Patent Application
20180002383
The invention relates to the use of the ubiquitous vertebrate glucose transporter GLUT1, or of fragments or sequences derived thereof, for the in vitro diagnosis of cancers, when used as a tumor marker, or for the screening of compounds useful for the preparation of drugs for the prevention or the treatment of pathologies linked to an infection of an individual with a primate T-lymphotropic virus (PTLV), or pathologies linked to an overexpression of GLUT1 on cell surfaces, or the in vitro detection of GLUT1 on cell surfaces. The invention also relates to pharmaceutical compositions containing GLUT1, or fragments or sequences derived thereof, and to their uses such as in the frame of the prevention or the treatment of pathologies linked to an infection of an individual with a PTLV.
The present application also relates to polypeptides derived from the soluble part of the glycoprotein of a Primate T-cell leukemia virus (PTLV), or fragments or variants thereof named receptor binding domain ligands (RBD) selected for their ability to bind specifically to the Glucose Transporter 1 (GLUT1).
The human T-cell leukemia virus (HTLV) is associated with leukemia and neurological syndromes. The role of viral envelopes in HTLV physiopathology is unclear and the envelope receptor, found in all vertebrate cell lines, remains unidentified.
HTLV envelope glycoproteins induce syncytium formation in vitro but their physiopathological effects are unclear. All vertebrate cell lines express functional HTLV envelope receptors, including cells resistant to HTLV envelope-mediated syncytium formation.
The Applicant found that expression of the HTLV receptor-binding domain decreased lactate production due to diminished glucose consumption whereas binding-defective envelope mutants did not alter glucose metabolism. Glucose starvation increased HTLV receptor expression, reminiscent of nutrient sensing responses. Accordingly, overexpression of Glucose Transporter 1 (GLUT1), the ubiquitous vertebrate glucose transporter, specifically increased HTLV envelope binding and GLUT1 colocalized with HTLV envelopes. Moreover, HTLV envelope binding was highest in human erythrocytes, where GLUT1 is abundantly expressed and is the sole glucose transporter isoform.
In the present invention, the Applicant identified specific fragments of PTLV envelope protein that bind to GLUT1.
The present application relates to an isolated polypeptide wherein said polypeptide is a soluble receptor binding domain (RBD) ligand derived from the soluble part of the glycoprotein of a primate T-lymphotropic virus binding to the Glucose Transporter 1 (GLUT1).
The present application also relates to an isolated polypeptide comprising human T-cell leukemia virus (HTLV) 2.RBD and comprising the amino acid sequence SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 43 or fragments or variants thereof.
The present application also relates to an isolated polypeptide comprising HTLV1.RBD and comprising the amino acid sequence SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 17 or SEQ ID NO: 19 or SEQ ID NO: 21 or fragments or variants thereof.
The present application also relates to an isolated polypeptide comprising HTLV4.RBD and comprising the amino acid sequence SEQ ID NO: 22 or SEQ ID NO: 23 or SEQ ID NO: 51 or fragments or variants thereof.
The present application also relates to an isolated polypeptide comprising HTLV3.RBD and comprising the amino acid sequence SEQ ID NO: 53 or fragments or variants thereof.
The present application also relates to an isolated polypeptide comprising simian T-cell leukemia virus (STLV) 1.RBD and comprising the amino acid sequence SEQ ID NO: 25 or fragments or variants thereof.
The present application also relates to an isolated polypeptide comprising STLV2.RBD and comprising the amino acid sequence SEQ ID NO: 27 or fragments or variants thereof.
The present application also relates to an isolated polypeptide comprising STLV3.RBD and comprising the amino acid sequence SEQ ID NO: 29 or SEQ ID NO: 55 or fragments or variants thereof.
In one embodiment, the isolated polypeptide further comprises a Tag or being fused to an antibody constant fragment or to a fluorescent protein.
The present application also relates to a composition comprising at least one isolated polypeptide wherein said polypeptide is a soluble receptor binding domain (RBD) ligand derived from the soluble part of the glycoprotein of a primate T-lymphotropic virus binding to the Glucose Transporter 1 (GLUT1).
In one embodiment, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient.
In another embodiment, the composition is a medicament.
The present application also relates to a method for diagnosing a GLUT1 related disease comprising:
In one embodiment, the GLUT1 related disease is GLUT1 deficiency syndrome. In another embodiment, the GLUT1 related disease is a cancer disease.
The present application also relates to a kit of parts comprising at least one isolated polypeptide wherein said polypeptide is a soluble receptor binding domain (RBD) ligand derived from the soluble part of the glycoprotein of a primate T-lymphotropic virus binding to the Glucose Transporter 1 (GLUT1).
In the present invention, the following terms have the following meanings:
The present application relates to peptides or polypeptides derived from the soluble part of the glycoprotein of a Primate T-cell leukemia virus (PTLV), or fragments or variants thereof, said polypeptides binding specifically to the ubiquitous vertebrate glucose transporter GLUT1 as set forth SEQ ID NO: 2. In one embodiment, said polypeptides are selected for their ability to bind specifically to the ubiquitous vertebrate glucose transporter GLUT1 as set forth SEQ ID NO: 2.
The present application thus relates to isolated polypeptides that are receptor binding domain ligands, wherein said receptor binding domain (RBD) ligands comprise or consist of a part or the totality of a receptor binding domain derived from the soluble part of a glycoprotein of an enveloped virus that binds to the glucose transporter GLUT1.
The term “derived from the soluble part of the glycoprotein of an enveloped virus” means that the ligand is a fragment or a part of a glycoprotein contained in the envelope of a virus and can be obtained, for example, by cloning.
The retroviruses of the invention encode an envelope (Env) glycoprotein present in mature retroviral virions. The Env protein is synthesized in the form of a propeptide, which is cleaved in Golgi apparatus by furine peptidase, resulting in two polypeptides: the transmembrane (TM) and the cell surface (SU) components. The SU domain contains two major subdomains: a domain of interaction with the TM domain and the RBD, the second being liable to interact with host cell membrane receptors.
An example of such envelope is represented in
In one embodiment, the isolated peptide of the invention comprises the SU domain of the glycoprotein envelope of a virus or a fragment of the SU domain, such as, for example, the RBD.
In another embodiment, the isolated peptide of the invention does not comprise the TM domain of the glycoprotein envelope of a virus. Therefore, in one embodiment of the invention, the isolated peptide of the invention is a soluble peptide, such as, for example, a soluble RBD. As used herein, the term “soluble peptide” refers to a peptide which is not anchored within a membrane, such as, for example, by a transmembrane domain.
The term “glycoprotein” is to be understood as meaning an envelope glycoprotein, a coat glycoprotein or a fusion glycoprotein”, wherein the term “glycoprotein” refers to a protein containing oligosaccharide chains covalently attached to polypeptide side-chains.
The term “binds to”, “binding to” means that the glycoprotein is liable to recognize the glucose transporter GLUT1 present on the surface of the cell. In one embodiment, a ligand that hinds to GLUT1 will thus form a complex with glucose transporter GLUT1, which complex may be detected by a method described herein. This complex can be detected if the ligand has been for example, but not limited to, covalently coupled with a detectable molecule such as an antibody constant fragment (Fc) or a fluorescent compound (e.g. Cyanine dye, Alexa dye, Quantum dye, etc). The complex can also be detected if the ligand has been tagged with different means well known to the person skilled in the art. For example, but without limitation, a tag used with the invention can be a tag selected from the group comprising or consisting of Hemaglutinin Tag, Poly Arginine Tag, Poly Histidine Tage, Myc Tag, Strep Tag, S-Tag, HAT Tag, 3× Flag Tag, Calmodulin-binding peptide Tag, SBP Tag, Chitin binding domain Tag, GST Tag, Maltose-Binding protein Tag, Fluorescent Protein Tag, T7 Tag, V5 Tag and Xpress Tag. The use of the ligand therefore allows on the one hand the identification and detection of the cell surface nutrient transporter depending on the ligand used, and on the other hand the quantification of the complex formed.
