AGENTS FOR CANCER THERAPY

Field of the Invention

This invention relates to agents that bind proline-rich protein 11 ("PRR11") , and to methods of using such agents including in

therapeutic methods for treating proliferative disorders such as cancer .

Background to the Invention

Proliferative diseases such as cancer exhibit abnormal cell growth, which in the case of cancer is caused by genetic changes to

oncogenes and tumour suppressor genes, ultimately leading to 'cell transformation' and uncontrolled cell division. Cell transformation is usually accompanied by abnormal expression of certain proteins, which can therefore be useful as biomarkers in cancer detection and/or therapy applications.

Some biomarker proteins are secreted by the cancer, which leads to them being useful in non-invasive diagnostic applications. In contrast, cancer biomarkers that are present in a significant amount on the cancer cell membrane are useful as therapeutic targets;

allowing therapeutic agents to be specifically directed to the cancer cells. Cancer is responsible for about 15% of all human deaths (World Cancer Report 2014 Chapter 1.1; World Health

Organization) . Hence, a need exists for more effective treatments. The identification of new biomarkers, present in a significant amount on the cancer cell surface, represents a key part of the search for novel cancer therapeutics.

Proline-rich protein 11 (PRR11) was identified as a gene implicated in cell cycle progression and lung cancer, and was found to contain one bipartite nuclear localization signal, two proline-rich regions and one zinc-finger domain (Ying Ji et. al . , Int. J. Biochemistry & Cell Biology, 45, 645-656(2012)) .

Proline-rich motifs are known to bind SH3 domains to mediate protein-protein interactions involved in cellular signalling. Zinc- finger domains are known to bind double stranded DNA and modulate gene transcription. Although the precise molecular mechanisms were not fully elucidated, Ying Ji et. al. found that PRRll expression was cell cycle-dependent and this was confirmed in further studies that showed PRRll levels increasing from Gl to G2/ -phase (Larance et. al., Am. Soc. for Biochemistry and Molecular Biology, 12.3, 638- 650) .

Preliminary data from Ying Ji et. al. indicated that PRRll was located in the cell nucleus, as well as in the cytoplasm in some cell lines. Larance et. al. performed immunoblotting and

immunofluorescence on permeablised cancer cells and found that PRRll was predominantly located in the cytoplasm. The extent to which different cancer types express PRRll has not previously been determined.

PRRll-specific antibodies are commercially available, e.g. from Abeam (Cambridge, UK; www.abcam.com) . However, these commercial antibodies are indicated "Target cellular localization: Cytoplasm" and the relevant protocols specify cell permeabilization steps before the cell is contacted with the antibody.

Further, more effective cancer treatments are the subject of ongoing research and development and are urgently needed. Summary of the Invention

The present invention is based in part on the unexpected finding that PRRll is present on the cell membrane of cancer cells, allowing it to be targeted by PRRll-binding agents. Hence, broadly, the present invention provides therapeutic PRRll-binding agents, which can be used in therapy for proliferative diseases, and methods for producing the same. Furthermore, the present inventors have surprisingly discovered that PRRll is cycled from the cell surface (internalised) . The present inventors have demonstrated that PRRll- binding molecules can be used to target therapeutically active moieties to PRRll-expressing cells: A therapeutically active moiety can be delivered into a PRRll-expressing cell by PRRll targeting. Hence, in one aspect, the invention provides a molecule that specifically binds proline-rich protein 11 (PRR11), wherein said molecule is conjugated to a therapeutically active moiety.

In a related aspect, the invention provides a conjugate having the formula I:

A-(L-D)P (I)

or a pharmaceutically acceptable salt or solvate thereof,

wherein :

A is a molecule that specifically binds proline-rich protein 11 ( PRR11 ) ;

L is a linker;

D is a therapeutically active moiety; and

p is 1 to 10.

The features disclosed herein can be applied to any aspect of the invention. In any aspect, the molecule that specifically binds proline-rich protein 11 can be referred to as the PRRll-binding molecule, and/or simply as "A". Similarly, the therapeutically active moiety can be referred to as such, and/or simply as "D". The Linker may be referred to as such, and/or simply as "L".

The therapeutically active moiety may be selected from the group consisting of a cytotoxic moiety, a chemotherapeutic agent, a molecularly targeted agent, a catalytic moiety that converts a prodrug into an active drug, a nano-particle , an immunostimulatory molecule and an immunomodulatory agent. In some embodiments, the cytotoxic moiety or chemotherapeutic agent is selected from the group consisting of a ribosome-inactivating protein, an alkylating agent, an alkaloid, a platinum coordination complex, a cytotoxic peptide or a radioactive atom.

The conjugate of the invention may be for use in a method of treating the human or animal body by therapy.

The conjugate of the invention may be for use in a method of treating a cancer in a mammalian subject, the method comprising administering the conjugate to the mammalian subject. In some embodiments, the cancer is a carcinoma. The carcinoma may be selected from: pancreatic cancer, cholangiocarcinoma, head and neck cancer, bladder cancer, lung cancer, breast cancer and ovarian cancer. In some embodiments, the cancer is pancreatic cancer.

PRRll may be present or expressed externally of the cell at the cell membrane, or be associated with the cellular membrane. In some embodiments, PRRll is cycled from the cell membrane to the cytosol.

In some embodiments, the molecule that specifically binds PRRll is selected from the group consisting of an antibody, an aptamer, and an affinity protein. Where the PRRll-binding molecule is an antibody molecule, it may be selected from: a monoclonal antibody, an intrabody, a complete antibody, a single domain antibody, a nanobody, a Fab fragment, a F(ab')2 fragment, a scFv, a diabody, a triabody, a human antibody, a humanised antibody, a bispecific antibody and a chimeric antibody or a molecule comprising or containing at least one immunoglobulin variable domain or parts of a immunoglobulin variable domain or any fragments variants modification or derivatives of a immunoglobulin domai . In some embodiments, the molecule that specifically binds PRRll specifically binds to an epitope from one or more of the following peptides and peptide fragments:

(A) SEQ ID NO:l, or a fragment having at least 80% sequence identity thereto;

(B) SEQ ID NO: 2, or a fragment having at least 80% sequence identity thereto;

(C) SEQ ID NO: 3, or a fragment having at least 80% sequence identity thereto;

(D) SEQ ID NO: , or a fragment having at least 80% sequence identity thereto;

(E) SEQ ID NO: 5, or a fragment having at least 80% sequence identity thereto ; (F) SEQ ID NO: 6, or a fragment having at least 80% sequence identity thereto;

(G) SEQ ID NO: 7, or a fragment having at least 80% sequence identity thereto;

(H) SEQ ID NO: 8, or a fragment having at least 80% sequence identity thereto; and/or

(I) SEQ ID NO: 9, or a fragment having at least 80% sequence identity thereto;

or parts or combinations thereof, wherein each fragment is at least 10 amino acids in length and wherein sequence identity is determined across the full length of the fragment.

In some embodiments, the molecule that specifically binds PRRll specifically binds to an epitope from: amino acid residues 125-360 of SEQ ID NO:l, or amino acid residues 150-329 of SEQ ID NO:l, or amino acid residues 288-337 of SEQ ID NO:l, or amino acid residues 200-280 of SEQ ID NO: 1.

In some embodiments, the PRRll-binding molecule specifically binds to PRRll at the D-box corresponding to amino acid residues 296-304 of the human PRRll sequence, at the KEN box corresponding to residues 316-318 of the human PRRll sequence, at the phosphodegron motif corresponding to residues 285-291 of the human PRRll sequence, and/or at the phosphodegron motif corresponding to residues 325-330 of the human PRRll sequence. Additionally or alternatively, the PRRll-binding molecule may specifically bind to the phosphor-ser residue (s) corresponding to residues 40 and/or 344 of the human PRRll sequence, and/or the antibody molecule specifically binds to the phosphor-Thr residue (s) corresponding to residues 287, 346 and/or 348 of the human PRRll sequence. Preferably, the PRRll- binding molecule binds human PRRll; however, the skilled person will understand that sequence alignment tools can be used to readily identify corresponding features in homologous PRRll proteins from other species.

In some embodiments, the linker (L) of the compound of formula (I) is an amino acid linker. The amino acid linker may comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In some embodiments, the amino acid linker consists of at least 50%, at least 60%, at least 70%, at least 80% or at least 90% glycine and serine residues.

In some embodiments, the linker comprises a valine-citrulline moiety. Valine-citrulline linkers are amenable to cleavage by proteases such as cathepsins. The valine-citrulline linker may also comprise a para-aminobenzylcarbonate (PABC) spacer.

In some embodiments, the linker is a hydrazone, disulfides or thioether. Hydrazones provide stability in serum but may be degraded in acidic conditions within the cell. Disulfides may be cleaved by intracellular thiols such as glutathiones via disulfide exchange. Thioesters may be proteolytically cleaved within the cell.

In some embodiments, the linker is a covalent bond. In other embodiments, the linker comprises a chain of atoms covalently bonded together .

The linker (L) may be covalently attached to A and/or covalently attached to D. The linker (L) may be covalently attached to A by a cysteine bridge and/or covalently attached to D by a cysteine bridge .

The linker (L) may comprise a cleavable protease recognition site (protease cleavage site) .

As will be apparent from the present disclosure, the invention provides a molecule that specifically binds proline-rich protein 11 (PRR11) for use in a method of treating a cancer in a mammalian subject. Preferably, the molecule is selected from the group consisting of an antibody, an aptamer, an affinity protein, a T cell receptor and a chimeric receptor. In some embodiments, the cancer is a carcinoma, i.e. a cancer of epithelial origin. The carcinoma may be selected from pancreatic cancer, cholangiocarcinoma, bladder cancer, lung cancer, breast cancer and ovarian cancer. The cancer may be a PRRll-driven cancer. PRR11 is associated with several cancer types and, without wanting to be bound by theory, PRR11 expression may be linked to carcinogenesis. Cancers in which PRR11 expression or overexpression contributes to carcinogenesis may be termed PRRll-driven cancers.

Preferably, the cancer expresses PRR11. In some embodiments, the cancer overexpresses PRR11 relative to non-cancer cells.

Preferably, the cancer expresses PRR11 at the cell surface or associated with the cellular membrane, e.g. at least 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, substantially all, or all of the PRRll in the cancer cell is present at the cell surface or is associated with the cellular membrane.

In some embodiments, the mammalian subject exhibits elevated PRRll expression levels. This can be determined by comparing the PRRll level to non-cancerous tissue (e.g. from the mammalian subject) and/or to a pre-determined control level optionally derived from (a) disease-free subject(s), e.g. in a subject's sample to the PRRll level in a control sample, e.g. using the methods described herein. The mammalian subject may have undergone treatment for cancer by surgery and/or chemotherapy and/or radiation and/or a molecular targeted agent. In some embodiments, the mammalian subject's PRRll expression levels are monitored following treatment by surgery and/or chemotherapy and/or radiation. The mammalian subject may be selected for treatment on the basis of PRRll expression levels that are elevated relative to non-cancerous tissue from the subject or relative to a pre-determined control level derived from a disease- free (control) subject or subjects. In some embodiments, the mammalian subject's PRRll expression levels are monitored following treatment with the PRR11-binding molecule. The PRRll may be detected at the cell surface. PRRll expression level may be

monitored following the use of the molecule in the method of treating the cancer. Molecules that specifically bind PRR11 may cause a reduction in the growth, proliferation, invasion, migration, adhesion and/or cell number of cancer cells in vitro and/or in an in vivo as compared with cancer cells grown under identical conditions, but in the absence of the PRRll-binding molecule. Preferably, the PRR11- binding molecule causes at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% reduction in one or more of these features as compared with cancer cells grown under identical conditions, but in the absence of said molecule.

