WO2011080401A1

WO2011080401A1 - Receptor tyrosine kinase-binding compositions

Info

Publication number: WO2011080401A1
Application number: PCT/FI2010/051101
Authority: WO
Inventors: Kari Alitalo; Andrey Anisimov; Denis Tvorogov; Veli-Matti LEPPÄNEN
Original assignee: Licentia Ltd
Priority date: 2009-12-31
Filing date: 2010-12-31
Publication date: 2011-07-07

Abstract

Provided herein are compositions comprising a first binding construct which specifically binds to a first epitope of a Receptor Tyrosine Kinase (RTK) and a second binding construct which binds to a second epitope of the RTK, wherein the second epitope is different from the first epitope, wherein each of the first and second binding constructs reduces ligand-induced activation of the RTK. In specific embodiments, the RTK is a receptor for a Vascular Endothelial Growth Factor (VEGF) or a Platelet-Derived Growth Factor (PDGF). Further provided herein are methods of inhibiting cellular activities, including inhibiting cell growth (e.g., tumor or cancer cell growth), which activities are mediated by the RTK. The methods comprise contacting an RTK-expressing cell with the composition of the present disclosures.

Description

RECEPTOR TYROSINE KINASE-BINDING COMPOSITIONS

BACKGROUND OF THE INVENTION

[0001] Vascular endothelial growth factor receptor-3 (VEGFR-3/FLT4) belongs to the receptor tyrosine kinase (RTK) family comprising VEGFR-1/Flt-1 and VEGFR-2/KDR/Flk-1 . Each member of this family transduces angiogenic signals in cells upon the appropriate ligand binding to the receptor and subsequent receptor autophosphorylation. Mice lacking the VEGFR3 gene die in utero due to the abnormal development of the blood vasculature, leading to cardiovascular failure, while mice deficient in the VEGFR-3 ligand, VEGF-C, results in embryonic lethality due to the inability of lymphatic vessel formation. In the developing embryo, the VEGFR-3 gene is expressed in the whole vasculature, whereas in non-embryonic tissues, e.g., adult tissues, the expression of this gene becomes restricted to the lymphatic system and a few specialized fenestrated blood endothelia. In tumor tissues, however, the expression of the VEGFR-3 gene is re-induced in the angiogenic blood vascular endothelium.

[0002] Several studies have shown that interfering with VEGFR-3 function inhibits tumor lymphangiogenesis and metastasis in mice. Blocking VEGFR-3 function also improves the tumor growth inhibition achieved by other anti- angiogenic therapies (Tammela T et al., Nature, 2008; 454(7204):656-60). Inhibition of VEGFR-3 can be achieved with small molecular weight tyrosine kinase inhibitors (Heckman CA, Cancer Res., (2008) 68(12):4754-62), soluble extracellular domain of VEGFR-3 for trapping VEGF-C/D; Thelen A et al., Int. J. Cancer., 2008; 122(1 1 ):2471 -81 ; or with monoclonal antibodies that bind to VEGFR-3 (Pytowski B, et al., J Natl Cancer Inst, 2005; 97(1 ):14-21 .).

[0003] Antibodies that block vascular endothelial growth factor (VEGF) have become an integral part of anti-angiogenic tumor treatment. Antibodies targeting ligands and receptors of the VEGFA/EGFR family are now in clinical trials. A common mechanism of these receptor-targeting antibodies is to bind to the receptor at or near the ligand binding site, thereby preventing the ligand from binding to the receptor to activate downstream signaling. Currently, antibodies that block VEGFR-2 and/or VEGFR-3 are directed against the ligand binding domain of this recepto. No other types of inhibitory or blocking antibodies against these angiogenic RTKs have been described.

SUMMARY OF THE INVENTION

[0004] The invention includes materials and methods for interfering with receptor tyrosine kinase (such as VEGFR) signaling through an apparently new mechanism. Described herein are binding constructs that specifically bind to an RTK and reduce or inhibit its ligand-induced activation via a mechanism different from (or in addition to) inhibition of ligand binding. Accordingly, the binding constructs reduce ligand-induced activation of the RTK, even in the presence of high ligand concentrations. Furthermore, when used in combination with a binding construct that inhibits ligand binding to the RTK, the binding constructs described herein are particularly effective at inhibiting the survival and growth of cells expressing the RTK.

[0005] Accordingly, one aspect of the invention is a composition comprising a first binding construct which specifically binds to a first epitope of a RTK and a second binding construct which binds to a second epitope of the RTK, wherein the second epitope is different from the first epitope and wherein each of the first binding construct and second binding construct reduces ligand- induced activation of the RTK. In specific aspects of the present disclosures, the RTK is a receptor for a Vascular Endothelial Growth Factor (VEGF) or a Platelet-Derived Growth Factor (PDGF), includ ing , but not l im ited to, a VEGFR-1 , VEGFR-2, VEGFR-3, PDGFR-a, and PDGFR-β. In some variations, the RTK is a human RTK. The invention is exemplified here with respect to VEGFR-3. In preferred variations, the inhibition is cooperative, where the binding constructs provide greater inhibition together than individually. In some variations, the inhibitant is synergistic.

[0006] In some embodiments of the invention, the first binding construct comprises an antibody, or an antigen binding fragment thereof, which specifically binds to the first epitope. In alternative or additional aspects, the second binding construct comprises an antibody, or an antigen binding fragment thereof, which specifically binds to the second epitope. In certain aspects, both of the first binding construct and second binding construct are antibodies, e.g., monoclonal antibodies, humanized or human antibodies; or fragment of such antibodies; or polypeptides that comprise antigen-binding domains of such antibodies.

[0007] In specific aspects, the first binding construct reduces ligand-induced activation of the RTK by inhibiting binding between the RTK and its ligand. In certain aspects, the first epitope bound by the first binding construct is a portion of the ligand binding domain of the RTK, or sufficiently close to the ligand binding domain to cause steric inhibition of ligand binding. In certain aspects, the second binding construct does not inhibit the binding of the RTK to its ligand. In particular aspects, the second binding construct reduces RTK activation by inhibiting dimerization of RTK monomers.

[0008] In variations in which the first and second binding constructs bind to VEGFR-3, or similar RTK, the first epitope preferably comprises at least a portion of the Ig-like domain D1 , D2, or D3, or a combination thereof, of the extracellular domain (ECD) of the receptor. For example, the first epitope may be the epitope which is recognized and bound by antibody 3C5. In exemplary embodiments, the first binding construct comprises antibody 3C5, or an antigen binding fragment thereof. In some variations, the second epitope is not located within any of Ig-like domains D1 , D2, or D3, of the VEGFR-3 ECD, or similar RTK. In some aspects, the second binding construct binds to a conformational epitope that requires a disulfide bond within VEGFR-3 or like RTK, e.g., the disulfide bond between Cys445 and Cys 534 of VEGFR-3 (SEQ ID NO: 6). In specific aspects, the second epitope comprises at least a portion of Ig-like domain D5 of the ECD of VEGFR-3, or similar RTK. In particular embodiments, the second epitope is the epitope of antibody 2E1 1 D1 1 (also referred to herein as 2E1 1 ). Accordingly, in some aspects, the second binding construct comprises 2E1 1 or an antigen binding fragment thereof.

[0009] Also provided herein are kits comprising (i) a first binding construct which specifically binds to a first epitope of an RTK, (ii) a second binding con- struct which binds to a second epitope of the RTK, wherein the second epitope is different from the first epitope, and (iii) instructions for use. The binding constructs of the kits provided herein may be in accordance with any of the teachings on binding constructs of the present disclosures. For example, the first binding construct may comprise antibody 3C5, or an antigen binding portion thereof, and the second binding construct may comprise antibody 2E1 1 , or an antigen binding portion thereof. Also, the first and second binding constructs may be packaged together or separately.

[0010] The invention also includes a kit or a unit dose comprising (i) a first binding construct which specifically binds to a first epitope of an RTK; and, (ii) a second binding construct which binds to a second epitope of the RTK, wherein the second epitope is different from the first epitope; wherein the first and second binding constructs are packaged together but not in admixture. The binding constructs of the kits or unit doses are in accordance with the teachings and description herein pertaining to binding constructs. In some variations, the first or second binding construct (or both binding constructs) are formulated with a pharmaceutically acceptable diluent or carrier.

[0011] Other aspects of the invention include therapeutic methods and uses for the binding constructs described herein.

[0012] The binding constructs, compositions, and kits described here are useful in methods of inhibiting cellular activity which is mediated through the RTK to which each of the first and second binding constructs bind. In specific aspects, the cellular activity is cellular growth (proliferation) and/or migration. In some embodiments, the cellular growth which is inhibited by the binding constructs is mediated via VEGFR-3, and the first binding construct and second binding construct bind to VEGFR-3 and block the ligand-induced activation of VEGFR-3. In certain aspects, the methods of the present disclosure inhibit the growth of a diseased cell, and in specific aspects, the diseased cell is a hyperproliferative cell. In additional aspects, the diseased cell (e.g., the hyper- proliferative cell) is in a mammal and the mammal has a disease which is treatable upon inhibiting VEGFR-3-mediated cellular activities, e.g., VEGFR-3- mediated cell growth. Therefore, provided herein are methods of treating a disease in a mammal in need thereof comprising administering to the mammal a composition, e.g., a pharmaceutical composition, of the present disclosures in an amount effective to treat the disease in the mammal.

[0013] The VEGFR and PDGFR RTK's are involved in the growth of blood and/or lymphatic vessels. In neoplastic disorders, such blood vessels supply oxygen, nutrients and other growth factors to neoplastic cells, facilitating and/or stimulating their growth; and serve as pathways for cell migration (e.g., tumor metastases). Thus, inhibition of RTK-mediated growth (and/or migration) of blood or lymphatic cells (e.g., endothelial cells or smooth muscle cells) according to methods of the invention is useful for inhibiting tumor growth and for inhibiting tumor metastases.

[0014] Thus, other aspects of the invention include methods of inhibiting RTK signaling; or inhibiting tumor growth; or inhibiting tumor metastases; such methods comprising administering to a mammalian subject in need thereof the binding constructs described herein. In some variations, the first and second binding constructs are administered together in a single composition. In some variations, the first and second binding constructs are separately administered, in any order. The separate administration can be essentially simultaneously, e.g., within a few seconds or minutes of each other; or can be administered at different times. The binding constructs are administered in an amount effective to achieve a desired therapeutic effect, such as inhibition of blood or lymphatic vessel growth; inhibition of tumor cell growth; and/or inhibition of tumor cell migration (metastases).

[0015] In other aspects of the invention, the binding constructs serve as targeting moieties that localize a heterologous moiety, e.g., a cytotoxic agent, to which one of the binding constructs is attached, to the cell expressing the RTK. In certain aspects, the cell is a diseased cell which expresses the RTK, as further described herein. In further aspects, the diseased cell is in a mammal and the mammal has a disease which is treatable upon localization of the heterologous moiety, e.g., the cytotoxic agent, to the diseased cell. Therefore, the pre- sent disclosures encompass methods of treating a disease in a mammal comprising delivering a heterologous moiety to a diseased cell which expresses the RTK to which the first and second binding constructs bind. In related aspects, the cell that is targeted is a proliferating blood or lymphatic vessel cell. The cytotoxic agent inhibits the growth of the cell and thereby inhibits angiogenesis or lymphangiogenesis.

[0016] Exemplary embodiments of the invention are characterized by the following numbered sub-paragraphs:

1 . A composition comprising a first binding construct that binds to a first epitope of a Receptor Tyrosine Kinase (RTK) extracellular domain (ECD) and a second binding construct that binds to a second epitope of the RTK ECD that differs from the first epitope, wherein each of the first binding construct and second binding construct reduces ligand-induced activation of the RTK (e.g., in a cell that expresses the RTK and is in environment where the cell is exposed to the ligand).

2. The composition of embodiment 1 , further comprising a pharmaceutically acceptable diluent or carrier. Exemplary diluents and carriers and related formulating agents are set forth below, e.g., in the pharmaceutical compositions and formulations section of the description. Compositions suitable for injection or intravenous administration are especially contemplated.

3. A kit comprising a first binding construct that binds to a first epitope of a Receptor Tyrosine Kinase (RTK) extracellular domain (ECD) and a second binding construct that binds to a second epitope of the RTK ECD that differs from the first epitope, wherein each of the first binding construct and second binding construct reduces ligand-induced activation of the RTK, wherein the first and second binding constructs are packaged together but not in admixture. The kit optionally includes other therapeutic agents, especially other therapeutic antibodies and/or anti-neoplastic agents; and optionally further includes instructions for dosing and/or formulating the agents.

4. A method of inhibiting ligand-induced activiation of a receptor tyrosine kinase (RTK), the method comprising contacting the RTK with a first binding construct that binds to a first epitope of a Receptor Tyrosine Kinase (RTK) extracellular domain (ECD) and with a second binding construct that binds to a second epitope of the RTK ECD that differs from the first epitope, wherein each of the first binding construct and second binding construct reduces ligand-induced activation of the RTK. In some variations, the method is targeted to cells, and both ex vivo and in vivo variations are contemplated. In preferred vaiations, synergistically effective amounts of the binding constructs are used, to obtain a level of inhibition that is not achieved with either construct alone; and/or to achieve a desired level of inhibition with fewer side effects, and/or with less total binding construct administration (smaller total dosage), and/or with less frequent dosing, for example.

5. The method of embodiment 4, wherein the RTK is expressed on the cells of a mammalian subject, and the contacting comprises administering the first and second binding constructs to the mammalian subject in amounts effective to inhibit ligand-induced activation of the RTK. Preferred subjects include dogs, cats, primates, and humans.

6. A use of a first binding construct that binds to a first epitope of a Receptor Tyrosine Kinase (RTK) extracellular domain (ECD) and a second binding construct that binds to a second epitope of the RTK ECD that differs from the first epitope, wherein each of the first binding construct and second binding construct reduces ligand-induced activation of the RTK, in a combination therapy for inhibiting ligand-induced activation of a receptor tyrosine kinase (RTK) in cells of a mammalian subject. Generally speaking, descriptions herein relating to methods of treatment are applicable to "uses" of the invention, and vice versa.

7. The composition, kit, method, or use of any one of embodiments 1 to 6, wherein the first binding construct comprises an antibody that binds the first epitope, or an antigen binding fragment thereof. The terms antibody and antigen binding fragment are intended to include molecules that include additional moieties attached to the antibody or fragment that do not eliminate the antigen binding properties of the antibody or fragment. 8. The composition, kit, method, or use of any one of embodiments 1 to 7, wherein the second binding construct comprises an antibody that binds the second epitope, or antigen binding fragment thereof.

9. The composition, kit, method, or use of embodiment 8, wherein the antibodies are monoclonal antibodies.

10. The composition, kit, method, or use of embodiment 8 or 9, wherein the antibodies are humanized or human antibodies.

1 1 . The composition, kit, method, or use of any one of embodiments 1 to 10, wherein each of the first and second binding constructs is an antigen binding fragment of an antibody, the antigen binding fragment selected from the group consisting of: fab, f(ab)2', fab3, scFv, diabody, triabody, tetrabody, minibody, and single-domain antibody. The terms diabody, triabody, and tetrabody refer to small bivalent, trivalent, or tetravalent antibody fragments. Such constructs can be expressed in a variety of cell types including bacteria and yeast in a functional form. For example, a diabody can comprise a heavy (VH) chain variable domain connected to a light chain variable domain (VL) on the same polypeptide chain, preferably connected to each other with a short peptide linker. This facilitates paring with the complementary domains of another chain and promotes the assembly of a dimeric molecule with two functional antigen binding sites. Exemplary descriptions of minibodies (e.g., single chain Fv-CH3 constructs) are found in Hu et al., Cancer Res., 1996; 56:3055-61 ; Tramontano et ai, J. Mol. Recognit., 1994; 7(1 ): 9-24; and Orita et al. Blood, 2005; 105(2): 562-66, all incorporated herein by reference. Domain antibody refers to the smallest functional binding unit of an antibody, e.g., corresponding to the variable region of either the heavy or light chains of a human antibody.

12. The composition, kit, method, or use of any one of embodiments 1 to 1 1 , wherein the first binding construct reduces activation of the RTK by inhibiting binding between the RTK and a ligand that binds to the RTK.

13. The composition, kit, method, or use of embodiment 12, wherein the first epitope is a portion of a ligand binding domain of the RTK. 14. The composition, kit, method, or use of any one of embodiments 12 to 13, wherein the second binding construct does not inhibit binding between the RTK and the ligand.

15. The composition, kit, method, or use of any one of embodiments 12 to 14, wherein the second epitope is an extracellular epitope that does not participate in ligand binding.

16. The composition, kit, method, or use of any one of embodiments 1 to 15, wherein the second binding construct inhibits RTK dimeri- zation.

17. The composition, kit, method, or use of any one of embodiments 1 to 16, wherein the RTK is selected from the group consisting of Vascular Endothelial Growth Factor Receptors -1 , -2, and -3 (VEGFR-1 , VEGFR-2, and VEGFR-3) and Platelet-Derived Growth Factor Receptors - alpha and -beta (PDGFR-a, and PDGFR-β).

18. The composition, kit, method, or use of any one of embodiments 1 to 16, wherein the RTK is human VEGFR-3.

19. The composition, kit, method, or use of embodiment 18, wherein the first epitope comprises at least a portion of the Immunoglobulin (Ig) homology domain D1 , the Ig homology domain D2, the Ig homology domain D3, or a combination thereof, of the VEGFR-3 ECD.

20. The composition, kit, method, or use of embodiment 19, wherein the first epitope is the epitope of antibody 3C5 (Imclone).

21 . The composition, kit, method, or use of embodiment 20, wherein the first binding construct comprises antibody 3C5 (Imclone), or an antigen binding fragment thereof.

22. The composition, kit, method, or use of any one of embodiments 18 to 21 , wherein the second epitope is not located within any of D1 to D3 of VEGFR-3.

23. The composition, kit, method, or use of any one of embodiments 18 to 22, wherein the second epitope is a conformational epitope, and wherein high affinity binding between the RTK and the ligand requires a disulfide bond between the Cys445 and Cys534 of VEGFR-3 (SEQ ID NO: 6). 24. The composition, kit, method, or use of any one of embodiments 18 to 23, wherein the second epitope comprises at least a portion of the Ig homology domain D5 of the VEGFR-3 ECD.

25. The composition, kit, method, or use of any of embodiments 18 to 24, wherein the second epitope is the epitope of antibody 2E1 1 (Accession No. 01083129).

26. The composition, kit, method, or use of embodiment 25, wherein the second binding construct comprises antibody 2E1 1 , or an antigen binding fragment thereof.

27. The composition, kit, method, or use of any one of embodiments 1 to 26, wherein at least one of the first binding construct and the second binding construct is conjugated to a heterologous moiety selected from the group consisting of: a polymer, a cytokine and a cytotoxic agent.

28. The method of any one of embod iments 5 and 7-27, wherein the mammalian subject has a neoplastic disorder, and the binding constructs are administered in an amount effective to inhibit neoplastic cell growth.

29. The use according to any one of embodiments 6 to 27, wherein the therapy is for a neoplastic disorder.

30. A method of inhibiting ligand-induced activation of a RTK in a cell, the method comprising contacting the cell with a composition of any one of embodiments 1 -2 and 7-27, in an amount effective to inhibit ligand- induced activation of the RTK.

31 . The method of embodiment 30, wherein the cell is a lymphatic endothelial cell, a blood endothelial cell, or a hematopoietic progenitor cell.

32. The method of any one of embodiments 5 to 28 and 30- 31 , wherein the first and second binding constructs are administered separately to the mammalian subject.

33. The method or use of any preceding method or use embodiment, wherein the mammal is a human. 34. Use of the composition of any of embodiments 1 -2 and 6- 27 in the preparation of a medicament for treating a neoplastic disorder in a mammal.

35. An isolated monoclonal antibody, or antigen binding fragment thereof, that binds to the extracellular domain (ECD) of a receptor tyrosine kinase (RTK) selected from the group consisting of VEGFR-1 , VEGFR-2, PDGFR-alpha, and PDGFR-beta, wherein the antibody or fragment permits a ligand of the RTK to bind the RTK but inhibits ligand-mediated phosphorylation of the RTK.

36. The antibody or antibody fragment according to embodiment 35 that is human or humanized.

37. A composition comprising the antibody of embodiment 35 or 36 and a pharmaceutically acceptable carrier.

38. A method for inhibiting cell growth in a mammalian organism, the method comprising: administering to a mammalian organism the composition of embodiment 37, wherein the organism has cells that express the RTK, and the antibody or fragment is present in the composition in an amount effective to inhibit ligand-mediated phosphorylation of the RTK, thereby inhibiting growth of the cells that express the RTK.

39. The composition of embodiment 37, further comprising a second monoclonal antibody, or fragment thereof, that binds to the RTK and inhibits the ligand from binding to the RTK.

40. A method for inhibiting cell growth in a mammalian organism, the method comprising: administering to a mammalian organism the composition of embodiment 39, wherein the organism has cells that express the RTK, and the antibodies or fragments are present in the composition in amounts effective to inhibit l igand-mediated phosphorylation of the RTK, thereby inhibiting growth of the cells that express the RTK.

