Protein aggregates

Abstract

The invention relates to a method of detecting cancer in a subject based on the level of p53 protein aggregates in a body fluid sample from the subject. The invention also relates to corresponding methods of determining the risk of cancer in a subject, monitoring the progression or onset of cancer, and / or determining the efficacy of a therapeutic intervention for treating cancer in a subject. The invention further relates to kits for use said methods.

Description

[0001] PROTEIN AGGREGATES

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The invention relates to a method of detecting cancer in a subject based on the level of p53 protein aggregates in a body fluid sample from the subject. The invention also relates to corresponding methods of determining the risk of cancer in a subject, monitoring the progression or onset of cancer, and / or determining the efficacy of a therapeutic intervention for treating cancer in a subject. The invention further relates to kits for use said methods.

[0004] BACKGROUND OF THE INVENTION

[0005] Screening of biomarkers is important for the early diagnosis of cancer at a stage when cancer may cause no or only mild symptoms. Detecting biomarkers at an early stage of cancer is often essential to improving the outcomes and promoting survival in cancer patients. Moreover, early-stage cancer treatments are often more effective, cost efficient, and less affected by side effects. A well-known cancer biomarker is the p53 protein. p53 (encoded by gene TP53) is a crucial tumour suppressor protein that protects genome integrity by regulating a wide range of cellular processes, such as DNA repair, cell cycle arrest, and apoptosis. Given its broad tumour-suppressing functions, TP53 is the most frequently mutated gene in human cancer, and the production of mutant p53 protein is intimately associated with oncogenesis. Wild-type (WT) p53 and some mutant forms of p53, such as the R248Q and R175H mutants, can form amyloid aggregates that lose the tumour-suppressing functions of p53 and exert tumour-promoting activities. p53 aggregation has been documented in various cancers, including neuroblastoma, breast cancer, and ovarian cancer. For example, glioblastoma (GB) is one of the most aggressive and recurrent brain tumours, with patients having an average survival of 14.6 months, and a five-year survival of 7.2%. Although there is currently no cure for GB, detection of GB in its nascent, low-grade form may improve patient survival, underscoring the urgent need for an effective diagnostic biomarker. The ARF-p53-MDM2 pathway, an important suppressor pathway, is dysregulated in 84% of GB patients and 94% of GB cell lines. Overexpression of mutated p53, including aggregation-prone mutants, is prevalent in GB and may lead to the accumulation of p53, and subsequently, the formation of intracellular p53 aggregates. Moreover, p53 aggregates have been found to promote chemoresistance in GB cells, suggesting a potential relationship between p53 aggregation and GB malignancy. While intracellular p53 aggregates may serve as a promising biomarker for the diagnosis and prognosis, or prediction of GB and other cancers associated with p53 aggregates, existing detection methods typically rely on cell based (e.g. biopsy-based) methods which are invasive, time consuming and costly. There exists an urgent and unmet need for methods of detecting cancer, particularly early stage cancer, that are reliable while being less invasive, less time consuming and less costly than existing cell-based methods.

[0006] SUMMARY OF THE INVENTION

[0007] The present invention is based on the surprising discovery that body fluid samples (such as blood samples) obtained from subjects with cancer exhibit significantly higher levels of p53 aggregates than body fluid samples (such as blood samples) obtained from subjects who do not have cancer. Moreover, the inventors found that the level of p53 aggregates in body fluid samples provides a reliable and powerful diagnostic tool for cancer. Advantageously, by employing body fluid samples, the methods of the invention are less invasive, less time consuming and less costly than the existing gold standard methods which typically rely on invasive pathological sections and / or CT or MRI scans.

[0008] The invention provides a method of detecting cancer in a patient, the method comprising: (a) comparing the level of p53 aggregates in a body fluid sample obtained from the patient to a threshold level; and (b) determining whether the patient has cancer based on the comparison performed in step (a).

[0009] The invention also provides a method of determining the risk of cancer in a patient, the method comprising: (a) comparing the level of p53 aggregates in a body fluid sample obtained from the patient to a threshold level; and (b) determining the risk of the patient having cancer based on the comparison performed in step (a).

[0010] The invention also provides a method for monitoring the progression of cancer or the onset of cancer in a patient, the method comprising: (a) comparing: (i) the level of p53 aggregates in a body fluid sample obtained from the patient at a first time point with (ii) the level of p53 aggregates in a body fluid sample obtained from the patient at a second subsequent time point; and (b) determining whether the cancer has progressed or onset of cancer has occurred between the first and second time point based on the comparison performed in step (a).

[0011] The invention also provides a method for determining the efficacy of a therapeutic intervention in a patient having cancer, the method comprising: (a) comparing: (i) the level of p53 aggregates in a body fluid sample obtained from the patient prior to administration of the therapeutic intervention with (ii) the level of p53 aggregates in a body fluid sample obtained from the patient after administration of the therapeutic intervention; and (b) determining the efficacy of the therapeutic intervention based on the comparison performed in step (a).

[0012] In one embodiment, the cancer is selected from glioblastoma, astrocytoma, oligodendroglioma and metastatic cancer, optionally metastatic breast cancer, metastatic lung cancer, or metastatic adenocarcinoma. In some embodiments, the cancer is selected from ovarian cancer, oesophageal cancer, lung cancer, breast cancer, and adenocarcinoma. In some embodiments, the metastatic cancer is metastatic ovarian cancer or metastatic oesophageal cancer. In some embodiments, the lung cancer is late-stage lung cancer.

[0013] In one embodiment, the method further comprises quantifying the level of p53 aggregates in the body fluid sample(s).

[0014] In one embodiment, the method comprises contacting the body fluid sample with a p53 aggregate-specific binding agent.

[0015] In one embodiment, the method comprises: (a) contacting the body fluid sample with a p53 capture binding agent to provide an enriched sample; (b) contacting the enriched sample with a p53 detection binding agent; and (c) detecting binding of the p53 aggregate-specific binding agent.

[0016] In one embodiment, the p53 detection binding agent is selected from an antibody or an antigen binding fragment thereof and an aptamer.

[0017] In one embodiment, the p53 detection binding agent binds to amino acid residues: (a) 211- 217 of SEQ ID NO: 1 , optionally wherein the p53 detection binding agent is PAb240 or an antigen binding fragment thereof; (b) 181-190 of SEQ ID NO: 1 , optionally wherein the p53 detection binding agent is DO-11 or an antigen binding fragment thereof; or (c) 256-270 of SEQ ID NO: 1 , optionally wherein the p53 detection binding agent is DO-12 or an antigen binding fragment thereof.

[0018] In one embodiment, the p53 capture binding agent is selected from an antibody or an antigen binding fragment thereof and an aptamer.

[0019] In one embodiment, the p53 capture binding agent binds to the N-terminal domain of p53, the central domain of p53 and / or to the C-terminal domain of p53.

[0020] In one embodiment, the p53 detection binding agent binds to the N-terminal domain of p53, the central domain of p53, and / or to the C-terminal domain of p53.

[0021] In one embodiment, the p53 capture binding agent is bound to a solid support, optionally where in solid support is selected from a bead, a solid surface, and a membrane.

[0022] In one embodiment the p53 capture binding agent binds to amino acid residues: (a) 20 to 25 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is DO-1 or an antigen binding fragment thereof; (b) 18 to 25 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb242 or an antigen binding fragment thereof; (c) 9 to 25 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb242 or an antigen binding fragment thereof; (d) 18 to 27 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb242 or an antigen binding fragment thereof; or (e) 46 to 55 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb1801 or an antigen binding fragment thereof.

[0023] In one embodiment, the p53 capture binding agent binds to amino acid residues: (a) 370 to 378 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb421 or an antigen binding fragment thereof or PAb122 or an antigen binding fragment thereof; (b) 371 to 380 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb421 or an antigen binding fragment thereof or PAb122 or an antigen binding fragment thereof; or (c) 388- 393 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is ICA-9 or an antigen binding fragment thereof.

[0024] In one embodiment, the body fluid sample is selected from a urine sample, a blood sample, a serum sample, a cerebrospinal fluid sample, a plasma sample, a saliva swab and a nasal swab.

[0025] The invention also provides a for detecting the level of p53 aggregate in a sample, the kit comprising: (a) a p53 detection binding agent; and (b) a p53 detection binding agent.

[0026] In one embodiment, the p53 capture binding agent binds to the N-terminal domain of p53 and / or the C-terminal domain of p53.

[0027] In one embodiment, the kit further comprises a solid support to which the p53 capture binding agent is bound, optionally wherein the solid support is selected from a bead, a solid surface and a membrane.

[0028] In one embodiment, the p53 aggregate-specific binding agent is conjugated to a solid support, optionally wherein the solid support is selected from a bead, a solid surface and a membrane.

[0029] In one embodiment, the p53 detection binding agent and / or the p53 capture binding agent are selected from an antibody or an antigen binding fragment thereof and an aptamer.

[0030] In one embodiment, (a) the p53 detection binding agent binds to amino acid residues 211-217 of SEQ ID NO: 1 , optionally wherein the p53 detection binding agent is PAb240 or an antigen binding fragment thereof; (b) the p53 detection binding agent binds to amino acid residues181- 190 of SEQ ID NO: 1 , optionally wherein the p53 detection binding agent is DO-11 or an antigen binding fragment thereof; or (c) the p53 detection binding agent binds to amino acid residues 256-270 of SEQ ID NO: 1 , optionally wherein the p53 detection binding agent is DO- 12 or an antigen binding fragment thereof. In one embodiment, (a) the p53 capture binding agent binds to amino acid residues 20-25 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is DO-1 or an antigen binding fragment thereof; (b) the p53 capture binding agent binds to amino acid residues 18-27 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb242 or an antigen binding fragment thereof; or (c) the p53 capture binding agent binds to amino acid residues 18-25 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb242 or an antigen binding fragment thereof; (d) the p53 capture binding agent binds to amino acid residues 9-25 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb242 or an antigen binding fragment thereof; or (e) the p53 capture binding agent binds to amino acid residues 46-55 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb1801 or an antigen binding fragment thereof.

[0031] In one embodiment, (a) the p53 capture binding agent binds to amino acid residues: 370 to 378 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb421 or an antigen binding fragment thereof; or (b) the p53 capture binding agent binds to amino acid residues: 388-393 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is ICA- 9 or an antigen binding fragment thereof.

[0032] DESCRIPTION OF THE FIGURES

[0033] Figure 1 : Development of the SiMoA assay. (A) Schematic illustration of the SiMoA assay. (B) Antibody pair selection. The DO-1 / DO-1 pair (top) can only detect non-monomeric p53 because the same epitope cannot bind to two identical antibodies simultaneously. The DO- 1 / PAb240 pair (bottom) can detect p53 aggregates because the PAb240 antibody targets an epitope that only exposes in unfolded p53 (aa 211-217), which is present in p53 aggregates. (C-D) AEB values of recombinant WT (right) and R248Q (left) p53 aggregates with the D-D and D-P pairs. (E-F) AEB values of the GB (left) and control (right) plasma samples with the D-D and D-P pairs. The blank levels are indicated by the dotted lines. (G) AEB values of 10 GB patients and 10 controls using the D-D pair. (H) ROC curve of the D-D pair. (I) AEB values of 10 GB patients and 10 controls using the D-P pair. (J) ROC curve of the D-P pair.

[0034] Figure 2: Validation of the presence of p53 aggregates in GB patient plasma. (A) Diffraction-limited images of the plasma samples of three GB patients (P1 , P2, P3) and two controls (C1 , C2) on the SiMPull surface. Scale bar represents 20 pm. (B) Number of fluorescence spots per FOV in the diffraction-limited images. (C) Super-resolution images of the plasma samples of two GB patients (P1 , P2) and two controls (C1 , C2). The scale bars in the full images and the insets represent 10 pm and 1 pm, respectively. (D) Comparison between the total number of localisations, number of clusters, number of localisations per cluster, and ratio of localisations in clusters of 8 GB patients vs 9 controls. The data were normalised to the corresponding values of the blank wells on the same coverslip. (E) Comparison between the mean cluster area between the plasma samples and recombinant p53 monomers and aggregates. Each data point of the plasma sample represents one individual, whereas each data point of the recombinant protein sample represents one experimental replicate. The data were normalised to the corresponding values of the blank wells on the same coverslip.

[0035] Figure 3: Calibrator development and assay optimisation. (A) Illustration of the calibrator. The DO-1- and PAb240-binding peptides are conjugated to a silica nanoparticle to mimic a p53 aggregate. An amide bond is formed between the lysine residue on the peptide and the carboxyl group on the silica nanoparticle. (B) Microscopic image of the calibrator labelled with fluorescent PAb240-antibody. The scale bar represents 20 pm. (C) Calibration curves of three experimental replicates. The blank levels are indicated by the dotted lines of at the bottom of the graph. The error bars denote the standard deviations of three on-plate replicates. (D) Signal-to-background ratios (SBR) of different detector-SBG concentration combinations. (E- F) AEB values obtained from 5 pL and 20 pL sample volumes.

[0036] Figure 4: p53 aggregate concentrations across primary and metastatic brain cancers. (A) Fitted p53 aggregate concentrations of 190 GB patients vs 22 controls. (B) ROC curve plotted from fitted p53 aggregate concentrations. (C) Normalised p53 aggregate concentrations of 190 GB patients vs 22 controls. (D) ROC curve plotted from normalised p53 aggregate concentrations. (E-F) Representative results from longitudinal study of two postoperative GB patients, followed with serial MRI scans and plasma p53 levels. Initial rise in p53 aggregate levels prior to recurrence is followed by a reduction in p53 aggregate levels posttreatment. The left chart area represents the period post-surgery during which follow-up MRI scans showed no evidence of recurrence, while the right chart area represents the period following recurrence, including stable recurrence. Dotted lines depict the date of onset of chemotherapy (dotted line - at about 450 days in (E) and at about 500 days in (F)) and further surgery (dotted line - at about 350 days in (F)). (G) p53 aggregate concentrations in the control vs metastatic brain cancers. (H) p53 aggregate concentrations in the control vs IDH-mutant primary cancer (astrocytoma and oligodendroglioma).

[0037] Figure 5: p53 aggregate detection using different p53 capture binding agents

[0038] (A) AEB values obtained using DO-1 as p53 capture binding agent and PAb240 as p53 detection binding agent for 6 GB patients and 6 controls. (B) AEB values obtained using PAb242 as p53 capture binding agent and PAb240 as p53 detection binding agent for 6 GB patients and 6 controls. (C) AEB values obtained using PAb421 as p53 capture binding agent and PAb240 as p53 detection binding agent for 6 GB patients and 6 controls. (D) AEB values obtained using PAb1801 as p53 capture binding agent and PAb240 as p53 detection binding agent for 6 GB patients and 6 controls.

[0039] Figure 6: Detection limits and total protein concentrations

[0040] (A) The lower detection limit and AEB ranges for different detector-SBG combinations; (B) Total protein concentrations measured in GB patients and control samples.

[0041] Figure 7: Diagnostic accuracy, specificity, sensitivity, and Youden indices at different cut-offs

[0042] The optimal threshold was determined by the Youden index to be 1 .895 (a.u.), with which the diagnostic accuracy, sensitivity, and specificity were 90.57%, 90.53%, and 90.91 %, respectively.

[0043] Figure 8: Synthesis of the calibrator beads

[0044] Schematic diagram showing synthesis of calibrator beads comprising p53 peptides conjugated to a silica bead.

[0045] Figure 9: p53 aggregate concentrations in ovarian cancer.

[0046] (A) p53 aggregate concentrations in the plasma samples of controls and patients with ovarian cancer. The p53 aggregate concentration in each sample was calculated from the calibration curve obtained in the same experiment. (B) ROC curve of control vs ovarian cancer (AUC=0.902).

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] The inventors made the surprising discovery that body fluid samples, such as blood samples, obtained from subjects with cancer had significantly higher levels of p53 aggregates than body fluid samples obtained from subjects who did not have cancer. Moreover, the inventors demonstrate herein that the level of p53 aggregates in body fluid samples, such as blood samples, unexpectedly serve as a reliable biomarker for cancer with high diagnostic accuracy.

[0049] Using glioblastoma as a model system, the inventors surprisingly found that the level of p53 aggregates in body fluid samples may be used to detect cancer in subjects with a high diagnostic accuracy of >90%. The inventors also discovered that p53 aggregate levels in body fluid samples are associated with glioblastoma recurrence and treatment response, highlighting the prognostic value of p53 aggregates as a cancer biomarker. For example, the inventors identified an increase in p53 aggregate levels prior to tumour recurrence and so detection of this biomarker could be used as an early indicator of tumour recurrence. Moreover, an increase in body fluid sample p53 aggregate levels was also identified in subjects with cancer having, astrocytoma, oligodendroglioma, metastatic adenocarcinoma, lung cancer and breast cancer indicating that elevated p53 aggregate levels in the body fluid sample is not specific to glioblastoma. Elevated p53 aggregate levels were also identified in subjects having ovarian cancer (with no brain metastases), oesophageal cancer and latestage lung cancer.

