WO2015110606A1

WO2015110606A1 - In vitro mitochondrial toxicity assay

Info

Publication number: WO2015110606A1
Application number: PCT/EP2015/051412
Authority: WO
Inventors: Nicholas Thomas; Jianliang Li; Hayley TINKLER; Christelle COUSIN; Jawida TOUHAMI; Vincent Petit
Original assignee: Metafora Biosystems
Priority date: 2014-01-23
Filing date: 2015-01-23
Publication date: 2015-07-30
Also published as: GB201401153D0

Abstract

The invention relates in vitro screening methods and kits for detecting and quantifying agents which are toxic to cells. In one embodiment, the invention provides methods and kits for detecting and quantifying mitochondrial toxicity which do not require parallel screening in cell cultures containing different carbon sources.

Description

IN VITRO MITOCHONDRIAL TOXICITY ASSAY

TECHNICAL FIELD OF THE INVENTION

The invention relates to in vitro screening assays and kits for toxicity screening of an agent, such as, for example, a xenobiotic, for use in the pharmaceutical, biotechnological, agrochemical, food, environmental and cosmetic industries. In particular, the invention relates to in vitro methods and kits which can be used to detect agents which have a toxic effect on mitochondria.

BACKGROUND TO THE INVENTION

Mitochondria occur in the cytoplasm of almost all eukaryotic cells, including fungi, plants and animals, and serve as the key organelle for sugar metabolism and energy production. Consequently, any agents or chemicals which are toxic to mitochondria, such as drugs, pesticides or environmental pollutants, can have significant effects on human health.

Mitochondrial toxicity is a common cause of drug failure in the pharmaceutical industry, both in development and in clinical use, and improved methods for detection of drug impact on mitochondrial integrity are required (Dykens JA, Will Y. The significance of mitochondrial toxicity testing in drug development. Drug Disc. Today. 2007;12(17-18):777- 85). Drug-induced toxicity in heart and liver are the leading cause of post-market drug withdrawal and both tissues have a high content of mitochondria. In cardiac tissue, where energy demands for ATP production are high and tissue metabolism is constantly modified to maintain energy production under differing conditions of exercise, stress, disease and other factors (Taegtmeyer H, Golfman L, Sharma S, Razeghi P, van Arsdall M. Linking gene expression to function: metabolic flexibility in the normal and diseased heart. Ann N Y Acad Sci. 2004;1015:202-13), bioenergetic balance can significantly influence drug toxicity. Cell assays are commonly used to detect drug-induced toxicity in vitro. Some of the most commonly used assays include, but are not limited to, leakage of intracellular markers as determined by lactate dehydrogenase (LDH) (Vickers, A.E., 1994. Cell Biol. Toxicol. 10, 407-414.), glutathione S-transferase (GST) (Oberley et al., Toxicol. Appl. Pharmacol. 131, 94-107, 1995), and potassium, and the reduction of tetrazolium dyes such as MTT (Mosmann, J Immunol. Methods 65, 55-63, 1983; Denizot et al., J Immunol. Methods. 89, 271-277, 1986), XTT (Roehm et al., J. Immunol. Methods, 142, 257-265, 1991), Alamar Blue (Goegan et al., Toxicol. In vitro 9, 257-266. 1995), INT, and quantitation of the viability biomarker ATP (Cell-Titer Glo® Luminescent cell viability assay, provided for example by Promega). All are being used as indicators of cell injury. However cells cultured under standard tissue culture conditions using glucose based media as the carbon source rely predominantly on glycolysis for production of ATP, a phenomenon known as the Crabtree effect (Rodriguez-Enriquez S, Juarez O, Rodriguez-Zavala JS, Moreno-Sanchez R. Multisite control of the Crabtree effect in ascites hepatoma cells Eur J Biochem. 2001; 268(8):2512-9), and may subsequently be resistant to drug-induced toxicity mediated through mitochondria. Culture using galactose based media as the carbon source forces cells to shift metabolism from glycolysis to oxidative phosphorylation based ATP production (Marroquin LD, Hynes J, Dykens JA, Jamieson JD, Will Y. Circumventing the Crabtree effect: replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicol Sci. 2007; 97(2):539-47) with a corresponding increase in sensitivity to mitochondrial toxicity.

Performing drug toxicity screening using galactose media in addition to glucose media results in a doubling or more of costs associated with duplication of testing and time required for adaptation of cells to modified culture conditions. Dual screening in different media also imposes additional demands on laboratory and growth room space and chemical requirements. Such testing regimes and protocols are complex, costly and time consuming, so much so that specialist "Mitochondrial Toxicity Assessment" services are commercially available (e.g. Cyroptex, www.cyprotex.com). In addition, the vast majority of drugs (approximately 91 %) fails to successfully complete the three phases of drug testing in humans. Despite the fact that all of these drugs were tested in animal models and were found to be safe, a majority of those drugs have to be abandoned at late stages of clinical trials that are expensive and time- consuming. Therefore there are limits at using these toxicity assays and there remains a need in the art for the development of new toxicity assays that provide more useful toxicity information.

There is therefore a need for simplified, cost-effective in vitro methods that may be used with cells growing in standard culture media, such as, for example, in glucose based culture media, to directly detect drug impact on mitochondrial function and predict mitochondrial toxicity which may manifest under changing in vitro and in vivo bioenergetic conditions.

Moreover, the toxicity information must be obtained quickly and cost-effectively at early stages of the drug discovery process. Consequently, a need exists for toxicity screening assays that do not require the use of animals but that provide reliable information on relative toxicity and mechanism of toxicity.

The present invention addresses these needs and provides methods and kits for use in in vitro toxicity screens and assays. The inventors surprisingly showed that the expression profile of nutrient transporters is modified after exposure of cells grown in in vitro culture, particularly grown in a glucose based culture medium, to an agent with mitochondrial toxicity.

Therefore, the invention is based on the detection and/or quantification of the expression profile of nutrient transporters on cell surfaces.

The present invention thus provides improved methods and kits which can be used for screening for mitochondrial toxicity than previously known in the prior art. Profiling using ligand binding to cell surface nutrient transporters readily detects differences in cell metabolism triggered by agents and drugs in a single screen and provides predictivity of the relative toxicity of the compounds under shifted metabolic conditions. WO2010079208 describes the use of receptor binding domain (RBD) ligands derived from glycoproteins of enveloped viruses that interact with cellular receptors, for quantification of expression of membrane receptors present on the surface of target cells, where such quantification allows determination of a physiological state of the target cell. Cellular nutrient transporter proteins (Hediger, M.A.; Clemencon, B.; Burner, R.E. et al. The ABCs of membrane transporters in health and disease (SLC series): introduction. Mol. Aspects. Med. 2013, 34(2-3), 95-107) play a pivotal role in cell homeostasis and in metabolic adaptation and regulation induced by, or required for, a wide range of cellular processes and consequently analysis of expression of these regulators of nutrient flux provides a means to monitor cell metabolism under normal or perturbed conditions.

Therefore, the present invention relates in particular to the detection of the expression profile of nutrient transporters on cell surface using RBD.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provided an in vitro method for detecting and/or quantifying mitochondrial toxicity of an agent comprising the steps of:

a) growing at least one cell in an in vitro culture in the presence of an agent;

b) determining the expression profile of at least one cell surface nutrient transporter in said at least one cell; and

c) comparing the expression profile obtained in step b) with a reference expression profile.

In one embodiment, the at least one cell surface nutrient transporter is selected from the group consisting of glucose transporters, inorganic phosphate transporters, amino acid transporters, heme transporters, inositol transporters and riboflavin transporters. Preferably the at least one cell surface nutrient transporter is selected from the group consisting of ASCT1, ASCT2, CAT1, FLVCR1, GLUT1, PARI, PAR2, PiTl, PiT2, RFT1, RFT3, SMIT1 and XPR1. More preferably the cell surface nutrient transporter is GLUT1.

In another embodiment, the expression of the cell surface nutrient transporter is determined at the RNA level. In a further embodiment, the expression of the cell surface nutrient transporter is determined at the protein level. Preferably the expression profile of the cell surface nutrient transporter is determined by detecting and quantifying the binding of a first ligand to the cell surface nutrient transporter.

In one embodiment, said first ligand is an antibody specific to the cell surface nutrient transporter. In another embodiment, the first ligand is a receptor binding domain ligand comprising a part or the totality of a receptor binding domain (RBD) derived from the soluble part of a glycoprotein of an enveloped virus that interacts with a cell surface nutrient transporter. In one embodiment, the enveloped virus is a retrovirus such as, for example, gammaretro virus or deltaretro virus. In another embodiment, the receptor binding domain ligand comprises a sequence selected from the group comprising SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 36, 58, 59, 60, 62, 64, 66, 68 and 71, fragments or variants thereof, or a sequence presenting a sequence identity of at least 70% with the sequences SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 36, 58, 59, 60, 62, 64, 66, 68 and 71.

In a further embodiment, the receptor binding domain ligand is encoded by a DNA sequence selected from the group comprising SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 61, 63, 65, 67, 69 and 70, or sequences presenting a sequence identity of at least 70% with the sequences SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 61, 63, 65, 67, 69 and 70.

In one embodiment, the expression profile is determined as a binding value for the first ligand to the cell surface nutrient transporter and comparing said binding value with a first reference binding value. In another embodiment, the method further comprises determining a second binding value for the binding of a second ligand to a second cell surface nutrient transporter and comparing said second binding value with a second reference binding value.

In a further embodiment, the second cell surface nutrient transporter is selected from the group consisting of ASCT1, ASCT2, CAT1, FLVCR1, GLUTl, PARI, PAR2, PiTl, PiT2, RFT1, RFT3, SMIT1 and XPR1. In one embodiment, the first cell surface nutrient transporter is GLUTl and the second cell surface nutrient transporter is PiTl or FLVCR1.

