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Title:
CHEMERIN POLYPEPTIDE FOR THE TREATMENT OF CANCER-INDUCED CACHEXIA
Document Type and Number:
WIPO Patent Application WO/2017/042318
Kind Code:
A1
Abstract:
The present invention relates to methods and pharmaceutical compositions for the treatment of cancer-induced cachexia. In particular, the present invention relates to a method of treating cancer-induced cachexia in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a chemerin polypeptide or a nucleic acid molecule encoding thereof.

Inventors:
STOCKMANN CHRISTIAN (FR)
KLOSE RALPH (FR)
Application Number:
PCT/EP2016/071275
Publication Date:
March 16, 2017
Filing Date:
September 09, 2016
Export Citation:
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Assignee:
INSERM (INSTITUT NAT DE LA SANTÉ ET DE LA RECH MÉDICALE) (FR)
UNIVERSITÉ PARIS DESCARTES (FR)
ASS ROBERT DEBRÉ POUR LA RECH MÉDICALE (FR)
International Classes:
A61K38/17; A61K33/243; A61K39/395; A61P21/06; A61P35/00
Domestic Patent References:
WO2008028250A12008-03-13
Foreign References:
US20040086966A12004-05-06
US20100266589A12010-10-21
Other References:
NTIKOUDI E ET AL: "Hormones of adipose tissue and their biologic role in lung cancer", CANCER TREATMENT REVIEWS, vol. 40, no. 1, February 2014 (2014-02-01), pages 22 - 30, XP028765241
VICTORIA CATALÁN ET AL: "Increased levels of chemerin and its receptor, chemokine-like receptor-1, in obesity are related to inflammation: tumor necrosis factor-[alpha] stimulates mRNA levels of chemerin in visceral adipocytes from obese patients", SURGERY FOR OBESITY AND RELATED DISEASES, vol. 9, no. 2, 1 March 2013 (2013-03-01), NL, pages 306 - 314, XP055249060, ISSN: 1550-7289, DOI: 10.1016/j.soard.2011.11.001
ROH ET AL: "Chemerin-A new adipokine that modulates adipogenesis via its own receptor", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 362, no. 4, 15 September 2007 (2007-09-15), pages 1013 - 1018, XP022249546, ISSN: 0006-291X, DOI: 10.1016/J.BBRC.2007.08.104
J. L. ROURKE ET AL: "Gpr1 is an active chemerin receptor influencing glucose homeostasis in obese mice", JOURNAL OF ENDOCRINOLOGY, vol. 222, no. 2, 14 July 2014 (2014-07-14), GB, pages 201 - 215, XP055249101, ISSN: 0022-0795, DOI: 10.1530/JOE-14-0069
Attorney, Agent or Firm:
COLLIN, Matthieu (FR)
Download PDF:
Claims:
CLAIMS:

1. A method of treating cancer-induced cachexia in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a chemerin polypeptide or a nucleic acid molecule encoding thereof.

2. The method of claim 1 wherein the subject suffers from a cancer selected from the group consisting of bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.

3. The method of claim 1 wherein the chemerin polypeptide comprises an amino acid sequence having at least 90% of identity with SEQ ID NO: 1.

4. The method of claim 1 wherein the nucleic acid molecule is included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

5. The method of claim 1 wherein the chemerin polypeptide or the nucleic acid molecule encoding thereof is administered to the subject in combination with a chemotherapeutic agent.

6. The method of claim 1 wherein the chemerin polypeptide or the nucleic acid molecule encoding thereof is administered to the subject in combination with an anti-VEGF agent.

7. The method of claim 6 wherein the anti-VEGF agent is anti-VEGF antibody.

Description:
CHEMERIN POLYPEPTIDE FOR THE TREATMENT OF

CANCER-INDUCED CACHEXIA

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of cancer-induced cachexia.

BACKGROUND OF THE INVENTION:

Despite its frequent side-effects, chemotherapy generally represents the first course of treatment for cancer patients. The benefits of chemotherapeutic agents stem not only from direct effects on the tumour cell but also from influences on the tumour microenvironment, resulting in a robust immune response that can be crucial to the therapeutic outcome 1 . However, drug delivery poses a significant problem as the vasculature of tumours is inefficient 2 . Using mouse models, it was shown that specific deletion of VEGF in tumour- infiltrating myeloid cells leads to normalized tumour blood vessels and increased tumour cell apoptosis .

Cancer-induced cachexia is the immediate cause of death in about 15% of cancer patients 4"6 . It is characterized by involuntary weight loss that is resistant to nutritional supplementation 1 . Weight loss starts with the breakdown of white adipose tissue (WAT) mediated by the lipolytic enzymes adipose triglyceride lipase (Atgl) and hormone-sensitive lipase (Hsl) 8 as well as loss of skeletal muscle 1 . WAT lipolysis is believed to be induced by tumour-derived factors, such as tumour necrosis factor alpha (TNF-a) and interleukin (IL-) 6 9 ' 10 . After an initial reduction of tumour mass, treatment with chemotherapeutic agents frequently exacerbates cachexia, hampering further treatment and increasing mortality n,u . There is an urgent need for treatment regimens that counter the development of cachexia and thus allow continued chemotherapy.