[0050] The body fluid-based methods of the invention offer significant advantages over cell-based p53 aggregate detection methods. For example, the methods of the invention are significantly less invasive than cell-based methods which typically rely on tissue biopsies. Advantageously, the methods of the invention may be repeated (e.g. at multiple time points) without causing significant or repeated discomfort to the subject.

[0051] Prior to the present invention, to the best of the inventors’ knowledge, it was not known that cancer associated p53 aggregates are present in body fluid samples (e.g. blood samples, urine samples, CSF samples, saliva samples, nasal swabs, preferably blood samples) or that the level of p53 aggregates in body fluid samples can be used as a diagnostic and prognostic biomarker for cancer. The present invention provides a remarkable contribution to the technical field by providing a cancer diagnostic method that has significant clinical utility.

[0052] The invention provides a method of detecting cancer in a subject, the method comprising: (a) comparing the level of p53 aggregates in a body fluid sample obtained from the subject to a threshold level; and (b) determining whether the subject has cancer based on the comparison performed in step (a). The invention also provides use of the level of p53 aggregates in a body fluid sample obtained from a subject for detecting cancer.

[0053] As used herein, the term “detecting” may be used interchangeably with “determining” or “diagnosing” and refers to the identification, confirmation and / or characterisation of a disease state (e.g. the presence or absence of cancer, or the risk of developing cancer).

[0054] As used herein, the term “aggregates” refers to a collection of proteins that are grouped together and embraces all forms of protein entities that may be present during protein aggregation, including, but not limited to, oligomers, protofibrils, filamentous aggregates (fibrils), amorphous aggregates, tangles, plaques and Lewy bodies. The aggregates are a dynamic, heterogenous group of molecules. “Aggregation” as used herein refers to a soluble protein / peptide forming an aggregated protein in its normal biological environment. Aggregates may be insoluble or soluble.

[0055] The p53 aggregates may be stable assemblies of multiple p53 monomers. These monomers may have an abnormal conformation (e.g. the monomers may be unfolded or misfolded). Such an abnormal conformation of p53 monomers (e.g. due to unfolding or misfolding of monomers) may result in the exposure of epitopes that would otherwise be hidden in a normal p53 monomer conformation. Thus, p53 monomers within an aggregate may be unfolded or misfolded.

[0056] The p53 aggregates may comprise two or more p53 protein monomers. For example, the p53 aggregates may comprise three or more, four or more, five or more, or six or more p53 monomers.

[0057] The p53 aggregates may comprise wild-type p53 monomers, mutant p53 monomers or a combination of wild type p53 monomers and mutant p53 monomers. In some embodiments, the p53 aggregates comprise only wild-type p53 monomers. In some embodiments, the p53 aggregates comprise only mutant p53 monomers. In some embodiments, the p53 aggregates comprise a combination of wild type p53 monomers and mutant p53 monomers.

[0058] In some embodiments, the p53 aggregates comprise two or more wild-type p53 monomers. In some embodiments, the p53 aggregates comprise two or more mutant p53 monomers. In some embodiments, the p53 aggregates comprise at least one wild-type p53 monomer and at least one mutant p53 monomer.

[0059] As used herein, the term “wild-type p53 monomer” refers to p53 molecules having a wild-type amino acid sequence. The human wild-type p53 amino acid sequence is set forth in SEQ ID NO:1. Wild-type p53 monomers encompass unfolded and misfolded p53 proteins, as well as p53 monomers having a normal conformation. As used herein, “wild-type monomeric p53” may also be referred to as wild-type p53 monomers and refers to p53 monomers having a wild-type amino acid sequence (i.e. does not include any amino acid mutations relative to the wild type human p53 amino acid sequence set forth in SEQ ID NO: 1).

[0060] As used herein, the term “mutant p53 monomers” refers to p53 monomers having an amino acid sequence which differs from the wild-type p53 amino acid sequence. Some p53 mutations are known to show increased aggregation propensity compared with wild-type p53, including the amino acid substitution mutations R248Q, R248W and R175H (mutation are relative to human wild-type sequence as set forth in SEQ ID NO:1).

[0061] As used herein, references to “subject”, “individual” or “patient” are used interchangeably herein. The subject may be a mammal, e.g. selected from a human, a non-human primate, a mouse, a dog, a horse, or a cat. Preferably, the subject is a human.

[0062] In one embodiment, the subject is an individual who is considered to be at risk of developing cancer or suspected of having a cancer, e.g. the individual may display clinical symptoms associated with cancer or has a familial history of cancer. In one embodiment, the subject has not been diagnosed as having a cancer. The subject may display no clinical symptoms associated with cancer. The subject may display one or more clinical symptoms associated with cancer. Typically, the clinical symptoms associated with cancer will depend on the type of cancer. The clinical symptoms associated with cancer are well known in the art. The subject may have a family history of cancer or a known genetic predisposition of cancer e.g. the individual has a mutant gene known to be associated with a higher risk of cancer.

[0063] In one embodiment, the subject has not been previously diagnosed as having cancer. In one embodiment, the subject has previously been diagnosed as having cancer. In one embodiment, the subject is in remission from cancer.

[0064] As used herein, the term “threshold level” refers to the level of p53 aggregates in a body fluid which is indicative of a known disease state (e.g. presence or absence of disease) or risk of disease (e.g. risk of developing cancer). The threshold level is a level of p53 aggregates that may be used to differentiate between samples from subjects that have cancer and subjects that do not have cancer and / or to differentiate between samples from subjects that have a high risk of developing cancer and subjects that have a low risk of developing cancer.

[0065] Threshold levels can be determined using any suitable methods in the art. For example, the threshold level may be determined using the Youden index (defined in Youden 1950, "Index for rating diagnostic tests". Cancer. 3: 32-35) which identifies the value that maximizes the sensitivity and specificity of a continuous variable to determine a threshold value for a dichotomous diagnostic test. The threshold level may also be determined by the K-index using receiver operating characteristic (ROC) analysis (Irwin 2011 , “A principled approach to setting optimal diagnostic thresholds: where ROC and indifference curves meet”. European Journal of Internal Medicine. 22(3): 230-234). Specifically, the threshold value corresponding to the point on the ROC curve with the shortest distance to the point (0, 1) on the plot is considered the optimal threshold.

[0066] The threshold level may refer to the level of p53 aggregates, or a score derived from the measurement of p53 aggregates, obtained from a “control” (e.g. healthy individuals, individuals associated with a particular group, such as a prognostic group, for example recurrence of the disease or non-response of the disease to a particular therapeutic agent or combination thereof). In other words, the threshold level may be predetermined by analysing results from subjects and controls, and determining an appropriate value for detecting whether a subject as has a particular disease state e.g. whether the subject is at risk of disease or whether disease is present or absent.

[0067] In some embodiments, the threshold level is determined using the level of p53 aggregates in control subjects or group of control subjects. In some embodiments, the method comprises comparing the level of p53 aggregates in a body fluid sample obtained from the subject to the level of p53 aggregates in a control.

[0068] In some embodiments, the control is the level of p53 aggregates in a body fluid sample from one or more healthy subjects (i.e. one or more subjects without cancer). In this embodiment, (i) the same or a lower level of p53 aggregates in the body fluid sample from the subject compared to the level of p53 aggregates in the control is indicative of the absence of cancer; and (ii) a higher level of p53 aggregates in the body fluid sample from the subject compared to the level of p53 aggregates in the control is indicative of the presence of cancer.

[0069] In some embodiments, the control is the level of p53 aggregates in a body fluid sample from one or more subjects with cancer. In this embodiment, (i) the same or a higher level of p53 aggregates in the body fluid sample from the subject compared to the level of p53 aggregates in the control is indicative of the presence of cancer.

[0070] It will be clear to those skilled in the art that the control subject(s) may be selected on a variety of bases which may include, for example, subjects known to be free from cancer. Comparison with a control is well known in the field of diagnostics. The “control” may comprise healthy subjects without cancer and / or subjects with cancer.

[0071] It will be understood that it is not necessary to measure controls levels or determine a threshold level for comparative purposes on every occasion. For example, for healthy / non-diseased controls, once the ‘normal range’ is established it can be used as a benchmark for all subsequent tests. A normal range can be established by obtaining samples from multiple control subjects without cancer and testing for the level of biomarker. Results (i.e. biomarker levels) for subjects suspected to have cancer can then be examined to see if they fall within, or outside of, the respective normal range. Use of a ‘normal range’ is standard practice for the detection of disease.

[0072] The threshold level may be a predetermined threshold level which is indicative of the presence of or absence of cancer.

[0073] The threshold level may be a level of p53 aggregates that is known to be associated with the presence of cancer. In this embodiment, (i) the same or a higher level of p53 aggregates in the body fluid sample as compared to the threshold level is indicative of the presence of cancer; and (ii) a lower level of p53 aggregates in the body fluid sample as compared to the threshold level is indicative of the absence of cancer.

[0074] The threshold level may be a level of p53 aggregates that is known to be associated with the absence of cancer. In this embodiment, (i) the same or a lower level of p53 aggregates in the body fluid sample as compared to the threshold level is indicative of the absence of cancer; and (ii) a higher level of p53 aggregates in the body fluid sample as compared to the threshold level is indicative of the presence of cancer.

[0075] References herein to "level of p53 aggregates" or "level" refer to a measurement that is made using any analytical method that is suitable for detecting p53 aggregates in a biological sample and that indicates the presence, absence, absolute amount or concentration, relative amount or concentration, titer, a level, an expression level, a ratio of measured levels, or the like, of, for, or corresponding to the aggregates in the biological sample. The exact nature of the "level" depends on the specific design and components of the particular analytical method employed to detect the p53 aggregates.

[0076] The invention also provides a method of determining the risk of cancer in a subject, the method comprising: (a) comparing the level of p53 aggregates in a body fluid sample obtained from the subject to a threshold level; and (b) determining the risk of the subject having cancer based on the comparison performed in step (a). The invention also provides use of the level of p53 aggregates in a body fluid sample obtained from a subject for determining the risk of cancer in a subject.

[0077] As used herein, the term “risk of cancer” refers to the likelihood or chance that a subject will develop cancer.

[0078] In some embodiments, the threshold level is the level of p53 aggregates in one or more control subjects. In some embodiments, the method comprises comparing the level of p53 aggregates in a body fluid sample obtained from the subject to the level of p53 aggregates in a control.

[0079] In some embodiments, the control is the level of p53 aggregates in a body fluid sample from one or more healthy subjects with a low risk of developing cancer. Subjects with a low risk of developing cancer may be identified based the absence of known risk factors e.g. high alcohol intake, smoking, familial history of cancer, presence of mutations associated with cancer, etc. In this embodiment, (i) the same or a lower level of p53 aggregates in the body fluid sample from the subject compared to the level of p53 aggregates in the control is indicative of the same or lower risk of cancer compared to the control; and (ii) a higher level of p53 aggregates in the body fluid sample from the subject compared to the level of p53 aggregates in the control is indicative of a higher risk of developing cancer than the control.

[0080] In some embodiments, the control is the level of p53 aggregates in a body fluid sample from one or more subjects with a high risk of developing cancer. In this embodiment, (i) the same or a higher level of p53 aggregates in the body fluid sample from the subject compared to the level of p53 aggregates in the control is indicative of the same or higher risk of developing cancer compared to the control; and (ii) a lower level of p53 aggregates in the body fluid sample from the subject compared to the level of p53 aggregates in the control is indicative of a lower risk of developing cancer compared to the control.

[0081] The threshold level may be a predetermined threshold level of p53 aggregates which is indicative of a known risk of developing cancer. Thus, a lower level of p53 aggregates in the body fluid sample from the subject compared to the threshold level may be indicative that the subject has a lower risk of cancer than the risk level associated with the threshold level. The same level of p53 aggregates in the body fluid sample from the subject compared to the threshold level may be indicative that the subject has the same risk of cancer as the risk level associated with the threshold level. A higher level of p53 aggregates in the body fluid sample from the subject compared to the threshold level is indicative that the subject has a higher risk of cancer than the risk level associated with the threshold level.

[0082] In some embodiments, wherein the threshold is indicative of a low risk of cancer the same or lower level of p53 aggregates in the body fluid sample from the subject as compared to the threshold level is indicative that the subject has a low risk of cancer. In some embodiments, wherein the threshold is indicative of a low risk of cancer a higher level of p53 aggregates in the body fluid sample from the subject compared to the threshold level is indicative that the subject has a higher risk of cancer. In other words, the subject is more likely to develop cancer than subjects who have levels of p53 aggregates at the same level or lower than the threshold level.

[0083] In some embodiments, if the subject is identified as having cancer, the method further comprises identifying the type of cancer. In some embodiments, if the subject is identified as being at risk of cancer, the method further comprises identifying the type of cancer the subject is at risk of having. The type of cancer may be confirmed by any suitable method, e.g. biopsy, imaging, endoscopic examination, and / or detection of tumour markers. Imaging may comprise e.g. X-ray imaging, computed tomography (CT) scan, ultrasound and / or magnetic resonance imaging (MRI).

[0084] In some embodiments, if the subject is identified as having cancer, the method further comprises treating said subject for cancer. In some embodiments, if the subject is identified as being at risk of cancer, the method further comprises treating (e.g. prophylactically / preventatively) said subject for cancer. It will be understood that the treatment administered will depend on the type and severity of cancer that the subject has or is at risk of having. In some embodiments, said treatment comprises surgery, radiotherapy, chemotherapy, immunotherapy, hormone therapy, thermal ablation, photodynamic therapy and / or small molecule therapy. As used herein, the term “treating” or “treatment” may refer to prophylactic, ameliorative or curative treatment.

[0085] If the subject is identified as having cancer, the method may further comprise monitoring the progression of cancer in the subject by comparing the p53 levels in body fluid samples obtained from the subject at two or more timepoints (e.g. a first and second timepoint) and determining whether the cancer has progressed between the two or more timepoints (e.g. a first and second timepoint) based on the comparison. In some embodiments, the method further comprises monitoring the progression of cancer in the subject by comparing the p53 levels in body fluid samples obtained from the subject at a first timepoint and a second timepoint and determining whether the cancer has progressed between the first timepoint and the second timepoint based on the comparison.

[0086] It will be understood that cancer that has “progressed” typically exhibits increased severity at a later timepoint (e.g. the second timepoint) as compared to an earlier timepoint (e.g. the first time point). Cancer that has not “progressed” typically exhibits the same severity or reduced severity at a later timepoint (e.g. the second timepoint) as compared to an earlier timepoint (e.g. the first timepoint).

[0087] The same or a lower level of p53 aggregates in the body fluid sample at a later timepoint as compared to level of p53 aggregates in the body fluid sample at an earlier timepoint typically indicates that the cancer has not progressed between the timepoints and / or the cancer has reduced in severity at the later timepoint as compared to earlier timepoint. A higher level of p53 aggregates in the body fluid sample at a later timepoint as compared to the level of p53 aggregates in the body fluid sample at an earlier timepoint typically indicates that the cancer has progressed between the timepoints.

[0088] The same or a lower level of p53 aggregates in the body fluid sample at the second time point as compared to level of p53 aggregates in the body fluid sample at the first time point typically indicates that the cancer has not progressed between the first and second time points and / or the cancer has reduced in severity at the second time point as compared to the first time point. A higher level of p53 aggregates in the body fluid sample at the second time point as compared to the level of p53 aggregates in the body fluid sample at the first time point typically indicates that the cancer has progressed between the first and second time points.

[0089] If the subject is identified as being at risk of cancer, the method may further comprise monitoring for the onset of cancer in the subject by comparing the p53 levels in body fluid samples obtained from the subject at two or more timepoints and determining whether onset of cancer has occurred between the two or more timepoints based on the comparison. In some embodiments, the method further comprises monitoring for the onset of cancer in the subject by comparing the p53 levels in body fluid samples obtained from the subject at a first timepoint and a second timepoint and determining whether onset of cancer has occurred between the two timepoints based on the comparison.

[0090] It will be understood that “onset” of cancer typically involves the development of cancerous tissue or cancer symptoms at a later timepoint (e.g. the second timepoint) as compared to an earlier timepoint (e.g. the first timepoint). No onset of cancer typically involves no development of cancer symptoms or the growth of cancerous tissues.

[0091] In some embodiments wherein the subject does not have cancer and / or is at risk of developing cancer when the earlier timepoint is taken, the same or a lower level of p53 aggregates in the body fluid sample at a later timepoint as compared to level of p53 aggregates in the body fluid sample at an earlier timepoint indicates that the onset of cancer has not occurred between the timepoints. In some embodiments, wherein the subject does not have cancer and / or is at risk of developing cancer when the earlier timepoint is taken a higher level of p53 aggregates in the body fluid sample at a later timepoint as compared to the level of p53 aggregates in the body fluid sample at an earlier indicates that the onset of cancer has occurred between the timepoints.