Methods for determining and/or quantifying binding of a RBD ligand on GLUT1 on the surface of a cell are well known by the skilled artisan. They include but are not limited to: immunoprecipitation assay, enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, immunofluorescence or image analysis, for example high content analysis, fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), and surface plasmon resonance (SPR).
In one embodiment, the receptor binding domain ligand containing part or the totality of the RBD can be fused to a fluorescent protein such as GFP, to an antibody constant fragment (such as, for example, Fc fragment from rabbit or from mouse), and/or chemically modified to add a fluorochrome, or a fluorescent compound (e.g. Cyanine dye, Alexa dye, Quantum dye, etc), and/or other tagged protein (e.g. Hemaglutinin Tag, Poly Arginine Tag, Poly Histidine Tage, Myc Tag, Strep Tag, S-Tag, HAT Tag, 3× Flag Tag, Calmodulin-binding peptide Tag, SBP Tag, Chitin binding domain Tag, GST Tag, Maltose-Binding protein Tag, T7 Tag, V5 Tag and Xpress Tag).
RBDs are found, in particular, in glycoproteins of the envelope of viruses, therefore, the receptor binding domain ligand contains the total RBD or a fragment or part of the RBD.
In one embodiment, said virus is selected from the group comprising retroviruses primate T cell leukemia virus (PTLV) selected from human T cell leukemia virus (HTLV) and simian T cell leukemia virus (STLV) which comprise or consist of HTLV-1, HTLV-2, HTLV-3, HTLV-4, STLV-2, STLV-3.
In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Human T Leukemia Virus-2, and is herein referred as HTLV2.RBD.
In one embodiment, said HTLV2.RBD comprises or consists of the amino acid sequence SEQ ID NO: 4 or fragments thereof.
In one embodiment, said fragments comprise or consist of amino acids 19 to 224 of SEQ ID NO: 4, or comprise or consist of amino acids 20 to 224 of SEQ ID NO: 4 or comprises or consists of amino acids 21 to 224 of SEQ ID NO: 4.
In one embodiment, said fragments comprise or consist of amino acids 1 to 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222 or 223 of SEQ ID NO: 4.
In another embodiment, said fragments comprise or consist of amino acids 19, 20 or 21 to 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 2.11, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222 or 223 of SEQ ID NO: 4.
In another embodiment, said fragments comprise or consist of SEQ ID NO: 4, encoded by the DNA sequence SEQ ID NO: 3.
In another embodiment, said fragments comprise or consist of amino acids 19, 20 or 21 to 178 of SEQ ID NO: 4.
In one embodiment, said HTLV2.RBD comprises or consists of the amino acid sequence SEQ ID NO: 5 or fragments thereof, encoded by the DNA sequence SEQ ID NO: 6.
In one embodiment, said HTLV2.RBD comprises or consists of the amino acid sequence SEQ ID NO: 7 or fragments thereof.
In one embodiment, said HTLV2.RBD comprises or consists of the amino acid sequence SEQ ID NO: 43 or fragments thereof.
In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Human T Leukemia. Virus-1, and is herein referred as HTLV LRBD. In one embodiment, said HTLV1.RBD comprises or consists of the amino acid sequence SEQ ID NO: 9 (encoded by SEQ ID NO: 8) or fragments thereof.
In one embodiment, said HTLV1.RBD comprises or consists of the amino acid sequence SEQ ID NO: 10 or fragments thereof.
In one embodiment, said fragments comprise or consist of amino acids 1 to 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 or 208 of SEQ ID NO: 10.
In one embodiment, said fragments comprise or consist of amino acids 21 to 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 or 208 of SEQ ID NO: 10.
In another embodiment, said fragments comprise or consist in SEQ ID NO: 11 (corresponding to amino acids 1 to 182 of SEQ ID NO: 10).
In one embodiment, said fragments comprise or consist of amino acids 21 to 182 of SEQ ID NO: 10.
In one embodiment, said HTLV1.RBD comprises or consists of the amino acid sequence SEQ ID NO: 11 or fragments thereof.
In one embodiment, said HTLVLRBD comprises or consists of the amino acid sequence SEQ ID NO: 13 (encoded by SEQ ID NO: 12) or fragments thereof.
In one embodiment, said HTLV1.RBD comprises or consists of the amino acid sequence SEQ ID NO: 15 (encoded by the DNA sequence SEQ ID NO: 14) or fragments thereof.
In one embodiment, said HTL1.RBD comprises or consists of the amino acid sequence SEQ ID NO: 17 (encoded by the DNA sequence SEQ ID NO: 16) or fragments thereof.
In one embodiment, said HTLV1.RBD comprises or consists of the amino acid sequence SEQ ID NO: 19 (encoded by the DNA sequence SEQ ID NO: 18) or fragments thereof.
In one embodiment, said HTLV1.RBD comprises or consists of the amino acid sequence SEQ ID NO: 21 (encoded by the DNA sequence SEQ ID NO: 20) or fragments thereof.
In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Human T Leukemia Virus-4, and is herein referred as HTLV4.RBD. In one embodiment, said HTLV4.RBD comprises or consists of the amino acid sequence SEQ ID NO: 51 or fragments thereof.
In one embodiment, said fragments comprise or consist of amino acids 1 to 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, or 734 of SEQ ID NO: 51.
In one embodiment, said fragments comprise or consist of amino acids 24 to 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, or 734 of SEQ ID NO: 51.
In another embodiment, said fragments comprise or consist of amino acids 22 to 237 of SEQ ID NO: 51, or comprise or consist of amino acids 23 to 237 of SEQ ID NO: 51, or comprise or consist of amino acids 24 to 237 of SEQ ID NO: 51.
In another embodiment, said fragments comprise or consist of amino acids 1 to 236 of SEQ ID NO: 51. In another embodiment, said fragments comprise or consist of amino acids 24 to 236 of SEQ ID NO: 51.
In another embodiment, said fragments comprise or consist of SEQ ID NO: 51, encoded by the DNA sequence SEQ ID NO: 50.
In one embodiment, said HTLV4.RBD comprises or consists of the amino acid sequence SEQ ID NO: 22 or fragments thereof.
In one embodiment, said fragments comprises or consists of amino acids 1 to 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203 or 204 of SEQ ID NO: 22.
In one embodiment, said fragments comprise or consist of amino acids 21 to 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203 or 204 of SEQ ID NO: 22.
In another embodiment, said fragments comprise or consist in SEQ ID NO: 23 (corresponding to amino acids 1 to 178 of SEQ ID NO: 22).
In another embodiment, said fragments comprise or consist in amino acids 21 to 178 of SEQ ID NO: 22.
In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Human T Leukemia Virus-3, and is herein referred as HTLV3.RBD. In one embodiment, said HTLV3.RBD comprises or consists of the amino acid sequence SEQ ID NO: 53 or fragments thereof.
In one embodiment, said fragments comprises or consists of amino acids 1 to 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, or 492 of SEQ ID NO: 53 or fragments thereof.
In one embodiment, said fragments comprise or consist of amino acids 23 to 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, or 492 of SEQ ID NO: 53 or fragments thereof.
In another embodiment, said fragments comprise or consist of amino acids 1 to 180 of SEQ ID NO: 53. In another embodiment, said fragments comprise or consist of amino acids 23 to 180 of SEQ ID NO: 53.
In another embodiment, said fragments comprise or consist of SEQ ID NO: 53, encoded by the DNA sequence SEQ ID NO: 52.
In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Simian 1′ Leukemia Virus-1, and is herein referred as STLV1.RBD. In one embodiment, said STLV 1.RBD comprises or consists of the amino acid sequence SEQ ID NO: 25 or fragments thereof.