Chemerin was initially defined as an adipokine 1 but has received considerable interest as a chemoattractant for macrophages, dendritic cells and NK cells 14~16 . NK cells and cytotoxic T cells are particularly important in the immunosurveiUance and suppression of tumours 17 ' 18 and chemerin has been shown to improve NK cell-based tumour surveillance. Expression of the chemerin gene ((Rarres ( retinoic acid receptor responder) 2) is frequently downregulated in human solid tumours, including lung cancer and melanoma. Overexpression of chemerin in melanoma cells in mouse models results in increased NK cell recruitment and tumour suppression 19 .

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of cancer-induced cachexia. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Chemotherapy remains a mainstay of cancer treatment but its use is often limited by the development of adverse reactions. Severe involuntary loss of body weight (cachexia) is a frequent cause of death in cancer patients and is exacerbated by chemotherapy. The inventors show that genetic inactivation of Vascular Endothelial Growth Factor (VEGF)-A in myeloid cells prevents chemotherapy-induced cachexia by inhibiting the lipolysis of white adipose tissue. It also improves clearance of senescent tumour cells by natural killer cells and inhibits tumour regrowth after chemotherapy. The effects depend on the adipokine and chemoattractant chemerin, which is released by the tumour endothelium in response to chemotherapy. The findings define chemerin as a critical mediator of the immune response elicited by chemotherapy as well as an important inhibitor of cancer cachexia. Targeting VEGF signaling should impede the lipolysis and weight loss that is frequently associated with chemotherapy, thereby dramatically improving the therapeutic outcome.

Accordingly the present invention relates to a method of treating cancer-induced cachexia in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a chemerin polypeptide or a nucleic acid molecule encoding thereof.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

In particular, the method of the present invention is particularly suitable for inhibiting the lipolysis of white adipose tissue and the loss of skeletal muscle.

In some embodiments, the subject suffers from a cancer selected from the group consisting of bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating, lobular carcinoma, lobular carcinoma in, situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adenocarcinroma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, non- Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma).

As used herein, the term "chemerin" has its general meaning in the art and refers to a 14 kDa protein, highly expressed in adipose tissue, liver and lung. It is secreted as the inactive, 143 amino acid (AA) long, prochemerin and activated by proteolytic cleavage of a 6 AA C-terminal sequence by serine proteases involved in coagulation-, inflammation or fibrinolysis cascades. It acts as a ligand for CMKLRl, Gprl and CCRL2. An exemplary human amino acid sequence is represented by SEQ ID NO: 1.

SEQ ID NO:l : Chemerin homo sapiens

MRRLLIPLAL WLGAVGVGVA ELTEAQRRGL QVALEEFHKH PPVQWAFQET SVESAVDTPF PAGIFVRLEF KLQQTSCRKR DWKKPECKVR PNGRKRKCLA

CIKLGSEDKV LGRLVHCPIE1 TQVLREAEEH QETQCLRVQR AGEDPHSFYF PGQFAFSKAL PRS

Accordingly the term "chemerin polypeptide" has its general meaning in the art and includes naturally occurring chemerin and conservative function variants and modified forms thereof. Chemerin is a family of structurally related cytokines.

In some embodiments, the chemerin polypeptide comprises an amino acid sequence having at least 90% of identity with SEQ ID NO:l. According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar are the two sequences. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5: 151-153, 1989; Corpet et al. Nuc. Acids Res., 16: 10881-10890, 1988; Huang et al., Comp. Appls Biosci., 8:155-165, 1992; and Pearson et al, Meth. Mol. Biol., 24:307-31, 1994). Altschul et al., Nat. Genet., 6: 119-129, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. By way of example, the alignment tools ALIGN (Myers and Miller, CABIOS 4: 11-17, 1989) or LFASTA (Pearson and Lipman, 1988) may be used to perform sequence comparisons (Internet Program® 1996, W. R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996). ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet at the NCSA Website, for instance. Alternatively, for comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). The BLAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al, J. Mol. Biol, 215:403-410, 1990; Gish. & States, Nature Genet., 3:266-272, 1993; Madden et al. Meth. EnzymoL, 266: 131-141, 1996; Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997; and Zhang & Madden, Genome Res., 7:649-656, 1997.

In some embodiments, it is contemplated that the chemerin polypeptides of the invention used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.

A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications. In some embodiments, the chemerin polypeptide of the invention is fused a Fc domain of an immunoglobulin. Suitable immunoglobulins are IgG, IgM, IgA, IgD, and IgE. IgG and IgA are preferred IgGs are most preferred, e.g. an IgGl . Said Fc domain may be a complete Fc domain or a function-conservative variant thereof. The chemerin polypeptide of the invention may be linked to the Fc domain by a linker. The linker may consist of about 1 to 100, preferably 1 to 10 amino acid residues.

