MX2008007351A - Ligands that have binding specificity for egfr and/or vegf and methods of use therefor - Google Patents
Ligands that have binding specificity for egfr and/or vegf and methods of use thereforInfo
- Publication number
- MX2008007351A MX2008007351A MXMX/A/2008/007351A MX2008007351A MX2008007351A MX 2008007351 A MX2008007351 A MX 2008007351A MX 2008007351 A MX2008007351 A MX 2008007351A MX 2008007351 A MX2008007351 A MX 2008007351A
- Authority
- MX
- Mexico
- Prior art keywords
- seq
- dom16
- ligand
- egfr
- tar15
- Prior art date
Links
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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
LIGANDS WHICH HAVE SPECIFIC ISSUES FOR EGF R AND / OR VEGF. AND METHODS OF USE OF THE M ISMS
RELATED APPLICATION This application claims the benefit of the Provisional Patent Application of the United States of America Number 60/742, 992, filed on December 6, 2005, and the benefit of the Provisional United States Patent Application. Number 60 / 758,355, filed January 1, 2006. All teachings of the above applications are incorporated herein by reference. ANTEC EDENTS OF THE INVENTION Cancer is a primary cause of mortality and pathology. Approaches to the treatment of cancer include surgery to remove tumors and chemotherapy. These approaches can cure some patients successfully. However, even patients who seem to have healed often suffer from a recurrence of the cancer, requiring additional therapy. Chemotherapeutic agents in general are non-selective agents that are toxic to cells, such as proliferating cells. In accordance with the foregoing, these agents can effectively kill the cancer cells, but they will also kill the healthy cells, producing several 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. An approach to solving the drawbacks of chemotherapeutic and surgical approaches to cancer therapy and diagnosis involves targeting cancer cells, for example, using antibodies or fragments of antibodies that bind to proteins that are expressed or they are over-expressed in cancer cells. A number of these target proteins have been identified. Among these proteins is the epidermal growth factor receptor (EGFR). The EG FR is a member of the ErbB 1 family, and transduces the signals that lead cell proliferation and survival, and the elaboration of the growth and angiogenic factors after the link with the epidermal growth factor (EGF) and / or with transforming growth factor alpha (TGF-alpha). In accordance with the above, it has been shown that EG FR is involved in tumor growth, in metastasis, and in angiogenesis. In addition, many cancers express EGF R, such as bladder cancer, ovarian cancer, colorectal cancer, breast cancer, lung cancer (e.g., non-microcellular lung carcinoma), gastric cancer, pancreatic cancer, prostate cancer , cancer of the head and neck, kidney cancer, and gallbladder cancer. ERB ITUX (cetuximab; I mclone Systems I nc.) Is a mouse / human chimeric antibody that binds to the human EG FR, which has been
approved for the treatment of certain cancers that express EGFR, in combination with irinotecan. An important pathophysiological process that facilitates tumor formation, metastasis, and recurrence is tumor angiogenesis. This process is mediated by the elaboration of angiogenic factors by the tumor, such as vascular endothelial growth factor (VEGF), which induces the formation of blood vessels that supply nutrients to the tumor. In accordance with the above, another approach for the treatment of certain cancers is to inhibit tumor angiogenesis mediated by VEG F, thereby causing starvation of the tumor. AVASTI N (bevacizumab, Genentech, "I nc.) Is a humanized antibody that binds to human VEGF, which has been approved for the treatment of colorectal cancer." It is reported that an antibody referred to as Antibody 2C3 (ATCC, Number Access PTA 1 595) binds to VEG F and inhibits the binding of vascular endothelial growth factor to the epidermal growth factor receptor 2. The direction of EGFR or VEGF with currently available therapies is not effective at all. patients, or for all cancers (for example, cancers that express EGFR) Therefore, there is a need for better agents for the treatment of cancer and other pathological conditions SHORT DESCRIPTION OF THE INVENTION The invention refers to ligands that have specificity
linkage for VEGF (e.g., human VEGF), to ligands that have binding specificity for EGFR (e.g., human EG FR), and to ligands that have link 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 a binding specificity for VEGF, a polypeptide domain having a binding site with a binding specificity for EG FR, or it may comprise a binding domain. polypeptide having a binding site with a binding specificity for VEG F and a polypeptide domain having a binding site with a binding specificity for EG FR. In one aspect, the invention relates to a ligand having binding specificity for VEG F and for EGFR. These ligands comprise at least one protein fraction having a binding site with a binding specificity for VEGF, and at least one protein fraction having a binding site with a binding specificity for EGFR. The protein fraction having a binding site with a binding specificity for VEG F and the protein fraction having a binding site with a binding specificity for EGFR, each may be any suitable binding moiety. The protein fractions can be a peptide moiety, a polypeptide moiety, or a protein moiety. For example, protein fractions can be provided by an antibody fragment having a binding site with a binding specificity for VEG F or EGF R, such as a single variable domain of
immunoglobulin having binding specificity for VEGF or EGFR. The ligand may comprise a protein fraction having a binding site with a binding specificity for VEGF, competing for binding with VEGF, with AVASTIN (bevacizumab, Genentech, Inc.) and / or with the 2C3 antibody (ATCC Number Access PTA 1595). The ligand may comprise a protein fraction having a binding site with a binding specificity for EGFR, competing for binding to EGFR, with ERBITUX (cetuximab; Imclone Systems, Inc.) and / or VECTIBIX (panitumumab; Amgen, Inc.). In some embodiments, the ligand comprises a protein fraction having a binding site with a binding specificity for VEGF, competing for binding to VEGF, with bevacizumab and / or 2C3 antibody (ATCC Accession Number PTA 1595), and further comprises a protein fraction having a binding site with a binding specificity for EGFR, competing for the EGFR binding, with cetuximab. In some embodiments, the ligand comprises a protein fraction that has a binding site with a binding specificity for VEGF (eg, a single immunoglobulin variable domain) competing for binding with VEGF, with an anti-domain antibody. VEGF (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 D 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 TAR 15-26-547 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 ligand comprises a protein fraction that has a binding site with a binding specificity for VEGF (eg, a single immunoglobulin variable domain) that competes for binding with VEGF, with TAR15-
26-555 (SEQ ID NO: 704). Additionally, or in other embodiments, the ligand may comprise a protein fraction having a binding site with a binding specificity for EGFR (eg, a single immunoglobulin variable domain) competing for binding to EGFR, with an antibody of the anti-EGFR domain (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),
DO 16-38 (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), OD 16-44 (SEQ ID NO: 350), DOM16-45 (SEQ ID NO: 351), OD 16-46 (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), OD 16-39-90 (SEQ ID NO: 421 DOM16-39-96 (SEQ ID NO: 422), DOM 16-39-100 (SEQ ID NO: 423 DOM 16-39-101 (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), NB19 (SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 (SEQ ID NO: 471), and NB22 (SEQ ID NO: 472 ). In particular modalities, the ligand has specificity
of binding for VEGF and for EGFR, and comprises a protein fraction having a binding site with a binding specificity for VEGF, competing for binding with VEGF, with an anti-VEGF domain antibody (dAb) selected from of 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 fraction having a site of binding with a binding specificity for EGFR, competing for the EGFR binding, 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 particular embodiments, the ligand has binding specificity for VEGF and for EGFR, and comprises a protein fraction that has a binding site with a binding specificity for VEGF, competing for binding with VEGF, with a domain antibody. 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 also comprises a protein fraction having a binding site with a binding specificity for EGFR, competing for the EGFR binding, with an anti-EGFR domain (dAb) antibody selected from the group consisting of DOM16-39- 521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ.
ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39- 619 (SEQ ID NO: 622). In more particular embodiments, the ligand has binding specificity for VEGF and for EGFR, and comprises at least one unique immunoglobulin variable domain with a binding specificity for VEGF and at least one unique immunoglobulin variable domain with a binding specificity for EGFR, wherein a single variable immunoglobulin domain with a binding specificity for VEGF competes for binding with VEGF, with an 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), 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), and 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 single immunoglobulin variable domain with a binding specificity for VEGF can comprise an amino acid sequence having an amino acid sequence identity of at least about 85 percent 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 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
((SSEEQQ IIDD NNOO :: 119911)), 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), and 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 a single immunoglobulin variable domain with a binding specificity for VEGF and at least one unique immunoglobulin variable domain with a binding specificity for EGFR. , wherein a single immunoglobulin variable domain with a binding specificity for EGFR competes for the EGFR binding, 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 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), NB19 (SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 (SEQ ID NO: 471), and NB22 (SEQ ID NO: 472). In other particular embodiments, the ligand has binding specificity for VEGF and for EGFR, and comprises at least a single immunoglobulin variable domain with a binding specificity for VEGF and at least one unique immunoglobulin variable domain with a binding specificity for EGFR. , wherein a single immunoglobulin variable domain with a binding specificity for EGFR competes for the EGFR binding, with an anti-EGFR domain (dAb) antibody selected from the group consisting of DOM16-39-210 (SEQ ID. NO: 541), DOM16-39-211 (SEQ ID NO: 542), DOM16-39-212 (SEQ ID NO: 543), DOM16-39-213 (SEQ.
ID NO: 544), DOM16-39-214 (SEQ ID NO: 545, DOM16-39-215 (SEQ
ID NO: 546), DOM16-39-216 (SEQ ID NO: 547, DOM16-39-217 (SEQ
ID NO: 548), DOM16-39-218 (SEQ ID NO: 549, DOM16-39-219 (SEQ
ID NO: 550), DOM16-39-220 (SEQ ID NO: 551, DOM16-39-221 (SEQ
ID NO: 552), DOM16-39-222 (SEQ ID NO: 553, DOM16-39-223 (SEQ
ID NO: 554), DOM16-39-224 (SEQ ID NO: 555, DOM16-39-225 (SEQ
ID NO: 556), DOM16-39-226 (SEQ ID NO: 557, DOM16-39-227 (SEQ
ID NO: 558), DOM16-39-228 (SEQ ID NO: 559, DOM16-39-229 (SEQ
ID NO: 560), DOM16-39-230 (SEQ ID NO: 561, DOM16-39-231 (SEQ.
ID NO: 562), DOM16-39-232 (SEQ ID NO: 563, DOM16-39-233 (SEQ
ID NO: 564), DOM16-39-234 (SEQ ID NO: 565, DOM16-39-235 (SEQ
ID NO: 566), DOM16-39-500 (SEQ ID NO: 72), DOM16-39-502 (SEQ
ID NO: 726), DOM16-39-503 (SEQ ID NO: 567) DOM16-39-504 (SEQ
ID NO: 568), DOM16-39-505 (SEQ ID NO: 569 DOM16-39-506 (SEQ
ID NO: 570), DOM16-39-507 (SEQ ID NO: 571 DOM16-39-508 (SEQ
ID NO: 572), DOM16-39-509 (SEQ ID NO: 573 DOM16-39-510 (SEQ
ID NO: 574), DOM16-39-511 (SEQ ID NO: 575 DOM16-39-512 (SEQ
ID NO.576), DOM16-39-521 (SEQ ID NO: 577 DOM16-39-522 (SEQ
ID NO: 578), DOM16-39-523 (SEQ ID NO: 579 DOM16-39-524 (SEQ
ID NO: 580), DOM16-39-527 (SEQ ID NO: 581 DOM16-39-525 (SEQ
ID NO: 582), DOM16-39-526 (SEQ ID NO: 583 DOM16-39-540 (SEQ
ID NO: 584), DOM16-39-541 (SEQ ID NO: 585 DOM16-39-542 (SEQ
ID NO: 586), DOM16-39-543 (SEQ ID NO: 587 DOM16-39-544 (SEQ
ID NO: 588), DOM16-39-545 (SEQ ID NO: 589 DOM16-39-550 (SEQ
ID NO: 590), DOM16-39-551 (SEQ ID NO: 591 DOM16-39-552 (SEQ
ID NO: 592), DOM16-39-553 (SEQ ID NO: 593 DOM16-39-554 (SEQ
ID NO: 594), DOM16-39-555 (SEQ ID NO: 595 DOM16-39-561 (SEQ
ID NO: 596), DOM16-39-562 (SEQ ID NO: 597 DOM16-39-563 (SEQ
ID NO: 598), DOM16-39-564 (SEQ ID NO: 599 DOM16-39-571 (SEQ
ID NO: 600), DOM16-39-572 (SEQ ID NO: 601 DOM16-39-573 (SEQ
ID NO: 602), DOM16-39-574 (SEQ ID NO: 603 DOM16-39-580 (SEQ
ID NO: 604), DOM16-39-591 (SEQ ID NO: 605 DOM16-39-592 (SEQ
ID NO: 606), DOM16-39-593 (SEQ ID NO: 607 DOM16-39-601 (SEQ
ID NO: 608), DOM16-39-602 (SEQ ID NO: 609 DOM16-39-603 (SEQ
ID NO: 610), DOM16-39-604 (SEQ ID NO: 611 DOM16-39-605 (SEQ
ID NO: 612), DOM16-39-607 (SEQ ID NO: 613 DOM16-39-611 (SEQ
ID NO: 614), DOM16-39-612 (SEQ ID NO: 615 DOM16-39-613 (SEQ
ID NO: 616), DOM16-39-614 (SEQ ID NO: 617 DOM16-39-615 (SEQ
ID NO: 618), DOM16-39-616 (SEQ ID NO: 619) DOM16-39-617 (SEQ ID NO: 620), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). For example, the single immunoglobulin variable domain with a binding specificity for EGFR may comprise an amino acid sequence having an amino acid sequence identity of at least about 85 percent 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), DO M16-75 (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), 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 single immunoglobulin variable domain with a binding specificity for EGFR may comprise an amino acid sequence having an amino acid sequence identity of at least about 85 percent with the amino acid sequence of a dAb selected from the group consisting of DOM16-39-210 (SEQ ID NO: 541), DOM16-39-211 (SEQ ID NO: 542), DOM16-39-212 (SEQ ID NO: 543), DOM16-39-213 (SEQ ID NO: 544), DOM16-39-214 (SEQ ID NO: 545), DOM16-39-215 (SEQ ID NO: 546), DOM16-39-216 (SEQ ID NO: 547), DOM16-39-217 (SEQ ID NO: 548), DOM16-39-218 (SEQ ID NO: 549), DOM16-39-219 (SEQ ID NO: 550), DOM16-39-220 (SEQ ID NO: 551), DOM16-39 -221 (SEQ ID NO: 552), DOM16-39-222 (SEQ ID NO: 553), DOM16-39-
223 (SEQ ID NO: 554 DOM16-39-224 (SEQ ID NO: 555 DOM16-39-225 (SEQ ID NO: 556 DOM16-39-226 (SEQ ID NO: 557 DOM16-39-227 (SEQ ID NO: 558 DOM16-39-228 (SEQ ID NO: 559 DOM16-39-229 (SEQ ID NO: 560 DOM16-39-230 (SEQ ID NO: 561 DOM16-39-231 (SEQ ID NO: 562 DOM16-39-232 (SEQ ID NO: 563 DOM16-39-233 (SEQ ID NO: 564 DOM16-39-234 (SEQ ID NO: 565 DOM16-39-235 (SEQ ID NO: 566 DOM16-39-500 (SEQ ID NO: 725 DOM16-39-502 (SEQ ID NO: 726) DOM16-39-503 (SEQ ID NO: 567 DOM16-39-504 (SEQ ID NO: 568 DOM16-39-505 (SEQ ID NO: 569 DOM16-39-506 (SEQ ID NO: 570 DOM16-39-507 (SEQ ID NO: 571 DOM16-39-508 (SEQ ID NO: 572 DOM16-39-509 (SEQ ID NO: 573 DOM16-39-510 (SEQ ID NO: 574 DOM16-39-511 (SEQ ID NO: 575 DOM16-39-512 (SEQ ID NO: 576 DOM16-39-521 (SEQ ID NO: 577 DOM16-39-522 (SEQ ID NO: 578 DOM16-39-523 ( SEQ ID NO: 579 DOM16-39-524 (SEQ ID NO: 580 DOM16-39-527 (SEQ ID NO: 581 DOM16-39-525 (SEQ ID NO: 582 DOM16-39-526 (SEQ ID NO: 583 DOM16 -39-540 (SEQ ID NO: 584 DOM16-39-541 (SEQ ID NO: 585 DOM16-39-542 (SEQ ID NO: 586 DOM16-39-543 (SEQ ID NO: 587 DOM16-39-544 (SEQ ID NO: 588 DOM16-39-545 (SEQ ID NO: 589 DOM16-39-550 (SEQ ID NO: 590 DOM16-39-551 (SEQ ID NO: 591 DOM16-39-552 ( SEQ ID NO: 592 DOM16-39-553 (SEQ ID NO: 593 DOM16-39-554 (SEQ ID NO: 594 DOM16-39-555 (SEQ ID NO: 595 DOM16-39-561 (SEQ ID NO: 596 DOM16 -39-562 (SEQ ID NO: 597 DOM16-39-563 (SEQ ID NO: 598 DOM16-39-564 (SEQ ID NO: 599 DOM16-39-571 (SEQ ID NO: 600 DOM16-39-572 (SEQ ID NO: 601 DOM16-39-
573 (SEQ ID NO: 602), DOM16-39-574 (SEQ ID NO: 603), DOM16-39-580 (SEQ ID NO: 604), DOM16-39-591 (SEQ ID NO: 605), DOM16- 39-592 (SEQ ID NO: 606), DOM16-39-593 (SEQ ID NO: 607), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-602 (SEQ ID NO: 609), DOM16-39-603 (SEQ ID NO: 610), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-605 (SEQ ID NO: 612), DOM16-39-607 (SEQ ID NO: 613) ), DOM16-39-611 (SEQ ID NO: 614), DOM16-39-612 (SEQ ID NO: 615), DOM16-39-613 (SEQ ID NO: 616), DOM16-39-614 (SEQ ID NO: 617), DOM16-39-615 (SEQ ID NO: 618), DOM16-39-616 (SEQ ID NO: 619), DOM16-39-617 (SEQ ID NO: 620), DOM16-39-618 ( SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). In some embodiments, the ligand has binding specificity for VEGF and for EGFR, and comprises at least one unique immunoglobulin variable domain with a binding specificity for VEGF and at least one unique immunoglobulin variable domain with a binding specificity for EGFR, wherein a single variable immunoglobulin domain with a binding specificity for VEGF competes for binding with VEGF, with an 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), TAR15-30 (SEQ ID NO: 126 ), TAR15-6-500 (SEQ ID NO: 127), TAR15-6-50 1 (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- 50O (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), and 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 variable immunoglobulin domain with a binding specificity for EGFR competes for the EGFR binding, with an anti-EGFR domain (dAb) antibody 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 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), 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 the additional embodiments, the ligand has binding specificity for VEGF and for EGFR, and comprises at least a single immunoglobulin variable domain with a binding specificity for VEGF and at least a single immunoglobulin variable domain with a binding specificity for EGFR , wherein a single variable immunoglobulin domain with a binding specificity for VEGF competes for binding with VEGF, with an 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), 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 TAR15-226-522 (SEQ ID NO: 171 TAR15-26-523 (SEQ ID NO: 172 TAR15-226-524 (SEQ ID NO: 173 TAR15 -26-525 (SEQ ID NO: 174 TAR15-226-526 (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 TAR15-226-532 (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 TAR15-265-540 (SEQ ID NO: 189 TAR15-26-541 (SEQ E) NO: 190 TAR15-26-542 (SEQ E) NO: 191 TAR15 -26-543 (SEQ ID NO: 192 TAR15-226-544 (SEQ ID NO: 193 TAR15-26-545 (SEQ ID NO: 194 TAR15-26-546 (S EQ 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 variable immunoglobulin domain with a binding specificity for EGFR competes for binding to EGFR, with an anti-EGFR domain (dAb) antibody selected from the group consisting of DOM16-39-210 (SEQ ID. NO: 541), DOM16-39-211 (SEQ ID NO: 542), DOM16-39-
212 (SEQ ID NO: 543), DOM16-39-213 (SEQ ID NO: 544 DOM16-39-214 (SEQ ID NO: 545), DOM16-39-215 (SEQ ID NO: 546 DOM16-39- 216 (SEQ ID NO: 547), DOM16-39-217 (SEQ ID NO: 548 DOM16-39-218 (SEQ ID NO: 549), DOM16-39-219 (SEQ ID NO: 550 DOM16-39- 220 (SEQ ID NO: 551), DOM16-39-221 (SEQ ED NO: 552 DOM16-39-222 (SEQ ID NO: 553), DOM16-39-223 (SEQ ID NO: 554 DOM16-39- 224 (SEQ ID NO: 555) ,, DOM16-39-225 (SEQ ID NO: 556 DOM16-39-226 (SEQ ID NO: 557), DOM16-39-227 (SEQ ID NO: 558 DOM16-39- 228 (SEQ ID NO: 559) ,, DOM16-39-229 (SEQ ID NO: 560 DOM16-39-230 (SEQ ID NO: 561), DOM16-39-231 (SEQ ID NO: 562 DOM16-39- 232 (SEQ ID NO: 563) ,, DOM16-39-233 (SEQ ID NO: 564 DOM16-39-234 (SEQ ID NO: 565), DOM16-39-235 (SEQ ID NO: 566 DOM16-39-500 (SEQ ID NO: 725), DOM16-39-502 (SEQ ID NO: 726 DOM16-39-503 (SEQ ID NO: 567), DOM16-39-504 (SEQ ID NO: 568 DOM16-39-505 ( SEQ ID NO: 569), DOM16-39-506 (SEQ ID NO: 570 DOM16-39-507 (SEQ ID NO: 571), DOM16-39-508 (SEQ ID NO: 572 DOM16-39-509 ( SEQ ID NO: 573) ,, DOM16-39-510 (SEQ ID NO: 574 DOM16-39-511 (SEQ ID NO: 575) ,, DOM16-39-512 (SEQ ID NO: 576 DOM16-39-521 (SEQ ID NO: 577), DOM16-39-522 (SEQ ID NO: 578 DOM16-39-523 (SEQ ID NO: 579), DOM16-39-524 (SEQ ID NO: 580 DOM16-39-527 (SEQ ID NO: 581), DOM16-39-525 (SEQ ID NO: 582 DOM16-39-526 (SEQ ID NO: 583) ,, DOM16-39-540 (SEQ ID NO: 584 DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586 DOM16-39-543 (SEQ ID NO: 587), DOM16-39-544 (SEQ ID NO: 588 DOM16-39-545 (SEQ ID NO: 589), DOM16-39-550 (SEQ ID NO: 590 DOM16-39-
551 (SEQ ID NO: 591 DOM16-39-552 (SEQ ID NO: 592 DOM16-39-553 (SEQ ID NO: 593 DOM16-39-554 (SEQ ID NO: 594 DOM16-39-555 (SEQ ID NO: 595 DOM16-39-561 (SEQ ID NO: 596 DOM16-39-562 (SEQ ID NO: 597 DOM16-39-563 (SEQ ID NO: 598 DOM16-39-564 (SEQ ID NO: 599 DOM16-39-571 (SEQ ID NO: 600 DOM16-39-572 (SEQ ID NO: 601 DOM16-39-573 (SEQ ID NO: 602 DOM16-39-574 (SEQ ID NO: 603 DOM16-39-580 (SEQ ID NO: 604 DOM16-39-591 (SEQ ID NO: 605 DOM16-39-592 (SEQ ID NO: 606 DOM16-39-593 (SEQ ID NO: 607 DOM16-39-601 (SEQ ID NO: 608 DOM16-39-602 ( SEQ ID NO: 609 DOM16-39-603 (SEQ ID NO: 610 DOM16-39-604 (SEQ ID NO: 611 DOM16-39-605 (SEQ ID NO: 612 DOM16-39-607 (SEQ ID NO: 613 DOM16 -39-611 (SEQ ID NO: 614 DOM16-39-612 (SEQ ID NO: 615 DOM16-39-613 (SEQ ID NO: 616 DOM16-39-614 (SEQ ID NO: 617 DOM16-39-615 (SEQ ID NO: 618 DOM16-39-616 (SEQ ID NO: 619 DOM16-39-617 (SEQ ID NO: 620 DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622) For example, the ligand may comprise a single variable domain of immunoglobulin with a specific d of binding for VEGF comprising an amino acid sequence having an amino acid sequence identity of at least about 85 percent 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 I [SEQ ID NO: 182 TAR15-26-534 (SEQ ID NO: 183 TAR15-26-535 l [SEQ ID NO: 184 TAR15-26-536 (SEQ ID NO: 185 TAR15-26-537 I [SEQ ID NO : 186 TAR15-26-538 (SEQ ID NO: 187 TAR15-26-539 l [SEQ ID NO: 188 TAR15-26-540 (SEQ ID NO: 189 TAR15-26-541 i (SEQ ID NO: 190 TAR15- 26-542 (SEQ ID NO: 191 TAR15-26-543 i (SEQ ID NO: 192 TAR15-26-544 (SEQ ID NO: 193 TAR15-26-545 i (SEQ ID NO: 194 TAR15-2 6-546 (SEQ ID NO: 195 TAR15-26-547 l (SEQ ID NO: 196 TAR15-26-548 (SEQ ID NO: 197), and TAR15-26-549 (SEQ ID NO: 198 TAR15-26- 550 (SEQ ID NO: 539), and TAR15-26-551 (SEQ ID NO: 540) and may further comprise a single immunoglobulin variable domain with a binding specificity for EGFR comprising an amino acid sequence having a sequence identity of amino acids of at least about 85 percent with the amino acid sequence of a selected dAb
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), 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 ligand may comprise a single immunoglobulin variable domain with a binding specificity for VEGF comprising an amino acid sequence having an amino acid sequence identity of at least about 85 percent 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 ((SSEEQQ IIDD NNOO :: 113344)), 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 ((SSEEQQ IIDD NNOO :: 114444)), 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), TAR15-26-5O0 (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 may further comprise a single immunoglobulin variable domain with a binding specificity for EGFR comprising an amino acid sequence having a
amino acid sequence identity of at least about 85 percent with the amino acid sequence of a dAb selected from the group consisting of DOM16-39-210 (SEQ ID NO: 541) DOM16-39-211 (SEQ ID NO. : 542 DOM16-39-212 (SEQ ID NO: 543) DOM16-39-213 (SEQ ID NO: 544 DOM16-39-214 (SEQ ID NO: 545) DOM16-39-215 (SEQ ID NO: 546 DOM16- 39-216 (SEQ ID NO: 547) DOM16-39-217 (SEQ ID NO: 548 DOM16-39-218 (SEQ ID NO: 549) DOM16-39-219 (SEQ ID NO: 550 DOM16-39-220 ( SEQ ID NO: 551) DOM16-39-221 (SEQ ID NO: 552 DOM16-39-222 (SEQ ID NO: 553) DOM16-39-223 (SEQ ID NO: 554 DOM16- 39-224 (SEQ ID NO: 555) DOM16-39-225 (SEQ ID NO: 556 DOM16-39-226 (SEQ ID NO: 557) DOM16-39-227 (SEQ ID NO: 558 DOM16-39-228 (SEQ ID NO: 559) DOM16- 39-229 (SEQ ID NO: 560 DOM16-39-230 (SEQ ID NO: 561) DOM16-39-231 (SEQ ID NO: 562 DOM16-39-232 (SEQ ID NO: 563) DOM16-39-233 ( SEQ ID NO: 564 DOM16-39-234 (SEQ ID NO: 565) DOM16-39-235 (SEQ ID NO: 566 DOM16-39-500 (SEQ ID NO: 725), DOM16-39-502 (SEQ ID NO. : 726 DOM16-39-503 (SEQ ID NO: 567), DOM16-39-504 (SEQ ID NO: 568 DOM16-39-505 (SEQ ID NO: 569) DOM16-39-506 (SEQ ID NO: 570 DOM16-39-507 (SEQ ID NO: 571) DOM16-39-508 (SEQ ID NO: 572 DOM16-39-509 (SEQ ID NO: 573) DOM16-39-510 (SEQ ID NO: 574 DOM16-39-511 (SEQ ID NO: 575) DOM16- 39-512 (SEQ ID NO: 576 DOM16-39-521 (SEQ ID NO: 577) DOM16-39-522 (SEQ ID NO: 578 DOM16-39-523 (SEQ ID NO: 579) DOM16-39-524 ( SEQ ID NO: 580 DOM16-39-527 (SEQ ID NO: 581) DOM16-39-525 (SEQ ID NO: 582 DOM16-
39-526 (SEQ ID NO: 583), DOM16-39-540 (SEQ ID NO: 584 DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586 DOM16-39- 543 (SEQ ID NO: 587), DOM16-39-544 (SEQ ID NO: 588 DOM16-39-545 (SEQ ID NO: 589), DOM16-39-550 (SEQ ID NO: 590 DOM16-39-551 ( SEQ ID NO: 591), DOM16-39-552 (SEQ ID NO: 592 DOM16-39-553 (SEQ ID NO: 593), DOM16-39-554 (SEQ ID NO: 594 DOM16-39-555 (SEQ ID NO: 595), DOM16-39-561 (SEQ ID NO: 596 DOM16-39-562 (SEQ ID NO: 597), DOM16-39-563 (SEQ ID NO: 598 DOM16-39-564 (SEQ ID NO: 599), DOM16-39-571 (SEQ ID NO: 600 DOM16-39-572 (SEQ ID NO: 601), DOM16-39-573 (SEQ ID NO: 602 DOM16-39-574 (SEQ ID NO: 603) , DOM16-39-580 (SEQ ID NO: 604 DOM16-39-591 (SEQ ID NO: 605), DOM16-39-592 (SEQ ID NO: 606 DOM16-39-593 (SEQ ID NO: 607), DOM16 -39-601 (SEQ ID NO: 608 DOM16-39-602 (SEQ ID NO: 609), DOM16-39-603 (SEQ ID NO: 610 DOM16-39-604 (SEQ ID NO: 611), DOM16-39 -605 (SEQ ID NO: 612 DOM16-39-607 (SEQ ID NO: 613), DOM16-39-611 (SEQ ID NO: 614 DOM16-39-612 (SEQ ID NO: 615), DOM16-39-613 (SEQ ID NO: 616 DOM1 6-39-614 (SEQ ID NO: 617), DOM16-39-615 (SEQ ID NO: 618 DOM16-39-616 (SEQ ID NO: 619), DOM16-39-617 (SEQ ID NO: 620 DOM16- 39-618 (SEQ ID NO: 621)), and DOM16-39-619 (SEQ ID NO: 622). In some embodiments, the ligand has binding specificity for VEGF and for EGFR, and comprises at least one unique immunoglobulin variable domain with a binding specificity for VEGF and at least one unique immunoglobulin variable domain with a binding specificity for EGFR, in
where a single variable immunoglobulin domain with a binding specificity for VEGF competes for binding with VEGF, with an 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 E) 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 E) 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 E 'NO: 148), TAR15 • 26-500
(SEQ E) 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 E) 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), and 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 a binding specificity for EGFR competes for the EGFR binding with cetuximab. For example, the single variable domain of immunoglobulin with a binding specificity for VEGF may comprise an amino acid sequence having an amino acid sequence identity of at least about 85 percent with the amino acid sequence of a dAb selected from of 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: 195), TAR15-26-547 (SEQ ID NO: 196) , TAR15-26-548 (SEQ ID NO: 197), and 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 unique immunoglobulin variable domain with a binding specificity for VEGF and at least one unique immunoglobulin variable domain with a binding specificity for EGFR, wherein a single variable immunoglobulin domain with a binding specificity for VEGF competes for binding with VEGF, with bevacizumab and / or 2C3 antibody (ATCC Accession Number PTA 1595); and a single variable immunoglobulin domain with a binding specificity for EGFR competes for the EGFR binding, with an anti-EGFR domain (dAb) antibody 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 DOM 16-39-116 (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), 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 has binding specificity for VEGF and for EGFR, and comprises at least one unique immunoglobulin variable domain with a binding specificity for VEGF and at least one unique immunoglobulin variable domain with a binding specificity for EGFR, wherein a single variable immunoglobulin domain with a binding specificity for VEGF competes for binding with VEGF, with bevacizumab and / or 2C3 antibody (ATCC Accession Number PTA 1595); and a single variable immunoglobulin domain with a binding specificity for EGFR competes for binding to EGFR, with an anti-EGFR domain (dAb) antibody selected from the group consisting of DOM16-39-210 (SEQ ID NO: 541), DOM16-39-211 (SEQ ID NO: 542), DOM16-39-212 (SEQ ID NO: 543), DOM16-39-213 (SEQ ID NO: 544), DOM16-39-214 (SEQ.
ID NO: 545), DOM16-39-215 (SEQ ID NO: 546), DOM16-39-216 (SEQ
ID NO: 547), DOM16-39-217 (SEQ ID NO: 548), DOM16-39-218 (SEQ
ID NO: 549), DOM16-39-219 (SEQ ID NO: 550), DOM16-39-220 (SEQ
ID NO: 551), DOM16-39-221 (SEQ ID NO: 552), DOM16-39-222 (SEQ
ID NO: 553), DOM16-39-223 (SEQ ID NO: 554), DOM16-39-224 (SEQ
ID NO: 555), DOM16-39-225 (SEQ ID NO: 556), DOM16-39-226 (SEQ
ID NO: 557), DOM16-39-227 (SEQ ID NO: 558), DOM16-39-228 (SEQ
ID NO: 559), DOM16-39-229 (SEQ ID NO: 560), DOM16-39-230 < I KNOW THAT
ID NO: 561), DOM16-39-231 (SEQ ID NO: 562), DOM16-39-232 [SEQ.
ID NO: 563), DOM16-39-233 (SEQ ID NO: 564), DOM16-39-234 [SEQ.
ID NO: 565), DOM16-39-235 (SEQ ID NO: 566), DOM16-39-500 (SEQ.
ID NO: 725), DOM16-39-502 (i SEQ ID NO: 726), DOM16-39-503 (SEQ
ID NO: 567), DOM16-39-504 (SEQ ID NO: 568), DOM16-39-505 (SEQ.
ID NO: 569), DOM16-39-506 (SEQ ID NO: 570), DOM16-39-507 (SEQ
ID NO: 571), DOM16-39-508 (SEQ ID NO: 572), DOM16-39-509 (SEQ.
ID NO: 573), DOM16-39-510 (SEQ ID NO: 574), DOM16-39-511 (SEQ
ID NO: 575), DOM16-39-512 (SEQ ID NO: 576), DOM16-39-521 (SEQ
ID NO: 577), DOM16-39-522 (SEQ ID NO: 578), DOM16-39-523 (SEQ.
ID NO: 579), DOM16-39-524 (SEQ ID NO: 580), DOM16-39-527 (SEQ
ID NO: 581), DOM16-39-525 (SEQ ID NO: 582), DOM16-39-526 (SEQ
ID NO: 583), DOM16-39-540 (SEQ ID NO: 584), DOM16-39-541 (SEQ.
ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-543 (SEQ
ID NO: 587), DOM16-39-544 (SEQ ID NO: 588), DOM16-39-545 (SEQ
ID NO: 589), DOM16-39-550 (SEQ ID NO: 590), DOM16-39-551 (SEQ
ID NO: 591), DOM16-39-552 (SEQ ID NO: 592), DOM16-39-553 (SEQ
ID NO: 593 DOM16-39-554 (SEQ ID NO: 594 DOM16-39-555 (SEQ ID NO: 595 DOM16-39-561 (SEQ ID NO: 596 DOM16-39-562 (SEQ ID NO: 597 DOM16- 39-563 (SEQ ID NO: 598 DOM16-39-564 (SEQ ID NO: 599 DOM16-39-571 (SEQ ID NO: 600 DOM16-39-572 (SEQ ID NO: 601 DOM16-39-573 (SEQ ID NO: 602 DOM16-39-574 (SEQ ID NO: 603 DOM16-39-580 (SEQ ID NO: 604 DOM16-39-591 (SEQ ID NO: 605 DOM16-39-592 (SEQ ID NO: 606 DOM16-39 -593 (SEQ ID NO: 607 DOM16-39-601 (SEQ ID NO: 608 DOM16-39-602 (SEQ ID NO: 609 DOM16-39-603 (SEQ ID NO: 610 DOM16-39-604 (SEQ ID NO : 611 DOM16-39-605 (SEQ ID NO: 612 DOM16-39-607 (SEQ ID NO: 613 DOM16-39-611 (SEQ ID NO: 614 DOM16-39-612 (SEQ ID NO: 615 DOM16-39- 613 (SEQ ID NO: 616 DOM16-39-614 (SEQ ID NO: 617 DOM16-39-615 (SEQ ID NO: 618 DOM16-39-616 (SEQ ID NO: 619 DOM16-39-617 (SEQ ID NO: 620) DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622) For example, the single immunoglobulin variable domain with a binding specificity for EGFR may comprise a sequence of amino acids that has an identity of amino acid sequence of at least about 85 percent 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 ( S EQ 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), NB19 (SEQ ID NO: 469), NB20 (SEQ ID NO: 470), NB21 (SEQ ID NO: 471), and NB22 (SEQ ID NO: 472). For example, the single immunoglobulin variable domain with a binding specificity for EGFR may comprise an amino acid sequence having an amino acid sequence identity of at least about 85 percent with the amino acid sequence of a dAb selected from the group consisting of DOM16-39-210 (SEQ ID NO: 541), DOM16-39-211 (SEQ ID NO: 542), DOM16-39-212 (SEQ ID NO: 543), DOM16-39-213 (SEQ. ID NO: 544) DOM16-39-214 (SEQ ID NO: 545) DOM16-39-215 (SEQ ID NO: 546) DOM16-39-216 (SEQ ID NO: 547) DOM16-39-217 (SEQ ID NO. : 548) DOM16-39-218 (SEQ ID NO: 549) DOM16-39-219 (SEQ ID NO: 550) DOM16-39-220 (SEQ ID NO: 551) DOM16-39-221 (SEQ ID NO: 552 ) DOM16-39-222 (SEQ ID NO.553) DOM16-39-
223 (SEQ ID NO: 554 DOM16-39-224 (SEQ ID NO: 555), DOM16-39 225 (SEQ ID NO: 556 DOM16-39-226 (SEQ ID NO: 557), DOM16-39 227 (SEQ ID NO: 558 DOM16-39-228 (SEQ ID NO: 559), DOM16-39 229 (SEQ ID NO: 560 DOM16-39-230 (SEQ ID NO: 561), DOM16-39 231 (SEQ ID NO: 562 DOM16 -39-232 (SEQ ID NO: 563), DOM16-39
233 (SEQ ID NO: 564 DOM16-39-234 (SEQ ID NO: 565), DOM16-39 235 (SEQ ID NO: 566 DOM16-39-500 (SEQ ID NO: 725), DOM16-39 502 (SEQ ID NO: 726) DOM16-39-503 (SEQ ID NO: 567), DOM16-39 504 (SEQ ID NO: 568 DOM16-39-505 (SEQ ID NO: 569), DOM16-39 506 (SEQ ID NO: 570) DOM16-39-507 (SEQ ID NO: 571), DOM16-39
508 (SEQ ID NO: 572 DOM16-39-509 (SEQ ID NO: 573), DOM16-39 510 (SEQ ID NO: 574 DOM16-39-511 (SEQ ID NO: 575), DOM16-39 512 (SEQ ID NO: 576 DOM16-39-521 (SEQ ID NO: 577), DOM16-39 522 (SEQ ID NO: 578 DOM16-39-523 (SEQ ID NO: 579), DOM16-39 524 (SEQ ID NO: 580 DOM16 -39-527 (SEQ ID NO: 581), DOM16-39
525 (SEQ ID NO: 582 DOM16-39-526 (SEQ ID NO: 583), DOM16-39 540 (SEQ ID NO: 584 DOM16-39-541 (SEQ ID NO: 585), DOM16-39 542 (SEQ ID NO: 586 DOM16-39-543 (SEQ ID NO: 587), DOM16-39 544 (SEQ ID NO: 588 DOM16-39-545 (SEQ ID NO: 589), DOM16-39 550 (SEQ ID NO: 590 DOM16 -39-551 (SEQ ID NO: 591), DOM16-39 552 (SEQ ID NO: 592 DOM16-39-553 (SEQ ID NO: 593), DOM16-39 554 (SEQ ID NO: 594 DOM16-39-555 (SEQ ID NO: 595), DOM16-39 561 (SEQ ID NO: 596 DOM16-39-562 (SEQ ID NO: 597), DOM16-39 563 (SEQ ID NO: 598 DOM16-39-564 (SEQ ID NO. : 599), DOM16-39 571 (SEQ ID NO: 600 DOM16-39-572 (SEQ ID NO: 601), DOM16-39
573 (SEQ ID NO: 602), DOM16-39-574 (SEQ ID NO: 603) DOM16-39-580 (SEQ ID NO: 604), DOM16-39-591 (SEQ ID NO: 605) DOM16-39- 592 (SEQ ID NO: 606), DOM16-39-593 (SEQ ID NO: 607), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-602 (SEQ ID NO: 609), DOM16- 39-603 (SEQ ID NO: 610), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-605 (SEQ ID NO: 612), DOM16-39-607 (SEQ ID NO: 613), DOM16-39-611 (SEQ ID NO: 614), DOM16-39-612 (SEQ ID NO: 615), DOM16-39-613 (SEQ ID NO: 616), DOM16-39-614 (SEQ ID NO: 617) ), DOM16-39-615 (SEQ ID NO: 618), DOM16-39-616 (SEQ ID NO: 619), DOM16-39-617 (SEQ ID NO: 620), DOM16-39-618 (SEQ ID NO. : 621), and DOM16-39-619 (SEQ ID NO: 622). In other embodiments, the ligand having binding specificity for VEGF and for EGFR comprises a first single variable domain of immunoglobulin with a binding specificity for VEGF and a second single variable domain of immunoglobulin with a binding specificity for EGFR, wherein the first single variable domain of immunoglobulin competes for binding with VEGF, with bevacizumab and / or 2C3 antibody (ATCC Accession Number PTA 1595); and the second single variable domain of immunoglobulin competes for the linkage with EGFR, with cetuximab. In particular embodiments, the ligand has binding specificity for VEGF and for EGFR, and comprises at least one unique immunoglobulin variable domain with a binding specificity for VEGF and at least one unique immunoglobulin variable domain with a binding specificity for EGFR, in
wherein the ligand comprises a single immunoglobulin variable domain with a binding specificity for VEGF comprising an amino acid sequence having an amino acid sequence identity of at least 90 percent with the amino acid sequence of a selected anti-VEGF dAb 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 single variable immunoglobulin domain with a binding specificity for EGFR comprising an amino acid sequence having an amino acid sequence identity of at least 90 percent with an amino acid sequence 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), or DOM16-39-200 (SEQ
ID NO: 441). In particular embodiments, the ligand has binding specificity for VEGF and for EGFR, and comprises at least one unique immunoglobulin variable domain with a binding specificity for VEGF and at least one unique immunoglobulin variable domain with a binding specificity for EGFR. , wherein the ligand comprises a single immunoglobulin variable domain with a binding specificity for VEGF comprising an amino acid sequence having an amino acid sequence identity of at least 90 percent 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 single variable immunoglobulin domain with a binding specificity for EGFR comprising an amino acid sequence having an amino acid sequence identity of at least 90 percent with an amino acid sequence selected from the group consisting of at DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ
ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). The ligand that has binding specificity for VEGF and for EGFR can inhibit the binding of epidermal growth factor (EGF) and / or transforming growth factor alpha (TGF-alpha) with EGFR, can inhibit EGFR activity, and / or can inhibit EGFR activity without substantially inhibiting the binding of epidermal growth factor (EGF) and / or transforming growth factor alpha (TGF-alpha) with EGFR. In addition, or alternatively, the ligand having binding specificity for VEGF and for EGFR can inhibit the binding of vascular endothelial growth factor with the vascular endothelial growth factor receptor 1 (VEGFR1) and / or the factor receptor. vascular endothelial growth 2 (VEGFR2), can inhibit the activity of vascular endothelial growth factor and / or can
inhibit the activity of vascular endothelial growth factor without substantially inhibiting the binding of vascular endothelial growth factor with VEGFR1 and / or VEGFR2. The ligand having binding specificity for VEGF and for EGFR may contain a protein binding fraction (eg, single immunoglobulin variable domain) with a binding specificity for VEGF that binds to 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 may contain a protein binding fraction (eg, single immunoglobulin variable domain) with a binding specificity for EGFR that binds to 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 be linked to 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 be linked to EGFR with an affinity (KD) which is between about 100 nM and about 1 pM or
about 1.0 nM to about 1000 pM, as determined by surface plasmon resonance. The ligand having binding specificity for VEG F and for EGFR may comprise a single immunoglobulin variable domain with a binding specificity for VEGF which is a VH H and / or a single immunoglobulin variable domain with a binding specificity for EG FR. which is a VH H. The ligand having binding specificity for VEGF and for EGFR may comprise a single immunoglobulin variable domain with a binding specificity for VEGF and a single immunoglobulin variable domain with a binding specificity for EG FR, where the unique immunoglobulin domains they are selected from the group consisting of a human VH and a human VL. In some embodiments, the ligand having binding specificity for VEGF and for EGFR may be an IgG-like format comprising two unique immunoglobulin variable domains with a binding specificity for VEGF, and two unique immunoglobulin variable domains with a binding specificity. for EGFR. In some embodiments, the ligand having binding specificity for VEG F and for EG FR may comprise an Fc region of antibody. The invention also relates to a ligand that has binding specificity for VEGF, which comprises at least a single variable domain of immunoglobulin with a
binding specificity for VEGF, wherein a single immunoglobulin variable domain with a binding specificity for VEGF competes for binding with VEGF, with an 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), 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 D NO: 148), TAR15-26-500 (SEQ ID
NO149), 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
NO155), 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), and 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 variable immunoglobulin domain with a binding specificity for VEGF can comprise an amino acid sequence having an amino acid sequence identity of at least about 85 percent 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 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), and TAR15-26-549
(SEQ ID NO: 198), TAR15-26-550 (SEQ ID NO: 539), and TAR15-26-551 (SEQ ID NO: 540). The ligand that has binding specificity for VEGF can inhibit the binding of vascular endothelial growth factor with the vascular endothelial growth factor receptor 1 (VEGFR1) and / or the vascular endothelial growth factor receptor 2 (VEGFR2), can inhibit the activity of vascular endothelial growth factor, and / or can inhibit the activity of vascular endothelial growth factor without substantially inhibiting the binding of vascular endothelial growth factor with VEGFR1 and / or VEGFR2. The ligand having binding specificity for VEGF may contain a single immunoglobulin variable domain with a binding specificity for VEGF that binds to 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 can be linked to VEGF with an affinity (KD) which 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 a binding specificity for VEGF which is a VHH- The ligand having binding specificity for VEGF may comprise a single immunoglobulin variable domain with a specificity of link for VEGF that is selected from the group consisting of human VH and a human V. In some embodiments, the ligand having binding specificity for VEGF is an IgG type format comprising at least two unique immunoglobulin variable domains with a binding specificity for VEGF. In some embodiments, the ligand having binding specificity for VEGF comprises an Fc region of antibody. The invention also relates to a ligand having binding specificity for EGFR, which comprises at least one unique immunoglobulin variable domain with a binding specificity for EGFR, wherein a single immunoglobulin variable domain with a binding specificity for EGFR competes for the EGFR binding, 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 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), 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). The invention also relates to a ligand having binding specificity for EGFR, which comprises at least one unique immunoglobulin variable domain with a binding specificity for EGFR, wherein a single immunoglobulin variable domain with a binding specificity for EGFR competes for the EGFR binding, with an anti-EGFR domain antibody (dAb) selected from the group consisting of DOM16-39-210 (SEQ ID NO.541), DOM16-39-211 (SEQ ID NO: 542 ), DOM16-39-212 (SEQ ID NO: 543), DOM16-39-213 (SEQ ID NO: 544), DOM16-39-214 (SEQ ID NO: 545), DOM16-39-215 (SEQ ID NO. : 546), DOM16-39-216 (SEQ ID NO: 547), DOM16-39-217 (SEQ
ID NO: 548), DOM16-39-218 (SEQ ID NO: 549 DOM16- 39-219 (SEQ
ID NO: 550), DOM16-39-220 (SEQ ID NO: 551 DOM16- 39-221 (SEQ
ID NO: 552), DOM16-39-222 (SEQ ID NO: 553 DOM16- 39-223 (SEQ
ID NO: 554), DOM16-39-224 (SEQ ID NO: 555 DOM16- 39-225 (SEQ
ID NO: 556), DOM16-39-226 (SEQ ID NO: 557 DOM16- 39-227 (SEQ
ID NO: 558), DOM16-39-228 (SEQ ID NO: 559 DOM16- 39-229 (SEQ
ID NO: 560), DOM16-39-230 (SEQ ID NO: 561 DOM16- 39-231 (SEQ
ID NO: 562), DOM16-39-232 (SEQ ID NO: 563 DOM16- 39-233 (SEQ
ID NO: 564), DOM16-39-234 (SEQ ID NO: 565 DOM16- 39-235 (SEQ
ID NO: 566), DOM16-39-500 (SEQ ID NO: 725 DOM16-39-502 (SEQ
ID NO: 726), DOM16-39-503 (SEQ ID NO: 567 DOM16-39-504 (SEQ.
ID NO: 568), DOM16-39-505 (SEQ ID NO: 569 DOM16-39-506 (SEQ.
ID NO: 570), DOM16-39-507 (SEQ ID NO: 571 DOM16-39-508 (SEQ
ID NO: 572), DOM16-39-509 (SEQ ID NO: 573 DOM16-39-510 (SEQ
ID NO: 574), DOM16-39-511 (SEQ ID NO: 575 DOM16-39-512 (SEQ
ID NO: 576), DOM16-39-521 (SEQ ID NO: 577 DOM16-39-522 (SEQ
ID NO: 578), DOM16-39-523 (SEQ ID NO: 579 DOM16-39-524 (SEQ
ID NO: 580), DOM16-39-527 (SEQ ID NO: 581 DOM16-39-525 (SEQ
ID NO: 582), DOM16-39-526 (SEQ ID NO: 583 DOM16-39-540 (SEQ
ID NO: 584), DOM16-39-541 (SEQ ID NO: 585 DOM16-39-542 (SEQ
ID NO: 586), DOM16-39-543 (SEQ ID NO: 587 DOM16-39-544 (SEQ
ID NO: 588), DOM1639-545 (SEQ ID NO: 589 DOM16-39-550 (SEQ
ID NO: 590), DOM16 '• 39-551 (SEQ ID NO: 591 DOM16- • 39-552 (SEQ
ID NO: 592), DOM16-39-553 (SEQ ID NO: 593 DOM16-39-554 (SEQ
ID NO: 594), DOM16-39-555 (SEQ ID NO: 595 DOM16- • 39-561 (SEQ
ID NO.596), DOM16-39-562 (SEQ ID NO: 597), DOM16-39-563 (SEQ ID NO: 598), DOM16-39-564 (SEQ ID NO.599), DOM16-39-571 (SEQ ID NO.600), DOM16-39-572 (SEQ ID NO: 601), DOM16-39-573 (SEQ ID NO: 602), DOM16-39-574 (SEQ ID NO: 603), DOM16-39 -580 (SEQ ID NO: 604), DOM16-39-591 (SEQ ID NO: 605), DOM16-39-592 (SEQ.
ID NO: 606), DOM16-39-593 (SEQ ID NO: 607), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-602 (SEQ ID NO: 609), DOM16-39-603 (SEQ ID NO.610), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-605 (SEQ ID NO: 612), DOM16-39-607 (SEQ ID NO: 613), DOM16-39 -611 (SEQ ID NO: 614), DOM16-39-612 (SEQ ID NO: 615), DOM16-39-613 (SEQ ID NO: 616), DOM16-39-614 (SEQ ID NO: 617), DOM16 -39-615 (SEQ ID NO: 618), DOM16-39-616 (SEQ ID NO: 619), DOM16-39-617 (SEQ ID NO: 620), DOM16-39-618 (SEQ ID NO: 621) , and DOM16-39-619 (SEQ ID NO: 622). For example, the single immunoglobulin variable domain with a binding specificity for EGFR may compran amino acid sequence having an amino acid sequence identity of at least about 85 percent 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), DOM6-66 (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), 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 single immunoglobulin variable domain with a binding specificity for EGFR may comprise an amino acid sequence having an amino acid sequence identity of at least about 85 percent with the amino acid sequence of a dAb selected from the group consisting of DOM16-39-210 (SEQ ID NO: 541), DOM16-39-211 (SEQ ID NO: 542), DOM16-39-212 (SEQ ID NO: 543), DOM16-39-213 (SEQ ID NO: 544), DOM16-39-214 (SEQ ID NO: 545), DOM16-39-215 (SEQ ID NO: 546), DOM16-39-216 (SEQ.
ID NO: 547), DOM16-39-217 (SEQ ID NO: 548), DOM16-39-218 (SEQ ID NO: 549), DOM16-39-219 (SEQ ID NO: 550), DOM16-39-220 (SEQ ID NO: 551), DOM16-39-221 (SEQ ID NO: 552), DOM16-39-222 (SEQ ID NO: 553), DOM16-39-223 (SEQ ID NO: 554), DOM16-39 -224 (SEQ ID NO: 555), DOM16-39-225 (SEQ ID NO: 556), DOM16-39-226 (SEQ.
ID NO: 557 DOM16-39-227 (SEQ ID NO: 558) DOM16-39-228 (SEQ ID NO: 559 DOM16-39-229 (SEQ ID NO: 560) DOM16-39-230 (SEQ ID NO: 561 DOM16-39-231 (SEQ ID NO: 562) DOM16-39-232 (SEQ ID NO: 563 DOM16-39-233 (SEQ ID NO: 564) DOM16-39-234 (SEQ ID NO: 565 DOM16-39- 235 (SEQ ID NO: 566) DOM16-39-500 (SEQ ID NO: 725 DOM16-39-502 (SEQ ID NO: 726), DOM16-39-503 (SEQ ID NO: 567 DOM16-39-504 (SEQ ID NO: 568) DOM16-39-505 (SEQ ID NO: 569 DOM16-39-506 (SEQ ID NO: 570) DOM16-39-507 (SEQ ID NO: 571 DOM16-39-508 (SEQ ID NO: 572 ) DOM16-39-509 (SEQ ID NO: 573 DOM16-39-510 (SEQ ID NO: 574) DOM16-39-511 (SEQ ID NO: 575 DOM16-39-512 (SEQ ID NO: 576) DOM16-39 -521 (SEQ ID NO: 577 DOM16-39-522 (SEQ ID NO: 578) DOM16-39-523 (SEQ ID NO: 579 DOM16-39-524 (SEQ ID NO: 580) DOM16-39-527 (SEQ ID NO: 581 DOM16-39-525 (SEQ ID NO: 582) DOM16-39-526 (SEQ ID NO: 583 DOM16-39-540 (SEQ ID NO: 584) DOM16-39-541 (SEQ ID NO: 585 DOM16-39-542 (SEQ ID NO: 586) DOM16-39-543 (SEQ ID NO: 587 DOM16-39-544 (SEQ ID NO: 588) DOM16-39-545 (SEQ ID NO: 589 DOM16-39- 550 (SEQ ID NO: 590) DOM16-39-551 (SEQ ID NO: 591 DOM16-39-552 (SEQ ID NO: 592) DOM16-39-553 (SEQ ID NO: 593 DOM16-39-554 (SEQ ID NO: 594) DOM16-39-555 (SEQ ID NO: 595 DOM16-39-561 (SEQ ID NO: 596) DOM16-39-562 (SEQ ID NO: 597 DOM16-39-563 (SEQ ID NO: 598) DOM16-39- 564 (SEQ ID NO: 599 DOM16-39-571 (SEQ ID NO: 600) DOM16-39-572 (SEQ ID NO: 601 DOM16-39-573 (SEQ ID NO: 602) DOM16-39-574 (SEQ ID NO: 603 DOM16-39-580 (SEQ ID NO: 604) DOM16-39-591 (SEQ
ID NO: 605), DOM16-39-592 (SEQ ID NO: 606), DOM16-39-593 (SEQ ID NO: 607), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-602 (SEQ ID NO: 609), DOM16-39-603 (SEQ ID NO: 610), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-605 (SEQ ID NO: 612), DOM16-39 -607 (SEQ ID NO: 613), DOM16-39-611 (SEQ ID NO: 614), DOM16-39-612 (SEQ.
ID NO: 615), DOM16-39-613 (SEQ ID NO: 616), DOM16-39-614 (SEQ ID NO: 617), DOM16-39-615 (SEQ ID NO: 618), DOM16-39-616 (SEQ ID NO: 619), DOM16-39-617 (SEQ ID NO: 620), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). The ligand having binding specificity for EGFR can inhibit the binding of epidermal growth factor (EGF) and / or transforming growth factor alpha (TGF alpha) with EGFR, can inhibit EGFR activity, and / or can inhibit EGFR activity without substantially inhibiting the binding of epidermal growth factor (EGF) and / or transforming growth factor alpha (TGF alpha) with EGFR. The ligand having binding specificity for EGFR may contain a single immunoglobulin variable domain with a binding specificity for EGFR that binds to EGFR with an affinity (KD) that is between about 100 nM and about 1 pM or about 10 nM at approximately 100 pM, as determined by surface plasmon resonance. The ligand that has binding specificity for VEGF and for EGFR can be linked to EGFR with an affinity (KD) that is
between about 1 00 nM and about 1 pM or about 1 0 nM to about 1000 pM, as determined by surface plasmon resonance. The ligand having binding specificity for EGFR may comprise a single variable domain of immunoglobulin with a binding specificity for EG FR which is a VH H - The ligand having binding specificity for EGFR may comprise a single variable domain of immunoglobulin with a binding specificity for EGFR that is selected from the group consisting of a human VH and a human V. In some embodiments, the ligand having binding specificity for EGFR is an IgG type format comprising at least two unique immunoglobulin variable domains with a binding specificity for EGFR. In some embodiments, the ligand having binding specificity for EGFR comprises an antibody Fc region. In some embodiments, the ligand comprises a single variable immunoglobulin domain polypeptide that antagonizes (inhibits) the binding of human EGFR to a receptor, wherein this unique immunoglobulin variable domain polypeptide comprises a sequence of CDR3 that is the same CDR3 sequence of an anti-EGFR dAb disclosed herein. In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that is linked to EG FR, wherein the polypeptide has an amino acid sequence
which is identical to the amino acid sequence of an anti-EGFR dAb disclosed herein, or differs from the amino acid sequence of an anti-EG FR dAb disclosed herein in no more than 25 amino acid positions , and has a sequence of CDR1 which has an identity of at least 50 percent with the CDR1 sequence of the anti-EG FR dAb. In other embodiments, the ligand comprises 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 the anti-EGFR dAb disclosed herein. , or differs from the amino acid sequence of the anti-EGFR dAb disclosed herein at no more than 25 amino acid positions, and has a CDR2 sequence that has at least 50 percent identity to the CDR2 sequence of the anti-EGFR dAb. In other embodiments, the ligand comprises 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 the anti-EGFR dAb disclosed herein. , or differs from the amino acid sequence of the anti-EGFR dAb disclosed herein at no more than 25 amino acid positions, and has a CDR3 sequence that has at least 50 percent identity to the CDR3 sequence of the anti-EGFR dAb. In other embodiments, the ligand comprises a polypeptide of
unique variable domain of immunoglobulin 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 as disclosed herein, or differs from the amino acid sequence of a anti-EG FR dAb disclosed herein at no more than 25 amino acid positions, and has a CDR1 sequence that has at least 50 percent identity with the CDR1 sequence of the anti-EGFR dAb, and has a sequence of CDR2 having at least 50 percent identity with the CDR2 sequence of the anti-EGFR dAb. In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds to EG FR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-EGFR dAb disclosed in present, or differs from the amino acid sequence of an anti-EGFR dAb disclosed herein in no more than 25 amino acid positions, and has a CDR2 sequence that has an identity of at least 50 percent with the CDR2 sequence of the anti-EGFR dAb, and has a CDR3 sequence that has at least 50 percent identity with the sequence of CDR3 of the anti-EGFR dAb. In other embodiments, the ligand comprises 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-EG FR dAb given to
known herein, or differs from the amino acid sequence of an anti-EG FR dAb disclosed herein at no more than 25 amino acid positions, and has a sequence of CDR1 having at least 50 identity by hundred with the CDR1 sequence of the anti-EGFR dAb, and has a sequence of CDR3 having at least 50 percent identity with the CDR3 sequence of the anti-EGFR dAb. In other embodiments, the ligand comprises 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 disclosed in present, or differs from the amino acid sequence of an anti-EGFR dAb as disclosed herein at no more than 25 amino acid positions, and has a sequence of CDR1 having at least 50 percent identity to the sequence of CDR1 of the anti-EGFR dAb, and has a CDR2 sequence that has at least 50 percent identity to the CDR2 sequence of the anti-EGFR dAb, and has a CDR3 sequence that has at least one 50 percent with the CDR3 sequence of the anti-EGFR dAb. In another embodiment, the invention is an EGFR antagonist having a CDR1 sequence that has at least 50 percent identity to the CDR1 sequence of an anti-EG FR dAb disclosed herein. In another embodiment, the invention is an EGFR antagonist having a CDR2 sequence that has an identity of at least 50
percent with the CDR2 sequence of an anti-EGFR dAb disclosed herein. In another embodiment, the invention is an EGFR antagonist having a CDR3 sequence that has at least 50 percent identity with the CDR3 sequence of an anti-EG FR dAb disclosed herein. In another embodiment, the invention is an EGFR antagonist having a CDR 1 sequence that has at least 50 percent identity to the CDR1 sequence of an anti-EG FR dAb disclosed herein, and a CDR2 sequence having at least 50 percent identity with the CDR2 sequence of the anti-EG FR dAb. In another embodiment, the invention is an EGFR antagonist having a CDR2 sequence that has at least 50 percent identity to the CD R2 sequence of an anti-EG FR dAb disclosed herein, and a CDR3 sequence having an identity of at least 50 percent with the CDR3 sequence of the anti-EGFR dAb. In another embodiment, the invention is an EG FR antagonist having a CDR 1 sequence that has at least 50 percent identity with the CDR1 sequence of an anti-EGFR dAb disclosed herein, and a CDR3 sequence that has at least 50 percent identity with the CDR3 sequence of the anti-EGF dAb. In another embodiment, the invention is an EGFR antagonist.
having a CDR 1 sequence that has at least 50 percent identity to the CDR1 sequence of an anti-EG FR dAb disclosed herein, and a CDR2 sequence having at least one identity 50 percent with the CDR2 sequence of the anti-EG FR dAb, and a CDR3 sequence having at least 50 percent identity with the CDR3 sequence of the anti-EGFR dAb. In some embodiments, the ligand comprises a single variable immunoglobulin domain polypeptide that antagonizes (inhibits) the binding of human VEGF to a receptor, wherein this single immunoglobulin variable domain polypeptide comprises a sequence of CDR3 that is the same sequence of CDR3 of an anti-VEG F dAb disclosed herein. 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 an anti-VEG F dAb disclosed at the moment, or differs from the amino acid sequence of an anti-VEG F dAb disclosed herein in no more than 25 amino acid positions, and has a sequence of CDR1 having at least 50 percent identity with the CDR1 sequence of anti-VEGF dAb. In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds to VEG F, wherein the polypeptide has an amino acid sequence that is
identical to the amino acid sequence of the anti-VEGF dAb disclosed herein, or differs from the amino acid sequence of the anti-VEGF dAb disclosed herein in no more than 25 amino acid positions, and has a sequence of CDR2 having an identity of at least 50 percent with the CDR2 sequence of the anti-VEGF dAb. In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds to VEG F, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of the anti-VEGF dAb disclosed herein. , or differs from the amino acid sequence of the anti-VEG F dAb disclosed herein at no more than 25 amino acid positions, and has a CDR3 sequence that has at least 50 percent identity with the sequence of CD R3 of the 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 an anti-VEG F dAb disclosed in present, or differs from the amino acid sequence of an anti-VEGF dAb as disclosed herein in no more than 25 amino acid positions, and has a sequence of CDR1 having at least 50 percent identity with the CDR1 sequence of anti-VEG F dAb, and has a sequence of CDR2 having at least 50 percent identity with the CDR2 sequence of dAb
anti-VEGF. 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 an anti-VEGF dAb disclosed in present, or differs from the amino acid sequence of an anti-VEGF dAb as disclosed herein in no more than 25 amino acid positions, and has a sequence of CDR2 having at least 50 percent identity with the CDR2 sequence of the anti-VEG F dAb, and has a CD sequence R3 having at least 50 percent identity with the CDR3 sequence of the anti-VEGF dAb. In other embodiments, the ligand comprises a single immunoglobulin variable domain polypeptide that binds to VEG F, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-VEGF dAb disclosed in present, or differs from the amino acid sequence of an anti-VEG F dAb disclosed herein at no more than 25 amino acid positions, and has a sequence of CDR1 having at least 50 percent identity with the CDR 1 sequence of anti-VEGF dAb, and has a CDR3 sequence that has at least 50 percent identity to the CDR3 sequence of anti-VEG F dAb. In other embodiments, the ligand comprises a polypeptide of single variable domain of immunoglobulin that binds to VEG F,
wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of an anti-VEGF dAb as disclosed herein, or differs from the amino acid sequence of an anti-VEGF dAb disclosed herein in no more than 25 amino acid positions, and has a CDR1 sequence that has at least 50 percent identity with the anti-VEGF dAb CDR1 sequence, and has a CDR2 sequence that has at least one identity 50 percent with the CDR2 sequence of the anti-VEGF dAb and has a sequence of CDR3 having at least 50 percent identity with the CDR3 sequence of the anti-VEGF dAb. In another embodiment, the invention is a VEG F antagonist having a CDR 1 sequence that has at least 50 percent identity to the CDR1 sequence of an anti-VEG F dAb disclosed herein. In another modality, the invention is a VEGF antagonist having a CDR2 sequence having at least 50 percent identity with the CDR2 sequence of an anti-VEG F dAb disclosed herein. In another embodiment, the invention is a VEG F antagonist having a CDR3 sequence that has at least 50 percent identity with the CDR3 sequence of an anti-VEG F dAb disclosed herein. In another embodiment, the invention is a VEG F antagonist that has a CDR 1 sequence that has an identity of when
minus 50 percent with the CDR1 sequence of an anti-VEGF dAb disclosed herein, and a CDR2 sequence having at least 50 percent identity with the CDR2 sequence of the anti-VEG F dAb In another embodiment, the invention is a VEGF antagonist having a CDR2 sequence that has at least 50 percent identity with the CDR2 sequence of an anti-VEGF dAb disclosed herein, and a sequence of CDR3 having an identity of at least 50 percent with the CDR3 sequence of the anti-VEGF dAb. In another embodiment, the invention is a VEGF antagonist having a CDR 1 sequence that has at least 50 percent identity to the CDR1 sequence of an anti-VEGF dAb disclosed herein, and a sequence of CDR3 having an identity of at least 50 percent with the CDR3 sequence of the anti-VEGF dAb. In another embodiment, the invention is a VEGF antagonist having a CDR 1 sequence that has at least 50 percent identity to the CDR1 sequence of an anti-VEGF dAb disclosed herein, and a sequence of CDR2 having an identity of at least 50 percent with the CDR2 sequence of the anti-VEGF dAb, and a CDR3 sequence having at least 50 percent identity with the CDR3 sequence of the anti-VEGF dAb . In the additional modalities, any of the ligands
described herein further comprises a toxin, such as a cytotoxin, a free radical generator, antimetabolite, protein, polypeptide, peptide, photoactive agent, anti-sense compound, chemotherapeutic product, radionuclide, or intrabody. In particular embodiments, the toxin is a surface activity toxin (e.g., a free radical generator, a radionuclide). In other embodiments, the ligand further comprises a fraction that prolongs the half-life, such as a polyalkylene glycol fraction, serum albumin or a fragment thereof, transferrin receptor or a transferrin binding portion thereof, or a fraction that com turns on a binding site for a polypeptide that improves the half-life in vivo. In some embodiments, the fraction that prolongs the half-life is a fraction comprising a binding site for a polypeptide that improves the in vivo half-life selected from the group consisting of an affibody, an SpA domain, a receptor domain of LDL class A, an EG F domain, and an avimer. In other embodiments, the fraction that prolongs the half-life is an antibody or antibody fragment (eg, a single immunoglobulin variable domain) that comprises a binding site for the serum albumin receptor or neonatal Fc. In particular embodiments, the fraction that prolongs the half-life is a single variable immunoglobulin domain that comprises a binding site for serum albumin that competes
for binding to 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), 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). For example, the single immunoglobulin variable domain comprising a binding site for serum albumin may comprise an amino acid sequence having an identity
of amino acid sequence of at least 85 percent with the amino acid sequence of 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). The invention also relates to an isolated or recombinant nucleic acid encoding a ligand described herein, and to a vector (eg, a recombinant vector) comprising the acid
Recombinant nucleic The invention also relates to a host cell (eg, recombinant host cell, isolated host cell), which comprises a recombinant nucleic acid or a vector of the invention. The invention also relates to a method for producing a ligand, which comprises maintaining a host cell of the invention under conditions suitable for the expression of this nucleic acid or vector, whereby a ligand is produced. In some modalities, the method also comprises isolating the ligand. The invention also relates to a ligand of the invention for use in therapy or diagnosis, and to the use of a ligand of the invention for the manufacture of a medicament for the treatment, prevention, or suppression of a disease described herein. (for example, cancer). The invention also relates to a pharmaceutical composition for the treatment, prevention, or suppression of a disease described herein (eg, cancer), which comprises, as an active ingredient, a ligand of the invention. In some embodiments, the invention relates to a ligand for use in the treatment of cancer, or cancer cells that overexpress EG FR and / or VEGF. In other embodiments, the invention relates to the use of a ligand for the manufacture of a medicament for killing cells (e.g., to selectively kill cancer cells
on normal cells). In other embodiments, the invention relates to the use of a ligand for the manufacture of a medicament for the treatment of cancer cells that overexpress EGFR and / or VEGF. The invention also relates to therapeutic methods comprising administering a therapeutically effective amount of a ligand of the invention to a subject in need thereof. In one embodiment, the invention relates to a method for the treatment of cancer, which comprises administering to a subject in need thereof a therapeutically effective amount of a ligand of the 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 for treating cancer comprises administering to a subject in need thereof, a therapeutically effective amount of the ligand of the invention and an anti-neoplastic composition, wherein said antineoplastic composition comprises at least one chemotherapeutic agent. The myotherapeutic agent can be selected from the group consisting of alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-Asparaginase, topoisomerase inhibitor, interferons, platinum coordination complexes, anthracenedione substituted urea, methyl hydrazine derivatives,
adrenocortical suppressor, ad renocorticosteroides, progestins, estrogens, antiestrogen, androgens, antiandrogen, and gonadotropin-releasing hormone analogue. In some embodiments, the chemotherapeutic agent is selected from the group consisting of cisplatin, dicarbazine, dactinomycin, mechlorethamine, streptozocin, cyclophosphamide, capecitabine, carmustine, lomustine, doxorubicin, daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, fluoro-uracil, vi nblastine, vincristine, bleomycin, paclitaxel, docetaxel, doxetaxe, aldesleucine, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon-alpha, irinotecan, leuprolide, leucovorin, megestrol , melphalan, mercaptopurine, oxaliplatin, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, 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. In some embodiments, the method is a method for the treatment of a cancer selected from the group consisting of bladder cancer, ovarian cancer, colo-rectal cancer (colo-rectal carcinoma), breast cancer, lung cancer ( non-microcellular lung carcinoma), gastric cancer, pancreatic cancer, prostate cancer, head and neck cancer, kidney cancer, and gallbladder cancer.
The invention also relates to a method for administering to a subject an anti-VEGF treatment and an anti-EGFR treatment, the method comprising the simultaneous administration of an anti-VEGF treatment and an anti-EGFR treatment, by administration to this subject of a therapeutically effective amount of a ligand having binding specificity for VEG F and EGFR. The invention also relates to a composition (e.g., pharmaceutical composition), which comprises a ligand of the invention and a physiologically or pharmaceutically acceptable carrier. In some embodiments, the composition comprises a vehicle for intravenous, intramuscular, intraperitoneal, intra-arterial, intrathecal, intra-articular, subcutaneous, pulmonary, intranasal, vaginal, or rectal administration. The invention also relates to a drug delivery device, which comprises the composition (e.g., pharmaceutical composition) of the invention, or a ligand of the invention. In one embodiment, the drug delivery device is for the simultaneous administration to a subject of an anti-VEGF treatment and an anti-EG FR treatment, and the device comprises a ligand having binding specificity for VEG F and EG FR. In some embodiments, the drug device comprises a plurality of therapeutically effective doses of the ligand. In other embodiments, the drug delivery device is selected from the group consisting of a device for
parenteral delivery, an intravenous delivery device, an intramuscular delivery device, an intraperitoneal delivery device, a transdermal delivery device, a pulmonary delivery device, an intra-arterial delivery device, an intrathecal delivery device, a delivery device, intra-articular delivery, a subcutaneous delivery device, a device of its intranasal minister, a vagi nal delivery device, a rectal delivery device, a syringe, a transdermal delivery device, a capsule, a tablet, a nebulizer, a inhaler, an atomizer, an aerosolizer, a fine mist nebulizer, a dry powder injector, a metered dose inhaler, a metered dose sprayer, a metered dose nebulizer, a metered dose sprayer, a catheter. The invention also relates to a ligand that has binding specificity for vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR)., which comprises at least a protein fraction having a binding site with a binding specificity for VEGF, at least one protein fraction having a binding site with a binding specificity for EGFR, and an Fc region of a antibody. These ligands may consist of a single polypeptide. In other embodiments, two ligands containing Fc regions are linked together, for example through a disulfide bond (e.g., in the hinge region), to form a dimer.