In one embodiment, the reference expression profile or the reference binding value is derived from the measurement of the expression profile or of the binding value in a cell culture growing in the absence of said agent. The person skilled in the art will understand that any difference between the measured expression profile and the reference expression profile, or of the measured binding value and the reference binding value, is indicative of the mitochondrial toxicity of the agent. In one embodiment the reference expression profile or the reference binding value is derived from the measurement of the expression profile or of the binding value in an in vitro cell culture growing in the presence of a standard compound. Where the standard compound is a known mitochondrial toxicant, the person skilled in the art will understand that the absence of a difference between the measured expression profile and the reference expression profile, or of the measured binding value and the reference binding value, is indicative of the mitochondrial toxicity of the agent. Alternatively, where the standard compound is known to have no effect upon mitochondrial toxicity, the person skilled in the art will understand that any difference between the measured expression profile and the reference expression profile, or of the measured binding value and the reference binding value, is indicative of the mitochondrial toxicity of the agent.

In another embodiment, the at least one cell is growing in a glucose based medium. In a further embodiment, the method comprises:

a. growing at least one cell in an in vitro culture in a glucose based medium in the presence and the absence of an agent; and

b. detecting and/or quantifying binding of a first ligand to a first cell surface nutrient transporter in the presence and the absence of said agent; wherein a difference in said binding of said first ligand to said first cell surface nutrient transporter in the presence and the absence of the agent is indicative of mitochondrial toxicity.

In one embodiment, the at least one cell is a mammalian cell. In one embodiment, the cell is a heart or liver cell. In one embodiment, the cell is derived from a stem cell.

In another embodiment, the agent is a molecule selected from the group consisting of drug, new chemical entity, protein, nucleic acid, carbohydrate and lipid.

In a further embodiment, the detecting or quantifying binding is conducted by flow cytometry or image analysis. In one embodiment, growing cells in the absence of the agent and detecting and/or quantifying binding of the first ligand to the first cell surface nutrient transporter in the absence of the agent is carried out at a different point in time to growing cells in the presence of the agent and detecting and/or quantifying binding of the first ligand to the first cell surface nutrient transporter in the presence of the agent. In one embodiment, at least one value is attributed to the detecting and/or quantifying binding of the first ligand to the first cell surface nutrient transporter to cells growing in the absence of the agent and said at least one value is used to determine the difference in the binding of the first ligand to the first cell surface nutrient transporter growing in the presence of the agent. In one embodiment, the at least one value is stored on a database to provide a stored value and said stored value is used to determine the difference in binding of the first ligand to the first cell surface nutrient transporter growing in the presence of the agent. According to a second aspect of the present invention, there is provided a kit comprising one or more ligands for binding to a cell surface nutrient transporter for detecting and/or quantifying mitochondrial toxicity and instructions for use thereof in a method as described hereinbefore. In one embodiment, said one or more ligands are preferably one or more receptor binding domain ligands comprising a part or the totality of a receptor binding domain (RBD) derived from the soluble part of a glycoprotein of an enveloped virus that interacts with a cell surface nutrient transporter.

In one embodiment, the kit comprises means for measuring the expression profile of at least one cell surface nutrient transporter at the RNA level for detecting and/or quantifying mitochondrial toxicity and instructions for use thereof as described hereinbefore.

According to a third aspect of the present invention, there is provided a use of a kit comprising one or more ligands for binding to a cell surface nutrient transporter for detecting and/or quantifying mitochondrial toxicity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an in vitro method for detecting and/or quantifying mitochondrial toxicity of an agent comprising the steps of:

As used herein, the term "cell surface nutrient transporter" refers to a nutrient transporter anchored in the plasma membrane of a cell.

In one embodiment, the method of the invention comprises determining the expression profile of one cell surface nutrient transporter, or of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nutrient transporters. Mammalian cells take up necessary nutrients via "nutrient transporters" on the cell surface and expel catabolites and other components. Nutrients and metabolites or catabolites are, for example, carbohydrates, amino acids, inorganic phosphate, nucleosides, lipids, vitamins, heme, ions etc. Nutrient transporters may be divided based on passive or active mechanisms of function. Passive (or facilitated) transporters allow diffusion of solutes across membranes down their electrochemical gradient. Active transporters create solute gradients across membranes, utilizing diverse energy-coupling mechanisms, such as, for example, ATP synthesis or hydrolysis. In one embodiment, the cell surface nutrient transporter belongs to the SLC series, wherein SLC stands for Solute Linked Carriers.

In one embodiment, the cell surface nutrient transporter is a transporter of glucose, such as, for example, a glucose importer (such as, for example, Glutl); a transporter of inorganic phosphate, such as, for example, an inorganic phosphate importer (such as, for example, PiTl or PiT2) or an inorganic phosphate exporter (such as, for example, XPR1); a transporter of amino acids, such as, for example, a transporter of neutral amino acids (such as, for example, a neutral amino acids importer (such as, for example, ASCT1 or ASCT2)), or a transporter of cationic amino acids (such as, for example, CAT1); a transporter of heme (such as, for example, FLVCR1); a transporter of inositol, such as, for example, a transporter of myo-inositol (such as, for example, SMIT1); or a transporter of riboflavin, such as, for example, an importer of riboflavin (such as, for example, RFT1, RFT3, PARI or PAR2).

As used herein, the term "expression" may refer alternatively to the transcription of a cell surface nutrient transporter (i.e. expression of the RNA) or to the translation (i.e. expression of the protein) of a cell surface nutrient transporter. Methods for determining the expression profile are well-known from the skilled artisan, and include, without limitation, determining the transcriptome (in an embodiment wherein expression relates to transcription of a nutrient transporter) or proteome (in an embodiment wherein expression relates to translation of a nutrient transporter) of a cell cultured in presence of the tested agent. In one embodiment of the invention, the expression of the cell surface nutrient transporter is assessed at the RNA level. Methods for assessing the transcription level of a transporter are well known in the prior art. Examples of such methods include, but are not limited to, RT-PCR, RT-qPCR, Northern Blot, hybridization techniques such as, for example, use of microarrays, and combination thereof including but not limited to, hybridization of amplicons obtained by RT-PCR, sequencing such as, for example, next-generation DNA sequencing (NGS) or RNA-seq (also known as "Whole Transcriptome Shotgun Sequencing") and the like.

In one embodiment of the invention, the expression of the cell surface nutrient transporter is assessed at the protein level. Methods for determining a protein level in a sample are well-known in the art. Examples of such methods include, but are not limited to, immunohistochemistry, Multiplex methods (Luminex), western blot, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, fluorescent-linked immunosorbent assay (FLISA), enzyme immunoassay (EIA), radioimmunoassay (RIA) and the like.

In one embodiment of the invention, determining the expression profile of a cell surface nutrient transporter specifically corresponds to the detection and quantification of said nutrient transporter present on the cell surface. Methods for analyzing the presence of a protein on the cell surface are well-known to the skilled artisan and include, without limitation, FACS analysis, immunohistochemistry, western blot associated with cell fractionation, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, fluorescent-linked immunosorbent assay (FLISA), enzyme immunoassay (EIA), radioimmunoassay (RIA) or image analysis, for example high content analysis and the like. In one embodiment, determining the expression profile of at least one cell surface nutrient transporter corresponds to detecting and/or quantifying binding of a first ligand to a cell surface nutrient transporter. Preferably, said ligand is a receptor binding domain ligand and the method of the invention comprises detecting and/or quantifying a complex formed between said receptor binding domain ligand and a cell surface nutrient transporter. In another embodiment, said ligand is an antibody specific of said cell surface nutrient transporter, and the method of the invention comprises detecting and/or quantifying a complex formed between said antibody and said cell surface nutrient transporter.

The expression "detecting and/or quantifying binding of a ligand, such as, for example, a receptor binding domain ligand, to a cell surface nutrient transporter" means that when a cell surface nutrient transporter is present a complex is formed between the nutrient transporter and the ligand. That complex can be detected if the ligand has been for example, but not limited to, covalently coupled with a detectable molecule such as an antibody constant fragment (Fc) or a fluorescent compound (e.g. Cyanine dye, Alexa dye, Quantum dye, etc). The complex can also be detected if the ligand has been tagged with different means well known to the person skilled in the art. For example, but without limitation, a tag used with the invention can be a tag selected from the group consisting of Hemaglutinin Tag, Poly Arginine Tag, Poly Histidine Tag, Myc Tag, Strep Tag, S-Tag, HAT Tag, 3x Flag Tag, Calmodulin-binding peptide Tag, SBP Tag, Chitin binding domain Tag, GST Tag, Maltose-Binding protein Tag, Fluorescent Protein Tag, T7 Tag, V5 Tag and Xpress Tag. The use of the ligand therefore allows on the one hand the identification and detection of the cell surface nutrient transporter depending on the ligand used, and on the other hand the quantification of the complex formed. In one embodiment, detecting or quantifying binding is conducted by flow cytometry or image analysis, for example high content analysis. Suitable systems for conducting image analysis include the IN Cell Analyzer systems from GE Healthcare Life Sciences, such as the IN Cel Analyzer 2000.

As used herein, the term "ligand" is meant to mean any substance that forms a complex with a cell surface nutrient transporter. Typical ligands include, but are not limited to, polypeptides and proteins.

As used herein, a polypeptide refers to a linear polymer of amino acids (preferably at least 50 amino acids) linked together by peptide bonds. A protein specifically refers to a functional entity formed of one or more polypeptides, and optionally of non- polypeptides cof actors.

In a further aspect, the ligand is a receptor binding domain ligand, wherein said receptor binding domain ligand comprises a part or the totality of a receptor binding domain (RBD) derived from the soluble part of a glycoprotein of an enveloped virus that interacts with a cell surface nutrient transporter.

The expression "derived from the soluble part of the glycoprotein of an enveloped virus" means that the ligand is a fragment or a part of a glycoprotein contained in the envelope of a virus and can be obtained, for example, by cloning. The term "glycoprotein" is to be understood as meaning an envelope glycoprotein, a coat glycoprotein or a fusion glycoprotein", wherein the term "glycoprotein" refers to a protein containing oligosaccharide chains covalently attached to polypeptide side- chains.

The expression "that interacts with a cell surface nutrient transporter" means that the glycoprotein is liable to recognize a receptor present on the surface of the cell. In one embodiment, a ligand that interacts with a cell surface nutrient transporter will thus form a complex with said cell surface nutrient transporter, which complex may be detected by a method as hereinabove described.