In some embodiments, the method of treating the cancer in the mammalian subject (which comprises the use of molecules that specifically bind PRR11) further comprises administering or carrying out an adjunct therapy selected from the group consisting of: (i) an anticancer drug and/or prodrug; (ii) radiotherapy; (iii) surgery; (iv) chemotherapy; (v) a molecular targeted agent. In some cases the method of treating the cancer comprises administering an anticancer drug and/or prodrug and/or radiotherapy and/or

chemotherapy and/or surgery and/or a molecular targeted agent.

These elements of cancer therapy can be administered in any

combination that includes the use of molecules that specifically bind PRR11, for example (i) and (iii) with PRRll-binding molecules, (ii) and (v) with PRRll-binding molecules and the elements can be administered in any order. Molecular targeted agents include agents that bind VEGF, VEGF-R, CD20, CD30, CD33, CD52, ErbB, EpCAM, CEA, gpA33, mucins, integrins ανβ3 or α5β1, HER1, HER2, HER3, HER4, EGFR, MET, IGF1R, EPHA3, TRAILR1/2, RANKL, Tenascin, FAP, PMSA, CAIX, TAG- 72 and other molecular targets known in the art to be implicated in cancer.

In some embodiments, the subject has previously been administered with an anticancer drug and/or prodrug, and/or radiotherapy and/or chemotherapy and/or surgery and/or a molecular targeted agent.

Additionally or alternatively, the method of treating the cancer in the mammalian subject may comprise administering radiotherapy and/or a molecular targeted agent and/or chemotherapy to the subject as discussed herein, concurrently with treatment with the PRRll-binding molecule. The skilled person will understand that radiotherapy can be used in combination with any treatment-related embodiment of this invention. In some embodiments, the method of treatment includes treatment by surgery.

In some embodiments, the molecule that specifically binds PRR11 is conjugated or linked to a cytotoxic moiety and/or a catalytic moiety therapeutically active moiety, e.g. an immunostimulatory molecule. Preferably, the cytotoxic moiety is an alkylating agent, an

alkaloid, a platinum coordination complex, a cytotoxic peptide or a radioactive atom.

The molecule that specifically binds PRR11 may be a chimeric T cell receptor expressed by a T cell. A T cell expressing the PRRll- binding chimeric T cell receptor can be used in a method of treating a cancer in a mammalian subject, said treatment comprising

administering the T cell to the mammalian subject. The molecule that specifically binds PRR11 may be an antibody. The anti-PRRll antibody may trigger a host immune response to the cancer via antibody-dependent cell-mediated cytotoxicity (ADCC) . In ADCC, antibody binding causes the recruitment of immune cells, e.g.

macrophages, neutrophils, eosinophils and natural killer (NK) cells and/or initiates the complement system. Alternatively or

additionally, the anti-PRRll antibody may be an antibody which inhibits PRR11 function. In some embodiments, the antibody molecule is an antagonist antibody. In some embodiments, molecule that specifically binds PRR11

specifically binds to an epitope from one or more of the following peptides or peptide fragments:

(A) SEQ ID NO:l, or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto;

(B) SEQ ID NO:2, or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto; (C) SEQ ID NO: 3, or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto;

(D) SEQ ID NO: 4, or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto;

(E) SEQ ID NO:5, or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto;

(F) SEQ ID NO: 6, or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto;

(G) SEQ ID NO:7, or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto;

(H) SEQ ID NO: 8, or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto; and/or

(I) SEQ ID NO: 9, or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto;

or parts or combinations thereof. Each fragment is at least 10 amino acids in length and sequence identity is determined across the full length of the fragment.

In some embodiments, the molecule that specifically binds PRRll specifically binds to an epitope from: amino acid residues 125-360 of SEQ ID NO:l, or amino acid residues 150-329 of SEQ ID NO:l, or amino acid residues 288-337 of SEQ ID NO:l, or amino acid residues 200-280 of SEQ ID NO:l. In some embodiments, the PRRll-binding molecule specifically binds to PRRll at the D-box corresponding to amino acid residues 296-304 of the human PRRll sequence, at the KEN box corresponding to residues 316-318 of the human PRRll sequence, at the phosphodegron motif corresponding to residues 285-291 of the human PRRll sequence, and/or at the phosphodegron motif corresponding to residues 325-330 of the human PRRll sequence. Additionally or alternatively, the PRRll-binding molecule may specifically bind to the phosphor-ser residue (s) corresponding to residues 40 and/or 344 of the human PRRll sequence, and/or the antibody molecule specifically binds to the phosphor-Thr residue (s) corresponding to residues 287, 346 and/or 348 of the human PRRll sequence. Preferably, the PRRll- binding molecule binds human PRRll; however, the skilled person will understand that sequence alignment tools can be used to readily identify corresponding features in homologous PRR11 proteins from other species. Preferably, the PRRll-binding molecule is an antibody selected from: a polyclonal antibody, a monoclonal antibody, an intrabody, a complete antibody, a single domain antibody, a nanobody, a Fab fragment, a F(ab')2 fragment, a scFv, a diabody, a triabody, a human antibody, a humanised antibody, a bispecific antibody and a chimeric antibody.

In some embodiments, the antibody molecule is a bispecific antibody or a triabody. Bispecific antibodies and triabodies are able to trigger additional aspects of the immune response by binding targets on immune effector cells, e.g. T cells and NK cells, e.g. CD3 or CD8, as well as binding PRR11.

In some embodiments, the mammalian subject has had surgical and/or pharmaceutical treatment for a cancer, e.g. treatment with an anticancer drug and/or prodrug and/or radiotherapy and/or

chemotherapy and/or surgery and/or a molecular targeted agent, including for carcinomas such as pancreatic cancer,

cholangiocarcinoma, bladder cancer, lung cancer, breast cancer and ovarian cancer, e.g. serous adenocarcenoma . In some embodiments these treatments are administered before treatment with the PRRll- binding molecule, while in other embodiments, these treatments are administered concurrently with the PRRll-binding molecule. In some embodiments, treatment with the PRRll-binding molecule is performed prior to the treatments listed above e.g. to sensitise the mammalian subject to the anticancer drug and/or prodrug and/or radiotherapy and/or chemotherapy and/or surgery and/or a molecular targeted agent .

In a another aspect, this invention provides a method of treating a mammalian subject having a cancer, the method comprising

administering a therapeutically effective amount of an antibody molecule that specifically binds proline-rich protein 11 (PRR11) . Optionally, the cancer is a PRRll-driven cancer. In some embodiments, the cancer the cancer is a carcinoma, e.g. a carcinoma selected from pancreatic cancer, cholangiocarcinoma , bladder cancer, lung cancer, breast cancer and ovarian cancer. Preferably, the cancer expresses PRRll. In some embodiments, the cancer

overexpresses PRRll relative to non-cancer cells. Preferably, the cancer expresses PRRll at the cell surface or associated with the plasma membrane, e.g. at least 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, substantially all, or all of the PRRll in the cancer cell is present at the surface or associated with the plasma membrane. In some embodiments, the carcinoma is selected from head and neck cancer, pancreatic cancer, cholangiocarcinoma, bladder cancer, lung cancer, breast cancer and ovarian cancer. In some embodiments, the cancer is not lung cancer. In some embodiments, the carcinoma is not lung cancer.

In some embodiments, the mammalian subject is selected for treatment on the basis of detection of PRRll expression levels, optionally wherein the mammalian subject has undergone treatment for cancer by surgery and/or chemotherapy and/or radiation. The PRRll expression level may be detected prior to the use of the molecule in the method of treating the cancer. In some embodiments, PRRll expression is elevated compared to non cancerous tissue and/or a pre-determined control level derived from disease-free subject (s) .

PRRll may be detected at the cell surface. In some embodiments, PRRll expression level is monitored following the use of the PRRll- binding molecule in the method of treating the mammalian subject.

The antibody molecule may modulate PRRll biological function leading to a therapeutic effect (e.g. be an antagonist antibody) and/or it may be conjugated or linked to a therapeutically active and/or cytotoxic moiety. Preferably, the cytotoxic moiety is an alkylating agent, an alkaloid, a platinum coordination complex, a cytotoxic peptide or a radioactive atom. Alternatively or additionally, the antibodies disclosed herein can be chimeric antigen receptors expressed by T cells, e.g. cytotoxic T cells. This allows the T cells to be directed to, and react against, PRR11-expressing cells, e.g. cancer cells. Preferably, the chimeric antigen receptor will be a Fab fragment, a F(ab')2 fragment, a scFv, a diabody, or a triabody in chimeric antigen applications.

The anti-PRRll antibody may trigger a host immune response to the cancer via antibody-dependent cell-mediated cytotoxicity (ADCC) . In ADCC, antibody binding causes the recruitment of immune cells, e.g. macrophages, neutrophils, eosinophils and natural killer (NK) cells. Alternatively or additionally, the anti-PRRll antibody may be an antibody which inhibits PRRll function. In some embodiments, the antibody molecule modulates PRRll biological function leading to a therapeutic effect (e.g. is an antagonist antibody) . Optionally, the method further comprises administering an anticancer drug or prodrug, as described herein, to the mammalian subject. The method of treatment may also include surgery and/or administration of radiotherapy. In a further aspect, this invention provides a pharmaceutical preparation comprising a molecule (e.g. and antibody molecule) that specifically binds proline-rich protein 11 (PRRll) . The

pharmaceutical preparation may exclude glycerol, sodium azide, bovine serum albumin (BSA) , phosphates and/or surfactants.

The antibody molecule may modulate PRRll biological function leading to a therapeutic effect (e.g. be an antagonist antibody) and/or it may be conjugated or linked to a therapeutically active and/or cytotoxic moiety. Preferably, the cytotoxic moiety is an alkylating agent, an alkaloid, a platinum coordination complex, a cytotoxic peptide or a radioactive atom.

In another aspect, this invention provides an article of manufacture comprising molecule (e.g. an antibody molecule) that specifically binds proline-rich protein 11 (PRRll) as defined herein, and an instruction label having instructions for human dosage and/or animal dosage and/or human and/or animal administration. In yet another aspect, this invention provides a molecule (e.g. an antibody molecule) that specifically binds proline-rich protein 11 (PRR11) as defined herein, for use in a method of diagnosing a cancer in a mammalian subject, wherein the PRRll-binding molecule is conjugated to a detectable moiety. In this aspect, the method optionally comprises selecting the mammalian subject for cancer treatment .