In some embodiments, the ligand having a binding specificity for VEGF, may comprise a protein fraction having a binding site with a binding specificity for VEGF, and an Fc region of an antibody. In some embodiments, the protein fraction that has binding specificity for VEGF is fused to an Fc region of an antibody. In other embodiments, the ligand having binding specificity for EGFR, may comprise a protein fraction having a binding site with a binding specificity for EGFR, and an Fc region of an antibody. In some embodiments, the protein fraction having binding specificity for EG FR is fused to an Fc region of an antibody. For example, the ligand may comprise two protein fractions having binding sites with a binding specificity for EG FR and an Fc region of an antibody. Additionally, or in other embodiments, the ligand having binding specificity for VEGF and EGFR, comprises a single variable domain with a binding specificity for VEG F, a single variable domain with a binding specificity for EGFR, and optionally a linker. In these embodiments, the single variable domain with a binding specificity for EG FR can be linked via the linker to the single variable domain of immunoglobulin with a binding specificity for VEGF. Suitable linkers include SEQ I D NO: 706, SEQ I D NO: 707, S EQ I D NO: 708, S EQ I D NO: 709, SEQ I D NO: 71 0, SEQ I D NO: 71 1,
SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 723 and SEQ ID NO: 724. The can ligand may also comprise an Fc region of an antibody, if desired. When the ligand further comprises an Fc region of an antibody, a linker can bind an immunoglobulin variable domain to the Fc region. In other embodiments, two ligands containing Fc regions are linked together, for example, through a disulfide bond (e.g., in the arti culation region), to form a dimer. Additionally, or in other embodiments, the ligand having binding specificity for VEGF and EG FR, comprises a single variable domain with a binding specificity for VEGF directly fused to a single variable domain with a binding specificity for EG FR. In embodiments wherein the ligand comprises a single variable domain with a binding specificity for VEGF, and only variable domain with a binding specificity for EGFR, and optionally one or more linkers, the only variable domains can independently be a variable domain of light chain or a heavy chain variable domain. For example, the ligand may comprise: a) a single variable domain with a binding specificity for VEGF that is a heavy chain variable region, and the only immunoglobulin variable domain with a binding specificity for EG FR that is a variable region of light chain; b) a single variable domain with a binding specificity for VEGF that is a light chain variable domain, and a single domain
variable with a binding specificity for EG FR which is a heavy chain variable domain; c) a single variable domain with a binding specificity for VEGF which is a heavy chain variable domain, and a single variable domain with a binding specificity for EG FR which is a heavy chain variable domain; or d) a single variable domain with a binding specificity for VEGF which is a light chain variable domain, and a single variable domain with a binding specificity for EG FR which is a light chain variable domain. In particular embodiments, the heavy chain variable region is a VH or VH H - In the additional modalities, the VH is a human VH. In other embodiments, the heavy chain variable region is a V ?. In another aspect of the invention, a ligand having binding specificity for VEGF and EGFR, comprises at least one unique immunoglobulin variable domain with a binding specificity for VEGF, and at least one unique immunoglobulin variable domain with a binding specificity for EG FR, wherein the single immunoglobulin variable domain with a binding specificity for EGFR is linked via a disulfide bond to the single immunoglobulin variable domain with a binding specificity for VEGF. Alternatively, a ligand having binding specificity for VEGF and EGFR may comprise at least one unique immunoglobulin variable domain with a binding specificity for VEG F, and at least one single variable domain of immunoglobulin with one
binding specificity for EGFR, wherein the single immunoglobulin variable domain with a binding specificity for EGFR is fused directly with the single immunoglobulin variable domain with a binding specificity for VEGF (ie, a single polypeptide comprising two dAbs) . In other particular embodiments, the ligand is a fusion of a dAb with an anti-serum albumin dAb (a DOM7 dAb). For example, the ligand may have the structure, from the amino terminus to the carboxyl terminus, of DOM15-10-DOM16-39-dAb anti-DOM16-39-DOM15-10-dAb anti-serum albumin, DOM15-26 -501-DOM16-39-dAb anti-serum albumin, or DOM16-39-DOM15-26-501-dAb anti-serum albumin. In the ad hoc modalities, the ligand having a binding site with a binding specificity for EGFR can compete for the EGFR binding with cetuximab and / or panitumumab and fuses with an anti-serum albumin dAb. Additionally, or in other embodiments, the ligand may comprise two or more dAbs (e.g., anti-EGFR dAbs) fused with anti-serum albumin dAb. BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1E depict 27 nucleotide sequences encoding human domain antibodies (dAbs) (Homo sapiens) that specifically bind to human VEGF. The nucleotide sequences presented are SEQ ID NOs: 1-27, 535 and 536. FIG. Figures 2A-2C are an alignment of twelve
nucleotide sequences encoding human dAbs that bind to 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 to human VEGF. The nucleotide sequences presented with SEQ ID NO: 20 and SEQ ID NOS: 39-49. Figures 4A-4J are an alignment of 53 nucleotide sequences encoding human dAbs that bind to human VEGF. The nucleotide sequences presented are SEQ ID NOs: 24, 50-99, 537 and 538. Figures 5A-5C illustrate the amino acid sequences of the dAbs encoded by several of the nucleic acid sequences shown in Figures 1A1E. The amino acid sequences presented are SEQ ID NOs: 100-126. Figure 6 is an alignment of the amino acid sequences of the dAbs encoded by 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 by the nucleic acid sequences shown in Figures 3A-2D. The ~ symbol has been inserted in the TAR15-8-500 sequence to facilitate alignment. The amino acid sequences presented are the 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 by the nucleic acid sequences shown in Figures 4A-4J. the amino acid sequences presented are SEQ ID NOs: 123, 149-198, 539 and
540. Figures 9A-9O illustrate several nucleotide sequences encoding human domain antibodies (dAbs) (Homo sapiens) that specifically bind to human EGFR. The presented nucleotide sequences are SEQ ID NOs: 199-324. Figures 10A-10I illustrate the amino acid sequences of the dAbs encoded by the nucleic acid sequences shown in Figures 9A-9O. The amino acid sequences presented are SEQ ID NOS: 325-450. Figures 11A-11B illustrate the amino acid sequences of various camelid VHHS that bind to EGFR, which are disclosed in International Publication Number 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), NBU (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), 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 as MSA16, which is also referred to as DOM7m-16 (SEQ ID NO: 473), MSA 12, which is also referred to as DOM7m-12 (SEQ ID NO: 474), and MSA 26 , which is also referred to as DOM7m-26 (SEQ ID NO: 475). Figure 12B is an alignment of the six VKS amino acid sequences that bind to rat serum albumin (RSA). The aligned amino acid sequences are from the VKS designated as 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 to human serum albumin (HSA). The aligned amino acid sequences are from the VKS designated as 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 amino acid sequences of seven VHs that bind to human serum albumin, and a sequence in consensus (SEQ ID NO: 488). The aligned sequences are from the 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 to the human serum albumin and to rat serum albumin. The aligned amino acid sequences are from the 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 the VKS that bind to rat serum albumin (RSA). The illustrated sequences are from the 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 the VHs that bind to rat serum albumin (RSA). The illustrated sequences are from the 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 several
VHHS from camelid that bind to mouse serum albumin, which are disclosed in International Publication Number 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 to prepare IgG type formats. Figures 17A-17F illustrate amino acid sequences of human dAbs that bind to human EGFR. The amino acid sequences presented are SEQ ID NOS: 541-622, 725 and 726. The sequences are continuous, without gaps; the symbols ~, -, and, have been inserted to show the locations of the CDRs. The CDR1 is flanked by ~, the CDR2 is flanked by -, and the CDR3 is flanked by. Figures 18A-18L illustrate nucleotide sequences encoding the dAbs shown in Figures 17A-17F. The nucleotide sequences presented are SEQ ID NOS: 623-703, 727 and 728. Figure 19 illustrates the amino acid sequence (SEQ ID NO: 704)
of a human dAb that binds to VEG F, and a nucleotide sequence (SEQ I D NO: 705) that codes for dAb. The sequences are continuous without gaps, and the symbols ~, -, and, have been inserted to show the locations of the CDRs. The CDR1 is flanked by ~, the CDR2 is flanked by -, and the CDR3 is flanked by. DETAILED DESCRIPTION OF THE INVENTION Within this specification, modalities have been described in a manner in which it is possible to write a clear and concise descriptive memory, but it is intended and appreciated that the modalities may be combined differently or separated. without departing from the invention. As used herein, the term "ligand" refers to a compound that comprises at least one peptide, polypeptide, or protein fraction that has a binding site with a binding specificity for a desired endogenous target compound. Ligands according to the invention preferably comprise immunoglobulin variable domains having different binding specificities, and which 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, is a single immunoglobulin variable domain (eg, a single immunoglobulin heavy chain variable domain (eg VH, VH H) A single light chain variable domain of
immunoglobulin (e.g., VL)) that has binding specificity for a desired cell surface target (e.g., a membrane protein, such as a receptor protein). Each polypeptide domain that has a binding site that has binding specificity for a cell surface target can also comprise one or more complementarity determining regions (CDRs) of an antibody or antibody fragment (eg, a single immunoglobulin variable domain), having binding specificity for a desired cell surface target in a suitable format, such that the binding domain has binding specificity for the cell surface target. For example, CDRs can be grafted onto a suitable protein scaffold or skeleton, such as an affibody, a SPA scaffold, a class A LDL receptor domain, or an EGF domain. In addition, the ligand may be bivalent (heterobivalent) or multivalent (heteromultivalent), as described herein. 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 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 in an antibody format (e.g., IgG type format, scFv, Fab, Fab ', F (ab') 2), or a scaffolding
or suitable protein skeleton, such as an affibrant, a SPA scaffold, a class A LDL receptor domain, an EGF domain, an avimer, and multispecific ligands, as described herein. The polypeptide domain having a binding site having binding specificity for a cell surface target (ie, first or second cell surface target), can also be a protein domain comprising a binding site for a desired target, for example a protein domain selected from an affibrant, a SPA domain, an LDL class A receptor domain, an avimer (see, for example, the United States Patent Application Publications). Numbers 2005/0053973, 2005/0089932,
2005/01 64301). If desired, a "ligand" may further comprise one or more additional fractions, each independently capable of being a peptide, polypeptide, or protein fraction, or a non-peptide moiety (eg, a polyalkylene glycol, a lipid, a carbohydrate). For example, the ligand may further comprise a fraction that extends the half-life, as described herein (for example, a polyalkylene glycol fraction, a fraction comprising albumin, an albumin fragment or an albumin variant, a fraction comprising transferrin, a transferrin fragment or transferrin variant, a fraction that binds to albumin, a fraction that binds to the neonatal Fc receptor). As used herein, the phrase "objective" refers to
a biological molecule (e.g., peptide, polypeptide, protein, lipid, carbohydrate), with which a polypeptide domain having a binding site can be linked. 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, VH H, VL) that specifically binds to a target, antigen or epitope, independently of other V domains; however, as the term is used herein, a single immunoglobulin variable domain may be present in one format (eg, hetero-multimer) with other variable regions or variable domains, where the other regions or domains are not required. for antigen binding by the single immunoglobulin variable domain (i.e., wherein the single immunoglobulin variable domain binds to the antigen independently of the additional variable domains). Each "single immunoglobulin variable domain" encompasses not only a single variable domain polypeptide of isolated antibody, but also larger polypeptides comprising one or more monomers of a single variable antibody domain polypeptide sequence. A "domain antibody" or "dAb" is the same as a "single variable immunoglobulin domain" polypeptide, such as
the term is used in the present. A single immunoglobulin variable domain polypeptide, as used herein, refers to a variable single-domain polypeptide of mammalian immunoglobulin, preferably human, but also includes rodent (e.g., as given to know in the International Publication Number WO 00/29004, the content of which is incorporated herein by reference in its entirety), or the VH H camelid dAbs. As used herein, camelid dAbs are single-variable immunoglobulin domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, and comprise heavy chain antibodies naturally devoid of light chain : (VH H) - Similar dAbs can be obtained for single-chain antibodies from other species, such as nurse shark. Preferred ligands comprise at least two different polypeptides from a single immunoglobulin variable domain, or at least two different dAbs. A single immunoglobulin variable domain "human" (eg, dAb, VH, V, V ?, V?) Can be derived from an antibody of human origin, or from a library prepared using variable region genes of human antibody. For example, as described herein, the only human immunoglobulin variable domains have one or more structural regions that are encoded by a human germline antibody gene segment, or have up to 5 differences.
of amino acids in relation to the amino acid sequence encoded by a segment of the human germline antibody gene. Preferably, the amino acid sequences of FW1, FW2, FW3 and FW4 are each encoded by an antibody gene segment of the human germ line, or collectively contain up to 10 amino acid differences relative to the amino acid sequences of the corresponding structure regions encoded by the segment of the human germline antibody gene. As used herein, "vascular endothelial growth factor" (VEGF) refers to mammalian VEGF-A proteins that occur naturally or endogenously, and to proteins that have an amino acid sequence that is the same as that of a mammalian VEGF-A protein that occurs naturally or corresponding endogenously (e.g., recombinant proteins, synthetic proteins (that is, produced using the methods of synthetic organic chemistry)). In accordance with the above, as defined herein, the term includes the mature VEG FA protein, the polymorphic or allelic variants, and other isoforms of a VEG FA (eg, produced by alternating splicing or other cellular processes), and Modified or unmodified forms of the above (for example, lipidated, glycosylated). The alternating splicing of the 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-1 21, VEGF-1 65, VEGF-1 89 and VEG F-206 are produced in humans. (See, for example, Ferrara, N., Endocrine Reviews, 25 (4): 581-61 1 (2004)). These isoforms and other naturally occurring isoforms are expressly encompassed by the term "VEGF". VEGF-A that occurs naturally or endogenously includes wild-type proteins, such as mature VEGF-A, polymorphic or allelic variants and other isoforms that occur naturally in mammals (e.g., humans, non-human primates) . These 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 VEG F that occurs naturally or correspondingly endogenous, are referred to by the name of the corresponding mammal. For example, when the corresponding mammal is a human being, the protein is designated as a human VEGF. A ligand (eg, single immunoglobulin variable domain) that inhibits VEG F binding with VEGFR1 or VEGFR2, inhibits the binding in the VEGFR1 binding assay or in the VEG FR2 assay described herein, at least about 40 percent, at least about 50 percent, at least about 60 percent, at least about 70 percent, at least about 80 percent, at least about
85 percent, at least about 90 percent, or at least about 95 percent, when assaying the ligand at a concentration of approximately 1 nM, approximately 1.0 nM, approximately 50 nM, approximately 1000 nM , approximately 1 μM, approximately 1.0 μM or approximately 1000 μM. A ligand that inhibits the binding of VEGF with VEGFR1 or with VEGFR2, also, or alternatively, can inhibit binding in the VEGF binding assay R1 or in the binding assay of VEGFR2 with a C50 I of about 1 μM or less, of about 500 nM or less, of about 1 00 nM or less, of about 75 nM or less, of about 50 nM or less, of about 1 0 nM or less, or of about 1 nM or less. A ligand (e.g., single immunoglobulin variable domain) that inhibits VEGF activity, inhibits viability in the VEGF assay described herein, by at least about 20 percent, at least about 30 percent, when at least about 40 percent, at least about 50 percent, at least about 60 percent, at least about 70 percent, at least about 80 percent, at least about 85 percent, when less about 90 percent, or at least about 95 percent. A ligand (for example, only variable domain of
immunoglobulin) which does not substantially inhibit the binding of VEGF with VEGFR1 or with VEG FR2 does not significantly inhibit binding in the VEG FR1 binding assay or in the VEGFR2 assay described herein. For example, this ligand could inhibit the VEGF binding in the VEG FR1 binding assay or in the VEGFR2 assay described herein, with an IC50 of about 1 mM or more, or it can inhibit binding by not more than about 20 percent, no more than about 1 5 percent, no more than about 10 percent, or no more than about 5 percent. As used in the present "epidermal growth factor receptor" (EG FR, ErbB 1, H ER1), it refers to mammalian EGFR proteins that occur naturally or endogenously, and to proteins that have a sequence of amino acids that is the same as that of a mammalian EGFR protein that occurs naturally or corresponding endogenously (e.g., recombinant proteins, synthetic proteins (ie, produced using the methods of synthetic organic chemistry)). In accordance with the foregoing, as defined herein, the term includes mature EGFR protein, polymorphic or allelic variants, and other EGFR isoforms (eg, produced by alternating splicing or other cellular processes), and modified or modified forms. not modified from the previous ones (for example, lipidated, glycosylated). The EG FR that occurs naturally or endogenously includes proteins of type
wild type, such as mature FR FR, polymorphic or allelic variants, and other isoforms that occur naturally in mammals (eg, humans, non-human primates). These proteins can be recovered or isolated from a source that naturally produces EG FR, for example. These proteins, and proteins having the same amino acid sequence as an EG FR that occurs naturally or correspondingly endogenous, are referred to by the name of the corresponding mammal. For example, when the corresponding mammal is a human being, the protein is designated as a human EG FR. A ligand (e.g., single variable immunoglobulin domain) that inhibits the binding of EGFR and / or TGF-alpha to EGFR, inhibits binding in the EGFR binding assay, or in the kinase assay. EGFR described herein, with an I C50 of about 1 μM or less, of about 500 nM or less, of about 1 00 nM or less, of about 75 nM or less, of about 50 nM or less, of about 1 0 nM or less, or approximately 1 nM or less. A ligand (eg, single immunoglobulin variable domain) that inhibits EGFR activity, inhibits EGFR kinase activity in the EGFR kinase assay described herein, with an IC50 of about 1 μM or less , of about 500 nM or less, of about 1 00 nM or less, of about 75 nM or less, of about
50 nM or less, of about 1 0 nM or less, or of about 1 nM or less. A ligand (eg, single immunoglobulin variable domain) that does not substantially inhibit the binding of EGF or TG F-alpha to EGFR, does not significantly inhibit the binding of EGF and / or TGF-alpha to the EG FR in the receptor binding assay or in the kinase assay described herein. For example, this ligand could inhibit the binding of EGF or TG F-alpha to EG FR in the receptor binding assay or in the kinase assay described herein, with a C50 I of approximately 1 mM. or more. "Affinity" and "avidity" are terms of the technique that describe the strength of a bonding interaction. With respect to the ligands of the invention, avidity refers to the overall binding force between the targets (eg, first cell surface target and second cell surface target) on the cell and the ligand.
Avidity is more than the sum of individual affinities for individual goals. As used herein, "toxin fraction" refers to a fraction comprising a toxin. A toxin is an agent that has detrimental effects on, or that alters cellular physiology
(for example, it causes cell necrosis, apoptosis, or inhibits cell division). As used herein, the term "dose" refers to the amount of ligand administered to a subject all at once (dose
unitary), or in two or more administrations for a defined time interval. For example, the dose may refer to the amount of ligand (eg, the ligand comprising a single variable domain of immunoglobulin that binds to VEGF, and a single variable domain of immunoglobulin that binds to EGFR), administered to a subject during the course of 1 day (24 hours) (daily dose), two days, one week, two weeks, three weeks, or one or more months (for example, by a single administration, or by two or more administrations ). The interval between the doses can be any desired amount of time. As used herein, "complementary" refers to when two immunoglobulin domains belong to families of structures that form pairs or cognate groups, or that are derived from such families and retain this feature. For example, a VH domain and a VL domain of an antibody are complementary; two VH domains are not complementary, and two V domains are not complementary. The complementary domains can be found in other members of the immunoglobulin superfamily, such as the Va and Vß domains (or y and d) of the T-cell receptor. Domains that are artificial, such as domains based on protein scaffolds that do not bind epitopes unless designed to do so, are not complementary. In the same way, two domains based (for example) on an immunoglobulin domain and a fibronectin domain are not complementary.
As used herein, "immunoglobulin" refers to a family of polypeptides that retain the immunoglobulin fold characteristic of the antibody molecules, which contains two β-sheets, and usually, a conserved disulphide. Members of the immunoglobulin super family are involved in many aspects of cellular and non-cellular interactions in vivo, including roles widely extended in the immune system (eg, 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 platelet-derived growth factor receptor). The present invention is applicable to all molecules of the immunoglobulin super family that have binding domains. Preferably, the present invention relates to antibodies. As used herein, "domain" refers to a folded protein structure that retains its tertiary structure independently of the rest of the protein. In general terms, the domains are responsible for the functional properties separated from the proteins, and in many cases they can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or the domain. A single variable antibody domain means a folded polypeptide domain comprising characteristic sequences of the antibody variable domains. Therefore, it includes the variable domains of
whole antibodies and the modified variable domains, for example, wherein one or more cycles have been replaced by sequences that are not characteristic of the variable antibody domain, or antibody variable domains that have been truncated or that comprise N-extents. or C-terminals, as well as the folded fragments of the variable dominoes that retain at least in part the binding activity and specificity of the full-length domain. Accordingly, each ligand comprises at least two different domains. "Repertoire". A collection of various variants, for example polypeptide variants, which differ in their primary structure. A library used in the present invention will encompass a repertoire of polypeptides comprising at least 1,000 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 single polypeptide or nucleic acid sequence. To this degree, the library is synonymous with repertoire. The sequence differences between the members of the library are responsible for the diversity present in the library. The library can take the form of a simple mixture of polypeptides or nucleic acids, or it can be in the form of organisms or cells, for example bacteria, viruses, animal or plant cells, and the like, transformed with a nucleic acid library. Preferably, each organism or individual cell contains
only one or a limited number of members of the library. Conveniently, the nucleic acids are incorporated into expression vectors, in order to allow expression of the polypeptides encoded by the nucleic acids. In a preferred aspect, therefore, a 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 library member in the form of nucleic acid, which can be express to produce its corresponding polypeptide member. Therefore, the population of host organisms has the potential to encode a large repertoire of genetically diverse polypeptide variants. As used herein, an antibody refers to IgG, IgM, IgA, IgD or Ig E, or a fragment (such as a Fab, F (ab ') 2, Fv linked with disulfide, scFv, multispecific antibody of closed conformation, disulfide-bonded scFv, diabody), either derived from any species that naturally produces an antibody, or created by recombinant DNA technology; either isolated from serum, B-cells, hybrids, transfectomas, yeast, or bacteria. As described herein, an "antigen" is a molecule that is linked via a binding domain according to the present invention. Typically, the antigens are ligated by antibody ligands, and are capable of eliciting an antibody response in vivo. It can be a polypeptide, protein,
nucleic acid, or another molecule. In general terms, the specific double ligands according to the invention are selected for the specificity of the target against two particular targets (e.g., antigen). In the case of conventional antibodies and fragments thereof, the antibody binding site by the variable cycles (L1, L2, L3, and H1, H2, H3) is capable of binding to the antigen. An "epitope" is a unit structure conventionally linked by an immunoglobulin VH / VL pair. Epitopes define the minimum 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 unit structure linked by a variable domain in isolation.
"Universal structure" refers to a single sequence of antibody structure corresponding to the regions of an antibody conserved in the sequence, as defined by Kabat ("Sequence of Proteins Immunological Interest", as defined by the Department of Health. and Human Services of the United States), or corresponding to the immunoglobulin repertoire or structure of the human germinal line, as defined by Chothia and Lesk, J. Mol. Biol. 196: 910-91 7 (1987). The invention provides the use of a single structure, or of a set of these 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 for the serum concentration of the ligand to be reduced by 50 percent, in vivo, for example, due to degradation of the ligand and / or elimination or Sequestration of the specific double ligand by natural mechanisms. The ligands of the invention are stabilized in vivo, and their life is increased by their binding to molecules that resist degradation and / or elimination or sequestration. Typically, these molecules are naturally occurring proteins, which have a long half-life in vivo. The half-life of a ligand is increased if its functional activity persists, in vivo, for a longer period than a similar ligand that is not specific to the molecule that increases the half-life. Accordingly, a specific ligand for HSA and two target molecules are compared to the same ligand, where the specificity for HSA is not present, ie, it does not bind to HSA, but binds to another molecule. For example, you can link to a third objective on the cell. Typically, the half-life increases by 1 0 percent, 20 percent, 30 percent, 40 percent, 50 percent, or more. Increases in the range of 2x, 3x, 4x, 5x, 1 0x, 20x, 30x, 40x, 50x or more of the half-life are possible. In an alternative way, or in addition, increases in the range of up to 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 1 50x of the half-life are possible. As it is referred to in the present, the term "competes" means that the link of a first objective with its domain of
Cognate target link is inhi bido when a second target is linked to its cognate target link domain. For example, the link can be sterically inhibited, for example, by physically blocking a link domain, or by altering the structure or environment of a link domain, so that its affinity is networked. or greed for an objective. A protein fraction competes for binding with one target (eg, EGFR, VEGF, serum albumin) with another agent, when the protein fraction inhibits the binding of the other agent to the target in a competitive binding assay (e.g. , a competitive ELISA or other suitable binding assay). For example, the protein fraction can inhibit the binding of another agent that binds to a target (for example, EGFR, VEGF, serum albumin) in a competitive binding assay, by at least about 25 percent, at least about 30 percent, at least about 40 percent, at least about 50 percent, when less about 60 percent, at least about 70 percent, at least about 80 percent, at least about 90 percent, or at least about 95 percent. As used herein, the terms "low restraint," "medium restraint," "high restraint," or "very restrictive conditions," describe the conditions for hybridization and washing of nucleic acids. It can
find a guide carrying out the hybridization reactions in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1 989), 6.3.1 -6.3.6, which is incorporated herein by reference in its entirety. Aqueous and non-aqueous methods are described in that reference, and any of them can be used. The specific hybridization conditions referred to herein are as follows: (1) low-restriction hybridization conditions in 6X sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by two washes in 0.2X SSC, SDS at 0.1 percent at least at 50 ° C (the temperature of the washings can be increased to 55 ° C for conditions of low restraint); (2) conditions of hybridization of medium restraint in 6X SSC at approximately 45 ° C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60 ° C; (3) conditions of high-restriction hybridization in 6X SSC, at approximately 45 ° C, followed by one or more washes in 0.2X SSC, 0.1 percent SDS at 65 ° C; and preferably (4) very high stringency hybridization conditions, which are with 0.5M sodium phosphate, 7% SDS at 65 ° C, followed by one or more washes in 0.2X SSC, 1% SDS at 65 ° C. The conditions of very high restriction (4) are the preferred conditions, and those that should be used, unless otherwise specified. Similar or homologous sequences (eg, with a sequence identity of at least about 70 percent) to the sequences disclosed herein,
They are also part of the invention. In some embodiments, the sequence identity at the amino acid level can be about 80 percent, 85 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent or higher. At the nucleic acid level, the sequence entity id can be about 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent or higher. Alternatively, there is a substantial identity when the nucleic acid segments are hybridized under selective hybridization conditions (eg, very high stringency hybridization conditions) to the chain complement. The nucleic acids can be present in whole cells, in a cellular list, or in a partially purified or substantially pure one. The "homology" or "sequence identity" or "similarity" calculations between two sequences (the terms are used interchangeably in the present) are carried out as follows. The sequences are aligned for optimal comparison purposes (for example, gaps can be introduced into one or both of a first and second amino acid or nucleic acid sequence for optimal alignment, and non-homologous sequences are discarded for the purposes of the comparison ). In a preferred embodiment, the length of a reference sequence
aligned for comparison purposes is at least 30 percent, at least preferably 40 percent, more preferably at least 50 percent, still more preferably at least 60 percent, and still most preferably at least 70 percent, 80 percent, 90 percent, or 1 00 percent of the length of the reference sequence. The amino acid residues or nucleotides are then compared at the amino acid positions or at the corresponding nucleotide positions. When a position in a 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 used herein), "homology" of amino acids or nucleic acid is equivalent to "identity" of amino acids or nucleic acid). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of ch to hollow, which need to be introduced for optimal alignment of the two sequences. Alignments of amino acid and nucleotide sequences, and homology, similarity, or identity, as defined herein, are preferably prepared and determined using the BLAST 2 sequence algorithm, using the default parameters (Tatusova, TA and collaborators, FEMS Microbiol Lett, 1 74: 1 87-1 88 (1999)). In an alternative way, it
it uses the BLAST algorithm (version 2.0) for the alignment of sequences, with the parameters established in the default values. BLAST (Basic Local Aligment Search Tool) is the heuristic search algorithm used by the blastp, blastn, blastx, tblastn, and tblastx progr; These programs ascribe meaning to their discoveries using the statistical methods of Karlin and Altschul, 1 990, Proc. Nati Acad. Sci. USA 87 (6): 2264-8. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (eg, cell culture, molecular genetics, nucleic acid chemistry). , hybridization techniques, and biochemistry). Conventional techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Clone: A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. and Ausubel et al., Short Protocols in Molecular Biology (1 999) 4th Edition, John Wiley &Sons, I nc. Which are incorporated herein by reference), and chemical methods. The invention relates to ligands that have binding specificity for VEGF. { for example, human VEGF), to ligands that have binding specificity for EG FR. { for example, human EG FR), and to ligands that have binding specificity for VEG F and EG FR. { for example, human VEGF and human EGFR). For example,
The ligand may comprise a polypeptide domain having a binding site with a binding specificity for VEGF, a polypeptide domain that has a binding site with a binding specificity for EG FR, or may comprise a polypeptide domain that it has a binding site with a binding specificity for VEGF, and a polypeptide domain having a binding site with a binding specificity for EGFR. The ligands of the invention provide several advantages. For example, as described herein, the ligand can be tailored to have a desired in vivo serum half life. Accordingly, the ligands can be used to control, reduce, or eliminate the general toxicity of the therapeutic agents, such as the 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 a dAb provide advantages over conventional antibodies when administered to treat cancer, for example when targeting solid tumors. Moreover, many cancers overexpress in EGFR, and ligands that have binding specificity for EGFR and VEGF can be administered to direct the inhibitory activity of VEGF to tumors or to the environment of cancer cells. This approach provides two beneficial activities directly at the site of a tumor or a cancer, that is, an activity against cancer
direct, by binding to EGFR, and inhibition of ligand binding (eg, EGF, TGF-alpha) to the receptor, and inhibition of angiogenesis that supports tumor formation and development. In accordance with the above, ligands having link specificity for VEGF and EG FR can be administered to a cancer patient (eg, a cancer expressing EGFR), in order to provide superior therapy using a single therapeutic agent . In addition, signals transduced through EGFR can lead to the production of angiogenic factors, such as VEG F. 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. In accordance with the above, the ligands of the invention having binding specificity for VEG F and EGFR can be administered to a subject in order to direct the delivery of the VEGF inhibitory activity of the ligand to the cells that overexpress EG FR. In accordance with the foregoing, anti-angiogenic therapy can be delivered specifically to sites where VEGF is being produced (eg, to cells that overexpress EGFR). In some embodiments, the ligand has binding specificity for VEGF, and comprises (at least one) unique immunoglobulin variable domain with a binding specificity for VEGF. In other embodiments, the ligand has specificity of
linkage for EG FR, and comprises (at least one) unique immunoglobulin variable domain with a binding specificity for EGFR. In certain embodiments, the ligand has binding specificity for VEG F and EGFR, and comprises (at least one) unique immunoglobulin variable domain with a binding specificity for VEG F, and (at least one) unique immunoglobulin variable domain with a binding specificity for EGFR. The ligand of the invention can be formatted as described herein. For example, the ligand of the invention can be formatted to tailor the serum half-life in vivo. If desired, the ligand may further comprise a toxin or a toxin moiety as described herein. In some embodiments, the ligand comprises a toxin of surface activity, such as a free radical generator (eg, a toxin containing selenium), or a radionuclide. In other embodiments, the toxin or toxin fraction is a polypeptide domain (e.g., a dAb) that has a binding site with a binding specificity for intracellular targeting. In particular embodiments, the ligand is in an IgG-like format that has binding specificity for VEGF and EGFR (eg, human VEG F and human EG FR). Ligand Formats The ligand of the invention can be formatted as a monospecific, specific double, or multispecific ligand, as described herein. See also International Publication
No. WO 03/002609, the total teachings of which are incorporated herein by reference, with respect to the formatting of ligands. These specific double ligands comprise unique variable immunoglobulin domains having different binding specificities. These specific double ligands can comprise combinations of heavy and light chain domains. For example, the double specific ligand may comprise a VH domain and a VL domain, which may be linked together in the form of a scFv (e.g., using a suitable linker, such as Gly4Ser), or may be formatted in a bispecific antibody or in an antigen binding fragment thereof (eg, F (ab ') 2 fragment). The specific double ligands do not comprise complementary VH / V pairs that form a conventional two-chain antibody antigen binding site that binds to the antigen or epitope in a cooperative manner. Instead, the double format ligands comprise a complementary VH / V pair, wherein the V domains have different binding specificities. In addition, the specific double ligands may comprise one or more CH or CL domains, if desired. A joint region can also be included if desired. These combinations of domains, for example, can mimic natural antibodies, such as I gG or IgM, or fragments thereof, such as Fv, scFv, or F (ab ') 2 molecules. Other structures are envisaged, such as a single arm of an IgG molecule comprising the
domains VH, VL, VH1, and C. Preferably, the double specific ligand of the invention comprises only two variable domains, although several of these ligands can be incorporated together in the same protein, for example, two of these ligands can be incorporated into an IgG or a multimeric immunoglobulin. , such as IgM. Alternatively, in another embodiment, a plurality of specific double ligands are combined to form a multimer. For example, two different double specific ligands are combined to create a tetra-specific molecule. One of skill in the art will appreciate that the variable regions released and weighted from a double specific ligand produced according to the method of the invention, may be on the same polypeptide chain, or alternatively, on different polypeptide chains. In the case that the variable regions are on different polypeptide chains, then they can be linked by means of a linker, generally a flexible linker (such as a polypeptide chain), a chemical linker group, or any other method known in the art. In some embodiments, the linker can be a "natural linker" containing carboxy-terminal amino acids of an antibody variable domain, and amino-terminal amino acids of an antibody constant domain. For example, a natural linker may contain the carboxy-terminal amino acids of VK, and the amino-terminal amino acids of C K (eg, KVEIKRTVAAPS (SEQ ID NO: 706)). In other modalities, the
The linker may contain less Lys and Arg residues than the natural linker (eg, LVTVSSAST (SEQ ID NO: 707) or (LVTVSSGGGGSGGGS (SEQ ID NO: 708).) If desired, the linker may be mutated to replace some or all positively charged residues (eg, in a natural linker), such as Lys and / or Arg, with residues that are not positively charged at physiological pH For example, the Lys and / or Arg residues can be replaced with Asn, Leu, G ln, or Ser. This type of linker provides the advantage of reducing the sensitivity to the protease (e.g., serine protease, cysteine protease, matrix metalloprotease, pepsin, trypsin, elastase, chymotrypsin, carboxypeptidase, cathepsin G (eg, cathepsin G), proteinase 3) Examples of these linkers include, GQGTNVE IN RTVAAPS (SEQ ID NO: 71 0), GQGTNVE I NQTVAAPS (SEQ ID NO: 71 1), GQGTNVEIQRTVAAPS (SEQ ID NO : 712), or GQGTLVTVSSTVAAPS (SEQ ID NO: 713) Proteases (eg it, a serine protease, cysteine protease, matrix metalloprotease, pepsin, trypsin, elastase, chymotrypsin, carboxypeptidase, cathepsin (eg, cathepsin G), proteinase 3) function in the normal metabolism and change of proteins . However, in certain physiological states, such as inflammatory states (eg, chronic obstructive pulmonary disease), and in cancer, the amount of proteases present in a tissue, organ, or animal (eg, in the lung, in or adjacent to a tumor) may increase. This increase in
proteases can result in accelerated degradation and inactivation of the endogenous proteins and of the therapeutic and diagnostic peptides, polypeptides, and proteins that are administered. In fact, some agents that have potential to be used in vivo (for example, to be used in the treatment, diagnosis, or prevention of the disease), have only limited efficacy, because they are rapidly degraded and inactivated by the proteases. The invention relates to a ligand comprising a linker that is resistant to degradation by the protease. The protease resistant ligands of the invention provide several advantages. For example, a protease resistant ligand can be administered to a subject, and it can remain active in vivo for longer than protease sensitive agents. In accordance with the foregoing, the protease-resistant ligand will remain functional for a period of time sufficient to produce biological effects. A ligand or linker that is resistant to degradation by the protease is not substantially degraded by a protease when incubated with the protease under conditions suitable for protease activity for at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours,
at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 24 hours, at least about 36 hours, or at least about 48 hours. A ligand or linker is not substantially degraded when not more than about 25 percent, not more than about 20 percent, not more than about 1 5 percent, not more than about 14 percent, not more than about 13 percent, no more than about 1 2 percent, no more than about 1 1 percent, no more than about 10 percent, no more than about 9 percent, no more than about 8 percent percent, no more than about 7 percent, no more than about 6 percent, no more than about 5 percent, no more than about 4 percent, no more than about 3 percent, no more than about 2 percent, no more than about 1 percent, or substantially none of the ligand or linker is degraded by the protease after of incubation with the protease for at least about 2 hours. The degradation of the protein can be evaluated using any suitable method, for example by SDS-PAG E. The protease resistance can be assessed using any suitable method. For example, a protease can be added to a ligand or linker solution in a regulator
suitable (eg, phosphate buffered serum), to produce a ligand or linker / protease solution, such as a solution of at least about 0.01 percent (w / w) of protease, from about 0.01 percent to about 5 percent (w / w) of protease, from about 0.05 percent to about 5 percent (w / w) of protease, from about 0.1 percent to about 5 percent (w / w) of protease, from about 0.5 percent to about 5 percent (w / w) of protease, from about 1 percent to about 5 percent (w / w) of protease, of at least about 0.01 percent (weight / weight) of protease, from at least about 0.02 percent (w / w) of protease, of at least about 0.03 percent (w / w) of protease, of at least about 0.04 percent (weight / weight) of protease , from at least about 0.05 percent (weight / weight) of protease, from at least about 0.06 percent (w / w) of protease, from at least about 0.07 percent (w / w) of protease, at least about 0.08 percent (w / w) of protease, of at least about 0.09 percent (w / w) of protease, of at least about 0.1 percent (w / w) of protease, at least approximately 0.2 percent (w / w) of protease, at least
about 0.3 per cent (w / w) of protease, from at least about 0.4 percent (w / w) of protease, from at least about 0.5 percent (w / w) of protease, at least about 0.6 percent (w / w) of protease, from at least about 0.7 percent (w / w) of protease, from at least about 0.8 percent (w / w) of protease, to at least about 0.9 percent (w / w) of protease, from at least about 1 percent (w / w) of protease, of at least about 2 percent (w / w) of protease, of at least about 3 percent (weight / weight) protease, at least about 4 percent (w / w) protease, or about 5 percent (w / w) protease. The ligand or linker / protease mixture can be incubated at a temperature suitable for protease activity (e.g., at 337 ° C), and samples can be taken at time intervals (e.g., at 1 hour, etc.). ), and the protease reaction is stopped. The samples can then be analyzed for protein degradation using any suitable method, such as SDS-PAGE analysis. The results can be used to establish a time course of degradation. The ligands can be formatted as bi- or multi-specific antibodies or fragments of antibodies, or in structures other than bi- or multi-specific antibodies. The appropriate formats
include any suitable polypeptide structure, wherein a variable antibody domain or one or more of the CDRs thereof can be incorporated to confer antigen binding specificity on the structure. A variety of suitable antibody formats are known in the art, such as bispecific IgG type formats (eg, chimeric antibodies, humanized antibodies, human antibodies, single chain antibodies, heavy chain heterodimers and / or light chain antibodies). , antigen binding fragments of any of the foregoing (eg, an Fv fragment (e.g., single chain Fv (scFv), a disulfide linked Fv), a Fab fragment, a Fab 'fragment, a F fragment (ab ') 2), a single variable domain (for example, VH, V, VH H), a dAb, and modified versions of any of the above (for example, modified by the covalent attachment of polyalkylene glycol (eg, polyethylene glycol, polypropylene glycol, polybutylene glycol), or other suitable polymer). See PCT Publication Number PCT / GB03 / 002804, filed June 30, 2003, which is designated in the United States (International Publication Number WO 2004/081 026) with respect to the only PEGylated variable domains and dAbs , the appropriate methods for its preparation, the longer half-life in vivo of the only PEGylated variable domains, and the monomers and multimers of dAb, the appropriate PEGs, the preferred hydrodynamic sizes of the PEGs, and the preferred hydrodynamic sizes of the unique domains variables
PEGylated and from dAb monomers and multimers. All teaching of the Publication of the TCP Number PCT / GB03 / 002804 (International Publication Number WO 2004/081 026), including the portions referred to above, are incorporated herein by reference. 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, the ligands, including The dAb monomers, dimer, and trimers can be linked to an antibody Fc region, comprising 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 these polypeptides. In some embodiments, the ligand comprises one, two, or more of the dAbs of the same or different binding specificities, and CH2, CH3, CH2-CH3, -CH2-joint, -CH3-joint, -CH2-CH3-joint, a portion of articulation-CH2, a portion of articulation-CH3, or a portion articulation-CH2-CH3. In these modalities, CH2, CH3, CH2-CH3, CH2-joint, CH3-articulation, CH2-CH3-joint, the CH2-joint portion, the CH3-joint portion, and the articulation-portion. CH2-CH3 can be from any desired antibody, such as human IgG, such as human IgG1 or human IgG4. In some embodiments, the ligand of the invention comprises an anti-EG FR dAb or an anti-VEG F dAb that is fused (e.g.
directly or through a linker) with an Fc region of an antibody. In some embodiments, the ligand is a Fc fusion comprising an anti-VEG F dAb that is disulfide linked to an anti-EG FR dAb. In particular examples, the ligand comprises two or more dAbs (eg, two dAbs that bind to EGFR, two dAbs that bind to VEG F, one dAb that binds to EGFR, and one dAb that binds to VEGF) , and an Fc region, and the ligand has the structure, from the amino terminus to the carboxyl terminus, VH-VH-FC, V-VL-FC, VH-VL-FC, VL-VH-FC. For example, the ligand can have the structure VH-V? -articulation-CH2-CH3, V? -VH-joint-CH2-CH3, V? -V? -articulation-CH2-CH3, or VH-VH-joint- CH2-CH3. If desired, VH shown in any of the above formulas can be a VH H-DOS ligands containing Fc regions can be linked together to form a dimer, for example through a disulfide bond (e.g. the articulation region). In general terms the orientation of the polypeptide domains having a binding site with a binding specificity for a target (eg, dAbs), and if the ligand comprises a linker, is a matter of design choice. However, some orientations, with or without linkers, can provide the preferred link characteristics compared to other orientations. All orientations (e.g., dAb l -dAb2-Fc; dAb2-dAb1 -Fc) are encompassed by the invention, and ligands containing an orientation that provides the
The desired link characteristics can be easily identified by routine screening. The dAb ligands and monomers can also be combined and / or formatted into multiple ligand structures other than antibodies, to form multivalent complexes, which bind the target molecules to the same antigen, thereby providing superior avidity. For example, natural bacterial receptors, such as SpA, can be used as scaffolds for the grafting of CDRs, in order to generate ligands that specifically bind to one or more epitopes. Details of this procedure are described in U.S. Patent No. U.S. 5,831, 01. Other suitable scaffolds include those based on fibronectin and afibodies. Details of suitable procedures are described in International Publication No. WO 98/58965. Other suitable scaffolds include lipocalin and CTLA4, as described in van den Beuken et al., J. Mol. Biol. 31 0: 591-601 (2001), and scaffolds such as those described in International Publication Number WO 00/69907 (Medical Research Council), which are based, for example, on the ring structure of the Bacterial GroEL or other chaperone polypeptides. Protein scaffolds can be combined, for example, CDRs can be grafted onto a scaffold of CTLA4, and can be used together with the immunoglobulin VH and V domains to form a ligand. In the same way, they can be combined
fibronectin, lipocalin, and other scaffolds. A variety of methods suitable for the preparation of any desired format are known in the art. For example, antibody chains and formats can be prepared (eg, bispecific IgG type formats, chimeric antibodies, humanized antibodies, human antibodies, single chain antibodies, homodimers, and heterodimers of heavy and / or light chains of antibodies). , by expressing suitable expression constructs and / or culturing suitable cells (eg, hybridomas, heterohybridomas, recombinant host cells containing recombinant constructs encoding the format). In addition, formats such as antibody antigen binding fragment or antibody chains (eg, bispecific binding fragments, such as an Fv fragment (eg, single chain Fv (scFv), bound Fv) can be prepared. with disulfide), a Fab fragment, a Fab 'fragment, an F (ab') 2 fragment), by expression of suitable expression constructs, or by enzymatic digestion of the antibodies, for example using papain or pepsin. The ligand can be formatted as a multispecific ligand, for example, as described in International Publication Number WO 03/002609, the entire teachings of which are incorporated herein by reference. This multispecific ligand possesses more than one epitope binding specificity. In general terms, the multispecific ligand comprises two or more binding domains of
epitope, such as dAbs or non-antibody protein domain comprising a binding site for an epitope, for example an affinity, a domain SpA, a class A LDL receptor domain, an EGF domain, an avimer . The multispecific ligands can be further formatted as described herein. In some embodiments, the ligand is in an IgG type format. These formats have the conventional structure of four chains of one molecule of IgG (two heavy chains and two light chains), where one or more of the variable regions (VH and V) have been replaced with a dAb or with a single variable domain of a desired specificity. Preferably, each of the variable regions (two VH regions and two V regions) is replaced with a dAb or with a single variable domain. The dAb (s) or the only variable domains that are included in an 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 four dAbs that have the same specificity; bispecific, and comprises three dAbs that have the same specificity and another dAb that has a different specificity; bispecific and comprises two dAbs that have the same specificity and two dAbs that have a common but different specificity; trispecific and comprises first and second dAbs that
they have the same specificity, a third dAb with a different specificity, and a fourth dAb with a different specificity from the first, second, and third dAbs; or tetra-specific, and comprises four dAbs that each have a different specificity. Antigen binding fragments of IgG-like formats (eg, Fab, F (ab ') 2, Fab', Fv, scFv) can be prepared. In addition, a particular constant region of a portion of Fc (eg, of an IgG, such as IgG 1), a variant, or a portion thereof, may be selected in order to tailor the effector function . For example, if the complement activation and / or antibody-dependent cellular cytotoxicity (ADCC) function is desired, the ligand may be of a lgG 1-like format, if desired, the lgG 1 -type format may comprise a mutated constant region (variant IgG heavy chain constant region) to minimize binding to Fc receptors, and / or the ability to fix complement (see, eg, Winter et al., British Patent Number GB 2,209,757 B; Morrison et al., International Publication No. WO 89/071 42; Morgan et al., International Publication Number WO 94/29351, December 22, 1994). In some embodiments, the IgG-like formats may comprise an anti-EG FR dAb (eg, DOM 1 6-39-542, DOM 1 6-39-61 8, or DOM 1 6-39-61 9), a dAb anti-VEGF (eg, DOM 1 5-26-501), or an anti-EGFR dAb dAb, and an anti-VEGF dAb. The ligands of the invention can be formatted as a
fusion protein containing a first single variable domain of immunoglobulin that is directly fused with a second single variable domain of immunoglobulin. If desired, this format can also comprise a fraction that extends the half-life. For example, the ligand may comprise a first single variable domain of immunoglobulin that is directly fused with a second single variable domain of immunoglobulin that directly fuses with a single variable domain of immunoglobulin that binds to serum albumin. For example, the ligand can be an in-line fusion of two or more protein fractions that have a binding site with a binding specificity for EG FR that competes for the EGFR binding, with an anti-EGFR domain antibody ( example, any of the DOM 1 6 dAbs disclosed herein), and is fused with an anti-serum albumin dAb (e.g., any of the DOM7 dAbs disclosed herein). In some embodiments, protein fractions that have a binding site with a binding specificity for EG FR (eg, anti-EGFR dAbs), have different epitope specificities. In other examples, the ligand is an online fusion protein comprising a protein fraction having a binding site with a binding specificity for EGFR (eg, anti-EG FR dAb), a protein fraction having a linkage site with a binding specificity for VEGF, and an anti-serum albumin dAb. In particular modalities, this online merger
comprises DOM16-39-618 dAb and / or DOM16-39-619, and an anti-serum albumin dAb (eg, DOM16-39-618- DOM7h-14, DOM7h-14- DOM16-39-618, DOM16-39- 619- DOM7h-14, DOM7h-14 - DOM16-39-619). In other embodiments, the online fusion comprises a protein fraction (e.g., dAb) that has an amino acid sequence identity of at least about 80 percent, 85 percent, 87 percent, 90 percent , 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent with the amino acid sequence of DOM16-39-618, a protein fraction having an amino acid sequence identity of at least about 80 percent, 85 percent, 87 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent with the amino acid sequence of DOM16-39-619, and / or a protein fraction (eg, dAb) that has at least about 80 percent, 85 percent, 87 percent percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent percent with the amino acid sequence of an anti-serum albumin dAb disclosed herein, such as DOM7h-14. In other particular embodiments, the ligand comprises an anti-VEGF dAb, an anti-EGFR dAb, and an anti-serum albumin dAb.