[0092] In some embodiments wherein the subject does not have cancer and / or is at risk of developing cancer when the first timepoint is taken, the same or a lower level of p53 aggregates in the body fluid sample at the second time point as compared to level of p53 aggregates in the body fluid sample at the first time point indicates that the onset of cancer has not occurred between the two time points. In some embodiments, wherein the subject does not have cancer and / or is at risk of developing cancer when the first timepoint is taken a higher level of p53 aggregates in the body fluid sample at the second time point as compared to the level of p53 aggregates in the body fluid sample at the first time point indicates that the onset of cancer has occurred between the two timepoints.

[0093] In some embodiments, the method involves comparing the level of p53 aggregates in body fluid samples obtained from the subject at 3, 4, 5, 6 or more time points.

[0094] In some embodiments, the methods involve monitoring the progression or onset of cancer at intervals, e.g. every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 8 months, every 10 months, or every 12 months.

[0095] The invention also provides a method for monitoring the progression of cancer or the onset of cancer in a subject, the method comprising: (a) comparing: (i) the level of p53 aggregates in a body fluid sample obtained from the subject at a first time point with (ii) the level of p53 aggregates in a body fluid sample obtained from the subject at a second subsequent time point; and (b) determining whether the cancer has progressed or onset of cancer has occurred between the first and second time point based on the comparison performed in step (a). The invention also provides use of the level of p53 aggregates in a body fluid sample obtained from a subject for monitoring the progression of cancer.

[0096] It will be understood that cancer that has “progressed” typically exhibits increased severity at the second time point as compared to the first time point. Cancer that has not “progressed” typically exhibits the same severity or reduced severity at the second time point as compared to the first time point.

[0097] The body fluid samples may be taken prior to commencement of therapy, at a different stage of therapy, and / or at timepoints after the completion of therapy. The body fluid samples may also be taken after a subject has been identified as being at risk of cancer. The method is also useful for the longitudinal monitoring of an individual following the completion of therapy or following the subject being identified as being at risk of cancer. The method is also useful for monitoring for the onset of cancer in a subject who is at risk of developing cancer.

[0098] In some embodiments, wherein the subject has cancer when the first timepoint is taken, the same or a lower level of p53 aggregates in the body fluid sample at the second time point as compared to level of p53 aggregates in the body fluid sample at the first time point indicates that the cancer has not progressed between the first and second time points and / or the cancer has reduced in severity at the second time point as compared to the first time point. In some embodiments, wherein the subject does not have cancer and / or is at risk of developing cancer when the first timepoint is taken, the same or a lower level of p53 aggregates in the body fluid sample at the second time point as compared to level of p53 aggregates in the body fluid sample at the first time point indicates that the onset of cancer has not occurred between the first and second time points.

[0099] In some embodiments wherein the subject has cancer when the first timepoint is taken, a higher level of p53 aggregates in the body fluid sample at the second time point as compared to the level of p53 aggregates in the body fluid sample at the first time point indicates that the cancer has progressed between the first and second time points. In some embodiments wherein the subject does not have cancer and / or is at risk of developing cancer when the earlier timepoint is taken, a higher level of p53 aggregates in the body fluid sample at the second time point as compared to the level of p53 aggregates in the body fluid sample at the first time point indicates that the onset of cancer has occurred between the first and second time points.

[0100] In some embodiments, the subject has been diagnosed as having cancer and has started treatment at the first time point and: (i) the same or a lower level of p53 aggregates in the body fluid sample at the second point as compared to the level of p53 aggregates in the body fluid sample at the first time point indicates that the cancer has not progressed and / or the cancer has reduced in severity; and (ii) a higher level of p53 aggregates in the body fluid sample at the second time point as compared to the level of p53 aggregates in the body fluid sample at the first time point indicates that the cancer has progressed.

[0101] A subject who is in remission does not typically exhibit cancer associated symptoms. In some embodiments, the subject is in remission at the first time point and: (i) the same or a lower level of p53 aggregates in the body fluid sample at the second point as compared to the level of p53 aggregates in the body fluid sample at the first time point indicates that the subject remains in remission at the second time point; and (ii) a higher level of p53 aggregates in the body fluid sample at the second time point as compared to the level of p53 aggregates in the body fluid sample at the first time point indicates that the subject is no longer in remission (e.g. the subject may exhibit cancer associated symptoms and / or cancer has recurred).

[0102] In some embodiments, the subject is at risk of developing cancer at the first time point and: (i) the same or a lower level of p53 aggregates in the body fluid sample at the second point as compared to the level of p53 aggregates in the fluid sample at the first time point indicates that onset of cancer has not occurred at the second time point; and (ii) a higher level of p53 aggregates in the body fluid sample at the second time point as compared to the level of p53 aggregates in the body fluid sample at the first time point indicates that the onset of cancer has occurred (e.g. the subject may exhibit cancer associated symptoms and / or growth of cancerous tissue has occurred). A subject who is at risk of cancer does not typically exhibit cancer associated symptoms.

[0103] If the cancer has progressed between the first and second time points, the method may further comprise treating said subject for the cancer. This may involve the subject starting a new cancer therapy or increasing the level of treatment (e.g. to a higher or more frequent dose or a more aggressive treatment strategy). Where the subject was in remission, it may involve the subject restarting a treatment they have received previously and / or starting a new cancer therapy.

[0104] If the cancer has not progressed between the first and second timepoints, the method may further comprise maintaining the current course of cancer treatment or moving the subject to a maintenance treatment (e.g. a less aggressive treatment).

[0105] If cancer onset has occurred between the first and second timepoints, the method may further comprise identifying the type of cancer. The method may further comprise treating said subject for the cancer. If the onset of cancer has not occurred between the first and second timepoints, the method may further comprise treating the subject with a preventative / prophylactic therapy.

[0106] In some embodiments, the second subsequent time point is at least one week after the first time point. In some embodiments, the second subsequent time point is at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 8 months, at least 10 months, or at least 12 months after the first time point.

[0107] In some embodiments, the method further comprises comparing the level of p53 aggregates in a body fluid sample obtained from the subject at two or more time points. For example, the method may involve comparing the level of p53 aggregates in body fluid samples obtained from the subject at 3, 4, 5, 6 or more time points.

[0108] The invention also provides a method for determining the efficacy of a therapeutic intervention in a subject having cancer or at risk of having cancer, the method comprising: (a) comparing: (i) the level of p53 aggregates in a body fluid sample obtained from the subject prior to administration of the therapeutic intervention with (ii) the level of p53 aggregates in a body fluid sample obtained from the subject after administration of the therapeutic intervention; and (b) determining the efficacy of the therapeutic intervention based on the comparison performed in step (a). The invention also provides use of the level of p53 aggregates in a body fluid sample obtained from a subject for determining the efficacy of a therapeutic intervention in a subject having cancer.

[0109] For subjects who are identified as being at risk of having cancer, the therapeutic intervention typically comprises the introduction of preventive measures, such as a change of lifestyle (e.g. reduced consumption of alcohol and / or smoking) and / or removal of any benign lesion(s).

[0110] Methods for monitoring efficacy of a therapy may be used to monitor the therapeutic effectiveness of existing therapies and new therapies in human subjects and in non-human animals (e.g. in animal models). Such methods may also be used to optimise dose (e.g. increase or decrease dosage) depending on whether and the extent to which any change in p53 aggregate levels is observed.

[0111] It will be understood that the efficacy of the therapeutic intervention will depend on the type of cancer being treated and the type of therapeutic intervention used. For example, therapeutic interventions intended to reduce or eradicate cancer would be considered efficacious if the subject exhibits a reduced level of p53 aggregates at the second time point as compared to the level of p53 aggregates at the first time point. Therapeutic interventions intended to stop the progression of a cancer may be considered efficacious if the subject exhibits the same level or a reduced level of p53 aggregates at the second time point as compared with the level of p53 aggregates at the first time point. Therapeutic interventions intended to maintain remission (which may be referred to as maintenance therapies) would be considered efficacious if the subject exhibits the same or a reduced level of p53 aggregates at the second time point as compared to the level of p53 aggregates at the first time point. Wherein the subject exhibits an increased level of p53 aggregates at the second time point as compared to the level of p53 aggregate at the first time point, the therapeutic intervention is typically considered to have low efficacy. Therapeutic interventions intended to provide a preventative or prophylactic therapy (i.e. to delay or prevent the onset of cancer) would be considered efficacious if the same level or a reduced level of p53 aggregates at the second time point as compared with the level of p53 aggregates at the first time point.

[0112] In some embodiments, the therapeutic intervention comprises surgery, radiotherapy, chemotherapy, immunotherapy, hormone therapy, thermal ablation, photodynamic therapy and / or small molecule therapy.

[0113] In some embodiments, wherein the subject has cancer, a higher level of p53 aggregates in the body fluid sample obtained after administration of the therapeutic intervention as compared to the level of p53 aggregate in the body fluid sample obtained prior to administration of the therapeutic intervention is indicative that the therapeutic agent is not efficacious. Higher levels of p53 aggregates in the body fluid sample after administration of the therapeutic intervention typically indicate that the therapeutic intervention has not stopped progression of the cancer or the onset of cancer.

[0114] In some embodiments, wherein the subject has cancer, the same level of p53 aggregates in the body fluid sample obtained after administration of the therapeutic intervention as compared to the level of p53 aggregate in the body fluid sample obtained prior to administration of the therapeutic intervention is indicative that the therapeutic agent is not efficacious. In other words, the levels of p53 aggregates in a body fluid sample from the subject have stayed the same following administration of the therapeutic intervention. For example, the same levels of p53 aggregates in the body fluid sample typically indicate that the therapeutic intervention has not reduced the severity / level of the cancer.

[0115] In some embodiments, wherein the subject has cancer, the same level of p53 aggregates in the body fluid sample obtained after administration of the therapeutic intervention as compared to the level of p53 aggregate in the body fluid sample obtained prior to administration of the therapeutic intervention is indicative that the therapeutic agent is efficacious. For example, the same levels of p53 aggregates (i.e. no change in levels) in the body fluid sample may indicate that the therapeutic intervention has stopped progression of cancer. If the subject is at risk of developing cancer, no change in the levels of p53 aggregates may indicate that the therapeutic intervention is efficacious in delaying or preventing the onset of cancer.

[0116] In some embodiments, wherein the subject has cancer, a lower level of p53 aggregates in the body fluid sample obtained after administration of the therapeutic intervention as compared to the level of p53 aggregates in the body fluid sample obtained prior to administration of the therapeutic intervention is indicative that the therapeutic agent is efficacious. Lower levels of p53 aggregates in the body fluid sample after administration of the therapeutic intervention typically indicates that the therapeutic intervention has stopped progression of the cancer or reduced the severity of the cancer.

[0117] In some embodiments, such as wherein the therapeutic intervention is a maintenance therapy, the same level of p53 aggregates in the body fluid sample obtained after administration of the therapeutic intervention as compared to the level of p53 aggregates in the body fluid sample obtained prior to administration of the therapeutic agent is indicative that the therapeutic agent may be efficacious. In this embodiment, depending on the rate of progression of the cancer prior to the therapeutic intervention, no change in the level of p53 aggregates may indicate that the therapeutic intervention has prevented progression of cancer. If the subject is in remission, no change in the level of p53 aggregate in the body fluid sample may indicate that the therapeutic intervention is efficacious in maintaining a state of remission.

[0118] In some embodiments, such as wherein the therapeutic intervention is a preventative or prophylactic therapy for a subject at risk of developing cancer, the same level of p53 aggregate in the body fluid sample obtained after administration of the therapeutic intervention as compared to the level of p53 aggregate in the body fluid sample obtained prior to administration of the therapeutic agent may be indicative that the therapeutic agent is efficacious. In this embodiment, no change in the level of p53 aggregate may indicate that the therapeutic intervention has delayed or prevented onset of cancer.

[0119] In some embodiments, the body fluid sample obtained from the subject after administration of the therapeutic intervention is obtained at least one week after the administration of therapeutic intervention. In some embodiments, the body fluid sample obtained from the subject after administration of the therapeutic intervention is obtained at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 8 months, at least 10 months, or at least 12 months after administration of the therapeutic intervention.

[0120] In some embodiments, the method comprises monitoring the efficacy of the therapeutic intervention in samples obtained from the subject at multiple time points during administration of the therapeutic intervention. In some embodiments, the method comprises monitoring the efficacy of the therapeutic intervention every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 8 months, every 10 months, or every 12 months.

[0121] In some embodiments, the method further comprises monitoring the progression of cancer or the onset of cancer in the subject after completion of a therapeutic intervention e.g. by a method described herein.

[0122] In the methods of the invention the cancer is a cancer associated with p53 aggregates. In one embodiment, the cancer is selected from brain cancer (including glioblastoma), breast cancer, adenocarcinoma, lung cancer (small cell, non-small cell and mesothelioma), oesophageal cancer, prostate cancer, ovarian cancer, leukaemia, renal cancer, endometrial cancer, small bowel cancers (duodenal and jejunal), pancreatic cancer, hepatobiliary tumours, germ cell cancers, head and neck cancers, thyroid cancer, blood cancer, and melanoma. p53 aggregates are known to be associated with a range of cancers.

[0123] Cancers associated with p53 aggregates are often diagnosed using invasive or timeconsuming diagnostic methods e.g. involving tissue biopsies, or imaging techniques such as a MRI scan or a CT scan. The methods of the invention advantageously provide a diagnostic tool which relies on the use of body fluid samples. By relying on body fluid samples, the methods of the invention are less invasive, less time consuming and less costly than other screening methods. Such samples are also suitable for use in high-throughput screening, further reducing the time to diagnosis and associated costs. Further advantageously, the methods of the invention may be repeated (e.g. at multiple time points) without causing significant discomfort to the subject.

[0124] The cancer may be a metastatic cancer. In one embodiment, the metastatic cancer is selected from metastatic adenocarcinoma, metastatic breast cancer, metastatic lung cancer, oesophageal cancer, prostate cancer, ovarian cancer, leukaemia and metastatic brain cancer. The metastatic cancer may also be associated with brain metastasis. In some embodiments, the breast cancer is associated with brain metastasis. In some embodiments, the metastatic lung cancer is associated with brain metastasis.

[0125] The cancer may be a brain tumour. The brain tumour may be selected from glioma (such as glioblastoma multiforme, oligodendroglioma, ependymomas, brain stem glioma), craniopharyngioma, haemangioblastoma, malignant meningioma, pineal region tumours and vestibular schwannoma.

[0126] In one embodiment, the cancer is glioblastoma. Glioblastoma represents one of the most aggressive and recurrent forms of brain tumour with a survival rate less than 5% after 5 years. The methods of the invention advantageously provide a diagnostic tool that can detect glioblastoma at an early stage, such as early during the onset of disease or early during recurrence. Early detection advantageously allows therapeutic interventions to be initiated while the tumour is more responsive to first line treatments which may in turn improve subject survival rates.

[0127] Methods of the invention may be used to confirm a diagnosis of cancer. Therefore, in one embodiment, the subject is suspected of suffering from a cancer. The subject may display one or more clinical symptoms associated with cancer. Clinical symptoms of cancer and are well known to the skilled practitioner. Symptoms can include heavy night sweats or fever, fatigue, unexplained bleeding or bruising, unexplained pain or ache, unexplained weight loss, unusual lump or swelling, as well as many other possible symptoms depending on the cancer type.

[0128] In some embodiments, the subject is subjected to further diagnostic tests (e.g. to determine the type of cancer). Such tests may include imaging (such as magnetic resonance imaging, computed tomography (CT), bone scan, mammogram, a positron emission tomography (PET) scan, ultrasound, X-ray a single photon emission computed tomography (SPECT) scan), further laboratory tests (e.g. complete blood count, circulating tumour cell tests), endoscopy procedures (e.g. bronchoscopy, colonoscopy, cystoscopy, laparoscopy, laryngoscopy, thoracoscopy) referral to an oncologist, and / or a biopsy.

[0129] In some embodiments, the method comprises quantifying the level of p53 aggregates in the body fluid sample(s). Detecting the level of p53 aggregates in the body fluid sample(s) may comprise: (a) quantifying the concentration p53 aggregates in the body fluid samples(s); and / or (b) the determining the relative concentration of p53 aggregates in the body fluid samples(s). The relative concentration of p53 aggregates in the body fluid sample(s) may be determined relative to the total protein concentration in the body fluid sample(s). The relative concentration of p53 aggregates in the body fluid sample(s) may be determined relative to the concentration of an external calibrant.

[0130] The level of p53 aggregates in the body fluid sample(s) may be determined directly, e.g. by counting the number of p53 aggregates in the sample. The level of p53 aggregates in the body fluid sample(s) may be determined indirectly, e.g. by measuring a signal that correlates with the number of p53 aggregates in the sample.

[0131] In some embodiments, the method comprises contacting the body fluid sample with a p53 aggregate-specific binding agent. As used herein, a “p53 aggregate-specific binding agent” is a binding agent which binds to p53 aggregates but does not bind to individual wild-type monomeric p53. The p53 aggregate-specific binding agent may bind to an epitope which is exposed in p53 aggregates but not in individual wild-type p53 monomers (e.g. wild-type monomers in the normal conformation). As discussed above, p53 aggregate formation may result in the unfolding or misfolding of p53 molecules which can result in exposure of epitopes that are not normally accessible in the normal wild-type p53 monomer conformation or p53 monomers which are correctly folded.

