Method of treating cancer

Abstract

The present application provides a method of treating a cancer in a subject, the method comprising administering a DSB-inducing agent and / or radiotherapy to the subject, wherein the subject has a PTPN12-deficient cancer. Also provided are methods of selecting a patient having a cancer for treatment with a DSB-inducing agent and / or radiotherapy, predicting whether a patient having a cancer is likely to respond to such therapies, and selecting such agents for use as a cancer therapeutic. Also provided is a method of treating a cancer in a subject, the method comprising administering (i) a DSB-inducing agent and / or radiotherapy and (ii) an inhibitor of PTPN12 to the subject.

Description

[0001] Method of Treating Cancer

[0002] Technical field

[0003] The present invention relates to the treatment of cancer with a DSB-inducing agent, and biomarkers for selecting subjects for treatment with a DSB-inducing agent.

[0004] Background

[0005] Double strand break (DSB)-inducing agents, for example DNA damaging compounds and radiotherapy, have a long history of use in the treatment of cancer. A full understanding of the mechanisms by which they function is essential to balance efficacy and toxicity. Often the therapeutic window is small, and these therapies end up causing a significant amount of collateral damage to healthy tissue. Doses are therefore limited by the toxicity they cause, which, in combination with the development of resistance, can limit the utility of these therapies.

[0006] One approach for treating cancer with these therapies includes identifying target genes and biomarkers which identify cancer cells that may show increased sensitivity to DSBs, due to e.g. a reduced ability to repair them. However, few of these biomarkers exist, and even fewer are actually used in a clinical setting. In particular, there are no biomarkers of sensitivity to radiotherapy in routine clinical use, and none have been tested in advanced trials or are recommended for use in clinical guidelines (Bleaney et al., Cancers (Basel). 2024;16(10):1942. 20 May 2024). Research has identified multi-gene signatures that can be used to predict sensitivity to chemotherapy and radiotherapy, however these are complex and difficult to use in a clinical setting.

[0007] There is a need in the art for improved methods of selecting patients for treatment with DSB-inducing therapies, based on biomarkers that predict sensitivity to such therapies.

[0008] Summary

[0009] In a first aspect of the invention, there is provided a method of treating a cancer in a subject, the method comprising administering a DSB-inducing agent and / or radiotherapy to the subject, wherein the subject has a PTPN12-deficient cancer.

[0010] In a second aspect of the invention, there is provided a DSB-inducing agent and / or radiotherapy for use in a method of treating a cancer in a subject, wherein the subject has a PTPN12-deficient tumor.

[0011] In a third aspect of the invention, there is provided the use of a DSB-inducing agent in the manufacture of a medicament for use in a method of treating cancer in a subject, wherein the subject has a PTPN12-deficient cancer.In some embodiments of the first to third aspects of the invention, the subject has been determined to have a PTPN12-deficient cancer, optionally wherein the determination that the subject has a PTPN12-deficient cancer has been made by measuring expression of PTPN12 in a tumour sample obtained from the subject.

[0012] In some embodiments of the first to third aspects of the invention, the method comprises a step of determining that a sample obtained from the subject is PTPN12-deficient.

[0013] In some embodiments of the first to third aspects of the invention, the DSB-inducing agent induces DSBs either directly, or indirectly through e.g. altering DNA replication fork progression, impeding DNA topoisomerase activities, or by causing the accumulation of single-strand breaks (SSBs)

[0014] In some embodiments of the first to third aspects of the invention, the DSB-inducing agent is a TOP1 chemotherapeutic agent, a TOP2 chemotherapeutic agent, a PARP inhibitor, a platinum based chemotherapeutic agent, or a radioisotope.

[0015] In some embodiments of the first to third aspects of the invention:

[0016] (i) the TOP1 chemotherapeutic agent is selected from: camptothecin, camptothecin derivatives, topotecan, irinotecan / SN38, rubitecan, indenoisoquinolines, indotecan, indimitecan, lamellarin D, and belotecan;

[0017] (ii) the TOP2 chemotherapeutic agent is selected from: etoposide, doxorubicin, epirubicin, daunorubicin, idarubicin, mitoxantrone, amsacrine, teniposide, tafluposide, ICRF-193, genistein, ellipticines, aurintricarboxylic acid, and HU-331 ; or

[0018] (iii) the PARP inhibitor is selected from: olaparib, niraparib, rucaparib, veliparib, pamiparib, and talazoparib;

[0019] (iv) the platinum-based chemotherapeutic agent is selected from: carboplatin, cisplatin and oxaliplatin; or

[0020] (v) the radioisotope is selected from: Lu177 and Ac225.

[0021] In some embodiments of the first to third aspects of the invention, the method further comprises administering radiotherapy to the subject, and / or administering a second anti-cancer agent to the subject.

[0022] In some embodiments of the first to third aspects of the invention, the agent comprises:

[0023] (a) an antibody-drug conjugate (ADC), optionally wherein the ADC is sacituzumab govitecan (Trodelvy), trastuzumab deruxtecan (Enhertu) and datopotamab deruxtecan (Datroway); or

[0024] (b) a radioconjugate, optionally wherein the radioconjugate comprises lutetium 177 (e.g. lutetium 177 dotatate (Lutathera), lutetium 177 PMSA (Pluvitco)), or actinium 225.In some embodiments of the first to third aspects of the invention, the ADC comprises a payload, and wherein the payload comprises a DSB-inducing agent.

[0025] In some embodiments of the first to third aspects of the invention, the cancer is selected from breast cancer, with a notably exemplification for triple-negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), oesophageal squamous cell carcinoma (OSCC), colon cancer, renal cell carcinoma (RCC) or other cancer type that is found to have PTPN12 deficiency.

[0026] In some embodiments of the first to third aspects of the invention, the method further comprises administering an inhibitor of PTPN12.

[0027] In some embodiments of the first to third aspects of the invention, PTPN12 gene expression is downregulated in the cancer.

[0028] In some embodiments of the first to third aspects of the invention, the cancer does not express functional PTPN12 protein.

[0029] In some embodiments of the first to third aspects of the invention, gene expression of PTPN12 in the cancer is downregulated by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to PTPN12 expression in normal cells, and / or wherein at least 90%, at least 95% or at least 99% of the cancer cells do not express a functional PTPN12 protein.

[0030] In some embodiments of the first to third aspects of the invention, the cancer comprises a loss-of-function mutation in PTPN12 and / or a deletion of a PTPN12 gene.

[0031] In some embodiments of the first to third aspects of the invention, the method comprises administering a PTPN12 inhibitor to the subject.

[0032] In a fourth aspect of the invention, there is also provided a method of treating a cancer in a subject, the method comprising administering (i) a DSB-inducing agent and / or radiotherapy and (ii) an inhibitor of PTPN12 to the subject.

[0033] In some embodiments of first to the fourth aspects of the invention, the PTPN12 inhibitor is an inhibitory RNA, such as an siRNA, an anti-sense oligonucleotide (ASO), an aptamer, a small molecule inhibitor, a nanoparticle or a peptide inhibitor.

[0034] In a fifth aspect of the invention, there is also provided a method of selecting an agent for use as a cancer therapeutic, the method comprising:

[0035] (i) providing one or more DSB-inducing agents;(ii) measuring the cell viability of PTPN12-deficient cells treated with the, or each, DSB-inducing agent;

[0036] (Hi) measuring the cell viability of PTPN12-wildtype cells treated with the, or each, DSB-inducing agent; and

[0037] (iv) selecting the agent for use as cancer therapeutic agent when the cell viability of the PTPN12- wildtype cells is at least 30% greater than the cell viability of the PTPN12-deficient cells at the same dose of DSB-inducing agent.

[0038] In some embodiments of the fifth aspect of the invention, the method comprises selecting the agent for use as a cancer therapeutic for a PTPN12-deficient cancer.

[0039] In a sixth aspect of the invention, there is also provided a method of selecting a patient having a cancer for treatment with a DSB-inducing agent and / or radiotherapy, the method comprising detecting, in a sample obtained from the cancer, whether the cancer comprises a PTPN12 deficiency, and selecting the patient for treatment when the cancer is PTPN12-deficient.

[0040] In a seventh aspect of the invention, there is also provided a method of predicting whether a patient having a cancer is likely to respond to a DSB-inducing agent and / or radiotherapy, the method comprising detecting, in a sample obtained from the cancer, whether the cancer comprises a PTPN12 deficiency, wherein when the patient is characterized as having a PTPN12-deficient cancer, the patient is likely to respond to a DSB-inducing agent.

[0041] In an eighth aspect of the invention, there is also provided a method of treating a cancer in a subject determined to have a PTPN12-deficient cancer, the method comprising:

[0042] (i) determining whether a sample obtained from the subject is PTPN12-deficient according to a sixth aspect of the invention, or determining whether the patient is likely to respond to a DSB-inducing agent according to a seventh aspect of the invention; and

[0043] (ii) administering a DSB-inducing agent and / or radiotherapy to a patient if the sample is determined to be PTPN12-deficient or if the patient is determined to be likely respond to a DSB-inducing agent and / or radiotherapy.

[0044] In an ninth aspect of the invention, there is also provided a kit for use in the detection and / or treatment of a PTPN12-deficient cancer in a subject, the kit comprising: (i) one or more reagents for detection of PTPN12 and / or quantification of PTPN12 expression; and (ii) instructions for treatment of a PTPN12-deficient cancer with one or more DSB-inducing agents and / or radiotherapy.

[0045] In some embodiments, the kit further comprises at least one DSB-inducing agent, optionally wherein the at least one DSB-inducing agent is as defined herein in accordance with the first to the third aspects of the invention.In some embodiments, the one or more reagents for detection of PTPN12 and / or quantification of PTPN12 expression comprise:

[0046] at least one polynucleotide probe that selectively hybridizes to a polynucleotide that encodes PTPN12, optionally to a polynucleotide that encodes a mutant form of a PTPN12 gene, such as a loss of function mutant form of PTPN12; and / or

[0047] at least one antigen-binding molecule that selectively binds to a PTPN12 polypeptide, optionally to a mutant form of a PTPN12 polypeptide.

[0048] Other objects and embodiments of the invention will be apparent from the detailed description that follows.

[0049] Brief description of the drawings

[0050] For a further understanding of the various described implementations, reference should be made to the detailed description below, in conjunction with the following drawings in which like reference numerals refer to corresponding parts throughout the figures.

[0051] Figure 1 - Genome-wide CRISPR knockout screens identify PTPN12 as a potential NHEJ factor: (a) Schematic of genome-wide CRISPR screen approach with IR treatment, (b) Results of the screen outlined in (a) using the DrugZ “NormZ score” of the 1.5Gy doses relative to the untreated sample, with single dose on the x-axis and hyperfractionated dose on the y-axis . (c) Line plot of each gene’s NormZ score in each radiation dose relative to untreated and normalised within each gene to observe patterns across different dose regimens, (d) Rank plot of average NormZ scores in split doses relative to untreated for each of the clustered genes from (c).

[0052] Figure 2 - Genome-wide CRISPR knockout screens identifies PTPN12 as a potential NHEJ factor: (a) Genome wide CRISPR screen DrugZ “NormZ score” of the 0.9Gy doses relative to the untreated sample, with single dose on the x-axis and hyperfractionated dose on the y-axis. (b) Heatmap of per-gene NormZ scores in different IR treatment conditions relative to untreated cells, clustered into groups, (c) Gene ontology results of the gene groups defined in (b). (d) Relative homologous recombination efficiency in different siRNA depletions in the DR-GFP assay previously published8.

[0053] Figure 3 - PTPN12 promotes cell survival in response to DNA damage and limits HR: (a) Clonogenic survival of WT U2OS cells and PTPN120U2OS cells with increasing doses of four genotoxic treatments, left to right: ionising radiation, etoposide, camptothecin and olaparib (b) Western blot of WT U2OS and PTPN1^° U2OS cells following 5Gy of IR. (c) Representative images of immunofluorescence for yH2AX and RPA324 hours after 1Gy of IR. (d) Quantification of immunofluorescence in (c) showing number of yH2AX foci per nucleus (left) and number of RPA32 foci (right). Statistics done using unpaired two-sided Wilcoxon test, ***p<0.001.Figure 4 - PTPN12 enhances cell survival in response to DNA damage and acts to limit HR repair: (a) Alamar blue viability assay of WT HAP1 cells and three PTPN120HAP1 clones with increasing doses of four genotoxic treatments, left to right: ionising radiation, etoposide, camptothecin and olaparib. (b) Western blot of WT HAP1 cells and three PTPN12^° HAP1 clones following 5Gy IR. (c) Representative images of immunofluorescence for yH2AX and RPA32 at multiple timepoints following 1Gy of IR in WT U2OS and PTPN1^° U2OS cells, (d) Quantification of immunofluorescence in (c) showing number of yH2AX foci per nucleus. Statistics done between WT and PTPN1^° cells at each time point using unpaired two-sided Wilcoxon test, ***p<0.001. (e) Same as (d) but for RPA32 foci, (f) Flow cytometry gating strategy for the TLR assay of HR efficiency, (g) Relative HR efficiency in the TLR assay from (f) with different siRNA depletions. Statistics done between CTRL siRNA +ISce-l and other siRNA using a paired, two-sided t-test, **p<0.01 ***p<0.001.

[0054] Figure 5 - Loss of PTPN12 does not perturb cell-cycle progression: (a) Cell cycle analysis of WT U2OS and PTPN12^° U2OS cells using quantitative image-based cytometry (QIBC) of DAPI staining with an EdU pulse to label replicating cells, (b) Cell cycle analysis of WT HAP1 and three PTPN12^° clones using flow cytometry of DAPI and EdU. (c) Flow cytometry gating strategy for the assay in (b).

[0055] Figure 6 - Actin polymerisation factors are dephosphorylated in response to DNA damage in a PTPN12-dependent manner: (a) Volcano plot of differential phospho-tyrosine peptides in PTPN12 U2OS cells relative to WT U2OS cells, (b) Gene ontology results for differential phospho-tyrosine peptides in WT U2OS 1 hour after 5Gy IR relative to untreated cells, (c) Western blot of U2OS cells transfected with either control or PTPN12 siRNA, 2 hours after 5Gy IR. (d) Representative images of yH2AX 1 hour following 1 Gy IR with or without treatment with the WASP inhibitor wiskostatin in WT U2OS cells treated with either control siRNA or PTPN12 siRNA and nuclei outlined, (e) Quantification of the yH2AX foci per nucleus in (d). Statistics done using unpaired, two-sided Wilcoxon test ***p<0.001. (f) Same as (d) but for RPA32 foci, (g) Quantification of the RPA32 foci per nucleus in (f). Statistics done using unpaired, two-sided Wilcoxon test *p<0.05, **p<0.01.

[0056] Figure 7 - Actin polymerisation factors are dephosphorylated in response to DNA damage in a PTPN12-dependent manner: (a) Volcano plot of phospho-tyrosine proteomics in WT U2OS cells 1 hour after 5Gy IR. (b) Same as (a) but in PTPN120U2OS cells, (c) String network of significant genes in (a) with the cytoskeleton organisation network labelled, (d) Western blot of WT HAP1 cells and PTPN120clone #1 following 5Gy IR. (e) Representative images of yH2AX 1 hour following 1Gy IR with or without treatment with WASP inhibitor wiskostatin in WT U2OS or PTPN12^° cells, with nuclei outlined, (f) Quantification of the yH2AX foci per nucleus in (e) Statistics done using unpaired, two-sided Wilcoxon test ***p<0.001.

[0057] Figure 8 - PTPN12 loss causes increased actin polymerisation at DNA damage sites: (a) Representative immunofluorescence images of WT and PTPN12^° U2OS cells for yH2AX and p-actin 4 hours after 1 Gy IR. (b) Quantification of immunofluorescence in (a) for p-actin intensity per cell in either the cytoplasm or nucleus; statistics done using unpaired, two-sided Wilcoxon test ***p<0.001.(c) Quantification of immunofluorescence in (a) for p-actin positive yH2AX foci as a percentage of total yH2AX foci; statistics done using paired, two-sided t-tests *p<0.05 **p<0.01 . (d) Western blot of chromatin fractionation in WT HAP1 and PTPN12^° HAP1 cells at multiple times following 5Gy IR. (f) Representative immunofluorescence images of proximity ligation between Ku80 and RPA70 in WT U2OS cells 4 hours treatment with 20pM etoposide for 30 minutes, (g) Quantification of PLA foci per nucleus in (f) statistics done using unpaired, two-sided Wilcoxon test **p<0.01 ***p<0.001.

[0058] Figure 9 - PTPN12 loss causes increased actin polymerisation at sites of DNA damage: (a) Western blot of chromatin fractionation in WT HAP1 cells and three PTPN120HAP1 clones 2 hours following 5Gy IR. (b) Western blot of chromatin fractionation in WT U2OS cells transfected with control or PTPN12 siRNA 4 hours following 5Gy IR. (c) Western blot of WT U2OS cells transfected with control, PTPN12 or Ku70 siRNA and 4 hours after treatment with 20pM etoposide for 30 minutes, (d) Representative immunofluorescence images of proximity ligation between Ku80 and RPA70 in WT U2OS cells transfected with control, PTPN12 or Ku70 siRNA and 4 hours after treatment with 20pM etoposide for 30 minutes. Statistics done using unpaired, two-sided Wilcoxon tests **p<0.01 ***p<0.001.

[0059] Figure 10 - PTPN12 restricts actin driven DSB movement and clustering: (a) Representative images of U2OS cells expressing 53BP1-GFP over time with magnified section (bottom right), starting at 2 hours post 5pM etoposide for 30 minutes with transfection of control or PTPN12 siRNA. Lines represent the movement of tracked foci colour scaled to time post treatment, (b) Mean square displacement (MSD) of 53BP1-GFP foci from (a); lines represent mean of replicate MSDs and error bars are standard error of replicate means, (c) Percentage of foci that cluster together from experiment in (a), (d) Representative images of yH2AX immunofluorescence with nuclei outlined in WT U2OS and PTPN12^° cells with or without treatment with 10pM wiskostatin. (e) Quantification of yH2AX foci size from (d). (f) Representative images of yH2AX immunofluorescence with nuclei outlined in cells treated with either control or PTPN12 siRNA and with or without treatment with 10pM wiskostatin. (g) Quantification of yH2AX foci size from (f). (h) Quantification of yH2AX foci per nucleus in U2OS cells transfected with either control siRNA (CTRLsi) or PTPN12 siRNA (PTPsi), following 2 hours of 100nM camptothecin treatment, statistics done using unpaired, two-sided Wilcoxon test *p<0.05 **p<0.01 ***p<0.001. (i) Same as (h) but for RPA32 foci per nucleus, (j) Same as (h) but for yH2AX foci size, (k) Same as (h) but for RPA32 foci size.

