[go: up one dir, main page]

MX2008008854A - Ligands that have binding specificity for vegf and/or egfr and methods of use therefor - Google Patents

Ligands that have binding specificity for vegf and/or egfr and methods of use therefor

Info

Publication number
MX2008008854A
MX2008008854A MX/A/2008/008854A MX2008008854A MX2008008854A MX 2008008854 A MX2008008854 A MX 2008008854A MX 2008008854 A MX2008008854 A MX 2008008854A MX 2008008854 A MX2008008854 A MX 2008008854A
Authority
MX
Mexico
Prior art keywords
seq
tar15
dom16
ligand
vegf
Prior art date
Application number
MX/A/2008/008854A
Other languages
Spanish (es)
Inventor
Holmes Steve
Sepp Armin
Ignatovich Olga
Beckmann Roland
M T De Wildt Rudolf
Original Assignee
Domantis Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Domantis Limited filed Critical Domantis Limited
Publication of MX2008008854A publication Critical patent/MX2008008854A/en

Links

Abstract

Disclosed are ligands that have binding specificity for vascular endothelial growth factor (VEGF), for epidermal growth factor receptor (EGFR), or for VEGF and EGFR. Also disclosed are methods of using these ligands. In particular, the use of these ligands for cancer therapy is described.

Description

LINKS THAT HAVE LIABILITY SPECIFICITY FOR VEGF AND / OR EGFR AND METHODS OF USE THEREOF Related Requests This application is a continuation of the Request No. 11 / 331,415, filed January 11, 2006, which is a continuation in part of the North American Application No. 11 / 098,758, filed on April 4, 2005, which is a continuation in part of the North American Application No. 10 / 925,366, filed on August 24, 2004. All teachings of the above Requests are incorporated herein by reference. Background of the Invention Cancer is a leading cause of mortality and morbidity. Methods to treat cancer include surgical intervention to remove tumors and chemotherapy. These methods can cure some patients successfully. However, even patients who appear to have been cured frequently suffer from a recurrence of cancer, which necessitates additional therapy. Chemotherapeutic agents are generally non-selective agents that are toxic to cells, such as proliferating cells. Accordingly, said agents can effectively kill cancer cells but also kill healthy cells, which produces various deleterious side effects. Certain cancer cells express or overexpress certain cellular components, such as cell surface proteins, or express different cellular components when compared to normal cells. The method for addressing the drawbacks of surgical and chemotherapeutic methods for cancer therapy and diagnosis involves targeting cancer cells, for example using antibodies or antibody fragments that bind to protein that are expressed or overexpressed in cancer cells. The number of said proteins have been identified. Among these proteins is the epidermal growth factor receptor (EGFR). EGFR is a member of the ErbB1 family and translates signals that lead to cell proliferation and survival, and to the elaboration of mortality, growth and angiogenic factors at the time of linkage. Epidermal growth factor (EGF) or transformation of alpha growth factor (TGF alpha). Consequently, EGFR has been shown to be involved in tumor growth, metastasis and angiogenesis. In addition, many cancers express EGFR, such as bladder cancer, ovarian cancer, colorectal cancer, breast cancer, lung cancer (e.g., non-small cell lung carcinoma), gastric cancer, pancreatic cancer, prostate cancer , cancer of the head and neck, kidney cancer and gallbladder cancer. ERBITUX (cetuximab; Imclone Systems Inc.) is a chimeric mouse / human antibody that binds to human EGFR that has been approved to treat certain cancers that express EGFR in combination with irinotecan. An important pathophysiological process that facilitates the formation, metastasis and recurrence of the tumor, is tumor angiogenesis. This process is transmitted by the elaboration of angiogenic factors through the tumor, such as vascular endothelial growth factor (VEGF), which induces the formation of blood vessels that supply nutrients to the tumor. Accordingly, another method for treating certain cancers is to inhibit tumor angiogenesis transmitted by VEGF, thus leaving the tumor without food. AVASTIN (Bevacizumab, Genetech, Inc.) is a humanized antibody that binds to human VEGF that has been approved to treat colorectal cancer. An antibody referred to as the 2C3 antibody (Access ATCC No. PTA 1595) is supported by binding to VEGF and inhibiting the binding of VEGF to the epidermal growth factor receptor 2. The targeting of EGFR or VEGF with currently available therapeutics is not effective in all patients, or for all types of cancer (eg, cancers that express EGFR). Therefore, there is a need for improved agents to treat cancer and other pathological conditions.
Brief Description of the Invention The present invention relates to ligands that have binding specificity for VEGF (eg, human VEGF), ligands that have binding specificity for EGFR (eg, human EGFR), and ligands that have binding specificity for VEGF and EGFR (e.g., human VEGF and human EGFR). For example, the ligand may comprise a polypeptide domain having a binding site with binding specificity for VEGF, a polypeptide domain having a binding site with binding specificity for EGFR, or comprises a polypeptide domain having a binding site with binding specificity for VEGF and a polypeptide domain having a binding site with EGFR binding specificity. In one aspect, the present invention relates to a ligand having binding specificity for VEGF and for EGFR. Said ligands comprise at least a portion of protein having a binding site with binding specificity for VEGF and at least a portion of protein having a binding site with binding specificity for EGFR. The protein portion having a binding site with binding specificity for VEGF and the protein portion having a binding site with binding specificity for EGFR can each be any suitable binding moiety. The protein portions can be a peptide portion, or polypeptide portion or protein portion. For example, portions of the protein can be provided through an antibody fragment having a binding site with binding specificity for VEGF or EGFR, such as a single immunoglobulin variable domain having binding specificity for VEGF or EGFR. The ligand may comprise a portion of the protein having a binding site with binding specificity for VEGF that competes to bind VEGF with AVASTIN (bevacizumab, Genentech, Inc.) and / or 2C3 antibody (ATCC Access No. PTA 1595). The ligand may comprise a portion of a protein having a binding site with binding specificity for EGFR that competes to bind EGFR with ERBITUX (cetuximab; Imclone Systems, Inc.). In some embodiments, the ligand comprises a portion of a protein having a binding site with binding specificity for VEGF that competes to bind to VEGF with bevacizumab and / or antibody 2C3 (Access ATCC No. PTA 1595), and further comprises a protein portion that has a binding site with binding specificity for EGFR that competes to bind EGFR with cetuximab. In some embodiments, the ligand comprises a portion of a protein having a binding site with binding specificity for VEGF (eg, a single immunoglobulin variable domain) competing to bind to VEGF with the anti-VEGF domain antibody ( dAb) selected from the group consisting of TAR15-1 (SEQ ID NO: 100), TAR15-3 (SEQ ID NO: 101), TAR15-4 (SEQ ID NO: 102), TAR15-9 (SEQ ID NO: 103 ), TAR15-10 (SEQ ID NO: 104), TAR15-11 (SEQ ID NO: 105), TAR15-12 (SEQ ID NO: 106), TAR15-13 (SEQ ID NO: 107), TAR15-14 ( SEQ ID NO.108), TAR15-15 (SEQ ID NO: 109), TAR15-16 (SEQ ID NO: 110), TAR15-17 (SEQ ID NO: 111), TAR15-18 (SEQ ID NO: 112) , TAR15-19 (SEQ ID NO: 113), TAR15-20 (SEQ ID NO: 114), TAR 15-22 (SEQ ID NO: 115), TAR15-5 (SEQ ID NO: 116), TAR15-6 ( SEQ ID NO: 117), TAR15-7 (SEQ ID NO: 118), TAR15-8 (SEQ ID NO: 119), TAR15-23 (SEQ ID NO: 120), TAR15-24 (SEQ ID NO: 121) , TAR15-25 (SEQ ID NO: 122), TAR15-26 (SEQ ID NO: 123), TAR15-27 (SEQ ID NO: 124), TAR15-29 (SEQ ID NO: 125), TA R15-30 (SEQ ID NO: 126), TAR15-6-500 (SEQ ID NO: 127), TAR15-6-501 (SEQ ID NO: 128), TAR15-6-502 (SEQ ID NO: 129), TAR15-6-503 (SEQ ID NO: 130), TAR15-6-504 (SEQ ID NO: 131), TAR15-6-505 (SEQ ID NO: 132), TAR15-6-506 (SEQ ID NO: 133 ), TAR15-6-507 (SEQ ID NO: 134), TAR15-6-508 (SEQ ID NO: 135), TAR15-6-509 (SEQ ID NO: 136), TAR15-6-510 (SEQ ID NO. : 137), TAR15-8-500 (SEQ ID NO: 138), TAR15-8-501 (SEQ ID NO: 139), TAR15-8-502 (SEQ ID NO: 140), TAR15-8-503 (SEQ ID NO: 141), TAR15-8-505 (SEQ ID NO: 142), TAR15-8-506 (SEQ ID NO: 143). TAR15-8-507 (SEQ ID NO: 144), TAR15-8-508 (SEQ ID NO: 145), TAR15-8-509 (SEQ ID NO: 146), TAR15-8-510 (SEQ ID NO: 147) ), TAR15-8-511 (SEQ ID NO: 148), TAR15-26-500 (SEQ ID NO: 149), TAR15-26-501 (SEQ ID NO: 150), TAR15-26-502 (SEQ ID NO. : 151), TAR15-26-503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153), TAR15-26-505 (SEQ ID NO: 154), TAR15-26-506 (SEQ ID NO: 155), TAR15-26-507 (SEQ ID NO: 156), TAR15-26-508 (SEQ ID NO: 157), TAR15-26-509 (SEQ ID NO: 158), TAR15-26-510 (SEQ ID NO: 159), TAR15-26-511 (SEQ ID NO: 160), TAR15-26-512 (SEQ ID NO: 161), TAR15-26-513 (SEQ ID NO: 162), TAR15-26 -514 (SEQ ID NO: 163), TAR15-26-515 (SEQ ID NO: 164), TAR15-26-516 (SEQ ID NO: 165), TAR15-26-517 (SEQ ID NO: 166), TAR15 -26- 518 (SEQ ID NO: 167), TAR15-26-519 (SEQ ID NO: 168), TAR15-26-520 (SEQ ID NO: 169), TAR15-26-521 (SEQ ID NO: 170) , TAR15-26-522 (SEQ ID NO: 171), TAR15-26-523 (SEQ ID NO: 172), TAR15-26-524 (SEQ ID NO: 173), TAR15-26-525 (SEQ ID NO: 174), TAR15-26-526 (SEQ ID NO: 175), TAR15-26-527 (SEQ ID NO: 17 6), TAR15-26-528 (SEQ ID NO: 177), TAR15-26-529 (SEQ ID NO: 178), TAR15-26-530 (SEQ ID NO: 179), TAR15-26-531 (SEQ ID NO: 180), TAR15-26-532 (SEQ ID NO: 181), TAR15-26-533 (SEQ ID NO: 182), TAR15-26-534 (SEQ ID NO: 183), TAR15-26-535 ( SEQ ID NO: 184), TAR15-26-536 (SEQ ID NO: 185), TAR15-26-537 (SEQ ID NO: 186), TAR15-26-538 (SEQ ID NO: 187), TAR15-26- 539 (SEQ ID NO: 188), TAR15-26-540 (SEQ ID NO: 189), TAR15-26-541 (SEQ ID NO: 190), TAR15-26-542 (SEQ ID NO: 191), TAR15- 26-543 (SEQ ID NO: 192), TAR15-26-544 (SEQ ID NO: 193), TAR15-26-545 (SEQ ID NO: 194), TAR15-26-546 (SEQ ID NO: 195), TAR15-26-547 (SEQ ID NO: 196), TAR15-26-548 (SEQ ID NO: 197), TAR15-26-549 (SEQ ID NO: 198), TAR15-26-550 (SEQ ID NO: 539 ), and TAR15-26-551 (SEQ ID NO: 540). In addition, or in other embodiments, the ligand may comprise a portion of a protein having a binding site with binding specificity for EGFR (eg, a single immunoglobulin variable domain) that competes to bind EGFR with an anti-domain antibody. -EGFR (dAb) selected from the group consisting of DOM16-17 (SEQ ID NO: 325 DOM16-18 (SEQ ID NO: 326 DOM16-19 (SEQ ID NO: 327 DOM16-20 (SEQ ID NO: 328 DOM16-21 (SEQ ID NO: 329 DOM16-22 (SEQ ID NO: 330 DOM16- 23 (SEQ ID NO: 331 DOM16-24 (SEQ ID NO: 332 DOM16-25 (SEQ ID NO: 333 DOM16-26 (SEQ ID NO: 334 DOM16-27 (SEQ ID NO: 335 DOM16-28 (SEQ ID NO. : 336 DOM16-29 (SEQ ID NO: 337 DOM16-30 (SEQ ID NO: 338 DOM16-31 (SEQ ID NO: 339 DOM16-32 (SEQ ID NO: 340 DOM16-33 (SEQ ID NO: 341 DOM16-35 (SEQ ID NO: 342 DOM16-37 (SEQ ID NO: 343 DOM16-38 (SEQ ID NO: 344 DOM16-39 (SEQ ID NO: 345 DOM16-40 (SEQ ID NO: 346 DOM16-41 (SEQ ID NO: 347 DOM16-42 (SEQ ID NO: 348 DOM16-43 (SEQ ID NO: 349 DOM16-44 (SEQ ID NO: 350 DOM16-45 (SEQ ID NO: 351 DOM16-46 (SEQ ID NO: 352 DOM16-47 ( SEQ ID NO: 353 DOM16-48 (SEQ ID NO: 354 DOM16-49 SEQ ID NO: 355 DOM16-50 (SEQ ID NO: 356 DOM16-59 SEQ ID NO: 357 DOM16-60 (SEQ ID NO: 358 DOM16- 61 SEQ ID NO: 359 DOM16-62 (SEQ ID NO: 360 DOM16-63 SEQ ID NO: 361 DOM16-64 (SEQ ID NO: 362 DOM16-65 SEQ ID NO: 363 DOM16-66 (SEQ ID NO: 364 DOM16 -67 SEQ ID NO: 365 DOM16-68 (SEQ ID NO: 366 DOM16-69 SEQ I D NO: 367 DOM16-70 (SEQ ID NO: 368 DOM16-71 SEQ ID NO: 369 DOM16-72 (SEQ ID NO: 370 DOM16-73 SEQ ID NO: 371 DOM16-74 (SEQ ID NO: 372 DOM16-75 SEQ ID NO: 373 DOM16-76 (SEQ ID NO: 374 DOM16-77 SEQ ID NO: 375 DOM16-78 (SEQ ID NO: 376 DOM16-79 SEQ ID NO: 377 DOM16-80 (SEQ ID NO: 378 DOM16- 81 SEQ ID NO: 379 DOM16-82 (SEQ ID NO: 380 DOM16-83 SEQ ID NO: 381 DOM16-84 (SEQ ID NO: 382 DOM16-85 SEQ ID NO: 383 DOM16-87 (SEQ ID NO: 384 DOM16 -88 SEQ ID NO: 385 DOM16-89 (SEQ ID NO: 386 DOM16-90 SEQ ID NO: 387 DOM16-91 (SEQ ID NO: 388 DOM16-92 SEQ ID NO: 389 DOM16-94 (SEQ ID NO: 390 DOM16-95 SEQ ID NO: 391 DOM16-96 (SEQ ID NO: 392 DOM16-97 SEQ ID NO: 393 DOM16-98 (SEQ ID NO: 394 DOM16-99 SEQ ID NO: 395 DOM16-100 (SEQ ID NO: 396 DOM16-101 SEQ ID NO: 397 DOM16-102 (SEQ ID NO: 398 DOM16-103 SEQ ID NO: 399 DOM16-104 (SEQ ID NO: 400 DOM16-105 SEQ ID NO: 401 DOM16-106 (SEQ ID NO. : 402 DOM16-107 SEQ ID NO: 403), DOM16-108 (SEQ ID NO: 404), DOM16-109 (SEQ ID NO: 405), DOM16-110 (SEQ ID NO: 406), DOM16-111 (SEQ. ID NO: 407), DOM16-112 (SEQ ID NO: 408), DOM16-113 (SEQ ID NO: 409), DOM16-114 (SEQ ID NO: 410), DOM16-115 (SEQ ID NO: 411), DOM16-116 (SEQ ID NO: 412), DOM16-117 (SEQ ID NO: 413), DOM16-118 (SEQ ID NO: 414), DOM16-119 (SEQ ID NO: 415), DOM16-39-6 (SEQ ID NO: 416), DOM16-39-8 (SEQ ID NO: 417), DOM16-39-34 (SEQ ID NO: 418), DOM16-39-48 (SEQ ID NO: 419), DOM16-39-87 (SEQ ID NO: 420), DOM 16-39-90 (SEQ. ID NO: 421), DOM 16-39-96 (SEQ ID NO: 422), DOM16-39-100 (SEQ ID NO: 423), DOM16-39-101 (SEQ ID NO: 424), DOM16-39- 102 (SEQ ID NO: 425), DOM16-39-103 (SEQ ID NO: 426), DOM16-39-104 (SEQ ID NO: 427), DOM16-39-105 (SEQ ID NO: 428), DOM16- 39-106 (SEQ ID NO: 429), DOM 16-39-107 (SEQ ID NO: 430), DOM16-39-108 (SEQ ID NO: 431), DOM16-39-109 (SEQ ID NO: 432) , DOM16-39-110 (SEQ ID NO: 433), DOM16-39-111 (SEQ ID NO: 434), DOM16-39-112 (SEQ ID NO: 435), DOM16-39-113 (SEQ ID NO: 436), DOM16-39-114 (SEQ ID NO: 437), DOM16-39-115 (SEQ IDNO: 438), DOM16-39-116 (SEQ ID NO: 439), DOM16-39-117 (SEQ ID NO. : 440), DOM16-39-200 (SEQ ID NO: 441), DOM16-39-201 (SEQ ID NO: 442), DOM16-3 9-202 (SEQ ID NO: 443), DOM16-39-203 (SEQ ID NO: 444), DOM16-39-204 (SEQ ID NO: 445), DOM16-39-205 (SEQ ID NO: 446), DOM16-39-206 (SEQ ID NO: 447), DOM16-39-207 (SEQ ID NO: 448), DOM16-39-209 (SEQ ID NO: 449), DOM16-52 (SEQ ID NO: 450), NB1 (SEQ ID NO: 451), NB2 (SEQ ID NO: 452), NB3 (SEQ ID NO: 453), NB4 (SEQ ID NO: 454), NB5 (SEQ ID NO: 455), NB6 (SEQ ID NO : 456), NB7 (SEQ ID NO: 457), NB8 (SEQ ID NO: 458), NB9 (SEQ ID NO: 459), NB10 (SEQ ID NO: 460), NB11 (SEQ ID NO: 461), NB12 (SEQ ID NO: 462), NB13 (SEQ ID NO: 463), NB14 (SEQ ID NO: 464), NB15 (SEQ ID NO: 465), NB16 (SEQ ID NO: 466), NB17 (SEQ ID NO: 467), NB18 (SEQ ID NO: 468), NB19 (SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 (SEQ ID NO: 471), and NB22 (SEQ ID NO: 472). In particular embodiments, the ligand has binding specificity for VEGF and for EGFR and comprises a portion of protein having a binding site with binding specificity for VEGF that competes to bind VEGF with the anti-VEGF domain antibody (dAb) selected from the group consisting of TAR15-6 (SEQ ID NO: 117), TAR15-8 (SEQ ID NO: 119), and TAR15-26 (SEQ ID NO: 123), and further comprises a protein portion having a binding site with binding specificity for EGFR that competes to bind EGFR with an anti-EGFR domain antibody (dAb) selected from the group consisting of DOM16-39 (SEQ ID NO.345), DOM16-39-87 (SEQ ID NO: 420), DOM16-39-100 (SEQ ID NO: 423), DOM16-39-107 (SEQ ID NO: 430), DOM16-39-109 (SEQ ID NO: 432), DOM16-39-115 (SEQ ID NO: 438), and DOM16-39-200 (SEQ. ID NO: 441). In more particular embodiments, the ligand has binding specificity for VEGF and for EGFR and comprises at least one variable domain of simple immunoglobulin with binding specificity for VEGF and at least one variable domain of single immunoglobulin with binding specificity for EGFR, wherein a simple immunoglobulin variable domain with binding specificity for VEGF competes to bind VEGF with an anti-VEGF (dAb) domain antibody selected from the group consisting of TAR15-1 (SEQ ID NO: 100 TAR15-3 (SEQ ID NO: 101), TAR15-4 (SEQ ID NO: 102 TAR15-9 (SEQ ID NO: 103), TAR15-10 (SEQ ID NO: 104 TAR15-11 (SEQ ID NO : 105), TAR15-12 (SEQ ID NO: 106 TAR15-13 (SEQ ID NO: 107), TAR15-14 (SEQ ID NO: 108 TAR15-15 (SEQ ID NO: 109), TAR15-16 (SEQ ID NO: 110 TAR15-17 (SEQ ID NO: 111), TAR15-18 (SEQ ID NO: 112 TAR15-19 (SEQ ID NO: 113), TAR15-20 (SEQ ID NO: 114 TAR 15-22 (SEQ ID NO: 115), TAR15-5 (SEQ ID NO: 116 TAR15-6 (SEQ ID NO: 117), TAR15-7 (SEQ ID NO: 118 TAR15-8 (SEQ ID NO: 119), TAR15-23 (SEQ ID NO: 120 TAR15-24 (SEQ ID NO: 121), TAR15-25 (SEQ ID NO: 122 TAR15-26 (SEQ ID NO: 123), TAR15-27 (SEQ ID NO: 124 TAR15-29 (SEQ ID NO: 125), TAR15-30 (SEQ ID NO: 126 TAR15-6-500 (SEQ ID NO: 127), TAR15-6-501 (SEQ ID NO: 128 TAR15-6-502 (SEQ ID NO: 129) , TAR15-6-503 (SEQ ID NO: 130 TAR15-6-504 (SEQ ID NO: 131), TAR15-6-505 (SEQ ID NO: 132 TAR15-6-506 (SEQ ID NO: 133), TAR15 -6-507 (SEQ ID NO: 134), TAR15-6-508 (SEQ ID NO: 135), TAR15-6-509 (SEQ ID NO: 136), TAR15-6-510 (SEQ ID NO: 137) , TAR15-8-500 (SEQ ID NO: 138), TAR15 -8-501 (SEQ ID NO: 139), TAR15-8-502 (SEQ ID NO: 140), TAR15-8-503 (SEQ ID NO: 141), TAR15-8-505 (SEQ ID NO: 142) , TAR15-8-506 (SEQ ID NO: 143), TAR15-8-507 (SEQ ID NO: 144), TAR15-8-508 (SEQ ID NO: 145), TAR15-8-509 (SEQ ID NO: 146), TAR15-8-510 (SEQ ID NO: 147), TAR15-8-511 (SEQ ID NO: 148), TAR15-26-500 (SEQ ID NO: 149), TAR15-26-501 (SEQ ID NO: 150), TAR15-26-502 (SEQ ID NO: 151), TAR15-26-503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153), TAR15-26-505 ( SEQ ID NO: 154), TAR15-26-506 (SEQ ID NO: 155), TAR15-26-507 (SEQ ID NO: 156), TAR15-26-508 (SEQ ID NO: 157), TAR15-26- 509 (SEQ ID NO: 158), TAR15-26-510 (SEQ ID NO: 159), TAR15-26-511 (SEQ ID NO: 160), TAR15-26-512 (SEQ ID NO: 161), TAR15- 26-513 (SEQ ID NO: 162), TAR15-26-514 (SEQ ID NO: 163), TAR15-26-515 (SEQ ID NO: 164), TAR15-26-516 (SEQ ID NO: 165), TAR15-26-517 (SEQ ID NO: 166), TAR15-26-518 (SEQ ID NO: 167), TAR15-26-519 (SEQ ID NO: 168), TAR15-26-520 (SEQ ID NO: 169) ), TAR15-26-521 (SEQ ID NO: 170), TAR15-26-522 (SEQ ID NO: 171), TAR15-26-523 (SEQ ID N O: 172), TAR15-26-524 (SEQ ID NO: 173), TAR15-26-525 (SEQ ID NO: 174), TAR15-26-526 (SEQ ID NO: 175), TAR15-26-527 ( SEQ ID NO: 176), TAR15-26-528 (SEQ ID NO: 177), TAR15-26-529 (SEQ ID NO: 178), TAR15-26-530 (SEQ ID NO: 179), TAR15-26- 531 (SEQ ID NO: 180), TAR15-26-532 (SEQ ID NO: 181), TAR15-26-533 (SEQ ID NO: 182), TAR15-26-534 (SEQ ID NO: 183), TAR15- 26-535 (SEQ ID NO: 184), TAR15-26-536 (SEQ ID NO: 185), TAR15-26-537 (SEQ ID NO: 186), TAR15-26-538 (SEQ ID NO: 187), TAR15-26-539 (SEQ ID NO: 188), TAR15-26-540 (SEQ ID NO: 189), TAR15-26-541 (SEQ ID NO: 190), TAR15-26-542 (SEQ ID NO: 191) ), TAR15-26-543 (SEQ ID NO: 192), TAR15-26-544 (SEQ ID NO: 193), TAR15-26-545 (SEQ ID NO: 194), TAR15-26-546 (SEQ ID NO. : 195), TAR15-26-547 (SEQ ID NO: 196), TAR15-26-548 (SEQ ID NO: 197), TAR15-26-549 (SEQ ID NO: 198), TAR15-26-550 (SEQ ID NO: 539), and TAR15-26-551 (SEQ ID NO: 540). For example, the variable domain of simple immunoglobulin with binding specificity for VEGF may comprise an amino acid sequence having at least about 85% amino acid sequence identity with the amino acid sequence of a dAb selected from the group consisting of TAR15. -1 (SEQ ID NO: 100), TAR15-3 (SEQ ID NO: 101), TAR15-4 (SEQ ID NO: 102), TAR15-9 (SEQ ID NO: 103), TAR15-10 (SEQ ID NO. : 104), TAR15-11 (SEQ ID NO: 105), TAR15-12 (SEQ ID NO: 106), TAR15-13 (SEQ ID NO: 107), TAR15-14 (SEQ ID NO: 108), TAR15- 15 (SEQ ID NO: 109), TAR15-16 (SEQ ID NO: 110), TAR15-17 (SEQ ID NO: 111), TAR15-18 (SEQ ID NO: 112), TAR15-19 (SEQ ID NO: 113), TAR15-20 (SEQ ID NO: 114), TAR 15-22 (SEQ ID NO: 115), TAR15-5 (SEQ ID NO: 116), TAR15-6 (SEQ ID NO: 117), TAR15- 7 (SEQ ID NO: 118), TAR15-8 (SEQ ID NO: 119), TAR15-23 (SEQ ID NO: 120), TAR15-24 (SEQ ID NO: 121), TAR15-25 (SEQ ID NO: 122), TAR15-26 (SEQ ID NO: 123), TAR15-27 (SEQ ID NO: 124), TAR15-29 (SEQ ID NO: 125), TAR15-30 (SEQ ID NO: 126), TAR15-6-500 (SEQ ID NO: 127), TAR15-6501 (SEQ D NO: 128) TAR15-6-502 (SEQ ID NO: 129 TAR15 6-503 (SEQ ID NO: 130) TAR15-6-504 (SEQ ID NO: 131 TAR15- 6-505 (SEQ ID NO: 132) TAR15-6-506 (SEQ ID NO: 133 TAR15- 6-507 (SEQ ID NO: 134 ) TAR15-6-508 (SEQ ID NO: 135 TAR15- 6-509 (SEQ ID NO: 136) TAR15-6-510 (SEQ ID NO: 137 TAR15-8-500 (SEQ ID NO: 138) TAR15-8 -501 (SEQ ID NO: 139 TAR15- 8-502 (SEQ ID NO: 140) TAR15-8-503 (SEQ ID NO: 141 TAR15- 8-505 (SEQ ID NO: 142) TAR15-8-506 (SEQ ID NO: 143 TAR15- 8-507 (SEQ ID NO: 144) TAR15-8-508 (SEQ ID NO: 145 TAR15- 8-509 (SEQ ID NO: 146) TAR15-8-510 (SEQ ID NO: 147 TAR15- 8-511 (SEQ ID NO: 148), TAR15-26-500 (SEQ ID NO: 149), TAR15-26-501 (SEQ ID NO: 150), TAR15-26-502 (SEQ ID NO: 151), TAR15-26-503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153), TAR15-26-505 (SEQ ID NO: 154), TAR15-26-506 (SEQ ID NO: 155), TAR15-26-507 (SEQ ID NO: 156), TAR15-26-508 (SEQ ID NO: 157), TAR15-26-509 (SEQ ID NO: 158), TAR15-26-510 (SEQ ID NO: 159), TAR15-26-511 ( SEQ ID NO: 160), TAR15-26-512 (SEQ ID NO: 161), TAR15-26-513 (SEQ E) NO: 162), TAR15-26-514 (SEQ ID NO: 163), TAR15-26 -515 (SEQ ID NO: 164), TAR15-26-516 (SEQ ID NO: 165), TAR15-26-517 (SEQ ID NO: 166), TAR15-26-518 (SEQ ID NO: 167), TAR15 -26-519 (SEQ ID NO: 168), TAR15-26-520 (SEQ ID NO: 169), TAR15-26-521 (SEQ ID NO: 170), TAR15-26-522 (SEQ ID NO: 171) , TAR15-26-523 (SEQ ID NO: 172), TAR15-26-524 (SEQ ID NO: 173), TAR15-26-525 (SEQ ID NO: 174), TAR15-26-526 (SEQ ID NO: 175), TAR15-26-527 (SEQ ID NO: 176), TAR15-26-528 (SEQ ID NO: 177), TAR15-26-529 (SEQ ID NO: 178), TAR15-26-530 (SEQ ID NO: 179), TAR15-26-531 (SEQ ID NO: 180), TAR15-26-532 (SEQ ID NO: 181), TAR15-2 6-533 (SEQ ID NO: 182), TAR15-26-534 (SEQ ID NO: 183), TAR15-26-535 (SEQ ID NO: 184), TAR15-26-536 (SEQ ID NO: 185), TAR15-26-537 (SEQ ID NO: 186), TAR15-26-538 (SEQ ID NO: 187), TAR15-26-539 (SEQ ID NO: 188), TAR15-26-540 (SEQ ID NO: 189 ), TAR15-26-541 (SEQ ID NO: 190), TAR15-26-542 (SEQ ID NO: 191), TAR15-26-543 (SEQ ID NO: 192), TAR15-26-544 (SEQ ID NO. : 193), TAR15-26-545 (SEQ ID NO: 194), TAR15-26-546 (SEQ ID NO: 195), TAR15-26-547 (SEQ ID NO: 196), TAR15-26-548 (SEQ ID NO: 197), TAR15-26-549 (SEQ ID NO: 198), TAR15-26-550 (SEQ ID NO: 539), and TAR15-26-551 (SEQ ID NO: 540). In other particular embodiments, the ligand has binding specificity for VEGF and for EGFR and comprises at least one variable domain of simple immunoglobulin with binding specificity for VEGF and at least one variable domain of single immunoglobulin with binding specificity for EGFR, wherein a single immunoglobulin variable domain with binding specificity for EGFR competes to bind EGFR with an anti-EGFR domain antibody (dAb) selected from the group consisting of DOM16-17 SEQ ID NO: 325 DOM16-18 SEQ ID NO: 326 DOM16-19 SEQ ID NO: 327 DOM16-20 SEQ ID NO: 328 DOM16-21 SEQ ID NO: 329 DOM16-22 SEQ ID NO: 330 DOM16-23 SEQ ID NO: 331 DOM16-24 SEQ ID NO: 332 DOM16- SEQ ID NO: 333 DOM16-26 SEQ ID NO: 334 DOM16-27 SEQ ID NO: 335 DOM16-28 SEQ ID NO: 336 DOM16-29 SEQ ID NO: 337 DOM16-30 SEQ ID NO: 338 DOM16-31 SEQ ID NO: 339 DOM16-32 SEQ ID NO: 340 DOM16-33 SEQ ID NO: 341 DOM16-35 SEQ ID NO: 342 DOM16-37 SEQ ID NO: 343 DOM16-38 SEQ ID NO: 344 DOM16-39 SEQ ID NO : 345 DOM16-40 SEQ ID NO: 346 DOM16-41 SEQ ID NO: 347 DOM16-42 SEQ ID NO: 348 DOM16-43 SEQ ID NO: 349 DOM16-44 SEQ ID NO: 350 DOM16-45 SEQ ID NO: 351 DOM16-46 SEQ ID NO : 352 DOM16-47 SEQ ID NO: 353 DOM16-48 SEQ ID NO: 354 DOM16-49 SEQ ID NO: 355 DOM16-50 SEQ ID NO: 356 DOM16-59 SEQ ID NO: 357 DOM16-60 SEQ ID NO: 358 DOM16-61 SEQ ID NO: 359 DOM16-62 SEQ ID NO: 360 DOM16-63 SEQ ID NO: 361 DOM16-64 (SEQ ID NO: 362), DOM16-65 (SEQ ID NC-.363), DOM16-66 (SEQ ID NO: 364), DOM16-67 (SEQ ID NO: 365), DOM16-68 (SEQ ID NO: 366), DOM16-69 (SEQ ID NO: 367 DOM16-70 (SEQ ID NO: 368), DOM16-71 (SEQ ID NO: 369 DOM16-72 (SEQ ID NO: 370), DOM16-73 (SEQ ID NO: 371 DOM16-74 (SEQ ID NO: 372), DOM16-75 (SEQ ID NO: 373 DOM16 -76 (SEQ ID NO: 374), DOM16-77 (SEQ ID NO: 375 DOM16-78 (SEQ ID NO: 376), DOM16-79 (SEQ ID NO: 377 DOM16-80 (SEQ ID NO: 378), DOM16-81 (SEQ ID NO: 379 DOM16-82 (SEQ ID NO: 380), DOM16-83 (SEQ ID NO: 381 DOM16-84 (SEQ ID NO: 382), DOM16-85 (SEQ ID NO: 383 DOM16 -87 (SEQ ID NO: 384), DOM16-88 (SEQ ID NO: 385 DOM16-89 (SEQ ID NO: 386), DO M16-90 (SEQ ID NO: 387 DOM16-91 (SEQ ID NO: 388), DOM16-92 (SEQ ID NO: 389 DOM16-94 (SEQ ID NO: 390), DOM16-95 (SEQ ID NO: 391 DOM16 -96 (SEQ ID NO: 392), DOM16-97 (SEQ ID NO: 393 DOM16-98 (SEQ ID NO: 394), DOM16-99 (SEQ ID NO: 395 DOM16-100 (SEQ ID NO: 396), DOM16-101 (SEQ ID NO: 397 DOM16-102 (SEQ ID NO: 398), DOM16-103 (SEQ ID NO: 399 DOM16-104 (SEQ ID NO: 400), DOM16-105 (SEQ ID NO: 401 DOM16 -106 (SEQ ID NO: 402), DOM16-107 (SEQ ID NO: 403 DOM16-108 (SEQ ID NO: 404), DOM16-109 (SEQ ID NO: 405 DOM16-110 (SEQ ID NO: 406), DOM16-111 (SEQ ID NO: 407 DOM16-112 (SEQ ID NO: 408), DOM16-113 (SEQ ID NO: 409 DOM16-114 (SEQ ID NO: 410), DOM16-115 (SEQ ID NO: 411 DOM16-116 (SEQ ID NO: 412), DOM16-117 (SEQ ID NO: 413 DOM16-118 (SEQ ID NO: 414) , DOM16-119 (SEQ ID NO: 415 DOM16-39-6 (SEQ ID NO: 416), DOM16-39-8 (SEQ ID NO: 417), DOM16-39-34 (SEQ ID NO: 418), DOM16 -39-48 (SEQ ID NO: 419), DOM16-39-87 (SEQ ID NO: 420), DOM16-39-90 (SEQ ID NO: 421), DOM16-39-96 (SEQ ID NO: 422) , DOM16-39-100 (SEQ ID NO: 423), DOM16-39-101 (SEQ ID NO: 424), DOM16-39-102 (SEQ ID NO: 425), DOM16-39-103 (SEQ ID NO: 426), DOM16-39-104 (SEQ ID NO: 427), DOM16-39-105 (SEQ ID NO: 428), DOM16-39-106 (SEQ ID NO: 429), DOM16-39-107 (SEQ ID NO: 430), DOM16-39-108 (SEQ ID NO: 431), DOM16-39-109 (SEQ ID NO: 432), DOM16-39-110 (SEQ ID NO: 433), DOM16-39-111 ( SEQ ID NO: 434), DOM16-39-112 (SEQ ID NO: 435), DOM16-39-113 (SEQ ID NO: 436), DOM16-39-114 (SEQ ID NO: 437), DOM16-39- 115 (SEQ ID NO: 438), DOM16-39-116 (SEQ ID NO: 4 39), DOM16-39-117 (SEQ ID NO: 440), DOM16-39-200 (SEQ ID NO: 441), DOM16-39-201 (SEQ ID NO: 442), DOM16-39-202 (SEQ ID NO: 443), DOM16-39-203 (SEQ ID NO: 444), DOM16-39-204 (SEQ ID NO: 445), DOM16-39-205 (SEQ ID NO: 446), DOM16-39-206 ( SEQ ID NO: 447), DOM16-39-207 (SEQ ID NO: 448), DOM16-39-209 (SEQ ID NO: 449), DOM16-52 (SEQ ID NO: 450), NB1 (SEQ ID NO: 451), NB2 (SEQ ID NO: 452), NB3 (SEQ ID NO: 453), NB4 (SEQ ID NO: 454), NB5 (SEQ ID NO: 455), NB6 (SEQ ID NO: 456), NB7 ( SEQ ID NO: 457), NB8 (SEQ ID NO: 458), NB9 (SEQ ID NO: 459), NB10 (SEQ ID NO: 460), NB11 (SEQ ID NO: 461), NB12 (SEQ ID NO: 462 ), NB13 (SEQ ID NO: 463), NB14 (SEQ ID NO: 464), NB15 (SEQ ID NO: 465), NB16 (SEQ ID NO: 466), NB17 (SEQ ID NO: 467), NB18 (SEQ ID NO: 468), NB19 (SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 (SEQ ID NO: 471), and NB22 (SEQ ID NO: 472). For example, the variable domain of single immunoglobulin with binding specificity for EGFR may comprise an amino acid sequence having at least about 85% amino acid sequence identity with the amino acid sequence of a dAb selected from the group consisting of DOM16 -17 (SEQ ID NO: 325 DOM16-18 (SEQ ID NO: 326 DOM16-19 [SEQ ID NO: 327 DOM16-20 (SEQ ID NO: 328 DOM16-21 [SEQ ID NO: 329 DOM16-22 (SEQ ID NO: 330 DOM16- 23; SEQ ID NO: 331 DOM16-24 (SEQ ID NO: 332 DOM16-25; SEQ ID NO: 333 DOM16-26 (SEQ ID NO: 334 DOM16-27 [SEQ ID NO: 335 DOM16-28 (SEQ ID NO : 336 DOM16-29 [SEQ ID NO: 337 DOM16-30 (SEQ ID NO: 338 DOM16-31 [SEQ ID NO: 339 DOM16-32 (SEQ ID NO: 340 DOM16-33; SEQ ID NO: 341 DOM16-35 (SEQ ID NO: 342 DOM16-37 [SEQ ID NO: 343 DOM16-38 (SEQ ID NO: 344 DOM16-39; SEQ ID NO: 345 DOM16-40 (SEQ ID NO: 346 DOM16-41 [SEQ ID NO: 347 DOM16-42 (SEQ ID NO: 348 DOM16-43; SEQ ID NO: 349 DOM16-44 (SEQ ID NO: 350 DOM16-45; SEQ ID NO: 351 DOM16-46 (SEQ ID NO: 352 DOM16-47 [ SEQ ID NO: 353 DOM16-48 (SEQ ID NO: 354 DOM16-49; SEQ ID NO: 355 DOM16-50 (SEQ ID NO: 356 DOM16-59; SEQ ID NO: 357 DOM16-60 (SEQ ID NO: 358 DOM16-61 SEQ ID NO: 359 [DOM16-62 SEQ ID NO: 360 DOM16-63 SEQ ID NO: 361 DOM16-64 SEQ ID NO: 362 DOM16-65 SEQ ID NO: 363 [DOM16-66 SEQ ID NO: 364 DOM16-67 SEQ ID NO: 365 [DOM16-68 SEQ ID NO: 366 DOM16-69 SEQ ID NO: 367 [DOM16-70 SEQ ID NO: 368 DOM16-71 SEQ ID NO: 369 [DOM16-72 SEQ ID NO: 370 DOM16-73 SEQ ID NO: 371 DOM16-74 SEQ ID NO: 372 DOM16-75 SEQ ID NO: 373 DOM16-76 SEQ ID NO: 374 DOM16-77 SEQ ID NO: 375 DOM16-78 SEQ ID NO: 376 DOM16-79 SEQ ID NO: 377 DOM16-80 SEQ ID NO: 378 DOM16-81 SEQ ID NO : 379 DOM16-82 SEQ ID NO: 380 DOM16-83 SEQ ID NO: 381 DOM16-84 SEQ ID NO: 382 DOM16-85 SEQ ID NO: 383 DOM16-87 SEQ ID NO: 384 DOM16-88 SEQ ID NO: 385 DOM16-89 SEQ ID NO: 386 DOM16-90 SEQ ID NO: 387 DOM16-91 SEQ ID NO: 388 DOM16-92 SEQ ID NO: 389 DOM16-94 SEQ ID NO: 390 DOM16-95 SEQ ID NO: 391 DOM16- 96 SEQ ID NO: 392 DOM16-97 SEQ ID NO: 393: DOM16-98 SEQ ID NO: 394 DOM16-99 SEQ ID NO: 395 [DOM16-100 SEQ ID NO: 396 DOM16-101 SEQ ID NO: 397; DOM16-102 SEQ ID NO: 398 DOM16-103 SEQ ID NO: 399 [DOM16-104 SEQ ID NO: 400 DOM16-105 SEQ ID NO: 401 DOM16-106 SEQ ID NO: 402 DOM16-107 SEQ ID NO: 403 [ DOM16-108 SEQ ID NO: 404 DOM16-109 SEQ ID NO: 405; DOM16-110 SEQ ID NO: 406 DOM16-111 SEQ ID NO: 407 [DOM16-112 SEQ ID NO: 408 DOM16-113 SEQ ID NO: 409) DOM16-114 (SEQ ID NO: 410), DOM16-115 (SEQ. ID NO: 411), DOM16-116 (SEQ ID NO: 412), DOM16-117 (SEQ ID NO: 413), DOM16-118 (SEQ ID NO: 414), DOM16-119 (SEQ ID NO: 415), DOM16 -39-6 (SEQ ID NO: 416), DOM16-39-8 (SEQ ID NO: 417), DOM16-39-34 (SEQ ID NO: 418), DOM16-39-48 (SEQ ID NO: 419) , DOM16-39-87 (SEQ ID NO: 420), DOM16-39-90 (SEQ ID NO: 421), DOM16-39-96 (SEQ ID NO: 422), DOM16-39-100 (SEQ ID NO: 423), DOM16-39-101 (SEQ ID NO: 424), DOM16-39-102 (SEQ ID NO: 425), DOM16-39-103 (SEQ ID NO: 426), DOM16-39-104 (SEQ ID NO: 427), DOM16-39-105 (SEQ ID NO: 428), DOM16-39-106 (SEQ ID NO: 429), DOM16-39-107 (SEQ ID NO: 430), DOM16-39-108 ( SEQ ID NO: 431), DOM16-39-109 (SEQ ID NO: 432), DOM16-39-110 (SEQ ID NO: 433), DOM16-39-111 (SEQ ID NO: 434), DOM16-39- 112 (SEQ ID NO: 435), DOM16-39-113 (SEQ ID NO: 436), DOM16-39-114 (SEQ ID NO: 437), DOM16-39-115 (SEQ ID NO: 438), DOM16- 39-116 (SEQ ID NO: 439), DOM16-39-117 (SEQ ID NO: 440), DOM16-39-200 (SEQ ID NO: 441), DOM16-39-201 (SEQ ID NO: 442), DOM16-39-202 (SEQ ID NO: 443), DOM16-39-203 (SEQ ID NO: 444), DOM16-39-204 (SEQ ID N O: 445), DOM16-39-205 (SEQ ID NO: 446), DOM16-39-206 (SEQ ID NO: 447), DOM16-39-207 (SEQ ID NO: 448), DOM16-39-209 ( SEQ ID NO: 449), DOM16-52 (SEQ ID NO: 450), NB1 (SEQ ID NO: 451), NB2 (SEQ ID NO: 452), NB3 (SEQ ID NO: 453), NB4 (SEQ ID NO : 454), NB5 (SEQ ID NO: 455), NB6 (SEQ ID NO: 456), NB7 (SEQ ID NO: 457), NB8 (SEQ ID NO: 458), NB9 (SEQ ID NO: 459), NB10 (SEQ ID NO: 460), NB11 (SEQ ID NO: 461), NB12 (SEQ ID NO: 462), NB13 (SEQ ID NO: 463), NB14 (SEQ ID NO: 464), NB15 (SEQ ID NO: 465), NB16 (SEQ ID NO: 466), NB17 (SEQ ID NO: 467), NB18 (SEQ ID NO: 468), NB19 (SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 ( SEQ ID NO: 471), and NB22 (SEQ ID NO: 472). In some embodiments, the ligand has binding specificity for VEGF and for EGFR and comprises at least one variable domain of simple immunoglobulin with binding specificity for VEGF and at least one variable domain of single immunoglobulin with binding specificity for EGFR, wherein the single immunoglobulin variable domain with binding specificity for VEGF competes to bind VEGF with the anti-VEGF domain antibody (dAb) selected from the group consisting of TAR15-1 (SEQ ID NO: 100), TAR15-3 (SEQ ID NO: 101), TAR15-4 (SEQ ID NO: 102), TAR15-9 (SEQ ID NO: 103), TAR15-10 (SEQ ID NO: 104), TAR15-11 (SEQ ID NO: 105), TAR15-12 (SEQ ID NO: 106), TAR15-13 (SEQ ID NO: 107), TAR15-14 (SEQ ID NO: 108), TAR15-15 (SEQ ID NOr 109), TAR15 -16 (SEQ ID NO: 110), TAR15-17 (SEQ ID NO: 111), TAR15-18 (SEQ ID NO: 112), TAR15-19 (SEQ ID NO: 113), TAR15-20 (SEQ ID NO. : 114), TAR 15-22 (SEQ ID NO: 115), TAR15-5 (SEQ ID NO: 116), TAR15-6 (SEQ ID NO: 117), TAR15-7 (SEQ ID NO: 118), TAR15 -8 (SEQ ID NO: 119), TAR15-23 (SEQ ID NO: 120), TAR15-24 (SEQ ID NO: 121), TAR15-25 (SEQ ID NOr 122), TAR15-26 (SEQ ID NO: 123), TAR15-27 (SEQ ID NO: 124) TAR15-29 (SEQ ID NO: 125), TAR15-30 (SEQ ID NO: 126) TAR15-6-500 (SEQ ID NO: 127), TAR15-6 -501 (SEQ ID NO: 128) TAR15-6-502 (SEQ ID NO: 129) TAR15-6 503 (SEQ ID NO: 130) TAR15-6-504 (SEQ ID NO: 131) TAR15-6-505 ( SEQ ID NO: 132) TAR15-6-506 (SEQ ID NO: 133) TAR15-6-507 (SEQ ID NO: 134) TAR15-6-508 (SEQ ID NO: 135) TAR15-6509 (SEQ ID NO: 136) TAR15-6-510 (SEQ ID NO: 137) TAR15-8-500 (SEQ ID NO: 138) TAR15-8-501 (SEQ ID NO: 139) TAR15-8-502 (SEQ ID NO: 140) TAR15-8-503 (SEQ ID NO: 141) TAR15-8-505 (SEQ ID NO : 142) TAR15-8-506 (SEQ ID NO: 143) TAR15-8-507 (SEQ ID NO: 144) TAR15-8-508 (SEQ ID NO: 145) TAR15-8-509 (SEQ ID NO: 146 ) TAR15-8-510 (SEQ ID NO: 147) TAR15-8-511 (SEQ ID NO: 148) TAR15-26-500 (SEQ ID NO: 149), TAR15-26-501 (SEQ ID NO: 150), TAR15-26-502 (SEQ ID NO: 151), TAR15-26- '503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153), TAR15-26-505 (SEQ ID NO: 154), TAR15-26-506 (SEQ ID NO: 155), TAR15-26-507 (SEQ ID NO: 156), TAR15-26-508 (SEQ ID NO: 157), TAR15-26-509 (SEQ ID NO: 158), TAR15-26-510 (SEQ ID NO: 159), TAR15-26-511 (SEQ ID NO: 160), TAR15-26-512 (SEQ ID NO: 161), TAR15-26-513 ( SEQ ID NO: 162), TAR15-26-514 (SEQ ID NO: 163), TAR15-26-515 (SEQ ID NO: 164), TAR15-26-516 (SEQ ID NO: 165), TAR15-26- 517 (SEQ ID NO: 166), TAR15-26-518 (SEQ ID NO: 167), TAR15-26-519 (SEQ ID NO: 168), TAR15-26-520 (SEQ ID NO: 169), TAR15- 26-521 (SEQ ID NO: 170), TAR15-26-522 (SEQ ID NO: 171), TAR15-26-523 (SEQ ID NO: 172), TAR15-26-524 (SEQ ID NO: 173), TAR15-26-525 (SEQ ID NO: 174), TAR15-26-526 (SEQ ID NO: 175), TAR15-26-527 (SEQ ID NO: 176), TAR15-26-528 (SEQ ID NO: 177) ), TAR15-26-529 (SEQ ID NO: 178), TAR15-26-530 (SEQ ID NO: 179), TAR15-26-5 31 (SEQ ID NO: 180), TAR15-26-532 (SEQ ID NO: 181), TAR15-26-533 (SEQ ID NO: 182), TAR15-26-534 (SEQ ID NO: 183), TAR15- 26-535 (SEQ ID NO: 184), TAR15-26-536 (SEQ ID NO: 185), TAR15-26-537 (SEQ ID NO: 186), TAR15-26-538 (SEQ ID NO: 187), TAR15-26-539 (SEQ ID NO: 188), TAR15-26-540 (SEQ ID NO: 189), TAR15-26-541 (SEQ ID NO: 190), TAR15-26-542 (SEQ ID NO: 191) ), TAR15-26-543 (SEQ ID NO: 192), TAR15-26-544 (SEQ ID NO: 193), TAR15-26-545 (SEQ ID NO: 194), TAR15-26-546 (SEQ ID NO. : 195), TAR15-26-547 (SEQ ID NO: 196), TAR15-26-548 (SEQ ID NO: 197), TAR15-26-549 (SEQ ID NO: 198), TAR15-26-550 (SEQ ID NO: 539), and TAR15-26-551 (SEQ ID NO: 540); and wherein a single immunoglobulin variable domain with binding specificity for EGFR competes for binding to EGFR with an anti-EGFR domain antibody (dAb) selected from the group consisting of DOM16-17 (SEQ ID NO: 325), DOM16- 18 (SEQ ID NO: 326), DOM16-19 (SEQ ID NO: 327), DOM16-20 (SEQ ID NO: 328), DOM16-21 (SEQ ID NO: 329), DOM16-22 (SEQ ID NO: 330), DOM16-23 (SEQ ID NO: 331), DOM16-24 (SEQ ID NO: 332), DOM16 -25 (SEQ ID NO 333), DOM16 -26 (SEQ ID NO: 334), DOM16-27 (SEQ ID NO 335), DOM16-28 (SEQ ID NO 336), DOM16 -29 (SEQ ID NO 337), DOM16 -30 (SEQ ID NO 338), DOM16 -31 (SEQ ID NO 339), DOM16 -32 (SEQ ID NO 340), DOM16 -33 (SEQ ID NO 341), DOM16 -35 (SEQ ID NO 342), DOM16 -37 (SEQ ID NO 343), DOM16 -38 (SEQ ID NO 344), DOM16 -39 (SEQ ID NO 345), DOM16 -40 (SEQ ID NO 346), DOM16 -41 (SEQ ID NO 347), DOM16 -42 (SEQ ID NO 348), DOM16 -43 (SEQ ID NO 349), DOM16 -44 [SEQ ID NO 350), DOM16 -45 (SEQ ID NO 351), DOM16 -46 (SEQ ID NO 352), DOM16 -47 (SEQ ID NO 353), DOM16 -48 (SEQ ID NO 354), DOM16 -49 [SEQ ID NO 355), DOM16 -50 [SEQ ID NO 356), DOM16- -59 < [SEQ ID NO 357), DOM16- -60 [SEQ ID NO 358), DOM16-61 [SEQ ID NO 359), DOM16- -62 [SEQ ID NO 360), DOM16-63, SEQ ID NO 361), DOM16- -64 SEQ ID NO 362), DOM16-65 ([SEQ ID NO 363), DOM16-66 (, SEQ ID NO 364), DOM16- -67 | [SEQ ID NO 365), DOM16- -68 I [SEQ ID NO 366), DOM16-69 (SEQ ID NO 367), DOM16-70 (SEQ ID NO 368), DOM16-71 (SEQ ID NO 369), DOM16-72 (SEQ ID NO 370), DOM16-73 ([SEQ ID NO 371), DOM16- -74 (SEQ ID NO 372), DOM16-75 (SEQ ID NO 373), DOM16-76 (SEQ ID NO 374), DOM16-77 (SEQ ID NO 375), DOM16-78 (SEQ ID NO 376), DOM16- • 79 (SEQ ID NO 377), DOM16- -80 (SEQ ID NO 378), DOM16-81 (SEQ ID NO 379), DOM16-82 (SEQ ID NO 380), DOM16-83 (SEQ ID NO: 381), DOM16-84 (SEQ ID NO 382), DOM16-85 (SEQ ID NO: 383), DOM16-87 (SEQ ID NO: 384), DOM16-88 (SEQ ID NO. : 385), DOM16-89 (SEQ ID NO: 386), DOM16-90 (SEQ ID NO: 387), DOM16-91 (SEQ ID NO: 388), DOM16-92 (SEQ ID NO: 389), DOM16- 94 (SEQ ID NO: 390), DOM16-95 (SEQ ID NO: 391), DOM16-96 (SEQ ID NO: 392), DOM16-97 (SEQ ID NO: 393), DOM16-98 (SEQ ID NO: 394), DOM16-99 (SEQ ID NO: 395), DOM16-100 (SEQ ID NO: 396), DOM16-101 (SEQ ID NO: 397), DOM16-102 (SEQ ID NO: 398), DOM16-103 (SEQ ID NO: 399), DOM16-104 (SEQ ID NO: 400), DOM16-105 (SEQ ID NO: 401), DOM16-106 (SEQ ID NO: 402), DOM16-107 (SEQ ID NO: 403), DOM16-108 (SEQ ID NO: 404), DOM16-109 (SEQ ID NO: 405), DOM16-110 (SEQ. ID NO: 406), DOM16-111 (SEQ ID NO: 407), DOM16-112 (SEQ ID NO: 408), DOM16-113 (SEQ ID NO: 409), DOM16-114 (SEQ ID NO: 410), DOM16-115 (SEQ ID NO: 411), DOM16-116 (SEQ ID NO: 412), DOM16-117 (SEQ ID NO: 413), DOM16-118 (SEQ ID NO: 414), DOM16-119 (SEQ ID NO: 415), DOM16-39-6 (SEQ ID NO: 416), DOM16-39-8 (SEQ ID NO: 417), DOM16-39-34 (SEQ ID NO: 418), DOM16-39-48 ( SEQ ID NO: 419), DOM16-39-87 (SEQ ID NO: 420), DOM16-39-90 (SEQ ID NO: 421), DOM16-39-96 (SEQ ID NO: 422), DOM16-39- 100 (SEQ ID NO: 423), DOM16-39-101 (SEQ ID NO: 424), DOM16-39-102 (SEQ ID NO: 425), DOM16-39-103 (SEQ ID NO: 426), DOM16- 39-104 (SEQ ID NO: 427), DOM16-39-105 (SEQ ID NO: 428), DOM16-39-106 (SEQ ID NO: 429), DOM16-39-107 (SEQ ID NO: 430), DOM16-39-108 (SEQ ID NO: 431), DOM16-39-109 (SEQ ID NO: 432), DOM16-39-110 (SEQ ID NO: 433), DOM16-39-111 (SEQ ID NO: 434) ), DOM16-39-112 (SEQ ID NO: 435), DOM16-39-113 (SEQ ID NO: 436) ), DOM16-39-114 (SEQ ID NO: 437), DOM16-39-115 (SEQ ID NO: 438), DOM16-39-116 (SEQ ID NO: 439), DOM16-39-117 (SEQ ID NO. : 440), DOM16-39-200 (SEQ ID NO: 441), DOM16-39-201 (SEQ ID NO: 442), DOM16-39-202 (SEQ ID NO: 443), DOM16-39-203 (SEQ ID NO: 444), DOM16-39-204 (SEQ ID NO: 445), DOM16-39-205 (SEQ ID NO: 446), DOM16-39-206 (SEQ ID NO: 447), DOM16-39-207 (SEQ ID NO: 448), DOM16-39-209 (SEQ ID NO: 449), DOM16-52 (SEQ ID NO: 450), NB1 (SEQ ID NO: 451), NB2 (SEQ ID NO: 452), NB3 (SEQ ID NO: 453), NB4 (SEQ ID NO: 454), NB5 (SEQ ID NO: 455), NB6 (SEQ ID NO: 456), NB7 (SEQ ID NO: 457), NB8 (SEQ ID NO : 458), NB9 (SEQ ID NO: 459), NB10 (SEQ ID NO: 460), NB11 (SEQ ID NO: 461), NB12 (SEQ ID NO: 462), NB13 (SEQ ID NO: 463), NB14 (SEQ ID NO: 464), NB15 (SEQ ID NO: 465), NB16 (SEQ ID NO: 466), NB17 (SEQ ID NO: 467), NB1 8 (SEQ ID NO: 468), NB 19 (SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 (SEQ ID NO: 471), and NB22 (SEQ ID NO: 472). For example, the ligand may comprise a variable domain of a simple immunoglobulin with binding specificity for VEGF comprising an amino acid sequence having at least about 85% amino acid sequence identity with an amino acid sequence of a dAb selected from the group consisting of TAR15-1 (SEQ ID NO: 00), TAR15-3 (SEQ ID NO: 101), TAR15-4 (SEQ ID NO: 102 TAR15-9 (SEQ ID NO: 103), TAR15-10 (SEQ ID NO: 104 TAR15-11 ( SEQ ID NO: 105), TAR15-12 (SEQ ID NO: 106 TAR15-13 (SEQ ID NO: 107), TAR15-14 (SEQ ID NO: 108 TAR15-15 (SEQ ID NO: 109), TAR15-16 (SEQ ID NO: 110 TAR15-17 (SEQ ID NO: 111), TAR15-18 (SEQ ID NO: 112 TAR15-19 (SEQ ID NO: 113), TAR15-20 (SEQ ID NO: 114 TAR15-22 ( SEQ ID NO: 115), TAR15-5 (SEQ ID NO: 116 TAR15-6 (SEQ ID NO: 117), TAR15-7 (SEQ ID NO: 118 TAR15-8 (SEQ ID NO: 119), TAR15-23 (SEQ ID NO: 120 TAR15-24 (SEQ ID NO: 121), TAR15-25 (SEQ ID NO: 122 TAR15-26 (SEQ ID NO: 123), TAR15-27 (SEQ ID NO: 124 TAR15-29 ( SEQ ID NO: 125), TAR15-30 (SEQ ID NO: 126 TAR15-6-500 (SEQ ID NO: 127), TAR15-6-501 (SEQ ID NO: 128), TAR15-6-502 (SEQ ID NO: 129) TAR15-6-503 (SEQ ID NO: 130), TAR15-6-504 (SEQ ID NO: 131) TAR15-6-505 (SEQ ID NO: 132), TAR15-6-506 (SEQ ID NO: 133) TAR15-6-507 (SEQ ID NO: 134), TAR15-6-508 (SEQ ID NO: 135) TAR15-6-509 (SEQ ID NO: 136), TAR15-6-510 (SEQ ID NO: 137) TAR15-8-500 (SEQ ID NO: 138), TAR15-8-501 (SEQ ID NO: 139) TAR15-8-502 (SEQ ID NO: 140), TAR15-8-503 (SEQ ID NO: 141) TAR15-8-505 (SEQ ID NO: 142), TAR15-8-506 (SEQ ID NO: 143) TAR15-8-507 (SEQ ID NO: 144), TAR15-8-508 (SEQ ID NO: 145) TAR15-8-509 (SEQ ID NO: 146), TAR15-8-510 (SEQ ID NO: 147) TAR15-8-511 (SEQ ID NO: 148), TAR15-26-500 (SEQ ID NO: 149), TAR15-26-501 (SEQ ID NO: 150), TAR15-26-502 (SEQ ID NO: 151), TAR15-26-503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153), TAR15-26-505 (SEQ ID NO: 154), TAR15-26-506 (SEQ ID NO: 155), TAR15-26 -507 (SEQ ID NO: 156), TAR15-26-508 (SEQ ID NO: 157), TAR15-26-509 (SEQ ID NO: 158), TAR15-26-510 (SEQ ID NO: 159), TAR15 -26-511 (SEQ ID NO: 160), TAR15-26-512 (SEQ ID NO: 161), TAR15-26-513 (SEQ ID NO: 162) ), TAR15-26-514 (SEQ ID NO: 163), TAR15-26-515 (SEQ ID NO: 164), TAR15-26-516 (SEQ ID NO: 165), TAR15-26-517 (SEQ ID NO. : 166), TAR15-26-518 (SEQ ID NO: 167), TAR15-26-519 (SEQ ID NO: 168), TAR15-26-520 (SEQ ID NO: 169), TAR15-26-521 (SEQ ID NO: 170), TAR15-26-522 (SEQ ID NO: 171), TAR15-26-523 (SEQ ID NO: 172), TAR15-26-524 (SEQ ID NO: 173), TAR15-26-525 (SEQ ID NO: 174), TAR15-26-526 (SEQ ID NO: 175), TAR15-26-527 (SEQ ID NO: 176), TAR15-26-528 (SEQ ID NO: 177), TAR15-26 -529 (SEQ ID NO: 178), TAR15-26-530 (SEQ ID NO: 179), TAR15-26-531 (SEQ ID NO: 180), TAR15-26-532 (SEQ ID NO: 181), TAR15 -26-533 (SEQ ID NO: 182), TAR15-26-534 (SEQ ID NO: 183), TAR15-26-535 (SEQ ID NO: 184), TAR15-26-536 (SEQ ID NO: 185) , TAR15-26-537 (SEQ ID NO: 186), TAR15-26-538 (SEQ ID NO: 187), TAR15-26-539 (SEQ ID NO: 188), TAR15-26-540 (SEQ ID NO: 189), TAR15-26-541 (SEQ ID NO: 190), TAR15-26-542 (SEQ ID NO: 191), TAR15-26-543 (SEQ ID NO: 192), TAR15-26-544 (SEQ ID NO: 193), TAR15-26-545 (SEQ ID NO: 194), TAR15-26-546 (SEQ ID NO: 195), TAR15- 26-547 (SEQ ID NO: 196), TAR15-26-548 (SEQ ID NO: 197), TAR15-26-549 (SEQ ID NO: 198), TAR15-26-550 (SEQ ID NO: 539), and TAR15-26-551 (SEQ ID NO: 540); and further comprising a variable domain of simple immunoglobulin with binding specificity for EGFR comprising an amino acid sequence having at least about 85% amino acid sequence identity with the amino acid sequence of a dAb selected from the group consisting of DOM16 -17 (SEQ ID NO 325 DOM16-18 (SEQ ID NO: 326) DOM16-19 (SEQ ID NO 327 DOM16-20 (SEQ ID NO: 328) DOM16-21 (SEQ ID NO. 329 DOM16-22 (SEQ ID NO. : 330) DOM16-23 (SEQ ID NO: 331 DOM16-24 (SEQ ID NO: 332) DOM16-25 (SEQ ID NO: 333 DOM16-26 (SEQ ID NO: 334) DOM16-27 (SEQ ID NO: 335 DOM16-28) (SEQ ID NO: 336) DOM16-29 (SEQ ID NO 337 DOM16-30 (SEQ ID NO: 338) DOM16-31 (SEQ ID NO 3393 DOM16-32 (SEQ ID NO: 340) DOM16-33 (SEQ ID NO. 341 DOM16-35 (SEQ ID NO: 342) DOM16-37 (SEQ ID NO 343 DOM16-38 (SEQ ID NO: 344) DOM16-39 (SEQ ID NO 345 DOM16-40 (SEQ ID NO: 346) DOM16-41 (SEQ ID NO 347 DOM16-42 (SEQ ID NO: 348) DOM16-43 (SEQ ID NO 349 DOM16-44 (SEQ ID NO: 350) DOM16-45 (SEQ ID NO 351 DOM16-46 (SEQ ID NO: 352 DO M16-47 (SEQ ID NO 353 DOM16-48 (SEQ ID NO: 354) DOM16-49 (SEQ ID NO 355 DOM16-50 (SEQ ID NO: 356) DOM16-59 (SEQ ID NO: 357 DOM16-60 (SEQ ID NO: 358) DOM16-61 (SEQ ID NO: 359 DOM16-62 (SEQ ID NO: 360) DOM16-63 (SEQ ID NO: 361 DOM16-64 (SEQ ID NO: 362) DOM16-65 (SEQ ID NO: 363 DOM16-66 (SEQ ID NO: 364) DOM16-67 (SEQ ID NO: 365 DOM16-68 (SEQ ID NO: 366) DOM16-69 (SEQ ID NO: 367 DOM16-70 (SEQ ID NO: 368) DOM16-71 (SEQ ID NO: 369 DOM16-72 (SEQ ID NO: 370) DOM16-73 (SEQ ID NO: 371 DOM16-74 (SEQ ID NO: 372) DOM16-75 (SEQ ID NO: 373 DOM16-76 (SEQ ID NO: 374) DOM16 -77 (SEQ ID NO: 375 DOM16-78 (SEQ ID NO: 376) DOM16-79 (SEQ ID NO: 377 DOM16-80 (SEQ ID NO: 378) DOM16-81 (SEQ ID NO: 379 DOM16-82 ( SEQ ID NO: 380) DOM16-83 (SEQ ID NO: 381 DOM16-84 (SEQ ID NO: 382) DOM16-85 (SEQ ID NO: 383 DOM16-87 (SEQ ID NO: 384) DOM16-88 (SEQ ID NO: 385 DOM16-89 (SEQ ID NO: 386) DOM16-90 (SEQ ID NO: 387 DOM16-91 (SEQ ID NO: 388) DOM16-92 (SEQ ID NO: 389 DOM16-94 (SEQ ID NO: 390 ) DOM16-95 (SEQ ID NO: 391 DOM16-96 (SEQ ID NO: 392) DOM16-97 (SEQ ID NO: 393 DOM16-98 (SEQ ID NO: 394) DOM16-99 (SEQ ID NO: 395) DOM16 -100 (SEQ ID NO: 396) DOM16-101 (SEQ ID NO: 397 DOM16-102 (SEQ ID NO: 398) DOM16-103 (SEQ ID NO: 399 DOM16-104 (SEQ ID NO: 400) DOM16-105 (SEQ ID NO: 401 DOM16-106 (SEQ ID NO: 402) DOM16-107 (SEQ ID NO: 403 DOM16-108 (SEQ ID NO: 404) DOM16- 109 (SEQ ID NO: 405 DOM16-110 (SEQ ID NO: 406) DOM16-111 (SEQ ID NO: 407), DOM16-112 (SEQ ID NO: 408), DOM16-113 (SEQ ID NO: 409), DOM16-114 (SEQ ID NO: 410), DOM16-115 (SEQ ID NO: 411), DOM16-116 (SEQ. ID NO: 412), DOM16-117 (SEQ ID NO: 413), DOM16-118 (SEQ ID NO: 414), DOM16-119 (SEQ ID NO: 415), DOM16-39-6 (SEQ ID NO: 416) ), DOM16-39-8 (SEQ ID NO: 417), DOM16-39-34 (SEQ ID NO: 418), DOM16-39-48 (SEQ ID NO: 419), DOM16-39-87 (SEQ ID NO. : 420), DOM16-39-90 (SEQ ID NO: 421), DOM16-39-96 (SEQ ID NO: 422), DOM16-39-100 (SEQ ID NO: 423), DOM16-39-101 (SEQ ID NO: 424), DOM16-39-102 (SEQ ID NO: 425), DOM16-39-103 (SEQ ID NO: 426), DOM16-39-104 (SEQ ID NO: 427), DOM16-39-105 (SEQ ID NO: 428), DOM16-39-106 (SEQ ID NO: 429), DOM16-39-107 (SEQ ID NO: 430), DOM16-39-108 (SEQ ID NO: 431), DOM16-39 -109 (SEQ ID NO: 432), DOM16-39-110 (SEQ ID NO: 433), DOM16-39-111 (SEQ ID NO: 434), DOM16-39-112 (SEQ ID NO: 435), DOM16 -39-113 (SEQ ID NO: 436), DOM16-39-114 (SEQ ID NO: 437), DOM16-39-115 (SEQ ID NO: 438), DOM16-39-116 (SEQ ID NO: 439) , DOM16-39-117 (SEQ ID NO: 440), DOM16-39-200 (SEQ ID NO: 441), DOM16-39-2 01 (SEQ ID NO: 442), DOM16-39-202 (SEQ ID NO: 443), DOM16-39-203 (SEQ ID NO: 444), DOM16-39-204 (SEQ ID NO: 445), DOM16- 39-205 (SEQ ID NO: 446), DOM16-39-206 (SEQ ID NO: 447), DOM16-39-207 (SEQ ID NO: 448), DOM16-39-209 (SEQ ID NO: 449), DOM16-52 (SEQ ID NO: 450), NB1 (SEQ ID NO: 451), NB2 (SEQ ID NO: 452), NB3 (SEQ ID NO: 453), NB4 (SEQ ID NO: 454), NB5 (SEQ ID NO: 455), NB6 (SEQ ID NO: 456), NB7 (SEQ ID NO: 457), NB8 (SEQ ID NO: 458), NB9 (SEQ ID NO: 459), NB10 (SEQ ID NO: 460) , NB11 (SEQ ID NO: 461), NB12 (SEQ ID NO: 462), NB13 (SEQ ID NO: 463), NB14 (SEQ ID NO: 464), NB15 (SEQ ID NO: 465), NB16 (SEQ ID NO: 466), NB17 (SEQ ID NO: 467), NB18 (SEQ ID NO: 468), NB19 (SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 (SEQ ID NO: 471), and NB22 (SEQ ID NO: 472). In some embodiments, the ligand has binding specificity for VEGF and for EGFR and comprises at least one variable domain of simple immunoglobulin with binding specificity for VEGF and at least one variable domain of single immunoglobulin with binding specificity for EGFR, wherein a single immunoglobulin variable domain with binding specificity for VEGF competes to bind to VEGF with the anti-VEGF domain antibody (dAb) selected from the group consisting of TAR15-1 (SEQ ID NO: 100), TAR15-3 (SEQ ID NO: 101), TAR15-4 (SEQ ID NO: 102), TAR15-9 (SEQ ID NO: 103), TAR15-10 (SEQ ID NO: 104), TAR15-11 (SEQ ID NO: 105), TAR15 -12 (SEQ ID NO: 106), TAR15-13 (SEQ ID NO: 107), TAR15-14 (SEQ ID NO: 108), TAR15-15 (SEQ ID NO: 109), TAR15-16 (SEQ ID NO. : 110), TAR15-17 (SEQ ID NO: 111), TAR15-18 (SEQ ID NO: 112), TAR15-19 (SEQ ID NO.