WO2014177662A1

WO2014177662A1 - In vitro method for determining if a subject suffering from cancer is responsive to a treatment comprising administering an effective amount of cxcl4l1 or an inhibitor of cxcl4l1

Info

Publication number: WO2014177662A1
Application number: PCT/EP2014/058931
Authority: WO
Inventors: Andreas Bikfalvi
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Universite de Bordeaux
Priority date: 2013-04-30
Filing date: 2014-04-30
Publication date: 2014-11-06

Abstract

The invention relates to an in vitro method for determining if a subject suffering from cancer is responsive to a treatment comprising administering an effective amount of CXCL4L1 or an inhibitor of CXCR3 or CXCL4L1 comprising the step of determining the presence or the absence of tumor cells expressing CXCR3 wherein the presence of tumor cells expressing CXCR3 is indicative that the subject is responsive to a treatment comprising administering an effective amount of an inhibitor of CXCL4L1 or an inhibitor of CXCR3, the absence of tumor cells expressing CXCR3 is indicative that the subject is not responsive to a treatment comprising administering an effective amount of an inhibitor of CXCL4L1 or an inhibitor of CXCR3.

Description

In vitro method for determining if a subject suffering from cancer is responsive to a treatment comprising administering an effective amount of CXCL4L1 or an inhibitor of CXCL4L1 FIELD OF THE INVENTION:

The present invention relates to an in vitro method for determining if a subject suffering from cancer is responsive to a treatment comprising administering an effective amount of CXCL4L1 or an inhibitor of CXCR3 or CXCL4L1 .

BACKGROUND OF THE INVENTION:

Angiogenesis and invasion are closely linked, playing an important role in physiological processes such as wound healing and in diseases such as cancer, diabetic retinopathy and various inflammatory disorders (Folkman, 1995). In particular, the expansion of solid tumors and other cancers critically depends on angiogenesis (Folkman, 1971 ) making anti-angiogenesis strategies relevant for cancer therapy (Folkman, 2001 ).

So, it has been shown that the chemokine CXCL4 and peptides derived from its carboxyl-terminal domain known to display significant antiangiogenic activity in vitro (Hagedorn et al., 2001 ; Jouan et al., 1999; Maione et al., 1990) and in vivo (Hagedorn et al., 2001 ; Maione et al., 1990; Sharpe et al., 1990) also suppress growth of various tumors (Maione et al., 1991 ; Tanaka et al., 1997) and metastasis (Kolber et al., 1995) in vivo. This effect is related to their antiangiogenic action (Kolber et al., 1995; Maione et al., 1991 ; Sharpe et al., 1990; Tanaka et al., 1997).

CXCL4L1 is a chemokine closely homologous to CXCL4 with only 34 % differences in the amino-terminus encoding the signal sequence and 4.3 % difference in the mature protein.

CXCL4L1 has been shown to be at least 50 times more potent than CXCL4 on cell migration (Struyf et al., 2004). Recombinant CXCL4L1 is 43 times more potent than CXCL4 in inhibiting endothelial cell proliferation, but 500-times more potent than CXCL4 in inhibiting endothelial cell migration (Dubrac et al., 2010).

Furthermore, inhibition of tumor development was observed in tumors derived from implantation of A549, LLC and B16 cells in mice, treated with recombinant CXCL4L1 (Struyf et al., 2007, WO2006/029487). On contrary, in the patent application WO 2010/040766, it has been shown that when coupled with a cytotoxic agent an antibody anti-CXCL4L1 can be useful in the treatment of cancer.

Although CXCL4L1 appears to play an important in cancer, the underlying mechanism is still unknown and does not allow defining a method of treatment of cancer based on this mechanism.

SUMMARY OF THE INVENTION: Now, the inventors have shown a better understanding of CXCL4L1 in tumor growth.

In particular, the inventors have shown that in tumor cells the pathway of CXCL4L1 is dependent on CXCR3.

More precisely, the inventors have discovered that CXCL4L1 specifically inhibits growth of tumor which cells do not express CXCR3 whereas inhibitors of CXCL4L1 inhibit tumor growth which cells express CXCR3.

These results are summarized in the table 1 below:

Table 1 : Summary of effect of various treatments depending on the presence of CXCR3

A subject of the present invention is therefore an in vitro method for determining if a subject suffering from cancer is responsive to a treatment comprising administering an effective amount of:

-an inhibitor of CXCL4L1 or an inhibitor of CXCR3

or

-a CXCL4L1

comprising the step of determining the presence or the absence of tumor cells expressing wherein:

-the presence of tumor cells expressing CXCR3 is indicative that:

-the subject is responsive to a treatment comprising administering an effective amount of an inhibitor of CXCL4L1 or an inhibitor of CXCR3

-the subject is not responsive to a treatment comprising administering an effective amount of a CXCL4L1 ,

-the absence of tumor cells expressing CXCR3 is indicative that:

-the subject is not responsive to a treatment comprising administering an effective amount of an inhibitor of CXCL4L1 or an inhibitor of CXCR3

-the subject is responsive to a treatment comprising administering an effective amount of a CXCL4L1 .

Further to a method for determining to which treatment will be responsive a subject, the present invention also relates to the treatment itself.

Therefore, the invention also relates to an inhibitor of CXCL4L1 for use in the treatment of a cancer wherein tumor cells expressing CXCR3 are present and wherein:

-the inhibitor of CXCL4L1 is

-an antibody or an aptamer directed against CXCL4L1

or

-a RNA complementary to CXCL4L1 mRNA.

Indeed, the inventors have shown that in cancer expressing CXCR3, inhibitors of CXCL4L1 notably decrease the growth of tumor cells.

The inventors have further shown that the inhibitors of CXCL4L1 also decrease invasiveness of cancer. Therefore, the role of inhibitors of CXCL4L1 in the treatment of cancer not only concerns primary cancer but also cancer metastasis.

Anti-cancer compounds that are already known or commercially marketed exert their anticancer properties through various ways, including through direct action on cancer cells. A number of the anti-cancer agents that act directly on cancer cells block cancer cell proliferation or are cytotoxic for cancer cells.

However, even after complete removal or treatment of a primary cancer, a malignant tumour often metastasizes. A metastatic malignant tumour is formed at a location distant from the primary lesion as a result of the metastasis of the primary tumor. This is one of the most important concerns in cancer therapy. Specifically, even if a primary lesion is treated, a patient may die because of the growth of a tumor that has metastasized to another organ. In the case of many types of clinically diagnosed solid cancer (a type of tumor that is a primary lesion resulting from the local growth of cancer), surgical obliteration is thought to be the first means for treatment. However, primary cancer cell metastasis is often observed after surgical operation. Cancer infiltration at a metastatic site spreads over the whole body, so that the patient will die due to the growth of metastatic cancer. It has been reported that for individual bodies having resectable tumors, primary tumor growth or local recurrence are often causes of death. It is thus currently considered that almost 40% of cancer victims with operable tumors will finally die because of metastatic disease following surgical operation. The effect of inhibitors of CXCL4L1 in the treatment of cancer is directed both to the treatment of primary cancer and to the prevention and treatment of metastasis.

The invention also relates to an inhibitor of CXCR3 for use in the treatment of a cancer wherein tumor cells expressing CXCR3 are present and wherein:

-the inhibitor of CXCR3 is

- an antagonist of CXCR3

or

- a nucleic acid molecule.

The invention also relates to CXCL4L1 for use in the treatment of a cancer wherein tumor cells expressing CXCR3 are absent. The invention also relates to a kit for determining if a subject suffering from cancer is responsive to a treatment comprising administering an effective amount of CXCL4L1 or an inhibitor of CXCR3 or CXCL4L1 comprising means for determining the presence or the absence of tumor cells expressing CXCR3.

The present invention also relates to a method of determining the prognosis of a subject suffering from cancer comprising the step of detecting the level of expression of CXCL4L1 in cancer cells obtained from said subject, wherein a high expression of CXCL4L1 indicates that the subject has a poor prognosis.

DETAILED DESCRIPTION OF THE INVENTION: Definitions:

As used therein, the term "cancer" refers to or describes the physiological condition in subjects that is typically characterized by unregulated cell growth or death. Examples of cancer include, but are not limited to, pancreatic cancer, colon carcinoma, renal cell carcinoma, glioma, glioblastoma and osteosarcoma. Pancreatic cancer may be pancreatic carcinomas notably pancreatic adenocarcinomas (e.g., pancreatic ductal adenocarcinomas) as well as other tumors of the exocrine pancreas (e.g., serous cystadenomas), acinar cell cancers, and pancreatic neuroendocrine tumors (such as insulinomas).

As used herein, the terms "subject" and "patient" are used interchangeably.

The "subject" and "subjects" may be a mammal, preferably a primate and more preferably a human.