[0060] Figure 11 - PTPN12 loss increases DSB toxicity and chromosomal translocations: (a) Experimental design to map genomic structural variants through NGS. (b) Heatmap of quantified mutations in WT or PTPN12^° HAP1 cells as an uncentred row z-score. (c) Inter-chromosomal translocations per million reads in inviable cells following 1Gy IR or in untreated cells, (d) Deletions per million reads in inviable cells 3 days after 1Gy IR delta to UT live cells and relative to WT HAP1 cells, (e) Chord diagram of inter-chromosomal translocations per million reads in WT HAP1 untreated cells or 3 days after 1Gy IR in WT HAP1 or PTPN12^° cells, (f) Heatmap of inter-chromosomal translocations between differentchromosomes per million reads per average megabase length of chromosomes, (g) Metaphase spreads with FISH of CENPB and TeloG, representative images (left) and quantification of aberrant chromosomes (right). Statistics done using a paired t-test between WT HAP1 and PTPN12^° cells, **p<0.01.

[0061] Figure 12 - PTPN12 loss increases DSB toxicity and chromosomal translocations: (a) Agarose gel of genomic DNA extracted from live or dead cells following 1Gy IR treatment, (b) Tapestation of sonicated DNA extracted from dead or live cells 1 day after 1 Gy IR treatment, (c) Genomic variants per million reads in WT HAP1 and PTPN120cells without treatment, (d) Heatmap of mutations per million reads in WT HAP1 or PTPNI^0. (e) Heatmap of deletions in live or dead WT HAP1 or PTPN1^° cells following 1 gy IR as a delta to WT HAP1 live untreated cells, (f) H3k36me3 levels against H3k27me3 levels at every gene in the genome determined by ChlP-seq in HAP1 cells22, correlation using Pearson method, (g) Boxplot of Log2 fold change between H3k36me3 and H3k27me3 at either all genes in the genome or at loci of inter-chromosomal translocations in dead WT HAP1 or PTPN12*0cells 3 days after 1 Gy IR.

[0062] Description

[0063] The inventors have identified that PTPN12 functions in the prevention of toxic DSB repair outcomes. In particular, PTPN12 was found to downregulate the phosphorylation of tyrosine residues on multiple targets regulating actin cytoskeleton polymerization, increasing the movement and clustering of DSBs and thus promoting toxic chromosomal rearrangements. The inventors found that because of this reduced ability to repair DSBs correctly, PTPN12-deficient cells (where PTPN12 had been knocked out or at least partially knocked down) showed increased sensitivity to agents that cause DSBs. This was observed for agents that cause DSBs directly, such as ionizing radiation, as well as agents that cause DSBs indirectly, such as PARP inhibitors, demonstrating that the sensitivity was not specific to a particular type of DSB-inducing agent or a particular DNA repair pathway. The inventors identified that PTPN12-deficiency could therefore be exploited to identify and treat cancer patients that show enhanced sensitivity to DSB-inducing agents, to improve the overall response to therapy.

[0064] PTPN12

[0065] Protein tyrosine phosphatase non-receptor type 12 (PTPN12; also known as PTP-PEST and proteintyrosine phosphatase G1 (PTPG1)), encodes a tyrosine phosphatase (Uniprot ID: Q05209, NCBI Gene ID: 5782). PTPN12 is a member of the proline-, glutamic acid-, serine- and threonine-rich (PEST) family of PTPs, and is a regulator of cell migration, adhesion, immunity, and survival. PTPN12 has been characterized as a tumor suppressor which regulates EGFR / HER3 signalling. PTPN12 is required for embryonic development, as PTPN12-knockout mice are embryonic lethal. At least 18 PTPN12 substrates have been identified, including HER2, FAK, PYK2, PSTPIP, WASP, p130Cas, paxillin, She, catenin, c-Abl, ArgBP2, p190RhoGAP, RhoGDI, cell adhesion kinase beta (CAKp), and Rho GTPase (Li et al., Cell Reports (2016), 15(6):1345-1358). PTPN12 has been found to bedownregulated in triple negative breast cancer, non-small cell lung cancer, and colorectal cancer. In breast cancer, PTPN12 functions as a master regulator of receptor tyrosine kinases (RTKs), and loss of PTPN12 was found to activate several RTKs including MET, PDGFRb, HER2, and EGFR, mediated by the Hippo signaling pathway effectors YAP and TAZ. Loss of PTPN12 was shown to increase cell proliferation in a Hippo-dependent manner (Emami et al., Int. J. Mol. Sci.

[0066] 2024;25(7):4064. Published 2024 Apr 5). Bioinformatics analysis of exome-wide association data identified a missense mutation in colorectal cancer, PTPN12 rs3750050, which increased the risk of CRC by 19% (Shen et al., Cancer Epidemiol. 2019;59:109-114). Biochemical assays illustrated that rs3750050 could impair the inhibitory effect of PTPN12 on Ras / MEK / ERK signaling by impeding SHC dephosphorylation, increase the expression of cyclin D1 and ultimately lead to aberrant cell proliferation, thus contributing to CRC pathogenesis.

[0067] DSB-inducinq agents & Radiotherapy

[0068] The present invention relates to the treatment of subjects with a DSB-inducing agent. As used herein, a “DSB-inducing agent” is any agent that causes DSBs in DNA, either directly or indirectly. For example, a DSB-inducing agent may have the direct effect of interstrand crosslinking, which indirectly causes DSBs through replication fork stalling. The DSB-inducing agent may be an agent that induces single strand breaks (SSBs) in DNA, which are then converted to DSB during DNA replication. Such agents thus induce an accumulation of DSBs in cells, particularly, for example, in rapidly dividing cells that are actively undergoing DNA replication, such as cancer cells.

[0069] The DSB may be a double-ended DSB or a single-ended DSB. A “double-ended DSB” is a DSB caused by a direct DSB in the middle of a double-stranded section of DNA, e.g. by ionising radiation or endonucleases. A small amount of DNA sequence may be inserted or deleted at the location of the DSB, ranging in size from 1 bp up to entire chromosome arms. The small amount of sequence may be of any length. For example, the small amount of sequence may be 2, 5, 10, 20, 30, 40, 50, 60, 80, 100, 150, or 200 bp. A “single-ended DSB” is where only one unique and isolated double-stranded DNA end is present. Single-ended breaks, in contrast to double-ended DSBs, arise from replication problems.

[0070] This can involve stalling of a fork at a replication-blocking lesion followed by replication fork collapse and breakage of one of the two sister chromatids formed behind the replication fork. A single-ended break can also arise when the replication machinery encounters a single-strand break and, upon unwinding of the DNA at the fork site, causes the disconnection of one of the two chromatids. The repair of single-ended breaks is arguably more difficult than the repair of two-ended DSBs and represents a particular challenge for the mechanisms devoted to maintaining genome stability, since both major DSB repair pathways, NHEJ and HR, rely on connecting two break ends (either without or with the potential to restore the sequence information that was lost at the break site). Moreover, broken replication forks, from which one-ended DSBs arise, cannot be rebuilt to resume replication since the required components such as the replication pre-initiation complex are no longer available(Ensminger M, Lobrich M. Br J Radiol. 2020;93(1115) :20191054). Unprotected telomeres can also be considered natural single-ended DSBs, i.e. with a unique double-strand end.

[0071] The present disclosure relates to the administration of a DSB-inducing agent and / or radiotherapy to a subject, wherein the subject has a PTPN12-deficient cancer. Subjects having a PTPN12-deficient cancer show sensitivity to DSB-inducing agents and / or radiotherapy. As used herein, “sensitivity” or “sensitized” refer to a cellular state wherein the cancer cells are more susceptible to killing by the DSB-inducing agent and / or radiotherapy. PTPN12-deficient cancer cells may show at least 10% increased cell death compared to PTPN12-proficient cancer cells when treated with the DSB-inducing agent and / or radiotherapy. PTPN12-deficient cancer cells may show at least 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or greater increased cell death compared to PTPN12-proficient cancer cells when treated with the DSB-inducing agent and / or radiotherapy. PTPN12-proficient cancer cells may show at least 10% increased cell viability compared to PTPN12-deficient cancer cells when treated with the DSB-inducing agent and / or radiotherapy. PTPN12-proficient cancer cells may show at least 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or greater increased cell viability compared to PTPN12-deficient cancer cells when treated with the DSB-inducing agent and / or radiotherapy. Subjects having a PTPN12-deficiency may therefore be able to receive a lower dose of the DSB-inducing agent and / or radiotherapy, while experiencing an equivalent treatment efficacy compared to a subject that does not have a PTPN12-deficient cancer and who is treated with a higher dose of the DSB-inducing agent and / or radiotherapy. In other words, the therapeutic window for the DSB-inducing agent and / or radiotherapy is increased when treating subjects having a PTPN12-deficient cancer compared to subjects having a PTPN12-proficient cancer.

[0072] The DSB-inducing agent and / or radiotherapy may be delivered in a targeted way. Targeted delivery refers to the administration of a therapy such that the cancer receives a higher dose of the therapy compared to non-cancerous tissue. Targeted delivery of the DSB-inducing agent may be achieved by using antibody-drug conjugates (ADCs) and / or radioconjugates, as described in more detail hereinbelow. Targeted delivery of radiotherapy may be achieved through standard protocols that concentrate the radiation beams at the site of the cancer, or by using radioactive materials that are placed directly within or at the site of the cancer, as described in more detail hereinbelow. Targeted delivery of the DSB-inducing agent and / or radiotherapy further increases the therapeutic window that can be achieved for these agents, thus further reducing the toxicity of these agents, especially when used in the treatment of subjects with PTPN12-deficient cancers.

[0073] The DSB-inducing agent may be a topoisomerase I (TOPI) chemotherapeutic agent or a topoisomerase II (TOP2) chemotherapeutic agent. Chemotherapeutic agents may include poisons and inhibitors of toposimerase I and / or II. Poisons may be a compound that targets the topoisomerase-DNA complex, rather than inhibits the topoisomerase. Suitable topoisomerase I chemotherapeutic agents include camptothecin, camptothecin derivatives, topotecan,irinotecan / SN38, rubitecan, indenoisoquinolines, indotecan, indimitecan, lamellarin D, and belotecan. Suitable topoisomerase II chemotherapeutic agents include etoposide, doxorubicin, epirubicin, daunorubicin, idarubicin, mitoxantrone, amsacrine, teniposide, tafluposide, ICRF-193, genistein, ellipticines, aurintricarboxylic acid, and HU-331 .

[0074] The DSB-inducing agent may be a PARP (poly ADP ribose polymerase) inhibitor. The PARP inhibitor may be a natural compound, synthetic compound, DNA, RNA, peptide, enzyme, ligand, cell extract, or secretion of a mammal, which inhibits the activity of PARP protein. The PARP inhibitor may be selected from one or more of iniparib (BSI 201), olaparib (AZD-2281), rucaparib (AG014699, PF-01367338) and veliparib (ABT-888), niraparib (MK-4827), pamiparib (BGB-290), talazoparib (BMN-603), CEP-9722, E7016(GPI- 21016), LT-673, MP-124, NMS-P118, E7449 (2X-121 ), saruparib (AZD-5305), fuzuloparib (SHR3162), amelparib (JPI-289). .

[0075] Platinum-based chemotherapies and their derivatives such as carboplatin, cisplatin, oxaliplatin, picoplatin, nedaplatin, triplatin tetranitrate (BBR3464), heptaplatin and satraplatin are broadly-used anticancer agents applicable to a wide range of tumor types including lung, ovarian, bladder, colorectal, oesophageal, head and neck, testicular, breast, cervix and gastric cancers. Such agents work by causing DNA breaks and triggering cell death in tumor cells. The DSB-inducing agent may be a platinum-based chemotherapeutic agent. The DSB-inducing agent may be a platinum-based chemotherapeutic agent selected from: carboplatin, cisplatin, oxaliplatin, picoplatin, nedaplatin, triplatin, nedaplatin, triplatin tetranitrate (BBR3464), heptaplatin and satraplatin.

[0076] The DSB-inducing agent may be an agent that induces interstrand cross-linking. Such agents bind covalently bind to two nucleotide residues of different DNA strands. During DNA replication, this causes replication fork stalling, which can result in a DSB. Interstrand cross-linking agents therefore indirectly induce DSBs. Suitable interstrand-crosslinking agents are nitrogen mustards and derivatives (including cyclophosphamide, melphalan, chlorambucil, ifosfamide, mechlorethamine and uramustine), nitrosoureas (including carmustine, lomustine, semustine and streptozotocin), alkyl sulfonates (including busulfan), ethylenimines (including thiotepa and altretamine), triazenes (including procarbazine, dacarbazine and temozolomide), platinums (including cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin and triplatin), mitomycin C and psoralens.

[0077] The DSB-inducing agent may be an alkylating agent. Alkylating agents mediate the transfer of an alkyl group from one molecule to DNA. The alkyl group may be transferred as an alkyl carbocation, a free radical, a carbanion or a carbine (or their equivalents). Alkylating agents can also cause replication stress, and indirect accumulation of DSBs. Illustrative, non-limitative examples of alkylating agents include nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates and triazenes, including, but not limited to, cyclophosphamide (Cytoxan™), busulfan, improsulfan, piposulfan, pipobroman, melphalan (L-sarcolysin), chlorambucil, mechlorethamine or mustine, uramustine or uracil mustard, novembichin, phenesterine, trofosfamide, ifosfamide, carmustine(BCNU), lomustine (CCNU), chlorozotocin, fotemustine, nimustine, ranimnustine, semustine (methyl-CCNU), streptozocin, thiotepa, triethylenemelamine, triethylenethiophosphoramine, procarbazine, altretamine, dacarbazine, mitozolomide and temozolomide.

[0078] The DSB-inducing agent may be an intercalating agent. Intercalating agents are capable of binding by insertion between DNA base-pairs. When intercalating agents bind to the DNA near the topoisomerase II enzyme during DNA replication, they prevent the resealing of double-strand breaks, causing the breaks to persist and accumulate. Intercalating agents therefore indirectly induce DSBs. Suitable DNA intercalating agents include berberine, ethidium bromide, proflavine, daunomycin, doxorubicin, thalidomide, ethidium homodimer, mitonafide, ametantrone; 1 ,10-phenanthroline; elliptinium; gilvocarcin V; amonafide; diminazene aceturate; DAPI; ferroin; amsacrine; propidium; nogalamycin; acriflavine; 9-amino-6-chloro-2-methoxyacridine; angelicin; 4-((3-bromophenyl)amino)-6,7-dimethoxyquinazoline; 1 ,1 '-(4,4,7,7-tetramethyl -4,7-diazaundecamethylene) bis-4-(3-methyl-2,3-dihydro (benzo-1 ,3-thiazole) -2-methylidene) quinolinium; 1,1'- ((4,4,7,7-tetramethyl) -4,7-diazaundecamethylene) bis- 4-(3-methyl-2,3-dihydro(benzo-1 ,3-oxazole) -2-methylidene) quinolinium; NSC 366140 and ditercalinium.

[0079] The DSB-inducing agent may be an anti-metabolic agent. Suitable anti-metabolic agents include fluropyrimidines, azathioprine and mercaptopurine. The additional DSB-inducing agent may be an agent that acts as a nucleoside analogue or nucleobase analogue. Analogues of physiological pyrimidine and purine nucleosides that have cytotoxic activity through their incorporation into DNA, by interfering with various enzymes involved in synthesis of nucleic acids or by modifying the metabolism of physiological nucleosides. Suitable nucleoside analogues include purine analogues, like fludarabine and cladribine; pyrimidine analogues, like decitabine, cytarabine and gemcitarabine, and fluoropyrimidines, like fluorouracil and capecitabine.

[0080] In some embodiments, the DSB-inducing agent is selected from the list comprising: etoposide, camptothecin, olaparib and carboplatin.

[0081] Antibody-drug Conjugates

[0082] The DSB-inducing agent may be provided as an ADC. That is, the DSB-inducing agent may be the payload of an ADC, conjugated to an antigen-binding molecule via a linker moiety. ADCs achieve targeted delivery of their payload to the site of the antigen. ADCs comprising an antigen-binding molecule that targets a cancer cell antigen can therefore achieve targeted delivery of a payload (e.g. a DSB-inducing agent) to the cancer.

[0083] Antigen-binding molecules suitable for use in ADCs comprising a DSB-inducing agent according to the invention refers to molecules that can bind to cancer-specific antigens to achieve targeted deliveryof a payload {e.g. the DSB-inducing agent according to the invention), so that the concentration of the payload is increased in the target cancer cells compared to healthy tissue.

[0084] Antigen-binding moieties may comprise, or may be derived from, antibodies (i.e. immunoglobulins (Igs)) and antigen-binding fragments of antibodies. Antigen-binding moieties also include target antigen-binding aptamers, e.g. a nucleic acid aptamers (reviewed, for example, in Zhou and Rossi, Nat Rev Drug Discov. (2017) 16(3) :181 -202). In some embodiments, an antigen-binding moiety comprises or consists of an antigen-binding peptide / polypeptide, e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody {i.e. a single-domain antibody (sdAb)), affilin, armadillo repeat protein (ArmRP), OBody or fibronectin - reviewed e.g. in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082-1101 , which is hereby incorporated by reference in its entirety (see also e.g. Boersma etal., J Biol Chem (2011) 286:41273-85 and Emanuel etal., Mabs (2011) 3:38-48).

[0085] Antigen-binding molecules also extend to non-antibody based targeting molecules, including, but not limited to, ligands that bind to cancer specific targets, such as somatostatin; peptides, such as lectins; nanoparticles, such as gold nanoparticles (GNPs); and carbohydrates, such as glucose.

[0086] Antigen-binding molecules suitable for use in ADCs comprising a DSB-inducing agent according to the invention include antigen-binding molecules that bind to an antigen expressed on the surface of a cancer cell. Preferably the antigen is expressed at a higher level on cancer cells compared to healthy cells. In some embodiments the antigen is expressed exclusively on the surface of cancer cells. In some embodiments, the tumor antigen may be selected from: HER2, EGFR, EG FRvll I , c-MET, FGFR2, FGFR3, EpCAM, BCMA, TROP-2, AXL, HER3, CD166, CEACAM5, GPNMB, Mesothelin, CD70, CD30, CD79b, Nectin-4, BCMA, FRa, CD33, CD22, CD19, Trop-2, gastric carcinoma antigen, cancer type-specific tumor associated antigens, preferably carcinoembryonic antigen (CEA), mucin (MUC), prostate-specific membrane antigen (PSMA), melanoma antigen, gplOO, BRCA Ras, WT1, MAGE, MAGE-A3, NY-ESO-1, CEA, MARTI , GP100, Proteinase3, Tyrosinase, Survivin, hTERT or EphA2. The tumor antigen may be a universal tumor antigen selected from the group of Survivin and TERT, or mixtures thereof.