41 . The composition of embodiment 37 or 39, further comprising a third monoclonal antibody, or fragment thereof, that binds to the ligand and inhibits the ligand from binding to the RTK. For example, if the RTK is VEGFR-1 , R-2, or R-3, then the anti-ligand antibody would bind one of the VEGF ligands (e.g., VEGF-A, -B, -C, or -D) that has high affinity for the receptor.

42. In a method of treatment that includes administering to a mammalian subject a first antibody, or antigen binding fragment thereof, that inhibits a ligand from the PDGF or VEGF family of ligands from binding to a receptor tyrosine kinase for the ligand, an improvement comprising administering to the mammalian subject a second antibody, or antigen binding fragment thereof, that binds to the extracellular domain (ECD) of the RTK, wherein the second antibody or fragment inhibits dimerization of the RTK and inhibits ligand-mediated phosphorylation of the RTK.

43. The improvement of embodiment 42, wherein the first antibody binds to the ligand.

44. The improvement of embodiment 42, wherein the first antibody binds to the RTK.

45. A method of making a binding construct comprising: (a) screening a library of compounds to identify a candidate compound that binds the extracellular domain (ECD) of a receptor tyrosine kinase (RTK), permits a ligand of the RTK to bind to the RTK, and inhibits the RTK from dimerizing; and (b) making a binding compound containing the candidate compound identified in (a), or a fragment thereof that retains the binding and inhibition properties. Such methods can be performed with libraries of antibodies, antibody fragments, or antibody-like compounds; and also can be performed with small molecule libraries, for example. The binding constructs identified by such methods also are intended as an aspect of the invention.

46. The method of embodiment 45, wherein the library contains antibodies or antigen binding fragments of antibodies.

47. The method of embodiment 45 or 46, wherein the RTK is selected from the group consisting of VEGFR-1 , VEGFR-2, PDGFR-alpha, and PDGFR-beta.

48. The method of embodiment 45 or 46, wherein the RTK is VEGFR-3, and wherein the antibody binds to a different epitope of VEGFR-3 than the epitope recoginzied by antibody 2E1 1 . 49. A binding construct made by a method according to any one of embodiments 45 to 48.

50. An antibody or antigen binding fragment thereof that comprises the antigen binding domain of an antibody made according to the method of any one of embodiments 46 to 48.

51 . The composition of any preceding composition embodiment, further including a standard of care cancer therapeutic. The section below relating to heterologous moieties and therapeutic agents lists numerous agents that are considered standard of care therapeutics for various cancers.

52. A method for inhibiting ligand-induced sprouting of microvascular endothelial cells in a mammalian organism, the method comprising: administering to a mammalian organism the composition of embodiment 39, wherein the organism has cells that express the RTK, and the antibodies or fragments are present in the composition in amounts effective to inhibit ligand- induced sprouting of microvascular endothelial cells in a mammalian organism.

53. Use of the composition of any of embodiments 34, in the preparation of a medicament for treating a Kaposis's sarcoma.

[0017] All description herein relating to methods of inhibiting also should be understood to define "uses" of the materials described herein. For example, the invention includes use of a composition comprising a first binding construct and second binding construct as further described herein, for the inhibition of cellular growth, e.g., inhibition of cancer cell growth or tumor cell growth. The invention also includes use of a composition comprising a first binding construct and second binding construct as further described herein in the manufacture of a medicament for inhibiting cellular growth, e.g., inhibiting cancer cell growth or tumor cell growth.

[0018] The foregoing summary is not intended to define every aspect of the invention, and additional aspects are described in other sections, such as the Detailed Description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document.

[0019] In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above. With respect to aspects of the invention described as a genus, all individual species are individually considered separate aspects of the invention. With respect to aspects described as a range, all sub-ranges and individual values are specifically contemplated.

[0020] Although the applicant(s) invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass within their scope the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention. Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, and all such features are intended as aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWING

[0021] Figure 1 provides a characterization of the VEGFR-3 blocking antibodies. Part (A) depicts graphs of data from a VEGFR-3/BaF3 cell survival assay in the presence of the different anti-VEGFR3 antibodies and 25 ng/ml of full-length (FL) or proteolytically processed, mature (ANAC) VEGF-C. Part (B) depicts a Western blot showing antibody inhibition of VEGFR-3 phosphorylation in HDME cells stimulated with VEGF-C in presence or absence of the indicated antibodies. Lysates were precipitated with polyclonal VEGFR-3 antibodies and blotted with pTyr (pY) or VEGFR-3 antibodies, as shown. Part (C) depicts antibody-mediated inhibition of ligand binding to VEGFR-3. Wells were pre-coated with VEGF-C. Recombinant extracellular domain of VEGFR-3 with or without the indicated antibodies was applied, and the bound proteins were analyzed in western blotting with anti-VEGFR-3 antibodies. Part (D) is a graph summarizing data from a binding assay. Wells were precoated as above. Recombinant VEGFR-3-AP was preincubated with different concentrations of either 2E1 1 or 3C5 antibodies and applied for binding. After washes, alkaline phosphatase activity was measured at OD405.

[0022] Figure 2 depicts an analysis of VEGFR-3 ECD immunoglobulin homology domain 5 epitopes. Part (A) depicts an amino acid sequence alignment of portions of the extra cellular domains of human and mouse VEGFR-3 (SEQ ID NO: 49 and 50) and VEGFR-2 receptors (SEQ ID NO: 51 and 52), including D5. The predicted extra loop and proteolytic processing site have been marked, as are the deleted and swapped sequences, plus the AFL4 binding peptide. The cysteine residues are bold and the two N-linked glycosylate sites of human R3 are underlined. Part (B) depicts the results of an antibody binding assay, showing sensitivity of the antibody epitopes to reduction of disulfide bonds. VEGFR-3-streptag III was stably expressed in 293T cells, precipitated with streptactin sepharose and analyzed by blotting with 2E1 1 and 9D9 antibodies under reducing and non-reducing conditions. Part (C) depicts a three-dimensional VEGFR-3 D5 model (Phyre), based on the MyBP-C structure (PDB code 1 GXE), with the cysteine residues highlighted in yellow. Note that in VEGFR3 D5, C445 and C534 make a disulfide bridge typical for immunoglobulin (Ig) homology domains. C466 and C486 are far apart in the model but probably interact in VEGFR-3 D5. In MyBP-C, there are no counterparts for residues S473-Q480 (dotted line). R472-S473 is the identified protease cleavage site. Part (D) depicts a model in which the surface is colored according to the electrostatic potential (red = negative, blue = positive charge). Note that the acidic residues in the AFL4 antibody-binding site center around F510 (see also Figure 7). Notably, the loop area, including the residues missing from MyBP-C (SLRRRQQQ; (SEQ ID NO: 43)) is positively charged.

[0023] Figure 3 depicts the characterization of VEGFR-3 D5 mutants. Part (A) is a schematic presentation of mutations made in VEGFR-3 D5. Four point mutations: C445S, C466S, C486S, C534S, one double mutation: DS (C445S and C534S combined), loop deletion (LD) and loop swap (LS) were made. Part (B) depicts results from Western blotting experiments. VEGFR-3 wt and mutants were expressed in 293T cells, VEGFR-3 was precipitated and analyzed by western blotting with anti-pY or anti-VEGFR-3 antibodies. In part (C), 293T cells transfected with the indicated mutants were stained with the 2E1 1 antibodies and analysed in flow cytometry (red/line). Green/shading: mock transfected cells. In part (D), Transfected 293T cell lysates were precipitated with 2E1 1 or 9D9 antibodies and blotted with 9D9 antibody. In part (E), VEGFR-3 D5 was cloned into the pSectag vector and expressed in 293T cells (upper panel). Conditioned medium was precipitated either with 9D9, Afl4 or 2E1 1 antibodies and western blotted with Afl4 antibodies. The lower panel represents same samples immunoblotted with the secondary antibody only. ^* indicate the IgG light chain.

[0024] Figure 4 depicts the synergistic inhibition of VEGFR-3 activation by the combination of 2E1 1 and 3C5 antibodies. Shown are results of the VEGFR-3/BaF3 cell survival assay done using the different anti-VEGFR-3 antibodies in presence of 10 ng/ml (A) or 100 ng/ml (B) of proteolytically processed, mature VEGF-C. Note that the AFL4 antibody has no effect. Panel (C) depicts antibody inhibition of Erk1 ,2 phosphorylation in VEGFR-3/BaF3 cells stimulated with 25 ng/ml of VEGF-C in presence or absence of the indicated antibodies (2E1 1 , 3C5, 9D9). Lysates were blotted with pErkl ,2 or tubulin antibodies, as shown. Panel (D) depicts antibody inhibition of Erk1 ,2 phosphorylation in VEGFR-3/BaF3 cells stimulated with increasing concentrations of VEGF-C (ng/ml) in presence or absence of the indicated antibodies. Lysates were blotted with pErkl ,2 or tubulin antibodies, as shown. Panel (E) depicts antibody inhibition of VEGFR-3 phosphorylation in HDME cells stimulated with increasing concentrations of VEGF-C in presence or absence of the indicated antibodies. Lysates were precipitated with polyclonal antibodies against VEGFR-3 and blotted with pY or VEGFR-3 antibodies. Panel (F) depicts antibody inhibition of Erk1 ,2 phosphorylation in HDME cells stimulated with VEGF- C in presence or absence of the indicated antibodies. Lysates were blotted with pErk1 ,2 or Erk1 ,2 antibodies.

[0025] Figure 5 depicts the effect of blocking antibodies on VEGFR-2 activation and heterodimerization with VEGFR-3. Panel (A) depicts antibody inhibition of VEGFR-2A/EGFR-3 heterodimerization in HDME cells untreated (-) or stimulated (+) with VEGF-C. Lysates were precipitated with polyclonal VEGFR-3 antibod ies and blotted with VEGFR-2 or VEGFR-3 antibodies. Panel (B) depicts antibody inhibition of VEGFR-2 phosphorylation in HDME cells untreated (-) or stimulated (+) with VEGF-C. Lysates were precipitated with polyclonal VEGFR-2 antibodies and blotted with pY or VEGFR-2 antibodies. Panel (C) depicts the effects of anti-VEGFR-3 and anti-VEGFR-2 antibodies on VEGFR-2 phosphorylation in PAE-VEGFR-2 cells untreated (-) or stimulated (+) with VEGF-C. Lysates were precipitated with polyclonal VEGFR-2 antibodies and blotted with pY or VEGFR-2 antibodies.

[0026] Figure 6 depicts the epitope mapping of 2E1 1 , 9D9 and AFL4 antibodies. In order to detect the binding sites of antibodies 9D9, 2E1 1 and AFL4, peptide scan (SPOT) analysis of the whole extracellular domain of VEGFR-3 was performed. (A) Peptides of 20 aa length (SEQ ID NOS: 53-59) with a transition frame of +3 were spotted on cellulose membranes and binding of the antibodies to the membranes was assessed by immunoblotting. The 2E1 1 antibody did not display reactivity to a linear epitope (data not shown). For 9D9, strong binding was found to a linear epitope comprising peptides covering the region from E586 to A617, which is located in D6. To further define the residues involved in the binding of 9D9, alanine scan and staggered end deletion (N-terminal deletion, SEQ ID NOS: 60-70; C-terminal deletion, SEQ ID NOS: 71 -78) analysis were employed (described in detail in the "materials and methods"). According to these analyses L598HDAHGNP605 (SEQ ID NOS:53-78) turned out to be the minimal peptide epitope. The residues that, when mutated to alanine, are marked in bold. Residue H602 corresponds to Q602 in the corresponding mouse sequence, thus providing an explanation for the species specificity of this antibody. When tested, 9D9 did not recognize murine VEGFR-3 in immunohistochemical stainings or in peptide scan analysis (data not shown). Panel (B) depicts results from probing a SPOTS membrane containing mouse VEGFR-3 peptides (SEQ ID NOS: 79-86) with the AFL4 antibodies and found binding to the region from E491 to D525, which is located in D5. This region was identical in the human VEGFR-3 sequence except that position S506 in the mouse was T506 in the human sequence. SPOTS analysis of the extracellular domain of human VEGFR-3 indeed showed that the AFL4 antibody binds to the corresponding human sequence (data not shown). The Kd values for VEGFR-3 binding were 66.9±8.4 nM for 9D9 and 14.6±4.0 nM for AFL4 as determined by surface plasmon resonance analysis (data not shown). Panel (C) depicts experiments to map the 2E1 1 eptiope. Since it was not possible to locate the 2E1 1 epitope by the same SPOT analysis, we made VEGFR-3 receptor with deletion of the first three extracellular Ig-like domains (D1 -D3) named VEGFR-3 Δ1 -3. This construct was expressed in 293T cells along with WT VEGFR-3, both containing the Streptaglll at C-terminus. The proteins precipitated with streptactin beads, run on SDS-PAGE under non- reducing conditions and blotted with the 2E1 1 and 3C5 antibodies.

[0027] Figure 7 depicts the analysis of the AFL4 epitope in different species (SEQ ID NOS: 87-91 ). Panel (A) shows a species alignment of the AFL4 binding region. The alignment revealed that the rat sequence (SEQ ID NO: 88) (species of origin of the AFL4 antibodies) contained a single amino acid difference from both mouse (SEQ ID NOS: 87) and human sequences (SEQ ID NO: 89), as indicated in italics. Thirteen amino acid peptides were made based on the mouse and rat sequences centered around residue 510 and differing only in position F/S514. Panel (B) shows that when we used these peptides as competitors in an ELISA in which AFL4 was used to bind to either mouse or human extracellular domain of VEGFR-3, the mouse/human peptide was at least 50x more potent inhibitor of binding (IC50) than the corresponding rat peptide. We cannot calculate the exact difference in IC50s because the rat peptide did not give us a complete inhibition at the highest dose. However, this experiment indicates that a single residue (F493 in mouse and human) is crucial for binding of AFL4. [0028] Figure 8 depicts the effect of anti-VEGFR-3 and anti-VEGFR-2 antibodies on VEGF-C-induced migration or HDME cells in the presence of indicated VEGFR-3 and VEGFR-2 antibodies.

[0029] Figure 9 shows the result of an analysis of the HDME cells by anti- podoplanin immunofluorescence staining followed by flow cytometry. The percentages of positive (LEC) and negative (BEC) cells are indicated above the graph.

[0030] Figure 10 depicts the effect of blocking antibodies on VEGF-C induced migration, sprouting and intracellular signaling in hLEC and hBEC. Effect of anti-VEGFR-3 and anti-VEGFR-2 antibodies on VEGF-C-induced migration of LECs (A) and BECs (B), LEC sprouting (C), and intracellular signaling (D).

[0031] Figure 10 (A and B) shows the results of migration assays performed with isolated LECs and BECs, as well as the results of a LEC sprouting assay (C).

[0032] Figure 1 1 is a photograph showing extensive sprouting in response to VEGF-C in K-LEC spheroids (A), whereas (B) compares the sprouting inhibitory effect of 2E1 1 , 3C5 as well as a combination of them.

[0033] Figure 12 (A to F) summarizes the results of Example 6.

[0034] Figure 13 shows the results of the surface Plasmon resonance analy- sisn of the vinign of monomeric VEGF-T3D1 -7 to monoclonal antibodies 9D9, 2E1 1 and AFL4.

[0035] Figure 14 is a photograph showing the immunofluorescent staining of 293T cells transfected with WT, LD and LS VEGFR-3, respectively.

[0036] Figure 15 (A to C) summarizes the experiments related to expression, phosphorylation and inhibition of VEGFRs.

[0037] Figure 16 displays Wester blottings showing that the 2E1 1 antibody does not inhibit VEGF-A induced VEGFR-2 phosphorylation or induce VEGFR- 2 or VEGFR-3 downregulation. DETAILED DESCRIPTION OF THE INVENTION

[0038] Described herein are compositions comprising a first binding construct and a second binding construct, each of which binds to a distinct epitope of the same RTK and reduces ligand-induced activation of the RTK. The epitope bound by the first binding construct (the "first epitope") is different from the epitope bound by the second binding construct (the "second epitope").

[0039] All teachings and descriptions of binding constructs are independently applicable to each of the first binding construct, the second binding construct, and any subsequent binding constructs (e.g., a third binding construct, a fourth binding construct, etc.), except where context clearly dictates otherwise.

Binding Constructs

[0040] As used herein, the term "binding construct" refers to a molecule comprising one or more binding units (directly or indirectly) associated with each other by covalent or non-covalent bonds.

[0041] As used herein, the term "binding unit" refers to the portion of a binding construct which specifically binds to the RTK. In some embodiments, the binding unit specifically binds to the RTK with high affinity. The term "high affinity" is used in a physiological context pertaining to the relative affinity of the binding construct for the RTK in vivo in a mammal, e.g., a laboratory test animal, a domesticated farm or pet animal, or a human. In certain embodiments, the binding unit has a dissociation constant (K_D) for the RTK which is in the sub-nanomolar (e.g., picomolar), nanomolar range, or micromolar range. In some embodiments, the K_D is between about 0.0001 nM and about 100 nM. In some embodiments, the K_D is at least or about 0.0001 nM, at least or about 0.001 nM, at least or about 0.01 nM, at least or about 0.1 nM, at least or about 1 nM, or at least or about 10 nM . In some embodiments, the K_D is no more than or about 100 nM, no more than or about 75 nM, no more than or about 50 nM, or no more than or about 25 nM.

[0042] In certain aspects, each binding unit or at least one of the binding units of the binding construct comprises at least one peptide or polypeptide. In other aspects, the binding unit comprises multiple peptides or polypeptides, covalently or non-covalently joined together. In specific aspects, the binding unit comprises at least one antibody, or antigen binding fragment thereof, such that the binding construct comprises at least one antibody, or antigen binding fragment thereof. In some embodiments, the binding construct comprises more than one binding unit, and, in specific aspects, some or all of the binding units of the binding construct are antibodies, or antigen binding fragments thereof. Further descriptions of such embodiments are described herein. See, e.g., the section entitled Antibodies and Antigen Binding Fragments.

[0043] In certain aspects, the binding construct comprises one or more binding units that are not polypeptides comprising an antibody or antigen binding fragment thereof. In some embodiments, the binding construct comprises at least one binding unit which is not an antibody or antigen binding fragment thereof, e.g., a ligand of the RTK (e.g., VEGF-C), or an RTK-binding portion thereof. In alternative embodiments, the binding unit is neither a peptide nor a polypeptide. In exemplary specific aspects, the binding unit comprises one or more of: an organic small molecule, an aptamer, and combinations thereof.

[0044] In some embodiments, the binding units (e.g., peptide, polypeptide, or other) are directly joined together in the absence of a linker. In alternative aspects, the binding units of the binding construct are indirectly connected via one or more linkers. Whether directly joined together or indirectly joined together through a linker, the binding units may be connected through covalent bonds (e.g., a peptide, ester, amide, or sulfhydryl bond) or non-covalent bonds (e.g., via hydrophobic interaction, hydrogen bond, van der Waals bond, electrostatic or ionic interaction), or a combination thereof. The binding units may be connected via any means known in the art, including, but not limited to, any of those taught herein with regard to conjugation of a binding construct to a second moiety. See the section herein entitled "Conjugates."

Linkers

[0045] In some embodiments of the present disclosures, the binding construct comprises at least one linker that connects two or more binding units, e.g., an antigen binding fragment of antibody 2E1 1 linked to an aptamer. A linker in some aspects also links a binding unit to other substituents of the binding construct, as further described herein. See the section entitled "Conjugates."

[0046] The linker in some aspects is a protein, polypeptide, peptide, or amino acid. In some embodiments, the linker is a heterologous protein, polypeptide, peptide, or amino acid. By "heterologous" is meant that the protein, polypeptide, or peptide is separate and distinct from the binding unit, which, in some aspects, is a protein, polypeptide, or peptide. In some embodiments, the linker comprises a peptide that links the binding units to form a single continuous peptide that can be expressed (e.g., recombinantly expressed) as a single molecule.

[0047] Peptide linkers of at least one amino acid residue are contemplated. In some embodiments the linker is a dipeptide or tripeptide, while in other embodiments, the linker comprises more than 3 amino acids, e.g., more than X amino acids, wherein X is 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 250, 500, 750, or 1000. In some aspect, the linker has more than 10,000 residues. In some embodiments the linker has from 1 -1 0, 1 -50, 1 -100, 1 -1000, or 1 -10,000 amino acid residues. In some embodiments, the peptide linker comprises residues with relatively inert side chains. For example, the linker may comprise one or more Gly and/or Ala residues. Peptide linker amino acid residues need not be linked entirely or at all via alpha-carboxy and alpha-amino groups. That is, peptides may be linked via side chain groups of various residues. Accordingly, the peptide linker may be a linear peptide linker, the amino acids of which are bound via the alpha amino and alpha carboxylate groups, or the peptide linker may be a branched peptide linker, the amino acids of which may be bound via side chain groups. For example, the linker comprises one or more Glu residues that are linked via the alpha amino groups and the side chain carboxyl groups.