In another related aspect, this invention provides a method of obtaining an image of an interior portion of a mammalian subject, the method comprising administering to the mammalian subject a molecule that specifically binds proline-rich protein 11 (PRRll) as defined herein, wherein the molecule that specifically binds PRRll is conjugated to a detectable moiety, and using a detector to obtain the image.

In a related aspect, a method of selecting a mammalian subject for treatment is provided, comprising using a PRRll-binding molecule to determine the PRRll expression level in a sample obtained from the mammalian subject, and comparing the PRRll level in the sample to a control level, wherein the sample obtained from the mammalian subject is optionally a fluid sample a cancer biopsy or resected tissue .

In a further related aspect, a method of detecting or prognosing a cancer in a mammalian subject is provided, the method comprising administering a molecule that specifically binds PRRll, as described herein, wherein the antibody molecule is conjugated to a detectable moiety. The subject may have received, or may be concurrently receiving treatment comprising administration of anticancer drug and/or prodrug and/or radiotherapy and/or chemotherapy and/or surgery and/or a molecular targeted agent.

In another related aspect, a method of detecting or prognosing a cancer in a mammalian subject is provided, wherein the method comprises using an molecule that specifically binds PRRll, as described herein, to determine PRR11 expression level in a sample obtained from the mammalian subject (optionally a fluid sample, a cancer biopsy or resected tissue) , the method further comprising comparing the PRR11 level in the sample to a control level. In some embodiments of these aspects, increased PRR11 expression level, determined by PRRll-antibody binding, indicates a worse prognosis, e.g. decreased probability of recurrence-free survival, increased risk of metastasis and/or relapse and/or poor response to

conventional chemotherapy and/or radiotherapy.

In some embodiments, an increased PRR11 expression level, determined by PRRll-antibody binding, indicates decreased probability of recurrence-free survival. In some embodiments, the cancer is pancreatic cancer, cholangiocarcinoma, bladder cancer, lung cancer, breast cancer and ovarian cancer, preferably bladder cancer. In some embodiments, increased PRRll expression level, determined by PRRll-antibody binding, indicates decreased probability of

recurrence-free survival and/or decreased probability of overall survival for a period of 50 months, 100 months or 10 years. In some embodiments, a method of prognosing bladder cancer is provided, wherein increased PRRll expression is linked to an increased chance of recurrence or increased mortality following treatment or surgery. The parts of GB 1401679.4 filed 31 January 2014 that relate to prognosing cancers based on PRRll expression are hereby specifically referenced and expressly incorporated herein.

As discussed herein, the methods of treatment relating to the use of PRRll-binding molecules for administration to a mammalian subject with a cancer may comprise administering the PRRll-binding molecule to sensitise the cancer to subsequent treatment with an anticancer drug and/or prodrug and/or radiotherapy and/or chemotherapy and/or surgery and/or a molecular targeted agent. Preferably the cancer is pancreatic cancer. The treatment to which the cancer is sensitised is preferably an anticancer drug, e.g. a chemotherapeutic such as Gemcitabine . However, the skilled person will appreciate that sensitisation to all other DNA-damaging treatments (e.g. radiation therapy) is also possible. In another aspect, an in vitro method of screening candidate agents that specifically bind proline-rich protein 11 (PRRll) , the method comprising :

(i) providing a sample containing a cell that expresses PRRll at the cell surface or associated with the cell membrane,

(ii) adding to the sample a candidate molecule to allow the candidate molecule to contact PRRll,

(iii) performing one or more washing steps to remove unbound candidate molecule from the cell,

(iv) adding to the sample a detectable molecule that binds to the candidate molecule,

(v) performing one or more washing steps to remove unbound detectable molecule,

(vi) detecting the amount of detectable molecule in the sample, and

(vii) optionally selecting the candidate agent if the amount of detectable molecule in the sample exceeds a pre-determined threshold level.

In a further aspect, in vitro methods of screening for agents that specifically bind proline-rich protein 11 (PRRll) are provided, comprising :

(i) contacting one or more PRRll peptide fragments with a molecule known to specifically bind the one or more PRRll peptide fragments, in the presence of a candidate agent; and

(ii) determining the extent of binding between the one or more PRRll peptide fragments and said molecule; and

(iii) comparing the extent of binding in (ii) with the extent of binding between one or more PRRll peptide fragments identical to those in (i) and a molecule identical to that in (i) under identical conditions to those in (i), but in the absence of a candidate agent; and

optionally (iv) selecting the candidate agent if the extent of binding in (iii) is reduced with respect to the extent of binding in (ii) . The molecule known to specifically bind the one or more PRRll peptide fragments is preferably an PRRl 1-binding molecule, e.g. an antibody of the invention as described herein.

In a related aspect, in vitro methods of screening agents that inhibit: cell growth, proliferation, invasion, migration, adhesion and/or cell number of cancer cells expressing proline-rich protein 11 (PRRll) and/or tumour colonisation wherein the tumour expresses PRRll, are provided, the methods comprising:

(i) incubating a candidate agent with cancer cells that express PRRll, the candidate agent optionally having been previously selected according to the competition-based screening methods described herein; and

(ii) measuring growth of said cultured cancer cells in an in vitro cell assay as compared with the growth of cancer cells cultured under identical conditions, but in the absence of any candidate agent; and optionally (iii) selecting the agent if it reduces cancer cell growth, proliferation, invasion, migration, adhesion and/or cell number.

In a further related aspect, in vitro methods of screening candidate anti-cancer agents are provided, comprising:

(i) incubating a population of cancer cells that express proline-rich protein 11 (PRRll) with a candidate anti-cancer agent which optionally has been previously selected according to the other screening methods described herein;

(ii) using a PRRl 1-binding molecule to perform FACS or flow- cytometry to determine PRRll expression levels on cancer cells of (i) ;

(iii) using PRRll-binding molecule to perform FACS or flow- cytometry to determine PRRll expression levels on a control

population of cancer cells that have not been cultured in the presence of the candidate anti-cancer agent;

comparing the results of (ii) and (iii) ,

optionally wherein the expression level of one or more cancer markers is also determined and compared using FACS or flow- cytometry . In another related aspect, in vivo methods of screening agents that inhibit tumour growth and/or metastasis in mammalian subjects that have tumours expressing PRRll, the method comprising:

(i) administering a candidate agent to the mammalian subject; and subsequently

(ii) measuring tumour growth and/or size and/or the number of tumours and/or metastasis at one or more time-points; and

(iii) comparing the extent of tumour growth and/or size and/or the number of tumours and/or metastasis with that in a control mammalian subject that also has a tumour that expresses PRRll but has not been administered with the candidate agent; and optionally (iv) selecting the candidate agent if it reduces tumour growth, tumour size, the number of tumours and/or metastasis. Optionally, the candidate agent has been previously selected according to the other screening methods described herein. The mammalian subject is not a human.

In some embodiments of the screening methods disclosed herein, the candidate agent is a candidate antibody. The methods may also comprise isolating a selected candidate antibody; and optionally further comprise determining the sequence of at least the

complementarity determining regions (CDRs) of said selected

antibody .

The candidate agent in any of the screening methods described herein may be an antibody. Optionally, such a candidate antibody has been produced by phage display or by immunization of a non-human mammal, e.g. a rat, mouse, goat, cow or rabbit.

In some embodiments of the various aspects of the invention, the mammalian subject is a human. The subject may have been diagnosed as having or being susceptible to developing a cancer, including pancreatic cancer, cholangiocarcinoma , bladder cancer, lung cancer, breast cancer and ovarian cancer. In some cases the subject may have had surgical, radiological and/or pharmaceutical treatment for a cancer, including pancreatic cancer, cholangiocarcinoma, bladder cancer, lung cancer, breast cancer and ovarian cancer (e.g. the treatment in accordance with the present invention may be of a subject who or that has had surgical resection of an pancreatic cancer, cholangiocarcinoma, bladder cancer, lung cancer, breast cancer and ovarian cancer tumour, optionally in combination with the anticancer chemotherapies discussed herein and/or radiotherapy and/or surgery and/or a molecular targeted agent) .

Embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying figures. However various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

Where used herein, "and/or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific

disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

The present invention includes the combination of the aspects and preferred features described, except where such a combination is clearly impermissible or is stated to be expressly avoided. Section headings are used herein are for convenience only and are not to be construed as limiting in any way.

Unless context dictates otherwise, the descriptions and definitions of the features set out herein are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

Brief Description of the Figures

Figure 1 shows A) Western blots of whole cell lysate from mouse pancreatic cell line K8484, human HeLa cells, and human pancreatic lines MiaPaCa2, MiaPaCa3, Suit2, Panel and FA-6 cells stained with HPA023923, Abl31652, Ab84565, Ab67244, PA5-30760 anti-PRRll primary antibodies and anti- -actin primary antibodies. B) shows Western blots of lysed and sonicated HeLa cells (top panel) and Panel cells (bottom panel) . Wash/flow-through fractions following cell surface biotinylation and separation on avidin beads. PRRll was stained with HPA023923 primary antibody. C ) shows a schematic diagram of PRRll: 'Pro' denotes proline-rich regions; blue denotes

phosphodegrons ; orange denotes the KEN box; green denotes the D box; red diamonds denote phosphor-Ser residues; grey diamonds denote phosphor-Thr residues. Figure 2 shows FACS plots of viable, unpermeablized Panel cells and MiaPaCa2 cells treated with Ab84565, PA5-30760 and HPA023923 anti- PRR11 primary antibodies followed by Alexafluor 647-tagged anti- rabbit secondary antibody (upper quadrants); and Abl31652 and

Ab67244 anti-PRRll primary antibodies followed by Alexafluor 647- tagged anti-mouse secondary antibody (lower quadrants) . Unstained and secondary antibody-only control plots are also shown (all quadrants) .

Figure 3 shows A) immunohistochemistry of a pancreatic cancer duct stained for PRRll using HPA023923 primary antibody and

counterstained with hematoxylin to show cell nuclei in blue. B) shows immunohistochemistry of bladder cancer, ovarian cancer, breast cancer and lung cancer sections stained for PRRll using HPA023923 primary antibody and counterstained with hematoxylin to show cell nuclei in blue.

Figure 4 shows immunohistochemistry of HeLa cells stained for PRRll using HPA023923 following treatment with short interfering RNA (siRNA) . A) shows a high degree of PRRll staining in cells treated with siRNA not designed to target PRRll. B ) shows a reduced degree of PRRll staining in cells treated with siRNA designed to target PRRll. Cells are stained for PRRll using HPA023923 primary antibody and counterstained with hematoxylin to show cell nuclei in blue. C ) shows Western blots of whole cell lysate of (i) cells treated with siRNA not designed to target PRRll and (ii) cells treated with siRNA designed to target PRRll when stained for PRRll using HPA023923 (top) compared to anti-tubulin controls (bottom) . Figure 5 shows A) the intensity of PRRll immunohistochemical staining in normal pancreas cells, chronic pancreatitis cells and pancreatic tumour cells. None (no staining), weak (staining was inconsistent and/or weak), moderate (appreciable staining) or strong (very intense staining) . B) shows immunohistochemistry of normal pancreas cells (black arrows, top panel), chronic pancreatitis cells (red arrows, bottom panel) and pancreatic tumour cells (brown staining, both panels) stained for PRRll using HPA023923. Cells are stained for PRRll using HPA023923 primary antibody and

counterstained with hematoxylin to show cell nuclei in blue.