[0087] In some embodiments, the target antigen is expressed at a higher level in PTPN12-deficient cancers. That is, in some embodiments, the target antigen is associated or correlated with being amplified or over-expressed in PTPN12-deficient cancers, compared to cancers that are not PTPN12-deficient. In some embodiments, the target antigen is EGFR.

[0088] The ADC may bind to an antigen present in the tumor microenvironment. Antigens present in the tumor microenvironment include CD25 / IL2R, B7-H3, ANTXR1.ADC payloads suitable for use according to the present invention include agents that induce DSBs. Suitable payloads are described in (Fu, Z., Li, S., Han, S., Shi, C., & Zhang, Y. (2022). Signal transduction and targeted therapy, 7(1), 93, hereby incorporated by reference in its entirety). Suitable payloads may be selected from: Calicheamicins, Duocarmycins, Exatecans, Doxorubicin, and Pyrrolobenzodiazepines.

[0089] ADCs suitable for use in accordance with the present invention include ADCs comprising a payload that induces DSBs. The ADC may be approved for the treatment of cancer. Approved ADCs comprising a payload that induces DSBs are described in e.g. Liu, K., Li, M., Li, Y. et al. A review of the clinical efficacy of FDA-approved antibody-drug conjugates in human cancers. Mol Cancer 23, 62 (2024) hereby incorporated by reference in its entirety, and include for example Gemtuzumab Ozogamicin (Mylotarg), Inotuzumab Ozogamicin (Besponsa), Loncastuximab Tesirine (Zynlonta), Sacituzumab govitecan (Trodelvy), Trastuzumab deruxtecan (Enhertu). The ADC may be Sacituzumab govitecan (Trodelvy) and / or Trastuzumab deruxtecan.

[0090] The skilled person appreciates that the ADC will be selected based on the type of PTPN12-deficient cancer to be treated, for example, if the PTPN12-deficient cancer is breast cancer, then ADCs targeting tumor antigens expressed on breast cancer cells may be selected for use in accordance with the present invention. In particular, when the PTPN12-deficient cancer is triple negative breast cancer, the ADC for use according to the invention may be Sacituzumab govitecan or Trastuzumab deruxtecan. When the PTPN12 deficient cancer is non-small cell lung cancer, the ADC may be Trastuzumab deruxtecan or Telisotuzumab adizutecan. When the PTPN12-deficient cancer is colorectal cancer, the ADC may be Telisotuzumab adizutecan.

[0091] A linker moiety according to the present disclosure may be any moiety suitable for linking the payload moiety (e.g. the DSB-inducing agent) to the antigen-binding molecule. Accordingly, they generally comprise a group enabling connection to the payload moiety, a group connecting conjugation to the antigen-binding region of the antigen-binding molecule, and a linker core.

[0092] Linker moieties are described e.g. in Su etal., Acta Pharmaceutica Sinica B (2021) 11(12): 3889-3907, Fu etal., Signal Transduction and Targeted Therapy (2022) 7:93, which is hereby incorporated by reference in its entirety.

[0093] A linker moiety according to the present disclosure may be a cleavable linker moiety or a non-cleavable moiety.

[0094] Cleavable linkers typically utilise differences between the environment of systemic circulation and that in cancer cells / the tumor microenvironment to release the payload moiety in a targeted manner. Cleavable linkers include chemical cleavage linkers {e.g. acid-cleavable linkers, GSH-cleavablelinkers, Fe(ll)-cleavable linkers) and enzyme cleavage linkers (e.g. cathepsin-cleavable linkers, glycosidase-cleavable linkers, phosphatase-cleavable linkers, sulfatase-cleavable linkers).

[0095] Non-cleavable linkers remain inert in common chemical and enzymatic environments in the body, with the payload moiety being released following processing of the ADC by cellular lysosomal proteases. Non-cleavable linkers include linkers comprising thioether or maleimidocaproyl groups.

[0096] In some embodiments, a linker moiety further comprises a spacer moiety. Spacer moieties are sometimes required due to the bulky nature of payload moieties. Commonly employed spacer moieties include para-aminobenzyl carbamate (PABC), hemiaminal groups, PEG groups, polar acyl sulfamide groups, a polar carbamoyl sulfamide groups and HydraSpace (described e.g. in Verkade et al., Antibodies (Basel) (2018) 7(1 ):12 and WO 2016 / 053107 A1 , both of which are hereby incorporated by reference in their entirety). PABC is commonly employed as a spacer moiety in cathepsin-cleavable dipeptide linkers, p-glucuronidase-cleavable linkers, p-galactosidase-cleavable linkers and phosphatase cleavable linkers.

[0097] To produce ADCs, suitable antigen-binding molecules may be conjugated to linker-payload moieties comprising a DSB-inducing agent according to the disclosure by any suitable techniques, which are well known to the skilled person and routinely employed in the art.

[0098] General methods for the conjugation of antigen-binding polypeptides / polypeptide complexes to linkerpayload moieties are described e.g. in Chudasama et al., Nature Chemistry, (2016), 8:114-119, Baah eta!., Molecules. (2021) 26(10): 2943, and Walsh eta / ., Chem. Soc. Rev. (2021) 50:1305-1353, all of which are hereby incorporated by reference in their entirety. Conjugation of antigen-binding moieties and linker-payload moieties and purification of antigen-binding molecules produced by such conjugation is described e.g. in Beck etal., (2017) Nat Rev Drug Discov 16: 315-337; Peters and Brown Biosci Rep (2015) 35: art:e00225; McCombs and Owen, The AAPS Journal (2015) 17: 339-351 ; Jackson, Org Process Res Dev (2016) 20: 852-866; and Olivier and Hurvitz, Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical Outcome to Target Cancer (2016) Wiley. Other relevant disclosures relating to conjugation and linkers are: Tsuchikama and An, Protein Cell. (2018) 9(1 ): 33-46, Khongorzul et al., Mol Cancer Res (2020) 18 (1 ): 3-19 and Drago et al., Nature Reviews Clinical Oncology (2021) 18: 327-344, all of which are hereby incorporated by reference in their entirety.

[0099] Lysine amide coupling

[0100] Lysine-based conjugation is one of the most widely used non-specific conjugation strategies. Such conjugation occurs on reactive amine side chains of lysine residues due to their good nucleophilicity. Immunoglobulin scaffolds contain over 80 lysine residues, most of which are exposed on the surface of the molecule. Among the surface lysine residues, more than 20 have been shown as highly solvent-accessible and can serve as potential ADC conjugation sites. Lysine conjugation follows twomain strategies that result in the formation of a stable amide or amidine bond between the protein and the drug-linker complex. Generally speaking, activated esters on the drug-linker complexes, often O-succinimide reagents such as N-hydroxysuccinimidyl (NHS) orsulfo-NHS esters, react with the antibody lysine residues and achieve the conjugation via amide bonds. On the other hand, stable amidine bonds can be generated on an antibody by the reaction of imido ester compounds, such as Traut’s reagent, with antibody lysine residues.

[0101] A one-step conjugation of a drug-linker moiety containing an amine-reactive group to the antibody via amide bonds is known, as well as two-step conjugation, where in the first step, a small bi-functional reagent containing both an amine- and a thiol-reactive functional groups is reacted with the available lysine e-amino groups to serve as a chemical adaptor, leaving free thiol-reactive groups on the antibody. In the second step, the payload drugs or drug-linker complexes are attached to the thiolreactive groups introduced previously to form the ADC. The two-step approach is often used when the drug / drug-linker complex contains a thiol-reactive module or as an alternative route when introducing an amine-reactive module into the drug or drug-linker complex is proven to be difficult. Four small adaptors commonly used in the two-step conjugation: SPDB disulfide, MCC (maleimidomethyl cyclohexane-1 -carboxylate), sulfo-SPDB, and Hydrazine.

[0102] Cysteine coupling

[0103] Cysteine modification occurs most commonly by 1 ,4-conjugate addition to A / -substituted maleimides. Maleimides are particularly attractive reagents due to their synthetic accessibility and rapid reaction rates with cysteine under mild conditions. The resulting thiosuccinimide conjugates are inherently unstable, due to their propensity towards retro-Michael addition. This instability can be mitigated by forcing post-conjugation hydrolysis of the thiosuccinimide, creating a stable chemical linkage.

[0104] Accordingly, a number of “self-hydrolysing” maleimides have now been developed, with ring-opening catalysed by adjacent functional groups such as primary amine, polyethylene glycol (PEG) and A / -aryl amongst the most promising. Other reagents including a-halocarbonyls, palladium oxidative-addition complexes, ethynylphosphonamidates, vinylphosphonites and ethynylbenziodoxolones.

[0105] Some non-maleimide cysteine conjugations are summarised in Kang, et al., Chem Sci (2021) 12, 13613-13647, and include the use of:

[0106] (i) alkynyl carboxylic acid derivatives;

[0107] (ii) 5-methylene pyrrolone (5MP);

[0108] (Hi) 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB);

[0109] (iv) phenyloxadiazole sulfone (PODS);

[0110] (v) aza-dibenzocyclooctyne (DBCO);

[0111] (vi) phosphonamidite;

[0112] (vii) 3-arylpropionitrile (APN);

[0113] (viii) perfluoroarene;

[0114] (ix) ethynylbenziodoxolone (EBX);(x) bicyclo[1.1.0]butane (BCB) carboxylic amide; and

[0115] (xi) allenamide.

[0116] Another possible approach is described in Cheng, et al., Front. Oncol. 12:951589 where 2-methylsulfonyl pyrimidine is used instead of a maleimide.

[0117] Genetic modification of the number of accessible cysteine residues on an antibody surface is a method to achieve site-selective and homogeneous modification. For example, in THIOMABs, the engineered cysteine is installed on an anti-MUC16 antibody by mutation of heavy chain alanine 114 (HC-A114). Other approaches have engineered antibodies to contain cysteine mutations at D265C, S239C, E269C, K326C or A327C, or to insert additional cysteines before and after positions HC-S239, HC-A114, and LC-V205.

[0118] Non-natural amino acid incorporation by genetic engineering

[0119] Site-specific incorporation of non-canonical amino acids (ncAAs) into antibodies results in an efficient approach to the site-specific modification of antibodies, and therefore homogeneous ADCs.

[0120] NcAAs bearing unique functionalities, such as ketones, azides, cyclopropenes or diene functional groups, have been developed and incorporated into antibodies. Such ncAAs include p-acetylphenylalanine (pAcF), which has a ketone side chain which can participate in oxime ligation reactions; Ne-(1-methylcycloprop-2-enecarboxamido)-lysine (CpK), which has a cyclopropene side chain which can participate in IEDDA reactions; para-azidomethyl phenylalanine (pAMF), which has a an azide side chain which can undergo click reactions; spiro[2.4]hepta-4,6-diene-lysine (SCpHK), which has a spiro[2.4]hepta-4,6-diene side chain which can participate in Diels-Alder reactions; and N6-(2-azidoethoxy)-carbonyl-L-lysine (AzK), which has a an azide side chain which can undergo click reactions.

[0121] Azide-containing ncAAs can undergo rapid CuAAC or SPAAC reactions under physiological conditions, para-azidophenylalanine (pAzF) can undergo reactions with, for example, cyclooctyne-functionalised linkers and dibenzylcyclooctyne (DBCO)-functionalised linkers. A cyclopropene derivative of lysine (N e-[((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl]-l-lysine; CypK) can undergo a rapid and efficient inverse-electron demand Diels-Alder (IEDDA) reaction with a tetrazinefunctionalised linker.

[0122] Cyclopentadiene-containing ncAAs, spiro[2.4]hepta-4,6-diene-lysine (SCpHK) and cyclopentadienelysine (CpHK), can undergo irreversible Diels-Alder cycloadditions with maleimide-modified drugs.

[0123] Enzymatic conjugation

[0124] Enzymes can be used to achieve site-selective antibody modification due to their high specificity and mild reaction conditions. Enzymes can either directly attach a payload to a specific amino acidsequence or introduce a reactive functionality on the antibody that can be further functionalised with the desired payload.

[0125] Transpeptidation using sortase

[0126] Sortase-mediated antibody conjugation (SMAC) technology is an additional enzymatic ligation approach. SMAC-technology uses Staphylococcus aureus sortase A, which is a transpeptidase that cleaves the amide bond between threonine and glycine residues in the LPXTG (X = any amino acid) pentapeptide motif, and subsequently catalyses the attachment of glycine-functionalised payloads to the newly generated C-terminus. The sortase recognition motif and a Strep II tag, which is used to aid removal of unreacted antibody, can be fused to the light and heavy chain C-terminus of different antibodies. Sortase-mediated conjugation can then be used to attach a series of penta-glycine tagged payloads.

[0127] Transpeptidation using microbial transglutaminase

[0128] The use of bacterial transglutaminases is a powerful approach for site-specific incorporation of a payload onto an antibody. A transglutaminase derived from Streptomyces mobaraensis catalyzes transpeptidation where a primary amine-containing linker is covalently attached to the primary amide side chain of a specific glutamine (Q295) within deglycosylated antibodies, resulting in ADCs with a defined drug to antibody ratio (DAR) arising from the conjugation of 2 linker-payloads (one conjugation site per heavy chain). An N297Q mutation prior to this conjugation provides two more reaction sites (resulting in the conjugation of 4-linker-payloads). An alternative version uses a peptide sequence-specific transglutaminase. This enzyme recognizes and utilizes an LLQG motif that is genetically incorporated, resulting in site-specific antibody-drug conjugation. Another advantage of this LLQG-specific bacterial transglutaminase is that conjugation sites can be flexibly laid by inserting this short peptide motif within the antibody structure. Further alternative approaches allow for the use of transglutaminase without deglycosylation.

[0129] N-Glycan engineering

[0130] Asn297 (N297) within the Fc domain and the N-glycan on this residue are conserved in all IgG classes, making these components attractive reaction sites for broadly applicable ADC conjugation. Incorporation of an aldehyde group on the N-glycan terminus using p-1 ,4-galactosyltransferase (GalT) and a-2,6-sialyltransferase (SialT) introduce a sialic acid on each N-glycan terminus, which is subsequently converted into an aldehyde group using Nal04 under mild oxidation conditions. The aldehyde groups generated can then be used to conjugate aminooxy-functionalized payloads.

[0131] Another approach is to incorporate non-natural saccharides possessing orthogonal reaction handles into the antibody. A technology based on this strategy is GlycoConnect in which the glycan chain at Asn297 is trimmed using the endoglycosidase Endo S2 and then azide groups are introduced using a mutant galactosyl transferase GalT(Y289L) and N-azidoacetylgalactosamine (GalNAz). The azide handles can be used for a strain-promoted click reaction with payloads.In some embodiments, the linker-payload terminates in an amino group which is conjugated to the antigen-binding molecule using transglutaminase.

[0132] Radioconjugates

[0133] The DSB-inducing agent may be provided as a radioconjugate. The term “radioconjugate,” as used herein refers to a compound or conjugate that comprises a radioisotope or radionuclide. That is, the DSB-inducing agent may be or comprise a radioisotope, as described herein. The radioisotope or radionuclide exerts an anti-cancer effect by inducing DSBs in the DNA of cancer cells.

[0134] Radioconjugates comprise a targeting moiety such as an antibody, peptide, or small molecule. The skilled person appreciates that the targeting moiety of the radioconjugate will be selected based on the type of PTPN12-deficient cancer to be treated. The targeting molecule is linked to the radioisotope or radionuclide bound by a metal ion chelator via a linker. Radioconjugates are designed to bind specifically to cancer cells, delivering a dose of radiation in a targeted manner that reduces the dose of radiation that healthy cells are exposed to and delivers radiation to cancer cells both inside and outside of the main tumor site, such as metastases, unlike traditional beamed based radiotherapy. Radioconjugates suitable for use in accordance with the present invention are described in Shah, H. J., Ruppell, E., Bokhari, R., Aland, P., Lele, V. R., Ge, C., & McIntosh, L. J. (2023). European journal of radiology open, 10, 100477, hereby incorporated by reference in its entirety.

[0135] As used herein, the terms “radiometal,” “radioisotope,” “radiometal ion” and “radioactive metal ion” are used interchangeably and refer to one or more isotopes of the elements that emit particles and / or photons. Suitable radioisotopes that may be used for therapeutic applications in accordance with the present invention include, e.g., beta or alpha emitters, such as, e.g., 225 Ac, 177 Lu, 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 1, 134 Ce, 149 Tb, 152 Tb, 155 Tb, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 255 Fm and 227 Th. Other non-limiting examples of radioisotopes that may be used as imaging agents in accordance with the present invention include gamma-emitting radioisotopes, such as, e.g., 177 Lu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, and 111 In. In certain embodiments, the radiometal ion is a “therapeutic emitter,” meaning a radiometal ion that is useful in therapeutic applications. Examples of therapeutic emitters include, but are not limited to, beta or alpha emitters, such as, 132 La, 135 La, 134 Ce, 144 Nd, 149 Tb, 152 Tb, 155 Tb, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 177 Lu, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 255 Fm and 227 Th, 226 Th, 230 U. Preferably, a radiometal ion used in the invention is an alpha-emitting radiometal ion, such as actinium-225 (225 Ac).

[0136] It is noted that certain radiometals may be used as therapeutic agents (e.g., 225 Ac) and / or as imaging agents (e.g., 111 In). A suitable radiometal for use as a therapeutic agent is one that is capable of reducing or inhibiting the growth of, or in particular killing, a cancer cell, such as a breast cancer cell. In certain embodiments, radioconjugates of the present invention can deliver acytotoxic payload with the ability to emit alpha and / or beta particles in the vicinity of a tumor by binding onto cancer cells' surface antigens and initiating cell death.

[0137] The terms “225Ac,”, “225 Ac,” or “Ac-225” refer to actinium-225 which is an alpha-emitting radiometal. The approximate ten-day half-life of 225 Ac (about 9.9 days) is long enough to be able to prepare the compounds described herein, but short enough to match the circulation pharmacokinetics of the antibody that is conjugated to the radiometal complex, such as hi 1 B6. 225 Ac decays in a series of steps that ultimately emits four alpha particles before reaching a stable isotope, 209 Bi, thereby providing an increased potency of the compounds.

[0138] In embodiments wherein the targeting moiety is a peptide, the peptide may be selected from somatostatin, bombesin, cholecystokinin / gastrin, neurotensin and vasoactive intestinal peptide. In some embodiments, the radioconjugate is Lutetium 177 oxodotreotide (Lutathera). Lutetium oxodotreotide (also referred to herein as Lutetium 177 Dotatate) exerts a cytotoxic effect on cancer cells via its somatostatin peptide, which binds to cells expressing somatostatin receptors. As a result, such cells receive a lethal dose of radiation. In particular, wherein the PTPN12-deficient cancer is a neuroendocrine tumor, the radioconjugate may be lutetium oxodotreotide. Lutetium 177 may also be bound to prostate-specific membrane antigen (PMSA) (Pluvitco), which targets PSMA positive cancers, and Radium 223 may be bound to an anti-HER2 antibody to target HER2 amplified cancers.