[0048] Linker peptides may be designed to have sequences that permit desired characteristics. For example, the use of glycyl residues allow for a rela- tively large degree of conformational freedom, whereas a proline would tend to have the opposite effect. Peptide linkers may be chosen so that they achieve particular secondary and tertiary structures, e.g., alpha helices, beta sheets or beta barrels. Quaternary structure can also be utilized to create linkers that join two binding units together non-covalently. For example, fusing a protein domain with a hydrophobic face to each binding unit may permit the joining of the two binding units via the interaction between the hydrophobic interfaces of the two molecules. In some embodiments, the linker may provide for polar interactions. For example, a leucine zipper domain of the proto-oncoproteins Myc and Max, respectively, may be used. Luscher and Larsson, Oncogene 18:2955-2966 (1999). In some embodiments, the linker allows for the formation of a salt bridge or disulfide bond. Linkers may comprise non-naturally occurring amino acids, as well as naturally occurring amino acids that are not naturally incorporated into a polypeptide. In some embodiments, the linker comprises a coordination complex between a metal or other ion and various residues from the multiple peptides joined thereby.

[0049] In alternative aspects, the linker is not a protein, polypeptide, peptide, or amino acid. Polysaccharides or other moieties also may be used to link binding units to form a binding construct. The binding units, for example, may be connected via chemical cross-linkages or intramolecular bridges, e.g., disulfide bridges.

[0050] Linkers may be chosen such that they are less likely to induce an allergic or immunological reaction. The linker may be selected for optimal conformational (steric) freedom between the various ligand binding units to allow them to interact with each other if desired, e.g., to form dimers, or to allow them to interact with ligand. The linker may be linear, such that consecutive binding units are linked in series, or the linker may serve as a scaffold to which various binding units are attached, e.g., a branched linker. A linker may also have multiple branches, e.g ., as d isclosed in Tarn, J . Immunol . Methods (1996); 196(1 ):17-32. Binding units may be attached to each other or to the linker scaffold via N-terminal amino groups, C-terminal carboxyl groups, side chains, chemically modified groups, side chains, or other means.

[0051] More than one type of linker may be used per binding construct. In some embodiments, a binding construct may comprise two or more different types of linker. Suitable linkers may also comprise the chemical modifications discussed below, e.g., in the section entitled Conjugates.

Antibodies and Antigen Binding Fragments Thereof

[0052] In some aspects of the present disclosures, the binding construct is an antibody, or antigen binding fragment thereof, which specifically binds to an RTK in accordance with the disclosures herein. The antibody can be any type of immunoglobulin that is known in the art. For instance, the antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc. The antibody can be monoclonal or polyclonal. The antibody can be a naturally-occurring antibody, e.g., an antibody isolated and/or purified from a mammal, e.g., mouse, rabbit, goat, horse, chicken, hamster, human, and the like. In this regard, the antibody may be considered as a mammalian antibody, e.g., a mouse antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, human antibody, and the like. The term "isolated" as used herein means having been removed from its natural environment. The term "purified," as used herein relates to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment and means having been increased in purity as a result of being separated from other components of the original composition. It is recognized that "purity" is a relative term, and not to be necessarily construed as absolute purity or absolute enrichment or absolute selection. In some aspects, the purity is at least or about 50%, is at least or about 60%, at least or about 70%, at least or about 80%, or at least or about 90% (e.g., at least or about 91 %, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99% or is approximately 100%. [0053] The antibody can have any level of affinity or avidity for the RTK. The dissociation constant (K_D) may be any of those exemplary dissociation constants described herein with regard to binding units. Binding constants, including dissociation constants, may be determined by methods known in the art, including, for example, methods which utilize the principles of surface plasmon resonance, e.g., methods utilizing a Biacore™ system. In accordance with the foregoing, in some embodiments, the antibody is in monomeric form, while in other embodiments, the antibody is in polymeric form. In certain embodiments in which the antibody comprises two or more distinct antigen binding regions fragments, the antibody is considered bispecific, trispecific, or multi-specific, or bivalent, trivalent, or multivalent, depending on the number of distinct epitopes that are recognized and bound by the binding construct.

[0054] Because the binding constructs reduce the ligand-induced activation of the RTK to which they bind, in some embodiments, the antibody is considered as a blocking antibody or neutralizing antibody. In some aspects, the K_D of the binding construct is about the same as the K_D of the native ligand of the RTK. In some aspects, the K_D of the binding construct is lower (e.g., at least 0.5-fold lower, at least 1 -fold lower, at least 2-fold lower, at least 5-fold lower, at least 10-fold lower, at least 25-fold lower, at least 50-fold lower, at least 75- fold lower, at least 100-fold lower) than the K_D of the native ligand of the RTK.

[0055] In some embodiments, the antibody can be a genetically-engineered antibody, e.g., a single chain antibody, a humanized antibody, a chimeric antibody, a CDR-grafted antibody, an antibody which includes portions of CDR sequences specific for VEGFR-3 (e.g., an antibody which includes portions of CDR sequences of antibody 2E1 1 D1 1 ), a humaneered antibody, a bispecific antibody, a trispecific antibody, and the like. Genetic engineering techniques also provide the ability to make fully human antibodies in a non-human source.

[0056] In some aspects, the antibody is a chimeric antibody. The term "chimeric antibody" is used herein to refer to an antibody containing constant domains from one species and the variable domains from a second, or more generally, containing stretches of amino acid sequence from at least two species.

[0057] In some aspects, the antibody is a humanized antibody. The term "humanized" when used in relation to antibodies is used to refer to antibodies having at least CDR regions from a nonhuman source which are engineered to have a structure and immunological function more similar to true human antibodies than the original source antibodies. For example, humanizing can involve grafting CDR from a non-human antibody, such as a mouse antibody, into a human antibody. Humanizing also can involve select amino acid substitutions to make a non-human sequence look more like a human sequence.

[0058] Use of the terms "chimeric or humanized" herein is not meant to be mutually exclusive, and rather, is meant to encompass chimeric antibodies, humanized antibodies, and chimeric antibodies that have been further humanized. Except where context otherwise indicates, statements about (properties of, uses of, testing, and so on) chimeric antibodies of the present disclosures apply to humanized antibodies of the present disclosures, and statements about humanized antibodies of the present disclosures pertain also to chimeric antibodies. Likewise, except where context dictates, such statements also should be understood to be applicable to antibodies and antigen binding fragments of such antibodies of the present disclosures.

[0059] In some aspects of the present disclosures, the binding construct is an antigen binding fragment of an antibody, which specifically binds to an RTK in accordance with the disclosures herein. The antigen binding fragment (also referred to herein as "antigen binding portion") may be an antigen binding fragment of any of the antibodies described herein. The antigen binding fragment can be any part of an antibody that has at least one antigen binding site, including, but not limited to, Fab, F(ab')2, dsFv, sFv, diabodies, triabodies, bis- scFvs, fragments expressed by a Fab expression library, domain antibodies, VhH domains, V-NAR domains, VH domains, VL domains, and the like. Antibody fragments of the invention, however, are not limited to these exemplary types of antibody fragments. [0060] A domain antibody comprises a functional binding unit of an antibody, and can correspond to the variable regions of either the heavy (V_H) or light (V_L) chains of antibodies. A domain antibody can have a molecular weight of approximately 13 kDa, or approximately one-tenth of a full antibody. Domain antibodies may be derived from full antibodies such as those described herein. The antigen binding fragments in some embodiments are monomeric or polymeric, bispecific or trispecific, bivalent or trivalent.

[0061] Antibody fragments that contain the antigen binding, or idiotype, of the antibody molecule may be generated by techniques known in the art. For example, such fragments include, but are not limited to, the F(ab' )2 fragment which may be produced by pepsin digestion of the antibody molecule; the Fab' fragments which may be generated by reducing the disulfide bridges of the F(ab')2 fragment, and the two Fab' fragments which may be generated by treating the antibody molecule with papain and a reducing agent.

[0062] A single-chain variable region fragment (sFv) antibody fragment, which consists of a truncated Fab fragment comprising the variable (V) domain of an antibody heavy chain linked to a V domain of a light antibody chain via a synthetic peptide, can be generated using routine recombinant DNA technology techniques (see, e.g., Janeway et al., supra). Similarly, disulfide-stabilized variable region fragments (dsFv) can be prepared by recombinant DNA technology (see, e.g., Reiter et al., Protein Engineering, 7, 697-704 (1994)).

[0063] Recombinant antibody fragments, e.g., scFvs, can also be engineered to assemble into stable multimeric oligomers of high binding avidity and specificity to different target antigens. Such diabodies (dimers), triabodies (trimers) or tetrabodies (tetramers) are well known in the art, see e.g., Kortt et al., Bio- mol Eng. 2001 18:95-108, (2001 ).

[0064] Bispecific antibodies (bscAb) are molecules comprising two single- chain Fv fragments joined via a glycine-serine linker using recombinant methods. The V light-chain (V_L) and V heavy-chain (V_H) domains of two antibodies of interest in exemplary embodiments are isolated using standard PCR methods. The V_L and V_H cDNA's obtained from each hybridoma are then joined to form a single-chain fragment in a two-step fusion PCR. Bispecific fusion proteins are prepared in a similar manner. Bispecific single-chain antibodies and bispecific fusion proteins are antibody substances included within the scope of the present invention. Exemplary bispecific antibodies are taught in U.S. Patent Application Publication No. 2005-0282233A1 and International Patent Application Publication No. WO 2005/087812, both applications of which are incorporated herein by reference in their entirety.

[0065] Suitable methods of making antibodies are known in the art. For instance, standard hybridoma methods are described in, e.g., Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA. Janeway et al . (eds.), Immunobiology, 5^th Ed., Garland Publishing, New York, NY (2001 )).

[0066] Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. In some aspects, an animal used for production of anti-antisera is a non-human animal including rabbits, mice, rats, hamsters, goat, sheep, pigs or horses. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies. In an exemplary method for generating a polyclonal antisera immunoreactive with the chosen VEGFR-3 epitope, 50 g of VEGFR-3 antigen is emulsified in Freund's Complete Adjuvant for immunization of rabbits. At intervals of, for example, 21 days, 50 g of epitope are emulsified in Freund's Incomplete Adjuvant for boosts. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.

[0067] Monoclonal antibodies for use in the invention may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein the human B-cell hybridoma technique. [0068] Briefly, in exemplary embodiments, to generate monoclonal antibodies, a mouse is injected periodically with recombinant VEGFR-3 against which the antibody is to be raised (e.g., 10-20 g emulsified in Freund's Complete Adjuvant). The mouse is given a final pre-fusion boost of a VEGFR-3 polypeptide containing the epitope that allows specific recognition of lymphatic endothelial cell in PBS, and four days later the mouse is sacrificed and its spleen removed. The spleen is placed in 10 ml serum-free RPMI 1640, and a single cell suspension is formed by grinding the spleen between the frosted ends of two glass microscope slides submerged in serum-free RPM I 1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 g/ml streptomycin (RPMI) (Gibco, Canada). The cell suspension is filtered through sterile 70-mesh Nitex cell strainer (Becton Dickinson, Parsip- pany, N.J.), and is washed twice by centrifuging at 200 g for 5 minutes and re- suspending the pellet in 20 ml serum-free RPMI. Splenocytes taken from three naive Balb/c mice are prepared in a similar manner and used as a control. NS- 1 myeloma cells, kept in log phase in RPMI with 1 1 % fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for three days prior to fusion, are centrifuged at 200 g for 5 minutes, and the pellet is washed twice.

[0069] Spleen cells (1 x 1 0⁸) are combined with 2.0 x 1 0⁷ NS-1 cells and centrifuged, and the supernatant is aspirated. The cell pellet is dislodged by tapping the tube, and 1 ml of 37 °C. PEG 1500 (50% in 75 mM Hepes, pH 8.0) (Boehringer Mannheim) is added with stirring over the course of 1 minute, followed by the addition of 7 ml of serum-free RPMI over 7 minutes. An additional 8 ml RPMI is added and the cells are centrifuged at 200 g for 10 minutes. After discarding the supernatant, the pellet is resuspended in 200 ml RPMI containing 15% FBS, 100 μΜ sodium hypoxanthine, 0.4 μΜ aminopterin, 16 μΜ thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1 .5 x 10⁶ splenocytes/ml and plated into 10 Corning flat-bottom 96-well tissue culture plates (Corning, Corning N.Y.).

[0070] On days 2, 4, and 6, after the fusion, 100 μΙ of medium is removed from the wells of the fusion plates and replaced with fresh medium. On day 8, the fusion is screened by ELISA, testing for the presence of mouse IgG binding to VEGFR-3 as follows. Immulon 4 plates (Dynatech, Cambridge, Mass.) are coated for 2 hours at 37° C. with 100 ng/well of VEGFR-3 diluted in 25 mM Tris, pH 7.5. The coating solution is aspirated and 200 μΙ/well of blocking solution (0.5% fish skin gelatin (Sigma) diluted in CMF-PBS) is added and incubated for 30 min. at 37° C. Plates are washed three times with PBS with 0.05% Tween 20 (PBST) and 50 μΙ culture supernatant is added. After incubation at 37° C. for 30 minutes, and washing as above, 50 μΙ of horseradish peroxidase conjugated goat anti-mouse IgG(fc) (Jackson ImmunoResearch, West Grove, Pa.) diluted 1 :3500 in PBST is added. Plates are incubated as above, washed four times with PBST, and 100 μΙ substrate, consisting of 1 mg/ml o-phenylene diamine (Sigma) and 0.1 μΙ/ml 30% H₂O₂ in 1 00 mM Citrate, pH 4.5, are added. The color reaction is stopped after 5 minutes with the addition of 50 μΙ of 15% H₂SO₄. A₄₉₀ is read on a plate reader (Dynatech).

[0071] Selected fusion wells are cloned twice by dilution into 96-well plates and visual scoring of the number of colonies/well after 5 days. The monoclonal antibodies produced by hybridomas are isotyped using the Isostrip system (Boehringer Mannheim, Indianapolis, Ind.).

[0072] When the hybridoma technique is employed, myeloma cell lines may be used. Such cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). For example, where the immunized animal is a mouse, one may use P3- X63/Ag8, P3-X63-Ag8.653, NS1 /1 .Ag 4 1 , Sp210-Ag14, FO, NSO/U, MPC-1 1 , MPC1 1 -X45-GTG 1 .7 a n d S 1 94/ 1 5XX0 B u i ; fo r rats , o n e m ay u se R210.RCY3, Y3-Ag 1 .2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with cell fusions. It should be noted that the hybridomas and cell lines produced by such techniques for producing the monoclonal antibodies are contemplated to be novel compositions of the present disclosures. [0073] Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli Calmette- Guerin) and Corynebacterium parvum are potentially useful human adjuvants.

[0074] Alternatively, other methods, such as EBV-hybridoma methods (Haskard and Archer, J. Immunol. Methods, 74(2), 361 -67 (1984),and Roder et al.5 Methods Enzymol., 121 , 140-67 (1986)), and bacteriophage vector expression systems (see, e.g., Huse et al., Science, 246, 1275-81 (1989)) are known in the art. Further, methods of producing antibodies in non-human animals are described in, e.g., U.S. Patents 5,545,806, 5,569,825, and 5,714,352, and U.S. Patent Application Publication No. 2002/0197266 Al).

[0075] Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (Proc Natl Acad Sci 86: 3833-3837; 1989), and Winter G and Milstein C (Nature 349: 293-299, 1991 ).

[0076] Phage display furthermore can be used to generate the antibody of the present disclosures. In this regard, phage l ibraries encoding antigen- binding variable (V) domains of antibodies can be generated using standard molecular biology and recombinant DNA techniques (see, e.g., Sambrook et al . (eds.), Molecular Cloning, A Laboratory Manual, 3^rd Edition, Cold Spring Harbor Laboratory Press, New York (2001 )), Phage encoding a variable region with the desired specificity are selected for specific binding to the desired antigen, and a complete or partial antibody is reconstituted comprising the selected variable domain. Nucleic acid sequences encoding the reconstituted antibody are introduced into a suitable cell line, such as a myeloma cell used for hybridoma production, such that antibodies having the characteristics of monoclonal antibodies are secreted by the cell (see, e.g., Janeway et al., supra, Huse et al., supra, and U.S. Patent 6,265,150). Related methods also are described in U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,571 ,698; U.S. Pat. No. 5,837,500; U.S. Pat. No. 5,702,892. The techniques described in U.S. Pat. No. 5,780,279; U .S. Pat. No. 5,821 ,047; U.S. Pat. No. 5,824,520; U .S. Pat. No. 5,855,885; U .S. Pat. No. 5,858,657; U.S. Pat. No. 5,871 ,907; U .S. Pat. No. 5,969,108; U.S. Pat. No. 6,057,098; U.S. Pat. No. 6,225,447.

[0077] Antibodies can be produced by transgenic mice that are transgenic for specific heavy and light chain immunoglobulin genes. Such methods are known in the art and described in, for example U.S. Patents 5,545,806 and 5,569,825, and Janeway et al., supra.

[0078] Methods for generating humanized antibodies are well known in the art and are described in detail in, for example, Janeway et al., supra, U.S. Patents 5,225,539, 5,585,089 and 5,693,761 , European Patent No. 0239400 Bl, and United Kingdom Patent No. 2188638. Humanized antibodies can also be generated using the antibody resurfacing technology described in U.S. Patent 5,639,641 and Pedersen et al., J. Mol. Biol, 235, 959-973 (1994).

[0079] Techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al., Proc Natl Acad Sci 81 : 6851 -6855, 1984; Neuberger et al., Nature 312: 604-608, 1984; Takeda et al., Nature 314: 452-454; 1985). Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce VEGFR-3-specific single chain antibodies.

[0080] A preferred chimeric or humanized antibody has a human constant region, while the variable region, or at least a CDR, of the antibody is derived from a non-human species. Methods for humanizing non-human antibodies are well known in the art. (see U.S. Patent Nos. 5,585,089, and 5,693,762). Generally, a humanized antibody has one or more amino acid residues introduced into its framework region from a source which is non-human. Humani- zation can be performed, for example, using methods described in Jones et al. (Nature 321 : 522-525, 1986), Riechmann et al., (Nature, 332: 323-327, 1988) and Verhoeyen et al. (Science 239:1534-1536, 1988), by substituting at least a portion of a rodent complementarity-determining region (CDRs) for the corresponding regions of a human antibody. Numerous techniques for preparing engineered antibodies are described, e.g., in Owens and Young, J. Immunol. Meth., 168:149-165 (1994). Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.

[0081] Likewise, using techniques known in the art to isolate CDRs, compositions comprising CDRs are generated. Complementarity determining regions are characterized by six polypeptide loops, three loops for each of the heavy or light chain variable regions. The amino acid position in a CDR is defined by Kabat et al., "Sequences of Proteins of Immunological Interest," U.S. Department of Health and Human Services, (1983), which is incorporated herein by reference. For example, hypervariable regions of human antibodies are roughly defined to be found at residues 28 to 35, from 49-59 and from residues 92-103 of the heavy and light chain variable regions (Janeway and Travers, Immunobiology, 2^nd Edition, Garland Publishing, New York, (1996)). The murine CDR also are found at approximately these amino acid residues. It is understood in the art that CDR regions may be found within several amino acids of these approximated residues set forth above. An immunoglobulin variable region also consists of four "framework" regions surrounding the CDRs (FR1 - 4). The sequences of the framework regions of different light or heavy chains are highly conserved within a species, and are also conserved between human and murine sequences.

[0082] Compositions comprising one, two, and/or three CDRs of a heavy chain variable region or a light chain variable region of a monoclonal antibody are generated. For example, using antibody 2E1 1 D1 1 , polypeptide compositions comprising 2E1 1 D1 1 CDRs are generated. Polypeptide compositions comprising one, two, three, four, five and/or six complementarity determining regions of a 2E1 1 D1 1 antibody are also contemplated. Using the conserved framework sequences surrounding the CDRs, PCR primers complementary to these consensus sequences are generated to amplify the 2E1 1 D1 1 CDR se- quence located between the primer regions. Techniques for cloning and expressing nucleotide and polypeptide sequences are well-established in the art (see e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^nd Edition, Cold Spring Harbor, New York (1989)). The amplified CDR sequences are ligated into an appropriate plasmid . The plasmid comprising one, two, three, four, five and/or six cloned CDRs optionally contains additional polypeptide encoding regions linked to the CDR.

[0083] It is contemplated that modified polypeptide compositions comprising one, two, three, four, five, and/or six CDRs of a heavy or light chain of a 2E1 1 D1 1 antibody are generated, wherein a CDR is altered to provide increased specificity or affinity or avidity to the target RTK. Sites at locations in the antibody 2E1 1 D1 1 CDRs are typically modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid substituted for a non-identical hydrophobic amino acid) and then with more dissimilar choices (e.g., hydrophobic amino acid substituted for a charged amino acid), and then deletions or insertions may be made at the target site.

[0084] Framework regions (FR) of a murine antibody are humanized by substituting compatible human framework regions chosen from a large database of human antibody variable sequences, including over twelve hundred human V_H sequences and over one thousand V_L sequences. The database of antibody sequences used for comparison is downloaded from Andrew C. R. Martin's KabatMan web page (http://www.rubic.rdg.ac.uk/abs/). The Kabat method for identifying CDR provides a means for delineating the approximate CDR and framework regions from any human antibody and comparing the sequence of a murine antibody for similarity to determine the CDRs and FRs. Best matched human V_H and V_L sequences are chosen on the basis of high overall framework matching, similar CDR length, and minimal mismatching of canonical and V_H / V_L contact residues. Human framework regions most similar to the murine sequence are inserted between the murine CDR. Alternatively, the murine framework region may be modified by making amino acid substitutions of all or part of the native framework region that more closely resemble a framework region of a human antibody.

