Improved GLUT1-binding polypeptides

Abstract

IMPROVED GLUT1-BINDING POLYPEPTIDES The present invention relates to a glucose transporter protein type 1 (GLUT1)-binding polypeptide comprising, or consisting of, an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 22 to 26, and variants or fragments thereof, wherein the sequences of said variants or fragments comprise a serine (S) residue at position corresponding to position 95 of SEQ ID NO: 1, 23 and 25, or to position 97 of SEQ ID NO: 22, 24 and 26. The present invention further relates to in vitro and in vivo methods of specifically detecting or / and measuring the level of GLUT1, and to the use of said polypeptides in diagnosis and therapy.

Description

IMPROVED GLUT1-BINDING POLYPEPTIDESFIELD OF INVENTION

[0001] The present invention relates to a glucose transporter protein type 1 (GLUT1)- binding polypeptide comprising, or consisting of, an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 22 to 26, and variants or fragments thereof, wherein the sequences of said variants or fragments comprise a serine (S) residue at position corresponding to position 95 of SEQ ID NO: 1, 23 and 25, or to position 97 of SEQ ID NO: 22, 24 and 26. The present invention further relates to in vitro and in vivo methods of specifically detecting or / and measuring the level of GLUT1, and to the use of said polypeptides in diagnosis and therapy.BACKGROUND OF INVENTION

[0002] Glucose transporter 1 (GLUT1), also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1), is a uniporter protein encoded by the SLC2A1 gene in humans. GLUT1 facilitates the transport of glucose across the plasma membranes of mammalian cells. This gene encodes a facilitative glucose transporter that is highly expressed in erythrocytes and endothelial cells, including cells of the blood-brain barrier, as well as in a wide range of cell types including immune cells and tumor cells. The encoded protein is found primarily in the cell membrane and on the cell surface, where it also acts as a receptor for human T-cell leukemia (HTLV) viruses.

[0003] Mutations in this gene can cause various syndromes and diseases such as e.g. GLUT1 deficiency syndrome, also called “De Vivo disease”. De Vivo disease is a rare and debilitating neurological disease that is relatively unknown to the medical profession but for which there is a treatment. According to estimates, more than 30,000 people are affected by De Vivo's disease in Europe and the USA only, and more than 90% of them are not diagnosed to date. In affected patients, the transporter that allows brain cells tocapture glucose malfunctions. The drop in glucose in the brain leads to epileptic seizures, bouts of abnormal movements and developmental delays. However, unlike many genetic diseases, De Vivo disease has a treatment in the form of a diet rich in lipids (called a ketogenic diet) which allows a significant improvement in patients' symptoms. Furthermore, molecules aimed at circumventing the deficit linked to the lack of glucose supply to the brain are currently being evaluated. A major challenge is therefore to diagnose the disease as early as possible to implement the appropriate treatment.

[0004] Besides De Vivo disease, a dysregulation of the level of the GLUT1 transporter occurs in a wide range of diseases including e.g. various cancers and pre-malignant disorders, inflammatory diseases, immune and auto-immune diseases, disorders of the central nervous system...

[0005] Accordingly, methods and tools allowing specific detection or / and accurate measurement of the level of GLUT1 are highly needed. Among those tools, GLUT1- binding compounds have been developed. However, currently available compounds capable of binding, targeting and inhibiting GLUT1 are limited.

[0006] GLUT 1 -binding compounds , which allow measuring of the level of GLUT 1 , may be used for diagnosis.

[0007] GLUTl-binding compounds may also be used as a drug or for therapeutic purposes in a wide range of diseases in which the level of GLUT1 is dysregulated.

[0008] Therefore, GLUT 1 represents an interesting target for the diagnosis and treatment of pathologies such as cancers and pre-malignant disorders, inflammatory diseases, immune and auto-immune diseases, disorders of the central nervous system.

[0009] GLUTl-binding polypeptides have been developed, but the production of those polypeptides is hampered by the fact that the GLUTl-binding polypeptides aggregate, which markedly decreases the polypeptide purity and yield, and can occasion non-specific binding, and / or decreased detection sensitivity.

[0010] Therefore, there is still a need for GLUTl-binding compounds, in particular GLUTl-binding polypeptides, that may be industrially produced in large scale.

[0011] The Inventors managed to engineer improved GLUT 1 -binding polypeptides that maximize the binding specificity to GLUT1, avoid non-specific binding to the cell surface, avoid polypeptide aggregates formation, exhibit a high purity, high production yield as well as high stability, and may be produced in large scale.

[0012] Such GLUT 1 -binding polypeptides represent attractive tools for specifically detecting or / and measuring the level of GLUT1 in a biological sample, as well as valuable diagnostic and therapeutic tools for treating GLUTl-associated diseases and disorders.SUMMARY

[0013] This invention thus relates to a glucose transporter protein type 1 (GLUT1)- binding polypeptide comprising, or consisting of, an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 22 to 26, and variants or fragments thereof, wherein the sequences of said variants or fragments comprise a serine (S) residue at position corresponding to position 95 of SEQ ID NO: 1, 23 and 25, or to position 97 of SEQ ID NO: 22, 24 and 26.

[0014] In some embodiments, said polypeptide is coupled or fused to a fluorescent label, such as a fluorescent protein, a fluorescent dye, or a quantum dot; a peptidic tag, such as a Fc fragment; a radioactive label; a paramagnetic metal; or a contrast agent.

[0015] In some embodiments, said polypeptide is coupled or fused to a superfolder green fluorescent protein (sfGFP) or a Fc fragment.

[0016] In some embodiments, said polypeptide further comprises a signal peptide, preferably an IL-2 signal peptide.

[0017] The invention also relates to a nucleic acid encoding the polypeptide according to the invention, or an expression vector comprising said nucleic acid, or a cell comprising said nucleic acid or said expression vector.

[0018] The invention is further directed to a diagnostic or pharmaceutical composition comprising the polypeptide according to the invention, or the nucleic acid, the expressionvector or the cell according to the invention, and at least one pharmaceutically acceptable excipient.

[0019] Another object of the invention is an in vitro method of specifically detecting or / and measuring the level of GLUT1 in a sample, wherein said method comprises the steps of: a) contacting said sample with the polypeptide according to the invention, and b) detecting and / or measuring the binding of said polypeptide to GLUT1.

[0020] A further object of the invention is the polypeptide according to the invention, for use for specifically detecting or / and measuring the level of GLUT1 in vivo.

[0021] In some embodiments, the in vitro method according to the invention, or the polypeptide for use according to the invention, is for diagnosing, prognosing or monitoring a GLUT 1 -associated disease in a subject, or for stratifying patients suffering from a GLUT 1 -associated disease.

[0022] In some embodiments, the in vitro method according to the invention, or the polypeptide for use according to the invention, is for diagnosing, prognosing or monitoring in a subject a cancer, or for stratifying patients suffering from a cancer, said cancer being selected from the group consisting of a brain cancer, a hypopharyngeal cancer, a breast cancer, a cervical cancer, an ovarian cancer, a lung cancer, a pancreatic cancer, a liver cancer, a colon cancer, squamous cell carcinoma, multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, nonHodgkin lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma (CTCL); a pre- malignant disorder, including myelodysplastic syndrome; an inflammatory disease, including psoriasis; an immune or auto-immune disease selected from the group consisting of autoimmune myocarditis, systemic lupus erythematous, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and Sjogren’s syndrome; or a disorder of the central nervous system, including GLUT1 deficiency syndrome.

[0023] The invention also relates to the polypeptide according to the invention, or the nucleic acid, the expression vector or the cell according to the invention, or the pharmaceutical composition according to the invention, for use as a medicament.

[0024] The invention further relates to polypeptide according to the invention, or the nucleic acid, the expression vector or the cell according to the invention, or the pharmaceutical composition according to the invention, for use in the prevention or treatment of a disease selected from the group consisting of a cancer, an inflammatory disease, an immune or auto-immune disease, and a disorder of the central nervous system.

[0025] In some embodiments, the disease is a cancer selected from the group consisting of a brain cancer, a hypopharyngeal cancer, a breast cancer, a cervical cancer, an ovarian cancer, a lung cancer, a pancreatic cancer, a liver cancer, a colon cancer, squamous cell carcinoma, multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, cutaneous T- cell lymphoma (CTCL); a pre-malignant disorder, including myelodysplastic syndrome; an inflammatory disease, including psoriasis; or an immune or auto-immune disease selected from the group consisting of autoimmune myocarditis, systemic lupus erythematous, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and Sjogren’s syndrome.

[0026] The invention is further directed to a coupled or fused polypeptide according to the invention, for use as a probe for medical imagery.

[0027] Another object of the invention is a kit of parts comprising the polypeptide according to the invention and at least one receptor-binding domain (RBD) derived from the soluble part of the glycoprotein of an enveloped virus selected from the group consisting of Amphotropic Murine Leukemia Retrovirus (amphoMLV), Feline Endogenous Virus (RD 114), Xenotropic Murine Leukemia Virus (NZB, Xeno), Gibbon Ape Leukemia Virus (GALV), Vesicular Stomatitis Virus (VSV), Feline Leukemia Virus C (FeLVC), Koala Retrovirus (KoRV), Porcine Endogeneous Retrovirus-A (Perv A), Porcine Endogeneous Retrovirus-B (Perv B), Bovine Leukemia Virus (BLV), HumanEndogenous Retrovirus W (HERV-W), Baboon Endogenous Virus (BaEV), Spleen Necrosis Virus (SNV), Simian Retrovirus (SRV), Mason-Pfizer Monkey Virus (MPMV), Human Endogenous Retrovirus T (HERV-T), and Feline Endogenous Retrovirus ERV- DC14 (DC-14).DEFINITIONS

[0028] In the present invention, the following terms have the following meanings:

[0029] The term “about” preceding a figure means plus or less 10% of the value of said figure. It is to be understood that the figure to which the term “about” refers is itself also specifically, and preferably, disclosed.

[0030] The term “amino acid” as used herein, refers to both natural and synthetic amino acids, and both D and L amino acids. “Standard amino acid” or “naturally occurring amino acid” means any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid residue” means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source. For example, naphtlylalanine can be substituted for tryptophan to facilitate synthesis. Other synthetic amino acids that can be substituted include, but are not limited to, L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, alphaamino acids such as L-alpha-hydroxylysyl and D-alpha-methylalanyl, L-alpha- methylalanyl, beta-amino acids, and isoquinolyl. The term “amino acid” also encompasses chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (such as amides), and substitutions.

[0031] The term “cancer”, as used herein, refers to any member of a class of diseases or disorders characterized by uncontrolled division of cells and the ability of these cells to invade other tissues, either by direct growth into adjacent tissue through invasion or by implantation into distant sites by metastasis. Metastasis is defined as the stage in which cancer cells are transported through the bloodstream or lymphatic system.

[0032] The term “diagnosis” as used herein, refers to medical diagnosis, the process of determining which disease explain the symptoms of a subject.

[0033] The term “diagnostic composition” refers to a composition that may be used to perform a diagnosis. It may be a composition to be used in vitro in order to perform an in vitro diagnosis, or a composition to be administered to a subject in order to perform an in vivo diagnosis.

[0034] 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 (z.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 Arthur M. Lesk, Computational Molecular Biology: Sources and Methods for Sequence Analysis (New- York: Oxford University Press, 1988); Douglas W. Smith, Biocomputing: Informatics and Genome Projects (New-York: Academic Press, 1993); Hugh G. Griffin and Annette M. Griffin, Computer Analysis of Sequence Data, Part 1 (New Jersey: Humana Press, 1994); Gunnar von Heinje, Sequence Analysis in Molecular Biology: Treasure Trove or Trivial Pursuit (Academic Press, 1987); Michael Gribskov and John Devereux, Sequence Analysis Primer (New York: M. Stockton Press, 1991); and Carillo et al., 1988. SIAM J. Appl. Math. 48(5): 1073-1082. 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., 1984. Nucl. Acid. Res. 12(1 Pt l):387-395; Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, WI), BLASTP, BLASTN, TBLASTN and FASTA (Altschul et al., 1990. J. Mol. Biol. 215(3):403-410). 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., 1990. J. Mol. Biol. 215(3):403- 410). The well-known Smith Waterman algorithm may also be used to determine identity.

[0035] The term “ligand” as used herein refers to a small molecule (including but not limited to proteins, peptides, peptidomimetic compounds and other small molecule compounds) that binds specifically to another molecule.

[0036] The term “monitoring” as used herein refers to the determination of the severity of a disease in a subject at different timepoints, such as for example before, during and / or after a therapy against said disease.

[0037] The term “patient stratification” refers to the division of a patient population into distinct subgroups based on established criteria, such as e.g. the presence or absence of particular disease characteristics. Preferably, the established criteria are not the treatment given or to be given. Patient stratification may help identifying patients who will benefit most from a particular treatment. Patient stratification may help determining if a patient is likely to respond, or to be responsive, to a treatment, and / or is likely to have adverse events when treated with said treatment. Patient stratification may also relate to a risk of disease progression, or disease relapse. Patient stratification may be implemented in connection with a future clinical trial or a subsequent stage of a clinical trial.

[0038] The term “polypeptide” refers to a linear polymer of amino acids (preferably at least 50 amino acids) linked together by peptide bonds.

[0039] The term “prevention” refers to prophylactic or preventive measures, which object is to prevent or avoid the occurrence of symptom. The term “prevention” may refer to a secondary prevention, i.e. to the prevention of the re-occurrence of a symptom or a relapse of the disease. It may also refer, when the disease is cancer, to the occurrence of metastases after the treatment and / or the removal of a tumor.

[0040] The term “prognosis” refers to the statistical prediction of the occurrence of death or of the occurrence of a disease-related event (or of the first disease-related event) over a specific time period for a subject. The term “prognosis” thus relates to the assessment of a specific risk of death or of a disease-related event for a subject.

[0041] The term “protein” refers to a functional entity formed of one or more polypeptides, and optionally of non-polypeptides cofactors.

[0042] The term “sample”, as used herein, refers to any biological material obtained via suitable methods known to the person skilled in the art from a subject. The sample may be collected in a clinically acceptable manner, e.g. , in a way that cells, nucleic acids (such as DNA and RNA), proteins and / or metabolites are preserved. A “sample” may include body tissue and / or bodily fluids.

[0043] The term “therapeutically effective amount” means level or amount of agent that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of a GLUT 1 -associated disease; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of a GLUTl-associated disease; (3) bringing about ameliorations of the symptoms of a GLUTl-associated disease; (4) reducing the severity or incidence of a GLUTl-associated disease; or (5) curing a GLUTl-associated disease. A therapeutically effective amount may be administered prior to the onset of a GLUTl-associated disease, for a prophylactic or preventive action. Alternatively, or additionally, the therapeutically effective amount may be administered after initiation of a GLUTl-associated disease, for a therapeutic action.

[0044] The term “treatment” refers to therapeutic or curative treatment, which object is to prevent or slow down (lessen) a GLUTl-associated disease. Those in need of treatment include those already with a GLUTl-associated disease as well as those prone to have a GLUTl-associated disease or those in whom a GLUTl-associated disease is to be prevented. A subject or mammal is successfully “treated” for a disease if, after receiving a therapeutic amount of a compound, the patient shows observable and / or measurable reduction in or absence of one or more of the following: reduction in the number of pathogenic cells; reduction in the percentage of total cells that are pathogenic; and / or relief to some extent, of one or more of the symptoms associated with the specific disease or condition; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

[0045] The term “subject”, as used herein, refers to an animal, preferably a mammal, more preferably a human. In some embodiments, the subject is a patient, i.e., a recipient of health care services, who / which is awaiting the receipt of, or is receiving medical careor was / is / will be the object of a medical procedure, or is monitored for the development of a disease.DETAILED DESCRIPTION

[0046] “Glucose transporter protein type 1” or “Glucose transporter 1” or “GLUT1”, also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1) refers to a glucose transporter, belonging to the SLC series, wherein SLC stands for SoLute Linked Carriers. In humans, GLUT1 is encoded by the SLC2A1 gene. GLUT1 is notably used as a receptor for the envelope glycoproteins (Env) from the human T-cell leukemia virus (HTLV), including HTLV-1, HTLV-2, HTLV-3 and HTLV-4.

[0047] Preferably, GLUT1 is human GLUT1. A reference amino acid sequence of GLUT1 is SEQ ID NO: 2 (UniProtKB accession number Pl 1166) encoded by SEQ ID NO: 3. GLUT1 may comprise or consist of an amino acid sequence presenting a sequence identity of at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more with SEQ ID NO: 2. GLUT1 may be encoded by a nucleotide sequence presenting a sequence identity of at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more with SEQ ID NO: 3.

[0048] The Inventors have engineered improved GLUT 1 -binding polypeptides that:- maximize the binding specificity to GLUT1, avoid non-specific binding to the cell surface,- avoid polypeptide aggregates formation,- exhibit a high purity,- exhibit high production yields,- exhibit high stability, and- may be produced in large scale.

[0049] Thus, a first object of the invention is glucose transporter protein type 1 (GLUT 1) -binding polypeptide comprising, or consisting of, an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 22 to 26, and variantsor fragments thereof, wherein the sequences of said variants or fragments comprise a serine (S) residue at position corresponding to position 95 of SEQ ID NO: 1, 23 and 25, or to position 97 of SEQ ID NO: 22, 24 and 26.

[0050] Unless indicated otherwise, the numbering of the amino acids used throughout herein is given with reference to the amino acid sequence set forth in SEQ ID NO: 1.

[0051] The polypeptide of the invention may be derived from a part of the soluble part of the glycoprotein of an enveloped virus, in particular a human T-cell leukemia virus (HTLV), including HTLV-1, HTLV-2 and HTLV-3 or a simian T-cell leukemia virus (STLV), including STLV-1, STLV-3 and STLV-5. HTLVs are human Deltaretroviruses and STLVs are simian Deltaretroviruses . In particular, the polypeptide of the invention is derived from a part of a receptor-binding domain (RBD) derived from the soluble part of the glycoprotein of an HTLV, preferably HTLV-2.

