Methods and uses of DDX3y specific antibodies

Abstract

Disclosed herein is a method of detecting a DDX3Y in a sample using antibodies that bind to DDX3Y. Also disclosed herein is a method of detecting a disease in a subject indicated by the presence of DDX3Y. Further disclosed herein is a method of identifying subjects with poor disease prognosis as indicated by the presence of DDX3Y.

Description

METHODS AND USES OF DDX3Y SPECIFIC ANTIBODIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Singapore provisional application no. 10202303463W, filed 08 December 2023, the contents of it being hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of molecular biology. In particular, the present invention relates to methods and uses of antibodies.STATEMENT OF DEPOSIT

[0003] The following hybridoma cell lines were deposited in CellBank Australia with the following accession numbers: CBA20230043 (11DDX3Y-44E3); CBA20230044 (hDDX3Y- 43D1); CBA20230045 (hDDX3Y-41H3E9); CBA20230046 (hDDX3Y-43E5A6); CBA20230047 (hDDX3Y-56A9A5); and CBA20230048 (hDDX3Y-72E8A4).BACKGROUND OF THE INVENTION

[0004] DDX3Y is a DNA / RNA helicase encoded by the DDX3Y gene found on the Y chromosome and its homolog, DDX3X, is encoded by the DDX3X gene found on the X chromosome. Although DDX3Y and DDX3X are homologous, they are believed to have very different physiological roles. DDX3Y is only expressed in significant amounts in the male testes and is believed to have a limited physiological role in sperm maturation, whereas DDX3X is ubiquitously expressed in cells of both male and female tissues and is thought to have multiple physiological roles.

[0005] No commercially available antibodies that specifically bind DDX3Y are known in the art. Due to the homologous nature of the DDX3Y and DDX3X protein sequences, having about 92% homology, all commercially available antibodies that bind DDX3 Y also recognize and crossreact with DDX3X. This lack of specificity is a major drawback in existing antibodies.

[0006] Without specific antibodies, it becomes difficult to study the expressions of DDX3X and DDX3Y independently of each other and it is difficult to study DDX3Y in large numbers ofhuman tissue specimens. Therefore, there is an unmet need for an antibody that is capable of differentiating between DDX3Y and DDX3X.SUMMARY

[0007] Described herein arc methods of using antigen-specific antibodies that can bind DDX3Y and have little to no not cross-react with DDX3X to detect the presence of DDX3Y.

[0008] In one aspect, the present disclosure refers to a method of detecting cancer in a subject, the method comprising contacting a sample obtained from the subject with an antibody capable of binding specifically to DDX3Y, wherein increased binding of the antibody to DDX3Y in the sample indicates the presence of cancer in the subject.

[0009] In another aspect, the present disclosure refers to a method of identifying subjects with poor disease prognosis, the method comprising contacting a sample obtained from the subject with an antibody capable of binding specifically to DDX3 Y, wherein increased binding of the antibody to DDX3 Y in the sample identifies the subject of having poor disease prognosis.

[0010] In yet another aspect, the present disclosure refers to a method of identifying DDX3Y in a sample, the method comprising applying an antibody capable of binding specifically to DDX3Y to the sample, detecting binding of the antibody in the sample, thereby identifying DDX3Y in the sample.

[0011] In a further aspect, the present disclosure refers to a semi-quantitative method of identifying DDX3Y in a sample, the method comprising applying an antibody capable of binding specifically to DDX3Y to the sample, detecting binding of the antibody in the sample, thereby identifying DDX3Y in the sample.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0013] Fig, 1 shows immunohistochemistry (IHC) analysis of tumour tissues collected from 159 prostate adenocarcinoma cases using anti-DDX3Y. Out of these 159 tumour samples, 48 (30%) had high levels of DDX3Y expression (29% with ++ and 1% with +++). Tumours with Gleason score GS9 (n=7) had significantly higher IHC score compared to tumours with GS6 (n=40) or GS7 (n=102). Statistical comparisons between groups in terms of p-values, as analysed by two-way ANOVA, are indicated.

[0014] Fig. 2 shows results indicating that high DDX3Y expression correlates with worse overall survival (OS) in prostate cancer patients (n=159).