[0085] "Conservative" amino acid substitutions are made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine (Ala, A), leucine (Leu, L), isoleucine (lie, I), valine (Val, V), proline (Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W), and methionine (Met, M); polar neutral amino acids include glycine (Gly, G), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y), aspar- agine (Asn, N), and glutamine (Gin, Q); positively charged (basic) amino acids include arginine (Arg, R), lysine (Lys, K), and histidine (His, H); and negatively charged (acidic) amino acids include aspartic acid (Asp, D) and glutamic acid (Glu, E). "Insertions" or "deletions" are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids. The variation may be introduced by systematically making substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity. Nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions). Methods for expressing polypeptide compositions useful in the invention are described in greater detail below.

[0086] Additionally, another useful technique for generating antibodies for use in the present invention may be one which uses a rational design type approach. The goal of rational design is to produce structural analogs of biologically active polypeptides or compounds with which they interact (agonists, antagonists, inhibitors, peptidomimetics, binding partners, etc.). In this case, the active polypeptides are 2E1 1 D1 1 antibodies discussed herein throughout. By creating such analogs, it is possible to fashion additional antibodies which are more immunoreactive than the native or natural 2E1 1 D1 1 molecules. In one approach, one would generate a three-dimensional structure for the antibodies or an epitope binding fragment thereof. This could be accomplished by x-ray crystallography, computer modeling or by a combination of both approaches. An alternative approach, "alanine scan," involves the random replacement of residues throughout molecule with alanine, and the resulting affect on function determined.

[0087] It also is possible to solve the crystal structure of the specific antibodies. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of anti-idiotype would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate additional antibodies from banks of chemically- or biologically-produced peptides.

[0088] Chemically constructed bispecific antibodies may be prepared by chemically cross-linking heterologous Fab or F(ab')2 fragments by means of chemicals such as heterobifunctional reagent su ccin im idyl -3-(2-pyridyld ith iol )- propionate (SPDP, Pierce Chemicals, Rockford, III .). The Fab and F(ab' )2 fragments can be obtained from intact antibody by digesting it with papain or pepsin, respectively (Karpovsky et al., J. Exp. Med. 160:1686-701 , 1984; Titus et al., J. Immunol., 138:4018-22, 1987).

[0089] Methods of testing antibodies for the ability to bind to the epitope of the RTK regardless of how the antibodies are produced are known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002/0197266 Al).

[0090] Selection of antibodies from an antibody population for purposes herein also include using blood vessel endothelial cells to "subtract" those antibodies that cross-react with VEGFR-3 or other epitopes on such cells. The remaining antibody population is enriched in antibodies preferential for lymphatic endothelial cell epitopes. Aptamers

[0091] Recent advances in the field of combinatorial sciences have identified short polymer sequences (e.g., oligonucleic acid or peptide molecules) with high affinity and specificity to a given target. For example, SELEX technology has been used to identify DNA and RNA aptamers with binding properties that rival mammalian antibodies, the field of immunology has generated and isolated antibodies or antibody fragments which bind to a myriad of compounds and phage display has been utilized to discover new peptide sequences with very favorable binding properties. Based on the success of these molecular evolution techniques, it is certain that molecules can be created which bind to any target molecule. A loop structure is often involved with providing the desired binding attributes as in the case of: aptamers which often utilize hairpin loops created from short regions without complimentary base pairing, naturally derived antibodies that utilize combinatorial arrangement of looped hyper- variable regions and new phage display libraries utilizing cyclic peptides that have shown improved results when compared to linear peptide phage display results. Thus, sufficient evidence has been generated to suggest that high affinity ligands can be created and identified by combinatorial molecular evolution techniques. For the present disclosures, molecular evolution techniques can be used to isolate binding constructs specific for the RTKs described herein. For more on aptamers, see, generally, Gold, L, Singer, B., He, Y. Y., Brody. E., "Aptamers As Therapeutic And Diagnostic Agents," J. Biotechnol. 74:5-13 (2000). Relevant techniques for generating aptamers may be found in U.S. Pat. No. 6,699,843, which is incorporated by reference in its entirety.

[0092] In some embodiments, the aptamer may be generated by preparing a library of nucleic acids; contacting the library of nucleic acids with a growth factor, wherein nucleic acids having greater binding affinity for the growth factor (relative to other library nucleic acids) are selected and amplified to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to the growth factor. The processes may be repeated, and the selected nucleic acids mutated and rescreened, whereby a growth fac- tor aptamer is be identified. Nucleic acids may be screened to select for molecules that bind to more than target RTK. Binding more than one target RTK can refer to binding more than one RTK simultaneously or competitively. In some embodiments a binding construct will comprise at least one aptamer, wherein a first binding unit binds a first epitope of an RTK and a second binding unit binds a second epitope of the RTK.

Epitopes

[0093] Each binding construct of the composition of the present disclosures binds to an epitope of an RTK. By "epitope of an RTK" as used herein is meant the region of or within the RTK which is bound by the binding unit(s) of the binding construct. In some embodiments, the epitope is a linear epitope. By "linear epitope" as used herein refers to the region of or within the RTK which is bound by the binding unit(s) of the binding construct, which region is composed of contiguous amino acids of the amino acid sequence of the RTK. The amino acids of a linear epitope are located in close promity to each other in the primary structure of the antigen and the secondary and/or tertiary structure^) of the antigen . For example, when the antigen, e.g., RTK, is in its properly folded state (e.g., its native conformation), the contiguous amino acids of the linear epitope are located in close proximity to one another.

[0094] In other aspects, the epitope of the binding construct is a conformational epitope. By "conformational epitope" is meant an epitope which is composed of amino acids which are located in close proximity to one another only when the RTK is in its properly folded state, but are not contiguous amino acids of the amino acid sequence of the RTK.

[0095] In particular aspects, the first epitope (which is specifically bound by the first binding construct) and the second epitope (which is specifically bound by the second binding construct) are linear epitopes. In other aspects, both of the first and second epitopes are conformational epitopes. In yet alternative aspects, only one of the first and second epitopes is a linear epitope while the other is a conformation epitope. In specific aspects, the first epitope is a linear epitope and the second epitope is a conformational epitope. Receptor Tyrosine Kinases

[0096] As reviewed in Schlessinger, Cell 103: 21 1 -225 (2000), RTKs are cell surface receptors comprising intrinsic protein tyrosine kinase activity. RTKs contain an extracellular ligand binding domain (that, in some instances, is glycosylated) which is connected to the cytoplasmic domain via a single trans- membrance helix. Within the cytoplasmic domain is a conserved protein tyrosine kinase core and additional regulatory sequences that are subjected to autophosphorylation and phosphorylation by heterologous protein kinases. RTKs exist in the cellular membrane as monomers, although, upon ligand binding, RTKs dimerize and autophosphorylate (e.g., transautophosphorylate), resulting in a cytoplasmic domain comprising a phosphorylated tyrosyl residue.

[0097] Multiple RTKs are known in the art and include any member of the RTK families set forth in Table 1 .

TABLE 1

Muscle Specific Kinase (MuSK)

Receptor Family

[0098] With regard to the binding constructs of the compositions of the present disclosures, ligand-induced activation of the RTK is reduced upon binding of the binding construct to the RTK. As used herein, the term "reduce" as well as like terms, e.g., "inhibit," do not necessarily imply 100% or a complete reduction or inhibition. Rather, there are varying degrees of reduction or inhibition of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. Accordingly, in some embodiments, ligand- induced activation of the RTK is completely abolished. In some embodiments, ligand-induced activation is substantially reduced, e.g., reduced by about 10% (e.g., by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, by about 90%) or more, as compared to ligand-induced activation of the RTK when the binding construct is absent or not bound to the RTK. Methods of measuring ligand-induced activation of an RTK are known in the art, and include, for example, measurement of tyrosine phosphorylation. See, for example, Example 1 .

[0099] With regard to the present disclosures, the binding constructs specifically bind to an RTK, which can be any RTK known in the art, including any of the family members of the RTK families of Table 1 . In certain aspects, the first binding construct reduces the binding of the RTK to its ligand. Accordingly, in some aspects, the first epitope is a portion of a ligand binding domain of the RTK. In some embodiments, the second binding construct does not reduce the binding of the RTK to its ligand. Accordingly, in some aspects, the second epitope is not a portion of a ligand binding domain of the RTK. In some embodiments, the second binding construct reduces dimerization of the RTK. In some aspects, the second binding construct reduces homodimerization of the RTK. In some aspects, the second binding construct reduces heterodimeriza- tion of the RTK. For example, the second binding construct may reduce the dimerization between VEGFR-3 and another VEGFR-3 or with a VEGFR-2. VEGF and PDGF Families

[00100] In particular aspects of the present disclosures, the RTK is a receptor for a Vascular Endothelial Growth Factor (VEGF) or a Platelet-Derived Growth Factor (PDGF), including, but not limited to, any of the growth factors set forth in Table 2.

TABLE 2

[00101] Other VEGF growth factors members include snake venom VEGFs (e.g., EMBL. AY033151 , AY033152, and AY42981 ), various VEGF-E (orf virus VEGF homologs, some of which are presented in Table 1 B) molecules including VEGF-E NZ2 (S67520), VEGF-E NZ7, VEGF-E D1701 , VEGF-E Orf-1 1 , and VEGF-E OV-IA82. (See generally, international patent publication no. WO 00/25085.)

[00102] Members of the PDGF/VEGF family are characterized by a number of structural motifs including a conserved PDGF motif defined by the sequence: P-[PS]-C-V-X(3)-R-C-[GSTA]-G-C-C (S EQ I D NO : 44) where the brackets indicate a variable position that can be any one of the amino acids within the brackets. The number contained within the parentheses indicates the number of amino acids that separate the "V" and "R" residues. This conserved motif falls within a large domain of 70-150 amino acids defined in part by eight highly conserved cysteine residues that form inter- and intramolecular disulfide bonds. This domain forms a cysteine knot motif composed of two disulfide bonds which form a covalently linked ring structure between two adjacent .beta, strands, and a third disulfide bond that penetrates the ring (see for example, FIG. 1 in Muller et al., Structure 5:1325-1338 (1997)), similar to that found in other cysteine knot growth factors, e.g., transforming growth factor- beta (TGF-β). The amino acid sequence of all known PDGF/VEGF proteins, with the exception of VEGF-E, contains the PDGF domain. The PDGF/VEGF family proteins are predominantly secreted glycoproteins that form either disul- fide-linked or non-covalently bound homo- or heterodimers whose subunits are arranged in an anti-parallel manner (Stacker and Achen, Growth Factors 17:1 - 1 1 (1999); Muller et al., Structure 5:1325-1338 (1997)). Binding constructs of the present disclosures include those that bind VEGF/PDGF growth factor monomers, homodimers, and heterodimers.

[00103] The VEGF subfamily is composed of members that share a VEGF homology domain (VHD) characterized by the sequence: C-X(22-24)-P-[PSR]- C-V-X(3)-R-C-[GSTA]-G-C-C-X(6)-C-X(32-41 )-C. (SEQ ID: 45). The VHD domain, determined through analysis of the VEGF subfamily members, comprises the PDGF motif but is more specific. The VEGF subfamily of growth factors and receptors regulate the development and growth of the vascular endothelial system.

[00104] VEGF family members include, but are not limited to: PDGF-A (see e.g ., GenBank Acc. No. X06374), PDGF-B (see e.g. GenBank Acc. No. M12783), VEGF (see e.g., GenBank Acc. No. Q16889 referred to herein for clarity as VEGF-A or by particular isoform), PIGF (see e.g., GenBank Acc. No. X54936 placental growth factor), VEGF-B (see e.g . , GenBank Acc. No. U48801 ; also known as VEGF-related factor (VRF)), VEGF-C (see e.g., GenBank Acc. No. X94216; also known as VEGF related protein (VRP)), VEGF-D (also known as c-fos-induced growth factor (FIGF); see e.g., Genbank Acc. No. AJ000185), VEGF-E (also known as NZ7 VEGF or OV NZ7; see e.g ., GenBank Acc. No. S67522), NZ2 VEGF (also known as OV NZ2; see e.g ., GenBank Acc. No. S67520), D1701 VEGF-like protein (see e.g ., GenBank Acc. No. AF106020; Meyer et al., EMBO J 18:363-374), and NZ10 VEGF-like protein (described in International Patent Appl ication PCT/US99/25869) (Stacker and Achen, Growth Factors 17:1 -1 1 (1999); Neufeld et al., FASEB J 13:9-22 (1999); Ferrara, J Mol Med 77:527-543 (1999)).

[00105] VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF (Li, X. and U. Eriksson, "Novel VEGF Family Members: VEGF-B, VEGF-C and VEGF-D," Int. J. Biochem. Cell. Biol., 33(4):421 -6 (2001 ))] Other VEGFs are bacterial or viral, the "VEGF-Es." Other VEGFs are derived from snake venom, the "NZ" series. (See e.g., Komori, et al. Biochemistry, 38(36):1 1796-803 (1999); Gasmi, et al., Biochem Biophys Res Commun, 268(1 ):69-72 (2002); Gasmi, et al ., J Biol Chem; 277(33):29992-8 (2002); de Azevedo, et al . , J . Biol . Chem., 276: 39836-39842 (2001 )).

[00106] VEGF-C, comprises a VHD that is approximately 30% identical at the amino acid level to VEGF-A. VEGF-C is originally expressed as a larger precursor protein, prepro-VEGF-C, having extensive amino- and carboxy- terminal peptide sequences flanking the VHD, with the C-terminal peptide containing tandemly repeated cysteine residues in a motif typical of Balbiani ring 3 protein. Prepro-VEGF-C undergoes extensive proteolytic maturation involving the successive cleavage of a signal peptide, the C-terminal pro-peptide, and the N-terminal pro-peptide. Secreted VEGF-C protein consists of a non- covalently-linked homodimer, in which each monomer contains the VHD. The intermediate forms of VEGF-C produced by partial proteolytic processing show increasing affinity for the VEGFR-3 receptor, and the mature protein is also able to bind to the VEGFR-2 receptor. (Joukov et al., EMBO J, 16:(13):3898- 391 1 (1997)). It has also been demonstrated that a mutant VEGF-C, in which a single cysteine at position 156 is either substituted by another amino acid or deleted, loses the ability to bind VEGFR-2 but remains capable of binding and activating VEGFR-3 (International Patent Publication No. WO 98/33917). In mouse embryos, VEGF-C mRNA is expressed primarily in the allantois, jugular area, and the metanephros. (Jou kov et al . , J. Cell. Physiol. 173:21 1 -215 (1997)). VEGF-C is involved in the regulation of lymphatic angiogenesis: when VEGF-C was overexpressed in the skin of transgenic mice, a hyperplastic lymphatic vessel network was observed, suggesting that VEGF-C induces lymphatic growth (Jeltsch et al., Science, 276:1423-1425 (1997)). Continued expression of VEGF-C in the adult also indicates a role in maintenance of differentiated lymphatic endothelium (Ferrara, J Mol Med 77 :527-543 (1999)). VEGF-C also shows angiogenic properties: it can stimulate migration of bovine capillary endothelial (BCE) cells in collagen and promote growth of human endothelial cells (see, e.g., International Patent Publication No. WO 98/33917, incorporated herein by reference). VEGF-Ci₅6s is a VEGF-C cysteine deletion variant that binds to VEGFR-3 but demonstrates reduced binding (relative to VEGF-C) to VEGFR-2. VEGF-Cises and related ligands specific for VEGFR-3 that may be used in accordance with the present disclosures are described in U.S. Pat. No. 6,130,071 , which is specifically incorporated by reference in its entirety. VEGF-C materials and methods are described in U .S. Pat. Nos. 6,245,530 and 6,221 ,839, incorporated herein by reference.

[00107] VEGF-D is structurally and functionally most closely related to VEGF-C (see International Patent Publication No. WO 98/07832 and U.S. Pat. No. 6,235,713, each incorporated herein by reference). Like VEGF-C, VEGF- D is initially expressed as a prepro-peptide that undergoes N-terminal and C- terminal proteolytic processing , and forms non-covalently l inked dimers. VEGF-D stimulates mitogenic responses in endothelial cells in vitro. During embryogenesis, VEGF-D is expressed in a complex temporal and spatial pattern, and its expression persists in the heart, lung, and skeletal muscles in adults. Isolation of a biologically active fragment of VEGF-D designated VEGF-DANAC, is described in International Patent Publ ication No. WO 98/07832, incorporated herein by reference. VEGF-DANAC consists of amino acid residues 93 to 201 of VEGF-D linked to the affinity tag peptide FLAG®. [00108] VEGF-A was originally purified from several sources on the basis of its mitogenic activity toward endothelial cells, and also by its ability to induce microvascular permeability, hence it is also called vascular permeability factor (VPF). VEGF-A has subsequently been shown to induce a number of biological processes including the mobilization of intracellular calcium, the induction of plasminogen activator and plasminogen activator inhibitor-1 synthesis, promotion of monocyte migration in vitro, induction of antiapoptotic protein expression in human endothelial cells, induction of fenestrations in endothelial cells, promotion of cell adhesion molecule expression in endothelial cells and induction of nitric oxide mediated vasodilation and hypotension (Ferrara, J Mol Med 77: 527-543 (1999); Neufeld et al., FASEB J 13: 9-22 (1999); Zachary, Intl J Biochem Cell Bio 30: 1 169-1 174 (1998)).

[00109] VEGF-A is a secreted, disulfide-linked homodimeric glycoprotein composed of 23 kD subunits. Five human VEGF-A isoforms of 121 , 145, 165, 1 89 or 206 amino acids in length (VEGFi₂i-206), encoded by distinct mRNA splice variants, have been described, all of which are capable of stimulating mitogenesis in endothelial cells. However, each isoform differs in biological activity, receptor specificity, and affinity for cell surface- and extracellular matrix-associated heparan-sulfate proteoglycans, which behave as low affinity receptors for VEGF-A. VEGF121 does not bind to either heparin or heparan- sulfate; VEGFi₄₅ and VEGFi₆₅, (GenBank Acc. No. M32977) are both capable of binding to heparin; and VEGFi₈₉ and VEGF206 show the strongest affinity for heparin and heparan-sulfates. VEGF121 , VEGFi₄₅, and VEGF165, are secreted in a soluble form, although most of VEGF16₅ is confined to cell surface and extracellular matrix proteoglycans, whereas VEGFi₈₉ and VEGF206 remain associated with extracellular matrix. Both VEGFi₈₉ and VEGF206 can be released by treatment with heparin or heparinase, indicating that these isoforms are bound to extracellular matrix via proteoglycans. Cell-bound VEGFi₈₉ can also be cleaved by proteases such as plasmin, resulting in release of an active soluble VEGFno. Most tissues that express VEGF are observed to express several VEGF isoforms simultaneously, although VEGF121 , and VEGF16₅ are the predominant forms, whereas VEGF206 is rarely detected (Ferrara, J Mol Med 77:527-543 (1999)). VEGFi₄₅ differs in that it is primarily expressed in cells derived from reproductive organs (Neufeld et al . , FASE B J 1 3 :9-22 (1999)).

[00110] The pattern of VEGF-A expression suggests its involvement in the development and maintenance of the normal vascular system, and in angio- genesis associated with tumor growth and other pathological conditions such as rheumatoid arthritis. VEGF-A is expressed in embryonic tissues associated with the developing vascular system, and is secreted by numerous tumor cell lines. Analysis of mice in which VEGF-A was knocked out by targeted gene disruption indicate that VEGF-A is critical for survival, and that the development of the cardiovascular system is highly sensitive to VEGF-A concentration gradients. Mice lacking a single copy of VEGF-A die between day 1 1 and 12 of gestation. These embryos show impaired growth and several developmental abnormalities including defects in the developing cardiovasculature. VEGF- A is also required post-natally for growth, organ development, regulation of growth plate morphogenesis and endochondral bone formation. The requirement for VEGF-A decreases with age, especially after the fourth postnatal week. In mature animals, VEGF-A is required primarily for active angiogenesis in processes such as wound healing and the development of the corpus lu- teum (Neufeld et al ., FASEB J 13:9-22 (1999); Ferrara, J Mol Med 77:527-543 (1999)). VEGF-A expression is influenced primarily by hypoxia and a number of hormones and cytokines including epidermal growth factor (EGF), TGF-β, and various interleukins. Regulation occurs transcriptionally and also post- transcriptionally such as by increased mRNA stability (Ferrara, J Mol Med 77:527-543 (1999)).