-113), TAR15-20 (SEQ ID NO: 114), TAR 15-22 (SEQ ID NO: 115), TAR15-5 (SEQ ID NO: 116 TAR15-6 (SEQ ID NO: 117), TAR15-7 (SEQ ID NO: 118 TAR15-8 (SEQ ID NO: 1 19), TAR15-23 (SEQ ID NO: 120 TAR15-24 (SEQ ID NO: 121), TAR15-25 (SEQ ID NO: 122 TAR15-26 (SEQ ID NO: 123), TAR15-27 (SEQ ID NO. : TAR15-29 (SEQ ID NO: 125), TAR15-30 (SEQ ID NO: 126 TAR15 6-500 (SEQ ID NO: 127 TAR15-6-501 (SEQ ID NO: 128 TAR15- 6-502 (SEQ ID NO: 129 TAR15-6-503 (SEQ ID NO: 130 TAR15- 6-504 (SEQ ID NO: 131 TAR15-6-505 (SEQ ID NO: 132 TAR15- 6-506 (SEQ ID NO: 133 TAR15- 6-507 (SEQ ID NO: 134 TAR15- 6-508 (SEQ ID NO: 135 TAR15-6-509 (SEQ ID NO: 136 TAR15- 6-510 (SEQ ID NO: 137 TAR15-8-500 (SEQ ID NO: 138 TAR15- 8-501 (SEQ ID NO: 139 TAR15-8-502 (SEQ ID NO: 140 TAR15- 8-503 (SEQ ID NO: 141 TAR15-8-505 (SEQ ID NO: 142 TAR15- 8 -506 (SEQ ID NO: 143 TAR15-8-507 (SEQ ID NO: 144 TAR15- 8-508 (SEQ ID NO: 145 TAR15-8-509 (SEQ ID NO: 146 TAR15-8-510 (SEQ ID NO : TAR15-8-511 (SEQ ID NO: 148 TAR15-26-500 (SEQ ID NO: 149), TAR15-26-501 (SEQ ID NO: 50), TAR15-26-502 (SEQ ID NO: 151) ), TAR15-26-503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153), TAR15-26-505 (SEQ ID NO: 154), TAR15-26-506 (SEQ ID NO. : 155), TAR15-26 -507 (SEQ ID NO: 156), TAR15-26-508 (SEQ ID NO: 157), TAR15-26-509 (SEQ ID NO: 158), TAR15-26-510 (SEQ ID NO: 159), TAR15 -26-511 (SEQ ID NO: 160), TAR15-26-512 (SEQ ID NO: 161). TAR15-26-513 (SEQ ID NO: 162), TAR15-26-514 (SEQ ID NO: 163), TAR15-26-515 (SEQ ID NO: 164), TAR15-26- 516 (SEQ ID NO: 165 ), TAR15-26-517 (SEQ ID NO: 166), TAR15-26-518 (SEQ ID NO: 167), TAR15-26-519 (SEQ ID NO: 168), TAR15-26-520 (SEQ ID NO: 169), TAR15-26-521 (SEQ ID NO: 170), TAR15-26-522 (SEQ ID NO: 171), TAR15-26-523 (SEQ ID NO: 172), TAR15-26-524 (SEQ ID NO: 173), TAR15-26-525 (SEQ ID NO: 174), TAR15-26-526 (SEQ ID NO: 175), TAR15-26-527 (SEQ ID NO: 176), TAR15-26-528 ( SEQ ID NO: 177), TAR15-26-529 (SEQ ID NO: 178), TAR15-26-530 (SEQ ID NO: 179), TAR15-26-531 (SEQ ID NO: 180), TAR15-26- 532 (SEQ ID NO: 181), TAR15-26-533 (SEQ ID NO: 182), TAR15-26-534 (SEQ ID NO: 183), TAR15-26-535 (SEQ ID NO: 184), TAR15- 26-536 (SEQ ID NO: 185), TAR15-26-537 (SEQ ID NO: 186), TAR15-26-538 (SEQ ID NO: 187), TAR15-26-539 (SEQ ID NO: 188), TAR15-26-540 (SEQ ID NO: 189), TAR15-26-541 (SEQ ID NO: 190), TAR15-26-542 (SEQ ID NO: 191), TAR15-26-543 (SEQ ID NO: 192 ), TAR15-26-544 (SEQ ID NO: 193), TAR15-26-545 (SEQ ID NO: 194), TAR15-26-546 (SEQ ID NO: 195), TAR15-26-547 (SEQ ID NO. : 196), TAR15-26-548 (SEQ ID NO: 197), TAR15-26-549 (SEQ ID NO: 198), TAR15-26 -550 (SEQ ID NO: 539), and TAR15-26-551 (SEQ ID NO: 540); and a single immunoglobulin variable domain with binding specificity for EGFR competes to bind EGFR with cetuximab. For example, the variable domain of simple immunoglobulin with binding specificity for VEGF may comprise an amino acid sequence having at least about 85% amino acid sequence identity with the amino acid sequence of a dAb selected from the group consisting of TAR15. -1 (SEQ ID NO: 100) TAR15-3 (SEQ ID NO: 101), TAR15-4 (SEQ ID NO: 102) TAR15-9 (SEQ ID NO: 103), TAR15-10 (SEQ ID NO: 104) TAR15-11 ( SEQ ID NO: 105), TAR15-12 (SEQ ID NO: 106) TAR15-13 (SEQ ID NO: 107), TAR15-14 (SEQ ID NO: 108) TAR15-15 (SEQ ID NO: 109), TAR15 -16 (SEQ ID NO: 110) TAR15-17 (SEQ ID NO: 111), TAR15-18 (SEQ ID NO: 112) TAR15-19 (SEQ ID NO: 113), TAR15-20 (SEQ ID NO: 114) ) TAR 15-22 (SEQ ID NO: 115), TAR15-5 (SEQ ID NO: 116) TAR15-6 (SEQ ID NO: 117), TAR15-7 (SEQ ID NO: 118) TAR15-8 (SEQ ID NO: 119), TAR15-23 (SEQ ID NO: 120) TAR15-24 (SEQ ID NO: 121), TAR15-25 (SEQ ID NO: 122) TAR15-26 (SEQ ID NO: 123), TAR15-27 (SEQ ID NO: 124) TAR15-29 (SEQ ID NO: 125), TAR15-30 (SEQ ID NO: 126) TAR15-6-500 (SEQ ID NO: 127), TAR15- 6-501 (SEQ ID NO: 128), TAR15-6-502 (SEQ ID NO: 129), TAR15-6-503 (SEQ ID NO: 130), TAR15-6-504 (SEQ ID NO: 131), TAR15-6-505 (SEQ ID NO: 132), TAR15-6-506 (SEQ ID NO: 133), TAR15-6-507 (SEQ ID NO.134), TAR15-6-508 (SEQ ID NO: 135), TAR15-6 -509 (SEQ ID NO: 136), TAR15-6-510 (SEQ ID NO: 137), TAR15-8-500 (SEQ ID NO: 138), TAR15-8-501 (SEQ ID NO: 139), TAR15 -8-502 (SEQ ID NO: 140), TAR15-8-503 (SEQ ID NO: 141), TAR15-8-505 (SEQ ID NO: 142), TAR15-8-506 (SEQ ID NO: 143) , TAR15-8-507 (SEQ ID NO: 144), TAR15-8-508 (SEQ ID NO: 145), TAR15-8-509 (SEQ ID NO: 146), TAR15-8-510 (SEQ ID NO: 147), TAR15-8-511 (SEQ ID NO: 148), TAR15-26-500 (SEQ ID NO: 149), TAR15-26-501 (SEQ ID NO: 150), TAR15-26-502 (SEQ ID NO: 151), TAR15-26-503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153), TAR15-26-505 (SEQ ID NO: 154), TAR15-26-506 ( SEQ ID NO: 155), TAR15-26-507 (SEQ ID NO: 156), TAR15-26-508 (SEQ ID NO: 157), TAR15-26-509 (SEQ ID NO: 158), TAR15-26- 510 (SEQ ID NO: 159), TAR15-26-511 (SEQ ID NO: 160), TAR15-26-512 (SEQ ID NO: 161), TAR15-26-513 (SEQ ID NO: 162), TAR15-26-514 (SEQ ID NO: 163), TAR15-26-515 (SEQ ID NO: 164), TAR15-26-516 (SEQ ID NO: 165), TAR15-26-517 (SEQ ID NO: 166), TAR15-26-518 (SEQ ID NO: 167), TAR15-26-519 (SEQ ID NO: 168), TAR15-26-520 (SEQ ID NO: 169), TAR15-26 -521 (SEQ ID NO: 170), TAR15-26-522 (SEQ ID NO: 171), TAR15-26-523 (SEQ ID NO: 172), TAR15-26-524 (SEQ ID NO: 173), TAR15 -26-525 (SEQ ID NO: 174), TAR15-26-526 (SEQ ID NO: 175), TAR15-26-527 (SEQ ID NO: 176), TAR15-26-528 (SEQ ID NO: 177) , TAR15-26-529 (SEQ ID NO: 178), TAR15-26-530 (SEQ ID NO: 179), TAR15-26-531 (SEQ ID NO: 180), TAR15-26-532 (SEQ ID NO: 181), TAR15-26-533 (SEQ ID NO: 182), TAR15-26-534 (SEQ ID NO: 183), TAR15-26-535 (SEQ ID NO: 184), TAR15-26-536 (SEQ ID NO: 185), TAR15-26-537 (SEQ ID NO: 186), TAR15-26-538 (SEQ ID NO: 187), TAR15-26-539 (SEQ ID NO: 188), TAR15-26-540 ( SEQ ID NO: 189), TAR15-26-541 (SEQ ID NO: 190), TAR15-26-542 (SEQ ID NO.191), TAR15-26-543 (SEQ ID NO: 192), TAR15-26- 544 (SEQ ID NO: 193), TAR15-26-545 (SEQ ID NO: 194), TAR15-26-546 (SEQ ID NO: 19) 5), TAR15-26-547 (SEQ ID NO: 196), TAR15-26-548 (SEQ ID NO: 197), TAR15-26-549 (SEQ ID NO: 198), TAR15-26-550 (SEQ ID NO: 539), and TAR15-26-551 (SEQ ID NO: 540). In other embodiments, the ligand has binding specificity for VEGF and for EGFR and comprises at least one variable domain of simple immunoglobulin with binding specificity for VEGF and at least one variable domain of single immunoglobulin with binding specificity for EGFR, wherein the single immunoglobulin variable domain with binding specificity for VEGF competes to bind to VEGF with bevacizumab and / or 2C3 antibody (ATCC Access No. PTA 1595); and a single immunoglobulin variable domain with binding specificity for EGFR that competes to bind to EGFR with an anti-EGFR domain antibody (dAb) selected from the group consisting of DOM16-17 (SEQ ID NO: 325), DOM16-18 (SEQ ID NO: 326), DOM16-19 (SEQ ID NO: 327), DOM16-20 (SEQ ID NO: 328), DOM16-21 (SEQ ID NO: 329), DOM16-22 (SEQ ID NO: 330) ), DOM16-23 (SEQ ID NO: 331), DOM16-24 (SEQ ID NO: 332), DOM16-25 (SEQ ID NO: 333), DOM16-26 (SEQ ID NO: 334), DOM16-27 ( SEQ ID NO: 335), DOM16-28 (SEQ ID NO: 336), DOM16-29 (SEQ ID NO: 337), DOM16-30 (SEQ ID NO: 338), DOM16-31 (SEQ ID NO: 339) , DOM16-32 (SEQ ID NO: 340), DOM16-33 (SEQ ID NO: 341), DOM16-35 (SEQ ID NO 342), DOM16-37 (SEQ ID NO: 343), DOM16-38 (SEQ ID NO: 344), DOM16-39 (SEQ ID NO 345), DOM16-40 (SEQ ID NO 346), DOM16-41 (SEQ ID NO 347), DOM16-42 (SEQ ID NO 348), DOM16-43 (SEQ ID NO .349), DOM16-44 (SEQ ID NO 350), DOM16-45 (SEQ ID NO 351), DOM16-46 (SEQ ID NO 352), DOM16-47 (SEQ ID NO 353), DOM16-48 (SEQ ID NO 354), DOM16-49 (SEQ ID NO 355), DOM16-50 (SEQ ID NO 356), DOM16-59 (SEQ ID NO 357), DOM16-60 (SEQ ID NO 358), DOM16-61 [SEQ ID NO 359), DOM16-62 (SEQ ID NO 360), DOM16-63 [SEQ ID NO 361), DOM16-64 (SEQ ID NO 362), DOM16-65 SEQ ID NO 363), DOM16-66 [SEQ ID NO 364), DOM16-67 < SEQ ID NO 365), DOM16-68 [SEQ ID NO 366), DOM16-69 (SEQ ID NO 367), DOM16-70 SEQ ID NO 368), DOM16-71 (SEQ ID NO 369), DOM16-72 (SEQ ID NO 370), DOM16-73 (SEQ ID NO 371), DOM16-74 ('SEQ ID NO 372), DOM16-75 (SEQ ID NO 373), DOM16-76 (SEQ ID NO 374), DOM16-77 (SEQ ID NO 375), DOM16-78 (SEQ ID NO 376), DOM16-79 (SEQ ID NO 377), DOM16-80 (SEQ ID NO 378), DOM16-81 (SEQ ID NO 379), DOM16-82 (SEQ ID NO 380), DOM16-83 (SEQ ID NO 381), DOM16-84 (SEQ ID NO 382), DOM16-85 (SEQ ID NO 383), DOM16-87 (SEQ ID NO 384), DOM16-88 (SEQ ID NO: 385), DOM16-89 (SEQ ID NO: 386), DOM16-90 (SEQ ID NO: 387), DOM16-91 (SEQ ID NO: 388), DOM16-92 (SEQ ID NO: 389 DOM16-94 (SEQ ID NO: 390), DOM16-95 (SEQ ID NO: 391 DOM16-96 ( SEQ ID NO.392), DOM16-97 (SEQ ID NO: 393 DOM16-98 (SEQ ID NO: 394), DOM16-99 (SEQ ID NO: 395 DOM16-100 (SEQ ID NO: 396), DOM16-101 (SEQ ID NO: 397 DOM16-102 (SEQ ID NO: 398), DOM16-103 (SEQ ID NO: 399 DOM16-104 (SEQ ID NO: 400), DOM16-105 (SEQ ID NO: 401 DOM16-106 ( SEQ ID NO: 402), DOM16-107 (SEQ ID NO: 403 DOM16-108 (SEQ ID NO: 404), DOM16-109 (SEQ ID NO: 405 DOM16-110 (SEQ ID NO: 406), DOM16-111 (SEQ ID NO: 407 DOM16-112 (SEQ ID NO: 408), DOM16-113 (SEQ ID NO: 409 DOM16-114 (SEQ ID NO: 410), DOM16-115 (SEQ ID NO: 411 DOM16-116 ( SEQ ID NO: 412), DOM16-117 (SEQ ID NO: 413 DOM16-118 (SEQ ID NO: 414), DOM16-119 (SEQ ID NO: 415 DOM16-39-6 (SEQ ID NO: 416), DOM16 -39-8 (SEQ ID NO: 417 DOM16-39-34 (SEQ ID NO: 418), DOM16-39-48 (SEQ ID NO: 419), DOM16-39-87 (SEQ ID NO: 420), DOM16 -39-90 (SEQ ID NO: 421), DOM16-39-96 (SEQ ID NO: 422), DOM16-39-100 (SEQ ID NO: 423), DOM16-39-101 (SEQ ID NO: 424) , D OM16-39-102 (SEQ ID NO: 425), DOM16-39-103 (SEQ ID NO: 426), DOM16-39-104 (SEQ ID NO: 427), DOM16-39-105 (SEQ ID NO: 428) ), DOM16-39-106 (SEQ ID NO: 429), DOM16-39-107 (SEQ ID NO: 430), DOM16-39-108 (SEQ ID NO: 431), DOM16-39-109 (SEQ ID NO. : 432), DOM16-39-110 (SEQ ID NO: 433), DOM16-39-111 (SEQ ID NO: 434), DOM16-39-112 (SEQ ID NO: 435), DOM16-39-113 (SEQ. ID NO: 436), DOM16-39-114 (SEQ ID NO: 437), DOM16-39-115 (SEQ ID NO: 438), DOM16-39-116 (SEQ ID NO: 439), DOM16-39-117 (SEQ ID NO: 440), DOM16-39-200 (SEQ ID NO: 441), DOM16-39-201 (SEQ ID NO: 442), DOM16-39-202 (SEQ ID NO: 443), DOM16-39 -203 (SEQ ID NO: 444), DOM16-39-204 (SEQ ID NO: 445), DOM16-39-205 (SEQ ID NO: 446), DOM16-39-206 (SEQ ID NO: 447), DOM16 -39-207 (SEQ ID NO: 448), DOM16-39-209 (SEQ ID NO: 449), DOM16-52 (SEQ ID NO: 450), NB1 (SEQ ID NO: 451), NB2 (SEQ ID NO. : 452), NB3 (SEQ ID NO: 453), NB4 (SEQ ID NO: 454), NB5 (SEQ ID NO: 455), NB6 (SEQ ID NO: 456), NB7 (SEQ ID NO: 457), NB8 (SEQ ID NO: 458), NB9 (SEQ ID NO: 459), NB10 (SEQ ID NO: 460), NB11 (SEQ ID NO: 461), NB12 (SEQ ID NO: 462), NB13 (SEQ ID NO: 463), NB14 (SEQ ID NO: 464), NB15 (SEQ ID NO: 465), NB16 (SEQ ID NO: 466), NB17 (SEQ ID NO: 467), NB18 (SEQ ID NO: 468), NB 19 (SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 (SEQ ID NO: 471), and NB22 (SEQ ID NO: 472). For example, the variable domain of single immunoglobulin with binding specificity for EGFR may comprise an amino acid sequence having at least about 85% amino acid sequence identity with the amino acid sequence of a dAb selected from the group consisting of DOM16 -17 (SEQ ID NO: 325), DOM16-18 (SEQ ID NO: 326), DOM16-19 (SEQ ID NO: 327), DOM16-20 (SEQ ID NO: 328), DOM16-21 (SEQ ID NO. : 329), DOM16-22 (SEQ ID NO: 330), DOM16-23 (SEQ ID NO: 331), DOM16-24 (SEQ ID NO: 332), DOM16-25 (SEQ ID NO: 333), DOM16-26 (SEQ ID NO: 334), DOM16-27 (SEQ ID NO: 335), DOM16-28 (SEQ ID NO: 336), DOM16-29 (SEQ ID NO: 337), DOM16-30 (SEQ ID NO • 338), DOM16-31 (SEQ ID NO 339), DOM16-32 (SEQ ID NO 340), DOM16-33 (SEQ ID NO: 341), DOM16-35 (SEQ ID NO 342), DOM16-37 (SEQ ID NO 343), DOM16-38 (SEQ ID NO 344), DOM16-39 (SEQ ID NO 345), DOM16-40 (SEQ ID NO 346), DOM16-41 (SEQ ID NO 347), DOM16-42 (SEQ ID NO 348), DOM16-43 (SEQ ID NO 349), DOM16-44 (SEQ ID NO 350), DOM16-45 (SEQ ID NO 351), DOM16-46 (SEQ ID NO 352), DOM16-47 [SEQ ID NO 353), DOM16-48 (SEQ ID NO 354), DOM16-49 [SEQ ID NO 355), DOM16-50 [SEQ ID NO 356), DOM16-59 ([SEQ ID NO 357), DOM16-60 [SEQ ID NO 358), DOM16-61 (SEQ ID NO 359), DOM16-62 [SEQ ID NO 360), DOM16-63 (SEQ ID NO 361), DOM16-64 (SEQ ID NO 362), DOM16-65 (SEQ ID NO 363), DOM16-66 (SEQ ID NO 364), DOM16-67 (SEQ ID NO 365), DOM16-68 (SEQ ID NO 366), DOM16-69 (SEQ ID NO 367), DOM16-70 (SEQ ID NO 368), DOM16-71 (SEQ ID NO 369), DOM16-72 (SEQ ID NO 370), DOM16-73 (SEQ ID NO 371), DOM16-74 (SEQ ID NO 372), DOM16-75 (SEQ ID NO 373), DOM16-76 (SEQ ID NO 374), DOM16-77 (SEQ ID NO: 375), DOM16-78 (SEQ ID NO: 376), DOM16-79 (SEQ ID NO- 377), DOM16-80 (SEQ ID NO: 378), DOM16-81 (SEQ ID NO: 379), DOM16-82 (SEQ ID NO: 380), DOM16-83 (SEQ ID NO: 381 DOM16-84 (SEQ ID NO: 382 DOM16-85 (SEQ ID NO: 383 DOM16-87 (SEQ ID NO: 384 DOM16-88 (SEQ ID NO: 385 DOM16-89 (SEQ ID NO: 386 DOM16-90 (SEQ ID NO: 387 DOM16-91 (SEQ ID NO: 388 DOM16-92 (SEQ ID NO: 389 DOM16- 94 (SEQ ID NO: 390 DOM16-95 (SEQ ID NO: 391 DOM16-96 (SEQ ID NO: 392 DOM16-97 (SEQ ID NO: 393 DOM16-98 (SEQ ID NO: 394 DOM16-99 (SEQ ID NO. : 395 DOM16-100 (SEQ ID NO: 396 DOM16-101 (SEQ ID NO: 397 DOM16-102 (SEQ ID M): 398] DOM16-103 (SEQ ID NO: 399 DOM16-104 (SEQ ID NO: 400 DOM16 -105 (SEQ ID NO: 401 DOM16-106 (SEQ ID NO: 402 DOM16-107 (SEQ ID NO: 403 DOM16-108 (SEQ ID NO: 404 DOM16-109 (SEQ ID NO: 405 DOM16-110 (SEQ ID NO: 406 DOM16-111 (SEQ ID NO: 407 DOM16-112 (SEQ ID NO: 408 DOM16-113 (SEQ ID NO: 409 DOM16-114 (SEQ ID NO: 410 DOM16-115 (SEQ ID NO: 411 DOM16- 116 (SEQ ID NO: 412 DOM16-117 (SEQ ID NO: 413 DOM16-118 (SEQ ID NO: 414 DOM16-119 (SEQ ID NO: 415 DOM16-39-6 (SEQ ID NO: 416 DOM16-39-8 (SEQ ID NO: 417 DOM16-39-34 (SEQ ID NO: 418), DOM16-39-48 ( SEQ ID NO: 419), DOM16-39-87 (SEQ ID NO: 420), DOM16-39-90 (SEQ ID NO: 421), DOM16-39-96 (SEQ ID NO: 422), DOM16-39- 100 (SEQ ID NO: 423), DOM16-39-101 (SEQ ID NO: 424), DOM16-39-102 (SEQ ID NO: 425), DOM16-39-103 (SEQ ID NO: 426), DOM16- 39-104 (SEQ ID NO: 427), DOM16-39-105 (SEQ ID NO: 428), DOM16-39-106 (SEQ ID NO: 429), DOM16-39-107 (SEQ ID NO: 430), DOM16-39-108 (SEQ ID NO: 431), DOM16-39-109 (SEQ ID NO: 432), DOM16-39-110 (SEQ ID NO: 433), DOM16-39-111 (SEQ ID NO: 434) ), DOM16-39-112 (SEQ ID NO: 435), DOM16-39-113 (SEQ ID NO: 436), DOM16-39-114 (SEQ ID NO: 437), DOM16-39-115 (SEQ ID NO. : 438), DOM16-39-116 (SEQ ID NO: 439), DOM16-39-117 (SEQ ID NO: 440), DOM16-39-200 (SEQ ID NO: 441), DOM16-39-201 (SEQ ID NO: 442), DOM16-39-202 (SEQ ID NO: 443), DOM16-39-203 (SEQ ID NO: 444), DOM16-39-204 (SEQ ID NO.445), DOM16-39-205 (SEQ ID NO: 446), DOM16-39-206 (SEQ ID NO: 447), DOM16-39-207 (SEQ ID NO.448), DOM16-39-209 (SEQ ID NO: 449), DOM16-52 (SEQ ID NO: 450), NB1 (SEQ ID NO: 451), NB2 (SEQ ID NO: 452), NB3 (SEQ ID NO: 45 3), NB4 (SEQ ID NO: 454), NB5 (SEQ ID NO: 455), NB6 (SEQ ID NO: 456), NB7 (SEQ ID NO: 457), NB8 (SEQ ID NO: 458), NB9 ( SEQ ID NO: 459), NB10 (SEQ ID NO: 460), NB11 (SEQ ID NO: 461), NB12 (SEQ ID NO: 462), NB13 (SEQ ID NO: 463), NB14 (SEQ ID NO: 464 ), NB15 (SEQ ID NO: 465), NB 16 (SEQ ID NO: 466), NB 17 (SEQ ID NO: 467), NB 18 (SEQ ID NO: 468), NB19 (SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 (SEQ ID NO: 471), and NB22 (SEQ ID NO: 472). In other embodiments, the ligand having binding specificity for VEGF and for EGFR comprises a first variable domain of single immunoglobulin with binding specificity for VEGF and comprises a second variable domain of single immunoglobulin with binding specificity for EGFR, wherein the first single immunoglobulin variable domain competes to bind VEGF with bevacizumab and / or a 2C3 antibody (ATCC Access No. PTA 1595); and the second variable domain of simple immunoglobulin competes to bind EGFR with ceruximab. In particular embodiments, the ligand has binding specificity for VEGF and for EGFR and comprises at least one variable domain of simple immunoglobulin with binding specificity for VEGF and at least one variable domain of single immunoglobulin with binding specificity for EGFR, wherein the ligand comprises a single immunoglobulin variable domain with binding specificity for VEGF comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence of an anti-VEGF dAb selected from the group consisting of TAR15-6 (SEQ ID NO: 117), TAR15-8 (SEQ ID NO: 119), and TAR15-26 (SEQ ID NO: 123), and further comprises a variable domain of simple immunoglobulin with binding specificity for EGFR that comprises an amino acid sequence having at least 90% amino acid sequence identity with an amino acid sequence selected from the group consisting of DOM16-39 (SEQ ID NO: 345), OD M16-39-87 (SEQ ID NO: 420), DOM16-39-100 (SEQ ID NO: 423), DOM16-39-107 (SEQ ID NO: 430), DOM16-39-109 (SEQ ID NO: 432 ), DOM16-39-115 (SEQ ID NO: 438), or DOM16-39-200 (SEQ ID NO: 441). The ligand having binding specificity for VEGF and for EGFR can inhibit the binding of epidermal growth factor (EGF) and / or transform the growth factor alpha (TGF alpha) to EGFR, inhibit EGFR activity, and / or inhibit EGFR activity without substantially inhibiting the binding of epidermal growth factor (EGF) and / or transforming growth factor alpha (TGF alpha) to EGFR. In addition, or alternatively, the ligand having binding specificity for VEGF and for EGFR can inhibit the binding of VEGF to vascular endothelial growth factor receptor 1 (VEGFRI) and / or vascular endothelial growth factor receptor 2 ( VEGFR2), inhibits VEGF activity and / or inhibits VEGF activity without substantially inhibiting the binding of VEGF to VEGFR1 and / or VEGFR2. The ligand having binding specificity for VEGF and for EGFR may contain a protein binding portion (eg, single immunoglobulin variable domain) with binding specificity for VEGF that binds VEGF with an affinity (KD) that is between about 100 nM and approximately 1 pM, as determined by surface plasmon resonance. The ligand having binding specificity for VEGF and for EGFR may contain a protein binding portion (eg, single immunoglobulin variable domain) with binding specificity for EGFR that binds EGFR with an affinity (KD) that is between about 100 nM and approximately 1 pM or approximately 10 nM to approximately 100 pM, as determined by surface plasmon resonance. The ligand having binding specificity for VEGF and for EGFR can bind VEGF with an affinity (KD) that is between about 100 nM and about 1 pM, as determined by surface plasmon resonance. The ligand having binding specificity for VEGF and for EGFR can bind EGFR with an affinity (KD) that is between about 100 nM and about 1 pM or about 10 nM to about 100 pM, as determined by surface plasmon resonance . The ligand having binding specificity for VEGF and for EGFR may comprise a single immunoglobulin variable domain with binding specificity for VEGF which is a VHH and / or a single immunoglobulin variable domain with binding specificity for EGFR which is a VHH. The ligand having binding specificity for VEGF and for EGFR may comprise a single immunoglobulin variable domain with binding specificity for VEGF and a single immunoglobulin variable domain with binding specificity for EGFR, wherein the single immunoglobulin domains are selected from the group consisting of human VH and human VL. In some modalities,, the ligand having binding specificity for VEGF and for EGFR can be an IgG type format comprising two variable domains of single immunoglobulin with binding specificity for VEGF, and two variable domains of single immunoglobulin with binding specificity for EGFR. In some embodiments, the ligand having binding specificity for VEGF and for EGFR may comprise an Fc antibody region. The present invention also relates to a ligand having binding specificity for VEGF, comprising at least one variable domain of single immunoglobulin with binding specificity for VEGF, wherein a single immunoglobulin variable domain with binding specificity for VEGF competes for link to VEGF with an anti-VEGF (dAb) domain antibody selected from the group consisting of TAR15-1 (SEQ ID NO: 100), TAR15-3 (SEQ ID NO: 101), TAR15-4 (SEQ ID NO: 102), TAR15-9 (SEQ ID NO: 103), TAR15-10 (SEQ ID NO: 104), TAR15-11 (SEQ ID NO: 105), TAR15-12 (SEQ ID NO: 106), TAR15-13 (SEQ ID NO: 107), TAR15-14 (SEQ ID NO: 108), TAR15-15 (SEQ ID NO: 109), TAR15-16 (SEQ ID NO: 110), TAR15-17 (SEQ ID NO: 111) ), TAR15-18 (SEQ ID NO: 112) TAR15-19 (SEQ ID NO: 113), TAR15-20 (SEQ ID NO: 114) TAR 15-22 (SEQ ID NO: 115), TAR15-5 (SEQ ID NO: 116) TAR15-6 (SEQ ID NO: 117), TAR15-7 (SEQ ID NO: 118) TAR15-8 (SEQ ID NO: 119), TAR15-23 (SEQ ID NO: 120) TAR15-24 (SEQ ID NO: 121), TAR15-25 (SEQ ID NO: 122) TAR15-26 (SEQ ID NO: 123), TAR15-27 (SEQ ID NO: 124) TAR15-29 (SEQ ID NO: 125), TAR15-30 (SEQ ID NO: 126) TAR15-6-500 (SEQ ID NO: 127), TAR15-6-501 (SEQ D NO: 128) TAR15-6-502 (SEQ ID NO: 129 TAR15-6-503 (SEQ ID NO: 130) TAR15-6-504 (SEQ ID NO: 131 TAR15-6-505 (SEQ ID NO: 132 ) TAR15-6-506 (SEQ ID NO: 133 TAR15-6-507 (SEQ ID NO: 134) TAR15-6-508 (SEQ ID NO: 135 TAR15-6-509 (SEQ ID NO: 136) TAR15-6 -510 (SEQ ID NO: 137 TAR15-8-500 (SEQ ID NO: 138) TAR15-8-501 (SEQ ID NO: 139 TAR15-8-502 (SEQ ID NO: 140) TAR15-8-503 (SEQ ID NO: 141 TAR15-8-505 (SEQ ID NO: 142) TAR15-8-506 (SEQ ID NO: 143 TAR15-8-507 (SEQ ID NO: 144) TAR15-8-508 (SEQ ID NO: 145 TAR15-8-509 (SEQ ID NO: 146) TAR15-8-510 (SEQ ID NO: 147 TAR15-8-511 (SEQ ID NO: 148), TAR15-26-500 (SEQ ID NO: 149), TAR15 -26-501 (SEQ ID NO: 150), TAR15-26-502 (SEQ ID NO: 151), TAR15-26-503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153) , TAR15-26-505 (SEQ ID NO: 154), TAR15-26-506 (SEQ ID NO: 155), TAR15-26-507 (SEQ ID NO: 156), TAR15-26-508 (SEQ ID NO: 157), TAR15-26-509 (SEQ ID NO: 158), TAR15-26-510 (SEQ ID NO: 159), TAR15-26-511 (SEQ ID NO: 160), TAR15-26-512 (SEQ ID NO: 161), TAR15-26-513 (SEQ ID NO: 162), TAR15-26-514 (SEQ ID NO: 163), TAR15-26-515 (SEQ ID NO: 164), TAR15-26-516 (SEQ ID NO: 165), TAR15-26-517 (SEQ ID NO: 166), TAR15-26-518 (SEQ ID NO: 167), TAR15-26-519 (SEQ ID NO: 168), TAR15-26-520 (SEQ ID NO: 169), TAR15-26-521 (SEQ ID NO: 170), TAR15-26 -522 (SEQ ID NO: 171), TAR15-26-523 (SEQ ID NO: 172), TAR15-26-524 (SEQ ID NO: 173), TAR15-26-525 (SEQ ID NO: 174), TAR15 -26-526 (SEQ ID NO: 175), TAR15-26-527 (SEQ ID NO: 176), TAR15-26-528 (SEQ ID NO: 177), TAR15-26-529 (SEQ ID NO: 178) , TAR15-26-530 (SEQ ID NO: 179), TAR15-26-531 (SEQ ID NO: 180), TAR15-26-532 (SEQ ID NO: 181), TAR15-26-533 (SEQ ID NO: 182), TAR15-26-534 (SEQ ID NO: 183), TAR15-26-535 (SEQ ID NO: 184), TAR15-26-536 (SEQ ID NO: 185), TAR15-26-537 (SEQ ID NO: 186), TAR15-26-538 (SEQ ID NO: 187), TAR15-26-539 (SEQ ID NO: 188), TAR15-26-540 (SEQ ID NO: 189), TAR15-26-541 ( SEQ ID NO: 190), TAR15-26-542 (SEQ ID NO: 191), TAR15-26-543 (SEQ ID NO: 192), TAR15-26-544 (SEQ ID NO: 193), TAR15-26- 545 (SEQ ID NO: 194), TAR15-26-546 (SEQ ID NO: 195), TAR15-26-547 (SEQ ID NO: 196) ), TAR15-26-548 (SEQ ID NO: 197), TAR15-26-549 (SEQ ID NO: 198), TAR15-26-550 (SEQ ID NO: 539), and TAR15-26-551 (SEQ ID NO: 540). For example, a single immunoglobulin variable domain with binding specificity for VEGF may comprise an amino acid sequence having at least about 85% amino acid sequence identity with the amino acid sequence of a dAb selected from the group consisting of TAR15. -1 (SEQ ID NO: 100 TAR15-3 (SEQ ID NO: 101), TAR15-4 (SEQ ID NO: 102 TAR15-9 (SEQ ID NO: 103), TAR15-10 (SEQ ID NO: 104 TAR15-11 (SEQ ID NO : 105), TAR15-12 (SEQ ID NO: 106 TAR15-13 (SEQ ID NO: 107), TAR15-14 (SEQ ID NO: 108 TAR15-15 (SEQ ID NO: 109), TAR15-16 (SEQ ID NO: 110 TAR15-17 (SEQ ID NO: 111), TAR15-18 (SEQ ID NO: 112 TAR15-19 (SEQ ID NO: 113), TAR15-20 (SEQ ID NO: 114 TAR 15-22 (SEQ ID NO: 115), TAR15-5 (SEQ ID NO: 116 TAR15-6 (SEQ ID NO: 117), TAR15-7 (SEQ ID NO: 118 TAR15-8 (SEQ ID NO: 119), TAR15-23 (SEQ ID NO: 120 TAR15-24 (SEQ ID NO: 121), TAR15-25 (SEQ ID NO: 122 TAR15-26 (SEQ ID NO: 123), TAR15-27 (SEQ ID NO: 124 TAR15-29 (SEQ ID NO: 125), TAR15-30 (SEQ ID NO: 126 TAR15-6-500 (SEQ ID NO: 127), TAR15- 6-501 (SEQ ID NO: 128), TAR15-6-502 (SEQ ID NO: 129), TAR15-6-503 (SEQ ID NO: 130), TAR15-6-504 (SEQ ID NO: 131), TAR15-6-505 (SEQ ID NO: 132), TAR15-6-506 (SEQ ID NO: 133), TAR15-6-507 (SEQ ID NO: 134), TAR15-6-508 (SEQ ID NO: 135), TAR15-6-509 (SEQ ID NO: 136), TAR15-6-510 ( SEQ ID NO: 137), TAR15-8-500 (SEQ ID NO: 138), TAR15-8-501 (SEQ ID NO: 139), TAR15-8-502 (SEQ ID NO: 140), TAR15-8-503 (SEQ ID NO: 141), TAR15-8-505 (SEQ ID NO: 142) ), TAR15-8-506 (SEQ ID NO: 143), TAR15-8-507 (SEQ ID NO: 144), TAR15-8-508 (SEQ ID NO: 145), TAR15-8-509 (SEQ ID NO. : 146), TAR15-8-510 (SEQ ID NO: 147), TAR15-8-511 (SEQ ID NO: 148), TAR15-26-500 (SEQ ID NO: 149), TAR15-26-501 (SEQ ID NO: 150), TAR15-26-502 (SEQ ID NO: 151), TAR15-26-503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153), TAR15-26-505 (SEQ ID NO: 154), TAR15-26-506 (SEQ ID NO: 155), TAR15-26-507 (SEQ ID NO: 156), TAR15-26-508 (SEQ ID NO: 157), TAR15-26-509 (SEQ ID NO: 158), TAR15-26-510 (SEQ ID NO: 159), TAR15-26-511 (SEQ ID NO: 160), TAR15-26-512 (SEQ ID NO: 161), TAR15-26-513 (SEQ ID NO: 162), TAR15-26-514 (SEQ ID NO: 163), TAR15-26-515 (SEQ ID NO: 164), TAR15-26-516 (SEQ ID NO: 165), TAR15-26-517 (SEQ ID NO: 166), TAR15-26-518 (SEQ ID NO: 167), TAR15-26-519 (SEQ ID NO: 168), TAR15-26-520 ( SEQ ID NO: 169), TAR15-26-521 (SEQ ID NO: 170), TAR15-26-522 (SEQ ID NO: 171), TAR15-26-523 (SEQ ID NO: 172), TAR15-26- 524 (SEQ ID NO: 173), TAR15-26-525 (SEQ ID NO: 174), TAR15-26-526 (SEQ ID NO: 175), TAR15-26-527 (SEQ ID NO: 176), TAR15- 26-528 (SEQ ID NO: 177), TAR15-26-529 (SEQ ID NO: 178), TAR15-26-530 (SEQ ID NO: 179), TAR15-26-531 (SEQ ID NO: 180), TAR15-26-532 (SEQ ID NO: 181), TAR15-26-533 (SEQ ID NO: 182), TAR15-26-534 (SEQ ID NO: 183), TAR15-26-535 (SEQ ID NO: 184 ), TAR15-26-536 (SEQ ID NO: 185), TAR15-26-537 (SEQ ID NO: 186), TAR15-26-538 (SEQ ID NO: 187), TAR15-26-539 (SEQ ID NO. : 188), TAR15-26-540 (SEQ ID NO: 189), TAR15-26-541 (SEQ ID NO: 190), TAR15-2 6-542 (SEQ ID NO: 191), TAR15-26-543 (SEQ ID NO: 192), TAR15-26-544 (SEQ ID NO: 193), TAR15-26-545 (SEQ ID NO: 194), TAR15-26-546 (SEQ ID NO: 195), TAR15-26-547 (SEQ ID NO: 196), TAR15-26-548 (SEQ ID NO: 197), TAR15-26-549 (SEQ ID NO: 198) ), TAR15-26-550 (SEQ ID NO: 539), and TAR15-26-551 (SEQ ID NO: 540). The ligand having binding specificity for VEGF can inhibit the binding of VEGF to vascular endothelial growth factor receptor 1 (VEGFR1) and / or vascular endothelial growth factor receptor 2 (VEGFR2), inhibits VEGF activity and / or inhibits VEGF activity without substantially inhibiting the binding of VEGF to VEGFR1 and / or VEGFR2. The ligand having binding specificity for VEGF can contain a single immunoglobulin variable domain with binding specificity for VEGF that binds VEGF with an affinity (KD) that is between about 100 nM and about 1 pM, as determined by resonance of plasmon of surface. The ligand having binding specificity for VEGF can bind VEGF with an affinity (KD) that is between about 100 nM and about 1 pM, as determined by surface plasmon resonance. The ligand having binding specificity for VEGF may comprise a single immunoglobulin variable domain with binding specificity for VEGF which is a VHH- The ligand having binding specificity for VEGF may comprise a single immunoglobulin variable domain with binding specificity for VEGF that is selected from the group consisting of human VH and human V. In some embodiments, the ligand having binding specificity for VEGF is an IgG type format comprising at least two single immunoglobulin variable domains with binding specificity for VEGF. In some embodiments, the ligand having binding specificity for VEGF comprises an Fc antibody region. The present invention also relates to a ligand having binding specificity for EGFR comprising at least one variable domain of single immunoglobulin with binding specificity for EGFR, wherein a single immunoglobulin variable domain with binding specificity for EGFR competes to bind to EGFR with an anti-EGFR domain antibody (dAb) selected from the group consisting of DOM16-17 (SEQ ID NO: 325), DOM16-18 (SEQ ID NO: 326), DOM16-19 (SEQ ID NO: 327) ), DOM16-20 (SEQ ID NO: 328), DOM16-21 (SEQ ID NO: 329) DOM16-22 (SEQ ID NO: 330), DOM16-23 (SEQ ID NO: 331) DOM16-24 (SEQ ID NO: 332), DOM16-25 (SEQ ID NO: 333) DOM16-26 (SEQ ID NO: 334), DOM16-27 (SEQ ID NO: 335) DOM16-28 (SEQ ID NO: 336), DOM16 -29 (SEQ ID NO 337), DOM16- -30 (SEQ ID NO: 338), DOM16 -31 (SEQ ID NO 339), DOM16- -32 (SEQ ID NO 340), DOM16 -33 (SEQ ID NO 341), DOM16- -35 (SEQ ID NO 342), DOM16 -37 (SEQ ID NO 343), DOM16 -38 (SEQ ID NO • 344), DOM16 -39 (SEQ ID NO 345), DOM16 -40 (SEQ ID NO 346), DOM16-41 (SEQ ID NO 347), DOM16-42 (SEQ ID NO 348), DOM16-43 (SEQ ID NO 349), DOM16-44 (SEQ ID NO .350), DOM16 -45 (SEQ ID NO 351), DOM16- -46 (SEQ ID NO: 352), DOM16 -47 (SEQ ID NO 353), DOM16- -48 (SEQ ID NO 354), DOM16-49 (SEQ ID NO 355), DOM16-50 [SEQ ID NO 356), DOM16- -59 (SEQ ID NO 357), DOM16- -60 (SEQ ID NO 358), DOM16-61 (SEQ ID NO 359), DOM16-62 (SEQ ID NO 360), DOM16-63 (SEQ ID NO 361), DOM16- -64 (SEQ ID NO 362), DOM16-65 (SEQ ID NO 363), DOM16-66 (SEQ ID NO 364), DOM16-67 [SEQ ID NO 365), DOM16-68 [SEQ ID NO 366], DOM16-69 [SEQ ID NO 367), DOM16-70 [SEQ ID NO 368], DOM16-71 (SEQ ID NO 369), DOM16-72 [SEQ ID NO 370), DOM16-73 (SEQ ID NO 371), DOM16- -74 [SEQ ID NO 372), DOM16- -75 [SEQ ID NO 373), DOM16- -76 [SEQ ID NO 374), DOM16-77 [SEQ ID NO 375), DOM16-78 I [SEQ ID NO 376), DOM16- -79 [SEQ ID NO 377), DOM16- -80 [SEQ ID NO 378), DOM16- -81 [SEQ ID NO 379), DOM16- -82 [SEQ ID NO 380), DOM16-83 (SEQ ID No. 381), DOM16-84 ([SEQ ID No. 382), DOM16-85 (SEQ ID NO 383), DOM16-87 (SEQ ID NO 384), DOM16-88 (SEQ ID NO 385), DOM16-89 (SEQ ID NO 386), DOM16-90 (SEQ ID NO: 387), DOM16-91 (SEQ ID NO: 388) DOM16-92 (SEQ ID NO: 389) ), DOM16-94 (SEQ ID NO: 390) DOM16-95 (SEQ ID NO: 391), DOM16-96 (SEQ ID NO: 392) DOM16-97 (SEQ ID NO: 393), DOM16-98 (SEQ ID NO: 394) DOM16-99 (SEQ ID NO: 395), DOM16-100 (SEQ ID NO: 396) DOM16-101 (SEQ ID NO: 397), DOM16-102 (SEQ ID NO: 398) DOM16-103 ( SEQ ID NO: 399), DOM16-104 (SEQ ID NO: 400) DOM16-105 (SEQ ID NO: 401), DOM16-106 (SEQ ID NO: 402) DOM16-107 (SEQ ID NO: 403), DOM16 -108 (SEQ ID NO: 404) DOM16-109 (SEQ ID NO: 405), DOM16-110 (SEQ ID NO: 406) DOM16-111 (SEQ ID NO: 407), DOM16-112 (SEQ ID NO: 408) ) DOM16-113 (SEQ ID NO: 409), DOM16-114 (SEQ ID NO: 410) DOM16-115 (SEQ ID NO: 411), DOM16-116 (SEQ ID NO: 412) DOM16-117 (SEQ ID NO. : 413), DOM16-118 (SEQ ID NO: 414) DOM16-119 (SEQ ID NO: 415), DOM16-39-6 (SEQ ID NO: 416) DOM16-39-8 (SEQ ID NO: 417), DOM16-39-34 (SEQ ID NO: 418) DOM16-39-48 (SEQ ID NO: 419), DOM16-39-87 (SEQ ID NO: 420), DOM16-39-90 (SEQ ID NO: 421) , DOM16-39-96 (SEQ ID NO: 422), DOM16-39-100 (SEQ ID NO: 423), DOM16-39-101 (SEQ ID NO: 424), DOM16-39-102 (SEQ ID NO: 425), DOM16-39 -103 (SEQ ID NO: 426), DOM16-39-104 (SEQ ID NO: 427), DOM16-39-105 (SEQ ID NO: 428), DOM16-39-106 (SEQ ID NO: 429), DOM16 -39-107 (SEQ ID NO: 430), DOM16-39-108 (SEQ ID NO: 431), DOM16-39-109 (SEQ ID NO: 432), DOM16-39-110 (SEQ ID NO: 433) , DOM16-39-111 (SEQ ID NO: 434), DOM16-39-112 (SEQ ID NO: 435), DOM16-39-113 (SEQ ID NO: 436), DOM16-39-114 (SEQ ID NO: 437), DOM16-39-115 (SEQ ID NO: 438), DOM16-39-116 (SEQ ID NO: 439), DOM16-39-117 (SEQ ID NO: 440), DOM16-39-200 (SEQ ID NO: 441), DOM16-39-201 (SEQ ID NO: 442), DOM16-39-202 (SEQ ID NO: 443), DOM16-39-203 (SEQ ID NO: 444), DOM16-39-204 ( SEQ ID NO: 445), DOM16-39-205 (SEQ ID NO: 446), DOM16-39-206 (SEQ ID NO: 447), DOM16-39-207 (SEQ ID NO: 448), DOM16-39- 209 (SEQ ID NO: 449), DOM16-52 (SEQ ID NO: 450), NB1 (SEQ ID NO: 451), NB2 (SEQ ID NO: 452), NB3 (SEQ ID NO: 453), NB4 (SEQ ID NO: 454), NBS (SEQ ID NO: 455), NB6 (SEQ ID NO: 456), NB7 (SEQ ID NO: 457) , NB8 (SEQ ID NO: 458), NB9 (SEQ ID NO: 459), NB10 (SEQ ID NO: 460), NB11 (SEQ ID NO: 461), NB12 (SEQ ID NO: 462), NB13 (SEQ ID NO: 463), NB14 (SEQ ID NO: 464), NB15 (SEQ ID NO: 465), NB16 (SEQ ID NO: 466), NB17 (SEQ ID NO: 467), NB18 (SEQ ID NO: 468), NB19 (SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 (SEQ ID NO: 471), and NB22 (SEQ ID NO: 472). For example, the variable domain of single immunoglobulin with binding specificity for EGFR may comprise an amino acid sequence having at least about 85% amino acid sequence identity with the amino acid sequence of a dAb selected from the group consisting of DOM16 -17 (SEQ ID NO: 325), DOM16-18 (SEQ ID NO: 326), DOM16-19 (SEQ ID NO: 327 DOM16-20 (SEQ ID NO: 328), DOM16-21 SEQ ID NO: 329 DOM16-22 (SEQ ID NO 330), DOM16-23 SEQ ID NO: 331 DOM16-24 (SEQ ID NO: 332), DOM16-25 SEQ ID NO: 333 DOM16-26 (SEQ ID NO 334), DOM16-27 SEQ ID NO: 335 DOM16-28 (SEQ ID NO 336), DOM16-29 SEQ ID NO: 337 DOM16-30 (SEQ ID NO 338), DOM16-31 SEQ ID NO: 339 DOM16-32 (SEQ ID NO 340), DOM16-33 SEQ ID NO: 341 DOM16-35 (SEQ ID NO 342), DOM16-37 SEQ ID NO: 343 DOM16-38 (SEQ ID NO 344), DOM16-39 SEQ ID NO: 345 DOM16-40 (SEQ ID NO 346), DOM16-41 SEQ ID NO: 347 DOM16-42 (SEQ ID NO 348), DOM16-43 SEQ ID NO: 349 DOM16-44 (SEQ ID NO 350), DOM16-45 SEQ ID NO: 351 DOM16-46 (SEQ ID NO 352), DOM16-47 SEQ ID NO: 353 DOM16-48 (SEQ ID NO 354), DOM16-49 SEQ ID NO: 355 DOM16-50 (SEQ ID NO 356), DOM16-59 SEQ ID NO: 357 DOM16-60 (SEQ ID NO 358), DOM16-61 SEQ ID NO: 359 DOM16-62 (SEQ ID NO 360), DOM16-63 SEQ ID NO: 361 DOM16-64 (SEQ ID NO 362), DOM16-65 SEQ ID NO: 363 DOM16-66 (SEQ ID NO 364), DOM16-67 SEQ ID NO: 365 DOM16-68 (SEQ ID NO 366), DOM16-69 SEQ ID NO: 367 DOM16-70 (SEQ ID NO 368), DOM16-71 SEQ ID NO: 369 DOM16-72 (SEQ ID NO 370), DOM16-73 SEQ ID NO: 371 DOM16-74 (SEQ ID NO ß72), DOM16-75 SEQ ID NO: 373 DOM16-76 (SEQ ID NO 374), DOM16-77 SEQ ID NO: 375 DOM16-78 (SEQ ID NO 376), DOM16-79 SEQ ID NO: 377 DOM16-80 (SEQ ID NO: 378 DOM16-81 SEQ ID NO: 379 DOM16-82 (SEQ ID NO: 380 DOM16-83 SEQ ID NO: 381 DOM16-84 (SEQ ID NO: 382 DOM16-85 SEQ ID NO: 383 DOM16-87 (SEQ ID NO: 384 DOM16-88 SEQ ID NO: 385 DOM16-89 (SEQ ID NO: 386 DOM16-90 SEQ ID NO: 387 DOM16-91 (SEQ ID NO: 388 DOM16-92 SEQ ID NO: 389 DOM16-94 (SEQ ID NO: 390 DOM16-95 SEQ ID NO: 391 DOM16-96 (SEQ ID NO: 392 DOM16-97 SEQ ID NO: 393 DOM16-98 (SEQ ID NO: 394 DOM16-99 SEQ ID NO: 395 DOM16-100 (SEQ ID NO: 396 DOM16-101 SEQ ID NO: 397 DOM16-102 (SEQ ID NO: 398 DOM16-103 SEQ ID NO: 399 DOM16-104 (SEQ ID NO: 400 DOM16-105 SEQ ID NO: 401 DOM16-106 (SEQ ID NO: 402 DOM16-107 SEQ ID NO: 403 DOM16-108 (SEQ ID NO: 404 DOM16-109 SEQ ID NO: 405 DOM16-110 (SEQ ID NO: 406 DOM16-111 SEQ ID NO: 407 DOM16-112 (SEQ ID NO: 408 DOM16-113 SEQ ID NO: 409 DOM16-114 (SEQ ID NO: 410 DOM16-115 SEQ ID NO: 411 DOM16-116 (SEQ ID NO: 412 DOM16-117 SEQ ID NO: 413 DOM16-118 (SEQ ID NO: 414 DOM16-119 SEQ ID NO: 415 DOM16-39-6 (SEQ ID NO: 416 DOM16-39-8 SEQ ID NO: 417 DOM16-39-34 (SEQ ID NO: 418), DOM16-39-48 (SEQ ID NO: 419), DOM16-39-87 (SEQ ID NO: 420), DOM16-39-90 (SEQ ID NO: 421), DOM16-39-96 (SEQ ID NO: 422), DOM16-39-100 (SEQ ID NO: 423), DOM16-39-101 (SEQ ID NO: 424), DOM16-39-102 ( SEQ ID NO: 425), DOM16-39-103 (SEQ ID NO: 426), DOM16-39-104 (SEQ ID NO: 427), DOM16-39-105 (SEQ ID NO: 428), DOM16-39- 106 (SEQ ID NO: 429), DOM16-39-107 (SEQ ID NO: 430), DOM16-39-108 (SEQ ID NO: 431), DOM16-39-109 (SEQ ID NO: 432), DOM16- 39-110 (SEQ ID NO: 433), DOM16-39-111 (SEQ ID NO: 434), DOM16-39-112 (SEQ ID NO: 435), DOM16-39-113 (SEQ ID NO: 436), DOM16-39-114 (SEQ ID NO: 437), DOM16-39-115 (SEQ ID NO: 438), DOM16-39-116 (SEQ ID NO: 439), DOM16-39-117 (SEQ ID NO: 440) ), DOM16-39-200 (SEQ ID NO: 441), DOM16-39-201 (SEQ ID NO: 442), DOM16-39-202 (SEQ ID NO: 443), DOM16-39-203 (SEQ ID NO. : 444), DOM16-39-204 (SEQ ID NO: 445), DOM16-39-205 (SEQ ID NO: 446), DOM16-39-206 (SEQ ID NO: 447), DOM16-39-207 (SEQ ID NO: 448), DOM16-39-209 (SEQ ID NO: 449), DOM16-52 (SEQ ID NO: 450), NB1 (SEQ I D NO: 451), NB2 (SEQ ID NO: 452), NB3 (SEQ ID NO: 453), NB4 (SEQ ID NO: 454), NB5 (SEQ ID NO: 455), NB6 (SEQ ID NO: 456) , NB7 (SEQ ID NO: 457), NB8 (SEQ ID NO: 458), NB9 (SEQ ID NO: 459), NB10 (SEQ ID NO: 460), NB11 (SEQ ID NO: 461), NB12 (SEQ ID NO: 462), NB13 (SEQ ID NO: 463), NB14 (SEQ ID NO: 464), NB15 (SEQ ID NO: 465), NB16 (SEQ ID NO: 466), NB17 (SEQ ID NO: 467), NB18 (SEQ ID NO: 468), NB19 (SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 (SEQ ID NO: 471), and NB22 (SEQ ID NO: 472). The ligand having binding specificity for EGFR can inhibit the binding of epidermal growth factor (EGF) and / or transform growth factor alpha (TGF alpha) to EGFR, inhibit EGFR activity, and / or inhibit the activity of EGFR without substantially inhibiting the binding of epidermal growth factor (EGF) and / or transforming growth factor alpha (TGF alpha) to EGFR. The ligand having binding specificity for EGFR may contain a single immunoglobulin variable domain with binding specificity for EGFR that binds EGFR with an affinity (KD) that is between about 100 nM and about 1 pM or about 10 nM to about 100 pM, as determined by surface plasmon resonance. The ligand having binding specificity for VEGF and for EGFR can bind EGFR with an affinity (KD) that is between about 100 nM and about 1 pM or about 10 nM to about 100 pM, as determined by surface plasmon resonance . The ligand having binding specificity for EGFR may comprise a single immunoglobulin variable domain with binding specificity for EGFR which is a VHH- The ligand having binding specificity for EGFR may comprise a single immunoglobulin variable domain with binding specificity for EGFR that is selected from the group consisting of human VH and a human VL.
In some embodiments, the ligand having binding specificity for EGFR is an IgG type format comprising at least two variable domains of single immunoglobulin with binding specificity for EGFR. In some embodiments, the ligand having binding specificity for EGFR "comprises an Fc antibody region. In some embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that antagonizes (inhibits) the binding of human EGFR to a receptor, wherein the single immunoglobulin variable domain polypeptide comprises a CDR3 sequence that is the same CDR3 sequence of an anti-EGFR dAb described herein In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-EGFR dAb described herein, or differs from the amino acid sequence of an anti-EGFR dAb described herein in no more than 25 amino acid positions and has a CDR1 sequence having at least 50% identity with the CDR1 sequence of the anti-EGFR dAb In other embodiments, the ligand comprises and a single immunoglobulin variable domain polypeptide that binds to EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-EGFR dAb described herein, or differs from the amino acid sequence of a dAb anti-EGFR described herein in no more than 25 amino acid positions and has a CDR2 sequence that has at least 50% identity with the CDR2 sequence of the anti-EGFR dAb. In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-EGFR dAb described herein, or differs from the sequence of amino acids of an anti-EGFR dAb described herein in no more than 25 amino acid positions and to a CDR3 sequence having at least 50% identity with the CDR3 sequence of the anti-EGFR dAb. In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-EGFR dAb described herein, or differs from the sequence of amino acids of an anti-EGFR dAb described herein in no more than 25 amino acid positions and has a CDR1 sequence that has at least 50% identity with the CDR1 sequence of the anti-EGFR dAb and has a CDR2 sequence that has at least 50% identity with the CDR2 sequence of the anti-EGFR dAb.
In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-EGFR dAb described herein, or differs from the sequence of amino acids of an anti-EGFR dAb described herein in no more than 25 amino acid positions and has a CDR2 sequence that has at least 50% identity with the CDR2 sequence of the anti-EGFR dAb and has a CDR3 sequence having at least 50% identity with the CDR3 sequence of the anti-EGFR dAb. In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-EGFR dAb described herein, or differs from the sequence of amino acids of an anti-EGFR dAb described in the present invention in no more than 25 amino acid positions and has a CDR1 sequence that has at least 50% identity with the CDR1 sequence of the anti-EGFR dAb and has a CDR3 sequence that it has at least 50% identity with the CDR3 sequence of the anti-EGFR dAb. In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-EGFR dAb described herein, or differs from the amino acid sequence of an anti-EGFR dAb described herein in no more than 25 amino acid positions and has a CDR1 sequence that has at least 50% identity with the CDR1 sequence of the anti-EGFR dAb and has a CDR2 sequence that has at least 50% identity with the CDR2 sequence of the anti-EGFR dAb and has a sequence CDR3 having at least 50% identity with the CDR3 sequence of the anti-EGFR dAb. In another embodiment, the present invention is an EGFR antagonist having a CDR1 sequence that has at least 50% identity with the CDR1 sequence of an anti-EGFR dAb described herein. In another embodiment, the present invention is an EGFR antagonist having a CDR2 sequence that has at least 50% identity with the CDR2 sequence of an anti-EGFR dAb described herein. In another embodiment, the present invention is an EGFR antagonist having a CDR3 sequence that has at least 50% identity with the CDR3 sequence of an anti-EGFR dAb described herein. In another embodiment, the present invention is an EGFR antagonist having a CDR1 sequence having at least 50% identity with the CDR1 sequence of an anti-EGFR dAb described herein, and a CDR2 sequence having at least 50% identity with the CDR2 sequence of the anti-EGFR dAb. In another embodiment, the present invention is an EGFR antagonist having a CDR2 sequence that has at least 50% identity with the CDR2 sequence of an anti-EGFR dAb described herein and a CDR3 sequence having at least 50% identity. with the CDR3 sequence of the anti-EGFR dAb. In another embodiment, the present invention is an EGFR antagonist having a CDR1 sequence that has at least 50% identity with the CDR1 sequence of an anti-EGFR dAb described herein, and a CDR3 sequence having at least 50% identity with the CDR3 sequence of the anti-EGFR dAb. In another embodiment, the present invention is an EGFR antagonist having a CDR1 sequence that has at least 50% identity with the CDR1 sequence of an anti-EGFR dAb described herein and a CDR2 sequence having at least 50% identity. with the CDR2 sequence of the anti-EGFR dAb and a CDR3 sequence having at least 50% identity with the CDR3 sequence of the anti-EGFR dAb. In some embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that antagonizes (inhibits) human VEGF that binds to a receptor, wherein the single immunoglobulin variable domain polypeptide comprises a CDR3 sequence that is the same CDR3 sequence of the anti-VEGF dAb described herein. In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds VEGF, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-VEGF dAb described herein, or differs from the sequence of amino acids of an anti-VEGF dAb described herein in no more than 25 amino acid positions and has a CDR1 sequence having at least 50% identity with the CDR1 sequence of anti-VEGF dAb. In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds to VEGF, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of anti-VEGF dAb described herein, or differs from the sequence of amino acids of the anti-VEGF dAb described herein in no more than 25 amino acid positions and has a CDR2 sequence having at least 50% identity with the CDR2 sequence of the anti-VEGF dAb. In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds VEGF, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-VEGF dAb described herein, or differs from the sequence of amino acids of the anti-VEGF dAb described herein in no more than 25 amino acid positions and has a CDR3 sequence having at least 50% identity with the CDR3 sequence of the anti-VEGF dAb. In other modalities, the ligand comprises a single immunoglobulin variable domain polypeptide that binds VEGF, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-VEGF dAb described herein, or differs from the amino acid sequence of an anti-VEGF dAb described herein in no more than 25 amino acid positions and has a CDR1 sequence that has at least 50% identity with the CDR1 sequence of anti-VEGF dAb, and has a CDR2 sequence that is at least 50 % identity with the CDR2 sequence of the anti-VEGF dAb.
In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds VEGF, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-VEGF dAb described herein, or differs from the sequence of amino acids of an anti-VEGF dAb described herein in no more than 25 amino acid positions and has a CDR2 sequence that has at least 50% identity with the CDR2 sequence of the anti-VEGF dAb and has a CDR3 sequence that has at least 50% identity with the CDR3 sequence of the anti-VEGF dAb.