As used herein, the term "effective amount" refers to the amount of a therapy (e. g. a prophylactic or therapeutic agent) which is sufficient to reduce or ameliorate the severity, duration and/or progression of cancer or one or more symptoms thereof, ameliorate one or more symptoms of cancer, prevent the advancement of cancer, cause regression of cancer, prevent the recurrence, development, or onset of cancer or one or more symptoms thereof, or enhance or improve the prophylactic or therapeutic effect (s) of another therapy (e. g., prophylactic or therapeutic agent).

As used herein, the terms "treat", "treatment" and "treating" cancer refer to the reduction or amelioration of the progression, severity, and/or duration of cancer, that results from the administration of one or more therapies (e.g., one or more prophylactic and/or therapeutic agents).

As used herein, the percentage of sequence identity refers to comparisons among amino acid sequences, and is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the amino acid sequence in the comparison window may comprise additions or deletions (i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical amino acid residue occurs in both sequences or an amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences.

As used herein, the terms "primer" and "probe" refer to the function of the oligonucleotide. A primer is typically extended by polymerase or ligation following hybridization to the target but a probe typically is not. A hybridized oligonucleotide may function as a probe if it is used to capture or detect a target sequence, and the same oligonucleotide may function as a primer when it is employed as a target binding sequence in an amplification primer. It will therefore be appreciated that any of the target binding sequences disclosed herein for amplification, detection or quantisation of the nucleic acid molecule encoding the toxin or the antitoxin of the invention may be used either as hybridization probes or as target binding sequences in primers for detection or amplification, optionally linked to a specialized sequence required by the selected amplification reaction or to facilitate detection.

Probes or primers may contain at least 10, 15, 20 or 30 nucleotides. Their length may be shorter than 400, 300, 200 or 100 nucleotides.

As used herein, the term "CXCR3" relates to all the isoforms of CXCR3. In one particular embodiment, "CXCR3" relates to the isoform CXCR3-A only. Method for determining if a patient is responsive to a treatment

The present invention relates an in vitro method for determining if a subject suffering from cancer is responsive to a treatment comprising administering an effective amount of:

-an inhibitor of CXCL4L1 or an inhibitor of CXCR3

or

-CXCL4L1

comprising the step of determining the presence or the absence of tumor cells expressing CXCR3

wherein:

-the presence of tumor cells expressing CXCR3 is indicative that:

-the subject is not responsive to a treatment comprising administering an effective amount of CXCL4L1 ,

-the absence of tumor cells expressing CXCR3 is indicative that:

-the subject is not responsive to a treatment comprising administering an effective amount of an inhibitor of CXCL4L1 or an inhibitor of CXCR3 -the subject is responsive to a treatment comprising administering an effective amount of CXCL4L1 .

CXCR3 is a well-known chemokines receptor, also called G protein-coupled receptor 9 (GPR9) or CD183 (Clark-Lewis I, et al. 2003).

Three variants of CXCR3 are known: CXCR3-A and CXCR3-B (Lasagni L, et al.

(2003) and CXCR3-alt.

Preferably, the step of determining whether tumor cells in said subject express a CXCR3 receptor is performed on a tumoral sample derived from a patient. For example, the sample can be a biopsy of the patient's tumor, a cell or tissue culture, etc.

Therefore, in one embodiment of the invention, the method comprises the step of providing a tumoral sample from the patient.

The expression of a CXCR3 may be detected at the nucleic acid level or at the protein level. Various techniques known in the art may be used to detect or quantify CXCR3 at the nucleotide level, including sequencing, hybridization, and amplification. Suitable methods include Southern blot (for DNAs), Northern blot (for RNAs), and fluorescent in situ

hybridization (FISH).

In this specific embodiment, the method comprises the step of:

-contacting the sample with the nucleic acid probe or primer that selectively hybridizes to the nucleic acid molecule encoding CXCR3,

-detecting the hybridization of the nucleic acid probe or primer with the nucleic acid molecule. CXCR3 isoforms expression levels may be measured by real-time quantitative polymerase chain reaction (qRT-PCR), for example using gene expression assays of Applied Biosystems on StepOne (Applied Biosystems) according to the manufacturer's protocol. The expression levels of interest transcripts may be normalized to S16 and HPRT1 housekeeping gene transcripts.

When the expression of CXCR3 is measured at the nucleic acid level, it is considered that tumor cells express CXCR3 when CXCR3 gene is expressed at a significative level that is to say at a level sufficient to lead, after translation, to a level of CXCR3 protein detectable by usual protein detection means.

In a preferred embodiment, the expression of CXCR3 is detected at the protein level for example by immunostaining. The detection or quantification of CXCR3 at the protein level may be achieved by well-known methods including, but not limited to, gel migration, ELISA, radio-immunoassays (RIA) immuno-enzymatic assays (IEMA), Western Blot or another method for detecting a bound ligand.

Protein detection methods require the use of a binding partner capable of selectively interacting with CXCR3.

In this specific embodiment, the method of the invention comprises contacting a sample from the patient with a binding partner specific for CXCR3, and determining the presence of the complex binding partner-CXCR3.

Preferably, the binding partner is an antibody, an aptamer or a double-stranded RNA molecule. Most preferably, the binding partner is an antibody.

This antibody may be polyclonal or monoclonal, as well as fragments or derivatives thereof having substantially the same antigen specificity. Fragments include Fab, Fab'2, CDR regions, etc. Derivatives include single-chain antibodies, humanized antibodies, poly- functional antibodies, etc.

Polyclonal antibodies directed against CXCR3 can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production.

Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against CXCR3 can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler et al. Nature. 1975; 256(5517):495-7; the human B-cell hybridoma technique (Cote et al Proc Natl Acad Sci U S

A. 1983;80(7):2026-30); and the EBV-hybridoma technique (Cole et al., 1985, in "Monoclonal

Antibodies and Cancer Therapy," Alan R. Liss, Inc. pp. 77-96).

CXCR3-specific antibodies suitable for use in the present invention are commercially available.

The binding of the binding partner may be directly detected if the binding partner is labeled with, for example, a fluorescent dye, a radioisotope or another detectable moiety.

Alternatively, the binding of the binding partner to CXCR3 may be indirectly detected through the use of a labeled second reagent that binds to the binding partner. Such reagents include labeled anti-immunoglobulin antibodies and enzyme-linked anti-immunoglobulin antibodies that bind to the anti-CXCR3 antibody, and labeled nucleic acids that can bind to one or both strands of a dsRNA molecule. In a specific embodiment, the method of the invention comprises contacting a sample from the patient with an antibody specific for CXCR3 and determining the presence of an immune complex.

The aforementioned assays generally involve the bounding of the binding partner (ie. antibody or aptamer) in a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against CXCR3. A body fluid sample containing or suspected of containing CXCR3 is then added to the coated wells. After a period of incubation sufficient to allow the formation of binding partner-CXCR3 complexes, the plate(s) can be washed to remove unbound material and a labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

Different immunoassays, such as radioimmunoassay or ELISA, have been described in the art.

Method for determining the presence of CXCR3 cells using a monoclonal antibody is disclosed in Garcia-Lopez et al. 2001 .

In a preferred embodiment, the cancer is selected from the group consisting of pancreatic cancer, in particular pancreatic ductal adenocarcinoma, colon carcinoma, renal carcinoma, in particular clear cell renal cell carcinoma, glioma, glioblastoma and osteosarcoma.

In a more preferred embodiment, the cancer is a pancreatic cancer or an osteosarcoma. Some subtypes of pancreatic ductal adenocarcinoma, clear cell renal cell carcinoma, glioma, glioblastoma and osteosarcoma express CXCR3.

Examples of tumor cells which express CXCR3 are Panc-1 cells or MG63.

Examples of tumor cells which do not express CXCR3 are BxPC3 cells. Inhibitor of CXCL4L1 or inhibitor of CXCR3

In one embodiment, the treatment comprises administering an effective amount of an inhibitor of CXCL4L1 or an inhibitor of CXCR3 and

-the presence of tumor cells expressing CXCR3 is indicative that the subject is responsive to a treatment comprising administering an effective amount of an inhibitor of CXCL4L1 or an inhibitor of CXCR3 -the absence of tumor cells expressing CXCR3 is indicative that the subject is not responsive to a treatment comprising administering an effective amount of an inhibitor of CXCL4L1 or an inhibitor of CXCR3. Indeed, the inventors have shown that administering monoclonal antibody or siRNA that inhibits CXCL4L1 or CXCR3 leads to an inhibition of growth when tumor cells express CXCR3 whereas the tumor growth is increased when tumor cells do not express CXCR3.

The inventors have also shown that inhibitor of CXCL4L1 blocks not only CXCL4L1 but also the interaction with CXCR3 receptors in cells which express CXCR3 such as monocytes, lymphocytes and tumors expressing CXCR3.

As disclosed above, CXCL4L1 is a paralog of CXCL4, also known as platelet factor variant-1 , PFAvl , PF4var1 , PF4ALT or SCYB4V1 . It was identified in 1989 (Struyf et al., 2007; Struyf et al., 2004). The amino acid sequence of CXCL4L1 is set forth as SEQ ID NO:1 .