(for example, DOM 1 5-1 0 - DOM 1 6-39 - dAb anti-serum albumin, DOM 1 6-39- DOM 1 5-1 0- dAb anti-serum albumin, DOM 1 5-26- 501 - DOM 16-39- dAb anti-serum albumin, DOM 1 6-39-DOM 1 5-26-501 - dAb anti-serum albumin). In other embodiments, the inline fusion comprises a protein fraction (eg, dAb) having an amino acid sequence identity of at least about 80 percent, 85 percent, 87 percent, 90 percent, percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent or 99 percent with the sequence of amino acids of an anti-VEGF dAb disclosed herein, such as DOM 1 5-1 0 or DOM 1 5-25-501, and / or a protein fraction having an amino acid sequence identity of at least about 80 percent, 85 percent, 87 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent , 98 percent or 99 percent with the amino acid sequence of an anti-EGFR dAb disclosed herein, such as DOM 1 6-39, and / or a protein fraction (e.g., dAb) that has an amino acid sequence identity of at least about 80 percent, 85 percent, 87 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent , 95 percent, 96 percent, 97 percent, 98 percent or 99 percent with the amino acid sequence of a serum anti-serum albumin dAb disclosed herein, such
as DOM7h- 14. In general terms, the orientation of polypeptide domains having a binding site with a binding specificity for 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-dAbl) are encompassed by the invention, and ligands containing an orientation that provides the desired link characteristics by screening can be easily identified. Formats that prolong the vine in half. The dAb ligands and monomers disclosed herein can be formatted to extend their serum half life in vivo. The longer half-life in vivo is useful in in vivo applications of immunoglobulins, especially antibodies, and more especially fragments of small-sized antibodies, such as dAbs. These fragments (Fvs, Fvs linked by d isulfide, Fvs, Fabs, scFvs, dAbs) are rapidly eliminated from the body, which may limit the clinical applications. A ligand can be formatted as a larger antigen binding fragment 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 hydrominodynamic size
larger, for example, by linking a polyalkylene glycol group (eg, a polyethylene glycol (PEG) group, polypropylene glycol, polybutylene glycol), serum albumin, transferrin, transferrin receptor, or at least the transferrin binding portion thereof , an Fc region of antibody, or by conjugation with an antibody domain. In some embodiments, the ligand (e.g., dAb monomer) is PEGylated. Preferably, the PEGylated ligand (e.g., dAb monomer) binds to VEGF and / or EGFR with substantially the same affinity or avidity as the same ligand that is not PEGylated. For example, the ligand may be a PEGylated ligand comprising a dAb that binds to VEGF or to EGFR with an affinity or avidity that differs from the avidity of the ligand in a non-PEGylated form by not more than a factor of about 1. , 000, preferably not greater than a factor of about 1 00, more preferably not greater than a factor of about 10, or with an affinity or avidity substantially unchanged in relation to the non-PEGylated form. See TCP Publication PCT Number / GB03 / 002804, filed June 30, 2003, which designates the United States (International Publication Number WO 2004/081 026), with respect to the only PEGylated variable domains and dAbs, the suitable methods for its preparation, the longer in vivo half-life of the only PEGylated variable domains and the dAb monomers and multimers, the appropriate PEGs, the preferred hydrodynamic sizes of the PEGs, and the sizes
preferred hydrodynamics of the only PEGylated variable domains and the dAb monomers and multimers. All teaching of the PCT Publication Number PCT / GB03 / 002804 (International Publication Number WO 2004/081 026), including the portions referred to above, is incorporated herein by reference. The hydrodynamic size of the ligands (e.g., dAb monomers and multimers) of the invention can be determined using methods that are well known in the art. For example, gel filtration chromatography can be used to determine the hydrodynamic size of a ligand. Gel filtration matrices suitable for determining 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 fraction attached to a dAb monomer) can be varied depending on the desired application. For exampleWhen it is intended that the ligand leaves the circulation and enters the peripheral tissues, it is desirable to maintain the hydrodynamic size of the ligand low to facilitate extravasation from the bloodstream. Alternatively, when it is desired to cause the ligand to remain 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 by the addition of a PEG fraction of 30 to 60 kDa (for example, linear or branched PEG, PEG of 30 to 40 kDa, such as the addition of two
fractions of PEG of 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 to reduce the secular effects of the toxic agents. The hydrodynamic size of the ligand (e.g., dAb monomer) and its serum half-life can also be increased by conjugation or binding of the ligand to a binding domain (e.g., antibody or antibody fragment) that is linked with an antigen or epitope that increases the half-life in vivo, as described herein. For example, the ligand (e.g., the dAb monomer) can be conjugated or linked to an antibody or an antibody or anti-Fc neonatal receptor fragment (e.g., an anti-SA or an anti-Fc receptor dAb). neonatal, Fab, Fab ', or scFv) or to an anti-SA poster or an anti-neonatal Fc receptor. Examples of albumin, albumin fragments, or albumin variants suitable for use in a ligand according to the invention are described in International Publication No. WO 2005 / 077042A2, which is incorporated herein by reference in its entirety. . In particular, the following albumins, fragments of albumin, or albumin variants can be used in the present invention: SEQ ID NO: 1 as disclosed in International Publication Number WO 2005 / 077042A2 (this sequence being explicitly incorporated) to the present disclosure by reference);
Fragment or variant of albumin comprising or consisting of amino acids 1 to 387 of SEQ ID NO: 1 of International Publication Number 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 of International Publication Number WO 2005 / 077042A2; (b) amino acids 76 to 89 of SEQ ID NO: 1 of International Publication Number WO 2005 / 077042A2; (c) amino acids 92 to 100 of SEQ ID NO: 1 of International Publication Number WO 2005 / 077042A2; (d) amino acids 170 to 176 of SEQ ID NO: 1 of International Publication Number WO 2005 / 077042A2; (e) amino acids 247 to 252 of SEQ ID NO: 1 of International Publication Number WO 2005 / 077042A2; (f) amino acids 266 to 277 of SEQ ID NO: 1 of International Publication Number WO 2005 / 077042A2; (g) amino acids 280 to 288 of SEQ ID NO: 1 of International Publication Number WO 2005 / 077042A2; (h) amino acids 362 to 368 of SEQ ID NO: 1 of International Publication Number WO 2005 / 077042A2; (i) amino acids 439 to 447 of SEQ ID NO: 1 of International Publication Number WO 2005 / 077042A2; (j) amino acids 462 to 475 of SEQ ID NO: 1 of International Publication Number WO 2005 / 077042A2; (k) amino acids 478 to 486 of SEQ ID NO: 1 of International Publication Number WO 2005 / 077042A2; and (I) amino acids 560 to 566 of SEQ ID NO: 1 of WO International Publication Number
2005 / 077042A2. Other examples of albumins, fragments, and analogs suitable for use in a ligand according to the invention are described in International Publication Number WO 2005 / 077042A2, which is incorporated herein by reference in its entirety. In particular, the following albumins, fragments, or variants may be used in the present invention: Human serum albumin as described in International Publication Number WO 03 / 076567A2, for example in Figure 3 (this sequence information being incorporated explicitly to the present disclosure by reference); Human serum albumin (HA), which consists of a single non-glycosylated polypeptide chain of 585 amino acids, with a molecular weight of the formula of 66,500 (see Meloun, et al., FEBS Letters 55: 136 (1975); Behrens, and collaborators, Fed. Proc. 34: 591 (1975); Lawn, et al., Nucleic Acids Research 9: 6102-6114 (1981); Minghetti, et al., J. Biol Chem.261: 6141 (1986)); A polymorphic variant or an analogue or fragment of albumin, as described in Weitkamp, et al., Ann. Hum. Genet.37: 219 (1973); A fragment or variant of albumin as described in European Patent Number 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 European Patent Number EP 399666, for example HA (1 -1 77) and HA (1 -200), and fragments between HA (1 -X), wherein X is any number from 1 78 to 1 99. When one (one or more) fraction that prolongs the half-life (eg, albumin, transferrin, and fragments and analogs thereof) is used in the ligands of the invention, it is can be conjugated to the ligand using any suitable method, such as by direct fusion to the target binding moiety (eg, dAb or antibody fragment), for example, using a single nucleotide construct that encodes a protein fusion, wherein the fusion protein is encoded as a single polypeptide chain with a fraction that extends the N- or C-terminally localized half-life for the cell surface target binding fractions. Alternatively, conjugation can be achieved by using a peptide linker between the fractions, for example, a peptide linker as described in International Publication Number WO 076567A2 or International Publication Number WO 2004/00301 9 (these being incorporated disclosures of linkers by reference to the present disclosure to provide the Examples for use in the present invention). Typically, a polypeptide that improves serum half-life in vivo, is a polypeptide that occurs naturally in vivo, and that resists degradation or removal by endogenous mechanisms that remove the material
unwanted organism (for example, the human being). For example, a polypeptide that enhances the vine to serum media in vivo can be selected from extracellular matrix proteins, proteins found in the blood, proteins found in the blood-brain barrier, or the neural tissue, the proteins located in the kidney, liver, lung, heart, skin, or bone, the tension proteins, the disease-specific proteins, or the proteins involved in the transport of Fc. Suitable polypeptides that improve serum half-life in vivo include, for example, transferrin-specific receptor-specific neuropharmaceutical fusion proteins (see U.S. Patent No. 5,977,307, the teachings of which are incorporated herein by reference). the present as a reference), the brain capillary endothelial cell receptor, transferrin, transferrin receptor (eg, soluble transferrin receptor, insulin, insulin-like growth factor receptor-1 (IGF-1), factor receptor Growth type insulin-2 (IGF-2), insulin receptor, blood coagulation factor X, s1-antitrypsin, and HN F-1 a .. Suitable polypeptides that improve serum half-life also include alpha-1 glycoprotein (orosomucoid, AAG), alpha-1 antichymotrypsin (ACT), alpha-1 microglobulin (HC protein, AIM), antithrombin II I (AT III), apolipoprotein A-1 (Apo A-1), apolipoprotein B (Apo B), ceruloplasmin (Cp), complement component C3 (C3), complement component C4 (C4), esterase inhibitors C 1
(C 1 INH), C-reactive protein (CRP), ferritin (FE R), hemopexin (H PX), lipoprotein (a) (Lp (a)), mannose binding protein (MBP), myoglobin (Myo) , prealbumin (transpiretin; PAL), retinol binding protein (RBP), and rheumatoid factor (RF). Suitable proteins from the extracellular matrix include, for example, collagens, laminins, integrins, and fibronectin. Collagens are the main proteins of the extracellular matrix. Approximately 1 5 types of collagen molecules are currently known, which are found in different parts of the body, for example type I collagen (which accounts for 90 percent of body collagen (found in bone, skin, tendon, ligaments, cornea, internal organs, or collagen type I I found in cartilage, spinal disc, notochord, and vitreous humor of the eye. Suitable proteins in the blood include, for example, plasma proteins (eg, fibrin, α-2 macroglobulin, serum albumin, fibrinogen (eg, fibrinogen A, fibrinogen B), serum amyloid protein A, haptoglobin, profilin , ubiquitin, uteroglobulin, and β-2 microglobulin), enzymes, and enzyme inhibitors (eg, plasminogen, lysozyme, cystatin C, anti-trypsin alpha-1, and the pancreatic trypsin inhibitor), immune system proteins, such as proteins of immunoglobulin na (e.g., IgA, IgD, IgE, IgG, IgM, immunoglobulin light chains (kappa / lambda)), transport proteins (e.g., retinol binding protein, α-1 microglobulin),
defensins (for example, beta-defensin 1, neutrophil defensin 1, neutrophil defensin 2, and neutrophil defensin 3), and the like. Suitable proteins found in the blood-brain barrier or neural tissue include, for example, melanocortin receptor, honey, ascorbate carrier, and the like. Suitable polypeptides that improve serum half-life in vivo also include proteins located in the kidney (eg, polycystin, type IV collagen, K1 organic anion transporter, Heymann antigen), proteins located in the liver ( for example, dehydrogenase rogenase, G250), proteins located in the lung (eg, secretory component, which binds to IGA), proteins located in the heart (eg, HSP27, which is associated with dilated cardiomyopathy ), proteins located in the skin (for example, keratin), bone-specific proteins, such as morphogenic proteins (BM Ps), which are a subset of the transforming growth factor protein super-family. β, which demonstrate osteogenic activity (eg, BM P-2, BM P-4, BM P-5, BM P-6, BM P-7, BM P-8), tumor-specific proteins (e.g. trophoblast antigen, recept or herceptin, estrogen receptor, cathepsins (for example, cathepsin B, which can be found in the liver and spleen)). The specific proteins of suitable diseases
they include, for example, antigens that are expressed only on activated T cells, including LAG-3 (lymphocyte activation gene), osteoprotegerin ligand (OPGL, see Nature 402, 304-309 (1999)), OX40 (a member of the family of TNF receptors, expressed on activated T cells, and specifically amplified in the cells producing human type I T-cell leukemia virus (HTLV-I), see Immunol. 65 (1): 263-70 (2000)). Suitable disease-specific proteins also include, for example, metalloproteases (associated with arthritis / cancers), including Drosophila CG651 2, human paraplegina, human FtsH, human AFG3L2, murine FtsH; and angiogenic growth factors, including acid fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), vascular endothelial growth factor / vascular permeability factor (VEGF / VPF), growth factor transformant-a (TG Fa), necrosis factor your moral-a (TN Fa), angiogenin, interleukin-3 (I L-3), interleukin-8 (I L-8), platelet-derived endothelial growth factor ( PD-ECGF), placental growth factor (PIG F), platelet-derived growth factor of midchin-B B (PDG F), and fractalkine. Suitable polypeptides that improve serum half-life in vivo also include strain proteins, such as heat shock proteins (HSPs). Heat shock proteins are usually found intracellularly. When they are found extracellularly, this is an indicator that a
cell has died and has spilled its contents outwards. This unscheduled cell death (necrosis) occurs when, as a result of trauma, disease, or injury, extracellular heat shock proteins trigger a response from the immune system. The binding to the extracellular heat shock protein may result in localization of the compositions of the invention at a disease site. Suitable proteins involved in Fc transport include, for example, the Brambell receptor (also known as FcRB). This Fc receiver has two functions, both of which are potentially useful for the supply. The functions are: (1) transport of IgG from the mother to the child through the placenta, (2) protection of IgG from degradation, thus prolonging its half-life in serum. It is thought that the receptor recycles IgG from the endosomes. (See Holliger et al., Nat. Biotechnol. 1 5 (7): 632-6 (1 997)). The methods for pharmacokinetic analysis and the determination of the half-life of the ligand will be familiar to those skilled in the art. Details can be found in Kenneth, A et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists, and in Peters et al., Pharmacokinetic analysis: A Practical Approach (1 996). Reference is also made to "PharmacokÃnetics", M Gibaldi &; D Perron, published by Marcel Dekker, Second Review, extra edition (1 982), which describes pharmacokinetic parameters, such as
half-lives of t-alpha and t-beta, and the area under the curve (AUC). Ligands containing a Fraction of Toxi na or a Toxi na The invention also relates to ligands comprising a toxin moiety or a toxin. Suitable toxin fractions comprise a toxin (eg, surface activity toxin, cytotoxin). The toxin or toxin fraction can be linked or conjugated to the ligand using any suitable method. For example, the toxin or toxin fraction can be covalently linked to the ligand directly or through a suitable linker. Suitable linkers can include non-dissociable or dissociable linkers, for example, pH-releasable linkers that comprise a dissociation site for the cellular enzyme (e.g., cellular esterases, cellular proteases such as cathepsin B). These dissociable linkers can be used to prepare a ligand that can release a toxin fraction or a toxin after the ligand is internalized. A variety of methods can be employed to bind or conjugate a toxin or toxin moiety with a ligand. The particular method selected will depend on the fraction of toxin or toxin and the ligand to be bound or conjugated. If desired, linkers containing terminal functional groups can be used to bind the ligand and the toxin or toxin moiety.
In general terms, conjugation is carried out by the reaction of the toxin or toxin fraction containing a reactive functional group (or which is modified to contain a group
reactive functional) with a linker, or directly with a ligand. Covalent bonds formed by the reaction of a toxin or toxin fraction that contains (or is modified to contain) a chemical or chemical functional group that, under the appropriate conditions, can react with a second chemical group, forming this way a covalent bond. If desired, a suitable reactive chemical group can be added to the ligand or linker using any suitable method. (See, for example, Hermanson, G.T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996)). Many combinations of suitable reactive chemical groups are known in the art, for example, an amine group can react with an electrolyte group, such as tosylate, methylate, halogen (chlorine, bromine, fluorine, iodine), N-hydroxy-succinimidyl-ester (NHS), and the like. The thiols can react with maleimide, iodoacetyl, acryloyl, pyridyl disulfides, thiol of 5-thiol-2-nitro-benzoic acid (TNB-thiol), and the like. An aldehyde functional group can be coupled with molecules containing amine or hydrazide, and an azide group can react with a trivalent phosphorus group to form phosphoramidate or phosphorimide bonds. Suitable methods for introducing activating groups into molecules are known in this field (see, for example, Hermanson, G.T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996)). Suitable toxin fractions and toxins include, for example, a maytansinoid (eg, maytansinol, for example
DM 1, DM4), a taxane, a calicheamicin, a duocarmycin, or derivatives thereof. The maytansinoid may be, for example, maytansinol an maytansinol analogue. Examples of the maytansinol analogues include those having a modified aromatic ring (eg, CD-1 9 -dechloro, C-20-demethoxy-C-20-acyloxy), and those having modifications in other positions (e.g. , C-9-CH, -C 1 4-alkoxymethyl, C-1 4-hydroxymethyl, or aceloxymethyl, C-1 5-hydroxyl / acyloxy, C-1 5-methoxy, C-1 8-N-demethyl, 4,5-deoxy). Maytansinol and maytansinol analogs are described, for example, in U.S. Patent Nos. 5,208,020 and 6,333.41, the content of which is incorporated herein by reference. Maytansinol can be coupled with antibodies and antibody fragments using, for example, a N-succinimidyl 3- (2-pyridyl-dithio) -propionate (also known as N-succinimidyl 4- (2-pyridyl-dithio) -pentanoate, or SPP), 4-succinimidyl-oxycarbonyl- a- (2-pyridyl-dithio) -toluene (SM PT), 3- (2-pyridyl-dithio) -butyrate of N-succinimidyl (SDPB), 2-imino-thiolane, or S-acetyl-succinic anhydride. The taxane can be, for example, a taxol, taxotere, or a novel taxane (see, for example, International Publication Number WO 01/3831 8). The calicheamicin can be, for example, a calcium amylamino complexing with bromine (for example, an alpha-, beta-, or gamma-bromine complex), a calicheamycin complexed with iodine (for example, an alpha- complex, beta-, or gamma-iodine), or analogs and mimetics thereof. The
Calicheamicins forming complex with bromine include 11-BR, 12-BR, I3-BR, I4-BR, J1-BR, J2-BR, and K1-BR. The complex forming calicheamicins include 11-1, 12-1, 13-1, J1-I, J2-I, L1-I, and K1-BR. Calicheamicin and the mutants, analogs, and mimetics thereof, are described, for example, in U.S. Patent Nos. 4,970,198; 5,264,586; 5,550,246; 5,712,374, and 5,714,586, the content of each of which is incorporated herein by reference. Duocarmycin analogs (e.g., KW-2189, DC88, DC89 CB1-TMI), and derivatives thereof, are described, for example, in U.S. Patent No. 5,070,092, in the U.S. Pat. United States of America Number 5,187,186, in U.S. Patent Number 5,641,780, U.S. Patent Number 5,641,780, U.S. Patent Number 4,923,990, and U.S. Pat. United States of America Number 5,101,038, the content of each of which is incorporated herein by reference. Examples of other toxins include, but are not limited to, anti-metabolites (e.g., methotrexate, 6-mercapto-purine, 6-thioguanine, cytarabine, 5-fluoro-uracil, decarbazine), alkylating agents (e.g., mechlorethamine) , thiotepa-chlorambucil, CC-1065 (see U.S. Patent Nos. 5,475,092, 5,585,499, 5,846,545), melphalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan, dibromo-
mannitol, streptozotocin, mitomycin C, and cis-dichloro-diamine / platinum (II) (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin , mitramycin, mitomycin, puromycin, anthramycin (AMC), duocarmycin and analogues or derivatives thereof, and anti-mitotic agents (eg, vincristine, vinblastine, taxol, auristatins (eg, auristatin E), and maytansinoids, and the like or homologs thereof The toxin may also be a surface-active toxin, such as a toxin that is a generator of free radicals (eg, toxin fractions containing selenium), or a fraction containing radionuclide. contain suitable radionuclides include, for example, the fractions containing radioactive iodine (131l or 125l), yttrium (90Y), lutetium (177Lu), actinium (225Ac), praseodymium, astatine (211At), rhenium (186Re), bismuth (212Bi or 213Bi), indium (111ln), technetium ("mTc), 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, a polypeptide, or a peptide, from bacterial sources, for example diphtheria toxin, Pseudomonas exotoxin (PE), and plant proteins, for example, ricin A chain (RTA), ribosome inactivating proteins (RIPs), gelonin, antiviral protein of ivy, saporin, and
dodecandrona, which can be contemplated to be used as toxins. Anti-sense nucleic acid compounds designed to bind, disable, promote degradation or prevent production of the mRNA responsible for the generation of a particular target protein can also be used, they can also be used as a toxin. Anti-sense compounds include anti-sense, single-stranded or double-stranded RNA or DNA, oligonucleotides, or analogs thereof, which can hybridize specifically to the individual mRNA species and prevent transcription and / or processing of RNA of the mRNA species, and / or la- translating the encoded polypeptide, and thereby effecting a reduction in the amount of the respective encoded polypeptide. Ching, et al., Proc. Nati Acad. Sci. USA. 86: 1 0006-1 001 0 (1,989); Broder, and collaborators, Ann. Int. Med. 1 1 3: 604-618 (1990); Loreau, et al., FEBS Letters 214: 53-56 (1990); Useful anti-sense therapeutics include, for example: Veglin â„¢ R (VasGene) and OGX-01 1 (Oncogenix). Toxins can also be photoactive agents. Suitable photoactive agents include the porphyrin-based materials, such as porfimer-sodium, the green porphyrins, chlorin E6, the hematoporphyrin derivative itself, phthalocyanines, etiopurpuri nas, texaphrine, and the like. The toxin may 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. These antibodies or antibody fragments (dAbs) can be directed to defined sub-cellular compartments or targets. For example, antibodies or antibody fragments (dAbs) can be linked to an intracellular target selected from erbB2, EGFR, BCR-ABL, p21Ras, Caspase 3, Caspase 7, Bcl-2, p53, Cyclin E, ATF- 1 / CREB, HPV16 E7, HPI, collagenase type IV, cathepsin L, as well as others described in Kontermann, RE, Methods, 34: 163-170 (2004), incorporated herein by reference in its entirety. Polypeptide Domains Linking with VEGF The invention provides polypeptide domains (eg, unique immunoglobulin variable domains, dAb monomers), which has a binding site with a binding specificity for VEGF. In preferred embodiments, the polypeptide domain (e.g., dAb) binds to VEGF with an affinity (KD;
(kd) / Kact, Vado (ka)) from 300 nM to 1 pM (ie, 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 from 1 nM to 1 pM, for example a KD of 1 x 10"7 M or less, preferably 1 x 10 ~ 8 M or less, more preferably 1 x 10 ~ 9 M or less. less, in a convenient manner of 1 x 10"10 M or less and most preferably 1 x 10" 11 M or less; and / or a KdeSacted index constant 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 s" \ more preferably 5 x 10"3 s" 1 to 1 x 10"5 s" \ for example 5 X 10"1 s" 1
or less, preferably 1 x 10"2 s" 1 or less, conveniently 1 x 103 s "1 or less, more preferably 1 x 10 ~ 4 s" 1 or less, still 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 a specificity of link for VEGF competes for the link with 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 (SEO 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 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), TAR1526-503 (SEQ ID NO: 152)
TAR15-26-504 SEQ ID NO 153), TAR1526-505 (SEQ ID NO: 154)
TAR15-26-506 SEQ ID NO 155), TAR1526-507 (SEQ ID NO: 156)
TAR15-26-508 < 'SEQ ID NO 157), TAR1526-509 (SEQ ID NO: 158)
TAR15-26-510 (, SEQ ID NO 159), TAR1526-511 (SEQ ID NO: 160)
TAR15-26-512 I SEQ ID NO 161), TAR1526-513 (SEQ ID NO: 162)
TAR15-26-514 SEQ ID NO 163), TAR1526-515 (SEQ ID NO: 164)
TAR15-26-516, SEQ ID NO 165), TAR1526-517 (SEQ ID NO: 166)
TAR15-26-518 [SEQ ID NO 167), TAR1526-519 (SEQ ID NO: 168)
TAR15-26-520; SEQ ID NO: 169) TAR1526-521 (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) TAR1526-525 (SEQ ID NO: 174)
TAR15-26-526 [SEQ ID NO 175), TAR1526-527 (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 some embodiments, the polypeptide domain having a binding site with a binding specificity for VEGF competes for binding with VEGF, with TAR15-26-555 (SEQ ID NO: 704). In some embodiments, the polypeptide domain having a binding site with a binding specificity for VEGF comprises an amino acid sequence having an amino acid sequence identity of at least about 80 percent, at least about 85 percent , at least about 90 percent, at least about 91 percent, at least about 92 percent, at least about 93 percent, at least about 94 percent, at least about 95 percent, when less about 96 percent, at least about 97 percent, at least about 98 percent, or at least about 99 percent 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) TAR15-265-541 (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 TAR15-265-549 (SEQ ID NO: 198), TAR15-26-550 (SEQ ID NO: 539), i / TAR15-26 -551 (SEQ ID NO: 540). In some embodiments, the polypeptide domain that has
a binding site with a binding specificity for VEGF comprises a sequence of amino acids having an amino acid sequence identity of at least about 80 percent, at least about 85 percent, at least about 90 percent at least about 91 per cent, at least about 92 per cent, at least about 93 per cent, at least about 94 per cent, at least about 95 per cent, at least about 96 per cent, at least about 97 percent, at least about 98 percent, or at least about 99 percent with the amino acid sequence of TAR1 5-26-555 (SEQ ID NO: 704). In preferred embodiments, the polypeptide domain having a binding site with a binding specificity for VEGF comprises an amino acid sequence having an amino acid sequence identity of at least about 90 percent, at least about 91 percent , at least about 92 percent, at least about 93 percent, at least about 94 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, or at least about 99 percent, 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), and TAR15-26 (SEQ ID NO: 123). For example, the polypeptide domain having a binding site with a 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 a binding specificity for VEGF, competes with any of the dAbs disclosed herein by the linkage to VEGF. Preferably the polypeptide domain having a binding site with a binding specificity for VEGF is a single immunoglobulin variable domain. The polypeptide domain having a binding site with a 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, a polypeptide domain having a binding site with a binding specificity for VEGF comprises a universal structure, as described herein. The universal structure can be a V (V? Or VK) structure, such as a structure comprising the amino acid sequences of structure encoded by the segment of the immunoglobulin gene DPK1, DPK2, DPK3, DPK4, DPK5, DPK6, DPK7, DPK8 , DPK9, DPK10, DPK12, DPK13, DPK15, DPK16, DPK18,
DPK1 9, DPK20, DPK21, DPK22, DPK23, DPK24, D PK25, DPK26, or DPK28 of the human germinal line. If desired, the VL structure may further comprise the amino acid sequence of structure encoded by the segment of the immunoglobulin gene J? 1, J? 2, J? 3, J? 4, or J? 5 of the human germinal line. In other embodiments, the universal structure can be a VH structure, such as a structure comprising the amino acid sequences of structure encoded by the segment of the immunoglobulin gene DP4, DP7, DP8, DP9, DP 10, DP31, DP33, DP38 , DP45, DP46, DP47, DP49, DP50, DP51, DP53, DP54, DP65, DP66,
DP67, DP68, or DP69 of the human germinal line. If desired, the VH structure may further comprise the amino acid sequence of structure encoded by the segment of the immunoglobulin gene JH 1, J H2, JH3, JH4, JH4b, JH5, and JH6 of the human germline. In certain embodiments, the polypeptide domain having a binding site with a 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 genetic segment of the human germinal line antibody, or the amino acid sequence of one or more of these structure regions collectively comprise up to five amino acid differences relative to the amino acid sequence of this correspondingly encoded structure region
by a genetic segment of the human germinal line antibody. In other embodiments, the amino acid sequences of FW1, FW2, FW3, and FW4 of the polypeptide domain having a binding site with a binding specificity for VEG F, are the same as the amino acid sequences of the corresponding structure regions encoded by a genetic segment of the human germline antibody, 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 the corresponding structure regions encoded by that genetic segment of the human germinal line antibody. In other embodiments, the polypeptide domain having a binding site with a binding specificity for VEGF comprises the FW1, FW2, and FW3 regions, and the amino acid sequence of these regions FW1, FW2, and FW3 are the same as the sequences of amino acids of the corresponding structure regions encoded by the genetic segments of the human germline antibody. In particular embodiments, the polypeptide domain having a binding site with a binding specificity for VEGF, comprises structure V DPK9, or a VH structure selected from the group consisting of DP47, DP45, and DP38. The polypeptide domain having a binding site with a specificity of
linkage for CD38 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 a binding specificity for VEG F, is substantially resistant to accumulation. For example, in some embodiments, less than about 10 percent is accumulated, less than about 9 percent, less than about 8 percent, less than about 7 percent, less than about 6 percent. , less than about 5 percent, less than about 4 percent, less than about 3 percent, less than about 2 percent, or less than about 1 percent of the polypeptide domain that has a site of link with a binding specificity for VEGF, when a solution of 1 to 5 milligrams / milliliter, of 5 to 10 milligrams / milliliter, of 10 to 20 milligrams / milliliter, of 20 to 50 milligrams / milliliter, of 50 to 100 milligrams / milliliter, 1 00 to 200 milligrams / milliliter, or 200 to 500 milligrams / milliliter of ligand or dAb in a solvent that is routinely used for drug formulation, such as serum, regulated serum, citrate regulatory serum, water, u n emulsion, and any of these solvents with an acceptable excipient, such as those approved by the FDA, is maintained at approximately 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, 1 5-20 ° C, 1 0-1 5 ° C, 5-1 0 ° C, 2 -5 ° C, 0-2 ° C, -10 ° C to 0 ° C, -20 ° C to -10 ° C, -40 ° C to -20 ° C, -60 ° C
at -40 ° C, or -80 ° C to -60 ° C, for a period of time, for example, of approximately 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. Accumulation can be evaluated using any suitable method, such as, by microscopy, evaluating the turbidity of a solution by visual inspection or spectroscopy, or by any other suitable method. Preferably, the accumulation is evaluated by dynamic light scattering. Polypeptide domains that have a binding site with a binding specificity for VEGF that are resistant to accumulation provide several advantages. For example, these polypeptide domains having a binding site with a binding specificity for VEGF, can be easily produced in a high yield 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 accumulation and loss of activity. In addition, the polypeptide domain having a binding site with a binding specificity for VEG F that is resistant to accumulation, can be produced more economically than other antigen or epitope binding polypeptides (e.g. conventional antibodies). For example, in general terms, the preparation of the antigen binding peptides
or epitopes intended for in vivo applications includes processes (e.g., gel filtration) that remove accumulated polypeptides. Failure to remove these aggregates can result in a preparation that is not suitable for in vivo application, due, for example, to the fact that aggregates of an antigen binding polypeptide that is intended to act as an antagonist, can function as an agonist through the induction of cross-linking or aggregating 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 adhere. In contrast, the accumulation-resistant polypeptide domain having a binding site with a binding specificity for VEGF of the invention, can be prepared for in vivo applications, without the need to include process steps that remove the aggregates, and it can be used in in vivo applications if n the aforementioned drawbacks caused by the polypeptide aggregates. In some embodiments, the polypeptide domain having a binding site with a binding specificity for VEG F, which is deployed in a reversible manner when heated to a temperature (Ts), and cooled to a temperature (Te), wherein Ts is greater than the melting temperature (Tm) of the polypeptide domain having a binding site with a binding specificity for VEG F, and Te is lower than the fusion temperature of the domain of
polypeptide having a binding site with a binding specificity for VEG F. For example, the polypeptide domain having a binding site with a binding specificity for VEGF can be displayed reversibly when heated to 80 ° C and cooled to about room temperature. A polypeptide that is deployed in a reversible manner loses its function when it is deployed, but gains the function again when it replicates. These polypeptides are distinguished from polypeptides that accumulate when they unfold, or that retract inappropriately (poorly folded polypeptides), that is, they do not regain function. The deployment and refolding of the polypeptide can be evaluated, for example, by directly or indirectly detecting the structure of the polypeptide, using any suitable method. For example, the structure of the polypeptide can be detected by circular dichroism (CD) (eg, UV-far CD, near-UV CD), fluorescence (eg, fluorescence of the side chains of tryptophan), susceptibility to proteolysis, nuclear magnetic resonance (RM N), or by detecting or measuring a polypeptide function that depends on an appropriate fold (e.g., binding to a target ligand, binding to a generic ligand). In one example, the deployment of the polypeptide is evaluated using a functional assay, wherein the loss of the binding function (e.g., the binding of a generic and / or target ligand, the binding of a substrate) indicates that the polypeptide is unfolded.