In one embodiment, said fragments comprises or consists of amino acids 1 to 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, or 487 of SEQ ID NO: 25 or fragments thereof.
In one embodiment, said fragments comprises or consists of amino acids 21 to 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, or 487 of SEQ ID NO: 25 or fragments thereof.
In another embodiment, said fragments comprise or consist of amino acids 1 to 180 of SEQ ID NO: 25. In another embodiment, said fragments comprise or consist of amino acids 21 to 180 of SEQ ID NO: 25.
In another embodiment, said fragments comprise or consist of SEQ ID NO: 25, encoded by the DNA sequence SEQ ID NO: 24.
In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Simian T Leukemia Virus-2, and is herein referred as STLV2.RBD. In one embodiment, said STLV2.RBD comprises or consists of the amino acid sequence SEQ ID NO: 27 or fragments thereof.
In one embodiment, said fragments comprises or consists of amino acids 1 to 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, or 486 of SEQ ID NO: 27 or fragments thereof.
In one embodiment, said fragments comprises or consists of amino acids 21 to 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, or 486 of SEQ ID NO: 27 or fragments thereof.
In another embodiment, said fragments comprise or consist of amino acids 1 to 175 of SEQ ID NO: 27. In another embodiment, said fragments comprise or consist of amino acids 21 to 175 of SEQ ID NO: 27.
In another embodiment, said fragments comprise or consist of SEQ ID NO: 27, encoded by the DNA sequence SEQ ID NO: 26.
In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Simian T Leukemia Virus-2, and is herein referred as STLV3.RBD. In one embodiment, said STLV3.RBD comprises or consists of the amino acid sequence SEQ ID NO: 55 or fragments thereof.
In one embodiment, said fragments comprises or consists of amino acids 1 to 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 489, 490, or 491 of SEQ ID NO: 55 or fragments thereof.
In one embodiment, said fragments comprises or consists of amino acids 22 to 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 489, 490, or 491 of SEQ ID NO: 55 or fragments thereof.
In another embodiment, said fragments comprise or consist of amino acids 1 to 181 of SEQ ID NO: 55. In another embodiment, said fragments comprise or consist of amino acids 22 to 181 of SEQ ID NO: 55.
In another embodiment, said fragments comprise or consist of SEQ ID NO: 55, encoded by the DNA sequence SEQ ID NO: 54.
In one embodiment, the soluble receptor binding domain ligand is isolated from the glycoprotein of Simian T Leukemia Virus-3, and is herein referred as STLV3.RBD. In one embodiment, said STLV3.RBD comprises or consists of the amino acid sequence SEQ ID NO: 29 or fragments thereof.
In another embodiment, said fragments comprise or consist of SEQ ID NO: 29, encoded by the DNA sequence SEQ ID NO: 28.
In one embodiment, the isolated polypeptides of the invention are selected from the group comprising the sequences SEQ ID NO: 4, 5, 7, 9, 10, 11, 13, 15, 17, 19, 21, 22, 23, 25, 27, 29, 43, 51, 53, and 55 fragments and variants thereof. According to another embodiment, the isolated polypeptides of the invention are encoded by a DNA sequence selected from the group comprising the sequences SEQ ID NO: 3, 6, 8, 12, 14, 16, 18, 20, 24, 26, 28, 50, 52, and 54.
In one embodiment, the isolated polypeptide of the invention comprises or consists of a sequence presenting a sequence identity of at least 70% with one of the sequences SEQ ID NO: 4, 5, 7, 9, 10, 11, 13, 15, 17, 19, 21, 22, 23, 25, 27, 29, 43, 51, 53, and 55, preferably a sequence identity of at least about 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more with one of the sequences SEQ ID NO: 4, 5, 7, 9, 10, 11, 13, 15, 17, 19, 21, 22, 23, 25, 27, 29, 43, 51, 53, and 55.
In another embodiment, the isolated polypeptide of the invention is encoded by a DNA sequence presenting a sequence identity of at least 70% with one of the sequences 3, 6, 8, 12, 14, 16, 18, 20, 24, 26, 28, 50, 52 and 54 preferably a sequence identity of at least about 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more with one of the sequences SEQ ID NO: 3, 6, 8, 12, 14, 16, 18, 20, 24, 26 28, 50, 52 and 54.
In one embodiment, the isolated polypeptide of the invention is a variant of one of the polypeptide having the sequences SEQ ID NO: 4, 5, 7, 9, 10, 11, 13, 15, 17, 19, 21, 22, 23, 25, 27, 29, 43, 51, 53, and 55.
A polypeptide “variant” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of techniques well known in the art. Modifications may be made in the structure of polypeptides and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics.
When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant or portion of a ligand of the invention, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence. For example, certain amino acids may be substituted by other amino acids in a protein structure without appreciable loss of its ability to bind cell surface nutrient transporters. Since it is the binding capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with similar properties. It is thus contemplated that various changes may be made in the peptide sequences, or corresponding DNA sequences that encode said peptides without appreciable loss of their biological utility or activity, In many instances, a polypeptide variant will contain one or more conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted by another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of 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.
In one embodiment, the isolated polypeptide of the invention is a fusion protein comprising a part or the totality of a receptor binding domain fused to a detection tag, such as, for example, a Fc fragment or a fluorescent protein such as GFP. Examples of Fc fragments include, but are not limited to, rabbit Fc fragment (amino acid sequence SEQ ID NO: 30, encoded by SEQ ID NO: 31), and mouse Pc fragment (amino acid sequence SEQ ID NO: 32, encoded by SEQ ID NO: 33).
In one embodiment, the isolated polypeptide of the invention is HTLV2.RBD fused to a mouse Fe fragment (encoded by the DNA sequence SEQ ID NO: 34).
In one embodiment, the isolated polypeptide of the invention 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. In one embodiment, the sequence of the receptor binding domain ligand is fused in N-terminal to a peptide signal sequence allowing the secretion of said receptor binding domain ligand. Examples of peptide signal sequences include, but are not limited to, human IL-2 peptide signal (SEQ ID NO: 44), human albumin peptide signal (SEQ ID NO: 45), human chymotrypsinogen peptide signal (SEQ ID NO: 46), human trypsinogen-2 peptide signal (SEQ ID NO: 47), gaussia luciferase peptide signal (SEQ ID NO: 48), and mouse IgM peptide signal (SEQ ID NO: 49).
The present invention also relates to a composition comprising at least one of the isolated polypeptide of the present invention.
The present invention also relates to a pharmaceutical composition comprising at least one of the isolated polypeptide of the invention and at least one pharmaceutically acceptable excipient.
The term “pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
The present invention also relates to a medicament, comprising at least one of the isolated polypeptide of the invention.
Preferably, the composition, the pharmaceutical composition or the medicament of the invention comprises a therapeutically effective amount of at least one of the isolated polypeptide of the invention.
It will be understood that the total daily usage of at least one of the isolated polypeptide of the invention, composition, pharmaceutical composition and medicament of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific RBD employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific RBD employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the RBD at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from about 10 to about 10,000 mg per adult per day, preferably 100 to about 5,000, more preferably from about 200 to about 2,000 mg per adult per day. Preferably, the compositions contain 10, 50, 100, 250, 500, 1000 and 2,000 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 10 to about 10,000 mg of the active ingredient, preferably 100 to about 5,000, more preferably from about 200 to about 2,000 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.1 mg/kg to about 100 mg/kg of body weight per day, preferably from about 1 mg/kg to 40 mg/kg of body weight per day, more preferably from about 2 mg/kg to 20 mg/kg of body weight per day.
In one embodiment, the composition, pharmaceutical composition or medicament comprises sustained-release matrices, such as biodegradable polymers.
