12 displayed a much higher susceptibility (CC50=7.031 ng / mL) towards Hla than non-cancerous HaCaT (CC50=1004 ng / mL) (Figure 5C). The selectivity index (SI) of Hla on SCC-12 was 142.8.

[0151] To understand the mechanism behind Hla toxicity in SCC-12, the role of Hla-cell surface ligand interaction was investigated. In human tissues, Hla specifically binds to cell surface ligand a disintegrin and metaHoprotease 10 (ADAM 10) at low concentrations. The essentiality of this interaction in Hla- induced SCC-12 cell death was investigated through short interfering RNA (siRNA) knockdown of ADAM 10. Reverse-transfection of lOnM ADAM10 Dicer- substrate siRNAs (DsiRNAs) resulted in approximately 80% reduction in ADAM10 mRNA expression 48 h-post transfection (results not shown). This is sufficient to attenuate SCC-12 susceptibility towards 2.5 ug / mL SA29213 BCM challenge, a concentration lethal towards untransfected SCC-12 (Figure 5D). Similarly, ADAM10 knockdown resulted in approximately 20-fold increase in tolerance towards purified Hla in SCC-12 (CC50untransfected=7.031 ng / mL, CC50transfcctcd= 139.8 ng / mL) (Figure 5E). This suggest that Hla-ADAMIO interaction, while not essential, allows Hla to exert its toxicity with ADAM 10- dependent cell type- specificity. The essentiality of Hla oligomerisation and transmembrane pore formation was also explored. Hla with a single amino acid modification H35L, a mutation which results in reduced pore-forming and oligomerisation abilities, was used in this study. Purified SA29213 Hla H35L oligomerisation-deficient mutant was produced in collaboration with NTU Protein Purification Platform (NTU-PPP). The loss of oligomerisation and pore-forming abilities drastically reduced Hla toxicity towards SCC-12. H35L is approximately 100 times less toxic than WT Hla towards SCC-12 as assessed through live / dead florescence microscopy (CC50WT=7.031 ng / mL, CC50H35L= 722.3 ng / mL) (Figure 5G). This strongly suggest that Hla oligomerisation and pore formation is important for efficient death induction in SCC-12.

[0152] Example 4

[0153] Identification of SCC-12-Specific SA29213 Protein Toxin

[0154] To identify the SCC-12-specific protein toxin present in SA29213 BCM, a 13,025- membered SA29213 transposon mutant library was constructed. Transposition was conducted using transposon bursa aurealis based on a protocol that was previously established. Transposon mutants were individually screened for the loss of SCC-12- specific toxicity in BCM generated using a modified high-throughput 96-well protocol. Initial screen identified 65 mutants to have reduced toxicity towards SCC-12, of which 37 were false-positives and 10 had reduced growth rates. Of the remaining 18 mutants, 9 produced BCM with low protein concentrations. BCM was generated for the remaining 9 mutants using a modified 6-well protocol and the toxicity of the mutant BCMs towards SCC-12 were verified. D3 BCM of all mutants except mutant 4 and 6 showed negligible toxicity towards SCC-12 24 h post-treatment when applied at 3 μg / mL (Figure 6A). S', aureus strain HG001 which produce BCM with low and no toxicity towards SCC-12, were included as controls.

[0155] LC-MS / MS of wild type (WT) SA29213 BCM ion-exchange fast protein liquid chromatography (FPLC) fractionation identified several proteins to be present in the fraction retaining toxicity towards SCC-12. The mRNA expression level of the corresponding genes in each mutant were quantified through RT-qPCR using RNA isolated from D3 bacteria biofilms. Of the genes of interest studied, the expression of alpha hemolysin (Hla) was significantly reduced in most of the identified mutants (Figure 6B). Western-blotting of mutant BCM also showed a reduction of Hla in mutants generating BCM with low toxicity towards SCC-12 (Figure 6C). hi addition, protein expression of Hla increases with prolonged culturing in WT SA29213, with expression notably high in BCMs after 4 days of culturing (Figure 6D). The addition of anti- staphylococcal Hla attenuates BCM-induced cellular toxicity, with a rescue of metabolic activity to 90.21% of untreated control (Figure 6E) and near-complete eradication of cell death (Figure 6F).

[0156] Example 5

[0157] Conclusion

[0158] A highly efficient and cost-effective way of generating .S'. aureus BCM for the study of S. aureus biofilm-associated soluble products has been described. The protocol allows for good accumulation of biofilm that is easily observable and quantifiable. The use of easily attainable aquaria filter wool as scaffold for the enrichment of biofilm makes the protocol much more accessible compared to the use of trans-well inserts and flow reactor systems in previously published protocols. In addition, this system allows effective enrichment of biofilm associated macromolecules and structures including extracellular nucleic acids and microvesicles, which could exert synergistic effects to secreted toxins.