Figure 6 shows A) the intensity of PRRll immunohistochemical staining in bladder cancer at different stages of disease. B) shows intensity of PRRll immunohistochemical staining in bladder cancer at different disease grades. None (no staining), weak (staining was inconsistent and/or weak) , moderate (appreciable staining) or strong (very intense staining) . C) shows a Kaplan-Meier survival estimate showing recurrence-free survival probability over time for bladder cancer patients with high or low PRRll expression.

Figure 7 shows A) the intensity of PRRll immunohistochemical staining in normal lung tissue, adenocarcinoma lung cancer, and squamous cell lung cancer. B) shows the intensity of PRRll

immunohistochemical staining in normal ovarian tissue, benign serous cystadenocarcinoma and serous adenocarcinoma. None (no staining), weak (staining was inconsistent and/or weak) , moderate (appreciable staining) or strong (very intense staining) .

Figure 8 shows A) the intensity of PRRll immunohistochemical staining in normal (benign) cells from biliary brushings compared with cholangiocarcinoma cells. None (no staining), weak (staining was inconsistent and/or weak) , moderate (appreciable staining) or strong (very intense staining) . B) shows immunohistochemistry of biliary tract brush specimens stained for PRRll using HPA023923. Figure 9 shows the results of investigations into the cooperative effect of PRRll knockdown with Gemcitabine . A) shows Western blots of whole cell lysate of cells treated with Gemcitabine (+) or not treated with Gemcitabine (-) and stained for PRRll (using HPA023923) (top), and p21, cyclin D3, cyclin Dl, phosphorylated CHK2 , total

CHK2 , phospho-PTEN (p-PTEN) , integrin (3-4 and a-tubulin. Expression of PRRll is undetectable in columns 2 and 4, which represent samples from cells treated with PRRll-knockdown siRNA. B ) shows the results of an MTT assay: absorbance at 490nm of pancreatic cancer cells following Gemcitabine treatment at 0.25 μΜ, 0.50 μΜ, 1.0 μΜ, and 2.0 μΜ. Upper plot represents cells treated with scrambled siRNA (NT) while the lower plot represents cells treated with PRRll-knockdown siRNA. Cancer cell viability following Gemcitabine treatment is substantially reduced in cells treated with PRRll-knockdown siRNA, showing that reduced PRRll expression sensitises cancer cells to chemotherapy .

Figure 10 presents bar-charts (upper 3 rows of panels) showing the intensity of PRRll immunohistochemical staining in normal spleen, head & neck, and colon cells (left-hand panels; left-hand bars) compared with staining in tumour spleen, head & neck, and colon cells (left-hand panels; right-hand bars) . Also shown is the intensity of PRRll immunohistochemical staining in normal liver cells (top-right panel) and normal appendix, lymph-node, spleen, thymus and tonsil cells (mid-right panels, taking the bars from left-to-right) . The lower-right panel shows the intensity of PRRll immunohistochemical staining in normal, NAT (normal adjacent to tumour) and tumour skin cells (taking the bars from left-to-right) . The bottom panels show (left) immunohistochemistry of normal urothelium - expression in basal layer high but low in epithelium and umbrealla cells (n=9) . Right: Skin - expression low in

keratinocytes but moderate/high at the base of hair follicle and sebaceous glands. Figure 11 presents a bar chart showing (from left-to-right) the intensity of PRRll immunohistochemical staining in normal lung, NAT (normal adjacent to tumour), adenocarcinoma, squamous cell tumour cells and small cell tumour cells.

Figure 12 presents FACS data plotted as histograms showing surface PRRll expression at 4C and 37C. The histograms of Figure 12 include data using unstained cells and secondary-only controls, in different cell lines. A: Panc-1 cells. B: HeLa cells. C: MiaPaCa2 cells. Top-left of each of A-C: Sigma HPA 023923 Rabbit anti-PRRll primary antibody; top-right of each: Santacruz SC-161167 goat anti-PRRll primary antibody; bottom-left of each: Abeam Abl31652 mouse anti- PRRll primary antibody; bottom-right of each: Abeam AB84565 Rabbit monoclonal anti-PRRll antibody.

Figure 13 shows results of a surrogate-ADC ZAP assay evaluating the cytotoxic activity of the commercial PRRll antibodies following binding of a Saporin conjugated secondary antibody. Saporin is a ribosome inhibitor. The ZAP assay was optimized using mouse transferrin as a positive control. Detailed Description

PRRll

The human PRRll protein has NCBI accession number NP_060774.2 and NCBI nucleotide sequence number NM_018304.3 (the entire contents fo which are expressly incorporated herein by reference) . The amino acid sequence for human PRRll is shown below.

PRRll has a predicted MW of 40kDa.

Human PRRll

1 mpkfkqrrrk lkakaerlfk kkeashfqsk litpppppps pervgissid

51 isqsrswlts swnfnfpnir daiklwtnrv wsiyswcqnc itqslevlkd

101 tifpsrichr elysvkqqfc ilesklcklq ealktisess sepsegqtch

151 msgkltnvpa cvlitpgdsk avlpptlpqp ashfpppppp pplppppppl

201 apvllrkpsl akalqagplk kdgpmqitvk dlltvklkkt qsldekrkli

251 pspkarnplv tvsdlqhvtl kpnskvlstr vtnvlitpgk sqmdlrkllr

301 kvdverspgg tpltnkenme tgtgltpvmt qalrrkfqla hprsptptlp

351 lstssfdeqn (SEQ ID NO: 1) The nucleotide and amino acid sequences for PRRll genes of other species can be found on the NCBI and similar public databases, e.g. by searching for sequences showing homology to the above human sequence.

Human PRRll has proline-rich regions at amino acid (aa) positions 34-41 and at aal85-202. Human PRRll also has two phosphodegron motifs (aa285-291 and 325-330), a D-box (aa296-304) and a KEN box (aa316-318), all suggesting it may be actively degraded by the proteasome. Ser40 and Ser344 are phosphor-ser . Thr287, Thr346 and Thr348 are phosphor-Thr .

Methods of diagnosing and prognosing cancers based on PRRll

expression are described in GB 1401679.4 filed 31 January 2014; the contents of which are expressly incorporated herein by reference.

PRRll Expression

Expression of PRRll in a sample can be determined by any method well-known in the art. For example, RT-PCR or qPCR can be used to determine the level of PRRll mRNA in a cell sample. Typically, this will involve the use of nucleotide primers specific to parts of the PRRll sequence, e.g. as in the NCBI accession number mentioned above. Each nucleotide primer may have a nucleotide sequence that would be translated into a part of any one of SEQ ID NOs:l-8 or combinations thereof, if the primer's nucleotide sequence formed part of an expression vector.

Other means of determining PRRll expression are provided herein: The present inventors have found PRRll to be present on the cell surface, so anti-PRRll antibodies, such as those discussed herein, can be used in FACS or flow-cytometry applications of live cells, e.g. as described herein. Alternatively, anti-PRRll antibodies can be used for FACS or flow-cytometry of permeablised cells. Other means of determining PRRll expression include western blot,

immunohistochemistry (both described herein) , ELISA and Southern blot and other means of detecting PRRll can be used in various embodiments of this invention. Many of these methods allow the amount of PRRll expression to be quantified. The PRRll expression level in a cancer cell can then be compared to that in non-cancer cells qualitatively or quantitatively. In most embodiments of this invention, the cancer will overexpress PRRll, e.g. relative to non- cancer cells.

Methods such as FACS, iimmnohistochemistry and western blotting can be used to determine the extent of PRRll at the cell surface, for example as described below. The extent of cell surface expression can be determined qualitatively or quantitatively, e.g. by

performing western blots of various cell fractions, as described herein. If a quantitative value for surface expression of PRRll is desired, the skilled person can use any one of the methods well- known in the art, for example methods for quantifying the intensity of western blot bands.

In some embodiments of the invention, PRRll expression levels are detected in cancer samples, e.g. via the methods outlined above. It may also be possible to detect PRRll expression levels in a cancer non-invasively using bioimaging strategies, for example MRI, PET, SPECT, bioluminescence/ fluorescence imaging or single positron emission computed tomography following administration of key imaging factors conjugated to the PRRll-binding molecules of the invention. Such applications utilize PRRll-binding molecules conjugated to a detectable moiety or PRRll-binding molecules conjugated to a moiety that interacts with or generates a detectable moiety. In other embodiments, PRRll expression levels may be determined from a patient biopsy, resected tissue or fluid sample, e.g. blood, serum, urine or a pancreatic cyst. These PRRll expression level-detection methods on patient samples or whole body ( imaging-type methods) discussed above can be used in methods of diagnosing cancer, or in methods of evaluating cancers e.g. to provide a prognosis.

PRRll-binding agents

The present invention provides agents that bind PRRll proteins and fragments thereof, and uses of the PRRll-binding molecules such as in therapeutic methods of treating mammalian subjects having cancer, particularly cancers that overexpress PRR11.

Binding kinetics and affinity (expressed as the equilibrium

dissociation constant Kd) of the molecule that specifically binds PRR11 may be determined using standard techniques, such as surface plasmon resonance, e.g. using BIAcore analysis.

The molecule that specifically binds PRR11 may have a dissociation constant for PRRll of less than 50nM, less than 40nM, less than 30nM, less than 20nM, less than ΙΟηΜ, or less than InM. For example, the molecule that specifically binds PRRll may have an affinity for PRRll of 1 to 20 nM. Preferably the molecule that specifically binds PRRll has and affinity constant (K _D ) of less than 10 nM, more preferably less than 5 nM and most preferably less than 2 nM. The affinity constants for binding to PRRll or a peptide fragment of PRRll, e.g. a peptide fragment defined below, can be determined using techniques well known in the art such as Biacore SPR analysis.

All types of binding molecules are encompassed as part of the invention, for example antibodies, oligonucleotide aptamers and peptide aptamers. The binding molecule may recognise and bind epitopes arising from any part of the PRRll protein. For example, the binding molecule may specifically bind one or more epitopes arising from one or more of the following human PRRll-derived peptide fragments, and/or parts and/or combinations thereof:

SEQ ID NO: 2: mpkfkqrrrklkakaerlfkkkeashfqsklitppppppspervg, or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto;

SEQ ID NO: 3: issidisqsrswltsswnfnfpnirdaiklwtnrvwsiyswcqnc, or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto;

SEQ ID NO: 4: itqslevlkdtifpsrichrelysvkqqfcilesklcklqealkt, or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto; SEQ ID NO: 5: isessscpscgqtchmsgkltnvpacvlitpgdskavlpptlpqp, or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto;

SEQ ID NO: 6: ashfpppppppplpppppplapvllrkpslakalqagplkkdgpm, or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto;

SEQ ID NO: 7: qitvkdlltvklkktqsldekrklipspkarnplvtvsdlqhvtl , or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto;

SEQ ID NO: 8: kpnskvlstrvtnvlitpgksqmdlrkllrkvdverspggtpltn, or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto; and/or

SEQ ID NO: 9: kenmetgtgltpvmtqalrrkfqlahprsptptlplstssfdeqn or a fragment having at least 80%, 90%, 95% or 99% sequence identity thereto.