The degree of unfolding and refolding of a polypeptide domain having a binding site with a binding specificity for VEGF, can be determined using a deployment and denaturation curve. A deployment curve can be produced by plotting the temperature as the ordinate, and the relative concentration of the folded polypeptide as the abscissa. The relative concentration of a folded polypeptide domain having a binding site with a binding specificity for VEG F can be determined directly or indirectly using any suitable method (e.g., CD, fluorescence, binding assay). For example, a solution of a polypeptide domain having a binding site with a binding specificity for VEGF can be prepared, and the ellipticity of the solution can be determined by circular dichroism. The ellipticity value obtained represents a relative concentration of the folded ligand or dAb monomer of 1 00 percent. The polypeptide domain that has a binding site with a binding specificity for VEG F in the solution, it is then deployed by increasing the temperature of the solution, and the ellipticity is determined in suitable increments (for example, after each increase of a degree in temperature). The polypeptide domain having a binding site with a binding specificity for VEGF in solution, is then refolded by increasingly reducing the temperature of the solution, and the ellipticity is determined in suitable increments. The data can be graphed to produce
a deployment curve and a fallback curve. The unfolding and retracting curves have a characteristic sigmoid shape that includes a portion where molecules of the polypeptide domain that have a binding site with a binding specificity for VEGF, a folding / unfolding transition, are folded. of the polypeptide domain having a binding site with a binding specificity for VEGF are deployed to different degrees, and a portion where the polypeptide domain that has a binding site with a binding specificity for VEGF is deployed. The intercept of the y-axis of the refolding curve is the relative amount of the refolded polypeptide domain having a binding site with a binding specificity for recovered VEGF. A recovery of at least about 50 percent, or at least about 60 percent, or at least about 70 percent, or at least about 80 percent, or at least about 85 percent , or at least about 90 percent, or at least about 95 percent, indicates that the ligand or dAb monomer is displayed in a reversible manner. In a preferred embodiment, the reversibility of the deployment of a polypeptide domain having a binding site with a binding specificity for VEGF is determined by preparing a solution of the polypeptide domain having a binding site with a binding specificity for VEGF , and graphing the curves of
unfolding and retreating with the heat. The solution of the polypeptide domain having a binding site with a binding specificity for VEG F can be prepared in any suitable solvent, such as an aqueous buffer having a suitable pH to allow a polypeptide domain having a binding site with a binding specificity for VEGF (eg, a pH that is approximately three units greater or less than the isoelectric point (pl)). The solution of the polypeptide domain having a binding site with a binding specificity for VEGF is sufficiently concentrated to allow deployment / folding to be detected. For example, the dAb monomer or ligand solution can be from about 0.1 μM to about 1000 μM, or preferably from about 1 μM to about 1.0 μM. If the melting temperature (Tm) of a polypeptide domain having a binding site with a binding specificity for VEGF is known, the solution can be heated to about 10 ° C below the Tm (Tm-1 0 ), and the fold can be evaluated by ellipticity or fluorescence (eg, UV-distant CD scan from 200 nanometers to 250 nanometers, CD of fixed wavelength at 235 nanometers or 225 nanometers, fluorescent emission spectra of tryptophan from 300 to 450 nanometers with excitation at 298 nanometers), to provide 1 00 percent relative dAb ligand or monomer. Then the solution heats up to at least 10 degrees
above the Tm (Tm + 10) in previously determined increments (for example, increments of approximately 0.1 to approximately 1 degree), and ellipticity or fluorescence is determined in each increment. Then, the polypeptide domain having a binding site with a binding specificity for VEG F is replicated by cooling to at least Tm-1 0 in previously determined increments, and the ellipticity or fluorescence at each increment is determined. If the melting temperature of a polypeptide domain having a binding site with a binding specificity for VEG F is not known, the solution can be deployed by heating increasingly from about 25 ° C to about 1000 ° C, and then Fold increasingly cooling to at least approximately 25 ° C, and the ellipticity or fluorescence is determined in each increment of heating and cooling. The obtained data can be graphed to produce a deployment curve and a fallback curve, where the y-axis intercept in the fallback curve is the relative amount of recovered refolded protein. In some embodiments, the polypeptide domain having a binding site with a binding specificity for VEGF does not comprise a variable domain of camelid immunoglobulin, or one or more structure amino acids that are unique to the immunoglobulin variable domains encoded by the genetic segments of the germinal lineage of camelid.
Preferably, the polypeptide domain having a binding site with a binding specificity for VEGF is secreted in an amount of at least about 0.5 milligrams / liter when expressed in E. coli, or in Pichia species (e.g. , P. pastoris). In other preferred embodiments, a polypeptide domain having a binding site with a binding specificity for VEGF is secreted in an amount of at least about 0.75 milligram / liter, of at least about 1 milligram / liter, of at least about 4 milligrams / liter, of at least about 5 milligrams / liter, of at least about 10 milligrams / liter, of at least about 15 milligrams / liter, of at least about 20 milligrams / liter, of at least about 25 milligrams / liter, of at least about 30 milligrams / liter, of at least about 35 milligrams / liter, of at least about 40 milligrams / liter, of at least about 45 milligrams / liter, or of at least about 50 milligrams / liter, or at least about 1 00 milligrams / liter, or at least about 200 milligrams / liter, or at least about 300 milligrams / liter, or at least approximately 400 milligrams / liter, or at least approximately 500 milligrams / liter, or at least
about 600 milligrams / liter, or at least about 700 milligrams / liter, or at least about 800 milligrams / liter, at least about 900 milligrams / liter, or at least about 1 gram / liter, when expressed in E. coli or in Pichia species (for example, P. pastoris). In other preferred embodiments, a polypeptide domain having a binding site with a binding specificity for VEGF, is secreted in an amount of at least about 1 milligram / liter to at least about 1 gram / liter, of at least about 1 milligram / liter to at least about 750 milligrams / liter, from at least about 1 00 milligrams / liter to at least about 1 gram / liter, from at least about 200 milligrams / liter to at least about 1 gram / liter, when less about 300 milligrams / liter to at least about 1 gram / liter, from at least about 400 milligrammes / liter to at least about 1 gram / liter, from at least about 500 milligrams / liter to at least about 1 gram / liter, from at least about 600 milligrams / liter to at least about 1 gram / liter, of at least about 700 milligrams masters / liter to at least about 1 gram / liter, from at least about 800 milligrams / liter to at least about 1 gram / liter,
or from at least about 900 milligrams / liter to at least about 1 gram / liter, when expressed in E. coli or in Pichia species (eg, P. pastoris). Although a polypeptide domain having a binding site with a binding specificity for VEGF described herein can be secreted when expressed in E. coli or in Pichia species (eg, P. pastoris), they can be produced using any suitable method, such as synthetic chemical methods or biological production methods that do not employ E. coli or Pichia species. Polypeptide Domains That Bind with EGFR The invention provides polypeptide domains (e.g., dAb) that have a binding site with a binding specificity for EGFR. In preferred embodiments, the polypeptide domain (e.g., dAb) binds to the EGFR with an affinity (KD; KD = KdeSactave (kd) / Kactave (ka)) from 300 nM to 1 pM (ie say, 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 a KD of 1 x 10"7 M or less, preferably 1 x 10" 8 M or less, more preferably of about 1 x 10 ~ 9 M or less, of 1 x 10"10 M or less. less, or 1 x 10"11 M or less; and / or an indicative constant KdeSactv left 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" \ 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
~ 2 s "1 or less, conveniently 1 x 10" 3 s "or less, more preferably 1 x 10" 4 s "1 or less, still more preferably 1 x 10" 5 s "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 a binding specificity for EGFR competes for the same. link 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), 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 polypeptide domain having a binding site with a binding specificity for EGFR, competes for the EGFR binding, with a dAb selected from the group consisting of: DOM16-39-210 (SEQ ID NO. : 541 DOM16-39-211 (SEQ ID NO: 542 DOM16-39-212 (SEQ ID NO: 543 DOM16-39-213 (SEQ ID NO: 544 DOM16-39-214 (SEQ ID NO: 545 DOM16-39- 215 (SEQ ID NO: 546 DOM16-39-216 (SEQ ID NO: 547 DOM16-39-217 (SEQ ID NO: 548 DOM16-39-218 (SEQ ID NO: 549 DOM16-39-219 (SEQ ID NO: 550 DOM16-39-220 (SEQ ID NO: 551 DOM16-39-221 (SEQ ID NO: 552 DOM16-39-222 (SEQ ID NO: 553 DOM16-39-223 (SEQ ID NO: 554 DOM16-39-224 (SEQ ID NO: 555 DOM16-39-225 (SEQ ID NO: 556 DOM16-39-226 (SEQ ID NO: 557 DOM16-39-227 (SEQ ID NO: 558 DOM16-39-228 (SEQ ID NO: 559 DOM16-39-229 (SEQ ID NO: 560 DOM16-39-230 (SEQ ID NO: 561 DOM16-39-231 (SEQ ID NO: 562 DOM16-39-232 (SEQ ID NO: 563 DOM16-39-233 ( SEQ ID NO: 564 DOM16-39-234 (SEQ ID NO: 565 DOM16-39-235 (SEQ ID NO: 566 DOM16-39-500 (SEQ ID NO: 725 DOM16-39-502 (SEQ ID NO: 726) DOM16-39-503 (SEQ ID NO: 567 DOM16-39-504 (SEQ ID NO: 568 DOM16-39-505 (SEQ ID NO: 569 DOM16-39-506 (SEQ ID NO: 570 DOM16-39-507 ( SEQ ID NO: 571 DOM16-39-508 (SEQ ID NO: 572 DOM16-39-509 (SEQ ID NO: 573 DOM16-39-510 (SEQ ID NO: 574 DOM16-39-511 (SEQ ID NO: 575 DOM16 -39-512 (SEQ ID NO: 576 DOM16-39-521 (SEQ ID NO: 577 DOM16-39-522 (SEQ ID NO: 578 DOM16-39-523 (SEQ ID NO: 579 DOM16-
9-524 (SEQ ID NO: 580 DOM16-39-527 (SEQ ID NO: 581 DOM16-39-525 (SEQ ID NO: 582 DOM16-39-526 (SEQ ID NO: 583 DOM16-39-540 (SEQ ID NO: 584 DOM16-39-541 (SEQ ID NO: 585 DOM16-39-542 (SEQ ID NO: 586 DOM16-39-543 (SEQ ID NO: 587 DOM16-39-544 (SEQ ID NO: 588 DOM16-39 -545 (SEQ ID NO: 589 DOM16-39-550 (SEQ ID NO: 590 DOM16-39-551 (SEQ ID NO: 591 DOM16-39-552 (SEQ ID NO: 592 DOM16-39-553 (SEQ ID NO. : 593 DOM16-39-554 (SEQ ID NO: 594 DOM16-39-555 (SEQ ID NO: 595 DOM16-39-561 (SEQ ID NO: 596 DOM16-39-562 (SEQ ID NO: 597 DOM16-39- 563 (SEQ ID NO: 598 DOM16-39-564 (SEQ ID NO: 599 DOM16-39-571 (SEQ ID NO: 600 DOM16-39-572 (SEQ ID NO: 601 DOM16-39-573 (SEQ ID NO: 602 DOM16-39-574 (SEQ ID NO: 603 DOM16-39-580 (SEQ ID NO: 604 DOM16-39-591 (SEQ ID NO: 605 DOM16-39-592 (SEQ ID NO: 606 DOM16-39-593 (SEQ ID NO: 607 DOM16-39-601 (SEQ ID NO: 608 DOM16-39-602 (SEQ ID NO: 609 DOM16-39-603 (SEQ ID NO: 610 DOM16-39-604 (SEQ ID NO: 611 DOM16-39-605 (SEQ ID NO: 612 DOM16-39-607 (SEQ ID NO: 613 DOM16-39-611 (SEQ ID NO: 614 DOM16-39-612 (SEQ ID NO: 615 DOM16-39-613 (SEQ ID NO: 616 DOM16-39-614 (SEQ ID NO: 617 DOM16-39-615 (SEQ ID NO: 618 DOM16-39-616 (SEQ ID NO: 619 DOM16-39-617 ( SEQ ID NO: 620), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). In some embodiments, the polypeptide domain having a binding site with a binding specificity for EGFR, comprises an amino acid sequence having an identity of
amino acid sequence of at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 91 percent, at least about 92 percent, at least about 93 percent , at least about 94 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, or at least about 99 percent 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), DOM1 6-29 (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), 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 polypeptide domain having a binding site with a binding specificity for EGFR comprises an amino acid sequence having an amino acid sequence identity of at least about 80 percent, at least about 85 percent , at least about 90 percent, at least about 91 percent, at least about 92 percent, at least about 93 percent, at least about 94 percent, at least about 95 percent, when less about 96 percent, at least about 97 percent, at least about 98 percent, or at least about 99 percent with the amino acid sequence of a dAb selected from the group consisting of: DOM16-39-210 (SEQ ID NO: 541), DOM16-39-211 (SEQ ID NO.542), DOM16-39-212 (SEQ ID NO: 543), DOM16-39-213 (SEQ ID NO: 544), DOM16-39-214 ( SEQ ID NO: 545), DOM16-39-215 (SEQ ID NO: 546), DOM16-39-216 (SEQ ID NO: 547), DOM16-39-217 (SEQ ID NO: 548), DOM16-39- 218 (SEQ ID NO: 549), DOM16-39-219 (SEQ ID NO: 550), DOM16-39-220 (SEQ ID NO: 551), DOM16-39-221 (SEQ ID NO: 552), DOM16- 39-222 (SEQ ID NO: 553), DOM16-39-223 (SEQ ID NO: 554), DOM16-39-224 (SEQ ID NO: 555), DOM16-39-225 (SEQ ID NO: 556), DOM16-
39-226 (SEQ ID NO: 557, DOM16-39-227 (SEQ ID NO: 558 DOM16-9-228 (SEQ ID NO: 559, DOM16-39-229 (SEQ ID NO: 560 DOM16-39-230 ( SEQ ID NO: 561, DOM16-39-231 (SEQ ID NO: 562 DOM16-39-232 (SEQ ID NO: 563, DOM16-39-233 (SEQ ID NO: 564 DOM16- 39-234 (SEQ ID NO: 565, DOM16-39-235 (SEQ ID NO: 566 DOM16-39-500 (SEQ ID NO: 725, DOM16-39-502 (SEQ ID NO: 726 DOM16-39-503 (SEQ ID NO: 567, DOM16- 39-504 (SEQ ID NO: 568 DOM16-39-505 SEQ ID NO: 569, DOM16-39-506 (SEQ ID NO: 570 DOM16-39-507 [SEQ ID NO: 571, DOM16-39-508 (SEQ ID NO: 572 DOM16-39-509 [SEQ ID NO: 573, DOM16-39-510 (SEQ ID NO: 574 DOM16-39-511 [SEQ ID NO: 575, DOM16-39-512 (SEQ ID NO: 576 DOM16-39-521 [SEQ ID NO: 577, DOM16-39-522 (SEQ ID NO: 578 DOM16-39-523 [SEQ ID NO: 579, DOM16-39-524 (SEQ ID NO: 580 DOM16-39- 527 [SEQ ID NO: 581, DOM16-39-525 (SEQ ID NO: 582 DOM16-39-526 [SEQ ID NO: 583, DOM16-39-540 (SEQ ID NO: 584 DOM16-39-541 (SEQ ID NO: 585, DOM16-39-542 (SEQ ID NO: 586 DOM16-39-543 [SEQ ID NO: 587, DOM16-39-544 (SEQ ID NO: 588 DOM16-39-545 [SEQ ID NO: 589, DOM16-39-550 (SEQ ID NO: 590 DOM16-39-551 [SEQ ID NO: 591, DOM16-39-552 (SEQ ID NO: 592 DOM16-39- 553 (SEQ ID NO: 593, DOM16-39-554 (SEQ ID NO: 594 DOM16-39-555 (SEQ ID NO: 595, DOM16-39-561 (SEQ ID NO: 596 DOM16-39-562 (SEQ ID NO: 597, DOM16-39-563 (SEQ ID NO: 598 DOM16-39-564 (SEQ ID NO: 599, DOM16-39-571 (SEQ ID NO: 600 DOM16-39-572 (SEQ ID NO: 601, DOM16-39-573 (SEQ ID NO: 602 DOM16-39-574 (SEQ ID NO: 603, DOM16-39-580 (SEQ ID NO: 604 DOM16-
39-591 (SEQ ID NO: 605), DOM16-39-592 (SEQ ID NO: 606), DOM16-39-593 (SEQ ID NO: 607), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-602 (SEQ ID NO: 609), DOM16-39-603 (SEQ ID NO: 610), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-605 (SEQ ID NO: 612) ), DOM16-39-607 (SEQ ID NO: 613), DOM16-39-611 (SEQ ID NO: 614), DOM16-39-612 (SEQ ID NO: 615), DOM16-39-613 (SEQ ID NO. : 616), DOM16-39-614 (SEQ ID NO: 617), DOM16-39-615 (SEQ ID NO: 618), DOM16-39-616 (SEQ ID NO: 619), DOM16-39-617 (SEQ ID NO: 620), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). In the preferred embodiments, the polypeptide domain having a binding site with a binding specificity for EGFR comprises an amino acid sequence having an amino acid sequence identity of at least about 90 percent, at least about 91 percent. percent, at least about 92 percent, at least about 93 percent, at least about 94 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, or at least about 99 percent with the amino acid sequence of DOM16-39 (SEQ ID NO: 345). For example, the polypeptide domain having a binding site with a 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 other preferred embodiments, the polypeptide domain having a binding site with a binding specificity for EGFR, comprises an amino acid sequence having an amino acid sequence identity of at least about 90 percent, at least about 91 percent. percent, at least about 92 percent, at least about 93 percent, at least about 94 percent, at least about 95 percent, at least about 96 percent, at least about '97 percent, at least about 98 percent, or at least about 99 percent with the amino acid sequence of: DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585) , DOM16-39-542 (SEQ ID
NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). In some embodiments, the polypeptide domain having a binding site with a binding specificity for EGFR competes with any of the dAbs disclosed herein for the EGFR linkage. Preferably, the polypeptide domain having a binding site with a binding specificity for EGFR is a single immunoglobulin variable domain. The polypeptide domain that
has a binding site with a binding specificity for EG FR, can comprise any suitable immunoglobulin variable domain, and preferably comprises a human variable domain,
0 a variable domain comprising the regions of human structure. In certain embodiments, the polypeptide domain having a binding site with a binding specificity for EG FR, comprises a universal structure, as described herein. In certain embodiments, the polypeptide domain having a binding site with a binding specificity for EG FR, resists accumulation, is deployed in a reversible manner, comprises
- a region of structure, and / or is secreted as described above for the polypeptide domain having a binding site with a binding specificity for VEG F. dAb Monomers That Bind with Serum Albumin The ligands of the invention they may further comprise a dAb monomer that binds to serum albumin (SA) with a Kd of 1 nM to 500 μM (i.e., 1 x 1 0"9 to 5 x 1 0-4), preferably 1 00 nM to 1 0 μM Preferably, for a ligand comprising an anti-serum albumin dAb, the linkage (eg, Kd and / or Kdesactad, as measured by surface plasmon resonance, for example using BiaCore) of the ligand with its objectives, it is
1 to 1 00,000 times (preferably 1 00 to 1 00,000, more preferably 1,000 to 1,000,000, or 1,000,000 to 1,000,000 times) stronger than for serum albumin. Preferably, the
Serum albumin is human serum albumin (HSA). In one embodiment, the first dAb (or a dAb monomer) is linked to serum albumin (e.g., HSA) with a Kd of about 50, preferably 70, and most preferably 100, 150, or 200 nM. In certain embodiments, the dAb monomer that binds to the serum albumin resists accumulation, unfolds in a reversible manner, and / or comprises a region of structure as described above for the dAb monomers that bind to CD38. In particular embodiments, the antigen binding fragment of an antibody that binds to serum albumin is a dAb that binds to human serum albumin. In certain embodiments, dAb binds to human serum albumin, and competes for binding to 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, dAb binds to human serum albumin, and comprises an amino acid sequence having an amino acid sequence identity of at least about 80 percent, or at least about 85 percent, or at least about 90 percent, or at least about 95 percent, or at least about 96 percent, or at least about 97 percent, or at least about 98 percent, or at least about 99 percent with the amino acid sequence of 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-2V (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). For example, the dAb that binds to human serum albumin can comprise an amino acid sequence having an amino acid sequence identity of at least about 90 percent, or at least about 95 percent, or at least about 96 percent, or at least about 97 percent, or at least
approximately 98 percent, or at least approximately 99 percent 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 identity of amino acid sequences is preferably determined using a suitable sequence alignment algorithm, and the default parameters, such as BLAST P (Karlin and Altschul, Proc. Nati. Acad. Sci. USA 87 (6): 2264- 2268 (1990)). In the most particular modalities, the dAb is a dAb V? which binds to 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: 497), 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 modalities, the
An antigen binding fragment of an antibody that binds to serum albumin is a dAb that binds to human serum albumin, and comprises the CDRs of any of the above amino acid sequences. Suitable camelid VHH that binds to serum albumin includes those disclosed in International Publication Number WO 2004/041862 (Ablynx NV), and herein, 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, camelid VHH binds to human serum albumin, and comprises an amino acid sequence having an amino acid sequence identity of at least about 80 percent, or at least about 85 percent, or at least about 90 percent, or at least about 95 percent, or at least about 96 percent, or at least about 97 percent, or at least about 98 percent, or at least
approximately 99 percent with any of the SEQ I D N Os: 51 8-534. The identity of the amino acid sequences is preferably determined using an appropriate sequence alignment algorithm, and the default parameters, such as BLAST P (Karlin and Altschul, Proc. Nati. Acad. Sci. USA 57 (6): 2264 -2268 (1990)). In some embodiments, the ligand comprises an anti-serum albumin dAb that competes with any anti-serum albumin dAb as disclosed herein, for binding to serum albumin (e.g., human serum albumin). Nucleic Acid, Vector, and Cell Nucleic Acid Molecules The invention also provides isolated and / or recombinant nucleic acid molecules encoding the ligands (e.g., specific double ligands) and multispecific ligands) that are described herein . Nucleic acids referred to herein as "isolated" are nucleic acids that have been separated from nucleic acids from genomic DNA or cellular RNA from their source of origin (eg, as it exists in cells or in a mixture of nucleic acids, such as a library), and include nucleic acids obtained by the methods described herein, or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis, by combinations of methods biological and chemical, and recombinant nucleic acids that are isolated
(See, for example, Daugherty, B. L. et al., Nucleic Acids Res., 1 9 (9): 2471 -2476 (1 991), Lewis, AP and J.S. Crowe, Gene, 1 01: 297 -302 (1 991)). Nucleic acids referred to herein as "recombinants", are nucleic acids that have been produced by recombinant DNA methodology, including nucleic acids that are generated by methods that rely on an artificial recombination method, such as the reaction in polymerase chain (PCR), and / or cloning in a vector using restriction enzymes. In certain embodiments, the isolated and / or recombinant nucleic acid comprises a nucleotide sequence encoding a ligand, as described herein, wherein this ligand comprises an amino acid sequence having an amino acid sequence identity of at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 91 percent, at least about 92 percent, at least about 93 percent, at least about 94 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, or at least about 99 percent with the amino acid sequence of a dAb which is linked to VEG F disclosed herein, or
a dAb that binds to EGFR disclosed herein. For example, in some embodiments, the isolated and / or recombinant nucleic acid comprises a nucleotide sequence encoding a ligand that has binding specificity for VEGF, as described herein, wherein this ligand comprises an amino acid sequence that has an amino acid sequence identity of at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 91 percent, at least about 92 percent, at least about 93 percent, at least about 94 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, or at least about 99 percent with the amino acid sequence of a dAb selected from the group consisting of: TA R15-1 (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 E) 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 E> 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, in some embodiments, isolated and / or recombinant nucleic acid comprises a nucleotide sequence that encodes a ligand that has binding specificity for VEGFA, as described herein, wherein this ligand comprises an amino acid sequence having an amino acid sequence identity of at least about 80 percent, at least about 85 percent, at least about 90 percent percent, at least about 91 percent, at least about 92 percent,
at least about 93 percent, at least about 94 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, or when less about 99 percent with the amino acid sequence of SEQ ID NO: 705 (TAR1 5-26-555). In other embodiments, the isolated and / or recombinant nucleic acid comprises a nucleotide sequence encoding a ligand that has binding specificity for EG FR, as described herein, wherein this ligand comprises an amino acid sequence that has an identity. of amino acid sequence of at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 91 percent, at least about 92 percent, at least about 93 percent, at least about 94 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, or at least about 99 percent percent with the amino acid sequence of a dAb selected from the group consisting of: DOM 1 6-1 7 (SE QID NO: 325), DOM 1 6-1 8 (SEQ ID NO: 326), DOM 1 6-1 9 (SEQ ID NO: 327), DOM 1 6-20 (SEQ ID NO: 328), DOM 1 6 -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), DO M16-70 (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), 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 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 this ligand comprises an amino acid sequence having an identity of amino acid sequence of at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 91 percent, at least about 92 percent, at least about 93 percent hundred, at least
about 94 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, or at least about 99 percent with a sequence of amino acids selected from the group consisting of the S EQ ID NOs: 623-703, 727 and 728. In other embodiments, the isolated and / or recombinant nucleic acid encoding a ligand having binding specificity for VEGF, as described herein, it comprises a nucleotide sequence having a nucleotide sequence identity of at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 91 percent, at least about 92 percent, at least about 93 percent, at least about 94 percent, at least approximately 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, or at least about 99 percent with a nucleotide sequence that encodes an anti-VEGF dAB selected from the group consisting of: TAR 1 5-1 (SEQ ID NO: 1), TAR1 5-3 (SEQ ID NO: 2), TAR 1 5-4 (SEQ ID NO: 3), TAR1 5- 9 (SEQ ID NO: 4), TAR 1 5-1 0 (SEQ ID NO: 5), TAR1 5-1 1 (SEQ ID NO: 6), TAR1 5-1 2 (SEQ ID NO: 7),
TAR15-13 (SEQ ID NO: 8), TAR15-14 (SEQ ID NO: 9), TAR15-15 (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) , TAR15-6-510 (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), T AR15-8-508 (SEQ ID NO: 46), TAR15-8-509 (SEQ ID NO: 47), R15-8-510 (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 TAR15-26-522 (SEQ ID NO: 72 TAR15-226-523 (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 TAR15-226-531 (SEQ ID NO: 81 TAR15-26-532 (SEQ ID NO: 82 TAR15-226-533 (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 TAR15-226-537 (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- 5 48 (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 identity of the nucleotide sequence is determined over the entire length of the nucleotide sequence encoding the selected anti-VEGF dAb. In other embodiments, nucleic acid isolated and / or
recombinant encoding a ligand having a binding specificity for VEG F, as described herein, comprises a nucleotide sequence having a nucleotide sequence identity of at least about 80 percent, at least about 85 percent percent, at least about 90 percent, at least about 91 percent, at least about 92 percent, at least about 93 percent, at least about 94 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, or at least about 99 percent with a nucleotide sequence that encodes TAR1 5-26-555 (SEQ IDNO: 705 ). In other embodiments, the isolated and / or recombinant nucleic acid encoding a ligand having binding specificity for EGFR, as described herein, comprises a nucleotide sequence having a nucleotide sequence identity of at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 91 percent, at least about 92 percent, at least about 93 percent, at least about 94 percent , at least approximately 95 per cent, at least approximately 96 per cent,
at least about 97 percent, at least about 98 percent, or at least about 99 percent with a nucleotide sequence encoding an anti-VEGF dAb selected from the group consisting of: DOM16-17 (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 (SEQ 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 identity of the nucleotide sequence is determined over the entire length of the nucleotide sequence encoding the selected anti-EGFR dAb In other embodiments, the isolated and / or recombinant nucleic acid encoding a ligand having binding specificity for EGFR, as described herein, comprises a nucleotide sequence having a nucleotide sequence identity of at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 91 percent, at least about 92 percent, at least about 93 percent, at least
about 94 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, or at least about 99 percent with a sequence of nucleotides encoding an anti-EGFR dAb selected from the group consisting of SEQ ID NOs: 623-703, 727 and 728. The invention also provides a vector comprising a recombinant nucleic acid molecule of the invention. In certain embodiments, the vector is an expression vector that comprises one or more expression control elements, or sequences that are operably linked to the recombinant nucleic acid of the invention. The invention also provides a recombinant host cell comprising a recombinant nucleic acid molecule or a vector of the invention. Vectors (e.g., plasmids, phagemids), expression control elements, host cells, and methods suitable for producing the recombinant host cells of the invention, are well known in the art, and the examples are further described herein . Suitable expression vectors may contain a number of components, for example, a replication origin, a selectable marker 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 a leader sequence, and
Similar. The expression control elements and a signal sequence, if present, may be provided by 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 to 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, an antibody chain, or a portion thereof, such as to dictate 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., early or late promoter from simian virus 40, terminal repeat promoter). long of Rous sarcoma virus, cytomegalovirus promoter, adenovirus late promoter.) In addition, expression vectors typically comprise a selectable marker for the selection of host cells bearing the vector, and in the case of a vector of Replicable expression, a replication origin Genes that encode products that confer resistance to antibiotics or drugs are common selectable markers, and can be used in prokaryotic cells (eg, lactamase gene (ampicillin resistance), Tet gene for resistance to tetracycline), and in eukaryotic cells (eg, neomycin resistance genes (G41 8 or
geneticin), gpt (mycophenolic acid), ampicillin, or hygromycin). The dihydrofolate reductase marker genes allow selection with methotrexate in a variety of hosts. Genes encoding the genetic product of host auxotrophic markers (eg, LEU2, URA3, HIS3) are frequently used as sellable markers in yeast. The use of viral (e.g., baculovirus) or phage vectors, and vectors that are capable of integrating into the genome of the host cell, such as retroviral vectors, is also contemplated. Expression vectors suitable for expression in mammalian cells and in prokaryotic cells (E. coli), insect cells (Schnieder S2 cells from Drosophila, Sf9) ', and in yeast (P. methanolica, P. pastoris, S cerevisiae), are well known in this field. Suitable host cells can be prokaryotic, including bacterial cells, such as E. coli, B. subtilis, and / or other suitable bacteria; eukaryotic cells, such as fungal or yeast cells (e.g., Pichia pastoris, Aspergillus sp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa), and other lower eukaryotic cells, and higher eukaryotic cells, such as those of insects (e.g. Examples are Schnieder S2 cells from Drosophila, insect cells S9 (International Publication No. WO 94/26087 (O'Connor)), from mammals (eg COS cells, such as COS-1 (ATCC, Access Number). CRL-1 650), and COS-7 (ATCC, Access Number
CRL-1651), CHO (eg, ATTC, Accession Number CRL-9096, CHO DG44 (Urlaub, G. and Chasin, LA, Proc. Nati. Acad. Sci. USA, 77 (7): 4216-4220 ( 1980))), 293 (ATCC, Accession Number CRL-1573), HeLa (ATCC, Accession Number CCL-2), CV1 (ATCC, Accession Number CCL-70), WOP (Dailey, L., et al. , J. Virol., 54: 739-749 (1985), 3T3, 293T (Pear WS, et al., Proc. Nati, Acad. Sci. USA, 90: 8392-8396 (1993)), NSO cells, SP2 / 0, HuT 78 cells, and the like, or plants (eg, tobacco). (See, for example, Ausubel, FM et al., Editors, Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley &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-host cell. The invention also provides a method for producing a ligature (for example, specific double ligand, multispecific ligand) of the invention, which comprises maintaining a recombinant host cell comprising a recombinant nucleic acid of the invention, under conditions suitable for expression of the recombinant nucleic acid, whereby, expresses the recombinant nucleic acid, and a ligand is produced. In some embodiments, the method further comprises isolating the ligand. Preparation of Immunoglobulin-based Ligands. Ligands (for example, specific double ligands,
multispecific ligands) according to the 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 designed antibody molecules. Techniques for the preparation of antibodies, for example, are described in the following reviews and references cited therein: Winter and Milstein, (1991) Nature 349: 293-299; Pluckthun (1992) Immunological Reviews 130: 151-188; Wright et al., (1992) Crit. Rev. Immunol. 12: 125-168; Holliger, P. and Winter, G. (1993) Curr. Op. Biotechn. 4,
446-449; Carter, et al. (1995) J. Hematother. 4, 463-470; Chester, K. A. and Hawkins, R. E. (1995) Trends Biotechn. 13, 294-300; Hoogenboom, H. R. (1997) Nature Biotechnol. ' 15, 125-126; Fearon, D. (1997) Nature Biotechnol. 15, 618-619; Plückthun, A. and Pack, P. (1997) Immunotechnology 3, 83-105; Cárter, P. and Merchant, A. M. (1997) Curr. Opin. Biotechnol. 8, 449-454; Holliger, P. and Winter, G. (1997) Cancer Immunol. Immunother.45,128-130. Suitable techniques employed for the selection of variable domains of antibodies with a desired specificity employ libraries and selection procedures that are known in the art. Natural libraries (Marks et al. (1991) J. Mol. Biol, 222: 581; Vaughan et al. (1996) Nature Biotech., 14: 309), using reconfigured V genes harvested from human B-cells, are well known by the experts in the field. Synthetic libraries (Hoogenboom and
Winter (1992) J. Mol. Biol, 227: 381; Barbas et al. (1992) Proc. Nati Acad. Sci. USA, 89: 4457; Nissim et al. (1994) EMBO J, 1 3: 692; Griffiths et al. (1 994) EMBO J., 1 3: 3245; De Kruif et al. (1995) J. Mol Biol, 248: 97), are prepared by cloning immunoglobulin V genes, usually using polymerase chain reaction. Errors in the polymerase chain reaction process can lead to a high degree of random selection. The VH and / or VL libraries can be screened against the target antigens or epitopes separately, in which case, the single-domain link is directly selected, or together. Library Vectors Systems A variety of selection systems are known in the art, which are suitable for use in the present invention. The examples of these systems are described below. The bacteriophage lambda expression systems can be screened directly as bacteriophage plaques or as colonies of lysogens, as previously described (H use et al. (1989; Science, 246: 1 275; Cato and Koprowski (1990) Proc). Nati Acad. Sci. USA, 87; Mullinax and collaborators (1 990) Proc. Nati, Acad. Sci. USA, 87: 8095; Persson et al. (1991) Proc. Nati. Acad. Sci. E UA, 88 : 2432), as they are for use in the invention, although these expression systems can be used to track up to 106 different members of a library, they are not really suitable for tracking larger numbers (more than 1 06
members). The screening display systems, which make it possible for a nucleic acid to be linked to the polypeptide it expresses. As used herein, a selection display system is a system that allows the selection, by suitable means of display, of the individual members of the library, through the link of the generic and / or the objective. Selection protocols for the isolation of desired members of large libraries are known in this field, as typified by phage display techniques. These systems, where various sequences of peptides are displayed on the surface of the filamentous bacteriophage (Scott and Smith (1990) Science, 249: 386), have proven useful for the creation of libraries of antibody fragments (and nucleotide sequences). which encode them), for the in vitro selection and amplification of specific antibody fragments that bind to a target antigen (McCafferty et al., International Publication No. WO 92/01 047). The nucleotide sequences encoding the variable regions are linked to the genetic fragments encoding the leader signals that direct them towards the periplasmic space of E. coli, and as a result, the resulting antibody fragments are displayed on the surface of the bacteriophage, typically as fusions with bacteriophage coating proteins (e.g., plll or pVI II). In an alternative way, the antibody fragments are displayed
externally on the capsids of lambda phages (phagobodies). An advantage of phage-based display systems is that, due to their biological systems, selected members of the library can be amplified simply by growing the phage containing the selected member of the library, in bacterial cells. Additionally, because the nucleotide sequence encoding the polypeptide member of the library is contained on a phage or phagemid vector, sequencing, expression, and subsequent genetic manipulation are relatively straightforward. 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 et al. (1991) Nature, 352: 624; Lowman et al. (1991) Biochemistry, 30: 10832; Burton et al. (1991) Proc. Nati Acad. Sci. USA, 88: 10134; Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133; Chang et al (1991) J. Immunol, 147: 3610; Breitling et al. (1991) Gene, 104: 147; Marks et al. (1991) supra; Barbas et al. (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 approach has been the use of scFv phage 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 et al. (1990) supra; Clackson et al. (1991) Nature, 352: 624; Marks et al. (1991) J. Mol. Biol, 222: 581; Chiswell et al. (1992) Trends Biotech., 10:80, Marks et al. (1992) J. Biol. Chem., 267). Different modalities of scFv libraries displayed on bacteriophage coating proteins have been described. Refinements of the phage display approaches are also known, for example, as described in International Publications Numbers WO WO96 / 06213 and WO92 / 01047 (Medical Research Council and co-workers), and WO97 / 08320 (Morphosys), which are they are incorporated herein by reference. Other systems for generating polypeptide libraries involve the use of cell-free enzymatic machine for the in vitro synthesis of the members of the library. In one method, RNA molecules are selected by alternating rounds of selection against a target, and amplification with polymerase chain reaction (Tuerk and Gold (1990) Science, 249: 505; Ellington and Szostak (1990) Nature, 346: 818). A similar technique can be used to identify DNA sequences that bind to a previously determined human transcription factor (Thiesen and Bach (1990) Nucleic Acids Res., 18: 3203; Beaudry and Joyce (1992)
Science, 257: 635; Publication is International Nos. WO 92/05258 and WO92 / 1 4843). In a similar manner, in vitro translation can be used to synthesize polypeptides as a method to generate large libraries. These methods, which generally comprise stabilized polysome complexes, are further described in International Publications Nos. WO88 / 08453, WO90 / 05785, WO90 / 07003, WO91 / 02076,
WO91 / 05058, and WO92 / 02536. Alternative delivery systems that are not phage based, such as those disclosed in International Publication Nos. WO95 / 22625 and WO95 / 1 1 922 (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 linking of a gene with its genetic product. For example, a selection system is described wherein nucleic acids encoding desirable gene products can be selected in microcapsules formed by water-in-oil emulsions, in International Publications Nos. WO99 / 02671, WO00 / 4071 2, and in Tawfik and Griffiths (1 998) Nature Biotechnol. 1 6 (7), 652-6. Genetic elements that encode a genetic product having a desired activity are compartmentalized into microcapsules, and then transcribed and / or translated to produce their respective gene products (RNA or protein) within the microcapsules. Subsequently, the elements are classified
genetic products that produce the genetic product that has the desired activity. This approach selects the genetic products of interest by detecting the desired activity by a variety of means. Library Construction The libraries intended for selection can be built using techniques known in this field, for example, as stipulated above, or can be purchased from commercial sources. Libraries that are useful in the present invention are described, for example, in International Publication Number WO99 / 20749. Once a vector system is selected, and one or more nucleic acid sequences encoding the polypeptides of interest are cloned into the library vector, diversity can be generated within the cloned molecules by undertaking mutagenesis prior to expression.; in an alternative manner, the encoded proteins can be expressed and selected, as described above, before mutagenesis, and additional rounds of selection are carried out. The mutagenesis of nucleic acid sequences encoding structurally optimized polypeptides is carried out by conventional molecular methods. Particularly useful is the polymerase chain reaction, or PCR (Mullis and Faloona (1987) Methods Enzymol, 1 55: 335, incorporated herein by reference). The polymerase chain reaction, which uses multiple cycles of DNA replication catalyzed by a DNA-dependent DNA polymerase,
Thermostable, to amplify the target sequence of interest, is well known in the art. The construction of different antibody libraries has been discussed in Winter et al. (1994) Ann. Rev. Immunology 1 2, 433-55, and references cited therein. The polymerase chain reaction is carried out using template DNA (at least 1 fg, more usefully 1 to 1,000 ng), and at least 25 picomoles of oligonucleotide primers; it may be convenient to use a larger amount of primer when the pool of primers is heavily heterogeneous, because each sequence is represented only by a small fraction of the molecules in the pool, and the quantities become limiting in subsequent amplification cycles. . A typical reaction mixture includes: 2 microliters of DNA, 25 picomoles of oligonucleotide primer, 2.5 microliters of 10X 1 PCR regulator (Perkin-Elmer, Foster City, CA), 0.4 microliters of dNTP 1.25 μM, 0.1 5 microliters (or 2.5 units) of Taq DNA polymerase (Perki n-Elmer, Foster City, CA), and deionized water to a total volume of 25 microliters. Mineral oil is superposed, and the polymerase chain reaction is carried out using a programmable thermal cycler. The duration and temperature of each step of a polymerase chain reaction cycle, as well as the number of cycles, are adjusted according to the current restriction requirements. The tempering temperature and the time are both determined by the efficiency with the
which is expected to quench a primer to a template, and the degree of mismatch that will be tolerated; obviously, when nucleic acid molecules are amplified and mutated simultaneously, a mismatch is required, at least in the first round of synthesis. The ability to optimize the restriction of the priming conditions of primers is well within the knowledge of a moderate expert in this field. A tempering temperature of between 30 ° C and 72 ° C is used. The initial denaturation of the template molecules normally occurs between 92 ° C and 99 ° C for 4 minutes, followed by 20 to 40 cycles consisting of denaturation (94 ° C to 99 ° C for 1 5 seconds to 1 minute). , temperature (the temperature is determined as discussed above, from 1 to 2 minutes), and extension (72 ° C for 1 to 5 minutes, depending on the length of the amplified product). The final extension is usually for 4 minutes at 72 ° C, and can be followed by an indefinite step (from 0 to 24 hours) at 4 ° C. Combination of Unique Variable Domains The domains useful in the invention, once selected, can be combined by a variety of methods known in the art, including covalent and non-covalent methods. Preferred methods include the use of polypeptide linkers, as described, for example, in relation to scFv molecules (Bird et al. (1988) Science 242: 423-426). The discussion of suitable linkers is provided in Bird and
collaborators, Science 242, 423-426; Hudson et al., Journal Immunol Methods 231 (1 999) 1 77-1 89; Hudson et al., Proc. Nat. Acad. Sci. USA 85, 5879-5883. The linkers are preferably flexible, allowing the two unique domains to interact. An example of a linker is a linker (Gly4Ser) n, where n = from 1 to 8, for example 2, 3, 4, 5, or 7. The linkers used in diabodies, which are less flexible, can also be employ (Holliger et al. (1 993) Proc. Nat. Acad. Sci. USA 90: 6444-6448). In one embodiment, the linker employed is not an immunoglobulin articulation region. Variable domains can be combined using methods other than linkers. For example, the use of disulphide bridges, provided through naturally occurring or designed cysteine residues, can be exploited to stabilize the VH-VH, VL-VL, or VH-VL dimer (Reiter et al. (1 994) Protein Eng. 7: 697-704) or by remodeling the interface between the variable domains to improve the "fit", and therefore, the stability of the interaction (Ridgeway et al. (1996) Protein. Eng. 7: 61 7-621; Zhu et al. (1 997) Protein Science 6: 781-788). Other techniques can be employed to unite or stabilize the variable domains of immunoglobulins, and in particular the VH domains of antibodies, as appropriate.