According to the invention, the polypeptide of the invention may be produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and making proteins are well known to those of skill in the art. The polypeptides of the invention may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols as described in Stewart and Young; Tarn et al., 1983; Merrifield, 1986 and Barany and Merrifield, Gross and Meienhofer, 1979. The polypeptides of the invention may also be synthesized by solid-phase technology employing an exemplary peptide synthesizer such as a Model 433 A from Applied Biosystems Inc. The purity of any given protein; generated through automated peptide synthesis or through recombinant methods may be determined using reverse phase HPLC analysis. Chemical authenticity of each peptide may be established by any method well known to those of skill in the art. As an alternative to automated peptide synthesis, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a protein of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides. A variety of expression vector/host systems may be utilized to contain and express the peptide or protein coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al, 1999); insect cell systems infected with virus expression vectors (e.g., baculovirus, see Ghosh et al., 2002); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid; see e.g., Babe et al., 2000); or animal cell systems. Those of skill in the art are aware of various techniques for optimizing mammalian expression of proteins, see e.g., Kaufman, 2000; Colosimo et al., 2000. Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC 12, K562 and 293 cells.

As used herein, the term "nucleic acid molecule" has its general meaning in the art and refers to a DNA or RNA molecule. However, the term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8- hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-

(carboxyhydroxylmethyl) uracil, 5-fiuorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, -uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

In some embodiments, the nucleic acid molecule of the present invention is included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector. Typically, the vector is a viral vector which is an adeno-associated virus (AAV), a retrovirus, bovine papilloma virus, an adenovirus vector, a lentiviral vector, a vaccinia virus, a polyoma virus, or an infective virus. In some embodiments, the vector is an AAV vector. As used herein, the term "AAV vector" means a vector derived from an adeno- associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. Retroviruses may be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special cell- lines. In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line is constructed containing the gag, pol, and/or env genes but without the LTR and/or packaging components. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV). Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are known in the art, see, e.g.. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference. In general, the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell. The gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest. Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference. This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest. The env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species. Typically, the nucleic acid molecule or the vector of the present invention include "control sequences'", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. Another nucleic acid sequence, is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters".

By a "therapeutically effective amount" is meant a sufficient amount of the chemerin polypeptide or the nucleic acid molecule encoding thereof to treat cancer-induces cachexia at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

According to the invention, the chemerin polypeptide or the nucleic acid molecule (inserted or not into a vector) of the present invention is administered to the subject in the form of a pharmaceutical composition. Typically, the chemerin polypeptide or the nucleic acid molecule (inserted or not into a vector) of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral- route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The chemerin polypeptide or the nucleic acid molecule (inserted or not into a vector) of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In some embodiments, the chemerin polypeptide or the nucleic acid molecule encoding thereof is administered to the subject in combination with a chemotherapeutic agent. The term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Intl. Ed. Engl. 33:183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including mo holino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PS ®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and phannaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)- imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and phannaceutically acceptable salts, acids or derivatives of any of the above.

In some embodiments, the chemerin polypeptide or the nucleic acid encoding thereof is administered sequentially (i.e. before or after) or concomitantly with the chemotherapeutic agent.

In some embodiments, the chemerin polypeptide or the nucleic acid molecule encoding thereof is administered to the subject in combination with an anti-VEGF agent. As used herein an "anti-VEGF agent" refers to a molecule that inhibits VEGF -mediated angiogenesis, vasculogenesis, or undesirable vascular permeability. For example, an anti- VEGF therapeutic may be an antibody to or other antagonist of VEGF. An "anti-VEGF antibody" is an antibody that binds to VEGF with sufficient affinity and specificity to be useful in a method of the invention. An anti-VEGF antibody will usually not bind to other VEGF homologues such as VEGF- B or VEGF-C, or other growth factors such as P1GF, PDGF or bFGF. A preferred anti- VEGF antibody is a monoclonal antibody that binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC® HB 10709 and is a high-affinity anti-VEGF antibody. A "high-affinity anti-VEGF antibody" has at least 10-fold better affinity for VEGF than the monoclonal anti-VEGF antibody A4.6.1. Preferably the anti-VEGF antibody is a recombinant humanized anti-VEGF monoclonal antibody fragment generated according to WO 98/45331, including an antibody comprising the CDRs or the variable regions of Y0317. More preferably, anti-VEGF antibody is the antibody fragment known as ranibizumab (LUCENTIS ®). The anti-VEGF antibody ranibizumab is a humanized, affinity-matured anti-human VEGF Fab fragment. Ranibizumab is produced by standard recombinant technology methods in E. coli expression vector and bacterial fermentation. Ranibizumab is not glycosylated and has a molecular mass of -48,000 daltons. See W098/45331 and U.S. 2003/0190317. Anti-VEGF agents include but are not limited to bevacizumab (rhuMab VEGF, Avastin®, Genentech, South San Francisco Calif), ranibizumab (rhuFAb V2, Lucentis®, Genentech), pegaptanib (Macugen®, Eyetech Pharmaceuticals, New York N.Y.), sunitinib maleate (Sutent®, Pfizer, Groton Conn.).

In some embodiments, the chemerin polypeptide or the nucleic acid molecule encoding thereof is administered sequentially (i.e. before or after) or concomitantly with the anti-VEGF agent. The invention will be further illustrated by the following figures and examples.

However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Loss of myeloid cell VEGF- A delays tumor growth and ameliorates cancer cachexia after chemotherapy.

(A) Schematic representation of experimental procedure to study tumour regrowth of LLC and B 16F 10 isografts in WT and mutant mice. 10 7 cells of the tumour cell line were subcutanously injected into mice and 8 mg kg c i s-d i am m i n cd i ch 1 o to p 1 a t i n u m( 11 ) (cisplatin or CDDP) was administered by intraperitoneal injection (i.p.) at the indicated time-points. Tumours were allowed to grow until the maximum size was reached .