The binding molecule may specifically bind an epitope (e.g. an immunogen/immunogenic peptide) derived from, comprising or

consisting of a peptide consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,

34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids from SEQ ID NO:l; and/or

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids from SEQ ID NO: 2; and/or

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids from SEQ ID NO: 3;

and/or

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,

44, or 45 amino acids from SEQ ID NO: 4;

and/or

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,

44, or 45 amino acids from SEQ ID NO: 5;

and/or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,

44, or 45 amino acids from SEQ ID NO: 6;

and/or

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,

44, or 45 amino acids from SEQ ID NO: 7;

and/or

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,

44, or 45 amino acids from SEQ ID NO: 8;

and/or

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids from SEQ ID NO: 9; or combinations thereof.

In some embodiments, the binding molecule specifically binds to one or more epitopes derived from a PRRll-derived peptide which is a combination of 2 or 3 or 4 or 5 or 6 or 7 or 8 of SEQ ID NOS:l-9, or parts thereof. Sometimes just 1, 2, 3, 4, 5, 6, 7, 8 or 9 residues from one of the fragments listed will be present in the immunogenic peptide, especially wherein that peptide comprises 1, 2, 3, 4, 5, 6, 7, 8 or 9 residues from another fragment, e.g. listed herein. In one non-limiting example of the invention, anti- PRR11 antibody "ab84565" is used in a method of treating cancer in a mammalian subject. In this example, the antibody specifically binds to one or more epitopes arising from the following PRRll-derived peptide:

"pgksqmdlrkllrkvdverspggtpltnkenmetgtgltpvmtqalrrkf" (aa 288-337) . In this example, therefore, the antibody binds to epitopes arising from a PRRll-derived peptide that is a combination of parts of SEQ ID NOS : 8 and 9. More particularly, ab84565 binds to epitopes from a peptide consisting of 28 amino acids of SEQ ID NO: 8 and 22 amino acids from SEQ ID NO: 9.

In another non-limiting example of the invention, anti-PRRll antibody "abl31652" is used in a method of treating cancer in a mammalian subject. In this example, the antibody binds to one or more epitopes arising from the following PRRl 1-derived peptide:

"lcklqealktisessscpscgqtchmsgkltnvpacvlitpgdskavlpptlpqpa shfpppppppp lpppppplapvllrkpslakalqagplkkdgpmqitvkdlltvklkktqsldekrklips pkarnplv tvsdlqhvtlkpnskvlstrvtnvlitpgksqmdlrkllrkvdverspggtpltnkenme tgtgltpv mtqalrrkfqlahprsptptlplstssfdeqn" (aa 125-360) .

In this example, therefore, the antibody binds to one or more epitopes arising from a PRRll-derived peptide that is a combination of SEQ ID NOs:4-9. More particularly, abl31652 binds to epitopes from a peptide consisting of 10 amino acids of SEQ ID NO: and all 45 amino acids from each of fragments SEQ ID NOs:5-9.

In a further non-limiting example of the invention, anti-PRRll antibody "HPA023923" is used in a method of treating cancer in a mammalian subject. In this example, the antibody binds to one or more epitopes arising from the following PRRll-derived peptide:

"lapvllrkpslakalqagplkkdgpmqitvkdlltvklkktqsldekrklipspka rnplvtvsdlq hvtlkpnskvlstr" (aa 200-280)

In this example, therefore, the antibody binds to one or more epitopes arising from a PRRll-derived peptide that is a combination of SEQ ID NOs:6-8. More particularly, HPA023923 binds to epitopes from a peptide consisting of 26 amino acids from SEQ ID NO: 6, all 45 amino acids from SEQ ID NO: 7 and 10 amino acids from SEQ ID NO: 8. In another non-limiting example of the invention, anti-PRRll antibody "Ab67244" is used in a method of treating cancer in a mammalian subject. In this example, the antibody binds to one or more epitopes arising from the full-length human PRR11 protein of SEQ ID NO:l (aa 1-360), because Ab67244 is a polyclonal antibody raised against the full length protein.

In a further non-limiting example of the invention, anti-PRRll antibody "PAS-30760" is used in a method of treating cancer in a mammalian subject. In this example, the antibody binds to one or more epitopes arising from the following PRRll-derived peptide: "hmsgkltnvpacvlitpgdskavlpptlpqpashfpppppppplpppppplapvllrkp slakalqa gplkkdgpmqitvkdlltvklkktqsldekrklipspkarnplvtvsdlqhvtlkpnskv lstrvtnv litpgksqmdlrkllrkvdverspggtpltnkenmetgtgltpvm" (aa 150-329) .

In this example, therefore, the antibody binds to one or more epitopes arising from a PRRll-derived peptide that is a combination of SEQ ID NOs:5-9. More particularly, PAS-30760 binds to epitopes from a peptide consisting of 31 amino acids from SEQ ID NO: 5, all 45 amino acids from SEQ ID NOs:6-7, and 14 amino acids from SEQ ID NO: 8.

In other non-limiting embodiments, anti-PRRll antibodies that bind the phosphodegron motifs (aa285-291 and 325-330), the D-box (aa296- 304) and/or the KEN box (aa316-318) are used in a method of treating cancer in a mammalian subject. Antibodies that bind these features of PRR11 may be used when elevated PRR11 expression is detected, as described herein. In some embodiments of the invention, antibodies that bind phosphor-ser residues Ser40 and/or Ser344 and/or phosphor- Thr residues Thr287 and/or Thr346 and/or Thr348 are used. Antibodies that specifically bind the above features can be used to modulate PRR11 function. For example, antibodies which bind to these features may prevent degradation of PRR11, which can lead to PRR11 accumulation in cells. Hence, antibodies that specifically bind the above features may be used to cause PRR11 build-up to promote cell death, e.g. in cancer cells.

Although the two non-limiting examples of the invention mentioned above involve antibodies that bind to epitopes arising from

contiguous PRR11 fragments (e.g. SEQ ID NOs : 3, 4, 5, 6, 7, 8), in other embodiments, the binding member specifically binds to epitopes arising from non-contiguous PRR11 fragments (e.g. SEQ ID NOs : 3 and 6) . This is possible because binding members such as antibodies recognise three-dimensional epitopes present on the folded protein. These epitopes can be derived from parts of the polypeptide that are adjacent in the three-dimensional folded protein despite being from distant parts of the linear amino acid sequence. Antibody molecules according to the invention may be anti-PRRll antibody molecules that test positive as an inhibitor of cancer cell growth, including growth, proliferation and/or cell number. This functional property of the antibody molecule may be assessed using any suitable assay, whether in vitro or in vivo. The antibody molecule may cause cell death of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99%, or a percentage-reduction in the growth (e.g. growth rate), proliferation and/or cell number of cultured cancer cells as compared with cultured cancer cells grown under identical

conditions, but in the absence of said antibody molecule at the percentages shown. The cultured cancer cells may comprise primary cancer culture and/or an established cancer cell line (e.g. K8484 cells, HeLa cells, MiaPaCa2 cells, MiaPaCa3 cells, Suit2 cells,

Panel cells or FA-6 cells) . The reduction in cell proliferation, growth rate, invasion (e.g. invasion rate and/or invasion directon) , migration rate, adhesion, metastatic capacity and/or cell number; and/or induction of cell death via apoptosis or necrosis, may be assessed over any suitable period, e.g. 24, 48, 96, 120 or more hours. Methods for assessing cell proliferation, growth rate, invasion, migration rate, adhesion, metastatic capacity and/or cell number, and/or cell death, are well known in the art. These methods are typically used to produce quantitative results.

The antibody molecule may be selected from: a polyclonal antibody, a monoclonal antibody, a complete antibody, a single domain antibody, a nanobody, a Fab fragment, a F(ab')2 fragment, a scFv, a diabody, a triabody, a human antibody, a humanised antibody, a bispecific antibody, antigen binding domains comprising only a heavy chain variable domain (VH) (e.g. camelid or shark antibodies) and a chimeric antibody. For example, the antibody molecule of the present invention may comprise a monoclonal antibody generated using HuCAL technology (AbD Serotec) , in particular, using HuCAL

technology directed against a peptide derived from the peptide fragments those listed herein. The skilled person will also understand that antibodies can also be generated by administering peptide fragments, e.g. those listed herein, in combination with an adjuvant to antibody-producing mammals such as mice, goats or rats.

Besides ^Λ intrabodies ' which are a special type of antibody that are expressed by the target cell and remain within the target cell, antibodies are unable to enter the target cell. The present invention may include the use of an intrabody in certain

embodiments. However, preferably, the antibody of the invention does not enter the cell.

As an alternative or supplement to immunising a mammal with a peptide, an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous

bacteriophage which display functional immunoglobulin binding domains on their surfaces. The library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with any of the proteins (or fragments) , or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.

In the present invention, the methods described herein may be employed to screen for further anti-PRRll antibodies and other agents that bind PRR11, and/or biologically modify properties of PRR11 (e.g. have antagonistic properties) . After production and/or isolation, the biological activity of an anti-PRRll antibody molecule may be tested. For example, the ability of the antibody molecule to inhibit growth and/or metastasis of a cancer cell line or primary or secondary tumour cell may be assessed in vitro or in vivo .

Competition between antibody molecules and other agents, or between antibody molecules other antibodies, may be assayed easily in vitro, for example using ELISA and/or by tagging a specific reporter molecule to one antibody molecule which can be detected in the presence of one or more other untagged antibody molecules, to enable identification of antibody molecules which bind the same epitope or an overlapping epitope. Such methods are readily known to one of ordinary skill in the art.

As stated above, in some embodiments the antibody is a bispecific antibody which is engineered to bind PRRll as well as binding one or more other targets e.g. immune receptors, which help to trigger an immune response. Bispecific antibodies according to the invention may bind targets on T cells or NK Cells, e.g. CD3 or CD8, as well as binding PRRll. Several types of bispecific antibodies are known to the skilled person, e.g. chemically-linked F(ab')2 fragment

antibodies in which one Fab fragment binds PRRll and the other Fab fragment binds an immune molecule.

As stated above, in some embodiments the antibody is a triabody. Triabodies have three binding domains, allowing them to bind three distinct targets. Triabodies of the invention bind PRRll as well as another tumour associated antibody and an immune target.

Antibody molecules disclosed herein may be used in chimeric antigen receptor strategies in which the PRRll-specific antibody is

expressed by cytotoxic T cells resulting in the T cells being directed to, and reacting against, PRRll-expressing cells, e.g.

cancer cells. Preferably, the antibody will be a Fab fragment, a F(ab')2 fragment, a scFv, a diabody, or a triabody.