[00111] Four additional members of the VEGF subfamily have been identified in poxviruses, which infect humans, sheep and goats. The orf virus- encoded VEGF-E and NZ2 VEGF are potent mitogens and permeability enhancing factors. Both show approximately 25% amino acid identity to mammalian VEGF-A, and are expressed as disulfide-liked homodimers. Infection by these viruses is characterized by pustular dermititis which may involve en- dothelial cell proliferation and vascular permeability induced by these viral VEGF proteins (Ferrara, J Mol Med 77:527-543 (1999); Stacker and Achen, Growth Factors 17:1 -1 1 (1999)). VEGF-like proteins have also been identified from two add itional strains of the orf virus, D1 701 (GenBank Acc. No. AF106020; described in Meyer et al., EMBO J. 18:363-374 (1999)) and NZ10 (described in International Patent Application PCT/US99/25869, incorporated herein by reference). These viral VEGF-like proteins have been shown to bind VEGFR-2 present on host endothelium, and this binding is important for development of infection and viral induction of angiogenesis (Meyer et al., EMBO J 18:363-374 (1999); International Patent Application PCT/US99/25869).

[00112] At least seven cell surface receptors that interact with PDGF/VEGF family members have been identified. These include PDGFR-a (See e.g., GenBank Acc. No. NM006206; Swiss Prot No. P16234), PDGFR-β (See e.g., GenBank Acc. No. NM002609; Swiss Prot. No. P09619), VEGFR-1/Flt-1 (fms- like tyrosine kinase-1 ; hereinafter "R-1 ") (GenBank Acc. No. X51602; De Vries, et al., Science 255:989-991 (1992)); VEGFR-2/KDR/Flk-1 (kinase insert domain containing receptor/fetal liver kinase-1 , hereinafter "R-2") (GenBank Acc. Nos. X59397 (Flk-1 ) and L04947 (KDR); Terman, et al., Biochem. Biophys. Res. Comm. 187:1579-1586 (1992); Matthews, et al., Proc. Natl . Acad. Sci. USA 88:9026-9030 (1991 )); VEGFR-3/Flt4 (fins-like tyrosine kinase 4; hereinafter "R-3") (U .S. Pat. No. 5,776,755 and GenBank Acc. No. X68203 and S66407; Pajusola et al., Oncogene 9:3545-3555 (1994); Hughes, et al., J. Mol. Evol 52(2):77-79 (2001 ); Pajusola, et al ., Oncogene 8(1 1 ):2931 -37) (1993); Borg, et al., Oncogene 10(5):973-984 (1995)), neuropilin-1 (Gen Bank Acc. No. NM003873), and neuropil in-2 (Gen Bank Acc. No. NM003872; SwissProt 060462). The two PDGF receptors mediate signaling of PDGFs. Non-human VEGF and PDGF receptors may also be employed as part of the invention, e.g., chicken VEGFR-1 may be used alone or in hybrid form with human R-1 for improved expression.

[00113] VEGF121 , VEGF165, VEGF-B, PIGF-1 and PIGF-2 bind VEGF-R1 ; VEGF1 21 , VEGF145, VEGF1 65, (fully processed mature) VEGF-C, (fully processed mature) VEGF-D (processed mature), VEGF-E, and NZ2 VEGF bind VEGF-R2; VEGF-C and VEGF-D bind VEGFR-3; VEGF165, VEGF-C, PIGF-2, and NZ2 VEGF bind neuropilin-1 ; and VEGF165 and VEGF-C binds neuropilin-2. (Neufeld, et al., FASEB. J. 13:9-22 (1999); Stacker and Achen, Growth Factors 17:1 -1 1 (1999); Ortega, et al., Fron. Biosci. 4:141 -152 (1999); Zachary, Intl. J. Biochem. Cell. Bio. 30:1 169-1 174 (1998); Petrova, et al., Exp. Cell . Res. 253: 1 1 7-130 (1999); U .S. Pat. Appl . Pub. No. 200301 1 3324). PDGF-A, PDGF-B, and PDGF-C bind PDGFR-a, PDGF-B and PDGF-D bind PDGF-β.

[00114] In accordance with the foregoing, in certain embodiments, the RTK is a receptor for a Vascular Endothelial Growth Factor (VEGF) or Platelet- Derived Growth Factor (PDGF). Exemplary human nucleotide and amino acid sequences of such receptors are set forth in the sequence listing as summarized below in Table 3:

TABLE 3

[00115] Both the ligands and the receptors generally exist as dimers, including both homodimers and heterodimers. Such dimers can influence binding. For example, for the PDGFs, PDGF-AA binds PDGFR-α/α. PDGF-AB and PDGF-CC bind PDGFR- α/α and PDGFR-α/β. PDGFR-BB binds both of the homodimers and the heterodimeric PDGF receptor. PDGF-DD binds PDGF receptor heterodimers and beta receptor homodimers. (See, e.g., Pietras, et al., Cancer Cell, 3:439-443 (2003)). VEGF-A can heterodimerize with VEGF-B and PIGF. The VEGFs, PDGFs, and PIGFs, may exist as two or more iso- forms, e.g., splice variants, and not all isoforms of a particular growth factor will share the same binding profile, or ability to dimerize with particular molecules. Certain isoforms of the same growth factor may also dimerize with each other. For example the 1 67 and 1 86 isoforms of VEGF-B can heterodimerize with each other.

[00116] As described above, RTKs generally comprise three principal domains: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain binds ligands, the transmembrane domain anchors the receptor to a cell membrane, and the intracellular domain possesses one or more tyrosine kinase enzymatic domains and interacts with downstream signal transduction molecules. The vascular endothelial growth factor receptors (VEGFRs) and platelet derived growth factor receptors (PDGFRs) bind their ligand through their extracellular domains (ECDs), which are comprised of multiple immunoglobulin-like or Ig-homology domains (Ig- domains). Ig-domains are identified herein using the designation "D#." For example "D1 " refers to the first Ig-like domain of a particular receptor ECD. "D1 -3" refers to a construct containing at least the first three Ig-like domains, and intervening sequence between domains 1 and 2 and 2 and 3, of a particular construct. Table 4 defines the boundaries of the Ig-domains for VEGFR-1 , VEGFR-2, and VEGFR-3 of the invention. These boundaries are significant as the boundaries chosen can be used to form constructs, and so can influence the binding properties of the resulting constructs. The complete ECD of PDGFRs and VEGFRs is not required for ligand (growth factor) binding. The ECD of VEGFR-1 (R-1 ) and VEGFR-2 (R-2) consists of seven Ig-like domains and the ECD of VEGFR-3 (R-3) has six intact Ig-like domains-D5 of R-3 is cleaved post-translationally into disulfide linked subunits leaving VEGFR-3. Veikkola, T., et al., Cancer Res. 60:203-212 (2000). In general, receptor fragments of at least the first three Ig-domains for this family are sufficient to bind ligand. The PDGFRs have five Ig-domains. The immunoglobulin-like domains for VEGFR-1 , VEGFR-2 AND VEGFR-3 are described in Table 4. TABLE 4

[00117] In certain aspects of the invention, the RTK that is the target of the binding constructs is VEGFR-3. Production of antibodies specific for VEGFR-3 is detailed in U.S. Pat. No. 6,107,046, which is incorporated herein by reference in its entirety.

[00118] In certain aspects in which the RTK is a VEGFR or a PDGFR, e.g., a VEGFR-3, the first epitope comprises at least a portion of Ig homology domain D1 , Ig homology domain D2, Ig homology domain D3, or a combination thereof, of a VEGFR or PDGFR, e.g., VEGFR-3. In particular aspects, the first epitope is the epitope of the antibody 3C5, which is further described herein. See, teachings under EXAMPLES. In specific aspects, the first binding construct comprises antibody 3C5, or an antigen binding fragment thereof. Antibody 3C5, as demonstrated herein, is a blocking antibody for VEGFR-3. Antibody 3C5 is being developed by Imclone Systems Incorporated (antibody IMC- 3C5), and ImClone's hF4-3C5 antibody has been the subject of multiple scientific publications. (See, e.g., Pytowski et al., J. National Cancer Inst., 2005; 97(1 ): 14-21 ; Zhang X, et al ., J Biol Chem. 2005 Jul 15; 280(28):2621 6-24; Jimenez et al., Mol. Cancer. Ther., 2005; 4: 427; and Goldman et al., FASEB J., 2007; 21 : 1003-1012) According to one published report, the antibody hF4- 3C5 was obtained by panning a human phage display library on soluble human VEGFR-3. The binding affinity constant of hF4-3C5 significantly exceeds that of the interaction of VEGFR-3 with VEGF-C. The antibody hF4-3C5 strongly inhibits the binding of soluble VEGFR-3 to immobilized VEGF-C and abolishes the VEGF-C-mediated mitogenic response of cells that expresses a chimeric human VEGFR-3-cFMS receptor. In fluorescence experiments, hF4- 3C5 reactivity is observed with human lymphatic endothelial cells (LECs) and human umbilical vein endothelial cells (HUVECs). See Persaud et al., Journal of Cell Science, 2004, 1 17 (13): 2745-2756. Each of these documents is incorporated herein by reference in its entirety.

[00119] More generally, other ligand blocking antibodies against RTK may be developed using routine techniques, which include determination of the receptor part responsible for ligand binding by expression of fragments of the receptor and testing them their direct binding of ligand e.g. in the receptor-Fc protein precipitation assay, or their ability to block ligand binding to full-length receptor extracellular domain e.g. in the BaF2 cell assay described herein. This task can be achieved by site-directed mutagenesis or by resolving crystal structure of receptor-ligand complex. Next step represents screening of a monoclonal antibody against the part of the receptor responsible for ligand binding. Appropriate aproaches include, but are not limited to, screening of a (human) phage display library or immunization of mice or rabbits. In a third step these antibodies are tested in a functional assay for their ability to inhibit cellular effects induced by ligand (growth factor). The BaF3 assay described in the experimental part of the present application represents an example of such an assay but other assays allowing one to screen antibody library for functional effects are available. Further steps of antibody development include extensive biochemical and in vivo characterization. More specifically, one would select antibodies that inhibit VEGF-C stimulated mitogenesis and survival of BaF3 VEGFR-3 cells as in Example 1 , Fig. 1 A, and antibodies that inhibit receptor extracellular domain binding to VEGF-C coated surface as in Example 1 , Fig. 1 C. The 3C5 antibody against VEGFR-3 is an example of a blocking antibody which were developed using an antibody phage display library with soluble VEGFR-3.

[00120] In certain embodiments, the second epitope, which is different from the first epitope, is not located within any of D1 , D2, and D3 of a VEGFR or PDGFR, e.g., VEGFR-3. In exemplary embodiments, the second epitope is a conformational eptitope and binding of the second binding construct to the VEGFR or PDGFR, e.g., VEGFR-3, requires at least one disulfide bond within the receptor to maintain the epitope. For example, if the VEGFR is VEGFR-3, the disulfide bond can be the disulfide bond between Cys at position 445 and the Cys at position 534 of VEGFR-3. In particular aspects, the second epitope comprises at least a portion of Ig domain D5 of the VEGFR-3, or the analogous region of another VEGFR or PDGFR, e.g., VEGFR-2. In particular aspects of the present disclosures, the second binding construct comprises antibody 2E1 1 D1 1 (also referred to herein as 2E1 1 ) which has been deposited with the European Collection of Cell Cultures, Center for Applied Microbiology and Research, Porton Down, Salisbury, SP4 0JG, U.K. on August 31 , 2001 as Accession No. 01083129 and is further described in U.S. Patent Application Publication No. 2008-0317723A1 ; U.S. Patent 6,107,046; Jussila et al., Cancer Res. 58:1599-1604, 1998; and International Patent Application Publication No. W) 95 33772 (all incorporated herein by reference in their entirety), or an antigen binding fragment thereof. These documents also further characterize the 9D9 antibody described herein, which was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1 b, D- 38124 Braunschweig, Germany (Deposit date 1995-03-23; Acc No. DSM ACC2210).

[00121] In specific embodiments, the second binding construct comprises an antibody which is different from 2E1 1 but the antibody recognizes and binds to the same epitope of of antibody 2E1 1 D1 1 . One of skill in the art is able to produce such additional antibodies that recognize the specific epitope or epitopes recognized by antibody 2E1 1 D1 1 via methods described herein and in the art. Suitable methods include, but are not limited to CDR-grafting techniques and phage display. The characterization of the 2E1 1 epitope herein facilitates selecting additional antibodies that recognize the same or a similar epitope. Moreover, the discovery that the 2E1 1 D1 1 antibody preferentially recognizes VEGFR-3 expressed on lymphatic endothelial cells over VEGFR-3 expressed on blood vessel endothelial cells demonstrates the feasibility of isolating such antibodies using conventional immunization and screening techniques (see e.g., Harlow and Lane, ANTIBODES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988). A population of VEGFR-3 antibodies can be screened for binding specificity or cross-reactivity against different cell populations described in U.S. Application Publication No. 2008- 0317723, which is incorporated herein by reference in its entirety. Additionally, antibodies that bind to the target RTKs can be screened for the ability to inhibit receptor dimerization (homodimerization or hetrodimerization), to select the desired antibodies.

Conjugates

[00122] In some embodiments, the binding construct of the composition of the present disclosures is attached or linked or conjugated to a second moiety (e.g., a heterologous moiety, a conjugate moiety). As used herein, the term "heterologous moiety" is synonomous with "conjugate moiety" and refers to any molecule (chemical or biochemical, naturally-occurring or non-coded) which is different from the binding constructs of the presently disclosed compositions. Exemplary heterologous moieties include, but are not limited to, a polymer, a carbohydrate, a lipid, a nucleic acid, an oligonucleotide, a DNA or RNA, an amino acid, peptide, polypeptide, protein, therapeutic agent, (e.g., a cytotoxic agent, cytokine), or a diagnostic agent.

[00123] In some embodiments, the binding constructs act as a targeting agent which localizes the heterologous moiety to a target cell or target tissue which expresses the RTK to which the binding constructs bind.

[00124] The binding constructs in some embodiments are chemically modified with various substituents. In some embodiments, the chemical modifications impart additional desirable characteristics as discussed herein. Chemical modifications in some aspects take a number of different forms such as heterologous peptides, polysaccarides, lipids, radioisotopes, non-standard amino acid resides and nucleic acids, metal chelates, and various cytotoxic agents.

[00125] The binding constructs in some embodiments are fused to heterologous peptides to confer various properties, e.g., increased solubility and/or stability and/or half-life, resistance to proteolytic cleavage, modulation of clearance, targeting to particular cell or tissue types. In some embodiments, the binding construct is linked to a Fc domain of IgG or other immunoglobulin. In some embodiments, the binding construct is fused to alkaline phosphatase (AP). Methods for making Fc or AP fusion constructs are found in WO 02/060950. By fusing the binding construct with protein domains that have specific properties (e.g. half life, bioavailability) it is possible to confer these properties to the the binding construct.

[00126] When the binding constructs are polypeptides, they can be modified, for instance, by glycosylation, amidation, carboxylation, or phosphorylation, or by the creation of acid addition salts, amides, esters, in particular C-terminal esters, and N-acyl derivatives. The proteins also can be modified to create peptide derivatives by forming covalent or noncovalent complexes with other moieties. Covalently bound complexes can be prepared by linking the chemical moieties to functional groups on the side chains of amino acids comprising the peptides, or at the N- or C-terminus.

[00127] Polypeptides can be conjugated to a reporter group, including, but not limited to a radiolabel, a fluorescent label, an enzyme (e.g., that catalyzes a calorimetric or fluorometric reaction), a substrate, a solid matrix, or a carrier (e.g., biotin or avidin). Examples of analogs are described in WO 98/28621 and in Olofsson, et al, Proc. Nat'l. Acad. Sci. USA, 95:1 1709-1 1714 (1998), U.S. Pat. Nos . 5 ,51 2 ,545 , and 5,474 , 982 ; U . S . Patent Appl ication Nos . 20020164687 and 20020164710.

[00128] Cysteinyl residues most commonly are reacted with haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carbocyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, .alpha.-bromo- .beta.(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuhbenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1 ,3-diazole.

[00129] Histidyl residues are derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

[00130] Lysinyl and amino terminal residues are reacted with succinic or car- boxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatiz- ing . alpha. -amino-containing residues include imidoesters such as methyl pi- colinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitroben- zenesulfonic acid; O-methylissurea; 2,4 pentanedione; and transaminase catalyzed reaction with glyoxylate.

[00131] Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylg lyoxal , 2 ,3-butanedione, 1 ,2- cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

[00132] The specific modification of tyrosyl residues per se has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to form O- acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using .sup.1251 or .sup.131 1 to prepare labeled proteins for use in radioimmunoassay.

[00133] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R1 ) such as 1 -cyclohexyl-3-(2-morpholinyl-(4- ethyl) carbodiimide or 1 -ethyl-3 (4 azonia 4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glu- taminyl residues by reaction with ammonium ions.

[00134] Derivatization with bifunctional agents is useful for crosslinking the binding construct to water-insoluble support matrixes. Such derivation may also provide the linker that may connect adjacent binding elements in a binding construct, or a binding elements to a heterologous peptide, e.g., a Fc fragment. Commonly used crosslin king agents include, e.g . , 1 , 1 -bis(diazoacetyl)-2- phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homo-bifunctional imidoesters, including disuc- cinimidyl esters such as 3,3'-dithiiobis(succinimidylpropioonate), and bifunctional maleimides such as bis-N-maleimido-1 ,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl) dithio] propioimidate yield photoactivatable intermediates that are capable of forming cross links in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide- activated carbohydrates and the reactive substrates described in U .S. Pat. Nos. 3,969,287; 3,691 ,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440, incorporated herein by reference, are employed for protein immobilization.

[00135] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

[00136] Other mod ifications include hydroxylation of prol ine and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the .alpha. -amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecule Properties, W. H. Freeman & Co., San Francisco, pp. 79-86,1983), acetylation of the N-terminal amine, and, in some instances, amidation of the C-terminal carboxyl groups. Such derivatives are chemically modified polypeptide compositions in which the binding construct polypeptide is linked to a polymer. [00137] In general, chemical derivatization may be performed under any suitable condition used to react a protein with an activated polymer molecule. Methods for preparing chemical derivatives of polypeptides will generally comprise the steps of (a) reacting the polypeptide with the activated polymer molecule (such as a reactive ester or aldehyde derivative of the polymer molecule) under conditions whereby the binding construct becomes attached to one or more polymer molecules, and (b) obtaining the reaction product(s). The optimal reaction conditions will be determined based on known parameters and the desired result. For example, the larger the ratio of polymer mole- cules:protein, the greater the amount of attached polymer molecule. In one embodiment, the binding construct polypeptide derivative may have a single polymer molecule moiety at the amino terminus. (See, e.g ., U .S. Pat. No. 5,234,784).

[00138] Derivatized binding constructs disclosed herein may have additional activities, enhanced or reduced biological activity, or other characteristics, such as increased or decreased half-life, as compared to the non-derivatized molecules.

[00139] In some embodiments, the binding construct is directly joined to a conjugate moiety in the absence of a linker. In alternative aspects, the binding construct is indirectly connected to the conjugate moiety via one or more linkers. Whether directly joined together or indirectly joined together through a linker, the binding construct may be connected through covalent bonds (e.g., a peptide, ester, amide, or sulfhydryl bond) or non-covalent bonds (e.g., via hydrophobic interaction, hydrogen bond, van der Waals bond, electrostatic or ionic interaction), or a combination thereof. The binding construct and conjugate moiety may be connected via any means known in the art, including, but not limited to, via a linker of any of the present disclosures. See, for example, the section herein entitled "Linkers."

Conjugates: Fc Fusions

[00140] For substituents such as an Fc region of human IgG, the fusion can be fused directly to a binding construct or fused through an intervening se- quence. For example, a human IgG hinge, CH2 and CH3 region may be fused at either the N-terminus or C-terminus of a binding construct to attach the Fc region. The resulting Fc-fusion construct enables purification via a Protein A affinity column (Pierce, Rockford, III.). Peptide and proteins fused to an Fc region can exhibit a substantially greater half-life in vivo than the unfused counterpart. A fusion to an Fc region allows for dimerization/multimerization of the fusion polypeptide. The Fc region may be a naturally occurring Fc region, or may be modified for superior characteristics, e.g., therapeutic qualities, circulation time, reduced aggregation. As noted above, in some embodiments, the binding constructs are conjugated, e.g., fused to an immunoglobulin or portion thereof (e.g ..variable region, CDR, or Fc region). Known types of immunoglobulins (Ig) include IgG, IgA, IgE, Ig D or IgM . The Fc region is a C- terminal region of an Ig heavy chain, which is responsible for binding to Fc receptors that carry out activities such as recycling (which results in prolonged half-life), antibody dependent cell-mediated cytotoxicity (ADCC), and complement dependent cytotoxicity (CDC).