As used therein, an inhibitor of CXCL4L1 is an antibody or an aptamer directed against CXCL4L1 that blocks or reduces substantially the activity of CXCL4L1 or a RNA complementary to CXCL4L1 mRNA that blocks or reduces substantially the expression of CXCL4L1 .

Typically, an inhibitor of CXCL4L1 blocks the anti-angiogenic activity of CXCL4L1 which can be assessed by the FGF-2 stimulated proliferation of bovine aortic endothelial (BAE) cells assay as described in WO 2010/040766).

In one embodiment, the inhibitor of CXCL4L1 is a RNA complementary to CXCL4L1 mRNA and that inhibits its translation.

The complementarity with CXCL4L1 may be complete or partial.

More preferably, the nucleic acid molecule is a siRNA.

As used herein, the term "siRNA" refers to a ribonucleic acid (RNA) or RNA analog comprising between about 10 to 50 nucleotides (or nucleotide analogs) capable of directing or mediating the RNA interference pathway. These molecules can vary in length and can contain varying degrees of complementarity to their target messenger RNA (mRNA) in the antisense strand. The term "siRNA" includes duplexes of two separate strands, i.e. double stranded RNA, as well as single strands that can form hairpin structures comprising of a duplex region. The siRNA may have a length of between about 10 to 50 nucleotides, or between about 15 to 50 nucleotides, or between about 20 to 50 nucleotides, or between about 25 to 50 nucleotides, or between about 30 to 50 nucleotides, or between about 35 to 50 nucleotides, or between about 40 to 50 nucleotides, or between about 10 to 45 nucleotides, or between about 10 to 40 nucleotides, or between about 10 to 35 nucleotides, or between about 10 to 30 nucleotides, or between about 10 to 25 nucleotides, or between about 10 to 20 nucleotides, or between about 15 to 50 nucleotides, or between about 15 to 35 nucleotides, or between about 15 to 30 nucleotides, or between about 15 to 25 nucleotides.

In one embodiment, the siRNA has a length of between 15 to 30 nucleotides.

The application of siRNA to down-regulate the activity of its target mRNA is known in the art. In some embodiments, mRNA degradation occurs when the anti-sense strand, or guide strand, of the siRNA directs the RNA-induced silencing complex (RISC) that contains the RNA endonuclease Ago2 to cleave its target mRNA bearing a complementary sequence. Accordingly, the siRNA may be complementary to any portion of varying lengths on the CXCL4L1 gene. The siRNA may also be complementary to the sense strand and/or the anti- sense strand of the CXCL4L1 gene. Accordingly, siRNA treatment may be used to silence the CXCL4L1 gene, thereby depleting the CXCL4L1 protein downstream.

In another embodiment, inhibitor of CXCL4L1 is an aptamer.

In a preferred embodiment, inhibitor of CXCL4L1 is an antibody.

In one embodiment, the antibody recognizes the histidine residue at position 67 of the mature PF4v1 protein.

An antibody that recognizes the histidine residue at position 67 and blocks the activity of CXCL4L1 is disclosed in WO2010/040766.

Antibodies may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination.

Antibodies may be derived from a number of species including, but not limited to, rodent (mouse, rat, rabbit, guinea pig, hamster, and the like), porcine, bovine, equine or primate and the like.

Procedures for raising "polyclonal antibodies" are well known in the art. For example, anti-CXCL4L1 polyclonal antibodies can be obtained from serum of an animal immunized against CXCL4L1 or a fragment thereof, which may be produced by genetic engineering for example or by peptide synthesis according to standard methods well-known by one skilled in the art. Typically, such antibodies can be raised by administering CXCL4L1 or a fragment thereof subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The antigens can be injected at a total volume of 100 μΙ per site at six different sites. Each injected material may contain adjuvants with or without pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times at six weeks' interval. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. This and other procedures for raising polyclonal antibodies are disclosed by Harlow et al., 1988.

Laboratory methods for preparing monoclonal antibodies are well known in the art (see, for example, Harlow et al., 1988). Monoclonal antibodies may be prepared by immunizing a mammal such as mouse, rat, primate and the like, with CXCL4L1 or a fragment thereof. The antibody-producing cells from the immunized mammal are isolated and fused with myeloma or heteromyeloma cells to produce hybrid cells (hybridoma). The hybridoma producing the monoclonal antibodies are utilized as a source of the desired monoclonal antibody. This standard method of hybridoma culture is described in (Kohler and Milstein, 1975).

Alternatively, the immunoglobulin genes may be isolated and used to prepare a library for screening for specifically reactive antibodies. Many such techniques including recombinant phage and other expression libraries are known to one skilled in the art.

In one embodiment, the treatment comprises administering an effective amount of an inhibitor of CXCL4L1 and

-the presence of tumor cells expressing CXCR3 is indicative that the subject is responsive to a treatment comprising administering an effective amount of an inhibitor of CXCL4L1

-the absence of tumor cells expressing CXCR3 is indicative that the subject is not responsive to a treatment comprising administering an effective amount of an inhibitor of CXCL4L1 .

An inhibitor of CXCR3 is a molecule that blocks or reduces substantially the expression or the activity of CXCR3.

Typically, an inhibitor of CXCR3 substantially blocks the subsequent cascade associated of biochemical events associated with CXCR3 activation in vivo.

As used therein, an inhibitor of CXCR3 is an antagonist of CXCR3 or an aptamer that blocks or reduces substantially the activity of CXCR3 or a RNA complementary to CXCR3 mRNA that blocks or reduces substantially the expression of CXCR3. In one embodiment, the inhibitor of CXCR3 is a RNA complementary to CXCR3 mRNA and that inhibits its translation.

The complementarity may be complete or partial.

Preferably, in this embodiment, the RNA complementary to CXCR3 mRNA is a siRNA.

Anti-CXCR3 siRNAs are commercially available.

In one embodiment, the inhibitor of CXCR3 is an aptamer directed against CXCR3. In one embodiment, the inhibitor of CXCR3 is an antagonist of CXCR3.

Many different classes of small molecule CXCR3 antagonists have been disclosed (for example see JE, Horuk R. (2009), Wijtmans M, et al. (2008), Du X et al. (2009), Hayes ME et al. (2008)).

For example, the CXCR3 antagonists may be a chemical compound named SCH 546738 which binds potently CXCR3 and antagonizes receptor activation by CXCR3 ligands (Jenh CH, et al. BMC Immunol. 2012 Jan 10;13(1 ):2). The small molecule SCH 54678 is of particular interest because it is described to have equivalent binding affinity as monoclonal antibodies (Mab).

In one embodiment, the treatment comprises administering an effective amount of an inhibitor of CXCR3 and

-the presence of tumor cells expressing CXCR3 is indicative that the subject is responsive to a treatment comprising administering an effective amount of an inhibitor of CXCR3

-the absence of tumor cells expressing CXCR3 is indicative that the subject is not responsive to a treatment comprising administering an effective amount of an inhibitor of CXCR3

CXCL4L1 :

In one embodiment, the treatment comprises administering an effective amount of CXCL4L1

and

wherein:

- the presence of tumor cells expressing CXCR3 is indicative that the subject is not responsive to a treatment comprising administering an effective amount of CXCL4L1 -the absence of tumor cells expressing CXCR3 is indicative that the subject is responsive to a treatment comprising administering an effective amount of CXCL4L1 .

Indeed the inventors have shown that CXCL4L1 promote the growth of tumor cells expressing CXCR3 whereas CXCL4L1 has no direct effect on tumor cells not expressing CXCR3 but inhibits tumor growth by inhibiting angiogenesis.

In the context of therapy, the term "CXCL4L1 " should be interpreted broadly and encompasses CXCL4L1 and variants thereof.

Examples of variants of CXCL4L1 used in therapy according to the invention include those described in the international patent application WO201 1012585.

Typically a variant of CXCL4L1 is a protein having at least 96% identity with amino acid sequence set forth as SEQ ID NO: 1 and having substantially the same activity as CXCL4L1 .

The activity of CXCL4L1 may be assessed by measuring inhibition of angiogenesis. This test may be done by the FGF-2 stimulated proliferation of bovine aortic endothelial (BAE) cells assay as described in WO 2010/040766). Alternatively, chemotaxic assays may be used with monocyte migration as read-out.

In one embodiment, the variant of CXCL4L1 has an amino acid sequences having at least 96%, 97%, 98% or 99% identity with SEQ ID NO: 1 .

In a preferred embodiment, the variant of CXCL4L1 comprises histidine 67 of CXCL4L1 .

In a preferred embodiment, the variant of CXCL4L1 comprises the amino acids 58 to 67 of CXCL4L1 .

The fragment of amino acids 58 to 67 of CXCL4L1 is set forth as SEQ ID NO: 2.