[0015] Fig. 3 shows that DDX3 Y is significantly highly expressed in prostate cancer cell lines DU145, 22Rvl and LNCaP, compared to the non-tumorigenic cell line RWPE-1. In other words, the data shows that the DDX3Y protein, but not DDX3X, is aberrantly expressed in prostate cancer cell lines, as analysed by Western blotting. An immortalized non-tumorigenic cell line RWPE-1 and the female-derived cell line HeLa were used as comparative controls. Statistical comparisons between groups in terms of p values, as analysed by ordinary one-way ANOVA, are indicated.

[0016] Fig. 4 shows Western blot analysis demonstrating the presence of binding to and therefore presence of DDX3Y. A) Western blot showing DDX3Y expression in the male NK / TCL cell lines, NKS1, NK92, and KAI-3. No specific staining is seen in female cell lines. Male cell lines SNK6, MEC04, and YT are also negative. B) Western blots for DDX3Y in aggressive B-cell lymphoma and multiple myeloma cell lines. Expression is noted in cell lines HT, MC-116, and U266. No expression is seen in female cell lines. Male cell lines SC-1, KMS-18, and RPMI 8226 showed no expression. C) Western blot showing DDX3Y expression in male acute myeloid leukaemia cell lines. Positive staining is noted in cell lines KG1, HEL, M0LM13, MV4-11, and M0LM14, but not Thpl.DEFINITIONS

[0017] As used herein, the term “antibody” (Ab) or “antibodies” refers to an immunoglobulin molecule or fragments of an immunoglobulin molecule that bind to a target antigen. Antibodies as described herein include within its scope molecules such as, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multi-specific antibodies (for example, including but not limited to, bispecific or tri-specific antibodies), and fragments and derivatives thereof (for example, including but not limited to a variable fragment (Fv), a single chain variable fragment (scFv), an antigen-binding fragment (Fab), an antigen-binding fragment arm 1 (Fabl), a monovalent antigen-binding fragment containing one Fab fragment and a portion of the hinge region (F(ab’)), a single chain antigen-binding fragment (scFab), a bivalent antigenbinding fragment including two Fab fragments linked by a disulfide bridge at the hinge region (F(ab’)2), an antigen-binding fragment aim 2 (Fab2), diabodies, triabodies, single-chain antibody molecules (for example, including but not limited to scFv), tctrabodics, linear antibodies, a single chain variable fragment conjugated to a constant fragment (scFv-Fc), minibodies, and single domain antibodies (for example, including but not limited to, VuH)). Antibodies as disclosedherein can specifically bind to the relevant targets disclosed herein such as DDX3Y (for example, including but not limited to, human DDX3Y).

[0018] As used herein, the term “anti-DDX3 Y antibody” or “DDX3 Y -specific antibody” refers to an antibody that binds DDX3Y.

[0019] As used herein, the term “specific” or “specifically” in reference to antibody binding, refers to the situation in which an antibody will not show any statistically significant binding to molecules other than its target. For example, in the context of the present disclosure, antibodies that specifically bind DDX3Y are capable of binding to DDX3Y and do not substantially bind molecules other than DDX3Y (for example, including but not limited to, DDX3X).

[0020] As used herein, the term “does not substantially bind”, in reference to an antibody, refers to the absence of binding of an antibody disclosed herein to a particular antigen, including but not limited to absence of off-site binding or unintended binding of the antibody to targets other than the intended target. The term “substantially” refers to the fact that an antibody will bind mainly to its target, but that some degree of off-site binding would be acceptable.

[0021] As used herein, the term “companion diagnostic” refers to a clinical test performed on a patient (or patient sample) that supports or further characterises an already existing / known disease state or pathological condition or is done in conjunction (either simultaneously or separately) with other methods know in the ail for identifying a disease or pathological condition. In one example, where the diagnosis of cancer is already known, a companion diagnostic would provide information on what type of genetic mutations may aid in identifying potential treatments for said cancer or would provide information of the severity of the cancer in question. As disclosed herein as an example, the companion diagnostic is a method of detecting DDX3 Y in a sample from a patient, whereby the patient may already have received a diagnosis of having cancer. In another example, the patient may be undergoing a method of detecting DDX3 Y in a sample as a companion diagnostic, whereby the diagnosis of cancer has not been received yet.