In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds VEGF, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-VEGF dAb described herein, or differs from the sequence of amino acids of an anti-VEGF dAb described herein in no more than 25 amino acid positions and has a CDR1 sequence that has at least 50% identity with the CDR1 sequence of the anti-VEGF dAb and has a CDR3 sequence that has at least 50% identity with the CDR3 sequence of the anti-VEGF dAb. In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds VEGF, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-VEGF dAb described herein, or differs from the sequence of amino acids of an anti-VEGF dAb described herein in no more than 25 amino acid positions and has a CDR1 sequence that has at least 50% identity with the CDR1 sequence of the anti-VEGF dAb and has a CDR2 sequence that has at least 50% identity with the CDR2 sequence of the anti-VEGF dAb and has a CDR3 sequence having at least 50% identity with the CDR3 sequence of the anti-VEGF dAb. In another embodiment, the present invention is a VEGF antagonist having a CDR1 sequence that has at least 50% identity with the CDR1 sequence of the anti-VEGF dAb described herein. In another embodiment, the present invention is a VEGF antagonist having a CDR2 sequence that has at least 50% identity with the CDR2 sequence of the anti-VEGF dAb described herein. In another embodiment, the present invention is a VEGF antagonist having a CDR3 sequence that has at least 50% identity with the CDR3 sequence of the anti-VEGF dAb described herein. In another embodiment, the present invention is a VEGF antagonist having a CDR1 sequence having at least 50% identity with the CDR1 sequence of an anti-VEGF dAb described herein, and a CDR2 sequence having at least 50% identity with the CDR2 sequence of the anti-VEGF dAb. In another embodiment, the present invention is a VEGF antagonist having a CDR2 sequence that has at least 50% identity with the CDR2 sequence of an anti-VEGF dAb described herein, and a CDR3 sequence having at least 50% identity with the CDR3 sequence of the anti-VEGF dAb. In another embodiment, the present invention is a VEGF antagonist having a CDR1 sequence that has at least 50% identity with the CDR1 sequence of an anti-VEGF dAb described herein, and a CDR3 sequence having at least 50% identity with the CDR3 sequence of the anti-VEGF dAb.
In another embodiment, the present invention is a VEGF antagonist having a CDR1 sequence having at least 50% identity with the CDR1 sequence of an anti-VEGF dAb described herein, and a CDR2 sequence having at least 50% identity with the CDR2 sequence of the anti-VEGF dAb and a CDR3 sequence having at least 50% identity with the CDR3 sequence of the anti-VEGF dAb. In further embodiments, in any of the ligands described herein, it further comprises a toxin, such as a cytoxin, free radical generator, antimetabolite, protein, polypeptide, peptide, photoactive agent, anti-sense compound, chemotherapeutic agent, radionuclide or intrabody. In particular modalities, the toxin is a surface active toxin (for example, a free radical generator, a radionuclide). In other embodiments, the ligand further comprises a half-life extension portion, such as a polyalkylene glycol moiety, serum albumin or a fragment thereof, a transferrin receptor or a transferrin binding portion thereof, or a portion thereof. comprises a binding site for a polypeptide that increases the half-life in vivo. In some embodiments, the half-life extension portion is a portion comprising a binding site of a polypeptide that increases the in vivo half-life selected from the group consisting of an antibody, an SpA domain, an A domain of the receptor class. LDL, an EGF domain, and an avimer. In other embodiments, the half-life extension portion is an antibody or an antibody fragment (e.g., a single immunoglobulin variable domain) that comprises a binding site for serum albumin or neonatal Fc receptor. In particular embodiments, the half-life extension portion is a single immunoglobulin variable domain comprising a binding site for serum albumin that competes to bind human serum albumin with a dAb selected from the group consisting of DOM7m-16 ( SEQ ID NO: 473 DOM7m-12 (SEQ ID NO: 474 DOM7m-26 (SEQ ID NO: 475 DOM7r-1 (SEQ ID NO: 476 DOM7r-3 (SEQ ID NO: 477 DOM7r-4 (SEQ ID NO: 478 DOM7r- 5 (SEQ ID NO: 479 DOM7r-7 (SEQ ID NO: 480 DOM7r-8 (SEQ ID NO: 481 DOM7h-2 (SEQ ID NO: 482 DOM7h-3 (SEQ ID NO: 483 DOM7h-4 (SEQ ID NO : 484 WED7h-6 (SEQ ID NO: 485 WED7h-1 (SEQ ID NO: 486 WED7h-7 (SEQ ID NO: 487 WED7h-22 (SEQ ID NO: 489, WED7h-23 (SEQ ID NO: 490 WED7h- 24 (SEQ ID NO: 491, DOM7h-25 (SEQ ID NO: 492 DOM7h-26 (SEQ ID NO: 493, DOM7h-21 (SEQ ID NO: 494 DOM7h-27 (SEQ ID NO: 495), DOM7h-8 (SEQ ID NO: 496 DOM7r-13 (SEQ ID NO: 497, DOM7r-14 (SEQ ID NO: 498 DOM7r-15 (SEQ ID NO: 499, DOM7r-16 (SEQ ID NO: 500 DOM7r-17 (SEQ ID NO: 501, DOM7r-18 (SEQ ID NO: 502), DOM7r-19 (SEQ ID NO: 503), DOM7r-20 (SEQ ID NO: 504), DOM7r-21 (SEQ ID NO: 505), DOM7r- 22 (SEQ ID NO: 506), DOM7r-23 (SEQ ID NO: 507), DOJvl7r-24 (SEQ ID NO: 508), DOM7r-25 (SEQ ID NO: 509), DOM7r-26 (SEQ ID NO: 510), DOM7r-27 (SEQ ID NO: 511), DOM7r-28 (SEQ ID NO: 512), DOM7r-29 (SEQ ID NO: 513), DOM7r-30 (SEQ ID NO: 514), DO M7r-31 (SEQ ID NO: 515), DOM7r-32 (SEQ ID NO: 516), and DOM7r-33 (SEQ ID NO: 517). For example, the single immunoglobulin variable domain comprising a binding site for serum albumin may comprise an amino acid sequence having at least 85% amino acid sequence identity with the amino acid sequence of a dAb selected from the group consists of DOM7m-16 (SEQ ID NO: 473), DOM7m-12 (SEQ ID NO: 474), DOM7m-26 (SEQ ID NO: 475), DOM7r-1 (SEQ ID NO: 476), DOM7r-3 ( SEQ ID NO: 477), DOM7r-4 (SEQ ID NO: 478), DOM7r-5 (SEQ ID NO: 479), DOM7r-7 (SEQ ID NO: 480), DOM7r-8 (SEQ ID NO: 481) , DOM7h-2 (SEQ ID NO: 482), DOM7h-3 (SEQ ID NO: 483), DOM7h-4 (SEQ ID NO: 484), DOM7h-6 (SEQ ID NO: 485), DOM7h-1 (SEQ ID NO: 486), DOM7h-7 (SEQ ID NO: 487), DOM7h-22 (SEQ ID NO: 489), DOM7h-23 (SEQ ID NO: 490), DOM7h-24 (SEQ ID NO: 491), DOM7h-25 (SEQ ID NO: 492), DOM7h-26 (SEQ ID NO: 493), DOM7h-21 (SEQ ID NO: 494), DOM7h-27 (SEQ ID NO: 495), DOM7h-8 (SEQ ID NO: 496), DOM7r-13 (SEQ ID NO: 497) DOM7r-14 (SEQ ID NO: 498) DOM7r-15 (SEQ ID NO: 499) DOM7r-16 (SEQ ID NO: 500) DOM 7r-17 (SEQ ID NO: 501) DOM7r-18 (SEQ ID NO: 502) DOM7r-19 (SEQ ID NO: 503) DOM7r-20 (SEQ ID NO: 504) DOM7r-21 (SEQ ID NO: 505) DOM7r-22 (SEQ ID NO: 506) DOM7r-23 (SEQ ID NO: 507) DOM7r-24 (SEQ ID NO: 508) DOM7r-25 (SEQ ID NO: 509) DOM7r-26 (SEQ ID NO: 510) DOM7r-27 (SEQ ID NO: 511) DOM7r-28 (SEQ ID NO: 512) DOM7r-29 (SEQ ID NO: 513) DOM7r-30 (SEQ ID NO: 514) DOM7r-31 (SEQ ID NO: 515) DOM7r-32 (SEQ ID NO: 516), and DOM7r-33 (SEQ ID NO: 517). The present invention also relates to a recombinant or isolated nucleic acid encoding a ligand described herein, and to a vector (eg, recombinant vector) comprising the recombinant nucleic acid. The present invention also relates to a host cell (e.g., recombinant host cell, isolated host cell) comprising a recombinant nucleic acid or vector of the present invention. The present invention also relates to a method for producing a ligand, characterized in that it comprises maintaining a host cell of the present invention under conditions suitable for expression of the nucleic acid or vector, whereby a ligand is produced. In some embodiments, the method further comprises isolating the ligand. The present invention also relates to a ligand of the present invention for use in therapy or diagnosis, and to the use of a ligand of the present invention for the manufacture of a medicament for the treatment, prevention or suppression of a disease described herein ( example, cancer). The present invention also relates to a pharmaceutical composition for the treatment, prevention or suppression of a disease described herein (eg, cancer) comprising as an active ingredient a ligand of the present invention. In some embodiments, the present invention relates to a ligand for use in the treatment of cancer, or cancer cells that overexpress EGFR and / or VEGF. In other modalities, the present invention relates to the use of a ligand for the manufacture of a medicament for killing cells (for example, selectively killing cancer cells with respect to normal cells). In other embodiments, the present invention relates to the use of a ligand for the manufacture of a medicament for treating cancer cells that overexpress EGFR and / or VEGF. The present invention also relates to therapeutic methods comprising administering a therapeutically effective amount of a ligand of the present invention to a subject in need thereof.
In one embodiment, the present invention relates to a method of treating cancer, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a ligand of the present invention. In some embodiments, the method further comprises administering to the subject a chemotherapeutic agent (e.g., in a low dose). In other embodiments, the method of treating cancer comprises administering to a subject in need thereof a therapeutically effective amount of a ligand of the present invention and an antineoplastic composition, wherein the antineoplastic composition comprises at least one chemotherapeutic agent. The chemotherapeutic agent can be selected from the group consisting of alkylating agents, antimetabolites, folic acid analogues, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodopilotoxins, antibiotics, L-asparaginase, topoisomerase inhibitor, interferons, platinum coordination complexes, anthracenedione substituted urea, methyl hydrazine derivatives, adrenocortical suppressors, adrenocorticosteroids, progestins, estrogens, anti-estrogens, androgens, anti-androgens, and gonadotropin-releasing hormone analogues. In some embodiments, the chemotherapeutic agent is selected from the group consisting of cisplatin, dicarbazine, dactinomycin, meclorethamin, streptozocin, cyclophosphamide, capecitabine, carmustine, lomustine, doxorubicin, daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, rhetotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel, docetaxol, doxetaxo, aldesleucin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, alpha interferon, irinotecan, leuprolide, leucovorin, megestrol, melphalan, mercaptopurine, oxaliplatin, plicamicin , mitotane, pegaspargase, pentostatin, pipobroman, plicamicin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, mustard uracil, vinorelbine, chlorambucil, taxol, an additional growth factor receptor antagonist and a combination of any of the above . In some embodiments, the method is a method of treating a cancer selected from the group consisting of bladder cancer, ovarian cancer, colorectal cancer (colorectal carcinoma), breast cancer, lung cancer (non-small cell lung carcinoma). ), gastric cancer, pancreatic cancer, prostate cancer, head and neck cancer, kidney cancer and gallbladder cancer. The present invention also relates to a method for administering to a subject anti-VEGF treatment and anti-EGFR treatment, wherein the method comprises the simultaneous administration of an anti-VEGF treatment and an anti-EGFR treatment by administering to the subject an amount therapeutically effective of a ligand that has binding specificity for VEGF and EGFR. The present invention also relates to a composition (e.g., pharmaceutical composition) comprising a ligand of the present invention and a physiologically or pharmaceutically acceptable carrier. In some embodiments, the composition comprises a vehicle for intravenous, intramuscular, intraperitoneal, intra-arterial, intrathecal administration, intra-articular, pulmonary, intranasal, vaginal, or rectal subcutaneous administration. The present invention also relates to a drug delivery apparatus comprising the composition (e.g., pharmaceutical composition) of the present invention or a ligand thereof, in one embodiment, the drug delivery apparatus is provided for administration in simultaneously with a subject anti-VEGF treatment and anti-EGFR treatment, and the apparatus comprises a ligand having binding specificity for VEGF and EGFR. In some embodiments, the drug apparatus comprises a plurality of therapeutically effective dose of the ligand. In other modalities, the drug delivery apparatus is selected from the group consisting of a parenteral delivery apparatus, intravenous delivery apparatus, intramuscular delivery apparatus, intraperitoneal delivery apparatus, intraperitoneal delivery apparatus, transdermal delivery apparatus, pulmonary delivery apparatus, intra-arterial delivery apparatus, intrathecal delivery apparatus, intra-articular delivery apparatus, subcutaneous delivery apparatus, intranasal delivery apparatus, vaginal delivery apparatus, rectal delivery apparatus, a syringe, a transdermal delivery apparatus, a capsule , a tablet, a nebulizer, an inhaler, an atomizer, an aerosol, a nebulizer, an inhaler in powder form, a metered dose inhaler, a metered dose sprayer, a metered dose nebulizer, a metered dose sprayer, a catheter Brief Description of the Drawings Figures 1A-1E illustrate twenty-seven nucleotide sequences encoding human domain antibodies (Homo sapiens) (dAbs) that specifically bind human VEGF. The presented nucleotide sequences are SEQ ID NOS: 1-27, 535 and 536. Figures 2A-2C are an alignment of twelve nucleotide sequences encoding human dAbs that bind human VEGF. The nucleotide sequences presented are SEQ ID NO: 18 and SEQ ID NOS: 28-38. Figures 3A-3D are an alignment of twelve nucleotide sequences encoding human dAbs that bind human VEGF. The presented nucleotide sequences are SEQ ID NO: 20 and SEQ ID NOS: 39-49.
Figures 4A-4J, are an alignment of fifty-three nucleotide sequences encoding human dAbs that bind to human VEGF. The nucleotide sequences presented are SEQ ID NO: 24, 50-99, 537 and 538. Figures 5A-5C illustrate the amino acid sequences of dAbs encoded through various nucleic acid sequences shown in the figures' 1 A- 1E.The amino acid sequences presented are SEQ ID NOS. 100-126. Figure 6 is an alignment of amino acid sequences of the dAbs encoded through the nucleic acid sequences shown in Figures 2A-2C. The amino acid sequences presented are SEQ ID NO: 117 and SEQ ID NOS: 127-137. Figures 7A-7B are an alignment of the amino acid sequences of the dAbs encoded through the nucleic acid sequences shown in Figures 3A-3D. The ~ symbol has been inserted into the sequence of TAR15-8-500 to facilitate alignment. The amino acid sequences presented are SEQ ID NO: 119 and SEQ ID NOS: 138-148. Figures 8A-8D are an alignment of the amino acid sequences of the dAbs encoded through the nucleic acid sequences shown in Figures 4A-4J. The amino acid sequences presented are SEQ ID NO: 123, 149-198, 539 and 540. Figures 9A-9O illustrate several nucleotide sequences encoding human-domain antibody (Homo sapiens) (dAbs) that specifically bind human EGFR . The nucleotide sequences presented are SEQ ID NOS: 199-324. Figures 10A-101, illustrate the amino acid sequences of the dAbs encoded through the nucleic acid sequences shown in Figures 9A-9O. The amino acid sequences presented herein are SEQ ID NOS: 325-450. Figures 11A to 11B illustrate the amino acid sequences of various Camelid VHHS that bind EGFR which are described in Publication WO 2005/044858. NB1 (SEQ ID NO: 451), NB2 (SEQ ID NO: 452), NB3 (SEQ ID NO: 453), NB4 (SEQ ID NO: 454), NB5 (SEQ ID NO: 455), NB6 (SEQ ID NO : 456), NB7 (SEQ ID NO: 457), NB8 (SEQ ID NO: 458), NB9 (SEQ ID NO: 459), NB10 (SEQ ID NO: 460), NB11 (SEQ ID NO: 461), NB12 (SEQ ID NO: 462), NB13 (SEQ ID NO: 463), NB14 (SEQ ID NO: 464), NB15 (SEQ ID NO: 465), NB16 (SEQ ID NO: 466), NB17 (SEQ ID NO: 467), NB1 8 (SEQ ID NO: 468), NB19 (SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 (SEQ ID NO: 471), NB22 (SEQ ID NO: 472). Figure 12A is an alignment of the amino acid sequences of three VKS that bind to mouse serum albumin (MSA). The aligned amino acid sequences are from VKS designated MSA16, which are also referred to as DOM7m-16 (SEQ ID NO: 473), MSA 12, which are also referred to as DOM7m-12 (SEQ ID NO: 474), and MSA 26, which are also referred to as DOM7m-26 (SEQ ID NO: 475). Figure 12B is an alignment of amino acid sequences of six VKS that bind to rat serum albumin (RSA). The aligned amino acid sequences are from VKS designated DOM7r-1 (SEQ ID NO: 476), DOM7r-3 (SEQ ID NO: 477), DOM7r-4 (SEQ ID NO: 478), DOM7r-5 (SEQ ID NO: 479), DOM7r-7 (SEQ ID NO: 480), and DOM7r-8 (SEQ ID NO: 481). Figure 12C is an alignment of the six VKS amino acid sequences that bind human serum albumin (HSA). The aligned amino acid sequences are from VKS designated DOM7h-2 (SEQ ID NO: 482), DOM7h-3 (SEQ ID NO: 483), DOM7h-4 (SEQ ID NO: 484), DOM7h-6 (SEQ ID NO: 485), DOM7h-1 (SEQ ID NO: 486), and DOM7h-7 (SEQ ID NO: 487). Figure 12D is an alignment of the seven HSV amino acid sequences that bind human serum albumin and a consensus sequence (SEQ ID NO: 488). The aligned sequences come from VHs designated as DOM7h-22 (SEQ ID NO: 489), DOM7h-23 (SEQ ID NO: 490), DOM7h-24 (SEQ ID NO: 491), DOM7h-25 (SEQ ID NO: 492), DOM7h-26 (SEQ ID NO: 493), DOM7h-21 (SEQ ID NO: 494), and DOM7h-27 (SEQ ID NO: 495). Figure 12E is an alignment of the amino acid sequences of three VKS that bind human serum albumin and rat serum albumin. The aligned amino acid sequences are from VKS designated as DOM7h-8 (SEQ ID NO: 496), DOM7r-13 (SEQ ID NO: 497), and DOM7r-14 (SEQ ID NO: 498). Figure 13 is an illustration of the amino acid sequences of VKS that bind rat serum albumin (RSA). The illustrated sequences are from VKS designated as DOM7r-15 (SEQ ID NO: 499), DOM7r-16 (SEQ ID NO: 500), DOM7r-17 (SEQ ID NO: 501), DOM7r-18 (SEQ ID NO: 502) ), DOM7r-19 (SEQ ID NO: 503). Figures 14A-14B are an illustration of the amino acid sequences of VHs that bind rat serum albumin (RSA). The illustrated sequences are from VHs designated as DOM7r-20 (SEQ ID NO: 504), DOM7r-21 (SEQ ID NO: 505), DOM7r-22 (SEQ ID NO: 506), DOM7r-23 (SEQ ID NO: 507) ), DOM7r-24 (SEQ ID NO: 508) DOM7r-25 (SEQ ID NO: 509), DOM7r-26 (SEQ ID NO: 510) DOM7r-27 (SEQ ID NO: 511), DOM7r-28 (SEQ ID NO: 512) DOM7r-29 (SEQ ID NO: 513), DOM7r-30 (SEQ ID NO: 514), DOM7r-31 (SEQ ID NO: 515), DOM7r-32 (SEQ ID NO: 516), and DOM7r-33 (SEQ ID NO: 517). Figure 15 illustrates the amino acid sequences of various Camelid VHHS binding mouse serum albumin which are described in Publication WO 2004/041862. Sequence A (SEQ ID NO: 518), Sequence B (SEQ ID NO: 519 Sequence C (SEQ ID NO: 520), Sequence D (SEQ ID NO: 521 Sequence E (SEQ ID NO: 522), Sequence F (SEQ ID NO: 523 Sequence G (SEQ ID NO: 524), Sequence H (SEQ ID NO: 525 Sequence I (SEQ ID NO: 526), Sequence J (SEQ ID NO: 527 Sequence K (SEQ ID NO: 528), Sequence L (SEQ ID NO: 529 Sequence M (SEQ ID NO: 530), Sequence N (SEQ ID NO: 531 Sequence O (SEQ ID NO: 532), Sequence P (SEQ ID NO: 533 Sequence Q (SEQ ID NO: 534) Figure 16 is a map of a vector used for preparing IgG type format Detailed Description of the Invention Within the present specification, modalities have been described in a form that allows a clear and concise specification to be written, although it is intended and it will be appreciated that the modalities may be combined or separated. in various forms without departing from the present invention. As used in the present invention, the term "ligand" refers to a compound comprising at least one peptide, polypeptide or protein portion having a binding site with binding specificity for a desired endogenous target compound. Ligands according to the present invention preferably comprise immunoglobulin variable domains having different binding specificities, and do not contain pairs of variable domains having the same specificity. Preferably, each domain has a binding site that has binding specificity for a cell surface target that is a single immunoglobulin variable domain (i.e., single immunoglobulin heavy chain variable domain (e.g., VH, VHH) variable domain single immunoglobulin light chain (e.g., V)) having binding specificity for a desired cell surface target (e.g., a membrane protein, such as a receptor protein). Each polypeptide domain having a binding site that has binding specificity for a cell surface target can also comprise one or more complementary determination regions (CDRs) of an antibody or antibody fragment (e.g., a variable domain). of simple immunoglobulin) having binding specificity for a desired cell surface target in a suitable format, so that the binding domain has binding specificity for the cell surface target. For example, CDRs can be grafted onto a suitable protein scaffold or scaffold, such as an affibody, an SpA scaffold, an LDL class A receptor domain, or an EGF domain. In addition, the ligand may be bivalent (heterobivalent) or multivalent (heteromultivalent) as described in the present invention. The first and second domains lack domains that share the same specificity. Therefore, "ligands" include polypeptides comprising two dAbs wherein each dAb binds to a different cell surface target. The ligands may also include polypeptides comprising at least two dAbs that bind to different cell surface targets (or the CDRs of a dAbs) in a suitable format, such as an antibody format (e.g., IgG, scFv, Fab format). , Fab ', F (ab') 2) or a suitable scaffold or protein skeleton, such as an affibody, a SpA scaffold, an LDL class A receptor domain, an EGF domain, avimer and multispecific ligands as described in the present invention. The polypeptide domain having a binding site that has binding specificity for a cell surface target (e.g., first or second cell surface targets) may also be a protein domain comprising a binding site for a desired target, eg, a protein domain of an affibody, an SpA domain, an LDL receptor domain class is selected A, an avimer (see for example, US Patent Application Publication Nos. 2005/0053973, 2005/0089932, 2005/0164301). If desired, a "ligand" can further comprise one or more additional portions, which can each be independently a peptide, polypeptide or protein portion or a non-peptide portion (eg, a polyalkylene glycol, a lipid, a carbohydrate ). For example, the ligand may further comprise a half-life extension portion as described in the present invention (eg, a polyalkylene glycol portion, a portion comprising albumin, an albumin fragment or an albumin variant, a portion that it comprises transferin, a fragment of transferin or a variant of transferin, a portion that binds albumin, a portion that binds to a neonatal receptor Fc). As used in the present invention, "objective" refers to a biological molecule (e.g., peptide, polypeptide, protein, lipid, carbohydrate) to which a polypeptide domain having a binding site can bind. The objective may be, for example, an intracellular target (eg, an intracellular protein target) or a cell surface target (eg, a membrane protein, a receptor protein). Preferably, the target is VEGF or EGFR. The phrase "single immunoglobulin variable domain" refers to a variable region of antibody (VH, VHH5 VL) that binds specifically to a target, antigen or epitope independently of other V domains.; however, as the term is used in the present invention, a single immunoglobulin variable domain can be in a format (eg, hetero-multimer) with other variable regions or variable domains where the other regions or domains are not required for the binding of antigens through a single immunoglobulin variable domain (ie, wherein the variable domain of single immunoglobulin binds to the antigen independently of the additional variable domains). Each "single immunoglobulin variable domain" comprises not only an isolated single antibody variable domain polypeptide, but also larger polypeptides comprising one or more monomers of a single antibody variable domain polypeptide sequence. A "domain antibody" or "dAb" is the same as a "single immunoglobulin variable domain" polypeptide as used in the present invention. A single immunoglobulin variable domain polypeptide, as used in the present invention, refers to a mammalian immunoglobulin variable domain polypeptide, preferably human, but also includes rodents ("for example, as described in Publication. WO 00/29004, the contents of which are incorporated herein by reference in their entirety), or dAbs camelid VHH- As used in the present invention, camelid dAbs are single immunoglobulin variable domain polypeptides that are derived from species which include camel, llama, alpaca, camel, and guanaco, and comprise heavy chain antibodies naturally devoid of light chain (VHH). Similar dAbs can be obtained from single chain antibodies from other species, such as nurse shark. Preferred ligands comprise at least two different single immunoglobulin variable domain polypeptides or at least two different dAbs. As used in the present invention, "vascular endothelial growth factor" (VEGF) refers to endogenous or naturally occurring mammalian VEGF-A proteins and to proteins having an amino acid sequence that is the same as that of a corresponding endogenous or naturally occurring mammalian VEGF-A protein (eg, recombinant proteins, synthetic proteins (i.e., produced using synthetic organic chemistry methods) Accordingly, as defined in the present invention, the term includes protein Mature VEGF-A, polymorphic or allelic variants, and other isoforms of a VEGF-A (eg, produced by alternative division processes or other cellular processes) and modified or unmodified forms of the above (eg, lipidated, glycosylated) The alternative division of RNA encoding human VEGF-A (Homo sapiens) produces several isoforms of human VEGF-A that differ in the number of amino acids in the protein sequence. For example, the isoforms referred to as VEGF-121, VEGF-165, VEGF-189 and VEGF-206 are produced in humans. (See, for example, Ferrara Publication, N., Endocrine Reviews, 25 (4): 581-611 (2004).). These isoforms and other isoforms that occur naturally are expressly included in the term "VEGF". Endogenous or naturally occurring VEGF-A includes type proteins such as mature VEGF-A, polymorphic or allelic variants and other isoforms that occur naturally in mammals (eg, human primates, non-human primates). Said proteins can be recovered or isolated from a source that naturally produces VEGF-A, for example. These proteins and proteins having the same amino acid sequence as a corresponding endogenous or naturally occurring VEGF are referred to by the name of the corresponding mammal. For example, when the mammal is a human, the protein is designated as human VEGF. A ligand (e.g., single immunoglobulin variable domain) that inhibits VEGF binding to VEGFR1 or VEGFR2 inhibits binding in the VEGFR1 binding assay or in the VEGFR2 assay described herein by at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% when the ligand is assayed at a concentration of about 1 nM, about 10 nM, about 50 nM, about 100 nM, about 1 μM, about 10 μM or about 100 μM. A ligand that inhibits the binding of VEGF to VEGFR1 or VEGFR2 can also, or alternatively, inhibit binding in the VEGFR1 binding assay or VEGFR2 assay with an IC50 of about 1 μM or less, about 500 nM or less, about 100 nM or less, approximately 75 nM or less, approximately 50 nM or less, approximately 10 nM or less or approximately 1 nM or less. A ligand (e.g., single immunoglobulin variable domain) that inhibits VEGF activity inhibits viability in the VEGF bioassay described in the present invention by at least about 20%, at least about 30%, at least about 40%. %, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% %. A ligand (e.g., single immunoglobulin variable domain) that does not substantially inhibit the binding of VEGF to VEGFR1 or VEGFR2 does not significantly inhibit binding in the VEGFR1 binding assay or VEGFR2 assay described herein. For example, said ligand must inhibit the VEGF binding in the VEGFR1 binding assay or VEGFR2 assay described herein with an IC50 of about 1 mM or more, or inhibit the binding by no more than about 20%, no more than about 15%. %, no more than about 10% or no more than about 5%. As used in the present invention, the term "epidermal growth factor receptor" (EGFR, ErbB1, HER1) refers to mammalian EGFR proteins that occur naturally or endogenously and to proteins that have an amino acid sequence that is the same to that of the corresponding endogenous or naturally occurring mammalian EGFR protein (e.g., recombinant proteins, synthetic proteins (e.g., proteins using synthetic organic chemistry methods).) Accordingly, as defined in the present invention, the term includes mature EGFR protein, polymorphic, or allelic variants, and other isoforms of an EGFR (eg, produced by alternative cleavage or other cellular processes), and modified or unmodified forms of the foregoing (eg, lipidated, glycosylated). endogenous or naturally occurring proteins include natural type proteins, such as mature EGFR, polymorphic or allelic variants and other isoforms that occur naturally in mammals (eg, human primates, non-human primates). Said proteins can be recovered or isolated from a naturally occurring source, for example, EGFR. These proteins and proteins having the same amino acid sequence as the corresponding endogenous or naturally occurring EGFRs are referred through the corresponding mammals. For example, when the corresponding mammal is a human, the protein is designated as a human EGFR. A ligand (e.g., single immunoglobulin variable domain) that inhibits the binding of EGF and / or TGF alpha to EGFR inhibits binding in the EGFR binding assay or EGFR kinase assay described herein with an IC50 of about 1 μM or less , about 500 nM or less, about 100 nM or less, about 75 nM or less, about 50 nM or less, about 10 nM or less, or about 1 nM or less. A ligand (e.g., 'single immunoglobulin variable domain) that inhibits EGFR activity inhibits EGFR kinase activity in the EGFR kinase assay described in the present invention with an IC50 of about 1 μM or less, about 500 nM or less, about 100 nM or less, about 75 nM or less, about 50 nM or less, about 10 nM or less, or about 1 nM or less. A ligand (e.g., single immunoglobulin variable domain) that does not substantially inhibit the binding of EGF or TGF alpha to EGFR, does not significantly inhibit the binding of EGF and / or TGF alpha to EGFR in the receptor binding or assay assay. kinase described here. For example, said ligand must inhibit the binding of EGF or TGF alpha to EGFR in the receptor binding assay or kinase assay described herein with an IC50 of about 1 mM or more. "Affinity" and "avidity" are terms in the art that describe the strength of a bonding interaction. With respect to the ligands of the present invention, avidity refers to the general strength of the link between the targets (eg, first cell surface target and second cell surface target) in the cell and the ligand. Avidity is more than the sum of individual affinities for individual goals. As used in the present invention, "toxin portion" refers to a portion comprising a toxin. A toxin is an agent that has detrimental effects on, or alters cellular physiology (e.g., causes necrosis, cellular apoptosis or inhibits cell division). As used in the present invention, the term "dose" refers to the quality of a ligand administered to a subject all at one time (unit dose), or in two or more administrations during a defined time interval. For example, the dose may refer to the amount of ligand (eg, ligand comprising a single immunoglobulin variable domain that binds VEGF and a single immunoglobulin variable domain that binds EGFR) administered to a subject in the course of a day (24 hours) (daily dose), two days, one week, two weeks, three weeks or one or more months (for example, through a single administration or through two or more administrations). The interval between the doses can be any desired amount of time. As used in the present invention, the term "complementary" refers to when two immunoglobulin domains belong to families of structures that form pairs or cognate groups or are derived from said families and retain this characteristic. For example, a VH domain and a VL domain of an antibody are complementary; two VH domains are not complementary, and two VL domains are not complementary. The complementary domains can be found in other members of the immunoglobulin superfamily such as the Va and Vp (or? And d) domains of the T cell receptor. The domains that are artificial, such as domains based on protein scaffolds that do not bind epitopes Unless they are constructed in this way, they are non-complementary. Likewise, two domains based on (for example) an immunoglobulin domain and a fibronectin domain are not complementary. As used in. The present invention, "immunoglobulin" refers to a family of polypeptides that retain the immunoglobulin fold characteristic of antibody molecules, which contain two β-sheets and normally, a conserved disulfide bond. Members of the immunoglobulin superfamily are involved in many aspects of cellular and non-cellular interactions in vivo, including broad roles in the immune system (e.g., antibodies, T cell receptor molecules and the like) involvement in cell adhesion (e.g. ICAM molecules) and intracellular signaling (e.g., receptor molecules, such as PDGF receptor). The present invention applies to all molecules of the immunoglobulin superfamily that possess binding domains. Preferably, the present invention relates to antibodies. As used in the present invention "domain" refers to a "multiplied" protein structure that retains its tertiary structure independently of the rest of the protein. Generally, the domains are responsible for the independent functional properties of the proteins, and in many cases they can be added or eliminated or transferred to other proteins without loss of function of the rest of the protein and / or the domain. By the term "single antibody variable domain" is meant a bent polypeptide domain comprising characteristic sequences of variable domains of the antibody. Accordingly, it includes complete variable domains of antibodies and modified variable domains, for example where one or more circuits have been replaced by sequences that are not characteristic of variable domains of antibodies or variable domains of antibodies that have been truncated or comprise N- or C-terminals, as well as multiplied fragments of variable domains that retain at least in part the binding activity and specificity of the total length domain. Therefore, each ligand comprises at least two different domains. "Repertoire" a collection of various variants, for example polypeptide variants that differ in their primary sequence. A library used in the present invention comprises a repertoire of polypeptides comprising at least 1000 members. "Library". The term "library" refers to a mixture of heterogeneous polypeptides or nucleic acids. The library is composed of members, each of which has a simple polypeptide or nucleic acid sequence. To this degree, the library is synonymous with repertoire. The differences in sequences among library members are responsible for the diversity found in the library. The library can take the form of a simple mixture of polypeptide or nucleic acids, or it can be in the form of organisms or cells, for example cells of bacteria, viruses, animals or plants or the like, transformed with a nucleic acid library. Preferably, each individual organism or cell contains only a limited number of library members. Conveniently, the nucleic acids are incorporated into expression vectors, in order to allow the expression of the polypeptides encoded by the nucleic acids. In a preferred aspect, therefore, the library can take the form of a population of host organisms, each organism containing one or more copies of an expression vector containing a single member of the library in the form of a nucleic acid that can be expressed to produce its corresponding polypeptide member. Therefore, the population of host organisms has the potential to encode a broad repertoire of genetically diverse polypeptide variants. As used in the present invention, an antibody refers to IgG, IgM, IgA, IgD or IgE or a fragment (such as a Fab, F (ab ') 2, Fv, Fv linked with disulfide, scFv, multi antibody -specific closed conformation, scFv linked by disulfide, diabody) either derived from any species that naturally produces an antibody, or created by recombinant DNA technology; either isolated from serum, B cells, hybridomas, transfectomas, yeast or bacteria. As used in the present invention, an "antigen" is a molecule that is linked by a binding domain according to the present invention. Normally, the antigens are ligated by antibody ligands and have the ability to raise an immune response in vivo. It can be a polypeptide, protein, nucleic acid or other molecule. Generally, the specific double ligands according to the present invention are selected for objective specificity against two particular targets (e.g., oxygen). In the case of antibodies and conventional fragments thereof, the antibody binding site defined by the variable circuits (L1, L2, L3 and H1, H2, H3) have the ability to bind the antigen. An "epitope" is a structure unit linked in a conventional manner by an immunoglobulin VH / VL pair. The epitopes define the minimal binding site for an antibody, and therefore represent the specificity target of an antibody. In the case of a single domain antibody, an epitope represents the structure unit linked by a variable domain in the isolation. The term "universal structure" refers to a sequence of simple antibody structure corresponding to regions of an antibody preserved in sequences as defined in the Kabat Publication ("Sequence of Immunological Proteins", US Department of Health and Human Services) or which corresponds to the repertoire or structure of human germline immunoglobulin as defined in Chothia and Lesk Publication, (1987) J. Mol. Biol. 196: 910-917. The present invention provides the use of a simple structure or a group of such structures, which has been found to allow the derivation of virtually any binding specificity through variation in the hyper-variable regions alone. The phrase "half-life" refers to the time it takes the serum concentration of ligand to reduce 50%, in vivo, for example due to degradation of the ligand and / or clearance or sequestration of the double specific ligand by natural mechanisms. The ligands of the present invention are stabilized in vivo and their half-life increased by binding to molecules that resist degradation and / or clearance or sequestration. Normally, these molecules are naturally occurring proteins that by themselves have a long half-life in vivo. The half-life of a ligand increases if its functional activity persists, in vivo, for a longer period than a similar ligand which was not specified to increase the half-life of the molecule. Therefore a specific ligand for HSA and two specific molecules is compared to the same ligand where the specificity with HSA is not present, that is, it does not bind to HAS although it binds to another molecule. For example, you can link a third objective in the cell. Normally, the half-life is increased by 10%, 20%, 30%, 40%, 50% or more. Increases in the range of 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, 5x or more of the half-life are also possible. As an alternative, or in addition, increments within the range of up to 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x of the half-life are also possible.