Kit for determining if a patient is responsive to a treatment

A further aspect of the invention relates to a kit for determining if a subject suffering from cancer is responsive to a treatment comprising administering an effective amount of CXCL4L1 or an inhibitor of CXCR3 or CXCL4L1 comprising means for determining the presence or the absence of tumor cells expressing CXCR3.

In one embodiment, the presence of tumor cells expressing CXCR3 may be detected at the nucleic acid level.

Then, the means for detecting the presence of tumor cells expressing CXCR3 are nucleic acid probes or primers that hybridize specifically to the nucleic acid molecule encoding CXCR3. In one embodiment, the presence of tumor cells expressing CXCR3 may be detected at the protein level.

Then, the means for detecting the presence of tumor cells expressing CXCR3 comprise a binding partner able of selectively interacting with CXCR3.

Binding partners specific of CXCR3 are disclosed above.

Therapeutic methods of the invention:

The present invention also relates to an inhibitor of CXCL4L1 for use in the treatment of a cancer wherein tumor cells expressing CXCR3 are present and wherein:

-the inhibitor of CXCL4L1 is:

-an antibody or an aptamer directed against CXCL4L1

or

- a RNA complementary to CXCL4L1 mRNA.

Another object of the present invention is a method for treating a cancer in a subject in need thereof comprising administering to said subject a therapeutically effective amount of an inhibitor of CXCL4L1 wherein said cancer comprises tumor cells expressing CXCR3 and wherein the inhibitor of CXCL4L1 is an antibody or an aptamer directed against CXCL4L1 or a RNA complementary to CXCL4L1 mRNA.

Another object of the present invention is a use of an inhibitor of CXCL4L1 for the manufacture of a medicament for treating a cancer wherein tumor cells expressing CXCR3 are present and wherein the inhibitor of CXCL4L1 is an antibody or an aptamer directed against CXCL4L1 or a RNA complementary to CXCL4L1 mRNA.

Inhibitors of CXCL4L1 are disclosed above.

In one embodiment, the inhibitor of CXCL4L1 is an antibody.

In one embodiment, the inhibitor of CXCL4L1 is a RNA complementary to CXCL4L1 mRNA, more preferably a SiRNA.

Preferably, the cancer is selected from the group consisting of pancreatic cancer, in particular pancreatic ductal adenocarcinoma, colon carcinoma, renal carcinoma, in particular clear cell renal cell carcinoma, glioma, glioblastoma and osteosarcoma.

Most preferably, the cancer is pancreatic cancer or osteosarcoma.

In one embodiment, the cancer is a primary cancer.

In other embodiment, the inhibitor of CXCL4L1 is for use in the prevention and the treatment of cancer metastasis wherein tumor cells expressing CXCR3 are present. In one embodiment, the method for treating cancer is a method for preventing and treating cancer metastasis wherein tumor cells expressing CXCR3 are present in a subject in need thereof comprising administering to said subject a therapeutically effective amount of an inhibitor of CXCL4L1 .

In one embodiment, the use of an inhibitor of CXCL4L1 is for the manufacture of a medicament for preventing and treating cancer metastasis wherein tumor cells expressing CXCR3 are present.

Preferably, cancer metastasis is selected from the group consisting of pancreatic cancer, in particular pancreatic ductal adenocarcinoma, colon carcinoma, renal carcinoma, in particular clear cell renal cell carcinoma, glioma, glioblastoma and osteosarcoma.

More preferably, cancer metastasis is selected from the group consisting of colon carcinoma, renal cell carcinoma, glioblastoma and osteosarcoma metastasis.

Most preferably, cancer metastasis is osteosarcoma metastasis. The present invention also relates to an inhibitor of CXCR3 for use in the treatment of a cancer wherein tumor cells expressing CXCR3 are present and wherein:

-the inhibitor of CXCR3 is

- an antagonist of CXCR3

- an aptamer

or

- a RNA complementary to CXCR3 mRNA.

Another object of the present invention is a method for treating a cancer in a subject in need thereof comprising administering to said subject a therapeutically effective amount of an inhibitor of CXCR3 wherein tumor cells expressing CXCR3 are present and wherein the inhibitor of CXCR3 is an antagonist of CXCR3, an aptamer, or a RNA complementary to CXCR3 mRNA. .

Another object of the present invention is a use of an inhibitor of CXCR3 for the manufacture of a medicament for treating a cancer wherein tumor cells expressing CXCR3 are present and wherein the inhibitor of CXCR3 is

- an antagonist of CXCR3

-an aptamer

or

- a RNA complementary to CXCR3 mRNA.

Inhibitors of CXCR3 are disclosed above.

In one embodiment, the inhibitor of CXCR3 is an antagonist of CXCR3. In one embodiment, the inhibitor of CXCR3 is a RNA complementary to CXCR3 mRNA, preferably a SiRNA.

Most preferably, the cancer is pancreatic cancer or osteosarcoma.

In one embodiment, the cancer is a primary cancer.

The present invention also relates to CXCL4L1 for use in the treatment of a cancer wherein tumor cells expressing CXCR3 are absent.

Another object of the present invention is a method for treating a cancer in a subject in need thereof comprising administering to said subject a therapeutically effective amount of CXCL4L1 , wherein said cancer does not comprise tumor cells expressing CXCR3.

Another object of the present invention is a use of CXCL4L1 for the manufacture of a medicament for treating a cancer wherein tumor cells expressing CXCR3 are absent.

As disclosed above, in the context of therapy, the term CXCL4L1 should be interpreted broadly and encompasses CXCL4L1 and variant thereof.

Preferably, the cancer is selected from the group consisting of pancreatic cancer, colon carcinoma, renal carcinoma, glioma, glioblastoma and osteosarcoma.

Most preferably, the cancer is pancreatic cancer or osteosarcoma.

Method of determining the prognosis of a subject suffering from cancer

The present invention also relates to a method of determining the clinical outcome of patients suffering from a cancer or, in other words, to assess the prognosis of a cancer in these patients.

Indeed, the inventors have shown that a high expression of CXCL4L1 indicates a less favourable clinical outcome.

Thus, the present invention also relates to a method of determining the prognosis of a subject suffering from cancer comprising the step of detecting the level of expression of CXCL4L1 in cancer cells obtained from said subject, wherein a high expression level of CXCL4L1 indicates that the subject has a poor prognosis.

On contrary a low expression level of CXCL4L1 indicates that the subject has a good prognosis. In particular, the inventors have shown that the five years overall survival rate is lower for patient with high expression of CXCL4L1 .

Therefore, a high expression level of CXCL4L1 indicates that the subject has a low five years overall survival rate. On contrary a low expression level of CXCL4L1 indicates that the subject has a high five years overall survival rate.

Typically, a high expression level of CXCL4L1 is intended by comparison to a reference value.

Said reference value may be determined in regard to the level of the expression of CXCL4L1 in tumor tissue samples obtained from cancer whose clinical outcome is known.

Once the level of expression of CXCL4L1 quantified for each tumor tissue sample, a reference value permitting the discrimination between good and bad outcome prognosis may be obtained by using statistical analysis in correlation with the clinical outcome of each corresponding cancer patient. In one embodiment, the method according to the present invention comprises the step of comparing said level of CXCL4L1 to a reference value wherein a high level of CXCL4L1 compared to said reference value is predictive that the subject has a poor prognosis.

In a more preferred embodiment, the cancer is selected from the group consisting of colon carcinoma, renal carcinoma, in particular clear cell renal cell carcinoma, glioma, glioblastoma and osteosarcoma.

In a most preferred embodiment, the cancer is osteosarcoma.

Typically the expression level of CXCL4L1 may be measured for example by RT-PCR or immunohistochemistry performed on a sample obtained by biopsy. In a preferred embodiment, the expression level of CXCL4L1 is measured by immunohistochemistry.

A method of prognosis according to the invention may be used in combination with any other methods already used for the prognostic assessment including stage, demographic and anthropometric parameters, results of routine clinical or laboratory examination.

Method of evaluating the risk of a subject to develop metastasis.

In most of solid tumor cancers, metastases are the main cause of death, with a significant reduction of the 5 year survival. More effective treatments are needed in patient metastasis or with a high risk to develop metastasis.

The present invention also enables the evaluation of the risk of a subject to develop metastasis.

Indeed, the inventors have shown that high expression of CXCL4L1 was statistically associated with lower five year metastasis free survival rate.

Therefore, the present invention also relates to a method of evaluating the risk of a subject suffering from cancer to develop metastasis comprising the step of detecting the level of expression of CXCL4L1 in cancer cells obtained from said subject, wherein a high expression level of CXCL4L1 indicates that the subject has an increased risk of metastasis.

On contrary, a low expression of CXCL4L1 indicates that the subject has a decreased risk of metastasis.

Said reference value may be determined in regard to the level of the expression of CXCL4L1 in tumor tissue samples obtained from cancer whose risk to develop metastasis is known.