In the pharmaceutical compositions of the present invention, the polypeptide of the invention, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
Preferably, the composition, pharmaceutical composition or medicament contains vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
Solutions comprising at least one of the isolated polypeptide of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The polypeptide of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the polypeptides in the required amount in the appropriate solvent with one or several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the isolated polypeptide plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. The at least one isolated polypeptide of the invention may be formulated within a therapeutic mixture to comprise about 10 to 10,000 milligrams, preferably from about 100 to 4,000 milligrams, more preferably from about 200 to 2,000 per dose or so. Multiple doses can also be administered.
In addition, the at least one isolated polypeptide of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.
In one embodiment, the at least one isolated polypeptide of the invention is topically administered. Examples of formulations adapted to topical administration include, but are not limited to, drops, solutions or topical gels.
In a preferred embodiment, the at least one isolated polypeptide of the invention is systemically administered, such as, for example, orally administered, intranasally administered or injected (including, for example, intraperitoneal, intravenously or intramuscularly injected).
The invention relates to the use of the ubiquitous vertebrate glucose transporter GLUT1 represented by SEQ ID NO: 2, or of fragments or sequences derived thereof, for the in vitro diagnosis of GLUT1 related diseases, such as, for example, cancers (when used as a tumor marker) or GLUT1 deficiency syndrome, or for the screening of compounds useful for the preparation of drugs for the prevention or the treatment of pathologies linked to an infection of an individual with a primate T-lymphotropic virus (PTLV), or pathologies linked to an overexpression or down-expression of GLUT1 on cell surfaces, or the in vitro detection and/or quantification of GLUT1 on cell surfaces.
The present application relates to a method for diagnosing GLUT1 related diseases comprising:
Examples of sample include, but are not limited to, blood, plasma, serum, cerebrospinal fluid, saliva, lymph, ascetic fluid, cystic fluid, urine, bile, nipple exudate, synovial fluid, bronchoalveolar lavage fluid, sputum, amniotic fluid, chorionic villi, peritoneal fluid, pleural fluid, pericardial fluid, semen, saliva, sweat and alveolar macrophages.
In one embodiment, the sample collected comprises red blood cells, preferably peripheral red blood cells.
In one embodiment, the sample collected is a drop of blood.
Methods for determining a protein expression in a sample are well-known in the art. Examples of such methods include, but are not limited to, Multiplex methods (Luminex), western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry, sandwich ELISA, fluorescent-linked immunosorbent assay (FLISA), enzyme immunoassay (EIA), radioimmunoassay (RIA) and the like.
As used herein, the term “reference” broadly encompasses any suitable reference expression level which may be used as a basis for comparison with respect to the measured expression level.
In one embodiment, the reference value is a personalized reference, determined earlier in a sample provided by the same subject.
In one embodiment, a reference value can be relative to an expression value derived from population studies, including without limitation, such subjects having similar age range, subjects in the same or similar ethnic group, condition history and the like.
In one embodiment, the reference value is constructed using algorithms and other methods of statistical and structural classification.
In one embodiment of the invention, the reference value is derived from the measurement of the expression value in a control sample derived from one or more substantially healthy subjects, wherein substantially healthy subjects are not affected and/or diagnosed with the GLUT1 related disease.
In another embodiment of the invention, the reference value is derived from the measurement of the expression value of the GLUT1 expression in a reference sample, preferably a reference sample, derived from a reference population.
In one embodiment, the reference population comprises substantially healthy subjects, preferably at least 100, more preferably at least 250, more preferably at least 500 substantially healthy subjects.
In another embodiment, the reference population comprises subjects having a GLUT1 related disease, preferably at least 10, more preferably at least 20, more preferably at least 50, more preferably at least 100, more preferably at least 250, more preferably at least 500 subjects having a GLUT1 related disease.
Examples of GLUT1 related diseases include but are not limited to: Glucose transporter type 1 (GLUT1) deficiency syndrome (DS), and cancer (such as, for example, breast cancer, Adenocarcinoma, Squamous cell carcinoma, Hepatocellular carcinoma, Glioblastoma, Rhabdomyosarcoma, tumor cells, lung cancer, vulvar squamous cell carcinoma, gastric cancer, and esophageal cancer).
Glucose transporter type 1 (GLUT1) deficiency syndrome (DS) is characterized by an encephalopathy marked by childhood epilepsy that is refractory to treatment, deceleration of cranial growth leading to microcephaly, psychomotor retardation, spasticity, ataxia, dysarthria and other paroxysmal neurological phenomena often occurring before meals, Symptoms appear between the age of 1 and 4 months, following a normal birth and gestation. The prevalence is unknown.
Diagnosis is based on the clinical picture and biochemical analysis of the cerebrospinal fluid (CSF). In the majority of cases the disease is associated with de novo mutations in the SLC2A1 gene. GLUT1 DS is transmitted as an autosomal dominant trait and in these cases the affected parent presents with a mild form of the disease.
The biochemical signature of GLUT1 DS is the presence of low glucose level and low lactate concentration in the cerebrospinal fluid (CSF) absence of hypoglycemia.
Other diseases were originally given other names and are now recognized as variants of GLUT1 DS. These include, but are not limited to, mitochondrial diseases, intracranial infection and subarachnoid hemorrhage neuroblastoma because of the opsoclonus-like eye movement abnormalities in early infancy, infantile-onset metabolic encephalopathies, infantile-onset seizures, developmental delay and deceleration of head growth, including chronic hypoglycemic syndromes and disorders of amino acid and organic acid metabolism, Rett syndrome, Angelman syndrome, cerebral palsy.
In one embodiment of the invention, the subject is a male. In another embodiment of the invention, the subject is a female.
In one embodiment, the subject of the invention is a human embryo. In another embodiment, the subject of the invention is a human fetus. In another embodiment, the subject of the invention is a new born child.
In one embodiment of the invention, the subject is a young child. As used herein, the term “young child” refers to a child from 0; 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11 months old; 1 year old; 1 year and 3 months old; 1 year and 6 months old; 1 year and 9 months old; 2 years; 2 years and 3 months old; 2 years and 6 months old; 2 years and 9 months old; 3 years old.
In one embodiment of the invention, the subject is a child. The term “child” may refer to subjects aged from 0 to 12, preferably from 3 to 12. More generally, the term child refers to a subject which is not yet an adolescent.
In another embodiment of the invention, the subject is an adolescent. In one embodiment, the term “adolescent” may refer to subjects aged from about 12 to 17, but the skilled artisan will appreciate that the length of adolescence may vary from one individual to another.
In another embodiment, the subject is an adult. In one embodiment, the term “adult” may refer to subjects of more than 17 years old. More generally, the term adult refers to a subject which is no more an adolescent.
In another embodiment, the subject of the invention has risk of developing a cancer described here above. In another embodiment, the subject of the invention has a predisposition of developing a cancer described here above.
In another embodiment, the subject of the invention has risk of developing a GLUT1 DS. In another embodiment, the subject of the invention has a predisposition of developing a GLUT1 DS. In one embodiment, said subject presents a familial history of GLUT1 DS.
The expression “determining and/or detecting and/or quantifying the binding of a ligand, such as, for example, a receptor binding domain ligand, to GLUT1” means that when GLUT1 is present a complex is formed between GLUTI and the ligand. This complex can be detected if the ligand has been for example, but not limited to, covalently coupled with a detectable molecule such as an antibody constant fragment (Fc) or a fluorescent ligand (e.g. Cyanine dye, Alexa dye, Quantum dye, etc). The complex can also be detected if the ligand has been tagged with different means well known to the person skilled in the art. For example, but without limitation, a tag used with the invention can be a tag selected from the group comprising or consisting of fluorescent proteins such as GFP, Hemaglutinin Tag, Poly Arginine Tag, Poly Histidine Tage, Myc Tag, Strep Tag, S-Tag, HAT Tag, 3× Flag Tag, Calmodulin-binding peptide Tag, SBP Tag, Chitin binding domain Tag, GST Tag, Maltose-Binding protein Tag, Fluorescent Protein Tag, T7 Tag, V5 Tag and Xpress Tag.