As referred to in the present invention, the term "competes" means that the binding of a first target to its cognate target binding domain is inhibited when a second target is linked to its cognate target binding domain. For example, the linkage can be sterically inhibited, for example by physical blocking of a binding domain by altering the structure or environment of the binding domain, so that its avidity affinity for the target is reduced. As used in the present invention, the terms "low stringency", "medium stringency", "high stringency", or "very high stringency conditions", describe conditions for hybridization and washing of nucleic acid. The guide for carrying out hybridization reactions can be found in the Current Protocols in Molecular Biology Publication, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated in its entirety to the present invention as a reference. Aqueous and non-aqueous methods are described in said references and any of them may be used. The specific hybridization conditions referred to herein are as follows: (1) low stringency hybridization conditions in sodium chloride / 6X sodium citrate (SSC) at a temperature of about 45C, followed by two washes in 0.2X SSC , 0.1% SDS at a temperature of at least 50C (the temperature of the washes can be increased to 55C for conditions of low stringency); (2) medium stringency hybridization conditions in 6X SSC at a temperature of approximately 45C, followed by one or more washes in 0.2X SSC, 0.1% SDS at a temperature of 60C; (3) high stringency hybridization conditions in 6X SSC at a temperature of approximately 45C, followed by one or more washes in 0.2X SSC, 0.1% SDS at a temperature of 65C; and preferably (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at a temperature of 65 C, followed by one or more washes in 0.2X SSC, 1% SDS at a temperature of 65C. The conditions of very high stringency (4) are the preferred conditions and should be used unless otherwise specified. Similar or homologous sequences (eg, at least about 70% sequence identity) to the sequences described herein are also part of the present invention. In some embodiments, the sequence identity at an amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. At the level of nucleic acid, the sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or greater. Alternatively, there is substantial identity when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., very high stringency hybridization conditions) for the complement of the strand. The nucleic acids can be found in whole cells, in a cell lysate or in a partially purified or substantially pure form. The calculations of "homology" or "sequence identity" or "similarity" between two sequences (the terms are used interchangeably in the present invention), are carried out as indicated below. The sequences are aligned for optimal comparison purposes (for example, gaps may be introduced into one or both of a first or second amino acid or nucleic acid sequence for optimal alignment and may be ignored for comparison purposes). In a preferred embodiment, the length of an aligned reference sequence for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at less 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides in the corresponding amino acid portions or nucleotide positions, they are compared later. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical in that position (as the term "homology" of acid is used in the present invention). nucleic or amino acid is equivalent to the "identity" of nucleic acid or amino acid). The percent identity and the two sequences is a function of the number of identical positions shared by the sequences, taking finds the number of openings, and the length of each opening, that need to be entered for optimal alignment of the two sequences. The alignments, homology and sequence or identity of the amino acid or nucleotide sequence, as defined in the present invention, are preferably prepared and determined using the BLAST 2 algorithm sequences, using default parameters (Tatusova, * TA and associates, FEMS Microbiol Lett, 174: 187-188 (1999)). As an alternative, the BLAST algorithm (version 2.0) is used for the sequence alignment, with parameters adjusted to default values. BLAST (Basic Local Alignment Search Tool) is a search algorithm and heuristic used through the programs blastp, blastn, blastx, tbiastn, and tbiastx; these programs assign importance to their discoveries using the statistical methods of Karlin and AltschuL 1990, Proc. Nati Acad. Sel E.U.A. 87 (6): 2264-8. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art (e.g., in cell culture techniques, molecular genetics, nucleic acid chemistry, hybridization and biochemistry). Standard techniques are used for molecular genetic and biochemical methods (see generally Publications, Sambrook and Associates, Molecular Cloning: A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Springer Harbor, NY and Subusubel and associates, Short Protocols in Molecular Biology (1999) 4th Edition, John Wiley &Sons, Inc. which are incorporated herein by reference) and chemical methods. The present invention relates to ligands that have binding specificity for VEGF (e.g., human VEGF), ligands that have binding specificity for EGFR (e.g., human EGFR), and to ligands that have binding specificity for VEGF and EGFR ( for example, human VEGF and human EGFR). For example, the ligand may comprise a polypeptide domain having a binding site with binding specificity for VEGF, a polypeptide domain that has a binding site with binding specificity for EGFR, or comprises a polypeptide domain having a binding site with binding specificity for VEGF and a polypeptide domain having a binding site with binding specificity for EGFR. The ligands of the present invention pde several advantages. For example, as described in the present invention, the ligand can be custom-made to have a desired in vivo serum half-life. Therefore, the ligands can be used to control, reduce, or eliminate general toxicity of therapeutic agents, such as cytotoxin used to treat cancer. In addition, dAbs are much smaller than conventional antibodies, and can be administered to achieve better tissue penetration than conventional antibodies. Therefore, dAbs and ligands comprising dAb pde advantages over conventional antibodies, when administered to treat cancer, for example to treat solid tumors. In addition, many cancers overexpress EGFR, and ligands that have binding specificity for EGFR and VEGF can be administered to direct VEGF inhibitory activity to tumors or to the environment of cancer cells. This method pdes two beneficial activities directly at the site of a tumor or cancer, i.e. direct anti-cancer activity, by binding to EGFR and inhibiting the binding of ligands (eg, EGF, TGF alpha) to the receptor, and inhibition of angiogenesis which supports the formation and development of the tumor. Accordingly, ligands that have binding specificity for VEGF and EGFR can be administered to a cancer patient (eg, cancer expressing EGFR) to pde superior therapy using a single therapeutic agent. In addition, signals transduced through EGFR can lead to the production of angiogenic factors, such as VEGF. Cancer cells (eg, in a tumor) that express or overexpress EGFR can produce a high level of VEGF that acts locally to induce the formation of the tumor vasculature. Accordingly, the ligands of the present invention having binding specificity for VEGF and EGFR can be administered to a subject to direct the delivery of VEGF inhibitory activity of ligand to cells that overexpress EGFR. Accordingly, anti-angiogenic therapy can be delivered specifically to sites where VEGF is being produced (eg, to cells that overexpress EGFR). In some modalities, the ligand has binding specificity for VEGF and comprises a (at least one) variable domain of simple immunoglobulin with binding specificity for VEGF. In other embodiments, the ligand has binding specificity for EGFR and comprises a single immunoglobulin variable domain (at least one) with binding specificity for EGFR. In certain embodiments, the ligand has binding specificity for VEGF and EGFR, and comprises a (at least one) variable domain of single immunoglobulin with binding specificity for VEGF and one (at least one) variable domain of single immunoglobulin with binding specificity for EGFR. The ligand of the present invention can be formatted as described in the present invention. For example, the ligand of the present invention can be formatted to tailor the average serum life in vivo. If desired, the ligand may further comprise a toxin or a toxin moiety as described in the present invention. In some embodiments, the ligand comprises a surface active toxin, such as a free radical generator (eg, selenium-containing toxin) or a radionuclide. In other embodiments, the toxin or toxin portion is a polypeptide domain (eg, a dAb) that has a binding site with binding specificity for an intracellular target. In particular embodiments, the ligand is an IgG-like format that has binding specificity for VEGF and EGFR (eg, human VEGF and human EGFR). Ligand Formats The ligand of the present invention can be formatted as a monospecific, specific dual or multi-specific ligand, as described herein. See also Publication WO 03/002609, the entire teachings of which are incorporated herein by reference, refer to the formatting of ligands. Said specific double ligands comprise variable domains of single immunoglobulin having different binding specificities. Said specific double ligands may comprise combinations of heavy and light chain domains. For example, the double specific ligand may comprise a VH domain and a V domain, which may be linked together in the form of a scFv (e.g., using a suitable linker such as Gly4Ser), or formatted in a biospecific antibody or fragment. of antigen binding 'thereof (eg, F (ab') 2 fragment). The specific double ligands do not comprise complementarity VH / V pairs that form a binding site for two standard antigen antibody antigens that bind the antigen or epitope operatively together. Rather, the double format ligands comprise a VH / VL complementarity pair wherein the V domains have different binding specificities. In addition, the specific double ligands may comprise one or more CH or C domains if desired. It can also be included if a joint region is desired. Such combinations of domains may for example be natural mimetic antibodies such as IgG or IgM, or fragments thereof, such as Fv, scFv, Fab or F (ab ') 2 molecules. Other structures, such as a single arm of an IgG molecule comprising VH, V, CH1 and C domains, are also considered. Preferably, the dual specific ligand of the present invention comprises only two of variable domains although several of said ligands can be incorporated together in the same protein, for example two of said ligands can be incorporated into an IgG or multimeric immunoglobulin, such as IgM. Alternatively, in another embodiment a plurality of double specific ligands combine to form a multimer. For example, two dual specific ligands are combined to create a tetra-specific molecule. Those skilled in the art will appreciate that the light and heavy variable regions of a double specific ligand produced according to the method of the present invention, they can be in the same polypeptide chain, or alternatively in different polypeptide chains. In case the variable regions are in different polypeptide chains, then they can be linked through a linker, generally a flexible linker (such as a polypeptide chain), a chemical linking group or any other method known in the art. technique. The ligands can be formatted as bi or multispecific antibodies or antibody fragments or in bi or multi-specific antibody structures. Suitable formats include, any suitable polypeptide structure in which the variable antibody domain or one or more of the CDRs thereof can be incorporated for the antigen in the structure. A variety of suitable antibody formats are known in the art, such as bispecific IgG type formats (e.g., chimeric antibodies, humanized antibodies, single chain antibodies, heavy chain and / or light chain antibody heterodimers, link fragments). of antigen from any of the foregoing (eg, an Fv fragment (eg, a single chain Fv (scFv), a disulfide linked Fv), a Fab fragment, a Fab 'fragment, an F (ab') fragment 2), a single variable domain (eg, VH, VL, VHH), a dAb, and modified versions of any of the foregoing (eg, modified through the polyalkylene glycol covalent injury (eg, polyethylene glycol, polypropylene glycol, polybutylene glycol) or another suitable polymer.) See PCT Publication / GB03 / 002804, filed on June 30, 2003, which is designated in the United States (WO 2004/081026) with respect to the pegylation of domains. simple ariables and dAbs, suitable methods for preparing them, increased in vivo half-life, of the PEGylated simple variable domains and dAb monomers and multimers, PEGs, suitable sizes of preferred hydrodynamic PEGs and sizes of single variable domains PEGylated hydrodynamic and monomers and dAb multimers. The total teachings of PCT / GB03 / 002804 (WO 2004/081026), including the preferred parts above, are incorporated by reference into the present invention. The ligand can be formatted using a suitable linker such as (Gly4Ser) n, where n = from 1 to 8, for example, 2, 3, 4, 5,6 or 7. If desired, including ligands, monomers, dimers and dAb trimers may be linked to an Fc antibody region, which comprises one or both of the CH2 and CH3 domains, and optionally a hinge region. For example, vectors encoding ligands linked as a single nucleotide sequence to an Fc region can be used to prepare said polypeptides. The dAb ligands and monomers can also be combined and / or formatted into multiligand structures without antibodies to multivalent complexes, which bind target molecules with the same antigen thus providing higher avidity. For example, natural bacterial receptors such as SpA can be used as scaffolds for the grafting of CDRs to generate ligands that specifically bind to one or more epitopes. The details of this procedure are described in the Patent 5,831,012. Other suitable scaffolds include those that are based on fibronectin and affibodies. The details of suitable procedures are described in Publication 98/58965. Other suitable scaffolds include lipocalin and CTLA4, as described in the publication by van den Beuken et al., J. Mol. Biol 310: 591-601 (2001), and scaffolds such as described in Publication WO 00/69907 (Medical Research Council), which are based for example on the bacterial GroEL ring structure or other chaperone polypeptides . Protein scaffolds can be combined, for example, CDRs can be grafted onto a CTLA4 scaffold used together with immunoglobulin VH or VL domains, to form a ligand. In the same way, fibronectin, lipocalin and other scaffolds can be combined. A variety of suitable methods for preparing any desired format is known in the art. For example, the antibody and format chains for example, bispecific IgG type format, chimeric antibodies, humanized antibodies, human antibodies, single chain antibodies, homodimers and heavy chain heterodimers and / or antibody light chains) can be prepared through of the expression of suitable expression constructs and / or culture of suitable cells (for example, hybrids, heterohybridomas, recombinant host cells containing recombinant constructs encoding the format). In addition, formats such as antibody antigen binding fragments or antibody chains (e.g., bispecific binding fragments such as an Fv fragment (e.g., single chain Fv (scFv), an Fv linked with disulfide), a fragment Fab, a Fab 'fragment, an F (ab') 2 fragment), can be prepared by expressing suitable expression constructs or by enzymatic digestion of antibodies, for example using papain or pepsin. The ligand can be formatted as a multi-specific ligand, for example as described in Publication WO 03/002609, the entire teachings of which are incorporated herein by reference. Said multi-specific ligand possesses more than one epitope binding specificity. Generally, the multi-specific ligand comprises two or more epitope binding domains such as dAbs or antibody protein domain comprising a binding site for an epitope, for example, an affibody, an SpA domain, an LDL receptor domain class A, an EGF domain, an avimer. The multispecific ligands can be formatted additionally as described in the present invention. In some embodiments, the ligand is an IgG type format. Said formats have the conventional structure of four chains of an IgG molecule (2 heavy chains and two light chains), wherein one or more of the variable regions (V H or V L) have been replicated with a single variable domain or dAb of a specificity desired. Preferably, each of the variable regions (2 VH regions and 2 V regions) is replaced with a dAb or simple variable domain. The dAb (s) or simple variable domain (s) that are included in the IgG type format can have the same specificity or different specificities. In some embodiments, the IgG type format is tetravalent and may have one, two, three or four specificities. For example, the IgG type format can be monospecific and comprises 4 dAbs that has the same specificity; bispecific and comprises 3 dAbs that has the same specificity as another dAb that has a different specificity; the bispecifics and comprising two dAbs that have the same specificity and two dAbs that have a common but different specificity; trispecific and comprising first and second dAbs that have the same specificity, a third dAb with a different specificity and a fourth dAb with a different specificity of the first, second and third dAbs; or tetraespecific and comprises four dAbs each having different specificity. Antigen binding fragments of IgG type formats (eg, Fab, F (ab ') 2, Fab', Fv, scFy) can be prepared. In addition, a particular constant region of an Fc part (for example, of an IgG, such as lgG1), variant or part thereof can be selected in order to tailor the effector function. For example, if an antibody-dependent cell complement and / or cytoxicity activation (ADCC) function is desired, the ligand can be a lgG1-like format. If desired, the IgG type format can comprise a mutated constant region (heavy chain constant region IgG variant) to minimize binding to Fc receptors and / or the ability to fix the complement (for example, see Wínter Publication and associates , GB 2,209,757 B, Morrison and associates, WO 89/07142, Morgan and associates, WO 94/29351, December 22, 1994). The ligands of the present invention can be formatted as a fusion protein containing a first variable domain of single immunoglobulin that is directly fused to a second variable domain of single immunoglobulin. If such a format is desired, it may further comprise an extension portion of half-life. For example, the ligand may comprise the first variable domain of single immunoglobulin that is fused directly to a second variable domain of single immunoglobulin that is directly fused to a variable domain of single immunoglobulin that binds to serum albumin. Generally targeting the polypeptide domains having a binding site with binding specificity to a target, and if the ligand comprises a linker, is a matter of design choice. However, some orientations, with or without linkers, can provide better link characteristics than other orientations. All orientations (e.g., dAbl-linker-dAb2; dAb2-linker-dAb1) are encompassed by the present invention and are ligands that contain an orientation that provides desired binding characteristics that can be easily identified by classification. Extended Medium Life Formats "The dAb ligands and monomers described herein can be formatted to extend their serum half life in vivo. The insed in vivo half-life is useful in in vivo applications of immunoglobulins, especially antibodies and most antibody fragments, especially small size such as dAbs. These fragments (Fvs, Fvs linked with disulfide, Fabs, scFvs, dAbs) are rapidly cleared from the body, which may inse clinical applications. A ligand can be formatted as a binding fragment of larger antigens of an antibody or as an antibody (eg, formatted as a Fab, Fab ', F (ab) 2, F (ab') 2, IgG, scFv) which has a larger hydrodynamic size. The ligands can also be formatted to have a larger hydrodynamic size, for example, by adhesion of a polyalkylene glycol group (eg, polyethylene glycol or (PEG) group, polypropylene glycol, polibutyl glycol), serum albumin, transferin, transferin receptor or at least the transfer part of transferin, an antibody region Fc, or by conjugation to an antibody domain. In some embodiments, the ligand (e.g., monomer dAb) is PEGylated. Preferably the PEGylated ligand (e.g., monomer dAb) binds VEGF and / or EGFR with substantially the same affinity or avidity as the same ligand that is not PEGylated. For example, the ligand can be a PEGylated ligand comprising a dAb that binds VEGF or EGFR with an avidity that differs from the avidity of the ligand in a PEGylated form by not more than a factor of about 1000, preferably not more than one factor. about 100, more preferably not more than a factor of about 10, or with avidity affinity substantially unchanged with respect to the PEGylated form. See Publication, PCT / GB03 / 002804, filed on June 30, 2003, which is designated in the United States (WO 2004/081026) with respect to simple variable domains PEGylated and dAbs, suitable methods to prepare them, life increased in vivo media of the PEGylated single variable domains and dAb monomers and multimers, suitable PEGs, preferred hydrodynamic sizes of PEGs, and preferred hydrodynamic sizes of PEGylated single variable domains and dAb monomers and multimers. The total teachings of PCT / GB03 / 002804 (WO 2004/081026), including the parts referred to above, are incorporated by reference into the present invention. The hydrodynamic size of the ligands (e.g., monomers and dAb multimers) of the present invention can be determined using methods that are known in the art. For example, gel filtration chromatography can be used to determine the hydrodynamic size of a ligand. Suitable gel filtration matrices for determining the hydrodynamic sizes of ligands, such as cross-linked agarose matrices, are well known and readily available. The size of a ligand format (e.g., the size of a PEG portion adhered to a dAb monomer) may vary depending on the desired application. For example, when the ligand is projected to leave the circulation and enter the peripheral tissues, it is desirable to keep the hydrodynamic size of the ligand low to facilitate emptying of the bloodstream. Alternatively, when it is desired to have a ligand that remains in the systemic circulation for a longer period of time, the size of the ligand can be increased, for example by formatting as an Ig-like protein or through the addition of a PEG-30 portion. at 60 kDa (eg, PEG of 30 to PEG 40 kDa, such as the addition of two PEG 20 kDa). The size of the ligand format can be tailored to achieve a desired in vivo serum half life, for example to control exposure to a toxin and / or reduce the side effects of toxic agents. The hydrodynamic size of the ligand (e.g., the dAb monomer) and its serum half-life can also be increased by conjugate or binding the ligand to a binding domain (e.g., antibody or antibody fragment) that binds to an antigen or epitope that increases the half-life in vivo, as described in the present invention. For example, the ligand (e.g., the dAb monomer) can be conjugated or bound to an anti-serum albumin or anti-neonatal Fc receptor antibody or antibody fragment, e.g., an anti-SA or anti-neonatal receptor dAb Fc, Fab, Fab 'or scFv, or an anti-SAo antibody or an anti-neonatal Fc receptor affinitor. Suitable examples of albumin, albumin fragments or albumin variants for use in a ligand according to the present invention are described in WO 2005/077042A2, which is incorporated herein by reference in its entirety. In particular, the following variants of albumin, albumin or albumin fragments can be used in the present invention: SEQ ID NO: 1 as described in WO 2005 / 077042A2, (this sequence is explicitly incorporated in the present invention as reference); • Fragment of albumin or albumin variant comprising or consisting of amino acids 1-387 of SEQ ID NO: 1 in Publication WO 2005 / 077042A2; • Albumin, or fragment or variant thereof, comprising an amino acid sequence selected from the group consisting of: (a) amino acids 54 to 61 of SEQ ID NO: 1 in Publication WO 2005 / 077042A2; (b) amino acids 76 to 89 of SEQ ID NO: 1 in Publication WO 2005 / 077042A2; (c) amino acids 92 to 100 of SEQ ID NO: 1 in Publication WO 2005 / 077042A2; (d) amino acids 170 to 176 of SEQ ID NO: 1 in Publication WO 2005 / 077042A2; (e) amino acids 247 to 252 of SEQ ID NO: 1 in Publication WO 2005 / 077042A2; (f) amino acids 266 to 277 of SEQ ID NO: 1 in Publication WO 2005 / 077042A2; (g) amino acids 280 to 288 of SEQ ID NO: 1 in Publication WO 2005 / 077042A2; (h) amino acids 362 to 368 of SEQ ID NO: 1 in Publication WO 2005 / 077042A2; (i) amino acids 439 to 447 of SEQ ID NO: 1 in Publication WO 2005 / 077042A2 O) amino acids 462 to 475 of SEQ ID NO: 1 in Publication WO 2005 / 077042A2; (k) amino acids 478 to 486 of SEQ ID NO: 1 in Publication WO 2005 / 077042A2; and (I) amino acids 560 to 566 of SEQ ID NO: 1 in Publication WO 2005 / 077042A2. Additional examples of albumin, fragments and analogues for use in a ligand according to the present invention are described in WO 03/076567A2, which is incorporated herein by reference in its entirety. In particular, the following albumin, fragments or variants can be used in the present invention: • Human serum albumin as described in Publication WO 03/076567 A2, for example, in Figure 3 (this information of sequence is explicitly incorporated by reference in the present invention); • Human serum albumin (HA) consisting of a single non-glycosylated polypeptide chain of 585 amino acids with a formula molecular weight of 66,500 (see Meloun Publication, and associates, FEBS Letters 55: 136 (1975); Behrens, and associates, Fed. Proc. 34: 591 (1975); Lawn, et al., Nucleic Acids Research P: 6102-6114 (1981); Minghetti, et al., J. Biol. Chem. 261: 6747 (1986)); • Polymorphic or analogous variant or fragment of albumin as described in the Publication of Weitkamp, and associates, Ann. Hum. Genet 37: 219 (1973); • A fragment or variant of albumin as described in EP 322094, for example, HA (1-373., HA (1-388), HA (1-389), HA (1-369), and HA (1 -419) and fragments between 1-369 and 1-419 • A fragment or variant of albumin as described in EP 399666, for example, HA (1-177) and HA (1-200) and fragments between HA ( IX), wherein X is any number from 178 to 199. Wherein one (one or more) half-life extension portion (e.g., albumin, transferin and fragments and analogs thereof) is used in the ligands of the present invention, can be conjugated to ligands using any suitable method such as by direct fusion to the target binding moiety (eg, dAb or antibody fragment), using for example a simple nucleotide moiety encoding a fusion protein, in wherein the fusion protein is encoded as a single polypeptide chain with a half-life extension portion located at N or C terminal e n the target cell surface binding portions. Alternatively, conjugation can be accomplished using a peptide linker between portions, a peptide linker as described in Publication WO 03/076567A2 or WO 2004/003019 (these linker descriptions are incorporated herein by reference for provide examples to be used in it). Typically, a polypeptide that improves the serum half-life in vivo is a polypeptide that occurs naturally in vivo and that resists degradation or elimination by endogenous mechanisms that eliminate unwanted material from the organism (eg, human). For example, a polypeptide that increases the half-life in vivo can be selected from proteins from the extracellular matrix, proteins found in the blood, proteins found in the blood-brain barrier or a neural tissue, proteins located in the kidney, liver, lung , heart, skin or bones, tension proteins, disease-specific proteins, or proteins involved in transport Fc. Suitable polypeptides that increase the half-life in vivo include, for example, fusion proteins of receptor-ligand-specific neuropharmaceuticals of transferrin receptor (see U.S. Patent No. 5,977,307, the teachings of which are incorporated herein by reference, receptor capillary endothelial cell of the brain, transferin, transferin receptor (eg, soluble transferrin receptor), insulin, insulin-like growth factor-1 receptor (IGF 1), insulin-like growth factor-2 receptor (IGF 2), insulin receptor, blood coagulation factor X, a1-antitrypsin and HNF 1a Suitable polypeptides that increase serum half-life also include alpha-1 glycoprotein (orosomucoid); AAG), alpha-1 antichymotrypsin (ACT), alpha-1 microglobulin (HC protein, AIM), anti-thrombin III (AT III), apolipoprotein A-1 (Apo A-1), apolipoprotein B (Apo B), ceruloplasmin (Cp), complement component C3 (C3), complement component C4 (C4), esterase inhibitor C1 (C1 INH), C-reactive protein (CRP), ferritin (FER), hemopexin (HPX), lipoprotein (Lp) (a)), handy binding protein (MBP), myoglobin (Myo), prealbumin (transthyretin; PAL), retinol binding protein (RBP), and rheumatoid factor (RF). Suitable proteins of the extracellular matrix include, for example, collagen and laminites, integrins and fibronectin. Collagens are the important proteins of the extracellular matrix. Approximately 15 types of collagen molecules are currently known, found in different parts of the body, for example, type I collagen (which covers 90% of the body's collagen) found in bones, skin, tendons, ligaments, cornea, internal organs, or type II collagen found in cartilage, vertebral discs, cord noto and vitreous humor of the eyes. Suitable proteins in the blood include, for example, plasma protein (e.g., fibrin, α-2 macroglobulin, serum albumin, fibrinogen (e.g., fibrinogen A, fibrinogen B), serum A amyloid protein, haptoglobin, profilin , ubiquitin, uteroglobulin and β-2 microglobulin), enzymes, enzyme inhibitors (eg, plasminogen, lysozyme, cystatin C, alpha-1-antitrypsin and pancreatic trypsin inhibitor), immune system proteins such as immunoglobulin proteins (eg, example, IgA, IgD, IgE, IgG, IgM, immunoglobulin light chains (kappa / lambda)), transport proteins (e.g., retinol binding protein, a-1 microglobulin), defensins (e.g., beta-defensin 1, neutrophil defensin 1, neutrophil defensin 2 and neutrophil defensin 3) and the like. Suitable proteins encintrated in the blood-brain or neural tissue barrier include, for example, melanocortin receptor, myelin, ascorbate transporter and the like. Polypeptides that increase serum half-life in vivo also include proteins located in the kidney (eg, polycystin, type IV collagen, organic anion transporter, Heymann's antigen), protein located in the liver (eg, alcohol dehydrogenase) , G250), proteins located in the lung (for example, secretion component that binds to IgA), proteins located in the heart (for example, HSP 27, which is associated with dilated cardiomyopathy), proteins located in the skin (for example , keratin), bone specific proteins such as morphogenic proteins (BMPs), which are a subgroup of the transforming growth factor superfamily of ß -reflecting proteins that demonstrate ostreogénica activity (eg, BMP-2, BMP-4). , BMP-5, BMP-6, BMP-7, BMP-8), tumor-specific proteins (e.g., tropoblast antigen, herceptin receptor, estrogen receptor, cathepsins (e.g., cathepsin B, which can be found in the liver and spleen)). Suitable disease-specific proteins include for example antigens expressed only on activated T cells, including LAG-3 (lymphocyte activation gene), osteoprotegerin ligands (OPGL, Nature 402, 304-309 (1999)), OX40 (a member of the TNF receptor family expressed on activated T cells and specifically activated on cells that produce human type I T cell leukemia virus (HTLV-1), see Immunol Publication 165 (l): 263-70 (2000) )). Suitable disease-specific proteins also include, for example, metalloproteases (associated with arthritis / breast cancer) including drosophila CG6512, human paraplegina, human FtsH, human AFG3L 2, murine ftsH; and angiogenic growth factor, including acid fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), vascular endothelial growth factor / vascular permeability factor (VEGF / VPF), growth-transforming factor-a (TGF a), tumor necrosis factor-alpha (TNF-a), angiogenin, interleukin-3 (IL-) 3), interleukin-8 (IL-8), platelet-derived endothelial growth factor (PD-ECGF), placental growth factor (P1GF), MD-platelet-derived growth factor-BB (PDGF), and fractalcin . Suitable polypeptides that increase serum half life in vivo also include strain proteins such as heat shock proteins (HSPs). HSPs are usually found intracellularly. When they are in extracellular form, it is an indicator that the cell has died and its contents are spilled. This unscheduled cell death (necrosis) occurs when as a result of trauma, disease or injury, extracellular HSPs are activated in response to the immune system. The binding to extracellular HSP can result in localization of the compositions of the present invention in a diseased site. Suitable proteins involved in Fc transport include, for example, Brambell receptor (also known as FcRB). This Fc receptor has two functions, both of which are potentially useful for administration. The functions are (1) transport of IgG from the mother to the child through the placenta (2) protection of IgG in terms of degradation, prolonging its half-life in this way. The receptor is considered to recycle IgG from the endosomes. (See Publication by Holliger and associates, Nat Biotechnol 15 (7): 632-6 (1997).) Methods for analysis and pharmacokinetic determination of half-life of ligand will be familiar to those skilled in the art. Publication by Kenneth, A. and associates: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters and Associates, Pharmacokinetc analysis: A Practical Approach (1996). Reference is also made to the Publication of "Pharmacokinetics", M Gibaldi &; D Perron, published by Marcel Dekker, 2nd Revision (1982), which describes pharmacokinetic parameters such as half-lives alpha t and beta t and urea under the curve (AUC). c Ligands Containing a Portion of Toxin or Toxin The present invention also relates to ligands comprising a toxin or toxin portion. Portions of suitable toxins comprise a toxin (e.g., active surface toxin, cytotoxin). The toxin or toxin portion can be linked or conjugated to the ligand using any suitable method. For example, toxin or toxin portion can be covalently bound to the ligand directly or through the appropriate linker. Suitable linkers can include non-dissociable or dissociable linkers, for example, pH-releasable linkers comprising a dissociation site for a cellular enzyme (eg, cellular esterases, cellular proteases such as cathepsin B). Said dissociable linkers can be used to prepare a ligand that can release a toxin or toxin portion after the ligand is internalized. A variety of methods can be used to bind or conjugate a portion of toxin or toxin to a ligand. The particular method selected will depend on the toxin or toxin portion and the ligand that will be bound conjugate. If desired, linkers containing terminal functional groups can be used to link the ligand and the toxin or toxin portion. Generally, conjugation is achieved by reacting the toxin portion to toxin containing a reactive functional group (or modified to contain a reactive functional group) as a linker or directly with a ligand. The covalent bonds formed by reacting a toxin to toxin portion containing (or modifying to contain) a chemical moiety or functional group which, under suitable conditions, can react with a second chemical group thereby forming a covalent bond. If desired, a suitable reactive chemical group can be added to a ligand or a linker using any suitable method. (See, for example, Hermanson Publication, GT, Bioconjugate Techniques, Academic Press: San Diego, CA.) Many combinations of reactive chemical groups suitable in the art are known, for example an amine group can react with an electrophilic group. such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidylester (NHS), and the like .. Thiols can react with maleimide, iodoacetyl, acryloyl ilo, pyridyl disulfides, thiol of 5-thiol acid -2-nitrobenzoic acid (TNB-thiol), and the like A functional group of aldehyde-containing molecules containing amine or hydrazide can be coupled, and an azide group can react with a trivalent phosphorus group to form phosphide amidate or phosphorus imide ligations. Suitable methods for introducing molecule activation groups are known in the art (see for example, Hermanson Publication, GT, Bioconjugate Techniques, Academic Press: San Diego, CA (1996)). Portions of suitable toxins include, for example, a maytansinoid (eg, maytansinol, eg, DM1, DM4), a taxane, a calicamicin, a duocarmycin, or derivatives thereof. The maytansinoid may be, for example, maytansinol or a maytansinol analogue. Examples of maytansinol analogs include those having a modified aromatic ring (eg, C-19-dechloro, C-20-demethoxy, C-20-acyloxy) and those having modifications in other positions (eg, C- 9-CH, C-14-alkoxymethyl, C-14-hydroxymethyl or acyloxymethyl, C-15-hydroxy / acyloxy, C-15-methoxy, C-18-N-demethyl, 4,5-deoxy). Maytansinol and maytansinol analogues are described, for example, in US Patent Nos. 5,208,020 and 6,333,410, the contents of which are incorporated herein by reference. Maitansinol can be coupled to antibodies and antibody fragments using, for example, a N-succinimidyl proprionate 3- (2-pyridyldithio) (also known as pentanoate N-succinimidyl 4- (2-pyridyldithio) or SPP), 4-succinimidyl-oxycarbonyl-a- (2-pyridyldithio) - toluene (SMPT), N-succinimidyl-3- (2-pyridyldithio) butyrate "(SDPB), 2-iminothiolane, or S-acetylsuccinic anhydride The taxane may be, for example, a taxol, taxoter, or novel taxane (see below). example, Publication WO 01/38318.) Calicheamycin can be, for example, a bromo complex calicheamicin (eg, a bromo complex alpha, beta or gamma), a complex-iodine calicheamycin (eg complex alpha, beta or gamma iodine), or analogs and imitations thereof Bromine complex calicheamicins include H-BR.12-BR, 13-BR, 14-BR, J1-BR, J2-BR and K1-BR The iodine complex calicheamicins include 11-1, 12-1, 13-1, J1-I, J2-I, L1-I and K1-BR Calicheamycin and mutants, analogues and mimetics are described for example in the US Patent Nos. 4,970,198; 5,264,586; 5,550,246; 5,712,374, and 5,714,586, the contents of which are incorporated herein by reference. Duocarmycin analogs (eg, KW-2189, DC88, DC89 CBI-TMI, and derivatives thereof) are described, for example, in U.S. Patent No. 5,070,092, U.S. Patent No. 5,187,186, U.S. Patent No. 5,641,780, US Patent No. 5,641,780, US Patent No. 4,923,990, and US Patent No. 5,101,038, the contents of which are incorporated herein by reference. Examples of other toxins include, but are not limited to antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, chlorambucil of tíoepa, CC- 1065 (see US Patent Nos. 5,475,092, 5,585,499, 5,846,545), melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine (II) platinum (DDP) cisplatin ), anthracyclines (e.g., daunorubicin (principally daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (primarily actinomycin), bleomycin, mithramycin, mitomycin, puromycin anthramycin (AMC)), duocarmycin, and analogs or derivatives thereof, agents and anti-mitotic agents (for example, vincristine, vinblastine, taxol, auristatins (for example, auristatin E) and maytansinoids, and analogues or homologs thereof The toxin can also be a to active surface toxin, such as a toxin that is a free radical generator (eg, portions of selenium-containing toxin) or a portion containing radionuclide. Portions containing suitable radionuclides include, for example, portions containing radioactive iodine (131l or 125l), yttrium (90Y), lutetium (177Lu), actinium (225Ac), praseodymium, astatine (211At), rhenium (186Re), bismuth (212Bi 213Bi), indium (111ln), technetium ("rnTc), phosphorus (32P), rhodium (188Rh), sulfur (35S), carbon (14C), tritium (3H), chromium (51Cr), chlorine (36CI) , cobalt (57Co or 58Co), iron (59Fe), selenium (75Se), or gallium (67Ga) The toxin can be a protein, polypeptide or peptide, from bacterial sources, for example, diphtheria toxin, pseudomonas exotoxin (PE) and plant proteins, for example ricin A chain (RTA), ribosome deactivating proteins (RIPs) gelonin, pokeweed antiviral protein, saporin, and dodecandron are contemplated to be used as toxins. antisense nucleic acid designed to bind disable, promote degradation or prevent the production of responsible mRNA of generating a particular target protein can also be used as a toxin. Antisense compounds include single or double stranded RNA or DNA, oligonucleotides or their analogs that can hybridize specifically to mRNA species and prevent transcription and / or RNA processing of mRNA species and / or translation of encoded polypeptide and thereby effect a reduction in the amount of the respective polypeptide. Ching, and associates, Proc. Nati Acad. Sci. E.U.A. 86: 10006-10010 (1989); Broder, and associates, Ann. Int. Med. 113: 604-618 (1990); Loreau, and associates, FEBS 'Letters 214: 53-56 (1990); Useful antisense therapeutics include for example: Vegli ™ (VasGene) and OGX-011 (Oncogenix). Toxins can also be photoactive agents. Suitable photoactive agents include porphyrin-based materials such as porfimer sodium, green porphyrins, E6 chlorin, the hematoporphyrin derivative itself, phthalocyanines, ethiopurpurines, texaprin, and the like. The toxin can be an antibody or an antibody fragment that binds to an intracellular target (e.g., an intrabody), such as a dAb that binds to an intracellular target. Said antibodies or antibody fragments (dAbs) can be directed to defined or objective subcellular compartments. For example, antibodies or antibody fragments (dAbs) can bind an intracellular target selected from erbB2, EGFR, BCR-ABL, p21Ras, Caspase3, Caspase7, Bcl-2, p53, cyclin E, ATF-1 / CREB, HPV16 E7, HP1 , collagenases type IV, cathepsin L as well as others described in the Publications of Kontermann, RE, Methods, 34: 163-170 (2004), incorporated herein by reference Domains of VEGF-Linking Polypeptides The present invention provides polypeptide domains (e.g., single immunoglobulin variable domains, dAb monomers) that have a binding site with binding specificity for VEGF. In preferred embodiments, the polypeptide domain (e.g., dAb) binds to VEGF with an affinity (KD); KD = Koff (kd) / Kon (ka)) from 300 nM to 1 pM (for example, 3 x 10"7 to 5 x 10" 12M), preferably 50 nM to 1 pM, more preferably 5 nM to 1 pM and most preferably 1 nM to 1 pM, for example and KD of 1 X 10"7 M or less, preferably 1 x 108 M or less, more preferably 1 x 109 M or less, conveniently 1 x 10" 10 M or less and more preferably 1 x 10 ~ 11 M or less; and / or a constant of rank K0ff of 5 x 10 ~ 1 s "1 to 1 x 10" 7 s "\ preferably 1 x 10 ~ 2 s" 1 to 1 x 10"6 more preferably 5 x 10" 3 s " 1 to 1 x 10"5 s" 1, for example 5 x 10"1 s" 1 or less, preferably 1 x 10"2 s" 1 or less, conveniently 1 x 10 -3 or less, more preferably 1 x 10"4 s" 1 or less, still preferably 1 x 10"5 s" 1 or less, and more preferably 1 x 10"6 s" 1 or less as determined by surface plasmon resonance In some embodiments, the dOminium of polypeptide having a binding site with binding specificity for VEGF competes to bind VEGF with a dAb selected from the group consisting of TAR15-1 (SEQ ID NO: 100), TAR15-3 (SEQ ID NO: 101), TAR15-4 (SEQ ID NO: 102), TAR15-9 (SEQ ID NO: 103), TAR15-10 (SEQ ID NO: 104), TAR15-11 (SEQ ID NO: 105), TAR15-12 (SEQ ID NO: 106), TAR15-13 (SEQ ID NO: 107), TAR15-14 (SEQ ID NO: 108), TAR15-15 (SEQ ID NO: 109), TAR15-16 (SEQ ID NO: 110), TAR15 -17 (SEQ ID NO: 111), TAR15-18 (SEQ ID NO: 112), TAR15-19 (SEQ ID NO: 113), TAR15-20 (SEQ ID NO: 114), TAR 15-22 (SEQ ID NO: 115), TAR15-5 (SEQ ID NOrl l), TAR15-6 (SEQ ID NO: 117) ), TAR15-7 (SEQ ID NO: 118), TAR15-8 (SEQ ID NO: 119), TAR15-23 (SEQ ID NO: 120), TAR15-24 (SEQ ID NO: 121), TAR15-25 ( SEQ ID NO: 122), TAR15-26 (SEQ ID NO: 123), TAR15-27 (SEQ ID NO: 124), TAR15-29 (SEQ ID NO: 125), TAR15-30 (SEQ ID NO: 126) , TAR15-6-500 (SEQ ID NO: 127), TAR15-6-501 (SEQ ID NO: 128), TAR15-6-502 (SEQ ID NO: 129), TAR15-6-503 (SEQ ID NO: 130), TAR15-6-504 (SEQ ID NO: 131), TAR15-6-505 (SEQ ID NO: 132), TAR15-6-506 (SEQ ID NO: 133), TAR15-6-507 (SEQ ID NO: 134), TAR15-6-508 (SEQ ID NO: 135), TAR15-6-509 (SEQ ID NO: 136), TAR15-6-510 (SEQ ID NO: 137), TAR15-8-500 ( SEQ ID NO: 138), TAR15-8-501 (SEQ ID NO: 139), TAR15-8-502 (SEQ ID NO: 140), TAR15-8-503 (SEQ ID NO: 141), TAR15-8- 505 (SEQ ID NO: 142), TAR15-8-506 (SEQ ID NO: 143), TAR15-8-507 (SEQ ID NO: 144), TAR15-8-508 (SEQ ID NO: 145), TAR15- 8-509 (SEQ ID NO: 146), TAR15-8-510 (SEQ ID NO: 147), TAR15-8-511 (SEQ ID NO: 148), TAR15-2 6-500 (SEQ ID NO: 149), TAR15-26-501 (SEQ ID NO: 150), TAR15-26-502 (SEQ ID NO: 151), TAR15-26-503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153), TAR15-26-505 (SEQ ID NO: 154), TAR15-26-506 (SEQ ID NO: 155), TAR15-26-507 (SEQ ID NO: 156) ), TAR15-26-508 (SEQ ID NO: 157), TAR15-26-509 (SEQ ID NO: 158), TAR15-26-510 (SEQ ID NO: 159), TAR15-26-511 (SEQ ID NO. : 160), TAR15-26-512 (SEQ ID NO: 161), TAR15-26-513 (SEQ ID NO: 162), TAR15-26-514 (SEQ ID NO: 163), TAR15-26-515 (SEQ ID NO: 164), TAR15-26-516 (SEQ ID NO: 165), TAR15-26-517 (SEQ ID NO: 166), TAR15-26-518 (SEQ ID NO: 167), TAR15-26-519 (SEQ ID NO: 168), TAR15-26-520 (SEQ ID NO: 169), TAR15-26-521 (SEQ ID NO: 170), TAR15-26-522 (SEQ ID NO: 171), TAR15-26 -523 (SEQ ID NO: 172), TAR15-26-524 (SEQ ID NO: 173), TAR15-26-525 (SEQ ID NO: 174), TAR15-26-526 (SEQ ID NO: 175), TAR15 -26-527 (SEQ ID NO: 176), TAR15-26-528 (SEQ ID NO: 177), TAR15-26-529 (SEQ ID NO: 178), TAR15-26-530 (SEQ ID NO: 179) , TAR15-26-531 (SEQ ID NO: 180), TAR15-26-532 (SEQ ID NO: 181), TAR15-26-533 (SEQ ID NO: 182), TAR15-26-534 (SEQ ID NO: 183), TAR15-26-535 (SEQ ID NO: 184), TAR15-26-536 (SEQ ID NO: 185), TAR15-26-537 (SEQ ID NO: 186), TAR15-26-53 (SEQ ID NO: 187), TAR15-26-539 (SEQ ID NO: 188), TAR15-26-540 (SEQ ID NO: 189), TAR15-26 -541 (SEQ ID NO: 190), TAR15-26-542 (SEQ ID NO: 191), TAR15-26-543 (SEQ ID NO: 192), TAR15-26-544 (SEQ ID NO: 193), TAR15 -26-545 (SEQ ID NO: 194), TAR15-26-546 (SEQ ID NO: 195), TAR15-26-547 (SEQ ID NO: 196), TAR15-26-548 (SEQ ID NO: 197) , TAR15-26-549 (SEQ ID NO: 198), TAR15-26-550 (SEQ ID NO: 539), and TAR15-26-551 (SEQ ID NO: 540). In some embodiments, the polypeptide domain that has a binding site with binding specificity for VEGF, comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with the amino acid sequence of a dAb selected from the group consisting of TAR15-1 (SEQ ID NO: 100), TAR15-3 (SEQ ID NO: 101), TAR15-4 (SEQ ID NO: 102), TAR15-9 (SEQ ID NO: 103), TAR15-10 (SEQ ID NO: 104), TAR15-11 (SEQ ID NO: 105), TAR15-12 (SEQ ID NO: 106), TAR15-13 (SEQ ID NO: 107), TAR15-14 (SEQ ID NO: 108), TAR15-15 (SEQ ID NO: 109), TAR15-16 (SEQ ID NO: 110 TAR15-17 (SEQ ID NO: 111), TAR15-18 (SEQ ID NO: 112 TAR15-19 (SEQ ID NO: 113), TAR15-20 (SEQ ID NO: 114 TAR 15-22 (SEQ ID NO: 115), TAR15-5 (SEQ ID NO: 116) TAR15-6 (SEQ ID NO: 117), TAR15-7 (SEQ ID NO: 118 TAR15-8 (SEQ ID NO: 119), TAR15-23 (SEQ ID NO: 120 TAR15-24 (SEQ ID NO: 121) , TAR15-25 (SEQ ID NO: 122 TAR15-26 (SEQ ID NO: "123), TAR15-27 (SEQ ID NO: 124 TAR15-29 (SEQ ID NO: 125), TAR15-30 (SEQ ID NO: 126 TAR15-6-500 (SEQ ID NO: 127), TAR15-6-501 (SEQ ID NO: 128 TAR15-6-502 (SEQ ID NO: 129), TAR15-6-503 (SEQ ID NO: 130 TAR15 -6-504 (SEQ ID NO: 131), TAR15-6-505 (SEQ ID NO: 132 TAR15-6-506 (SEQ ID NO: 133), TAR15-6-507 (SEQ ID NO: 134 TAR15-6 -508 (SEQ ID NO: 135), TAR15-6-509 (SEQ ID NO: 136 TAR15-6-510 (SEQ ID NO: 137), TAR15-8-500 (SEQ ID NO: 138 TAR15-8-501 (SEQ ID NO: 139), TAR15-8-502 (SEQ ID NO: 140 TAR15-8-503 (SEQ ID NO: 141), TAR15-8-505 (SEQ ID NO: 142 TAR15-8-506 (SEQ ID NO: 143), TAR15-8-507 (SEQ ID NO: 144 TAR15-8-508 (SEQ ID NO: 145), TAR15-8-509 (SEQ ID NO: 146 TAR15-8-510 (SEQ ID NO : 147), TAR15-8-511 (SEQ ID NO: 148 TAR15-26-500 (SEQ ID NO: 149), TAR15-26-501 (SEQ ID NO: 50), TAR15-26-502 (SEQ ID NO : 151), TAR15-26-503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153), TAR15-26-505 (SEQ ID NO: 154), TAR15-26-506 (SEQ ID NO: 155), TAR15-26 -507 (SEQ ID NO: 156), TAR15-26-508 (SEQ ID NO: 157), TAR15-26-509 (SEQ ID NO: 158), TAR15-26-510 (SEQ ID NO: 159), TAR15 -26-511 (SEQ ID NO: 160), TAR15-26-512 (SEQ ID NO: 161), TAR15-26-513 (SEQ ID NO: 162), TAR15-26-514 (SEQ ID NO: 163) , TAR15-26-515 (SEQ ID NO: 164), TAR15-26-516 (SEQ ID NO: 165), TAR15-26-517 (SEQ ID NO: 166), TAR15-26-518 (SEQ ID NO: 167), TAR15-26-519 (SEQ ID NO: 168), TAR15-26-520 (SEQ ID NO: 169), t TAR15-26-521 (SEQ ID NO: 170), TAR15-26-522 (SEQ. ID NO: 171), TAR15-26-523 (SEQ ID NO: 172), TAR15-26-524 (SEQ ID NO: 173), TAR15-26-525 (SEQ ID NO: 174), TAR15-26-526 (SEQ ID NO: 175), TAR15-26-527 (SEQ ID NO: 176), TAR15-26-528 (SEQ ID NO: 177), TAR15-26-529 (SEQ ID NO: 178), TAR15-26 -530 (SEQ ID NO: 179), TAR15-26-531 (SEQ ID NO: 180), TAR15-26-532 (SEQ ID NO: 181), TAR15-26-533 (SEQ ID NO: 182), TAR15 -26-534 (SEQ ID NO: 183), TAR15-26-535 (SEQ ID NO: 184), TAR15-26-536 (SEQ I) D NO: 185), TAR15-26-537 (SEQ ID NO: 186), TAR15-26-538 (SEQ ID NO: 187), TAR15-26-539 (SEQ ID NO: 188), TAR15-26-540 (SEQ ID NO: 189), TAR15-26-541 (SEQ ID NO: 190), TAR15-26-542 (SEQ ID NO: 191), TAR15-26-543 (SEQ ID NO: 192), TAR15-26 -544 (SEQ ID NO: 193), TAR15-26-545 (SEQ ID NO: 194), TAR15-26-546 (SEQ ID NO: 195), TAR15-26-547 (SEQ ID NO: 196), TAR15 -26-548 (SEQ ID NO: 197), TAR15-26-549 (SEQ ID NO: 198), TAR15-26-550 (SEQ ID NO: 539), and TAR15-26-551 (SEQ ID NO: 540) ). In preferred embodiments, the polypeptide domain having a binding site with binding specificity for VEGF, comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at less about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity of amino acid sequence with the amino acid sequence of a dAb selected from the group that you harbored in TAR15-6 (SEQ ID NO: 117), TAR15-8 (SEQ ID NO: 119), and TAR15-26 (SEQ ID NO: 123) ). For example, the polypeptide domain having a binding site with binding specificity for VEGF may comprise TAR15-6 (SEQ ID NO: 117), TAR15-8 (SEQ ID NO: 119), or TAR15-26 (SEQ ID NO: 123). In some embodiments, the polypeptide domain having a binding site with binding specificity for VEGF competes with any of the dAbs described herein for binding to VEGF. Preferably the polypeptide domain having a binding site with binding specificity for VEGF is a single immunoglobulin variable domain. The polypeptide domain having a binding site with binding specificity for VEGF can comprise any suitable immunoglobulin variable domain, and preferably comprises a human variable domain or a variable domain comprising regions of human structure. In certain embodiments, the polypeptide domain having a binding site with binding specificity for VEGF * comprises a universal structure, as described in the present invention. The universal structure can be a structure V (V? O VK), such as a structure comprising the amino acid sequences of the structure encoded by the immunoglobulin gene segment DPK1, DPK2, DPK3, DPK4, DPK5, DPK6, DPK7, DPK8, DPK9, DPK10, DPK12, DPK13, DPK15, DPK16, DPK18, DPK19, DPK20, DPK21, DPK22, DPK23, DPK24, DPK25, DPK26 or DPK 28 from the human germinal line. If desired, the VL structure may further comprise the amino acid sequence of the structure encoded by the immunoglobulin gene segment J? 1, J? 2, J "3, J? 4, or J? 5 of the germline. human In other embodiments, the universal structure may be a VH structure, such as a structure comprising the structure amino acid sequences encoded by the immunoglobulin gene segment DP4, DP7, DP8, DP9, DPIO, DP31, DP33, DP38, DP45 , DP46, DP47, DP49, DP50, DP51, DP53, DP54, DP65, DP66, DP67, DP68 or DP69 of the human germ line. If desired, the VH structure can further comprise the amino acid sequence structure encoded by the human germline immunoglobulin gene segment JH1, JH2, JH3, JH4, J? 4b, JH5 and JH6.