[0022] As used herein, the term “sample” refers to biological materials taken from a subject. In one example, the sample is a tissue sample or a cell sample. In another example, the sample is a protein sample. In yet another example, the sample is a tissue lysate or a cell lysate.DETAILED DESCRIPTION

[0023] DDX3Y and DDX3X arc DNA / RNA helicases with high protein sequence homology, showing about 92% homology to each other. In spite of their homology, DDX3Y and DDX3X have different physiological roles and different expression profiles. However, studying theirphysiological roles and expression profiles individually has been difficult because hitherto, no commercially available antibodies specific for DDX3Y had been available. Due to their high protein sequence homologies, all commercially available antibodies that bind DDX3Y cross-react with DDX3X. Accordingly, it had not been possible previously to comprehensively and reliably study the physiological role and expression profile of DDX3Y independently of DDX3X.

[0024] It had previously been shown that DDX3Y is expressed in certain cancers. Therefore, the present application also describes the use of DDX3Y in diagnosis or detection of certain cancers using the described antibody.

[0025] Before development of the monoclonal antibody that is specific for DDX3Y over its highly homologous paralog DDX3X, there had been no way to reliably identify tumours in which DDX3Y is upregulated.

[0026] DDX3Y-specific antibodies described herein have been used to study DDX3Y expression in cancer and non-cancerous tissue. These studies demonstrate the differential expression of DDX3Y in prostatic adenocarcinoma and allow clinicians to stratify patients who may be responsive to therapy (e.g., by using an antibody-based companion diagnostic).

[0027] In the present disclosure, the differential expression of DDX3Y in prostatic adenocarcinoma and its correlation with various clinicopathologic parameters in a cohort of 159 patients is shown using a DDX3Y-specific antibody (also referred to herein as an anti-DDX3Y antibody). The data presented herein shows that specifically DDX3Y is upregulated in about 30% of prostatic adenocarcinomas (Fig. 1 ), as well as being correlated with poor prognosis (Fig. 2).

[0028] Specifically, 48 out of 159 cases (30%) had high level of DDX3Y expression (29% 2+, 1% 3+) (Fig. 1). Median overall survival in DDX3Y-high tumours was 65.8 months, as compared to 101.6 months in DDX3Y-low tumours. DDX3Y-high tumours also correlated with worse overall survival (p=0.0332) (Fig. 2).

[0029] The experimental data provided herein demonstrates that high levels of DDX3Y expression in subjects with prostatic adenocarcinoma correlate with worse disease outcome (Fig. 2). Conversely, it was shown that subjects with low levels of DDX3Y expression had a better disease outcome (Fig. 2). Thus, DDX3Y is shown to be a prognostic marker in guiding the risk stratification and treatment of subjects with prostatic adenocarcinoma.

[0030] Together, these findings show the clinical application of a companion diagnostic using a DDX3Y-spccific antibody, for example, in prostate cancer (also referred to as prostatic adenocarcinoma), lymphomas, and other cancers.

[0031] Thus, disclosed herein, in one example, is the use of an anti-DDX3Y antibody in detecting cancer. In one example, the method using an anti-DDX3Y antibody is used as a companion diagnostic. In one example, disclosed herein is a method of using the anti-DDX3Y antibody to detect the presence of DDX3Y in a sample. In another example, disclosed herein is a method of using an anti-DDX3Y antibody to detect the level of DDX3 Y present in a sample.

[0032] In one example, the method disclosed herein comprises applying an antibody capable of binding specifically to DDX3 Y to the sample, detecting binding of the antibody to the sample, thereby identifying DDX3Y in the sample. In another example, a semi-quantitative method of identifying DDX3 Y in a sample, the method comprising applying an antibody capable of binding specifically to DDX3Y to the sample, detecting binding of the antibody in the sample, thereby identifying DDX3Y in the sample, is disclosed.

[0033] The methods as disclosed herein can be, but are not limited to, qualitative, semi- qualitative, and quantitative methods. As used herein, the term “qualitative methods” refers to methods to identify the presence or absence of a constituent element in a sample. In one example, the qualitative method is to detect the presence or absence of DDX3 Y in a sample. As used herein, the term “semi-quantitative methods” refers to methods to estimate an approximate concentration of a constituent element in a sample. In one example, the semi-quantitative method is to score the approximate amount of staining for DDX3 Y in a sample. In another example, the semi-quantitative method refers to a score of (minus),and “+++”, each of which reflects an approximate percentage of cell staining observed in a sample. As used herein, the term “quantitative methods” refers to methods to accurately determine the concentration of a constituent element in a sample. In one example, the quantitative method is to measure the concentration of DDX3Y in a sample.