Once the level of expression of CXCL4L1 quantified for each tumor tissue sample, a reference value permitting the discrimination between high and bad low risk to develop metastasis may be obtained by using statistical analysis in correlation with, for example, the five year metastasis free survival rate of each corresponding cancer patient. In one embodiment, the method according to the present invention comprises the step of comparing said level of CXCL4L1 to a reference value wherein a high level of CXCL4L1 compared to said reference value is predictive that the subject has an increased risk to develop metastasis. In a preferred embodiment, the cancer is selected from the group consisting of pancreatic cancer, in particular pancreatic ductal adenocarcinoma, colon carcinoma, renal carcinoma, in particular clear cell renal cell carcinoma, glioma, glioblastoma and osteosarcoma.

In a most preferred embodiment, the cancer is osteosarcoma.

The invention will further be illustrated in view of the following figures and examples. FIGURES:

Figure 1 shows the expression of CXCR3 in pancreatic adenocarcinoma. Presence of CXCR3 was measured by qRT-PCR for CXCR3 isoforms in pancreatic cancer cell lines in vitro (A) and in primary tumors from BxPC3 and PANC-1 cells implanted into mice (B) Figure 2 shows the in vitro effect of recombinant CXCL4L1 (GST-CXCL4L1 ) on proliferation and invasion of BXPC3 cells.

Figure 3 shows the in vitro effects of CXCL4L1 and CXCR3 knock-down or antibody treatment on the proliferation of Panc-1 cells. Cells were transfected with the specific siRNAs or control siRNAs, grown in complete medium and treated or not with the monoclonal anti- CXCL4L1 antibody over 120 h. Figure 4 shows the effect of anti-CXCL4L1 antibody on in vivo pancreatic tumor development on BxPC3 cells. Tumors were implanted subcutaneously in Rag2 γ/c mice and injected with Mabl_1 or with the control antibody by injection through the tail vein. Tumor growth was monitored over a two month period (A). On the day of sacrifice, tumors were excised and volumes determined (B).

Figure 5 shows the effect of anti-CXCL4L1 antibody on in vivo pancreatic tumor development on Panc-1 cells.Tumors were implanted subcutaneously in Rag2 γ/c mice and injected with Mabl_1 or with the control antibody by injection through the tail vein. Tumor growth was monitored over a three month period (A). On the day of sacrifice, tumors were excised and volumes determined (B).

Figure 6 shows a comparison of CXCL4L1 expression in osteosarcoma tissues by RT-qPCR (A) and expression level of CXCL4L1 in osteosarcoma by histological type (B). Tissue sections from normal bone marrow, benign-tumor and osteosarcoma were stained with an anti-CXCL4L1 (Mab-L1 ).

Figure 7 shows the expression level of CXCL4L1 in osteosarcoma metastasis by histological type. Tissue sections from osteosarcoma metastasis were stained with Mab-L1 .

Figure 8 shows Kaplan-Meier analysis of overall survival (A) and Kaplan-Meier analysis of metastasis-free survival (B) regarding immunohistochemical expression of CXCL4L1 in osteosarcoma. Figure 9 shows immunohistochemical expression of CXCR3 in osteosarcoma tissue samples and clinical outcomes. Tissue sections osteosarcoma were stained with anti- CXCR3. Expression level of CXCR3 in osteosarcoma by histological type (A). Kaplan-Meier analysis of overall survival (B). Kaplan-Meier analysis of metastasis-free survival (C) are shown.

Figure 10 shows effect of monoclonal Mabl_1 on tumor growth in the chick chorioallantoic membrane (CAM) model. MG63.2 cells are implanted into the CAM and grown for 5 days in the presence or absence of Mabl_1 . Tumor size is measured by biomicrocopy. Figure 11 shows RT-qPCR analysis of CXCL4L1 (A) and CXCR3 (B) in MG63.2 cell line and xenograft tumor. Tumor expression of CXCL4L1 and CXCR3 were confirmed by immunostaining. Figure 12 shows that CXCL4L1 blocking antibody inhibits growth of sub-cutaneous osteosarcoma tumor. It is shown tumor weigh (A) and growth (B) treated or not with CXCL4L1 blocking antibody and quantification of hypoxia probe (C), CD34 (D) and Ki-67 (D) of tumor treated or not with CXCL4L1 blocking antibody. Figure 13 shows that CXCL4L1 blocking antibody inhibits growth of orthotopic osteosarcoma tumor. Tumor growth of mice treated or not with CXCL4L1 blocking antibody was analyzed by bioluminescence (B) and IRM (A). The RT-qPCR analysis of the expression of CXCL4L1 and CXCR3 in orthotopic tumors were confirmed by immunostaining. Hypoxia probe (C) and Ki-67 (D) of tumor treated or not with CXCL4L1 blocking antibody was quantified.

EXAMPLES:

Materials and methods

Human tumor samples

Human adenocarcinoma samples were provided by Prof Martin Schilling (Klinik fur

Allgemeine Chirurgie, Viszeral-, GefaB- und Kinderchirurgie, Homburg, Germany). Fresh tumor tissues were obtained during surgery and directly snap-frozen in liquid nitrogen.

Cell culture, PDAC-CAM model, treatment and transfection

Pancreatic tumor cell lines (BxPC3, PANC-1 ) were cultivated in DMEM (1 g/l glucose) (Invitrogen, Cergy Pontoise, France). Human osteosarcoma MG63 cells (ATCC CRL-1427) were grown in MEM medium (Life Technologies, Inc. BRL). All media were supplemented with 10% fetal bovine serum, antibiotics (penicillin/streptomycin), and L-glutamine. Human umbilical vein endothelial cells (HUVEC, Lonza, Levallois-Perret, France) were maintained in EBM-2 (Lonza) supplemented with EGM-2 SingleQuots (Lonza), which contain 2% FBS. Human hepatic myofibroblasts were grown in DMEM that contained 5% fetal calf serum, 5% pooled human serum and 5 ng/mL epidermal growth factor (EGF). Cultures were incubated at 37 ^<C in 5% C02.

Fertilized chicken eggs (Gallus gallus) were handled as previously described. At embryonic day 10 (E10), 4 millions BxPC3 cells diluted in serum free medium in a final volume of 40μΙ were deposited as a thin layer on the intact chick chorioallantoic membrane (CAM) surface. Luciferase-tagged BxPC3 and PANC-1 tumor cells were established by transducing cells with recombinant lentiviral vectors expressing firefly luciferase.

To study the involvement of epigenetic events in the CXCL4L1 silencing in pancreatic BxPC3 cell line, cells were treated with 5'-aza (1 μΜ) for at least 15 days and/or TSA (150 nM) for the last 24 hours. The MG63 cell line was treated with 10ng/ml of IL-1 β during 48 hours.

Expression of CXCR3 in pancreatic adenocarcinoma

CXCR3 isoforms expression levels were measured by real-time quantitative polymerase chain reaction (qRT-PCR) using gene expression assays (Applied Biosystems) on StepOne (Applied Biosystems), according to the manufacturer's protocol. The expression levels of interest transcripts were normalized to S16 and HPRT1 housekeeping gene transcripts. The inventors next analysed CXCR3 protein expression by immunostaining in pancreatic tumor cells, in subcutaneously or orthotopically tumors implanted into mice and in human samples. Primary antibodies used for CXCR3 and CXCR3B staining were: Mouse Anti- human CXCR3 (clone 2ar1 ) ref ab64714, Abeam and Mouse Anti-CXCR3B Monoclonal Antibody, Cat. No. CAB010, Creative Biomart respectively.

Cell proliferation and invasion assay

Cell proliferation was evaluated by the WST-1 assay (Roche, Neuilly sur Seine Cedex, France) using standard procedures. Briefly, cells were seeded in 96-well plates at a concentration of 1 ^χ 10⁴ (HUVECs) or 3 ^χ 10³ (BxPC3 and PANC-1 ) cells/well and allowed to adhere overnight. Complete medium was replaced by serum-free medium with or without FGF-2, CXCL4L1 proteins and Mabl_1 blocking antibody at indicated concentrations for 24 (HUVECs), 48 (BxPC3 cells) or 120 (PANC-1 cells) hours.

Cell invasion assays were carried out using 24-well microchemotaxis Boyden chambers precoated with 10 μg Matrigel (BD Biosciences, Le Pont-de-Claix, France). Cells (1 x 1 0⁵) were seeded to the upper chamber and FGF-2 or 1 % FCS were used as a chemoattractant for HUVEC and BxPC3 cells respectivelly. Cells were incubated at 37 ^<C for 24 h, then, cells on the lower face of the filter were fixed, stained with crystal violet and counted under a microscope.