[0132] The N- and / or C- termini of p53 are intrinsically disordered and so they are typically available for binding regardless of whether the protein is aggregated. As such, the N- and / or C- termini of p53 are typically the preferred binding sites for the p53 capture binding agent. Aggregation typically exposes the central domain of p53, and so the central domain of p53 is typically the preferred binding target for the p53 aggregate specific binding agent.

[0133] In some embodiments, the p53 aggregate specific binding agent binds to the N-terminal domain of p53, the central domain of p53, and / or the C-terminal domain of p53. For example, in some embodiments, the p53 aggregate specific binding agent binds to the N-terminal domain of p53. In some embodiments, the p53 aggregate specific binding agent binds to the C-terminal domain of p53. In some embodiments, the p53 aggregate specific binding agent binds to the central domain of p53.

[0134] Typically, the p53 aggregate-specific binding agent is an antibody or antigen binding fragment thereof.

[0135] In some embodiments, the p53 aggregate-specific binding agent binds an epitope located within residues 98-303 of SEQ ID NO: 1.

[0136] In some embodiments, the p53 aggregate-specific binding agent binds an epitope located at residues 211-217 of SEQ ID NO: 1.

[0137] In some embodiments, the p53 aggregate-specific binding agent binds an epitope located within residues 181-190 of SEQ ID NO: 1.

[0138] In some embodiments, the p53 aggregate-specific binding agent binds an epitope located within residues 256-270 of SEQ ID NO: 1.

[0139] In some embodiments, the p53 aggregate-specific binding agent is PAb240 or an antigen binding fragment thereof. PAb240 is a commercially available antibody (Novus Biologicals, Cat. No. NB200-103) which binds to an epitope (amino acid residues 211-217) in the central DNA-binding region of p53. Biotinylated PAb240 is also commercially available (Novus Biologicals, Cat. No. NB200-103B). Other p53 aggregate-specific binding agents include, but are not limited to, DO-11 (BIO-RAD, Cat. No. MCA1704) which binds to an epitope (amino acid residues 181-190) in the central DNA-binding region of p53; and DO-12 (CancerTools, Cat. No. 153403) which binds to an epitope (amino acid residues 256-270) in the central DNA- binding region of p53.

[0140] As used herein, the term “binding agent” may comprise any molecule which can specifically bind the target antigen or epitope (e.g. p53 molecules or p53 aggregates). In one embodiment, the binding agent, such as the antibody, specifically binds to the target biomarker (e.g. p53 molecules or p53 aggregates). The specificity of a binding agent (e.g. an antibody) is the ability of the binding agent (e.g. antibody) to recognize a particular antigen as a unique molecular entity and distinguish it from another. A binding agent (e.g. an antibody) that “specifically binds” to an antigen or an epitope is a term well understood in the art. A binding agent is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and / or with greater affinity with a particular target antigen or epitope, than it does with alternative targets. A binding agent (e.g. an antibody) “specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances.

[0141] A binding agent may be a naturally occurring or chemically synthesised molecule, capable of specific binding to the desired target. A binding agent may include a peptide, an antibody or a fragment thereof, aptamer or oligonucleotide.

[0142] Non-limiting exemplary binding agents include aptamers, antibodies, adnectins, ankyrins, other antibody mimetics and other protein scaffolds, autoantibodies, chimeras, small molecules, nucleic acids, lectins, ligand-binding receptors, imprinted polymers, avimers, peptidomimetics, hormone receptors, cytokine receptors, synthetic receptors, and modifications and fragments thereof.

[0143] In some embodiments, the p53 binding agent is selected from an aptamer and an antibody. The p53 binding agent is typically an antibody.

[0144] The term “antibody”, and its plural form “antibodies”, includes, inter alia, polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding portions / fragments, such as F(ab')2, Fab proteolytic fragments, and single chain variable region fragments (scFvs). Thus, in one embodiment an antibody herein is an antigen-binding portion of an antibody. An antibody fragment suitable for use in the methods of the invention is one which retains the ability to bind to the target biomarker so that the biomarker (e.g. p53 molecules or p53 aggregates) may be detected. In the context of the present invention, an antibody fragment retains the ability to bind p53 molecules as described herein. Suitable antibody fragments may include F(ab')2, Fab proteolytic fragments, and single chain variable region fragments (scFvs). In a preferred embodiment, the binding agents are antibodies (preferably a monoclonal antibody) or an antigen binding fragment thereof. The antibody may be an IgG antibody e.g. lgG1 , lgG2, lgG3 or lgG4.

[0145] Detection of the level of p53 aggregates in the blood body fluid sample may involve the use of two p53 binding agents - (i) a p53 capture binding agent; and (ii) a p53 detection binding agent. In this embodiment, the same binding agent is not used as the p53 capture and detection binding molecules. In other words, the p53 capture and detection binding agents are different binding agents which specifically bind to different epitopes of the p53 molecule. Thus, the p53 capture binding agent and the p53 detection binding agent do not bind to the same epitope on a p53 molecule. The use of a capture binding agent (e.g. an antibody) allows for the enrichment of p53 aggregates from the body fluid samples, prior to detection by the detection binding agent. In some embodiments, enrichment of p53 aggregates comprises removing proteins from the sample proteins which are not bound by the p53 capture binding agent (e.g. by washing), thereby providing an enriched sample. In some embodiments, enrichment of p53 aggregates comprises removing from the sample proteins which are bound by the p53 capture binding agent (e.g. by affinity), thereby providing a separate enriched sample. Enrichment of p53 aggregates advantageously allows for the detection of p53 aggregates in body fluid samples, even when present at low concentrations in the body fluid sample. This provides a method that has a high diagnostic accuracy, by providing high sensitivity and specificity to p53 aggregates. As demonstrated herein, such methods may have a diagnostic accuracy of greater than 90%, a sensitivity of greater than 90% and a specificity of greater than 80%.

[0146] Thus, in some embodiments, the method comprises: (a) contacting the body fluid sample with a p53 capture binding agent (e.g. a binding agent that binds to the N-terminal domain of p53 and / or the C-terminal domain of p53) to provide an enriched sample; (b) contacting the enriched sample with a p53 detection binding agent; and (c) detecting binding of the p53 detection binding agent.

[0147] As used herein, an “enriched sample” refers to a body fluid sample from a subject which has been contacted with a p53 capture binding agent (e.g. a binding agent that binds to the N- terminal domain of p53 and / or the C-terminal domain of p53). Contacting the body fluid sample with said capture binding agent results in p53 molecules present in the sample being concentrated thus providing for an enriched sample.

[0148] The term “p53 capture binding agent” refers to binding agents which are capable of binding to p53 species. Typically, the p53 capture binding agent is capable of binding to any type of p53 species. p53 capture binding agents may include a binding agent that binds to the N-terminal domain of p53, the central domain of p53, and / or the C-terminal domain of p53. As used herein, the term ”N-terminal domain of p53” or “p53 N-terminal domain” refers to the N-terminus of p53 and encompasses amino acid residues 1-97 of wild-type p53 (as set out in SEQ ID NO:1). Amino acids 1-97 include two distinct transcription-activation domains (TADs) of p53 - TAD1 and TAD2, encompassing amino acid residues 1-42 and 43-67, respectively; and a proline rich domain encompassing amino acid residues 68-97.

[0149] As used herein, the term “C-terminal domain of p53” or “p53 C-terminal domain” refers to the C-terminus of p53 and encompasses amino acid residues 304-393 of wild-type p53 (as set out in SEQ ID NO:1). Amino acids 304-393 includes the nuclear localisation domain (“NLS”, encompassing amino acid residues 304-322), the oligomerisation domain (“OD”, encompassing amino acid residues 323-363), and the basic domain (“BD”, encompassing amino acid residues 364-393).

[0150] As used herein, the term “central domain of p53” or “p53 central domain” refers to the domain located between the N- and C-terminal domains. The central domain of p53 encompasses amino acid residues 98-303 of wild-type p53 (as set out in SEQ ID NO: 1). The central domain includes the central DNA binding domain (DBD), encompasses amino acid residues 98-303.

[0151] The term “p53 detection binding agent” refers to a binding agent that specifically binds to p53 aggregates and is capable of being detected. Such binding agents may also be referred to as a p53 aggregate-specific binding agent. The p53 detection binding agent is specific for p53 aggregates and binds to an epitope (or multiple epitopes) which are only present or exposed in p53 aggregates. For example, the p53 detection binding agent may bind to an epitope which is only accessible following p53 aggregate formation. The epitope may become exposed to or accessible by the p53 detection antigen following unfolding or misfolding of p53 molecules. p53 detection binding agents may include a binding agent that binds to the N-terminal domain of p53, the central domain of p53, and / or the C-terminal domain of p53. In one embodiment, the p53 detection binding agent binds the central binding domain of p53.

[0152] The p53 detection binding agent may be labelled to allow for detection. In other words, the detection binding agent may be labelled with a detectable marker. Suitable labels / detectable markers may include a luminescent, fluorescent, enzyme or radioactive marker. In one embodiment, the detection binding agent is labelled with a fluorescent marker. The fluorescent marker is a fluorescent dye, in particular a fluorescent dye with photophysical properties. In another embodiment, the detection binding agent is labelled with a nucleic acid reporter. In this embodiment, a nucleic acid sequence acts as a label (a “nucleic acid reporter”) to indicate that the detection binding agent is present in the sample. The nucleic acid reporter may then be detected by qPCR, digital PCR, or next generating sequencing (NGS). In a further embodiment, the nucleic acid reporter comprises a target ID or barcode sequence which may be used to identify the presence of the reporter. In one embodiment, the detection binding agent is labelled with an affinity tag, e.g. a biotin, avidin, streptavidin or His (e.g. hexa-His) tag. In one embodiment, the p53 detection binding agent is labelled with biotin.

[0153] In some embodiments, the p53 capture binding agent and the p53 detection binding agent are antibodies.

[0154] In some embodiments, the p53 capture binding agent binds to the N-terminal domain of p53, the central domain of p53, and / or the C-terminal domain of p53. In one embodiment, the p53 capture binding agent binds to the N-terminal domain of p53 and / or the C-terminal domain of p53. In one embodiment, the p53 capture binding agent binds to the N-terminal domain of p53. In one embodiment, the p53 capture binding agent binds to the C-terminal domain of p53. In one embodiment, the p53 capture binding agent binds to the central domain of p53.

[0155] In some embodiments, the p53 capture binding agent binds to both p53 aggregates and wildtype p53 monomers. Beneficially, the p53 capture binding agent acts to capture and enrich (or facilitate enrichment of) p53 species present in the sample. When the sample is subsequently contacted with a p53 detection binding agent, an enhanced signal is produced.

[0156] In some embodiments, the p53 detection binding agent binds to the N-terminal domain of p53, the central domain of p53, and / or the C-terminal domain of p53. In one embodiment, the p53 detection binding agent binds to the N-terminal domain of p53. In one embodiment, the p53 detection binding agent binds to the C-terminal domain of p53. In one embodiment, the p53 detection binding agent binds to the central domain of p53.

[0157] In some embodiments, (i) the p53 capture binding agent binds to the N-terminal domain of p53, the central domain of p53, and / or the C-terminal domain of p53; and (ii) the p53 detection binding agent binds to the N-terminal domain of p53, the central domain of p53, and / or the C- terminal domain of p53.

[0158] In some embodiments, the p53 capture binding agent binds to the N-terminal domain of p53 and the p53 detection binding agent binds to the central domain of p53. In some embodiments, the p53 capture binding agent binds to the C-terminal domain of p53 and the p53 detection binding agent binds to the central domain of p53.

[0159] In some embodiments, the p53 capture binding agent (e.g. a binding agent that binds to the N-terminal domain of p53 and / or the C-terminal domain of p53) is bound to a solid support. The p53 capture binding agent may be indirectly or directly bound to a solid support. The p53 capture binding agent may be conjugated to the solid support. In some embodiments, the p53 capture binding agent binds the N-terminal domain of p53 and / or the C-terminal domain of p53 and is bound to a solid support. The p53 capture binding agent which binds the p53 N- terminal domain and / or the p53 C-terminal domain may be indirectly or directly bound to a solid support. In some embodiments, the solid support is selected from a bead, a solid surface, and a membrane. In some embodiments, the solid support is a magnetic bead (e.g. a paramagnetic bead).

[0160] In some embodiments, after contacting the body fluid sample with the p53 capture binding agent, the method comprises removing unbound proteins (i.e. proteins not bound by the p53 capture binding agent) to provide an enriched sample, or by removing bound proteins from the sample to provide a separate enriched sample. Unbound proteins may be removed by separating the p53 capture binding agent from the body fluid sample, e.g. by removing the body fluid sample and washing (e.g. with a buffer) the p53 capture binding agents to remove unbound protein. Alternatively, the p53 capture binding agent may be removed from the body fluid sample (e.g. by affinity).

[0161] Binding of the p53 detection binding agent to p53 aggregates may be determined through use of a detectable marker or label.

[0162] In some embodiments, binding of the p53 detection binding agent to p53 aggregates is determined by immunofluorescence. In some embodiments, the p53 detection binding agent is conjugated to a fluorescent dye. In some embodiments, the p53 detection binding agent is conjugated to an enzyme capable of hydrolysing a fluorogenic substrate to produce a fluorescent signal. In some embodiments, binding of the p53 detection binding agent to p53 aggregates is determined by contacting the sample with a further binding agent which (i) binds to p53 aggregate-specific binding agent; and (ii) is conjugated to a fluorescent dye.

[0163] In some embodiments, the p53 detection binding agent is associated with p-galactosidase. In other words, the p53 detection binding agent is indirectly or directly linked (e.g. covalently or non-covalently) to a p-galactosidase. The p53 detection binding agent may be indirectly associated with p-galactosidase. For example, another molecule (e.g. streptavidin) or a linker may be present between the p53 detection binding agent and the p galactosidase. In some embodiments, the p53 detection binding agent is conjugated to p-galactosidase (e.g. directly or indirectly via a streptavidin molecule). In some embodiments, the p53 detection binding agent is directly conjugated to p-galactosidase. In some embodiments, the p53 detection binding agent is indirectly conjugated to p-galactosidase via a streptavidin molecule.

[0164] In some embodiments, the p53 detection binding agent is associated with biotin (e.g. conjugated to biotin) and the method further comprises contacting the enriched sample with streptavidin-p-galactosidase. In these embodiments, the method further comprises contacting the enriched sample with a substrate of p-galactosidase. p-galactosidase is a hydrolase enzyme which catalyses the hydrolysis of p-galactosides in substrates. In some embodiments, the substrate of p-galactosidase is a fluorogenic substrate which produces a detectable fluorophore when hydrolysed by p-galactosidase. In some embodiments, the substrate of p- galactosidase is resorufin p-D-galactopyranoside (RGP) and binding of the p53 detection binding agent is detected by detecting formation of resorufin.

[0165] In some embodiments, the p53 detection binding agent is associated with horseradish peroxidase and the method further comprises contacting the enriched sample with 3, 3', 5,5'- tetramethylbenzidine, 3,3'-Diaminobenzidine and / or 2,2'-azino-di-[3-ethylbenzthiazoline-6- sulfonic acid].

[0166] In some embodiments, the p53 detection binding agent is selected from an antibody or an antigen binding fragment thereof and an aptamer. Typically, the p53 detection binding agent is an antibody or antigen binding fragment thereof.

[0167] In some embodiments, the p53 detection binding agent binds an epitope located within residues 98-303 of SEQ ID NO: 1 .

[0168] In some embodiments, the p53 detection binding agent binds an epitope located within residues 211-217 of SEQ ID NO: 1.

[0169] In some embodiments, the p53 detection binding agent binds an epitope located within residues 181-190 of SEQ ID NO: 1.

[0170] In some embodiments, the p53 detection binding agent binds an epitope located within residues 256-270 of SEQ ID NO: 1.

[0171] In some embodiments, the p53 detection binding agent is PAb240 or an antigen binding fragment thereof. PAb240 is a commercially available antibody (Novus Biologicals, Cat. No. NB200-103) which binds to an epitope (amino acid residues 211-217) in the central DNA- binding region of p53. Other p53 detection binding agents include, but are not limited to, DO- 11 (BIO-RAD, Cat. No. MCA1704) which binds to an epitope (amino acid residues 181-190) in the central DNA-binding region of p53; and DO-12 (CancerTools, Cat. No. 153403) which binds to an epitope (amino acid residues 256-270) in the central DNA-binding region of p53.

[0172] In some embodiments, the p53 capture binding agent (e.g. a binding agent that binds to the N-terminal domain of p53 and / or the C-terminal domain of p53) is selected from an antibody or an antigen binding fragment thereof and an aptamer. Typically, the p53 capture binding agent is an antibody or antigen binding fragment thereof.

[0173] In some embodiments, the p53 capture binding agent binds to the p53 N-terminal domain. In some embodiments, the p53 capture binding agent which binds to the p53 N-terminal domain is selected from an antibody or an antigen binding fragment thereof and an aptamer. Typically, the p53 capture binding agent which binds to the p53 N-terminal domain is an antibody or antigen binding fragment thereof.