In certain embodiments, the polypeptide domain having a binding site with binding specificity for VEGF comprises one or more structure regions comprising an amino acid sequence that is the same as the amino acid sequence of a corresponding structure region encoded by a segment of the human germline antibody gene, or the amino acid sequences of one or more of the structure regions collectively comprise up to 5 amino acid differences relative to the amino acid sequence of the corresponding structure region encoded by a gene segment of human germ line antibody. In other embodiments, the amino acid sequences of FW1, FW2, FW3 and FW4 of the polypeptide domain having a binding site with binding specificity for VEGF are the same as the amino acid sequences of the corresponding structure regions encoded by a segment. of human germline antibody gene, or the amino acid sequences of FW1, FW2, FW3 and FW4 collectively contain up to 10 amino acid differences relative to the amino acid sequences of corresponding structure regions encoded by the line antibody gene segment human germinal. In other embodiments, the polypeptide domain having a binding site with binding specificity for VEGF, comprises regions FW1, FW2 and FW3 and the amino acid sequences of regions FW1, FW2 and FW3 are the same to the amino acid sequences of the corresponding structure regions encoded the human germline antibody gene segments. "In particular embodiments, the polypeptide domain having a binding site with binding specificity for VEGF comprises the DPK9 VL structure, or a VH structure selected from the group consisting of DP47, DP45 and DP38. The polypeptide domain having a binding site with binding specificity for VEGF, may comprise a binding site for a generic ligand, such as protein A, protein L and protein G. In certain embodiments, the polypeptide domain having a binding site with binding specificity for VEGF is substantially resistant to aggregation. or, in some embodiments, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2% or less of about 1% of the polypeptide domain having a binding site with binding specificity for VEGF, is added when a solution of 1-5 mg / ml, 5-10 mg / ml, 10-20 mg / ml, 20-50 mg / ml, 50-100 mg / ml, 100-200 mg / ml or 200-500 mg / ml ligand or dAb in a solvent that is used in a Routine for a drug formulation, such as saline, buffered saline, citrated buffered saline, water, an emulsion and any of these solvents with an acceptable excipient such as those approved by the FDA, is maintained at a temperature of about 22 ° C, 22-25 ° C, 25-30 ° C, 30-37 ° C, 37-40 ° C, 40-50 ° C, 50-60 ° C, 60-70 ° C, 70-80 ° C C, 15-20 ° C, 10-15 ° C, 5-10 ° C, 2-5 ° C, 0-2 ° C, -10 ° C to 0 ° C, -20 ° C to -10 ° C , -40 ° C to -20 ° C, -60 ° C to -40 ° C, or -80 ° C to -60 ° C, for a period of time for example 10 minutes, 1 hour, 8 hours, 24 hours , 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 1 year, or 2 years. Aggregation can be evaluated using any suitable method, such as, by microscopy, evaluating the turbid of a solution by visual inspection or spectroscopy or any other suitable method. Preferably, the aggregation is evaluated by dynamic light scattering. Polypeptide domains that have a binding site with binding specificity for VEGF that are resistant to aggregation provide several advantages. For example, said polypeptide domains having a binding site with binding specificity for VEGF can be easily produced in high yields as soluble proteins by expression, using a suitable biological production system, such as E. coli, and can be formulated and / or stored at higher concentrations than conventional polypeptides, and with less aggregation and loss of activity. In addition, the polypeptide domain having a binding site with binding specificity for VEGF that are resistant to aggregation can produce more economically than other antigen or epitope binding polypeptides (eg, conventional antibodies). For example, generally, the preparation of the antigen or epitope binding polypeptides projected for in vivo applications, include processes (eg, gel filtration) that eliminate aggregated polypeptides. Failure to eliminate such aggregates may result in a preference that is not suitable for in vivo applications due, for example, to that the aggregates of an antigen binding polypeptide that is projected to act as an antagonist may function as an agonist inducing cross-linking and agglomeration of the target antigen. "Protein aggregates can also reduce the efficacy of the therapeutic polypeptide by inducing an immune response in the subject to which they are administered. In contrast, the aggregation-resistant polypeptide domain having a binding specific binding site for VEGF of the present invention can be prepared for in vivo applications, without the need to include process steps that eliminate aggregates, and can be used in in vivo applications without the aforementioned disadvantages caused by the polypeptide aggregates. In some embodiments, the polypeptide domain having a binding site with binding specificity for VEGF, is reversibly split when heated to a temperature (Ts) and cooled to a temperature (Te), where Ts is greater at the melting temperature (Tm) of the polypeptide domain having a binding site with binding specificity for VEGF, and Te is lower than the fusion temperature of the polypeptide domain having a binding site with binding specificity for VEGF . For example, a polypeptide domain having a binding site with binding specificity for VEGF can be reversibly split when heated to a temperature of 80 ° C and cooled to approximately room temperature. A polypeptide that unfolded reversibly loses function when it unfolds but regains its function when it is redoubled. Said polypeptides are distinguished from polypeptides that are added when they unfold or that they redouble in an inadequate manner (badly bent polypeptides) that is, they do not gain again the function. The cleavage and redoubling of polypeptide can be evaluated, for example, by direct or indirect detection of the polypeptide structure using any suitable method. For example, the polypeptide structure can be detected by circular dichroism (CD) (e.g., far-UV CD, near-UV CD), fluorescence (e.g., side chain fluorescence of tryptophan), susceptibility to proteolysis, magnetic resonance Nucleic acid (NMR), or by detecting or measuring a polypeptide function that depends on the proper fold (binding to a target ligand, binding to a generic ligand). In one example, cleavage of the polypeptide is evaluated using a functional assay in which loss of binding function (eg, binding to a generic and / or target ligand, binding to a substrate) indicates that the polypeptide is unfolded. The degree of cleavage and redoubling of a polypeptide domain having a binding site with binding specificity for VEGF can be determined using a curve of unfolding or denaturation curve. A splitting curve can be produced by plotting temperature as the ordinate and the relative concentration of folded polypeptide as the abscissa. The relative concentration of the bent polypeptide domain having a binding site with binding specificity for VEGF, can be determined directly or indirectly using any suitable method (for example, CD, fluorescence, binding assay). For example, a polypeptide domain having a binding site with binding specificity for VEGF can be prepared and the ellipticity of the solution determined by CD. The ellipticity value obtained represents a relative concentration of bent ligand (e.g., monomer dAb) of 100%. The polypeptide domain having a binding site with binding specificity for VEGF, in the solution is subsequently unfolded by increasing the temperature of the solution in increments and the ellipticity is determined in appropriate increments (eg, after each increment of one degree in temperature). The polypeptide domain having an eplace site with binding specificity for VEGF, in the solution, is subsequently redoubled by reducing the solution temperature in increments and the ellipticity is determined in appropriate increments. The data can be plotted to produce a splitting curve and a bending curve. The doubling and redoubling curves have a characteristic sigmoid shape that includes a part in which the polypeptide domain having a binding site with binding specificity for VEGF molecules is doubled, a cleavage / doubling transition in which the domain of polypeptide having a binding site with binding specificity for VEGF molecules are split into various degrees, and a part in which the polypeptide domain having a binding site with binding specificity for VEGF, is split. The intercept of the y-axis of the redoubling curve is the relative amount of the redoubled polypeptide domain having a binding site with binding specificity for recovered VEGF. A recovery of at least about 50%, or at least about 60%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, is indicative that the dAb ligand or monomer is reversibly split. In a preferred embodiment, the ability to reverse the unfolding of a polypeptide domain having a binding site with binding specificity for VEGF is determined by preparing a polypeptide domain having a binding site with binding specificity for a VEGF solution. , and that traces bending curves and bending doubled. The polypeptide domain having a binding site with binding specificity for a VEGF solution can be prepared in any suitable solvent, such as an aqueous buffer having a suitable pH to allow the polypeptide domain having a binding site binding specificity for VEGF, dissolves (eg, a pH that is approximately 3 units above below the isoelectric point (pi)). The polypeptide domain having a binding site with binding specificity for a VEGF solution is sufficiently concentrated to allow splitting and / or bending to be detected. For example, the ligand solution The monomer dAb may be about 0.1 μM to about 100 μM, or preferably about 1 μM to about 10 μM. If the melting temperature (Tm) of a polypeptide domain having a binding site with binding specificity for VEGF is known, the solution can be heated to approximately ten degrees below the Tm (Tm-10) and the fold assessed. by ellipticity or fluorescence (eg, distant-UV CD scan from 200 nm to 250 nm, fixed length CD at 235 nm or 225 nm, fluorescent emission spectrum from tryptophan at 300 to 450 nm with excitation at 298 nm) to provide a dua ligand or monomer bent 100% relative. The solution is then heated to ten degrees above Tm (Tm + 10) in predetermined increments (e.g., increments of about 0.1 to about 1 degree), and the ellipticity or fluorescence is determined at each increment. Subsequently, the polypeptide domain having a binding site with binding specificity for VEGF is redoubled by cooling to at least Tm-10 in predetermined increments and an ellipticity or fluorescence determined at each increment. If the melting temperature of a polypeptide domain having a binding site with binding specificity for VEGF is not known, the solution can be split by increasing heating at a temperature of about 25 ° C to about 100 ° C and subsequently redoubled by cooling the increase to a temperature of at least about 25 ° C, and ellipticity or fluorescence at each increase in heating and cooling will be determined. The obtained data can be processed to produce a splitting curve and a redoubling curve, where the intercept of the y-axis of the roll-up curve is the relative amount of redoubled protein recovered. In some embodiments, the polypeptide domain having a binding site with binding specificity for VEGF, does not comprise a Camelia immunoglobulin variable domain, or one or more structure amino acids that are unique to the immunoglobulin variable domains encoded by the Gene segments of germline antibodies of Camelid.
Preferably, the polypeptide domain having a binding site with binding specificity for VEG is secreted in an amount of at least about 0.5 mg / L when expressed in E. coli or Pichia species (eg, P. pastoris ). In other preferred embodiments, the polypeptide domain having a binding site with binding specificity for VEGF, is secreted in an amount of at least about 0.75 mg / L, at least about 1 mg / L, at least about 4 mg / L. l, at least about 5 mg / l, at least about 10 mg / l, at least about 15 mg / l, at least about 20 mg / l, at least about 25 mg / l, at least about 30 mg / l, at least about 35 mg / l, at least about 40 mg / l, at least about 45 mg / l, or at least about 50 mg / l, or at least about 100 mg / l, or at least about 200 mg / l , or at least about 300 mg / l, or at least about 400 mg / l, or at least about 500 mg / l, or at least about 600 mg / l, or at least about 700 mg / l, or at least about 800 mg / l, at least approximately 900 mg / l, or at least approximately Ig / I when expressed in specific is E. coli or in Pichia (for example, P. pastoris). In other preferred embodiments, a polypeptide domain having a binding site with binding specificity for VEGF, is secreted in an amount of at least about 1 mg / L to at least about Ig / I, at least about 1 mg / L. to at least about 750 mg / l, at least about 100 mg / l to at least about 1 g / l, at least about 200 mg / l to at least about 1 g / l, at least about 300 mg / l to at least about 1 g / l, at least about 400 mg / l to at least about 1 g / l, at least about 500 mg / l to at least about Ig / I, at least about 600 mg / l to at least about 1 g / l, at least about 700 mg / l to at least about 1 g / l, at least about 800 mg / l to at least about Ig / l, or at least about 900 mg / L to at least about Ig / I when expressed in E. coli or Pichia species (eg, P. pastoris). Although a polypeptide domain having a binding site with binding specificity for VEGF, described herein may be secretable when expressed in E. coli species or in Pichia species (eg, P. pastoris), they may be produced using any suitable method , such as synthetic chemical methods or biological production methods that do not use E. coli or Pichia species. EGFR Binding Polypeptide Domain The present invention provides polypeptide domains (e.g., dAb) that have a binding site with binding specificity for EGFR. In preferred embodiments, the polypeptide domain (e.g., dAb) binds to EGFR with an affinity (KD); KD = Koff (kd) / Kon (ka)) from 300 nM to 1 pM (for example, 3 x 10"7 to 5 x 10 ~ 12M), preferably 100 nM to 1 pM, or 50 nM to 10 pM, more preferably 10 nM to 100 pM and most preferably about 1 nM, for example and K of 1 X 10"7 M or less, preferably 1 x 10" 8 M or less, more preferably about 1 x 10"9 M or less , 1 x 10"10 M or less or 1 x 10 ~ 11 M or less, and / or a Koff constant of 5 x 10" 1 s "1 to 1 x 10" 7 s "1 preferably 1 x 10" 2 s "1 to 1 x 10" 6 s "1, more preferably 5 x 1 O" 3 s "1 to 1 x 10 ~ 5 s" \ for example 5 x 10"1 s" 1 or less, preferably 1 x 102 s "1 or less, conveniently 1 x 10" 3 s "1 or less, more preferably 1 x 10 ~ 4 s" 1 or less, even more preferably 1 x 10"5 s" 1 or less, and most preferably 1 x 10"6 s" 1 or less as determined by surface plasmon resonance. In some embodiments, the polypeptide domain having a binding site with binding specificity for EGFR competes to bind to EGFR with a dAb selected from the group consisting of DOM16-17 (SEQ ID NO: 325), DOM16-18 (SEQ. ID NO: 326), DOM16-19 (SEQ ID NO: 327), DOM16-20 (SEQ ID NO: 328), DOM16-21 (SEQ ID NO: 329), DOM16-22 (SEQ ID NO: 330), DOM16-23 (SEQ ID NO.331), DOM16-24 (SEQ ID NO: 332), DOM16-25 (SEQ ID NO: 333), DOM16-26 (SEQ ID NO: 334), DOM16-27 (SEQ ID NO: 335), DOM16-28 (SEQ ID NO: 336), DOM16-29 (SEQ ID NO: 337), DOM16-30 (SEQ ID NO: 338), DOM16-31 (SEQ ID NO: 339), DOM16 -32 (SEQ ID NO: 340), DOM16-33 (SEQ ID NO: 341), DOM16-35 (SEQ ID NO: 342), DOM16-37 (SEQ ID NO: 343), DOM16-38 (SEQ ID NO. : 344), DOM16-39 (SEQ ID NO: 345), DOM16-40 (SEQ ID NO: 346), DOM16-41 (SEQ ID NO: 347), DOM16-42 (SEQ ID NO: 348 DOM16-43 ( SEQ ID NO: 349 DOM16-44 SEQ ID NO: 350 DOM16-45 (SEQ ID NO: 351 DOM16-46 SEQ ID NO: 352 DOM16-47 (SEQ ID NO: 353 DOM16-48 SEQ ID NO: 354 DOM16-49 (SEQ ID NO.355 DOM16-50 SEQ ID NO: 356 DOM16-59 (SEQ ID NO: 357 DOM16-60 SEQ ID NO: 358 DOM16-61 (SEQ ID NO: 359 DOM16-62 SEQ ID NO: 360 DOM16-63 (SEQ ID NO: 361 DOM16-64 SEQ ID NO: 362 DOM16-65 (SEQ ID NO: 363 DOM16-66 SEQ ID NO: 364 DOM16-67 (SEQ ID NO: 365 DOM16-68 SEQ ID NO: 366 DOM16-69 (SEQ ID NO: 367 DOM16-70 SEQ ID NO: 368 DOM16-71 (SEQ ID NO: 369 DOM16-72 SEQ ID NO: 370 DOM16-73 (SEQ ID NO: 371 DOM16-74 SEQ ID NO: 372 DOM16-75 (SEQ ID NO: 373 DOM16- 76 SEQ ID NO: 374 DOM16-77 (SEQ ID NO: 375 DOM16-78 SEQ ID NO: 376 DOM16-79 (SEQ ID NO: 377 DOM16-80 SEQ ID NO: 378 DOM16-81 (SEQ ID NO: 379 DOM16 -82 SEQ ID NO: 380 DOM16-83 (SEQ ID NO: 381 DOM16-84 SEQ ID NO: 382 DOM16-85 (SEQ ID NO: 383 DOM16-87 SEQ ID NO: 384 DOM16-88 (SEQ ID NO: 385 DOM16-89 SEQ ID NO: 386 DOM16-90 (SEQ ID NO: 387 DOM16-91 SEQ ID NO: 388 DOM16-92 (SEQ ID NO: 389 DOM16-94 SEQ ID NO: 390 DOM16-95 (SEQ ID NO: 391 DOM16-96 SEQ ID NO: 392 DOM16-97 (SEQ ID NO: 393 DOM16-98 SEQ ID NO: 394 DOM16-99 (SEQ ID NO: 395), DOM16-100 SEQ ID NO: 396 DOM16-101 (SEQ ID NO: 397), DOM16-102 'SEQ ID NO: 398 DOM16-103 (SEQ ID NO: 399), DOM16-104 (SEQ ID NO: 400 DOM16-105 (SEQ ID NO: 401), DOM16-106 (SEQ ID NO: 402 DOM16-107 (SEQ ID NO: 403 ), DOM16-108 (SEQ ID NO: 404 DOM16-109 (SEQ ID NO: 405), DOM16-110 (SEQ ID NO: 406 DOM16-111 (SEQ ID NO: 407), DOM16-112 (SEQ ID NO: 408 DOM16-113 (SEQ ID NO: 409), DOM16-114 (SEQ ID NO: 410 DOM16-115 (SEQ ID NO: 411), DOM16-116 (SEQ ID NO: 412 DOM16-117 (SEQ ID NO: 413 ), DOM16-118 (SEQ ID NO: 414 DOM16-119 (SEQ ID NO: 415), DOM16-39-6 (SEQ ID NO: 416 DOM16-39-8 (SEQ ID NO: 417), DOM16-39- 34 (SEQ ID NO: 418 DOM16-39-48 (SEQ ID NO: 419), DOM16-39-87 (SEQ.
ID NO: 420), DOM16-39-90 (SEQ ID NO: 421), DOM16-39-96 (SEQ ID NO: 422), DOM16-39-100 (SEQ ID NO: 423), DOM16-39- 101 (SEQ ID NO: 424), DOM16-39-102 (SEQ ID NO: 425), DOM16-39 -103 (SEQ ID NO: 426), DOM16-39-104 (SEQ ID NO: 427), DOM16-39-105 (SEQ ID NO: 428), DOM16-39-106 (SEQ.
ID NO: 429), DOM16-39-107 (SEQ ID NO: 430), DOM16-39-108 (SEQ ID NO: 431), DOM16-39-109 (SEQ ID NO: 432), DOM16-39-110 (SEQ ID NO: 433), DOM16-39-111 (SEQ ID NO: 434), DOM16-39 -112 (SEQ ID NO: 435), DOM16-39-113 (SEQ ID NO: 436), DOM16-39-114 (SEQ ID NO: 437), DOM16-39-115 (SEQ.
ID NO: 438), DOM16-39-116 (SEQ ID NO: 439), DOM16-39-117 (SEQ ID NO: 440), DOM16-39-200 (SEQ ID NO: 441), DOM16-39-201 (SEQ ID NO: 442), DOM16-39-202 (SEQ ID NO: 443), DOM16-39 -203 (SEQ ID NO: 444), DOM16-39-204 (SEQ ID NO: 445), DOM16-39-205 (SEQ ID NO: 446), DOM16-39-206 (SEQ ID NO: 447), DOM16 -39-207 (SEQ ID NO: 448), DOM16-39-209 (SEQ ID NO: 449), DOM16-52 (SEQ ID NO: 450), NB1 (SEQ ID NO: 451), NB2 (SEQ ID NO : 452), NB3 (SEQ ID NO: 453), NB4 (SEQ ID NO: 454), NB5 (SEQ ID NO: 455), NB6 (SEQ ID NO: 456), NB7 (SEQ ID NO: 457), NB8 (SEQ ID NO: 458), NB9 (SEQ ID NO: 459), NB10 (SEQ ID NO: 460), NB11 (SEQ ID NO: 461), NB 12 (SEQ ID NO: 462), NB13 (SEQ ID NO : 463), NB14 (SEQ ID NO: 464), NB15 (SEQ ID NO: 465), NB 16 (SEQ ID NO: 466), NB 17 (SEQ ID NO: 467), NB18 (SEQ ID NO: 468) , NB19 (SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 (SEQ ID NO: 471), and NB22 (SEQ ID NO: 472). In some embodiments, the polypeptide domain having a binding site with binding specificity for EGFR comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91% , at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with the amino acid sequence of a dAb selected from the group consisting of DOM16-17 (SEQ ID NO: 325), DOM16-18 (SEQ ID NO: 326), DOM16-19 (SEQ ID NO: 327) ), DOM16-20 (SEQ ID NO: 328), DOM16-21 SEQ ID NO: 329), DOM16-22 (SEQ ID NO • 330), DOM16-23 SEQ ID NO 331), DOM16-24 (SEQ ID NO: 332), DOM16-25 SEQ ID NO .333), DOM16-26 (SEQ ID NO 334), DOM16-27 SEQ ID NO 335), DOM16-28 (SEQ ID NO 336), DOM16-29 SEQ ID NO: 337), DOM16-30 (SEQ ID NO 338), DOM16-31 SEQ ID NO .339), DOM16-32 (SEQ ID NO 340), DOM16-33 SEQ ID NO 341), DOM16-35 (SEQ ID NO 342), DOM16-37 SEQ ID NO 343), DOM16-38 (SEQ ID NO 344), DOM16-39 SEQ ID NO 345), DOM16-40 (SEQ ID NO 346), DOM16-41 SEQ ID NO 347), DOM16-42 [SEQ ID NO 348), DOM16-43 SEQ ID NO 349), DOM16-44 [SEQ ID NO 350), DOM16-45 SEQ ID NO 351), DOM16-46 | [SEQ ID NO 352), DOM16-47 SEQ ID NO 353), DOM16-48. { SEQ ID NO 354), DOM16-49 SEQ ID NO 355), DOM16-50 (SEQ ID NO 356), DOM16-59 SEQ ID NO 357), DOM16-60 (SEQ ID NO 358), DOM16-61 SEQ ID NO 359), DOM16-62 (SEQ ID NO 360), DOM16-63 SEQ ID NO 361), DOM16-64 (SEQ ID NO 362), DOM16-65 SEQ ID NO 363), DOM16-66 (SEQ ID NO 364), DOM16-67 SEQ ID NO 365), DOM16-68 (SEQ ID NO 366), DOM16-69 SEQ ID NO 367), DOM16-70 (SEQ ID NO 368), DOM16-71 SEQ ID NO 369), DOM16-72 (SEQ ID NO 370), DOM16-73 SEQ ID NO371), DOM16-74 (SEQ ID NO: 372), DOM16-75 SEQ ID DO NOT. 373), DOM16-76 (SEQ ID NO: 374), DOM16-77 SEQ ID NO: 375), DOM16-78 (SEQ ID NO: 376), DOM16-79 SEQ ID NO: 377 DOM16-80 (SEQ ID NO. : 378 DOM16-81 SEQ ID NO: 379 DOM16-82 (SEQ ID NO: 380 DOM16-83 SEQ ID NO: 381 DOM16-84 (SEQ ID NO: 382 DOM16-85 SEQ ID NO: 383 DOM16-87 (SEQ ID NO: 384 DOM16-88 SEQ ID NO: 385 DOM16-89 (SEQ ID NO: 386 DOM16-90 SEQ ID NO: 387 DOM16-91 (SEQ ID NO: 388 DOM16-92 SEQ ID NO: 389 DOM16-94 (SEQ ID NO: 390 DOM16-95 SEQ ID NO: 391 DOM16-96 (SEQ ID NO: 392 DOM16-97 SEQ ID NO: 393 DOM16-98 (SEQ ID NO: 394 DOM16-99 SEQ ID NO: 395 DOM16-100 ( SEQ ID NO: 396 DOM16-101 SEQ ID NO: 397 DOM16-102 (SEQ ID NO: 398 DOM16-103 SEQ ID NO: 399 DOM16-104 (SEQ ID NO: 400 DOM16-105 SEQ ID NO: 401 DOM16-106 (SEQ ID NO: 402 DOM16-107 SEQ ID NO: 403 DOM16-108 (SEQ ID NO: 404 DOM16-109 SEQ ID NO: 405 DOM16-110 (SEQ ID NO: 406 DOM16-111 SEQ ID NO: 407 DOM16- 112 (SEQ ID NO: 408 DOM16-113 SEQ ID NO: 409 DOM16-114 (SEQ ID NO: 410 DOM16-115 SEQ ID NO: 411 DOM16-116 (SEQ ID NO: 412 DOM16-117 SEQ ID NO: 413 DOM16 -118 (SEQ ID NO: 4 14 DOM16-119 SEQ ID NO: 415 DOM16-39-6 (SEQ ID NO: 416 DOM16-39-8 SEQ ID NO: 417 DOM16-39-34 (SEQ ID NO: 418), DOM16-39-48 (SEQ ID NO: 419), DOM16-39-87 (SEQ ID NO: 420), DOM16-39-90 (SEQ ID NO: 421), DOM16-39-96 (SEQ ID NO: 422), DOM16-39-100 (SEQ ID NO: 423), DOM16-39-101 (SEQ ID NO: 424), DOM16-39-102 (SEQ ID NO: 425), DOM16-39-103 (SEQ ID NO: 426), DOM16-39-104 (SEQ ID NO: 427), DOM16-39-105 (SEQ ID NO: 428), DOM16-39-106 (SEQ ID NO.429), DOM16-39-107 ( SEQ ID NO: 430), DOM16-39-108 (SEQ ID NO: 431), DOM16-39-109 (SEQ ID NO: 432), DOM16-39-110 (SEQ ID NO: 433), DOM16-39- 111 (SEQ ID NO: 434), DOM16-39-112 (SEQ ID NO: 435), DOM16-39-113 (SEQ ID NO: 436), DOM16-39-114 (SEQ ID NO: 437), DOM16- 39-115 (SEQ ID NO: 438), DOM16-39-116 (SEQ ID NO: 439), DOM16-39-117 (SEQ ID NO: 440), DOM16-39-200 (SEQ ID NO: 441), DOM16-39-201 (SEQ ID NO: 442), DOM16-39-202 (SEQ ID NO: 443), DOM16-39-203 (SEQ ID NO: 444), DOM16-39-204 (SEQ ID NO: 445) ), DOM16-39-205 (SEQ ID NO: 446), DOM16-39-206 (SEQ ID NO: 447), DOM16-39-207 (SEQ ID NO: 448), DOM16-39-209 (SEQ ID NO. : 449), DOM16-52 (SEQ ID NO: 450), NB1 (SEQ ID NO: 451), NB2 (SEQ ID NO: 452), NB3 (SEQ ID NO: 453), NB4 (SEQ ID NO: 454), NB5 (SEQ ID NO: 455), NB6 (SEQ ID NO: 456), NB7 (SEQ ID NO: 457), NB8 (SEQ ID NO: 458), NB9 (SEQ ID NO: 459), NB10 (SEQ ID NO: 460), NB11 (SEQ ID NO: 461), NB 12 (SEQ ID NO: 462), NB 13 (SEQ ID NO: 463), NB14 (SEQ ID NO: 464), NB15 (SEQ ID NO: 465), NB16 (SEQ ID NO: 466), NB 17 (SEQ ID NO: 467), NB 18 (SEQ ID NO: 468), NB 19 ( SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 (SEQ ID NO: 471), and NB22 (SEQ ID NO: 472). In preferred embodiments, the polypeptide domain having a binding site with binding specificity for EGFR comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93% , at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with the amino acid sequence of DOM16 -39 (SEQ ID NO: 345). For example, the polypeptide domain having a binding site with binding specificity for EGFR may comprise the amino acid sequence of DOM16-39-87 (SEQ ID NO: 420), DOM16-39-100 (SEQ ID NO: 423 ), DOM16-39-107 (SEQ ID NO: 430), DOM16-39-109 (SEQ ID NO: 432), DOM16-39-115 (SEQ ID NO: 438), or DOM16-39-200 (SEQ ID NO: 441). In some embodiments, the polypeptide domain having a binding site with binding specificity for EGFR, competes with any of the dAbs described herein for binding to EGFR. Preferably, the polypeptide domain having a binding site with binding specificity for EGFR is a single immunoglobulin variable domain. The polypeptide domain having a binding site with binding specificity for EGFR may comprise any suitable immunoglobulin variable domain, and preferably comprises a human variable domain or a variable domain comprising regions of human structure. In certain embodiments, the polypeptide domain having a binding site with binding specificity for EGFR, comprises a universal structure, as described in the present invention. In certain embodiments, the polypeptide domain having a binding site with binding specificity for EGFR resists aggregation, reversibly unfolds, comprises a region of structure and is secreted as described above for the polypeptide domain having a binding site with binding specificity for VEGF. DAB Abrasive Linkers to Serum Albumin The ligands of the present invention may further comprise a dAb monomer that binds serum albumin (SA) with a Kd of 1 nM to 500 μM (eg, x 10"9 to 5 x 10" 4), preferably 100 nM to 10 μM. Preferably, for a ligand comprising an anti-SA dAb, the linkage (e.g., Kd and / or K0ff as measured by resonance to surface plasmon, e.g. using BiaCore) of the ligand to its target (s) is 1 to 100000 times (preferably 100 to 100000, more preferably 1000 to 100000, or 10000 to 100000 times) stronger than SA passes. Preferably, serum albumin is human serum albumin (HSA). In one embodiment, the first dAb (or dAb monomer) links SA (e.g., HSA) with a Kd of about 50, preferably 70, and more preferably 100, 150 or 200 nM. In certain embodiments, the dAb monomer linking SA resists aggregation, reversibly unfolded and / or comprises a structure region as described above for dAb monomers that bind VEGF. In particular embodiments, the antigen binding fragment of an antibody binding serum albumin is a dAb that binds human serum albumin. In certain embodiments, dAb binds human serum albumin and competes to bind albumin with a dAb selected from the group consisting of DOM7m-16 (SEQ ID NO: 473), DOM7m-12 (SEQ ID NO: 474), DOM7m-26 (SEQ ID NO: 475), DOM7r-1 (SEQ ID NO: 476), DOM7r-3 (SEQ ID NO: 477), DOM7r-4 (SEQ ID NO: 478), DOM7r-5 (SEQ ID NO: 479) ), DOM7r-7 (SEQ ID NO: 480), DOM7r-8 (SEQ ID NO: 481), DOM7h-2 (SEQ ID NO: 482), DOM7h-3 (SEQ ID NO: 483), DOM7h-4 ( SEQ ID NO: 484), DOM7h-6 (SEQ ID NO: 485), DOM7h-1 (SEQ ID NO: 486), DOM7h-7 (SEQ ID NO: 487), DOM7h-22 (SEQ ID NO: 489) , DOM7h-23 (SEQ ID NO: 490), DOM7h-24 (SEQ ID NO: 491), DOM7h-25 (SEQ ID NO: 492), DOM7h-26 (SEQ ID NO: 493), DOM7h-21 (SEQ ID NO: 494), DOM7h-27 (SEQ ID NO: 495), DOM7h-8 (SEQ ID NO: 496), DOM7r-13 (SEQ ID NO: 497), DOM7r-14 (SEQ ID NO: 498), DOM7r-15 (SEQ ID NO: 499), DOM7r-16 (SEQ ID NO: 500), DOM7r-17 (SEQ ID NO: 501), DOM7r-18 (SEQ ID NO: 502), DOM7r-19 (SEQ ID NO: 503), DOM7r-20 (SEQ ID NO: 504), DOM7r-21 (SEQ ID NO: 505), DOM7r-22 (SEQ ID NO: 506), DOM7r-23 (SEQ ID NO: 507) DOM7r-24 (SEQ ID NO: 508), DOM7r-25 (SEQ ID NO: 509) DOM7r-26 (SEQ ID NO: 510), DOM7r-27 (SEQ ID NO: 511) DOM7r -28 (SEQ ID NO: 512), DOM7r-29 (SEQ ID NO: 513) DOM7r-30 (SEQ ID NO: 514), DOM7r-31 (SEQ ID NO: 515) DOM7r-32 (SEQ ID NO: 516) ), and DOM7r-33 (SEQ ID NO: 517). In certain embodiments, the dAb binds human serum albumin and comprises an amino acid sequence having at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% amino acid sequence identity with the amino acid sequence of a dAb selected from the group consisting of in DOM7m-16 (SEQ ID NO: 473), DOM7m-12 (SEQ ID NO: 474), DOM7m-26 (SEQ ID NO: 475), DOM7r-1 (SEQ ID NO: 476), DOM7r-3 (SEQ ID NO: 477), DOM7r-4 (SEQ ID NO: 478), DOM7r-5 (SEQ ID NO: 479), DOM7r-7 (SEQ ID NO: 480), DOM7r-8 (SEQ ID NO: 481), DOM7h-2 (SEQ ID NO: 482), DOM7h-3 (SEQ ID NO: 483), DOM7h-4 (SEQ ID NO: 484), DOM7h-6 (SEQ ID NO: 485) DOM7h-1 (SEQ ID NO: 486), DOM7h-7 (SEQ ID NO: 487) DOM7h-22 (SEQ ID NO: 489), DOM7h-23 (SEQ ID NO: 490) DOM7h-24 ( SEQ ID NO: 491), DOM7h-25 (SEQ ID NO: 492) DOM7h-26 (SEQ ID NO: 493), DOM7h-21 (SEQ ID NO: 494 DOM7h-27 (SEQ ID NO: 495), DOM7h- 8 (SEQ ID NO: 496 DOM7r-13 (SEQ ID NO: 497), DOM7r-14 (SEQ ID NO: 498 DOM7r-15 (SEQ ID NO: 499), DOM7r-16 (SEQ ID NO: 500 DOM7r-17 (SEQ ID NO: 501), DOM7r-18 (SEQ ID NO: 502 DOM7r-19 (SEQ ID NO: 503), DOM7r-20 (SEQ ID NO: 504 DOM7r-21 (SEQ ID NO: 505), DOM7r- 22 (SEQ ID NO: 506 DOM7r-23 (SEQ ID NO: 507), DOM7r-24 (SEQ ID NO: 508 DOM7r-25 (SEQ ID NO: 509), DOM7r-26 (SEQ ID NO: 510 DOM7r-27 (SEQ ID NO: 511), DOM7r-28 (SEQ ID NO: 512 DOM7r-29 (SEQ ID NO: 513), DOM7r-30 (SEQ ID NO: 514 DOM7r-31 (SEQ ID NO: 515), DOM7r- 32 (SEQ ID NO: 516 and DOM7r-33 (SEQ ID NO: 5 7) For example, the dAb binding human serum albumin can comprise an amino acid sequence having at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% amino acid sequence identity with DOM7h-2 (SEQ ID NO: 482), DOM7h- 3 (SEQ ID NO: 483), DOM7h-4 (SEQ ID NO: 484), DOM7h-6 (SEQ ID NO: 485), DOM7h-1 (SEQ ID NO: 486), DOM7h-7 (SEQ ID NO: 487), DOM7h-8 (SEQ ID NO: 496), DOM7r-13 (SEQ ID NO: 497), DOM7r-14 (SEQ ID NO: 498), DOM7h-22 (SEQ ID NO: 489), DOM7h-23 (SEQ ID NO: 490), DOM7h-24 (SEQ ID NO: 491), DOM7h-25 (SEQ ID NO: 492), DOM7h-26 (SEQ ID NO: 493), DOM7h -21 (SEQ ID NO: 494), and DOM7h-27 (SEQ ID NO: 495). The amino acid sequence identity is determined using preferably an appropriate sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, Proc. Nati, Acad. Sel. E.U.A. 87 (6): 226-2268 (1990)). In more particular modalities, the dAb is a V? dAb which binds human serum albumin and has an amino acid sequence selected from the group consisting of DOM7h-2 (SEQ ID NO: 482), DOM7h-3 (SEQ ID NO: 483), DOM7h-4 (SEQ ID NO: 484 ), DOM7h-6 (SEQ ID NO: 485), DOM7h-1 (SEQ ID NO: 486), DOM7h-7 (SEQ ID NO: 487), DOM7h-8 (SEQ ID NO: 496), DOM7r-13 ( SEQ ID NO: 4 £ 7), and DOM7r-14 (SEQ ID NO: 498), or a VH dAb having an amino acid sequence selected from the group consisting of: DOM7h-22 (SEQ ID NO: 489), DOM7h -23 (SEQ ID NO: 490), DOM7h-24 (SEQ ID NO: 491), DOM7h-25 (SEQ ID NO: 492), DOM7h-26 (SEQ ID NO: 493), DOM7h-21 (SEQ ID NO. : 494), DOM7h-27 (SEQ ID NO: 495). In other embodiments, the antigen binding fragment has antibody that binds to serum albumin is a dAb that binds human serum albumin and comprises the CDRs of any of the above amino acid sequence. Suitable Camelid VHH that binds serum albumin includes those described in Publication WO 2004/041862 (Ablynx NV) and in the present invention, such as Sequence A (SEQ ID NO: 518), Sequence B (SEQ ID NO: 519), Sequence C (SEQ ID NO: 520), Sequence D (SEQ ID NO: 521), Sequence E (SEQ ID NO: 522), Sequence F (SEQ ID NO: 523), Sequence G (SEQ ID NO: 524), Sequence H (SEQ ID NO: 525), Sequence I (SEQ ID NO: 526), Sequence J (SEQ ID NO: 527), Sequence K (SEQ ID NO: 528), Sequence L (SEQ ID NO: 529), Sequence M (SEQ ID NO: 530), Sequence N (SEQ ID NO.; 531), Sequence O (SEQ ID NO: 532), Sequence P (SEQ ID NO: 533), Sequence Q (SEQ ID NO.534). In certain embodiments, the Camelid VHH binds human serum albumin and comprises an amino acid sequence having at least about 80%, or at least about 85%, or at least about 90%, "or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% amino acid sequence identity with any of SEQ ID NOS: 518-534. The amino acid is preferably determined using an appropriate sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, Proc. Nati, Acad. Sel. EUA 87 (6): 2264-2268 (1990)). embodiments, the ligand comprises an anti-serum albumin dAb that competes with an anti-serum albumin dAb described in the present invention for binding to serum albumin (e.g., human serum albumin). Nucleic Acid Molecules, Vector and Host Cell The present invention also provides isolated and / or recombinant nucleic acid molecules encoding ligands (eg, specific double ligands and multi-specific ligands) as described in the present invention. The nucleic acids referred to in the present invention, as "isolated" are nucleic acids that have been separated from the nucleic acids of the genomic DNA or cellular RNA from their source of origin (eg, as they exist in cells or in a mixture of nucleic acids. such as a library), and include nucleic acids obtained through the methods described herein or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis through combinations of biological and chemical methods and recombinant nucleic acids which they are isolated (see for example, Daugherty, BL and associates, Nucleic Acids Res., 19 (9): 2471-2476 (1991), Lewis, AP and JS Crowe, Gene, 101: 297-302 (1991)). The nucleic acids referred to herein as "recombinant" are nucleic acids that have been produced by recombinant DNA methodology, including nucleic acids that are generated by methods that depend on an artificial recombination method, such as the polymerase chain reaction (PCR) ) and / or cloning into a vector using restriction enzymes. In certain embodiments, the isolated and / or recombinant nucleic acid comprises a nucleotide sequence encoding a ligand, such as described in the present invention, wherein the ligand comprises an amino acid sequence having at least about 80%, less about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with the amino acid sequence of a dAb that binds VEGF described herein, or a dAb that binds EGFR described here. For example, in some embodiments, the isolated and / or recombinant nucleic acid comprises a nucleotide sequence encoding a ligand having binding specificity for VEGF, as described herein, wherein the ligand comprises an amino acid sequence having the less about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with the amino acid sequence of a dAb "selected from the group consisting of TAR15-1 (SEQ ID NO: 100), TAR15-3 (SEQ ID NO: 101 TAR15-4 (SEQ ID NO: 102), TAR15-9 (SEQ ID NO: 103 TAR15-10 (SEQ ID NO: 104), TAR15-11 (SEQ ID NO: 105) TAR15-12 (SEQ ID NO: 106), TAR15-13 (SEQ ID NO: 107 TAR15-14 (SEQ ID NO: 108), TAR15-15 (SEQ ID NO: 109 TAR15-16 (SEQ ID NO: 110) , TAR15-17 (SEQ ID NO: 111 TAR15-18 (SEQ ID NO: 112), TAR15-19 (SEQ ID NO: 113 TAR15-20 (SEQ ID NO: 114), TAR 15-22 (SEQ ID NO: 115 TAR15-5 (SEQ ID NO: 116), TAR15-6 (SEQ ID NO: 117 TAR15-7 (SEQ ID NO: 118), TAR15-8 (SEQ ID NO: 119 TAR15-23 (SEQ ID NO: 120) ), TAR15-24 (SEQ ID N0: 121 TAR15-25 (SEQ ID NO: 122), TAR15-26 (SEQ ID NO: 123 TAR15-27 (SEQ ID NO: 124), TAR15-29 (SEQ ID NO. : TAR15-30 125 (SEQ ID NO: 126), TAR15-6-500 (SEQ ID NO: 127 TAR15-6-501 (SEQ ID NO: 128), TAR15-6-502 (SEQ ID NO: 129 TAR15- 6-503 (SEQ ID NO: 130), TAR15-6-504 (SEQ ID N 0: 131 TAR15-6-505 (SEQ ID NO: 132), TAR15-6-506 (SEQ ID NO: 133), TAR15 -6-507 (SEQ ID NO: 134), TAR15-6-508 (SEQ ID NO: 135), TAR15-6-509 (SEQ ID NO: 136), TAR15-6-510 (SEQ ID NO: 137) , TAR15-8-500 (SEQ ID NO: 138), TAR15 -8-501 (SEQ ID NO: 139), TAR15-8-502 (SEQ ID NO: 140), TAR15-8-503 (SEQ ID NO: 141), TAR15-8-505 (SEQ ID NO: 142) , TAR15-8-506 (SEQ ID NO: 143), TAR15-8-507 (SEQ ID NO: 144), TAR15-8-508 (SEQ ID NO: 145), TAR15-8-509 (SEQ ID NO: Í46), TAR15-8-510 (SEQ ID NO: 147), TAR15-8-511 (SEQ ID NO: 148), TAR15-26-500 (SEQ ID NO: 149), TAR15-26-501 (SEQ ID NO: 150), TAR15-26-502 (SEQ ID NO: 151), TAR15-26-503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153), TAR15-26-505 ( SEQ ID NO: 154), TAR15-26-506 (SEQ ID NO: 155), TAR15-26-507 (SEQ ID NO: 156), TAR15-26-508 (SEQ ID NO: 157), TAR15-26- 509 (SEQ ID NO: 158), TAR15-26-510 (SEQ ID NO: 159), TAR15-26-511 (SEQ ID NO: 160), TAR15-26-512 (SEQ ID NO: 161), TAR15- 26-513 (SEQ ID NO: 162), TAR15-26-514 (SEQ ID NO: 163), TAR15-26-515 (SEQ ID NO: 164), TAR15-26-516 (SEQ ID NO: 165), TAR15-26-517 (SEQ ID NO: 166), TAR15-26-518 (SEQ ID NO: 167), TAR15-26-519 (SEQ ID NO: 168), TAR15-26-520 (SEQ ID NO: 169 ), TAR15-26-521 (SEQ ID NO: 170), TAR15-26-522 (SEQ ID NO: 171), TAR15-26-523 (SEQ ID NO: 172), TAR15-26-524 (SEQ ID NO: 173), TAR15-26-525 (SEQ ID NO: 174), TAR15-26-526 (SEQ ID NO: 175), TAR15-26-527 (SEQ ID NO: 176), TAR15-26-528 (SEQ ID NO: 177), TAR15-26-529 (SEQ ID NO: 178), TAR15-26-530 (SEQ ID NO: 179), TAR15-26-531 ( SEQ ID NO: 180), TAR15-26-532 (SEQ ID NO: 181), TAR15-26-533 (SEQ ID NO: 182), TAR15-26-534 (SEQ ID NO: 183), TAR15-26- 535 (SEQ ID NO: 184), TAR15-26-536 (SEQ ID NO: 185), TAR15-26-537 (SEQ ID NO: 186), TAR15-26-538 (SEQ ID NO: 187), TAR15- 26-539 (SEQ ID NO: 188), TAR15-26-540 (SEQ ID NO: 189), TAR15-26-541 (SEQ ID NO: 190), TAR15-26-542 (SEQ ID NO: 191), TAR15-26-543 (SEQ ID NO: 192), TAR15-26-544 (SEQ ID NO: 193), TAR15-26-545 (SEQ ID NO: 194), TAR15-26-546 (SEQ ID NO: 195 ), TAR15-26-547 (SEQ ID NO: 196), TAR15-26-548 (SEQ ID NO: 197), TAR15-26-549 (SEQ ID NO: 198), TAR15-26-550 (SEQ ID NO. : 539), and TAR15-26-551 (SEQ ID NO: 540). In other embodiments, the isolated and / or recombinant nucleic acid comprises a nucleotide sequence encoding a ligand having binding specificity for EGFR, as described herein, wherein the ligand comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95 %, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with the amino acid sequence of a dAb selected from the group consisting of DOM16-17 ID NO 325), DOM16-18 (SEQ ID NO: 326) DOM16-19 ID NO 327), DOM16-20 (SEQ ID NO: 328) DOM16-21 ID NO 329), DOM16-22 (SEQ ID NO: 330) DOM16-23 ID NO 331), DOM16-24 (SEQ ID NO.332) DOM16-25 ID NO 333), DOM 16-26 (SEQ ID NO: 334) DOM16-27 ID NO 335), DOM16-28 (SEQ ID NO: 336) DOM16-29 ID NO 337), DOM16-30 (SEQ ID NO: 338) DOM16-31 ID NO 339), DOM16-32 (SEQ ID NO: 340) DOM16-33 ID NO 341), DOM16-35 (SEQ ID NO: 342) DOM16-37 ID NO 343), DOM16-38 (SEQ ID NO: 344) DOM16-39 ID NO 345), DOM16-40 (SEQ ID NO: 346) DOM16-41 ID NO 347), DOM16-42 (SEQ ID NO: 348) DOM16-43 ID NO 349), DOM16-44 (SEQ ID NO: 350) DOM16-45 ID NO 351), DOM16-46 (SEQ ID NO: 352 ) DOM16-47 ID NO 353), DOM16-48 (SEQ ID NO: 354) DOM16-49 ID NO 355), DOM16-50 (SEQ ID NO: 356) DOM16-59 ID NO 357), DOM16-60 (SEQ ID NO: 358) DOM16-61 ID NO 359), DOM16-62 (SEQ ID NO: 360) DOM16-63 ID NO 361), DOM16-64 (SEQ ID NO: 362) DOM16-65 ID NO 363), DOM16 -66 (SEQ ID NO: 364) DOM16-67 ID NO 365), DOM16-68 (SEQ ID NO: 366) DOM16-69 ID NO 367), DOM16-70 (SEQ ID NO: 368) DOM16-71 ID NO 369), DOM16-72 (SEQ ID NO: 370) DOM16-73 ID NO 371), DOM16-74 (SEQ ID NO: 372) DOM16-75 ID NO 373), DOM16-76 (SEQ ID NO: 374) DOM16 77 (SEQ ID NO: 375 DOM16-78 (SEQ ID NO: 376) DOM16-79 (SEQ ID NO: 377 DOM16-80 (SEQ ID NO: 378) DOM16-81 (SEQ ID NO: 379 DOM16-82 ( SEQ ID NO: 380) DOM16-83 (SEQ ID NO: 381 DOM16-84 (SEQ ID NO: 382) DOM16-85 (SEQ ID NO: 383 DOM16-87 (S EQ ID NO: 384) DOM16-88 (SEQ ID NO: 385) DOM16-89 (SEQ ID NO: 386) DOM16-90 (SEQ ID NO: 387 DOM16-91 (SEQ ID NO: 388) DOM16-92 (SEQ ID NO: 389 DOM16-94 (SEQ ID NO: 390) DOM16-95 (SEQ ID NO: 391 DOM16-96 (SEQ ID NO: 392) DOM16-97 (SEQ ID NO: 393 DOM16-98 (SEQ ID NO: 394) DOM16-99 (SEQ ID NO: 395) DOM / 16-100 (SEQ ID NO: 396) DOM16- 101 (SEQ ID NO: 397 DOM16-102 (SEQ ID NO: 398) DOM16-103 (SEQ ID NO. : 399 DOM16-104 (SEQ ID NO: 400) DOM16-105 (SEQ ID NO: 401 DOM16-106 (SEQ ID NO: 402) DOM16- 107 (SEQ ID NO: 403 DOM16-108 (SEQ ID NO: 404) DOM16- 109 (SEQ ID NO: 405 DOM16-110 (SEQ ID NO: 406) DOM16- 111 (SEQ ID NO: 407 DOM16-112 (SEQ ID NO: 408) DOM16- 113 (SEQ ID NO: 409 DOM16-114 (SEQ ID NO: 410) DOM16- 115 (SEQ ID NO: 411 DOM16-116 (SEQ ID NO: 412) DOM16- 117 (SEQ ID NO: 413 DOM16-118 (SEQ ID NO: 414) DOM16- 119 (SEQ ID NO: 415 DOM16-39-6 (SEQ ID NO: 416) DOM16-39-8 (SEQ ID NO: 417 DOM16-39-34 (SEQ ID NO: 418) DOM16-39-48 (SEQ ID NO: 419), DOM16-39-87 (SEQ ID NO: 420), DOM16-39-90 (SEQ ID NO: 421), DOM16-39-96 (SEQ ID NO: 422) ), DOM16-39-100 (SEQ ID NO: 423), DOM16-39-101 (SEQ ID NO: 424), DOM16-39-102 (SEQ ID NO: 425), DOM16-39-103 (SEQ ID NO. : 426), DOM16-39-104 (SEQ ID NO: 427), DOM16-39-105 (SEQ ID NO: 428), - DOM16-39-106 (SEQ ID NO: 429), DOM16-39-107 ( SEQ ID NO: 430), DOM16-39-108 (SEQ ID NO: 431), DOM16-39-109 (SEQ ID NO: 432), DOM16-39-110 (SEQ ID NO: 433), DOM16-39- 111 (SEQ ID NO: 434), DOM16-39-112 (SEQ ID NO: 435), DOM16-39-113 (SEQ ID NO: 436), DOM16-39-114 (SEQ ID NO: 437), DOM16- 39-115 (SEQ ID NO: 438), DOM16-39-116 (SEQ ID NO: 439), DOM16-39-117 (SEQ ID NO: 440), DOM16-39-200 (SEQ ID NO: 441), DOM16-39-201 (SEQ ID NO: 442), DOM16-39-202 (SEQ ID NO: 443), DOM16-39-203 (SEQ ID NO: 444), DOM16-39-204 (SEQ ID NO: 445) ), DOM16-39-205 (SEQ ID NO: 446), DOM16-39-206 (SEQ ID NO: 447), DOM16-39-207 (SEQ ID NO: 448), DOM16-39-209 (SEQ ID NO. : 449), DOM16-52 (SEQ ID NO: 450), NB1 (SEQ ID NO: 451), NB2 (SEQ ID NO: 452), NB3 (SEQ ID NO: 453), NB4 (SEQ ID NO: 454), NB5 (SEQ ID NO: 455), NB6 (SEQ ID NO: 456), NB7 (SEQ ID NO: 457), NB8 (SEQ ID NO: 458), NB9 (SEQ ID NO: 459), NB10 (SEQ ID NO: 460), NB11 (SEQ ID NO: 461), NB12 (SEQ ID NO: 462), NB13 (SEQ ID. NO: 463), NB14 (SEQ ID NO: 464), NB15 (SEQ ID NO: 465), NB16 (SEQ ID NO: 466), NB17 (SEQ ID NO: 467), NB18 (SEQ ID NO: 468), NB 19 (SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 (SEQ ID NO: 471), and NB22 (SEQ ID NO: 472). In other embodiments, the isolated and / or recombinant nucleic acid encoding a ligand having binding specificity for VEGF, as described herein, wherein the nucleic acid comprises a nucleotide sequence having at least about 80%, less about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% nucleotide sequence identity with a nucleotide sequence encoding an anti-VEGF dAb selected from the group consisting of TAR15-1 (SEQ ID NO: 1), TAR15-3 (SEQ ID NO: 2), AR15-4 (SEQ ID NO: 3), TAR15-9 (SEQ ID NO: 4), TAR15-10 (SEQ ID NO: 5), TAR15-11 (SEQ ID NO: 6), TAR15-12 (SEQ ID NO: 7), TAR15-13 (SEQ ID NO: 8), TAR15-14 (SEQ ID NO: 9), TAR1 5-15 (SEQ ID NO: 10), TAR15-16 (SEQ ID NO: 11), TAR15-17 (SEQ ID NO: 12), TAR15-18 (SEQ ID NO: 13), TAR15-19 (SEQ ID NO: 14), TAR15-20 (SEQ ID NO: 15), TAR 15-22 (SEQ ID NO: 16), TAR15-5 (SEQ ID NO: 17), TAR15-6 (SEQ ID NO: 18), TAR15-7 (SEQ ID NO: 19), TAR15-8 (SEQ ID NO: 20), TAR15-23 (SEQ ID NO: 21), TAR15-24 (SEQ ID NO: 22), TAR15-25 (SEQ ID NO: 23), TAR15-26 (SEQ ID NO: 24), TAR15-27 (SEQ ID NO: 25), TAR15-29 (SEQ ID NO: 26), TAR15-30 (SEQ ID NO: 27), TAR15 -6-500 (SEQ ID NO: 28), TAR15 -6-501 (SEQ ID NO: 29 TAR15-6-502 (SEQ ID NO.30), TAR15 -6-503 (SEQ ID NO: 31 TAR15-6 -504 (SEQ ID NO: 32), TAR15 -6-505 (SEQ ID NO: 33 TAR15-6-506 (SEQ ID NO: 34), TAR15 -6-507 (SEQ ID NO: 35 TAR15-6-508 (SEQ ID NO: 36), TAR15 -6-509 (SEQ ID NO: 37 TAR ° 15-6-510 (SEQ ID NO: 38), TAR15-8-500 (SEQ ID NO: 39 TAR15-8-501 (SEQ ID NO: 40), TAR15 -8-502 (SEQ ID NO: 41 TAR15-8-503 (SEQ ID NO: 42), TAR15 -8-505 (SEQ ID NO: 43 TAR15-8-506 (SEQ ID NO: 44), TAR15 -8-507 (SEQ ID NO: 45 TAR15-8-508 (SEQ ID NO: 46), TAR15 8-509 (SEQ ID NO: 47), Rl 5-8-510 (SEQ ID NO: 48), TAR15 8-511 (SEQ ID NO: 49), TAR15-26-500 (SEQ ID NO: 50), TAR15 • 26-501 (SEQ ID NO: 51 TAR15 -26-502 (SEQ ID NO: 52), TAR15 26-503 (SEQ ID NO: 53 TAR15-26-504 (SEQ ID NO: 54), TAR15 26-505 (SEQ ID NO: 55 TAR15-26-506 (SEQ ID NO: 56), TAR15 -26-507 (SEQ ID NO: 57 TAR15-26-508 (SEQ ID NO: 58), TAR15 • 26-509 (SEQ ID NO.59 TAR15-26-510 (SEQ ID NO: 60), TAR15 • 26-511 (SEQ ID NO: 61 TAR15-26-512 (SEQ ID NO: 62), TAR15 26-513 (SEQ ID NO: 63 TAR15-26-514 (SEQ ID NO: 64), TAR15 26-515 (SEQ ID NO: 65 TAR15-26-516 (SEQ ID NO: 66), TAR15 26-517 (SEQ ID NO: 67 TAR15-26-518 (SEQ ID NO.68), TAR15 26-519 (SEQ ID NO: 69 TAR15-26-520 (SEQ ID NO: 70), TAR15 26-521 (SEQ ID NO: 71 TAR'15-26-522 (SEQ ID NO: 72), TAR15 26- 523 (SEQ ID NO: 73 TAR15-26-524 (SEQ ID NO: 74), TAR15 26-525 (SEQ ID NO: 75 TAR15-26-526 (SEQ ID NO: 76), TAR15 26-527 (SEQ ID NO.77 TAR15-26-528 (SEQ ID NO: 78), TAR15-26-529 (SEQ ID NO: 79) TAR15-26-530 (SEQ ID NO: 80), TAR15-26-531 (SEQ ID NO: 81), TAR15-26-532 (SEQ ID NO: 82), TAR15-26-533 (SEQ ID NO: 83), TAR15-26-534 (SEQ ID NO: 84) ), TAR15-26-535 (SEQ ID NO: 85), TAR15-26-536 (SEQ ID NO: 86), TAR15-26-537 (SEQ ID NO.87), TAR15-26-538 (SEQ ID NO. : 88), TAR15-26-539 (SEQ ID NO: 89), TAR15-26-540 (SEQ ID NO: 90), TAR15-26-541 (SEQ ID NO: 91), TAR15-26-542 (SEQ ID NO: 92), TAR15-26-543 (SEQ ID NO: 93), TAR15-26-544 (SEQ ID NO: 94), TAR15-26-545 (SEQ ID NO: 95), TAR15-26-546 (SEQ ID NO: 96), TAR15-26-547 (SEQ ID NO: 97), TAR15-26-548 (SEQ ID NO: 98), TAR15-26-549 (SEQ ID NO: 99), TAR15-21 (SEQ ID NO: 535), TAR15-2 (SEQ ID NO.536), TAR15-26-550 (SEQ ID NO: 537), and TAR15 -26-551 (SEQ ID NO: 538). Preferably, the nucleotide sequence identity is determined throughout the length of the nucleotide sequence encoding the selected anti-VEGF dAb. In other embodiments, the isolated and / or recombinant nucleic acid encoding a ligand having binding specificity for EGFR, as described herein, wherein the nucleic acid encoding a ligand having binding specificity for EGFR, as herein described, comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, %, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% nucleotide sequence identity with a nucleotide sequence encoding an anti-EGFR dAb selected from the group consisting of DOM16-7 (SEQ ID NO: 199), DOM16-18 (SEQ ID NO.200) DOM16-19 SEQ ID NO: 201 DOM16-20 (SEQ ID NO: 202) DOM16-21 SEQ ID NO: 203 DOM16-22 (SEQ ID NO.204) DOM16-23 SEQ ID NO: 205 DOM16-24 (SEQ ID NO: 206) DOM16-25 SEQ ID NO: 207 DOM16-26 (SEQ ID NO: 208) DOM16-27 SEQ ID NO: 209 DOM16-28 (SEQ ID NO: 210) DOM16-29 SEQ ID NO: 211 DOM16-30 (SEQ ID NO: 212) DOM16-31 SEQ ID NO: 213 DOM16- 32 (SEQ ID NO: 214) DOM16-33 SEQ ID NO: 215 DOM16-35 (SEQ ID NO: 216) DOM16-37 SEQ ID NO: 217 DOM16-38 (SEQ ID NO: 218) DOM16-39 SEQ ID NO : 219 DOM16-40 (SEQ ID NO: 220) DOM16-41 SEQ ID NO: 221 DOM16-42 (SEQ ID NO: 222) DOM16-43 SEQ ID NO: 223 DOM16-44 (SEQ ID NO: 224) DOM16- 45 SEQ ID NO: 225 DOM16-46 (SEQ ID NO: 226) DOM16-47 SEQ ID NO: 227 DOM16-48 (SEQ ID NO: 228) DOM16-49 SEQ ID NO: 229 DOM16-50 (SEQ ID NO: 230) DOM16-59 SEQ ID NO: 231 DOM16-60 (SEQ ID NO: 232) DOM16-61 SEQ ID NO: 233 DOM16-62 (SEQ ID NO: 234) DOM16-63 SEQ ID NO: 235 DOM16-64 ( SEQ ID NO: 236) DOM16-65 (SEQ ID NO: 237 DOM16-66 (SEQ ID NO: 238) DOM16-67 (SEQ ID NO: 239 DOM16-68 (SEQ ID NO: 240) DOM16-69 (SEQ ID NO: 241 DOM16-70 (SEQ ID NO: 242) DOM16-71 (SEQ ID NO: 243 DOM16-72 (SE Q ID NO: 244) DOM16-73 (SEQ ID NO: 245 DOM16-74 (SEQ ID NO: 246) DOM16-75 (SEQ ID NO: 247 DOM16-76 (SEQ ID NO: 248) DOM16-77 (SEQ ID NO: 249 DOM16-78 (SEQ ID NO: 250) DOM16-79 (SEQ ID NO: 251 DOM16-80 (SEQ ID NO: 252) DOM16-81 (SEQ ID NO: 253 DOM16-82 (SEQ ID NO: 254 ) DOM16-83 (SEQ ID NO: 255 DOM16-84 (SEQ ID NO: 256) DOM16-85 (SEQ ID NO: 257 DOM16-87 (SEQ ID NO: 258) DOM16-88 (SEQ ID NO: 259 DOM16- 89 (SEQ ID NO: 260) DOM16-90 (SEQ ID NO: 261 DOM16-91 (SEQ ID NO: 262) DOM16-92 (SEQ ID NO: 263 DOM16-94 (SEQ ID NO: 264) DOM16-95 ( SEQ ID NO: 265 DOM16-96 (SEQ ID NO: 266) DOM16-97 (SEQ ID NO: 267 DOM16-98 (SEQ ID NO: 268) DOM16-99 (SEQ ID NO: 269) DOM16-100 (SEQ ID NO: 270) DOM16-101 (SEQ ID NO: 271 DOM16-102 (SEQ ID NO: 272) DOM16-103 (SEQ ID NO: 273 DOM16-104 (SEQ ID NO: 274) DOM16-105 (SEQ ID NO: 275 DOM16-106 (SEQ ID NO: 276) DOM16-107 (SEQ ID NO: 277 DOM16-108 (SEQ ID NO: 278) DOM16-109 (SEQ ID NO: 279 DOM16-110 (SEQ ID NO: 280) DOM16 -111 (SEQ ID NO: 281 DOM16-112 (SEQ ID NO: 282) DOM16-113 (SEQ ID NO: 283 DOM16-114 (SEQ ID NO: 284) DOM16-115 (SEQ ID NO: 285 DOM16-116 (SEQ ID NO: 286) DOM16-117 (SEQ ID NO: 287), DOM16-118 (SEQ ID NO: 288), DOM16-119 (SEQ. ID NO: 289), DOM16-39-6 (SEQ ID NO: 290), DOM16-39-8 (SEQ ID NO: 291), DOM16-39-34 (SEQ ID NO: 292), DOM16-39-48 (SEQ ID NO: 293), DOM16-39-87 (SEQ ID NO: 294), DOM16-39-90 (SEQ ID NO: 295), DOM16-39-96 (SEQ ID NO: 296), DOM16-39-100 (SEQ ID NO: 297), DOM16-39-101 (SEQ ID NO: 298), DOM16-39-102 (SEQ ID NO: 299), DOM16-39-103 (SEQ ID NO: 300), DOM16-39-104 (SEQ ID NO: 301), DOM16-39-105 (SEQ ID NO: 302), DOM16-39-106 ( SEQ ID NO: 303), DOM16-39-107 (SEQ ID NO: 304), DOM16-39-108 (SEQ ID NO: 305), DOM16-39-109 (SEQ ID NO: 306), DOM16-39- 110 (SEQ ID NO: 307), DOM16-39-111 (SEQ ID NO: 308), DOM16-39-112 (SEQ ID NO: 309), DOM16-39-113 (SEQ ID NO: 310), DOM16- 39-114 (SEQ ID NO: 311), DOM16-39-115 (SEQ ID NO: 312), DOM16-39-116 (SEQ ID NO: 313), DOM16-39-117 (SEQ ID NO: 314), DOM16-39-200 (SEQ ID NO: 315), DOM16-39-201 (SEQ ID NO: 316), DOM16-39-202 (SEQ ID NO: 317), DOM16-39-203 (SEQ ID NO: 318) ), DOM16-39-204 (SEQ ID NO: 319), DOM16-39-205 (SEQ ID NO: 320), DOM16-39-206 (SEQ ID NO: 321), DOM16-39-207 (SEQ ID NO. : 322), DOM16-39-209 (SEQ ID NO: 323), and DOM16-52 (SEQ ID NO: 324). Preferably, the nucleotide sequence identity is determined throughout the length of the nucleotide sequence encoding the selected anti-EGFR dAb. The present invention also provides a vector comprising a recombinant nucleic acid molecule of the present invention. In certain embodiments, the vector is an expression vector comprising one or more expression control elements or sequences that are operably linked to the recombinant nucleic acid of the present invention. The present invention also provides a recombinant host cell comprising a recombinant nucleic acid molecule or vector of the present invention. Suitable vectors (eg, plasmids, phagmids), expression control elements, host cells and methods for producing recgining host cells of the present invention are well known in the art, and the examples are further described in the present invention. . Suitable expression vectors may comprise a number of components, for example, a replication origin, a selectable labeled gene, one or more expression control elements, such as a transcription control element (e.g., promoter, enhancer, terminator) and / or one or more translation signals, a signal sequence or leader sequence and the like. The expression control elements and a signal sequence, if found, can be provided through the vector or another source. For example, transcriptional and / or translational control sequences of a cloned nucleic acid encoding an antibody chain can be used for direct expression.