[0052] The term “receptor-binding domain” (RBD) designates a part or fragment of the soluble part of a glycoprotein of an enveloped virus that interacts with a cell surface receptor, such as a nutrient transporter, an integral protein, a GPI-anchored protein, a polysaccharide, a hetero- or proteoglycan or any other component of the extracellular matrix, used by the virus as a viral receptor.

[0053] The expression “derived from the soluble part of the glycoprotein of an enveloped virus” means that the polypeptide is a fragment or a part of a glycoprotein contained in the envelope of a virus and can be obtained, for example, by cloning or gene synthesis. A polypeptide “derived from the soluble part of the glycoprotein of an enveloped virus” also designates a variant of a fragment, or a variant of a part of a glycoprotein contained in the envelope of a virus.

[0054] The term “glycoprotein” refers to a protein containing oligosaccharide chains covalently attached to polypeptide side-chains.

[0055] The expression “that interacts with a cell surface receptor” means that the polypeptide or glycoprotein is liable to recognize a receptor present on the surface of thecell. A polypeptide that interacts with a cell surface receptor can thus form a complex with said cell surface receptor.

[0056] In some embodiments, the polypeptide is soluble, i.e. it does not comprise a transmembrane domain. Therefore, in some embodiments of the invention, the polypeptide of the invention is a soluble polypeptide. As used herein, the term “soluble polypeptide” refers to a polypeptide which is not anchored within a membrane, such as, for example, by a transmembrane or a GPI anchor domain.

[0057] The inventors have surprisingly shown that substituting the cysteine residue originally present at position 95 of SEQ ID NO: 1 for a serine residue avoided polypeptide aggregates formation and greatly increased the yield as well as the purity of the GLUT1- binding polypeptide of the invention. This in particular allows for large-scale production. This substitution also maximizes the binding specificity to GLUT1, avoiding non-specific binding to the cell surface. In addition, the GLUT 1 -binding polypeptide of the invention presents high stability overtime (e.g., at 4°C or 37°C, as shown in the example part).

[0058] Thus, the polypeptide of the invention is a GLUTl-binding polypeptide comprising, or consisting of, an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 22 to 26, and variants or fragments thereof, wherein the sequences of said variants or fragments comprise a serine (S) residue at position corresponding to position 95 of SEQ ID NO: 1, 23 and 25, or to position 97 of SEQ ID NO: 22, 24 and 26.

[0059] In a subject sequence, the “position corresponding to” a specific position in a reference sequence may be easily identified by aligning the subject sequence with the reference sequence.

[0060] In some embodiments, the subject sequence is a variant of the reference sequence having the same length as the reference sequence, and the “position corresponding to” position X of the reference sequence is position X in the subject sequence. For example, in some embodiments, in a variant of SEQ ID NO: 1 having the same length as SEQ ID NO: 1 (i.e. 160 amino acids), the position corresponding to position 95 of SEQ ID NO: 1 is position 95 in the variant of SEQ ID NO: 1.

[0061] In some embodiments, the polypeptide of the invention is a GLUTl-binding polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO: 1, a variant or fragment thereof, wherein the sequence of said variant or fragment comprises a serine (S) residue at position corresponding to position 95 of SEQ ID NO: 1.

[0062] In some embodiments, the polypeptide of the invention is a GLUTl-binding polypeptide which comprises, or consists of, the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polypeptide of the invention is a GLUTl-binding polypeptide which comprises, or consists of, the amino acid sequence of SEQ ID NO: 23. In some embodiments, the polypeptide of the invention is a GLUTl-binding polypeptide which comprises, or consists of, the amino acid sequence of SEQ ID NO: 25.

[0063] The sequences of SEQ ID NO: 1, 23 and 25 contain a serine (S) residue at position 95.

[0064] In some embodiments, the polypeptide of the invention is a GLUTl-binding polypeptide comprising, or consisting of, a variant or a fragment of the amino acid sequence of SEQ ID NO: 1, provided that the sequence of said variant or fragment comprises the serine (S) residue at position 95 of SEQ ID NO: 1. In some embodiments, the polypeptide of the invention is a GLUTl-binding polypeptide comprising, or consisting of, a variant or a fragment of the amino acid sequence of SEQ ID NO: 23, provided that the sequence of said variant or fragment comprises the serine (S) residue at position 95 of SEQ ID NO: 23. In some embodiments, the polypeptide of the invention is a GLUTl-binding polypeptide comprising, or consisting of, a variant or a fragment of the amino acid sequence of SEQ ID NO: 25, provided that the sequence of said variant or fragment comprises the serine (S) residue at position 95 of SEQ ID NO: 25.

[0065] Thus, the sequence of the variant or fragment of the sequence of SEQ ID NO: 1 comprises a serine (S) residue at position 95, this position being defined with reference to the amino acid sequence of SEQ ID NO: 1. The sequence of the variant or fragment of the sequence of SEQ ID NO: 23 comprises a serine (S) residue at position 95, this position being defined with reference to the amino acid sequence of SEQ ID NO: 23. Thus, the sequence of the variant or fragment of the sequence of SEQ ID NO: 25 comprises a serine(S) residue at position 95, this position being defined with reference to the amino acid sequence of SEQ ID NO: 25.

[0066] In some embodiments, the polypeptide of the invention is a GLUTl-binding polypeptide which comprises, or consists of, the amino acid sequence of SEQ ID NO: 22. In some embodiments, the polypeptide of the invention is a GLUTl-binding polypeptide which comprises, or consists of, the amino acid sequence of SEQ ID NO: 24. In some embodiments, the polypeptide of the invention is a GLUTl-binding polypeptide which comprises, or consists of, the amino acid sequence of SEQ ID NO: 26.

[0067] The sequences of SEQ ID NO: 22, 24 and 26 contain a serine (S) residue at position 97.

[0068] In some embodiments, the polypeptide of the invention is a GLUTl-binding polypeptide comprising, or consisting of, a variant or a fragment of the amino acid sequence of SEQ ID NO: 22, provided that the sequence of said variant or fragment comprises the serine (S) residue at position 97 of SEQ ID NO: 22. In some embodiments, the polypeptide of the invention is a GLUTl-binding polypeptide comprising, or consisting of, a variant or a fragment of the amino acid sequence of SEQ ID NO: 24, provided that the sequence of said variant or fragment comprises the serine (S) residue at position 97 of SEQ ID NO: 24. In some embodiments, the polypeptide of the invention is a GLUTl-binding polypeptide comprising, or consisting of, a variant or a fragment of the amino acid sequence of SEQ ID NO: 26, provided that the sequence of said variant or fragment comprises the serine (S) residue at position 97 of SEQ ID NO: 26.

[0069] Thus, the sequence of the variant or fragment of the sequence of SEQ ID NO: 22 comprises a serine (S) residue at position 97, this position being defined with reference to the amino acid sequence of SEQ ID NO: 22. The sequence of the variant or fragment of the sequence of SEQ ID NO: 24 comprises a serine (S) residue at position 97, this position being defined with reference to the amino acid sequence of SEQ ID NO: 24. The sequence of the variant or fragment of the sequence of SEQ ID NO: 26 comprises a serine (S) residue at position 97, this position being defined with reference to the amino acid sequence of SEQ ID NO: 26.

[0070] The polypeptide of the invention may comprise, or consists of, a variant of the amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 22 to 26.

[0071] 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.

[0072] When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant 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 receptor, preferably 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 or size. 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 histidine, 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) Vai, He, Leu, Met, Ala, Phe; (4) Lys, Arg, His; and (5) Phe, Tyr, Trp, His.

[0073] As used herein, the term “conservative amino acid substitution” may further be defined as an amino acid exchange within one of the following five groups:I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly,II. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gin,III. Polar, positively charged residues: His, Arg, Lys,IV. Large, aliphatic, nonpolar residues: Met, Leu, He, Vai, Cys,V. Large, aromatic residues: Phe, Tyr, Trp.

[0074] A variant may also, or alternatively, contain non-conservative changes. For example, variant polypeptides may differ from a native sequence by substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. 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.

[0075] In some embodiments, the variant is a functional variant. A “functional variant” of a polypeptide is a variant of said polypeptide which exhibits a same function or activity as said polypeptide, for example the same capacity of binding to a particular protein or receptor, in particular GLUT1.

[0076] According to the invention, the variants of SEQ ID NO: 1 or SEQ ID NO: 22 to 26 specifically bind to GLUT1.

[0077] In some embodiments, the variant of SEQ ID NO: 1 is capable of binding to GLUT1 with an affinity at least equivalent to the one of SEQ ID NO: 1. In some embodiments, the variant of SEQ ID NO: 22 is capable of binding to GLUT1 with an affinity at least equivalent to the one of SEQ ID NO:22. In some embodiments, the variant of SEQ ID NO: 23 is capable of binding to GLUT1 with an affinity at least equivalent to the one of SEQ ID NO: 23. In some embodiments, the variant of SEQ ID NO: 24 is capable of binding to GLUT1 with an affinity at least equivalent to the one of SEQ ID NO: 24. In some embodiments, the variant of SEQ ID NO: 25 is capable of binding to GLUT1 with an affinity at least equivalent to the one of SEQ ID NO: 25. In some embodiments, the variant of SEQ ID NO: 26 is capable of binding to GLUT1 with an affinity at least equivalent to the one of SEQ ID NO: 26.

[0078] In some embodiments, the variant of the sequence SEQ ID NO: 1 has a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence SEQ ID NO: 1. In some embodiments, the variants of the sequences SEQ ID NO: 22 to 26 have a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence SEQ ID NO: 22 to 26, respectively.

[0079] In some embodiments, the variant of the sequence SEQ ID NO: 1 comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence SEQ ID NO: 1. In some embodiments, the variants of the sequences SEQ ID NO: 22 to 26 comprise or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence SEQ ID NO: 22 to 26, respectively.

[0080] Preferably, the variant of the sequence SEQ ID NO: 1 comprises or consists of a sequence at least 85%, preferably at least 90%, more preferably at least 95% identical to the sequence SEQ ID NO: 1. Preferably, the variants of the sequences SEQ ID NO: 22 to26 comprise or consist of a sequence at least 85%, preferably at least 90%, more preferably at least 95% identical to the sequence SEQ ID NO: 22 to 26, respectively.

[0081] Preferably, the variant of SEQ ID NO: 1 has the same length as SEQ ID NO: 1. Preferably, the variant of SEQ ID NO: 22 has the same length as SEQ ID NO: 22.Preferably, the variant of SEQ ID NO: 23 has the same length as SEQ ID NO: 23.Preferably, the variant of SEQ ID NO: 24 has the same length as SEQ ID NO: 24.Preferably, the variant of SEQ ID NO: 25 has the same length as SEQ ID NO: 25.Preferably, the variant of SEQ ID NO: 26 has the same length as SEQ ID NO: 26.

[0082] In some embodiments, the variant of anyone of SEQ ID NO: 1 and SEQ ID NO: 22 to 26 may have a size of at least 159, 158, 157, 156, 155, 154, 153, 152, 151, 150, 149, 148, 147, 146, 145, 144, 143, 142, 141, 140, 139, 138, 137, 136, 135, 134, 133, 132, 131, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60 amino acids.

[0083] In some embodiments, the variant of anyone of SEQ ID NO: 1 and SEQ ID NO: 22 to 26 has a size of at least 150 amino acids, 140 amino acids, 130 amino acids, 120 amino acids, 110 amino acids, or 100 amino acids.

[0084] In some embodiments, the length of the variant of anyone of SEQ ID NO: 1 and SEQ ID NO: 22 to 26 is of less than 160, 159, 158, 157, 156, 155, 154, 153, 152, 151, 150, 149, 148, 147, 146, 145, 144, 143, 142, 141, 140, 139, 138, 137, 136, 135, 134, 133, 132, 131, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60 amino acids.

[0085] In some embodiments, the length of the variant of anyone of SEQ ID NO: 1 and SEQ ID NO: 22 to 26 is of less than 160 amino acids, less than 150 amino acids, less than 140 amino acids, less than 130 amino acids, less than 120 amino acids, less than 110 amino acids, or less than 100 amino acids.

[0086] In some embodiments, the variant of SEQ ID NO: 1 comprises or consists of amino acids 37 to 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 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 of SEQ ID NO: 1. In some embodiments, the variants of SEQ ID NO: 22 to 26 comprise or consist of amino acids 37 to 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 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 of SEQ ID NO: 22 to 26, respectively.

[0087] The polypeptide of the invention may comprise, or consists of, a fragment of the amino acid sequence of SEQ ID NO: 1. The polypeptide of the invention may comprise, or consists of, a fragment of the amino acid sequence of anyone of SEQ ID NO: 22 to 26.

[0088] By “fragment” of a reference sequence is meant herein a sequence constituted by a chain of consecutive amino acids of a reference sequence and whose size is smaller than the size of the reference sequence.

[0089] In the context of the invention, the fragment of anyone of SEQ ID NO: 1 and SEQ ID NO: 22 to 26 may for example have a size of at least 159, 158, 157, 156, 155, 154, 153, 152, 151, 150, 149, 148, 147, 146, 145, 144, 143, 142, 141, 140, 139, 138, 137,136, 135, 134, 133, 132, 131, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119,118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101,100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78,77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60 amino acids.

[0090] Preferably, the fragment of anyone of SEQ ID NO: 1 and SEQ ID NO: 22 to 26 has a size of at least 150 amino acids, 140 amino acids, 130 amino acids, 120 amino acids, 110 amino acids, or 100 amino acids.

[0091] In some embodiments, the length of the fragment of anyone of SEQ ID NO: 1 and SEQ ID NO: 22 to 26 is of less than 160, 159, 158, 157, 156, 155, 154, 153, 152, 151,150, 149, 148, 147, 146, 145, 144, 143, 142, 141, 140, 139, 138, 137, 136, 135, 134, 133,132, 131, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, 115,114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96,95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72,71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60 amino acids.

[0092] In some embodiments, the length of the fragment of anyone of SEQ ID NO: 1 and SEQ ID NO: 22 to 26 is of less than 160 amino acids, less than 150 amino acids, less than 140 amino acids, less than 130 amino acids, less than 120 amino acids, less than 110 amino acids, or less than 100 amino acids.

[0093] In some embodiments, the fragment of SEQ ID NO: 1 comprises or consists of amino acids 37 to 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 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 of SEQ ID NO: 1. In some embodiments, the fragment of SEQ ID NO: 22 to 26 comprises or consists of amino acids 37 to 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 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 of SEQ ID NO: 22 to 26, respectively.

[0094] The polypeptide of the invention may also consist of a fragment of a variant of the sequence SEQ ID NO: 1. For example, the polypeptide of the invention may consist of a fragment of a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence SEQ ID NO: 1.

[0095] The polypeptide of the invention may also consist of a fragment of a variant of anyone of the sequences SEQ ID NO: 22 to 26. For example, the polypeptide of the invention may consist of a fragment of a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to anyone of sequences SEQ ID NO: 22 to 26.

[0096] The polypeptide of the invention may be modified by means well-known in the art, for instance by the addition of one or more functional group such as a phosphate, acetate, lipid or carbohydrate group, and / or by the addition of one or more protecting group. For example, the polypeptide can be modified by the addition of one or more functional groups such as phosphate, acetate, or various lipids and carbohydrates. The polypeptide of the invention can also exist as a polypeptide derivative. The term “polypeptide derivative” refers to compound having an amino group (— NH— ), and more particularly, a peptide bond. Polypeptides may be regarded as substituted amides. Like the amide group, the peptide bond shows a high degree of resonance stabilization. The C- -N single bond in the peptide linkage has typically about 40 percent double-bond character and the C=O double bond about 40 percent single-bond character. “Protecting groups” are those groups that prevent undesirable reactions (such as proteolysis) involving unprotected functional groups. Specific examples of amino protecting groups include formyl; trifluoroacetyl; benzyloxycarbonyl; substituted benzyloxycarbonyl such as (ortho- or para-) chlorobenzyloxycarbonyl and (ortho- or para-) bromobenzyloxycarbonyl; and aliphatic oxycarbonyl such as t-butoxycarbonyl and t- amiloxycarbonyl. The carboxyl groups of amino acids can be protected through conversion into ester groups. The ester groups include benzyl esters, substituted benzyl esters such as methoxybenzyl ester; alkyl esters such as cyclohexyl ester, cycloheptyl ester or t-butyl ester. The guanidino moiety may be protected by nitro; or arylsulfonyl such as tosyl, methoxybenzensulfonyl or mesitylenesulfonyl, even though it does not need a protecting group. The protecting groups of imidazole include tosyl, benzyl and dinitrophenyl. The indole group of tryptophan may be protected by formyl or may not be protected.

[0097] The modification of the polypeptide of the invention may aim at improving its lifetime in vivo. One type of modification is the addition to the N- or C-termini of the polypeptide of polyethylene glycol (PEG). PEG is known by the person skilled in the art to have many properties that make it an ideal carrier for polypeptides such as high watersolubility, high mobility in solution and low immunogenicity. This modification also protects the polypeptides from exopeptidases and therefore increases their overall stability in vivo. Other modifications used to prevent degradation of the polypeptides byendopeptidases or exopeptidases include N-terminal modifications such as acetylation or glycosylation, C-terminal modifications such as amidation and use of unnatural amino acids (P-amino and oc-trifluoromethyl amino acids) at particular sites within the polypeptides. Another alternative to increase polypeptide molecular size is the genetic fusion of the polypeptide to the Fc domain of human immunoglobulin (including, for example, IgA, IgM and IgG) or the fusion of the polypeptide to albumin.

[0098] According to the invention, the polypeptides may be modified chemically or enzymatically to improve their stability or bioavailability. Mention may be made of the following modifications but they are not limited thereto: modifications of the C-terminal or N-terminal end of the polypeptides such as N-terminal deamination or acylation (preferably acetylation) or such as C-terminal amidation or esterification; modifications of the amide bond between two amino acids, such as acylation (preferably acetylation) or alkylation at the nitrogen or alpha carbon; changes in chirality, such as the substitution of a natural amino acid (L-enantiomer) by the corresponding D-enantiomer; this modification may optionally be accompanied by inversion of the side chain (from the C-terminal end to the N-terminal end); changes to azapeptides, in which one or more alpha carbons are replaced by nitrogen atoms; and / or changes to betapeptides, in which one or more carbons are added on the N-alpha side or on the C-alpha side of the main chain.