[0034] The antibody disclosed herein can be used for qualitative, semi-quantitative, and quantitative methods, as well as analytical methods, such as, but not limited to, Western immunoblotting, immunohistochemistry (IHC), immunoprecipitation (IP), immunostaining for immunofluorescence microscopy, enzyme-linked immunosorbent assays (ELISAs), enzyme- linked immunospots (ELISPOTs), immunocytochemistry (ICC), flow cytometry, and fluorescence-activated cell sorting (FACS). The methods disclosed herein can be performed by a person skilled in the art using, for example, standard lab protocols. In one example, the antibody disclosed herein is used in immunohistochemical and / or immunofluorescence studies of DDX3Y expression in diseased tissue or a diseased sample. In another example, the method is an ex vivo or in vitro method.

[0035] The methods described herein comprise measuring DDX3Y in, for example but not limited to, two or more distinct samples. In one example, DDX3Y is measured in a sample and compared to DDX3Y measured in another sample, for example, a reference sample. In another example, DDX3Y is measured in a patient-derived sample and is compared to the measurement of DDX3Y in a reference sample. In yet another example, the level of DDX3Y measured using a DDX3Y-specific antibody in a diseased sample is increased compared to binding of the same antibody to a reference sample. In another example, the increase in binding is as compared to binding of the same antibody to a non-diseased sample. In yet another example, the comparison is based on a sample obtained from a healthy subject. In other words, the term “reference sample”, as used herein is used to refer to samples that have been obtained from, for example, healthy subjects, non-diseased subjects, laboratory reference samples, negative controls, and combinations thereof.

[0036] In one example, the antibody disclosed herein is used in immunohistochemical and / or immunofluorescence studies of DDX3Y expression in diseased tissue or a diseased sample.

[0037] Disclosed herein are uses for DDX3Y-specific antibodies. In one example, the use is to provide a subject or patient with information about their possible prognosis following diagnosis with a disease or disorder. In one example, the disease or disorder is cancer. In another example, the cancer is prostate cancer. In another example, the disease or disorder is a DDX3Y-related disease or disorder. In a further example, the disease or disorder is a DDX3Y-rclatcd cancer. In yet another example, the disease or disorder is prostatic adenocarcinoma. In yet another example, the disease or disorder is a DDX3Y-related prostatic adenocarcinoma.

[0038] In another example, the method of detecting cancer as disclosed herein is used as a companion diagnostic method in conjunction with existing methods in a subject. Examples of such existing methods of detecting cancer include, but are not limited to, methods such as immunohistochemistry, biomarker analysis, biopsy analysis and similar methods employed by a person skilled in the art. Thus, in one example, the method of detecting cancer in a subject as disclosed herein is performed as a companion diagnostic with an existing method of detecting cancer.

[0039] In another example, there is disclosed a method of identifying subjects with poor disease prognosis. The method can comprise the steps of contacting a sample obtained from the subject with an antibody capable of binding specifically to DDX3Y and determining the amount of DDX3Y present in the sample. In one example, an increased binding of the antibody to DDX3Y in the sample identifies the subject of having poor disease prognosis.

[0040] In another example, an increase in the amount of DDX3Y in a sample (detected using any of the methods disclosed herein) is indicative of a poor prognosis. That is to say, there is an inverse correlation present between the amount or concentration of DDX3Y in a sample and the prognosis of the subject from which the sample had been obtained. By way of an example, a high amount of DDX3Y detected in a sample means that the subject is less likely to survive and recover from a disease or disorder, including but not limited to cancer. Basis for comparison of the levels of DDX3 Y as disclosed herein can be the amount of DDX3Y in a healthy subject or a subject that does not have the disease or disorder.

[0041] In the methods disclosed herein, the subject can be a subject or patient previously identified as having cancer. In some examples, the subject had been previously identified as having cancer. In some examples, the subject had not been previously identified as having cancer. This is, for example, when the methods used herein are used in conjunction with existing tools of initial diagnosis. Thus, the data disclosed herein demonstrates that the absence of DDX3Y in normal prostate in conjunction with presence of DDX3Y in prostate cancer (PCa) illustrates the role of DDX3Y in promoting malignancy and validating the prognostic value of DDX3Y.