Immunoprecipitation

3 pooled T6 implanted CAM or 1 x10^s BxPC3 cells were collected in 500μΙ lysis buffer containing 10% glycerol, 50 mM Tris-HCI, 0.1 mM EDTA, 5 mM sodium fluoride, 1 mM sodium pyrophosphate, 1 mM sodium vanadate, a protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany), 1 % (vol/vol) Nonidet P-40, 0.1 % SDS, and 0.1 % deoxycholate; pH7.5. Lysates were incubated for 1 hour at 4°C on a rotating mixer, and centrifuged at 13,000 rpm for 10 min at 4^<C. Approximately 20μΙ of Protein G slurry (Invitrogen) previously incubated overnight at 4^<C with 2 μg of Mabl_1 antibody, was added to each sample, adjusted to 1 mg protein/ml lysis buffer and incubated for 1 hour at 4°C. After centrifugation to remove non-specific binding, proteins bound to the antibody were eluted with 50 μΙ of loading buffer containing β-mercaptoethanol, boiled at 95^ for 10 min, and analyzed by Western blot. Membranes were immunoblotted with Mabl_1 antibody and proteins were visualized with enhanced chemiluminescence reagent (Pierce, Rockford, IL).

RT-PCR, RNA extraction and semi-quantitative RT-PCR

Total RNA was extracted from cells, mice tumors and human tumors using the TriZol reagent protocol (Invitrogen). After DNAse treatment (DNA free, Ambion) and quantification, reverse transcription was done with 2 μg of total RNA using the RevertAid H Minus First strand cDNA Synthesis kit (Fermentas) and random hexamers.

CXCR3 expression levels were measured by real-time quantitative polymerase chain reaction (qRT-PCR) using gene expression assays (Applied Biosystems) on StepOne (Applied Biosystems), according to the manufacturer's protocol. The expression levels of interest transcripts were normalized to S16 and HPRT1 housekeeping gene transcripts. RNAs from normal human tissues were obtained from Strategene.

Histology and immunohistochemistry and tissue microarray

Frozen tissues were cut into 8 μΓη-thick sections and stained with hematoxylin-eosin for histological analyses, localization of metastasis and selection of most representative tumor areas. Primary antibodies used were rabbit anti-human cytokeratin 19 (ab8591 ; Abeam, Cambridge), rabbit anti-mouse NG2 Chondroitin Sulfate Proteoglycan (AB5320, Millipore, Temecula), rat anti-mouse CD31 (557355, BD Biosciences, San Jose, California), mouse anti-human Ki-67 (M7240, DakoCytomaton, Glostrup, Denmark), mouse anti-human CXCR3 (ab64714, Abeam), chicken anti-human VAMP-2 (AB5625, Millipore), rabbit anti- human a-amylase (sc-25562, Santa Cruz) and mouse monoclonal antibody for CXCL4 (Mabl_4, clone 2G2-3E3-3C1 1 ) and for CXCL4L1 (Mabl_1 , clone 9E1 1 -2D5-2G1 ). As a negative control, the primary antibody was substituted with a IgG control antibody (Jackson ImmunoResearch laboratory, West Grove, Pennsylvania). Corresponding secondary antibodies were Alexa Fluor 488- or 546- coupled antibodies (Invitrogen) or HRP-coupled antibodies (Dakocytomation). Cell nuclei were stained with dapi dye. Imaging was carried out using a Nikon DXM Eclipse E600 microscope. Tissue microarray was done as follows. Paraffin sections from tissue microarrays (TMA) built up using several human pancreatic tissue specimens originated from different types of pancreas cancers lesions including lymph node metastases and from pancreatitis specimens were obtained from BioCat GMbH (Heidelberg, Germany). The TMA contain each 96 spots with 1 .5 mm diameter. The tissues were fixed in 4% paraformaldehyde in PBS (Cat.N ⁰ : PACA0909-2-OL). The TMA exact composition can be obtained at www.biocat.com/bc/pdf/Pancreas%20Carcinoma%20TMA. TMAs (4 μηι thick) were de-paraffinized in xylene and hydrated serially in 100%, 95%, and 80% ethanol. Endogenous peroxidase was quenched in 3% H₂0₂ in PBS for 1 hour, then slides were incubated with Mabl_1 monoclonal antibody diluted at 1 /1000 overnight at 4^<C. Sections were washed three times in PBS, and antibody binding was revealed using the Ultra- Vision Detection System anti-Polyvalent HRP/DAB kit according to the manufacturer's instructions (Lab Vision). Finally, the slides were counterstained with Mayer's hematoxylin and washed in distilled water. After dehydration and mounting, expression of CXCL4L1 or CXCR3 was evaluated by two independent investigators.

The expression of CXCR3 was analyzed on 70 human pancreatic adenocarcinomas, included in 3 tissue microarray blocks obtained from 70 patients who underwent pancreatic surgical resection at Beaujon Hospital between 1997 and 2004. The cores were taken at random sites inside the tumors and each tumour specimen was represented by 4 x 1 -mm cores on the tissue microarray. The following histopathological and clinical data were available: tumor size (10-105 mm; median 30 mm), tumor differentiation (well-differentiated n=35, moderately differentiated n=27 or poorly differentiated n=8), lymph node metastasis (positive in 44/70), perineural (present in 47/59 available) and vascular invasion (present in 44/60 available), pT (T1 n=1 , T2 n=10 or T3 n=59) classification according to TNM/UICC, disease-free and overall survival. Immunostaining of a 3μηι slice was performed as indicated. The immunohistochemical staining was evaluated in a semi-quantitative fashion. A score was calculated, obtained by multiplying the intensity (negative scored as 0, weak scored as 1 , moderate scored at 2 and strong scored as 3) by percentage of stained cells (0 to 100%). The mean score of the 4 cores which represented each tumour was taken into account.

Labeling of MabL1

Monoclonal antibody against CXCL4L1 (MabL1 ) was labeled with IRDye 800CW (Protein Labeling Kit-HighMW#928-38040, LI-COR®, Lincoln, NE, USA) or with biotin using the kit sulfo-NHS-LC-Biotin (PIERCE, Rockford, IL, USA) following the manufacturer's instructions. The conjugates were dialyzed extensively against phosphate buffered saline to remove excess of non-reacted dyes. MabL1 antibody was labeled without loss of their initial activities.

The IRdye-MabL1 and biotinylated-MabL1 were used in bio-distribution, pharmacokinetics and tolerance studies in mice and the IRdye-MabV1 in the study of targeting tumors expressing CXCL4L1 . Statistical analysis

Results are presented as mean ± the standard error of the mean. Statistical significance was determined by a one-tailed, unpaired Student's t test using Prism 5.03 GraphPad Software. For the tissue microarrays, the statistical analysis was performed by using a Kruskal-Wallis test, followed by Dunns multiple comparison procedure. P-values <0.05 were considered significant (^***, <0.001 ; ^**, <0.01 ; ^*, <0.05).

In vivo mouse models

Male RAG-y/c mice were housed and treated in the animal facility of Bordeaux 1 University ("Animalerie Mutualisee Bordeaux I"). All animal procedures were done according to institutional guidelines.

Clearance kinetic of MabL1 conjugated to IRDve or Biotin: To determine the clearance rate and possible non-specific binding of the antibody, RAG-y/c mice (n=50) received an intravenous injection of 25μg of labeled antibodies into the tail vein. At different times post-injection, animals were imaged with the Odyssey Imaging System (LI-COR®) equipped with the MousePODTM until there was no detectable signal above background. After imaging, blood was collected intracardially and the organs were removed, scanned on the Odyssey Imaging System and homogenized for determination of Mabl_1 concentration by an indirect ELISA assay. Data were fitted to two-phase exponential decay by using Prism software (GraphPad, San Diego, CA, USA).

ELISA assays

To evaluate the half-life and clearance of Mabl_1 antibody in mice, we perform a direct ELISA assay: recombinant CXCL4L1 protein diluted in a coating solution is immobilized on a microplate. After several steps of washing and blocking, plasma samples and organs lysates are added to the plate. For samples containing MabL1 conjugated to biotin, the result is obtained directly after adding streptavidin-HRP and a substrate solution (TMB) on a microplate reader set to 450nm. While for samples containing IRdye-MabV1 an additional step with a biotinylated anti-mouse antibody is required.

Xenograft models:

Eight weeks old mice were anaesthetized with intraperitoneal injection of ketamine (150mg/kg) and xylazine (15mg/kg) and xenografted with 3x10⁶ and 3.75x10⁵ BxPC3 cells in 10ΟμΙ serum free medium by subcutaneous and intra-spleenic injection respectively.

Treatment with MabL1 antibody:

In order to evaluate the role of endogenous CXCL4L1 in tumor development, anti- CXCL4L1 neutralizing antibodies were injected into mice. RAG-y/c mice were divided in two groups: one which received 5C^g IgG control antibody (Jackson ImmunoResearch Laboratories; control group, n=16), the other 5C^g of anti-CXCL4L1 neutralizing antibody (group Mabl_1 , n=16). Treatment started at day 14 and antibodies were injected twice a week. Tumor dimensions were measured each week and tumor volumes were calculated using the formula: 4/3 x Π a x b² (where a and b are the largest and the smallest radius respectively). Mice were sacrificed after 7 weeks of treatment; tumors were removed, measured and stored in liquid nitrogen before immunohistochemistry studies.