The receptor binding domain ligand containing part or the totality of the RBD can be fused to an antibody constant fragment (such as, for example, Fc fragment from rabbit or from mouse), and/or chemically modified to add a fluorochrome, or a fluorescent compound (e.g. Cyanine dye, Alexa dye, Quantum dye, etc).

RBD are found, in particular, in glycoproteins of the envelope of viruses, therefore, the receptor binding domain ligand contains the total RBD or a fragment or part of the RBD.

In one embodiment, said virus is selected from the group consisting of retroviruses, such as, for example, (i) gammaretroviruses such as for example, murine (MLV), feline (FeLV) or gibbon ape leukaemia virus (GaLV); and (ii) deltaretroviruses such as, for example, primate T cell leukaemia virus (such as, for example, human T cell leukaemia virus (HTLV) and simian T cell leukaemia virus (STLV)) or bovine leukaemia virus (BLV). The gamma and deltaretroviruses encode an Env glycoprotein present in mature retrovirus virons. The Env protein is synthesized in the form of a propeptide, which is clived in Golgi apparatus by furine peptidase, resulting in two polypeptides: the transmembrane (TM) and the cell surface (SU) components. The SU domain contains two major subdomains: a domain of interaction with the TM domain and the RBD, the further being liable to interact with host cell membrane receptors.

An example of an envelope protein is represented in Figure 6.

In one embodiment, the ligand of the invention comprises the SU domain of the glycoprotein envelope of a virus or a fragment of the SU domain, such as, for example, the RBD. In another embodiment, the ligand of the invention does not comprise the TM domain of the glycoprotein envelope of a virus. Therefore, in one embodiment of the invention, the ligand of the invention is a soluble peptide, such as, for example, a soluble RBD. As used herein, the term "soluble peptide" refers to a peptide which is not anchored within a membrane, such as, for example, by a transmembrane domain.

In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Amphotropic MLV, and is herein referred as Ampho.RBD.

In one embodiment, said Ampho.RBD comprises or consists of the amino acid sequence SEQ ID NO: 1 or fragments thereof.

In one embodiment, said fragment comprises or consists of amino acids 31 to 328 of SEQ ID NO: l, preferably amino acids 33 to 328 of SEQ ID NO: 1. In one embodiment, said fragment comprises or consists of amino acids 1 to 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, or 327 of SEQ ID NO: 1.

In another embodiment, said fragment comprises or consists of amino acids 31, preferably 33, to 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, or 327 of SEQ ID NO: 1. In another embodiment, said fragment comprises or consists of SEQ ID NO: 2, encoded by the DNA sequence SEQ ID NO: 3.

In another embodiment, said fragment comprises or consists of amino acids 31 to 244 of SEQ ID NO: 2.

In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Feline endogenous virus, and is herein referred as RD114.RBD.

In one embodiment, said RD114.RBD comprises or consists of the amino acid sequence SEQ ID NO: 4 or fragments thereof.

In one embodiment, said fragment comprises or consists of amino acids 19 to 239 of SEQ ID NO: 4. In one embodiment, said fragment comprises or consists of amino acids 1 to 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238 of SEQ ID NO: 4.

In another embodiment, said fragment comprises or consists of amino acids 19 to 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 or 238 of SEQ ID NO: 4.

In another embodiment, said fragment comprises or consists of SEQ ID NO: 5, encoded by the DNA sequence SEQ ID NO: 6. In another embodiment, said fragment comprises or consists of amino acids 19 to 222 of SEQ ID NO: 5.

In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Xenotropic Murine Leukaemia Virus, and is herein referred as Xeno.RBD.

In one embodiment, said Xeno.RBD comprises or consists of the amino acid sequence SEQ ID NO: 7 or fragments thereof.

In one embodiment, said fragment comprises or consists of amino acids 36 to 316 of SEQ ID NO: 7. In one embodiment, said fragment comprises or consists of amino acids 1 to 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314 or 315 of SEQ ID NO: 7.

In another embodiment, said fragment comprises or consists of amino acids 36 to 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314 or 315 of SEQ ID NO: 7.

In another embodiment, said fragment comprises or consists of SEQ ID NO: 8, encoded by the DNA sequence SEQ ID NO: 9.

In another embodiment, said fragment comprises or consists of amino acids 36 to 296 of SEQ ID NO: 8. In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Feline Leukaemia Virus C, and is herein referred as FeLVC.RBD.

In one embodiment, said FeLVC.RBD comprises or consists of the amino acid sequence SEQ ID NO: 10 or fragments thereof.

In one embodiment, said fragment comprises or consists of amino acids 34 to 312 of SEQ ID NO: 10, or comprises or consists of amino acids 35 to 312 of SEQ ID NO: 10.

In one embodiment, said fragment comprises or consists of amino acids 1 to 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310 or 311 of SEQ ID NO: 10.

In another embodiment, said fragment comprises or consists of amino acids 34 or 35 to 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310 or 311 of SEQ ID NO: 10. In another embodiment, said fragment comprises or consists of SEQ ID NO: 11, encoded by the DNA sequence SEQ ID NO: 12.

In another embodiment, said fragment comprises or consists of amino acids 34 or 35 to 232 of SEQ ID NO: 11.

In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Env Koala Retrovirus, and is herein referred as KoRV.RBD.

In one embodiment, said KoRV.RBD comprises or consists of the amino acid sequence SEQ ID NO: 13 or fragments thereof.

In one embodiment, said fragment comprises or consists of amino acids 36 to 331 of SEQ ID NO: 13. In one embodiment, said fragment comprises or consists of amino acids 1 to 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329 or 330 of SEQ ID NO: 13.

In another embodiment, said fragment comprises or consists of amino acids 36 to 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329 or 330 of SEQ ID NO: 13.

In another embodiment, said fragment comprises or consists of SEQ ID NO: 14, encoded by the DNA sequence SEQ ID NO: 15. In another embodiment, said fragment comprises or consists of amino acids 36 to 253 of SEQ ID NO: 14.

In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Env Porcine Endogeneous Retrovirus-B, and is herein referred as PervB.RBD. In one embodiment, said PervB.RBD comprises or consists of the amino acid sequence SEQ ID NO: 16 or fragments thereof.

In one embodiment, said fragment comprises or consists of amino acids 44 to 326 of SEQ ID NO: 16.

In one embodiment, said fragment comprises or consists of amino acids 1 to 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324 or 325 of SEQ ID NO: 16.

In another embodiment, said fragment comprises or consists of amino acids 44 to 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324 or 325 of SEQ ID NO: 16.

In another embodiment, said fragment comprises or consists of SEQ ID NO: 17, encoded by the DNA sequence SEQ ID NO: 18. In another embodiment, said fragment comprises or consists of amino acids 44 to 239 of SEQ ID NO: 17.

In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Human T Leukaemia Virus-2, and is herein referred as HTLV2.RBD. In one embodiment, said HTLV2.RBD comprises or consists of the amino acid sequence SEQ ID NO: 19 or fragments thereof.

In one embodiment, said fragment comprises or consists of amino acids 19 to 224 of SEQ ID NO: 19, or comprises or consists of amino acids 20 to 224 of SEQ ID NO: 19 or comprises or consists of amino acids 21 to 224 of SEQ ID NO: 19. In one embodiment, said fragment comprises or consists of amino acids 1 to 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222 or 223 of SEQ ID NO: 19.

In another embodiment, said fragment comprises or consists of amino acids 19, 20 or 21 to 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222 or 223 of SEQ ID NO: 19.

In another embodiment, said fragment comprises or consists of SEQ ID NO: 20, encoded by the DNA sequence SEQ ID NO: 21. In another embodiment, said fragment comprises or consists of amino acids 19, 20 or 21 to 178 of SEQ ID NO: 20.

In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Env Bovine Leukaemia Virus, and is herein referred as BLV.RBD.

In one embodiment, said BLV.RBD comprises or consists of the amino acid sequence SEQ ID NO: 22 or fragments thereof.

In one embodiment, said fragment comprises or consists of amino acids 34 to 215 of SEQ ID NO: 22. In one embodiment, said fragment comprises or consists of amino acids 1 to 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 or 214 of SEQ ID NO: 22. In another embodiment, said fragment comprises or consists of amino acids 34 to 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 or 214 of SEQ ID NO: 22.

In another embodiment, said fragment comprises or consists of SEQ ID NO: 23, encoded by the DNA sequence SEQ ID NO: 24.

In another embodiment, said fragment comprises or consists of amino acids 34 to 181 of SEQ ID NO: 23.

In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Env Porcine Endogeneous Retrovirus-A, and is herein referred as PervA.RBD.

In one embodiment, said PervA.RBD comprises or consists of the amino acid sequence SEQ ID NO: 25 or fragments thereof.

In one embodiment, said fragment comprises or consists of amino acids 44 to 329 of SEQ ID NO: 25. In one embodiment, said fragment comprises or consists of amino acids 1 to 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327 or 328 of SEQ ID NO: 25.

In another embodiment, said fragment comprises or consists of amino acids 44 to 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327 or 328 of SEQ ID NO: 25.

In another embodiment, said fragment comprises or consists of SEQ ID NO: 26, encoded by the DNA sequence SEQ ID NO: 27. In another embodiment, said fragment comprises or consists of amino acids 44 to 309 of SEQ ID NO: 26.

In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Human endogenous retrovirus W, and is herein referred as HERV- W.RBD.

In one embodiment, said HERV-W.RBD comprises or consists of the amino acid sequence SEQ ID NO: 28 or fragments thereof.

In one embodiment, said fragment comprises or consists of amino acids 21 to 189 of SEQ ID NO: 28. In one embodiment, said fragment comprises or consists of amino acids 1 to 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187 or 188 of SEQ ID NO: 28.

In another embodiment, said fragment comprises or consists of amino acids 21 to 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187 or 188 of SEQ ID NO: 28.

In another embodiment, said fragment comprises or consists of SEQ ID NO: 29, encoded by the DNA sequence SEQ ID NO: 30.