[0042] In one example, the anti-DDX3Y antibody used in the methods disclosed herein is a polyclonal antibody. In another example, the anti-DDX3 Y antibody used in the methods disclosed herein is a monoclonal antibody. In one example, the antibody disclosed herein is an antibody obtained from a hybridoma cell line with a deposition number of CBA20230043, CBA20230044, CBA20230045, CBA20230046, CBA20230047, or CBA20230048. In another example, the antibody is an antigen- specific antibody that binds specifically to DDX3Y in the presence of DDX3X. This means that there is minimal to no cross-binding of the DDX3Y-specific antibody to DDX3X. In another example, the DDX3Y-specific antibody is an antibody obtained from the hybridoma cell line with the deposition number CBA20230043 (44E3), or CBA20230044 (43D1). In one example, the DDX3Y-specific antibody is an antibody obtained from the hybridoma cell line 34G11. In another example, the DDX3Y- specific antibody is an antibody comprising SEQ ID Nos: 1 to 6 (34Gl l).

[0043] In one example, the disease disclosed herein is cancer. In another example, the cancer is lymphoma, leukaemia or prostate cancer. In another example, the lymphoma is B-cell lymphoma, diffuse large B-cell lymphoma, or non-Hodgkins’ s lymphoma.

[0044] In one example, the subject identified to have cancer is to be treated or is treated with an anti-cancer drug.

[0045] In one example, the anti-cancer drug as disclosed herein for treating prostate cancer can be, but is not limited to, abiraterone acetate, Akeega (niraparib tosylate monohydrate and abiraterone acetate), apalutamide, bicalutamide, cabazitaxel, Casodex (bicalutamide), darolutamide, degarelix, docetaxel, Eligard (leuprolide acetate), enzalutamide, Erleada (apalutamide), Firmagon (degarelix), flutamidc, goscrclin acetate, Jcvtana (cabazitaxel), leuprolide acetate, Lupron Depot (leuprolide acetate), lutetium Lu177vipivotide tetraxetan, Lynparza (olaparib), mitoxantrone hydrochloride, Nilandron (nilutamide), nilutamide, niraparib tosylate monohydrate and abiraterone acetate, Nubeqa (darolutamide), olaparib, Orgovyx (relugolix), Pluvicto (lutetium Lu177vipivotide tetraxetan), Provenge (sipuleucel-T), radium 223 dichloride, relugolix, Rubraca (rucaparib camsylate), rucaparib camsylate, sipuleucel-T, Taxotere (docetaxel), talazoparib tosylate, Talzenna (talazoparib tosylate), Xofigo (radium 223 dichloride), Xtandi (enzalutamide), Yonsa (abiraterone acetate), Zoladex (goserelin acetate), Zytiga (abiraterone acetate), and combinations thereof.