Characterization of Ligands The binding of a specific double ligand with the cell, or the binding of each binding domain with each specific target, can be tested by methods that will be familiar to those skilled in the art, and include ELI SA. In a preferred embodiment of the invention, the binding is tested using the monoclonal phage EL ISA. The phage ELISA can be carried out according to any suitable procedure: an example protocol is stipulated below. Phage populations produced in each round of selection can be screened to determine the binding by ELISA with the selected o-epitope antigen, in order to identify "polyclonal" phage antibodies. The phages from the individual infected bacterial colonies can then be screened from these populations by EL I SA, to identify "monoclonal" phage antibodies. It is also desirable to screen the fragments of soluble antibodies for binding to the antigen or epitope, and this can also be undertaken by ELI SA, using reagents, for example, against a C- or N-terminal tag (see, for example, Winter et al. (1 994) Ann. Rev. Immunology 12, 433-55, and references cited therein).
The diversity of the selected phage monoclonal antibodies can also be evaluated by gel electrophoresis of the polymerase chain reaction products (Marks et al., 1 991, supra; Nissim et al., 1 994, supra),
probing (Tomlinson et al. (1992) J. Mol. Biol. 221, 776), or by sequencing the vector DNA. Structure of the Ligands In the case that each variable term is selected from the V-gene repertoires, it is selected, for example, using the phage display technology as described herein, then these variable domains comprise a region of universal structure, such 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 International Publication Number WO99 / 20749. When V-gene repertoires are used, the variation in the polypeptide sequence is preferably located within the structural cycles of the variable domains. The polypeptide sequences of any variable domain can be altered by mixing the DNA or by mutation, in order to improve the interaction of each variable domain with its complementary pair. The DNA mixture is known in the art, and is taught, for example, by Stemmer, 1994, Nature 370: 389-391, and in the Patent of the
United States of America Number 6, 297.053, both of 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 constructions of vectors required for selection can be constructed,
preparation, and formatting of the specific double ligands and can be manipulated as stipulated in conventional laboratory manuals, such as Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, E UA. The manipulation of the nucleic acids useful in the present invention is typically carried out in recombinant vectors. As used herein, "vector" refers to a separate element that is used to introduce heterologous DNA into cells for expression and / or replication thereof. The methods by which they are selected and constructed, and subsequently these vectors are used, are well known to one of ordinary skill in the art. Numerous vectors are publicly available, including bacterial plasmids, bacteriophages, artificial chromosomes, and episomal vectors. These vectors can be used for simple cloning and mutagenesis; in an alternative way, the gene expression vector is used. A useful vector according to the invention can be selected to accommodate a polypeptide coding sequence of a desired size, typically 0.25 kilobases (kb) to 40 kilobases or more in length. A suitable host cell is transformed with the vector after the in vitro cloning manipulations. Each vector contains different functional components, which generally include a cloning site (or "polylinker"), a replication origin, and at least one selectable marker gene. If a given vector is an expression vector, it additionally possesses one or more of the
following: an enhancer element, promoter, transcription termination, and signal sequences, each positioned in the vicinity of the cloning site, such that they are operably linked to the gene encoding a specific double ligand according to the invention . Both the cloning and expression vectors generally contain nucleic acid sequences that make it possible for the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is one that makes it possible for the vector to replicate independently of the chromosomal DNA of the host, and includes replication origins or sequences that replicate autonomously. 'These sequences are well known for a variety of bacteria, yeast, and viruses. The replication origin of plasmid pB R322 is suitable for most of the gram-negative bacteria, the origin of the plasmid of 2 micras is suitable for the yeast, and different viral origins (eg SV 40, adenovirus) are useful for the cloning vectors in mammalian cells. Generally speaking, replication origin is not needed for mammalian expression vectors, unless they are used in mammalian cells capable of replicating high levels of DNA, such as COS cells. Conveniently, a cloning or expression vector may contain a selection gene, also referred to as a selectable marker. This gene encodes a protein
necessary for the survival or growth of transformed host cells cultured in a selective culture medium. Accordingly, host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, for example, ampicillin, neomycin, methotrexate, or tetracycline, auxotrophic complement deficiencies, or supply the critical nutrients not available in the culture medium. Because the replication of vectors encoding a specific double ligand according to the present invention is most conveniently carried out in E. coli, a selectable marker of £ is useful. coli, for example, the ß-lactamase gene that confers resistance to the antibiotic ampicillin. These can be obtained from E. coli plasmids, such as pBR322, or a pUC plasmid, such as pUC18 or pUC1 9. Expression vectors usually contain a promoter that is recognized by the host organism, and is operably linked to the coding sequence of interest. This promoter can be inducible or constitutive. The term "operatively linked" refers to a juxtaposition wherein the described components are in a relationship that allows them to function in their intended manner. A control sequence "operably linked" with a coding sequence is ligated in such a way that the expression of the sequence of
coding 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 the hybrid promoters, such as the tac promoter. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence operably linked to the coding sequence. Preferred vectors are expression vectors that make possible the expression of a nucleotide sequence corresponding to a member of the polypeptide library. Accordingly, selection with the first and / or the second antigen or epitope can be carried out by separate propagation and expression of a single clone expressing the polypeptide library member, or by using any screening display system. As described above, the preferred screening display system is the display of bacteriophages. Therefore, phage or phagemid vectors can be used, for example, plT1 or plT2. The leader sequences useful in the invention include pel B, stll, ompA, phoA, bla, and pelA. An example is the vectors of phagemids that have a replication origin of E. coli (for double-stranded replication), and also a replication origin of the phage (for the production of single-stranded DNA). The manipulation and expression of these vectors are well known in
the matter (Hoogenboom and Winter (1992) supra; Nissim et al. (1 994) supra). Briefly stated, the vector contains a β-lactam asa gene for conferring selectivity to the phagemid, and a lac promoter upstream of an expression cassette consisting (N- to C-terminal) in a leader sequence of the pellet. B (which directs the expressed polypeptide to the periplasmic space), a multiple cloning site (for the cloning of the nucleotide version of the library member), optionally one or more peptide tags (for detection), optionally one or more stop codons TAG, and phage protein pl ll. Accordingly, using different suppressor and non-suppressor strains of E. coli, and with the addition of glycoal, isopropyl thio-β-D-galactoside (IPTG), or of an auxiliary phage, such as VCS M 1 3, the The vector can be replicated as a plasmid without expression, can produce large amounts of the polypeptide library member alone, or can produce phages, some of which contain at least one copy of the polypeptide-pll fusion on its surface. The construction of vectors encoding double specific ligands according to the invention employs conventional ligation techniques. Isolated vectors or DNA fragments are dissociated, tailored, and religated in the desired form to generate the required vector. If desired, an analysis can be carried out to confirm that the correct sequences are present in the constructed vector, in a known manner.
Suitable methods for constructing expression vectors, for the preparation of in vitro transcripts, for the production of DNA in host cells, and for carrying out the analyzes in order to evaluate expression and function, are known to the experts in this field. The presence of a genetic sequence in a sample is detected, or its amplification and / or expression is quantified, by conventional methods, such as Southern or Northern analysis, Western blot, staining of DNA, RNA or protein spots, in situ hybridization. , immunocytochemistry, or sequence analysis of nucleic acid or protein molecules. Those skilled in the art will readily foresee the manner in which these methods can be modified, if desired. Skeletons Skeletons can be based on immunoglobulin molecules, or can be of a non-immunoglobulin origin, as stipulated above. Each domain of the specific double ligand can be a different skeleton. Preferred immunoglobulin backbones, as defined herein, include any one or more of those selected from the following: an immunoglobulin molecule that comprises at least: (i) the CL domain (kappa or lambda subclass) of an antibody; or (ii) the CH 1 domain of an antibody heavy chain; an immunoglobulin molecule comprising the CH 1 and CH 2 domains of an antibody heavy chain; an immunoglobulin molecule comprising the
CH 1, CH 2, and CH 3 domains of an antibody heavy chain; or any of the subset (ii) in conjunction with the CL domain (kappa or lambda subclass) of an antibody. You can also include a region of articulation domain. These combinations of domains, for example, can mimic natural antibodies, such as IgG or IgM, or fragments thereof, such as Fv, scFv, Fab, or F (ab ') 2 molecules. Experts in this field 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 domain-specific interaction with one or more epitopes. Conveniently, an epitope binding domain according to the present invention comprises three complementarity determining regions. Suitable protein scaffolds include any of those selected from the group consisting of the following: those based on immunoglobulin domains, those based on fibronectin, those based on affibodies, those based on CTLA4, those based on chaperones, such as GroEL, those based on lipocalin, and those based on the bacterial Fc receptors SpA and SpD. Those skilled in the art will appreciate that this list is not intended to be exhaustive. Scaffolding for Use in the Construction of Ligands Selection of the Conformation of the Main Chain Members of the immunoglobulin super family
they all 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 the sequences and crystallographic structures has revealed that, contrary to expectation, five of the six antigen binding cycles of the antibodies (H 1, H2, L1, L2, L3) adopt a limited number of conformations of the main chain, or canonical structures (Chothia and Lesk (1987) J. Mol. Biol, 1 96: 901; Chothia et al., (1989) Nature, 342: 877). The analysis of the lengths of the cycles and the key residues has made it possible, therefore, to predict the conformations of the main chain of H 1, H2, L1, L2, and L3 that are "in the majority of human antibodies (Chothia et al. (1992) J. Mol. Biol, 227: 799; Tomlinson et al. (1,995) EMBO J, 1 4: 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 cycle lengths, which they depend on the length and the presence of particular residues, or types of residues, at key positions in the cycle and in the structure of the antibody (Martin et al., (1 996) J. Mol. Biol, 263: 800; Shirai et al., (1 996) FEBS Letters, 399: 1.) Ligand libraries and / or libraries can be designed. link domains, where certain cycle lengths have been chosen and
key waste, to ensure that the conformation of the main chain of members is known. Conveniently, these are real conformations of the molecules of the immunoglobulin super family that are found in nature, to minimize the chances that they are not functional, as discussed in the foregoing. The segments of the germline V gene serve as a basic structure suitable for building libraries of antibodies or T-cell receptors; Other sequences are also useful. 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 its function. "The canonical structure theory is also useful for evaluating the number of different conformations of the main chain encoded by the ligands, to predict the conformation of the main chain based on the sequences of the specific double bonds, and to choose the residues for the diversification that do not affect the canonical structure. , in the V? domain, the L1 cycle can adopt one of four canonical structures, the L2 cycle has a single canonical structure, and 90 percent of the human V? domains adopt one of four or five canonical structures for the cycle L3 (Tomlinson et al. (1995) supra), therefore, in the V? domain only, different canonical structures can be combined to create a range of different conformations of the main chain. Since the domain V?
codifies a different range of canonical structures for cycles L1 .L2, and L3, and that domains V? and V? can be paired with any VH domain that can encode various canonical structures for the H 1 and H2 cycles, the number of combinations of canonical structures observed for these five cycles is very large. This implies that the generation of diversity in the conformation of the main chain can be essential for the production of a wide range of binding specificities. However, by constructing an antibody library based on a single conformation of the known main chain, it has been found that, contrary to expectation, diversity in the conformation of the main chain is not required to generate sufficient diversity for address substantially all antigens. Still in a more surprising way, the individual conformation of the main chain does not need to be a consensus structure - one single conformation can be used that naturally occurs as the basis for the entire library. Accordingly, in a preferred aspect, the ligands of the invention possess a conformation of a single known backbone. The conformation of a single main chain that is selected is preferably common among the molecules of the immunoglobulin super family type in question. A conformation is a common place when it is observed that a significant number of naturally occurring molecules adopt it. In accordance with the foregoing, in a preferred aspect of the invention,
the natural presentation of the different conformations of the primary chain for each binding cycle of an immunoglobulin domain is considered, and then a variable domain that occurs naturally is selected, which possesses the desired combination of conformations of the main chain for the different cycles. If none are available, you can choose the nearest equivalent. It is preferable that the desired combination of main chain conformations for the different cycles be created by selecting the genetic segments of the germline that encode the desired main chain conformations. It is more preferable that the genetic segments of the selected germline are frequently expressed in nature, and it is highly preferable that they are those that are most frequently expressed from all the natural germline genetic segments. In the design of ligands (e.g., ds-dAbs) or library thereof, the case of the different conformations of the main chain for each of the six antigen binding cycles can be considered separately. For H 1, H 2, L 1, L 2, and L 3, a given conformation is chosen that is adopted between 20 percent and 1,00 percent of the antigen binding cycles of the naturally occurring molecules. Typically, its observed incidence is greater than 35 percent (that is, between 35 percent and 1 percent), and ideally, greater than 50 percent, or even greater than 65 percent. Because the
Most of the H3 cycles do not have canonical structures, it is preferable to select a confo rmation of the main chain that is a common place among these cycles, which do exhibit canonical structures. For each of the cycles, therefore, the conformation most frequently observed in the natural repertoire is selected. In human antibodies, the most popular canonical structures (CS) for each cycle are as follows: H 1 -CS 1 (79 percent of the expressed repertoire), H2 - CS 3 (46 percent), L 1 - CS 2 of VK (39 percent), L2 - CS 1 (1 00 percent), L3 - CS 1 of V? (36 percent) (the calculation assumes a?:? Ratio of 70: 30, Hood et al. (1967) Cold Spring Harbor Symp. Quant. Biol, 48: 1 33). For the H3 cycles that have canonical structures, one length of the C DR3 (Kabat et al. (1 991) Sequences of proteins of immunological interest, Department of Health and Human Services of the United States) of seven residues with a salt bridge from residue 94 to residue 01, it seems to be the most common. There are at least 16 human antibody sequences in the EM BL data library with the length of H3 required and key residues to form this conformation, and at least two crystallographic structures in the protein data bank that can be used as a protein. base for the modeling of the antibodies (2cgr and 1 tet). The genetic segments of the germinal line most frequently expressed in this combination of canonical structures are segment VH 3-23 8DP-47), segment JH JH4b, segment V? O2 / O12 (DPK9), and the
segment J? J? 1 - The segments VH DP45 and DP38 are also suitable. These segments, therefore, can be used in combination as a basis for constructing a library with the conformation of the desired individual main chain. In an alternative way, instead of deselecting the conformation of the individual main chain based on the natural presentation of the different conformations of the main chain for each of the link cycles in isolation, the natural presentation of the conformation combinations is used. of the main chain as the basis for choosing the conformation of the individual main chain. In the case of antibodies, for example, the natural presentation of combinations of canonical structures for any two can be determined., three, four, five, or the six cycles of antigen binding. Here, it is preferable that the selected conformation be a common site in naturally occurring antibodies, and more preferably that it is more frequently observed in the natural repertoire. Therefore, in human antibodies, for example, when natural combinations of the five cycles of antigen binding, H 1, H 2, L 1, L 2, and L 3 are considered, the most frequent combination of canonical structures is determined, and then combined with the most popular conformation for the H3 cycle, as a basis for selecting the conformation of the individual backbone.
Diversification of the Canonical Sequence Having selected several known main chain conformations, or preferably a single conformation of the known backbone, specific double ligands (eg, ds-dAbs) or library libraries can be constructed for use in the invention, varying each binding site of the molecule, in order to generate a repertoire with structural and / or functional diversity. This means that variants are generated, in such a way that they have sufficient diversity in their structure and / or function, in such a way that they are able to provide a range of activities. The desired diversity is typically generated by varying the selected molecule in one or more positions. The positions to be changed can be randomly selected, or selected in a preferable manner. Then the variation can be achieved either by random selection, during which the resident amino acid is replaced by any amino acid or analogue thereof, natural or synthetic, producing a very large number of variants, or by replacement of the resident amino acid with one or more of a defined subset of amino acids, producing a more limited number of variants. Different methods have been reported to introduce this diversity. Polymerase chain reaction susceptible to error can be used (Hawkins et al. (1992) J. Mol. Biol, 226: 889), chemical mutagenesis (Deng et al., (1 994) J. Biol. Chem., 269: 9533), or bacterial mutant strains (Low and
collaborators (1996) J. Mol. Biol., 260: 359), to introduce random mutations in the genes that code for the molecule. Methods for mutating the selected positions are also well known in the art, and include the use of mismatched oligonucleotides or degenerate oligonucleotides, with or without the use of the polymerase chain reaction. For example, several libraries of synthetic antibodies have been created by directing mutations toward antigen binding cycles. The H3 region of a human tetanus toxoid binding Fab has been randomly selected to create a range of novel binding specificities (Barbas et al. (1992) Proc. Nati, Acad. Sci. USA, 89: 4457). The random or semi-random H3 and L3 regions have been appended to the segments of the germline V gene to produce large libraries with unmutated structure regions (Hoogenboom and Winter (1992) J. Mol. Biol, 227: 381; Barbas et al. (1 992) Proc. Nati, Acad. Sci. USA, 89: 4457; Nissim et al. (1 994) EMBO J, 1 3: 692; Griffiths et al. (1 994) EMBO J, 13: 3245; De Kruif et al. (1995) J. Mol. Biol, 248: 97). This diversification has been extended to include some or all of the other antigen binding sites (Crameri et al. (1 996) Nature Med., 2: 1 00; Riechmann et al. (1 995) Bio / Technology, 1 3: 475; Morphosys, International Publication Number WO97 / 08320, supra). Because the random selection of cycles has the potential to create approximately more than 1 01 5 structures for H3
only, and similarly a large number of variants for the other five cycles, it is not feasible, using current transformation technology, or even employing systems without cells, to produce a library that represents all possible combinations. For example, in one of the largest libraries constructed to date, 6x1010 different antibodies were generated, which is only a fraction of the potential diversity for a library of this design (Griffiths et al. (1994), supra). Preferably, only residues that are directly involved in the creation or modification of 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 objective, and therefore, the diversity must be concentrated in the target link site, while avoiding the changing residues that are crucial for the global packaging of the molecule, or to maintain the conformation of the selected main chain. Diversification of the Canon Sequence as applied to the Antibody Domains. In the case of antibody-based ligands (e.g., ds-dAbs), the binding site for each target is most often the antigen binding site. Accordingly, preferably only the residues at the antigen binding site are varied. These residues are extremely diverse in the repertoire of
human antibodies, and they are known to make contacts in the high resolution antibody / antigen complexes. For example, in L2, it is known that the positions are 50 and 53 are diverse in naturally occurring antibodies, and it is observed that they make contact with the antigen. In contrast, the conventional approach would have been to diversify all the residues in the corresponding complementarity determining region (CDR1), as defined by Kabat et al. (1991), some seven residues, comparing with the two diversified in the library for used according to the invention. This represents a significant improvement in terms of the functional diversity required to create a range of antigen binding specificities. In nature, the diversity of antibodies is the result of two processes: somatic recombination of the genetic segments of the germinal line V, D, and J, to create a pure primary repertoire (the so-called diversity of the germinal line and of binding), eh somatic hyper-mutation of the resulting reconfigured V genes. The analysis of human antibody sequences has shown that the diversity in the primary repertoire is focused on the center of the antigen binding site, while the somatic hyper-mutation extends the diversity to regions at the periphery of the binding site of the antigen. antigen, which are highly conserved in the primary repertoire (see Tomlinson et al., (1 996) J. Mol. Biol., 256: 81 3). This complementarity has probably evolved as a
efficient strategy to search for sequence space, and, although apparently unique to antibodies, it can be easily applied to other polypeptide repertoires. The wastes that are varied are a subset of those that form the liaison site for the objective. Different subsets (including overlaps) of residues in the target link site are diversified at different stages during the selection, if desired. In the case of a repertoire of antibodies, an initial "pure" repertoire can be created, where some, but not all, residues in the antigen binding site are diversified. As used herein in this context, the term "pure" refers to antibody molecules that do not have a previously determined purpose. These molecules resemble those that are encoded by the immunoglobulin genes of an individual that has not undergone immu ne diversification, as is the case with fetal and newborn individuals, whose immune systems have not yet been assaulted by an immune system. wide variety of antigenic stimuli. Then this repertoire is selected against a range of antigens or epitopes. If required, additional diversity can then be introduced outside the diversified region in the initial repertoire. This mature repertoire can be selected for a modified function, specificity, or affinity. The pure repertoires of binding domains for the construction of double specific ligands, wherein some or all of the residues at the antigen binding site are varied are
known in this field. (See International Publications Nos. WO 2004/058821, WO 2004/003019, and WO 03/002609). The "primary" library mimics the natural primary repertoire, with diversity restricted to residues in the center of the antigen binding site that are diverse in the V gene segments of the germline (diversity of the germline) , or diversify during the recombination process (binding diversity). These diversifying residues include, but are not limited to, H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97, H98, L50, L53, L91, L92, L93, and L94. L96. In the "somatic" library, diversity is restricted to the residues that are diversified during the recombination process (union diversity), or that are highly somatically mutated. These diversifying residues include, but are not limited to: H31, H33, H35, H95, H96, H97, H98, L30, L31, L32, L34 and L96. It is known that all the residues listed above as suitable for diversification in these libraries make contacts in one or more antibody-antigen complexes. Because in both libraries not all residues of the antigen binding site are varied, additional diversity is incorporated during selection by varying the remaining residues, if desired to do so in this manner. It will be apparent to one skilled in the art that any subset of any of these residues (or additional residues comprising the antigen binding site) can be used for the initial and / or subsequent diversification of the antigen binding site.
In the construction of the libraries for use in the invention, the diversification of the selected positions is typically achieved at the level of the nucleic acid, by altering the coding sequence that specifies the polypeptide sequence, so that a number of nucleic acids can be incorporated. possible amino acids (all 20 or a subset of them) in that position. Using the nomenclature of the U PAC, the most versatile codon is N N K, which codes for all amino acids, as well as the stop codon TAG. The N NK codon is preferably used in order to introduce the required diversity. Other codons that achieve the same ends are also useful, including codon NN N, which leads to the production of 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 10 most diverse positions is added in each of the VH, V? And V? Regions, more than 76 percent of the side chain diversity comes from only seven different residues, these being serine (24 percent), tyrosine (1.4 percent), asparagine (1.1 percent), glycine (9 percent), alanine (7 percent), aspartate (6 percent), and threonine (6 percent) ). This inclination towards the hydrophilic residues and towards the small residues that can provide flexibility of the main chain, reflects the evolution of the surfaces that are
predisposed to binding with a wide range of antigens or epitopes, and can help explain the required promiscuity of antibodies in the primary repertoire. Because it is preferable to mimic this amino acid distribution, the distribution of amino acids at the positions to be varied preferably is similar to that seen at the antigen binding site of the antibodies. This bias in amino acid substitution that allows the selection of certain polypeptides (not only polypeptide antibodies) against a range of target antigens, is easily applied to any repertoire of polypeptides. There are different methods for varying the inclination of the amino acid distribution in the position (including the use of tri-nucleotide mutagenesis, see International Publication Number WO97 / 08320), of which the preferred method, due to the ease of synthesis, is the use of conventional degenerate codons. By comparing the amino acid profile encoded by all combinations of degenerate codons (with individual, double, triple, and quadruple degeneration in equal proportions at each position) with the use of natural amino acids, 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, respectively, using the nomenclature l UPAC - are those that they are closer to the desired amino acid profile: they encode 22 percent serine and 1 1 percent tyrosine, asparagine, glycine, alanine,
Aspartate, threonine, and cysteine. Accordingly, libraries are preferably constructed using any of the DVT, DVC, or DVY codons in each of the diversified positions. Therapeutic and Diagnostic Compositions, and Uses. The invention provides compositions comprising the ligands of the invention, and a pharmaceutically acceptable carrier, diluent, or excipient, and therapeutic and diagnostic methods employing the ligands or compositions of the invention. The ligands according to the method of the present invention can be used in therapeutic and prophylactic applications in vivo, in in vivo diagnostic applications, and the like. The therapeutic and prophylactic uses of the ligands of the invention involve the administration of the ligands according to the invention to a recipient mammal, just like a human being. The ligands bind to the targets with high affinity and / or avidity. In some embodiments, such as with the IgG ligands, the ligands may allow the recruitment of cytotoxic cells to mediate the annihilation of cancer cells, for example, by antibody-dependent cellular cytotoxicity. Substantially pure ligands of a homogeneity of at least 90 to 95 percent are preferred for administration to a mammal, and homogeneity is more preferred.
from 98 to 99 percent or more for pharmaceutical uses, especially when the mammal is a human being. Once purified, partially or until homogeneous as desired, the ligands can be used diagnostically or therapeutically (including extracorporeally), or in the development and performance of immunofluorescent staining, test procedures, 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 typically find use in the prevention, suppression, or treatment of disease states. For example, the ligands may be administered to treat, suppress, or prevent a chronic inflammatory disease, allergic hypersensitivity, cancer, bacterial or viral infection, autoimmune disorders (which include, but are not limited to, type I diabetes, asthma, sclerosis multiple, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, spondylarthropathy (eg, ankylosing spondylitis), systemic lupus erythematosus, inflammatory bowel disease (eg, Crohn's disease, ulcerative colitis), myasthenia gravis, and Behcet's syndrome), psoriasis, endometriosis, and abdominal adhesions (for example, after abdominal surgery). The ligands are useful for the treatment of infectious diseases wherein the cells infected with an infectious agent contain higher levels of cell surface EGFR than the uninfected cells, or which contain one or more
cell surface targets that are not present in uninfected cells, such as a protein that is encoded by the infectious agent (e.g., bacteria, virus). Ligands according to the invention that are capable of binding to EG FR, can be internalized by EGFR-expressing cells (eg, endocytosed), and can deliver therapeutic agents (eg, a toxin) intracellularly (e.g. , provide a dAb that is linked to an intracellular target). In addition, the ligands provide an element by which a binding domain (eg, a dAb monomer) that is capable of specifically binding an intracellular target to an intracellular environment can be delivered. This strategy requires, for example, a link domain with physical properties that make it possible to remain functional within the cell. Alternatively, if the intracellular compartment of final destination is oxidant, a good fold ligand may not be required to be free of disulfide. In the present application, the term "prevention" involves the administration of the protective composition prior to the induction of the disease. "Suppression" refers to the administration of the composition after an inductive event, but before the clinical appearance of the disease. "Treatment" involves the administration of the protective composition after the symptoms of the disease are manifested. The treatment includes
the improvement of the symptoms associated with the disease, and also the prevention or delay of the establishment of the disease, and also the reduction of the severity or frequency of the symptoms of the disease. The term "cancer" refers to the pathological condition in mammals that is typically characterized by poorly regulated proliferation or survival. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia, and lymphoid malignancies. More particular examples of cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer (e.g., microcellular lung carcinoma, non-microcellular lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung), cancer of the peritoneum, hepatocellular cancerGastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, gallbladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, cancer colo-rectal, multiple myeloma, chronic myelogenous leukemia, acute myelogenous leukemia, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, carcinoma of the penis, cancer of the head and neck, and the like. Cancers characterized by the expression of EG FR on the surface of cancer cells (cancers expressing EG FR) include, for
example, bladder cancer, ovarian cancer, colo-rectal cancer, breast cancer, lung cancer (e.g., non-microcellular lung carcinoma), gastric cancer, pancreatic cancer, prostate cancer, head and neck cancer, renal cancer, and gallbladder cancer. Animal model systems that can be used to evaluate the efficacy of the ligands of the invention to prevent, treat, or suppress the disease (eg, cancer) are available. Suitable cancer models include, for example, models of human xenograft and orthotopic cancers in animal models, such as the SCI D-hu myeloma model (Epstein J, and Yaccoby, S., Methods Mol. Med. 1 1 3: 1 83-90 (2005), Tassone P, et al., Clin Cancer Res. 1 1 (11): 4251-8 (2005)), mouse models of human lung cancer (e.g., Meuwissen R. et al. Berns A, Genes Dev. 19 (6): 643-64 (2005)), and mouse models of metastatic cancers (eg, Kubota T., J. Cell Biochem. 56 (1): 4-8 (1 994 )). In general terms, the present ligands will be used in a purified form together with pharmacologically appropriate vehicles. Typically, these vehicles include solutions, emulsions, or aqueous or alcoholic / aqueous suspensions, including serum and / or regulated media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, and lactated Ringer. Suitable physiologically acceptable adjuvants, if necessary to maintain a complex
of polypeptide in suspension, can be selected from thickeners, such as carboxy-methyl-cellulose, polyvinyl-pyrrolidone, gelatin, and alginates. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. There may also be preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases present (Mack (1981) Remington's Pharmaceutical Sciences, 1st 6th Edition). A variety of suitable formulations, including sustained release formulations, can be used. The ligands of the present invention can be used as compositions administered separately, or in conjunction 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 after administration of the additional agent. In general terms, the ligand and the additional agent are administered in a manner that provides an overlap of the therapeutic effect. Additional agents that may be administered or formulated with the invention ligand include, for example, different immunotherapeutic drugs, such as cyclosporin, methotrexate, adriamycin, or cisplatin, antibiotics, antifungals, anti-viral agents, and immunotoxins. For example, when the antagonist is
administered to prevent, suppress, or treat lung inflammation or a respiratory disease, it may be administered in conjunction with phosphodiesterase inhibitors (eg, phosphodiesterase 4 inhibitors), bronchodilators (eg, beta2 agonists, anticholinergics, theophylline), agonists- short-acting beta (for example, albuterol, salbutamol, bambuterol, fenoterol, isoeterin, isoproterenol, levalbuterol, metaproterenol, pirbuterol, terbutaline, and tyrlato), long-acting beta-agonists (eg formoterol and salt meterol), anticholinergic action short (for example, ipratropium bromide and oxitropium bromide), long-acting anticholinergics (eg, tiotropium), theophylline (eg, short-acting formulations, long-acting formulations), steroid-inhaled (eg, beclomethasone, budesonide, flunisolide, fluticasone propionate, and triamcinolone), oral steroids (eg, methyl-prednisolone, prednisolone, prednisolone, and pred nisone), short-acting beta-agonists with combined anticholinergics (eg, albuterol / salbutamol / ipratropium, and fenoterol / ipratropium), agonists long-acting beta with combined inhaled steroids (eg, salmeterol / fluticasone, and formoterol / budesonide), and mucolytic agents (eg, erdostein, acetyl-cysteine, bromhecism, carbocysteine, guiafenesin, and iodinated glycerol). The ligands of the invention can be co-administered (for example, to treat cancer, an inflammatory disease, or another disease), with one with a variety of suitable co-therapeutic agents, including cytokines, analgesics / antipyretics,
antiemetics, and chemotherapy products. Other suitable co-therapeutic agents include immunosuppressive agents selected from the group consisting of cyclosporin, azathioprine, mycophenolic acid, mycophenolate-mofetil, corticosteroids, methotrexate, gold salts, sulfazalazine, antimalarial agents, brequinar, lefl unomide, mizoribine, 1 5-deoxy-spergualin, 6-mercapto-purine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, and anti-thymocyte globulin, anti-inflammatory agents selected from the group consisting of aspirin, other salicylates, steroid drugs, NSAI Ds (non-steroidal anti-inflammatory drugs), Cox-2 inhibitors, and DMARDs (anti-rheumatic drugs modifying the disease); anti-psoriasis agents selected from the group consisting of coal tar, vitamin A, anthralin, calci potriene, terazoten, corticosteroids, methotrexate, retinoids, cyclosporine, etanercept, alefacept, etaluzimab, 6-thioguapine, mycophenolate-mofetil, tacrolimus ( FK-506), and hydroxy urea. Cytokines include, without limitation, a lymphokine, tumor necrosis factors, tumor necrosis factor-like cytokine, lymphotoxin, interferon, macrophage inflammatory protein, granulocyte monocyte colony stimulating factor, interleukin (including, without limitation, interleukin-1) , interleukin-2, interleukin-6, interleukin-1 2, interleukin-1 5, interleukin-1 8), growth factors, which include, without limitation (eg, growth hormone, insulin-like growth factor)
1 and 2 (IGF-1 and IGF-2), granulocyte colony stimulating factor (GCSF), platelet-derived growth factor (PGDF), epidermal growth factor (EGF), and agents for the stimulation of erythropoiesis, for example recombinant human erythropoietin (Epoetin alfa), EPO, a hormonal agonist, hormone antagonists (e.g., flutamide, tamoxifen, leuprolide acetate (LUPRON), and steroids (e.g., dexamethasone, retinoid, betamethasone, cortisol, cortisone, prednisone , dehydrotestosterone, glucocorticoid, mineralocorticoid, estrogen, testosterone, progestin.) Analgesics / antipyretics may include, without limitation, aspirin, acetaminophen, ibuprofen, naproxen-sodium, buprenorphine hydrochloride, propoxyphene hydrochloride, propoxyphene napsylate, meperidine hydrochloride, hydromorphone hydrochloride, morphine sulfate, oxycodone hydrochloride, codeine phosphate, dihydrocodeine bitartrate, pentazocine hydrochloride, beta bitartrate drocodone, levorphanol tartrate, diflunisal, trolamine salicylate, nalbuphine hydrochloride, mefenamic acid, butorphanol tartrate, choline salicylate, butalbital, phenyl-toloxamine citrate, diphenhydramine citrate, methotrimeprazine, cinamedrin hydrochloride, meprobamate, and the like. Antiemetics can also be co-administered to prevent or treat nausea and vomiting, for example, suitable antiemetics include meclizine hydrochloride, nabilone, prochlorperazine, dimenhydrinate, promethazine hydrochloride, triethylperazine,
scopolamine, and the like. Chemotherapeutic agents, as this term is used herein, include, but are not limited to, for example, anti-microtubule agents, e.g., taxol (pacl itaxel), taxotere (docetaxel); alkylating agents, for example, cyclophosphamide, carmustine, lomustine, and chlorambucil; cytotoxic antibiotics, for example dacti nomicin, doxorubicin, mitomycin C, and bleomycin; anti-metabolites, for example cytarabine, gemcitabine, methotrexate, and 5-fl uoro-u-racyl; antimitotics, for example, vincristine, vinca alkaloids, e.g. etoposide, vinblastine, and vincristine; and others, such as cisplatin, dacarbazine, procarbazine, and hydroxy urea; and combinations thereof. The ligands of the invention can be used to treat cancer in combination with another therapeutic agent. For example, a ligand of the invention can be administered in combination with a chemotherapeutic agent or with an antineoplastic composition comprising (at least one) chemotherapeutic agent. In a convenient manner, in this therapeutic approach, the amount of chemotherapeutic agent that must be administered to be effective can be reduced. Accordingly, the invention provides a method for the treatment of cancer, which comprises administering to a patient in need thereof, a therapeutically effective amount of a ligand of the invention and a chemotherapeutic agent, wherein the chemotherapeutic agent is administered in a low dose. In general terms, the amount of
The chemotherapeutic agent that is co-administered with a ligand of the invention is about 80 percent, or about 70 percent, or about 60 percent, or about 50 percent, or about 40 percent. percent, or about 30 percent, or about 20 percent, or about 1 0 percent or less, of the dose of chemotherapeutic agent alone that is normally administered to a patient. Accordingly, 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 may include "cocktails" of different cytotoxic agents or other agents in conjunction with the ligands of the present invention, or even combinations of ligands according to the present invention having different specificities, such as the ligands selected using different antigens or target epitopes, whether they are clustered or not before administration. The route of administration of the pharmaceutical compositions according to the invention can be any suitable route, such as any of those commonly known to those of ordinary skill in the art. For therapy, including, without imitation, immunotherapy, the ligands of the invention can be administered to any patient according to conventional techniques. The
administration can be by any appropriate mode, including parenterally (eg, intravenous, intramuscular, intraperitoneal, intra-articular, intrathecal), transdermally, by the pulmonary route, or also, appropriately, by direct infusion with a catheter. The dosage and the frequency of administration will depend on the age, sex, and condition of the patient, the concurrent administration of other drugs, contraindications, and other parameters that should be taken into account by the clinic. Administration may be local (eg, local delivery to the lung by pulmonary administration (eg, intranasal administration) or local injection directly into a tumor), or systemically, as indicated. The ligands of this invention can be lyophilized for storage, and can be reconstituted in a suitable vehicle before use. This technique has been shown to be effective with conventional immunoglobulins, and lyophilization and reconstitution techniques known in the art can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitution can lead to different degrees of loss of antibody activity (eg, with conventional immunoglobulins, IgM antibodies tend to have a greater loss of activity than antibodies. of IgG), and that you may have to adjust the levels of use upwards to compensate. The compositions containing the ligands can be administered for prophylactic and / or therapeutic treatments. In
certain therapeutic applications, an amount suitable to carry out at least a partial inhibition, suppression, modulation, annihilation, or some other measurable parameter, of a population of selected cells, 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, but in general will be in the range of 0.005 to 5.0 milligrams of ligand per kilogram of body weight, most commonly using doses of 0.05 to 2.0 milligrams / kilogram / dose. For prophylactic applications, compositions containing the present ligands or cocktails thereof at similar or slightly lower dosages may also be administered to prevent, inhibit, or delay the establishment of the disease (e.g., to sustain remission or passivity, or to prevent the acute phase). The skilled person will be able to determine the appropriate dosage range for treating, suppressing, or preventing the disease. When a ligand is given 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, from about 10 micrograms / kilogram to about 80 milligrams / kilogram, from about 1000 micrograms / kilogram to about 80 milligrams / kilogram, from about 1 thousand kilograms / kilogram to about 80
milligrams / kilogram, from about 1 milligram / kilogram to about 70 milligrams / kilogram, from about 1 milligram / kilogram to about 60 milligrams / kilogram, from about 1 milligram / kilogram to about 50 milligrams / kilogram, of about 1 milligram / kilogram at about 40 milligrams / kilogram, from about 1 milligram / kilogram to about 30 milligrams / kilogram, from about 1 milligram / kilogram to about 20 milliliters per kilogram, of about 1 milligram / kilogram to about 10 milligrams / kilogram, of about 1 0 micrograms / kilogram to about 10 milligrams / kilogram, from about 1 0"micrograms / kilogram to about 5 milligrams / kilogram, from about 1 micrograms / -kilogram to about 2.5 milligrams / kilogram, of about 1 milligram / kilogram , of approximately 2 milligrams / kilogram, of approximately 3 milligrams / kilogram, about 4 milligrams / kilogram, about 5 milligrams / kilogram, about 6 milligrams / kilogram, about 7 milligrams / kilogram, about 8 milligrams / kilogram, about 9 milligrams / kilogram, or about 1 0 milligrams / kilogram 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 10 micrograms / kilogram to about 10 hours.