[0099] In this respect, it is possible to modify one or more of the lysine amino acids (K) of the polypeptides, notably by: amidation: this modification is simple to achieve, the positive charge of the lysine being substituted by hydrophobic groups (for example acetyl or phenylacetyl); amination: by formation of secondary amide from the primary amine R = (CH2)4-NH3+, for example by forming N-methyl, N-allyl or N-benzyl groups; andby formation of N-oxide, N-nitroso, N-dialkyl phosphoryl, N-sulfenyl, or N-glycoside groups.

[0100] It is also or alternatively possible to modify one or more threonine (T) and / or serine (S) amino acids of the polypeptides, notably by adding an ester or ether group at the OH group of the side chain of threonine and / or serine. Esterification, a simple operation, can be performed using a carboxylic acid, an anhydride, by bridging, etc, to form acetates or benzoates. Etherification, which gives more stable compounds, can be performed using an alcohol, a halide, etc. to form a methyl ether for example or an O- glycoside.

[0101] It is also or alternatively possible to modify one or more glutamine (Q) amino acids for example by amidation, by forming secondary or tertiary amines, in particular with groups of methyl, ethyl type, whether or not functionalized.

[0102] It is also or alternatively possible to modify one or more glutamate (E) and / or aspartate (D) amino acids, for example: by esterification, to form methyl esters, whether or not substituted, ethyl esters, benzyl esters, thiols (activated esters); and by amidation, notably to form N,N dimethyl groups, nitroanilides, pyrrolidinyls.

[0103] In some embodiments, the polypeptide according to the invention is glycosylated. In other embodiments, the polypeptide according to the invention is not glycosylated.

[0104] The polypeptide of the invention, which is a GLUT 1 -binding polypeptide comprising, or consisting of, an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 22 to 26, and variants or fragments thereof, specifically binds to GLUTE

[0105] The expression "specifically binds to", as used herein, refers to the binding specificity and affinity of a molecule or a domain thereof for a particular target or epitope, or a domain thereof, even in the presence of a heterogeneous population of other proteins and biological molecules. Thus, in some embodiments, under designated assay conditions, the polypeptide of the invention binds preferentially to its target and does notbind in a significant amount to other components present in a test sample or subject. In some embodiments, such a polypeptide shows high affinity binding to its target with an equilibrium dissociation constant equal or below 1 x 10'6M (e.g., at least 0.5 x 10'6, 1 x IO’7, 1 x 10'8, 1 x 10'9, 1 x IO'10and less). Standard assays to evaluate the binding ability of two biological molecules are known in the art, including for example, ELISAs, Western blots, RIAs and flow cytometry. The binding kinetics (e.g., binding affinity) of the molecules also can be assessed by standard assays known in the art, such as by Biacore analysis.

[0106] Typically, when flow cytometry (FACS) is used to determine if a polypeptide of the invention specifically binds to GLUT1, on cells naturally expressing GLUT1 or on GLUT 1 -transfected cells with a 100% transfection efficacy, the polypeptide of the invention is typically considered to specifically bind to GLUT1 if at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the cells are positively bound and / or marked with the polypeptide of the invention.

[0107] Typically, when flow cytometry (FACS) is used to determine if a polypeptide of the invention specifically binds to GLUT1, on cells naturally expressing GLUT1 or on GLUT 1 -transfected cells with a 100% transfection efficacy, the polypeptide of the invention is typically considered to specifically bind to GLUT1 if at least 10%, preferably 40%, even more preferably 70%, of the cells are positively bound and / or marked with the polypeptide of the invention.

[0108] Alternatively, a polypeptide may be considered to specifically bind to GLUT1 if, when compared by FACS to a negative control (for instance the secondary antibody used in the FACS assay), on cells expressing GLUT1, the ratio of the MFI binding between the tested polypeptide over the negative control is higher than 5, 10, 20, 30, 40, 50, 100, 150, 200, 500, 1000 or 1500.

[0109] In some embodiments, the polypeptide of the invention is coupled or fused to a detectable label.

[0110] In some embodiments, the polypeptide of the invention is coupled or fused to a fluorescent label, such as a fluorescent protein, a fluorescent dye, or a quantum dot; apeptidic tag, such as a Fc fragment; a radioactive label; a paramagnetic metal; or a contrast agent.

[0111] In some embodiments, the polypeptide of the invention is coupled or fused to a fluorescent protein, a fluorescent dye, a quantum dot, a peptidic tag, a radioactive label, a paramagnetic metal, or a contrast agent.

[0112] In some embodiments, the polypeptide of the invention is coupled or fused to a fluorescent label. Examples of fluorescent label include, but are not limited to, fluorescent proteins, fluorescent organic dyes, and quantum dots. These polypeptides may be useful as optical imaging probes.

[0113] Examples of fluorescent proteins include, but are not limited to, BFP, CFP, GFP, eGFP, sfGFP, mCherry, tdTomato, mPlum, mStrawberry, J-Red, DS-Red, mOrange, mCitrine, Venus, Ypet, YFP and Emerald. Another example of fluorescent protein is phycoerythrin. The fluorescent protein may be fused to the polypeptide by techniques of molecular cloning well known in the art.

[0114] The inventors have shown that superfolder green fluorescent protein (sfGFP) greatly increases the yield of the polypeptides of the invention.

[0115] Thus, preferably, the polypeptide of the invention is coupled or fused to sfGFP.

[0116] A reference sequence of sfGFP is SEQ ID NO: 4. In the context of the invention, the sequence of the sfGFP may be at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence SEQ ID NO: 4.

[0117] The sfGFP protein may be encoded by the reference sequence of SEQ ID NO: 5. The sequence encoding the sfGFP may be at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence SEQ ID NO: 5.

[0118] Example of fluorescent organic dyes include but are not limited to, commercial Alexa Fluor® dyes, fluorescein, rhodamine, or Cy® dyes (such as Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5).

[0119] In some embodiments, the polypeptide of the invention is coupled or fused to a peptidic tag.

[0120] Example of peptidic tags include, but are not limited to, an antibody crystallizable region (Fc), Enzymes (alkaline phosphatase or horseradish peroxidase), Hemagglutinin 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 X-press tag. The peptidic tag may be fused to the polypeptide by techniques of molecular cloning well known in the art or covalently attached to the polypeptide.

[0121] In some embodiments, the polypeptide of the invention, is fused to a Fc fragment. Examples of Fc fragments include, but are not limited to, rabbit Fc fragment or mouse Fc fragment.

[0122] In some embodiments, the polypeptide of the invention is a fusion protein.

[0123] In some embodiments, the polypeptide of the invention is a fusion protein, wherein the polypeptide is fused to a detection tag or a fluorescent protein. In some embodiments, the polypeptide of the invention is fused to a Fc fragment or a GFP.

[0124] In some embodiments, the polypeptide of the invention is coupled or fused to a radioactive label. Examples of radioactive labels include, but are not limited to, non- metallic radioisotopes and radioactive metals.

[0125] Examples of non-metallic radioisotopes comprise, but are not limited to, 1-125, 1-123, 1-131, C-l l, F-18, Br-75, Br-76, Br-77, Br-80, and At-211. The non-metallic radioisotopes may be conjugated covalently to either terminus of the polypeptide, functional groups of amino acid side chains, be part of a linear stabilized peptide as an additional substituent, e.g., in an amino acid phenylalanine or tyrosine carrying fluorine, bromine or iodine, or as an additional substituent carboxy or methyl, or as a replacement of any regular carbon atom in the polypeptide. These radioisotopes are useful in polypeptides as positron emission tomography (PET) probes or as single-photon emission computed tomography (SPECT) probes.

[0126] Examples of radioactive metals include, but are not limited to, Cu-64, Cu-67, Ga- 67, Ga-68, Zr-89, Y-90, Tc-99m, In-111, Tb-161, Lu-177, Re-186, Re-188, and Bi-213. The radioactive metals may be covalently attached to the polypeptide, directly connected to the polypeptide or through a spacer.

[0127] In some embodiments, the polypeptide of the invention is coupled or fused to paramagnetic metals.

[0128] Examples of paramagnetic metals comprise, but are not limited to, Gd, Fe, Mn. The paramagnetic metals may be covalently attached to the polypeptides, directly connected to the polypeptides or through a spacer. These polypeptides are useful as magnetic resonance imaging (MRI) probes.

[0129] In some embodiments, the polypeptide of the invention is coupled with at least one contrast agent. Examples of contrast agents are well-known by the skilled person. In some embodiments, the contrast agent is 1-125.

[0130] As used herein, the term “contrast agent” refers to an agent used to improve the visibility of internal bodily structures in medical imaging technics.

[0131] The polypeptide of the invention coupled or fused to a detectable label or a contrast agent may be used as a probe for medical imaging.

[0132] In some embodiments, the polypeptide of the invention coupled or fused to a detectable label or a contrast agent is for use as a probe for medical imagery.

[0133] The term “medical imaging” as used herein refers to imaging techniques suitable to visualize in vivo a subject’s internal structures (z.e. tissues or organs). Such techniques include but are not limited to, computed tomography (CT scan), endoscopic ultrasound (EUS), magnetic resonance imaging (MRI), positron-emission tomography (PET), single photon emission tomography (SPECT), magnetic resonance cholangiopancreatography, fluorimetry, fluorescence, and near-infrared (NIR) fluorescent imaging. In the context of the invention the polypeptide of the invention coupled or fused to a detectable label may be used as a probe, to localize in vivo GLUT 1 -expressing cells in a subject’s internal structures.

[0134] In some embodiments of the invention, the polypeptide of the invention coupled or fused to a detectable label is for use as a tracer. The present invention thus further relates to the use as a tracer of a polypeptide coupled or fused to a detectable label.

[0135] The term “tracer”, as used herein, refers to a recognition agent providing insight into GLUTl-associated disease location, progression and / or structure for pre-, intra- and post-operative surgery.

[0136] Methods for coupling at least one detectable label to a polypeptide are well known in the state of the art. For instance, the at least one detectable label may be bound covalently or non-covalently.

[0137] Techniques to couple polypeptides to 1-125 are well known in the state of the art. A non-limited example of such a method is the following: iodine present in a reduced form (Nal) reacts with the phenol group of a tyrosine or with the side chain of a histidine residue. These groups are pre-oxidized with an oxidizing agent (iodogen). The peptides preparation (100 pg for ImCi = 37 MBq) is then added to an iodogen solution and incubated for 10 minutes at 4°C. The reaction is stopped using a stop solution comprising for example 200 pL of PBS with sodium azide per marking. In parallel, a mouse serum is added onto a PD10 column. Then the reaction solution is added onto the PD10 column and the peptide coupled with the iodine is collected.

[0138] In embodiments concerning detectable labels encoded by a nucleic acid sequence, the detectable label may be fused to the polypeptide of the invention, by techniques of molecular cloning well known in the art.

[0139] In some embodiments, the polypeptide of the invention is labeled with, coupled or fused to sfGFP and does not comprise a signal peptide.

[0140] In some embodiments, the polypeptide of the invention is labeled with, coupled or fused to sfGFP and comprises a signal peptide.

[0141] In some embodiments, the polypeptide of the invention comprises a signal peptide.

[0142] A “signal peptide”, also referred to as “signal sequence”, “targeting signal”, “localization signal”, “localization sequence”, “transit peptide”, “leader sequence” or “leader peptide” is a short peptide, e.g. 15-40 amino acids long, usually present at the N- terminus of most newly synthesized proteins that are destined toward the secretory pathway. A signal peptide may target the protein which comprises it for transfer to a specific organelle (such as the endoplasmic reticulum, Golgi or endosomes), or for insertion into a cellular membrane, or for secretion.

[0143] Examples of signal peptide sequences include, but are not limited to, HTLV envelope glycoprotein signal peptide, human IL-2 signal peptide, human albumin signal peptide, human chymotrypsinogen signal peptide, human trypsinogen-2 signal peptide, Gaussia luciferase signal peptide, and mouse IgM signal peptide.

[0144] The inventors have shown that human IL-2 signal peptide greatly increases the yield of the polypeptides of the invention.

[0145] Thus, preferably, the polypeptide of the invention comprises an IL-2 signal peptide, more preferably a human IL-2 signal peptide.

[0146] A reference amino acid sequence of human IL-2 signal peptide is SEQ ID NO: 6. In the context of the invention, the sequence of the human IL-2 signal peptide may be at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence SEQ ID NO: 6.

[0147] A reference nucleic acid sequence encoding human IL-2 signal peptide is SEQ ID NO: 7. In the context of the invention, the nucleic acid sequence encoding human IL- 2 signal peptide may be at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence SEQ ID NO: 7.

[0148] The polypeptide described herein can be produced synthetically by chemical synthesis or enzymatic synthesis as it is well known in the art. Alternatively, polypeptide of the invention may be produced by recombinant DNA technology, wherein nucleotide sequences encoding the polypeptide of the invention can be introduced into a protein expression vector and produced, transformed or transfected in a suitable host organism orcell (e.g., bacteria, insect cells, etc.), cultivated under conditions suitable for expression and then purified.

[0149] A variety of expression vector / host systems may be utilized to contain and express the peptide or protein coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus, see Ghosh et al., 2002); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.

[0150] In some embodiments, the polypeptide is obtained by a cloning method, such as, for example, using any production system known in the art, such as, for example, E. coli, yeast, baculovirus-insect cell, or mammalian cells such as HEK or CHO expression system.

[0151] Conventional methods for preparing and purifying recombinant proteins may be used for producing the polypeptides of the invention. The polypeptide of the invention can, for example, be obtained by culturing genetically transformed cells of the invention and recovering the polypeptide expressed by said cell, from the culture. If necessary, the polypeptide may then be purified by conventional procedures, for example by fractional precipitation, in particular ammonium sulfate precipitation, electrophoresis, gel filtration, or affinity chromatography.

[0152] Thus, the present disclosure also relates to a method for producing a recombinant host cell expressing a polypeptide of the invention, said method comprising the steps consisting of: i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described above into a competent host cell, ii) culturing in vitro or ex vivo the recombinant host cell obtained and iii) optionally, selecting the cells which express and / or secrete the polypeptide of the invention.

[0153] Such recombinant host cells can be used for the production of polypeptides and fusions proteins of the present invention.

[0154] The disclosure thus further relates to a method of producing a polypeptide or fusion protein of the invention, which comprises the steps of: i) culturing a transformed host cell according to the invention under conditions suitable to allow expression of said polypeptide or fusion protein; and ii) recovering the expressed polypeptide or fusion protein.

[0155] The invention further relates to a nucleic acid encoding the polypeptide according to the invention.

[0156] In some embodiments, the nucleic acid sequence encoding the polypeptide of the invention comprises or consists of a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 22 to 26, and variants or fragments thereof, wherein the amino acid sequences of said variants or fragments comprises a serine (S) residue at position corresponding to position 95 of SEQ ID NO: 1, 23 and 25, or to position 97 of SEQ ID NO: 22, 24 and 26.

[0157] In some embodiments, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1 consists of the nucleic acid sequence of SEQ ID NO: 8.

[0158] In some embodiments, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1 or a variant or fragment thereof consists of a nucleic acid sequence presenting a sequence identity of at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the nucleic acid sequence SEQ ID NO: 8.

[0159] An additional polypeptide (“tag”) can be added on for the purpose of identifying or purifying the polypeptide. Protein tags make it possible, for example, for the polypeptide to be adsorbed, with high affinity, to a matrix, and for the matrix then to be washed stringently with suitable buffers without the complex being eluted to any significant extent, and for the adsorbed complex subsequently to be eluted selectively. Examples of protein tags which are known to the skilled person are a (His)e tag, a Myc tag, a FLAG tag, a hemagglutinin tag, a glutathione transferase (GST) tag, intein havingan affinity chitin- binding tag or maltose-binding protein (MBP) tag. These protein tags can be located N- terminally, C -terminally and / or internally.

[0160] In some embodiments, the sequence of the polypeptide is fused in N-terminal to a signal peptide sequence allowing the secretion of said polypeptide. Examples of signal peptide sequences include, but are not limited to, HTLV envelope glycoprotein signal peptide, human IL-2 signal peptide, human albumin signal peptide, human chymotrypsinogen signal peptide, human trypsinogen-2 signal peptide, Gaussia luciferase signal peptide, and mouse IgM signal peptide.

[0161] Preferably, the nucleic acid encoding the polypeptide of the invention comprises a nucleic acid sequence encoding IL-2 signal, more preferably a nucleic acid sequence encoding human IL-2 signal.

[0162] A reference nucleic acid sequence encoding human IL-2 signal peptide is SEQ ID NO: 7. In the context of the invention, the nucleic acid sequence encoding human IL- 2 signal peptide may be at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence SEQ ID NO: 7.

[0163] The invention also pertains to an expression vector comprising a nucleic acid encoding the polypeptide according to the invention.

[0164] The expression vector may be any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0165] The expression vector may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said polypeptide upon administration to a subject. The vectors may further comprise one or several origins of replication and / or selectable markers.

[0166] The promoter region may be homologous or heterologous with respect to the coding sequence, and provide for ubiquitous, constitutive, regulated and / or tissue specific expression, in any appropriate host cell, including for in vivo use. Examples of promoters include bacterial promoters (T7, pTAC, Trp promoter, etc.), viral promoters (LTR, TK, CMV-IE, etc.), and mammalian gene promoters (albumin, PGK, etc).

[0167] Examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR. Examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, and 293 cells.

[0168] The invention further relates to a cell comprising a nucleic acid encoding the polypeptide according to the invention, or an expression vector comprising a nucleic acid encoding the polypeptide according to the invention.

[0169] Mammalian cells that are useful in recombinant polypeptide production include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells.

[0170] Mammalian host systems for the expression of recombinant polypeptides also are well known to those of skill in the art. Host cell strains may be chosen for a particular ability to process the expressed polypeptide or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing which cleaves a “prepro” form of the polypeptide may also be important for correct insertion, folding and / or function.

[0171] Different host cells such as CHO, HeLa, MDCK, 293, or WI38 have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign polypeptide.

[0172] The present invention also relates to a composition comprising, consisting essentially of, or consisting of at least one polypeptide, nucleic acid, expression vector or cell according to the invention.