[0139] In embodiments wherein the targeting moiety is an antigen-binding molecule, the antigen-binding molecule that binds to a tumor antigen. The tumor antigen may be any tumor antigen expressed on the surface of cancer cells. Preferably, the tumor antigen is expressed at a higher level on cancer cells compared to normal cells. In some embodiments, the tumor antigen is expressed exclusively on the surface of cancer cells. The tumor antigen may be any tumor antigen as described above, as suitable for use in ADCs in accordance with the present invention.

[0140] In some embodiments, the DSB-inducing agent and / or radiotherapy is selected from the list comprising: etoposide, camptothecin, Olaparib, carboplatin and Lu 177- Dotatate.

[0141] Radiotherapy

[0142] The present invention relates to methods of treating cancer using radiotherapy. Radiotherapy is a key treatment for cancer, being used in approximately 50% of cancer treatments in the developed world. Radiotherapy can be used to cure cancer. It is estimated that radiotherapy is primary treatment modality used in 16% of patients who are cured of their cancer. By comparison, chemotherapy is the primary modality in only 2% of cancer cures. Radiotherapy involves the delivery of ionizing radiation to cancer cells to induce DSBs, which are toxic to the cancer cells. X-rays, gamma rays and charged particles (e.g. electrons, protons, positrons, alpha particles) are examples of types of radiation used for cancer treatment. Radiotherapy relies on the production of reactive oxygen species (ROS), alsoknown as free radicals. Free radicals are highly reactive chemical species containing oxygen which can act to destroy cellular components such as DNA and membranes. Enough free radical damage will induce apoptosis, or cell death. Radiotherapy can be given in two different ways: from outside the body (known as external beam radiotherapy or external radiotherapy) or from inside the body (known as internal radiotherapy). The radiotherapy may be delivered by a machine outside the body (external radiotherapy), or it may come from radioactive material placed in the body, within or near cancer cells (internal radiotherapy). Alternatively, internal radiation therapy may be performed using a radiopharmaceutical, i.e. a radioactive drug, which is typically swallowed or administered parenterally. Another method is to use radioactive embolization particles. The radiotherapy may be targeted to the tumor location. In methods according to the invention wherein the subject is treated with radiotherapy, the cancer may be a solid tumor.

[0143] Typically, the radiotherapy uses a source of energy of more than 50 keV, for instance a source of energy equal to or greater than 60 keV. It is typically greater than 60 keV, for instance it may be equal to or greater than 70 keV, for instance equal to or greater than 80 keV, or equal to or greater than 100 keV. The radiotherapy may for instance use a source of energy equal to or greater than 200 keV, for instance equal to or greater than 400 keV.

[0144] The radiotherapy may for instance comprise supplying X-ray or gamma ray photons with an incident energy of more than 50 keV, for instance with an incident energy equal to or greater than 60 keV, for example equal to or greater than 70 keV, or equal to or greater than 80 keV.

[0145] The radiotherapy may comprise supplying X-ray or gamma ray photons with an incident energy equal to or greater than 100 keV, for instance with an incident energy equal to or greater than 200 keV, or equal to or greater than 400 keV. The photons may for instance have an incident energy of from 0.05 MeV (50 keV) to 10 MeV, for instance from 0.06 MeV (60 keV) to 10 MeV, or for instance from 0.08 MeV (80 keV) to 10 MeV, for example from 0.1 MeV (100 keV) to 1 MeV. The photons may for instance have an incident energy of from 0.2 MeV (200 keV) to 10 MeV, for instance from 0.4 MeV (400 keV) to 10 MeV.

[0146] Alternatively, the radiotherapy may comprise supplying electrons, positrons or protons with an incident energy that is equal to or greater than 10 MeV, for instance with an incident energy that is equal to or greater than 50 MeV. The incident energy may for instance be from 60 MeV to 300 MeV, for example from 70 MeV to 250 MeV. The treatment may for instance use, proton beam radiation (proton beam therapy) wherein the incident energy is thus defined, for example is from 70 MeV to 250 MeV.

[0147] When the radiotherapy comprises irradiating a site of the cancer with radiation from an external source, the radiotherapy may employ X-rays, gamma rays, electrons or protons. For instance, the radiotherapy may be selected from conformal radiotherapy, intensity modulated radiotherapy (IMRT), image guided radiotherapy (IGRT), 4-dimensional radiotherapy (4D-RT), stereotacticradiotherapy and radiosurgery, proton therapy, electron beam radiotherapy, and adaptive radiotherapy.

[0148] Conventionally, external radiotherapy uses a single external beam, usually of X-rays, with the patient being exposed from several directions such as front and back or side to side. Although the technology is very well established it is limited in its ability to spare normal tissue from excessive dose. Recent developments have included stereotactic radiosurgery (SRS) in which highly focused beams are used to target well defined tumor regions typically in the brain or spine. It is claimed that the ability to accurately target tumor regions and use shorter treatment regimes enhances the treatment efficacy. A typical example of a SRS system is Cyberknife™, which has had FDA clearance for treatment of tumors in any part of the body since 2001. The radiotherapy source is mounted on a robot arm and can deliver a pencil thin beam of radiation at 6-8Gy per minute. Again, the main rationale for this approach is to increase the dose accuracy to the tumor and deliver dose escalation. Intensity modulated radiation therapy (IMRT) utilises multiple radiation beams to deliver maximum energy into fields that accurately map even complex tumor structures such as those wrapping around blood vessels. Medical professionals are required to map the structure one image at a time prior to devising a treatment protocol. There is increasing evidence of advanced survival using both SRS and IMRT techniques and reduced toxicity and normal tissue damage.

[0149] Proton therapy uses an external beam of protons to target the tumor site, the advantage being an ability to target a tumor mass more easily than using X-ray radiotherapy. This is due to the protons having limited side scatter due to their high mass and a well-defined penetration depth. In a similar fashion to X-ray based treatments, the protons my either directly damage DNA by scattering or indirectly by free radical generation.

[0150] Often, when the radiotherapy comprises irradiating a site of the cancer with radiation from an external source, X-ray radiation is used.

[0151] After administering the particles to a subject, a period of time sufficient to allow the particles to accumulate at the site of the cancer or tumor is usually allowed to elapse before directing X-ray radiation to the cancer. The time period between administration of the particles and irradiation with X-rays will depend on, amongst other things, the mode of administration, whether there is a targeting moiety attached to the particles and the nature of the cancer.

[0152] The step of directing X-ray radiation to a site of the cancer or tumor tissue may be carried out at least 3 hours, especially at least 6 hours, typically 9 to 48 hours, particularly 12 to 24 hours, after administering, typically orally or parenterally (including but not limited to intratumoral injection), the particle or the pharmaceutical composition to the subject.When the pharmaceutical composition comprising the plurality of particles is used in combination with radiotherapy comprising irradiating a site of the cancer with radiation from an external source, the dose of radiation will depend on the type of radiation and the area of the body on which it is being deployed. Typically, the maximum dose that can be applied is 70-74Gy. In radiosensitive organs this may be reduced. The external radiotherapy can be administered in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) throughout the course of the treatment. Single or multiple doses can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.

[0153] Generally, the subject is exposed to a total X-ray dose of from 20 to 70 Gy, such as for example 40 to 50 Gy.

[0154] Typically, a treatment or method for treating cancer of the invention comprises directing a 1 .0 to 3.0 Gy, typically 1.5 to 2.5 Gy dose, more typically a 1.8 to 2.0 Gy dose of X-ray radiation to a site of the cancer or tumor tissue. Such small frequent doses are intended to allow healthy cells time to grow to repair any damage caused by the radiation.

[0155] Typically, the X-ray radiation has an incident energy of more than 50 keV, for instance an incident energy equal to or greater than 60 keV, for example equal to or greater than 70 keV, or equal to or greater than 80 keV. The X-ray radiation may for instance have an incident energy that is equal to or greater than 100 keV, for instance an incident energy equal to or greater than 200 keV, or equal to or greater than 400 keV. The X-ray radiation may for instance have an incident energy of from 0.05 MeV (50 keV) to 10 MeV, for instance from 0.06 MeV (60 keV) to 10 MeV, or for instance from 0.08 MeV (80 keV) to 10 MeV, for example from 0.1 MeV (100 keV) to 1 MeV. The X-ray radiation may for instance have an incident energy of from 0.2 MeV (200 keV) to 10 MeV, for instance from 0.4 MeV (400 keV) to 10 MeV.

[0156] In some embodiments, the DSB-inducing agent and / or radiotherapy is selected from the list comprising: etoposide, camptothecin, Olaparib, carboplatin, sacituzumab govitecan, Lu177-Dotatate and X-Ray. In some embodiments, the DSB-inducing agent and / or radiotherapy is etoposide. In some embodiments, the DSB-inducing agent and / or radiotherapy is camptothecin. In some embodiments, the DSB-inducing agent and / or radiotherapy is Olaparib. In some embodiments, the DSB-inducing agent and / or radiotherapy is carboplatin. In some embodiments, the DSB-inducing agent and / or radiotherapy is Lu177-Dotatate. In some embodiments, the DSB-inducing agent and / or radiotherapy is X-Ray. In some embodiments, the DSB-inducing agent and / or radiotherapy is sacituzumab govitecan.

[0157] Pharmaceutical Combinations

[0158] In some embodiments the methods of the present invention comprise administering a DSB-inducing agent in combination with radiotherapy and / or a second anti-cancer agent.Radiotherapy may be administered to the subject before the DSB-inducing agent. In some embodiments, the radiotherapy is administered as a debulking intervention, e.g., before receiving surgery. Radiotherapy may be administered at the same time as the DSB-inducing agents.

[0159] Radiotherapy and chemotherapy have been used in combination in the clinic to treat various cancers as they show synergy due to the complementary mechanisms of action, i.e. , through the induction of DNA damage such as DSBs. The combination of radiotherapy and a DSB-inducing agent for treating PTPN12-deficient cancers according to the present invention may therefore be expected to provide an even greater efficacy, as patients with a PTPN12-deficient cancer already show sensitivity to both agents.

[0160] The DSB-inducing agent may be administered in combination with one or more additional anti-cancer agents. The DSB-inducing agent may be administered as part of a chemotherapy regimen. For example, many different chemotherapy regimens using multiple agents are established treatments well known in the art. Lists of such regimens can be found online, for example at https: / / www.ebmt.org / ebmt / documents / list-chemotherapy-drugs-agents-and-regimens-v2 (published 5 June 2024; hereby incorporated by reference in its entirety).

[0161] The DSB-inducing agent may be administered in combination with a targeted therapy. The targeted therapy may be a kinase inhibitor or an antibody that has a target that is unique to the cancer cells. For example, the targeted therapy may be a small molecule that binds to a mutant kinase expressed only by cancer cells. Small molecule targeted therapies are known in the art, and are described in e.g. Liu GH et al., (2020). 2022;3(4):e181. Published 2022 Oct 13, hereby incorporated by reference in its entirety. In other examples, the targeted therapy may be an antibody that binds to a protein expressed only on the surface of cancer cells. Antibody-based targeted therapies are known in the art, and are described in e.g. Goydel RS, Rader C. Oncogene. 2021 ;40(21):3655-3664, hereby incorporated by reference in its entirety.

[0162] The DSB-inducing agent may be administered in combination with an immunotherapy.

[0163] Immunotherapies are known in the art. The immunotherapy be selected from: therapeutic antibodies, cancer vaccines, checkpoint inhibitor therapy, and immune system modulators (e.g. interferons and interleukins). The immunotherapy may be selected from pembrolizumab, nivolumab, atezolizumab, durvalumab and ipilimumab. Suitable immunotherapies are described in e.g. Rui R, Zhou L, He S. Front Immunol. 2023;14:1212476. Published 2023 Aug 24, hereby incorporated by reference in its entirety.

[0164] Provided herein are methods of treating cancer comprising administering (i) a DSB-inducing agent and (ii) an inhibitor of PTPN12 to the patient. In some embodiments, the subject has a PTPN12-deficient cancer. In some embodiments, the patient does not have a PTPN12-deficient cancer. Insome embodiments, the patient has a PTPN12-proficient cancer. Each agent may be formulated for administration separately, i.e., both agents are not present in a single composition.

[0165] In some embodiments the invention relates to compositions (e.g. pharmaceutical combinations) comprising a DSB-inducing agent and a PTPN12 inhibitor, i.e., both agents are present in a single composition.

[0166] The combinations / compositions described herein may be characterized by reference to certain functional properties. In some embodiments, a combination described herein may possess one or more of the following properties:

[0167] inhibits tumor growth and / or reduces tumor size / volume, to an extent which is greater than the tumor growth inhibition / reduction in tumor size / volume observed when a constituent agent of the combination / composition is used alone;

[0168] increases survival of subjects having a cancer;

[0169] increases survival of subjects having a cancer, to an extent which is greater than the increase in survival observed when a constituent agent of the combination / composition is used alone; synergistically inhibits tumor growth and / or synergistically reduces tumor size / volume, relative to the tumor growth inhibition / reduction in tumor size / volume observed when a constituent agent of the combination / composition is used alone; and / or

[0170] synergistically increases survival of subjects having a cancer, relative to the increase in survival observed when a constituent agent of the combination / composition is used alone.

[0171] In some embodiments, a combination / composition of the present disclosure displays anticancer activity. In some embodiments, the combination / composition increases killing of cancer cells. In some embodiments, the combination / composition causes a reduction in the number of cancer cells in vivo, e.g. as compared to an appropriate control condition. The cancer may be a cancer as described herein.

[0172] It will be appreciated that a given combination / composition may display more than one of the properties recited in the preceding paragraphs. A given combination / composition may be evaluated for the properties recited in the preceding paragraphs using suitable assays. For example, the assays may be e.g. in vitro assays, optionally cell-based assays or cell-free assays. In some embodiments, the assays may be e.g. in vivo assays, i.e. performed in non-human animals. In some embodiments, the assays may be e.g. ex vivo assays, i.e. performed using cells / tissue / an organ obtained from a subject.

[0173] Where assays are cell-based assays, they may comprise treating cells with a combination / composition in order to determine whether the combination / composition displays one or more of the recited properties. Assays may employ species labelled with detectable entities in order to facilitate their detection. Assays may comprise evaluating the recited properties following treatment ofcells separately with a range of quantities / concentrations of a given combination / composition {e.g. a dilution series).

[0174] Analysis of the results of such assays may comprise determining the concentration at which 50% of the maximal level of the relevant activity is attained. The concentration of a given agent at which 50% of the maximal level of the relevant activity is attained may be referred to as the ‘half-maximal effective concentration’ of the agent in relation to the relevant activity, which may also be referred to as the ‘EC50’. Depending on the property, the EC50 may also be referred to as the ‘half-maximal inhibitory concentration’ or ‘IC50’, this being the concentration of the agent at which 50% of the maximal level of inhibition of a given property is observed.

[0175] Cell killing can be investigated, for example, using any of the methods reviewed in Zaritskaya etal., Expert Rev Vaccines (2011 ), 9(6):601 -616, hereby incorporated by reference in its entirety. Examples of in vitro assays of cytotoxicity / cell killing assays include release assays such as the51Cr release assay, the lactate dehydrogenase (LDH) release assay, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) release assay, and the calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on the detection of factors released from lysed cells. Cell killing of a given test cell type by a given effector immune cell type can be analyzed e.g. by coculturing the test cells with the effector immune cells, and measuring the number / proportion of viable / dead {e.g. lysed) test cells after a suitable period of time. Other suitable assays include the xCELLigence real-time cytolytic in vitro potency assay described in Cerignoli et al., PLoS One. (2018) 13(3): e0193498 (hereby incorporated by reference in its entirety).

[0176] A “PTPN12 inhibitor” or an “inhibitor of PTPN12” refers to an agent that reduces a biological activity of PTPN12. The biological activity may be completely inhibited such that it is reduced by 100%, or the biological activity may be partially inhibited, such that it is reduced by about 50%, about 60%, about 70%, preferably about 80%, more preferably about 90%, and most preferably about 95%. The biological activity may be tyrosine phosphatase activity. For example, the PTPN12 inhibitor may be a small molecule that binds to PTPN12, an inhibitory RNA that reduces expression of PTPN12 (for example an siRNA or miRNA), an anti-sense oligonucleotide (ASO), an aptamer, a nanoparticle or a peptide-based inhibitor. A PTPN12 inhibitor may be a pan-PTP inhibitor that binds to other members of the PTP family, or it may be a specific inhibitor of PTPN12.

[0177] In some embodiments, the PTPN12 inhibitor is an siRNA.

[0178] In some embodiments, the PTPN12 inhibitor is a selective inhibitor of PTPN12. A “selective PTPN12 inhibitor” is an inhibitor that binds to PTPN12 with a Kd that is lower than the Kd for binding to a protein other than PTPN12. In some embodiments, a selective PTPN12 inhibitor binds to PTPN12 with a Kd that is at least about 20 fold lower, preferably at least about 30 fold lower, more preferably at least about 50 fold lower and most preferably at least about 70 fold lower than the Kd for binding ofany other protein. In some embodiments, the PTPN12 inhibitor binds to PTPN12 with a Kd of less than 10 nM, less than 5nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM, or less than 0.01 nM.

[0179] Suitable PTPN12 inhibitors are described in Yang CF et al., Cardiovasc Res. 2020;116(5):1032-1046, and Johnson D et al., Chem Biol Drug Des. 2025;105(2):e70058, hereby incorporated by reference in their entirety. The PTPN12 inhibitor may be Auranofin or J1 -65.

[0180] In some embodiments, a combination / composition of the present disclosure achieves a synergistic therapeutic and / or prophylactic effect. That is, in some embodiments, the combination / composition achieves a treatment effect that is synergistic {i.e. super-additive), relative to what is observed when a constituent of the combination / composition is employed as a monotherapy.

[0181] As used herein, a ‘synergistic’ or ‘super-additive’ level of a relevant effect {e.g. cancer growth inhibition, reduction in tumor size / volume, reduction in the number / proportion of cancer cells, increase in survival) for a given combination / composition refers to a level of the effect which is greater than the sum of the effects observed for the individual components of the combination / composition.