In another embodiment, said fragment comprises or consists of amino acids 21 to 121 of SEQ ID NO: 29.

In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Human endogenous retrovirus R, and is herein referred as HERV- R.RBD. In one embodiment, said HERV-R.RBD comprises or consists of the amino acid sequence SEQ ID NO: 31 or fragments thereof.

In one embodiment, said fragment comprises or consists of amino acids 23 to 295 of SEQ ID NO: 31. In one embodiment, said fragment comprises or consists of amino acids 1 to 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293 or 294 of SEQ ID NO: 31.

In another embodiment, said fragment comprises or consists of amino acids 23 to 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293 or 294 of SEQ ID NO: 31.

In another embodiment, said fragment comprises or consists of SEQ ID NO: 32, encoded by the DNA sequence SEQ ID NO: 33.

In another embodiment, said fragment comprises or consists of amino acids 23 to 281 of SEQ ID NO: 32.

In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Gibbon Ape Leukemia virus, and is herein referred as GALV.RBD. In one embodiment, said GALV.RBD comprises or consists of the amino acid sequence SEQ ID NO: 34 or fragments thereof.

In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Human T Leukaemia Virus-1, and is herein referred as HTLV1.RBD.

In one embodiment, said HTLV1.RBD comprises or consists of the amino acid sequence SEQ ID NO: 35 or fragments thereof.

In one embodiment, said fragments comprise or consist of amino acids 1 to 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 or 208 of SEQ ID NO: 35.

In one embodiment, said fragments comprise or consist of amino acids 21 to 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 or 208 of SEQ ID NO: 35.

In another embodiment, said fragments comprise or consist in SEQ ID NO: 71 (corresponding to amino acids 1 to 182 of SEQ ID NO: 35). In one embodiment, said fragments comprise or consist of amino acids 21 to 182 of SEQ ID NO: 35.

In one embodiment, said HTLV1.RBD comprises or consists of the amino acid sequence SEQ ID NO: 71 or fragments thereof. In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Human T Leukaemia Virus-4, and is herein referred as HTLV4.RBD.

In one embodiment, said HTLV4.RBD comprises or consists of the amino acid sequence SEQ ID NO: 36 or fragments thereof.

In one embodiment, said fragments comprises or consists of amino acids 1 to 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203 or 204 of SEQ ID NO: 36.

In one embodiment, said fragments comprise or consist of amino acids 21 to 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203 or 204 of SEQ ID NO: 36. In another embodiment, said fragments comprise or consist in SEQ ID NO: 59 (corresponding to amino acids 1 to 178 of SEQ ID NO: 36).

In another embodiment, said fragments comprise or consist in amino acids 21 to 178 of SEQ ID NO: 36.

In another embodiment, said HTLV4.RBD comprises or consists of the amino acid sequence SEQ ID NO: 58 or fragments thereof.

In one embodiment, said fragments comprise or consist of amino acids 1 to 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,

375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391,

392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,

409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425,

426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,

443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459,

460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476,

477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493,

494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510,

511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527,

528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544,

545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561,

562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578,

579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595,

596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612,

613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629,

630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646,

647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663,

664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680,

681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697,

698, 699, 700, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723,

724, 725, 726, 727, 728, 729, 730, 731, 732, 733, or 734 of SEQ ID NO: 58.

In one embodiment, said fragments comprise or consist of amino acids 24 to 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391,

724 725, 726, 727, 728, 729, 730, 731, 732, 733, or 734 of SEQ ID NO: 58.

In another embodiment, said fragments comprise or consist of amino acids 22 to 237 of SEQ ID NO: 58, or comprise or consist of amino acids 23 to 237 of SEQ ID NO: 58, or comprise or consist of amino acids 24 to 237 of SEQ ID NO: 58.

In another embodiment, said fragments comprise or consist of amino acids 1 to 236 of SEQ ID NO: 58. In another embodiment, said fragments comprise or consist of amino acids 24 to 236 of SEQ ID NO: 58.

In another embodiment, said fragments comprise or consist of SEQ ID NO: 58, encoded by the DNA sequence SEQ ID NO: 70. In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Human T Leukemia Virus-3, and is herein referred as HTLV3.RBD. In one embodiment, said HTLV3.RBD comprises or consists of the amino acid sequence SEQ ID NO: 60 or fragments thereof. In one embodiment, said fragments comprises or consists of amino acids 1 to 181, 182,

183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,

200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,

217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,

234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250,

251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,

268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,

285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301,

302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,

319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,

336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,

353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,

370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386,

387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,

404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420,

421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437,

438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,

455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471,

472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488,

489, 490, 491, or 492 of SEQ ID NO: 60 or fragments thereof. In one embodiment, said fragments comprise or consist of amino acids 23 to 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, or 492 of SEQ ID NO: 60 or fragments thereof.

In another embodiment, said fragments comprise or consist of amino acids 1 to 180 of SEQ ID NO: 60. In another embodiment, said fragments comprise or consist of amino acids 23 to 180 of SEQ ID NO: 60.

In another embodiment, said fragments comprise or consist of SEQ ID NO: 60, encoded by the DNA sequence SEQ ID NO: 61.

In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Simian T Leukemia Virus-1, and is herein referred as STLV1.RBD. In one embodiment, said STLV1.RBD comprises or consists of the amino acid sequence SEQ ID NO: 62 or fragments thereof.

In one embodiment, said fragments comprises or consists of amino acids 1 to 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, or 487 of SEQ ID NO: 62 or fragments thereof.

In one embodiment, said fragments comprises or consists of amino acids 21 to 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, or 487 of of SEQ ID NO: 62 or fragments thereof.

In another embodiment, said fragments comprise or consist of amino acids 1 to 180 of SEQ ID NO: 62. In another embodiment, said fragments comprise or consist of amino acids 21 to 180 of SEQ ID NO: 62.

In another embodiment, said fragments comprise or consist of SEQ ID NO: 62, encoded by the DNA sequence SEQ ID NO: 63.

In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Simian T Leukemia Virus-2, and is herein referred as STLV2.RBD. In one embodiment, said STLV2.RBD comprises or consists of the amino acid sequence SEQ ID NO: 64 or fragments thereof.

In one embodiment, said fragments comprises or consists of amino acids 1 to 176, 177,

178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,

195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,

212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,

229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,

246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,

263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,

280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,

297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313,

314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,

331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347,

348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,

365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,

382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,

399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415,

416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432,

433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449,

450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, or 486 of SEQ ID NO: 64 or fragments thereof.

In one embodiment, said fragments comprises or consists of amino acids 21 to 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, or 486 of SEQ ID NO: 64 or fragments thereof.

In another embodiment, said fragments comprise or consist of amino acids 1 to 175 of SEQ ID NO: 64. In another embodiment, said fragments comprise or consist of amino acids 21 to 175 of SEQ ID NO: 64.

In another embodiment, said fragments comprise or consist of SEQ ID NO: 64, encoded by the DNA sequence SEQ ID NO: 65.

In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Simian T Leukemia Virus-3, and is herein referred as STLV3.RBD. In one embodiment, said STLV3.RBD comprises or consists of the amino acid sequence SEQ ID NO: 66 or fragments thereof.

In one embodiment, said fragments comprises or consists of amino acids 1 to 182, 183,

473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 489, 490, or 491 of SEQ ID NO: 66 or fragments thereof.

In one embodiment, said fragments comprises or consists of amino acids 22 to 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 489, 490, or 491 of SEQ ID NO: 66 or fragments thereof.

In another embodiment, said fragments comprise or consist of amino acids 1 to 181 of SEQ ID NO: 66. In another embodiment, said fragments comprise or consist of amino acids 22 to 181 of SEQ ID NO: 66. In another embodiment, said fragments comprise or consist of SEQ ID NO: 66, encoded by the DNA sequence SEQ ID NO: 67.

In another embodiment, said STLV3.RBD comprises or consists of the amino acid sequence SEQ ID NO: 68 or fragments thereof.

According to a preferred embodiment, receptor binding domain ligands are selected from the group comprising the sequences SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 36, 58, 59, 60, 62, 64, 66, 68 and 71, fragments and variants thereof, more preferably selected from the group comprising the sequences SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 34, 35, 36, 58, 59, 60, 62, 64, 66, 68 and 71, fragments and variants thereof. According to another embodiment, receptor binding domain ligands are encoded by a DNA sequence selected from the group comprising the sequences SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 61, 63, 65, 67, 69 and 70.

In one embodiment, the receptor binding domain ligand comprises or consists of a sequence presenting a sequence identity of at least 70% with one of the sequences SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 36, 58, 59, 60, 62, 64, 66, 68 and 71, preferably a sequence identity of at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more with one of the sequences SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35,

36, 58, 59, 60, 62, 64, 66, 68 and 71. In another embodiment, the receptor binding domain ligand is encoded by a DNA sequence presenting a sequence identity of at least 70% with one of the sequences SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 61, 63, 65, 67, 69 and 70, preferably a sequence identity of at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more with one of the sequences SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 61, 63, 65, 67, 69 and 70.

As used herein; the term "identity", when used in a relationship between the sequences of two or more polypeptides or of two or more DNA sequences, refers to the degree of sequence relatedness between polypeptides or DNA sequences respectively, as determined by the number of matches between strings of two or more amino acid residues or of two or more nucleotides respectively. "Identity" measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related polypeptides or DNA sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988). Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. \2, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith Waterman algorithm may also be used to determine identity.

In one embodiment, the receptor binding domain ligand is a variant of one of the polypeptide having the sequences SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 36, 58, 59, 60, 62, 64, 66, 68 and 71. A polypeptide "variant" as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of techniques well known in the art. Modifications may be made in the structure of polypeptides and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics.