In one embodiment, determining and/or detecting and/or quantifying binding is conducted by flow cytometry, immunofluorescence or image analysis, for example high content analysis.
For illustration purpose, the polypeptide of the invention can be selected for its ability to bind specifically to said GLUT1, or fragments of GLUT1, according to the following method using a EGFP-tagged. GLUT1-binding component derived from PTLV RBD (receptor binding domain) as an example of such polypeptide that is able to bind to GLUT1.
A EGFP-tagged Glut 1-binding component derived from PTLV RBD is applied onto live or fixed suspension or attached cells. After washes with appropriate buffer, cells are incubated for 30 min at RT, washed and analyzed or quantified as attached on an appropriate support on a fluorescent microscope or as individual cell suspension on a fluorescent analysis cell sorter (FACS). Alternatively, a non-fluorescent GLUT1-binding component derived from PTLV RBD is applied as described above and revealed with a secondary fluorochrome-tagged reagent such as a fluorochrome-tagged secondary antibody directed against the PTLV RBD or against a non fluorochrome tag attached to the said PTLV RBD component.
The invention relates more particularly to the use as defined above, of fragments of GLUT1 chosen among the followings:
These fragments of GLUT1 correspond to the predicted extracellular loops of human GLUT1 as described by Mueckler, M., and C. Makepeace. 1997. Identification of an amino acid residue that lies between the exofacial vestibule and exofacial substrate-binding site of the GLUT1 sugar permeation pathway. J Biol Chem. 272(48):30141-6.
The invention also relates to the use of an isolated polypeptide of the invention, for the preparation of drugs for the prevention or the treatment of pathologies linked to an overexpression of GLUT1 on cell surfaces, such as:
The invention relates more particularly to the use for the preparation of drugs for the prevention or the treatment of pathologies linked to an overexpression of GLUT1 on cell surfaces, of compounds chosen among the followings:
The invention relates more particularly to the use of polypeptides corresponding to the envelope proteins of PTLV, or fragments or sequences derived thereof, said polypeptides being selected for their ability to bind specifically to the ubiquitous vertebrate glucose transporter GLUT1 represented by SEQ ID NO: 2, or of nucleotide sequences encoding said polypeptides, for the preparation of drugs for the prevention or the treatment of pathologies linked to an overexpression of GLUT1 on cell surfaces, and the in vitro diagnosis of said pathologies.
The invention relates more particularly to the use as defined above, of polypeptides able to bind to at least one of the above mentioned fragments of GLUT1 corresponding to SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42.
The invention relates more particularly to the use as defined above, of polypeptides able to bind to at least the fragment of GLUT1 corresponding to SEQ ID NO: 42.
The invention relates more particularly to the use as defined above, of GLUT1 binding polypeptides mentioned above chosen among the followings:
the polypeptide corresponding to the envelope protein of a variant of HTLV-1, said polypeptide having the following sequence SEQ ID NO: 15,
the polypeptide corresponding to the envelope protein of a variant of HTLV-1, said polypeptide having the following sequence SEQ ID NO: 17,
the polypeptide corresponding to the envelope protein of a variant of HTLV-1, said polypeptide having the following sequence SEQ ID NO: 19,
the polypeptide corresponding to the envelope protein of a variant of HTLV-1, said polypeptide having the following sequence SEQ ID NO: 21,
the polypeptide corresponding to the envelope protein of a variant of HTLV-2, said polypeptide having the following sequence SEQ ID NO: 7,
The invention relates more particularly to the use of mentioned above of GLUT1 binding polypeptides as defined above, characterized in that the treated or detected pathologies are the followings:
The invention relates more particularly to the use of polypeptides selected for their ability to bind specifically to GLUT1 as mentioned above, and more particularly GLUT1 binding polypeptides as defined above, for the in vitro detection of GLUT1 on cell surfaces in the frame of processes for the in vitro diagnosis of pathologies linked to an overexpression or down-expression of GLUT1 on cell surfaces, such as pathologies defined above, said processes comprising the following steps:
The invention relates more particularly to the use of polypeptides as defined above for the in vitro diagnosis of cancers or of GLUT1 DS, characterized in that the polypeptides used are chosen among the polypeptides defined above selected for their ability to bind specifically to GLUT1.
The invention relates more particularly to the use as defined above, of GLUT1 binding polypeptides, or of nucleotide sequences encoding said polypeptides, for the preparation of drug vectors containing at their surface said polypeptides, said vectors being useful for targeting GLUT1 overexpressing cells for the prevention or the treatment of pathologies linked to an overexpression of GLUT1 on cell surfaces, said vectors containing molecules active against said pathologies, or containing genes in the frame of gene therapy of these pathologies.
The invention relates more particularly to the use as defined above, of GLUT1 binding polypeptides, or of nucleotide sequences encoding said polypeptides, for the preparation of drug vectors containing at their surface GLUT1 binding polypeptides, said vectors being useful for targeting GLUT1 overexpressing tumor cells, or cells involved in the inflammatory mechanism, or activated cells of the immune system, or cells of the central nervous system, for the prevention or the treatment of related pathologies as defined above.
The invention concerns more particularly the use of GLUT1 binding polypeptides, or of nucleotide sequences encoding said polypeptides, for the preparation of drug vectors as defined above, wherein the molecules active against the pathologies are antitumor molecules, or molecules against inflammatory conditions, immune or auto-immune diseases, or disorders of the central nervous system.
The invention also relates to the use of nucleotide sequences encoding polypeptides compounds selected for their ability to bind specifically to GLUT1 as defined above, such as nucleotide sequences encoding the polypeptides defined above, or fragments thereof, for the preparation, by substitution of one or several nucleotides of said nucleotide sequences, of mutant nucleotide sequences encoding corresponding mutant polypeptides unable to bind to GLUT1.
The invention also relates to the use of mutant polypeptides unable to bind to GLUT1 as defined above:
The invention relates more particularly to the use as defined above, of mutant polypeptides corresponding to the polypeptides defined above, wherein:
The invention also relates to the use of mutant nucleotide sequences encoding corresponding mutant polypeptides unable to bind to GLUT1 as defined above, for the preparation of transgenic mammal cells expressing said mutant polypeptides, said cells having a negative transdominant effect with regard to PTLV, thus preventing infection and dissemination of this latter in the organism.
The invention also relates to pharmaceutical compositions containing GLUT1 represented by SEQ ID NO: 2, or fragments or sequences derived thereof, said fragments or derived sequences being able to bind to the envelope proteins of the primate T-cell leukemia viruses (PTLV), in association with a pharmaceutically acceptable carrier.
The invention relates more particularly to pharmaceutical compositions containing mutant polypeptides corresponding to the polypeptides defined above, wherein:
The invention also relates to transgenic mammal cells expressing mutant polypeptides unable to bind to GLUT1 as defined above, said cells having a negative transdominant effect with regard to PTLV, thus preventing infection and dissemination of this latter in the organism.
The invention relates more particularly to pharmaceutical compositions containing transgenic mammal cells as defined above, in association with a pharmaceutically acceptable carrier.
The invention also relates to therapeutic vectors useful for targeting GLUT1 overexpressing cells in pathologies linked to an overexpression of GLUT1 on cell surfaces, such as defined above, said vectors containing at their surface GLUT1 binding polypeptides chosen among those defined above, and containing molecules active against said pathologies, as defined above, or containing genes in the frame of gene therapy.
The invention relates more particularly to pharmaceutical compositions containing therapeutic vectors as described above, in association with a pharmaceutically acceptable carrier.