[00141] For example, according to some definitions the human IgG heavy chain Fc region stretches from Cys226 to the C-terminus of the heavy chain. The "hinge region" generally extends from Glu216 to Pro230 of human lgG1 (hinge regions of other IgG isotypes may be aligned with the lgG1 sequence by aligning the cysteines involved in cysteine bonding). The Fc region of an IgG includes two constant domains, CH2 and CH3. The CH2 domain of a human IgG Fc region usually extends from amino acids 231 to amino acid 341 . The CH3 domain of a human IgG Fc region usually extends from amino acids 342 to 447. References made to amino acid numbering of immunoglobulins or immunoglobulin fragments, or regions, are all based on Kabat et al. 1991 , Sequences of Proteins of Immunological Interest, U .S. Department of Public Health, Bethesda, Md. In a related embodiments, the Fc region may comprise one or more native or modified constant regions from an immunoglobulin heavy chain, other than CH1 , for example, the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4 regions of IgE. [00142] Suitable conjugate moieties include portions of immunoglobulin sequence that include the FcRn binding site. FcRn, a salvage receptor, is responsible for recycling immunoglobulins and returning them to circulation in blood. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379). The major contact area of the Fc with the FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain. The major contact sites include amino acid residues 248, 250- 257, 272, 285, 288, 290-291 , 308-31 1 , and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain.

[00143] Some conjugate moieties may or may not include FcyR binding site(s). FcyR are responsible for ADCC and CDC. Examples of positions within the Fc region that make a direct contact with FcyR are amino acids 234- 239 (lower hinge region), amino acids 265-269 (B/C loop), amino acids 297- 299 (CVE loop), and amino acids 327-332 (F/G) loop (Sondermann et al., Nature 406: 267-273, 2000). The lower hinge region of IgE has also been implicated in the FcRI binding (Henry, et al., Biochemistry 36, 15568-15578, 1997). Residues involved in IgA receptor binding are described in Lewis et al., (J Immunol. 175:6694-701 , 2005). Amino acid residues involved in IgE receptor binding are described in Sayers et al. (J Biol Chem. 279(34):35320-5, 2004).

[00144] Amino acid modifications may be made to the Fc region of an immunoglobulin. Such variant Fc regions comprise at least one amino acid modification in the CH3 domain of the Fc region (residues 342-447) and/or at least one amino acid modification in the CH2 domain of the Fc region (residues 231 - 341 ). Mutations believed to impart an increased affinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al . 2001 , J . Biol . Chem. 276:6591 ). Other mutations may reduce binding of the Fc region to FcyRI, FcyRI IA, FcyRI IB, and/or FcyRIIIA without significantly reducing affinity for FcRn. For example, substitution of the Asn at position 297 of the Fc region with Ala or another amino acid removes a highly conserved N-glycosylation site and may result in reduced immunogenicity with concomitant prolonged half-life of the Fc region, as well as reduced binding to FcyRs (Routledge et al. 1995, Transplantation 60:847; Friend et al . 1999, Transplantation 68: 1632; Shields et al. 1995, J. Biol. Chem. 276:6591 ). Amino acid modifications at positions 233-236 of lgG1 have been made that reduce binding to FcyRs (Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol . 29:261 3). Some exemplary amino acid substitutions are described in US Patents 7,355,008 and 7,381 ,408, each incorporated by reference herein in its entirety.

Heterologous Moieties: Polymers, Carbohydrates, and Lipids

[00145] In some embodiments, the heterologous moiety is a polymer. The polymer may be branched or unbranched. The polymer may be of any molecular weight. The polymer in some embodiments has an average molecular weight of between about 2 kDa to about 100 kDa (the term "about" indicating that in preparations of a water soluble polymer, some molecules will weigh more, some less, than the stated molecular weight). The average molecular weight of the polymer is in some aspect between about 5 kDa and about 50 kDa, between about 1 2 kDa to about 40 kDa or between about 20 kDa to about 35 kDa.

[00146] In some embodiments, the polymer is modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization may be controlled. The polymer in some embodiments is water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. In some embodiments, when, for example, the composition is used for therapeutic use, the polymer is pharmaceutically acceptable. Additionally, in some aspects, the polymer is a mixture of polymers, e.g ., a co-polymer, a block co-polymer.

[00147] In some embodiments, the polymer is selected from the group consisting of: polyamides, polycarbonates, polyalkylenes and derivatives thereof including, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polymers of acrylic and methacrylic esters, including poly(methyl methacry- late), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacry- l ate) , poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate), polyvinyl polymers including polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinyl acetate), and polyvinylpyrrolidone, polyglycolides, polysilox- anes, polyurethanes and co-polymers thereof, celluloses including alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxy- lethyl cellulose, cellulose triacetate, and cellulose sulphate sodium salt, polypropylene, polyethylenes including poly(ethylene glycol), poly(ethylene oxide), and poly(ethylene terephthalate), and polystyrene.

[00148] In some aspects, the polymer is a biodegradable polymer, including a synthetic biodegradable polymer (e.g., polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone)), and a natural biodegradable polymer (e.g., alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins (e.g., zein and other prolamines and hydrophobic proteins)), as well as any copolymer or mixture thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.

[00149] In some aspects, the polymer is a bioadhesive polymer, such as a bioerodible hydrogel described by H. S. Sawhney, C. P. Pathak and J. A. Hub- bell in Macromolecules, 1993, 26, 581 -587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

[00150] In some embodiments, the polymer is a water-soluble polymer or a hydrophilic polymer. Suitable water-soluble polymers are known in the art and include, for example, polyvinylpyrrolidone, hydroxypropyl cellulose (HPC; Klu- cel), hydroxypropyl methylcellulose (HPMC; Methocel), nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropyl butylcellulose, hydroxypropyl pentyl- cellulose, methyl cellulose, ethylcellulose (Ethocel), hydroxyethyl cellulose, various alkyl celluloses and hydroxyalkyl celluloses, various cellulose ethers, cellulose acetate, carboxymethyl cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, vinyl acetate/crotonic acid copolymers, poly- hydroxyalkyl methacrylate, hydroxymethyl methacrylate, methacrylic acid copolymers, polymethacryl ic acid , polymethylmethacrylate, mal e i c a n hydride/methyl vinyl ether copolymers, poly vinyl alcohol, sodium and calcium polyacrylic acid, polyacrylic acid, acidic carboxy polymers, carboxypolymethyl- ene, carboxyvinyl polymers, polyoxyethylene polyoxypropylene copolymer, po- lymethylvinylether co-maleic anhydride, carboxymethylamide, potassium methacrylate divinylbenzene co-polymer, polyoxyethyleneglycols, polyethylene oxide, and derivatives, salts, and combinations thereof. In some aspects, the water soluble polymers or mixtures thereof include, but are not limited to, N- linked or O-linked carbohydrates, sugars, phosphates, carbohydrates; sugars; phosphates; polyethylene glycol (PEG) (including the forms of PEG that have been used to derivatize proteins, including mono-(C1 -C 10) alkoxy- or aryloxy- polyethylene glycol); monomethoxy-polyethylene glycol; dextran (such as low molecular weight dextran, of, for example about 6 kD), cellulose; cellulose; other carbohydrate-based polymers, poly-(N-vinyl pyrrolidone)polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol. Also encompassed by the present invention are bifunctional crosslinking molecules which may be used to prepare covalently attached multimers. [00151] A particularly preferred water-soluble polymer for use herein is polyethylene glycol (PEG). As used herein, polyethylene glycol is meant to encompass any of the forms of PEG that can be used to derivatize other proteins, such as mono-(C1 -C10) alkoxy- or aryloxy-polyethylene glycol. PEG is a linear or branched neutral polyether, available in a broad range of molecular weights, and is soluble in water and most organic solvents. PEG is effective at excluding other polymers or peptides when present in water, primarily through its high dynamic chain mobility and hydrophibic nature, thus creating a water shell or hydration sphere when attached to other proteins or polymer surfaces. PEG is nontoxic, non-immunogenic, and approved by the Food and Drug Administration for internal consumption.

[00152] Proteins or enzymes when conjugated to PEG have demonstrated bioactivity, non-antigenic properties, and decreased clearance rates when administered in animals. F. M. Veronese et al ., Preparation and Properties of Monomethoxypoly(ethylene glycol)-modified Enzymes for Therapeutic Applications, in J. M. Harris ed., Poly(Ethylene Glycol) Chemistry-Biotechnical and Biomedical Applications, 127-36, 1 992, incorporated herein by reference. These phenomena are due to the exclusion properties of PEG in preventing recognition by the immune system. In addition, PEG has been widely used in surface modification procedures to decrease protein adsorption and improve blood compatibility. S. W. Kim et al., Ann. N.Y. Acad. Sci. 516: 1 16-30 1987; Jacobs et al., Artif. Organs 12: 500-501 , 1988; Park et al., J. Poly. Sci, Part A 29:1725-31 , 1991 , incorporated herein by reference. Hydrophobic polymer surfaces, such as polyurethanes and polystyrene can be modified by the grafting of PEG (MW 3,400) and employed as nonthrombogenic surfaces. Surface properties (contact angle) can be more consistent with hydrophilic surfaces, due to the hydrating effect of PEG. More importantly, protein (albumin and other plasma proteins) adsorption can be greatly reduced, resulting from the high chain motility, hydration sphere, and protein exclusion properties of PEG.

[00153] PEG (MW 3,400) was determined as an optimal size in surface immobilization studies, Park et al., J. Biomed. Mat. Res. 26:739-45, 1992, while PEG (MW 5,000) was most beneficial in decreasing protein antigenicity. (F. M. Veronese et al ., In J . M . Harris, et al ., Poly(Ethylene Glycol) Chemistry- Biotechnical and Biomedical Applications, 127-36.).

[00154] Methods for preparing pegylated binding construct polypeptides may comprise the steps of (a) reacting the polypeptide with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby the binding construct polypeptide becomes attached to one or more PEG groups, and (b) obtaining the reaction product(s). In general, the optimal reaction conditions for the acylation reactions will be determined based on known parameters and the desired result. For example, the larger the ratio of PEG: protein, the greater the percentage of poly-pegylated product. In some embodiments, the binding construct will have a single PEG moiety at the N- terminus. See U.S. Pat. No. 8,234,784, herein incorporated by reference.

[00155] In some embodiments, the heterologous moiety is a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (a starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, ga- lactomannan.

[00156] In some embodiments, the heterologous moiety is a lipid. The lipid, in some embodiments, is a fatty acid, eicosanoid, prostaglandin, leukotriene, thromboxane, N-acyl ethanolamine), glycerolipid (e.g . , mono-, di-, tri- substituted glycerols), glycerophospholipid (e.g., phosphatidylcholine, phos- phatidylinositol, phosphatidylethanolamine, phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterol lipid (e.g., steroid, cholesterol), prenol lipid, saccharolipid, or a polyketide, oil, wax, cholesterol, sterol, fat-soluble vitamin, monoglyceride, diglyceride, triglyceride, a phospholipid.

Heterologous Moieties: Therapeutic Agents

[00157] In some embodiments, the heterologous moiety is a therapeutic agent. The therapeutic agent may be any of those known in the art. Examples of therapeutic agents that are contemplated herein include, but are not limited to, natural enzymes, proteins derived from natural sources, recombinant proteins, natural peptides, synthetic peptides, cyclic peptides, antibodies, receptor agonists, cytotoxic agents, immunoglobins, beta-adrenergic blocking agents, calcium channel blockers, coronary vasodilators, cardiac glycosides, antiarrhythmics, cardiac sympathomemetics, angiotensin converting enzyme (ACE) inhibitors, diuretics, inotropes, cholesterol and triglyceride reducers, bile acid sequestrants, fibrates, 3-hydroxy-3-methylgluteryl (HMG)-CoA reductase inhibitors, niacin derivatives, antiadrenergic agents, alpha-adrenergic blocking agents, centrally acting antiadrenergic agents, vasodilators, potassium-sparing agents, thiazides and related agents, angiotensin II receptor antagonists, peripheral vasodilators, antiandrogens, estrogens, antibiotics, retinoids, insulins and analogs, alpha-glucosidase inhibitors, biguanides, meglitinides, sulfonylureas, thizaolidinediones, androgens, progestogens, bone metabolism regulators, anterior pituitary hormones, hypothalamic hormones, posterior pituitary hormones, gonadotropins, gonadotropin-releasing hormone antagonists, ovulation stimulants, selective estrogen receptor modulators, antithyroid agents, thyroid hormones, bulk forming agents, laxatives, antiperistaltics, flora modifiers, intestinal adsorbents, intestinal anti-infectives, antianorexic, anticachexic, antibulimics, appetite suppressants, antiobesity agents, antacids, upper gastrointestinal tract agents, anticholinergic agents, aminosalicylic acid derivatives, biological response modifiers, corticosteroids, antispasmodics, 5-HT partial agonists, antihistamines, cannabinoids, dopamine antagonists, serotonin antagonists, cytoprotectives, histamine H2-receptor antagonists, mucosal protective agent, proton pump inhibitors, H. pylori eradication therapy, erythropoieses stimulants, hematopoietic agents, anemia agents, heparins, antifibrinolytics, hemostatics, blood coagulation factors, adenosine diphosphate inhibitors, glycoprotein receptor inhibitors, fibrinogen-platelet binding inhibitors, thromboxane^ inhibitors, plasminogen activators, antithrombotic agents, glucocorticoids, mineralcorticoids, corticosteroids, selective immunosuppressive agents, antifungals, drugs involved in prophylactic therapy, AIDS-associated infections, cytomegalovirus, non-nucleoside reverse transcriptase inhibitors, nucleoside analog reverse transcriptse inhibitors, protease inhibitors, anemia, Kaposi's sarcoma, aminoglycosides, carbapenems, cephalosporins, glycopoptides, lin- cosamides, macrolies, oxazolidinones, penicillins, streptogramins, sulfonamides, trimethoprim and derivatives, tetracyclines, anthelmintics, amebicies, biguanides, cinchona alkaloids, folic acid antagonists, quinoline derivatives, Pneumocystis carin i i therapy, hyd razides, im idazoles, triazoles, n itro- imidzaoles, cyclic amines, neuraminidase inhibitors, nucleosides, phosphate binders, cholinesterase inhibitors, adjunctive therapy, barbiturates and derivatives, benzodiazepines, gamma aminobutyric acid derivatives, hydantoin derivatives, iminostilbene derivatives, succinimide derivatives, anticonvulsants, ergot alkaloids, antimigrane preparations, biological response modifiers, car- bamic acid eaters, tricyclic derivatives, depolarizing agents, nondepolarizing agents, neuromuscular paralytic agents, CNS stimulants, dopaminergic reagents, monoamine oxidase inhibitors, COMT inhibitors, alkyl sulphonates, ethylenimines, imidazotetrazines, nitrogen mustard analogs, nitrosoureas, platinum-containing compounds, antimetabolites, purine analogs, pyrimidine analogs, urea derivatives, antracyclines, actinomycinds, camptothecin derivatives, epipodophyllotoxins, taxanes, vinca alkaloids and analogs, antiandro- gens, antiestrogens, nonsteroidal aromatase inhibitors, protein kinase inhibitor antineoplastics, azaspirodecanedione derivatives, anxiolytics, stimulants, monoamind reuptake inhibitors, selective serotonin reuptake inhibitors, antidepressants, benzisooxazole derivatives, butyrophenone derivatives, dibenzodi- azepine derivatives, dibenzothiazepine derivatives, diphenylbutylpiperidine derivatives, phenothiazines, thienobenzodiazepine derivatives, thioxanthene derivatives, allergenic extracts, nonsteroidal agents, leukotriene receptor antagonists, xanthines, endothelin receptor antagonist, prostaglandins, lung surfactants, mucolytics, antimitotics, uricosurics, xanthine oxidase inhibitors, phosphodiesterase inhibitors, metheamine salts, nitrofuran derivatives, quinolones, smooth muscle relaxants, parasympathomimetic agents, halogenated hydrocarbons, esters of amino benzoic acid, amides (e.g. lidocaine, articaine hydrochloride, bupivacaine hydrochloride), antipyretics, hynotics and sedatives, cyclopyrrolones, pyrazolopyrimidines, nonsteroidal anti-inflammatory drugs, opioids, para-aminophenol derivatives, alcohol dehydrogenase inhibitor, hepa- rin antagonists, adsorbents, emetics, opoid antagonists, cholinesterase reactivators, nicotine replacement therapy, vitamin A analogs and antagonists, vitamin B analogs and antagonists, vitamin C analogs and antagonists, vitamin D analogs and antagonists, vitamin E analogs and antagonists, vitamin K analogs and antagonists.

[00158] The binding constructs of the presently disclosed compositions may be conjugated to one or more cytokines and growth factors that are effective in inhibiting tumor metastasis, and wherein the cytokine or growth factor has been shown to have an antiproliferative effect on at least one cell population. Such cytokines, lymphokines, growth factors, or other hematopoietic factors include, but are not limited to: M-CSF, GM-CSF, TNF, IL-1 , IL-2, IL-3, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-9, IL-10, IL- , IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL- 1 8, IFN, TNFa, TNF1 , TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin. Additional growth factors for use in pharmaceutical compositions of the invention include angiogenin, bone morphogenic protein-1 , bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-1 1 , bone morphogenic protein-12, bone morphogenic protein-13, bone morphogenic protein-14, bone morphogenic protein-15, bone morphogenic protein receptor IA, bone morphogenic protein receptor IB, brain derived neurotrophic factor, ciliary neutrophic factor, ciliary neutrophic factor receptor a, cytokine-induced neutrophil chemotactic factor 1 , cytokine-induced neutrophil, chemotactic factor 2 a, cytokine-induced neutrophil chemotactic factor 2 β, β endothelial cell growth factor, endothelin 1 , epithelial-derived neutrophil attrac- tant, glial cell line-derived neutrophic factor receptor a 1 , glial cell line-derived neutrophic factor receptor a 2, growth related protein, growth related protein a, growth related protein β, growth related protein γ, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor a, nerve growth factor nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor a, transforming growth factor β, transforming growth factor β1 , transforming growth factor β1 .2, transforming growth factor β2, transforming growth factor β3, transforming growth factor β5, latent transforming growth factor β1 , transforming growth factor β binding protein I, transforming growth factor β binding protein II, transforming growth factor β binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, and chimeric proteins and biologically or immunologically active fragments thereof.

[00159] In some embodiments, the conjugate comprises a binding construct as described herein and a cytotoxic agent. The cytotoxic agent is any molecule (chemical or biochemical) which is toxic to a cell. In some aspects, when a cytotoxic agent is conjugated to a binding construct of the invention, the results obtained are synergistic. That is to say, the effectiveness of the combination therapy of a binding construct and the cytotoxic agent is synergistic, i.e., the effectiveness is greater than the effectiveness expected from the additive individual effects of each. Therefore, the dosage of the cytotoxic agent can be reduced and thus, the risk of the toxicity problems and other side effects is concomitantly reduced. In some embodiments, the cytotoxic agent is a che- motherapeutic agent. Chemotherapeutic agents are known in the art and include, but not limited to, platinum coordination compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids and difluoronucleosides, as described in U.S. Pat. No. 6,630,124.

[00160] In some embodiments, the chemotherapeutic agent is a platinum coordination compound. The term "platinum coordination compound" refers to any tumor cell growth inhibiting platinum coordination compound that provides the platinum in the form of an ion. In some embodiments, the platinum coordi- n a t i o n c o m p o u n d i s cis-diamminediaquoplatinum (ll)-ion; chloro(diethylenetriamine)-platinum(ll)chloride; dichloro(ethylenediamine)- platinum(l l), diamnnine(1 ,1 -cyclobutanedicarboxylato) platinum(l l) (carboplatin); spiroplatin; iproplatin; diamnnine(2-ethylnnalonato)-platinunn(l l); ethylenedia- minennalonatoplatinunn(l l); aqua(1 ,2-diaminodyclohexane)-sulfatoplatinunn(l l); (1 ,2-d iam inocyclohexane)nnalonatoplatin unn ( l l ) ; (4-caroxyphthalato)(1 ,2- diaminocyclohexane)platinunn(l l); (1 ,2-diaminocyclohexane)-

(isocitrato)platinum(l l); (1 ,2-diaminocyclohexane)cis(pyruvato)platinunn(l l); (1 ,2-diaminocyclohexane)oxalatoplatinunn(l l); ormaplatin; and tetraplatin.

[00161] In some embodiments, cisplatin is the platinum coordination compound employed in the compositions and methods of the present invention. Cisplatin is commercially available under the name PLATINOL™ from Bristol Myers-Squibb Corporation and is available as a powder for constitution with water, sterile sal ine or other suitable vehicle. Other platinum coordination compounds suitable for use in the present invention are known and are available commercially and/or can be prepared by conventional techniques. Cisplatin , or cis-dichlorodiammineplatinum II, has been used successfully for many years as a chemotherapeutic agent in the treatment of various human solid malignant tumors. More recently, other diamino-platinum complexes have also shown efficacy as chemotherapeutic agents in the treatment of various human solid malignant tumors. Such diamino-platinum complexes include, but are not limited to, spiroplatinum and carboplatinum. Although cisplatin and other diamino-platinum complexes have been widely used as chemotherapeutic agents in humans, they have had to be delivered at high dosage levels that can lead to toxicity problems such as kidney damage.

[00162] In some embodiments, the chemotherapeutic agent is a topoisom- erase inhibitor. Topoisomerases are enzymes that are capable of altering DNA topology in eukaryotic cells. They are critical for cellular functions and cell proliferation. Generally, there are two classes of topoisomerases in eukaryotic cells, type I and type II. Topoisomerase I is a monomeric enzyme of approximately 100,000 molecular weight. The enzyme binds to DNA and introduces a transient single-strand break, unwinds the double helix (or allows it to unwind), and subsequently reseals the break before dissociating from the DNA strand. Various topoisomerase inhibitors have recently shown clinical efficacy in the treatment of humans afflicted with ovarian, cancer, esophageal cancer or non-small cell lung carcinoma.