[0174] In some embodiments, the p53 capture binding agent binds to the p53 N-terminal domain at an epitope located within residues 1-97 of p53 (SEQ ID NO: 1).

[0175] In some embodiments, the p53 capture binding agent which binds to the p53 N-terminal domain binds to amino acid residues 20 to 25 of SEQ ID NO: 1. In some embodiments, the p53 capture binding agent which binds to the p53 N-terminal domain is DO-1 or an antigen binding fragment thereof. DO-1 is a commercially available antibody (Abeam, Cat. No. Ab1101) which binds to an epitope close to the N-terminal (amino acid residues 20-25) of p53 that is present in both wild-type and mutant (unfolded or misfolded) conformations.

[0176] In some embodiments, the p53 capture binding agent which binds to the p53 N-terminal domain binds to amino acid residues 18 to 27 of SEQ ID NO: 1. In some embodiments, the p53 capture binding agent which binds to the p53 N-terminal domain binds to amino acid residues 18 to 25 of SEQ ID NO: 1. In some embodiments, the p53 capture binding agent which binds to the p53 N-terminal domain binds to amino acid residues 9 to 25 of SEQ ID NO: 1. In some embodiments, the p53 capture binding agent which binds to the p53 N-terminal domain is PAb242 or an antigen binding fragment thereof. PAb242 is a commercially available antibody (Novus Biologicals, Cat. No. NBP2-53113) which binds to an epitope in the N-terminal region (amino acid residues 18-27) of p53 that is present in both native and mutant conformations.

[0177] In some embodiments, the p53 capture binding agent binds to the p53 N-terminal domain binds to amino acid residues 46 to 55 of SEQ ID NO: 1. In some embodiments, the p53 capture binding agent which binds to the p53 N-terminal domain is PAb1801 or an antigen binding fragment thereof. PAb1801 is a commercially available antibody (Abeam, Cat. No. Ab28) which binds to an epitope in the N-terminal region (amino acid residues 46-55) of p53 that is present in both native and mutant conformations.

[0178] In some embodiments, the p53 capture binding agent binds to the p53 C-terminal domain. In some embodiments, the p53 capture binding agent which binds to the p53 C-terminal domain is selected from an antibody or an antigen binding fragment thereof and an aptamer. Preferably, the p53 capture binding agent which binds to the C-terminal domain is an antibody or antigen binding fragment thereof.

[0179] In some embodiments, the p53 capture binding agent binds to a p53 C-terminal domain at an epitope located within residues 304-393 of p53 (SEQ ID NO: 1). In some embodiments, the p53 capture binding agent which binds to the p53 C-terminal domain binds to amino acid residues 370 to 378 of SEQ ID NO: 1. In some embodiments, the p53 capture binding agent which binds to the C-terminal domain is PAb421 or an antigen binding fragment thereof. PAb421 is a commercially available antibody (Abeam, Cat. No. Ab245685) which binds to an epitope in the C-terminal region (amino acid residues 370-378) of p53 that is present in both native and mutant conformations. In some embodiments, the p53 capture binding agent which binds to the C-terminal domain is PAb122 or an antigen binding fragment thereof. PAb122 is a commercially available antibody (Abeam, Cat. No. Ab90363) which binds to an epitope in the C-terminal region (amino acid residues 370-378) of p53 that is present in both native and mutant conformations.

[0180] In some embodiments, the p53 capture binding agent which binds to the p53 C-terminal domain binds to amino acid residues 388 to 393 of SEQ ID NO: 1 . In some embodiments, the p53 capture binding agent which binds to the C-terminal domain-specific binding agent is ICA- 9 or an antigen binding fragment thereof. ICA-9 is a monoclonal antibody (described by Hupp and Lane 1995 Cell Biology and Metabolism vol 270, 30, p18165-18174) which binds to an epitope in the C-terminal region (amino acid residues 388-393) of p53 that is present in both native and mutant conformations.

[0181] Any suitable body fluid sample may be used for the methods of the invention including without limitation blood, plasma, serum, urine, cerebrospinal fluid (CSF), saliva swabs, nasal swabs, or an extract or purification therefrom, or dilution thereof. Body fluid samples also include specimens from a live subject or taken post-mortem. The samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner. Advantageously, by relying on body fluid samples, the methods of the invention are less invasive, less time consuming and less costly than screening methods which rely on detection of intracellular p53 aggregates, imaging techniques such as MRI scans or CT scans, and methods which involve tissue biopsies.

[0182] In some embodiments, the body fluid sample is selected from urine, blood e.g. serum or plasma, cerebrospinal fluid, saliva swab, and nasal swab. In one embodiment, the body fluid sample is a blood sample. In one embodiment, the blood sample is a plasma sample.

[0183] In some embodiments, the method further comprises obtaining the body fluid sample(s) from the subject. Any suitable method way be used to obtain the sample. For example, blood samples may be obtained e.g. by venipuncture or by finger prick. A cerebrospinal fluid sample may be obtained e.g. through a lumbar puncture (spinal tap), a cisternal puncture, a ventricular puncture or a ventricular drain. References to acts carried out on a body fluid sample “obtained” from a subject are intended to encompass acts carried out only on a body fluid sample already obtained or “obtainable” from a subject and vice versa.

[0184] The invention also provides a method for determining whether a subject has, or is at risk of developing a cancer, the method comprising: a) contacting the body fluid sample with a plurality of beads comprising a p53 capture binding agent to provide an enriched sample; b) contacting the enriched sample with a p53 detection binding agent; c) individually isolating the beads into separate isolations; d) detecting the presence of the p53 detection binding agent in the separate isolations to determine the number of separate isolations containing a p53 aggregate bound by the capture binding agent and quantify the level of p53 aggregates present in the sample.

[0185] The above method is particularly advantageous for use in the present invention because it provides sensitivity which is much greater than traditional immunoassays and can enable the detection of single molecules in a sample. In brief, the technology involves performing a paramagnetic microbead-based sandwich ELISA, followed by isolation of individual capture beads in arrays of femtoliter-sized reaction wells. Singulation of capture beads within microwells permits buildup of fluorescent product from an enzyme label, so that signal from a single immunocomplex can be detected with a charge-coupled device (CCD) camera in 30 seconds. At very low analyte concentrations, Poisson statistics dictate that bead-containing microwells in the array will contain either a single labelled analyte molecule or no analyte molecules, resulting in a digital signal of either “active” or “inactive” wells. Data collection involves counting active wells corresponding to single enzyme labels. At higher analyte concentrations, digital measurements transition to analog measurements of total fluorescence intensity. Data is usually reported as Average Enzymes per Bead (AEB).

[0186] The method may use a single-molecular array (SIMOA) assay. The SIMOA assay, developed and commercialised by Quanterix Inc, makes use of antibody-coated paramagnetic beads for capture of the target protein in the sample, which in turn are then incubated with a biotinylated detector antibody in a diluent, and then streptavidin beta-galactosidase (SBG) to form an immunocomplex. These beads are then drawn into a flow cell consisting of a large array of microwells each of which has sufficient space for one bead only, where they are loaded magnetically into the array. Resorufin p-D-galactopyranoside (RGP) is added, and the array is sealed with oil to prevent contamination between microwells. Any wells containing an immuno-complexed bead will ultimately show as fluorescent (“ON”) when the flow cell is imaged. Other wells containing an empty bead (or no bead) will remain dark (“OFF”). The paramagnetic beads themselves are also fluorescent at a different wavelength, such that during readout the instrument can determine which microwells contain a bead and which do not. This information together with the number of “ON” beads is used to obtain the fraction of beads which have detected a protein of interest - which can through use of a calibration curve, be used to obtain estimate the analyte concentration in the sample. The SIMOA assay is very sensitive for low concentrations where it works in a digital fashion, where each capture bead only has 1 or 0 immunocomplex formed on it.

[0187] In one embodiment, the p53 aggregates are quantified by measuring brightness distribution of the protein complexes present in the sample. This embodiment is based on the principle that larger protein aggregates will bind more antibodies and hence be brighter and produce a larger fluorescence signal. It is therefore possible to use the brightness of the capture complexes as a proxy for size and measure the fraction of complexes brighter than a certain threshold.

[0188] To determine the number of p53 aggregates within a sample and also to reduce experimental variation and further improve consistency in results, the inventors developed a calibrator standard which is specific to onp53 epitopes (e.g. p53 monomers and / or p53 aggregates) used in the methods of the invention and targeted by the p53 binding agents used to detect p53 aggregates. The calibrator standard advantageously ensures consistency and reliability of the methods regardless of variance among experiments. Thus, in one embodiment the methods of the invention comprise comparing the level of p53 aggregates in the sample to a calibrator standard, wherein the calibrator standard comprises one or more p53 aggregate epitopes, such as two or more, three or more, four or more, five or more, p53 epitopes. The epitopes may be the same (i.e. multiple of the same epitope are used) or different (i.e. the multiple epitopes may be derived from different parts of the same protein, or derived from different proteins).

[0189] When the calibration standard comprises more than one p53 epitopes, said epitopes can be coupled together. This helps to stabilise the epitope for use in the assay. The number of epitopes complexed together can be chosen to model the size of the aggregate to be detected. For example, the calibration standard may comprise two p53 epitopes as a dipeptide. Furthermore, the p53 epitopes may be the same or different. For example, multiple antibody epitopes may be joined together. Using multiple epitopes can increase the versatility of the calibrator and / or more closely resemble p53 aggregates to be captured if different epitopes are exposed. In a further embodiment, the p53 epitope comprises a first p53 peptide or protein. In a further embodiment, the p53 epitope further comprises a second p53 peptide or protein.

[0190] In one embodiment, the first p53 peptide or protein comprises a p53 aggregate epitope (e.g. in the DNA-binding region of p53) to which the p53 detection binding agent binds. In one embodiment, the first peptide or protein comprises amino acids 211-217 of p53. In one embodiment, the first peptide or protein comprises amino acids 181-190 of p53. In one embodiment, the first peptide or protein comprises amino acids 256-270 of p53.

[0191] In one embodiment, the second p53 peptide or protein comprises a p53 N-terminal domain epitope or a p53 C-terminal; domain epitope to which the p53 capture binding agent binds. In one embodiment, the second peptide or protein comprises amino acids 20-25 of p53. In one embodiment, the second peptide or protein comprises a p53 N-terminal domain epitope, in particular the second peptide or protein comprises amino acids 18-27 of p53. In one embodiment, the second peptide or protein comprises a p53 N-terminal domain epitope, in particular the second peptide or protein comprises amino acids 18-25 of p53. In one embodiment, the second peptide or protein comprises a p53 N-terminal domain epitope, in particular the second peptide or protein comprises amino acids 9-25 of p53. In one embodiment, the second peptide or protein comprises a p53 N-terminal domain epitope, in particular the second peptide or protein comprises amino acids 46-55 of p53.

[0192] In one embodiment, the second peptide or protein comprises a p53 C-terminal domain epitope, in particular the second peptide or protein comprises amino acids 371-380 of p53. In one embodiment, the second peptide or protein comprises a p53 C-terminal domain epitope, in particular the second peptide or protein comprises amino acids 388-393 of p53.

[0193] In some embodiments, the calibrator standard comprises a solid support to which the p53 epitopes are bound. In some embodiments, the solid support is selected from a bead, a solid surface and a membrane. In some embodiments, the solid support is a silica bead or nanoparticle. In some embodiments, the calibrator standard mimics a fixed size aggregate with multiple binding peptides of each antibody.

[0194] In some embodiments, the p53 detection binding agent binds to amino acid residues 211-217 of SEQ ID NO: 1 ; and the first p53 peptide or protein has an amino acid sequence comprising amino acid residues 211-217 of SEQ ID NO: 1. In some embodiments, the first peptide or protein has an amino acid sequence comprising K(polyethylene glycol (PEG))8TFRHSVV.

[0195] In some embodiments, the p53 capture binding agent binds to amino acid residues 20-25 of SEQ ID NO: 1 and the second p53 peptide or protein has an amino acid sequence comprising amino acid residues 20-25 of SEQ ID NO: 1. In some embodiments, the second p53 peptide or protein has an amino acid sequence comprising K(PEG)sTFSDLWKLLP.

[0196] In some embodiments, the p53 capture binding agent binds to amino acid residues 18-27 of SEQ ID NO: 1 and the second p53 peptide or protein has an amino acid sequence comprising amino acid residues 18-27 of SEQ ID NO: 1.

[0197] In some embodiments, the p53 capture binding agent binds to amino acid residues 18-25 of SEQ ID NO: 1 and the second p53 peptide or protein has an amino acid sequence comprising amino acid residues 18-25 of SEQ ID NO: 1.

[0198] In some embodiments, the p53 capture binding agent binds to amino acid residues 18-27 of SEQ ID NO: 1 and the second p53 peptide or protein has an amino acid sequence comprising amino acid residues 9-25 of SEQ ID NO: 1.

[0199] In some embodiments, the p53 capture binding agent binds to amino acid residues 46-55 of SEQ ID NO: 1 and the second p53 peptide or protein has an amino acid sequence comprising amino acid residues 46-55 of SEQ ID NO: 1.

[0200] In some embodiments, the p53 capture binding agent binds to amino acid residues 371-380 of SEQ ID NO: 1 ; and the second p53 peptide has an amino acid sequence comprising amino acid residues 371-380 of SEQ ID NO: 1 . In some embodiments, the p53 capture binding agent is PAb421 or an antigen binding fragment thereof.

[0201] In some embodiments, the p53 capture binding agent binds to amino acid residues 371-380 of SEQ ID NO: 1 ; and the second p53 peptide has an amino acid sequence comprising amino acid residues 388-3393 of SEQ ID NO: 1.

[0202] The invention also provides a kit for detecting the level of p53 aggregate in a sample, the kit comprising: (a) a p53 capture binding agent and (b) a p53 detection binding agent.

[0203] In one embodiment, the kit further comprises instructions for use in: (i) diagnosing cancer; (ii) determining the risk of developing cancer; (iii) monitoring progression or onset of cancer; and / or (iv) determining the efficacy of a therapeutic intervention in subject with cancer.

[0204] The p53 capture binding agent, and the p53 detection binding agent may be as defined above in the context of the methods of the invention.

[0205] In some embodiments, the kit comprises: (a) a p53 detection binding molecule which binds an epitope having an amino acid sequence comprising amino acid residues 211-217 of SEQ ID NO: 1 ; and (b) a p53 capture binding agent which binds an epitope having an amino acid sequence comprising amino acid residues 20-25 of SEQ ID NO: 1. In some embodiments, the kit comprises a calibrator. The calibrator may be as defined above in the context of the methods of the invention.

[0206] In some embodiments, the kit further comprises a solid support to which the p53 capture binding agent is bound. The solid support may be selected from a bead, a solid surface and a membrane.

[0207] According to a further aspect, there is provided a method of treating a cancer in a subject, wherein the method comprises: (a) comparing the level of p53 aggregates in a body fluid sample obtained from the subject to a threshold level; (b) determining whether the subject has cancer based on the comparison performed in step (a) ; and (c) administering a treatment to the subject if the subject is determined to have a cancer.

[0208] The method may further comprise detecting the level of p53 aggregates in the body fluid sample. The level of p53 aggregates may be determined as defined above in the context of the other methods of the invention.

[0209] Any suitable treatment may be used. The treatment may be as defined above in the context of the other methods of the invention.

[0210] According to a further aspect, there is provided a method of treating a subject at risk of developing a cancer, wherein the method comprises: (a) comparing the level of p53 aggregates in a body fluid sample obtained from the subject to a threshold level; (b) determining whether the subject has cancer based on the comparison performed in step (a) ; and (c) administering a treatment to the subject if the subject is determined to be at risk of developing a cancer.

[0211] The method may further comprise detecting the level of p53 aggregates in the body fluid sample. The level of p53 aggregates may be determined as defined above in the context of the other methods of the invention.

[0212] The threshold level may be a threshold level as defined herein. For example, the threshold level may a threshold level of p53 aggregates which is indicative of a known risk of cancer being present in the subject. Thus, a lower level of p53 aggregates in the body fluid sample as compared to the threshold level may be indicative that the subject has a lower risk of cancer as compared to the risk level associated with the threshold level. The same level of p53 aggregates in the body fluid sample as compared to the threshold level may be indicative that the subject has the same risk of cancer as the risk level associated with the threshold level. A higher level of p53 aggregates in the body fluid sample as compared to the threshold level may be indicative that the subject has a higher risk of cancer as compared to the risk level associated with the threshold level. In some embodiments, the same or lower level of p53 aggregates in the body fluid sample as compared to the threshold level may be indicative that the subject has a low risk of cancer. In other words, the subject may be less likely to develop cancer in the future than subjects who have levels of p53 aggregates above the threshold level. In some embodiments, a higher level of p53 aggregates in the body fluid sample as compared to the threshold level may be indicative that the subject has a high risk of cancer. In other words, the subject may be more likely to develop cancer in the future than subjects who have levels of p53 aggregates at the same level or lower the threshold level.