The invention also relates to a method for the screening of compounds useful for:
The invention relates more particularly to a method for the screening of compounds useful for the prevention or the treatment of pathologies linked to an overexpression of GLUT1 on cell surfaces, and the in vitro diagnosis of said pathologies, comprising the steps described above:
The invention also relates to a method for the in vitro diagnosis pathologies linked to an overexpression of GLUT1 on cell surfaces, characterized in that it comprises:
The invention also relates to a method for the in vitro diagnosis pathologies linked to a down-expression of GLUT1 on cell surfaces (such as, for example, GLUT1 DS), characterized in that it comprises:
The invention relates more particularly to a method as defined above for the in vitro diagnosis of pathologies mentioned above.
The invention also relates to a kit for research use or for in vitro diagnosis, such as for example in vitro diagnosis of pathologies linked to an overexpression of GLUT1 on cell surfaces as described above, comprising at least one polypeptide of the invention as defined above, said at least one polypeptide being optionally labeled, and, if necessary reagents for the detection of the binding of said at least one polypeptide to GLUT1 initially present on cell surfaces in the biological sample.
The invention also relates to a kit for the in vitro diagnosis of pathologies linked to an down-expression of GLUT1 on cell surfaces as described above, comprising at least one polypeptide of the invention as defined above, said at least one polypeptide being optionally labeled, and, if necessary reagents for the detection of the binding of said at least one polypeptide to GLUT1 initially present on cell surfaces in the biological sample.
The present invention also relates to a kit of parts for detecting and/or determining the expression level of GLUT1 nutrient transporter on the cell surface, comprising at least one polypeptide of the invention.
The present invention also relates to a kit of parts for detecting and/or quantifying the binding of the RBD ligand of the present invention to GLUT1 nutrient transporter, comprising at least one polypeptide of the invention.
In one embodiment, the expression level of GLUT1 nutrient transporter is assessed at the protein level, and the kit of the invention may further comprises means for detecting the expression level of at least one normalization protein.
The present invention also relates to a kit of parts for diagnosing a disease related to GLUT1, comprising at least one polypeptide of the invention or a composition comprising at least one polypeptide of the invention.
In one embodiment, the kit comprises other elements, such as, for example, instructions for use; vials, containers or other storage vessels containing each of the unit doses; delivery devices such as needles, catheters, syringes, tubing and the like; and/or packaging suitable for safely and conveniently storing and/or transporting the kit. Preferably the instructions for use are a label or package insert, wherein the label or package insert indicates that the composition of the invention.
A “package insert” refers to instructions included in commercial packages of the compositions, that contains information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such compositions.
For the purposes herein, a “vial” refers to a container which holds the composition of the invention. The vial may be sealed by a stopper pierceable by a syringe. Generally, the vial is formed from a glass material.
The composition in the vial can be in various states including liquid, lyophilized, frozen etc. The fixed dosage composition is preferably stable as a liquid. Stability may be measured by any means known in the art, although turbidity is a preferred measure. Turbidity level of below about 10, 15, 20, or 30 Nephelometric Turbidity Unit (NTU) can generally be considered a stable fixed dosage composition. Turbidity measurements can be taken by incubating the fixed dosage compositions over time periods such as 0 h, 2 h, 4h, 6 h, 12 h, 18 h, 24 h, 36 h, 72 h, 7 days and 14 days at storage temperatures such as room temperature or 37° C. Preferably the fixed dosage composition is considered to be stable as a liquid when it is stored for 14 days at room temperature and exhibits a turbidity of less than about 15 NTU.
The invention is further illustrated with the detailed description hereafter of the determination of GLUT1 as a specific receptor for PTLV RBD.
The human T-cell leukemia virus (HTLV) type 1 and 2 are present in all areas of the world as endemic or sporadic infectious agents [Slattery, 1999]. The etiological role of HTLV-1 in adult T cell leukemia (ATL) and tropical spastic paraparesis/HTLV-associated myelopathy (TSP/HAM) has been well established [Poiesz, 1980; Yoshida, 1982; Gessain, 1985; Osame, 1986]. The apparently restricted tropism of HTLV to T lymphocytes in infected patients [Cavrois, 1996; Hanon, 2000] contrasts with the ability of the viral-encoded envelope glycoprotein (Env) to bind to and direct entry into all vertebrate cell types tested in vitro [Sutton, 1996; Trejo, 2000; Kim, 2003]. Retroviral infections depend on early interactions between Env and cellular receptors. Identification of cellular receptors and coreceptors for other retroviral envelopes have helped to elucidate certain aspects of retrovirus physiopathology as well as their transmission and spreading within organisms and populations [Berger, 1999; Clapham, 2001; Weiss, 2002]. However, no clear association between HTLV Env and HTLV-associated diseases has been established and the identity of the receptor(s) for HTLV-1 and HTLV-2 Env has remained elusive.
Numerous cell surface components have been shown to play a role in HTLV Env-mediated syncytia formation [Niyogi, 2001; Daenke, 1999; Hildreth, 1997]. Nevertheless, HTLV Env-dependent cell membrane fusion and syncytia formation appear to be distinct from receptor binding per se [Denesvre, 1996; Daenke, 2000; Kim, 2000; Kim, 2003]. The search for HTLV Env receptor has been hindered in part by its ubiquitous presence [Sutton, 1996; Trejo, 2000; Jassal, 2001; Kim, 2003]. Additionally, the induction of rampant syncytium formation in cell culture upon expression of HTLV Env [Hoshino, 1983; Nagy, 1983] has prevented efficient and persistent Env expression. Based on our observation that the HTLV Env amino terminal domain shares striking structural and functional homology with that of murine leukemia viruses (MLV), we defined HTLV Env receptor-binding domain (RBD) and derived HTLV Env-based tools that overcome the problem of syncytia formation [Kim, 2000; Kim, 2003]. We were thus able to follow specific interactions between the Env RBD and a primary HTLV receptor. Using these tools, we have previously shown that the HTLV receptor is expressed on the surface on T lymphocytes, the major HTLV reservoir in vivo, only following T cell receptor activation [Manel, 2003].
Here we describe striking metabolic alterations in cell cultures following expression of HTLV envelopes as well as HTLV receptor binding domains. These alterations are characterized by a defect in the acidification of the cell culture medium associated with a decreased lactate production and a decline in glucose consumption and uptake. These observations as well as the knowledge that Env receptors for the related MLV and most of the gammaretrovirus belong to the family of multiple-membrane spanning transporters [Overbaugh, 2001] prompted us to test ubiquitous lactate and glucose transport-associated molecules as receptors for HTLV Env. We show that the ubiquitous GLUT-1 glucose transporter, present in all vertebrates, is an essential and specific component of the receptor for HTLV. Moreover, interaction of GLUT-1 with the entire HTLV-1 and HTLV-2 envelopes as well as the truncated HTLV-1 and HTLV-2 RBDs alters glucose metabolism.
c, 2-deoxyglucose and fructose uptake following transfection of 293T with an irrelevant DNA (control), H1RBD, H2RBD or ARBD expression vectors. Control cells were also incubated with glucose transporter inhibitors cytochalasin and phloretin. Data are the means of triplicate measures and are representative of two to three independent experiments.
The present invention is further illustrated by the following examples.