[0173] The present invention also relates to a pharmaceutical composition comprising, consisting essentially of, or consisting of at least one polypeptide, nucleic acid, expression vector or cell according to the invention, and at least one pharmaceutically acceptable excipient.

[0174] The present invention also relates to a diagnostic composition comprising, consisting essentially of, or consisting of at least one polypeptide, nucleic acid, expression vector or cell according to the invention, and at least one pharmaceutically acceptable excipient.

[0175] As used herein, the term “consisting essentially of’, with reference to a composition, means that the at least one polypeptide, nucleic acid, expression vector or cell of the invention is the only one agent, therapeutic agent or diagnostic agent, with a biologic activity within said composition.

[0176] The present invention also relates to a diagnostic composition comprising or consisting essentially of at least one labeled polypeptide of the invention, and at least one pharmaceutically acceptable excipient.

[0177] As used herein, the term “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a subject, especially a human, as appropriate. Hence, “Pharmaceutically acceptable excipient” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a subject, especially a human, as appropriate. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. For human administration, preparations should meet pyrogenicity, sterility, general safety and purity standards as required by regulatory offices, such as, for example, FDA Office or EMA. A pharmaceutically acceptable carrier or excipient may thus refer to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0178] Pharmaceutically acceptable excipients include water, saline, Ringer's solution, dextrose solution, and solutions of ethanol, glucose, sucrose, dextran, mannose, mannitol,sorbitol, polyethylene glycol (PEG), phosphate, acetate, gelatin, collagen, Carbopol®, or vegetable oils. One may additionally include suitable preservatives, stabilizers, antioxidants, antimicrobials, and buffering agents, such as, for example, BHA, BHT, citric acid, ascorbic acid, or tetracycline.

[0179] Other examples of pharmaceutically acceptable excipients that may be used in the composition of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, poly acrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat.

[0180] In addition, pharmaceutically acceptable excipients may comprise some excipients, such as, for example, surfactants (e.g. hydroxypropylcellulose); suitable carriers, such as, for example, solvents and dispersion media containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils, such as, for example, peanut oil and sesame oil; isotonic agents, such as, for example, sugars or sodium chloride; coating agents, such as, for example, lecithin; agents delaying absorption, such as, for example, aluminum monostearate and gelatin; preservatives, such as, for example, benzalkonium chloride, benzethonium chloride, or chlorobutanol, thimerosal; buffers, such as, for example, boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, sodium biphosphate; tonicity agents, such as, for example, dextrose, potassium chloride, propylene glycol, sodium chloride; antioxidants and stabilizers, such as, for example, sodium bisulfite, sodium metabisulfite, sodium thiosulfite, thiourea; nonionic wetting or clarifying agents, such as, for example, polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol; viscosity modifying agents, such as, for example dextran 40, dextran 70, gelatin, glycerin, hydroxyethylcellulose, hydroxymethylpropylcellulose, lanolin, methylcellulose,petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulo se .

[0181] In some embodiments, the diagnostic composition of the invention is for diagnosing a GLUT 1 -associated disease, such as a disease in which GLUT1 expression on cells is affected or dysregulated, using a method of the invention as described hereinabove.

[0182] In some embodiments, the polypeptide, nucleic acid, expression vector or cell according to the invention is encapsulated.

[0183] The techniques of encapsulation are well known in the state of the art. Examples of capsule include, but are not limited to, phospholipids, polymers and liposomes.

[0184] In some embodiments, the polypeptide, nucleic acid, expression vector or cell according to the invention is encapsulated with a detectable label.

[0185] The present invention also relates to a medicament composition comprising, consisting essentially of, or consisting of at least one polypeptide, nucleic acid, expression vector or cell according to the invention.

[0186] Another object of the present invention is a kit for implementing the methods of the invention, wherein said kit comprises at least one polypeptide, nucleic acid, expression vector or cell according to the invention.

[0187] In some embodiments, the kit of the invention further comprises cells displaying GLUT1 at the cell surface for use as a reference.

[0188] Another object of the present invention is a kit of parts comprising the polypeptide according to the invention and at least one receptor-binding domain (RBD) derived from the soluble part of the glycoprotein of an enveloped virus selected from the group consisting of Amphotropic Murine Leukemia Retrovirus (amphoMLV), Feline Endogenous Virus (RD 114), Xenotropic Murine Leukemia Virus (NZB, Xeno), Gibbon Ape Leukemia virus (GALV), Vesicular Stomatitis Virus (VSV), Feline Leukemia Virus C (FeLVC), Koala Retrovirus (KoRV), Porcine Endogeneous Retrovirus-A (Perv A),Porcine Endogeneous Retrovirus-B (Perv B), Bovine Leukemia Virus (BLV), Human Endogenous Retrovirus W (HERV-W), Baboon Endogenous Virus (BaEV), Spleen Necrosis Virus (SNV), Simian Retrovirus (SRV), Mason-Pfizer Monkey Virus (MPMV), Human Endogenous Retrovirus T (HERV-T), and Feline Endogenous Retrovirus ERV- DC14 (DC-14).

[0189] Non-limiting examples of receptor-binding domain (RBD) derived from the soluble part of the glycoprotein of an enveloped virus includes polypeptides comprising or consisting of the amino acid sequences of SEQ ID NO: 27 (amphoMLV), SEQ ID NO: 28 (GALV), SEQ ID NO: 29 (RD114), SEQ ID NO: 30 (VSV), SEQ ID NO: 31 (NZB, Xeno), SEQ ID NO: 32 (FeLVC), SEQ ID NO: 33 (KoRV), SEQ ID NO: 34 (Perv A), SEQ ID NO: 35 (Perv B), SEQ ID NO: 36 (BLV), SEQ ID NO: 37 (HERV-W), SEQ ID NO: 38 (BaEV), SEQ ID NO: 39 (SNV), SEQ ID NO: 40 (SRV), SEQ ID NO: 41 (MPMV), SEQ ID NO: 42 (HERV-T), SEQ ID NO: 43 (ERV-DC14), or fragments thereof.

[0190] Thus, the kit of parts may comprise the polypeptide according to the invention and at least one receptor-binding domain (RBD) comprising, or consisting of, an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a sequence selected from the group consisting of SEQ ID NO: 27 (amphoMLV), SEQ ID NO: 28 (GALV), SEQ ID NO: 29 (RD114), SEQ ID NO: 30 (VSV), SEQ ID NO: 31 (NZB, Xeno), SEQ ID NO: 32 (FeLVC), SEQ ID NO: 33 (KoRV), SEQ ID NO: 34 (Perv A), SEQ ID NO: 35 (Perv B), SEQ ID NO: 36 (BLV), SEQ ID NO: 37 (HERV-W), SEQ ID NO: 38 (BaEV), SEQ ID NO: 39 (SNV), SEQ ID NO: 40 (SRV), SEQ ID NO: 41 (MPMV), SEQ ID NO: 42 (HERV-T), SEQ ID NO: 43 (ERV-DC14), and fragments thereof.

[0191] Said at least one receptor-binding domain (RBD) derived from the soluble part of the glycoprotein of an enveloped virus may for instance bind to at least one receptor selected from CAT1, PiT2, XPR1, SMIT1, PiTl, ASCT1, ASCT2, FLVCR, PARI, PAR2, SMVT.

[0192] The invention further relates to an in vitro method of specifically detecting or / and measuring the level of GLUT1 in a sample, wherein said method comprises the steps of:a) contacting said sample with the polypeptide according to the invention, and b) detecting and / or measuring the binding of said polypeptide to GLUT1.

[0193] The invention further relates to the polypeptide according to the invention, for use for specifically detecting or / and measuring the level of GLUT1 in vivo.

[0194] In some embodiments, the term “level of GLUT1” refers to the amount of GLUT1 present at the surface of a cell and / or within the cell.

[0195] In some embodiments, the method of the invention is for assessing the expression level of GLUT1 present on the cell surface. In other embodiments, the method of the invention is for assessing the expression level of GLUT1 present within the cell.

[0196] In some embodiments, the in vitro method of the invention is for diagnosing or monitoring a GLUT 1 -associated disease in a subject.

[0197] In some embodiments, the sample is a biological sample.

[0198] Examples of biological samples include, but are not limited to, body fluids, cell samples, tissue samples, biopsy samples.

[0199] In some embodiments, the biological sample is a body fluid. Examples of body fluids include, but are not limited to, blood, plasma, serum, lymph, ascetic fluid, cystic fluid, urine, bile, synovial fluid, bronchoalveolar lavage fluid, sputum, amniotic fluid, peritoneal fluid, cerebrospinal fluid, pleural fluid, pericardial fluid, semen, saliva, sweat and milk.

[0200] In some embodiments the biological sample is a tissue sample. Examples of tissues include, but are not limited to, placenta, intestine, brain, liver, lung, kidney, cornea, retina, heart breast, cervix, kidney, pancreas, ovary, skin, nerve, spleen, thymus, esophagus, stomach, testis, hair, skin, bone, uterus, bladder and spinal cord.

[0201] In some embodiments, the biological sample is a biopsy sample. In some embodiments, the biological sample is a fine-needle aspirate sample. In some embodiments, the biological sample is a resection sample.

[0202] In some embodiments, the biological sample is a cell sample. Examples of cell samples include, without being limited to, red blood cells, peripheral blood mononuclear cells (PBMC), peripheral white blood cells, cell samples obtained from bone marrow aspirates, tissue biopsies such as tumor biopsies, lymph nodes biopsies, intestinal or synovial biopsies, or cell sample obtained from broncho-alveolar lavage or cerebrospinal fluid, cell culture sample.

[0203] In some embodiments, the methods according to the present invention comprise a step of providing a biological sample from a subject.

[0204] In some embodiments, the sample was previously taken from the subject, i.e., the in vitro methods of the invention do not comprise a step of recovering a sample from the subject. Consequently, according to this embodiment, the in vitro methods of the invention are non-invasive methods.

[0205] The methods of the invention comprise the step of detecting and / or measuring the binding of a polypeptide of the invention, to GLUT1 in a sample.

[0206] Techniques to measure the binding of a polypeptide, or of a ligand, to its receptor are known in the art and may imply the detection and measure of the amount of the ligandreceptor complexes. In the context of the present invention, such techniques could for example rely on the detection and measure of the number of complexes formed by the polypeptide of the invention with GLUT1 present in the sample, such as, for example, at the cell surface of cells present in a biological sample.

[0207] Example of such technique include, but are not limited to, flow cytometry analysis, immunohistochemistry, western blot associated or not with cell fractionation, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, fluorescent-linked immunosorbent assay (FLISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), image analysis, for example high content analysis, computed tomography (CT scan), endoscopic ultrasound (EUS), magnetic resonance imaging (MRI), positron-emission tomography (PET), single photon emission tomography (SPECT), magnetic resonance cholangiopancreatography, fluorimetry, fluorescence, and near-infrared (NIR) fluorescent imaging.

[0208] Examples of such techniques amenable to an in vitro use 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), flow cytometry (FACS).

[0209] In some embodiments, the step of detecting and / or measuring the binding of a polypeptide of the invention to GLUT1 comprises the use of an antibody directed against the polypeptide of the invention. For example, an antibody directed against the polypeptide of the invention may bind to a polypeptide of the invention bound to GLUT1, thus allowing detecting and / or measuring the binding of a polypeptide of the invention to GLUTE Said antibody directed against the polypeptide of the invention may be coupled or fused to a detectable label, tag or contrast agent. Said antibody directed against the polypeptide of the invention may also be detected by a secondary antibody.

[0210] In some embodiments, the methods of the invention comprise a step of detecting and / or measuring the binding of a polypeptide of the invention to GLUT1 in vivo.

[0211] Techniques to measure the binding of a polypeptide, or of a ligand, to its receptor in vivo are known in the art and may imply the detection and measure of the amount of the ligand-receptor complexes. In the context of the present invention, such techniques could for example rely on the detection and measure of the number of complexes formed by the polypeptide of the invention with GLUT1 present in vivo, such as, for example, at the cell surface of cells.

[0212] Examples of such techniques suitable for an in vivo use include, but are not limited to, computed tomography (CT scan), endoscopic ultrasound (EUS), magnetic resonance imaging (MRI), positron-emission tomography (PET), single photon emission tomography (SPECT), magnetic resonance cholangiopancreatography, fluorimetry, fluorescence, and near-infrared (NIR) fluorescent imaging.

[0213] The present invention thus also relates to an in vivo method for specifically detecting or / and measuring the level of GLUT1, using a polypeptide according to the invention.

[0214] The present invention also relates to a polypeptide according to the invention, for use in an in vivo method for specifically detecting or / and measuring the level of GLUT1 in a subject.

[0215] The present invention also relates to the use of a polypeptide according to the invention, for the manufacture of a kit for specifically detecting or / and measuring the level of GLUT1 in a subject.

[0216] In some embodiments, said method comprises the detection and / or measure of the level of GLUT1 within the body of a subject, such as, for example, in a specific organ or tissue.

[0217] The present application also relates to an in vivo method for specifically detecting or / and measuring the level of GLUT1 in a subject, comprising: a) contacting a polypeptide according to the invention with a cell, a sample, a tissue or an organ, and b) detecting and / or quantifying the polypeptide bound to GLUT1 present in the cell, sample, tissue or organ within said subject.

[0218] The present invention also relates to a polypeptide according to the invention, for use in an in vivo method for specifically detecting or / and measuring the level of GLUT1 in a subject, comprising: a) contacting a polypeptide according to the invention with a cell, a sample, a tissue or an organ, and b) detecting and / or quantifying the polypeptide bound to GLUT1 present in the cell, sample, tissue or organ within said subject.

[0219] The present application also relates to an in vivo method for specifically detecting or / and measuring the level of GLUT1 in a subject, comprising: a) administering to the subject a polypeptide according to the invention, and b) detecting and / or quantifying the binding of said polypeptide to GLUT1 within said subject, for example by medical imaging.

[0220] The present invention also relates to a polypeptide according to the invention, for use in an in vivo method for specifically detecting or / and measuring the level of GLUT1 in a subject, comprising: a) administering to the subject a polypeptide according to the invention, and b) detecting and / or quantifying the binding of said polypeptide to GLUT1 within said subject, for example by medical imaging.

[0221] In some embodiments, the polypeptide according to the invention is coupled with at least one detectable label.

[0222] In some embodiments, the step of detecting and / or measuring or quantifying the binding of a polypeptide of the invention to GLUT1 comprises the use of an antibody directed against the polypeptide of the invention. For example, an antibody directed against the polypeptide of the invention may bind to a polypeptide of the invention bound to GLUT1, thus allowing detecting and / or measuring the binding of a polypeptide of the invention to GLUT1. Said antibody directed against the polypeptide of the invention may be coupled or fused to a detectable label, tag or contrast agent. Said antibody directed against the polypeptide of the invention may also be detected by a secondary antibody.

[0223] In some embodiments, the polypeptide of the invention may be used for in vivo diagnosis by medical imaging.

[0224] In some embodiments, the polypeptide administered to the subject is comprised in a composition, preferably a pharmaceutical composition or a diagnostic composition.

[0225] In some embodiments, said method comprises the detection and / or measure of the level of GLUT1 using medical imaging techniques.

[0226] Examples of specific medical imaging techniques that may be used are well known to the skilled artisan and include, but are not limited to, computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), positron emission tomography (PET) or single-photon emission computed tomography (SPECT) and are described in Boonstra et al. (2015. Oncotarget. 6(16): 14260-73).

[0227] The present invention thus relates to an in vivo method for specifically detecting or / and measuring the level of GLUT1 in a subject, comprising the steps of: a) administering to said subject a labeled polypeptide according to the invention; and, b) detecting and / or measuring the binding of said labeled polypeptide to GLUT1 using medical imaging.

[0228] The present invention also relates to a polypeptide according to the invention, for use in an in vivo method for specifically detecting or / and measuring the level of GLUT1 in a subject, comprising the steps of: a) administering to said subject a labeled polypeptide according to the invention; and, b) detecting and / or measuring the binding of said labeled polypeptide to GLUT1 using medical imaging.

[0229] In some embodiments, the methods of the invention are for diagnosing or monitoring a GLUT 1 -associated disease in a subject.

[0230] In some embodiments, the methods of the invention are for prognosing a GLUT1- associated disease.

[0231] In some embodiments, the methods of the present invention are for identifying a subject as presenting a risk of developing a GLUT 1 -associated disease.

[0232] In some embodiments, the methods of the invention are for assessing the severity of a GLUT 1 -associated disease in a subject.

[0233] In some embodiments, the methods of the invention are for stratifying patients suffering from a GLUT 1 -associated disease for treatment responsiveness, for disease progression or for risk of relapse.

[0234] Therefore, the present invention relates to a method for diagnosing a GLUT1- associated disease in a subject, for identifying a subject as presenting a risk of developing a GLUT 1- associated disease, for assessing the severity of a GLUT 1 -associated disease in a subject, for prognosing a GLUTl-associated disease in a subject, or for monitoring aGLUTl-associated disease in a subject, for stratifying a patient suffering from a GLUT1- associated disease for treatment responsiveness, for disease progression or for risk of relapse, said method comprising specifically detecting or / and measuring the level of GLUT1, using a polypeptide according to the invention.

[0235] The present invention also relates to a polypeptide according to the invention, for use in a method of diagnosing a GLUTl-associated disease in a subject, of identifying a subject as presenting a risk of developing a GLUTl-associated disease, of assessing the severity of a GLUTl-associated disease in a subject, of prognosing a GLUTl-associated disease in a subject, of monitoring a GLUTl-associated disease in a subject, or of stratifying a patient suffering from a GLUTl-associated disease for treatment responsiveness, for disease progression or for risk of relapse, said method comprising detecting or / and measuring the level of GLUT1, using a polypeptide according to the invention.

[0236] The present invention also relates to the use of a polypeptide according to the invention for detecting or / and measuring the level of GLUT1, in the manufacture of a kit for the diagnosis, prognosis or monitoring of a GLUTl-associated disease, or for the stratification of a patient suffering from a GLUTl-associated disease for treatment responsiveness, for disease progression or for risk of relapse.