Antibody conjugates

Alternatively or additionally, the antibody molecules of the present invention may be conjugated or linked to a therapeutically active moiety, for example a moiety that is cytotoxic. Such antibodies may be useful for targeting cancer that is spreading or prone to spread and delivering the therapeutically active moiety to cancer cells.

A further class of groups that can be incorporated into the

antibodies of the present invention are affinity tags that can be introduced into the antibodies to enable them to be manipulated or detected in one or more subsequent steps. A wide range of affinity tags are known in the art suitable affinity tags include members of specific binding pairs, antibodies and antigens, biotin which binds to streptavidin and avidin, polyhistidine (e.g. hexa-His or tri-His tags) or amino di- or tri-carboxylates which bind to metal ions such as Ni ²⁺ or Co ^2~ , Flag or Glu epitopes which bind to anti-Flag antibodies, S-tags which bind to streptavidin, calmodulin binding peptide which binds to calmodulin in the presence of Ca2+;

ribonuclease S which binds to aporibonuclease S; and c-Myc which recognises anti-c-Myc antibody. Examples of other affinity tags that can be used in accordance with the present invention will be apparent to those skilled in the art. Antibodies including these affinity tags can be easily purified and manipulated.

The term "therapeutically active moiety" encompasses a moiety having beneficial, prophylactic and/or therapeutic properties.

In one embodiment the therapeutically active moiety is a cytotoxic chemotherapeutic agent. Cytotoxic chemotherapeutic agents are well known in the art and include anti-cancer agents such as:

Alkylating agents including nitrogen mustards such as

mechlorethamine (HN2) , cyclophosphamide, ifosfamide, melphalan (L- sarcolysin) and chlorambucil; 10 ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonates such as busulfan; nitrosoureas such as carmustine (BCNU) , lomustine (CCNLJ) , semustine (methyl-CCN-U) and streptozoein ( streptozotocin) ; and triazenes such as decarbazine (DTIC;

dimethyltriazenoimidazolecarboxamide) ;

Antimetabolites including folic acid analogues such as methotrexate (amethopterin) ; pyrimidine analogues such as fluorouracil (5- fluorouracil ; 5-FU) , floxuridine ( fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside) ; and purine analogues and related inhibitors such as mercaptopurine ( 6-mercaptopurine ; 6-MP) ,

thioguanine ( 6-thioguanine; TG) and pentostatin (2'- deoxycofonnycin) . Natural Products including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin

(actinomycin D) , daunorabicin (daunomycin; rubidomycin) ,

doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin Q; enzymes such as L-asparaginase ; and biological response modifiers such as interferon alphenomes. Miscellaneous agents include platins, i.e. platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin anthracenedione such as mitoxantrone and antbracycline; substituted urea such as

hydroxyurea; methyl hydrazine derivative such as procarbazine (N- methylhydrazine, MIH) ; and adrenocortical suppressant such as mitotane (o, p'-DDD) and aminoglutethimide ; taxol and

analogues /derivatives ; and hormone agonists/antagonists such as flutamide and tamoxifen.

Methods of conjugating antibodies to therapeutic agents are well known in the art. In a further embodiment, the cytotoxic moiety is a cytotoxic peptide or polypeptide moiety by which we include any moiety which leads to cell death.

Cytotoxic peptide and polypeptide moieties are well known in the art and include, for example, ricin, abrin, Pseudomonas exotoxin, RNase, tissue factor and the like.

The use of ricin as a cytotoxic agent is described in Burrows & Thorpe, P.N.A.S. USA 90: 8996-9000, 1993, incorporated herein by reference, and the use of tissue factor, which leads to localised blood clotting and infarction of a tumour, has been described by Ran et al., Cancer Res. 58: 4646-4653, 1998 and Huang et al., Science 275: 25 547-550, 1997. Tsai et al . , Dis . Colon Rectum 38: 1067- 1074, 1995 describes the abrin A chain conjugated to a monoclonal antibody and is incorporated herein by reference. Other ribosome inactivating proteins are described as cytotoxic agents in WO

96/06641. Pseudomonas exotoxin may also be used as the cytotoxic polypeptide moiety (see, for example, Aiello et al, P.N.A.S. USA 92: 10457-10461, 1995.

Certain cytokines, such as TNF and IL- 2, may also be useful as cytotoxic and/or therapeutic agents. Certain radioactive atoms may also be cytotoxic if delivered in sufficient doses. Thus, the cytotoxic moiety may comprise a radioactive atom which, in use, delivers a sufficient quantity of radioactivity to the target site so as to be cytotoxic. Suitable radioactive atoms include phosphorus-32 , iodine-125, iodine-131, indium-Ill, rhenium-186, rhenium- 188 or yttrium-90, or any other isotope which emits enough energy to destroy neighbouring cells, organelles or nucleic acid. Preferably, the isotopes and density of radioactive atoms in the antibody of the invention are such that a dose of more than 4000 cGy, and more preferably at least 6000, 8000 or 10000 cGy, is delivered to the target site and, preferably, to the cells at the target site and their organelles, particularly the nucleus . The radioactive atom may be attached to the binding moiety in known ways. For example, EDTA or another chelating agent may be attached to the binding moiety and used to attach lllln or 90Y. Tyrosine residues may be labelled with 125 I or 1311. Alternatively, any of these systems can be incorporated into a prodrug system. Such prodrug systems are well known in the art and include ADEPT systems in which an antibody according to the present invention is conjugated or conjugatable or fused to an agent capable of converting a prodrug to a cytotoxic moiety is an enzyme for use in antibody directed enzyme prodrug therapy.

In some cases, the anti-PRRll antibody is not conjugated to a cytotoxic agent. In some embodiments, the therapeutic effect of such unconjugated antibodies is derived from the antibody's ability to act by biologically modifying properties of PRRll (e.g. as a PRRll antagonist) ; reducing PRRll expression and/or downstream activity, which can be measured by assaying the appropriate second messengers. In some embodiments, the therapeutic effect of such unconjugated antibodies is derived from the antibody's ability to recruit the immune complement system or to recruit immune effector cells such as antigen presenting cells, T-cells and/or NK cells. Pharmaceutical compositions

The anti-PRRll antibody molecules of the present invention may be comprised in pharmaceutical compositions with a pharmaceutically acceptable excipient.

A pharmaceutically acceptable excipient may be a compound or a combination of compounds entering into a pharmaceutical composition which does not provoke secondary reactions and which allows, for example, facilitation of the administration of the anti-PRRll antibody molecule, an increase in its lifespan and/or in its efficacy in the body or an increase in its solubility in solution. These pharmaceutically acceptable vehicles are well known and will be adapted by the person skilled in the art as a function of the mode of administration of the anti-PRRll antibody molecule.

In some embodiments, anti-PRRll antibody molecules may be provided in a lyophilised form for reconstitution prior to administration. For example, lyophilised antibody molecules may be re-constituted i sterile water and mixed with saline prior to administration to an individual .

Anti-PRRll antibody molecules will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the antibody molecule. Thus

pharmaceutical compositions may comprise, in addition to the anti- PRRll antibody molecule, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the anti-PRRll antibody molecule The precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route, as discussed below.

In some embodiments, the pharmaceutical preparation will exclude water and/or exclude glycerol and/or exclude sodium azide and/or exclude bovine serum albumin (BSA) and/or exclude phosphates and/or exclude surfactants. For intra-venous administration, e.g. by injection, the

pharmaceutical composition comprising the anti-PRRll antibody molecule may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers,

antioxidants and/or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine;

preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium

chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol ; 3'- pentanol; and m-cresol); low molecular weight polypeptides;

proteins, such as serum albumin, gelatin or immunoglobulins;

hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagines, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol;

salt-forming counter-ions, such as sodium; metal complexes (e.g. Zn- protein complexes); and/or non-ionic surfactants, such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG) .

A pharmaceutical composition comprising an anti-PRRll antibody molecule may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

An anti-PRRll antibody molecule as described herein may be used in a method of treatment of the human or animal body, including

prophylactic treatment (e.g. treatment before the onset of a condition in an individual to reduce the risk of the condition occurring in the individual; delay its onset; or reduce its severity after onset) . The method of treatment may comprise administering an anti-PRRll antibody molecule to an individual in need thereof.

Administration is normally in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated. Appropriate doses of antibody molecules are well known in the art (Ledermann J. A. et al . (1991) Int. J. Cancer 47: 659-664; Bagshawe K.D. et al . (1991) Antibody, Immunoconj ugates and Radiopharmaceuticals 4: 915-922) . Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used. A therapeutically effective amount or suitable dose of an antibody molecule may be determined by comparing its in vitro activity and in vivo activity in an animal model.

Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the antibody is for

prevention or for treatment, the size and location of the area to be treated, the precise nature of the antibody (e.g. whole antibody, fragment) and the nature of any detectable label or other molecule attached to the antibody.

A typical antibody dose will be in the range 100 g to 1 g for systemic applications, and 1 ]ig to 1 mg for topical applications. An initial higher loading dose, followed by one or more lower doses, may be administered. Typically, the antibody will be a whole antibody, e.g. the IgGl or IgG4 isotype. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. Treatments may be every two to four weeks for subcutaneous administration and every four to eight weeks for intra-venous administration. Treatment may be periodic, and the period between administrations is about two weeks or more, e.g.

about three weeks or more, about four weeks or more, or about once a month. Treatment may be given before, and/or after surgery, and/or may be administered or applied directly at the anatomical site of surgical treatment or invasive procedure. Suitable formulations and routes of administration are described above. In some preferred embodiments, the therapeutic effect of the anti- PRRll antibody molecule may persist for several half-lives,

depending on the dose. For example, the therapeutic effect of a single dose of anti-PRRll antibody molecule may persist in an individual for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more.

Screening Methods

The anti-PRRll antibody molecules of the present invention may be used in screening methods for identifying other PRR11-binding molecules, e.g. further PRRll antibodies. Screening methods can also be used to assess the capacity of candidate compounds to inhibit cancer cell growth and/or reduce tumour growth, invasion, migration, adhesion, metastatic capacity and/or cell number. In vitro and in vivo methods are provided.

PRRll-binding agents can be identified via competition binding assays; the binding of candidate agents is determined by observing the ability of the candidate agent to block or displace anti-PRRll antibodies from binding PRRll molecules or fragments. Such assays are very well-known in the art. Some such assays use a single sub- maximal concentration of radiolabeled antibodies (which may be as described herein) in competition with the candidate agent at various concentrations to determine the ICsc of the candidate agent, giving an indication of the candidate agent's affinity for the PRRll molecules or fragment. PRRll-binding agents identified in this way can then be used in other aspects of the invention.

PRRll-binding agents, e.g. anti-PRRll antibodies, can be screened in vitro and/or in vivo for their ability to inhibit cancer cell growth and/or reduce tumour growth. These screening methods are described herein, and broadly rely on a comparison of cancer cell or tumour growth, invasion, migration, adhesion, metastatic capacity and/or cell number in the presence of the PRRll-binding agent, with growth, invasion, migration, adhesion, metastatic capacity and/or cell number in the absence of the PRRll-binding agent. Although such screening methods in general are known to the skilled person, the present invention provides for the first time a method of screening that relies on binding to PRRll present at the surface of cancer cells .