Cell proliferation in standard culture media is accompanied by acidification of the milieu that translates into a color change from red to yellow tones in the presence of the phenol-red pH indicator. Upon transfection of either highly syncytial HTLV-1 and HTLV-2 envelopes, or a non-syncytial chimeric envelope that harbors the HTLV-1 RBD in a MLV Env backbone (H183FEnv), culture medium did not readily acidify, and harbored red tones for several days post-transfection (
Decrease of pH in cell culture is primarily due to extracellular accumulation of lactate [Warburg, 1956]. Lactate is the major byproduct of anaerobic glycolysis in vitro and its excretion is mediated by an H+/lactate symporter [Halestrap, 1999], We monitored lactate content in culture supernatants following transfection of various retroviral envelopes and RBD. Lactate accumulation was consistently 3-fold lower in H183FEnv- and HTLV RBD-transfected cells than in control- or MLV Env-transfected cells (
To examine whether a direct relationship exists between binding of the HTLV envelope receptor and diminished extracellular acidification and lactate accumulation, we attempted to generate HTLV-1 RBD (H1RBD) mutants with impaired receptor binding capacities. To this end, mutations resulting in single alanine substitutions were introduced at two different positions in H1RBD, D106 and Y114 which are highly conserved among primate T-lymphotropic viruses. Although both D106A and Y114A RBD mutants were expressed and secreted as efficiently as the wild-type H1RBD (
Extracellular lactate accumulates in cell cultures following its transport across cellular membranes by the MCT1 monocarboxylate transporter [Garcia, 1994]. Because HTLV and MLV share a common organization of the extracellular envelope [Kim, 2000]and the receptors for MLV Env are multispanning metabolite transporters [Overbaugh, 2001], we assessed whether the HTLV RBD bound to MCT1. Moreover, similar to our previous data concerning expression of the HTLV receptor on T cells [Manel, 2003], expression of MCT1 chaperone CD147 [Kirk, 2000] increases during T cell activation [Kasinrerk, 1992]. However, separate and combined overexpression of MCT1 and CD147 did not result in increased H1RBD binding, arguing against a role for these molecules as receptors for HTLV Env.
In addition to a decrease in extracellular lactate accumulation, expression of the HTLV RBD also led to decreased intracellular lactate content, indicative of metabolic alterations upstream of lactate transport. In cell cultures, lactate accumulation results from the degradation of glucose during anaerobic glycolysis. Therefore, we assessed whether the decreased accumulation of lactate observed upon expression of HTLV RBD was linked to glucose metabolism. We measured glucose consumption as normalized to cellular protein content. Glucose consumption of cells expressing an HTLV RBD within the context of the H183FEnv entire envelope or the H1RBD was significantly decreased as compared to control cells (
A simple model whereby the HTLV envelope inhibits glucose consumption via direct binding to a glucose transporter can explain the metabolic effects described above. Upon evaluation of the different glucose transporter candidates, GLUT-1 appears to be the only one encompassing all the known properties of the HTLV receptor. Indeed, GLUT-1 expression is increased upon glucose deprivation and is transports glucose in all vertebrate cells [Mueckler, 1985], while fructose is transported by GLUT-5. Furthermore, GLUT-1 is not expressed on resting primary T cells and its expression is induced upon T cell activation [Rathmell, 2000; Chakrabarti, 1994] with kinetics that are strikingly similar to what we have reported for the HTLV receptor [Manel, 2003]. Since human but not murine erythrocytes have been described to be the cells exhibiting the highest concentration of GLUT-1 [Mueckler, 1994], we evaluated HTLV receptor availability on freshly isolated red blood cells. Binding of H1RBD on human erythrocytes was strikingly efficient, reaching levels higher than those observed on any other tested cell type, whereas ARBD binding to erythrocytes was minimal (
In order to directly test the ability of HTLV envelopes to bind GLUT-1, we derived a tagged GLUT-1 expression vector and overexpressed this protein in HeLa cells. Both H1RBD and H2RBD binding was dramatically increased upon GLUT-1 overexpression (
Interaction of GLUT-1 with its ligand cytochalasin B inhibits glucose transport [Kasahara, 1977]. Since we showed that binding of HTLV envelopes to GLUT-1 inhibits glucose consumption and uptake, we tested whether cytochalasin B would abrogate HTLV RBD binding. Indeed, cytochalasin B treatment of Jurkat T cells dramatically inhibited binding of H1RBD, whereas binding of ARBD was not affected (
Viral receptor permits entry and thus infection. No cellular system currently exists that lacks GLUT-1 expression. Thus, we developed a system in which HTLV infection is specifically inhibited at the level of envelope-receptor interaction. In this system, over-expression of HTLV-2 RBD interferes with infecting incoming HTLV particles and specifically decreases HTLV titers by at least 2 logs, while no effect is detected on control A-MLV titers. To determine if GLUT-1 is an entry receptor for HTLV, we overexpressed GLUT-1, GLUT-3 or Pit2 in addition to the interfering H2RBD. While Pit2 and GLUT-3 had no effect on HTLV titers, GLUT-1 completely alleviated the interference to infection induced by H2RBD (
Here we show that HTLV-1 and -2 envelopes interact with GLUT-1 through their receptor binding domains. This interaction strongly inhibits glucose consumption and glucose uptake, leading to decreased lactate production and a block in extracellular milieu acidification. Mutations that specifically altered receptor binding of both HTLV-1 and 2 envelopes released the block in glucose consumption, indicative of a direct correlation between receptor binding determinants in the HTLV envelopes and glucose transport. Glucose starvation was rapidly followed by increased binding of HTLV envelopes, highlighting a nutrient-sensing negative feedback loop between glucose availability and cell surface HTLV receptor expression. Further evidence converged to identify GLUT-1 as the receptor, including increased binding of HTLV RBD upon overexpression of GLUT-1 but not GLUT-3, immunoprecipitation of GLUT-1 by H1RBD but not the receptor-binding mutant H1RBD Y114A, uppermost binding of HTLV RBD on human erythrocytes, where GLUT-1 is the major glucose transporter isoform, and no binding of HTLV RBD on human primary hepatocytes and murine erythrocytes, where GLUT-1 is minimally expressed. Finally, GLUT-1 could specifically alleviate interference to infection induced by HTLV RBD. GLUT-1 fits all other known properties of the HTLV receptor. Indeed, as previously demonstrated for the HTLV receptor [Manel, 2003], GLUT-1, but not the GLUT 2-4 isoforms, is not expressed on resting T lymphocytes [Chakrabarti, 1994; Korgun, 2002] and is induced upon immunological [Frauwirth, 2002; Yu, 2003] or pharmacological [Chakrabarti, 1994] activation. Moreover, GLUT-1 orthologues are highly conserved among vertebrates, but are highly divergent between vertebrates and insects [Escher, 1999].
GLUT-1 is thus a new member of the multimembrane spanning metabolite transporters that serve as receptors for retroviral envelopes. Interestingly, until now, all envelopes that recognize these receptors have been encoded by retroviruses that have a so-called simple genetic organization, such as MLV, feline leukemia viruses, porcine endogenous retrovirus and the gibbon ape leukemia virus [Overbaugh, 2001], whereas HTLV belongs to the so-called complex retroviruses which code for several additional regulatory proteins. However, we have shown that in contrast to the wide phylogenetic divergence of their genomic RNA, the envelopes of HTLV and MLV share a similar modular organization with some highly conserved amino acid motifs in their respective receptor binding domains [Kim, 2000].
Cell-to-cell contact appears to be required for HTLV transmission, and the cytoskeleton appears to play a major role in this process [Igakura, 2003]. Indeed, we observed that the HTLV receptor, despite pancellular expression, is specifically concentrated to mobile membrane regions and cell-to-cell contact areas. It should therefore be expected that the HTLV envelope receptor is associated to the cytoskeleton. Importantly, a cytoplasmic-binding partner of GLUT-1, GLUT1CBP, which encodes a PDZ domain, has been reported to link GLUT-1 to the cytoskeleton [Bunn, 1999]. It will therefore be interesting to evaluate the respective roles of the HTLV envelope, its cytoskeleton-associated cellular partners, such as GLUT-1, GLUT1CBP and their immediate interacting cell components.
Because expression of the HTLV receptor is induced upon glucose starvation, transmission of HTLV may be more efficient in cells that are locally starved for glucose, such as lymphocytes in lymph nodes [Yu, 2003]. Furthermore, the ability of circulating erythrocytes to dock HTLV, as shown here, might provide a means to distribute HTLV to such tissues.