A promoter can be provided for expression in a desired host cell. The promoters can be constitutive or inducible. For example, a promoter can be operably linked to a nucleic acid encoding an antibody, antibody chain or part thereof, so as to direct the transcription of the nucleic acid. A variety of suitable promoters are available for prokaryotic hosts (e.g., lac, tac, T3, T7 promoters for E. coli) and eukaryotes (e.g., simian 40 late early promoter, sarcoma virus long-terminal repeat promoter). de Rous, cytomegalovirus promoter, adenovirus late promoter). In addition, "expression" vectors usually comprise a selectable marker for the selection of host cells carrying the vector, and in the case of a replicable expression vector, an origin or replication.The genes encoding products that confer antibiotic resistance or Drugs are common selectable markers and can be used in prokaryotic cells (eg lactamase gene (ampicillin resistance) Tet gene for tetracycline resistance) and eukaryotic (eg neomycin (G418 or geneticin), gpt (mycophenolic acid), ampicillin, or hygromycin resistance genes.) Dihydrofolate reductase marker genes allow selection with methotrexate in a variety of hosts.The genes encoding the host auxotrophic marker gene product (eg LEU2, URA3, HIS3) frequently are used as selectable markers in yeast, the use of viral vectors (eg baculovirus) or phage, and vectors that have the ability to integrate into the genome of the host cell, such as retroviral vectors, are also contemplated. Such expression vectors for expression for expression in mammalian cells and eukaryotic cells (E. coli), insect cells (Drosophila S2 Schnieder cells, Sf9) and yeast (P. methanolica, P. pastoris, S. cerevisiae) are well known in the art. Suitable host cells can be prokaryotic, including bacterial cells such as £. coli, B. subtilis and / or other suitable bacteria; eukaryotic cells, such as fungal or yeast cells (eg, Pichia pastoris, Aspergillus sp., Saccharomyces cerevisiae, Schizosaccharotnyces pombe, Neurospora crassa), or other lower eukaryotic cells, and higher eukaryotic cells such as those from insects (S2 Drosophila Schnieder cells, Sf9 insect cells (WO 94/26087 (O'Connor)), mammals (e.g. , COS cells, such as COS-1 (ATCC Access Number CRL-1650) and COS-7 (Access ATCC Number CRL-1651), CHO (eg ATCC Access Number CRL-9096, CHO DG44 (Ur? aub, G. and Chasin, LA., Proc. Nati. Acac. Sci USA, 77 (7): 4216-4220 (1980))), 293 (ATCC Access Number CRL-1573), HeLa (ATCC Access Number CCL-2), CV1 (Access ATCC Number CCL-70), WOP (Dailey, L., and associates, J. Virol, 54: 739-749 (1985), 3T3, 293T (Pear, WS, and associates, Proc. Nati. Acad. Sci USA, 90: 8392-8396 (1993)) NSO cells, SP2 / 0 cells, HuT 78 and the like or plants (for example tobacco) (See, for example, Ausubel, FM and associates, eds, Current Protocols in Mol.ecular Biology, Greene Publishings Associates and John Wi Law &Sons Inc. (1993)). In some embodiments, the host cell is an isolated host cell and is not part of a multicellular organism (e.g., plant or animal). In preferred embodiments, the host cell is a non-human host cell. The present invention also provides a method for producing a ligand (for example, double specific ligand, multispecific ligand) of the present invention, comprising maintaining a recombinant host cell comprising a recombinant nucleic acid of the present invention under conditions suitable for expression of the acid recombinant nucleic acid, whereby the recombinant nucleic acid expresses and a ligand is produced. In some modalities, the method further comprises isolating the ligand. Preparation of immunoalobulin-based ligands Ligands (for example, double specific ligands, multispecific ligands) according to the present invention can be prepared according to previously established techniques, used in the field of antibody engineering, for the preparation of scFv, "phage" antibodies and other constructed antibody molecules. The techniques for antibody preparation are described, for example, in the following reviews and references herein: Winter &; Milstein, (1991) Nature 349: 293-299; Pluckthun (1992) Immunological Reviews 13 0: 151-188; Wright and associates, (1992) Crit. Rev. Immunol. 12: 125-168; Holliger, P. & Winter, G. (1993) Curr. Opin. Biotechnol. 4, 446-449; Carter, and associates (1995) J. Hematother. 4, 463-470; Chester, K.A. & Hawkins, R.E. (1995) Trends Biotechnol. 13, 294-300; Hoogenboom, H.R. (1997) Nat. Biotechnol. 15, 125-126; Fearon, D. (1997) Nat. Biotechnol. 15, 618-619; Pluckthun, A. & Pack, P. (1997) Immunotechnology 3, 83-105; Carter, P. & Merchant, A.M. (1997) Curr. Opin. Biotechnol. 8, 449-454; Holliger, P. & Winter, G. (1997) Cancer Immunol. Immunother. 45,128-130. Suitable techniques for selection of variable domains of antibody with desired specificity expand libraries and selection procedures that are known in the art. Natural libraries (Marks and associates (1991) J. Mol. Biol., 222: 581; Vaughan and associates (1996) Nature Biotech., 14: 309) which use V genes collected from human B cells that are known to the experts in the technique. Synthetic libraries (Hoogenboom &Winter (1992) J Mol.
Biol, 227: 381; Barbas and associates (1992) Proc. Nati Acad. Sci USA, 89: 4457; Nissim et al. (1994) EMBO J, 13: 692; Griffiths and associates (1994) EMBO J., 13: 3245; De Kruif et al. (1995) J. Mol. Biol., 248: 97) were prepared by cloning immunoglobulin V genes, usually using PCR. Errors in the PCR process can lead to a high degree of randomization. The VH and / or VL libraries can be screened against target antigens or epitopes separately, in which case a single domain link is directly selected for, or together. Library vector systems A variety of selection systems are known in the art which are suitable for use in the present invention. Examples of such systems are described below. The bacteriophage lambda expression systems can be classified directly as bacteriophage plaques or as colonies of lysogens, both as previously described (Huse et al. (1989, Science, 246: 1275; Cato and Koprowski (1990) Proc. Nati. Acad. Sci USA, 87; Mullínax and associates (1990) Proc. Nati Acad. Sci USA, 87: 8095; Persson and associates (1991) Proc. Nati, Acad. Sci USA, 88: 2432), and are of use in present invention Although such expression systems can be used to classify up to 106 different members of a library, they are not really suitable for classifying larger numbers (greater than 106 members). deployment of selection, which allows a nucleic acid to be linked to the polypeptide it expresses.As used in the present invention, a selection deployment system is a system that allows selection, through appropriate deployment means, of the individual members of the library, linking to the generic and / or objective. Selection protocols for isolating desired members of large libraries are known in the art, as typified by phage display techniques. Such systems, in which the various peptide sequences are displayed on the surface of filamentous bacteriophages (Scott and Smith (1990) Science, 249: 386), have proven useful in creating libraries of antibody fragments (and nucleotide sequences). encoding them) for the selection and in vitro amplification of specific antibody fragments that bind to a target antigen (McCafferty et al., WO 92/01047). The nucleotide sequences encoding the variable regions are linked to gene fragments encoding leader sequences that direct them to the periplasmic space of E. coli and as a result, the resulting antibody fragments are displayed on the surface of the bacteriophage, usually as fusions to Bacteriophage coating proteins (e.g. plll op VI II). Alternatively, antibody fragments in lambda phage capsids (fagebodies) are displayed externally. An advantage of phage-based display systems is that, because they are biological systems, the selected library members can be amplified simply by growing the phage containing the selected library member in bacterial cells. In addition, since the nucleotide sequence is that it encodes the polypeptide library member is contained in a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation is relatively simple. Methods for the construction of bacteriophage antibody display libraries and lambda phage display libraries are well known in the art (McCafferty et al. (1990) Nature, 348: 552; Kang et al. (1991) Proc. Nati. Acad. Sci USA, 88: 4363; Clackson and associates (1991) Nature, 352: 624; Lowman and associates (1991) Biochemistry, 30: 10832; Burton and associates (1991) Proc. Nati, Acad. Sci USA, 88: 10134; Hoogenboom and associates (1991) Nucleic Acids Res., 19: 4133; Chang et al. (1991) J. Immunol, 147: 3610; Breitling et al. (1991) Gene, 104: 147; Marks and associates (1991) supra; Barbas and associates (1992) supra; Hawkins and Winter (1992) J. Immunol, 22: 867; Marks et al., 1992, J. Biol. Chem., 267: 16007; Lerner et al. (1992) Science, 258: 1313, incorporated herein by reference. A particularly convenient method has been the use of phage-scFv libraries (Huston et al., 1988, Proc. Nati, Acad. Sci USA, 85: 5879-5883; Chaudhary et al. (1990) Proc. Nati Acad. Sci USA, 87 : 1066-1070; McCafferty and associates (1990) supra; Clackson and associates (1991) Nature, 352: 624; Marks and associates (199"1) J Mol. Biol, 222: 581; Chiswell and associates (1992) Trends Biotechnol , 10: 80; Marks et al. (1992) J. Biol. Chem., 267) Several modalities of scFv libraries deployed in bacteriophage coat proteins have already been described Refinements of phage display methods are also known. for example as described in WO96 / 06213 and WO92 / 01047 (Medical Research Council and associates) and WO97 / 08320 (Morphosys), which are incorporated herein by reference.Other systems for generating polypeptide libraries involve the use of cell-free enzymatic machinery for s in vitro synthesis of the library members. In one method, RNA molecules are selected by alternating turns of selection against a PCR target and amplification (Tuerk and Gold (1990) Science, 249: 505; Ellington and Szostak (1990) Nature, 346: 818). A similar technique can be used to identify DNA sequences which bind to a predetermined human transcription factor (Thiesen and Bach (1990) Nucleic Acids Res., 18: 3203; Beaudry and Joyce (1992) Science, 257: 635; WO92 / 05258 and WO92 / 14843). In a similar manner, in vitro translation can be used to synthesize polypeptides as a method for generating large libraries. These methods, which generally comprise stabilized polysome complexes, are described further in WO88 / 08453, WO90 / 05785, WO90 / 07003, WO91 / 02076, WO91 / 05058 and WO92 / 02536. Alternative non-phage display systems, such as those described in WO95 / 22625 and WO95 / 11922 (Affymax), use polysomes to display polypeptides for selection. A still further category of techniques involves the selection of repertoires in artificial compartments, which allow the ligation of a gene with its gene product. For example, a selection system in which nucleic acids encoding desirable gene products can be selected in microcapsules formed by water-in-oil emulsions is described in publications W099 / 02671, WO00 / 40712 and Tawfik & Griffiths (1998) Nature Biotechnol 16 (7), 652-6. Genetic elements that encode a gene product having a desired activity are compartmentalized into microcapsules and subsequently transcribed and / or translated to produce those respective gene products (RNA or protein) within the microcapsules. The genetic elements that produce gene products that have desired activity are classified subsequently. This method selects gene products of interest by detecting the desired activity through a variety of means. Construction of Libraries Libraries screened for selection may be constructed using techniques known in the art, for example as set forth above, or may be purchased from commercial sources. Libraries that are useful in the present invention are described, for example, in WO99 / 20749. A vector system is chosen and one or more nucleic acid sequences encoding polypeptides of interest are cloned into the library vector, a diversity can be generated between the cloned molecules by carrying out the mutagenesis before the expression; alternatively, the encoded proteins can be expressed and selected, as described above, before further mutagenesis and rounds of selection are carried out. The mutagenesis of nucleic acid sequences encoding structurally optimized polypeptides is carried out by standard molecular methods. Of particular interest is the polymerase chain reaction or PCR (Mullis and Faloona (1987) Methods Enzymol., 155: 335, incorporated herein by reference). PCR that uses multiple cycles of DNA replication catalyzed by DNA-dependent DNA polymerase, thermostable to amplify the target sequence of interest, is well known in the art. The construction of the various antibody libraries has been mentioned in the Winter and Associates publication. (1994) Ann. Rev. Immunology 12, 433-55, and the references therein mentioned. PCR is carried out using template DNA (at least 1fg, more usefully, 1-1000 ng) and at least 25 pMol. of oligonucleotide primers; it may be convenient to use a greater amount of primer when the primer group is highly heterogeneous, since each sequence is represented only by a small fraction of the group molecules, and the amounts become limiting in the subsequent amplification cycles. A typical reaction mixture includes two points: 2 μl of DNA, 25 pMol. of oligonucleotide primer, 2.5 μl of 10X PCR 1 buffer (Perkin-Elmer, Foster City, CA), 0.4 μl of 1.25 μM dNTP, 0.15 μl (or 2.5 units) of Taq DNA polymerase (Perkin Elmer, Foster City, CA ) and deionized water at a total volume of 25 μl. The mineral oils are covered and the PCR is carried out using a programmable thermal cycler. The length and temperature of each step of a PCR cycle, as well as numbers of cells, is adjusted according to the rigor requirements in effect. Hardening temperature and timing are determined, both through the efficiency with which the primer is expected to harden to a template and the degree of incompatibility that is tolerated; obviously, when nucleic acid molecules are simultaneously amplified and mutagenized, incompatibility is required, at least in the first round of synthesis. The ability to optimize the stringency of hardening conditions of the primer is also within the knowledge of one skilled in the art. A hardening temperature between 30 ° C and 72 ° C is what is normally used. Initial denaturation of the template molecules normally occurs at a temperature between 92 ° and 99 ° C for 4 minutes, followed by 20-40 cycles consisting of denaturation (94-99 ° C for 15 seconds at 1 minute), hardening (temperature determined as described above, 1-2 minutes) and extension (72 ° C for 1-5 minutes, depending on the length of the amplified product). The final extension is usually 4 minutes at a temperature of 72 ° C, and can be followed by an indefinite step (0-24 hours) at a temperature of 4 ° C. Combination of simple variable domains The domains useful in the present invention, once selected, can be combined through a variety of methods known in the art, including covalent and non-covalent methods. Preferred methods include the use of polypeptide linkers as described in the present invention, for example, in relation to scFV molecules (Bird et al., (1988) Science 242: 423-426). A description of suitable linkers is provided in the publication of Bird and associates. Science 242, 423-426; Hudson et al., Journal Immunol Methods 231 (1999) 177-189; Hudson et al., Proc Nat Acad Sd USA 85, 5879-5883. The linkers are preferably flexible, allowing two simple domains to interact. An example of a linker is a linker (Gly Ser) n, where n = 1 to 8, for example, 2, 3, 4, 5 or 7. Linkers used in diabodies, which are less flexible, can also be used (Holliger et al., (1993) Proc Nat Acad Sci USA 90: 6444-6448). In one embodiment, the linker employed is not an immunoglobulin articulation region. The variable domains can be combined using methods other than linkers. For example, the use of disulphide bridges, provided through naturally occurring or constructed cysteine residues, can be exploited to stabilize VH-VH, VL-VL or VH-V dimers (Reiter and associates, (1994) Protein Eng 7: 697-704) or by remodeling the interface between the variable domains to improve the "fit" and thus stability of the interaction (Ridgeway et al., (1996) Protein Eng. 7: 617-621; Zhu et al., (1997) Protein Science 6: 781-788). Other techniques for binding or stabilizing variable immunoglobulin domains, and in particular VH antibody domains, may be employed as appropriate. Characterization of ligands The binding of a specific double ligand to a cell or the binding of each binding domain to each specific target can be tested by methods familiar to those skilled in the art, and including ELISA. In a preferred embodiment of the present invention, the linkage is tested using monoclonal phage ELISA. The phage ELISA assay can be carried out according to any suitable procedure: an exemplary protocol is set forth below. The populations of phages produced in each round of selection can be classified for binding by ELISA to the selected antigen or epitope, to identify "polyclonal" phage antibodies. The phage of simple infected bacterial colonies from these populations can subsequently be classified by ELISA to identify "monoclonal" phage antibodies. It is also desirable to classify soluble antibody fragments for binding to an antigen or epitope, and this can also be carried out by ELISA assay using reagents, for example, against a C- or N-terminal label (see for example the publication of Winter and associates (1994) Ann. Rev. Immunology 12, 433-55 and the references therein mentioned.
The diversity of selected phage monoclonal antibodies can also be evaluated by gel electrophoresis of PCR products (Marks and associates, 1991, supra, Níssim and associates 1994 supra), by probing (Tomlinson and associates, 1992) J. Mol. Biol. 227, 776) or by sequencing the vector DNA. Ligand structure In the case where each variable domain is selected from V-gene repertoires, selected for example using phage display technology as described herein, then these variable domains comprise a region of universal structure, so that they can be recognized by a specific generic double specific ligand, as defined herein. The use of universal structures, generic ligands and the like is described in WO99 / 20749. When V-gene repertoires are used, the variation in the polypeptide sequence is preferably located within the structural circuits of the variable domains. The polypeptide sequences of any variable domain can be altered by DNA shuffling or by mutation in order to increase the interaction of each variable domain with its complementary pair. DNA rebar is known in the art and is considered, for example, in the publication by Stemmer, 1994, Nature 370: 389-391 and in US Patent No. 6,297,053, which are incorporated herein by reference. Other methods of mutagenesis are well known to those skilled in the art. In general, nucleic acid molecules and vector constructs for selection, preparation and formation of specific double ligands can be constructed and manipulated as set forth in standard laboratory manuals, such as Sambrook and associates. (1989) Mol.ecular Cloning: A Laboratory Manual, Cold Spring Harbor, USA. The nucleic acid manipulation used in the present invention is usually carried out in recombinant vectors. As used in the present invention, vector refers to an independent element that is used to introduce heterologous DNA into cells for expression and / or replication thereof. The methods by which these vectors are selected or constructed, and subsequently used, are well known to those skilled in the art. Numerous vectors are publicly available, including bacterial plasmids, bacteriophages, artificial chromosomes and episomal vectors. Said vectors can be used for cloning and simple mutagenesis; as an alternative, a gene expression vector is used. A vector to be used according to the present invention can be selected to accumulate a coding and polypeptide sequence of a desired size, typically 0.25 kilobase (kb) at 40 kb or more in length. An appropriate host cell is transformed with the vector after in vitro cloning manipulation. Each vector contains various functional components, which generally include a cloning site ("polylinker"), a replication origin and at least one selectable marker gene. If the determined vector is an expression vector, it also has one or more of the following: an enhanced element, promoter, transcription termination sequences and signal sequences, each placed in the vicinity of the cloning site, so that they are linked operatively to the gene encoding a specific double ligand according to the present invention. Both cloning and expression vectors generally contain nucleic acid sequences that allow the vector to replicate in one or more selected host cells. Typically in cloning vectors, this technique is one that allows the vector to replicate independently of the host chromosomal DNA and includes replication origins or replication sequences autonomously. These sequences are known by a variety of bacteria, yeasts and viruses. The replication origin of plasmid pBR322 is suitable for most Gram-negative bacteria, and the origin of plasmid of 2 micras is suitable for yeast, and several viral origins (for example SV40, adenovirus) are useful for cloning vectors in mammalian cells. Generally, replication origin is not needed for expression vectors unless they are used in mammalian cells with the ability to replicate high levels of DNA, such as COS cells. Conveniently, a cloning or expression vector may contain a selection gene also required as a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. The host cells not transformed with the vector containing the selection gene will therefore not survive in the culture medium. Typical selection genes encode proteins that contain resistance to antibiotics and other toxins, eg, ampicillin, neomycin, methotrexate or tetracycline, auxotrophic complement deficiencies or delivery in important nutrients not available in the growth medium. Since the vector replication encoding a specific double ligand according to the present invention is most conveniently carried out in E. coli, an E-selected marker. coli, for example, the ß-lactamase gene that confers resistance to antibiotic ampicillin, will be used. This can be obtained from E. coli plasmids, such as pBR322 or a pUC plasmid such as pUC18 or pUC19. Expression vectors typically contain a promoter that is recognized by the host organism and is operably linked to the coding sequence of interest. Said promoter can be inducible or constitutive. The term "linked in operable form" refers to a juxtaposition wherein the components described are in a relationship that allows them to function in their projected form. A control sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequences. Promoters suitable for use with prokaryotic hosts include, for example, the β-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence linked operably to the coding sequence. Preferred vectors are expression vectors that allow the expression of a nucleotide sequence corresponding to a polypeptide library member. Therefore, selection with the first and / or second epitope antigens can be carried out by a separate propagation and expression of a single clone expressing the member of the polypeptide library or through the use of any selection display system. As described above, the preferred selection deployment system is bacteriophage display. Therefore, phage or phagemid vectors can be used (eg plT1 or plT2) The leader sequences in the present invention include pelB, st 11, ompA, phoA, blah and pelA An example of phagemid vectors having a origin of E. coli replication (for double-stranded replication) and also a phage replication origin (for the production of single-stranded DNA) The manipulation and expression of said vectors is well known in the art (Hoogenboom and Winter ( 1992) supra; Nissim and associates. (1994) supra). Briefly, the vector contains a β-lactamase gene to confer selectivity in the phagemid and a lac promoter upstream of an expression band consisting (N to C terminal) in a pelB leader sequence (directing the polypeptide expressed to the periplasmic space), a multiple cloning site (to clone the nucleotide version of the library member) optionally, one or more peptide tags (for detection) optionally one or more stop codons TAG and the phage protein pIII. Therefore, using various suppressor and non-suppressor strains of E. coli and with the addition of glucose, iso-propyl thio-β-D-galactoside (IPTG) or an auxiliary phage, such as VCS M13, the vector has the ability to replicate as a plasmid without expression, produce large quantities of the polypeptide library member alone or produce phages, some of which contain at least one copy of the polypeptide-plll fusion on its surface. The construction of vectors encoding double specific ligands according to the present invention employs conventional ligation techniques. The isolated vectors or DNA fragments are dissociated, tailored and reigated in the desired form to generate the required vector. If desired, the analysis to confirm that the correct sequences are present in the constructed vector can be carried out in a known manner. Suitable methods for constructing expression vectors, preparing in vitro transfections, introducing DNA into host cells, carrying out assays to evaluate expression and function are known to those skilled in the art. The presence of a gene sequence in a sample is detected, or its amplification and / or expression is quantified by conventional methods, such as Southern or Northern analysis, Western spotting, spotting of DNA, RNA or protein, in-hybridization. Situ, immunocytochemistry or sequence analysis of nucleic acid or protein molecules. Those skilled in the art will readily consider how these methods can be "modified if desired.
Skeletons Skeletons can be based on immunoglobulin molecules or can be of non-immunoglobulin origin, as stated above. Each domain of the specific double ligand can be a different skeleton. Preferred immunoglobulin backbones as used in the present invention, include any of one or more of those selected from the following: an immunoglobulin molecule comprising at least (i) the CL domain (kappa or lambda subclass) of a antibody; or (ii) the CH1 domain of an antibody heavy chain; an immunoglobulin molecule comprising the CH1 and CH2 domains of an antibody heavy chain; an immunoglobulin molecule comprising the CH1, CH2 and CH3 domains of an antibody heavy chain; or any of subgroup (ii) together with the CL domain (kappa or lambda subclass) of an antibody. A joint region domain can also be included. Said combinations of domains may be, for example, natural antibodies mimicked such as IgG or IgM, or fragments thereof, such as Fv, scFv, Fab or F (ab ') 2 molecules. Those skilled in the art will be aware that this list is not intended to be exhaustive. Protein scaffolds Each binding domain comprises a protein scaffold and one or more CDRs that are involved in the specific interaction of the domain with one or more epitopes. Conveniently, an epitope linkage domain according to the present invention comprises three CDRs. Suitable protein scaffolds include any of those selected from the group consisting of the following: those that are based on immunoglobulin domains, those that are based on fibronectin, those that are based on antibodies, those that are based on CTLA4, those that they are based on chaperones such as GroEL, those based on lipocalin and those based on bacterial receptors Fc SpA and SpD. Those skilled in the art will appreciate that this list is not intended to be exhaustive. Scaffolds for use in ligand construction Selection of the main-chain conformation The members of the immunoglobulin superfamily share a similar fold for their polypeptide chain. For example, although the antibodies are highly diverse in terms of their primary sequence, the comparison of sequence and crystallographic structures have revealed that, contrary to what was expected, five of six antibody antigen binding circuits (H1, H2 , L1, L2, L3), adopt a limited number of main-chain conformations, or canonical structures (Chothia and Lesk (1987) J. Mol. Biol., 196: 901; Chothia and associates (1989) Nature, 342: 877). The analysis of circuit lengths and key residues has therefore allowed the anticipation of the main-chain conformations of H1, H2, L1, L2 and L3 found in most human antibodies (Chothia and associates. (1992) J Mol. Biol., 227: 799; Tomlinson et al. (1995) EMBO J., 14: 4628; Williams et al. (1996) J. Mol. Biol., 264: 220). Although the H3 region is much more diverse in terms of sequence, length and structure (due to the use of D segments) it also forms a limited number of main-chain conformations for short circuit lengths that depend on the length and the presence of particular residues, or residue types, at key positions in the circuit and antibody structure (Martin et al. (1996) J. Mol. Biol., 263: 800; Shirai et al. (1996) FEBS Letters, 399: 1 ). The ligand libraries and / or link domains can be designed so that certain lengths of circuits and key residues have been chosen to ensure that the main chain conformation of the members is known. Conveniently, these are actual conformations of molecules of the immunoglobulin superfamily found in nature, to minimize the opportunities for them to be nonfunctional, as described above, the germline V gene segments serve as an adequate basic structure to build antibodies or T-cell receptor libraries; other sequences are also used. Variations can occur at a low frequency, so that a small number of functional members can have an altered main-chain conformation, which does not affect their function. The canonical structure theory is also used to evaluate the number of different major-chain conformations encoded by the ligands, to anticipate the main-chain conformation based on double-specific ligand sequences and to choose residues for diversification that does not affect the canonical structure. It is known that in the V? Domain, the L1 circuit can adopt one of four canonical structures, the L2 circuit has a simple canonical structure and that 90% of the V? humans adopt one of four or five canonical structures of the L3 circuit (Tomlinson et al. (1995) supra); therefore in the V domain? alone, different canonical structures can be combined to create a range of different main chain conformations. Because the V? Domain encodes a different range of canonical structures for the L1, L2 and L3 circuits and that the V? Domains? and V? l can be paired with any VH domain that encodes various canonical structures for circuits H1 and H2, the number of combinations of canonical structures observed for these five circuits is very large. This implies that the generation of diversity in the main-chain conformation can be essential for the production of a wide range of binding specificities. However, by building an antibody library based on a single known backbone conformation it has been found that, contrary to expectation, the diversity in the main chain conformation is not required to generate sufficient diversity to address substantially all the antigens. More surprisingly, the simple main chain conformation does not need to be a consensus structure-a simple conformation, which occurs naturally can be used as the basis of an entire library. Therefore, in a preferred aspect, the ligands of the present invention possess a simple known backbone conformation. The single main chain conformation that is chosen is preferably common among the molecules of the immunoglobulin superfamily type in question. A conformation is common when a significant number of molecules that occur naturally are observed for adoption. Accordingly, in a preferred aspect of the present invention, the natural emergence of the different main chain conformations for each linker circuit of an immunoglobulin domain is considered and a naturally occurring variable domain having the desired combination of Main chain conformations for different circuits. If it is not available, you can choose the nearest equivalent. It is preferable that the desired combination of main chain conformations for the different circuits be created by selecting germline gene segments that encode the desired main chain conformations. It is more preferable that the selected germline gene segments are frequently expressed in nature, and most preferably they are the most frequently expressed of all the germline gene segments. In the design of ligands (for example ds-dAbs) or libraries thereof, the incidence of the different main chain conformations for each of the six antigen binding circuits can be considered separately. For H1, H2, L1, L2 and L3, a given conformation that is adopted between 20% and 100% of the antigen binding circuits of naturally occurring molecules is the one chosen. Normally, an incidence is observed above 35% (for example between 35% and 100%) and ideally above 50% or even above 65%. Since the vast majority of H3 circuits do not have canonical structures, it is preferred to select a main chain conformation that is common among the circuits displaying canonical structures. For each of the circuits, the conformation that is most frequently observed in natural repertoires is selected. In human antibodies, the most popular canonical structures (CS) for each circuit are the following: H1-CS1 (of expressed repertoire), H2-CS3 (46%), L1-CS2 of VK) (39%), L2 - CS 1 (100%), L3 - CS 1 of V ?, (36%) (the calculation assumes a ratio?:? Of 70:30, Hood and associates. (1967) Cold Spring Harbor Symp. Quant. Biol , 48: 133). For H3 circuits that have CDR3 structures, one length (Kabat et al. (1991) Sequences of proteins of immunological interest, US Department of Health and Human Services) of seven residues with a salt bridge from residue 94 to residue 101 appears to be more common. There are at least 16 human antibody sequences in the EMBL data libraries, with a required H3 length and key residues to form this conformation and at least two crystallographic structures in the protein data bank that can be used as a basis for modeling of antibodies (2cgr and 1 tet). The most frequently expressed germline gene segments due to this combination of canonical structures are segment VH 3-23 (DP-47) segment JH JH4b, segment V? O2 / O12 (DPK9) and segment J? J 1- The segments VH DP45 and DP38 are also suitable. These segments are therefore used in combination as a basis for building a library with the desired simple main chain conformation.