[0213] The treatment administered to the subject may be as defined above in the context of the other methods of the invention.

[0214] The amino acid sequence of human p53 is provided by SEQ ID NO: 1 (Uniprot P04637):

[0215] MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGP DEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAK SVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEWRRCPHHE RCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVWPYEPPEVGSDCTTIHYNYMCNSS CMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPG STKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSR AHSSHLKSKKGQSTSRHKKLMFKTEGPDSD (SEQ ID NO: 1)

[0216] EXAMPLES

[0217] The invention will be further clarified by the following examples, which are intended to be purely exemplary of the invention and are in no way limiting.

[0218] Example 1

[0219] Assay development and optimisation

[0220] The SiMoA assay relies on a pair of monoclonal antibodies to capture and detect p53 aggregates in plasma samples (Fig. 1A). Specifically, SiMoA utilises antibody-conjugated paramagnetic beads to capture target proteins in the sample, followed by incubation with a biotinylated detector antibody and streptavidin-p-galactosidase fusion protein (SBG), forming single immunocomplexes on the bead. The beads are then introduced to a microwell array, such that each microwell contains only one or no bead. The immunocomplex-carrying bead in the microwell can catalyse the hydrolysis of a fluorogenic substrate, resorufin p-D- galactopyranoside (RGP), enabling quantification of p53 aggregates. Two p53 antibodies, DO-1 (Abeam, Cat. No. Ab1101) and PAb240 (Novus Biologicals, Cat. No. NB200-103). DO-1 recognises an epitope close to the N-terminal (aa 20-25) of p53 that is present in both native and mutant conformations24. In contrast, PAb240 binds to an epitope (aa 211-217) in the central DNA-binding region of p53 that is only accessible in the mutant conformation of p5324. The inventors initially evaluated the performance of two capturedetector pairs, DO-1 capture / DO-1 detector (D-D) and DO-1 capture / PAb240 detector (D-P) (Fig. 1 B). The D-D pair cannot detect p53 monomers due to identical epitope binding. In contrast, the D-P pair lacks the selectivity of the D-D pair for multimers but can detect p53 aggregates as aggregation requires partial unfolding of the protein, exposing the PAb240- binding epitope25.

[0221] The performance of the D-D and D-P pairs was compared using recombinant p53 aggregates (Fig. 1C and 1 D) and plasma samples from a GB patient and a control (Fig. 1 E and 1 F). The readout value of the SiMoA platform is the average enzyme per bead (AEB), which is derived from the ratio of the number of fluorescent ‘on’ beads to the total number of beads in the assay26. Samples containing higher concentrations of the target protein will have more beads with immunocomplexes containing enzymes and hence more microwells will be fluorescent, leading to a higher AEB. The AEB value can be converted to a concentration using a calibration sample.

[0222] The AEB values were higher for recombinant aggregates with the D-D pair than the D-P pair (Fig. 1C and 1 D). Both pairs showed increased signal levels from the R248Q p53 aggregates compared to the WT p53 aggregates, since the R248Q p53 mutant has a higher aggregation propensity compared to WT p5327. The patient sample showed comparable signal with both pairs, whereas the control sample showed signal levels significantly higher than the background with the D-D pair but not the D-P pair (Fig. 1 E and 1 F). Reducing the sample volume lowered the AEB values for both pairs, with the control signal for the D-P pair retained at a background level. The D-P pair showed much better discrimination of the patient samples than the D-D pair.

[0223] The performance of both pairs was further compared using 10 control and 10 GB patient samples (Fig. 1G-J). The D-D pair showed no significant difference between the controls and GB patient samples (Fig. 1G) (Control vs GB (Median (IQR)): 0.303 (0.134, 0.502) vs 0.354 (0.268, 0.665), Mann-Whitney II test p-value = 0.385), whereas the D-P pair showed a significant difference (Fig. 11) (Control vs GB (Median (IQR)): 0.013 (0.011 , 0.017) vs 0.059 (0.045, 0.080), Mann-Whitney II test p-value = 0.003). A single control sample exhibited anomalously high AEB values with both D-D and D-P pairs. The receiver operating characteristic (ROC) curve demonstrated an area under the curve (AUC) of 0.62 and 0.90 for the D-D and D-P pairs, respectively (Fig. 1 H and 1 J), suggesting that the D-P pair has a superior diagnostic accuracy to the D-D pair for p53 aggregate detection in liquid biopsies.

[0224] Validating the presence of p53 aggregates using super-resolution imaging

[0225] To further characterise the p53 aggregates detected by the SiMoA assay, super-resolution imaging was utilised on plasma samples using a single-molecule pull-down (SiMPull) assay. SiM Pull captures the proteins of interest on an antibody-coated glass coverslip for microscopic characterisation28-29. Similar to the SiMoA assay, the inventors immobilized DO-1 antibodies on PEGylated coverslips to selectively ‘pull down’ p53 proteins in the plasma samples. The captured p53 aggregates were then labelled with fluorophore-conjugated PAb240 antibody for fluorescence imaging. The inventors imaged a total of 8 GB samples with high p53 concentrations as detected by SiMoA and 9 control samples showing no p53 aggregates.

[0226] Numerous fluorescent spots can be observed in the diffraction-limited images of patient plasma samples but not in the control samples (Fig. 2A), in agreement with the SiMoA results. Significantly more fluorescent spots per field-of-view (FOV) were observed in the GB plasma samples than the controls (Control vs GB (Median (IQR)): 9.75 (7.38, 14.00) vs 617.25 (15.00, 1960.13), Mann-Whitney II test p-value = 0.034) (Fig. 2B). Super-resolution imaging further demonstrated the presence of aggregates over 200 nm across in the patient samples (Fig. 2C). Cluster analysis of the localisation data showed significantly higher number of localisations per FOV (Control vs GB (Median (IQR)): 0.864 (0.716, 0.864) vs 9.576 (2.923, 49.859), Mann-Whitney II test p-value = 0.001), number of clusters per FOV (Control vs GB (Median (IQR)): 0.561 (0.316, 0.751) vs 6.925 (1.638, 30.780), Mann-Whitney U test p-value = 6.355x1 O'4), average number of localisations per cluster (Control vs GB (Median (IQR)): 0.592 (0.405, 0.808) vs 1.638 (1.353, 1.918), Mann-Whitney II test p-value = 0.008), and ratio of clustered localisations (Control vs GB (Median (IQR)): 5.269 (3.813, 10.119) vs 57.210 (36.153, 82.255), Mann-Whitney II test p-value = 0.008) in the GB plasma samples compared with controls (Fig. 2D). When comparing the mean cluster areas of plasma samples with those of recombinant WT and R248Q p53 monomer and aggregates at 1 nM, it was found that most control samples had similar cluster areas to those of monomeric WT or R248Q p53; while the GB patient samples exhibited higher cluster areas comparable to those of the 1 nM WT p53 aggregates (Fig. 2E). These data collectively suggest that more and larger p53 aggregates exist in the GB patients’ plasma, which may be responsible for the elevated signals in the SiMoA assays. Calibrator development and assay optimisation

[0227] The inventors next developed a calibrator for the SiMoA assay based on the D-P antibody pair, which enables calculation of p53 aggregate concentrations from the AEB values and can compensate for experimental variation and ensure consistency in the results. Since recombinant p53 is prone to aggregation and can form aggregates that are heterogeneous in size and structure27, it was not suitable as a calibration standard. The inventors therefore developed a calibrator composed of silica beads conjugated with the epitopes targeted by the DO-1 and PAb240 antibodies. The two peptides are covalently attached to the silica nanoparticle and are thus unlikely to inter- or intra-molecularly interact with each other to form aggregates. Hence, this single entity peptide-coated silica nanoparticle can mimic a fixed size aggregate with multiple antibody-binding peptides (Fig. 3A)27. The inventors first evaluated the aggregation propensity of the two peptides using the online amyloid prediction platform, PASTA2.0. Both peptides and their concatenated sequence were predicted not to form amyloid aggregates (amyloids = 0), suggesting a low likelihood of either self-aggregation or cross-aggregation. Fluorescence images of the calibrator beads also showed no significant aggregation (Fig. 3B). In SiMoA experiments, the calibrator bead showed highly consistent AEB values in three experimental replicates (Fig. 3C). The calibrator exhibited a large linear range spanning from approximately 4 pM to 3000 pM, covering AEB values between 0.01 and 1 , within which the SiMoA operates in the ‘digital’ mode for aggregate detection26.

[0228] To improve the sensitivity and diagnostic accuracy of the assay, the inventors further optimised the SiMoA assay conditions, including detector concentration, SBG enzyme concentration, and sample volume. The signal-to-background ratios (SBR) for various detector-SBG concentration combinations are shown in Fig. 3D. The lower detection limit and AEB range for all conditions were calculated and are shown in Fig. 6A. The three conditions with the highest SBR (0.3 pg / mL detector + 50 pM SBG; 0.5 pg / mL detector + 50 pM SBG; and 0.3 pg / mL detector + 150 pM SBG) had similar lower detection limits, whereas 0.3 pg / mL detector + 150 pM SBG had the largest AEB value at maximum calibrator bead concentration. A sample volume of 20 pL per well differentiated control and GB patient samples better than using 5 pL (Fig. 3E and 3F). Therefore, 0.3 pg / mL capture detector, 150 pM SBG, and 20 pL sample per well were considered to be optimal for detection of p53 aggregates.

[0229] Detection of p53 aggregates in GB patient plasma

[0230] A total of 22 control and 190 GB patient plasma samples were tested using the D-P antibody pair (see Table 1 for sample demographics). p53 aggregate concentrations were higher in the GB patients compared to controls (Control vs GB (Median (IQR)): 0 (0, 0) vs 126.100 (46.523, 308.072), Mann-Whitney II test p-value = 5.131 x10'1°, Fig. 4A). Notably, 18 of the 22 control samples (81.82%) had AEB values equal to or less than the blank AEB value on the same plate, and their concentrations were thus set to 0 pM. On the contrary, only 12 out of the 190 GB patients (6.32%) had undetectable p53 aggregate concentrations (0 pM). The ROC curve had an AUG of 0.905, demonstrating significant diagnostic accuracy (Fig. 4B). To ensure that the higher p53 aggregate concentrations detected in patient samples were not simply a result of elevated total protein concentrations, the fitted p53 aggregate concentration was normalised to the total protein concentrations (as measured from A28o, see Fig. 6B)(Fig. 4C). The difference was still significant after normalisation (Control vs GB (Median (IQR)): 0 (0, 0) vs 26.003 (9.388, 58.321), Mann-Whitney II test p-value = 3.056x1 O'10), while the corresponding ROC curve showed a slightly higher AUC of 0.910 (Fig. 4D). The optimal threshold was determined by the Youden index to be 1.895 (a.u.)31, with which the diagnostic accuracy, sensitivity, and specificity were 90.57%, 90.53%, and 90.91%, respectively (Fig. 7). Next, 25 patients with GB were identified for whom plasma samples were available at 2 or more post operative timepoints for inclusion in a retrospective longitudinal review. All patients underwent concurrent MRI enabling us to map the p53 aggregate measurement to the extent of radiological disease. p53 was detectable in all patient samples and radiological findings (stable disease vs tumour recurrence) were then mapped to subsequent treatment (surgery, radiotherapy and / or chemotherapy). All patients demonstrated clinical congruence with p53 levels, either through a rise in p53 levels prior to tumour recurrence (40%), a decline in p53 levels post-treatment (16%), or a combination of these two patterns (44%) (Fig. 4E-F).

[0231] Table 1. Sample demographics.

[0232] Controls GB patients

[0233] (n=22) (n=190)

[0234] Age (years, %)

[0235] 21-30 0 (0) 3 (1.6)

[0236] 31-40 2 (9.1) 8 (4.2)

[0237] 41-50 4 (18.2) 20 (10.5)

[0238] 51-60 5 (22.7) 64 (33.7)

[0239] 61-70 4 (18.2) 60 (31.6)

[0240] 71-80 5 (22.7) 32 (16.8)

[0241] 81-90 2 (9.1) 3 (1.6)

[0242] 91-100 0 (0) 0 (0)

[0243] Sex

[0244] Women (%) 9 (40.9) 69 (36.3) Detection of elevated p53 aggregate levels in the plasma of patients with IDH-mutant primary and metastatic brain cancers

[0245] Given the broad tumour-suppressing function of p53, it was investigated whether p53 aggregate levels were elevated in the plasma of other brain cancer patients, including IDH- mutant primary and metastatic brain cancers. Patients with metastatic lung cancer and breast cancer, all of whom had isolated brain metastatic lesions and stable disease elsewhere, showed comparable or higher plasma p53 aggregate concentrations than the GB samples (GB vs breast cancer vs lung cancer (Median (IQR)): 26.003 (9.388, 58.321) vs 95.160 (10.807, 213.070) vs 90.468 (44.214, 514.595)) (Fig. 4G), whereas patients with the lower grade IDH-mutant brain tumours, astrocytoma and oligodendroglioma, exhibited lower, but still significant, concentrations of p53 aggregates compared with GB (GB vs astrocytoma vs oligodendroglioma (Median (IQR)): 26.003 (9.388, 58.321) vs 23.568 (15.542, 43.751) vs 9.658 (5.780, 57.970)) (Fig. 4H). The plasma samples from patients with other brain metastases showed a variety of p53 aggregate concentrations; however, the sample sizes were too small to be considered statistically significant. These data suggest that elevated plasma p53 aggregate concentrations are not a specific indicator for GB and may be indicative of the presence of other cancers within the brain and possibly elsewhere in the body.

[0246] Detection of elevated p53 aggregate levels in ovarian cancer

[0247] The inventors next investigated whether p53 aggregate levels were elevated in the plasma of other cancer patients, including ovarian cancer patients without brain metastases. Plasma p53 aggregate concentrations were higher in ovarian patients compared to controls (Mann- Whitney II test p-value = 1.625x1 O'10, Fig. 9A). The ROC curve had an AUC of 0.902, demonstrating significant diagnostic accuracy (Fig. 9B). The optimal threshold was determined by the Youden index to be 10.89 pM31, with which the diagnostic accuracy, sensitivity, and specificity were 85.53%, 96%, and 83.47%, respectively. These data confirm that elevated plasma p53 aggregate concentrations may be indicative of the presence of cancers in parts of the body other than the brain.

[0248] Discussion

[0249] As demonstrated herein, the present inventors surprisingly discovered that body fluid samples (such as blood samples) obtained from cancer patients exhibit significantly higher levels of p53 aggregates than body fluid samples (such as blood samples) obtained from patients who do not have cancer. The inventors also surprisingly identified that the level of p53 aggregates in body fluid samples provides a reliable and powerful tool for diagnosis and prognosis of cancer. Thus, the inventors have demonstrated for the first time that p53 aggregates can be employed as a biomarker for cancers associated with p53 aggregates. Moreover, by using body fluid samples to detect p53 aggregates the inventors have developed improved methods for detecting cancer, determining the risk of cancer in a subject, monitoring the progression of cancer, and / or determining the efficacy of a therapeutic intervention for treating cancer in a subject which are less invasive, less time consuming and less costly than existing screening cell based methods which rely on detection of intracellular p53 aggregates.

[0250] The present inventors have also developed and optimised a SiMoA assay for quantifying p53 aggregates in human plasma samples. Using the DO-1 and PAb240 antibodies for capture and detection, the assay demonstrated a diagnostic accuracy of 90.90% in distinguishing between GB patients (n=190) and controls (n=22). The presence of p53 aggregates over 200 nm across was validated with super-resolution imaging using the same antibody pair. It is also demonstrated that p53 aggregate concentrations are associated with GB recurrence and treatment response, credibly showing that plasma p53 aggregate concentrations are a potential prognostic biomarker for GB. Moreover, increased p53 aggregate concentrations in cancer patients with brain metastases. The fact that p53 aggregates can be detected in IDH mutant primary and metastatic brain tumours indicates that the presence of p53 aggregate is not an exclusive biomarker for GB diagnosis and thus credibly demonstrate that p53 aggregates have diagnostic and prognostic applications beyond GB. Indeed, the detection of elevated p53 aggregate levels in ovarian cancer demonstrates that p53 aggregates in body fluid samples have diagnostic and prognostic applications in p53 aggregate associated cancers outside the brain.

[0251] The D-P antibody pair is the basis of the SiMoA assay. The DO-1 antibody-conjugated beads are thought to capture and enrich all p53 species, whereas the PAb240 antibody detects p53 aggregates that expose the unfolded DNA-binding domain. In contrast, while the D-D antibody pair is thought to detect multimeric p53, it did not show a clear separation between controls and GB patients, presumably due to the presence of p53 dimers and / or tetramers in the plasma of healthy individuals.