[0046] In another example, the anti-cancer drug as disclosed herein for treating a lymphoma or leukaemia can be an anticancer drug that is suitable for treating, for example, B-cell non-Hodgkin lymphoma. Examples of such anticancer drugs are, but is not limited to, acalabrutinib, Adcetris (brentuximab vedotin), Aliqopa (copanlisib hydrochloride), Arranon (nelarabine), asparaginase erwinia chrysanthemi (recombinant)-rywn, axicabtagene ciloleucel, Beleodaq (belinostat), bclinostat, bcndamustinc hydrochloride, Bcndcka (bcndamustinc hydrochloride), BiCNU (carmustine), bleomycin sulfate, bortezomib, brentuximab vedotin, brexucabtagene autoleucel, Breyanzi (lisocabtagene maraleucel), Brukinsa (zanubrutinib), Calquence (acalabrutinib), carmustine, chlorambucil, Columvi (glofitamab-gxbm), copanlisib hydrochloride, crizotinib, cyclophosphamide, dacarbazine, denileukin diftitox, dexamethasone, doxorubicin hydrochloride, epcoritamab-bysp, Epkinly (epcoritamab-bysp), Folotyn (pralatrexate), Gazyva (obinutuzumab), glofitamab-gxbm, ibritumomab tiuxetan, ibrutinib, Imbruvica (ibrutinib), Intron A (recombinant interferon alfa-2b), Istodax (romidepsin), Jaypirca (pirtobrutinib), Keytruda (pembrolizumab), Kymriah (tisagenlecleucel), lenalidomide, Leukeran (chlorambucil), lisocabtagene maraleucel, loncastuximab tesirine-lpyl, lomustine, Lunsumio (mosunetuzumab-axgb), Matulane (procarbazine hydrochloride), methotrexate sodium, mogamulizumab-kpkc, Monjuvi (tafasitamab-cxix), mosunetuzumab-axgb, Mozobil (plerixafor), nelarabine, nivolumab, Opdivo (nivolumab), Obinutuzumab, Ontak (dcnilcukin diftitox), Pcmazyrc (pcmigatinib), pembrolizumab, pemigatinib, pirtobrutinib, plerixafor, polatuzumab vedotin-piiq, Polivy (polatuzumab vedotin-piiq), Poteligeo (mogamulizumab-kpkc), pralatrexate, prednisone,procarbazine hydrochloride, recombinant interferon alfa-2b, Revlimid (lenalidomide), Riabni (rituximab), Rituxan (rituximab), Rituxan Hycela (rituximab and hyaluronidase human), rituximab, rituximab and hyaluronidase human, romidepsin, Ruxience (rituximab), Rylaze (asparaginase erwinia chrysanthemi [recombinant] -rywn), Selinexor, tafasitamab-cxix, tazcmctostat hydrobromidc, Tazvcrik (tazcmctostat hydrobromidc), Tccartus (brcxucabtagcnc autoleucel), tisagenlecleucel, Treanda (bendamustine hydrochloride), Trexall (methotrexate sodium), Truxima (rituximab), Velcade (bortezomib), Venclexta (venetoclax), venetoclax, vinblastine sulfate, vincristine sulfate, vorinostat, Xalkori (crizotinib), Xpovio (selinexor), Yescarta (axicabtagene ciloleucel), zanubrutinib, Zevalin (ibritumomab tiuxetan), Zolinza (vorinostat), Zynlonta (loncastuximab tesirine-lpyl), CHOP (cyclophosphamide, doxorubicin hydrochloride [Hydroxydaunomycin], vincristine sulfate [Oncovin], prednisone), CVP (Cyclophosphamide, vincristine sulfate, prednisone), EPOCH (etoposide phosphate, prednisone, vincristine sulfate [Oncovin], cyclophosphamide, doxorubicin hydrochloride [Hydroxydaunomycin]), Hyper-CVAD (cyclophosphamide, vincristine sulfate, doxorubicin hydrochloride [Adriamycin], dexamethasone), R-CHOP (rituximab, cyclophosphamide, doxorubicin hydrochloride [Hydroxydaunomycin], vincristine sulfate [Oncovin], prednisone), R- CVP (rituximab, cyclophosphamide, vincristine sulfate, prednisone), R-EPOCH (rituximab, etoposide phosphate, prednisone, vincristine sulfate [Oncovin], cyclophosphamide, doxorubicin hydrochloride [Hydroxydaunomycin]), R-ICE (rituximab, ifosfamidc, carboplatin, etoposide phosphate), ABVD (doxorubicin hydrochloride, bleomycin, vinblastine sulfate, and dacarbazine), ABVE (doxorubicin hydrochloride, bleomycin, vincristine sulfate, and etoposide phosphate), ABVE-PC (doxorubicin hydrochloride, bleomycin, vincristine sulfate, etoposide phosphate, prednisone, and cyclophosphamide), BEACOPP (bleomycin, etoposide phosphate, doxorubicin hydrochloride [Adriamycin], cyclophosphamide, vincristine sulfate [Oncovin], procarbazine hydrochloride, and prednisone), COPDAC (cyclophosphamide, vincristine sulfate [Oncovin], prednisone, and dacarbazine), COPP (cyclophosphamide, vincristine sulfate [Oncovin], procarbazine hydrochloride, and prednisone), COPP-ABV (cyclophosphamide, vincristine sulfate [Oncovin], procarbazine hydrochloride, prednisone, doxorubicin hydrochloride [Adriamycin], bleomycin, vinblastine sulfate), ICE (ifosfatnide, carboplatin, etoposide phosphate), MOPP (mechlorethamine hydrochloride, vincristine sulfate [Oncovin,], procarbazine hydrochloride, prednisone), OEPA (vincristine sulfate [Oncovin], etoposide phosphate, prednisone, doxorubicin hydrochloride [Adriamycin]), OPPA (vincristine sulfate [Oncovin], procarbazine hydrochloride, prednisone, doxorubicin hydrochloride [Adriamycin]), STANFORD V (mechlorethaminehydrochloride, doxorubicin hydrochloride, vinblastine sulfate, vincristine sulfate, bleomycin, etoposide phosphate, prednisone), VAMP (vincristine sulfate, doxorubicin hydrochloride [Adriamycin], methotrexate, prednisone), and combinations thereof.