When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant or portion of a polypeptide of the invention, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence. For example, certain amino acids may be substituted by other amino acids in a protein structure without appreciable loss of its ability to bind cell surface nutrient transporters. Since it is the binding capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with similar properties. It is thus contemplated that various changes may be made in the peptide sequences, or corresponding DNA sequences that encode said peptides without appreciable loss of their biological utility or activity. In many instances, a polypeptide variant will contain one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted by another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of 10 amino acids or fewer (i.e. 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid(s)). Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide. In one embodiment, the receptor binding domain ligand is a fusion protein comprising a part or the totality of a receptor binding domain fused to a detection tag, such as, for example, a Fc fragment or a GFP. Examples of Fc fragments include, but are not limited to, rabbit Fc fragment (amino acid sequence SEQ ID NO: 37, encoded by SEQ ID NO: 38), and mouse Fc fragment (amino acid sequence SEQ ID NO: 39, encoded by SEQ ID NO: 40). In one embodiment, the receptor binding domain ligand is selected from the group comprising HTLV2.RBD fused to a mouse Fc fragment (encoded by the DNA sequence SEQ ID NO: 41), PERVB.RBD fused to a mouse Fc fragment (encoded by the DNA sequence SEQ ID NO: 42), RD114.RBD fused to a mouse Fc fragment (encoded by the DNA sequence SEQ ID NO: 43), HERVW.RBD fused to a mouse Fc fragment (encoded by the DNA sequence SEQ ID NO: 44), KoRV.RBD fused to a mouse Fc fragment (encoded by the DNA sequence SEQ ID NO: 45), HERVR.RBD fused to a mouse Fc fragment (encoded by the DNA sequence SEQ ID NO: 46), Ampho.RBD fused to a rabbit Fc fragment (encoded by the DNA sequence SEQ ID NO: 47), FeLVC.RBD fused to a rabbit RBD (encoded by the DNA sequence SEQ ID NO: 48), BLV.RBD fused to a rabbit RBD (encoded by the DNA sequence SEQ ID NO: 49), PER VA. RBD fused to a rabbit Fc fragment (encoded by the DNA sequence SEQ ID NO: 50), and Xeno.RBD fused to a rabbit Fc fragment (encoded by the DNA sequence SEQ ID NO: 51). In one embodiment, the receptor binding domain ligand is obtained by a cloning method, such as, for example, using any production system known in the art, such as, for example, Escherichia coli, yeast, baculovirus-insect cell, or mammalian cells such as HEK or CHO, expression system. In one embodiment, the sequence of the receptor binding domain ligand is fused in N-terminal to a peptide signal sequence allowing the secretion of said receptor binding domain ligand. Examples of peptide signal sequences include, but are not limited to, human IL-2 peptide signal (SEQ ID NO: 52), human albumin peptide signal (SEQ ID NO: 53), human chymotrypsinogen peptide signal (SEQ ID NO: 54), human trypsinogen-2 peptide signal (SEQ ID NO: 55), gaussia luciferase peptide signal (SEQ ID NO: 56), and mouse IgM peptide signal (SEQ ID NO: 57).

In one embodiment, the receptor binding domain ligand comprises a part or the totality of HTLV2.RBD, HTLV1.RBD, HTLV3.RBD, HTLV4.RBD, STLV1.RBD, STLV2.RBD or STLV3.RBD and binds to the Glutl nutrient transporter. In one embodiment, the receptor binding domain ligand comprises a part or the totality of Ko.RBD, and binds to the PiTl nutrient transporter. In one embodiment, the receptor binding domain ligand comprises a part or the totality of RD114.RBD and binds to the ASCT2 nutrient transporter. In one embodiment, the receptor binding domain ligand comprises a part or the totality of Ampho.RBD and binds to the PiT2 nutrient transporter. In one embodiment, the receptor binding domain ligand comprises a part or the totality of FeLVC.RBD and binds to the FLVCR1 nutrient transporter. In one embodiment, the receptor binding domain ligand comprises a part or the totality of PERV-A.RBD and binds to the RFTl and/or the RFT3 nutrient transporter. In one embodiment, the receptor binding domain ligand comprises a part or the totality of Xeno.RBD and binds to the XPRl nutrient transporter. In one embodiment, the receptor binding ligand comprises a part or the totality of HERV-W.RBD and binds to the ASCT1 nutrient transporter. In one embodiment, the receptor binding domain ligand comprises a part or the totality of GALV.RBD and binds to the PiTl nutrient transporter.

In one embodiment, the first cell surface nutrient transporter is selected from the group consisting of ASCT1, ASCT2, CAT1, FLVCR1, GLUT1, PARI, PAR2, PiTl, PiT2, RFTl, RFT3, SMITl and XPRl. Preferably the cell surface nutrient transporter is GLUT1.

In one embodiment, the at least one cell surface nutrient transporter is selected from the group consisting of ASCT1, ASCT2, CAT1, FLVCR1, GLUT1, PARI, PAR2, PiTl, PiT2, RFTl, RFT3, SMITl and XPRl. Preferably, the cell surface nutrient transporter is GLUT1.

In one aspect, the method additionally comprises detecting and/or quantifying binding of a second ligand to a second cell surface nutrient transporter in the presence of said agent to be tested, thereby obtaining a second binding value, and comparing said second binding value with a second reference binding value, wherein the second reference binding value is preferably measured in the absence of said agent.

In one embodiment, the method comprises measuring the binding of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more ligands to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cell surface nutrient transporters. According to this embodiment, the method of the invention may be an in vitro method for detecting and/or quantifying mitochondrial toxicity of an agent in a cell culture comprising the steps of

a. detecting and/or quantifying binding of a first ligand, preferably of a first receptor binding domain ligand, to a first cell surface nutrient transporter in the presence of said agent, thereby obtaining a first binding value, and comparing the first binding value with a first reference binding value,

b. optionally detecting and/or quantifying binding of a second ligand, preferably of a second receptor binding domain ligand, to a second cell surface nutrient transporter in the presence of said agent, thereby obtaining a second binding value, and comparing the second binding value with a second reference binding value,

c. optionally detecting and/or quantifying binding of a 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th, 9^th, 10^th, 11^th, 12^th or more ligand, preferably of a 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th, 9^th, 10^th, 11^th, 12^th or more receptor binding domain ligand, to a 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th,

9^th, 10^th, 11^th, 12^th or more cell surface nutrient transporter in the presence of said agent, thereby obtaining a 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th, 9^th, 10^th, 11^th, 12^th or more binding value, and comparing the 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th, 9^th, 10^th, 11^th, 12^th or more binding value with a 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th, 9^th, 10^th, 11^th, 12^th or more reference binding value.

In one embodiment, a difference between at least one of the binding value and the corresponding reference binding value, preferably of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more binding values and the corresponding reference binding values, is indicative of the mitochondrial toxicity of said agent. In one embodiment, the absence of difference between at least one of the binding value and the corresponding reference binding value, preferably of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more binding values and the corresponding reference binding values, is indicative of the mitochondrial toxicity of said agent. In another aspect, the second, third, fourth,..., cell surface nutrient transporter is selected from the group consisting of ASCT1, ASCT2, CAT1, FLVCR1, GLUT1, PARI, PAR2, PiTl, PiT2, RFT1, RFT3, SMIT1 and XPRl.

In a further aspect, the second, third, fourth,..., ligand is a receptor binding domain ligand, wherein said receptor binding ligand comprises a part or the totality of a receptor binding domain (RBD) derived from the soluble part of a glycoprotein of an enveloped virus that interacts with a cell surface nutrient transporter.

In one embodiment, the at least two receptor binding domain ligands are liable to interact with at least two different cell surface nutrient transporters. In one embodiment, the method of the invention comprises detecting and/or quantifying the binding of ligands to GLUT1.

In one aspect, the first cell surface nutrient transporter is GLUT1 and the second cell surface nutrient transporter is PiTl or FLVCR1.

In another embodiment, the first cell surface nutrient transporter is PiTl and the second cell surface nutrient transporter FLVCR 1.

In one embodiment, the method of the invention comprises detecting and/or quantifying the binding of ligands to GLUT1, PiTl and FLVCR1. Preferably, said ligands are receptor binding domain ligands derived from HTLV1.RBD, HTLV2.RBD, HTLV3.RBD, HTLV4.RBD, STLVl.RBD, STLV2.RBD, or STLV3.RBD, preferably HTLV2.RBD; KoRV.RBD and FeLVC.RBD.

In one embodiment, the method of the invention comprises detecting and/or quantifying the binding of ligands to GLUT1 and PiT2. Preferably, said ligands are receptor binding domain ligands derived from HTLV1.RBD, HTLV2.RBD, HTLV3.RBD, HTLV4.RBD, STLVl.RBD, STLV2.RBD, or STLV3.RBD, preferably HTLV2.RBD; and Ampho.RBD. As used herein, the term "reference" broadly encompasses any suitable reference, such as, for example, any reference expression profile or any reference binding value, which may be used as a basis for comparison with respect to the measured expression profile or binding value, for example. Preferably, the standard reference is a personalized reference, determined using the same culture of cells as the one used for determining the expression profile or the binding value in the presence of the agent to be tested.

In one embodiment of the invention the reference expression profile is the expression profile measured in a culture of cells before, preferably just before, the addition of the agent to be tested within the culture medium. Accordingly, in one embodiment, the reference binding value is the binding value measured in a culture of cells before, preferably just before, the addition of the agent to be tested within the culture medium.

In one embodiment, the reference is an index value or is derived from one or more risk prediction algorithms or computed indices for the mitochondrial toxicity of tested agents. A reference can be relative to a number or value derived from cell population studies, preferably based on cells which are the same as the ones used for testing the mitochondrial toxicity of said agent, and which are cultured in the same culture medium with the same culture conditions.

In one embodiment, the reference is constructed using algorithms and other methods of statistical and structural classification. In another embodiment of the invention, the reference is derived from the measurement of the expression profile or of a binding value, in a control cell sample exposed to an agent (or "standard compound") known to not present a mitochondrial toxicity. According to this embodiment, a difference between the measured expression profile or binding value and the reference is indicative of the mitochondrial toxicity of the agent. In another embodiment of the invention, the reference is derived from the measurement of the expression profile or of a binding value, in a control cell sample exposed to an agent (or a "standard compound") known to present a mitochondrial toxicity. According to this embodiment, the absence of difference between the measured expression profile or binding value and the reference is indicative of the mitochondrial toxicity of the agent.