(B) Determination of speed of tumour growth of LLC isografts after chemotherapy. Tumour doubling time (DT) was calculated from the last treatment and the end volume for a total period of 6 days (WT, n = 17; Mut, n = 17).

(C) Graphical representation of regression of tumour growth kinetics of untreated and cisplatin-treated WT and mutant LLC isografts after s.c. injection of tumour cells into different cohorts of mice (WT control, n = 9; Mut control, n = 11; WT + CDDP, n = 17; Mut + CDDP, n = 17). (D) Tumor volumes of of cisplatin-treated WT and mutant LLC isografts at endpoint (day 18; WT + CDDP, n = 17; Mut + CDDP, n = 17).

(E) Body weight loss of untreated and cisplatin-treated LLC-bearing WT and mutant mice at endpoint. Weight loss is given as percentage of the original body weight (day 18; WT: n > 4; Mut: n > 7).

Figure 2: Chemerin protects Mut mice from chemotherapy-induced WAT lipolysis and weight loss.

(A) Amount of white adipose tissue (WAT) normalized to tibia length of untreated, cisplatin-treated and cisplatin + anti-chemerin-treated WT and Mut mice on day 18 (WT: n > 4; Mut: n > 7).

(B) Weight loss of LLC-tumour bearing WT and Mut mice without treatment and after administration of CDDP alone or with chemerin-neutralizing antibody on day 18. Weight loss is given as percentage of the original body weight (WT: n > 4, Mut: n > 7).

(C) N-fold change in gene-expression of Atgl in gonadal fat tissue from WT and Mut animals after treatment with CDDP ± chemerin-neutralizing antibody. Untreated animals served as controls (WT: n > 4; Mut: n > 7).

(D) N-fold change in gene-expression of Hsl in gonadal fat tissue from WT and Mut animals after treatment with CDDP ± chemerin-neutralizing antibody. Untreated animals served as controls (WT: n > 4; Mut: n > 7).

(E) Quantification of levels of Atgl transcripts in explant cultures of white adipose tissue from C57/B16J-WT mice. WAT explants were treated for 24 hours as indicated (n > 4).

(F) Colorimetric determination of free fatty acid (FFA) release in supernatants from WAT explants treated as described in (E) (n > 4).

Figure 3: Chemerin release and natural killer cell antitumour defense account for the improved growth restriction upon cisplatin treatment in Mut mice.

(A) Tumour volumes of WT and Mut mice treated with CDDP alone, CDDP and chemerin or CDDP and mAB P 136 (n > 9).

(B) Quantification of SA-B-gal-positive cells in (B) (untreated, n > 4, CDDP, n > 6). Figure 4: Graphic summary and proposed mechanism.

Proposed model for the improved outcome of chemotherapy and prevention of body weight loss by targeting VEGF in myeloid cells: increased levels of circulating chemerin due enhanced release of chemerin by the tumor endothelium improve NK cell recruitment to the tumor and prevent WAT lipolysis.

Figure 5: Chemerin prevents loss of skeletal muscle.

Gastrocnemius muscle weight of untreated, cisplatin-treated and cisplatin + anti- chemerin-treated WT and Mut mice on day 18 (WT: n > 4; Mut: n > 7).

EXAMPLE:

Material & Methods

Animals and procedures. The Animal Care and Use Committee of the Bezirksregierung Dusseldorf, Germany, approved all procedures performed on mice. Male mice at 10-12 weeks of age (C57B1/6J) were used. Chemotherapy was started eight days after subcutanous injection of 10 7 LLC cells and ten days after injection of 10 7 B16F10 cells. Three doses of cisplatin (Sigma, 8 mg/kg) were given by intraperitoneal injection (i.p.) every two days. Tumour size was monitored every two days using a caliper and the tumour volume was calculated as V = ji /6 * A x B2. Tumours were allowed to grow until the maximum permitted size was reached or ulcerations occured. Pimonidazole hydrochloride (Hypoxyprobe-1, HPI) was injected intraperitoneally (60 mg/kg body weight) 30 min before tumour removal and detected by the monoclonal antibody Mab- 1. Tumour doubling time was calculated as DT = (T - TO) x ln2/(lnV - InVO), where T - TO indicates the time between two measurements and V0 and V denote the tumour volume at these times.

Cell culture. Cells were cultured in DMEM High Glucose medium supplemented with 10 % fetal calf serum, 50 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere of 5 % CO2 in air. Depletion of Natural Killer cells. After the last cytotoxic treatment, randomized cohorts of WT and Mut mice were injected i.p. with anti-NKl .l mAb PK136 (4 mg/kg of body weight) at day 13 and 15. Control mice received i.p. injections of 100 μΐ PBS. Chemerin neutralization. Randomized cohorts of WT and Mut mice received intraperitoneal injections of 400 μg/kg body weight anti-chemerin (R&D Systems) on days 11 , 13 and 15. Control mice were injected i.p. with PBS.