[0237] The present invention thus also relates to a method for diagnosing a GLUTl- associated disease in a subject, for identifying a subject as being at risk of developing a GLUTl-associated disease, for assessing the severity of a GLUTl-associated disease in a subject, for prognosing a GLUTl-associated disease in a subject, or for monitoring a GLUTl-associated disease in a subject, or for stratifying a patient suffering from a GLUTl-associated disease for treatment responsiveness, for disease progression or for risk of relapse, wherein said method comprises the steps of: a) contacting a biological sample from said subject with a polypeptide according to the invention; and b) measuring the binding of said polypeptide to GLUT1.

[0238] The present invention also relates to a polypeptide according to the invention, for use in a method of diagnosing a GLUT 1- associated disease in a subject, identifying a subject as presenting a risk of developing a GLUTl-associated disease, assessing the severity of a GLUTl-associated disease in a subject, prognosing a GLUTl-associated disease in a subject, or monitoring a GLUTl-associated disease in a subject, or for stratifying a patient suffering from a GLUTl-associated disease for treatment responsiveness, for disease progression or for risk of relapse, wherein said method comprises the steps of: a) contacting a biological sample from said subject with a polypeptide according to the invention; and b) measuring the binding of said polypeptide to GLUTL

[0239] The present application also relates to a method for diagnosing a GLUTl- associated disease, for identifying a subject as being at risk of developing a GLUTl- associated disease, for assessing the severity of a GLUTl-associated disease in a subject, for prognosing a GLUTl-associated disease in a subject, or for monitoring a GLUTl- associated disease in a subject, or for stratifying a patient suffering from a GLUTl- associated disease for treatment responsiveness, for disease progression or for risk of relapse, said method comprising the steps of: a) contacting the polypeptide according to the invention to a cell, sample, tissue, and / or organ, b) detecting and / or measuring the binding of said polypeptide to GLUT1 in said cell, sample, tissue, and / or organ.

[0240] The present invention also relates to a polypeptide according to the invention, for use in a method of diagnosing a GLUTl-associated disease in a subject, identifying a subject as presenting a risk of developing a GLUTl-associated disease, assessing the severity of a GLUTl-associated disease in a subject, prognosing a GLUTl-associated disease in a subject, or monitoring a GLUTl-associated disease in a subject, or for stratifying a patient suffering from a GLUTl-associated disease for treatment responsiveness, for disease progression or for risk of relapse, said method comprising the steps of:a) contacting the polypeptide according to the invention to a cell, sample, tissue, and / or organ, b) detecting and / or measuring the binding of said polypeptide to GLUT1 in said cell, sample, tissue, and / or organ.

[0241] The term “GLUT 1 -associated disease” as used herein may refer to diseases wherein pathways involving glucose homeostasis and / or metabolism are dysregulated.

[0242] The GLUT 1 -associated disease may be due to a mutation within the SLC2A1 gene. Said mutation within the SLC2A1 gene may result in a partial or complete loss of function or partial or complete loss of expression.

[0243] The GLUT 1 -associated disease may be related to an overexpression or a dysregulation of GLUT1 on the cell surface.

[0244] In some embodiments, the GLUT 1 -associated disease is a cancer, an inflammatory disease, an immune or auto-immune disease, or a disorder of the central nervous system.

[0245] In some embodiments, the GLUT 1 -associated disease is a cancer.

[0246] Said cancer may be a solid tumor or a liquid tumor (such as e.g. a blood cancer).

[0247] In some embodiments, the cancer is a solid tumor, selected from the group consisting of a brain cancer, a hypopharyngeal cancer (e.g. hypopharyngeal carcinoma), a breast cancer, a cervical cancer (e.g. cervical carcinoma), an ovarian cancer (e.g. ovarian carcinoma), a lung cancer, a pancreatic cancer (e.g. pancreatic adenocarcinoma), a liver cancer (e.g. hepatocarcinoma), a colon cancer, and squamous cell carcinoma.

[0248] In some embodiments, the cancer is a liquid tumor selected from myeloma or leukemia. In some embodiments, the cancer is a liquid tumor, selected from the group consisting of multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, cutaneous T- cell lymphoma (CTCL).

[0249] Examples of cancers include, but are not limited to, tumors, sarcomas, carcinomas, leukemias, lymphomas, and metastasis.

[0250] In some embodiments, the GLUTl-associated disease is an inflammatory disease.

[0251] Examples of inflammatory diseases include, but are not limited to, psoriasis.

[0252] In some embodiments, the GLUTl-associated disease is an immune or autoimmune disease.

[0253] Examples of immune or auto-immune diseases include, but are not limited to, immune or auto-immune diseases in the frame of CD28 T-cell activation, or immune or auto-immune diseases in the frame of immunomodulation.

[0254] In some embodiments, the immune or auto-immune disease is selected from the group consisting of autoimmune myocarditis, systemic lupus erythematous, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and Sjogren’s syndrome.

[0255] In some embodiments, the GLUTl-associated disease is a disorder of the central nervous system.

[0256] Examples of disorders of the central nervous system include, but are not limited to, GLUT1 deficiency syndrome.

[0257] In some embodiments, the methods of the invention comprise a step of comparing the binding detected and / or measured at step b) with a reference binding value.

[0258] As used herein, the term “reference” broadly encompasses any suitable reference binding level which may be used as a basis for comparison with respect to the determined binding. In some embodiments, the reference is constructed using algorithms and / or other methods of statistical and hierarchical classification. In another aspect, the reference binding level is stored in a database to provide a stored binding level and the stored binding level is used to determine the difference in the binding level. The database may, for example, be stored on a computer or a server.

[0259] In some embodiments, the reference binding level is an index value or is derived from one or more risk prediction algorithms or computed indices for the presence of cells wherein the function of GLUT1 is altered (e.g., increased or decreased). A reference binding level can be relative to a number or value derived from population studies, including without limitation, such populations of subjects having similar age range, subjects in the same or similar ethnic group.

[0260] The term “cells wherein the function of GLUT1 is altered” as used herein refers to cells wherein glucose metabolism or influx is abnormally increased or decreased.

[0261] In some embodiments, the reference value is determined by measuring the binding of the polypeptide of the invention to GLUT1 in a reference population.

[0262] In some embodiments, the reference population refers to a population comprising at least 1, preferably at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100 or more substantially healthy subjects, i.e., subject who are not affected and / or who have not been diagnosed with the GLUT 1 -associated disease being considered. According to those embodiments, a determined binding level different from the reference binding level may be indicative of the presence of a GLUT 1 -associated disease.

[0263] In other embodiments, the reference population refers to a population comprising at least 1, preferably at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100 or more subjects affected (and preferably diagnosed) with the GLUT 1- associated disease being considered. According to those embodiments, a determined binding level different from the reference binding level may be indicative of the absence of a GLUTl-associated disease.

[0264] In the present invention, two numeric values, in particular two binding levels, are considered as different if the first numeric value is higher (such as, for example, the first numeric value is about 20% higher than the second one, preferably is about 30, 40, 50, 60, 70, 80, 90% or more higher than the second one) or lower than the second one(such as, for example, the first numeric value is about 20% lower than the second one, preferably is about 30, 40, 50, 60, 70, 80, 90% or more lower than the second one).

[0265] In some embodiments, two numeric values, in particular two binding levels, are considered as different if the first numeric value is increased by a factor of or above about 1.01, preferably by a factor of or above, about 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, more preferably the value is increased by a factor of or above about 1.1, 1.15, 1.20, 1.25, 1.30, 1.3, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, more preferably by a factor of or above about 2, 3, 4, 5 or more when compared to the second value or if the first numeric value is decreased by a factor of or below about 0.99, preferably by a factor of or below, about 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.92, 0.91, more preferably the value is decreased by a factor of or below about 0.90, 0.85, 0.80, 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.1 or less when compared to the second value.

[0266] In some embodiments, by implying a multitude of measures of the binding of the polypeptide of the invention to GLUT1 in the reference population, it is conceivable to use as reference value a mathematical representation of the binding of the polypeptide of the invention to GLUT1 such as for example, a mean or a median.

[0267] In some embodiments, the reference value is a personalized reference, determined at different time points in the same subject (such as, for example, before receiving a treatment for a GLUT 1- associated disease).

[0268] In some embodiments, the reference value is an internal reference value, determined in different part of the same subject, such as, for example, in different organs or tissues. The reference value may for example be determined in / on cells in / on which GLUT1 expression is not affected or dysregulated, in the same subject. This type of reference value is in particular useful in the implementation of methods based on medical imaging. This type of reference value may also be useful in the implementation of methods based on sample analysis, wherein the reference value may be determined in / on reference cells in / on which GLUT1 expression is not affected or dysregulated, said reference cells being comprised in the analyzed sample.

[0269] The present invention thus relates to an in vitro method for diagnosing a subject with, for prognosing a subject with, or identifying a subject as being at risk of developing a GLUT 1 -associated disease, wherein said method comprises the steps of: a) contacting a biological sample previously obtained from said subject with a polypeptide according to the invention; b) measuring the binding of said polypeptide to GLUT1; and c) comparing the binding measured at step b) with a reference value.

[0270] In some embodiments, the step of comparing the binding of the polypeptide of the invention to a reference value allows to diagnose a subject with, or at risk of developing, a GLUT 1- associated disease.

[0271] In some embodiments, the method of the invention is for diagnosing, for prognosing, or for assessing a risk of developing a GLUT 1 -associated disease associated with an increased GLUT1 level in a subject, and a binding of the polypeptide of the invention to GLUT1 measured at step b) higher than the binding of the polypeptide of the invention to GLUT1 measured in a reference population of substantially healthy subjects (or in a reference sample from a reference population) is indicative of the presence of said disease, or of a risk of developing said disease.

[0272] In some embodiments, the method of the invention is for diagnosing, for prognosing, or for assessing a risk of developing a GLUT 1 -associated disease associated with an increased GLUT1 level at the cell surface in a subject, and a binding of the polypeptide of the invention to GLUT1 measured at step b) higher than the binding of the polypeptide of the invention to GLUT1 measured in a reference population of substantially healthy subjects (or in a reference sample from a reference population) is indicative of the presence of said disease, or of a risk of developing said disease.

[0273] In some embodiments, the method of the invention is for diagnosing, for prognosing, or for assessing a risk of developing a GLUT 1 -associated disease associated with a decreased GLUT1 level in a subject, and a binding of the polypeptide of the invention to GLUT1 measured at step b) lower than the binding of the polypeptide of the invention to GLUT1 measured in a reference population of substantially healthy subjects(or in a reference sample from a reference population) is indicative of the presence or of a risk of developing said disease.

[0274] In some embodiments, the method of the invention is for diagnosing, for prognosing, or for assessing a risk of developing a GLUTl-associated disease associated with a decreased GLUT1 level at the cell surface in a subject, and a binding of the polypeptide of the invention to GLUT1 measured at step b) lower than the binding of the polypeptide of the invention to GLUT1 measured in a reference population of substantially healthy subjects (or in a reference sample from a reference population) is indicative of the presence or of a risk of developing said disease.

[0275] The present application also relates to an in vitro method for diagnosing a GLUTl-associated disease in a subject. The present application also relates to an in vitro method for prognosing a GLUTl-associated disease in a subject.

[0276] Another object is an in vitro method for diagnosing or prognosing in a subject a cancer selected from the group consisting of a brain cancer, a hypopharyngeal cancer, a breast cancer, a cervical cancer, an ovarian cancer, a lung cancer, a pancreatic cancer, a liver cancer, a colon cancer, squamous cell carcinoma, multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma (CTCL); a pre-malignant disorder, including myelodysplastic syndrome; an inflammatory disease, including psoriasis; an immune or auto-immune disease selected from the group consisting of autoimmune myocarditis, systemic lupus erythematous, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and Sjogren’s syndrome; or a disorder of the central nervous system, including GLUT1 deficiency syndrome.

[0277] The present application also relates to a polypeptide of the invention for use for diagnosing or prognosing a GLUTl-associated disease in a subject.

[0278] Another object is a polypeptide of the invention for use for diagnosing or prognosing in a subject a cancer selected from the group consisting of a brain cancer, a hypopharyngeal cancer, a breast cancer, a cervical cancer, an ovarian cancer, a lungcancer, a pancreatic cancer, a liver cancer, a colon cancer, squamous cell carcinoma, multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma (CTCL); a pre-malignant disorder, including myelodysplastic syndrome; an inflammatory disease, including psoriasis; an immune or auto-immune disease selected from the group consisting of autoimmune myocarditis, systemic lupus erythematous, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and Sjogren’s syndrome; or a disorder of the central nervous system, including GLUT1 deficiency syndrome.

[0279] In some embodiments, the method for the in vivo diagnosis or prognosing of a GLUTl-associated disease, comprises: a) contacting at least one polypeptide according to the invention with a cell, a sample, a tissue or an organ, and b) detecting and / or quantifying the at least one polypeptide according to the invention bound to GLUT1 present in the cell, sample, tissue or organ within said subject.

[0280] In some embodiments, the method for the in vivo diagnosis or prognosing of GLUTl-associated disease comprises: a) administering to a subject in need thereof at least one polypeptide according to the invention, and b) detecting and / or quantifying the binding of at least one polypeptide according to the invention within said subject, for example by medical imaging.

[0281] In some embodiments, the polypeptide according to the invention is coupled with at least one detectable label, and may be used for in vivo diagnosis or prognosis by medical imaging.

[0282] In some embodiments, the polypeptide administered to the subject is comprised in a diagnostic or prognostic composition.

[0283] The present invention also relates to a polypeptide according to the invention, for use in a in vivo diagnosis or prognosis method of a GLUTl-associated disease in a subject,wherein said method comprise the detection and / or measure of the level of GLUT1 using medical imaging techniques.

[0284] In some embodiments, the in vivo method for diagnosing or prognosing a subject with or identifying a subject at risk of developing a GLUTl-associated disease comprises the steps: a) administering to said subject a labeled polypeptide according to the invention; and, b) detecting and / or measuring the binding of said labeled polypeptide to GLUT1 using medical imaging; c) optionally comparing a binding measured at step b) with a reference value.

[0285] In another embodiment, the diagnosis or prognosis method of the invention is an in vitro or ex vivo method, i.e., the method of the invention is performed on a cell, sample, tissue and / or organ that was obtained from a patient prior to the implementation of the method of the invention. Consequently, in some embodiments, the method of the invention does not comprise obtaining a sample from the patient, i.e., the method of the invention is non-invasive.

[0286] Another object of the invention is an in vitro method for patient stratification, e.g. for treatment responsiveness, comprising specifically detecting or / and measuring the level of GLUT1 in a sample by using the polypeptide, nucleic acid, expression vector, cell, or pharmaceutical composition according to the invention.

[0287] In particular, it is herein disclosed in vitro method for patient stratification comprising the steps of: a) contacting said sample with the polypeptide according to the invention, and b) detecting and / or measuring the binding of said polypeptide to GLUT1.

[0288] In some embodiments, the method for patient stratification is for determining if a patient is likely to respond, or to be responsive, to a determined treatment.

[0289] In some embodiments, the method for patient stratification is for determining if a patient is likely to have adverse events when treated with a determined treatment.

[0290] In some embodiments, the method for patient stratification is for selecting a patient for treatment.

[0291] In some embodiments, the method for patient stratification is for determining whether or not to treat the patient using a determined treatment.

[0292] In some embodiments, the method for patient stratification may be implemented in connection with a clinical trial.

[0293] In some embodiments, the clinical trial is for testing the efficacy of a determined treatment, for example the polypeptide, nucleic acid, expression vector, cell, or pharmaceutical composition according to the invention against a disease.

[0294] In some embodiments, the method for patient stratification is for determining which patients will be enrolled in a clinical trial.

[0295] In some embodiments, the method for patient stratification is for determining which patients will be enrolled in a subsequent stage of the same clinical trial.

[0296] In some embodiments, the method for patient stratification is for determining which patients will be enrolled in a subsequent clinical trial.

[0297] In some embodiments, the determined treatment comprises, or consists of, a polypeptide, nucleic acid, expression vector, cell, or pharmaceutical composition according to the invention.

[0298] In some embodiments, the patient suffers from a GLUTl-associated disease.

[0299] In some embodiments, the patient suffers from a disease selected from the group consisting of a cancer, an inflammatory disease, an immune or auto-immune disease, and a disorder of the central nervous system.

[0300] In some embodiments, the patient suffers from a cancer, said cancer being selected from the group consisting of a brain cancer, a hypopharyngeal cancer, a breast cancer, a cervical cancer, an ovarian cancer, a lung cancer, a pancreatic cancer, a liver cancer, a colon cancer, squamous cell carcinoma, multiple myeloma, acute myeloidleukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma (CTCL); a pre-malignant disorder, including myelodysplastic syndrome; an inflammatory disease, including psoriasis; an immune or auto-immune disease selected from the group consisting of autoimmune myocarditis, systemic lupus erythematous, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and Sjogren’s syndrome; or a disorder of the central nervous system, including GLUT1 deficiency syndrome.

[0301] In some embodiments, the method for patient stratification comprises: a) contacting at least one polypeptide according to the invention with a cell, a sample, a tissue or an organ, and b) detecting and / or quantifying the at least one polypeptide according to the invention bound to GLUT1 present in the cell, sample, tissue or organ within said subject.

[0302] In some embodiments, the method for patient stratification comprises: a) administering to a subject in need thereof at least one polypeptide according to the invention, and b) detecting and / or quantifying the binding of at least one polypeptide according to the invention within said subject, for example by medical imaging.

[0303] In some embodiments, the polypeptide according to the invention is coupled with at least one detectable label, and may be used for in vivo stratification by medical imaging.

[0304] The present invention also relates to a polypeptide according to the invention, for use in a in vivo method of stratifying patients suffering from a GLUT 1 -associated disease, wherein said method comprises the detection and / or measure of the level of GLUT1 using medical imaging techniques.

[0305] In some embodiments, the in vivo method for stratifying patients suffering from a GLUT 1 -associated disease comprises the steps: a) administering to said subject a labeled polypeptide according to the invention; and,b) detecting and / or measuring the binding of said labeled polypeptide to GLUT1 using medical imaging; c) optionally comparing a binding measured at step b) with a reference value.