In one embodiment of the invention, the reference is derived from the measurement of the expression profile or of the binding value, in a control cell culture in the absence of the tested agent. According to this embodiment, a difference between the measured expression profile or binding value and the reference is indicative of the mitochondrial toxicity of the agent.

In one embodiment, a culture of cells is provided, and separated in two different culture batches, wherein the first culture batch is exposed to the agent to be tested (for measuring the expression profile or the binding value) and the second culture batch is not exposed to the agent to be tested (for measuring the reference expression profile or the reference binding value).

In a further aspect, growing cells in the absence of the agent and determining the expression profile or detecting and/or quantifying binding of the first ligand to the first cell surface nutrient transporter in the absence of the agent is carried out at a different point in time to growing cells in the presence of the agent and determining the expression profile or detecting and/or quantifying binding of the first ligand to the first cell surface nutrient transporter in the presence of the agent.

By a "different point in time" is meant a time point which may be before or after the point in time when the cells are grown in the presence of the agent and detecting and/or quantifying binding of the first ligand to the first surface nutrient transporter.

In one aspect, at least one value is attributed to the expression profile and/or to the detecting and/or quantifying binding of the first ligand to the first cell surface nutrient transporter to cells growing in the absence of the agent (i.e. said value corresponds to a reference expression profile and/or to a reference binding value) and the at least one value is used to determine the difference in the binding of the first ligand to the first cell surface nutrient transporter growing in the presence of the agent.

The value may, for example, be a number or symbol which has a relative worth. In another aspect, the at least one value is stored on a database to provide a stored value and the stored value is used to determine the difference in the expression profile or the difference in the binding of the first ligand to the first cell surface nutrient transporter growing in the presence of the agent. The database may, for example, be stored on a computer or a server.

In one embodiment of the invention, the expression profile and the reference expression profile are considered as different if the difference between both numerical values is considered as statistically significant, i.e. the p-value is lower than 0.05, preferably lower than 0.01. In one embodiment of the invention, an increase of the expression profile superior or equal to 10%, preferably superior or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100% or more compared to the reference expression profile indicates that the agent presents a mitochondrial toxicity. In another embodiment of the invention, a decrease of the expression profile superior or equal to 10%, preferably superior or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100% or more compared to the reference expression profile indicates that the agent presents a mitochondrial toxicity.

In one embodiment of the invention, the binding value and the reference binding value are considered as different if the difference between both numerical values is considered as statistically significant, i.e. the p-value is lower than 0.05, preferably lower than 0.01. In one embodiment of the invention, an increase of the binding value superior or equal to 10%, preferably superior or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100% or more compared to the reference binding value indicates that the agent presents a mitochondrial toxicity. In another embodiment of the invention, a decrease of the binding value superior or equal to 10%, preferably superior or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100% or more compared to the reference binding value indicates that the agent presents a mitochondrial toxicity.

In one embodiment, an increase of the expression of GLUTl, PiTl and/or FLVCRl, preferably an increase of the presence of GLUTl, PiTl and/or FLVCRl on the cell surface of a cell exposed to an agent as compared to a reference is indicative of the mitochondrial toxicity of said agent.

In one embodiment, an increase of the expression of GLUT1, combined with no change in the expression of PiT2, preferably an increase of the presence of GLUT1, combined with no change in the presence of PiT2 on the cell surface of a cell exposed to an agent as compared to a reference is indicative of the mitochondrial toxicity of said agent.

In one embodiment, the cell used in the present invention is rich in mitochondria, such as, for example, heart cells and liver cells. In one embodiment, a cell rich in mitochondria comprises at least about 100 mitochondria, preferably at least about 500 mitochondria, more preferably at least about 1000 mitochondria.

In another aspect, the cell is a mammalian cell, preferably a human cell. Examples of mammalian cells that may be used in the present invention include, but are not limited to, heart cells, liver cells, neurons and other neural cells, muscular cells, pancreatic cells and the like. Preferably the cell is a heart or liver cell. In a further aspect, the cell is derived from a stem cell.

The term "stem cells" as used herein concerns pluripotent or multipotent stem cells. Examples of stem cells include, but are not limited to, embryonic stem cells (ESC), adult stem cells, haematopoietic stem cells, neural stem cells, mesenchymal stem cells and induced pluripotent stem cells(iPS). In one particular embodiment of the invention, embryonic stem cells derived from human embryonic stem cells which were produced by a process that necessarily involved the destruction of the human embryo, may be excluded from the implementation of the invention.

In one embodiment, cells are cardiomyocytes, preferably human cardiomyocytes, such as, for example, CYTIVA™ cardiomyocytes provided by GE Heathcare, stem cells derived cardiomyocytes provided by Cellartis and ReproCELL, or iCell® cardiomyocytes provided by Cellular Dynamics. In another embodiment, cells are liver cells, preferably human liver cells, preferably primary hepatocytes isolated from liver. Examples of human liver cell lines include, but are not limited to HepaRG, C3a, Hep 3B, Hep G2, Hub 7, Smmc 7721, NCI-HI1755, THLE 3, SNU 387, NCI-H735, CFPAC-1, MCA-RH7777, NCI-N87, DMS 153, SNU 182, PLC/PRF/5, SK-HEP1, Capan-1, SU.86.86, THLE-2, SNU 398, SNU 475, NCI- HI 688 or SNU 423.

The term "in vitro culture" as used herein means any non in vivo artificial environment which sustains cell growth under defined conditions. Non limiting examples include culture or multiwall dishes containing solid media which provide all the nutrients, growth factors and vitamins required for cell growth. Alternatively, shake flasks or bioreactors containing liquid media can be used.

In one embodiment, the cells are cultured in a glucose based medium.

By "glucose based medium" is meant that glucose is the main energy source in the medium. Glucose is metabolised by the cells principally by glycolysis to form pyruvate and thus provides energy for the cells to grow (Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications by R. Ian Freshney, published (6th ed) by John Wiley & Sons, Feb 2, 2011). The glucose based medium may also comprise nutrients necessary for the survival of cultured cells, such as, for example, growth factors, amino acids, vitamins, antibiotics and the like. This medium is classically pH- buffered.

Examples of glucose based culture medium that may be used for the culture of cardiomyocytes include, but are not limited to, RPMI1640+B27 (provided by Invitrogen), sodium bicarbonate buffered Medium 199 (Cellgro M-199, Mediatech) optionally supplemented to make 'CCT' (Creatine-Carnitine-Taurine) with (mM): 5 mM creatine, 2 mM L-carnitine and 5 mM taurine, and Dulbeco's Modified Eagle Medium (DMEM). Examples of glucose based culture medium that may be used for the culture of liver cells include, but are not limited to, Dulbeco's Modified Eagle Medium (DMEM), phenol red-free Williams Medium E and the like.

In another embodiment, the cells are cultured in a galactose based medium, or in any culture medium adapted to the culture of said cells.

In one embodiment, classical culture conditions are applied, such as, for example, temperature of about 37 °C, C0₂ content of about 5% and humidity higher than about 80%.

In one aspect, the agent to be tested is a molecule selected from the group consisting of drug, agrochemical, pesticide, cosmetic, food additive, new chemical entity, protein, nucleic acid, carbohydrate and lipid.

By "new chemical entity" is meant a molecule which has been synthesised or developed by a pharmaceutical or biotech company as a potential drug. Typically a new chemical entity has a molecular weight less than 1000 Daltons, preferably less than 500 Daltons, most preferably less than 300 Daltons.

In one embodiment, the agent to be tested is a xenobiotic. As used herein, the term "xenobiotic" refers to an agent, preferably a chemical, which may be found in an organism but which is not produced by said organism, or which is not part of the normal diet of said organism. The term xenobiotic may also refer to an agent present in an organism in much higher concentrations than expected. For example, antibiotics are an example of xenobiotic in humans.

According to a second aspect of the present invention, there is provided a kit for detecting and/or quantifying mitochondrial toxicity of an agent, comprising means for measuring the expression profile of at least one cell surface nutrient transporter, and instructions for use thereof in a method as hereinbefore described for the first aspect of the invention. In one embodiment, the expression profile is measured at the RNA level, and the kit of the invention comprises means for total RNA extraction, means for reverse transcription of total RNA, and means for quantifying the expression of RNA corresponding to cell surface nutrient transporter(s). In one embodiment, the means for determining the expression of cell surface nutrient transporter(s) are PCR primers, preferably qPCR primers, specific for said cell surface nutrient transporter(s). Examples of PCR or qPCR primers that may be used for assessing the expression of GLUT1 include, but are not limited to, the following couple of primers: Forward primer: 5 ' -TCACTGTGCTCCTGGTTCTG-3 ' (SEQ ID NO: 72) - Reverse primer: 5'- CCTCGGGTGTCTTGTC ACTT-3 ' (SEQ ID NO: 73).

In one embodiment of the invention, the kit of the invention comprises means for total RNA extraction, means for reverse transcription of total RNA, and reagents for carrying out a quantitative PCR as hereinabove described (such as, for example, primers, buffers, enzyme, and the like). In one embodiment of the invention, the kit of the invention comprises DNA probes, which may be hybridized to the qPCR amplicons to detect said cell surface nutrient transporter(s).

In one embodiment, the means for determining the expression of the cell surface nutrient transporter is a microarray comprising probes specific for said cell surface nutrient transporters.

In one embodiment, said means for quantifying the expression of RNA corresponding to the cell surface nutrient transporters hereinabove described is a microarray. The present invention thus also relates to microarrays for measuring the RNA expression profile of cell surface nutrient transporters as hereinabove described, and/or for implementing the in vitro method of the invention.

In another embodiment of the invention, the expression profile is measured at the protein level, and the kit of the invention comprises means for total protein extraction, as well as antibodies or receptor binding domain ligands as hereinabove described for detecting cell surface nutrient transporter(s).

In one embodiment, the kit comprises one or more ligands, preferably one or more receptor binding domain ligands as hereinabove described, for binding to a cell surface nutrient transporter for detecting and/or quantifying mitochondrial toxicity.