As an alternative, instead of choosing the simple main chain conformation based on the natural emergence of the different main chain conformations of each link circuits in isolation, the natural emergence of combinations of main chain conformations is used as the basis for Choose the simple main chain conformation. In the case of antibodies, for example, the natural emergence of combinations of canonical structures for either two, three, four, five or for the six antigen binding circuits can be determined. Here it is preferred that the chosen conformation be common in antibodies that occur naturally and it is most preferable that it be observed more frequently in the natural repertoire. Therefore, in human antibodies, for example, when the natural combinations of the five antigen binding circuits H1, H2, L1, L2 and L3 are considered, the most common combination of canonical structures is determined and subsequently combined with most popular formation of the H3 circuit, as a basis for choosing the simple main chain conformation. Canonical Sequence Diversification Having selected known backbone conformations, or preferably a single known backbone conformation, double specific ligands (eg, ds-dAbs) or libraries can be constructed for use in the present invention, with each linker site varying from one to the other. the molecule in order to generate a repertoire with structural and / or functional diversity. This means that the variants are generated so that they possess sufficient diversity in their structure and / or function so that they have the capacity to provide a range of activities. The desired diversity is usually generated by varying the selected molecule in one or more positions. The positions that will be changed can be chosen randomly or are selected preferentially. The variation can then be achieved either by randomization during which the resident amino acid is replaced by any amino acid or analog thereof, natural or synthetic, producing a very large number of variants or replacing the resident amino acid with one or more of a defined subgroup of amino acids, producing a more limited number of variants. Several methods have been reported to introduce such diversity. PCR prone to errors (Hawkins and associates (1992) J. Mol .. Biol, 226: 889), chemical mutagenesis (Deng and associates (1994) J. Biol. Chem., 269: 9533) or bacterial mutation strains ( Low and associates (1996) J. Mol. Biol., 260: 359) can be used to introduce random mutations into the genes encoding the Molecule. Methods for mutating selected positions are also known in the art and include the use of incompatible oligonucleotides or degenerating oligonucleotides, with or without the use of PCR. For example, several libraries of synthetic antibodies have been created to direct mutations to the antigen binding circuits. The H3 region of a human tetanus toxoid binding Fab has been randomized to create a range of novel binding specificities (Barbas et al., (1992) Proc. Nati, Acad. Sci. USA, 89: 4457). The random or signaling H3 and L3 regions have been appended to segments of the germline V gene to produce large libraries with unmutated structure regions (Hoogenboom &Winter (1992) J. Mol. Biol., 227: 381; associates (1992) Proc. Nati, Acad. Sci. USA, 89: 4457; Níssim and associates. (1994) EMBO J., 13: 692; Griffiths and associates. (1994) EMBO J, 13: 3245; De Kruif and associates (1995) J. Mol. Biol., 248: 97). Such amusement has been extended to include some of the other antigen binding circuits (Crameri et al. (1996) Nature Med., 2: 100; Riechmann et al. (1995) Bio / Technology, 13: 475; Morphosys, WO97 / 08320, supra). Since circuit scrambling has the potential to create approximately more than 1015 structures for H3 alone, and a similarly large number of variants for the other five circuits, it is not feasible to use current transformation technology or even use cell-free systems to produce a library that represents all possible combinations. For example, in one of the largest libraries constructed to date, 6 x 1010 different antibodies, which is only a fraction of the potential diversity of a library of this design, were generated (Griffiths et al. (1994) supra) . Preferably, only residues that are directly involved in creating or modifying the desired function of each domain of the double specific ligand molecule are diversified. For many molecules, the function of each domain will be linked to an object and therefore the diversity must be concentrated in the target binding site, avoiding at the same time changing residues that are crucial for the general packaging of the molecule or to maintain the conformation of the main chain chosen. Diversification of canonical sequences as applied to antibody domains In the case of antibody-based ligands (for example ds-dAbs) the binding site for each target is most often the antigen binding site. Therefore, only the residues at the antigen binding site are preferably varied. These residues are extremely diverse in the repertoire of human antibodies and are known to make contacts in high resolution antibody / antigen complexes. For example, in L2 it is known that positions 50 and 53 are diverse in antibodies that occur naturally and are observed to make contact with the antigen. In contrast, the conventional method may have been to diversify all residues in the Complementarity Determination Region (CDR1) as defined in the Kabat and associates publication. (1991, supra), some of the seven residues compared with the two diversified in the library to be used in accordance with the present invention. This represents a significant improvement in terms of functional diversity required to create a range of antigen binding specificities. By nature, the diversity of antibodies is the result of two processes: somatic recombination of germline V, D, and J gene segments to create a primary naive repertoire (called the germinal line and union diversity) and somatic hypermutation of V genes resulting readjustments. Analysis of human antibody sequences have shown that the diversity in the primary repertoire is focused on the center of the antigen binding site, whereas somatic hypermutation disperses the diversity to regions at the periphery of the antigen binding site that are highly conserved in the primary antigen repertoire (see Tomlinson and associates. (1996) J. Mol. Biol., 256: 813). This complementarity has been implicated in a proven way as an efficient strategy to search for space in the sequence, and although apparently unique for antibodies, it can be easily applied to other polypeptide repertoires. The residues that are varied are a subset of those that form the liaison site for the objective. The different subgroups (including overlaps) of residues in the effective link site are diversified at different stages during the selection, if desired. In the case of an antibody repertoire, an initial "naive" repertoire can be created where some, although not all residues in the antigen binding site are diversified. As used within this context, the term "naive" refers to antibody molecules that do not have a predetermined purpose. These molecules resemble those that are encoded by the immunoglobulin genes of an individual who has not undergone immune diversification, as is the case with fetal and newborn individuals whose immune system has not yet been stimulated by a wide variety of immune systems. antigenic stimuli. Subsequently this repertoire is selected against a range of antigens or epitopes. If required, then additional diversity can be introduced outside the diversified region in the initial repertoire. This matured repertoire can be selected for function, specificity or modified affinity. The naive repertoires of binding domains for the construction of dual specific ligands wherein some or all of the residues at the antigen binding site are varied and are known in the art. (See publications WO 2004/058821, WO 2004/003019 and WO 03/002609). The "primary" library mimics the natural primary repertoire, with a diversity restricted to residues in the center of antigen binding sites that are diverse in the germ line V (germline diversity) or diversified segments during recombination processes (union diversity). The residues that are diversified include but are not limited to H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97, H98, L50, L53, L91, L92, L93, L94 and L96. In the "somatic" library, diversity is restricted to residues that are diversified during the process of "recombination (union diversity) or are highly somatic mutated." The residues that are diversified include but are not limited to: H31, H33 , H35, H95, H96, H97, H98, L30, L31, L32, L34 and L96 All the residues described above as suitable for diversification in these libraries are known to make contacts in one or more antibody-antigen complexes. that in both libraries, not all the residues in the antigen binding site are varied, additional diversity is incorporated during selection, the remaining residues varying, if one wishes to do so.It will be appreciated by one skilled in the art that any subgroup of any of these residues (or additional residues that comprise the antigen binding site) can be used for initial and / or subsequent diversification of the site ce of antigens. In the construction of libraries for use in the present invention, the diversification of chosen positions is normally achieved at the level of nucleic acid, by altering the coding sequence that specifies the polypeptide sequence so that the number of possible amino acids can be incorporated ( 20 or a subgroup thereof) in said position. Using the lUPAC nclature, the most versatile codon is NNK, which encodes all amino acids as well as the TAG stop codon. The NNK codon is preferably used in order to introduce the required diversity. Other codons that achieve the same ends are also used, including the NNN codon, which leads to the additional stop codons TGA and TAA. A characteristic of the side chain diversity at the antigen binding site of human antibodies is a pronounced tilt that favors certain amino acid residues. If the amino acid composition of the ten most diverse positions in each of the regions VH, V? and V? is added, more than 76% of the side chain diversity comes only from seven different residues, being these, serine (24%), tyrosine (24%), asparagine (11%), glycine (9%), alanine (7) %), aspartate (6%) and threonine (6%). This inclination towards hydrophilic residues and small residues that provide main chain flexibility probably reflects the evolution of surfaces that are predisposed to bind a high range of antigens or epitopes and may help explain the required promiscuity of the antibodies in the primary repertoire. Since it is preferable to mimic this distribution of amino acids, the distribution of amino acids in the positions that will be varied, preferentially mimics that observed in the binding site of the antibodies. Said bias in the substitution of amino acids that allows the selection of certain (not only antibody polypeptides) against a range of target antigens, is easily applied to any polypeptide repertoire. These are various methods to tilt the amino acid distribution in the position that will be varied (including the use of tri-nucleotide mutagenesis)., see publication WO97 / 08320), which is the preferred method, due to the ease of synthesis, and the use of conventional degeneracy codons. By comparing the amino acid profile encoded by all codon combinations of degeneration (with single, double triple and quadruple degeneration in equal proportions in each position) with the use of the natural amino acid it is possible to calculate the most representative codon. The codons (AGT) (AGC) T, (AGT) (AGC) C and (AGT) (AGC) (CT) that is, DVT, DVC and DVY, which respectively use lUPAC nomenclature - are closest to the desired amino acid profile : they encode 22% serine and 11% tyrosine, asparagine, glycine, ala-nina, aspartate, threonine and cysteine. Preferably, therefore, the libraries are constructed using either the DVT, DVC or DVY codon in each of the diversified positions. Therapeutic and Diagnostic Compositions and Uses The present invention provides compositions comprising the ligands of the present invention and a pharmaceutically acceptable carrier, diluent or excipient, and therapeutic and diagnostic methods employing the ligands or compositions of the present invention. The ligands according to the method of the present invention can be used in therapeutic and prophylactic in vivo applications, in vivo diagnostic applications and the like. The therapeutic and prophylactic uses of ligands of the present invention involve the administration of ligands according to the present invention to a recipient mammal, such as a human. The ligand linkage is directed with high affinity and / or avidity. In some embodiments, such as IgG-like ligands, the ligands may allow the recruitment of cytotoxic cells to transmit the killing of cancer cells, for example by antibody-dependent cellular cytotoxicity. Substantially pure ligands of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more • homogeneity is most preferred for pharmaceutical uses, especially when the mammal is a human. Once purified, partially or for homogeneity as desired, the ligands can be used for diagnosis or in therapeutic form (including extracorporeal) or in the development and performance of assay procedures, immunofluorescent staining and the like (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY). For example, the ligands of the present invention will normally find use in the prevention, suppression or treatment of disease states. For example, the ligands may be administered to treat suppressing or preventing chronic inflammatory disease, allergic hypersensitivity, cancer, bacterial or viral infections, autoimmune disorders (which include but are not limited to type I diabetes, asthma, multiple sclerosis, rheumatoid arthritis, arthritis juvenile rheumatoid arthritis, psoriatic arthritis, spondyloarthropathy (eg, alkylosing spondylitis), systemic lupus erythematosus, inflammatory bowel disease (eg, Crohn's disease, ulcerative colitis) myasthenia gravis and Behcet's syndrome, psoriasis, endometriosis, and abdominal adhesions (eg post abdominal surgery.) Ligands are useful for treating infectious diseases in which cells infected with an infectious agent contain higher levels of cell surface EGFR than uninfected cells or containing one or more cell surface targets that are not found in cells not infected, such as a protein that is encoded by the infectious agent (eg, bacteria, virus). Ligands according to the present invention that have the ability to bind to EGFR can be interned by cells expressing EGFR (for example endocytosed) and can deliver therapeutic agents (for example a toxin) in intracellular form (for example supplying a dAb that links to an intracellular target). further, the ligands provide a means through which a binding domain (eg a dAb monomer) having the specific ability to bind to an intracellular target can be delivered to an intracellular environment. This strategy requires, for example, a link domain with physical properties that allow it to remain functional within the cell. Alternatively, if the intracellular compartment of final destination is oxidized, a ligand multiplied as disulfide free may not be necessary. In the present application, the term "prevention" implies the administration of the protective composition before the induction of the disease. "Deletion" refers to the administration of the composition after an inductive agent, although prior to the clinical emergence of the disease. The "treatment" involves administration of the protective composition after the symptoms of the disease manifest. The work includes reducing symptoms associated with the disease, and also preventing or delaying the generation of the disease and also decreasing the severity or frequency of the symptoms of the disease. The term "cancer" refers to a pathological condition in mammals that is typically characterized by proliferation or deregulated cell survival. Examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, leukemia, and lymphoid malignancies. More particular examples of cancers include squamous cell cancers (e.g., epithelial squamous cellulose cancer), lung cancers (e.g., small cell lung carcinoma, non-small cell lung carcinoma, lung carcinoma, squamous cell carcinoma of the lung). , cancer of the peritoneum, hepatocellular cancer, gastric cancer of the stomach including gastrointestinal cancer, pancreatic cancer, glioblastoma, hepatoma, cervical cancer, ovarian cancer, liver cancer, cancer of the bladder, gallbladder cancer, breast cancer, colon cancer , rectal cancer, colorectal cancer, multiple myeloma, chronic myelogenous leukemia, acute meelagenous leukemia, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal carcinoma , carcinoma of the penis, cancer of the head, neck and the like. Cancers characterized by the expression of EGFR on the surface of cancer cells (eg, cancers expressing EGFR) include for example bladder cancer, ovarian cancer, colorectal cancer, breast cancer, lung cancer (eg, lung carcinoma) of non-small cell), gastric cancer, pancreatic cancer, prostate cancer, head cancer of the neck, kidney cancer and gallbladder cancer. Animal model systems that can be used to evaluate the efficacy of the ligands of the present invention for preventing, treating or suppressing the disease (eg, cancer) are available. Suitable cancer models include for example xenoinj "erto and orthotopic models of human cancers in animal models such as the SCID-hu myeloma model (Epstein J, and Yaccoby, S., Methods Mol Med. 113: 183-90 (2005)). , Tassone P, and associates, Clin Cancer Res. 11 (11): 4251-8 (2005)), mouse models of human lung cancer (e.g. Meuwissen R and Berns A, Genes Dev. 19 (6): 643 -64 (2005)), and mouse models of metastatic cancers (e.g. Kubota T., J Cell Biochem. 56 (1): 4-8 (1994)). Generally, the ligands of the present invention will be used in purified form together with pharmacologically suitable carriers. Typically, these carriers include aqueous or alcoholic / aqueous solutions, emulsions or suspensions including saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride and lactated Ringer. Suitable physiologically acceptable adjuvants, if it is necessary to maintain a polypeptide complex in the suspension, they can be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates. Intravenous vehicles include fluid and nutrient fillers and electrolyte fillers, such as those based on Ringer dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases may be present (Mack (1982) Remington's Pharmaceutical Sciences, sixteenth edition). A variety of suitable formulations can be used, including prolonged release formulations. The ligands of the present invention can be used as compositions administered separately or together with other agents. The ligands can be administered and / or formulated together with one or more additional therapeutic or active agents. When a ligand is administered with an additional therapeutic agent, the ligand can be administered before, simultaneously with or subsequent to the administration of the additional agent. Generally, the ligand and the additional agent is administered in a form that provides a Overlapping therapeutic effect. Additional agents that can be administered and formulated with the ligand of the present invention include, for example, various immunotherapeutic drugs such as cyclosporin, methotrexate, adriamcin or cisplatin, antibiotics, antifungals, anti-viral agents and immunotoxins. For example, when the antagonist is administered to prevent suppress or treat lung inflammation or respiratory disease, may be administered together with phosphodiesterase inhibitors (for example inhibitors of phosphodiesterase-4), bronchodilators (e.g. beta 2 agonists, anticholinergics, theophylline) Agonists short-acting beta (eg albuterol, salbutamol, bambuterol, fenoterol, isoeterina, isoproterenol, levalbuterol, metaproterenol, pirbuterol, terbutaline and tornlato) agonists long-acting beta (for formoterol example and salmeterol), short-acting anticholinergics j [by example ipratropium bromide and oxitropium bromide), long-acting anticholinergics (eg tiotropium), theophylline (e.g. short acting, long acting formulation), inhaled steroids (e.g. beclomethasone, beclomethasone, budesonide, flunisolide, propionate fluticasone and triamcinolone), oral steroids (for example tilprednisolona, prednisolone, prednisolone and prednisone) agonists short-acting beta combined with anticholinergics (eg albuterol / salbutamol / ipratropium and fenoterol / ipratropium), beta-agonists, long-acting combined with inhaled steroids (eg "salmeterol / fluticasone, and formoterol / budenoside) and mucolytic agents (for example erdostein, acetylcysteine, bromheksin, carbocysteine, guifenesin and iodinated glycerol). The ligands of the present invention can be administered together (for example to treat cancer, an inflammatory disease or another disease) with a variety of suitable co-therapeutic agents, including cytokines, analgesics / antipyretics, antiemetics and chemotherapeutics. Further suitable co-therapeutic agents include immunosuppressants selected from the group consisting of cyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil, corticosteroids, methotrexate, gold salts, sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine, 15-deoxyspergualine, 6-mercaptopurine agents , cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-thymocyte globulin and, antiinflammatory agents selected from the group consisting of aspirin, other salicylates, steroidal drugs, NSAIDs (NSAIDs), Cox-2 inhibitors , and DMARDs (antirheumatic drugs for disease modification); anti-psoriasis agents selected from the group consisting of coal tar, vitamin A, anthralin, calcipotriene, tarazotene, corticosteroids, methotrexate, retinoids, cyclosporine, etanercept, alefacept, efaluzimab, 6-thioguanine, mycophenolate mofetil, tacrolimus (FK-506 ), and hydroxyurea. Cytokines include, without limitation, a lymphokine, tumor necrosis factors, cytokine type tumor necrosis factor, lymphotoxin, interferon, macrophage, inflammatory protein, granulocyte monocyte colony stimulation factor, interleukin (including without interleukin limitation) -1, interleukin-2, interleukin-6, interleukin-12, interleukin-15, interleukin-18), growth factors including without limitation (eg growth hormone, insulin-like growth factor 1 and 2 (IGF-1 and IGF-2), granulocyte colony stimulation factor (GCSF), platelet-derived growth factor (PGDF), epidermal growth factor (EGF), and agents for erythropoiesis stimulation, for example recombinant human erythropoietin (Epoetin alpha), EPO, a hormonal agonist, hormonal antagonists (eg flutamide, tamoxifen, leuprolide acetate (LUPRON)), and steroids (eg, dexamethasone, retinoid, betamethasone, cortisone) l, cortisone, prednisone, dehydrotestosterone, glucocorticoid, mineralocorticoid, estrogen, testosterone, progestin). Analgesics / antipyretics may include, without limitation, for example aspirin, acetaminophen, buprofen, naproxen sodium, buprenorphine hydrochloride, propoxyphene hydrochloride, propoxyphene napsylate, meperidine hydrochloride, morphine hydromorphone sulfate hydrochloride, oxycodone hydrochloride, codeine phosphate, dihydrocodeine bitartrate, pentazocine hydrochloride, hydrocodone bitartrate, levorphanol tartrate, diflunisal, trolamine salicylate, nalbuphine hydrochloride, mefenamic acid, butorphanol tartrate, colifon salicylate, butal bital, phenyltoloxamine citrate, citrate diphenhydramine, methotrimeprazine, cinnamedrine hydrochloride, meprobamate and the like. Antiemetics can also be administered together to prevent or treat nausea and vomiting (eg, suitable antiemetics include meclizine hydrochloride, nabilone, prochlorperazine, dimenhydrinate, promethazine hydrochloride, tiethylperazine, scopolamine and the like.) Chemotherapeutic agents, as used the term in the present invention, include but are not limited to for example antimicrotubule agents, for example taxol (paclitaxel), taxotere (docetaxel); alkylating agents (for example cyclophosphamide, carmustine, lomustine and chlorambucil; cytotoxic antibiotics for example dactinomycin, doxorubicin, mitomycin-C, and bleomycin, antimetabolites, for example cytarabine, gemcitatin, methotrexate and 5-fluorouracil, antimycotics for example vinca vincristine alkaloids, for example etoposide, vincblastine and vincristine, and others such as cisplatin, dacarbazine, procarbazine and hydroxyurea; and combinations thereof. of the present invention can be used to treat cancer in combination with another therapeutic agent. For example, a ligand of the present invention can be administered in combination with a chemotherapeutic agent or an antineoplastic composition comprising one (at least one) chemotherapeutic agent. Conveniently, in a therapeutic method, the amount of chemotherapeutic agent that must be administered to be effective can be reduced. The present invention therefore provides a method for treating cancer, wherein the method comprises administering to a patient in need thereof a therapeutically effective amount of a ligand of the present invention and a chemotherapeutic agent, wherein the chemotherapeutic agent is administered a low dose Generally, the amount of chemotherapeutic agent that is administered in conjunction with a ligand of the present invention is approximately * 80%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10% or less, of the dose of chemotherapeutic agent alone which is normally administered to a patient. Therefore, co-therapy is particularly convenient when the chemotherapeutic agent causes harmful or undesirable side effects which can be reduced or eliminated at lower doses. The pharmaceutical compositions can include "cocktails" of various cytotoxic agents or other agents together with ligands of the present invention, or even combinations of ligands according to the present invention having different specificities, such as ligands selected using different target antigens or epitopes, whether they are gathered or not before the administration. The route of administration of pharmaceutical compositions according to the present invention can be any suitable route, such as those commonly known to those skilled in the art. For therapy, including without limitation immunotherapy, the ligands of the present invention can be administered to any patient according to standard techniques. The administration can be any suitable mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, intrathecally, intraarticularly, through the pulmonary route or also in an appropriate manner by direct fusion (for example with a catheter). administration will depend on the age, sex and condition of the patient, concurrent administration to the drugs, contraindications and other parameters that will be taken into account by the specialist.The administration can be local (for example local delivery to the lung by pulmonary administration (for example intranasal administration) or local injection directly into a tumor) or systemically as indicated.The ligands of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use.This technique has been shown to be effective with conventional immunoglobulins and can be use lyophilization techniques and reconstitution known. Those skilled in the art will appreciate that lyophilization and reconstitution can lead to varying degrees of loss of antibody activity (for example with conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that levels of use may have to be adjusted upwards for compensation. The compositions comprising the ligands can be administered for prophylactic and / or therapeutic treatments. In certain therapeutic alterations, an amount adequate to achieve at least the inhibition, suppression, modulation, partial extermination or some other measurable parameter of a selected cell population is defined as a "therapeutically effective dose". The amounts needed to achieve this dosage will depend on the severity of the disease and the general health of the patient, although generally making 0.005 to 5 mg of ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg / kg / dose being the most commonly used. For prophylactic applications, the compositions containing the ligands of the present invention or cocktails thereof may also be administered in slightly lower doses or the like, to prevent, inhibit or erode the generation of the disease (e.g. to sustain remission or latency). , to prevent the acute phase). The specialist will have the ability to determine the proper dosage range to treat, suppress or prevent the disease. When a ligand is administered to treat, suppress or prevent a disease, it can be administered up to four times a day, twice a week, once a week, once every two weeks, once a month, or once every two months in a dose for example of about 10 μg / kg to about 80 mg / kg, about 100 μg / kg to about 80 mg / kg, about 1 mg / kg to about 80 mg / kg, approximately 1 mg / kg to approximately 70 mg / kg, approximately 1 mg / kg to approximately 60 mg / kg, approximately 1 mg / kg to approximately 50 mg / kg, approximately 1 mg / kg to approximately 40 mg / kg kg, about 1 mg / kg to about 30 mg / kg, about 1 mg / kg to about 20 mg / kg, about 1 mg / kg to about 10 mg / kg, about 10 μg / kg to about 10 mg / kg, about 10 μg / kg to about 5 mg / kg, about 10 μg / kg to about 2.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg or approximately 10 mg / kg. In particular embodiments, the specific double ligand is administered to treat, suppress or prevent a chronic inflammatory disease once every two weeks or once a month in a dose of about up to about 10 μg / kg to about 10 mg / kg (e.g. about 10 μg / kg, about 100 μg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about mg / kg, approximately 8 mg / kg, approximately 9 mg / kg or approximately 10 mg / kg). In particular embodiments, the ligand of the present invention is administered in a dose that provides EGFR saturation or a desired in vivo serum concentration. The specialist can determine the appropriate dosage to achieve saturation, for example by grinding the ligand and monitoring the amount of free binding sites in cells expressing EGFR or serum concentration of the ligand. Therapeutic regimens that involve administering a therapeutic agent to achieve the target saturation or a desired serum concentration of the agent are common in the art, particularly in the field of oncology. The treatment or therapy carried out using the compositions described herein is considered to be "effective" if one or more of the symptoms are reduced (for example by at least 10% or at least one point on a clinical evaluation scale), in relation to to the symptoms that are found before treatment, or in relation to the symptoms in an individual (human or animal model) not treated with said composition or other suitable control. The symptoms will obviously vary depending on the disease or disorder addressed, they can be measured by a person skilled in the art or specialist. Such symptoms can be measured, for example, by monitoring the level of one or more "biochemical indicators of the disease or disorder (eg, levels of an enzyme or metabolite correlated with the disease, numbers of affected cells, etc.) by monitoring physical manifestations ( for example, inflammation, tumor size, etc.) or by means of an accepted clinical evaluation scale, for example, the Expanded Disability Status (multiple sclerosis), the Irvine Inflammatory Bowel Disease Questionnaire (evaluation of 32 points that evaluates the quality of life with respect to to bowel function, systèmic symptoms, social function and emotional state - the score fluctuates, from 32 to 44, with the highest grades being an indication of a better quality of life), the Quality of Life Rheumatoid Arthritis Scale, or other scales of accepted clinical evaluation known in the art. A sustained reduction (for example one day or more, preferably more) of the symptoms of the disease or disorder by at least 10% or through one or more points a given clinical scale indicates the "effective" treatment. In a similar way, prophylaxis carried out using a composition as described herein is "effective" if the generation or severity of one or more symptoms is delayed, reduced or eliminated relative to the symptoms in a similar individual (human or animal model) not treated with the composition. A composition containing ligands according to the present invention can be used in prophylactic and therapeutic preparations to aid in the alteration, deactivation, extermination or elimination of a selected target population of cells in a mammal. In addition, selected ligands and repertoires of polypeptides described herein can be used in extracorporeal or in vitro form selectively to exterminate, drastically reduce or otherwise effectively eliminate a population of target cells from a heterogeneous cell collection. The blood of a mammal can be combined extracorporeally with the ligands, for example antibodies, cell surface receptors or binding proteins thereof whereby the unwanted cells are exterminated or otherwise eliminated from the blood to return to the blood. mammal according to standard techniques. In one embodiment, the present invention relates to a method of delivering anti-angiogenic therapy (anti-VEGF therapy) to a site containing cells that express or overexpress EGFR, wherein the method comprises administering an effective amount of a ligand that has binding specificity for VEGF and for EGFR to a subject in need thereof. The present invention also relates to the use of a ligand having binding specificity for VEGF and for EGFR to deliver therapy (anti-VEGF therapy) to a site containing cells that express or overexpress EGFR. The present invention also relates to the use of a ligand having binding specificity for VEGF and for EGFR for the manufacture of a medicament for delivering anti-angiogenic therapy (anti-VEGF therapy) to a site containing cells that express or overexpress EGFR , or to inhibit angiogenesis in a site that contains cells that express or overexpress EGFR. In particular embodiments, the present invention relates to a method of treating cancer, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a ligand, as described in the present invention, having specificity of link for VEGF and for EGFR. In particular embodiments, the patient has a cancer that expresses EGFR, such as bladder cancer, ovarian cancer, colorectal cancer, breast cancer, lung cancer (e.g., non-small cell lung carcinoma) gastric cancer, pancreatic cancer, prostate cancer, neck cancer, kidney cancer and gallbladder cancer. In other embodiments, the present invention relates to a method of treating cancer, wherein the method comprises administering to a subject in need thereof, a therapeutically effective amount of a ligand, as described herein (e.g., a ligand having binding specificity for VEGF, a ligand having binding specificity for EGFR, a ligand having binding specificity for VEGF and for EGFR) and an anti-neoplastic composition, wherein the anti-neoplastic composition comprises at least one selected chemotherapeutic agent from the group consisting of alkylating agents, antimetabolites, folic acid analogues, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodopiilotoxins, antibiotics, L-asparaginase, topoisomerase inhibitor, interferons, platinum coordination complexes, urea substituted with anthracenedione, methyl hydrazine derivatives, adrenocortic suppressors ales, adrenocorticosteroids, progestins, estrogens, antiestrogens, androgens, antiandrogens and gonadotropin-releasing hormone analogs. In some modalities, the chemotherapeutic agent is selected from the group consisting of cisplatin, cisplatin, dicarbazine, dactinomycin, mechlorethamine, streptozocin, cyclophosphamide, capecitabine, "carmustine, lomustine, doxorubicin, daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel, docetaxel, doxetaxe, aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alfa, irinotecan, leuprolide, leucovorin, megestrol, melphalan, mercaptopurine, oxaliplatin, plicamicin , mitotane, pegaspargase, pentostatin, pipobroman, plicamicin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, taxol, an additional growth factor receptor antagonist and a combination of any of the above . Assays for evaluating ligands The ligands of the present invention can be assayed using any suitable in vitro or in vivo assay. For example, using the receptor binding assays or bioassays described here *.
Bioensavo for VEGF activity: This bioassay measures the capacity of ligands (for example dAbs) to neutralize the proliferation of HUVE cells induced by VEGF (human vascular endothelial cells). Reductive HUVE cells in 96-well plates were incubated for 72 hours with pre-equilibrated VEGF and dAb protein. Subsequently the number of cells is measured using a cell viability ink. The test is carried out as indicated below. HUVE cells were trypsinized from a subconfluent vial of 172 cm2. The medium was aspirated, the cells were washed with 5 ml of trypsin and subsequently incubated with 2 ml of trypsin at room temperature for 5 minutes. The cells were gently removed from the base of the vial, eliminating against the hand. 8 ml of induction medium was subsequently added to the flask, pipetting the cells to disperse any lumps. Viable cells were counted using tripan blue staining. The cells were turned and washed 2X in an induction medium, rotating the cells downward and aspirating the medium after each wash. After the final aspiration, the cells were diluted to 10 5 cells / ml (the induction medium) and plated in 100 μl per deposit in a 96-well plate (10,000 cells / reservoir). The plate was incubated for > 2 h @ 37 ° C to allow adhesion of the cells. 60μl protein dAb 60μl and induction medium containing 40 ng / ml VEGF165 (for a final concentration of 10 ng / ml), was added to a 96-well plate with v-bottom and scaled with the film. The dAb / VEGF mixture was subsequently "incubated at a temperature of 37 ° C for 0.5-1 hour. The dAb / VEGF plate was removed from the incubator and 100 μl of the solution was added to each plate deposit that HUVEC (final volume of 200 μl). This plate was then returned to the incubator at a temperature of 37 ° C for a period of at least 72 hours. The control deposits include the following: deposits that contain cells but not VEGF; deposits containing cells, a positive control that neutralizes the anti-VEGF antibody and VEGF; and control deposits containing cells and VEGF only. Cell viability was evaluated by adding 20 μl per Celltier96 reagent reservoir and the plate was incubated at a temperature of 37 ° C for 2 to 4 hours until a brown color developed. The reaction was stopped by the addition of 20 μl per deposit of 10% (w / v) SDS. The absorbance was then read at 490 nm using a Wallac microplate vector. The absorbance of the control tanks without VEGF was subtracted from all values. The absorbance is proportional to the number of cells. Control reservoirs containing the anti-VEGF control antibodies should also exhibit minimal cell proliferation. Deposits containing VEGF alone must exhibit maximum cell proliferation. Examples Example 1. VEGF receptor binding assays VEGF is a specific mitogen for endothelial cells in vitro and a potent antigenic factor in vivo, with high levels of protein that is expressed in various types of tumors. It is a 45kDa glycoprotein that is active as a homodimer. Several isoforms have been described which arise through the division of alternative mRNA. Of these isoforms, VEGF-121 and VEGF-165 appear to be the most abundant. The specific action of VEGF on endothelial cells is mainly regulated by two types of receptor tyrosine (RTK) kinases, VEGF R1 (Flt-I) and VEGF R2 (KDR / Flk-1). However, it seems that VEGF activities such as mitogenicity, chemotaxis and induction and induction of morphological changes are transmitted by VEGF R2, even though both receptors undergo phosphorylation at the time of VEGF binding. VEGF receptor 2 binding assay This method describes a VEGF receptor binding assay for measuring the ability of ligands (e.g., dAbs) to prevent binding of VEGF-165 to the VEGF 2 receptor.
A recombinant human VEGF R2 / Fg chimera was used in this assay, comprising the extracellular domain of human VEGF R2 fused to the Fc region of human IgGi. In synthesis, the receptor was captured in an ELISA plate, subsequently the plate was blocked to avoid non-specific binding. Subsequently, a mixture of VEGF-165 and ligand was added, the plate was washed and the VEGF-165 bound by the receptor was detected using a biotinylated anti-VEGF antibody and an antibody (HRP) conjugated with anti-biotin horseradish peroxidase. The plate was developed using a colorimetric substrate and the OD was read at 450 nm. If the dAb blocked the VEGF link to the receiver, then no color was detected. The test was carried out as indicated below. A 96-well Nunc Maxisorp assay plate was coated overnight at a temperature of 4 ° C with 100 μl per tank of recombinant human R 2 / Fc VEGF (R & D Systems, Cat. No: 357-KD-050) a ß a concentration of 0.5 μg / ml in carbonate buffer. The deposits were washed 3 times with 0.05% Tween / PBS and 3 times with PBS. 200 μl per 2% deposit in PBS was added to block the plate and the plate was incubated for a minimum of 1 hour at room temperature. The deposits were washed (as indicated above), or subsequently 50 μl of ligand per deposit was added to each deposit. 50 μl VEGF at a concentration of 6ng / ml in diluent (for a final concentration of 3 ng / ml) was subsequently added to each tank and the plate was incubated for 2 hours at room temperature (for the supernatant assay; μl of the supernatant were added to each reservoir, then 20 μl of VEGF at a concentration of 15ng / ml). The following controls were included: Ong / ml VEGF (diluent only); 3 ng / ml VEGF (R &D Systems, Cat No: 293-VE-050); 3ng / ml VEGF with 0.1 μg / ml neutralization antibody VEGF (R & D Systems cat # MAB293). The plate was washed (as indicated above) and subsequently 10Oμ I of biotinylated anti-VEGF antibody (R & D Systems, Cat No: BAF293), 0.5μg / ml in diluent was added and incubated for 2 hours at room temperature. . The deposits were washed (as indicated above), or subsequently 100 μl of anti-biotin antibody conjugated with HRP (dilution 1: 5000 in diluent; Stratech, Cat No: 200-032-096) was added. The plate was subsequently incubated for 1 hour at room temperature. The plate was washed (as indicated above), ensuring that any traces of Tween-20 were removed to limit the background in the subsequent peroxidase assay and to aid in the prevention of bubbles in the test plate deposits that can provide OD readings not accurate. 10Oμl of a SureBlue 1-Component TMB MicroWell Peroxidase solution was added to each tank and the plate was left at room temperature for up to 20 minutes. The deep blue soluble product was developed as a conjugated labeling with bound HRP that reacted with the substrate. The reaction was stopped by the addition of 100 μl of 1M hydrochloric acid (the blue color turned yellow). The OD at 450 nm of the plate was read in a 96-well plate reader 30 minutes after the addition of acid. OD450nm is proportional to the amount of bound HRP-streptavidin conjugate. For some trials L. was added. The L protein lattice two dAb monomers. The expected results of the controls are as indicated below. Ong / ml VEGF should provide a low signal of < 0.15 OD; 3 ng / ml VEGF should provide a signal of > 0.5 OD; and 3 ng / ml VEGF previously incubated with 0.1 μg / ml neutralizing antibody should provide a signal of < 0.2 OD. VEGF receptor 1 binding assay This assay measures the binding of VEGF to VEGF R1 and the ability of the ligands to block this interaction. A recombinant human VEGF R1 / Fc chimera was used here, comprising the extracellular domain of VEGF R1 fused to the Fc region of IgG-i. The receptor was captured on an ELISA plate, subsequently the plate was blocked to avoid non-specific binding. Subsequently, a mixture of VEGF-165 and ligand was added, the plate was washed and the receptor-linked VEGF-165 was detected using a biotinylated anti-VEGF antibody and an anti-biotin antibody conjugated with HRP. The plate was developed using a colorimetric substrate and the OD was read at 450 nm. The test was carried out as indicated below.
A Nunc Maxisorp 96-tank assay plate was coated overnight at a temperature of 4 ° C with 10Oμ I per deposit of recombinant human VEGF R1 / Fc (R & D Systems, Cat No: 321-FL-050) @ 0.1 μg / ml in carbonate buffer. The deposits were washed 3 times with 0.05% Tween / PBS and 3 times with PBS. 200 μl per 2% BSA deposit in PBS was added to block the plate and the plate was incubated for a minimum of 1 hour at room temperature. The tanks were washed (as indicated above), then 50 μl of purified dAb protein per deposit was added to each tank. 50 μl of VEGF, a concentration of 1 ng / ml in diluent (for a final concentration of 500pg / ml), was subsequently added to each tank and the plate was incubated for 1 hour at room temperature (supernatant assay; 80μl of supernatant 20μl of VEGF @ 2.5ng / ml was added to each deposit). The following controls were included: Ong / ml VEGF (only diluents); 500pg / ml VEGF; and 500pg / ml VEGF with 1μg / ml of anti-VEGF antibody (R & D Systems cat # MAB293). The plate was washed (as indicated above) and subsequently 100 μl of biotinylated anti-VEGF antibody, 50 ng / ml in diluent was added and incubated for 1 hour at room temperature. The deposits were washed (as indicated above), then 10Oμl of anti-biotin antibody conjugated with HRP (dilution 1: 5000 in diluent) was added. The plate was subsequently incubated for 1 hour at room temperature. The plate was washed (as indicated above), ensuring that any traces of Tween-20 were removed to remove the background in the subsequent peroxidase assay and to aid in the prevention of bubbles in the test plate deposits that can provide OD readings not accurate. 10Oμ I of a solution was added to each tank SureBlue 1 -Component TMB MicroWell Peroxidase, and the plate was left at room temperature for up to 20 minutes. A deep blue soluble product developed as the conjugate labeled with linked HRP, reacted with the substrate. The reaction was stopped by the addition of 10Oμl of 1M hydrochloric acid. The OD, at 450 nm of the plate was read on a 96-well plate reader 30 minutes after the addition of acid. The OD450 is provided to the amount of bound streptavidin-HRP conjugate. Expected result of controls: 0 ng / ml VEGF should provide a low signal of < 0.15 OD; 500pg / ml VEGF should provide a signal of > 0.8 OD; and 500pg / ml VEGF previously incubated 1ug / ml neutralizing antibody should provide a signal of < 0.3 OD. Table 1 TAR15-1 had a Kd of 50-80 nM when tested in various concentrations on a low density BIAcore chip. Other VK dAbs were passed over the low density chip at a concentration (50 nM). Different dAbs showed different binding kinetics. * dAb assayed in 50nM VH was passed over the low density VEGF chip in a BIAcore at a concentration (50 nM). Different dAbs showed different binding kinetics. Example 2. EGFR linkage EGFR binding assay 25μl of ligand (eg dAb) was coated on a 96-well plate and then 25μl of Streptavidin-Alexa Fluor (1ug / ml) (Molecular Probes) and 25 μl of A431 cells were added. (ATCC No. CRL-1555) (8x105 / ml). All reagents were prepared in PBS / 1% BSA. The plate was incubated for 30 minutes at room temperature. Without disturbing the cells, 25μl of biotinylated EGF (Invitrogen) was added at 40ng / ml to each reservoir, and the plate was incubated for three hours at room temperature. Fluorescence was measured using the AB8200 Cellular Detection System (Applied Biosystems) detection system. Ligands (for example dAbs) that inhibited the binding of biotinylated EGF to EGFR expressed in A431 cells, resulted in lower fluorescence counts. Deposits without ligand provided a reference of maximum fluorescence (for example biotinylated EGF bond) and deposits without biotinylated ligand or EGF provided a baseline reference of fluorescence. These controls were included in all the trials. The results obtained in this trial using certain dAbs, are presented in Table 3. Kinase Assay In a 96-well plate, 5x10 4 A431 cells (ATCC No. CRL-1555) were plated by deposit in RPMI-1640 supplemented with 10% fetal calf serum. The plate was incubated overnight at a temperature of 37 ° C / 5% CO2 to allow adhesion of the cells, then the medium was replaced with RPMI-1640. The plate was incubated for 4 hours at a temperature of 37 ° C / 5% CO2. The ligand (prepared in RPMI-1640) was added to the tanks and the plate was incubated for 45 minutes at a temperature of 37 ° C / 5% CO2. EGF (Invitrogen) was added to the reservoirs to provide a final concentration of 100 ng / ml and the plate was incubated for 10 minutes at room temperature. The deposits were washed twice with PBS cooled with ice. Cold lysis buffer (1% NP-40, 20mM Tris, 137mM NaCl, 10% glycerol, 2mM EDTA, 1mM sodium orthovanadate, 10μg / ml aprotinin, 10μg / ml leupeptin) was added and the plate was incubated on ice during 10 minutes. The supernatants were transferred to an ELISA plate which had been coated overnight with anti-VEGF antibody (R & amp; amp; amp;; D Systems) in 1 μg / ml carbonate buffer. The ELISA plate was incubated for 2 hours at room temperature. The plate was washed three times with PBS / 0.05% Tween 20. Anti-phosphotyrosine antibody conjugated to horseradish peroxidase (Upstate Biotechnology) was added at 1ug / ml, and the plate was incubated for 1 hour at room temperature. The plate was washed three times with PBS / Tween and three times with PBS. The reaction was developed with a substrate micro-deposit peroxidase s-substrate SureBlue TMB 1 (KPL) and the reaction was stopped with 1 M HCl after 25 minutes. The absorbance was read using a Wallac plate reader. The results obtained in this test using certain dAbs, anti-VEGF are presented in Table 3.
Table 3 * The data presented are the lowest to the highest values obtained and the (average) Example 3. IgG type formats that have binding specificity for VEGF and EGFR The skeleton pBudCE4.1 (Invitrogen) was used to clone constant regions of ¡¡ immunoglobulin, such as in the heavy chain constant region lgG1 and the light chain kappa constant region (see figure 16 for review). An Ig Kappa chain leader was used to facilitate the secretion of the expressed protein. The constant Ig regions (IgG1 and human CK) were produced by GeneArt (Germany). The heavy chain constant region and the signal peptide were cloned into pBudCE4.1 as a Hind III / BglII fragment at the HindIII / BamHI restriction sites. The light chain constant region and the signal peptide were cloned into pBudCE4.1 as a NotI / MIul fragment. Cloning of dAb in IgG vectors and production of IgG type format VK dAb (specific for VEGF or EGFR) was cloned into the IgG vector as a Sall / BsiWI fragment. VH Ab (specific for VEGF or EGFR was cloned into the IgG vector as a BamHI / Xhol fragment.) The plasmid was subsequently transfected into cells HEK293T (ATCC CRL-11268) and IgG was expressed temporarily for five days. The produced IgG was purified using streamline protein A. The purified IgG was checked in a SDS gel of reduction and not reduction and bands of the expected size were observed. Several dAbs that bind to VEGF or EGFR were formatted in IgG type formats that have binding specificity for VEGF and EGFR. The IgG type formats were prepared by producing constructs that encoded an IgG heavy chain in which VH is a dAb and a light chain Kappa where VK is a dAb. The IgG type formats that were prepared are shown in Table 4, and the results obtained for some of the IgG type formats in assays are presented in Table 5 (Dummy VH and Dummy VK are germline sequences that do not bind to VEGF or EGFR).
Table 5 Although the present invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail may be made in the present invention without departing from the scope thereof comprised in the attached claims.