Primary antibodies. Rat anti-CD31 (Biolegend), mouse anti-SMA-a (Chemicon), rat anti-KI-67 (Abeam), rat anti-Cdl lc (Biolegend), rat anti-CD4 (Biolegend), rat anti-CD8 (Biolegend), mouse anti-NKl . l (Biolegend), rat anti-F4/80 (Serotec), rabbit anti-p53 (Santa Cruz), rabbit anti-p21 (Oncogene), goat anti-chemerin (R&D Systems), rabbit anti-S 100A4 (Fsp-1) (Dako), mouse anti-MAB-1 (HPI), rabbit anti-PPAR (Abeam), rabbit anti-fl-actin (Santa Cruz Biotechnology) and rabbit anti-NKI-A59 dilution (a gift from B. Floot, Netherlands Cancer Institute, Amsterdam, Netherlands) were used as primary antibodies.

RNA extraction and RT-qPCR. Total RNA was isolated by phenol/chloroform extraction. cDNA was synthesized from 3 μg of DNA-free total RNA using M-MLV Reverse Transcriptase (Promega) and oligo-dT primers (Life Technologies). Gene-specific transcription levels were determined using SYBR Green Mastermix (Promega) and an IQ5 real-time PCR machine (Bio-Rad). PCR conditions: 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Data were normalized to 16S mRNA levels.

Immunofluorescence/immunohistochemistry. OCT-embedded frozen sections (ΙΟμηι thick) were thawed at room temperature and fixed in methanol/acetone (1 : 1) for 5 min at -20 °C. Sections were blocked with 5 % normal goat serum (Sigma) for 60 minutes at room temperature and incubated with the primary antibody overnight at 4 °C. Species-specific fluorochrome-conjugated Alexa 488 and Alexa 568 (Invitrogen) were used as secondary antibodies for 30 min at room temperature. For staining with mouse-derived antibodies, Mouse on mouse (M. O.M.) Basic Kit (Vector Laboratories) was used in accordance with the manufacturer's instructions. Cell nuclei were stained with DAPI (Invitrogen) and cover slips were mounted with Mounting Medium (Dako). The TUNEL assay was performed in accordance with the manufacturer's instructions (Promega). Chemerin immunohistochemistry and chemerin/FSP-1 double immunofluorescence were performed with the CSA kit (DA O) after heat-induced antigen retrieval.

Senescence associated β-galactosidase staining. ΙΟμηι frozen sections of OCT- embedded tumours were stained for the senescence marker SA-P-galactosidase according to the manufacturer's instructions (Cell Signaling).

Flow cytometric analysis. Tumours were digested with 0.1 % collagenase type III (Worthington) and 1 U/ml DNAse I (Promega) and incubated at 37 °C for 1 h. 10 7 tumour cells were incubated with Fc-Block (BD Biosciences) before labeling with fluorochrome- conjugated antibodies (Biolegend). Data were acquired on a flow cytometer (LSR II, BD Biosciences) using the FACS Diva software. Samples were stained with 7-AAD before analysis to exclude non-viable cells. Isolation of endothelial cells. To isolate endothelial cells, tumours were digested in cell lysis buffer (DMEM + 2 mg/ml collagenase type III) for 1 h at 37 °C. 4xl0 7 cells were incubated with mouse CD31 microbeads (Miltenyi Biotec) and endothelial cells (EC) were separated to a purity of > 90 % by positive selection according to the manufacturer's instruction.

Determination of free fatty acids of isolated WAT. Fat tissue explants were obtained from gonadal white adipose tissue of C57/B16J-WT mice and cut into small pieces. The pieces were maintained for 18 hours in DMEM supplemented with 2 % BSA at 37 °C and 5 % CO2. Aliquots of medium were analysed for free fatty acids (FFA) using a commercial kit (Free Fatty Acid Quantification Kit, Abeam) following the manufacturer's instructions.

Quantitative analysis of histology markers. For quantitative analysis of blood vessels, five areas of each tumour section were randomly selected and photographed using a Nikon Eclipse E1000 microscope and the Nikon DS-Ril camera system. The area (number of pixels/px) marked by CD31 was measured using the ImageJ program (National Institutes of Health) and calculated as the percentage of the area covered by DAPI. Pericyte coverage was calculated as percentage of total number of blood vessels counted. To determine cell proliferation, apoptosis and cellular senescence, cells positive for the marker in question were counted in five randomly chosen tumour areas for each section and the mean value calculated.

ELISA. Concentrations of VEGF-A and chemerin in tumours and aliquots of medium were determined using commercial kits (Quantikine ELISA Immunoassay, R&D Systems) and expressed in pg/ml per mg of whole tissue protein. Serum levels of TNF-a and IL-6 were measured using mouse TNF-a and IL-6 quantikine ELISA kit (R&D Systems) and normalized to serum protein levels. Western Blot. Protein samples were separated using a 10 % polyacrylamide gel under reducing and denaturating conditions and transferred onto a PVDF membrane followed by ECL-detection of the antibody. For quantitative analysis, the membranes were scanned with the ImageQuant LAS 4000mini (GE Healtcare Life Sciences) and the integrated density was measured using the software ImageJ (National Institutes of Health).