[0159] The findings demonstrate S. aureus Hla to be the primary agent within SA29213 BCM causing SCC-12-specific cell death. Protein expression of Hla corresponds to the extent of toxicity towards SCC-12, and the addition of anti-Hla whole sera effectively neutralises SA29213 BCM toxicity. Hla is a beta-barrel pore forming toxin (PFT) that is responsible for various S. aurcus-associatcd malignancies.

[0160] Although bacterial toxins have been investigated for their potential application in various cancers, there is currently no reports of its use in skin cancer therapy. Unlike bacterial-mediated cancer therapy, the use of bacterial protein toxin eliminates the use of live bacteria, which can pose the risk of exaggerated infections. SA29213 BCM and Hla is effective at low concentrations (CC50=2.O33 μg / mL and 7.031 ng / mL, respectively), indicating it potency towards the SCC cell line. SA29213 BCM severely reduces metabolic activity of SCC- 12 and actively causes DNA damage and cell death.

Claims

CLAIMS1. A method of treating cancer in a subject, the method comprising administering a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof to the subject, wherein the cancer expresses a disintegrin and metalloprotease 10 (ADAM 10).

2. The method of claim 1, wherein the cancer overexpresses ADAM 10 as compared to a reference.

3. The method of claim 1 or claim 2, wherein the cancer is a skin carcinoma.

4. The method of any one of claims 1 to 3, wherein the cancer is squamous cell carcinoma (SCC).

5. The method of any one of claims 1 to 4, wherein the polypeptide is a wild-type Staphylococcus aureus HLA polypeptide.

6. The method of claim 5, wherein the polypeptide is provided in a Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof.

7. The method of claim 6, wherein the Staphylococcus aureus biofilm-conditioned media or a purified fraction thereof is from SA29213.

8. The method of any one of claims 1 to 7, wherein the method comprises topically or parenterally administering the polypeptide to the subject.

9. The method of any one of claims 1 to 8, wherein the polypeptide is provided at a dose of about 0.01 lg / mL to Ipg / ml.

10. A method of killing a cancer cell, the method comprising contacting the cancer cell with a polypeptide having at least 70%> sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof for a sufficient time and under conditions to kill the cancer cell, wherein the cancer expresses ADAM 10.

11. A polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof for use in treating cancer in a subject, wherein the cancer expresses ADAM 10.

12. Use of a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof in the manufacture of a medicament for treating cancer in a subject, wherein the cancer expresses ADAMI 0.

13. A pharmaceutical composition comprising a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof.

14. A pharmaceutical composition comprising a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof for use in treating cancer in a subject, wherein the cancer expresses ADAMI 0.

15. Use of a pharmaceutical composition comprising a polypeptide having at least 70% sequence identity to wild-type Staphylococcus aureus alpha hemolysin (Hla) polypeptide or a fragment thereof in the manufacture of a medicament for treating cancer in a subject, wherein the cancer expresses ADAM 10.

16. A method of treating cancer in a subject, the method comprising administering a pharmaceutical composition comprising a Staphylococcus aureus biofilm- conditioned medium or a purified fraction thereof to the subject, wherein the Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof comprises a wild-type Staphylococcus aureus HLA polypeptide, wherein the cancer expresses ADAM10.

17. A pharmaceutical composition comprising a Staphylococcus aureus biofilm- conditioned medium or a purified fraction thereof, wherein the Staphylococcusaureus biofilm-conditioned medium or a purified fraction thereof comprises a wild-type Staphylococcus aureus HLA polypeptide.

18. A pharmaceutical composition comprising a Staphylococcus aureus biofilm- conditioned medium or a purified fraction thereof for use in treating cancer in a subject, wherein the Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof comprises a wild-type Staphylococcus aureus HLA polypeptide, wherein the cancer expresses ADAM10.

19. Use of a pharmaceutical composition comprising a Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof in the manufacture of a medicament for treating cancer in a subject, wherein the Staphylococcus aureus biofilm-conditioned medium or a purified fraction thereof comprises a wild-type Staphylococcus aureus HLA polypeptide, wherein the cancer expresses ADAM 10.

20. A method of preparing Staphylococcus aureus biofilm-conditioned media (BCM), the method comprising a) culturing Staphylococcus aureus within a wool scaffold in a culture media for a sufficient time and under conditions to allow accumulation of Staphylococcus aureus biofilm, and b) isolating the culture medium to obtain Staphylococcus aureus BCM.

21. The method of claim 20, wherein the wool scaffold is aquaria filter wool.

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