milligrams / kilogram (eg, from about 10 micrograms / kilogram, from about 1000 micrograms / kilogram, of about 1 milligram / kilogram, of about 2 milligrams / kilogram, of about 3 milligrams / kilogram, of about 4 milligrams / kilogram , of approximately 5 milligrams / kilogram, of approximately 6 milligrams / kilogram, of approximately 7 thousand igrates / kilogram, of approximately 8 milligrams / kilogram, of approximately 9 milligrams / kilogram, or of approximately 10 milligrams /-kilogram). In particular embodiments, the ligand of the invention is administered in a dose that provides EGFR saturation or a desired serum concentration in vivo. The skilled physician can determine the appropriate dosage to achieve saturation, for example, by titrating the ligand and monitoring the amount of binding sites on the cells expressing EGFR, or the serum concentration of the ligand. Therapeutic regimens that involve the administration of a therapeutic agent to achieve 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 symptoms are reduced (for example, by at least 10%, or at least one point on the evaluation scale).
clinical), in relation to these symptoms present before treatment, or in relation to these symptoms in an individual (human or animal model) not treated with the com position or other adequate control. The symptoms will obviously vary depending on the disease or the disorder, if they can be measured by a clinician or technician ordinarily skilled. These symptoms can be measured, for example, by monitoring the level of one or more biochemical indicators of the disease or disorder (for example, the levels of an enzyme or metabolite correlated with the disease, the number of cells affected, etc. .), by monitoring physical manifestations (eg, inflammation, tumor size, etc.), or by a accepted clinical evaluation scale, for example, the Scale of State of Expanded Disability (for multiple sclerosis) , I rvine Inflammatory I Disease Questionnaire (the evaluation of 32 points evaluates the quality of life with respect to intestinal function, systemic symptoms, social function, and emotional state - the rating is in the range of 32 to 224, with higher grades indicating a better quality of life), the Rheumatoid Arthritis Scale of Quality of Life, or other standard clinical evaluation scale accepted, as is known in this field. A sustained reduction (for example, one day or more, preferably longer) in the symptoms of the disease or disorder by at least 10 percent, or by one or more points on a given clinical scale, indicates a treatment "cash". In a similar way, prophylaxis
carried out using a composition as described herein, is "effective" if the establishment or severity of one or more symptoms in relation to these symptoms is delayed, reduced, or eliminated in a similar individual (human model). or animal) not treated with the composition. A composition containing the ligands according to the present invention can be used in prophylactic and therapeutic settings to aid in the alteration, inactivation, annihilation, or removal of a selected target population of cells in a mammal. In addition, selected ligands and repertoires of polypeptides described herein, may be used extracorporeally or in vitro selectively to effectively kill, consume, or otherwise remove a population of target cells from a heterogeneous collection of cells. The blood of a mammal can be combined extracorporeally with the ligands, for example the antibodies, the cell surface receptors, or the lace proteins thereof, where the unwanted cells are annihilated or otherwise removed from the blood. re, to return to the mammal in accordance with conventional techniques. In one embodiment, the invention relates to a method for delivering anti-angiogenic therapy (anti-VEGF therapy) to a site containing cells that express or overexpress EGFR, which comprises administering an effective amount of a ligand that has binding specificity for VEG F and for EGFR, to a subject
I need it. The invention also relates to the use of a ligand having binding specificity for VEG F and for EG FR, to deliver an anti-angiogenic therapy (anti-VEGF therapy) to a site containing cells that express or overexpress EG FR. . The 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-VEG F therapy) to a site containing cells that express or overexpress EG FR, or to inhibit angiogenesis in a site that contains cells that express or overexpress EGFR. In particular embodiments, the invention relates to a method for the treatment of cancer, which comprises administering to a subject in need thereof, a therapeutically effective amount of a ligand, as described herein, having binding specificity for VEGF and for EG FR. 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-microcellular lung carcinoma), gastric cancer, cancer pancreatic cancer, prostate cancer, head and neck cancer, kidney cancer, and gallbladder cancer. In other embodiments, the invention relates to a method for the treatment of cancer, which comprises administering to a subject in need, a therapeutically effective amount of a
ligand, as described herein (eg, a ligand having binding specificity for VEGF, a ligand having binding specificity for EGFR, a ligand having binding specificity for VEGF and EGFR), and an anti-binding composition. neoplastic, wherein this anti-neoplastic composition comprises at least one chemotherapeutic agent selected from the group consisting of alkylating agents, anti-metabolites, analogs of phytic acid, pyrimidine analogs, purine analogues, and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthracenedione-substituted urea, methyl-hydrazine derivatives, adrenocortical suppressor, adrenocorticosteroids, progestins, estrogens, anti-estrogen, androgens, anti-androgen, and gonadotropin-releasing hormone analogue. In some modalities, the chemotherapeutic agent is selected from the group consisting of cisplatin, dicarbazine, dactinomycin, mechlorethamine, streptozocin, cyclophosphamide, capecitabine, carmustine, lomustine, doxorubicin, daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluoro-uracil , vinblastine, vincristine, bleomycin, paclitaxel, docetaxel, doxetaxe, aldesleucine, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon-alpha, irinotecan, leuprolide, leucovorin, megestrol, melphalan, mercapto -purine, oxaliplatin, plicamycin, mitotane, pegaspargase,
pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thiowa nina, thiotepa, uracil mustard, vinorel bina, chlorambucil, taxol, an additional growth factor receptor antagonist, and a combination of any of the foregoing. Assays to Evaluate Ligands The ligands of the invention can be assayed using any suitable in vitro or in vivo assay. For example, using the receptor binding assays or bioassays described herein. Bioassay to Determine VEGF Activity: This bioassay measures the ability of ligands (eg, dAbs) to neutralize proliferation of HVE cells (human vascular endothelial cells) induced by VEG F. HUVE cells applied to 96-well plates , incubated for 72 hours with previously balanced VEGF, and dAb protein. The number of cells is then measured using a cell viability dye. The test is carried out as follows. HVE cells are tri-syn- thesized from a sub-confluent 1 75 square centimeter flask. The medium is aspirated, the cells are washed with 5 milliliters of tri psine, and then incubated with 2 milliliters of trypsin at room temperature for 5 minutes. The cells are gently dislodged from the base of the flask, tapping against the hand. Then add 8 milliliters of induction medium to the flask, pipetting the cells to disperse any lumps.
Viable cells are counted using trypan blue dye. The cells are centrifuged and washed twice in an induction medium, centrifuging the cells, and aspirating the medium after each wash. After the final aspiration, the cells are diluted to 1 05 cells / milliliter (in the induction medium), and applied at 1000 microliters per well in a 96-well plate (1 0,000 cells / well). The plate is incubated for > 2 hours at approximately 37 ° C, to allow cell attachment. 60 microliters of dAb protein and 60 microliters of induction medium containing 40 nanograms / milliliter of
VEG F165 (for a final concentration of 1.0 nanograms / milliliter), to a 96 well bottom-V plate, and sealed with film. The dAb / VEGF mixture is then incubated at 37 ° C for 0.5 to 1 hour. The dAb / VEGF plate is removed from the incubator, and 1 00 microliters of solution (final volume of 200 microliters) are added to each well of the plate containing H UVEC. This plate is then returned to the incubator at 37 ° C for a period of at least 72 hours. Control wells include the following: wells that contain cells but do not contain VEGF; wells containing cells, a positive control anti-VEGF antibody and VEGF; and control wells containing cells and VEG F only.
Cell viability is assessed by adding 200 microliters per well of Celltiter 96 reagent, and the plate is incubated at 37 ° C for 2 to 4 hours, until a brown color develops. The reaction
stop by adding 20 microliters per well of S DS to 1 0 percent (weight / volume). The absorbance is then read at 490 nanometers, using a Wallac microplate reader. The absorbance of control wells without VEGF is subtracted from all other values. The absorbance is proportional to the number of cells. Control wells containing the anti-VEGF control antibodies should also exhibit minimal cel- lular proliferation. Wells that contain VEGF alone must exhibit maximum cell proliferation. EXAM PLOS Example 1. VEGF Receptor Linkage Assays. VEG F is a specific mitogen for endothelial cells in vitro, and a potent angiogenic factor in vivo, with high levels of protein that is expressed in different types of tumors. It is a 45 kDa glycoprotein that is active as a homodimer. Several isoforms have been described that occur through the splicing of alternating mRNA. Of these isoforms, VEGF-121 and VEGF-165 seem to be the most abundant. The specific action of VEG F on endothelial cells is mainly regulated by two types of receptor tyrosine kinase (RTK), VEGF R1 (Flt-1), and VEGF R2 (KDR / Flk-1). However, it appears that VEGF activities, such as mitogenicity, chemotaxis, and induction of morphological changes, are mediated by VEGF R2, even when both receptors undergo phosphorylation after VEGF binding.
VEGF Receptor Engagement Test 2. This method describes a VEGF receptor binding assay to measure the ability of ligands (e.g., dAbs) to prevent binding of VEG F-1 with the VEGF 2 receptor. A recombinant human VEGF R2 / Fc chimera was used in this assay, which comprises the extracellular domain of human VEGF R2 fused to the Fc region of human IgGi. Briefly, the receptor was captured on a ISA plate, then the plate was blocked to prevent the non-specific binding. Then a mixture of VEGF-1 65 and ligand was added, the plate was washed, and the VEGF-1 65 bound to the receptor was detected using a biotinylated anti-VEGF antibody, and an anti-biotin antibody conjugated with rad peroxidase. red icle (HRP). The plate was developed using a colorimetric substrate, and the OD was read at 405 nanometers. If the dAb blocked the VEGF link with the receptor, then no color was detected. The test was carried out as follows. A Nunc Maxisorp 96-well assay plate was coated overnight at 4 ° C with 100 microliters per well of recombinant human VEG F R2 / Fc (R &D Systems, Cat. No. 357-KD-050) in a concentration of 0.5 micrograms / milliliter, in a carbonate buffer. The wells were washed three times with 0.05 percent Tween / phosphate-buffered serum, and three times with phosphate-buffered serum. 200 microlitres per well of 2 percent bovine serum albumin in phosphate-buffered serum were added to block the plaque, and the plaque was
incubated for a minimum of 1 hour at room temperature. The wells were washed (as above), and then 500 microliters per well of ligand was added to each well. Then 50 microliters of VEG F, at a concentration of 6 nanograms / milliliter in diluent (for a final concentration of 3 nanograms / milliliter) were added to each well, and the plate was incubated for 2 hours at room temperature (for supernatants assay, 80 microliters of supernatant were added to each well, and then 20 microliters of VEGF in a concentration of 1 5 nanograms / milliliter). The following controls were included: 0 nanograms / milliliter of VEGF (diluent only); 3 nanograms / milliliter of VEG F (R &D Systems, Cat. No. 293-VE-050); 3 nanograms / milliliter of VEGF with 0.1 micrograms / milliliter of anti-VEGF neutralizing antibody (R & D Systems, Cat. No. MAB293). The plate was washed (as above), and then 1000 microliters of biotinylated anti-VEGF antibody (R & D Systems, Cat. No. BAF293), 0.5 micrograms / milliliter of diluent, was added and incubated for 2 hours at room temperature. ambient. The wells were washed (as above), and then 1000 microliters of anti-biotin antibody conjugated with H RP was added (dilution to 1: 5000 in diluent; Stratech, Cat. No .: 200-032-096). The plate was then incubated for 1 hour at room temperature. The plate was washed (as above), ensuring that
remove any traces of Tween 20, to limit the background, in the following peroxidase assay, and to aid in the prevention of bubbles in the wells of the assay plate, which could give inaccurate OD measurements. 1 00 microliters of peroxidase solution were added
SureBlue 1-Component TM B M icroWell to each well, and the plate was left at room temperature for up to 20 minutes. A soluble deep blue product was developed when the conjugate labeled with H RP reacted with the substrate. The reaction was stopped by the addition of 1000 microliters of 1 M hydrochloric acid (the blue color became yellow). The OD, at 450 nanometers, of the plate was read in a 96-well plate reader within 30 minutes after the acid addition. The OD at 450 nanometers is proportional to the amount of conjugated streptavidin-H RP bound. For some trials, L protein was added. Protein L crosslinks two dAb monomers. The expected results of the controls are as follows: 0 nanog bouquets / milliliter of VEG F should give a low signal of < 0.1 5 OD; 3 nanograms / milliliter of VEGF should give a signal of > 0.5 OD; and 3 nanograms / milliliter of VEGF previously incubated with 0.1 microgram / milliliter of neutralizing antibody, should give a signal < 0.2 OD. VEGF Receptor Binding Assay 1 This assay measures the binding of VEGF-1 65 to VEGF R1, and the
ability of ligands to block their interaction. Here a recombinant human VEG F R1 / Fc chimera was used, comprising the extracellular domain of human VEGF R1 fused to the Fc region of human IgGi. The receptor was captured on an ELISA plate, and then the plate was blocked to prevent non-specific binding. Then a mixture of VEGF-1 and ligand was added, the plate was washed, and the VEGF-165 bound to the receptor was detected using a biotinylated anti-VEGF antibody, and an anti-biotin antibody conjugated with H RP. The plate was developed using a colorimetric substrate, and the OD was read at 450 nanometers. The assay was carried out as follows: A Nunc Maxisorp 96-well assay plate was coated overnight at 4 ° C with 1000 μL per well of recombinant human VEG F R1 / Fc (R &D Systems, Cat. No 321 -FL-050), at approximately 0.1 nanograms / milliliter in carbonate buffer. The wells were washed three times with 0.05 percent Tween / phosphate-buffered serum, and three times with phosphate-buffered serum. 200 microliters per well of 2 percent bovine serum albumin in phosphate-buffered serum was added to block plaque, and the plate was incubated for a minimum of 1 hour at room temperature. The wells were washed (as above), and then 50 microliters per well of purified dAb protein was added to each well. Then 50 microliters of VEGF were added, at a concentration of 1 nanogram / milliliter in diluent (for a
final concentration of 500 picograms / milliliter) to each well, and the plate was incubated for 1 hour at room temperature (supernatants assay, 80 microliters of supernatant was added to each well, and then 20 microliters of VEGF at approximately 2.5 nanograms / milliliter ). The following controls were included: 0 nanograms / m ililiter of VEG F (diluent only); 500 picograms / milliliter of VEG F; and 500 picograms / milliliter of VEGF with 1 microgram / milliliter of anti-VEGF antibody (R & D Systems, Cat. No. MAB293). The plate was washed (as above), and then 1000 microliters of biotinylated anti-VEGF antibody, 50 nanograms / milliliter in diluent, was added and incubated for 1 hour at room temperature. The wells were washed (as above), and then 1000 microliters of anti-biotin antibody conjugated with H RP (dilution to 1: 5000 in diluent) was added. The plate was then incubated for 1 hour at room temperature. The plate was washed (as above), making sure that any traces of Tween 20 were removed to limit the background in the next peroxidase assay, and to help prevent bubbles in the wells of the assay plate, which could give inaccurate OD readings. One thousand microliters of SureBlue 1-Compound TM B M icroWell peroxidase solution was added to each well, and the plate was left at room temperature for up to 20 minutes. It developed
a soluble product of a deep blue color when the conjugate labeled with bound HRP reacted with the substrate. The reaction was stopped by the addition of 1000 microliters of 1 M hydrochloric acid. The OD, at 450 nanometers, of the plate was read in a 96-well plate reader within 30 minutes after the addition of acid. The OD at 450 nanometers is proportional to the amount of conjugated streptavidin-H RP bound. Expected result of the controls: 0 nanograms / milli liter of VEG F should give a low signal of < 0.1 5 OD; 00 picograms / milliliter of VEG F should give a signal of > 0.8 OD; and 500 picograms / milliliter of VEGF previously incubated with 1 microg bouquet / milliliter of neutralizing antibody, should give a signal of < 0.3 OD.
Table 1
TAR1 5-1 had a Kd of 50 to 80 nM when tested in different concentrations on a Biacore low density chip. Other VK dAbs were passed over the low density chip at a concentration (50 nM). Different dAbs showed different binding kinetics. Table 2
* The dAb was tested at 50 nanometers. The VH dAbs were passed over the low density VEG F chip in a Biacore at a concentration (50 nM). Different dAbs showed different binding kinetics.
Example 2. EGFR assay Enzyme assay of EGFR 25 microliters of ligand (e.g., dAb) were applied to a 96-well plate, and then 25 microliters of streptavidin-Alexa Fluor (1 microgram / milliliter) was added (Molecular
Probes), and 25 microliters of A431 cells (ATCC No. CRL-1 555) (8x1 05 cells / milliliter). All reagents were prepared in serum buffered with 1% bovine serum albumin / phosphate. The plate was incubated for 30 minutes at room temperature. Without altering the cells, 25 microliters of biotinylated EG F (I nvitrogen) was added at 400 na to nograms / milliliter to each well, and the plate was incubated for 3 hours at room temperature. Fluorescence was measured using an AB8200 Cell Detection System (Applied Biosystems). Ligands (for example, dAbs) that inhibited the binding of
Biotinylated EGF with EGFR expressed on A431 cells, resulted in lower fluorescence counts. Wells without ligand provided a reference of maximum fluorescence (ie, biotinylated EG F binding), and wells without ligand or with biotinylated EGF, provided a reference of the background level of the fluorescence. These controls were included in all the trials. The results obtained in this assay using certain anti-EGFR dAbs, are presented in Table 3. EGFR Kinase Assay In a 96-well plate, 5x1 04 A431 cells (ATCC No. CRL-1 555) were applied per well in RPM I-1 640 supplemented with 1 0 percent fetal calf serum. The plate was incubated overnight at 37 ° C / 5% CO2, to allow the cells to adhere, and then the medium was replaced with RPM I-1 640. The plate
it was incubated for 4 hours at 37 ° C / 5% CO2. The ligand (prepared in RPM I-1 640) was added to the wells, and the plate was incubated for 45 minutes at 37 ° C / 5% CO2. EGF (I nvitrogen) was added to the wells to give a final concentration of 1 00 nanograms / milliliter, and the plate was incubated for 10 minutes at room temperature. The wells were washed twice with ice-cold phosphate-buffered serum. Cold lysis buffer (1 percent NP-40, 20 mM Tris, 37 mM NaCl 1, 1 percent glycerol, 2 mM EDTA, 1 mM sodium ortho-vanadate, 10 micrograms / milliliter of aprotinin was added , 1 microgram / milliliter of leupeptin), and the plate was incubated on ice for 10 minutes. The supernatants were transferred to an ELISA plate, which had been coated overnight with an anti-EGFR antibody (R & D Systems) at 1 microgram / milliliter in carbonate buffer. The ELISA plate was incubated for 2 hours at room temperature. The plate was washed three times with 0.05 percent phosphate buffered serum / Tween 20. Anti-phosphotyrosine antibody conjugated with red radish peroxidase (Upstate Biotechnology) was added at 1 microgram / milliliter, and the plate was incubated for 1 hour at room temperature. The plate was washed three times with phosphate-regulated serum / Tween, and three times with phosphate-buffered serum. The reaction was developed with the SureBl peroxidase substrate TM B 1-Microwell component (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 anti-EGFR dAbs are presented in Table 3. Table 3
* The data presented are the lowest to highest values obtained, and the (average). Example 3. IgG type formats that have link specificity
VEGF and EGFR Vectors The base structure of pBudCE4.1 (invitrogen) was used to clone the immunoglobulin constant regions, such as the heavy chain constant region and the kappa light chain constant region of lgG1 (see Figure 16 to have a panorama).
An Ig Kappa leader chain was used to facilitate the secretion of the expressed protein. The Ig constant regions (human IgG1 and CK) were produced by GeneArt (Germany). The heavy chain constant region and the signal peptide were cloned into pBudCE4.1 as a HindIII / BglII fragment at the HindIII / BamHI restriction sites. The light chain constant region and the signal peptide are
cloned into pBudCE4.1 as a NotI / MIuI fragment. Cloning of dAb in the IgG Vectors and Prod uction of the
Format Ti po IgG. The dAb VK (specific for VEGF or EGFR) was cloned into the I gG vector as a Sall / BsiWI fragment. The VH dAb (specific for VEG F or EG FR) was cloned into the IgG vector as a BamH I / XhoI fragment. The purified IgG was verified on a reducing SDS gel and not a reductive, and bands of the expected size were observed. Several dAbs that are linked with VEGF or with EG FR were formatted in IgG-like formats that have binding specificity for VEGF and EGFR. The IgG type formats were prepared by producing constructs encoding an IgG heavy chain, where 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 the assays are presented in Table 5. (False VH and false VK are the germline sequences that they do not bind with VEGF or with EGFR).
Table 4
Table 5
Example 4. Double specific online formats. The domain antibodies that bind to VEGF and to EGFR were incorporated into fusion polypeptides containing an anti-VEGFR dAb and an anti-EGFR dAb in a single polypeptide chain. Some of the fusion polypeptides also included an antibody Fc region (-CH2-CH3 from human IgG1). Specific examples of fusion polypeptides that were cloned and expressed include TAR15-10 fused to DOM16-39-206 and Fc (SEQ ID NO.715); DOM16-39-206 merged with TAR15-10 and with Fc (SEQ ID NO: 716), DOM 16-39-206 merged with TAR15-26-501 and with Fc (SEQ ID NO: 717); TAR15-26-501 merged with DOM16-39-206 and with Fc (SEQ ID NO: 718); TAR15-10 merged with DOM16-39-206 (SEQ ID NO: 719); DOM16-39-206 merged with TAR15-10 (SEQ ID NO: 720); DOM16-39-206 merged with TAR15-26-501 (SEQ ID NO: 721); and TAR15-26-501 merged with DOM16-39-206 (SEQ ID NO: 722). The positions of the previous mergers are listed as they appear in the fusion proteins from the term
amino to the carboxyl terminus. The DNA encoding the dAbs was amplified by polymerase chain reaction, and cloned into the expression vectors using conventional methods. In-line fusion polypeptides were produced by expression of the expression vectors in Pichia (fusion that did not contain an Fc region), or in HEK293T cells (Fc region containing fusions). The in-line fusions were batch-linked, and affinity purified on the protein A in line and L-protein resins in line for the EK293T H cells (labeled with Fc), and for the constructs expressed by Pichia, respectively. Portions of various Fc-containing fusions are listed in Table 6, as they appear in the fusion proteins, from the terminus to the carboxyl terminus. In accordance with the above, the structure of the fusion proteins can be seen by reading the table from left to right. The first fusion protein presented in Table 6 has the structure, from the amino terminus to the carboxyl terminus, of DOM 1 5-1 0-Linker 1 -DOM 1 6-39-206-Linker 2-Fc. the binding activities of the fusions were evaluated using the EGFR binding assay described in Example 2, and the binding assay of the VEGF R 2 receptor described in Example 1. General robustness and resistance to degradation were tested by subjecting the fusions in line to proteolysis with trypsin. A double specific ligand solution was prepared and
trypsin (1/25 (w / w) of trypsin to the ligand), and incubated at 30 ° C. Samples were taken at 0 minutes (ie, before the addition of trypsin), at 60 minutes, at 180 minutes, and at 24 hours. At the given time points, the reaction was stopped by the addition of a complete protease inhibitor cocktail to a final concentration of 2X (Roche, Code: 11 836 145 001), with the PAGE loading dye, followed by flash freezing of the samples in liquid nitrogen. Samples were analyzed by SDS-PAGE, and protein bands were visualized to reveal a time course for protease degradation from fusions. These experiments demonstrated that in-line fusions that have a "natural" linker (KVEIKRTVAAPS (SEQ ID NO: 706), which contains the carboxy-terminal amino acids of Vk and the amino-terminal amino acids of Ck, were susceptible to rapid proteolysis, with obvious degradation as early as the 10-minute time point SDS-PAGE analysis revealed that degradation occurred in the linkers between the dAbs, and in the linkers between the dAb and Fc. New linkers were designed that contained less Lys and Arg residues, which are dissociation points for trypsin, and are abundant in the natural linker.Fusions containing the designed linkers (LVTVSSAST (SEQ ID NO: 707)) or (LVTVSSGGGGSGGGS (SEQ ID NO: 708) ) showed a much improved resistance to trypsin proteolysis.
Additional binding assays were carried out to evaluate the potency of the in-line fusions containing the designed linkers. The results revealed that the designed linkers had no substantial adverse effect on the power.
Table 6 Fusion Polypeptides Containing Fc
Example 5. Additional designed linkers. Several mutations designed to the C-terminal region of the Vk dAbs expressed on the light chain of the IgG type formats were introduced to reduce the sensitivity to the protease. He
"natural linker" was GQGTKVE I KRTVAAPS (SEQ I D NO: 709, which contains the carboxy-terminal amino acids of Vk and the amino-terminal amino acids of Ck). Linkers variants 1 to 3 were designed with amino acid replacements that replaced some or all of the positively charged residues in the natural linker, with more conservative substitutions that are not positively charged at physiological pH. It is possible that the arginine residue in the natural linker is less susceptible to alteration due to the ionic interactions it forms within the CL domain. The variant linker 1 (GQGTNVEI N RTVAAPS (S EQ I D NO: 71 0)) replaces both or sinas in the natural linker with asparagine. The linker vari before 1, and the variant linker 2 (GQGTNVEI NQTVAAPS (SEQ ID NO: 71 1)), which additionally changes the arginine in the natural linker to glutamine, introduces an N-glycosylation (NxT) site in the linker . The SDS-PAGE E analysis of the IgG-type formats containing the variant linker 1 or the variant linker 2 showed that the light chain had a higher molecular weight, consistent with an N-glycosylation event. The variant linker
(GQGTNVEIQ RTVAAPS (SEQ ID NO: 71 2)) removes the N-glycosylation site, while leaving the arginine in the natural linker at its place. The variant linker 4 (GQGTLVTVSSTVAAPS (SEQ ID NO: 71 3)) replaces the six C-terminal amino acids of the Vk domain with the corresponding residues from a VH domain, and
It is devoid of positive charges. The protease resistance (resistance to trypsin evaluated as described in Example 4) of the IgG-like formats containing the linkers varied from 1 to 4, revealed that the IgG-like formats containing the designed variant linkers were more resistant to the protease than an IgG type format that contained the natural linker. Example 6. Fusions of dAb DOM 16-dAb anti-serum albumin. DAb DOM 1 6-dAb fusions anti-serum albumin were designed, and were expressed as a fusion of an anti-EGFR dAb with an anti-serum albumin dAb (a DOM7 dAb). The portions of the fusions are listed in Table 7 as they appear in the fusion proteins, from the amino terminus to the carboxyl terminus. In accordance with the above, the structure of the fusion proteins can be seen by reading the table from left to right. The first fusion protein presented in Table 7 has the structure, from the amino terminus to the carboxyl terminus, of DOM 1 6-39-61 8 (S 1 2P) -linker-DOM7h-1 4. DOM 1 6 -39-61 8 contains a mutation of Serine up to Proline at position 1 2, which stops the link with protein L, and prevents the accumulation of the light chain. The DOM7 dAbs are mutated to abrogate the link to the pawnshop, and are therefore inactivated.
Table 7
Example 7. Mapping the EGFR Epitope. Epitope mapping was conducted in competitive binding assays using anti-EGFR, EGF, and ERBITUX dAbs (cetuximab, Imclone Systems). Link studies were carried out using a Biacore biosensor. DOM16-39-200 was used as a reference in this study. DOM16-39-200, and other dAbs designated as DOM16-39-X, are mature variants by affinity of DOM16-39. In accordance with the above, all dAbs in the DOM16-39 series will have substantially the same epitope specificity, because affinity maturation produces dAbs that bind with a higher affinity, but does not alter the specificity of dAb. The results showed that DOM16-72, DOM16-79, and DOM16-112 competed with DOM16-39-200 for the EGFR binding, indicating that these dAbs bind to overlapped epitopes. However, DOM16-32, DOM16-52, and DOM16-80 were shown to bind to a different epitope. It is known that ERBITUX (cetuximab; Imclone Systems) inhibits the binding of EGF with EGFR (overlapping epitopes that bind with cetuximab and EGF on EGFR). The results of this study also demonstrated that DOM16-39-200 and cetuximab competed for the link with EGFR, indicating that the DOM16-39-200 epitope overlapped with the cetuximab epitope, and with the binding site for EGF. Although this invention has been shown and described particularly with reference to its preferred embodiments, it will be
It is understood by those skilled in the art that different changes may be made in the form and in the details thereof without departing from the scope of the invention encompassed by the appended claims.