Statistical analysis. Statistical analysis was performed with the Prism 6.0 software (GraphPad Software). Statistical significance was determined by an unpaired students t-test. Statistical significance is indicated as * p<0.05, ** p<0.01, *** p<0.001. Results

Targeting of VEGF-A in myeloid cells delays tumor growth and ameliorates cancer cachexia after chemotherapy

We have previously crossed mice with a /oxP-flanked Vegfa allele to mice with the Cre recombinase under the control of the lysozyme M promoter. The VEGF-A gene is specifically deleted in the myeloid cells of the resulting mutant (Mut, LysMCre/VEGF f / f ) mice and the animals' response to chemotherapy is improved: the mice show vascular normalization and an increase in tumour cell apoptosis 3 . We subjected wild-type (WT, LysMCre-/VEGF+/+) and mutant mice carrying Lewis lung carcinomas (LLC) or B16 melanomas to three cycles of cisplatin treatment (CDDP, 8 mg/kg body weight, see scheme Fig. 1A). In LLC and B16 tumors, loss of VEGF-A (in Mut mice) significantly increased tumour-doubling time (Fig. IB and 1C) and was associated with significantly reduced endpoint tumour volumes (Fig. 1 D). In contrast, WT tumours reached endpoint volumes comparable to those of untreated tumours (Fig. 1C and D), indicative of treatment failure. Ulcerations in the mice injected with B16F10 melanoma cells forced termination of the control experiment ahead of schedule.

Treatment with cytotoxic agents frequently exacerbates cachexia and limits the outcome of therapy n,u . Untreated LLC-bearing WT and Mut mice had similarly reduced body weights at endpoint (Fig. IE). Upon chemotherapy, the loss of body weight depended on the presence of myeloid VEGF-A. LLC-bearing WT mice showed a significant drop in body weight that was mitigated in Mut mice by deletion of myeloid cell-derived VEGF-A (Fig. IE). Deletion of myeloid-derived VEGF-A leads to vessel normalization and enhanced drug delivery

Irrespective of the genotype, cisplatin treatment reduced levels of VEGF-A, lowered vascular density and increased pericyte coverage to varying degrees. These observations are consistent with the notion that chemotherapy induces vascular regression 20 . In line with previous results 3 , comparison of WT and Mut mice reveals that the loss of myeloid cell- derived VEGF results in lower levels of VEGF within the tumours as well as in vascular normalization, increased pericyte coverage and decreased tumour hypoxia. Although vascular normalization and improved oxygenation is associated with accelerated tumour growth in untreated Mut tumours, it paradoxically results in delayed tumour outgrowth after chemotherapy (Fig. 1C). The apparent contradiction arises from the augmented delivery of chemotherapeutic agent to the tumour leading to enhanced DNA damage responses, indicated by increased levels of cisplatin-DNA adducts and higher expression of p53 and p21. Thus, normalizing the vasculature by targeting VEGF-A exerts opposing effects on tumour growth kinetics pre- and post-chemotherapy.

Targeting VEGF-A in myeloid cells enhances chemotherapy-induced DNA damage and immune cell recruitment

Not only do chemotherapeutic agents drive the apoptosis of tumour cells, they may be associated with a wide range of other outcomes including so-called premature or therapy- induced senescence, which they promote by inducing DNA damage responses 21,22 . Untreated tumours show hardly any senescence irrespective of the genotype (data not shown, untreated). Deleting VEGF-A in myeloid cells increases tumour-cell death upon chemotherapy and promotes therapy-induced senescence, as shown by senescence-associated β-galactosidase (SA β-Gal) staining (data not shown, day 14, CDDP). The changes presumably stem at least partially from improved drug delivery and are accompanied by a decreased number of cells positive for the proliferation marker KI-67. Tumours from Mut mice show a decline in SA β- Gal positive cells at endpoint (day 18), whereas the number of senescent cells increases further in WT tumours (data not shown, day 18, CDDP). It thus seems likely that targeting VEGF-A in myeloid cells improves the clearance of senescent cells after chemotherapy.

Senescent cells remain viable and secrete a range of inflammatory cytokines 23 . The senescence-associated secretory phenotype (SASP) is believed to trigger an immune response involving macrophages and natural killer (NK) cells that facilitates immune cell-mediated tumour clearance 24~26 . We used flow cytometry to analyse tumour infiltration by immune cells following chemotherapy and found it to be enhanced (from day 14 until the endpoint at day 18) in the absence of myeloid cell-derived VEGF-A (data not shown). There were markedly higher numbers of NK cells (NKl .l) (data not shown) and significantly increased numbers of intratumoural macrophages (F4/80) and dendritic cells (CD 11c). The numbers of intratumoural T helper cells and cytotoxic T cells were unaffected. Cisplatin treatment caused transient increases in the expression of particular SASP cytokines (IL-6, Ccl2 and Vcaml) in tumours in Mut mice (day 14) and cannot explain the enhanced recruitment of immune cells after chemotherapy in Mut mice (data not shown).

Inactivation of VEGF-A in myeloid cells enhances endothelial expression of chemerin upon chemotherapy

A number of cytokines and chemokines are involved in the recruitment of immune cells to malignant tumours 27 ' 28 . The chemoattractant chemerin has a crucial role in immune cell trafficking 14~16 . Forced expression of chemerin in tumour cells gives rise to an NK cell- based antitumour response and restricts tumour growth, while low levels of chemerin are associated with tumour progression and a poor outcome 19 . Increasing the level of chemerin within the tumour may thus represent a promising therapeutic approach. We found that chemotherapy increases the level of chemerin in tumours in WT mice (data not shown). Surprisingly, the effect was significantly enhanced in Mut mice, showing that the absence of VEGF-A in myeloid cells stimulates the chemotherapy-evoked expression of chemerin (data not shown) and drastically increases the levels of circulating chemerin (data not shown).