[0182] Quantitative methods for assessing synergism are described e.g. in Tallarida, Genes Cancer. (2011) 2(11):1003-1008 and Chou, Cancer Res (2010) 70:440-446, both of which are hereby incorporated by reference in their entirety. Additive, synergistic and antagonistic effects may be evaluated in experiments in which a range of different doses of the combination / composition and the individual constituents thereof are evaluated for the relevant effect. Dose-response curves may be plotted, and evaluated in order to determine whether the combination / composition achieves a synergistic level of the relevant effect relative to the individual constituents of the combination / composition employed in isolation {i.e. as monotherapies). In some embodiments, synergy may be evaluated using combination index (Cl) values calculated using the Chou-Talalay method described in Chou, Cancer Res (2010) 70:440-446. According to the Chou-Talalay method, for a given combination Cl = 1 indicates an additive effect, Cl <1 indicates synergism, and Cl >1 indicates antagonism.

[0183] In some embodiments, a combination / composition of the present disclosure achieves a synergistic {i.e. super-additive) reduction in cancer growth, delay to cancer growth, prevention of cancer growth, reduction in the severity of one or more symptoms of the cancer, reduction in the number of cancer cells, reduction of the cancer burden, reduction of tumor size / volume and / or increase in survival of subjects having the cancer, relative to what is observed when a component of the combination / composition is employed alone. In some embodiments, a combination / composition achieves a synergistic {i.e. super-additive) reduction in cancer growth, delay to cancer growth, prevention of cancer growth, reduction in the severity of one or more symptoms of the cancer, reduction in the number of cancer cells, reduction of the cancer burden, reduction of tumor size / volume and / or increase in survival of subjects having the cancer, relative to what is observed when a constituent of the combination / composition is employed as a monotherapy.PTPN12-deficient Cancers

[0184] The terms “cancer” and “cancerous” refer to the physiological condition in mammals typically characterised by unregulated cell growth. A cancer may be any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor or increased risk of or predisposition to the unwanted cell proliferation, neoplasm, or tumor. The cancer may be benign or malignant.

[0185] As used herein, the term “cancer” refers to non-metastatic and metastatic cancers, including early stage and late-stage cancers. By “non-metastatic” is meant a cancer that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site. The term "metastatic cancer” refers to cancerthat has spread or is capable of spreading from one part of the body to another. Generally, a non-metastatic cancer is any cancer that is a Stage 0, 1 , or II cancer, and occasionally a Stage III cancer. A metastatic cancer, on the other hand, is usually a stage IV cancer.

[0186] Examples of tissues include the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g. renal epithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node (including abdominal lymph node, axillary lymph node, cervical lymph node, inguinal lymph node, mediastinal lymph node, pelvic lymph node, periaortic lymph node), lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentume, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, white blood cells.

[0187] A “cancer” can comprise any one or more of the following or a combination thereof: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adenocarcinoma (COAD), adrenocortical cancer, adrenocortical carcinoma (ACC), adrenal cancer, anal cancer, astrocytoma, bladder cancer, bladder urothelial carcinoma (BLCA), blood cancer, bone cancer, bone marrow tumor, brain tumor, brain lower grade glioma (LGG), brain stem nerve gliomas, breast cancer, breast invasive carcinoma (BRCA), cancer of the female genital system, cancer of the male genital system, central nervous system lymphoma, cervical cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), childhood rhabdomyosarcoma, childhood sarcoma, chronic or acute leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), cholangiocarcinoma (CHOL), CNS tumor, colon and rectal cancer, colon cancer, cutaneous or intraocular melanoma, endocrine cancer, endometrial cancer, endometrial sarcoma, oesophageal cancer, oesophageal carcinoma (ESCA), eye cancer, fallopian tube carcinoma, gallbladder cancer, gastric (or stomach) cancer, gastrointestinaltract cancer, glioma, glioblastoma multiforme (GBM), hairy cell leukemia, head and / or neck cancer, head and neck squamous cell carcinoma (HNSC), hepatocellular cancer, Hodgkin's disease, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), large intestinal cancer, laryngeal cancer, leukemia, liver cancer, liver hepatocellular carcinoma (LIHC), lung cancer, lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), lymphocytic lymphoma, lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), malignant fibrous histiocytoma, malignant thymoma, melanoma, mesothelioma (MESO), multiple myeloma, myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nervous system cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian serous cystadenocarcinoma (OV), pancreatic cancer, pancreatic adenocarcinoma (PAAD), parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma and paraganglioma (PCPG), pituitary adenoma, pituitary tumor, plasma cell neoplasm, primary CNS lymphoma, prostate cancer, prostate adenocarcinoma (PRAD), rectal or colorectal cancer, rectum adenocarcinoma (READ), renal cell carcinoma, renal pelvic carcinoma, respiratory system carcinoma, retinoblastoma, salivary gland cancer, sarcoma (SARC), skin cancer, skin cutaneous melanoma (SKCM), small cell lung cancer, small intestine cancer, soft tissue sarcoma, stomach cancer, squamous cell carcinoma, testicular cancer, testicular germ cell tumors (TGCT), thyroid cancer, thyroid carcinoma (THCA), thymoma (THYM), urinary system cancer, ureter cancer, urethral cancer, uterine sarcoma, uterine carcinosarcoma (UCS), uterine corpus endometrial carcinoma (UCEC), uveal melanoma (UVM, also known as intraocular melanoma), vaginal cancer, vascular system, vulval cancer, Waldenstrom's macroglobulinemia and Wilms' tumor. The cancer may be found in any location of the body.

[0188] The cancer may be selected from Uterine Corpus Endometrial Carcinoma, Stomach Adenocarcinoma, Ovarian Serous Cystadenocarcinoma, Colorectal Carcinoma, Uterine Carcinosarcoma, Skin Cutaneous Melanoma, Esophageal Adenocarcinoma, Esophageal Squamous Cell Carcinoma, Lung Squamous Cell Carcinoma, Non-Small Cell Lung Cancer, Cervical Squamous Cell Carcinoma, Liver Hepatocellular Carcinoma, Head and Neck Squamous Cell Carcinoma, Bladder Urothelial Carcinoma, Glioblastoma Multiforme, Lung Adenocarcinoma, Pancreatic Adenocarcinoma, Diffuse Large B-cell Lymphoma, Sarcoma, Breast Carcinoma, Testicular Cancer, Brain Lower Grade Glioma, Prostate Adenocarcinoma, and Kidney Renal Clear Cell Carcinoma.

[0189] In some embodiments, the cancer is selected from breast cancer, non-small cell lung cancer, and colorectal cancer. In some embodiments the breast cancer is triple negative breast cancer. PTPN12 has been shown to be downregulated in triple negative breast cancer, where it is implicated as a tumour suppressor gene (Sun T, Aceto N, Meerbrey KL, et al. Cell. 2011 ;144(5):703-718, hereby incorporated by reference in its entirety). PTPN12 has been shown to be downregulated in non-small cell lung cancer, where it was found to correlate with unfavourable survival duration in patients (Cao X, Chen YZ, Luo RZ, et al. Oncotarget. 2015;6(13):11704-11713, hereby incorporated by reference in its entirety). PTPN12 has been shown to be downregulated in colorectal carcinoma, where itsdownregulation contributed to increased motility of colon carcinoma cells (Espejo R, Rengifo-Cam W, Schaller MD, Evers BM, Sastry SK. Am J Physiol Cell Physiol. 2010;299(2):C454-C463, hereby incorporated by reference in its entirety).

[0190] In some embodiments, the cancer is non-invasive breast cancer, non-small cell lung cancer, or colorectal cancer, early invasive breast cancer, non-small cell lung cancer, or colorectal cancer, or invasive non-specific breast cancer, non-small cell lung cancer, or colorectal cancer. In some embodiments, the breast cancer is invasive non-specific breast cancer. In some embodiments, the cancer is advanced and / or metastatic breast cancer, non-small cell lung cancer, or colorectal cancer. In some embodiments, the cancer is primary metastatic breast cancer, non-small cell lung cancer, or colorectal cancer and / or secondary metastatic breast cancer, non-small cell lung cancer, or colorectal cancer.

[0191] In some embodiments, the breast cancer is ductal carcinoma, tubular carcinoma, classic lobular carcinoma, invasive cribriform carcinoma, neuroendocrine carcinoma, mucinous carcinoma, apocrine carcinoma, pleomorphic lobular carcinoma, medullary carcinoma, metaplastic Carcinoma or adenoid cystadenocarcinoma. In some embodiments, the breast cancer is ductal carcinoma of the breast.

[0192] In some embodiments, the breast cancer is luminal A subtype, luminal B subtype, HER-2(+) type (or HER-2 overexpression type), basal cell Type (basal-like subtype, BLBC, including triple negative breast cancer (TNBC)) or normal breast-like subtype (normal breast-like subtype). In some embodiments, the breast cancer is triple negative breast cancer.

[0193] In some embodiments, the breast cancer does not comprise a mutation in BRCA1 and / or BRCA2.

[0194] In some embodiments, the breast cancer is advanced and / or metastatic triple negative breast cancer.

[0195] In some embodiments, the colorectal cancer is colorectal adenocarcinoma, preferably, the colorectal adenocarcinoma is cribriform acne adenocarcinoma, medullary carcinoma, micropapillary carcinoma, mucinous adenocarcinoma, serrated adenocarcinoma or signet ring cell carcinoma.

[0196] In some embodiments, the NSCLC is squamous cell carcinoma (i.e., epidermoid carcinoma), large cell carcinoma, adenocarcinoma, adenosquamous carcinoma, carcinomas with pleomorphic, sarcomatoid, or sarcomatous elements, carcinoid tumor, or salivary gland carcinoma.

[0197] Cancers in accordance with the present invention are PTPN12-deficient cancers. PTPN12-deficient cancers are cancers comprising a PTPN12 deficiency. A PTPN12 deficiency is any reduction in the PTPN12 expression level and / or activity compared to that observed in non-cancerous tissue. The PTPN12 deficiency may be caused by one or more genetic alterations. The one or more genetic alterations may result in reduced expression of PTPN12 compared to that observed in non-canceroustissue. The one or more genetic alterations may be selected from: hypermethylation, deep deletion, mutation in the gene encoding PTPN12, or a combination thereof.

[0198] A PTPN12 deficiency may result in reduced expression of PTPN12 compared to that observed in non-cancerous tissue. In some cases, the cancer is a cancer with less than 70% of the level of expression of PTPN12 compared to the level of expression in the control sample. In some embodiments, the gene expression of PTPN12 is downregulated by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in the cancer, compared to the level of PTPN12 gene expression in the control sample. The level of PTPN12 expression may be the level of PTPN12 nucleic acid, such as mRNA encoding PTPN12, or the level of PTPN12 protein.

[0199] The one or more genetic alterations may be one or more genetic mutations. The one or more genetic alterations may be one or more mutations that cause partial or full loss of function of PTPN12, i.e., a loss of function mutation in PTPN12. The one or more mutations may be homozygous (i.e. present in both copies of the PTPN12 gene in the genome), or heterozygous (present in only one copy of the PTPN12 gene in the genome). In some embodiments, the one or more mutations comprises a nonsynonymous mutation (which results in a change to the encoded protein sequence). The one or more mutation may be a loss of function mutation. The one or more mutations may be adjacent to or proximal to the 5' end of the open reading frame of the gene encoding PTPN12. Without being bound by theory, a nonsynonymous mutation occurring adjacent to or proximal to the 5' end of the open reading frame has increased likelihood of generating a loss of function or inactivation of PTPN12.

[0200] In some embodiments, the one or more mutations are in the phosphatase domain of PTPN12 (i.e., the one or more mutations may be a mutation to one or more amino acids within residues 28-293 of SEQ ID NO: 1). The one or more mutation may abrogate the catalytic activity of PTPN12. The one or more mutations may be at a residue within the active site of PTPN12. Mutations within the phosphatase domain of PTPN12 are described in Nair, A., et al., (2018). Nature medicine, 24(4), 505-511 , hereby incorporated by reference in its entirety. For example, the one or more mutations may comprise a mutation of H230 or C231. The one or more mutations may comprise a substitution, for example, H230Y or C231 A. such mutations alter the spatial configuration of the PTPN12 catalytic cleft.

[0201] In some embodiments the one or more mutations comprise a missense mutation (a point mutation that results in a codon that encodes a different amino acid residue compared to the wild-type or nonmutated amino acid sequence). In some embodiments, the missense mutation is rs3750050 (Shen et al., Cancer Epidemiol. 2019;59:109-114).In some embodiments, the one or more mutations comprise a null mutation (leading to the deletion of the gene encoding PTPN12).

[0202] In some embodiments, the one or more mutations (e.g., the nonsynonymous mutation) comprises a nonsense mutation (point mutation in the gene sequence that results in a premature stop codon or nonsense codon on the transcribed mRNA that produces a translation product that is truncated or incomplete). In some embodiments, the nonsense mutation occurs adjacent to or proximal to the 5' end of the open reading frame of the gene encoding PTPN12.

[0203] In some embodiments, the one or more mutations (e.g., nonsynonymous mutation) comprises a nonstop mutation (point mutation occurring within translational stop codons that result in continued and inappropriate translation of mRNA transcript into the 3' untranslated region).

[0204] In some embodiments, the one or more mutations (e.g., nonsynonymous mutation) comprises an insertion of one or more nucleotides in the gene encoding PTPN12. In some embodiments, the insertion results in a frameshift mutation (change in the open reading frame of the gene encoding PTPN12).

[0205] In some embodiments, the one or more mutations (e.g., nonsynonymous mutation) comprises a deletion of one or more nucleotides. In some embodiments, the deletion results in a frameshift mutation. In some embodiments, the frameshift mutation results in a gene encoding an altered (e.g., inactivated) protein product.

[0206] In some embodiments, the one or more mutations comprises an inversion. In some embodiments, the one or more mutations comprises a deletion-insertion.

[0207] In some embodiments, the PTPN12-deficient cancer may be a cancer wherein at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of cells do not express functional PTPN12 protein.

[0208] In some cases, the PTPN12-deficient cancer may be a cancer with less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or less than 5% of the level of expression of PTPN12 expressed in a control sample, such as the level of expression of PTPN12 in a non-cancerous tissue. In some cases, the cancer is a cancer with less than 75% of the expression of PTPN12 than the level of expression in the control sample. The level of PTPN12 expression may be at the level of PTPN12 nucleic acid, such as PTPN12 encoding mRNA, or the level of PTPN12 protein.The cancer may be a cancer with less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or less than 5% of the amount of protein of PTPN12 than the amount of PTPN12 protein in a control sample, such as a sample of non-cancerous tissue from the patient. In some cases, the cancer is a cancer with less than 70% of the amount of PTPN12 protein than the amount of PTPN12 protein in the control sample. The level of PTPN12 expression may be the level of PTPN12 nucleic acid, such as PTPN12 encoding mRNA, or the level of PTPN12 protein.

[0209] The cancer may be a cancer with less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or less than 5% of the amount of mRNA of PTPN12 than the level of PTPN12 mRNA in control sample, such as a sample of non-cancerous tissue from the patient. In some cases, the cancer is a cancer with less than 70% of the amount of PTPN12 mRNA than the amount of PTPN12 mRNA in the control sample.

[0210] The amount of PTPN12 mRNA present in a control sample may be measured using any appropriate technique, for example, using quantitative reverse transcription PCR (RT-qPCR), or by measuring PTPN12 protein levels using western immunoblots or proteomics.

[0211] The cancer may be a cancer with less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or less than 5% of the PTPN12 activity of the level of PTPN12 activity in a control sample, such as a sample of non-cancerous tissue from the patient. In some cases, the cancer is a cancer with less than 70% of the PTPN12 activity than the amount of PTPN12 activity in the control sample. PTPN12A activity may be measured, for example, by examining the phosphorylation level of a known PTPN12 target, for example, by measuring the phosphorylation levels of BCAR1. Appropriate methods for measuring phosphorylation on target proteins are known to the skilled person and include, for example, western blots with phospho-specific antibodies, or mass spectrometry.

[0212] In some embodiments, the one or more mutations comprises a homozygous deletion. In some embodiments, the one or more mutations comprises a missense mutation in the gene encoding PTPN12, wherein the mutated gene is predicted to encode a nonfunctional protein. For example, the mutated gene is predicted to encode a nonfunctional protein using a SIFT algorithm (see, e.g., Nature Protocols (2016) 11 :1 -9), wherein the SIFT value is equal to or approximately zero. In some embodiments, the one or more mutations comprises a nonsense mutation in the gene encoding PTPN12, wherein the mutated gene encodes a truncated protein. In some embodiments, the one or more mutations comprises a nonstop mutation in the gene encoding PTPN12, wherein the mutated gene encodes a longer (e.g., non-functional or inactivated) protein. In some embodiments, the one ormore mutations is a duplication, a deletion, or an insertion. In some embodiments, the duplication, deletion, or insertion results in a frameshift mutation.

[0213] In some embodiments, the one or more mutations alters (e.g., increases or decreases) the expression of the gene encoding PTPN12. In some embodiments, the one or more mutations comprises a splice site mutation. In some embodiments, the one or more mutations (e.g., splice site mutation) results in altered splicing of a transcriptional product of the gene encoding PTPN12. In some embodiments, the one or more mutations results in a transcriptional product having impaired nuclear translocation. In some embodiments, the one or more mutations results in a transcriptional product having impaired translation. In some embodiments, the one or more mutations results in a translational product having a non-natural substitution of one amino acid for another. In some embodiments, the one or more mutations results in a translational product having a deletion or an insertion of one or more amino acid residues. In some embodiments, the one or more mutations results in a truncated translational product. In some embodiments, the one or more mutations results in translational product that is a fusion with another protein. In some embodiments, the translational product is inactive or has low activity relative to a translational product expressed from a wild-type gene encoding PTPN12.

[0214] In some embodiments, the one or more mutations (e.g., the nonsynonymous mutation) comprises a missense mutation (point mutation that results in a codon that encodes a different amino acid residue compared to the wild-type or non-mutated amino acid sequence). In some embodiments, the missense mutation occurs adjacent to or proximal to the 5' end of the open reading frame of the gene encoding PTPN12.

[0215] In some embodiments, the one or more mutations is a duplication, a deletion, or an insertion in the gene encoding PTPN12. In some embodiments, the duplication, deletion, or insertion results in a frameshift mutation.

[0216] In some embodiments, the expression level and / or activity of PTPN12 is decreased by a mutation in the gene encoding PTPN12. In some embodiments, the mutation is a deletion.

[0217] In some embodiments, a PTPN12 deficiency is measured in a tissue sample (e.g., cancer sample) obtained from the subject using a method of mutational detection analysis (e.g., next generation sequencing). In some embodiments, the tissue sample is a tumor biopsy sample (e.g., fresh or fixed tumor biopsy sample). In some embodiments, the tissue sample is a blood sample comprising circulating tumor DNA.