Examples

Example 1 - Detection of PRRll expression and localisation

Materials and Methods

Human tissue specimens

Appropriate informed consent was obtained from patients diagnosed with cancer. Tissues were collected prior to biopsy or surgery under Addenbrookes Hospital Tissue Bank Consent for research

Antibodies

Abl31652, Ab84565 and Ab67244 primary anti-PRRll antibodies were obtained from Abeam (Cambridge, UK) . HPA023923 primary anti-PRRll antibody was obtained from Sigma-Aldrich (Dorset, UK) . PA5-30760 primary anti-PRRll antibody was obtained from Thermo Fisher

Scientific Inc. (Illinois, USA) .

Cell Surface Biotinylation Biotinylation of cell surface proteins in Panel, LNCaP and HeLa cells were performed using Cell Surface Biotinylation Kit (Pierce, UK) . Cells were washed in ice cold PBS twice then incubated with biotin, on a rocker at 4°C. The biotin reaction was quenched by adding quenching buffer before scraping the cells. Cells were pelleted at 500g for three minutes, washed in TBS and lysed in lysis buffer. Lysed cells were sonicated for 30 minutes using a Biorupter at 4°C. Cell debris was removed by centrifugation at lOOOOg for 2 minutes. Cleared lysate was then incubated with agarose resin for an hour. The column was washed to remove non-specific protein and biotinylated proteins were eluted in SDS-PAGE buffer containing DTT.

Western blotting

Samples were prepared in sample buffer and run on 10% SDS-PAGE, transferred to nitrocellulose and probed using the following antibodies at the indicated dilutions; Beta-actin (1:3000), HSP70 (1:3000), IGFR (Cell Signalling, 1:1000) and B23 (Abeam, 1:20,000) . PRR11: HPA023923 (1:500), Abl31652 (1:1000), Ab 84565 (1:500), Ab67244 (1:500), PAS-30760 ( 1 : 500 ) .

FACS Analysis

Cells were grown until ~-80% confluent, washed twice in ice cold PBS, scraped and pelleted (3,500 rpm, 3 minutes) . Viable cells were counted using a haemocytometer and blocked in PBS containing 2% BSA for 15 minutes at 4°C. 2x106 cells were re-suspended in lOOul of PBS containing 0.5% BSA and incubated for 30 minutes at 4°C on a flatbed roller with primary antibody. After washing twice in PBS containing 0.5% BSA cells were incubated for 30 minutes at 4°C on a flat-bed roller with either a-mouse or a-rabbit Alexa Fluor 647 secondary antibody (Life Technologies, 1:1000) or a-goat APC secondary antibody (R&D, 1:200) . Cells were then washed twice as before and analysed on a FACS Calibur machine (BD Biosciences) .

Viable single cells were selected by gating the population on forward and side scatter. Alexafluor 488 and allophycocyanin (APC) fluorescence intensity was measured for each antibody and compared to secondary antibody only and unstained control populations. All conditions were tested on three independent biological replicates and a minimum of 50,000 events were counted each time.

Histology and Immunohistochemistry

Immunohistochemistry was performed on a pancreatic tissue microarray (TMA) comprised of 6mm cores. Samples were stained for PRRll using HPA023923 primary antibody and counterstained with hematoxylin to show cell nuclei in blue. Tissue was either provided by

Addenbrookes Hospital Tissue Bank or purchased from Stretton

Scientific or Insight Biotechnology. All immunohistochemistry (IHC) was performed using a Bondmax autostainer. PRRll was stained using 1.5M Tris EDTA, pH8.0 for antigen recovery and rabbit anti-PRRll antibody (1:150, Atlas Antibodies) diluted in a buffer containing 300mM Tris buffered saline, 1% donkey serum (Sigma Aldrich) and 0.05% Tween. Nuclei were counterstained with haematoxylin and slides coverslipped using DPX. All slides were imaged using an Aperio scanner at 20x magnification. siRNA-mediated PRRll knockdown

siRNA-mediated PRRll knockdown was completed using Smartpool siRNA

(Dharmacon) as directed by the manufacturer. Cells were incubated for 72 hours post transfection before harvesting.

Res lts

Antibodies

Of the five antibodies tested by Western blotting (HPA023923,

Abl31652, Ab84565, Ab67244 and PAS-30760), four gave bands of approximately the right height, i.e. around 40kDa (HPA023923,

Abl31652, Ab67244 and PAS-30760; see Figure 1A) . Many antibodies showed higher molecular weight bands which may represent post- translationally modified forms of PRRll or nonspecific bands.

Cell Surface PRRll Detection

Western blots of lysed and sonicated HeLa and Panel cells were used to demonstrate the cell-surface location of the PRRll protein.

Biotin does not cross the cell membrane, so protein biotinylation is limited to cell-surface proteins. Cell-surface proteins were biotinylated and the cells were lysed and sonicated, as described above. Following affinity-purification of the cell-surface fraction on avidin beads, PRR11 was detected via western blot using HPA023923 primary antibody. Almost all of the detected PRR11 protein is present in the cell surface fraction (Figure IB) .

The cell-surface location of PRR11 protein was confirmed by

performing FACS on viable, unpermeablized Panel cells and MiaPaCa2 cells. All five anti-PRRll primary antibodies (Ab84565, PA5-30760, HPA023923, Abl31652 and Ab67244) were able to achieve a large (over 10-fold) increase in staining with Alexafluor 647-tagged secondary antibodies of Panel and iaPaCa2 cells compared to cells treated with the secondary antibodies alone, indicating binding of the primary antibodies to the cell surface (Figure 2) .

Immunohistochemistry of pancreatic cancer ducts (Figure 3A) bladder cancer, ovarian cancer, breast cancer and lung cancer (Figure 3B) show strong staining. Discrete lines at the periphery of the cells are visible, particularly in at the high magnification of Figure 3A, indicating a membranous localisation.

Validation of PRR11 staining

Knockdown of PRR11 expression using siRNA results in a marked reduction in immunohistochemical signal (Figure 4A and 4B) , which is evident in the darker staining of cells in Figure 4A that had been treated with siRNA controls (not designed to target PRR11) compared with the markedly reduced staining of cells in Figure 4B that were treated with siRNA designed to target PRRll. Figure 4C shows a similar reduction in PRRll-staining by western blot of cells treated with siRNA designed to target PRRll (ii) compared to cells treated with siRNA not designed to target PRRll (i) . These data confirm that the anti-PRRll antibody is indeed detecting PRRll and also demonstrate that siRNA can be used as a way of reducing PRRll expression . Example 2 - Quantification of P R11 expression

Mate-rials and Methods

Tissue Samples and Immunohistochemistry

Patient samples were obtained and prepared as described in Example 1, above. Immunohistochemistry was performed, as described above, on pancreatic samples, including pancreatic cancer samples, bladder cancer samples, lung samples including lung cancer samples and ovarian samples including ovarian cancer samples.

PRRll expression scoring

Tissue staining was measured by intensity only and was scored as none (no staining) = 0, weak (staining was inconsistent and/or weak) = 1, moderate (appreciable staining) = 2, or strong (very intense staining) = 3. All grouped pvalues (n=~3) were calculated using a Kruskai-Wallis test. All pairwise comparisons were completed using a Mann-Whitney test. A p-value of <0.05 was classed as significant.

Results

PRRll was found to be overexpressed in pancreatic cancer. Figure 5A shows that PRRll was detected on almost all pancreatic cancer tissue compared to no detection in normal tissue. A very low proportion of chronic (non-tumour) pancreatitis tissue showed weak staining, with the remainder showing no PRRll staining. The difference in PRRll expression between pancreatic cancer tissue and either normal pancreas tissue or chronic pancreatitis tissue is highly significant (p < 0.0001) . Figure 5B shows the immunohistochemistry of normal pancreas tissue (black arrows, top panel), chronic pancreatitis tissue (red arrows, bottom panel) and pancreatic tumour tissue (brown staining, both panels) that exemplify the samples from which the scores in Figure 5A were taken.

PRRll was found to be overexpressed in bladder cancer, with

expression being detectable in the large majority of bladder cancer tissue (Figure 6) . The degree of expression changes significantly with stage (Figure 6A) and grade (Figure 6B) in bladder cancer, with high expression being linked to an increased chance of recurrence (data not shown) . Hence, PRRll expression levels provide prognostic indication. Figure 6C shows that patients with low PRRll expression levels (upper curve) have substantially higher recurrence-free survival probability than those with high PRRll expression (lower curve) . The survival probability of high-PRRll patients is

decreased relative to low-PRRll patients at every time point, and is particularly decreased after about 45 months.

PRRll was also found to be overexpressed in lung cancer and ovarian cancer (Figure 7) . In lung (Figure 7A) , the majority of squamous carcinoma tissue displayed strong staining whereas most normal lung tissue displayed no PRRll staining, with a minority showing weak staining. This represents a significant difference (p = 0.038) . In ovarian tissue (Figure 7B) , only ovarian cancer tissue (serous adenocarcinoma) showed PRRll staining with over 70% of ovarian cancer tissue displaying strong or moderate staining (normal ovarian tissue and benign serous cystadenocarcinoma showed no PRRll

staining, and this difference is highly significant; p < 0.001) .

PRRll was also found to be overexpressed in cholangiocarcinoma

(Figure 8) . In normal (benign) tissue, less than 50% of tissue displayed PRRll staining compared with almost all

cholangiocarcinoma, of which over 75% showed strong or moderate staining for PRRll (Figure 8A) . Figure 8B shows biliary tract brush specimens stained for PRRll using HPA023923, which exemplify the samples from which the scores in Figure 8A were taken.

These results show that PRRll expression is significantly increased across a range of different cancer types.

Example 3 - PRRll expression in normal tissues

Materials and Methods

Tissue Samples and Immunohistochemistry

6mm cores from the adrenal gland, bone marrow, breast, cerebellum tissue, cardiac muscle, cerebral grey matter, cerebral white matter, colon, endometrium, oesophagus, eye, hypophysis, kidney, larynx, liver, lung, mesothelium, ovary, pancreas, parathyroid gland, peripheral nerves, prostate, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus gland, tonsi 1 or uterine cervix were obtained from a total of 3 or 4 subjects for each sample type. Samples were obtained, prepared and

immunohistochemistry was performed as a performed on a tissue microarray (TMA) as described above in Example 1, above.

PRRll expression scoring

Tissue expression levels were measured by staining intensity and were scored as = "-" (no staining) ; weak (staining was inconsistent and/or weak) = "+"; moderate (appreciable staining) = "++"; or strong (very intense staining) = "+++".

Results

The intensity of PRRll staining for each tissue is presented below in Table 1.