The physiological source of chemerin within the tumor microenvironment is unknown. The tumour cells do not release chemerin as a consequence of chemotherapy: treatment of LLC cells in vitro with 3μg/ml cisplatin, a concentration that causes a significant DNA damage response, did not trigger chemerin release while cisplatin treatment of B 16F 10 cells produced no increase in the basal level of chemerin secreted. Consistently, immunohistochemical analysis of tumor sections revealed only subtle chemerin reactivity in untreated tumours of WT and Mut mice as well as in tumours from cisplatin-treated WT animals (data not shown). However, tumours from Mut mice showed strong chemerin immunoreactivity of the tumor vasculature upon chemotherapy (data not shown). The result indicates that tumour endothelial cells release chemerin in response to chemotherapy and that VEGF-A from myeloid cells suppresses the release.

To test this hypothesis we analysed the release of chemerin by the murine endothelial cell line bEnd3. Cisp latin treatment (3 μg/ml) (data not shown) caused a pronounced induction of chemerin release, accompanied by the accumulation of the transcription factor PPAR-γ, which stimulates chemerin expression 29 . The addition of exogenous murine VEGF- A suppresses the effect and blocks the increased production of chemerin (data not shown). Comparable results were obtained in endothelial cells isolated from tumours of both genotypes. PPAR-γ and chemerin were only expressed in endothelial cells (EC) of tumours derived from Mut mice after chemotherapy (data not shown), confirming that ablation of myeloid cell-derived VEGF-A significantly increases the expression of chemerin in response to chemotherapy (data not shown). The data verify the tumor endothelium as a major source of chemerin in response to chemotherapy and show that chemerin production is suppressed by myeloid cell-derived VEGF-A.

Chemerin protects Mut mice from chemotherapy-induced WAT lipolysis and weight loss

Chemerin was initially described as an adipokine with context-dependent pro- and antilipolytic effects on WAT 13 ' 30 . It seemed conceivable that changes in chemerin levels could affect lipid metabolism in our cancer models. It was initially important to assess the contribution of loss of adipose tissue to the overall weight loss associated with chemotherapy. We thus weighed gonadal adipose depots in mice subjected to chemotherapy. Consistent with the concept that cachexia involves breakdown of WAT, chemotherapy of WT mice resulted in a >60% reduction in gonadal WAT (Fig. 2A). The expression of the major lipolytic enzymes adipose triglyceride lipase (Atgl) and hormone-sensitive lipase (Hsl) in WAT isolated from cisplatin-treated WT mice was significantly upregulated (Fig. 2C and D). The increase in lipolytic enzymes depended on the presence of myeloid-derived VEGF-A: chemotherapy of Mut mice caused a far smaller loss of WAT (Fig. 2A). The data suggest that differences in chemerin release underlie not only the altered tumour immune cell infiltration but also the striking difference in weight loss between WT and Mut mice following chemotherapy. To test this interpretation we depleted chemerin by means of an anti-chemerin antibody. Remarkably, the antibody caused Mut mice to suffer the same loss of body weight and WAT as WT mice on cisplatin treatment (Fig. 2A and B). Furthermore, following chemotherapy the Atgl and Hsl genes were expressed at similar levels in WT mice and in Mut mice treated with the antibody (Fig. 2C and D). The differences in weight and WAT loss upon chemotherapy could not be accounted for by variations in food intake, which did not depend on genotype, although chemotherapy resulted in a reduced food intake in both WT and Mut mice. Likewise, serum levels of the lipid-mobilizing cytokines TNF-a and IL-6 were similar across genotypes and treatment regimens. The protection from chemotherapy-induced cachexia in Mut mice is thus associated with the loss of myeloid cell- derived VEGF-A and the resulting increase in the level of circulating chemerin.

The cause of weight loss associated with chemotherapy is poorly understood. Our findings suggest that cisplatin might have a direct lipolytic effect that is modulated by chemerin. To investigate the possibility, gonadal WAT explants from C57B16/j mice were treated with cisplatin, which was found to induce Atgl expression (Fig 2 E) and to stimulate release of fatty acids (Fig. 2 F). The expression of Hsl was also induced, although not significantly. Consistent with its context-specific role in enhancing or inhibiting lipolysis, chemerin increased Atgl expression and lipolysis in WAT explants (Fig. 2 E and F) but suppressed the increased expression of Atgl and WAT lipolysis caused by addition of cisplatin (Fig. 2 E and F). The experiment confirms that cisplatin directly stimulates WAT lipolysis and that the effect is negated by chemerin, which thereby protects against therapy- associated loss of body weight.