[0218] In some embodiments, the PTPN12 deficiency results in reduced expression of PTPN12. In some embodiments, reduced expression of PTPN12 in a diseased (e.g., cancerous) tissue sample indicates responsiveness of the disease (e.g., cancer) to a particular therapeutic intervention (e.g., administration of a DSB-inducing agent, or radiotherapy). In some embodiments, reduced expressionof PTPN12 is detected in a tissue sample obtained from a subject having cancer. In some embodiments, reduced expression of PTPN12 in a cancer is identified by comparing the level of expression of PTPN12 in the cancer with the level of expression of PTPN12 in non-diseased tissue. In some embodiments, reduced expression of PTPN12 is a predictive indicator that the subject’s cancer will respond or will likely respond to treatment with a DSB-inducing agent and / or radiotherapy.

[0219] PTPN12 expression may be determined at the protein or mRNA level. PTPN12 expression may determined in a bulk sample of the cancer, or in individual cells of the sample of the cancer (i.e., at the single cell level).

[0220] In some embodiments, the disclosure provides a biomarker comprising a mutation in the PTPN12 gene (e.g., loss of function mutation resulting in a decreased expression level and / or activity of PTPN12) in one or more human cancers. In some embodiments, the presence of a mutation in PTPN12 in a diseased (e.g., cancerous) tissue sample indicates responsiveness of the disease (e.g., cancer) to a particular therapeutic intervention (e.g., administration of a DSB-inducing agent, or radiotherapy). In some embodiments, the presence of a mutation in PTPN12 is detected in a tissue sample obtained from a subject having cancer. In some embodiments, the presence of a mutation in PTPN12 is a predictive indicator that the subject’s cancer will respond or will likely respond to treatment with a DSB-inducing agent and / or radiotherapy. In some embodiments, the presence of a mutation in PTPN12 that results in a loss of function of PTPN12 (e.g., decreased expression level and / or activity) is a predictive indicator that the subject’s cancer will respond or will likely respond to treatment with a DSB-inducing agent and / or radiotherapy.

[0221] A PTPN12-deficient cancer may be a cancer wherein all cells of the cancer comprise a PTPN12 deficiency. Alternatively, a PTPN12-deficient cancer may be a cancer wherein a subset of the cells of the cancer comprise a PTPN12 deficiency. Accordingly, in some embodiments, a PTPN12-deficient cancer may be a cancer wherein at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of cells comprise a PTPN12 deficiency. A deficiency in PTPN12 at the cellular level may be determined by performing single cell sequencing on a sample obtained from the cancer. A deficiency in PTPN12 results in reduced expression of PTPN12 level may be identified, at the cellular level, by performing single cell RNA sequencing on a sample obtained from the cancer.

[0222] The PTPN12 deficiency may arise from the same genetic alteration in all cancer cells. Alternatively, the cancer may comprise cells having a PTPN12 deficiency caused by different genetic alterations.

[0223] The cancer may comprise a PTPN12 deficiency as described herein. The cancer may be a PTPN12-deficient breast cancer, such as a PTPN12 deficient triple negative breast cancer. The cancer may be a PTPN12-deficinet lung cancer, such as PTPN12-deficient non-small cell lung cancer. The cancer may be a PTPN12-deficient colorectal cancer.Treatment of Cancer

[0224] Provided herein are methods of treating cancer comprising administering a DSB-inducing agent and / or radiotherapy to the subject, wherein the subject has a PTPN12-deficient cancer. In some embodiments, the DSB-inducing agent and / or the radiotherapy is administered in combination with a second anti-cancer agent, and / or a PTPN12 inhibitor.

[0225] Administration of the DSB-inducing agent, the radiotherapy, the PTPN12 inhibitor, and / or the additional therapeutic agent is preferably in a "therapeutically effective” or “prophylactically effective” amount, this being sufficient to show therapeutic or prophylactic benefit to the subject. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease / condition and the particular therapy / agent administered. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease / disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0226] Administration of the DSB-inducing agent and / or radiotherapy may be alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. The DSB-inducing agent, the radiotherapy, the PTPN12 inhibitor, and / or the additional therapeutic agent may be administered simultaneously or sequentially. The DSB-inducing agent, the radiotherapy, the PTPN12 inhibitor, and / or the additional therapeutic agent may be administered simultaneously, e.g., as a combined preparation or as separate preparations one administered immediately after the other. The DSB-inducing agent, the radiotherapy, the PTPN12 inhibitor, and / or the additional therapeutic agent may be administered separately and simultaneously, e.g., as separate preparations administered at the same time, or immediately after one another. Alternatively, they may be administered separately and sequentially, where one agent is administered and then the other administered later, after a predetermined time interval. Throughout the course of treatment, the DSB-inducing agent, the radiotherapy, the PTPN12 inhibitor, and / or the additional therapeutic agent may be administered separately on some days and simultaneously or sequentially on other days {e.g., on some days only one agent may be administered, while on other days both agents may be administered).

[0227] Simultaneous administration refers to administration of the DSB-inducing agent, the radiotherapy, the PTPN12 inhibitor, and / or the additional therapeutic agent together, for example as a pharmaceutical composition containing both agents (combined preparation), or immediately after each other and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel. Sequential administration refers to administration of one of the DSB-inducing agent, the radiotherapy,the PTPN12 inhibitor, and / or the additional therapeutic agent followed, after a given time interval, by separate administration of the other agent. The DSB-inducing agent, the PTPN12 inhibitor, and / or the additional therapeutic agent may be administered by one or more routes of administration, e.g.

[0228] parenteral, intravenous injection, oral, subcutaneous, intradermal, intraperitoneal or intratumoral. It is not required that the two agents are administered by the same route, although this is the case in some embodiments. If multiple doses of an agent are administered, it is not required that each dose is administered using the same administration route, although this is the case in some embodiments.

[0229] In some embodiments, the methods comprise additional therapeutic or prophylactic intervention, e.g. for the treatment / prevention of a cancer. In some embodiments, the therapeutic or prophylactic intervention is selected from chemotherapy, immunotherapy, surgery, vaccination and / or hormone therapy. In some embodiments, the therapeutic or prophylactic intervention comprises leukapheresis. In some embodiments the therapeutic or prophylactic intervention comprises a stem cell transplant.

[0230] A treatment may involve administration of more than one drug. A drug may be administered alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated. For example, the chemotherapy may be a co-therapy involving administration of two drugs, one or more of which may be intended to treat the cancer.

[0231] The chemotherapy may be administered by one or more routes of administration, e.g. parenteral, intravenous injection, oral, subcutaneous, intradermal or intratumoral.

[0232] The chemotherapy may be administered according to a treatment regime. The treatment regime may be a pre-determined timetable, plan, scheme or schedule of chemotherapy administration which may be prepared by a physician or medical practitioner and may be tailored to suit the patient requiring treatment. The treatment regime may indicate one or more of: the type of chemotherapy to administer to the patient; the dose of each drug or radiation; the time interval between administrations; the length of each treatment; the number and nature of any treatment holidays, if any etc. For a co-therapy a single treatment regime may be provided which indicates how each drug is to be administered.

[0233] Methods of Biomarker Detection

[0234] A number of methods that are suitable for detecting the presence of a loss of function mutation in a biomarker (e.g. PTPN12), or for detecting the level of gene expression or the level of a protein are known in the art and are suitable for use in conjunction with the methods described herein.

[0235] In some aspects described herein, the presence, absence or level of a protein is determined using immunoassays. An immunoassay is a biochemical test that uses antigen binding molecules such as antibodies or aptamers to detect and measure the amount of a protein in a sample. Examples of immunoassays include immunohistochemistry and ELISA.Immunohistochemistry (IHC) is a laboratory technique that uses antibodies to identify a protein of interest in a sample. For example, immunohistochemical (IHC) slide staining can be utilized to identify proteins in cells of a tissue section and hence is widely used in the study of different types of cells, such as cancerous cells and immune cells in biological tissue. Thus, IHC staining may be used in research to understand the distribution and localization of the differentially-expressed biomarkers in a cancerous tissue.

[0236] Other methods for measuring the amount of a protein include Enzyme Linked Immunonosorbent Assays (ELISA) and western blot. ELISA may involve contacting a sample with a surface that comprises immobilised antigen binding molecules specific to a protein of interest, such that the protein of interest binds to the immobilised antigen binding molecule. A further antigen binding molecule is then applied over the surface, such that it binds to the protein of interest that is bound to the immobilised antigen binding molecule. This antigen binding molecule is linked to an enzyme or radiolabel such that, after washing to remove any unbound antibody, detection of the enzyme or radiolabel is indicative of the presence of the protein of interest in the sample. Western blot involves the separation of the proteins from a sample using electrophoresis, transfer of the separated proteins to a solid support such as a membrane, and subsequent detection of the proteins using a radiolabelled antigen binding molecule such as an antibody.

[0237] Methods for determining the presence of a mutation of interest in a protein include in-situ hybridization (ISH). ISH is commonly used to look for the presence of a genetic abnormality or condition such as amplification of cancer-causing genes specifically in cells that, when viewed under a microscope, morphologically appear to be malignant. In situ hybridization (ISH) employs labeled DNA or RNA probe molecules that are anti-sense to a target gene sequence or transcript to detect or localize targeted nucleic acid target genes within a cell or tissue sample. ISH is performed by exposing a cell or tissue sample immobilized on a glass slide to a labeled nucleic acid probe which is capable of specifically hybridizing to a given target gene in the cell or tissue sample. Several target genes can be simultaneously analyzed by exposing a cell or tissue sample to a plurality of nucleic acid probes that have been labeled with a plurality of different nucleic acid tags. By utilizing labels having different emission wavelengths, simultaneous multicoloured analysis may be performed in a single step on a single target cell or tissue sample.

[0238] Methods for detecting the presence of a mutation of interest in a gene include sequencing technologies, such as Next Generation Sequencing (NGS). Such methods can be used to determine whether or not a sample has a particular sequence of genetic material, such as a particular DNA or mRNA sequence. NGS may involve the analysis of genomic DNA, mRNA, or cDNA. NGS methods include whole exome sequencing (WES), whole genome sequencing (WGS), or sequencing of captured genomic loci (targeted or panel sequencing), or array technologies, such as e.g. SNP arrays, or other molecular counting assays. In some embodiments, methods involve obtaining sequence data by DNA sequencing, and particularly next generation sequencing. In suchembodiments, the sequence data comprises sequencing reads, or information derived therefrom such as the count of the number of sequencing reads that have a particular sequence. When non-digital technologies are used, such as array technology, the sequence data may comprise a signal (e.g. an intensity value) that is indicative of the number of sequences in the sample that have a particular sequence, for example by comparison to an appropriate control. Information such as the count of sequencing reads that have a particular sequence can be derived from sequencing reads by mapping the sequence data to a reference sequence, for example a reference genome, using methods known in the art (such as e.g. Bowtie (Langmead, B., Trapnell, C., Pop, M. et al. Ultrafast and memoryefficient alignment of short DNA sequences to the human genome. Genome Biol 10, R25 (2009)) or BWA (Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009 Jul 15;25(14):1754-60). This may result in aligned sequencing reads, for example in the form of a SAM or BAM file. Thus, sequence data may be associated with a particular genomic location (where the “genomic location” refers to a location in the reference genome to which the sequence data was mapped).

[0239] The skilled person will appreciate that the level of activity of a protein may be determined by a variety of methods known in the art, which may be selected according to the nature of the activity that is to be determined. The activity may be indirectly determined, meaning that a downstream activity of another component of a signalling pathway is determined as an indicator of the level of activity of the protein. The activity may be determined by measuring the direct biochemical reaction catalysed by the protein of interest. For example, as is the case for the phosphatase PTPN12 of the present disclosure, where the protein of interest is a phosphatase, activity can be measured by detecting the level of phosphorylated substrate. For example, the activity of PTPN12 may be measured by detecting the phosphorylation of tyrosine on its substrates, such as MET and / or PDGFRp. In other examples, activity can be measured using a phosphatase assay that detects the dephosphorylation of a sensor substrate. Such reagents are commercially available, and include e.g. PhosphoSens Substrate Systems (Assay Quant Technologies), and EnzChek™ Phosphatase Assay Kit (ThermoFisher Scientific). A high level of PTPN12 activity may be indicated by low levels of phosphorylated substrate. A low level of PTPN12 activity may be indicated by a high level of phosphorylated substrate.

[0240] Methods according to the present invention may be performed, or products may be present, in vitro, ex vivo, or in vivo. The term “in vitro” is intended to encompass experiments with materials, biological substances, cells and / or tissues in laboratory conditions or in culture, whereas the term “in vivo” is intended to encompass experiments and procedures with intact multi-cellular organisms. “Ex vivo” refers to something present or taking place outside an organism, e.g. outside the human or animal body, which may be on tissue (e.g. whole organs) or cells taken from the organism.Methods of Selecting Patients

[0241] Provided herein is a method of selecting a patient having a cancer for treatment with a DSB-inducing agent and / or radiotherapy, the method comprising detecting, in a sample obtained from the cancer, whether the cancer comprises a PTPN12 deficiency, and selecting the patient for treatment when the cancer is PTPN12-deficient.

[0242] Also provided herein is a method of predicting whether a patient having a cancer is likely to respond to a DSB-inducing agent and / or radiotherapy, the method comprising detecting, in a sample obtained from the cancer, whether the cancer comprises a PTPN12 deficiency, wherein when the patient is characterized as having a PTPN12-deficient cancer, the patient is likely to respond to a DSB-inducing agent.

[0243] A PTPN12 deficiency is any reduction in the PTPN12 expression level and / or activity compared to that observed in non-cancerous tissue. The PTPN12 deficiency may be any PTPN12 deficiency as described herein. Accordingly, the step of detecting whether the cancer comprises a PTPN12 deficiency may comprise detecting the expression level of PTPN12, detecting the activity of PTPN12, and / or detecting the presence of a genetic mutation in PTPN12 in a sample obtained from the patient. Detection of PTPN12 may be performed by any method known in the art, including those described herein.

[0244] Methods of Selecting Agents

[0245] Provided herein is a method of selecting an agent for use as a cancer therapeutic, the method comprising: providing one or more DSB-inducing agents; measuring the cell viability of PTPN12-deficient cells treated with the, or each, DSB-inducing agent; measuring the cell viability of PTPN12-wildtype cells treated with the, or each, DSB-inducing agent; and selecting the agent for use as cancer therapeutic agent when the cell viability of the PTPN12-wildtype cells is at least 10% greater than the cell viability of the PTPN12-deficient cells at the same dose of DSB-inducing agent.

[0246] Such methods may be used to determine whether an agent is suitable for use as a DSB-inducing agent in accordance with the invention. In other words, the methods may be used to identify DSB-inducing agents to which PTPN12-deficient cells are sensitive. The agent may be selected for use as a cancer therapeutic when the cell viability of the PTPN12-wildtype cells is at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 35%, at least 40%, at least 45%, or at least 50% greater than the cell viability of the PTPN12-deficient cells at the same dose of DSB-inducing agent.

[0247] The methods may comprise treating cells with the, or each, DSB-inducing agent, and measuring cell viability after a period of time. For example, cell viability may be measured 6 hours, 12 hours, 24hours, 36 hours, 48 hours, or 72 hours after the cells were treated with the, or each, DSB-inducing agent.

[0248] The methods may comprise treating cells with various doses of the, or each, DSB-inducing agent. For example, the cells may be treated with, 2, 3, 4, 5, 6, or more doses of the, or each, DSB-inducing agent. The agent may be selected for use as cancer therapeutic agent when the cell viability of the PTPN12-wildtype cells is at least 10% greater than the cell viability of the PTPN12-deficient cells at the same dose of DSB-inducing agent, for at least one dose tested.

[0249] As used herein, “cell viability” refers to the proportion of live, healthy cells within a population. Cell viability can be measured by determining the proportion of dead cells, or by measuring specific markers of healthy cells, using methods known in the art. Cell killing can be investigated, for example, using any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (2011 ), 9(6):601 -616, hereby incorporated by reference in its entirety. Examples of in vitro assays of cytotoxicity / cell killing assays include release assays such as the51Cr release assay, the lactate dehydrogenase (LDH) release assay, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) release assay, and the calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on the detection of factors released from lysed cells. Cell killing of a given test cell type by a given effector immune cell type can be analyzed e.g. by co-culturing the test cells with the effector immune cells, and measuring the number / proportion of viable / dead {e.g. lysed) test cells after a suitable period of time. Other suitable assays include the xCELLigence real-time cytolytic in vitro potency assay described in Cerignoli et al., PLoS One. (2018) 13(3): e0193498 (hereby incorporated by reference in its entirety). Cell viability may be determined using a label to measure markers of healthy / live cells, and / or markers of dead / dying cells. For example, the label may be a commercially available label selected from: TO-PRO-3 iodide (PI), 7-AAD, SYTOX Nucleic Acid Stain, SYTOX AADvanced Dead Cell Stain Kit. The label may be a membrane integrity dye. Cell viability may be measured by labelling both live and dead cells, wherein the live and dead cells are labelled with different markers (e.g. different colour fluorophores). The label may be measured by flow cytometry, fluorescence microscopy, and / or microplate assay. Cell viability assays are described in Riss TL et al., Cell Viability Assays. 2013 May 1 [Updated 2016 Jul 1]. In: Markossian S, Grossman A, Arkin M, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004, hereby incorporated by reference in its entirety.

[0250] As used herein, “PTPN12-wildtype” refers to a cellular state wherein PTPN12 is expressed at normal levels and at functions with a normal activity (i.e. there is no genetic mutation in the PTPN12 gene that alters its function and / or activity level). A normal expression level of PTPN12 is that observed in healthy, non-cancerous tissue. A normal activity level of PTPN12 is that observed in healthy, non-cancerous tissue.Patients

[0251] The term “cancer patient” as used herein is used interchangeably with “patient”, “subject” or “individual”. The subject to be treated may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be a non-human animal, but is more preferably human. The subject may be male or female. The subject may be a patient. Therapeutic uses may be in humans or animals (veterinary use).

[0252] The subject may be any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the phylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and / or rhesus monkeys (Macaca mulatta) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish. In some embodiments, the subject is human.

[0253] In certain embodiments of the methods disclosed herein, the subject may be selected in accordance with one or more clinical parameters. For example, the one or more clinical parameters may include parameters related to the medical condition (such as disease subtype, for example tumor type, or disease progression status), or biomarker status.

[0254] In some embodiments, the patient is diagnosed with cancer. In some embodiments, the patient has a suspected and / or unconfirmed diagnosis of cancer. In some embodiments, the patient has been determined to be at increased risk of having or developing cancer.

[0255] The invention relates to subjects having a PTPN12-deficient cancer. In some embodiments, the healthy tissue of the subject does not have a PTPN12 deficiency, i.e., a PTPN12-deficiency is only found in the cancer of the subject. In some embodiments, the PTPN12 deficiency is only found in somatic cells. In some embodiments, the PTPN12 deficiency is cause by an alternation to somatic cells.