Table 1

Normal tissue score

Tissue Pathology (3 or 4 cores per

Hypophysis Normal/cancer adjacent normal -/-/- adenohypophysis tissue

Kidney Normal renal tissue -/-/-

Larynx cancer adjacent normal/normal +/+/- larynges tissue

Liver Normal hepatic tissue +/+/+

Lung Normal lung tissue +/-/-

Mesothelium Normal mesothelial/lung -/-/- tissue

Ovary Normal ovary tissue -/-/-

Pancreas Normal pancreatic tissue -/-/+

Parathyroid gland Normal parathyroid gland -/-/- tissue (thyroid gland tissue)

Peripheral nerves Normal peripheroneural tissue -/+/+

Prostate Normal prostatic tissue +/-/+

Salivary gland Normal/cancer adjacent normal +++/+++/+++

salivary gland tissue

Skeletal muscle Normal skeletal muscle tissue -/-/-

Skin Cancer adjacent normal/normal +/++/+

skin tissue

Small intestine Normal small intestine tissue +/-/-

Spleen Normal spleen tissue +/-/-

Stomach Normal gastric tissue (smooth +/+/+

muscle)

Testis Normal testis tissue +/+/+ (in spermatids only)

Thymus gland Normal thyroid gland tissue -/-/+

Thymus gland Normal thymic or haemorrhage -/++/+

tissue

Tonsil Normal tonsilar tissue -/-/-

Uterine cervix Cancer adjacent normal -/+/- cervical canals tissue Table 1 shows that, in contrast to the results obtained in Example 2, PRR11 expression is generally very weak in healthy tissue. Taken together, Examples 2 and 3 indicate that PRRll-binding agents preferentially bind to cancerous tissues.

Example 4 - Sensitisation of cancer cells to chemotherapy by PRR11 knockdown

Materials and Methods

Pancreatic cancer cells were treated with siRNA to knockdown PRR11 expression and were cultured in the presence or absence of

Gemcitabine. Negative controls were treated with scrambled siRNA (NT) instead of PRRll siRNA. Pancreatic cancer cells treated with 0.50 μΜ Gemcitabine or not treated with Gemcitabine were used for Western blot experiments. Pancreatic cancer cells treated with 0.25 μΜ, 0.50 μΜ, 1.0 μΜ or 2.0 μΜ Gemcitabine were used for cell death assays .

Western blotting

Pancreatic cancer cells were lysed and whole cell lysate of was stained for PRRll (using HPA023923) . Samples were prepared in sample buffer, run on 10% SDS-PAGE and transferred to

nitrocellulose. Anti-p21, anti-cyclin D3, anti-cyclin Dl, anti- phosphorylated CHK2 , anti-total CHK2, anti-phospho-PTEN (p-PTEN) , anti-integrin β-4 and anti-a-tubulin was used to blot for expression of these proteins.

Cell death assay

Pancreatic cancer cells were treated with siRNA to knockdown PRRll expression and were cultured in the presence or absence of

Gemcitabine at 0.25 μΜ, 0.50 μΜ, 1.0 μ or 2.0 μΜ. Negative controls were treated with scrambled siRNA (NT) instead of PRRll siRNA. siRNA-mediated PRR11 knockdown

siRNA-mediated PRR11 knockdown was completed using Smartpool siRNA ( Dharmacon) as directed by the manufacturer. Cells were incubated for 72 hours post transfection before harvesting.

Results

Expression of PRR11 was not detected by Western blot following PRRll-knockdown siRNA, as shown in Fig. 9A (columns 2 and 4) .

Expression of the other proteins is altered by Gemcitabine treatment to varying degrees, whereas PRRll-siRNA knockdown leaves their expression broadly unaffected.

Fig. 9B shows that PRRll-knockdown results in a substantial

reduction in the viability of pancreatic cancer cells exposed to Gemcitabine treatment at all concentrations of Gemcitabine that were used. This indicates that PRRll inhibition achieves sensitization of cancer cells to chemotherapy.

All references, including patent documents, disclosed herein are incorporated by reference in their entirety for all purposes, particularly for the disclosure referenced herein.

The aim of the following Examples was to identify the best

commercial antibodies that recognize PRRll - and to evaluate the tractability of PRRll in a surrogate ADC assays.

Example 5 - Immunohistochemistry

Obj ective

To assess PRRll expression in human tissue using TMA' s

specific to each tissue type.

Methodology

FDA approved human normal tissue TMA slides were analyzed by IHC. Slides were stained using Bondmax autostainer and scanned using the Aperio system. Results and Discussion

PRR-11 expression in normal and tumour tissues

Normal human tissue TMA slides from Insight Biotechnology were analyzed for PRR11 expression using IHC. Each case on the scanned image was carefully scored as below and the percentage was

calculated against total number of cases.

Zero: No expression

One: Weak expression

Two: Moderate expression

Three: High expression

The data is represented in the bar graphs as in Figure 10

PRR-11 expression in lung normal and cancer tissue

Human lung tissue TMA slides from Insight Biotechnology were analyzed for PRR11 expression using IHC. In each case, the scanned image was carefully scored and the percentage was calculated as above .

The data is represented in the bar graphs as in Figure 11. Conclusion

PRR11 is very weakly expressed in normal spleen and liver. All the lymph derived tissues showed no expressions except thymus and tonsils with a weak staining. In head and neck tissues PRR11 is expressed moderately in contrast to a very strong expression in the tumor tissues. Complete absence of PRR11 in colon of both normal and tumor tissue is observed. Interestingly, the base of hair follicle and sebaceous gland of skin showed moderate to high expression whereas low PRR11 expression was observed in keratinocytes .

PRR11 is very weakly expressed in normal lung tissue and in normal tissue adjacent to the tumour. PRR11 is moderately expressed in adenocarcinoma whereas moderate to strong expression was observed in squamous cell carcinoma. Most of the small cell carcinomatous tissue does not show PRR11 expression with the exception of few

carcinomatous cells that show weak to moderate expression. The data suggests that there is differential expression of PRRll between normal and tumor tissues. Such differential expression provides the therapeutically active moiety conjugated to a PRRll- targeting agent with a medically-useful therapeutic index, by only targeting tumor cells and not normal cells.

Example 6 - FACS Analysis

Obj ective

Develop FACS analysis using the commercial antibodies under different conditions.

Methodology

Stable cell lines (HeLa, Panc-1 and MiaPaCa2) were maintained and harvested after 70-80% confluency. The cells were scraped and incubated with primary and secondary antibodies (one hour each) after blocking with 2% BSA (30 min) and analyzed by FACS caliber. This method was applied across different cell lines using different antibodies . To determine the expression of PRRll, four different commercial antibodies were used on three different cell lines and analyzed by FACS. The primary antibodies used are as follows.

1. Polyclonal rabbit anti-PRRll (Sigma: HPA 023923)

2. Polyclonal goat anti-PRRll (Santacruz: SC-161167)

3. Polyclonal mouse anti-PRRll (Abeam: Abl31652)

4. Monoclonal rabbit anti-PRRll (Abeam: AB84565)

To look for the evidence of internalization the cells were incubated with primary and secondary antibodies at 37°C and compared with cells that were incubated with primary and secondary antibodies at 4°C. At 4°C the cell will have limited ability to cycle their cell surface proteins where at 37°C we would expect a greater degree of cell cycling of cell surface proteins. Hence a loss of a positive FACs signal at 37°C would suggest the target protein is

internalized. Results

PRRll expression at these two different temperatures in different cell lines is plotted as histograms and are shown in Figures 12A-C. Referring to Figure 12A (Panc-1 cells) : Compared to the secondary controls (blue peak) with all the commercial antibodies a clear shift in PRRll is observed (orange peak) suggesting that PRRll is expressed on the majority of Panc-1 cells. With HPA rabbit, Abeam mouse and Abeam rabbit antibodies 10 to 100 fold PRR-11 expression was observed. In contrast, around 10 fold difference was observed with Santa Cruz (SC) goat antibody.

Surprisingly, when the cells were incubated with antibodies at 37°C the peak shifts towards left side (green peak) of the graph

providing an indication of internalization of PRRll protein at 37°C. This is true for the four antibodies used.

Referring to Figure 12B (HeLa cells) : Compared to the secondary controls (blue peak) with all the commercial antibodies a clear shift in PRRll is observed (orange peak) suggesting that PRRll is expressed on the majority of HeLa cells. With HPA rabbit, Abeam mouse and Abeam rabbit antibodies 10 to 100 fold PRRll expression was observed. In contrast, around 10 fold difference was observed with Santa Cruz (SC) goat antibody.

Surprisingly, when the cells were incubated with antibodies at 37°C the peak shifts towards left side (green peak) of the graph

indicating the possibility of internalization of PRRll protein at 37°C. This is true for the four antibodies used.

Referring to Figure 12C (MiaPaCa2 cells) : Compared to the secondary controls (blue peak) with all the commercial antibodies a clear shift in PRRll is observed (orange peak) suggesting that PRRll is expressed on the majority of MiaPaCa2 cells. With HPA rabbit, Abeam mouse and Abeam rabbit antibodies 10 to 100 fold PRRll expression was observed. In contrast, around 10 fold difference was observed with Santa Cruz (SC) goat antibody. Surprisingly, when the cells were incubated with antibodies at 37°C the peak shifts towards left side (green peak) of the graph

indicating the possibility of internalization of PRRll protein at 37°C. This is true for the four antibodies used.

Conclusion

This data confirms the expression of PRRll in Panc-1, HeLa and MiaPaCa2 cell lines with four different antibodies. Importantly, there is a shift in the peak at 37°C which suggests internalization of PRRll.

Example 7 - ZAP Assay

Obj ective

To evaluate the activity of PRRll antibodies associated with a cytotoxic therapeutic moiety.

Methodology

ZAP cytotoxicity assay was developed to test the PRRll commercial antibodies.

In the ZAP assay, secondary antibody is conjugated to Saporin, a ribosome inhibitor. When the primary antibody recognizes target antigen, the secondary antibody binds to the primary antibody with the conjugate.

If the target antigen is internalized/endocytosed, Saporin protein is released in the cytosol to inactivate the ribosomes and thus kills the cells. Cells not expressing the target antigen do not internalize and are not affected. In this Example, the cell death was detected by measuring absorbance as the end point.

Surrogate ADC ZAP assay was optimized using mouse transferrin as a positive control. Briefly, HeLa cells 3,000 per well were seeded in a 96- well flat-bottom plate and incubated overnight at 37°C in the incubator. After the incubation, Fab-ZAP reagent (4.5μg/ml) was made in DMEM complete media. This media was used to dilute mouse transferrin with the starting concentration of μg/ml. The

transferrin antibody was further diluted.

Higher concentrations 4, 0.4 and 0.04 μg/ l of transferrin antibody was able to kill the cells with cytotoxicity 57.5, 60.3 and 50.2 respectively as calculated with no antibody ZAP control.

Percent cytotoxicity is calculated as:

% Cytotoxicity = Mean OP of ZAP control-Mean OP of sample _x ioo

Mean OP of ZAP control

Results and Discussion

ZAP assay in both HeLa cells and in Panc-1 confirms that the PRR11 protein is internalized and the cytotoxic Saproin is released resulting in cell death. This suggests that agents which bind PRR11 can be internalized and therapeutic moieties can be released internally of PRR11 expressing cells.