In one embodiment, the present invention thus relates to a kit comprising one or more receptor binding domain ligands as hereinabove described, wherein the one or more receptor binding domain ligand(s) comprise(s) a sequence selected from the group consisting of SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 36, 58, 59, 60, 62, 64, 66, 68 and 71, fragments or variants thereof, or a sequence presenting a sequence identity of at least 70% with any of said sequences.

In one embodiment, the kit of the invention comprises the following receptor binding domain ligand: HTLV2.RBD. In one embodiment, the kit of the invention comprises the following receptor binding domain ligands: HTLV2.RBD, KoRV.RBD and a FeLVC.RBD.

In another embodiment, the kit of the invention comprises the following receptor binding domain ligands: HTLV2.RBD and Ampho.RBD.

In a third aspect of the present invention, there is provided a use of a kit as hereinabove described, preferably comprising one or more ligands for binding to a cell surface nutrient transporter for detecting and/or quantifying mitochondrial toxicity in a method as hereinbefore described for the first aspect of the present invention.

BRIEF DESCIPTION OF THE DRAWINGS FIGURE 1: Cardiotoxicity of test drugs. Cell viability was determined by imaging of TOT03 staining of nuclei of non-viable cells following incubation of cardiomyocytes with the indicated concentrations of drugs for 72 hours in glucose (·) or galactose culture media (^■). Data are the means of 3 replicates.

FIGURE 2: Profiling of effects of drug treatment on RBD binding. Parallel coordinate axis analysis of immunofluorescence imaging data for RBD binding to cardiomyocytes treated 10 with increasing concentrations of drugs for 72 hours in glucose and galactose media (0.3μΜ, Ι.ΟμΜ, 3.0μΜ, Ι Ι.ΟμΜ, or 33.0μΜ). Cells showing significant toxicity following drug treatment (Antimycin A and Mubritinib in galactose medium and higher concentrations of Amiodarone, Imatinib, Sorafenib and Sunitinib in glucose and/or galactose media) were not analysed for RBD binding. Data are the means of 3 replicates.

FIGURE 3: RBD binding to metabolite transporters in drug treated cardiomyocytes in glucose media. Immunofluorescence intensity data for RBD binding to cardiomyocytes exposed to drugs at concentrations of 0.03-33. ΟμΜ in glucose medium for 72hours were analysed by hierarchical clustering. Drug concentrations causing significant toxicity were excluded from analysis. Data are the mean of 3 replicates.

FIGURE 4: Flow cytometry profiling of RBD binding to cardiomyocyte metabolite transporters. RBD binding was quantified by flow cytometry on cells treated with the indicated drug concentrations for 24 hours in (A) glucose and (B) galactose media, intensity values are shown relative to DMSO treated cells. Data are means of three independent experiments.

FIGURE 5: RBD binding to GLUT1 and PiT2 transporters in drug treated cardiomyocytes in glucose media. RBD binding to GLUT1 (black) and PiT2 (white) were measured by flow cytometry in cells exposed to the indicated drug concentrations for 24 hours. Mean and standard deviation of 3 experiments, *p<0.05, **p<0.01. FIGURE 6: schematic diagram of the HTLV-1 envelope glycoprotein (Env). Mature Env is constituted of two subunits formed after cleavage of the amino terminal signal peptide (SP) and cleavage of the Env polyprotein precursor into the extracellular SU and the membrane-anchored TM. SU comprises three distinct subdomains: an amino terminal receptor-binding domain (RBD), a central proline-rich region (PRR) and a carboxy terminal domain (C-term).

EXPERIMENTAL The invention will now be described with reference to the specific examples below. EXAMPLE 1

The following is a current state of the art example of detection of drug-induced mitochondrial toxicity detected in a dual media culture system. Cytiva™ cardiomyocytes derived from H7 human embryonic stem cells were seeded at 15,000 cells per well in RPMI1640+B27 medium (Life Technologies) into 384 well plates coated with Matrigel (BD Bioscences). Cells were cultured for 5 days in glucose media followed by 1 day in glucose or galactose media. Test compounds were diluted from DMSO stocks into glucose or galactose media to yield final assay concentrations in the range 0.05μΜ to ΙΟΟμΜ. Plates were incubated for 72 hours and Hoechst 33342 (ΙμΜ) and ΤΟΤΟ-3(1μΜ) (Invitrogen) were added for 1 hour prior to imaging. Cardiomyocytes were imaged on IN Cell Analyzer 2000 (GE Healthcare) using excitation and emission filters for Hoechst and TOTO-3. Automated image analysis was performed with IN Cell Investigator (GE Healthcare) to measure total cells (Hoechst staining) and non-viable cells (TOTO-3 staining). Data for drug effects on cell viability in glucose and galactose media are shown in Fig. 1.

Amiodarone, an anti-arrhythmic drug with known mitochondrial toxicity used as a positive toxic control, showed equivalent toxicity in both glucose and galactose media. Nifedipine, a calcium channel blocker used as a negative control, showed no impact on cell viability in either media at the concentrations tested. Antimycin A an inhibitor of cytochrome c reductase which interferes with the electron transport chain of oxidative phosphorylation, showed minimal toxicity in glucose medium and extreme toxicity in galactose medium, confirming a metabolic shift from glycolysis to oxidative phosphorylation following culture of cardiomyocytes in galactose medium. Among the five anti-cancer drugs tested three, Imatinib, Sunitinib and Tasocitinib, gave very similar results in both media, with Imatinib and Sunitinib showing equivalent toxicity and Tascocitinib showing minimal toxicity. Sorafenib and Mubritinib both showed sensitisation of toxicity in galactose medium with Sorafenib showing a 4 fold reduction in IC₅₀ between glucose and galactose media. In the case of Mubritinib sensitisation of toxicity was extremely pronounced, changing from minimal impact on viability in glucose medium to extreme toxicity in galactose medium. This dramatic increase in toxicity, associated with changing cardiomyocyte metabolism from glycolysis to oxidative phosphorylation, is a strong indication that Mubritinib toxicity is mediated through mitochondria.

EXAMPLE 2

To investigate whether monitoring metabolic changes occurring in drug treated cardiomyocytes could be predictive of metabolism dependent drug toxicity cell surface expression of a panel of seven nutrient transporters were analysed using RBD ligand binding analyzed by high content screening.

IgG-Fc tagged RBD fusion proteins were produced as follows. HTLV2.RBD.mFc (encoded by SEQ ID NO: 32), KoRV.RBD.mFc (encoded by SEQ ID NO: 36) and RD114.RBD.mFc (encoded by SEQ ID NO: 34) ligands were derived from the sequences encoding the first 178, 252 and 223 amino acids of the HTLV-2, KoRV, and RD114 Env, respectively. The corresponding RBD-encoding sequence was fused to the mouse IgGl-Fc fragment (mFc). Ampho.RBD.rFc (encoded by SEQ ID NO: 27), Xeno.RBD.rFc (encoded by SEQ ID NO: 31), FeLVC.RBD.rFc (encoded by SEQ ID NO: 28) and PERVA.RBD.rFc (encoded by SEQ ID NO: 30) ligands were derived from the sequences encoding the first 243, 293, 232 and 309 and amino acids of the Amphotropic-MLV, Xenotropic-MLV, FeLVC and PERVA Env, respectively. The corresponding RBD-encoding sequence was fused to the rabbit IgGl-Fc fragment (rFc).

All constructs were inserted into the eukaryotic pCSI expression vector and were transfected into 293T cells by calcium-phosphate precipitation. Cells were washed 16 hours later and incubated for another 48 hours in serum-free Optipro SFM medium (Invitrogen) supplemented with glutamine and non-essential amino acids. Conditioned media was harvested, filtered through 0.45 μιη filters and concentrated 100-fold by centrifugation at 3,500 G on 10 kDa cut-off Centricon Plus-70 (Millipore). Samples were aliquoted and stored at -80°C. Each preparation was verified for integrity by western blotting.

Cardiomyocytes were cultured and treated with test compound as described for Example 1 with 0.1% DMSO used as a control. Following 72 hour compound exposure media were aspirated, cells were blocked for 20 minutes at room temperature with PBS containing 5% FCS and O. lmg/ml human IgG and then RBD probes added in glucose or galactose culture media with ΙμΜ Hoechst and incubated for 15 min at 37°C. Cells were washed twice with 5% FCS and ΙμΜ Hoechst in PBS at room temperature and then incubated for 30 minutes at 4°C with Alexa Fluor 647 chicken anti-rabbit IgG (Invitrogen, 1:400) or R-PE labelled goat anti-mouse IgG (Invitrogen, 1:200) in 5% FCS and ΙμΜ Hoechst in PBS. Following antibody labelling cells were washed twice with 5% FCS in PBS with ΙμΜ Hoechst and fixed for 30 minutes at 4°C in 5% FCS in PBS with ΙμΜ Hoechst and 0.1% paraformaldehyde. Cardiomyocytes were imaged on IN Cell Analyzer 2200 (GE Healthcare) using excitation and emission filters for Hoechst, R-PE and Alexa Fluor 647. Automated image analysis of RBD staining intensity was performed with IN Cell Investigator (GE Healthcare). Analysis of RBD binding profiles (Fig. 2) showed a range of drug effects on cell surface nutrient transporter expression. In glucose media the kinase inhibitors Tascocitinib, Sunitinib, Imatinib and Sorafenib yielded similar profiles at sub-toxic drug concentrations while in galactose medium the same compounds produced individually diverse RBD binding signatures. The profiles of this class of anti-cancer drugs were different from the assay control compounds, Amiodarone, Nifedipine and Antimycin A in both glucose and galactose media.

The most significant finding was the dose-dependent changes in RBD signatures observed for Sorafenib and Mubritinib in glucose media (Fig. 2). In both cases these compounds, which had previously been shown to have sensitisation of toxicity in galactose media (Fig. 1), showed profile changes not observed with other kinase inhibitors showing equal toxicity in both media. Sorafenib at Ι ΙμΜ, a concentration producing significant toxicity in galactose medium but not in glucose medium, showed an increase in GLUTl and PiTl expression and a decrease in several other transporters. Mubritinib showed a marked change in RBD profiles with increasing drug concentrations with significant up regulation of GLUTl and the heme transporter FLVCRl at Ι ΙμΜ and 33μΜ, drug concentrations at which minimal impact on cell viability were observed (Fig. 1). Up regulation of FLVCRl may be indicative of loss of mitochondrial integrity leading to leaching of free heme into the cell cytoplasm and a subsequent increase in expression and/or mobilisation of the export transporter to remove the toxic heme from the cell.