[0256] Samples

[0257] A “sample” as used herein may be a cell or tissue sample, a biological fluid, an extract (e.g. a DNA extract obtained from a cell, tissue or fluid sample), from which genomic or proteomic material can be obtained for genomic, proteomic, or transcriptomic analysis, such as genomic sequencing (e.g. wholegenome sequencing, or targeted sequencing such as whole exome sequencing or targeted panel sequencing). The sample may be a cell, tissue or biological fluid sample obtained from a subject (e.g. a biopsy). The sample may be any sample comprising DNA, RNA, mRNA, or proteins. In particular, the sample may be a tumor sample, a biological fluid sample containing DNA or cells, a blood sample (including plasma or serum sample), a urine sample, a cervical smear, an ascites fluid sample, or a sample derived therefrom (e.g. after DNA purification). It has been found that urine, ascites fluid and cervical smears contain cells, and so may provide a suitable sample for use in accordance with the present invention. Other sample types suitable for use in accordance with the present disclosure include fine needle aspirates, lymph nodes samples (e.g. aspirates or biopsies), surgical margins, bone marrow or other tissue from a tumor microenvironment, where tumor tissue may be found or expected to be found. The sample may be one which has been freshly obtained from a subject or may be one which has been processed and / or stored prior to genomic / transcriptomic analysis (e.g. frozen, fixed or subjected to one or more purification, enrichment or extraction steps). The sample may be a cell or tissue culture sample. As such, a sample as described herein may refer to any type of sample comprising cells or genomic material derived therefrom, whether from a biological sample obtained from a subject, or from a sample obtained from e.g. a cell line. In embodiments, the sample is a sample obtained from a subject, such as a human subject. The sample is preferably from a mammalian (such as e.g. a mammalian cell sample or a sample from a mammalian subject, such as a cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit or guinea pig), preferably from a human (such as e.g. a human cell sample or a sample from a human subject).

[0258] A sample may be a tissue biopsy, such as a sample of fresh or fresh frozen tissue or a sample of formalin-fixed paraffin embedded (FFPE) tissue, or a sample of biological fluid (sometimes referred to as liquid biopsy). The samples used in methods of the present disclosure are typically samples comprising tumor cells (e.g. a tumor sample or sample comprising circulating tumor cells) or genetic material derived from tumor cells. A sample may be a “mixed” sample comprising cells with different genotypes or genetic material derived therefrom. For example, a sample may be a sample comprising tumor cells and normal cells, or DNA derived therefrom.

[0259] A “tumor sample” refers to a sample that contains tumor cells or genetic material derived therefrom. The tumor may be benign, precancerous or cancerous / malignant. The tumor sample may be a cell or tissue sample (e.g. a biopsy) obtained directly from a tumor. A tumor sample may be a sample that comprises tumor cell or genetic material derived therefrom, that has not been obtained directly from a tumor. For example, a tumor sample may be a sample comprising circulating tumor cells or circulating tumor DNA. Thus, a tumor sample may also be a biological fluid (e.g. a liquid biopsy such as a blood, urine, or cerebrospinal fluid biopsy). A sample comprising a mixture of tumor cells and other cells (or material genetic derived therefrom) may be subject to one or more processing steps, whether prior to or subsequent to the acquisition of sequence data, in order to identify sequence data that is representative of the genetic material from the tumor. For example, a sample comprising cells may be subject to one or more cell purification steps which selectively enrich the sample for tumor cells. Asanother example, a sample of genetic material may be subject to one or more capture and / or size selection steps to selectively enrich the sample for tumor-derived genetic material. Protocols for doing this are known in the art. As another example, sequence data may be subjected to one or more filtering steps to enrich the data for information that relates to tumor-derived genetic material.

[0260] Protocols for doing this are known in the art. In embodiments, the sample is a sample comprising tumor cells.

[0261] A ’’control sample” refers to a sample that contains cells or genetic material derived therefrom, wherein the cells have been obtained from a non-cancerous tissue of the patient. The non-cancerous tissue may be from a tissue that is proximal to the tissue from which the tumor sample has been obtained. The non-cancerous tissue is preferably a tissue of the same type as the tissue from which the tumor sample has been obtained. For example, where the tumor sample has been derived from the colon, the control sample is preferably also derived from non-cancerous tissue of the colon. In some cases, the control sample may have been obtained from the patient at an earlier time point, such as before the patient developed the cancer. However, in most cases, the control sample is obtained at the same or a similar time point to the time point at which the tumor sample is obtained. Preferably, the control sample has been subject to the same or similar processing steps as the tumor sample has been subject to. As such, the sample processing steps set out above with respect to a tumor sample also apply to a control sample. The control sample is normally obtained from the same patient as the tumor sample is obtained from. However, in some cases, the control sample may be obtained from another individual, such as an individual of the same family as the patient, or an individual with similar characteristics of age, gender, race or lifestyle characteristics as the patient. Methods described herein usually relate to samples that have been obtained from a patient. Such methods therefore do not involve surgery of the patient in order to obtain the sample.

[0262] Methods described herein may use a threshold value instead of a control sample. A “threshold value” is a value that has been determined as an indicator of level of expression or activity of the protein in a non-cancerous or “normal” tissue. The threshold value may be determined as the average of a plurality of samples, such as the mean, median or mode of plurality of samples derived from a population of individuals, such as a population of individuals with similar characteristics of age, gender, race or lifestyle characteristics as the patient.

[0263] As such, where a method described herein refers to a control sample, that reference may instead be substituted with reference to a threshold value.

[0264] Kits

[0265] In a further aspect, the present invention also provides a kit for use in the detection and / or treatment of a PTPN12-deficient cancer in a subject.In some embodiments, the kit comprises: (i) one or more reagents for detection of PTPN12 and / or quantification of PTPN12 expression.

[0266] In some embodiments, the kit comprises:

[0267] (i) one or more reagents for detection of PTPN12 and / or quantification of PTPN12 expression; and

[0268] (ii) instructions for treatment of a PTPN12-deficient cancer with one or more DSB- inducing agents and / or radiotherapy.

[0269] Kits according to the present disclosure may comprise a predetermined quantity of articles according to (i), as described in the preceding paragraphs. In some embodiments, articles according to (i) are provided in containers (e.g. in vials or bottles). In some embodiments, the kit may comprise (i) one or more reagents for detection of PTPN12 and / or quantification of PTPN12 expression, as described herein, each provided in a predetermined quantity in separate containers (e.g. in vials or bottles), and (ii) instructions (e.g. a protocol) for treatment of a PTPN12-deficient cancer with one or more DSB-inducing agents and / or radiotherapy. The instructions may take any suitable form, such as for example a product label, insert, information leaflet, or link (e.g. a QR code) to downloadable instructions hosted remotely.

[0270] In some embodiments, detection of PTPN12 refers to the detection of wildtype PTPN12. In some embodiments, the detection PTPN12 refers to the detection of PTPN12 comprising one or more genetic alternations. That is, in some embodiments, detection of PTPN12 refers to detection of mutant forms of PTPN12. The one or more genetic alterations may be one or more genetic mutations. The one or more genetic alterations may be one or more mutations that cause partial or full loss of function of PTPN12, i.e., a loss of function mutation in PTPN12, i.e. a loss of function mutation as described herein. In some embodiments, detection of PTPN12 may refer to the detection of PTPN12 comprising a loss of function mutation.

[0271] The one or more mutations may be homozygous (i.e. present in both copies of the PTPN12 gene in the genome), or heterozygous (present in only one copy of the PTPN12 gene in the genome). In some embodiments, the one or more mutations comprises a nonsynonymous mutation (which results in a change to the encoded protein sequence). The one or more mutation may be a loss of function mutation. The one or more mutations may be adjacent to or proximal to the 5' end of the open reading frame of the gene encoding PTPN12. In some embodiments, detection of PTPN12 refers to detection of PTPN12 comprising one or more mutations adjacent to or proximal to the 5’ end of the open reading frame of the gene encoding PTPN12.In some embodiments, the instructions for treatment of a PTPN12-deficient cancer with one or more DSB-inducing agents and / or radiotherapy are instructions for treating or preventing of a PTPN12-deficient cancer according to a treatment / prevention method as described herein.

[0272] In some embodiments, the kit further comprises at least one DSB-inducing agent. In some embodiments, the at least one DSB-inducing agent is as defined herein (for example, as defined in accordance with the first to third aspects of the present invention).

[0273] In some embodiments, the one or more reagents for detection of PTPN12 and / or quantification of PTPN12 expression comprise:

[0274] at least one polynucleotide probe that selectively hybridizes to a polynucleotide that encodes PTPN12, optionally to a polynucleotide that encodes a mutant form of a PTPN12 gene, such as a loss of function mutant form of PTPN12; and / or

[0275] at least one antigen-binding molecule that selectively binds to a PTPN12 polypeptide, optionally to a mutant form of a PTPN12 polypeptide.

[0276] In some embodiments, the kit further comprises reagents, buffers and / or standards required for execution of a method according to the present disclosure. Kits according to the present disclosure may include instructions for use, e.g. in the form of an instruction booklet or leaflet. The instructions may include a protocol for performing any one or more of the methods described herein.

[0277] In some embodiments, the components of a kit according to the present invention are arranged / packaged in such a way suitable for use in a method as described herein.

[0278] The present invention also provides the use of a kit as described herein in any of the methods described herein. That is, the present invention provides the use of a kit as described herein in a method of selecting patients, in a method of selecting agents, in a method of biomarker detection, or in a method of treatment and / or prevention of cancer, as described herein.

[0279] The manufacture of kits according to the present disclosure preferably follows standard procedures which are known to the person skilled in the art.

[0280] Sequences

[0281]

[0282]

[0283] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof. While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0284] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0285] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0286] Throughout this specification, including the claims which follow, unless the context requires otherwise, the words “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0287] Examples

[0288] Example 1: Genome-wide CRISPR screens identify PTPN12 as a potential NHEJ factor

[0289] To identify genetic determinants of radiosensitivity, the inventors performed genome-wide CRISPR-Cas9 gene knockout screens in non-transformed human RPE-1 TP53 cells treated with X-ray ionising radiation (IR). Cells were exposed to radiation either in a single dose, or in three hyperfractionated doses separate by 8-hour incubation periods (Fig. 1a). Several known radio-protective factors were thus identified, including ATM, members of the HIPPO signalling and CUL3 pathways7, and most components of the core NHEJ complex (Fig. 1b). Interestingly, whereas most genes showed similar results for single and hyperfractionated IR, NHEJ knockouts displayed increased sensitivity to hyperfractionated IR than to single-dose IR (Fig. 1b). Hierarchical clustering of all sensitisers generated several gene groups (Fig. 2a), with gene-ontology analysis of these groups showing that in the hyperfractionated sensitivity group the strongest gene enrichments were for NHEJ-related factors, such as DSB repair via NHEJ and V(D)J recombination (Fig. 2b).

[0290] To classify other genes in the genome-wide screen data that display a similar “NHEJ-like” sensitivityprofile, the inventors used correlation-based clustering, thereby identifying a further 73 genes as potential NHEJ-like factors (Fig. 1c). As well as additional known DNA-damage response (DDR) factors connected to DSB repair, including RNF8 and POLQ, this set also contained protein tyrosine phosphatase non-receptor type 12 (PTPN12) (Fig. 1c-d). Supporting data implying that PTPN12 promotes NHEJ repair, a literature search identified PTPN12 as a negative regulator of HR in a previously-published siRNA based screen dataset (Fig. 2c; but PTPN12 was not specifically mentioned or discussed in the publication)8.

[0291] Example 2: PTPN12 promotes cell survival in response to DNA damage and limits HR

[0292] To explore a role of PTPN12 in the DDR, the inventors generated human U2OS and HAP1 knockout cell lines, and discovered that compared to controls, they were hypersensitive to IR (Fig. 3a, Fig. 4a). To further investigate the mechanisms of PTPN12 in the DDR, the inventors also tested PTPN12K0viability with multiple other genotoxic agents: etoposide, a TOP2 poison that induces double-ended DSBs, camptothecin, a TOP1 poison that causes single-strand breaks (SSBs) resulting in single-ended DSBs through replication fork collapse, and olaparib, a poly(ADP-ribose) polymerase (PARP)inhibitor that causes the accumulation of SSBs and therefore also the formation of single-ended DSBs. PTPN12K0cells were hypersensitive to etoposide, camptothecin and olaparib (Fig. 3a, Fig. 4a), suggesting a broad role(s) for PTPN12 in maintaining genome stability in response to DSB induction. This result was surprising, as NHEJ repair of camptothecin or olaparib induced DNA damage is not thought to impact cell survival, and is actually toxic in certain contexts9.

[0293] Immunofluorescence studies of the DSB marker yH2AX and the single-stranded DNA (ssDNA) marker RPA following 1Gy IR revealed that compared to wild-type controls, PTPN12^° U2OS repaired DSBs slower and with higher levels of RPA foci formation (Fig. 3c-d, 4c-e) suggesting an increased preference for resection-dependent repair, such as HR. To assess HR proficiency directly, the cellbased Traffic Light Reporter (TLR) assay was used10, showing that siRNA mediated depletion of PTPN12 significantly increased HR efficiency (Fig. 4f-g), consistent with data in a previous report8(Fig. 2c). Importantly, no cell cycle defects of PTPN1^° cells in either the U2OS or HAP1 backgrounds were observed (Fig. 5a-c), indicating that the impact of PTPN12 on HR repair efficiency was not related to changes in cell cycle distribution.

[0294] For siRNA mediated depletion of PTPN12, siRNA targeting human PTPN12 were obtained from Horizon Discovery (horizondiscovery.com): ON-TARGETplus Human PTPN12 siRNA (Catalo ID: LQ-008064-00-0005). The siRNAs were used as an equimolar pool.

[0295] Example 3: Actin polymerisation factors are dephosphorylated by PTPN12 following DNA damage

[0296] Since PTPN12 is a tyrosine phosphatase, the inventors conducted phosphoproteomic studies with phospho-tyrosine enrichment in U2OS wild-type and PTPN12K0cells without or following IR. In untreated PTPN12K0cells, several known PTPN12 target proteins11 12had peptides with increased tyrosine phosphorylation compared to wild-type U2OS (Fig. 6a). Furthermore, following IR treatment, four peptides that had reduced tyrosine phosphorylation in WT U2OS (Fig. 7a) but displayed no significant shift in phosphorylation in PTPN12K0cells (Fig. 7b) were identified. Notably, these proteins, N-WASP, FLNB, AGFG1 and DOK1 are all associated with the promotion of actin cytoskeleton structures13 14, as observed through String and gene ontology analyses (Fig. 7c and Fig. 6b).

[0297] Western immunoblot analysis of N-WASP pY256 revealed IR-induced phosphorylation in both HAP1 cells (Fig. 7d) and U2OS cells (Fig. 6c) that was markedly higher with PTPN1 ^° or its siRNA depletion than in controls. Western blot analysis confirmed the mass spectrometry results, finding increased N-WASP pY256 following IR in HAP1 PTPN12K0cells (Fig. 7d) and U2OS cells with siRNA depletion of PTPN12 (Fig. 6c). N-WASP, and its haematopoietic-cell specific homolog WASP, are key regulators of ARP2 / 3 complex mediated actin polymerisation15 16. Previous reports have demonstrated a role for the N-WASP homolog WASP in maintaining genome stability217, but not N-WASP despite it being ubiquitously expressed15. Examination of the homologous phosphorylation to N-WASP Y256 on WASP, Y291 , revealed no change following IR in PTPN12^° cells (Fig. 7d) or with PTPN12 siRNA depletion (Fig. 6c).To investigate if over-activated N-WASP contributed to the defective DSB repair in PTPNI^0cells, immunofluorescence for yH2AX with and without wiskostatin, a potent chemical inhibitor of N-WASP and WASP18, was performed. Again, following I R, significantly higher numbers of yH2AX foci per cell in PTPN12^° cells than in control cells were observed, indicating delayed repair kinetics. By contrast, in the presence of wiskostatin, there was no statistically significant difference in yH2AX foci between WT and PTPN12*0U2OS cells.

[0298] Collectively, these data supported a role for PTPN12 in dephosphorylating actin polymerisation factors to facilitate efficient DNA DSB repair.

[0299] Example 4: PTPN12 loss causes increased actin polymerisation at DNA damage sites Given that IR-dependent targets of PTPN12 are strongly associated with actin polymerisation, the inventors next investigated the impact of PTPN12 on actin polymerisation. For this, immunofluorescence of p-actin combined with harsh pre-extraction was used to quantify levels of cellular filamentous actin (F-actin). Increased intensity of p-actin in PTPN12KO U2OS cells relative to WT cells specifically in the nucleus was observed, with no significant change in the cytoplasm (Fig. 8a-b). Co-staining p-actin with yH2AX allowed the inventors to analyse levels of actin accumulation specifically at DSBs, finding significantly more p-actin positive yH2AX foci with PTPN12KO at multiple time points following IR (Fig. 8a, c). These results were confirmed via chromatin fractionation in HAP1 cells, which revealed increased levels of p-actin on the chromatin following IR which was particularly pronounced in PTPN12KO cells (Fig. 8d). This was further validated in three separate HAP1 PTPN12KO clones (Fig. 9a) and U2OS cells following siRNA depletion of PTPN12 (Fig. 9b).

[0300] Interestingly, increased levels of Ku80 on chromatin in PTPN12KO cells was observed (Fig. 8d, Fig.

[0301] 9a), which was surprising since these cells show a shift towards slower HR repair and reduced NHEJ repair. Given that markers of increased DNA resection in PTPN12KO cells were observed, a process that should reduce Ku80 binding, the inventors investigated whether Ku80 was binding to resected DNA ends or if these two phenotypes are mutually exclusive at DSBs. Thus, the inventors employed the proximity ligation assay (PLA) with antibody probes to RPA70 and Ku80 to determine their colocalization following DNA damage. In these experiments, the inventors treated cells with etoposide as it logistically benefited the PLA approach, improving data consistency, but also to demonstrate that these DSB repair mechanisms are not specific to IR induced DNA damage. The inventors observed that RPA70-Ku80 PLA foci signals were significantly increased in WT U2OS cells following etoposide treatment (Fig. 8e-f), in a manner that was abrogated by siRNA depletion of Ku70 and dependent on the presence of both antibodies (Fig. 8e-f, Fig. 9d-e). Notably, the etoposide-induced increase in RPA70-Ku80 PLA foci was substantially enhanced upon PTPN12 siRNA depletion (Fig. 8e-f, Fig. 9d-e), strongly suggesting that in the absence of PTPN12, there is recruitment of Ku80 to resected DSBs.Together, these data demonstrated that PTPN12 restricts actin accumulation at sites of DNA damage, and in the absence of PTPN12 this leads to aberrant repair processes that could explain the hypersensitivity of PTPN12KO cells to DNA damaging agents.