[00163] In some aspects, the topoisomerase inhibitor is camptothecin or a camptothecin analog. Camptothecin is a water-insoluble, cytotoxic alkaloid produced by Cam ptotheca accu m inata trees ind igenous to Ch ina and Nothapodytes foetida trees indigenous to India. Camptothecin exhibits tumor cell growth inhibiting activity against a number of tumor cells. Compounds of the camptothecin analog class are typically specific inhibitors of DNA topoisomerase I. By the term "inhibitor of topoisomerase" is meant any tumor cell growth inhibiting compound that is structurally related to camptothecin. Compounds of the camptothecin analog class include, but are not limited to; topo- tecan, irinotecan and 9-amino-camptothecin.

[00164] In additional embodiments, the cytotoxic agent is any tumor cell growth inhibiting camptothecin analog claimed or described in: U.S. Pat. No. 5,004,758, issued on Apr. 2, 1991 and European Patent Application Number 8831 1366.4, published on Jun. 21 , 1989 as 20' Publication Number EP 0 321 122; U.S. Pat. No. 4,604,463, issued on Aug. 5, 1986 and European Patent Application Publication Number EP 0 137 145, published on Apr. 17, 1 985; U.S. Pat. No. 4,473,692, issued on Sep. 25, 1984 and European Patent Application Publication Number EP 0 074 256, published on Mar. 16, 1983; U .S. Pat. No. 4,545,880, issued on Oct. 8, 1985 and European Patent Application Publication Number EP 0 074 256, published on Mar. 1 6, 1983; European Patent Application Publication Number EP 0 088 642, published on Sep. 14, 1983; Wani et al., J. Med. Chem., 29, 2358-2363 (1986); Nitta et al., Proc. 14th International Congr. Chemotherapy, Kyoto, 1985, Tokyo Press, Anticancer Section 1 , p. 28-30, especially a compound called CPT-1 1 . CPT-1 1 is a camptothecin analog with a 4-(piperidino)-piperidine side chain joined through a carbamate linkage at C-1 0 of 1 0-hydroxy-7-ethyl camptothecin. CPT-1 1 is currently undergoing human clinical trials and is also referred to as irinotecan; Wani et al, J. Med. Chem., 23, 554 (1980); Wani et. al., J. Med. Chem., 30, 1774 (1987); U.S. Pat. No. 4,342,776, issued on Aug. 3, 1982; U.S. patent application Ser. No. 581 ,916, filed on Sep. 13, 1990 and European Patent Application Publication Number EP 418 099, published on Mar. 20, 1991 ; U.S. Pat. No. 4,513,138, issued on Apr. 23, 1985 and European Patent Application Publication Number EP 0 074 770, published on Mar. 23, 1983; U .S. Pat. No. 4,399,276, issued on Aug. 16, 1983 and European Patent Application Publication Number 0 056 692, published on Jul . 28, 1982; the entire disclosure of each of which is hereby incorporated by reference. All of the above-listed compounds of the camptothecin analog class are available commercially and/or can be prepared by conventional techniques including those described in the above-listed references. The topoisomerase inhibitor may be selected from the group consisting of topotecan, irinotecan and 9-aminocamptothecin.

[00165] The preparation of numerous compounds of the camptothecin analog class (including pharmaceutically acceptable salts, hydrates and solvates thereof) as well as the preparation of oral and parenteral pharmaceutical compositions comprising such a compounds of the camptothecin analog class and an inert, pharmaceutically acceptable carrier or diluent, is extensively described in U.S. Pat. No. 5,004,758, issued on Apr. 2, 1991 and European Patent Application Number 8831 1366.4, published on Jun. 21 , 1989 as Publication Number EP 0 321 122, the teachings of which are incorporated herein by reference.

[00166] In still yet another embodiment of the invention, the chemotherapeu- tic agent is an antibiotic compound. Suitable antibiotic include, but are not limited to, doxorubicin, mitomycin, bleomycin, daunorubicin and streptozocin.

[00167] In some embodiments, the chemotherapeutic agent is an antimitotic alkaloid. In general, antimitotic alkaloids can be extracted from Cantharanthus roseus, and have been shown to be efficacious as anticancer chemotherapy agents. A great number of semi-synthetic derivatives have been studied both chemically and pharmacologically (see, O. Van Tellingen et al, Anticancer Research, 12, 1699-1716 (1992)). The antimitotic alkaloids of the present invention include, but are not limited to, vinblastine, vincristine, vindesine, Taxol and vinorelbine. The latter two antimitotic alkaloids are commercially available from Eli Lilly and Company, and Pierre Fabre Laboratories, respectively (see, U.S. Pat. No. 5,620,985). In a preferred aspect of the present invention, the antimitotic alkaloid is vinorelbine.

[00168] In another embodiment of the invention, the chemotherapeutic agent is a difluoronucleoside. 2'-deoxy-2',2'-difluoronucleosides are known in the art as having antiviral activity. Such compounds are disclosed and taught in U.S. Pat. Nos. 4,526,988 and 4,808614. European Patent Application Publication 184,365 discloses that these same difluoronucleosides have oncolytic activity. In certain specific aspects, the 2'-deoxy-2',2'-difluoronucleoside used in the compositions and methods of the present invention is 2'-deoxy-2',2'- difluorocytidine hydrochloride, also known as gemcitabine hydrochloride. Gemcitabine is commercially available or can be synthesized in a multi-step process as disclosed and taught in U.S. Pat. Nos. 4,526,988, 4,808,614 and 5,223,608, the teachings of which are incorporated herein by reference.

Heterologous Moieties: Diagnostic agents

[00169] In some embodiments, the conjugate comprises a binding construct as described herein and a diagnostic agent. The diagnostic agent in some aspects is an imaging agent. Many appropriate imaging agents are known in the art, as are methods of attaching the labeling agents to the peptides of the invention (see, e.g., U.S. Pat. No. 4,965,392, U.S. Pat. No. 4,472,509, U.S. Pat. No. 5,021 ,236 and U.S. Pat. No. 5,037,630, incorporated herein by reference). The imaging agents are administered to a subject in a pharmaceutically acceptable carrier, and allowed to accumulate at a target site having the lymphatic endothelial cells. This imaging agent then serves as a contrast reagent for X-ray, magnetic resonance, sonographic or scintigraphic imaging of the target site. The binding constructs described herein are a convenient and important addition to the available arsenal of medical imaging tools for the diagnostic investigation of cancer, lymphedema and other lymphatic endothelial cell disorders. Of course, it should be understood that the imaging may be performed in vitro where tissue from the subject is obtained through a biopsy, and the presence of lymphatic endothelial cells is determined with the aid of the imaging agents described herein in combination with histochemical techniques for preparing and fixing tissues.

[00170] Paramagnetic ions useful in the imaging agents of the invention include for example chromium (I II), manganese (I I), iron (I II), iron (II), cobalt (I I), nickel (II) copper (II), neodymium (III), samarium (III), ytterbium(lll), gadolinium (III), vanadium (II), terbium (III), dysprosium (I II), holmium (I II) and erbium (III). Ions useful for X-ray imaging include but are not limited to lantanum (II I), gold (II I), lead (II) and particularly bismuth (II I). Radioisotopes for diagnostic applications include for example, ²¹1 astatine, ¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁶⁷copper, ¹⁵²Eu, ⁶⁷gallium, ³hydrogen, ¹²³iodine, ¹²⁵iodine, ^{1 1 1} indium, ⁵⁹iron, ³²phosphorus, ¹⁸⁶rhenium, ⁷⁵selenium, ³⁵sulphur, "mtechnicium, and ⁹⁰yttrium.

[00171] The binding constructs described herein in some aspects are labeled according to techniques well known to those of skill in the are. For example, the binding constructs can be iodinated by contacting the peptide with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite or an enzymatic oxidant such as lactoperoxidase. Antibodies are labeled in some instances with technetium-99m by ligand exchange, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to the column. These and other techniques for labeling proteins and peptides are well known to those of skill in the art.

Pharmaceutical compositions and formulations

[00172] In some embodiments of the present disclosures, the composition comprises a pharmaceutically-acceptable carrier, such that the composition is a pharmaceutical composition. In this regard, provided herein is a pharmaceutical composition comprising a first binding construct which specifically binds to a first epitope of a RTK and a second binding construct which specifically binds to a second epitope of the RTK, wherein the second epitope of the RTK is different from the first epitope, wherein each of the first binding construct and the second binding construct reduces ligand-induced activiation of the RTK, and a pharmaceutically acceptable carrier.

[00173] The pharmaceutically-acceptable carrier is any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active binding construct(s), and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. In one aspect the pharmaceutically acceptable carrier is one which is chemically inert to the active ingredient(s) of the pharmaceutical composition, e.g., the first binding construct and the second binding construct, and one which has no detrimental side effects or toxicity under the conditions of use. The carrier in some embodiments does not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. The pharmaceutical composition in some aspects is free of pyrogens, as well as other impurities that could be harmful to humans or animals. Pharmaceutically-acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agnets, isotonic and absorption delaying agents and the like; the use of which are well known in the art.

[00174] Acceptable carriers, excipients or stabilizers are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, ar- ginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG). [00175] Therapeutic formulations of the compositions useful for practicing the invention such as polypeptides, polynucleotides, or antibodies may be prepared for storage by mixing the selected composition having the desired degree of purity with optional physiologically pharmaceutically-acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, ed., Mack Publishing Company (1990)) in the form of a ly- ophilized cake or an aqueous solution. Pharmaceutical compositions may be produced by admixing with one or more suitable carriers or adjuvants such as water, mineral oil, polyethylene glycol, starch, talcum, lactose, thickeners, stabilizers, suspending agents, etc. Such compositions may be in the form of solutions, suspensions, tablets, capsules, creams, salves, ointments, or other conventional forms.

[00176] The composition to be used for in vivo administration should be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. Therapeutic compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In some cases the form should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The composition for parenteral administration ordinarily will be stored in lyophilized form or in solution.

[00177] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[00178] The choice of carrier will be determined in part by the particular type of binding constructs of the pharmaceutical composition, as well as by the particular route used to administer the pharmaceutical composition. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition.

[00179] The pharmaceutical composition of the present disclosures can comprise any pharmaceutically acceptable ingredient, including, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffering agents, chelating agents, coating agents, coloring agents, desiccants, detergents, diluents, disinfectants, disintegrants, dispersing agents, dissolution enhancing agents, dyes, emollients, emulsifying agents, emulsion stabilizers, fillers, film forming agents, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oleaginous vehicles, organic bases, pastille bases, pigments, plas- ticizers, polishing agents, preservatives, sequestering agents, skin penetrants, solubilizing agents, solvents, stabilizing agents, suppository bases, surface active agents, surfactants, suspending agents, sweetening agents, therapeutic agents, thickening agents, tonicity agents, toxicity agents, viscosity-increasing agents, water-absorbing agents, water-miscible cosolvents, water softeners, or wetting agents.

[00180] In some embodiments, the pharmaceutical composition comprises any one or a combination of the following components: acacia, acesulfame potassium, acetyltributyl citrate, acetyltriethyl citrate, agar, albumin, alcohol, de- hydrated alcohol, denatured alcohol, dilute alcohol, aleuritic acid, alginic acid, aliphatic polyesters, alumina, aluminum hydroxide, aluminum stearate, amy- lopectin, a-amylose, ascorbic acid, ascorbyl palmitate, aspartame, bacteriostatic water for injection, bentonite, bentonite magma, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, benzyl benzoate, bronopol, butylated hydroxyanisole, butylated hydroxytoluene, butylparaben, butylparaben sodium, calcium alginate, calcium ascorbate, calcium carbonate, calcium cyclamate, dibasic anhydrous calcium phosphate, dibasic dehydrate calcium phosphate, tribasic calcium phosphate, calcium propionate, calcium silicate, calcium sorbate, calcium stearate, calcium sulfate, calcium sulfate hemihydrate, canola oil, carbomer, carbon dioxide, carboxymethyl cellulose calcium, carboxymethyl cellulose sodium, β-carotene, carrageenan, castor oil, hydrogenated castor oil, cationic emulsifying wax, cellulose acetate, cellulose acetate phthalate, ethyl cellulose, microcrystalline cellulose, powdered cellulose, silicified microcrystalline cellulose, sodium carboxymethyl cellulose, ce- tostearyl alcohol, cetrimide, cetyl alcohol, chlorhexidine, chlorobutanol, chloro- cresol, cholesterol, chlorhexidine acetate, chlorhexidine gluconate, chlorhexidine hydrochloride, chlorodifluoroethane (HCFC), chlorodifluoromethane, chlorofluorocarbons (CFC)chlorophenoxyethanol, chloroxylenol, corn syrup solids, anhydrous citric acid, citric acid monohydrate, cocoa butter, coloring agents, corn oil, cottonseed oil, cresol, m-cresol, o-cresol, p-cresol, croscar- mellose sodium, crospovidone, cyclamic acid, cyclodextrins, dextrates, dextrin, dextrose, dextrose anhydrous, diazolidinyl urea, dibutyl phthalate, dibutyl se- bacate, diethanolamine, diethyl phthalate, difluoroethane (HFC), dimethyl-β- cyclodextrin, cyclodextrin-type compounds such as Captisol®, dimethyl ether, dimethyl phthalate, dipotassium edentate, disodium edentate, disodium hydrogen phosphate, docusate calcium, docusate potassium, docusate sodium, do- decyl gallate, dodecyltrimethylammonium bromide, edentate calcium disodium, edtic acid, eglumine, ethyl alcohol, ethylcellulose, ethyl gallate, ethyl laurate, ethyl maltol, ethyl oleate, ethylparaben, ethylparaben potassium, ethylparaben sodium, ethyl vanillin, fructose, fructose liquid, fructose milled, fructose pyro- gen-free, powdered fructose, fumaric acid, gelatin, glucose, liquid glucose, glyceride mixtures of saturated vegetable fatty acids, glycerin, glyceryl behen- ate, glyceryl monooleate, glyceryl monostearate, self-emulsifying glyceryl monostearate, glyceryl palmitostearate, glycine, glycols, glycofurol, guar gum, heptafluoropropane (HFC), hexadecyltrimethylammonium bromide, high fructose syrup, human serum albumin, hydrocarbons (HC), dilute hydrochloric acid , hyd rogenated vegetable oil , type I I , hyd roxyethyl cel l u lose, 2- hydroxyethyl- -cyclodextrin, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, 2-hydroxypropyl- -cyclodextrin, hydroxypropyl methyl- cellulose, hydroxypropyl methylcellulose phthalate, imidurea, indigo carmine, ion exchangers, iron oxides, isopropyl alcohol, isopropyl myristate, isopropyl palmitate, isotonic saline, kaolin, lactic acid, lactitol, lactose, lanolin, lanolin alcohols, anhydrous lanolin, lecithin, magnesium aluminum silicate, magnesium carbonate, normal magnesium carbonate, magnesium carbonate anhydrous, magnesium carbonate hydroxide, magnesium hydroxide, magnesium lauryl sulfate, magnesium oxide, magnesium silicate, magnesium stearate, magnesium trisilicate, magnesium trisilicate anhydrous, malic acid, malt, maltitol, mal- titol solution, maltodextrin, maltol, maltose, mannitol, medium chain triglycerides, meglumine, menthol, methylcellulose, methyl methacrylate, methyl oleate, methylparaben, methylparaben potassium, methylparaben sodium, microcrystaNine cellulose and carboxymethylcellulose sodium, mineral oil, light mineral oil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine, montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin, peanut oil, petrolatum, petrolatum and lanolin alcohols, pharmaceutical glaze, phenol, liquified phenol, phenoxyethanol, phenoxypropanol, phenylethyl alcohol, phenyl- mercuric acetate, phenylmercuric borate, phenylmercuric nitrate, polacrilin, po- lacrilin potassium, poloxamer, polydextrose, polyethylene glycol, polyethylene oxide, polyacrylates, polyethylene-polyoxypropylene-block polymers, polyme- thacrylates, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitol fatty acid esters, polyoxyethylene stearates, polyvinyl alcohol, polyvinyl pyrrolidone, potassium alginate, potassium benzo- ate, potassium bicarbonate, potassium bisulfite, potassium chloride, postas- sium citrate, potassium citrate anhydrous, potassium hydrogen phosphate, po- tassium metabisulfite, monobasic potassium phosphate, potassium propionate, potassium sorbate, povidone, propanol, propionic acid, propylene carbonate, propylene glycol, propylene glycol alginate, propyl gallate, propylparaben, propylparaben potassium, propylparaben sodium, protamine sulfate, rapeseed oil, Ringer's solution, saccharin, saccharin ammonium, saccharin calcium, saccharin sodium, safflower oil, saponite, serum proteins, sesame oil, colloidal silica, colloidal silicon dioxide, sodium alginate, sodium ascorbate, sodium benzoate, sodium bicarbonate, sodium bisulfite, sodium chloride, anhydrous sodium citrate, sodium citrate dehydrate, sodium chloride, sodium cyclamate, sodium edentate, sodium dodecyl sulfate, sodium lauryl sulfate, sodium metabisulfite, sodium phosphate, dibasic, sodium phosphate, monobasic, sodium phosphate, tribasic, anhydrous sodium propionate, sodium propionate, sodium sorbate, sodium starch glycolate, sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters (sorbitan fatty esters), sorbitol, sorbitol solution 70%, soybean oil, spermaceti wax, starch, corn starch, potato starch, pregelatinized starch, sterilizable maize starch, stearic acid, purified stearic acid, stearyl alcohol, sucrose, sugars, compressible sugar, confectioner's sugar, sugar spheres, invert sugar, Sugartab, Sunset Yellow FCF, synthetic paraffin, talc, tartaric acid, tar- trazine, tetrafluoroethane (HFC), theobroma oil, thimerosal, titanium dioxide, alpha tocopherol, tocopheryl acetate, alpha tocopheryl acid succinate, beta- tocopherol, delta-tocopherol, gamma-tocopherol, tragacanth, triacetin, tributyl citrate, triethanolamine, triethyl citrate, trimethyl- -cyclodextrin, trimethyltet- radecylammonium bromide, tris buffer, trisodium edentate, vanillin, type I hy- drogenated vegetable oil, water, soft water, hard water, carbon dioxide-free water, pyrogen-free water, water for injection, sterile water for inhalation, sterile water for injection, sterile water for irrigation, waxes, anionic emulsifying wax, carnauba wax, cationic emulsifying wax, cetyl ester wax, microcrystalline wax, nonionic emulsifying wax, suppository wax, white wax, yellow wax, white petrolatum, wool fat, xanthan gum, xylitol, zein, zinc propionate, zinc salts, zinc stearate, or any excipient in the Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1 980), which is incorporated by reference in its entirety, discloses various components used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional agent is incompatible with the pharmaceutical compositions, its use in pharmaceutical compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.

[00181] The pharmaceutical compositions may be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition may be at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, or at least 10.5 up to and including pH 1 1 , depending on the formulation and route of administration. In certain embodiments, the pharmaceutical compositions may comprise buffering agents to achieve a physiological compatible pH. The buffering agents may include any compounds capabale of buffering at the desired pH such as, for example, phosphate buffers (e.g., PBS), triethanola- mine, Tris, bicine, TAPS, tricine, HEPES, TES, MOPS, PIPES, cacodylate, MES, and others. In certain embodiments, the strength of the buffer is at least 0.5 mM, at least 1 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 120 mM, at least 150 mM, or at least 200 mM. In some embodiments, the strength of the buffer is no more than 300 mM (e.g., at most 200 mM, at most 100 mM, at most 90 mM, at most 80 mM, at most 70 mM, at most 60 mM, at most 50 mM, at most 40 mM, at most 30 mM, at most 20 mM, at most 10 mM, at most 5 mM, at most 1 mM).

[00182] In some embodiments, the foregoing component(s) may be present in the pharmaceutical composition at any concentration, such as, for example, at least A, wherein A is 0.0001 % w/v, 0.001 % w/v, 0.01 % w/v, 0.1 % w/v, 1 % w/v, 2% w/v, 5% w/v, 10% w/v, 20% w/v, 30% w/v, 40% w/v, 50% w/v, 60% w/v, 70% w/v, 80% w/v, or 90% w/v. In some embodiments, the foregoing component(s) may be present in the pharmaceutical composition at any concentration, such as, for example, at most B, wherein B is 90% w/v, 80% w/v, 70% w/v, 60% w/v, 50% w/v, 40% w/v, 30% w/v, 20% w/v, 10% w/v, 5% w/v, 2% w/v, 1 % w/v, 0.1 % w/v, 0.001 % w/v, or 0.0001 %. In other embodiments, the foregoing component(s) may be present in the pharmaceutical composition at any concentration range, such as, for example from about A to about B. In some embodiments, A is 0.0001 % and B is 90%.

Salts

[00183] In some embodiments, the binding construct is in the form of a salt, e.g., a pharmaceutically acceptable salt. As used herein the term "pharmaceutically acceptable salt" refers to salts of compounds that retain the biological activity of the parent compound, and which are not biologically or otherwise undesirable. Such salts can be prepared in situ during the final isolation and purification of the binding construct, or separately prepared by reacting a free base function with a suitable acid. Many of the binding constructs disclosed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.