Chemerin release and natural killer cell antitumour defense account for the improved growth restriction upon cisplatin treatment in Mut mice

Chemotherapy causes an increase in the intratumoural release of chemerin in Mut mice. Chemerin might thus be involved in the enhanced immune response in the absence of myeloid cell-derived VEGF-A, which is associated with the improved control of tumour growth. The interpretation was tested by means of an anti-chemerin antibody, which drastically diminished chemotherapy-induced recruitment of NK cells in WT and Mut mice (data not shown), whereas infiltration of DCs and macrophages was unaffected. The antibody completely blocked the clearance of senescent tumour cells after cytotoxic treatment in the absence of myeloid cell-derived VEGF-A, resulting in equal numbers of senescent cells in tumours from WT and Mut mice at endpoint (Fig. 3B). Blocking chemerin led to comparable outcomes in WT and Mut mice at endpoint (Fig. 3A). Comparable results were obtained by depleting NK cells (Fig. 3 A). In the absence of NK cells, senescent cells were not cleared and remained in Mut tumors upon tumor regrowth (Fig. 3B) and there was no delay in tumor growth after chemotherapy (Fig. 3A). The findings unequivocally link the improved outcome of chemotherapy in the absence of myeloid cell-derived VEGF-A to the release of the chemoattractant chemerin, which activates NK cell-based antitumor defenses. Discussion:

Targeting VEGF-A in myeloid cells leads to vascular normalization 3 . Here we show that targeting VEGF-A is also associated with an enhanced senescence response upon chemotherapy. In addition to improved drug delivery, the reduced tumour hypoxia in Mut tumours may contribute to the effect, as hypoxia has been reported to prevent cellular senescence 31 . While T cell-mediated immune responses are impaired by a lack of oxygen 32 , it is unknown how NK cells react under hypoxic conditions. It is attractive to speculate that the reduced hypoxia in Mut mice improves NK cell-mediated cytotoxicity.

In addition to shaping the tumour vasculature, VEGF-A modulates the performance of various immune cells 33 . It may have an effect on the migration and cytotoxicity of NK cells, although findings are inconsistent 34,35 . It clearly attracts regulatory T cells to the tumour microenvironment 36 and interferes with the maturation of dendritic cells 33 . The absence of myeloid cell-derived VEGF-A from the tumour microenvironment could thus improve antitumour immune responses.

The chemotherapeutic agent cisplatin reduces vascular density and increases pericyte coverage, consistent with its known anti-angiogenic properties 20 . The effect is independent of myeloid cell-derived VEGF-A, although the density of blood vessels before chemotherapy is higher in tumours from wild-type mice than in those from mutant mice lacking VEGF-A in their myeloid cells. The reduction in tumour blood vessels upon chemotherapy may thus be enhanced by VEGF-A. The effect may stem from improved drug delivery and/or be related to the presumably higher number of proliferating endothelial cells upon VEGF-A-driven angiogenesis. The proliferating cells in the vasculature would be more susceptible to cytotoxic damage than quiescent cells. Our study reveals that chemotherapy increases the level of PPAR-γ within tumour endothelial cells and stimulates them to release chemerin. Chemerin has opposing effects on lipid metabolism depending on the nutritional status and on other factors. In vitro experiments show that chemerin may have pro- or antilipolytic effects depending on the experimental conditions 13 ' 30 . In vivo evidence is limited, although treatment of fasted mice with chemerin is known to inhibit lipolysis and release of free fatty acids 0 . Consistently, we show that lipolysis and the release of free fatty acids are down-regulated by the addition of chemerin to WAT cultures after the chemotherapeutic induction of lipolysis. In contrast, chemerin treatment of WAT explants before chemotherapy induces lipolysis. We speculate that chemerin acts as a rheostat in the homeostasis of fat tissue, preventing excessive accumulation or depletion of fat reserves in the presence of powerful anti- or prolipolytic stimuli.

Tumour endothelial cells release chemerin in response to chemotherapy but the effect is suppressed by VEGF-A derived from myeloid cells. Lowering intratumoural levels of VEGF-A after chemotherapy thus has an additional important effect: as well as normalizing the vasculature, it also fosters the endothelial production of chemerin. It remains to be determined whether elimination of myeloid cell-derived VEGF-A will have a similarly stimulatory effect when cytotoxic agents other than cisplatin are used. Cisplatin is non- immunogenic 37 and it will be important to investigate the effects on chemerin release of other, immunogenic chemotherapeutics as well as of commonly used antibody- and tyrosine kinase inhibitor-based anti-VEGF strategies.

The tumouricidal effects of many chemotherapeutic agents may depend on the active contribution of immune cell effectors, especially those of the adaptive immune compartment l . In our particular tumor models, therapeutic success critically depends on NK cell-mediated tumour immune surveillance and tumour cell clearance. It is important to note that not all tumours are sensitive to NK cell-mediated tumour surveillance. Further work will be necessary to evaluate the outcome of drug-induced senescence and stromal chemerin release in tumor models that are controlled by T cells.

In summary, our study reveals that chemotherapy with cisplatin simulates tumour endothelial cells to release chemerin. We show further that chemerin is a critical mediator of NK cell-mediated antitumour defenses as well as of lipolysis and cachexia (Fig. 4). VEGF-A derived from myeloid cells suppresses the stimulation of endothelial chemerin release by chemotherapy. Hence, targeting VEGF signaling should impede the lipolysis and weight loss that is frequently associated with chemotherapy. Our study therefore offers novel therapeutic avenues to improve the outcome of chemotherapy. REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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