[0047] In some examples, treatment of the cancer described herein can also include chemotherapy and / or resection with administration of an anti-cancer drug. In another example, the treatment is a combination of an anti-cancer drug and chemotherapy. A person skilled in the art would appreciate that once a subject is identified as having a cancer, the appropriate course of treatment would be decided by the skilled medical professional.

[0048] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations arc considered to be within the scope of this invention.

[0049] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof.

[0050] As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically, + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0051] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., aswell as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0052] Certain embodiments may also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0053] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0054] Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.EXPERIMENTAL SECTIONAnalysis ofDDX3Y expression in prostatic adenocarcinoma

[0055] To analyse DDX3Y expression in prostatic adenocarcinoma, immunohistochemical (IHC) staining was performed using a tissue microarray (TMA) with 159 anonymized prostate adenocarcinoma tissue samples. These samples were stained with the DDX3Y- specific monoclonal antibody. Clinical follow-up information was available for 132 cases (83%). Gleason scoring (GS), tumour staging, and survival data were collected from patient files. Staining intensity and the proportion of tumour cells stained were scored independently by two pathologists and the results were compared. The DDX3Y expression based on the IHC staining was scored semi- quantitatively as follows: Score 0 (-) = no discernible specific staining; Score 1 (+) = faint staining in any number of tumour cells; Score 2 (++) = weak-to-moderate cytoplasmic granular staining in more than 50% of tumour cells and Score 3 (+++) = strong cytoplasmic granular staining in more than 50% of tumour cells (Fig. 1). Cases with scores of 0 or 1 were grouped together as “DDX3Y low” expression and the remainder as “DDX3Y high” expression. The relationship between clinicopathological parameters and the level of DDX3Y IHC expression was tested using a Chi- squared (x2) test. The DDX3Y expression was subsequently compared against patient clinical datato correlate the expression with survival or prognosis. Overall survival (OS) outcomes were estimated using the Kaplan-Meier analysis and compared between DDX3 Y low and DDX3Y high groups with the log-rank statistics (Fig. 2).Determination of endogenous expression levels of the DDX3Y protein in prostatic adenocarcinoma cells

[0056] For this, three different prostate cancer cell lines were used: DU145 (ATCC #HTB-81; androgen-insensitive), 22Rvl (ATCC #CRL-2505; androgen-sensitive), and LNCaP (ATCC #CRL-1740; androgen-sensitive). An immortalized non-tumorigenic cell line RWPE-1 and the female-derived cell line HeLa were used as comparative controls. DU145 and 22Rvl cells were cultured in MEMa medium whereas LNCaP cells were cultured in RPMI 1640 medium. Both media were supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. Relative expression of DDX3Y in the three prostate cancer cell lines and the two comparative control cell lines was determined by Western blotting (Fig. 3).Tissue microarray methodology - Origin of samples (Patients and Tumours)

[0057] The study was approved by the SingHealth Centralized Institutional Review Board, including a waiver for informed consent. The cohort comprised 159 prostate cancer cases diagnosed at the Department of Anatomical Pathology, Singapore General Hospital, from January 2005 to December 2008. Clinicopathological data including age, tumour size, Gleason score and the extent of tumour (pT stage) were retrieved from case records and pathological reports, and de- identified by Health Services Research Unit (HSRU).Tissue microarray (TMA ) construction

[0058] Tissue microarrays (TMAs) were constructed from radical prostatectomy specimens. Areas of interest were reviewed and circled by a pathologist on Hematoxylin and eosin (H&E)- stained slides, and corresponding areas were marked on the donor formalin-fixed paraffin- embedded (FFPE) tissue blocks and punched with the Manual Tissue Arrayer, Model MTA-1 (Beecher Instruments, Inc., Sun Prairie, Wl, USA) using a 1.0 mm punch. Two representative cores per case were transferred to two recipient blocks respectively, with at least one tonsil core as orientation marker.Staining

[0059] Immunohistochemistry was performed on 4 pm thick sections cut from tissue microarray blocks using the DDX3Y-1 antibody, clone 44E3 at 1:500 dilution and incubation at room temperature for 20 minutes. An automated staining platform (Leica Bond III) with a polymcr-bascd detection kit (BOND Polymer Refine Detection kit, cat no: DS9800) was used, and antigen retrieval was performed using the manufacturer’s Epitope Retrieval Solution 2(H2) at 100°C for 20 minutes.Scoring