Claims (35)

  1. CLAIMS 1. A ligand having binding specificity for vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) comprising at least one variable domain of simple immunoglobulin with binding specificity for VEGF and at least one domain "Simple immunoglobulin variable with binding specificity for EGFR, wherein each variable domain of single immunoglobulin with binding specificity for VEGF competes to bind VEGF with an anti-VEGF domain antibody (dAb) selected from the group consisting of: TAR15 -6 (SEQ ID NO: 117), TAR15-8 (SEQ ID NO: 119), TAR15-26 (SEQ ID NO: 123), TAR15-6-500 (SEQ ID NO: 127), TAR15-6-501 (SEQ ID NO.128), TAR15-6-502 (SEQ ID NO.129), TAR15-6-503 (SEQ ID NO: 130), TAR15-6-504 (SEQ ID NO: 131), TAR15-6 -505 (SEQ ID NO: 132), TAR15-6-506 (SEQ ID NO: 133), TAR15-6-507 (SEQ ID NO: 134), TAR15-6-508 (SEQ ID NO: 135), TAR15 -6-509 (SEQ ID NO: 136), TAR15-6-510 (SEQ ID NO: 137), TAR15-8-500 (SE Q ID NO: 138), TAR15-8-501 (SEQ ID NO: 139), TAR15-8-502 (SEQ ID NO: 140), TAR15-8-503 (SEQ ID NO: 141), TAR15-8- 505 (SEQ ID NO: 142), TAR15-8-506 (SEQ ID NO: 143), TAR15-8-507 (SEQ ID NO: 144), TAR15-8-508 (SEQ ID NO: 145), TAR15- 8-509 (SEQ ID NO: 146), TAR15-8-510 (SEQ ID NO: 147), TAR15-8-511 (SEQ ID NO: 148), TAR15-26-500 (SEQ ID NO: 149), TAR15-26-501 (SEQ ID NO: 150), TAR15-26-502 (SEQ ID NO: 151), TAR15-26-503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153 ), TAR15-26-505 (SEQ ID NO: 154), TAR15-26-506 (SEQ ID NO: 155), TAR15-26-507 (SEQ ID NO: 156), TAR15-26-508 (SEQ ID NO. : 157), TAR15-26-509 (SEQ ID NO: 158), TAR15-26-510 (SEQ ID NO: 159), TAR15-26-511 (SEQ ID NO: 160), TAR15-26-512 (SEQ ID NO: 161), TAR15-26-513 (SEQ ID NO: 162), TAR15-26-514 (SEQ ID NO: 163), TAR15-26-515 (SEQ ID NO: 164), TAR15-26-516 (SEQ ID NO: 165), TAR15-26-517 (SEQ ID NO: 166), TAR15-26-518 (SEQ ID NO: 167), TAR15-26-519 (SEQ ID NO: 168), TAR15-26 -520 (SEQ ID NO: 169), TAR15-26-521 (SEQ ID NO: 170), T AR15-26-522 (SEQ ID NO: 171), TAR15-26-523 (SEQ ID NO: 172), TAR15-26-524 (SEQ ID NO: 173), TAR15-26-525 (SEQ ID NO: 174 ), TAR15-26-526 (SEQ ID NO: 175), TAR15-26-527 (SEQ ID NO: 176), TAR15-26-528 (SEQ ID NO: 177), TAR15-26-529 (SEQ ID NO. : 178), TAR15-26-530 (SEQ ID NO: 179), TAR15-26-531 (SEQ ID NO: 180), TAR15-26-532 (SEQ ID NO: 181), TAR15-26-533 (SEQ ID NO: 182), TAR15-26-534 (SEQ ID NO: 183), TAR15-26-535 (SEQ ID NO: 184), TAR15-26-536 (SEQ ID NO: 185), TAR15-26-537 (SEQ ID NO: 186), TAR15-26-538 (SEQ ID NO: 187), TAR15-26-53 (SEQ ID NO: 188), TAR15-26-540 (SEQ ID NO: 189), TAR15-26 -541 (SEQ ID NO: 190), TAR15-26-542 (SEQ ID NO: 191), TAR15-26-543 (SEQ ID NO: 192), TAR15-26-544 (SEQ ID NO: 193), TAR15 -26-545 (SEQ ID NO: 194), TAR15-26-546 (SEQ ID NO: 195), TAR15-26-547 (SEQ ID NO: 196), TAR15-26-548 (SEQ ID NO: 197) , TAR15-26-549 (SEQ ID NO: 198), TAR15-26-550 (SEQ ID NO: 539) and TAR15-26-551 (SEQ ID NO: 540).
  2. 2. The ligand as described in claim 1, characterized in that each variable domain of simple immunoglobulin with binding specificity for VEGF comprises a sequence of an amino acid having at least about 85% sequence identity with the sequence of a dAb selected from the group consisting of TAR15-6 (SEQ ID NO: 117), TAR15-8 (SEQ ID NO: 119), TAR15-26 (SEQ ID NO: 123), TAR15-6-500 (SEQ ID NO: 127) ), TAR15-6-501 (SEQ ID NO: 128), TAR15-6-502 (SEQ ID NO: 129), TAR15-6-503 (SEQ ID NO: 130), TAR15-6-504 (SEQ ID NO. : 131), TAR15-6-505 (SEQ ID NO: 132), TAR15-6-506 (SEQ ID NO: 133), TAR15-6-507 (SEQ ID NO: 134), TAR15-6-508 (SEQ ID NO: 135), TAR15-6-509 (SEQ ID NO: 136), TAR15-6-510 (SEQ ID NO: 137), TAR15-8-500 (SEQ ID NO: 138), TAR15-8-501 (SEQ ID NO: 139), TAR15-8-502 (SEQ ID NO: 140), TAR15-8-503 (SEQ ID NO: 141), TAR15-8-505 (SEQ ID NO: 142), TAR15-8 -506 (SEQ ID NO: 143), TAR15-8-507 (SEQ ID NO: 144), TAR15-8-508 (SEQ ID NO: 145), TAR15-8-509 (SEQ ID NO: 146), TAR15-8-510 (SEQ ID NO: 147), TAR15-8-511 (SEQ ID NO: 148), TAR15-26-500 (SEQ ID NO: 149), TAR15-26-501 (SEQ ID NO: 150), TAR15-26-502 (SEQ ID NO: 151), TAR15-26-503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153), TAR15-26-505 (SEQ ID NO: 154), TAR15-26-506 (SEQ ID NO: 155), TAR15-26-507 (SEQ ID NO: 156), TAR15-26-508 (SEQ ID NO: 157), TAR15-26-509 (SEQ ID NO: 158), TAR15-26-510 (SEQ ID NO: 159), TAR15-26-511 ( SEQ ID NO: 160), TAR15-26-512 (SEQ ID NO: 161), TAR15-26-513 (SEQ ID NO: 162), TAR15-26-514 (SEQ ID NO: 163), TAR15-26- 515 (SEQ ID NO: 164), TAR15-26-516 (SEQ ID NO: 165), TAR15-26-517 (SEQ ID NO: 166), TAR15-26-518 (SEQ ID NO: 167), TAR15- 26-519 (SEQ ID NO: 168), TAR15-26-520 (SEQ ID NO: 169), TAR15-26-521 (SEQ ID NO: 170), TAR15-26-522 (SEQ ID NO: 171), TAR15-26-523 (SEQ ID NO: 172), TAR15-26-524 (SEQ ID NO: 173), TAR15-26-525 (SEQ ID NO: 174), TAR15-26-526 (SEQ ID NO: 175), TAR15-26-527 (SEQ ID NO: 176), TAR15-26-528 (SEQ ID NO: 177), TAR15-26-529 (SEQ ID NO: 178), TAR15-26- 530 (SEQ ID NO: 179), TAR15-26-531 (SEQ ID NO: 180), T AR15-26-532 (SEQ ID NO: 181), TAR15-26-533 (SEQ ID NO: 182), TAR15-26-534 (SEQ ID NO: 183), TAR15-26-535 (SEQ ID NO: 184 ), TAR15-26-536 (SEQ ID NO: 185), TAR15-26-537 (SEQ ID NO: 186), TAR15-26-538 (SEQ ID NO: 187), TAR15-26-539 (SEQ ID NO. : 188), TAR15-26-540 (SEQ ID NO: 189), TAR15-26-541 (SEQ ID NO: 190), TAR15-26-542 (SEQ ID NO: 191), TAR15-26-543 (SEQ ID NO: 192), TAR15-26-544 (SEQ ID NO: 193), TAR15-26-545 (SEQ ID NO: 194), TAR15-26-546 (SEQ ID NO: 195), TAR15-26-547 (SEQ ID NO: 196), TAR15-26-548 (SEQ ID NO: 197), TAR15-26-549 (SEQ ID NO: 198), TAR15-26-550 (SEQ ID NO: 539) and TAR15-26 -551 (SEQ ID NO: 540).
  3. 3. The ligand as described in claim 1 or 2, characterized in that the ligand inhibits the binding of epidermal growth factor (EGFR) and / or growth factor alpha (TGFalpha) to EGFR.
  4. 4. The ligand as described in claim 1 or 2, characterized in that the ligand inhibits EGFR activity.
  5. 5. The ligand as described in claim 1 or 2, characterized in that the ligand inhibits EGFR activity without substantially inhibiting the binding of epidermal growth factor (EGF) and / or high growth transforming factor (TGFalpha) to EGFR.
  6. 6. The ligand as described in claim 1 or 2, characterized in that the ligand inhibits the binding of VEGF to vascular endothelial growth factor receptor (VEGFR1) and / or vascular endothelial growth factor receptor 2 (VEGFR2). .
  7. 7. The ligand as described in claim 1 or 2, characterized in that the ligand inhibits the activity of VEGF.
  8. The ligand as described in claim 1 or 2, characterized in that the ligand inhibits the activity of VEGF without substantially inhibiting the binding of VEGF to vascular endothelial growth factor receptor (VEGFR1) 1 and / or factor 2 receptor. of vascular endothelial growth (VEGFR2).
  9. 9. The ligand as described in claim 1 or 2, characterized in that the variable domain of simple immunoglobulin having binding specificity for VEGF competes to bind VEGF with bevacizumab.
  10. 10. The ligand as described in claim 1 or 2, characterized in that each variable domain of single immunoglobulin having binding specificity for EGFR competes to bind EGFR with cetuximab.
  11. 11. The ligand as described in any one of claims 1 to 1, characterized in that the variable domain of simple immunoglobulin with binding specificity for VEGF, binds to VEGF with an affinity (KD) that is between about 100 nM and about 1 pM, as determined by surface plasmon resonance.
  12. 12. The ligand as described in any one of claims 1 to 11, characterized in that each variable domain of single immunoglobulin with binding specificity for EGFR binds to EGFR with an affinity (KD) that is between about 100 nM and approximately 1 pM, as determined by surface plasmon resonance.
  13. 13. The ligand as described in claim 12, characterized in that each * variable domain of single immunoglobulin with binding specificity for EGFR binds to EGFR with an affinity (KD) that is between about 10 nM and about 100 pM, such as it is determined by surface plasmon resonance.
  14. 14. The ligand as described in any one of claims 1 to 10, characterized in that the ligand binds to VEGF with an affinity (KD) that is between about 100 nM and about 1 pM, as determined by resonance of plasmon of surface.
  15. 15. The ligand as described in any of claims 1 to 10 and 14, characterized in that the ligand binds to EGFR with an affinity (KD) that is between about 100 nM and about 1 pM, as determined by surface plasmon resonance.
  16. 16. The ligand as described in claim 15, characterized in that the ligand binds to EGFR with an affinity (KD) that is between about 10 nM and about 100 pM, as determined by surface plasmon resonance.
  17. 17. The ligand as described in any one of claims 1 to 16, characterized in that the ligand comprises a single immunoglobulin variable domain with binding specificity for VEGF which is a VHH and / or a single immunoglobulin variable domain. with binding specificity for EGFR which is a VHH-
  18. 18. The ligand as described in any of claims 1 to 16, characterized in that it comprises a single immunoglobulin variable domain with binding specificity for VEGF and a variable domain of simple immunoglobulin with binding specificity for EGFR that are independently selected from the group consisting of human VH and a V.
  19. 19. The ligand as described in any one of claims 1 to 18, characterized in that the ligand is an IgG-like format comprising two variable domains of single immunoglobulin with binding specificity for VEGF, and two immunoglobulin variable domains simple with binding specificity for VEGF.
  20. 20. The ligand as described in any one of claims 1 to 19, characterized in that the ligand comprises an antibody region Fc.
  21. 21. A ligand having binding specificity for vascular endothelial growth factor (VEGF), characterized in that it comprises at least one variable domain of single immunoglobulin with binding specificity for VEGF, wherein each variable domain of single immunoglobulin with binding specificity for VEGF competes to bind to VEGF with an anti-VEGF domain antibody (dAb) selected from the group consisting of TAR15-6 (SEQ ID NO: 117), TAR15-8 (SEQ ID NO: 119), TAR15-26 ( SEQ ID NO: 123), TAR15-6-500 (SEQ ID NO: 127), TAR15-6-501 (SEQ ID NO: 128), TAR15-6-502 (SEQ ID NO: 129), TAR15-6- 503 (SEQ ID NO: 130), TAR15-6-504 (SEQ ID NO: 131), TAR15-6-505 (SEQ ID NO: 132), TAR15-6-506 (SEQ ID NO: 133), TAR15- 6-507 (SEQ ID NO: 134), TAR15-6-508 (SEQ ID NO: 135), TAR15-6-509 (SEQ ID NO: 136), TAR15-6-510 (SEQ ID NO: 137), TAR15-8-500 (SEQ ID NO: 138), TAR15-8-501 (SEQ ID NO: 139), TAR15-8-502 (SEQ ID NO: 140), TAR15-8-503 (SEQ ID NO.141) ), TAR15-8-505 (SEQ ID NO: 142), TAR 15-8-506 (SEQ ID NO: 143), TAR15-8-507 (SEQ ID NO: 144), TAR15-8-508 (SEQ ID NO: 145), TAR15-8-509 (SEQ ID NO: 146) ), TAR15-8- 510 (SEQ ID NO: 147), TAR15-8-511 (SEQ ID NO: 148), TAR15-26-500 (SEQ ID NO: 149), TAR15-26-501 (SEQ ID NO. : 150), TAR15-26-502 (SEQ ID NO: 151), TAR15-26-503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153), TAR15-26-505 (SEQ ID NO: 154) ), TAR15-26-506 (SEQ ID NO: 155), TAR15-26-507 (SEQ ID NO: 156), TAR15-26-508 (SEQ ID NO: 157), TAR15-26-509 (SEQ ID NO. : 158), TAR15-26-510 (SEQ ID NO: 159), TAR15-26-511 (SEQ ID NO: 160), TAR15-26-512 (SEQ ID NO: 161), TAR15-26-513 (SEQ ID NO: 162), TAR15-26-514 (SEQ ID NO: 163), TAR15-26-515 (SEQ ID NO: 164), TAR15-26-516 (SEQ ID NO: 165), TAR15-26-517 (SEQ ID NO: 166), TAR15-26-518 (SEQ ID NO: 167), TAR15-26-519 (SEQ ID NO: 168), TAR15-26-520 (SEQ ID NO: 169), TAR15-26-521 (SEQ ID NO: 170), TAR15-26-522 (SEQ ID NO: 171), TAR15-26-523 (SEQ ID NO: 172), TAR15-26-524 (SEQ ID NO: 173), TAR15-26-525 ( SEQ ID NO: 174), TAR15-26-526 (SEQ ID NO: 175), TAR15-26-527 (SEQ ID NO: 176), TAR15-26-528 (SEQ ID NO: 177), TAR15-26- 529 (SEQ ID NO: 178), TAR15-26-530 (SEQ ID NO: 179), TAR15-26-531 (SEQ ID NO: 180), TAR15-26-532 (SEQ ID NO: 181), TAR15- 26-533 (SEQ ID-NO: 182), TAR15-26-534 (SEQ ID NO: 183), TAR15-26-535 (SEQ ID NO: 184), TAR15-26-536 (SEQ ID NO: 185) , TAR15-26-537 (SEQ ID NO: 186), TAR15-26-538 (SEQ ID NO: 187), TAR15-26-539 (SEQ ID NO: 188), TAR15-26-540 (SEQ ID NO: 189), TAR15-26-541 (SEQ ID NO: 190), TAR15-26-542 (SEQ ID NO: 191), TAR15-26-543 (SEQ ID NO: 192), TAR15-26-544 (SEQ ID NO: 193), TAR15-26-545 (SEQ ID NO: 194), TAR1 5-26-546 (SEQ ID NO: 195), TAR15-26-547 (SEQ ID NO: 196), TAR15-26-548 (SEQ ID NO: 197), TAR15-26-549 (SEQ ID NO: 198) ), TAR15-26-550 (SEQ ID NO: 539) and TAR15-26-551 (SEQ ID NO: 540).
  22. The ligand as described in claim 21, characterized in that each variable domain of single immunoglobulin with binding specificity for VEGF comprises an amino acid sequence having at least about 85% amino acid sequence identity with the amino acid sequence of a dAb selected from the group consisting of TAR15-6 (SEQ ID NO: 117), TAR15-8 (SEQ ID NO: 119), TAR15-26 (SEQ ID NO: 123), TAR15-6-500 (SEQ ID NO: 127), TAR15-6-501 (SEQ ID NO: 128), TAR15-6-502 (SEQ ID NO: 129), TAR15-6-503 (SEQ ID NO: 130), TAR15-6-504 ( SEQ ID NO: 131), TAR15-6-505 (SEQ ID NO: 132), TAR15-6-506 (SEQ ID NO: 133), TAR15-6-507 (SEQ ID NO: 134), TAR15-6- 508 (SEQ ID NO: 135), TAR15-6-509 (SEQ ID NO: 136), TAR15-6-510 (SEQ ID NO: 137), TAR15-8-500 (SEQ ID NO: 138), TAR15- 8-501 (SEQ ID NO: 139), TAR15-8-502 (SEQ ID NO: 140), TAR15-8-503 (SEQ ID NO: 141), TAR15-8-505 (SEQ ID NO: 142), TAR15-8-506 (SEQ ID NO: 143), TAR15-8-507 (SEQ ID NO: 144), TAR15-8-50 8 (SEQ ID NO: 145), TAR15-8-509 (SEQ ID NO: 146), TAR15-8-510 (SEQ ID NO: 147), TAR15-8-511 (SEQ ID NO: 148), TAR15- 26-500 (SEQ ID NO: 149), TAR15-26-501 (SEQ ID NO: 150), TAR15-26-502 (SEQ ID NO: 151), TAR15-26-503 (SEQ ID NO: 152), TAR15-26-504 (SEQ ID NO: 153), TAR15-26-505 (SEQ ID NO: 154), TAR15-26-506 (SEQ ID NO: 155), TAR15-26-507 (SEQ ID NO: 156) ), TAR15-26-508 (SEQ ID NO: 157), TAR15-26-509 (SEQ ID NO: 158), TAR15-26-510 (SEQ ID NO: 159), TAR15-26-511 (SEQ ID NO. : 160), TAR15-26-512 (SEQ ID NO: 161), TAR15-26-513 (SEQ ID NO: 162), TAR15-26-514 (SEQ ID NO: 163), TAR15-26-515 (SEQ ID NO: 164), TAR15-26-516 (SEQ ID NO: 165), TAR15-26-517 (SEQ ID NO: 166), TAR15-26-518 (SEQ ID NO: 167), TAR15-26-519 (SEQ ID NO: 168), TAR15-26-520 (SEQ ID NO: 169), TAR15-26-521 (SEQ ID NO: 170), TAR15-26-522 (SEQ ID NO: 171), TAR15-26 -523 (SEQ ID NO: 172), TAR15-26-524 (SEQ ID NO: 173), TAR15-26-525 (SEQ ID NO: 174), TAR15-26-526 (SEQ ID NO: 175), TAR15 -26-527 (SEQ ID NO: 176), TAR15-26-528 (SEQ ID NO: 177), TAR15-26-529 (SEQ ID NO: 178), TAR15-26-530 (SEQ ID NO: 179), TAR15-26-531 (SEQ ID NO: 180), TAR15-26-532 ( SEQ ID NO: 181), TAR15-26-533 (SEQ ID NO: 182), TAR15-26-534 (SEQ ID NO: 183), TAR15-26-535 (SEQ ID NO: 184), TAR15-26- 536 (SEQ ID NO: 185), TAR15-26-537 (SEQ ID NO: 186), TAR15-26-538 (SEQ ID NO: 187), TAR15-26-539 (SEQ ID NO: 188), TAR15- 26-540 (SEQ ID NO: 189), TAR15-26-541 (SEQ ID NO: 190), TAR15-26-542 (SEQ ID NO: 191), TAR15-26-543 (SEQ ID NO: 192), TAR15-26-544 (SEQ ID NO: 193), TAR15-26-545 (SEQ ID NO: 194), TAR15-26-546 (SEQ ID NO: 195), TAR15-26-547 (SEQ ID NO: 196) ), TAR15-26-548 (SEQ ID NO: 197), TAR15-26-549 (SEQ ID NO: 198), TAR15-26-550 (SEQ ID NO: 539) and TAR15-26-551 (SEQ ID NO. : 540).
  23. 23. The ligand as described in claim 21 or 22, characterized in that the ligand inhibits the binding of VEGF to vascular endothelial growth factor receptor (VEGFR1) and / or vascular endothelial growth factor receptor 2 (VEGF2). .
  24. 24. The ligand as described in claim 21 or 22, characterized in that the ligand inhibits the activity of VEGF.
  25. 25. The ligand as described in claim 21 or 22, characterized in that the ligand inhibits the activity of VEGF without substantially inhibiting the binding of VEGF to the vascular endothelial growth factor receptor 1 (VEGFR1) and / or the factor 2 receptor. Vascular endothelial growth (VEGFR2).
  26. 26. The ligand as described in claim 21 or 22, characterized in that each variable domain of simple immunoglobulin with binding specificity for VEGF competes to bind to VEGF with bevacizumab.
  27. 27. The ligand as described in claim 21 or 22, characterized in that each variable domain of simple immunoglobulin with binding specificity for VEGF, binds to VEGF with an affinity (KD) that is between about 100 nM and about 1 pM, as determined by surface plasmon resonance.
  28. 28. The ligand as described in any of claims 21 to 26, characterized in that the ligand binds to VEGF with an affinity (KD) that is between about 100 nM and about 1 pM, as determined by resonance of plasmon of surface.
  29. 29. The ligand as described in any of claims 21 to 28, characterized in that the ligand comprises a single immunoglobulin variable domain with binding specificity for VEGF which is a VHH-
  30. 30. Ligand as described with any of claims 21 to 28, characterized in that the ligand comprises a single immunoglobulin variable domain with binding specificity for VEGF that is selected from the group consisting of human VH and human V.
  31. 31. The ligand as described in any of claims 21 to 30, characterized in that the ligand is a dAb monomer.
  32. 32. The ligand as described in any of claims 1 to 31, for use in therapy or diagnosis.
  33. 33. The ligand use of any of claims 1 to 31 for the manufacture of a medicament for treating cancer.
  34. 34. A composition comprising a ligand as described in any one of claims 1 to 31 and a physiologically acceptable carrier.
  35. 35. A drug delivery apparatus comprising the composition as described in claim 34. R E S U M E N We describe ligands that have binding specificity for vascular endothelial growth factor (VEGF), for epidermal growth factor receptor (EGFR), or for VEGF and EGFR. Methods for using these ligands are also described. In particular, the use of these ligands for cancer therapy is described.
MX/A/2008/008854A 2006-01-11 2008-07-09 Ligands that have binding specificity for vegf and/or egfr and methods of use therefor MX2008008854A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US11331415 2006-01-11

Publications (1)

Publication Number Publication Date
MX2008008854A true MX2008008854A (en) 2008-09-26

Family

ID=

Similar Documents

Publication Publication Date Title
US7696320B2 (en) Ligands that have binding specificity for VEGF and/or EGFR and methods of use therefor
US20130041136A1 (en) Ligands that have binding specificity for egfr and/or vegf and methods of use therefor
US20100021473A1 (en) Bispecific Ligands With Binding Specificity to Cell Surface Targets and Methods of Use Therefor
US20110159003A1 (en) Ligands That Bind Il-4 and/or Il-13
US20080241166A1 (en) Ligands that bind a receptor
WO2022179039A1 (en) Anti-human cd73 antibody and use thereof
MX2008008854A (en) Ligands that have binding specificity for vegf and/or egfr and methods of use therefor
MX2008007351A (en) Ligands that have binding specificity for egfr and/or vegf and methods of use therefor
MX2008007369A (en) Bispecific ligands with binding specificity to cell surface targets and methods of use therefor
MX2008009528A (en) Ligands that bind il-4 and/or il-13
HK1103238A (en) Compositions and methods for treating inflammatory disorders
HK1156053A (en) Ligand
HK1138017A (en) Ligand