In vivo tumor targeting:

To evidence tumor targeting of Mabl_1 , the antibody was labeled with IRdye and injected into the tail vein of mice with subcutaneous BxPC3 tumors and with metastasis after spreading of BxPC3 cells through portal vein following intra-spleenic injection. 6 days after injection, animals were euthanized and the tissues were removed and imaged on the Odyssey Imaging System. Immediately after imaging, organs were frozen and cut into 10 and 40μηι sections for immunohistochemistry studies and Odyssey imaging respectively.Cell proliferation and invasion assay

ln-vivo tumor growth imaging:

Mice bearing intra-pancreatic tumors were injected intraperitoneal^ with 200 μΙ of 25 mg/ml d-luciferin (Invitrogen) in PBS and were anesthetized during imaging using isoflurane through nose cone. Bioluminescence images were acquired using a Photon Imager (Biospace Lab, Paris, France).

Xenopus embryo model

Xenopus embryos and microinjection:

Eggs were obtained from Xenopus laevis females primed with human chorionic gonadotrophin, fertilized artificially and cultured in 0.1 X MMR [49]. Two blastomeres of 4-cell stage embryos were injected using a Nanoject system (Drummond Scientific). β-galactosidase and whole-mount in-situ hybridization were_carried out using current protocols. For the generation of anti-sense probe, the X-msr cDNA was linearized with Stul and transcribed with T7 RNA polymerase.

Plasmids, RNA synthesis: For expression in Xenopus embryos, the entire coding region of CXCL4L1 was cloned into the Kpnl and Bglll sites of pxT7 vector. For microinjection experiments, the capped mRNAs were synthesized in-vitro by using mMessage mMachine kit (Ambion).

Results

Expression of CXCR3 in pancreatic adenocarcinoma

The inventors have tested whether CXCR3 is expressed in BxPC3 and PANC-1 cells. Messenger RNA expression analysis (Fig. 1 ) and immunostaining showed high expression of CXCR3-A in PANC-1 cells but no expression in BXPC3 cells. Some CXCR3-B mRNA was detected in PANC-1 cells and low mRNA expression was also seen in BxPC3. However, no CXCR3 protein expression was seen in BxPC3 in contrast to PANC-1 .

When PANC-1 cells are implanted subcutaneously or orthotopically into mice, a 50- fold and 35-fold increase in expression was observed for CXCR3-A and CXCR-B respectively in comparison to cell cultures as measured by mRNA expression. In addition, positive immunoreactivity for CXCR3 was also evidenced in these tumors.

The inventors have next analyzed CXCR3 expression in human 70 PDAC samples included in tissue microarrays. The cytoplasmic and nuclear scores ranged from 20 to 300 (median: 175) and from 0 to 300 (median: 175) respectively. Twenty-two (31 %) of tumors presented a membranous staining pattern, some of which had cytoplasmic staining. CXCR3 nuclear and membranous expression correlated positively with tumor size (p=0.02 and p=0.06 respectively). CXCR3 membranous expression correlated with presence of perineural invasion (p=0.04). Functional studies with CXCL4L1 in pancreatic carcinoma cells and vasculature

In vitro effects of CXCL4L1

CXCL4L1 exhibited a much stronger inhibitory effect on endothelial cell proliferation than CXCL4 (43-fold higher, Dubrac et al.). However, no growth inhibitory effect of was observed in BxPC3 cells (Fig. 2A). Invasion of endothelial cells, but not that of BxPC3 cells, was significantly inhibited by CXCL4L1 . No effect on apoptosis on either tumor or endothelial cells was seen (data not shown). The inventors next compared the role of CXCL4L1 in another pancreatic cancer cell line, Panc-1 cells, which in contrast do express CXCR3 protein. This cell line already expresses CXCL4L1 in cell culture. The inventors therefore, investigated the effect of the modulation of CXCL4L1 activity on this pancreatic tumor cell line by blocking CXCL4L1 activity. Blocking by either monoclonal antibody or siRNA for CXCL4L1 or CXCR3 led to inhibition of cell proliferation (Fig. 3). The sequences of siRNA are given in table 2.

Target Target Sequence Oligos

Table 2 Combining blockade by monoclonal antibody and CXCR3 siRNA further decreased cell proliferation. This indicates that CXCL4L1 is a positive regulator of Panc-1 growth and that CXCR3 is also involved in the growth promoting activity.

Effect of CXCL4L1 on vascular development in vivo

In order to ascertain that CXCL4L1 is able to specifically act on blood vessel development in vivo, the inventors performed additional experiments using the Xenopus model, a well established in vivo model of vessel development. The non injected side of the embryos showed no disturbance of ISV formation as measured by in situ hybridization with X-msr. CXCL4L1 was efficiently expressed after transduction as measured by ELISA (136 ± 61 .6 pg/ng of total CXCL4L1 protein for 4 embryos). Results are shown in table 2 below.

Table 3

Overexpression of CXCL4L1 caused a specific alteration of the vascular phenotype. Endothelial cells do not align properly to form intersomitic vessels leading to a strong disorganization of the vessel. Most defects correspond to either reduced growth of ISV or their abnormal projection into adjacent somites. The non-injected side of the embryos showed no disturbance of ISV formation. Injected embryos showed no obvious defects in the vascular vitelline network, head vessels or posterior cardinal veins. Moreover, the formation of somites seems to occur normally in injected embryos. Quantification showed that 68-77 % of injected embryos with CXCL4L1 mRNA exhibited ISV alteration (Table 2). Together, these data suggest that CXCL4L1 regulates angiogenesis acting on guidance cues to migrating endothelial cells or vessel assembly.

Effect of endogenous CXCL4L1 on tumor development

RAG-y/c mice were inoculated subcutaneously with BxPC3 or PANC-1 cells and then treated with Mabl_1 antibody which is a monoclonal antibody specific of CXCL4L1 developed by the inventors. Prior to the tumor experiments, the inventors characterize the half-life, clearance and tumor targeting of this antibody in mice. Infrared imaging of whole animals and organs illustrate a rapid dispersion of unconjugated IRDye (the half life in the blood of the IRDye is 5.525 hours and Mabl_1 labeled with IRDye followed by complete clearance from mice 72 h after injection). A strong fluorescent signal was observed at the site of injection, in the liver and bladder 30 min post injection followed at 2 h by kidneys and lungs. Fluorescent intensities in these organs decreased then until 8 hours whereas the signal in liver was still detected at 48 h. The presence of the labeled antibody within the blood stream was evidenced by the blue fluorescent signal. Mabl_1 concentration was also quantified in in plasma and in organs. The half-life in the blood of the anti-CXCL4L1 antibody was 7.5 and 6.8 days when labeled with IRDye and biotin respectively. On day 8 post-injection, no detectable amounts of antibody in heart and lungs are measured and in liver, kidneys and spleen those reached respectively 15, 18 and 8% of the initial concentration determined at 4h.

The inventors next investigated whether Mabl_1 was able to target metastatic lesions in mice. BxPC3 cells were injected in the spleen (1 x10^s cells) in mice. Metastatic lesions usually occurred 2 month post-injection. Infrared-dye labelled Mabl_1 antibody was administered by i.v. injection. Mice were sacrificed at the end of the experiment and organs imaged. Imaging of organs revealed targeting by the antibody of metastatic lesions in the lung and kidney.

In BxPC3 tumor xenografts, injection of the anti-CXCL41 antibody in mice yields to an increase in tumor growth (Fig. 4). Mabl_1 (25μg) was injected 2-times a week and tumor growth was monitored during a 2-month period. Forty-eight days after implantation tumor size increased by 2-fold in Mabl_1 treated animals when compared to non-treated controls (2612 mm3 in Mabl_1 -treated mice versus 1 184 mm³ in control mice). The tumor growth in Mabl_1 treated mice was 81 .5 mm3/day in comparison to 34.9 mm3/day in untreated controls. Vessel density was quantified by estimating the number of small (<10 μηι²), medium (10-100 μηι²) or large CD31 -positive vessels (>100 μηι²). An increase in the density of small vessels was observed in treated tumors compatible with an angiogenesis-related effect. These results are compatible with an inhibitory effect of endogenous CXCL4L1 in pancreatic carcinoma that may be overridden during tumor progression by production of stimulatory angiogenesis factors.