Claims (196)
- CLAIMS 1. A ligand having a binding specificity for the vascular endothelial growth factor (VEGF) and for the epidermal growth factor receptor (EGFR), which comprises at least one protein fraction having a binding site with a specificity of linkage for VEGF, and at least a protein fraction having a binding site with a binding specificity for EGFR. The ligand of claim 1, wherein each said protein fraction having a binding site with a binding specificity for EGFR, competes for binding to EGFR, with an anti-EGFR domain (dAb) antibody selected from 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). 3. The ligand of claim 1, wherein each said protein fraction having a binding site with a binding specificity for EGFR, competes for binding to EGFR, with an anti-EGFR domain (dAb) antibody selected at from the group consisting of: DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39- 551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). The ligand of claim 1, wherein each said protein fraction having a binding site with a binding specificity for VEGF, competes for binding with VEGF, with a selected anti-VEGF domain (dAb) antibody to starting 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 wherein each protein fraction having a binding site with a binding specificity for EGFR, competes for the EGFR binding, with an anti-EGFR domain (dAb) antibody 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). The ligand of claim 1, wherein each protein fraction having a binding site with a binding specificity for VEGF, competes for binding with VEGF, with an anti-VEGF domain (dAb) antibody selected from 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 wherein each protein fraction having a binding site with a binding specificity for EGFR, competes for the EGFR binding, with an anti-EGFR domain (dAb) antibody selected from the group consisting of: DOM16- 39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39- 551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16 -39-619 (SEQ ID NO: 622). The ligand of claim 1, wherein each protein fraction having a binding site with a binding specificity for VEGF, competes for binding with VEGF, with bevacizumab. The ligand of claim 1 or claim 4, wherein each protein fraction having a binding site with a binding specificity for EGFR competes for the EGFR co-link with cetuximab. 8. The ligand of any one of claims 1 to 7, wherein each protein fraction having a binding site with a binding specificity for VEGF competes for binding with VEGF, with bevacizumab; and wherein each protein fraction having a binding site with a binding specificity for EGFR, competes for the EGFR binding, with cetuximab. The ligand of any one of claims 1 to 8, wherein each protein fraction having a binding site with a binding specificity for VEGF, and each protein fraction having a binding site with a binding specificity for VEGFR, are provided by an antibody fragment. 10. The ligand of claim 9, wherein said antibody fragment is a single immunoglobulin variable domain. 11. A ligand that has a binding specificity for vascular endothelial growth factor (VEGF) and for the epidermal growth factor receptor (EGFR), which comprises at least one unique immunoglobulin variable domain with a binding specificity for VEGF, and at least one unique immunoglobulin variable domain with a binding specificity for EGFR, wherein each unique immunoglobulin variable domain with a binding specificity for EGFR competes for EGFR binding, with an anti-EGFR domain (dAb) antibody. 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). 12. A ligand having a binding specificity for vascular endothelial growth factor (VEGF) and for the epidermal growth factor receptor (EGFR), which comprises at least one unique immunoglobulin variable domain with a binding specificity for VEGF, and at least one unique immunoglobulin variable domain with a binding specificity for EGFR, wherein each unique immunoglobulin variable domain with a binding specificity for EGFR competes for binding with EGFR, with an anti-domain (dAb) antibody -EGFR selected from the group consisting of: DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39- 542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), D0M16-39-604 (SEQ ID NO: 611), DOM16- 39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). The ligand of claim 11 or claim 12, wherein each unique immunoglobulin variable domain with a binding specificity for EGFR is fused to an Fc region of an antibody. The ligand of claim 11 or claim 12, wherein each unique immunoglobulin variable domain with a binding specificity for VEGF is fused to an Fc region of an antibody. The ligand of claim 11, wherein each unique immunoglobulin variable domain with a binding specificity for EGFR, comprises an amino acid sequence having an amino acid sequence identity of at least about 85 percent with the sequence of amino acids of a 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). The ligand of claim 12, wherein each unique immunoglobulin variable domain with a binding specificity for EGFR, comprises an amino acid sequence having an amino acid sequence identity of at least about 85 percent with the amino acid sequence of a dAb selected from the group consisting of: DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16- 39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). 17. A ligand having a binding specificity for vascular endothelial growth factor (VEGF) and for the epidermal growth factor receptor (EGFR), which comprises at least one unique immunoglobulin variable domain with a binding specificity for VEGF, and at least one unique immunoglobulin variable domain with a binding specificity for EGFR, wherein: each unique immunoglobulin variable domain with a binding specificity for VEGF competes for binding with VEGF, with a domain antibody (dAb) anti-VEGF 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 each unique immunoglobulin variable domain with a binding specificity for EGFR competes for binding to EGFR, with an anti-EGFR domain (dAb) antibody 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). 18. A ligand having binding specificity for vascular endothelial growth factor (VEGF) and for the epidermal growth factor receptor (EGFR), which comprises at least one unique immunoglobulin variable domain with a binding specificity for VEGF, and at least one unique immunoglobulin variable domain with a binding specificity for EGFR, wherein: each unique immunoglobulin variable domain with a binding specificity for VEGF competes for binding with 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), and TAR15-26 (SEQ ID NO: 123); and each unique immunoglobulin variable domain with a binding specificity for EGFR competes for binding to EGFR, with an anti-EGFR domain (dAb) antibody selected from the group consisting of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). The ligand of claim 17, wherein each unique immunoglobulin variable domain with a binding specificity for VEGF comprises an amino acid sequence having an amino acid sequence identity of at least about 85 percent 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), and TAR15-26 (SEQ ID NO: 123); and each unique immunoglobulin variable domain with a binding specificity for EGFR, competes for the EGFR binding, with an anti-EGFR domain (dAb) antibody 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). The ligand of claim 18, wherein each unique immunoglobulin variable domain with a binding specificity for VEGF, comprises an amino acid sequence having an amino acid sequence identity of at least about 85 percent with the sequence of amino acids of a 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 each unique immunoglobulin variable domain with a binding specificity for EGFR competes for the EGFR binding, with an anti-EGFR domain antibody (dAb), selected from the group consisting of DOM16-39-521 (SEQ ID NO. : 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ. ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM 16-39-61 9 (SEQ ID NO: 622). twenty-one . The ligand of any one of claims 1 to 1 9, wherein this ligand inhibits the binding of epidermal growth factor (EG F) and / or transforming growth factor-alpha (TG F-alpha) to EGFR. 22. The ligand of any of claims 1 to 1 9, wherein this ligand inhibits the activity of EG FR. 23. The ligand of any one of claims 1 to 1 9, wherein this ligand inhibits the activity of EG FR without substantially inhibiting the binding of epidermal growth factor (EGF), and / or transforming growth factor alpha (TGF). -alfa), with the EGFR. 24. The ligand of any of claims 1 to 23, wherein this ligand inhibits the binding of vascular endothelial growth factor to the vascular endothelial growth factor receptor 1 (VEGFR1) and / or to the endothelial growth factor receptor. vascular 2 (VEGFR2). 25. The ligand of any of claims 1 to 23, wherein this ligand inhibits the activity of VEGF. 26. The ligand of any one of claims 1 to 23, wherein this ligand inhibits the activity of vascular endothelial growth factor without substantially inhibiting the binding of vascular endothelial growth factor to the vascular endothelial growth factor receptor 1 (VEGFR1). and / or the vascular endothelial growth factor 2 receptor (VEG FR2). 27. The ligand of any one of claims 1 to 26, wherein each unique immunoglobulin variable domain with a binding specificity for VE GF, binds to VEG F with an affinity (KD) that is between about 1 00 nM and approximately 1 pM, as determined by surface plasmon resonance. The ligand of any of claims 10 to 26, wherein each unique immunoglobulin variable domain with a binding specificity for EG FR, binds to the EG FR with an affinity (KD) that is between about 1 00 nM and about 1 pM, as determined by surface plasmon resonance. 29. The ligand of claim 28, wherein each unique immunoglobulin variable domain with a binding specificity for EGFR, binds to the EG FR with an affinity (KD) that is between about 1.0 nM and about 1000 pM. , as determined by surface plasmon resonance. 30. The ligand of any of claims 1 to 26, wherein this ligand binds to VEGF with an affinity (KD) that is between about 1 00 nM and about 1 pM, as determined by surface plasmon resonance. 31 The ligand of any of claims 1 to 26 and 30, wherein this ligand binds to the EGFR with an affinity (KD) that is between about 1 00 nM and about 1 pM, as determined by surface plasmon resonance. 32. The ligand of claim 31, wherein this ligand binds to the EGFR with an affinity (KD) that is between about 1.0 nM and about 1000 pM, as determined by surface plasmon resonance. The ligand of any of claims 1 0-32, wherein this ligand comprises a single immunoglobulin variable domain with a binding specificity for VEGF that is a V H H and / or a single immunoglobulin variable domain with a specificity of linkage for EGFR which is a VH H - 34. The ligand of any of claims 1 0-32, wherein each unique immunoglobulin variable domain with a binding specificity for VEGF, and each unique immunoglobulin variable domain with a specificity of linkage for EGF R, are independently selected from the group consisting of a human VH and a human VL. 35. The ligand of any one of claims 1 to 34, wherein this ligand is an IgG-like format comprising two unique immunoglobulin domes, with a binding specificity for VEGF, and two unique immunoglobulin variable domains with a specificity. Link for EG FR. 36. The ligand of any of claims 1 to 35, wherein this ligand comprises an antibody Fc region. 37. A ligand having binding specificity for vascular endothelial growth factor (VEGF), and for the epidermal growth factor receptor (EG FR), which comprises at least one single immunoglobulin variable domain with a binding specificity for VEGF, and at least one single immunoglobulin variable domain with a binding specificity for EGFR, wherein: each unique immunoglobulin variable domain with a binding specificity for VEGF competes by binding with VEGF, with an anti-VEGF domain (dAb) antibody 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 each unique immunoglobulin variable domain with a binding specificity for EGFR competes for the linkage with EGFR, with cetuximab. 38. The ligand of claim 37, wherein each unique immunoglobulin variable domain with a binding specificity for VEGF, comprises an amino acid sequence having an amino acid sequence identity of at least about 85 percent with the sequence of amino acids of a 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). 39. A ligand that has a binding specificity for vascular endothelial growth factor (VEGF) and for the epidermal growth factor receptor (EGFR), which comprises at least one unique immunoglobulin variable domain with a binding specificity for VEGF, and at least one single Immunoglobulin variable domain with a binding specificity for EGFR, wherein: each unique immunoglobulin variable domain with a binding specificity for VEGF competes for binding with VEGF, with bevacizumab; and each unique immunoglobulin variable domain with a binding specificity for EGFR, competes for the EGFR binding, 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). 40. A ligand that has binding specificity for vascular endothelial growth factor (VEGF) and for the epidermal growth factor receptor (EGFR), which comprises at least one unique immunoglobulin variable domain with a binding specificity for VEGF , and at least one unique immunoglobulin variable domain with a binding specificity for EGFR, wherein: each unique immunoglobulin variable domain with a binding specificity for VEGF competes for binding with VEGF, with bevacizumab; and each unique immunoglobulin variable domain with a binding specificity for EGFR, competes for binding with EGFR, with an anti-EGFR domain antibody (dAb) selected from from the group consisting of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 ( SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39 -619 (SEQ ID NO: 622). 41. The ligand of claim 39, wherein each unique immunoglobulin variable domain with a binding specificity for EGFR comprises an amino acid sequence having an amino acid sequence identity of at least about 85 percent with the amino acid sequence of a 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) . 42. The ligand of claim 40, wherein each unique immunoglobulin variable domain with a binding specificity for EGFR, comprises an amino acid sequence having an amino acid sequence identity of at least about 85 percent with the sequence of amino acids of a dAb selected from the group consisting of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586) , DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). 43. A ligand that has a binding specificity for vascular endothelial growth factor (VEG F) and for the epidermal growth factor receptor (EGFR), which comprises a first single immunoglobulin variable domain with a binding specificity for VEGF , and a second single variable immunoglobulin domain with a binding specificity for EGFR, where: the first single variable domain of immunoglobulin competes for binding to VEG F, with bevacizumab; and the second single immunoglobulin variable domain competes for the link with EGFR, with cetuximab. 44. The ligand of any of claims 37 to 43, wherein this ligand inhibits the binding of epidermal growth factor (EGF), and / or transforming growth factor alpha (TGF-alpha), with EGFR. 45. The ligand of any of claims 37 to 43, wherein this ligand inhibits the activity of EG FR. 46. The ligand of any of claims 37 to 43, wherein this ligand inhibits EGFR activity without substantially inhibiting the binding of epidermal growth factor (EGF) and / or transforming growth factor alpha (TG F-alpha ) with the EG FR. 47. The ligand of any of claims 37 to 46, wherein this ligand inhibits the binding of vascular endothelial growth factor to the growth factor receptor. vascular endothelial 1 (VEG FR1) and / or vascular endothelial growth factor receptor 2 (VEGFR2). 48. The ligand of any of claims 37 to 46, wherein this ligand inhibits the activity of VEG F. 49. The ligand of any of claims 37 to 46, wherein this ligand inhibits the activity of endothelial growth factor. vascular without substantially inhibiting the binding of vascular endothelial growth factor with the vascular endothelial growth factor receptor 1 (VEGFR1) and / or with the vascular endothelial growth factor receptor 2 (VEGFR2). 50. The ligand of any of claims 37 to 49, wherein each unique immunoglobulin variable domain with a binding specificity for VEGF, binds to VEGF with an affinity (KD) that is between about 1 00 nM and about 1. pM, as determined by surface plasmon resonance. 51 The ligand of any of claims 37 to 50, wherein each unique immunoglobulin variable domain with a binding specificity for EG FR, binds to the EG FR with an affinity (KD) that is between about 1 00 nM and about 1 pM, as determined by surface plasmon resonance. 52. The ligand of claim 51, wherein each unique immunoglobulin variable domain with a binding specificity for EGFR, binds to the EGFR with an affinity (KD) that is between about 1 0 n M and about 1 00 pM, as determined by surface plasmon resonance. 53. The ligand of any of claims 37 to 49, wherein this ligand binds to VEGF with an affinity (KD) that is between about 1 00 nM and about 1 pM, as determined by surface plasmon resonance. 54. The ligand of any of claims 37 to 49 and 53, wherein this ligand binds to the EGFR with an affinity (KD) that is between about 1 00 nM and about 1 pM, as determined by plasmon resonance. superficial 55. The ligand of claim 54, wherein this ligand binds to the EGFR with an affinity (KD) that is between about 1.0 nM and about 1000 pM, as determined by surface plasmon resonance. 56. The ligand of any of claims 37 to 55, wherein this ligand comprises a single immunoglobulin variable domain with a binding specificity for VEGF that is a VH H. and / or a single immunoglobulin variable domain with a binding specificity for EG FR which is a VH H-57. The ligand of any of claims 37 to 55, wherein each unique immunoglobulin variable domain with a binding specificity for VEGF, and each unique immunoglobulin variable domain with a binding specificity for EG FR, are selected from the group consisting of a human VH and a human VL. 58. The ligand of any of claims 37-57, wherein this ligand is an IgG-like format comprising two unique immunoglobulin variable domains with a binding specificity for VEGF, and two unique immunoglobulin variable domains with a binding specificity for EG FR. 59. The ligand of any of claims 37 to 58, wherein this ligand comprises an antibody Fc region. 60. A ligand having binding specificity for the epidermal growth factor receptor (EGFR), which comprises at least one unique immunoglobulin variable domain with a binding specificity for EGFR, wherein each unique variable domain of immunoglobulin na with a binding specificity for EG FR competes for the EGFR binding, with an anti-EGFR domain antibody (dAb) selected from the group consisting of DOM 1 6-39 (SEQ ID NO: 345), DOM 1 6-39-87 (SEQ ID NO: 420), DOM 16-39-100 (SEQ ID NO: 423), DOM 16-39-1 07 (SEQ ID NO: 430), DOM 1 6-39-1 09 (SEQ ID: 432), DOM 1 6-39-1 1 5 (SEQ ID NO: 438), and DOM 16-39-200 (SEQ ID NO: 441). 61 A ligand having binding specificity for the epidermal growth factor receptor (EGFR), which comprises at least one unique immunoglobulin variable domain with a binding specificity for EGFR, wherein each unique immunoglobulin variable domain with a specificity of linkage for EG FR, competes for the EGFR binding, with an anti-EG FR domain antibody (dAb) selected from from the group consisting of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 ( SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39 -619 (SEQ ID NO: 622). 62. The ligand of claim 60 or claim 61, wherein this ligand inhibits the binding of epidermal growth factor (EGF) and / or transforming growth factor alpha (TGF-alpha) to EGFR. 63. The ligand of claim 60 or claim 61, wherein this ligand inhibits EGFR activity. 64. The ligand of claim 60 or claim 61, wherein this ligand inhibits EGFR activity without substantially inhibiting the binding of epidermal growth factor (EGF) and / or transforming growth factor alpha (TGF-alpha). with the EGFR. 65. The ligand of any of claims 60 to 64, wherein each unique immunoglobulin variable domain with a binding specificity for EGFR, binds to the EGFR with an affinity (KD) that is between about 100 nM and about 1 pM, as determined by surface plasmon resonance. 66. The ligand of claim 65, wherein each unique immunoglobulin variable domain with a binding specificity for EGFR, binds to the EGFR with an affinity (KD) that is between about 10 nM and about 1000 pM, as determined by surface plasmon resonance. 67. The ligand of any of claims 60 to 64, wherein this ligand binds to the EGFR with an affinity (KD) that is between about 1 00 nM and about 1 pM, as determined by surface plasmon resonance. 68. The ligand of claim 67, wherein this ligand binds to the EGFR with an affinity (KD) that is between about 1.0 nM and about 1000 pM, as determined by surface plasmon resonance. 69. The ligand of any of claims 60 to 68, wherein this ligand comprises a single immunoglobulin variable domain with a binding specificity for EGFR that is a VH H. 70. The ligand of any of claims 60 to 68, wherein each unique immunoglobulin variable domain with a binding specificity for EGFR is independently selected from the group consisting of a human VH and a human V. 71 The ligand of any of claims 1 to 70, wherein this ligand further comprises a toxin. 72. The ligand of claim 71, wherein said toxin is a surface activity toxin. 73. The ligand of claim 72, wherein said surface activity toxin comprises a generator of free radicals or a radionuclide. 74. The ligand of claim 73, wherein said toxin is a cytotoxin, a free radical generator, an anti-metabolite, protein, polypeptide, peptide, photoactive agent, anti-sense compound, chemotherapeutic product, radionuclide, or intrabody. . 75. The ligand of any of claims 1 to 74, wherein this ligand further comprises a fraction that extends the half-life. 76. The ligand of claim 75, wherein the fraction that prolongs the half-life is a polyalkylene glycol fraction, serum albumin or a fragment thereof, a transferrin receptor or a portion of the transferrin enol ace thereof, or a fraction comprising a binding site for a polypeptide that improves the half-life in vivo. 77. The ligand of claim 76, wherein the fraction that prolongs the aforementioned half-life is a fraction comprising a binding site for a polypeptide that improves the in vivo half-life selected from the group consisting of an affinity, a SpA domain, a class A LDL receptor domain, an EGF domain, and an avimer. 78. The ligand of claim 76, wherein the fraction that prolongs the aforementioned half-life is a polyethylene glycol fraction. 79. The ligand of claim 76, wherein the fraction which prolongs the aforementioned half-life is an antibody or antibody fragment comprising a binding site for serum albumin or for the neonatal Fc receptor. 80. The ligand of claim 79, wherein the antibody or antibody fragment comprising a binding site for serum albumin or for the neonatal Fc receptor, is an antibody fragment, and this antibody fragment is a single domain. Immunoglobulin variable comprising a binding site for serum albumin. 81. The ligand of claim 80, wherein the single immunoglobulin variable domain comprising a binding site for serum albumin, competes for binding to 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), 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). 82. The ligand of claim 80, wherein the single variable domain of immunoglobulin comprising a binding site for serum albumin, comprises an amino acid sequence having an amino acid sequence identity of at least 85 percent with the amino acid sequence of 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). 83. A ligand of any of claims 1 to 70, for use in therapy or diagnostics. 84. A ligand of any of claims 1 to 70, for use in the treatment of cancer. 85. A ligand of any of claims 1 to 70 for the treatment of cancer cells that overexpress EGFR and / or VEGF. 86. The use of the ligand of any of claims 1 to 70, for the manufacture of a medicament for the treatment of cancer. 87. The use of the ligand of any of claims 1 to 70, for the manufacture of a medicament for the treatment of cancer cells that overexpress EGFR and / or VEGF. 88. A method for the treatment of cancer, which comprises administering to a subject in need thereof, a therapeutically effective amount of the ligand of any of the claims 1 to 70. 89. A method for the treatment of cancer, which comprises administering to a subject in need thereof, a therapeutically effective amount of the ligand of any of claims 1 to 70, and to a chemotherapeutic agent. 90. A method for the treatment of cancer, which comprises administering to a subject in need thereof a therapeutically effective amount of the ligand of any of claims 1 to 70, and an anti-neoplastic composition, wherein said anti-neoplastic composition The neoplastic agent comprises at least one chemotherapeutic agent selected from the group consisting of alkylating agents, antimetabolites, folic acid analogues, pyrimidine analogs, purine analogs, and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-Asparaginase, inhibitor of topoisomerase, interferons, platinum coordination complexes, anthracenedione substituted urea, methyl hydrazine derivatives, adrenocortical suppressor, adrenocorticosteroids, progestins, estrogens, anti-estrogen, androgens, anti-androgen, and gonadotropin-releasing hormone analogue. 91 The method of claim 90, wherein the chemotherapeutic agent is selected from the group consisting of cisplatin, dicarbazine, dactinomycin, mechlorethamine, streptozocin, cyclophosphamide, capecitabine, carmustine, lomustine, doxorubicin, daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluoro-uracil, vinblastine, vincristine, bleomycin, paclitaxel, docetaxel, doxetaxe, aldesleucine, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxy urea, ifosfamide, interferon-alpha, irinotecan , leuprolide, leucovorin, megestrol, melphalan, mercaptopurine, oxaliplatin, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thiowa nina, thiotepa, uracil mustard, vinorelbine, chlorambucil, taxol, a additional growth factor receptor antagonist, and a combination of any of the foregoing. 92. The method of any of claims 88 to 91, wherein the cancer is bladder cancer, ovarian cancer, colorectal cancer, breast cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, head and neck cancer, kidney cancer, and gallbladder cancer. 93. The method of any of claims 88 to 91, wherein the cancer is non-microcellular lung carcinoma or colo-rectal carcinoma. 94. A method for administering to a subject an anti-VEGF treatment and an anti-EGFR treatment, the method comprising the simultaneous administration of an anti-VEGF treatment and an anti-EGFR treatment, by administering to this subject an amount therapeutically effective of a ligand of any one of claims 1 to 59. 95. A composition comprising a ligand of any of claims 1 to 70, and a physiologically acceptable carrier. 96. The composition of claim 95, wherein said composition comprises a vehicle for intravenous, intramuscular, intraperitoneal, intra-arterial, intrathecal, intra-articular, or subcutaneous administration. 97. The composition of claim 95, wherein said composition comprises a vehicle for pulmonary, intranasal, vaginal, or rectal administration. 98. A drug delivery device, which comprises the composition of claim 95. 99. A drug delivery device for the simultaneous administration to a subject of an anti-VEGF treatment and an anti-EGFR treatment, comprising the device a ligand of any of claims 1 to 59. 1 00. The drug delivery device of claim 98 or 99, wherein said device comprises a plurality of therapeutically effective doses of the ligand. 1 01. The drug delivery device of any of claims 98 to 1 00, wherein this drug delivery device is selected from the group consisting of a parenteral delivery device, a non-venous delivery device, a delivery device, and a delivery device. intramuscular supply, an intraperitoneal delivery device, a device for transdermal delivery, a pulmonary delivery device, an intra-arterial delivery device, an intrathecal delivery device, an intra-articular delivery device, a subcutaneous delivery device, an intranasal delivery device, a vaginal delivery device, and a rectal delivery device. 1 02. The a drug delivery device of claim 1, wherein the device is selected from the group consisting of a syringe, a transdermal delivery device, a capsule, a tablet, a nebulizer, an inhaler, an atomizer, an aerosolizer, a fine mist nebulizer, a dry powder inhaler, a metered dose inhaler, a metered dose sprayer, a metered dose nebulizer, a metered dose sprayer, a catheter. 1 03. The use of a ligand of any of claims 1 to 70, for the manufacture of a medicament for the selective annihilation of cancer cells on normal cells. 1 04. The use of a ligand of any one of claims 1-70 for the manufacture of a medicament for killing cells. 1 05. The use of claim 1 04, wherein said ligand comprises an antibody Fc region. 1 06. An isolated or recombinant nucleic acid encoding a ligand of any of claims 1 to 70. 107. A vector comprising the recombinant nucleic acid of claim 106. 108. A host cell comprising the recombinant nucleic acid of claim 106, or the vector of claim 107. 109. A method for producing a ligand, which comprises maintaining the host cell of claim 108 under conditions suitable for the expression of the aforementioned nucleic acid or vector, whereby a ligand is produced. 110. The method of claim 109, which further comprises isolating the ligand. 111. A composition comprising a single-domain antibody polypeptide construct that antagonizes binding of human EGFR to a receptor, wherein this single-domain antibody polypeptide construct comprises a sequence of CDR3 that is the same as the CDR3 sequence of a 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). 112. A composition comprising a single-domain antibody polypeptide construct that antagonizes the binding of human EGFR with a receptor, wherein this single-domain antibody polypeptide construct comprises a CDR3 sequence that is the same as the CDR3 sequence of a dAb selected from the group consisting of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585) , DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ. ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). 113. A composition of a single-domain antibody polypeptide construct that antagonizes the binding of human EGFR to a receptor, wherein this single-domain antibody polypeptide construct comprises an amino acid sequence selected from the group consisting of at 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), DOM16-39-200 (SEQ ID NO: 441), and a sequence at least 85 percent identical to any of the above. 114. A composition of a single-domain antibody polypeptide construct that antagonizes the binding of human EGFR with a receptor, wherein this single-domain antibody polypeptide construct comprises an amino acid sequence selected from the group consisting of at DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16- 39-618 (SEQ ID NO: 621), DOM16-39-619 (SEQ ID NO.622), and a sequence at least 85 percent identical to any of the foregoing. 115. A composition comprising a single-domain antibody polypeptide construct that antagonizes the binding of human EGFR with a receptor, wherein this single-domain antibody polypeptide construct comprises an amino acid sequence selected from the group that consists 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), DOM16-39-200 (SEQ ID NO: 441), and a sequence at least 85 percent identical to any of the above. 116. A composition comprising a single-domain antibody polypeptide construct that antagonizes the binding of human EGFR with a receptor, wherein this single-domain antibody polypeptide construct comprises an amino acid sequence selected from the group consisting of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591) ), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), DOM16-39-619 (SEQ ID NO. : 622), and a sequence at least 85 percent identical to any of the above. 117. A composition that. comprises a construction single-domain antibody polypeptide that antagonizes the binding of human EGFR with a receptor, wherein this single-domain antibody polypeptide construct comprises an amino acid sequence 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), DOM16-39-200 (SEQ ID NO: 441), and a sequence at least 92 percent identical to any of the foregoing. 118. A composition comprising a single-domain antibody polypeptide construct that antagonizes the binding of human EGFR with a receptor, wherein this single-domain antibody polypeptide construct comprises an amino acid sequence selected from the group that consists of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO. : 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), DOM16-39-619 (SEQ ID NO: 622), and a sequence at least 92 percent identical to any of the above. 119. A composition comprising a single-domain antibody polypeptide construct that antagonizes the binding of human EGFR with a receptor, wherein this single-domain antibody polypeptide construct comprises an amino acid sequence selected from the group consisting of D0M16-39 (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), DOM16-39-200 (SEQ ID NO: 441) ), and a sequence at least 94 percent identical to any of the above. 120. A composition comprising a single-domain antibody polypeptide construct that antagonizes the binding of human EGFR with a receptor, wherein this single-domain antibody polypeptide construct comprises an amino acid sequence selected from the group consisting of consists of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO. : 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), DOM16-39-619 (SEQ ID NO: 622), and a sequence at least 94 percent identical to any of the above. 121. A composition comprising a single-domain antibody polypeptide construct that antagonizes the binding of human EGFR with a receptor, wherein this single-domain antibody polypeptide construct comprises an amino acid sequence selected from the group consisting of consists 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), DOM16-39-200 (SEQ ID NO: 441), and a sequence at least 96 percent identical to any of the above. 122. A composition comprising a single-domain antibody polypeptide construct that antagonizes the binding of human EGFR with a receptor, wherein this single-domain antibody polypeptide construct comprises an amino acid sequence selected from the group consisting of consists of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO. : 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), DOM16-39-619 (SEQ ID NO: 622), and a sequence at least 96 percent identical to any of the foregoing. 123. A composition comprising a single-domain antibody polypeptide construct that antagonizes the binding of human EGFR with a receptor, wherein this single-domain antibody polypeptide construct comprises an amino acid sequence selected from the group consisting of consists 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), DOM16-39-200 (SEQ ID NO: 441), and a sequence at least 98 percent identical to any of the previous 124. A composition comprising a single-domain antibody polypeptide construct that antagonizes the binding of human EGFR with a receptor, wherein this single-domain antibody polypeptide construct comprises an amino acid sequence selected from the group that consists of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO. : 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), DOM16-39-619 (SEQ. ID NO: 622), and a sequence at least 98 percent identical to any of the above. 125. A composition comprising a single-domain antibody polypeptide construct that antagonizes the binding of human EGFR with a receptor, wherein this single-domain antibody polypeptide construct comprises an amino acid sequence selected from the group that consists 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), DOM16-39-200 (SEQ ID NO: 441), and a sequence at least 99 percent identical to any of the foregoing. 126. A composition comprising a single-domain antibody polypeptide construct that antagonizes the linkage of the human EGFR with a receptor, wherein this single-domain antibody polypeptide construct comprises an amino acid sequence selected from the group consisting of DOM16-39-521 (SEQ ID NO: 577), DOM16-39 -541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16 -39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), DOM16-39-619 (SEQ ID NO: 622), and a sequence at least 99 percent identical to either of the previous ones. 127. A composition of any of the claims 111 to 125 wherein the single-domain antibody polypeptide construct comprises a tetravalent double specific antibody polypeptide construct comprising: a) a first copy of a first fusion protein comprising a single-domain antibody polypeptide that it binds to a first epitope, fused to an IgG heavy chain constant domain; b) a second copy of the first fusion protein; c) a first copy of a second fusion protein comprising a single domain antibody polypeptide that binds to a second epitope, fused to a light chain constant domain; d) a second copy of the second fusion protein; where the first and second copies of the first fusion proteins are linked to one another by their respective heavy chain constant domains of IgG, and wherein the light chain constant domain of the first copy of the second fusion protein binds disulfide to the domain heavy chain constant of IgG from the first copy of the first fusion protein, and wherein the light chain constant domain of the second copy of the second fusion protein binds with disulfide with the heavy chain constant domain of IgG of the second copy of the first fusion protein, and wherein the construction of the polypeptide is linked to the first and second epitopes. 128. The composition of claim 111, wherein the first and / or the second epitope is an EGFR epitope. 129. An immunoglobulin variable single domain polypeptide that binds to EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence 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), or differs from the amino acid sequence 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 no several amino acid positions, and has a CDR1 sequence that has at least 50 percent identity with the CDR1 sequence 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). 130. An immunoglobulin variable single domain polypeptide that binds to EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16- 39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622), or differs from the amino acid sequence of DOM16- 39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611) ), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622), in no more than 25 amino acid positions, and has a sequence of CDR1 having an identity of when minus 50 percent with the CDR1 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621) ), and DOM16-39-619 (SEQ ID NO: 622). 131. An immunoglobulin variable single domain polypeptide that binds to EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of D0M16-39 (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), or differs from the amino acid sequence 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 DOM 16-39-200 (SEQ ID NO: 441), in no more than 25 amino acid positions, and has a CDR2 sequence that has an identity of at least 50 percent with the CDR2 sequence 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). 132. An immunoglobulin variable single domain polypeptide that binds to EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16- 39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622), or differs from the amino acid sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591) ), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622) in no more than 25 amino acid positions, and has a CDR2 sequence that has an identity of at least 50 percent with the CDR2 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16 -39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608) , DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). 133. An immunoglobulin variable single domain polypeptide that binds to EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence 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), or differs from the amino acid sequence 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 no more than 25 amino acid positions, and has a CDR3 sequence that has an identity of at least 50 percent with the CDR3 sequence 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). 134. An immunoglobulin variable single domain polypeptide that binds to EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16- 39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO.621), and DOM16-39-619 (SEQ ID NO: 622), or differs from the amino acid sequence of DOM16- 39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622) in no more than 25 amino acid positions, and has a CDR3 sequence that has an identity of at least 50 percent with the CDR3 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39 -541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SE Q ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). 135. An immunoglobulin variable single domain polypeptide that binds to EGFR, wherein the polypeptide has an amino acid sequence that is identical to the sequence of amino acids 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), or differs from the amino acid sequence 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 no more than 25 amino acid positions, and has a sequence of CDR1 that has an identity of at least 50 percent with the sequence of CDR1 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) and has a CDR2 sequence that has at least one percent identity with the CDR2 sequence 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). 136. An immunoglobulin variable single domain polypeptide that binds to EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16- 39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16- 39-619 (SEQ ID NO: 622), or differs from the amino acid sequence of DOM16-39-521 (SEQ ID NO: 577), - DOM16-39-541 (SEQ ID NO: 585), DOM16-39- 542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622) in no more than 25 amino acid positions, and has a CDR1 sequence that has an identity of at least 50 percent with the CDR1 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39 -541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16 -39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622) and has a sequence of CDR2 having an identity of when minus the percent with the CDR2 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16 -39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621) , and DOM16-39-619 (SEQ ID NO: 622). 137. A variable immunoglobulin single-domain polypeptide that binds to EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence 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), or differs from the amino acid sequence 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 no more than 25 positions of amino acids, and has a CDR2 sequence that has an identity of at least 50 percent with the CDR2 sequence 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) and has a CDR3 sequence that has an identity of at least 50 percent with the CDR3 sequence 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). 138. An immunoglobulin variable single domain polypeptide that binds to EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16- 39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39- 619 (SEQ ID NO: 622), or differs from the amino acid sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ. ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622) in no more than 25 amino acid positions, and has a CDR2 sequence that has an identity of at least 50 percent with the sequence of CDR2 of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622), and has a CDR3 sequence that has an identity at least the percent with the CDR3 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586) , DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO. 621), and DOM16-39-619 (SEQ ID NO: 622). 139. An immunoglobulin variable single domain polypeptide that binds to EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence 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), or differs from the amino acid sequence 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 no more than 25 positions of amino acids, and has a CDR1 sequence that has an identity of at least 50 percent with the CDR1 sequence 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) and has a CDR3 sequence that has an identity of at least 50 percent with the CDR3 sequence 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). 140. 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 DOM16-39-521 (SEQ ID NO: 577), DOM16-39 -541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16 -39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622), or differs from the amino acid sequence of DOM16-39 -521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611) ), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622) in no more than 25 amino acid positions, and has a CDR1 sequence that has at least one identity 50 percent with the CDR1 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16 -39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621) , and DOM16-39-619 (SEQ ID NO: 622) and has a CDR3 sequence that has an identity of at least 50 percent with the CDR3 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608) ), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). 141. An immunoglobulin variable single domain polypeptide that binds to EGFR, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence 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), or differs from the amino acid sequence 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 no more than 25 amino acid positions, and has a CDR1 sequence that has an identity of at least 50 percent with the CDR1 sequence of DOM16-39 (SEQ ID NO: 345), DOM16-39-87 (SEQ. ID NO: 420), DOM16-39-1 OO (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) and has a CDR2 sequence that has an identity of at least 50 percent with the CDR2 sequence of DOM16-39 (SEQ. ID NO: 345), DOM16-39-87 (SEQ ID NO: 420), DOM16-39-1 OO (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) and has a CDR3 sequence that has an identity of at least 50 percent with the CDR3 sequence of DOM16-39 (SEQ ID NO: 345), DOM16-39-87 (SEQ ID NO: 420), DOM16-39-100 (SEQ ID NO: 42 3), 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). 142. 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 DOM16-39-521 (SEQ ID NO: 577), DOM16-39 -541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16 -39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622), or differs from the amino acid sequence of DOM16-39-521 (SEQ. ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622) in no more than 25 amino acid positions, and has a CDR1 sequence that has an identity of at least 50 percent with the CDR1 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621) ), and DOM16-39-619 (SEQ ID NO: 622) and has a CDR2 sequence that has an identity of at least 50 percent with the CDR2 sequence of DOM16-39-521 (SEQ ID NO: 577) , DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622) and has a sequence of CDR3 having an identity of at least 50 percent with the CDR3 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID. NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 ( SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). 143. An EGFR antagonist having a CDR1 sequence that has at least 50 percent identity to the CDR1 sequence 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). 144. An EGFR antagonist having a CDR1 sequence that has at least 50 percent identity with the CDR1 sequence of D0M16-39-521 (SEQ ID NO: 577), D0M16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 ( SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). 145. An EGFR antagonist having a CDR2 sequence that has at least 50 percent identity to the CDR2 sequence 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). 146. An EGFR antagonist that has a sequence of CDR2 having an identity of at least 50 percent with the CDR2 sequence of D0M16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 ( SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39- 618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). 147. An EGFR antagonist having a CDR3 sequence that has at least 50 percent identity to the CDR3 sequence 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). 148. An EGFR antagonist having a CDR3 sequence that has at least 50 percent identity to the CDR3 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 ( SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). 149. An EGFR antagonist that has a sequence of CDR1 having at least 50 percent identity with the CDR1 sequence 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) and a CDR2 sequence that has at least 50 percent identity with the CDR2 sequence 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). 150. An EGFR antagonist having a CDR1 sequence that has at least 50 percent identity to the CDR1 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 ( SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622) and a CDR2 sequence having at least 50 percent identity with the CDR2 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 ( SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). 151. An EGFR antagonist that has a sequence of CDR2 having an identity of at least 50 percent with the CDR2 sequence 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) and a CDR3 sequence having at least 50 percent identity with the CDR3 sequence 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). 152. An EGFR antagonist having a CDR2 sequence that has at least 50 percent identity to the CDR2 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 ( SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622) and a CDR3 sequence that has an identity of at least 50 percent with the CDR3 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 ( SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). 153. An EGFR antagonist that has a sequence of CDR1 having at least 50 percent identity with the CDR1 sequence 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) and a CDR3 sequence having at least 50 percent identity with the CDR3 sequence 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). 154. An EGFR antagonist having a CDR1 sequence that has at least 50 percent identity to the CDR1 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 ( SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622) and a CDR3 sequence that has an identity of at least 50 percent with the CDR3 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 ( SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622). 155. An EGFR antagonist that has a sequence of CDR1 having at least 50 percent identity with the CDR1 sequence 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) and a CDR2 sequence that has at least 50 percent identity with the CDR2 sequence 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) and a CDR3 sequence that has at least 50 percent identity with the CDR3 sequence 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). 156. An EGFR antagonist having a CDR1 sequence that has at least 50 percent identity with the CDR1 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 ( SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622) and a CDR2 sequence having at least 50 percent identity with the CDR2 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 ( SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39-619 (SEQ ID NO: 622) and a CDR3 sequence that has an identity of at least 50 percent with the CDR3 sequence of DOM16-39-521 (SEQ ID NO: 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 ( SEQ ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39 -619 (SEQ ID NO: 622). 157. A ligand having binding specificity for the vascular endothelial growth factor (VEGF) and for the epidermal growth receptor (EGFR), which comprises at least one protein fraction that has a binding site with a binding specificity for VEGF , at least a protein fraction having a binding site with a binding specificity for EGFR, and an Fc region of an antibody. 158. The ligand of claim 157, wherein said protein fraction having a binding site with binding specificity for VEGF, is a domain antibody (dAb) that binds to VEGF. 159. The ligand of claim 158, wherein said dAb that binds to VEGF, competes for binding to VEGF, with 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). 160. The ligand of claim 158, wherein the dAb that binds to VEGF, competes for binding to VEGF, with TAR15-26-555 (SEQ ID NO: 704). 161. The ligand of any of claims 157 to 160, wherein the mentioned protein fraction having a binding site with binding specificity for EGFR is a domain antibody (dAb) that binds to EGFR. 162. The ligand of claim 161, wherein the aforementioned dAb that binds to EGFR, competes for binding to EGFR, with an anti-EGFR 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). 163. The ligand of claim 161, wherein said dAb which binds to EGFR, competes for the EGFR binding, with an anti-EGFR dAb selected from the group consisting of DOM16-39-521 (SEQ ID NO. : 577), DOM16-39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ. ID NO: 591), DOM16-39-601 (SEQ ID NO: 608), DOM16-39-604 (SEQ ID NO: 611), DOM16-39-618 (SEQ ID NO: 621), and DOM16-39- 619 (SEQ ID NO: 622). 164. A ligand having binding specificity for vascular endothelial growth factor (VEGF), which comprises at least one fraction of a protein having a binding site with binding specificity for VEGF, and an Fc region of an antibody. 165. The ligand of claim 164, wherein said protein fraction having a binding site with binding specificity for VEGF is a domain antibody (dAb). 166. The ligand of claim 165, wherein said dAb competes for binding with VEGF, with 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). 167. The ligand of claim 165, wherein said dAb competes for binding to VEGF, with TAR15-26-555 (SEQ ID NO: 704). 168. A ligand having binding specificity for the epidermal growth factor receptor (EGFR), which comprises at least one fraction of a protein having a binding site with binding specificity for EGFR, and an Fc region of an antibody . 169. The ligand of claim 168, wherein said protein fraction having a binding site with binding specificity for EGFR is a domain antibody (dAb). 170. The ligand of claim 169, wherein said dAb competes for binding to EGFR, with an anti-EGFR 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). 171. The ligand of claim 169, wherein said dAb competes for the EGFR binding, with an anti-EGFR dAb selected from the group consisting of DOM16-39-521 (SEQ ID NO: 577), DOM16- 39-541 (SEQ ID NO: 585), DOM16-39-542 (SEQ ID NO: 586), DOM16-39-551 (SEQ ID NO: 591), DOM16-39-601 (SEQ. IDNO: 608), DOM 1 6-39-604 (SEQ IDNO: 61 1), DOM 1 6-39-61 8 (SEQ ID NO: 621), and DOM 1 6-39-61 9 (SEQ ID NO: 622). 72. The ligand of claim 1, wherein this ligand comprises at least two protein fractions that have binding sites with binding specificity for EGFR and an Fc region of an antibody. 1 73. A ligand that has binding specificity for vascular endothelial growth factor (VEGF) and for the epidermal growth factor receptor (EGFR), which comprises at least one unique immunoglobulin variable domain with binding specificity for VEGF , at least one unique immunoglobulin variable domain with binding specificity for EG FR, and a linker, wherein the single immunoglobulin variable domain with binding specificity for EGFR is linked via the linker to the unique immunoglobulin variable domain with specificity Link for VEGF. 1 74. A ligand having binding specificity for vascular endothelial growth factor (VEGF) and for the epidermal growth factor receptor (EG FR), which comprises at least one unique immunoglobulin variable domain with binding specificity. for VEGF, at least one unique immunoglobulin variable domain with binding specificity for EGFR, where the single immunoglobulin variable domain with binding specificity for EGFR is fused directly fused to the single immunoglobulin variable domain with binding specificity for VEGF. 175. The ligand of claim 173, wherein said linker is selected from the group consisting of SEQ ID NO: 706, SEQ ID NO: 707, SEQ ID NO: 708, SEQ ID NO: 709, SEQ ID NO. : 710, SEQ ID NO: 711, SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 723 and SEQ ID NO: 724. 176. The ligand of claim 173 or 175, wherein this ligand further comprises an Fc region of an antibody. 177. The ligand of claim 176, wherein this ligand further comprises a second linker, and wherein one of the unique immunoglobulin variable domains is linked via the aforementioned second linker to the Fc region. 178. The ligand of claim 177, wherein said second linker is selected from the group consisting of SEQ ID NO: 706, SEQ ID NO: 707, SEQ ID NO: 708, SEQ ID NO: 709, SEQ ID NO: 710, SEQ ID NO: 711, SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 723 and SEQ ID NO: 724. 179. The ligand of claim 174, wherein this ligand further comprises a linker and an Fc region of an antibody, and wherein the single immunoglobulin variable domain is linked via the linker to the Fc region of an antibody. 180. The ligand of claim 179, wherein said linker is selected from the group consisting of SEQ ID NO: 706, SEQ ID NO: 707, SEQ ID NO: 708, SEQ ID NO: 709, SEQ ID NO. : 710, SEQ ID NO: 711, SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 723 and SEQ ID NO: 724. 181. The ligand of any of claims 173 to 180, wherein: a. the single immunoglobulin variable domain with a binding specificity for VEGF is a heavy chain variable domain, and the only immunoglobulin variable domain with a binding specificity for EGFR is a light chain variable domain; b. the single immunoglobulin variable domain with a binding specificity for VEGF is a light chain variable domain, and the only immunoglobulin variable domain with a binding specificity for EGFR is a heavy chain variable domain; c. the single immunoglobulin variable domain with a binding specificity for VEGF is a heavy chain variable domain, and the single immunoglobulin variable domain with a binding specificity for EGFR is a heavy chain variable domain; or d. the single immunoglobulin variable domain with a binding specificity for EGFR is a light chain variable domain, and the only immunoglobulin variable domain with a binding specificity for VEGF is a light chain variable domain. 182. The ligand of claim 181, wherein said heavy chain variable domain is VH or VHH- 183. The ligand of claim 181, wherein the heavy chain variable domain mentioned is a VH. 184. The ligand of claim 181, wherein said light chain variable domain is a V ?. 185. A ligand that has a binding specificity for vascular endothelial growth factor (VEGF) and / or for the epidermal growth factor receptor (EGFR), which comprises a first single variable domain of immunoglobulin, a second single domain immunoglobulin variable, and an Fc region of an antibody. 186. The ligand of claim 185, wherein this ligand has the formula, from the amino terminus to the carboxyl terminus, of dAb1-dAb2-Fc, where dAbl is the first single immunoglobulin variable domain, dAb2 is the second only immunoglobulin variable domain, and Fc is the Fc region of an antibody, wherein the dAbl is linked to the dAb2 directly or through a first linker, and the dAb2 is linked to the Fc directly or through a second linker. 187. The ligand of claim 186 wherein the dAbl and the dAb2 are the same. 188. The ligand of claim 186 or 187, wherein the dAbl and the dAb2 are each, a single light chain variable domain. 189. The ligand of claim 188, wherein the dAbl and the dAb2 are each Vk. 190. The ligand of claim 186 or 187, wherein the dAbl and the dAb2 are each, a single heavy chain variable domain. 191. The ligand of any of claims 185 to 190, wherein the first single variable domain of immunoglobulin and the second single variable domain of immunoglobulin each have a binding specificity for EGFR. 192. The ligand of any of claims 185 to 190, wherein the first single variable domain of immunoglobulin and the second single variable domain of immunoglobulin each have a binding specificity for VEGF. 193. The ligand of any of claims 186 and 188 to 190, wherein the dAbl has binding specificity for EGFR, and the dAb2 has binding specificity for VEGF. 194. The ligand of any of claims 186 and 188-190, where dAbl has binding specificity for VEGF, and dAb2 has binding specificity for EGFR. 195. A ligand, wherein this ligand is a dimer comprising a first ligand and a second ligand, wherein the first ligand and the second ligand are each as defined in any of claims 185 to 194. 196. The ligand of claim 195, wherein said dimer comprises a disulfide bond between the first ligand and the second ligand.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US60/742,992 | 2005-12-06 | ||
| US60/758,355 | 2006-01-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| MX2008007351A true MX2008007351A (en) | 2008-09-02 |
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