[0306] In another embodiment, the stratification method of the invention is an in vitro or ex vivo method, i.e., the method of the invention is performed on a cell, sample, tissue and / or organ that was obtained from a patient prior to the implementation of the method of the invention. Consequently, in some embodiments, the method of the invention does not comprise obtaining a sample from the patient, i.e., the method of the invention is non- invasive.

[0307] Another object of the invention is a polypeptide, nucleic acid, expression vector, cell, or pharmaceutical composition according to the invention for use for patient stratification.

[0308] In some embodiments, the polypeptide, nucleic acid, expression vector, cell, or pharmaceutical composition according to the invention is for use for patient stratification for determining if a patient is likely to respond, or to be responsive, to a determined treatment.

[0309] In some embodiments, the polypeptide, nucleic acid, expression vector, cell, or pharmaceutical composition according to the invention is for use for patient stratification for determining if a patient is likely to have adverse events when treated with a determined treatment.

[0310] In some embodiments, the polypeptide, nucleic acid, expression vector, cell, or pharmaceutical composition according to the invention is for use for patient stratification for selecting a patient for treatment.

[0311] In some embodiments, the polypeptide, nucleic acid, expression vector, cell, or pharmaceutical composition according to the invention is for use for patient stratification for determining whether or not to treat the patient using a determined treatment.

[0312] In some embodiments, the polypeptide, nucleic acid, expression vector, cell, or pharmaceutical composition according to the invention is for use for patient stratificationfor determining which patients will be enrolled in a clinical trial, in a subsequent stage of the same clinical trial, or in a subsequent clinical trial.

[0313] In some embodiments, the determined treatment comprises, or consists of, a polypeptide, nucleic acid, expression vector, cell, or pharmaceutical composition according to the invention.

[0314] In some embodiments, the patient suffers from a GLUT 1 -associated disease.

[0315] In some embodiments, the patient suffers from a disease selected from the group consisting of a cancer, an inflammatory disease, an immune or auto-immune disease, and a disorder of the central nervous system.

[0316] In some embodiments, the patient suffers from a cancer, said cancer being selected from the group consisting of a brain cancer, a hypopharyngeal cancer, a breast cancer, a cervical cancer, an ovarian cancer, a lung cancer, a pancreatic cancer, a liver cancer, a colon cancer, squamous cell carcinoma, multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma (CTCL),; a pre-malignant disorder, including and myelodysplastic syndrome; an inflammatory disease, including psoriasis; an immune or auto-immune disease selected from the group consisting of autoimmune myocarditis, systemic lupus erythematous, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and Sjogren’s syndrome; or a disorder of the central nervous system, including GLUT1 deficiency syndrome.

[0317] The present application also relates to an in vitro method for monitoring a GLUTLassociated disease in a subject.

[0318] The term “monitoring” as used herein may refer to the determination of the number of cells wherein glucose metabolism is dysregulated in the body of a subject as a function of time, such as, for example, before, during and after a therapy against a GLUTLassociated disease.

[0319] The term “therapy against a GLUTl-associated disease” as used herein may refer to GLUTl-binding polypeptides of the invention, glucose or derivatives (such as galactose, 2-fluorodeoxyglucose, 2-deoxyglucose, 3-O-metylglucose), glucose deprivation, chemotherapy, radiation, surgery, immunotherapy, androgenic steroids, cytochalasin B, forskolin, dipyridamole, isobutylmethylxanthine, ethanol, genistein, cadmium, barbiturate, dehydroascorbic acid, tricyclic antidepressants, estradiol, antiestrogens, faslodex (ICI 182780), tamoxifen, gamma agonists of peroxisome proliferator- activated receptors (PPAR) such as thiazolidinedione, troglitazone, pioglitazone, rosiglitazone, drugs known to the skilled artisan as drugs for treating a GLUTl-associated disease.

[0320] In some embodiments, the method of monitoring of the invention comprises comparing two binding levels, such as, for example, a binding determined before treatment with a binding level determined after treatment.

[0321] In some embodiments, a decreased binding level of the polypeptide of the invention after treatment is indicative of the efficacy of the treatment.

[0322] In some embodiments, a binding level after treatment equivalent or superior to the one determined before treatment is indicative of the absence of efficacy of the treatment.

[0323] The present application thus relates to an in vitro method for monitoring a GLUTl-associated disease in a subject.

[0324] Another object is an in vitro method for monitoring in a subject a cancer selected from the group consisting of a brain cancer, a hypopharyngeal cancer, a breast cancer, a cervical cancer, an ovarian cancer, a lung cancer, a pancreatic cancer, a liver cancer, a colon cancer, squamous cell carcinoma, multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma (CTCL); a pre-malignant disorder, including myelodysplastic syndrome; an inflammatory disease, including psoriasis; an immune or auto-immune disease selected from the group consisting of autoimmune myocarditis,systemic lupus erythematous, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and Sjogren’s syndrome; or a disorder of the central nervous system, including GLUT1 deficiency syndrome.

[0325] The present application also relates to a polypeptide of the invention for use for monitoring a GLUT 1 -associated disease in a subject.

[0326] Another object is a polypeptide of the invention for use for monitoring in a subject a cancer selected from the group consisting of a brain cancer, a hypopharyngeal cancer, a breast cancer, a cervical cancer, an ovarian cancer, a lung cancer, a pancreatic cancer, a liver cancer, a colon cancer, squamous cell carcinoma, multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, nonHodgkin lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma (CTCL); a pre- malignant disorder, including myelodysplastic syndrome; an inflammatory disease, including psoriasis; an immune or auto-immune disease selected from the group consisting of autoimmune myocarditis, systemic lupus erythematous, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and Sjogren’s syndrome; or a disorder of the central nervous system, including GLUT1 deficiency syndrome.

[0327] In some embodiments, the method for monitoring a GLUT 1 -associated disease in a subject comprises the steps of: a. contacting an effective amount of polypeptide of the invention, preferably coupled with at least one contrast agent, to a cell, sample, tissue, and / or organ of said subject, b. detecting and / or quantifying the binding of the polypeptide of the invention to GLUT1 in said cell, sample, tissue, and / or organ, preferably by medical imaging, c. treating the subject with a therapy against a GLUT1 -associated disease, d. contacting an effective amount of the polypeptide of the invention, preferably coupled with at least one contrast agent to a cell, sample, tissue, and / or organ of said subject, ande. detecting and / or quantifying the binding of the polypeptide of the invention to GLUT1 in said cell, sample, tissue, and / or organ.

[0328] In some embodiments, the method of the invention further comprises a step of comparing the binding determined in step e) with the binding determined in step b), thereby monitoring a GLUTl-associated disease.

[0329] In some embodiments, the absence or the decrease of detection of GLUT1 in a cell, sample, tissue, and / or organ after a therapy against a GLUTl-associated disease, is indicative of a remission.

[0330] In other embodiments, the presence or the increase of detection of GLUT1 in a cell, sample, tissue, and / or organ after a therapy against a GLUTl-associated disease, is indicative of a remission.

[0331] The invention also relates to the polypeptide according to the invention, for use as a medicament.

[0332] The invention further relates to the nucleic acid or expression vector according to the invention, for use as a medicament.

[0333] The invention further relates to the cell according to the invention, for use as a medicament.

[0334] The invention further relates to the pharmaceutical composition according to the invention, for use as a medicament.

[0335] The present invention also relates to a method for preventing or treating a subject in need thereof, said method comprising administering to said subject the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical composition according to the invention.

[0336] In some embodiments, said method specifically targets GLUT1 in the subject.

[0337] The present invention also relates to the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical composition according to the invention, for use in the prevention or the treatment of a GLUT 1 -associated disease.

[0338] In some embodiments, the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical composition according to the invention, are for use in the prevention or the treatment of a disease selected from the group consisting of a cancer, an inflammatory disease, an immune or auto-immune disease, and a disorder of the central nervous system.

[0339] In some embodiments, the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical composition according to the invention, are for use in the prevention or the treatment of a cancer selected from the group consisting of a brain cancer, a hypopharyngeal cancer, a breast cancer, a cervical cancer, an ovarian cancer, a lung cancer, a pancreatic cancer, a liver cancer, a colon cancer, squamous cell carcinoma, multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma (CTCL); a pre-malignant disorder, including myelodysplastic syndrome; an inflammatory disease, including psoriasis; or an immune or auto-immune disease selected from the group consisting of autoimmune myocarditis, systemic lupus erythematous, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and Sjogren’s syndrome.

[0340] In some embodiments, said treatment is by specifically targeting GLUT1.

[0341] The present invention also relates to a method of preventing or treating a GLUT 1 - associated disease in a subject in need thereof, said method comprising administering to said subject the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical composition according to the invention.

[0342] The present invention also relates to a method of preventing or treating a disease selected from the group consisting of a cancer, an inflammatory disease, an immune or auto-immune disease, and a disorder of the central nervous system, in a subject in need thereof, said method comprising administering to said subject the polypeptide, the nucleicacid, the expression vector, the cell or the pharmaceutical composition according to the invention.

[0343] The present invention also relates to a method of preventing or treating a cancer selected from the group consisting of a brain cancer, a hypopharyngeal cancer, a breast cancer, a cervical cancer, an ovarian cancer, a lung cancer, a pancreatic cancer, a liver cancer, a colon cancer, squamous cell carcinoma, multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma (CTCL); a pre-malignant disorder, including myelodysplastic syndrome; an inflammatory disease, including psoriasis; or an immune or auto-immune disease selected from the group consisting of autoimmune myocarditis, systemic lupus erythematous, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and Sjogren’s syndrome, in a subject in need thereof, said method comprising administering to said subject the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical composition according to the invention.

[0344] In some embodiments, said method specifically targets GLUT1 in the subject.

[0345] In some embodiments, a therapeutically effective amount of the polypeptide of the invention is administered to the subject.

[0346] In some embodiments, the administration of a polypeptide of the invention, modulates the flux of glucose through the GLUT1 receptor.

[0347] The present invention also relates to the use of the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical composition according to the invention, for the manufacture of a medicament for the prevention or treatment of a GLUTl-associated disease.

[0348] The present invention also relates to the use of the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical composition according to the invention, for the manufacture of a medicament for the prevention or treatment of adisease selected from the group consisting of a cancer, an inflammatory disease, an immune or auto-immune disease, and a disorder of the central nervous system.

[0349] The present invention also relates to the use of the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical composition according to the invention, for the manufacture of a medicament for the prevention or treatment of a cancer selected from the group consisting of a brain cancer, a hypopharyngeal cancer, a breast cancer, a cervical cancer, an ovarian cancer, a lung cancer, a pancreatic cancer, a liver cancer, a colon cancer, squamous cell carcinoma, multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma (CTCL); a pre-malignant disorder, including myelodysplastic syndrome; an inflammatory disease, including psoriasis; or an immune or auto-immune disease selected from the group consisting of autoimmune myocarditis, systemic lupus erythematous, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and Sjogren’s syndrome.

[0350] In some embodiments, said treatment is by specifically targeting GLUT1.

[0351] The present application also relates to a method for targeting cells, samples, tissues, and / or organs expressing GLUT1, wherein said method comprises the administration of a polypeptide of the invention.

[0352] For instance, in the case of cancer, cancerous or tumorous cells and / or immune cells may be targeted.

[0353] Such method may be used, for example, for targeting therapeutic agents to cells, samples, tissues, and / or organs in a subject in need thereof.

[0354] In some embodiments, the targeting method of the invention is for targeting anticancer drugs to cancer cells, in particular to GLUT 1 -expressing cancer cells.

[0355] In some embodiments, the polypeptide of the invention, is encapsulated with a therapeutic agent to be specifically administered to cells, samples, tissues or organs of a subject in need thereof.

[0356] In some embodiments, the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention is to be administered at a dose determined by the skilled artisan and personally adapted to each subject.

[0357] It will be understood that the usage of the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention will be decided by the attending physician within the scope of sound medical judgment. The specific effective amount for any particular patient will depend upon a variety of factors including the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and like factors well known in the medical arts.

[0358] In some embodiments, a therapeutically effective amount of the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention is administered at least once a day, twice a day, or at least three times a day.

[0359] In other embodiments, a therapeutically effective amount of the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention is administered every two, three, four, five, or six days.

[0360] In other embodiments, a therapeutically effective amount of the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention is administered every week, twice a week, every two weeks, or once a month.

[0361] In other embodiments, a therapeutically effective amount of the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention is administered every month for a period at least 2, 3, 4, 5, or 6 months.

[0362] In other embodiments, a therapeutically effective amount of the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention ranges from about 1 pg to 5 g.

[0363] In other embodiments, a therapeutically effective amount of the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention to be administered ranges from about 0.1 pg / kg to 1 g / kg.

[0364] In other embodiments, the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention is to be administered in combination with another treatment for a GLUTl-associated disease.

[0365] Examples of agents for treating a GLUTl-associated disease include, but are not limited to, GLUT 1 -binding polypeptides of the invention, glucose or derivatives (such as galactose, 2-fluorodeoxyglucose, 2-deoxyglucose, 3-O-metylglucose), glucose deprivation, chemotherapy, radiation, surgery, immunotherapy, androgenic steroids, cytochalasin B, forskolin, dipyridamole, isobutylmethylxanthine, ethanol, genistein, cadmium, barbiturate, dehydroascorbic acid, tricyclic antidepressants, estradiol, antiestrogens, faslodex (ICI 182780), tamoxifen, gamma agonists of peroxisome proliferator- activated receptors (PPAR) such as thiazolidinedione, troglitazone, pioglitazone, rosiglitazone, drugs known to the skilled artisan as drugs for treating a GLUTl-associated disease.

[0366] In some embodiments, the method for treating a GLUTl-associated disease in a subject in need thereof, comprises administering to the subject the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention prior to, concurrent to and / or posterior to, another treatment against a GLUTl-associated disease.

[0367] In some embodiments, the subject is affected, preferably is diagnosed with a GLUTl-associated disease. In other embodiments, the subject of the invention is at risk of developing a GLUTl-associated disease.

[0368] In some embodiments, the subject of the invention is in a remission stage following a GLUT 1 -associated disease.

[0369] In some embodiments, the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention is to be administered by injection, orally, topically, nasally, buccally, rectally, vaginally, intratracheally, by endoscopy, transmucosally, or by percutaneous administration.

[0370] In some embodiments, the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention is to be administered by injection, preferably is to be systemically injected. Examples of formulations adapted to systemic injections include, but are not limited to, liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection. Examples of systemic injections include, but are not limited to, intravenous, subcutaneous, intramuscular, intradermal, intravitreal, and intraperitoneal injection, or perfusion.

[0371] Preferably, when injected, the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention is sterile. Methods for obtaining a sterile polypeptide, nucleic acid, expression vector, cell or composition, include, but are not limited to, GMP synthesis (GMP stands for “Good manufacturing practice”).

[0372] In some embodiments, the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention are to be orally administered. Examples of formulations adapted to oral administration include, but are not limited to, solid forms, liquid forms and gels. Examples of solid forms adapted to oral administration include, but are not limited to, pill, tablet, capsule, soft gelatine capsule, hard gelatine capsule, caplet, compressed tablet, cachet, wafer, sugar-coated pill, sugar coated tablet, or dispersing / or disintegrating tablet, powder, solid forms suitable for solution in, or suspension in, liquid prior to oral administration and effervescent tablet. Examples of liquid forms adapted to oral administration include, but are not limited to,solutions, suspensions, drinkable solutions, elixirs, sealed phial, potion, drench, syrup and liquor.

[0373] In other embodiments, the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention are to be topically administered. Examples of formulations adapted to topical administration include, but are not limited to, sticks, waxes, creams, lotions, ointments, balms, gels, masks, leave-on washes and / or the like.

[0374] Depending on the cell(s), sample(s), tissue(s) and / or organ(s) targeted, the skilled artisan can determine the technology needed for the introduction of the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention in the targeted cell(s), sample(s), tissue(s) and / or organ(s).

[0375] In some embodiments, the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention are to be administered in a sustained-release form. In other embodiments, the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical or diagnostic composition according to the invention comprise a delivery system that controls the release of the agent.

[0376] The present invention further relates to a method for the treatment of cancer in a subject, preferably of a GLUTl-associated cancer, more preferably of a cancer selected from the group consisting of a brain cancer, a hypopharyngeal cancer (e.g. hypopharyngeal carcinoma), a breast cancer, a cervical cancer (e.g. cervical carcinoma), an ovarian cancer (e.g. ovarian carcinoma), a lung cancer, a pancreatic cancer (e.g. pancreatic adenocarcinoma), a liver cancer (e.g. hepatocarcinoma), a colon cancer, and squamous cell carcinoma, comprising the steps of: a) diagnosing said cancer using an in vitro or in vivo method according to the invention for diagnosing a cancer, b) treating said cancer.

[0377] Said cancer may for instance be treated by chemotherapy, radiotherapy or surgery.

[0378] In some embodiments, said cancer is a hypopharyngeal carcinoma, a cervical carcinoma, an ovarian carcinoma, a pancreatic adenocarcinoma, an hepatocarcinoma, or a squamous cell carcinoma.

[0379] Preferably, said cancer is a GLUT 1- associated cancer.

[0380] Examples of chemotherapies include, but are not limited to: a. alkylating agents that act mainly by forming covalent bonds between DNA bases, including, but not limited to, nitrogen mustards (e.g., cyclophosphamide), aziridines and epoxides (e.g., thiopeta), alkyl sulfonates (e.g., busulfan), nitro sureas (e.g., BCNU and CCNU), hydrazine and triazine derivatives (e.g., procarbazine and temozolomide); b. cisplatin and its analogs that act by forming DNA adducts which lead to intrastrand and inter- strand linking leading to the formation of DNA filaments, including, but not limited to, carboplatin, cisplatin and oxaliplatin; c. antimetabolites including but not limited to folate metabolism inhibitors (e.g., methotrexate, trimetrexate, tomudex), 5-fluoropyrimidines (e.g., 5-FU), oral fluoropyramidines (e.g., tegafur, uracil, capecitabine), nucleoside analogs (e.g., cytarabine), gemcitabine and 6-thiopurines (e.g., 6-MP and 6-TG); d. topoisomerase-interactive agents that affect the topologic states of DNA by interfering or modulating DNA cleavage, strand passage and re-ligation, including, but not limited to, epipodophyllotoxins (e.g. , etoposide and teniposide), camptothecin analogs, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin), mitoxantrone and losoxantrone, and dactinomycin; e. antimicro tubule agents, which interfere with the proper polymerization / depolymerization of microtubules, including, but not limited to, vinca alkaloids (e.g., vincristine, vinorelbine and vinblastine), taxanes (e.g., paclitaxel, docetaxel) and estramustine phosphate; andf. numerous miscellaneous agents exist which cannot be classified into any of the above groups, including but not limited to suramin, bleomycin, L-asparaginase and amifostine.