[0302] Example 5: PTPN12 restricts actin driven DSB movement and clustering

[0303] Previous studies into actin polymerisation at DSBs have characterised a mechanism of actin-driven break movement2-4. To explore this phenotype in the context of PTPN12, live imaging of 53BP1 -GFP to track movement of DSB foci in real time was performed. Depletion of PTPN12 via siRNA resulted in a significant increase in the mean-square displacement (MSD) of 53BP1-GFP foci following etoposide treatment (Fig. 10a-b). Tracking 53BP1-GFP focus movement also allowed the analysis of DSB clustering, finding a significant increase in clustering events in PTPN12 siRNA treated cells compared to control siRNA (Fig. 10c). An additional approach to analysing DSB clustering is via quantification of yH2AX focus morphology, such as size and intensity, as a relative measurement of the number of breaks per focus25. This revealed a significant increase in yH2AX foci size in PTPN12^° compared to WT U2OS cells, and this increase was reduced upon treatment with wiskostatin (Fig. 10d-e).

[0304] Combined with previous data, these findings demonstrated that in the absence of PTPN12, actin accumulation at DSBs increases, promoting DSB movement and clustering of independent DSBs in an N-WASP dependent manner.

[0305] Example 6: DSB clustering drives toxic genomic rearrangements in the absence of PTPN12 Recent work has demonstrated that actin-driven DSB motility and clustering actually drive chromosomal rearrangements34. Given that these phenotypes were observed in PTPN12^° cells and simultaneously observed recruitment of Ku80 to resected DSBs, the inventors hypothesised that the DNA damage sensitivity of PTPN12^° could be due to the toxic buildup of genomic rearrangements.

[0306] To investigate mutagenic outcomes of DNA damage and, specifically, to assess their relative toxicities, the inventors developed a next-generation sequencing (NGS) based approach to quantify mutations in inviable cells. Briefly, following treatment, HAP1 cells were either harvested from the culture plate via trypsinisation or from the culture medium as live or inviable cells respectively (Fig. 11 a). Samples were harvested each day following IR for a total of three days to temporally track mutations, and paired-end NGS was employed to map large-scale structural variants (Fig. 11a). As expected, DNA extracted from inviable cells showed significant degradation (Fig. 12a), consistent with DNA fragmentation during cell death processes19. All DNA samples were then fragmented via sonication to a consistent fragment size ready for NGS library preparation (Fig. 12b).

[0307] Utilising this approach allowed us to quantify IR induced deletions as either 40-1 OObp or structural >100bp events, as well as genomic rearrangements as either intra-chromosomal rearrangements or inter-chromosomal translocations (Fig. 11a). In untreated, live cells, low levels of these variants were detected with limited differences between WT HAP1 and PTPNI^0cells (Fig. 12c). Following IR, only deletions of 40-1 OObp were detectable as induced in live cells, while larger deletions andchromosomal rearrangements were not significantly induced (Fig. 11b, Fig. 12d). However, in inviable cells, there was a strong induction of all variants in both WT and PTPN120cells that peaked two days after IR but continued to three days after IR (Fig. 11b, Fig. 12d). Importantly, inviable samples harvested from untreated cells showed a much lower induction of variants relative to the IR treated samples, indicating that the variants observed were the result of IR induced damage (Fig. 11 b, Fig.

[0308] 12d).

[0309] While all variants were elevated in PTPN120cells relative to WT cells, this enrichment was strongest for inter-chromosomal translocations and particularly at three days after IR treatment (Fig. 11b-c, Fig.

[0310] 12d-e). Although deletions of 40-1 OObp were significantly increased in PTPN12^°, this differential skewed towards larger deletions, e.g. >=70bp (Fig. 11 d, Fig. 12e). Analysis of the locations of inter-chromosomal translocations found limited differences between WT and KO PTPN12^° cells, as rearrangements appeared to be randomly distributed across the genome (Fig. 11 e-f), consistent with the understanding that X-ray induced DSBs are essentially randomly distributed20. To validate these genomic rearrangement findings, metaphase spreads with fluorescent in-situ hybridisation (FISH) of centromere and telomere probes were performed (Fig. 11e). This again found a sharp increase in chromosome aberrations in IR-treated PTPN12^° cells compared to WT HAP1 cells. However, the sequencing data showed a subtle bias towards translocations on chromosome 19 in PTPN120cells (Fig. 11 e-f), which the inventors hypothesised could be related to the high gene-density and transcriptional activity of chromosome 1921’22.

[0311] To assess this the inventors analysed relative levels of the euchromatin marker H3K36me3 and the heterochromatin marker H3k27me3 from previously published HAP1 datasets23, as the ratio between these marks acts as a measure of chromatin compaction (Fig. 12f). Translocation loci in PTPN120cells exhibited a significantly increased H3k36me3 to H3k27me3 ratio (Fig. 12g), suggesting the translocations skew towards open, transcriptionally active regions of the genome. Interestingly, translocations in both WT HAP1 and PTPN12^° cells occurred at loci with very variable chromatin conformation, with the distribution of the H3k36me3 to K3k27me3 ratio at these loci actually being comparable to the ratio in all gene loci across the genome (Fig. 12g), suggesting impacts on a broad range of chromatin conformations. Therefore, although IR-induced translocations in PTPN120cells skewed towards open active chromatin, these translocations still occurred in a wide variety of chromatin contexts, which is again likely due to the essentially random distribution of X-ray induced DSBs20.

[0312] In sum, these data clearly demonstrated a role for PTPN12 in guarding the genome against deletions and genomic rearrangements at DSBs. Taken together with the other data, they suggest that PTPN12 limits translocations between different DSB ends by restricting actin-driven break motility from bringing multiple DSB ends into close proximity.Example 7: Discussion

[0313] PTPN12, also known as PTP-PEST or PTPG1 , is a tyrosine phosphatase with known functions in cell proliferation, development and cytoskeletal regulation24. Here, the inventors describe a novel function of PTPN12 in the maintenance of genome stability by preventing toxic DSB repair outcomes.

[0314] The inventors found that in response to IR treatment, multiple targets regulating actin cytoskeleton polymerisation were phosphorylated at tyrosine residues to promote actin polymerisation at DNA breaks via the Arp2 / 3 complex (Fig. 7a-d, Fig. 6b-c). Actin-polymer formation is central to the recently described mechanism of DSB mobility, in which broken DNA ends move within the nucleus and ultimately cluster together2-5. By dephosphorylating these phospho-tyrosine residues, PTPN12 likely downregulates this process, restricting DSB mobility and clustering (Fig. 7d-g, Fig. 10, Fig. 6c-g). The inventors found this process is critical in maintaining genome stability, as clustering of multiple DSBs is prone to causing genomic rearrangements that are highly toxic. Loss of PTPN12 therefore causes significantly increased DSB movement and clustering (Fig. 10), promoting genomic rearrangements (Fig. 11), primarily inter-chromosomal translocations (Fig. 11b, Fig. 12d), which ultimately increases cellular toxicity to a range of DNA damaging agents. Notably, PTPN12 is downregulated in multiple cancers, including triple-negative breast cancer and colon cancer2526. Whereas this has been attributed to a mechanism for promoting growth signals25, it could also provide a source of carcinogenic genome instability and could be an important biomarker for the use of genotoxic therapeutics strategies, such as radiotherapy.

[0315] Investigations into the toxicity of mutations not only found that PTPN12 loss increases radiation toxicity through increased genomic rearrangements, but also that these rearrangements are a key toxic outcome of DSBs (Fig. 11a-b, Fig. 12d). IR-induced deletions up to 10Obp were found to be tolerated in live cells (Fig. 11 b, Fig. 12e), at least in the short term, whereas IR-induced intra-chromosomal rearrangements and inter-chromosomal translocations were undetected in living cells, but substantially enriched in inviable cells (Fig. 11b, Fig. 12d). This supports the understanding that genomic rearrangements, although infrequent, are a highly deleterious outcome of DSBs and could be a primary driver of DSB toxicity. In addition, translocations were found to occur at a highly diverse set of loci, relatively irrespective of chromatin context (Fig. 12g). Although repair processes and outcomes have been shown to skew significantly based on genomic context, the present data suggest this may be more limited than previously thought1’6’27.References

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[0317] 2. Haarhuis, J. H. I. et al. A Mediator-cohesin axis controls heterochromatin domain formation. Nat. Common. 13, 754 (2022).

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[0323] 8. Zhao, Y., Shapiro, S. S. & Eto, M. F-actin clustering and cell dysmotility induced by the pathological W148R missense mutation of filamin B at the actin-binding domain. Am. J. Physiol. - Cell Physiol.310, C89 (2015).

[0324] 9. Catrina, I. E. et al. The temporally controlled expression of Drongo, the fruit fly homolog of AGFG1 , is achieved in female germline cells via P-bodies and its localization requires functional Rabi 1. RNA Biol. 13, 1117 (2016).

[0325] 10. Jain, N. & Thanabalu, T. Molecular difference between WASP and N-WASP critical for chemotaxis of T-cells towards SDF-1a. Sci. Rep.5, 15031 (2015).

[0326] 11. Prehoda, K. E., Scott, J. A., Mullins, R. D. & Lim, W. A. Integration of Multiple Signals Through Cooperative Regulation of the N-WASP-Arp2 / 3 Complex. Science 290, 801-806 (2000). 12. Schrank, B. R. et al. Nuclear ARP2 / 3 drives DNA break clustering for homology-directed repair. Natore 559, 61-66 (2018).

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Claims

Claims1. A method of treating a cancer in a subject, the method comprising administering a DSB-inducing agent and / or radiotherapy to the subject, wherein the subject has a PTPN12-deficient cancer.

2. A DSB-inducing agent and / or radiotherapy for use in a method of treating a cancer in a subject, wherein the subject has a PTPN12-deficient tumor.

3. Use of a DSB-inducing agent in the manufacture of a medicament for use in a method of treating cancer in a subject, wherein the subject has a PTPN12-deficient cancer.

4. The method according to claim 1 , the DSB-inducing agent and / or radiotherapy for use according to claim 2, or the use according to claim 3, wherein the subject has been determined to have a PTPN12-deficient cancer, optionally wherein the determination that the subject has a PTPN12-deficient cancer has been made by measuring expression of PTPN12 in a tumour sample obtained from the subject.

5. The method according to claim 1 , the DSB-inducing agent and / or radiotherapy according to claim 2, or the use according to claim 3, wherein the method comprises a step of determining that a sample obtained from the subject is PTPN12-deficient.

6. The method according to any one of claims 1 , 4, or 5, the DSB-inducing agent and / or radiotherapy according to any one of claims 2, 4, or 5, or the use according to any one of claims 3 to 5, wherein the DSB-inducing agent induces DSBs either directly, or indirectly through e.g. altering DNA replication fork progression, impeding DNA topoisomerase activities, or by causing the accumulation of single-strand breaks (SSBs)7. The method according to any one of claims 1 or 4 to 6, the DSB-inducing agent and / or radiotherapy according to any of claims 2 or 4 to 6, or the use according to any one of claims 3 to 6, wherein the DSB-inducing agent is a TOP1 chemotherapeutic agent, a TOP2 chemotherapeutic agent, a PARP inhibitor, a platinum based chemotherapeutic agent, or a radioisotope..

8. The method, the DSB-inducing agent and / or radiotherapy, or the use according to claim 7, wherein(vi) the TOP1 chemotherapeutic agent is selected from: camptothecin, camptothecin derivatives, topotecan, irinotecan / SN38, rubitecan, indenoisoquinolines, indotecan, indimitecan, lamellarin D, and belotecan;(vii)the TOP2 chemotherapeutic agent is selected from: etoposide, doxorubicin, epirubicin, daunorubicin, idarubicin, mitoxantrone, amsacrine, teniposide, tafluposide, ICRF-193, genistein, ellipticines, aurintricarboxylic acid, and HU-331 ; or56(viii) the PARP inhibitor is selected from: olaparib, niraparib, rucaparib, veliparib, pamiparib, and talazoparib;(ix) the platinum-based chemotherapeutic agent is selected from: carboplatin, cisplatin and oxaliplatin; or(x) the radioisotope is selected from: Lu177 and Ac225.

9. The method according to any one of claims 1 or 4 to 8, the DSB-inducing agent and / or radiotherapy according to any of claims 2 or 4 to 8, or the use according to any one of claims 3 to 8, wherein the method further comprises administering radiotherapy to the subject, and / or administering a second anti-cancer agent to the subject.

10. The method according to any one of claims 1 or 4 to 9, the DSB-inducing agent and / or radiotherapy according to any of claims 2 or 4 to 9, or the use according to any one of claims 3 to 9, wherein the agent comprises:(a) an antibody-drug conjugate (ADC), optionally wherein the ADC is sacituzumab govitecan (Trodelvy), trastuzumab deruxtecan (Enhertu) and datopotamab deruxtecan (Datroway); or(b) a radioconjugate, optionally wherein the radioconjugate comprises lutetium 177 (e.g. lutetium 177 dotatate (Lutathera), lutetium 177 PMSA (Pluvitco)), or actinium 225.

11. The method, the DSB-inducing agent and / or radiotherapy for use, or the use according to claim 10, wherein the ADC comprises a payload, and wherein the payload comprises a DSB-inducing agent.

12. The method according to any one of claims 1 or 4 to 11 , the DSB-inducing agent and / or radiotherapy according to any of claims 2 or 4 to 11 , or the use according to any one of claims 3 to 11 , wherein the cancer is selected from breast cancer, with a notably exemplification for triplenegative breast cancer (TNBC), non-small cell lung cancer (NSCLC), oesophageal squamous cell carcinoma (OSCC), colon cancer, renal cell carcinoma (RCC) or other cancer type that is found to have PTPN12 deficiency.

13. The method according to any one of claims 1 or 4 to 12, the DSB-inducing agent and / or radiotherapy according to any of claims 2 or 4 to 12, or the use according to any one of claims 3 to 12, wherein the method further comprises administering an inhibitor of PTPN12.

14. The method according to any one of claims 1 or 4 to 13, the DSB-inducing agent and / or radiotherapy according to any of claims 2 or 4 to 13, or the use according to any one of claims 3 to 13, wherein PTPN12 gene expression is downregulated in the cancer.5715. The method according to any one of claims 1 or 4 to 14, the DSB-inducing agent and / or radiotherapy according to any of claims 2 or 4 to 14, or the use according to any one of claims 3 to 14, wherein the cancer does not express functional PTPN12 protein.

16. The method according to any one of claims 1 or 4 to 15, the DSB-inducing agent and / or radiotherapy according to any of claims 2 or 4 to 15, or the use according to any one of claims 3 to 15, wherein gene expression of PTPN12 in the cancer is downregulated by at least 50% compared to PTPN12 expression in normal cells, and / or wherein at least 95% of the cancer cells do not express a functional PTPN12 protein.

17. The method according to any one of claims 1 or 4 to 16, the DSB-inducing agent and / or radiotherapy according to any of claims 2 or 4 to 16, or the use according to any one of claims 3 to 16, wherein the cancer comprises a loss-of-function mutation in PTPN12 and / or a deletion of a PTPN12 gene.

18. The method according to any one of claims 1 or 4 to 17, the DSB-inducing agent and / or radiotherapy according to any of claims 2 or 4 to 17, or the use according to any one of claims 3 to 17, wherein the method comprises administering a PTPN12 inhibitor to the subject.

19. A method of treating a cancer in a subject, the method comprising administering (i) a DSB-inducing agent and / or radiotherapy and (ii) an inhibitor of PTPN12 to the subject.

20. The method according to claim 18 or claim 19, or the method, DSB-inducing agent and / or radiotherapy, or the use according to any one of claims 13 to 17 wherein the PTPN12 inhibitor is an inhibitory RNA, such as an siRNA, an anti-sense oligonucleotide (ASO), an aptamer, a small molecule inhibitor, a nanoparticle or a peptide inhibitor.

21. A method of selecting an agent for use as a cancer therapeutic, the method comprising:(v) providing one or more DSB-inducing agents;(vi) measuring the cell viability of PTPN12-deficient cells treated with the, or each, DSB-inducing agent;(vii) measuring the cell viability of PTPN12-wildtype cells treated with the, or each, DSB-inducing agent; and(viii) selecting the agent for use as cancer therapeutic agent when the cell viability of the PTPN12-wildtype cells is at least 30% greater than the cell viability of the PTPN12-deficient cells at the same dose of DSB-inducing agent.

22. The method according to claim 20, wherein the method comprises selecting the agent for use as a cancer therapeutic for a PTPN12-deficient cancer.

23. A method of selecting a patient having a cancer for treatment with a DSB-inducing agent and / or radiotherapy, the method comprising detecting, in a sample obtained from the cancer, whether the cancer comprises a PTPN12 deficiency, and selecting the patient for treatment when the cancer is PTPN12-deficient.

24. A method of predicting whether a patient having a cancer is likely to respond to a DSB-inducing agent and / or radiotherapy, the method comprising detecting, in a sample obtained from the cancer, whether the cancer comprises a PTPN12 deficiency, wherein when the patient is characterized as having a PTPN12-deficient cancer, the patient is likely to respond to a DSB-inducing agent.

25. A method of treating a cancer in a subject determined to have a PTPN12-deficient cancer, the method comprising:(iii) determining whether a sample obtained from the subject is PTPN12-deficient according to claim 23, or determining whether the patient is likely to respond to a DSB-inducing agent according to claim 24; and(iv) administering a DSB-inducing agent and / or radiotherapy to a patient if the sample is determined to be PTPN12-deficient or if the patient is determined to be likely respond to a DSB-inducing agent and / or radiotherapy.

26. A kit for use in the detection and / or treatment of a PTPN12-deficient cancer in a subject, the kit comprising: (i) one or more reagents for detection of PTPN12 and / or quantification of PTPN12 expression; and (ii) instructions for treatment of a PTPN12-deficient cancer with one or more DSB-inducing agents and / or radiotherapy.

27. The kit of claim 26, wherein the kit further comprises at least one DSB-inducing agent, optionally wherein the at least one DSB-inducing agent is as defined in any one of claims 6 to 11.

28. The kit of claim 26 or claim 27, wherein the one or more reagents for detection of PTPN12 and / or quantification of PTPN12 expression comprise:at least one polynucleotide probe that selectively hybridizes to a polynucleotide that encodes PTPN12, optionally to a polynucleotide that encodes a mutant form of a PTPN12 gene, such as a loss of function mutant form of PTPN12; and / orat least one antigen-binding molecule that selectively binds to a PTPN12 polypeptide, optionally to a mutant form of a PTPN12 polypeptide.59

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