[0252] This study highlights the potential utility of plasma p53 aggregate concentrations for early detection, diagnosis and monitoring of patients with brain cancer. Earlier detection of both primary and recurrent disease in GB and IDH mutant glioma would enable rapid institution of treatment and thereby maximise the potential for patient benefit. There is preliminary evidence that earlier detection of GB, in its low-grade state, may improve survival32. Similarly, early detection of brain metastasis enables the rapid institution of a broad range of therapies that are not available when lesions are large and symptomatic. Although MRI remains the primary method for identifying brain tumours, including at recurrence, alternate lower cost approaches, such as liquid biopsy3334, that do not have the logistical challenges of MRI, and that avoid the high levels of patient anxiety whilst awaiting an MRI scan would be advantageous.

[0253] Glioma has been shown previously to be a particularly challenging cancer to detect through liquid biopsy and as such, these data may indicate broader applicability of this approach across cancers in which liquid biopsy is more straightforward. Thus, the prevalence of TP53 mutation across cancers and the broad applicability of p53 aggregate detection advantageously enables diagnosis of a broad range of cancer types.

[0254] Materials and Methods

[0255] Plasma sample collection and processing

[0256] Patients were recruited at Addenbrooke’s Hospital, Cambridge, UK as part of the Integrated Clinically-Augmented Repository for Universal Sampling (ICARUS) tissue collection (REC 18 / EE / 0172). Patients with suspected GB on pre-operative contrast-enhanced MRI were chosen for participation in the study. Sampling was performed during the initial surgery for a new diagnosis of glioma. Plasma from control patients undergoing non-cancer surgery was collected intra-operatively (matching conditions for the GB cohort). Follow up samples were collected during outpatient clinics with MRI being performed as per routine clinical paradigm. Informed consent was obtained from all subjects, and all experiments conform to the principles set out in the Declaration of Helsinki. Samples were centrifuged at 1 ,500 g at 4°C for 10 min before being aliquoted into 0.5-mL protein lo-bind microcentrifuge tubes (Eppendorf, Cat. No. 0030108094). The aliquoted samples were stored at -80 °C. All plasma samples underwent a maximum of three freeze-thaw cycles before being tested.

[0257] To determine the total protein concentration of the plasma samples, all samples were first diluted 10-fold in phosphate-buffered saline (PBS, Gibco, Cat. No. 10010023). The total protein concentrations were then determined by A280 (NanoDrop One, Thermo Scientific, Cat. No. ND-ONE-W).

[0258] Blood samples from subjects having ovarian cancer were collected as described in Crispin-Ortuzar, M., et al. (2023). Integrated radiogenomics models predict response to neoadjuvant chemotherapy in high grade serous ovarian cancer. Nat Commun 14, 6756.

[0259] Single Molecule array (SiMoA) bead conjugation

[0260] The antibody-conjugated SiMoA bead was prepared following the manufacturer’s instructions. Firstly, 100 pg of DO-1 antibody (Abeam, Cat. No. Ab1101) was buffer-exchanged into the Bead Conjugation Buffer (Quanterix, Cat. No. 101354) using an AmiconUltra-0.5 centrifugal filter (Merck, Cat. No. UFC500396, molecular weight cut-off 50 kDa). The buffer-exchanged antibody was then diluted with the Bead Conjugation Buffer to 0.2 mg / mL and kept on ice before use. Secondly, a total of 4.2 x 108carboxylated paramagnetic beads (Quanterix, Cat. No. 101354) were transferred to a 1.5 mL microcentrifuge tube (Eppendorf, Cat. No. 0030108116). The tube was then placed on a magnetic separator (Cytiva, Cat. No. 28-9489- 64) for 1 min, and the supernatant was removed. The beads were washed three times with the Bead Wash Buffer (Quanterix, Cat. No. 101354) and another three times with the Bead Conjugation Buffer. Subsequently, the washed beads were activated using a freshly prepared solution of 0.3 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, Thermo Scientific, Cat. No. A35391) in the Bead Conjugation Buffer at 4 °C for 30 min on a HulaMixer (Thermo Scientific, Cat. No. 15920D). Thirdly, the activated beads were washed once with the Bead Conjugation Buffer, followed by the addition of the buffer-exchanged antibody. The reaction mixture was incubated at 4 °C for 2 hours on the HulaMixer. Next, the antibody- conjugated beads were washed twice with the Bead Wash Buffer and blocked with the Bead Blocking Buffer (Quanterix, Cat. No. 101354) for 45 min at room temperature on the HulaMixer. Finally, the blocked beads were washed twice with the Bead Wash Buffer, resuspended with 300 pL of Bead Diluent (Quanterix, Cat. No. 101354), and stored at 4 °C until use.

[0261] Preparation of recombinant p53 aggregate

[0262] The recombinant p53 protein was purchased from GenScript (WT p53: 1.10 mg / mL, GenScript, Cat. No. U0276EA140-5 / P4EB001 ; R248Q p53: 1.41 mg / mL, GenScript, Cat. No. U0276EA140-9 / P4EB001). The recombinant p53 aggregates were prepared by diluting the stock proteins using the aggregation buffer (50 mM Tris, 150 mM NaCI, 5 mM 1 ,4-dithiothreitol (DTT, Roche, Cat. No. 10708984001), pH 7.2) to 1 pM. The protein samples were then incubated at 37 °C on a shaking incubator (Grant-bio) for 72 hours, sonicated in an ice-water bath for 5 min, and diluted with the aggregation buffer to the indicated concentrations.

[0263] SiMoA assay

[0264] The SiMoA assay was performed following the manufacturer’s instructions. Briefly, to each well of a 96-well plate (Quanterix, Cat. No. 103077), 20 pL of plasma and 80 pL of Homebrew Sample / Detector diluent (Quanterix, Cat. No. 101359) were loaded, followed by the addition of 25 pL of DO-1 -conjugated beads in Bead Diluent (2 x 107beads / mL). The maximum sample volume in the preliminary experiments was set to 25 pL per well. For assay optimisation, the sample consisted of either 20 pL of plasma mixed with 80 pL of diluent, or 5 pL of plasma and 95 pL of diluent. Plasma samples were sonicated in an ice-water bath for 5 min before being introduced to the plate. The samples and calibrators were tested in duplicates. The 96-well plate was then incubated on a shaking incubator for 30 min at 30 °C, 800 rpm. Subsequently, the plate was washed using a microplate washer (Quanterix). Meanwhile, the biotinylated PAb240 (Novus Biologicals, Cat. No. NB200-103B) detector antibody was diluted with the Sample / Detector diluent to 0.3 pg / mL. After the first wash, 100 pL of the detector antibody was introduced to each well. The plate was then incubated for 10 min on the shaking incubator before returned to the washer for the second wash. The SBG (Quanterix, Cat. No. 101361) was diluted with the SBG diluent (Quanterix, Cat. No. 101361) to 150 pM. Next, 100 pL of the SBG solution was introduced to each well and the plate was incubated for 10 min on the shaking incubator. Finally, the plate was washed again and loaded on the SiMoA SR-X detection system for analysis.

[0265] Preparation of the calibrator

[0266] The 15-nm 3-aminopropyl(3-oxobutanoic acid) functionalised silica nanoparticles (SiNaP- COOH, Merck, Cat. No. 660450, estimated concentration 11.47 pM) were centrifuged at 10,000 g at room temperature for 1 hour. The supernatant was discarded, and the pellet resuspended in an equal volume of 2-(N-morpholino)ethanesulfonic acid (MES) buffer (10 mM, pH 5.7). Meanwhile, 1 ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, ThermoFisher, Cat. No. A35391) and sulfo-N-hydroxysuccinimide (sulfo-NHS, ThermoFisher, Cat. No. A39269) were freshly dissolved in cold MES buffer (10 mM, pH 5.7) at 10 mg / mL and 20 mg / mL, respectively. These solutions were mixed with the SiNaP-COOH suspension to achieve final concentrations of 100 nM SiNaP-COOH, 400 pM EDC, and 100 pM sulfo-NHS. The mixture was sonicated for 30 min, centrifuged at 10,000 g for 1 hour at room temperature, and the pellet resuspended in 1 mL of 10 mM MES buffer at 200 nM. The DO-1-binding peptide (sequence: K(PEG)8TFSDLWKLLP, Cat. No. U577SIB280-5) and the PAb240-binding peptide (sequence: K(PEG)8TFRHSVV, Cat. No. U577SIB280-7) were separately dissolved in water to give 1 mM concentrations. To 1 mL of the activated SiNaP-COOH suspension, a mixture of 5 pL DO-1 -binding peptide solution and 5 pL PAb240-binding peptide solution was added. The reaction mixture was placed on a HulaMixer and incubated overnight at room temperature and centrifuged at 10,000 g for 1 hour at 4 °C. The pellet was resuspended in 1 mL of 1 :1 H2O:DMSO (v / v), sonicated for 5 min, and centrifuged at 5,000 g for 1 hour at 4 °C. Finally, the pellet was resuspended in 200 pL of 1 :1 H2O:DMSO (v / v), aliquoted, and stored at -20 °C until use. The stock concentration of the peptide-conjugated SiNaP was estimated to be 1 pM, assuming no loss during the reactions. The synthesis route is shown in Fig. 8.

[0267] Prediction of aggregation propensity

[0268] The aggregation propensities of the DO-1 -binding peptide (sequence: TFSDLWKLLP), PAb240 binding peptide (sequence: TFRHSVV), and their concatenated peptide (sequence: TFSDLWKLLPTFRHSW), were input into the PASTA2.0 website and analysed with default thresholds for peptides. The top pairing energy and the energy threshold were set to 1 and - 5, respectively.

[0269] Data analysis

[0270] The calibration curve was fitted using the four-parameter logistic (4PL) fit with 1 / y2 weighting. The p53 aggregate concentrations in the samples were back-calculated from the mean AEB values of two duplicate wells using the calibration curve. The lower limit of detection was back- calculated from the mean ± 2.5 standard deviation (SD) of the blank AEB. The normalised p53 aggregate concentrations were calculated by dividing the fitted concentration (pM) by the protein A280 of the same sample. Mann-Whitney II test was performed for datasets that are not normally distributed. P values below 0.05 are considered statistically significant. All analyses were performed using the OriginLab 2021b software (OriginLab Corporation, USA).

[0271] SiMPull of p53 aggregates in plasma

[0272] The SiMPull coverslips were prepared as previously described36’37. The coverslips were firstly cleaned by three 10-min sonication periods in 18.2 MQ cm water, acetone (Thermo Fisher, Cat. No. 10442631), and methanol (Thermo Fisher, Cat. No. 10675112). The coverslips were then sonicated in 1 M KOH solution for 20 min, thoroughly rinsed with methanol, water, and methanol, dried with a stream of nitrogen, and cleaned with argon plasma for 15 min (PDC- 002, Harrick Plasma). Subsequently, the coverslips were silanised with a 3:5:100 mixture of 3-aminopropyl triethoxysilane (Fisher Scientific UK, Cat. No. 10677502), acetic acid (Merck, Cat. No. 45726), and methanol, and sonicated in two cycles, each comprising a 60 s ‘ON’ period and a 10 min ‘OFF’ period. The silanised coverslips were again rinsed with excess methanol, water, and methanol before being dried with a stream of nitrogen. Next, a 50-well PDMS gasket (Merck, Cat. No. GBL103250) was carefully affixed to each coverslip. Meanwhile, a 100:1 aqueous mixture of methoxy-PEG-Succinimidyl Valerate (110 mg / mL, Laysan Bio Inc., Cat. No. MPEG-SVA-5000) and biotin-PEG-Succinimidyl Valerate (100 mg / mL, Laysan Bio Inc., Cat. No. Biotin-PEG-SVA-5000) was freshly prepared. Each well on the coverslip was passivated with 9 pL of the mixture and 1 pL of 1 M NaHCO3 solution (pH 8.5). The coverslips were then incubated in a humidity chamber overnight at room temperature, rinsed with excess 18.2 MQ cm water, and dried with a stream of nitrogen. Each well was further passivated by introducing a mixture of 9 pL of 10 mg / mL methyl-PEG4-NHS- Ester (Thermo Fisher, Cat. No. 22341) and 1 pL of 1 M NaHCO3 solution (pH 8.5). The coverslips were again incubated overnight in the humidity chamber, washed with 18.2 MQ cm water, and dried with a stream of nitrogen. Finally, the dried coverslips were stored in a desiccator at -20 °C until use. The SiMPull assay was performed as previously described36. Prior to the experiment, the following buffers and reagents were prepared and stored on ice: (1) PBST: PBS supplemented with 0.05% tween 20 (Merck, Cat. No. P1379-100ML); (2) PBS+1% tween: PBS supplemented with 1% tween 20; (3) PBS-BSA: PBS supplemented with 0.1 mg / mL bovine serum albumin (BSA, Thermo Fisher, Cat. No. B14). The PBST and PBS+1% tween buffers were filtered with a 0.02 pm filter (VWR, Anotop 25, Cat. No. 516-1501). In addition, the capture antibody (DO- 1 , Abeam, Cat. No. Ab1101) was biotinylated with sulfo-NHS-LC-LC-Biotin (Thermo Scientific, Cat. No. 21338) following the manufacturer’s instructions. The biotinylated capture antibody and fluorescently labelled imaging antibody (Alexa Fluor 647-conjugated PAb240, Novus Biologicals, Cat. No. BS200-103AF647) were diluted with PBS-BSA to 10 nM and 1 nM, respectively, and stored on ice before use.

[0273] To each well of the SiMPull coverslip, 10 pL of 0.2 mg / mL neutravidin (Thermo Fisher, Cat. No. 31000) in PBST was added and incubated for 10 min at room temperature. The wells were then washed twice with 10 pL of PBST and once with 10 pL of PBS+1% tween (three-step washing). Next, 10 pL of 10 nM biotinylated capture antibody in PBS-BSA was added, incubated for 10 min at room temperature, and washed using the same three-step washing method. The wells were then blocked with 1 mg / mL BSA in PBS for 15 min at room temperature and washed. Samples (10 pL / well) were then added and incubated overnight at 4 °C, followed by three-step washing. Plasma samples were sonicated for 5 min in an icewater bath and diluted 1 :1 in PBS. The recombinant p53 aggregates were prepared using the same protocol as in the SiMoA assay and diluted with the aggregation buffer to 1 nM. The monomeric WT and R248Q p53 samples were prepared by mixing the 1 pM protein solutions 1 :3 with 8 M guanidine hydrochloride solution (pH 8.5, Merck, Cat. No. G7294-100ML). The mixture was then incubated overnight at 4 °C and further diluted using the aggregation buffer to a final protein concentration of 1 nM. After the three-step washing, the wells were again blocked with 1 mg / mL BSA in PBS for 15 min and washed. Then, the 1 nM imaging antibody in PBS-BSA was introduced to all wells, incubated for 10 min, and removed by three-step washing. Finally, the dSTORM imaging buffer (50 mM cysteamine, 10% glucose, 50 mM Tris, 10 mM NaCI, 0.5 mg / mL glucose oxidase, 40 pg / mL catalase, pH 8.0) was freshly prepared and introduced to all wells38. The wells were sealed with a clean coverslip and imaged immediately. All incubation steps were performed in a humidity chamber.

[0274] Fluorescence imaging

[0275] Fluorescence imaging was performed on a home-built total internal reflection fluorescence (TIRF) microscope, using a flat-field illumination module with four lasers (405 nm: Oxxius, LBX-LD; 488 nm: Cobolt, MLD 488; 561 nm: Roithner LaserTechnik, RLTMLL 561 100 3; 638 nm: Cobolt, MLD 638)39. The laser beams were cleaned by the corresponding excitation filters (405 nm: FF01 -417 / 60-25, Semrock; 488 nm: LL01 -488-25, Semrock; 532 nm: FF01 -532 / 3- 25, Semrock; 638 nm: FF01 -640 / 14-25, Semrock) and coupled into a 70-pm square-core optical fibre (05806-1 Rev. A, CeramOptec) using an aspheric lens (C220TMD-A, Thorlabs). The optical fibre was agitated using a vibrational motor (304-111 , Precision Microdrives) operating at 1 .5 V for speckle removal. The laser output from the fibre was collimated with an adjustable collimator (C40FC-A, Thorlabs), cleaned by a quadband excitation filter (FF01- 390 / 482 / 563 / 640-25x36, Semrock), focused by an achromatic lens (AC254-125-A-ML, Thorlabs), and reflected by a pentaband dichroic beam splitter (R405 / 488 / 561 / 635 / 800-T1- 25x36, Semrock) into an oil-immersion objective (100x CFI Apo TIRF, NA 1.49, MRD01991 , Nikon) mounted on a microscope body (Ti-E Eclipse, Nikon). The fluorescence from the sample was collected by the objective and cleaned by a quadband emission filter (FF01- 446 / 523 / 600 / 677-25x36, Semrock) and a short-pass filter (FESH0750, Thorlabs). The fluorescence was further cleaned by corresponding emission filters (405 nm: FF01-480 / 40-25, Semrock; 488 nm: FF03-525 / 50-25, Semrock; 561 nm: FF01 -600 / 37-25, Semrock; 638 nm: LP02-647RU-25, Semrock) before being recorded on a sCMOS camera (Prime BSI Express, Teledyne Photometries). The pixel size was 87.21 nm under two-by-two binning. All devices were controlled using MicroManager (pManager v1 .4.22)40. Automated imaging was achieved using a home-written script. dSTORM imaging was performed under the illumination of 405 nm laser (43 mW) and 638 nm laser (110 mW), the power densities of which were approximately 430 and 1 ,100 W / cm2, respectively. For each well of a SiM Pull coverslip, two fields-of-view (FOVs) were imaged. For plasma samples, the imaging was performed once. For p53 monomer and aggregate samples, three replicates were used. A total of 5,000 frames were obtained for each FOV with 50 ms exposure time. The images were analysed using the ThunderSTORM plugin in lmageJ / FIJI41 ,42. Localisations with intensities below 200 and uncertainties above 20 nm were filtered out. The cluster analysis was performed using density-based spatial clustering of applications with noise (DBSCAN) with the E and min_sample set to 75 nm and 5, respectively43. All localisation data ware normalised to the blank (0.1 mg / mL BSA) on the same coverslip.