[0381] The present invention further relates to a method for the treatment of diseases characterized by a lower expression and / or function of GLUT1 in a subject, comprising the steps of: a) diagnosing said disease using an in vitro or in vivo method according to the invention for diagnosing a GLUT 1- associated disease; b) treating said disease, preferably by glucose supplementation.

[0382] The present application also relates to a method for specifically inhibiting GLUT1 activity in a subject in need thereof, wherein a therapeutically effective amount of the polypeptide, the nucleic acid, the expression vector, the cell or the pharmaceutical according to the invention is administered to said subject.

[0383] The term “inhibiting GLUT1 activity” as used herein may refer to inhibiting the flux of glucose transport within a cell, or across the cell membrane, by GLUT1.

[0384] Another object of the present invention is a screening method to identify compounds modulating the level of GLUT1, said method comprising the detection and / or measure of the level of GLUT1 in a sample using the in vitro or in vivo methods of the invention.

[0385] Hence, the present invention further relates to a screening method to identify compounds modulating the level of GLUT1, using a polypeptide of the invention, said method comprising the steps of: a) measuring the level of GLUT1 in a sample, preferably a biological sample from a subject, using an in vitro method of the invention; b) contacting said sample with the tested compound; c) measuring the level of GLUT1 in said sample using an in vitro method of the invention; and, d) comparing the levels of GLUT1 measured at step a) and c).

[0386] The present invention also relates to a screening method to identify compounds modulating the level of GLUT1 using a polypeptide of the invention, said method comprising the steps of: a) measuring the level of GLUT1 in a subject using an in vivo method of the 5 invention; b) contacting said subject with said compound; c) measuring the level of GLUT1 in said subject using an in vivo method of the invention; and, d) comparing the levels of GLUT1 measured at step a) and c).10TABLE OF SEQUENCESBRIEF DESCRIPTION OF THE DRAWINGS

[0387] Figure 1 is a histogram showing the GFP fluorescence intensity measured in the supernatants of expi293F cells transfected with a plasmid comprising the sequence of either (1) HTLV-2.RBD with its natural signal peptide, and coupled to EGFP (H2.EGFP), or (2) HTLV-2.RBD with the signal peptide of IL-2, and coupled to EGFP (IL2- H2.EGFP). Fluorescence was measured in the supernatants after 3 days of cell culture.

[0388] Figure 2 is a histogram showing the binding activity of H2.EGFP and IL2-H2.EGFP constructs, as indicated, on GLUT1 transporter expressed at the surface of red blood cells. Supernatants of expi293F cells transfected with a plasmid comprising either (1) the sequence of HTLV-2.RBD with its natural signal peptide, and coupled to EGFP (H2.EGFP), or (2) the sequence of HTLV-2.RBD with the signal peptide of IL-2, and coupled to EGFP (IL2-H2.EGFP), were incubated with whole blood. GFP fluorescence of stained red blood cells was measured and compared to fluorescence of unstained red blood cells to calculate the signal / noise of each supernatant.

[0389] Figure 3 is a histogram showing the GFP fluorescence intensity measured in the supernatants of expiCHO cells transfected with a plasmid comprising the sequence of(1) HTLV-2.RBD with its natural signal peptide, and coupled to EGFP (H2.EGFP),(2) HTLV-2.RBD C-terminally truncated of 20 amino acids, with the signal peptide of IL-2, and coupled to EGFP (IL2-H2.A20aa.EGFP), or (3) HTLV-2.RBD with the signal peptide of IL-2, and coupled to sfGFP (IL2-H2.sfGFP). Fluorescence was measured in the supernatants after 3 days of cell culture.

[0390] Figure 4 is a histogram showing the binding activity of H2.EGFP, IL2-H2.A20aa.EGFP, and IL2-H2. sfGFP constructs, as indicated, on GLUT1 transporter expressed at the surface of red blood cells. Supernatants of expiCHO cells transfected with a plasmid comprising the sequence of (1) HTLV-2.RBD with its natural signal peptide, and coupled to EGFP (H2.EGFP), (2) HTLV-2.RBD C-terminally truncated of 20 amino acids, with the signal peptide of IL-2, and coupled to EGFP (IL2-H2.A20aa.EGFP), or (3) HTLV-2.RBD with the signal peptide of IL-2, and coupled to sfGFP (IL2-H2. sfGFP) were incubated with whole blood. GFP fluorescence of stained red blood cells was measured and compared to fluorescence of unstained red blood cells to calculate the signal / noise of each supernatant.

[0391] Figure 5 is a histogram showing the GFP fluorescence intensity measured in the supernatants of expiCHO cells transfected with a plasmid comprising the sequence of (1) HTLV-2.RBD with the signal peptide of IL-2, and coupled to sfGFP C48S (IL2- H2.sfGFPC48S), (2) HTLV-2.RBD comprising a C100S substitution, with the signal peptide of IL-2, and coupled to sfGFP C48S (IL2-H2C100S.sfGFPC48S),(3) HTLV-2.RBD comprising a C95S substitution, with the signal peptide of IL-2, and coupled to sfGFP (IL2-H2C95S. sfGFP), (4) HTLV-2.RBD comprising a C135S substitution, with the signal peptide of IL-2, and coupled to sfGFP (IL2-H2C135S. sfGFP), (5) HTLV-2.RBD with the signal peptide of IL-2, coupled to sfGFP and a HisTag (IL2-H2. sfGFP. HisTag), or (6) HTLV-2.RBD with the signal peptide of IL-2, coupled to sfGFP and a StrepTag II (IL2-H2. sfGFP. StrepTagll). Fluorescence was measured in the supernatants after 3 days of cell culture.

[0392] Figure 6 is a histogram showing the binding activity of IL2-H2.sfGFPC48S, IL2- H2C100S.sfGFPC48S, IL2-H2C95S.sfGFP, IL2-H2C135S.sfGFP, IL2-H2. sfGFP.HisTag and IL2-H2.sfGFP.StrepTagII constructs, as indicated, on GLUT1 transporter expressed at the surface of red blood cells. Supernatants of expiCHO cells transfected with a plasmid comprising the sequence of (1) HTLV-2.RBD with the signal peptide of IL-2, and coupled to sfGFP C48S (IL2-H2.sfGFPC48S), (2) HTLV-2.RBD comprising a C100S substitution, with the signal peptide of IL-2, and coupled to sfGFP C48S (IL2-H2C100S.sfGFPC48S), (3) HTLV-2.RBD comprising a C95S substitution, with the signal peptide of IL-2, and coupled to sfGFP (IL2-H2C95S. sfGFP), (4) HTLV-2.RBD comprising a C135S substitution, with the signal peptide of IL-2, and coupled to sfGFP (IL2-H2C135S. sfGFP), (5) HTLV-2.RBD with the signal peptide of IL-2, coupled to sfGFP and a HisTag (IL2-H2. sfGFP.HisTag), or (6) HTLV-2.RBD with the signal peptide of IL-2, coupled to sfGFP and a StrepTag II (IL2-H2. sfGFP. StrepTagll) were incubated with whole blood. GFP fluorescence of stained red blood cells was measured and compared to fluorescence of unstained red blood cells to calculate the signal / noise of each supernatant.

[0393] Figure 7 is a photograph of an SDS-PAGE gel in heated and reduced conditions. Wells were loaded as follows:1. Ladder2. Rabbit IgG3. SV40i-IL2-H2. sfGFP construct4. IL2-H2.sfGFPC48S construct5. IL2-H2C100S.sfGFPC48S construct6. IL2-H2C95S. sfGFP construct7. IL2-H2C135S. sfGFP construct8. IL2-H2.sfGFP-HisTag construct9. IL2-H2.sfGFP-StrepTagII construct10. LadderThe square indicates the band of the IL2-H2C95S. sfGFP construct.

[0394] Figure 8 is a photograph of an SDS-PAGE gel in non-heated and non-reduced conditions. Wells were loaded as follows:1. Rabbit IgG2. Ladder3. SV40i-IL2-H2.sfGFP construct4. IL2-H2.sfGFPC48S construct5. IL2-H2C100S.sfGFPC48S construct6. IL2-H2C95S.sfGFP construct7. IL2-H2C135S.sfGFP construct8. IL2-H2.sfGFP-HisTag construct9. IL2-H2.sfGFP-StrepTagII construct10. LadderThe square indicates the band of the IL2-H2C95S.sfGFP construct.

[0395] Figure 9 is a graph showing the titration on red blood cells of the IL2-H2C95S- sfGFPC48S-StrepTagII protein produced and purified from culture supernatants from either expiCHO cells or expi293F cells, as indicated. 0.16 pL, 0.63 pL, 2.5 pL, lOpL or 40 pL of purified protein from either expiCHO cells or expi293F cells transfected with a plasmid comprising the sequence of HTLV-2.RBD comprising a C95S substitution, with the signal peptide of IL-2, coupled to sfGFP C48S and a StrepTag II (IL2-H2C95S- sfGFPC48S-StrepTagII) were incubated with whole blood. GFP fluorescence of stained red blood cells was measured and compared to fluorescence of unstained red blood cells to calculate the signal / noise of each supernatant.

[0396] Figure 10 is a graph showing the binding activity of the IL2-H2C95S- sfGFPC48S-StrepTagII protein produced and purified from culture supernatant from expiCHO cells, on WT HCT116 cells or GLUT1 KO HCT116 cells, as indicated. 0.16 pL, 0.63 pL, 2.5 pL, lOpL or 40 pL of purified protein from expiCHO cells transfected with a plasmid comprising the sequence of HTLV-2.RBD comprising a C95S substitution, with the signal peptide of IL-2, coupled to sfGFP C48S and a StrepTag II (IL2-H2C95S-sfGFPC48S-StrepTagII) were incubated with WT HCT116 cells or GLUT1 KO HCT116 cells. GFP fluorescence of stained cells was measured andcompared to fluorescence of unstained cells to calculate the signal / noise of each supernatant.

[0397] Figure 11 is a graph showing the binding activity of the IL2-H2C95S- sfGFPC48S-StrepTagII protein produced and purified from culture supernatant from expi293F cells, on WT HCT116 cells or GLUT1 KO HCT116 cells, as indicated. 0.16 pL, 0.63 pL, 2.5 pL, lOpL or 40 pL of purified protein from expi293F cells transfected with a plasmid comprising the sequence of HTLV-2.RBD comprising a C95S substitution, with the signal peptide of IL-2, coupled to sfGFP C48S and a StrepTag II (IL2-H2C95S-sfGFPC48S-StrepTagII) were incubated with WT HCT116 cells or GLUT1 KO HCT116 cells. GFP fluorescence of stained cells was measured and compared to fluorescence of unstained cells to calculate the signal / noise of each supernatant.

[0398] Figure 12 is a histogram showing the binding activity of the IL2-H2C95S- sfGFPC48S-StrepTagII protein on GLUT1 transporter expressed at the surface of red blood cells, after storage at 4°C or 37°C for 1 to 14 days. 12 pg / mL of purified protein from expi293F were incubated with diluted whole blood. GFP fluorescence of stained red blood cells was measured and compared to fluorescence of unstained red blood cells to calculate the signal / noise of each supernatant.EXAMPLES

[0399] The present invention is further illustrated by the following examples.Table 1: Sequences of the constructs of the examples.“sfGFP C48S” denotes a superfolder green fluorescent protein (sfGFP) in which the cysteine residue originally present at position 48 has been substituted for a serine residue. To shorten the name, this sequence is also called “sfGFP48S”.To be more concise (especially when the name of a construct is long), H2C95S, H2C100S, H2C135S are also called H2S95, H2S100, H2S135 respectively.Example 1:Materials and MethodsCloning & plasmid preparation

[0400] The coding sequences tested to improve H2.GFP bioproduction were cloned into the pcDNA3.4 plasmid backbone by GenScript. Industrial grade DNA preparations (90% supercoiled DNA ±10%, with endotoxin level < 0.01 EU / pg) were also made by GenScript. Plasmids were validated by digestion and sequencing at GenScript facilities.Cell culture and production (USP)

[0401] The expression plasmids were transfected into expiCHO-S (Thermo Scientific A29133) and / or expi293F cell lines (Thermo Scientific A14635) to assess the production and secretion of H2.GFP. These cell lines were cultured in suspension at 37°C, 8% CO2, 80% humidity and agitation. The optimized media used for cell culture and protein production were purchased from Thermo Scientific (ExpiCHO expression medium A2910001 & Expi293 expression medium A1435101). The plasmids were transfected with expif ectamine according to the manufacturer’ s protocol, feed and / or enhancers were added the day after. The cultures were harvested 72 hours post transfection.

[0402] At the end of the culture, 2 centrifugation steps were done: a first one at 300g,5min, +4°C to get rid of the cells and a second one at 3000g, 20 min, +4°C to pellet cell debris. The supernatants obtained were filtered through 0.2 pm membrane and stored at -20°C until the quality control steps were completed. Purification of H2.GFP or its variants was triggered only if the protein passed first quality control steps: fluorescence measurement in supernatant and binding test on red blood cells (RBC).Fluorescence measurement in the harvested supernatants

[0403] This first quality control step allowed the comparison of the quantities of H2.GFP or its variants produced and secreted in the culture medium by measuring fluorescence intensity of GFP.

[0404] Fluorescence intensity is assumed to correlate with the quantity of H2.GFP or its variants in the culture medium. The fluorescence levels were measured with a plate reader (Spectramax iD5, Molecular Devices). For this purpose, 100 pL of the supernatants were pipetted in a 96-well black / clear plate (Coming 354649) for fluorescence reading from top (Xex 482 nm, Xem 522 nm). 100 pL of supernatant from non-transfected cells or cells transfected with a non-fluorescent protein were used as a negative control. Proper fluorescence intensity should be above 1.108to pass this quality control step.Binding test with harvested supernatants

[0405] This second quality control step was used to evaluate binding activity of H2.GFP or its variants on GLUT1 transporter at the surface of red blood cells, resulting from correct folding of the protein.

[0406] Aliquots of 500 pL supernatants versus FACS buffer alone (DPBSlx without Ca / Mg + 0.33 mg / mL BSA + 0.09% NaN3 + 1 mM EDTA) were incubated 20 min at 37°C with 50 pL of whole blood diluted 1 / 3000 to detect binding of H2.GFP or its variants on GLUT1 transporter by flow cytometry. Cells were washed with DPBSlx and pelleted by centrifugation two times before resuspension in FACS buffer for acquisition with FACSLyric (BD Biosciences).

[0407] Data analysis was performed using FlowJo software (BD Biosciences). GFP fluorescence (GeoMean) of stained and unstained red blood cells was used to calculateSignal / Noise of each supernatant. For each plasmid tested in production, the increase of the Signal / Noise value allowed selection of positive modifications during the optimization process of H2.GFP bioproduction. Proper Signal / Noise on red blood cells should be above 300 to pass this quality control step.Binding test on red blood cells

[0408] This test was used to evaluate the evolution overtime of the binding activity of IL2-H2C95S-sfGFPC48S-StrepTagII protein in purified form on GLUT1 transporter at the surface of red blood cells, resulting from the stability the protein.

[0409] Aliquots of purified IL2-H2C95S-sfGFPC48S-StrepTagII were prepared from the same construct batch at 14, 7, 3 and 1 day before the experiment, and stored at 4°C or 37°C. On the same day, whole blood was diluted at 1 / 3000 in DPBSlx, incubated for 20 min at 37°C with 12 pg / mL of the IL2-H2C95S-sfGFPC48S-StrepTagII construct from prepared aliquots, then washed with DPBSlx and pelleted by centrifugation two times before resuspension in FACS buffer for acquisition with FACSLyric (BD Biosciences).

[0410] Data analysis was performed using FlowJo software (BD Biosciences). GFP fluorescence (GeoMean) of stained and unstained red blood cells was used to calculate Signal / Noise of each supernatant.SDS-PAGE

[0411] Electrophoresis was used to assess the quantity of H2.GFP or its variants present in the supernatants and to detect aggregates by comparison of migration profiles in “native” conditions (no DTT, no heating) and in denaturing conditions (50 mM DTT + 10 min at 95°C before loading the samples on the gel).

[0412] Precast gradient gels from Bio-Rad (Mini-PROTEAN® TGX™ Precast Gels, 456-109x) were used for migration in TGS lx (Tris, Glycine, SDS) Bio-Rad (1610732, lOx stock). To stain proteins after migration, ready-to-use Quick Coomassie Stain solution (NeoBiotech, NB-45-00078-1L) was used.Purification protocol (DSP)

[0413] The optimized version of H2.GFP was purified on an Akta Pure chromatography system (Cytiva) using a 2-step protocol: affinity purification on StrepTrap XT 1 mL column (Cytiva 29401317) thanks to the StrepTagll tag present in the construct and a Size Exclusion Chromatography step on Superdex 200 Increase 10 / 300 GL column (Cytiva 28990944). Purity of the purified protein was visually assessed by SDS-PAGE after each step in the purification process. To determine protein concentration of the purified protein, optical density at 280 nm was measured with a spectrophotometer (Spectramax iD5, Molecular Devices) and calculation was made thanks to the absorptivity value determined on the ProtParam tool website using protein sequence. Final yield (amount of purified protein / volume of culture) was then calculated.Results

[0414] By screening different expression plasmids, the inventors identified modifications having a positive impact on H2.GFP construct production and binding activity.Signal sequence

[0415] First, the natural signal sequence of HTEV-2.RBD was replaced with the signal sequence of IE-2 (Figure 1 and Figure 2). Expi293F cells were transfected with a plasmid comprising the sequence of either (1) HTEV-2.RBD with its natural signal peptide and coupled to EGFP (H2.EGFP), or (2) HTEV-2.RBD with the signal peptide of IE-2 and coupled to EGFP (IL2-H2.EGFP). As shown on Figure 1, the IL2-H2.EGFP construct, comprising the signal sequence of IL-2, exhibited a higher GFP fluorescence intensity than the H2.EGFP construct, comprising the natural signal sequence. The inventors thus demonstrated that the IL-2 signal sequence increased the amount of IL2-H2.EGFP construct produced by the expi293F cells in the culture supernatants after 3 days of culture, as compared to the H2.EGFP construct.