Hierarchical cluster analysis of RBD binding data in glucose media (Fig. 3) confirmed these observations and the predictive potential of RBD profiling to identify compounds with metabolic modulation of toxicity. All concentrations of Mubritinib, Antimycin A and Ι ΙμΜ Sorafenib clustered separately from lower concentrations of Sorafenib and from all doses of the compounds not showing metabolic sensitisation of toxicity, Imatinib, Sunitinib and Tascocitinib. These findings indicate that RBD binding analysis in glucose medium is predictive of bioenergetic modulation of toxicity, obviating the need for dual media screening of compounds. EXAMPLE 3

To confirm the observations of example 2, to determine whether these observations could be detected at a shorter drug treatment time, and to determine the utility of using an alternate analysis method (i.e. RBD ligands) to profile cardiomyocyte nutrient transporter expression, flow cytometry was used to measure RBD binding to cardiomyocytes treated for only 24 hours with a subset of the drugs used in examples 1 and 2.

Cardiomyocytes were seeded at 40,000 cells per well in RPMI1640+B27 (Invitrogen). 96 well plates were coated with MatrigelTM (BD Bioscences) and cells were cultured for 5 days in glucose media followed by 1 day in glucose or galactose media. Antimycin A, Nifedipine, Imatinib, Sorafenib and Mubritinib were diluted from DMSO stocks into glucose or galactose media to yield 1, 10 or 30 μΜ final assay concentrations. 0.1% DMSO was used as negative control. After 24 hours of drug treatment, cells were prepared for RBD binding to nutrient transporters. Cardiomyocytes were detached using trypsin 0.25% (Invitrogen) for 5 minutes at 37°C and transferred into a 96-well V-shape microplate. RBDs were premixed pairwise (one mouse Fc and one rabbit Fc fused RBD) in culture medium containing 0.1% sodium azide and ImM EDTA. RBDs were added to cardiomyocytes and incubated at 37 °C for 15 minutes. Cells were washed once with PBS/2% FCS and then incubated with Alexa 10 FluorTM 647 goat anti-rabbit IgG (Invitrogen, 1:800) and R-PE goat anti-mouse IgGl (Invitrogen, 1:200) antibodies in binding buffer (PBS/2% FCS/0.1% sodium azide/lmM EDTA) for 30 minutes at 4°C. Cells were washed twice with binding buffer and labeled with mouse anti-human CD90-FITC antibody (BD Biosciences, clone 5E10, 1:500) and 1 μg/mL DAPI to restrict RBD binding analysis to CD90 negative cells (mainly composed of cardiomyocytes, as evidenced by CD90/cardiac Troponin T labeling, data not shown). After 30 minutes of incubation at 4°C, cells were washed and resuspended in binding buffer before flow cytometry analysis. Fluorescent signals were acquired on a FACSVerse flow cytometer (BD Biosciences) with 405, 488 and 640 nm excitation, and data analysis was performed using Flowjo software (Tree Star Inc.). Dead cells and CD90 positive cells, representing non cardiomyocytes, were excluded from the analysis. Fluorescence minus one (FMO) controls were used to establish background levels in RBD channels (R-PE and AF647). Signals were converted into molecules of equivalent soluble fluorochrome (MESF) values using calibration beads (R-PE and AF647 MESF quantum beads; Bang Laboratories) according to the manufacturer's instructions. Clustering of flow cytometry data (Fig. 4) showed similar results to those obtained by high content screening with data from cardiomyocytes in glucose medium treated with drugs with metabolic regulated toxicity (ΙΟμΜ Sorafenib, ΙμΜ Antimycin A, ΙμΜ Mubritinib and 30μΜ Mubritinib) clustering separately from non-toxic and/or non- sensitised drug concentrations (Nifedipine, Imatinib and ΙμΜ Sorafenib). This data clearly indicates that drug impact on cellular metabolism and bioenergetics can be detected after only 24 hours exposure. As with analysis of drug effects at 72 hours by high content screening, flow cytometry data showed up regulation of GLUT1, although increase in the FLVCR1 heme transporter was not observed, possibly indicating that drug impact on mitochondrial integrity leading to heme release is a later event in drug induced cardiotoxicity. Analysis of RBD binding to GLUT1 by flow cytometry (Fig. 5) showed a significant increase in the cell surface expression of this key transporter correlating with energy substrate dependent toxicity of the kinase inhibitors Sorafenib and Mubritinib.

While preferred illustrative embodiments of the present invention are described, one skilled in the art will appreciate that the present invention can be practiced by other than the described embodiments, which are presented for purposes of illustration only and not by way of limitation. The present invention is limited only by the claims that follow.

Claims

An in vitro method for detecting and/or quantifying mitochondrial toxicity of an agent comprising the steps of:

a) growing at least one cell in an in vitro culture in the presence of an agent; b) determining the expression profile of at least one cell surface nutrient transporter in said at least one cell; and

The method according to claim 1, wherein said at least one cell surface nutrient transporter is selected from the group consisting of glucose transporter, inorganic phosphate transporter, amino acid transporter, heme transporter, inositol transporter and riboflavin transporter.

The method according to either of claims 1 or 2, wherein the at least one cell surface nutrient transporter is selected from the group consisting of ASCT1, ASCT2, CAT1, FLVCR1, GLUT1, PARI, PAR2, PiTl, PiT2, RFT1, RFT3, SMIT1 and XPRl.

The method according to claim 3, wherein the cell surface nutrient transporter is GLUT1.

The method according to any of claims 1 to 4, wherein the expression of the cell surface nutrient transporter is determined at the RNA level.

The method according to any of claims 1 to 4, wherein the expression of the cell surface nutrient transporter is determined at the protein level.

The method according to any of claims 1 to 6, wherein the expression profile of the cell surface nutrient transporter is determined by detecting and quantifying the binding of a first ligand to the cell surface nutrient transporter.

8. The method according to claim 7, wherein said first ligand is an antibody specific to the cell surface nutrient transporter.

9. The method according to claim 7, wherein said first ligand is a receptor binding domain ligand comprising a part or the totality of a receptor binding domain (RBD) derived from the soluble part of a glycoprotein of an enveloped virus that interacts with a cell surface nutrient transporter.

10. The method according to claim 9, wherein said enveloped virus is a retrovirus.

11. The method according to either of claims 9 or 10, wherein the receptor binding domain ligand comprises a sequence selected from the group consisting of SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32,

34, 35, 36, 58, 59, 60, 62, 64, 66, 68 and 71, fragments or variants thereof, or a sequence presenting a sequence identity of at least 70% with any of said sequences.

12. The method according to claim 9, wherein the receptor binding domain ligand is encoded by a DNA sequence selected from the group consisting of SEQ ID

NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 61, 63, 65, 67, 69 and 70, or sequences presenting a sequence identity of at least 70% with any of said sequences.

13. The method according to any one of claims 7 to 12 wherein the expression profile is determined as a binding value for the first ligand to the cell surface nutrient transporter and comparing said binding value with a first reference binding value.

14. The method of claim 13, further comprising determining a second binding value for the binding of a second ligand to a second cell surface nutrient transporter and comparing said second binding value with a second reference binding value.

15. The method according to claim 14, wherein said second cell surface nutrient transporter is selected from the group consisting of ASCT1, ASCT2, CAT1, FLVCR1, GLUT1, PARI, PAR2, PiTl, PiT2, RFT1, RFT3, SMIT1 and XPR1.

16. The method according to either of claims 14 or 15, wherein the first cell surface nutrient transporter is GLUT1 and the second cell surface nutrient transporter is PiTl or FLVCR1.

17. The method according to any one of claims 1 to 16, wherein the reference expression profile or the reference binding value is derived from the measurement of the expression profile or of the binding value in an in vitro cell culture growing in the absence of the agent.

18. The method according to any one of claims 1 to 16, wherein the reference expression profile or the reference binding value is derived from the measurement of the expression profile or of the binding value in an in vitro cell culture growing in the presence of a standard compound.

19. The method according to any one of claims 1 to 18, wherein the at least one cell is growing in a glucose based medium.

20. The in vitro method for detecting and/or quantifying mitochondrial toxicity in cell culture according to any one of claims 1 to 19 comprising the steps of

a. growing cells in an in vitro culture in a glucose based medium in the presence and the absence of an agent and

b. detecting and/or quantifying binding of a first ligand to a first cell surface nutrient transporter in the presence and the absence of said agent wherein a difference in said binding of said first ligand to said first cell surface nutrient transporter in the presence and the absence of the agent is indicative of mitochondrial toxicity.

21. The method according to any of claims 1 to 20, wherein the at least one cell is a mammalian cell.

22. The method according to any of claims 1 to 21, wherein the at least one cell is a heart or liver cell.

23. The method according to any of claims 1 to 22, wherein the cell is derived from a stem cell.

24. The method according to any of claims 7 to 23, wherein said detecting or quantifying binding is conducted by flow cytometry or image analysis.

25. A kit comprising one or more ligands for binding to a cell surface nutrient transporter for detecting and/or quantifying mitochondrial toxicity and instructions for use thereof in a method according to any of claims 1 to 24, wherein said one or more ligands are preferably one or more receptor binding domain ligands comprising a part or the totality of a receptor binding domain (RBD) derived from the soluble part of a glycoprotein of an enveloped virus that interacts with a cell surface nutrient transporter.

26. A kit comprising means for measuring the expression profile of at least one cell surface nutrient transporter at the RNA level for detecting and/or quantifying mitochondrial toxicity and instructions for use thereof in a method according to any of claims 5 to 25.

27. Use of a kit comprising one or more ligands for binding to a cell surface nutrient transporter for detecting and/or quantifying mitochondrial toxicity.