[0276] Example 2

[0277] As set out in Example 1 , the SiMoA assay relies on a pair of monoclonal antibodies to capture and detect p53 aggregates in plasma samples (Fig. 1A). The SiMoA assay set out in Example 1 was further tested using three further p53 capture antibodies together with PAb240 as the p53 detection antibody. Following the experimental methods from Example 1 , the following p53 capture antibodies were tested PAb242 which binds an epitope in the N-terminal domain TAD1 at amino acid residues9-25 (Novus Biologicals NBP2-53113), PAb421 which binds an epitope in the C- terminal domain at amino acid residues 371-380 (Abeam Ab245685) and PAb1801 which binds an epitope in the N-terminal domain TAD2 at amino acid residues 46-55 (Abeam Ab28). These antibodies were separately conjugated to paramagnetic beads following the same procedure as the DO-1 antibody.

[0278] A total of 6 controls and 6 GB samples were tested per antibody pair. All antibody pairs were able to detect p53 aggregates in the GB patient samples relative to controls (Figure 5). These data credibly demonstrate that any antibody targeting the N- or C-terminal domain of p53 can be used as the capture antibody can enrich the p53 in the samples and show similar results with the PAb240 detector antibody.

[0279] References

[0280] 1 . Janie, A., et al. DNA repair processes are critical mediators of p53-dependent tumor suppression. Nature Medicine 24, 947-953 (2018).

[0281] 2. Chen, J. The Cell-Cycle Arrest and Apoptotic Functions of p53 in Tumor Initiation and Progression. Cold Spring Harbor Perspectives in Medicine 6(2016).

[0282] 3. Fridman, J.S. & Lowe, S.W. Control of apoptosis by p53. Oncogene 22, 9030-9040 (2003).

[0283] 4. Olivier, M., Hollstein, M. & Hainaut, P. TP53 mutations in human cancers: origins, consequences, and clinical use. Cold Spring Harbor perspectives in biology 2, a001008 (2010).

[0284] 5. Kwan, K., Castro-Sandoval, O., Gaiddon, C. & Storr, T. Inhibition of p53 protein aggregation as a cancer treatment strategy. Current Opinion in Chemical Biology 72, 102230 (2023).

[0285] 6. Liu, Q., Li, L., Yu, Y. & Wei, G. Elucidating the Mechanisms of R248Q Mutation- Enhanced p53 Aggregation and Its Inhibition by Resveratrol. The Journal of Physical Chemistry B 127, 7708-7720 (2023).

[0286] 7. Lei, J., Cai, M., Shen, Y, Lin, D. & Deng, X. Molecular dynamics study on the inhibition mechanisms of ReACp53 peptide for p53-R175H mutant aggregation. Physical Chemistry Chemical Physics 23, 23032-23041 (2021).

[0287] 8. Xu, J., et al. Gain of function of mutant p53 by coaggregation with multiple tumor suppressors. Nature Chemical Biology 7, 285-295 (2011).

[0288] 9. Ostermeyer, A.G., Runko, E., Winkfield, B., Ahn, B. & Moll, U.M. Cytoplasmically sequestered wild-type p53 protein in neuroblastoma is relocated to the nucleus by a C-terminal peptide. 93, 15190-15194 (1996).

[0289] 10. Ferraz da Costa, D.C., et al. Resveratrol prevents p53 aggregation in vitro and in breast cancer cells. Oncotarget , 29112-29122 (2018).

[0290] 11. Iwahashi, N., et al. Cytoplasmic p53 aggregates accumulated in p53-mutated cancer correlate with poor prognosis. PNAS nexus 1 , pgac128 (2022).

[0291] 12. Wu, W., et al. Glioblastoma multiforme (GBM): An overview of current therapies and mechanisms of resistance. Pharmacological research 171 , 105780 (2021).

[0292] 13. Eckel-Passow, J.E., et al. Glioma Groups Based on 1 p / 19q, IDH, and TERT Promoter Mutations in Tumors. The New England journal of medicine 372, 2499-2508 (2015). 14. Zhang, Y, et al. The p53 Pathway in Glioblastoma. Cancers 10(2018).

[0293] 15. Olafson, L.R., Gunawardena, M., Nixdorf, S., McDonald, K.L. & Rapkins, R.W. The role of TP53 gain-of-function mutation in multifocal glioblastoma. Journal of neurooncology 147, 37-47 (2020).

[0294] 16. Pedrote, M.M., et al. Oncogenic Gain of Function in Glioblastoma Is Linked to Mutant p53 Amyloid Oligomers. iScience 23, 100820 (2020).

[0295] 17. Soragni, A., et al. A Designed Inhibitor of p53 Aggregation Rescues p53 Tumor Suppression in Ovarian Carcinomas. Cancer Cell 29, 90-103 (2016).

[0296] 18. Silva, J.L., Gallo, C.V.D.M., Costa, D.C.F. & Rangel, L.P. Prion-like aggregation of mutant p53 in cancer. Trends in Biochemical Sciences 39, 260-267 (2014).

[0297] 19. Rissin, D.M., et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nature Biotechnology 28, 595-599 (2010).

[0298] 20. Li, D. & Mielke, M.M. An Update on Blood-Based Markers of Alzheimer’s Disease Using the SiMoA Platform. Neurology and Therapy 8, 73-82 (2019).

[0299] 21. Wilson, D.H., et al. The Simoa HD-1 AnalyzerA Novel Fully Automated Digital Immunoassay Analyzer with Single-Molecule Sensitivity and Multiplexing. 21 , 533-547 (2016).

[0300] 22. Morasso, C., et al. Fast quantification of extracellular vesicles levels in early breast cancer patients by Single Molecule Detection Array (SiMoA). Breast Cancer Research and Treatment 192, 65-74 (2022).

[0301] 23. Schubert, S.M., et al. Ultra-sensitive protein detection via Single Molecule Arrays towards early stage cancer monitoring. Scientific Reports 5, 11034 (2015).

[0302] 24. Sabapathy, K. & Lane, D.P Understanding p53 functions through p53 antibodies. Journal of molecular cell biology 11 , 317-329 (2019).

[0303] 25. Palanikumar, L., et al. Protein mimetic amyloid inhibitor potently abrogates cancer- associated mutant p53 aggregation and restores tumor suppressor function. Nature Communications 12, 3962 (2021).

[0304] 26. Rissin, D.M., et al. Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range. Analytical chemistry 83, 2279-2285 (2011).

[0305] 27. Julian, L., et al. Characterization of full-length p53 aggregates and their kinetics of formation. Biophysical journal 121 , 4280-4298 (2022).

[0306] 28. Jain, A., et al. Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484-488 (2011).

[0307] 29. Danial, J.S.H. & Klenerman, D. Single molecule imaging of protein aggregation in Dementia: Methods, insights and prospects. Neurobiology of Disease 153, 105327 (2021).

[0308] 30. Walsh, I., Seno, F., Tosatto, S.C.E. & Trovato, A. PASTA 2.0: an improved server for protein aggregation prediction. Nucleic Acids Research 42, W301-W307 (2014).

[0309] 31. Fluss, R., Faraggi, D. & Reiser, B. Estimation of the Youden Index and its associated cutoff point. Biometrical journal. Biometrische Zeitschrift 47 , 458-472 (2005).

[0310] 32. Nakasu, S., Deguchi, S. & Nakasu, Y. IDH wild-type lower-grade gliomas with glioblastoma molecular features: a systematic review and meta-analysis. Brain tumor pathology 40, 143-157 (2023).

[0311] 33. Bui, K.T, Blinman, P, Kiely, B.E., Brown, C. & Dhillon, H.M. Experiences with scans and scanxiety in people with advanced cancer: a qualitative study. Supportive care in cancer : official journal of the Multinational Association of Supportive Care in Cancer 29, 7441-7449 (2021).

[0312] 34. Derry-Vick, H.M., et al. Scanxiety among Adults with Cancer: A Scoping Review to Guide Research and Interventions. Cancers 15(2023).

[0313] 35. Klee, G.G. Human anti-mouse antibodies. Archives of pathology & laboratory medicine 124, 921-923 (2000).

[0314] 36. Xia, Z., et al. A computational suite for the structural and functional characterization of amyloid aggregates. Cell Reports Methods 3, 100499 (2023). 37. Di Antonio, M., et al. Single-molecule visualization of DNA G-quadruplex formation in live cells. Nature Chemistry 12, 832-837 (2020).

[0315] 38. Jimenez, A., Friedl, K. & Leterrier, C. About samples, giving examples: Optimized Single Molecule Localization Microscopy. Methods (San Diego, Calif.) 174, 100-114 (2020).

[0316] 39. Lam, J.Y.L., et al. An economic, square-shaped flat-field illumination module for TIRF- based super-resolution microscopy. Biophysical Reports 2, 100044 (2022).

[0317] 40. Edelstein, A.D., et al. Advanced methods of microscope control using pManager software. Journal of biological methods 1(2014). 41. Ovesny, M., Kfizek, P., Borkovec, J., Svindrych, Z. & Hagen, G.M. ThunderSTORM: a comprehensive Imaged plug-in for PALM and STORM data analysis and superresolution imaging. Bioinformatics 30, 2389-2390 (2014).

[0318] 42. Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nature Methods 9, 676-682 (2012). 43. Ester, M., Kriegel, H.-P., Sander, J. & Xu, X. A density-based algorithm for discovering clusters in large spatial databases with noise, in kdd, Vol. 96226-231 (1996).

Claims

CLAIMS1 . A method of detecting cancer in a patient, the method comprising:(a) comparing the level of p53 aggregates in a body fluid sample obtained from the patient to a threshold level; and(b) determining whether the patient has cancer based on the comparison performed in step (a).

2. A method of determining the risk of cancer in a patient, the method comprising:(a) comparing the level of p53 aggregates in a body fluid sample obtained from the patient to a threshold level; and(b) determining the risk of the patient having cancer based on the comparison performed in step (a).

3. A method for monitoring the progression of cancer or the onset of cancer in a patient, the method comprising:(a) comparing: (i) the level of p53 aggregates in a body fluid sample obtained from the patient at a first time point with (ii) the level of p53 aggregates in a body fluid sample obtained from the patient at a second subsequent time point; and(b) determining whether the cancer has progressed or onset of cancer has occurred between the first and second time point based on the comparison performed in step (a).

4. A method for determining the efficacy of a therapeutic intervention in a patient having cancer, the method comprising:(a) comparing: (i) the level of p53 aggregates in a body fluid sample obtained from the patient prior to administration of the therapeutic intervention with (ii) the level of p53 aggregates in a body fluid sample obtained from the patient after administration of the therapeutic intervention; and(b) determining the efficacy of the therapeutic intervention based on the comparison performed in step (a).

5. The method of any one of the preceding claims, wherein the cancer is selected from glioblastoma, astrocytoma, oligodendroglioma, ovarian cancer, oesophageal cancer, lung cancer, breast cancer, adenocarcinoma, and metastatic cancer, optionally metastatic breast cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic oesophageal cancer, or metastatic adenocarcinoma.

6. The method of any one of the preceding claims, wherein the method further comprises quantifying the level of p53 aggregates in the body fluid sample(s).

7. The method of any one of the preceding claims, wherein the method comprises contacting the body fluid sample with a p53 aggregate-specific binding agent.

8. The method of any one of the preceding claims, wherein the method comprises:(a) contacting the body fluid sample with a p53 capture binding agent to provide an enriched sample;(b) contacting the enriched sample with a p53 detection binding agent; and(c) detecting binding of the p53 aggregate-specific binding agent.

9. The method of claim 8, wherein the p53 detection binding agent is selected from an antibody or an antigen binding fragment thereof and an aptamer.

10. The method of claim 8 or claim 9, wherein the p53 detection binding agent binds a peptide comprising amino acid residues:(a) 211-217 of SEQ ID NO: 1 , optionally wherein the p53 detection binding agent is PAb240 or an antigen binding fragment thereof;(b) 181-190 of SEQ ID NO: 1 ; optionally wherein the p53 detection binding agent is DO-11 or an antigen binding fragment thereof; or(c) 256-270 of SEQ ID NO: 1 , optionally wherein the p53 detection binding agent is DO-12 or an antigen binding fragment thereof.11 . The method of any of claims 8-10, wherein the p53 capture binding agent is selected from an antibody or an antigen binding fragment thereof and an aptamer.

12. The method of any of claims 8-11 , wherein the p53 capture binding agent binds to the N-terminal domain of p53, the central domain of p53, and / or the C-terminal domain of p53.

13. The method of any of claims 8-12, wherein the p53 detection binding agent binds to the N-terminal domain of p53, the central domain of p53, and / or the C-terminal domain of p53.

14. The method of any of claims 8-13, wherein the p53 capture binding agent is bound to a solid support, optionally where in solid support is selected from a bead, a solid surface, and a membrane.

15. The method of any of claim 8-14, wherein the p53 capture binding agent binds to amino acid residues:(a) 20 to 25 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is DO- 1 or an antigen binding fragment thereof;(b) 18 to 27 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb242 or an antigen binding fragment thereof;(c) 18 to 25 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb242 or an antigen binding fragment thereof;(d) 9 to 25 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb242 or an antigen binding fragment thereof; or(e) 46 to 55 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb1801 or an antigen binding fragment thereof.

16. The method of any of claim 8-14, wherein the p53 capture binding agent binds to amino acid residues:(a) 370 to 378 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb421 or an antigen binding fragment thereof or PAb122 or an antigen binding fragment thereof; or(b) 388 to 393 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is ICA-9 or an antigen binding fragment thereof.

17. The method of any preceding claim, wherein the body fluid sample is selected from a urine sample, a blood sample, a serum sample, a cerebrospinal fluid sample, a plasma sample, a saliva swab and a nasal swab.

18. A kit for detecting the level of p53 aggregate in a sample, the kit comprising:(a) a p53 capture binding agent; and(b) a p53 detection binding agent; and optionally a calibrator.

19. The kit of claim 18, wherein the p53 capture binding agent binds to the N-terminal domain of p53 and / or the C-terminal domain of p53.

20. The kit of claim 18 or claim 19, further comprising a solid support to which the p53 capture binding agent is bound, optionally wherein the solid support is selected from a bead, a solid surface and a membrane.

21. The kit of any of claims 18-20, wherein the p53 aggregate-specific binding agent is conjugated to a solid support, optionally wherein the solid support is selected from a bead, a solid surface and a membrane.

22. The kit of any of claims 18-21 , wherein the p53 detection binding agent and / or the p53 capture binding agent are selected from an antibody or an antigen binding fragment thereof and an aptamer.

23. The kit of any of claims 18-22, wherein:(a) the p53 detection binding agent binds to amino acid residues 211-217 of SEQ ID NO: 1 , optionally wherein the p53 detection binding agent is PAb240 or an antigen binding fragment thereof;(b) the p53 detection binding agent binds to amino acid residues 181-190 of SEQ ID NO: 1 , optionally wherein the p53 detection binding agent is DO-11 or an antigen binding fragment thereof; or(c) the p53 detection binding agent binds to amino acid residues 256-270 of SEQ ID NO: 1 , optionally wherein the p53 detection binding agent is DO-12 or an antigen binding fragment thereof.

24. The kit of any of claims 18-23, wherein:(a) the p53 capture binding agent binds to amino acid sequence comprising amino acid residues 20-25 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is DO-1 or an antigen binding fragment thereof;(b) the p53 capture binding agent binds to amino acid residues 18-27 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb242 or an antigen binding fragment thereof;(c) the p53 capture binding agent binds to amino acid residues 18-25 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb242 or an antigen binding fragment thereof;(d) the p53 capture binding agent binds to amino acid residues 9-25 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb242 or an antigen binding fragment thereof; or(e) the p53 capture binding agent binds to amino acid residues 46-55 of SEQ ID NO: 1 optionally wherein the p53 capture binding agent is PAb1801 or an antigen binding fragment thereof.

25. The kit of any of claims 18-23, wherein:(a) the p53 capture binding agent binds to amino acid residues: 370-378 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is PAb421 or an antigen binding fragment thereof; or(b) the p53 capture binding agent binds to amino acid residues: 388-393 of SEQ ID NO: 1 , optionally wherein the p53 capture binding agent is ICA-9 or an antigen binding fragment thereof.

Images