[0060] Staining intensity and the proportion of tumour cells stained were scored independently by two pathologists and the results were compared. The finding that yielded the highest scores was used in the analysis. Score 1 was defined as faint, barely perceptible staining, in any amount of tumour cells. Score 2 was defined as weak-to-moderate, cytoplasmic granular staining in more than 50% of tumour cells and score 3 was defined as strong cytoplasmic granular staining in more than 50% of tumour cells. Cases with score 0 and 1 were grouped together as DDX3Y low expression and the remainder as DDX3Y higher expression.Analysis ofDDX3Y expression in haematological malignancies

[0061] Expression of DDX3Y in NK / T-ccll lymphoma cell lines was determined by Western blotting (Fig. 4A). For this, six different male NK / T-cell lymphoma cell lines were used: NKS1 , SNK6 (RRID:CVCL_A673), NK92 (ATCC #CRL-2407), KAI-3 (RRID:CVCL_2969), MEC04 (Coppo et al., 2009) and YT (RR1D:CVCL_1797), NK92 (ATCC); and five different female NK / T-cell lymphoma cell lines were used: KHYG1 (RRID:CVCL_2976), SNK1 (RRID:CVCL_A671), HANK1 (RRID:CVCL_8226), NKYS (RRID:CVCL_8461), and SNT8 (RRID:CVCL_A677) (Fig. 4A).

[0062] Expression of DDX3Y in B-cell lymphoma cell lines was determined by Western blotting (Fig. 4B). For this, four different male B-cell lymphoma cell lines were used: HT (ATCC #CRL-2260), MC-116 (RRID:CVCL_1399), SC-1 (RRID:CVCL_1888), and Raji (ATCC #CCL- 86); and one female B-cell lymphoma cell line was used: U2932 (RRID:CVCL_1896)(Fig. 4B).

[0063] Expression of DDX3Y in multiple myeloma cell lines was determined by Western blotting (Fig. 4B). For this, three different male multiple myeloma cell lines were used: KMS-18 (RRID:CVCL_A637), U266 (ATCC #TIB-196), and RPMI 8226 (RRID:CVCL_0014); and one female multiple myeloma cell line was used: NCI H929 (ATCC #CRL-358O)(Fig. 4B).SEQUENCE LISTINGBUDAPEST TREATY DEPOSITS »:

Claims

CLAIMS1. A method of detecting cancer in a subject, the method comprising contacting a sample obtained from the subject with an antibody capable of binding specifically to DDX3Y, wherein increased binding of the antibody to DDX3Y in the sample indicates the presence of cancer in the subject.

2. A method of identifying subjects with poor disease prognosis, the method comprising contacting a sample obtained from the subject with an antibody capable of binding specifically to DDX3 Y, wherein increased binding of the antibody to DDX3 Y in the sample identifies the subject of having poor disease prognosis.

3. The method of any one of the preceding claims, wherein the increase in binding is as compared to binding of the same antibody to a non-diseased sample.

4. The method any one of the preceding claims, wherein the subject identified to have cancer is to be treated with an anticancer drug.

5. The method of any one of claims 3 to 4, wherein the subject had been previously identified as having cancer.

6. The method of any one of the preceding claims, wherein the method is performed as a companion diagnostic method in conjunction with an existing method.

7. The method of any one of the preceding claims, wherein the cancer or the disease is selected from the group consisting of prostate cancer, leukaemia, and lymphoma.

8. A method of identifying DDX3Y in a sample, the method comprising applying an antibody capable of binding specifically to DDX3 Y to the sample, detecting binding of the antibody in the sample, thereby identifying DDX3Y in the sample.

9. A semi-quantitative method of identifying DDX3Y in a sample, the method comprising applying an antibody capable of binding specifically to DDX3Y to the sample, detecting binding of the antibody in the sample, thereby identifying DDX3Y in the sample.

10. The method of any one of claims 8 to 9, wherein an increase in the amount of binding of the antibody to DDX3Y in the sample is indicative of a poor prognosis.

11. The method of any one of claims 8 to 10, wherein the method is an ex vivo or in vitro method.

12. The method of any one of the preceding claims, wherein the antibody is obtained from a hybridoma cell line with a deposition number selected from the group consisting of CBA20230043, CBA20230044, CBA20230045, CBA20230046, CBA20230047, and CBA20230048.

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