In Panc-1 tumor xenografts, the administration of CXCL4L1 antibody had an opposite effect (Fig. 5). Using the same treatment protocol as for BxPC3 cells, administration of the Mabl_1 antibody led to a reduction of tumor size in both subcutaneously and orthotopically implanted tumors (s.c. implantation: 1 140 mm³ in Mabl_1 -treated mice versus 1845 mm³ in control mice; orthotopic implantation: 2.8e+06 cpm in MabLI -treated mice versus 1 .25e+07 cpm in control mice). Orthotopic tumor growth was monitored using Luciferase biomuminescence. The inventors also determined tumor weight, lung metastasis, proliferation and angiogenesis. Tumor weight was reduced in both orthotopic and subcutaneous tumors in the presence of MabLI . Lung metastasis as measured by the presence of hS16mRNA was significantly reduced in orthotopic tumors. Furthermore, proliferation measured by hMIB1 expression was also decreased in orthotopic tumors after MabLI injection. However, no effect on vessel quantity measured by PCAM1 expression was seen.

Depending on the expression of CXCR3 receptor, the response to CXCL4L1 blockade was shown to be different. In BxPC3 tumor xenografts, which do not express CXCR3 protein, blockade of endogenous CXCL4L1 resulted in an increase in tumor development and angiogenesis. This is in agreement with an effect exclusively on the vasculature as documented by in vitro experiments and in vivo angiogenesis models. In contrast, in PANC-1 tumor xenografts, which express high levels of CXCR3, the blockade of endogenous CXCL4L1 led to a decreased tumor development. Furthermore, we observed that CXCR3A and B are up -regulated in PANC-1 cells in vivo. This is reinforced by functional studies that show that CXCR3 knock-down diminishes PANC-1 cell proliferation. This is in line with the observation that tumors cells, which express CXCR3-A, exhibit an increase in tumor cell survival and proliferation.

Taken together this suggests that tumor cells that express CXCR3 may override the inhibitory effect of CXCL4L1 on the vasculature, which may explain the effect of the antibody treatment. These datas point to an essential role of CXCR3-A in this context. Thus, tumor response to CXCL4L1 may depend on the expression of CXCR3 on the tumor cells. Analysis of CXCR3 expression in PDAC samples from patients is also in line with a pro-tumor effect. Indeed, when CXCR3 is expressed in tumor cells expression is correlated with tumor progression.

Role of CXCL4L1 in the metastatic spreading of osteosarcoma

The inventors have then investigated the role of CXCL4L1 in other type cancer: in osteosarcoma (OS) and particularly in OS metastasis.

In osteosarcoma, lung metastases are the main cause of death, with a significant reduction of the 5-year survival. More effective treatments are needed for patients with pulmonary metastases. Recent studies have demonstrated that the inhibition of the CXCR3 chemokine pathway down regulates the growth of OS lung metastasis.

Since as shown above CXCL4L1 chemokine angiostatic and chemotactic activities are mediated by CXCR3, the inventors have focused on deciphering the activity of the couple CXCL4L1/CXCR3 in vitro and in vivo together with the expression analysis of the couple, in 82 biopsies of patients with OS to investigate their relation with outcome.

A series of 82 human osteosarcomas were analysed by immunohistochemical staining of tissue microarrays for expression of the CXCL4L1 and the CXCR3 chemokines receptors.

In human patients the link between the expression of CXCL4L1 and CXCR3 chemokines receptors with metastasis free survival and overall survival was analysed, in uni and multivariate analysis.

CXCL4L1 P CXCR3 P

AGE AT DIAGNOSIS (CL) .2586 <18 57 ( 69.5) 22 ( 62.9) 35 ( 74.5)

>=18 25 ( 30.5) 13 (37.1) 12 (25.5)

SEX .8250 FEMALE 34(41.5) 15(42.9) 19 (40.4)

MALE 48 ( 58.5) 20 (57.1) 28 ( 59.6)

TUMOR SIZE (CL) .1642 <= 5CM 5 ( 6.9) 4 ( 12.5) 1 ( 2.5)

>5CM 67(93.1) 28 ( 87.5) 39 ( 97.5)

MISSING 10 3 7

HISTOLOGICAL TYPE .0644

OSOB 46 ( 57.5) 15(42.9) 31 ( 68.9)

Osoc 20 ( 25.0) 12 (34.3) 8 ( 17.8)

OTHERS 14 ( 17.5) 8 ( 22.9) 6 ( 13.3)

MISSING 2 0 2

Table 4: Prognostic factors for metastasis free survival and overall survival of patients with osteosarcoma

Quantitative RT-PCR showed a 2-fold increase in CXCL4L1 expression between MG63 and the highly metastatic MG63.2 OS cells line, and a 6-fold increase of CXCR3 expression in grafted mice. CXCL4L1 was not associated with patient age at surgery, tumor histological type or grade, the response to chemotherapy, in a multivariate or an univariate analysis

In univariate analysis, CXCL4L1 statistically associated with overall survival (p=0.0372). The estimated 5-years overall survival rates of 88.6% and 67.8% for patients with low and high expression of chemokine

Overexpression of CXCL4L1 was statistically associated with an increased risk of metastasis (p=0.001 ) CXCL4L1 expression correlates with invasiness.

Five years metastasis free survival rates of 86.7% and 53.9% in the group with low and high expression of CXCL4L1 .

According to the results in our mouse model, overexpression of CXCL4L1 in human OS was associated with an increased risk of metastasis with a HR of 2,24 (95% CI =[1 .39:3,62], p=0.001 ). Altogether, these results indicate for the first time, that CXCL4L1 could be a pro- tumoral factor that favors metastatic dissemination independently of its antiangiogenic properties.

In addition, in patients, it was shown that CXCL4L1 is a powerful independent prognostic factor in metastasis free survival. CXCL4L1 could be then considered as a new pronostic marker of OS metastatic risk, useful in adapting therapeutic strategies.

Further, by using a specific CXCL4L1 blocking-antibody activity, the effect of the blockade of this chemokine on tumor growth and the presence of lung metastasis were evaluated in a murine orthotopic model of osteosarcoma xenograft.

Inhibition of the CXCL4L1 activity with a specific blocking-antibody resulted in a decrease of tumor growth without significant impact on tumor angiogenesis. This effect was coupled with a reduction of pulmonary metastases in the OS murine model.

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Claims

CLAIMS:

1 . An in vitro method for determining if a subject suffering from cancer is responsive to a treatment comprising administering an effective amount of:

-an inhibitor of CXCL4L1 or an inhibitor of CXCR3

or

-CXCL4L1

wherein:

-the presence of tumor cells expressing CXCR3 is indicative that:

-the absence of tumor cells expressing CXCR3 is indicative that:

-the subject is responsive to a treatment comprising administering an effective amount of CXCL4L1 .

2. The method according to claim 1 :

wherein the treatment comprises administering an effective amount of an inhibitor of CXCL4L1 or an inhibitor of CXCR3 and

wherein:

-the presence of tumor cells expressing CXCR3 is indicative that the subject is responsive to a treatment comprising administering an effective amount of an inhibitor of CXCL4L1 or an inhibitor of CXCR3

-the absence of tumor cells expressing CXCR3 is indicative that the subject is not responsive to a treatment comprising administering an effective amount of an inhibitor of CXCL4L1 or an inhibitor of CXCR3.

3. The method according to claim 1 :

wherein the treatment comprises administering an effective amount of CXCL4L1 and

wherein: - the presence of tumor cells expressing CXCR3 is indicative that the subject is not responsive to a treatment comprising administering an effective amount of CXCL4L1

-the absence of tumor cells expressing CXCR3 is indicative that the subject is responsive to a treatment comprising administering an effective amount of CXCL4L1 .

4. The method according to any of claims 1 to 3 wherein the cancer is selected from the group consisting of pancreatic cancer, colon carcinoma, renal cell carcinoma, glioblastoma and osteosarcoma.

5. An inhibitor of CXCL4L1 for use in the treatment of a cancer wherein tumor cells expressing CXCR3 are present and wherein:

-the inhibitor of CXCL4L1 is:

-an antibody or an aptamer directed against CXCL4L1

or

-a RNA complementary to CXCL4L1 mRNA.

6. The inhibitor of CXCL4L1 according to claim 5 for use in the prevention and the treatment of cancer metastasis wherein tumor cells expressing CXCR3 are present.

7. The inhibitor of CXCL4L1 according to claim 5 or 6 wherein said inhibitor of CXCL4L1 is an antibody.

8. The inhibitor of CXCL4L1 according to claim 5 or 6 wherein said inhibitor of CXCL4L1 is a SiRNA.

9. CXCL4L1 for use in the treatment of a cancer wherein tumor cells expressing

CXCR3 are absent.

10. A method of determining the prognosis of a subject suffering from cancer comprising the step of detecting the level of expression of CXCL4L1 in cancer cells obtained from said subject, wherein a high expression level of CXCL4L1 indicates that the subject has a poor prognosis.

1 1 . A method of evaluating the risk of a subject suffering from cancer to develop metastasis comprising the step of detecting the level of expression of CXCL4L1 in cancer cells obtained from said subject, wherein a high expression level of CXCL4L1 indicates that the subject has an increased risk of metastasis.

12. The method according to claim 10 or 1 1 wherein the cancer is an osteosarcoma.