[0416] Then, the binding capacity of the H2.EGFP and IL2-H2.EGFP constructs was evaluated using red blood cells which express the GLUT1 transporter. The supernatants from expi293F cells transfected with a plasmid comprising either H2.EGFP or IL2- H2.EGFP construct, were incubated with whole blood. As shown on Figure 2, the IL2-H2.EGFP construct exhibited an increased binding towards red blood cells, as compared to the H2.EGFP construct. This confirmed the proper protein folding of both constructs. In addition, the inventors thus showed that the higher amount of IL2-H2.EGFP construct present in the supernatant directly led to an increased fluorescence due to the binding of the construct on red blood cells.

[0417] The inventors thus demonstrated that the IL-2 signal peptide has a positive impact on the construct quantity.Label

[0418] In a second step, the effect of super folder GFP (sfGFP) was compared to EGFP (Figure 3 and Figure 4). ExpiCHO cells were transfected with a plasmid comprising the sequence of (1) HTLV-2.RBD with its natural signal peptide, and coupled to EGFP (H2.EGFP), (2) HTLV-2.RBD C-terminally truncated of 20 amino acids, with the signal peptide of IL-2, and coupled to EGFP (IL2-H2.A20aa.EGFP), or (3) HTLV-2.RBD, with the signal peptide of IL-2, and coupled to sfGFP (IL2-H2. sfGFP). As shown on Figure 3, the IL2-H2. sfGFP construct exhibited a much higher GFP fluorescence signal than the H2.EGFP construct and the IL2-H2.A20aa.EGFP construct. These results demonstrate that truncation of the hydrophobic C-terminal sequence of HTLV-2 did not increase the amount of IL2-H2.A20aa.EGFP construct produced by the cells and detected in the cell culture supernatants, as compared to the reference H2.EGFP construct. On the other hand, the inventors thus showed that replacement of EGFP by sfGFP strongly increased the amount of IL2-H2. sfGFP construct produced by the cells and detected in the cell culture supernatants after 3 days culture, as compared to the reference H2.EGFP construct.

[0419] These results were confirmed by evaluation of the binding capacity of these constructs on red blood cells (Figure 4). The supernatants from expiCHO cells transfected with a plasmid comprising either H2.EGFP, IL2-H2.A20aa.EGFP, or IL2- H2. sfGFP construct, were incubated with whole blood. As shown on Figure 4, the IL2- H2. sfGFP construct exhibited an increased binding towards red blood cells, as compared to the H2.EGFP, and IL2-H2.A20aa.EGFP constructs. The inventors thus showed that the higher amount of IL2-H2. sfGFP construct in the supernatant of expiCHO cells led to anincreased fluorescence due to the binding of the IL2-H2.sfGFP construct on red blood cells, as compared to both H2.EGFP and IL2-H2.A20aa.EGFP constructs.

[0420] The inventors thus demonstrated that use of sfGFP or sfGFPC48S instead of EGFP has a positive impact on the construct quality.Engineering of improved polypeptide sequences

[0421] The inventors then tried to engineer GLUT 1 -binding polypeptide exhibiting an optimized purity and yield. To this aim, they undertook to mutate various amino acids in the polypeptide sequences. The inventors in particular tested the impact of substituting the different cysteines by a serine in the HTLV-2.RBD (Figure 5). The natural HTLV- 2.RBD sequence contain nine cysteines.

[0422] ExpiCHO cells were transfected with a plasmid comprising the sequence of (1) HTLV-2.RBD with the signal peptide of IL-2, and coupled to sfGFP C48S (IL2- H2.sfGFPC48S), (2) HTLV-2.RBD comprising a C100S substitution, with the signal peptide of IL-2, and coupled to sfGFP C48S (IL2-H2C100S.sfGFPC48S), (3) HTLV- 2.RBD comprising a C95S substitution, with the signal peptide of IL-2, and coupled to sfGFP (IL2-H2C95S. sfGFP), (4) HTLV-2.RBD comprising a C135S substitution, with the signal peptide of IL-2, and coupled to sfGFP (IL2-H2C135S. sfGFP), (5) HTLV- 2.RBD with the signal peptide of IL-2, coupled to sfGFP and a HisTag (IL2- H2.sfGFP.HisTag), or (6) HTLV-2.RBD with the signal peptide of IL-2, coupled to sfGFP and a StrepTag II (IL2-H2. sfGFP. StrepTagll).

[0423] In most cases, substituting cysteines by serines completely impacted the structure and conformation of the HTLV-2.RBD protein, and therefore abrogated its production and binding activity. For example, as shown on Figure 5, the C100S substitution (IL2- H2C100S.sfGFPC48S construct) or the C135S substitution (IL2-H2C135S. sfGFP construct) dramatically decreased the GFP fluorescence signal as compared to the reference construct IL2-H2.sfGFPC48S. The inventors thus showed that mutating certain cysteines such as the C100 and C135 was deleterious for the HTLV-2.RBD polypeptide.

[0424] On the opposite, the C95S substitution (IL2-H2C95S.sfGFP construct)surprisingly preserved the amount of GFP fluorescence signal, as compared to the reference construct IL2-H2.sfGFPC48S (Figure 5). Besides, addition of a HisTag (IL2- H2.sfGFP.HisTag construct) or a StrepTag II (IL2-H2.sfGFP.StrepTagII construct) did not impact the GFP fluorescence signal as compared to the reference construct IL2- H2.sfGFPC48S (Figure 5). The inventors thus surprisingly showed that the C95S substitution in the HTLV-2.RBD did not impact the amount of construct produced in the cell supernatants, neither did the addition of a HisTag or a StrepTag II.

[0425] The inventors thus demonstrated that, although most cysteine residues of the HTLV-2.RBD are crucial for H2.sfGFP production, the cysteine residue at position 95 can surprisingly be mutated to serine without protein denaturation or loss.

[0426] These results were confirmed by evaluation of the binding capacity of these constructs on red blood cells (Figure 6). The supernatants from expiCHO cells transfected with a plasmid comprising either IL2-H2.sfGFPC48S, IL2- H2C100S.sfGFPC48S, IL2-H2C95S.sfGFP, IL2-H2C135S.sfGFP, IL2- H2.sfGFP. HisTag, or IL2-H2.sfGFP.StrepTagII construct were incubated with whole blood.

[0427] As shown on Figure 6, IL2-H2C100S.sfGFPC48S and IL2-H2C135S.sfGFP constructs did not exhibit any significant binding towards red blood cells, as compared to the reference IL2-H2.sfGFPC48S construct. On the opposite, IL2-H2C95S.sfGFP, IL2- H2.sfGFP. HisTag, and IL2-H2.sfGFP.StrepTagII constructs exhibited a similar binding activity than the reference IL2-H2.sfGFPC48S construct (Figure 6). The inventors thus showed that the C95S substitution in the HTLV-2.RBD did not impact the binding capacity of the construct, neither did the addition of a HisTag or a StrepTag II.

[0428] The inventors thus demonstrated that, although most cysteine residues of the HTLV-2.RBD are crucial for H2.sfGFP production, the cysteine residue at position 95 can surprisingly be mutated to serine without binding activity decrease or loss.

[0429] Figure 7 shows a SDS-PAGE gel in heated and reduced conditions, and Figure 8 shows a SDS-PAGE gel in non-heated and non-reduced conditions. Lane 2 of Figure 7 and lane 1 of Figure 8 shows rabbit IgG, which is a positive control of production, anddemonstrates a visible overexpression and expected sizes of the bands on both gels (heavy chain around 55 kDa and light chain around 25 kDa on Figure 7 and IgG around 120 kDa on Figure 8). IL2-H2C100S.sfGFPC48S construct was loaded on lane 5, while IL2- H2C135S.sfGFP construct was loaded on lane 7 on both Figure 7 and Figure 8.

[0430] There was no visible band in either reducing or non-reducing condition, demonstrating that mutations C100S and C135S are deleterious for protein expression. SV40i-IL2-H2.sfGFP construct was loaded on lane 3, IL2-H2.sfGFPC48S construct was loaded on lane 4, IL2-H2.sfGFP-HisTag construct was loaded on lane 8, and IL2- H2.sfGFP-StrepTagII construct was loaded on lane 9 on both Figure 7 and Figure 8. All of these constructs were expressed in expiCHO cells, however bands were visible only in reduced conditions (Figure 7) meaning that proteins were aggregated in non-reducing conditions (Figure 8). IL2-H2C95S.sfGFP construct was loaded on lane 6 on both Figure 7 and Figure 8. IL2-H2C95S.sfGFP was the only construct which appeared identically either in reducing (Figure 7) or non-reducing (Figure 8) conditions. The inventors thus showed that the C95S substitution greatly decreased protein aggregation / multimerization.

[0431] The inventors thus surprisingly demonstrated that the C95S substitution in HTLV-2.RBD has a positive impact on the construct quality and production (i.e., greatly reduced aggregation). Moreover, they showed that the purification tag does not impact the construct production or binding.

[0432] The IL2-H2C95S-sfGFPC48S-StrepTagII construct was created by combining the IL-2 signal peptide, the C95S substitution, the sfGFPC48S label and the StrepTagll purification tag.

[0433] Construct IL2-H2C95S-sfGFPC48S-StrepTagII was produced after transfection of expiCHO cells or expi293F cells. The construct was then purified from expiCHO and expi293F cell supernatants first by affinity purification thanks to the StrepTagll present in the construct, and then by Size Exclusion Chromatography. Final yield was 18 mg / mL from expiCHO cells and 77 mg / mL from expi293F cells as reported in Table 2 below.

[0434] Table 2: Summary of purification of the IL2-H2C95S-sfGFPC48S-StrepTagII construct produced in expiCHO cells and expi293F cells.

[0435] Binding activity of the IL2-H2C95S-sfGFPC48S-StrepTagII protein produced and purified from culture supernatants from either expiCHO cells or expi293F cells was evaluated on red blood cells (Figure 9). 0.16 pL, 0.63 pL, 2.5 pL, lOpL or 40 pL of supernatants from either expiCHO or expi293F cells transfected with a plasmid comprising the sequence of HTLV-2.RBD comprising a C95S substitution, with the signal peptide of IL-2, coupled to sfGFP C48S and a StrepTag II (IL2-H2C95S- sfGFPC48S-StrepTagII) were incubated with whole blood.

[0436] As shown on Figure 9, the binding of the IL2-H2C95S-sfGFPC48S-StrepTagII construct, produced by expiCHO or expi293F cells, at the surface of red blood cells increased in a dose-dependent manner. No difference between the IL2-H2C95S- sfGFPC48S-StrepTagII construct produced by expiCHO cells and the one produced by expi293F cells was observed.

[0437] The binding activity of the IL2-H2C95S-sfGFPC48S-StrepTagII construct produced by expiCHO cells (Figure 10) or expi293F cells (Figure 11) was then evaluated on WT HCT116 cells and GLUT1 KO HCT116 cells. 0.16 pL, 0.63 pL, 2.5 pL, lOpL or 40 pL of the purified protein from expiCHO cells supernatant (Figure 10) or expi293F cells supernatant (Figure 11) transfected with a plasmid comprising the sequence of HTLV-2.RBD comprising a C95S substitution, with the signal peptide of IL-2, coupled to sfGFP C48S and a StrepTag II (IL2-H2C95S-sfGFPC48S-StrepTagII) were incubated with WT HCT116 cells or GLUT1 KO HCT116 cells.

[0438] As shown on Figure 10 and Figure 11, the binding of the IL2-H2C95S- sfGFPC48S-StrepTagII construct at the surface of WT HCT116 cells increased in a dosedependent manner, while there was no binding at the surface of GLUT1-KO HCT116cells. No difference between the IL2-H2C95S-sfGFPC48S-StrepTagII construct produced by expiCHO cells and the one produced by expi293F cells was observed.

[0439] The stability of the IL2-H2C95S-sfGFPC48S-StrepTagII construct produced by expi293F cells (Figure 12) was then evaluated by comparing its binding capacity on red blood cells after storage at 4°C or 37 °C for 1 to 14 days.

[0440] As shown in Figure 12, the binding of the IL2-H2C95S-sfGFPC48S-StrepTagII construct to red blood cells was highly stable upon storage at 4°C for 1 to 14 days. Storage at 37°C during 14 days of the IL2-H2C95S-sfGFPC48S-StrepTagII construct only marginally affected its binding capacity, demonstrating a very good stability of the construct even when stored at 37°C.Conclusion

[0441] The inventors thus engineered an optimized GLUT 1 -binding polypeptide IL2- H2C95S-sfGFPC48S-StrepTagII, which surprisingly exhibited a much higher production yield, a better purity and excellent binding activity on GLUT1 positive cells.

Claims

CLAIMS1. A glucose transporter protein type 1 (GLUTl)-binding polypeptide comprising, or consisting of, an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 22 to 26, and variants or fragments thereof, wherein the sequences of said variants or fragments comprise a serine (S) residue at position corresponding to position 95 of SEQ ID NO: 1, 23 and 25, or to position 97 of SEQ ID NO: 22, 24 and 26.

2. The polypeptide according to claim 1, wherein said polypeptide is coupled or fused to a fluorescent label, such as a fluorescent protein, a fluorescent dye, or a quantum dot; a peptidic tag, such as a Fc fragment; a radioactive label; a paramagnetic metal; or a contrast agent.

3. The polypeptide according to claim 1 or 2, wherein said polypeptide is coupled or fused to a superfolder green fluorescent protein (sfGFP) or a Fc fragment.

4. The polypeptide according to any one of claims 1 to 3, wherein said polypeptide further comprise a signal peptide, preferably an IL-2 signal peptide.

5. A nucleic acid encoding the polypeptide according to any one of claims 1 to 4, or an expression vector comprising said nucleic acid, or a cell comprising said nucleic acid or said expression vector.

6. A diagnostic or pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 4, or the nucleic acid, the expression vector or the cell according to claim 5, and at least one pharmaceutically acceptable excipient.

7. An in vitro method of specifically detecting or / and measuring the level of GLUT1 in a sample, wherein said method comprises the steps of: a) contacting said sample with the polypeptide according to any one of claims 1 to 4, and b) detecting and / or measuring the binding of said polypeptide to GLUTE8. The polypeptide according to any one of claims 1 to 4, for use for specifically detecting or / and measuring the level of GLUT1 in vivo.

9. The in vitro method according to claim 7, or the polypeptide for use according to claim8, for diagnosing, prognosing or monitoring a GLUT 1 -associated disease in a subject, or for stratifying patients suffering from a GLUT 1 -associated disease.

10. The in vitro method according to claim 7, or the polypeptide for use according to claim 8, for diagnosing, prognosing or monitoring in a subject a cancer, or for stratifying patients suffering from a cancer, said cancer being selected from the group consisting of a brain cancer, a hypopharyngeal cancer, a breast cancer, a cervical cancer, an ovarian cancer, a lung cancer, a pancreatic cancer, a liver cancer, a colon cancer, squamous cell carcinoma, multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma (CTCL); a pre-malignant disorder, including myelodysplastic syndrome; an inflammatory disease, including psoriasis; an immune or auto-immune disease selected from the group consisting of autoimmune myocarditis, systemic lupus erythematous, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and Sjogren’s syndrome; or a disorder of the central nervous system, including GLUT1 deficiency syndrome.

11. The polypeptide according to any one of claims 1 to 4, or the nucleic acid, the expression vector or the cell according to claim 5, or the pharmaceutical composition according to claim 6, for use as a medicament.

12. The polypeptide according to any one of claims 1 to 4, or the nucleic acid, the expression vector or the cell according to claim 5, or the pharmaceutical composition according to claim 6, for use in the prevention or treatment of a disease selected from the group consisting of a cancer, an inflammatory disease, an immune or autoimmune disease, and a disorder of the central nervous system.

13. The polypeptide, nucleic acid, expression vector, cell, or pharmaceutical composition for use according to claim 12, wherein the disease is a cancer selected from the groupconsisting of a brain cancer, a hypopharyngeal cancer, a breast cancer, a cervical cancer, an ovarian cancer, a lung cancer, a pancreatic cancer, a liver cancer, a colon cancer, squamous cell carcinoma, multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma (CTCL); a pre-malignant disorder, including myelodysplastic syndrome; an inflammatory disease, including psoriasis; or an immune or auto-immune disease selected from the group consisting of autoimmune myocarditis, systemic lupus erythematous, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and Sjogren’s syndrome.

14. The coupled or fused polypeptide according to claim 2, for use as a probe for medical imagery.

15. A kit of parts comprising the polypeptide according to any one of claims 1 to 4 and at least one receptor-binding domain (RBD) derived from the soluble part of the glycoprotein of an enveloped virus selected from the group consisting of Amphotropic Murine Leukemia Retrovirus (amphoMLV), Feline Endogenous Virus (RD 114), Xenotropic Murine Leukemia Virus (NZB, Xeno), Gibbon Ape Leukemia Virus (GALV), Vesicular Stomatitis Virus (VSV), Feline Leukemia Virus C (FeLVC), Koala Retrovirus (KoRV), Porcine Endogeneous Retrovirus-A (Perv A), Porcine Endogeneous Retrovirus-B (Perv B), Bovine Leukemia Virus (BLV), Human Endogenous Retrovirus W (HERV-W), Baboon Endogenous Virus (BaEV), Spleen Necrosis Virus (SNV), Simian Retrovirus (SRV), Mason-Pfizer Monkey Virus (MPMV), Human Endogenous Retrovirus T (HERV-T), and Feline Endogenous Retrovirus ERV-DC14 (DC- 14).

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