The identification of GLUT-1 as a receptor for HTLV envelopes provides additional clues as to the ubiquitous in vitro expression of the receptor on cell lines and the paradoxical restriction of HTLV tropism to T lymphocytes in vivo. Rapid and dramatic metabolic alterations associated with the blockade of glucose consumption are likely to take place upon expression of the HTLV envelope in vivo, early after infection. Therefore, we propose that in vivo, HTLV infection initially spreads with a large tropism, however early after infection the vast majority of cells that are highly dependent on GLUT-1 activity are rapidly eliminated. In contrast, resting T lymphocytes that have an extremely low metabolic rate and as such are much less dependent on glucose uptake, can tolerate this effect and are therefore maintained in vivo. Furthermore, local imbalances in the access to glucose following HTLV infection may lead to specific physiological alterations [Akaoka, 2001]. In this regard, it will be of interest to study the potential relationship between HTLV-associated neuropathologies and the specific dependence of neurons on GLUT-1 mediated glucose consumption [Siegel, 1998].
Cell culture, 293T human embryonic kidney and HeLa cervical carcinoma cells were grown in Dulbecco's modified Eagle medium (DMEM) with high glucose (4.5 g/l) and Jurkat T-cells were grown in RPMI supplemented with 10% fetal bovine serum (FBS) at 37° C. in a 5% CO2-95% air atmosphere. For glucose starvation experiments, cells were grown in either glucose-free DMEM (Life Technologies) or glucose-free RPMI (Dutscher) with 10% dialyzed FBS (Life Technologies) and glucose (1 g/l) was supplemented when indicated.
Expression vectors. Full length envelope expression vectors for HTLV-1 (pCEL/2[Denesvre, 1995]) and Friend ecotropic MLV (pCEL/F [Denesvre, 1995]), have been previously described. For the HTLV-2 envelope, a fragment from pHTE2 [Rosenberg, 1998] encompassing the tax, rex and env genes and the 3′ LTR was inserted in the pCSI [Battini, 1999] vector (pCSIX.H2). Full length envelope expression vectors for amphotropic MLV (pCSI.A), or devoid of its R peptide (pCSI.AΔR), and H183FEnv that contains the N-terminal 183 amino acids of the HTLV-1 receptor-binding domain in the F-MLV envelope background, as well as truncated envelope expression vectors, derived from pCSI and encoding either of the first 215 residues of HTLV-1 SU (H1RBD), the first 178 residues of HTLV2-SU (H2RBD) or the first 397 residues of the amphotropic marine leukemia virus (MLV) SU (ARBD), fused to a C-terminal rabbit IgG Fc tag (rFc) or to EGFP (H2 All point mutations introduced in HTLV-1 and -2 RBD constructs were generated using the quickchange site-directed mutagenesis method and mutations were verified by sequencing. Human Glut-1 and Glut-3 cDNA were amplified by PCR from the pLib HeLa cDNA library (Clontech), and inserted into pCHIX, a modified version of the pCSI vector that contains a cassette comprising a factor Xa cleavage site, two copies of the hemagglutinin (HA) tag, and a histidine tag. The resulting construct (pCHIX.hGLUT1) encodes a GLUT-1 protein with a HA-His tag at the C-terminal end. GLUT-1 and GLUT-3 were also inserted in a modified pCSI vector containing a DsRed2 C-terminal tag. Similarly, human CD147 was amplified from 293T total RNA by RT-PCR and inserted into the pCHIX backbone in frame with the HA-His tag (pCHIX.hCD147).
Envelope expression and metabolic measurements. 293T cells were transfected with the various envelope expression vectors using a modified version of the calcium phosphate method. After an overnight transfection, cells were washed in phosphate-buffered saline (PBS) and fresh medium was added. Media were harvested at the indicated time points, filtered through a 0.45 μm pore-size filter, and lactate and glucose were measured with enzymatic diagnostic kits (Sigma). Values were normalized to cellular protein content using the Bradford assay (Sigma) after solubilization of cells in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1.0% Nonidet P-40, 0.5% deoxycholate) and clarification by centrifugation,
Assay of hexose uptake. 2-deoxy-D[1-3H]glucose, D[U-14C] fructose and 3-O-[14C]methyl-D-glucose were obtained from Amersham. Hexose uptake assay were adapted from Harrison et al. 1991). After transfection, approximatively 250,000 were seeded/well in 24-well plates. The next day, cells were washed two times in PBS, incubated in serum-free DMEM, washed one time in serum-free glucose-free DMEM, and incubated for 20′ in 500 μl serum-free glucose-free DMEM modulo inhibitors (20 μM cytochalasin B, 300 μM phloretin; SIGMA). Uptake was initiated by adding labeled hexoses to a final concentration of 0.1 mM (2 μCi/ml for 2-2-deoxy-D[1-3H]glucose and 0.2 μCi/ml for D[U-14C] fructose and 3-O-[14C]methyl-D-glucose) and cells were incubated for 5′ additional minutes. Cells were then resuspended in 500 μl cold serum-free glucose-free DMEM, wash one time in serum-free glucose-free DMEM, and solubilized in 400 μl of 0.1% SDS. 3 μl was used for Bradford normalization, while the rest was used for detection of either 3H or 14C by liquid scintillation in a Beckman counter.
Western blots. Culture media (10 μl) from 293T cells expressing wild type or mutant HTLV-1 RBDs, and/or GLUT-1 for GLUT-3 expression vector. were subjected to electrophoresis on SDS-15% acrylamide gels, transferred onto nitrocellulose (Protran; Schleicher & Schuell), blocked in PBS containing 5% powdered milk and 0.5% Tween 20, probed with either a 1:5000 dilution of horseradish peroxidase-conjugated anti-rabbit immunoglobulin or 1:2000 dilution of anti-HA 12CA5 (Roche) monoclonal antibody followed by a 1:5000 dilution of horseradish peroxidase-conjugated anti-mouse immunoglobulin, and visualized using an enhanced chemiluminescence kit (Amersham).
Binding assays. Binding assays were carried out as previously described [Manel, 2003]. Briefly, 5×105 cells (293T, HeLa, Jurkat or freshly isolated human erythrocytes) were incubated with 500 μl of H2RBD, H2RBD or ARBD supernatants for 30 min at 37° C., washed with PBA (1% BSA, 0.1% sodium azide in PBS), and incubated with a sheep anti-rabbit IgG antibody conjugated to fluorescein isothiocyanate (Sigma). When indicated, cytochalasin. B (20 μM; Sigma) was added to cells for 1 hour prior to binding analyses. Binding was analyzed on a FACSCalibur (Becton Dickinson) and data analysis was performed using CellQuest (Becton Dickinson) and WinMDI (Scripps) softwares.
Infections. 293T cells were transfected in 6-wells plate, and one day after transfection, medium was replaced by high glucose DMEM supplemented with fructose (5 g/l) and non-essential amino acids. The next day, infection was initiated by adding supernatants containing MLV particles pseudotyped with either HTLV-2 or A-MLV envelopes. The following day, fresh medium was added, and 24 hours later cells were fixed and stained for alkaline phosphatase activity and dark focus of infection were counted. Viral particles were obtained by transfecting 293T cells with pLAPSN, pGagPol and either pCSIX.H2 or pCSI.A, and harvesting the 0.45 μm-filtered supernatants 24 hours later.
| Number | Date | Country | Kind |
|---|---|---|---|
| 03291067.1 | May 2003 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14575132 | Dec 2014 | US |
| Child | 15698661 | US | |
| Parent | 10555289 | Jul 2006 | US |
| Child | 12547924 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12547924 | Aug 2009 | US |
| Child | 14575132 | US |