[00184] Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphor sulfonate, diglu- conate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isothionate), lactate, maleate, methane sulfonate, nicotinate, 2-naphthalene sulfonate, oxalate, palmitoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p- toluene-sulfonic acid, salicylic acid, and the like. Examples of acids which can be employed to form pharmaceutically acceptable acid addition salts include, for example, an inorganic acid, e.g., hydrochloric acid, hydrobromic acid, sulphuric acid, and phosphoric acid, and an organic acid, e.g., oxalic acid, maleic acid, succinic acid, and citric acid.

[00185] Basic addition salts also can be prepared in situ during the final isolation and purification of the source of salicylic acid, or by reacting a carboxylic acid-containing moiety with a suitable base such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary, or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like, and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, me- thylammonium, dimethylammonium, trimethylammonium, triethylammonium, diethylammonium, and ethylammonium, amongst others. Other representative organic amines useful for the formation of base addition salts include, for example, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines.

[00186] Further, basic nitrogen-containing groups can be quaternized with the binding construct of the present disclosure as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; long chain halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; arylalkyi halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.

Routes of Administration

[00187] In some embodiments, the pharmaceutical composition comprising the binding constructs described herein is formulated for parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intrathecal administration, or inter- peritoneal administration. In other embodiments, the pharmaceutical composition is administered via nasal, spray, oral, aerosol, rectal, or vaginal admini- stration. The compositions may be administered by infusion, bolus injection or by implantation device.

[00188] The following discussion on routes of administration is merely provided to illustrate exemplary embodiments and should not be construed as limiting the scope in any way.

[00189] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the composition of the present disclosure dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, manni- tol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipi- ents, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and other pharmacologically compatible excipients. Lozenge forms can comprise the composition of the present disclosure in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the composition of the present disclosure in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to, such excipients as are known in the art.

[00190] The compositions of the disclosure, alone or in combination with other suitable components, can be delivered via pulmonary administration and can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propel- lants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations also may be used to spray mucosa. In some embodiments, the composition is formulated into a powder blend or into microparticles or nanoparticles. Suitable pulmonary formulations are known in the art. See, e.g., Qian et al., Int J Pharm 366: 218-220 (2009); Adjei and Garren, Pharmaceutical Research, 7(6): 565-569 (1990); Kawashima et al., J Controlled Release 62(1 -2): 279-287 (1999); Liu et al., Pharm Res 10(2): 228-232 (1993); International Patent Application Publication Nos. WO 2007/133747 and WO 2007/14141 1 .

[00191] Topical formulations are well-known to those of skill in the art. Such formulations are particularly suitable in the context of the invention for application to the skin.

[00192] In some embodiments, the pharmaceutical composition described herein is formulated for parenteral administration. For purposes herein, parenteral administration includes, but is not limited to, intravenous, intraarterial, intramuscular, intracerebral, intracerebroventricular, intracardiac, subcutaneous, intraosseous, intradermal, intrathecal, intraperitoneal, retrobulbar, in- trapulmonary, intravesical, and intracavernosal injections or infusions. Administration by surgical implantation at a particular site is contemplated as well.

[00193] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The term, "parenteral" means not through the alimentary canal but by some other route such as subcutaneous, intramuscular, intraspinal, or intravenous. The composition of the present disclosure can be administered with a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such as propylene glycol or polyeth- ylene glycol, dimethylsulfoxide, glycerol, ketals such as 2,2- dimethyl-153- dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hy- droxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[00194] Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral . Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[00195] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium hal- ides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers , (d ) am photeric detergents such as, for exam ple , al kyl-β- aminopropionates, and 2-alkyl -imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[00196] The parenteral formulations in some embodiments contain preservatives or buffers. In order to minimize or eliminate irritation at the site of injection, such compositions optionally contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[00197] Injectable formulations are in accordance with the invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

[00198] Additionally, the composition of the present disclosures can be made into suppositories for rectal administration by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

[00199] It will be appreciated by one of skill in the art that, in addition to the above- described pharmaceutical compositions, the composition of the disclosure can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.

[00200] The dose of the pharmaceutical composition also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular pharmaceutical composition. Typically, the attending physician will decide the dosage of the pharmaceutical composition with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, binding constructs of the pharmaceutical composition to be administered, route of administration, and the severity of the condition being treated. [00201] For purposes herein, the amount or dose of the pharmaceutical composition administered is sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject or animal over a reasonable time frame. For example, the dose of the pharmaceutical composition is sufficient to treat or prevent a disease or medical condition in a period of from about 12 hours, about 18 hours, about 1 to 4 days or longer, e.g., 5 days, 6 days, 1 week, 10 days, 2 weeks, 16 to 20 days, or more, from the time of administration. In certain embodiments, the time period is even longer. The dose is determined by the efficacy of the particular pharmaceutical composition and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.

[00202] Many assays for determining an administered dose are known in the art. In some embodiments, an assay which comprises comparing the extent to which the binding constructs block VEGFR-3-mediated cell growth upon administration of a given dose to a mammal among a set of mammals each of which is given a different dose of binding constructs is used to determine a starting dose to be administered to a mammal. The extent to which the binding constructs block VEGFR-3-mediated cell growth upon administration of a certain dose can be assayed by methods known in the art.

Dose

[00203] By way of example and not intending to limit the invention, the dose of the binding construct of the present disclosure can be about 0.0001 to about 1 g/kg body weight of the subject being treated/day, from about 0.0001 to about 0.001 g/kg body weight/day, or about 0.01 mg to about 1 g/kg body weight/day. The pharmaceutical composition in some aspects comprise the binding construct of the present disclosure at a concentration of at least A, wherein A is about about 0.001 mg/ml, about 0.01 mg/ml, about 0.1 mg/ml, about 0.5 mg/ml , about 1 mg/ml , about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 1 0 mg/ml, about 1 1 mg/ml, about 12 mg/ml, about 13 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 17 mg/ml, about 18 mg/ml, about 19 mg/ml, about 20 mg/ml, about 21 mg/ml, about 22 mg/ml, about 23 mg/ml, about 24 mg/ml, about 25 mg/ml or higher. In some embodiments, the pharmaceutical composition comprises the binding construct at a concentration of at most B, wherein B is about 30 mg/ml, about 25 mg/ml, about 24 mg/ml, about 23, mg/ml, about 22 mg/ml, about 21 mg/ml, about 20 mg/ml, about 1 9 mg/ml, about 18 mg/ml, about 17 mg/ml, about 16 mg/ml, about 15 mg/ml, about 14 mg/ml, about 13 mg/ml, about 12 mg/ml, about 1 1 mg/ml, about 10 mg/ml, about 9 mg/ml, about 8 mg/ml, about 7 mg/ml, about 6 mg/ml, about 5 mg/ml, about 4 mg/ml, about 3 mg/ml, about 2 mg/ml, about 1 mg/ml, or about 0.1 mg/ml. In some embodiments, the compositions may contain an analog at a concentration range of A to B mg/ml, for example, about 0.001 to about 30.0 mg/ml.

[00204] Additional dosing guidance can be guaged from other antibody therapeutics, such as bevacizumab (Avastin™ Genentech); Cetuximab (Exbi- tux™ Imclone), Panitumumab (Vectibix™ Amgen), and Trastuzumab (Her- ceptin™ Genetech).

Timing of Administration

[00205] The disclosed pharmaceutical formulations may be administered according to any regimen including, for example, daily (1 time per day, 2 times per day, 3 times per day, 4 times per day, 5 times per day, 6 times per day), every two days, every three days, every four days, every five days, every six days, weekly, bi-weekly, every three weeks, monthly, or bi-monthly. Timing, like dosing can be fine-tuned based on dose-response studies, efficacy, and toxicity data, and initially guaged based on timing used for other antibody therapeutics.

Controlled Release Formulations

[00206] The pharmaceutical composition is in certain aspects modified into a depot form, such that the manner in which the active ingredients of the pharmaceutical composition (e.g. the binding constructs) is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450,150). Depot forms in various aspects, include, for example, an implantable composition comprising a porous or non-porous material, such as a polymer, wherein the binding constructs are encapsulated by or diffused throughout the material and/or degradation of the non-porous material. The depot is then implanted into the desired location within the body and the binding constructs are released from the implant at a predetermined rate.

[00207] Accordingly, the pharmaceutical composition in certain aspects is modified to have any type of in vivo release profile. In some aspects, the pharmaceutical composition is an immediate release, controlled release, sustained release, extended release, delayed release, or bi-phasic release formulation. Methods of formulating peptides (e.g., peptide binding constructs) for controlled release are known in the art. See, for example, Qian et al., J Pharm 374: 46-52 (2009) and International Patent Application Publication Nos. WO 2008/1 30158, WO2004/033036; WO2000/032218; and WO 1 999/040942. Suitable examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481 ), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman, et al., Biopolymers, 22: 547-556 (1983)), poly (2- hydroxyethyl-methacrylate) (Langer, et al., J. Biomed. Mater. Res., 15:167-277 (1981 ) and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer, et al, supra) or poly-D(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release compositions also may include liposomes, which can be prepared by any of several methods known in the art (e.g., DE 3,218,121 ; Epstein, et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); Hwang, et al., Proc. Natl . Acad . Sci . USA, 77:4030-4034 (1 980); EP 52,322; EP 36,676; EP 88,046; EP 143,949).

Combinations

[00208] The compositions comprising a first and second binding construct of the present disclosures may be employed alone, or in combination with other agents. In some embodiments, more than one type of first binding construct or type of second construct are administered. For example, the administered composition , e.g . , pharmaceutical composition , may comprise antibody 2E1 1 D1 1 , an antibody which binds to the ligand binding domain of VEGFR-3, and a VEGF-C-binding portion of VEGFR-3, or an aptamer. In some embodiments, one or both of the first and second binding constructs are administered together with a therapeutic agent or diagnostic agent, including any of those described herein. Certain diseases, e.g., cancers, or patients may lend themselves to a treatment of combined binding construct and chemotherapeutic agent to achieve an additive or even a synergistic effect compared to the use of any one therapy alone.

Uses

[00209] The compositions, e.g., pharmaceutical compositions, comprising a first binding construct and second binding construct, each of which bind to a distinct epitope of an RTK and reduce ligand-induced activation of the RTK, may be employed in a number of applications.

Targeting Applications

[00210] As many of the RTKs known to date are associated with or implicated in one or more diseases (including, medical conditions, syndromes, and the like), the binding constructs of the present disclosures are useful as targeting moieties for the specific delivery or localization of a therapeutic agent or diagnostic agent to a cell expressing the RTK. In such embodiments, the binding construct of the composition is not the pharmaceutically active ingredient. Rather, the therapeutic agent or diagnostic agent which is attached to the binding construct is the active ingredient of the composition. The therapeutic or diagnostic agent delivered via the compositions of the present disclosures may be any of those described herein or known in the art. In some aspects, the therapeutic agent is one which is known as a therapeutic for a disease in which RTK-expressing cells are commonly targeted. [00211] Accordingly, provided herein are methods of treating or preventing or diagnosing a disease, comprising administering to a patient in need thereof a composition, e.g., a pharmaceutical composition, comprising a first binding construct which binds to a first epitope of an RTK and a second construct which binds to a second epitope of the RTK, wherein the second epitope is different from the first epitope, wherein each of the first binding construct and second binding construct reduce ligand-induced activiation of the RTK. In some embodiments, at least one of the first binding construct and second binding construct is (directly or indirectly via a linker) attached or conjugated or linked to a therapeutic or diagnostic agent. The attachment, conjugation, or linkage may be via covalent or non-covalent bonds, or via a combination of both types of bonds.

[00212] In some aspects, the therapeutic and diagnostic application of these disclosures can occur in vitro and/or ex vivo. In such embodiments, the method comprises contacting a cell expressing the RTK to which the first binding construct and second binding construct binds.

[00213] The amount of the composition contacted or administered is one which is effective to treat or prevent or diagnose the disease. Doses and administration schedules or regimens may be determined in accordance with the teachings herein.

[00214] For purposes of the present disclosures, the term "treat" and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment {e.g., cure) or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the methods of the present disclosures can provide any amount or any level of treatment or prevention of a disease in a patient, e.g., a human. Furthermore, the treatment or prevention provided by the method disclosed herein can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented . Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.

[00215] For purposes herein, the term "diagnose" and related terms refers to the determination or analysis of a patient for the existence of a disease. The binding constructs of the present disclosures also are useful to detect increases or decreases in VEGFR-3 proteins in tissue samples including samples for sites of a suspected diseased state.

Diseases

[00216] With regard to the methods disclosed herein, the disease is one of any number of diseases or medical conditions or syndromes currently known in the art, including, those described in the "International Classification of Diseases -10-Clinical Modification (ICD-10-CM) Official Guidelines for Coding and Report 2009" published by the World Health Organization, which is incorporated by reference into the present discslosures in its entirety. Accordingly, in some embodiments, the disease is any of the following: an infectious or parasitic disease, an inflammatory disease, an autoimmune disease, a hyperprolif- erative disease (e.g., a neoplasm, a tumor, a cancer), a neurodegenerative disease, a disease of the blood and blood-forming organs, an endocrine, nutritional, or metabolic disease, a mental or behavioural disease, a disease of the nervous system, sense organs, eye, adnexa, ear, mastoid process, circulatory system, respiratory system, digestive system, skin, subcutaneous tissue, musculoskeletal system, connective tissue, hypertension, diabetes, atherosclerosis, and the like. In some aspects, the disease is one selected from the group consisting of: rheumatoid arthritis, edemas (and other types of plasma leakage), cancer associated disorders such as cancer-associated ascites formation, diabetes, and inflammatory diseases such as psoriasis. In particular aspects, the disease is any disease associated with abnormally high levels of growth factor expression. Neoplasms

[00217] The materials and methods described herein are especially useful for inhibiting neoplastic cell growth or spread; particularly neoplastic cell growth for which the RTK targeted by the binding agents plays a role. For example, VEGFR-3 plays a role in neoplastic cell growth for some neoplastic cells that express VEGFR-3. VEGFR-3 also plays a role in neoplastic cell growth for a large variety of neoplasms that have blood vessels that express VEGFR-3. VEGFR-3 also plays a role in neoplastic cell spread through lymphatic vessels that express VEGFR-3. The presence and putative involvement of VEGFR-3 or another RTK in a particular neoplasm can be confirmed by imaging (in vivo or biopsy specimens) with imaging agents that recognize the RTK and/or imaging agents that recognize the growth factor ligand(s) that bind to and signal through the RTK. Because of the frequency with which cancers metastasize, the therapeutic agents of the invention may have prophylactic benefit even in the absence of direct evidence of pathogenic VEGFR-3 expression.

[00218] Neoplasms treatable by the composition of the present disclosures include solid tumors, for example, carcinomas and sarcomas. Carcinomas include malignant neoplasms derived from epithelial cells which infiltrate, for example, invade, surrounding tissues and give rise to metastases. Adenocarcinomas are carcinomas derived from glandular tissue, or from tissues that form recognizable glandular structures. Another broad category of cancers includes sarcomas and fibrosarcomas, which are tumors whose cells are embedded in a fibrillar or homogeneous substance, such as embryonic connective tissue. The invention also provides methods of treatment of cancers of myeloid or lymphoid systems, including leukemias, lymphomas, and other cancers that typically are not present as a tumor mass, but are distributed in the vascular or lymphoreticular systems. Further contemplated are methods for treatment of adult and pediatric oncology, growth of solid tumors/malignancies, myxoid and round cell carcinoma, locally advanced tumors, cancer metastases, including lymphatic metastases. The cancers listed herein are not intended to be limiting . Both age (child and adult), sex (male and female), primary and secon- dary, pre- and post-metastatic, acute and chronic, benign and malignant, anatomical location cancer embodiments and variations are contemplated targets. Cancers are grouped by embryonic origin (e.g., carcinoma, lymphomas, and sarcomas), by organ or physiological system, and by miscellaneous grouping. Particular cancers may overlap in their classification, and their listing in one group does not exclude them from another.

[00219] Carcinomas that may be targeted include adrenocortical, acinar, acinic cell, acinous, adenocystic, adenoid cystic, adenoid squamous cell, cancer adenomatosum, adenosquamous, adnexel, cancer of adrenal cortex, adrenocortical, aldosterone-producing, aldosterone-secreting, alveolar, alveolar cell, ameloblastic, ampullary, anaplastic cancer of thyroid gland, apocrine, basal cell, basal cell, alveolar, comedo basal cell, cystic basal cell, morphea- like basal cell, multicentric basal cell, nodulo-ulcerative basal cell, pigmented basal cell, sclerosing basal cell, superficial basal cell, basaloid, basosquamous cell, bile duct, extrahepatic bile duct, intrahepatic bile duct, bronchioalveolar, bronchiolar, bronchioloalveolar, bronchioalveolar, bronchioalveolar cell, bronchogenic, cerebriform, cholangiocelluarl, chorionic, choroids plexus, clear cell, cloacogenic anal, colloid, comedo, corpus, cancer of corpus uteri, cortisol- producing, cribriform, cylindrical, cylindrical cell, duct, ductal, ductal cancer of the prostate, ductal cancer in situ (DCIS), eccrine, embryonal, cancer en cui- rasse, endometrial, cancer of endometrium, endometroid, epidermoid, cancer ex mixed tumor, cancer ex pleomorphic adenoma, exophytic, fibrolamellar, cancer fibrosum, follicular cancer of thyroid gland, gastric, gelatinform, gelatinous, giant cell, giant cell cancer of thyroid gland, cancer gigantocellulare, glandular, granulose cell, hepatocellular, Hurthle cell, hypernephroid, infantile embryonal, islet cell carcinoma, inflammatory cancer of the breast, cancer in situ, intraductal, intraepidermal, intraepithelial, juvenile embryonal, Kulchitsky- cell, large cell, leptomeningeal, lobular, infiltrating lobular, invasive lobular, lobular cancer in situ (LCIS), lymphoepithelial, cancer medullare, medullary, medullary cancer of thyroid gland, medullary thyroid, melanotic, meningeal, Merkel cell, metatypical cell, micropapillary, cancer mol'le, mucinous, cancer muci'parum, cancer mucocellula're, mucoepidermoid, cancer muco'sum, mu- cous, nasopharyngeal, neuroendocrine cancer of the skin, noninfiltrating, non- small cell, non-small cell lung cancer (NSCLC), oat cell, cancer ossificans, osteoid, Paget's, papillary, papillary cancer of thyroid gland, periampullary, preinvasive, prickle cell, primary intrasseous, renal cell, scar, schistosomal bladder, Schneiderian, scirrhous, sebaceous, signet-ring cell, cancer sim'plex, small cel l , smal l cel l l ung cancer (SCLC), spindle cell , cancer spong io'sum , squamous, squamous cell, terminal duct, anaplastic thyroid, follicular thyroid, medullary thyroid, papillary thyroid, trabecular cancer of the skin, transitional cell, tubular, undifferentiated cancer of thyroid gland, uterine corpus, verrucous, villous, cancer villo'sum, yolk sac, squamous cell particularly of the head and neck, esophageal squamous cell, and oral cancers and carcinomas.

[00220] Sarcomas that may be targeted include adipose, alveolar soft part, ameloblastic, avian, botryoid, sarcoma botryoi'des, chicken, chloromatous, chondroblastic, clear cell sarcoma of kidney, embryonal, endometrial stromal, epithelioid, Ewing's, fascial, fibroblastic, fowl, giant cell, granulocytic, heman- gioendothelial, Hodgkin's, idiopathic multiple pigmented hemorrhagic, immunoblastic sarcoma of B cells, immunoblastic sarcoma of T cells, Jensen's, Kaposi's, kupffer cell, leukocytic, lymphatic, melanotic, mixed cell, multiple, lymphangio, idiopathic hemorrhagic, multipotential primary sarcoma of bone, osteoblastic, osteogenic, parosteal, polymorphous, pseudo-kaposi, reticulum cell, reticulum cell sarcoma of the brain, rhabdomyosarcoma, rous, soft tissue, spindle cell, synovial, telangiectatic, sarcoma (osteosarcoma)/malignant fibrous histiocytoma of bone, and soft tissue sarcomas.

[00221] Lymphomas that may targeted include AIDS-related, non-Hodgkin's, Hodgkin's, T-cell, T-cell leukemia/lymphoma, African, B-cell, B-cell monocy- toid, bovine malignant, Burkitt's, centrocytic, lymphoma cu'tis, diffuse, diffuse, large cell, diffuse, mixed small and large cell, diffuse, small cleaved cell, follicular, follicular center cell, follicular, mixed small cleaved and large cell, follicular, predominantly large cell, follicular, predominantly small cleaved cell, giant follicle, giant follicular, granulomatous, histiocytic, large cell, immunoblastic, large cleaved cell, large nocleaved cell, Lennert's, lymphoblastic, lymphocytic, in- termediate; lymphocytic, intermediately differentiated, plasmacytoid; poorly differentiated lymphocytic, small lymphocytic, well differentiated lymphocytic, lymphoma of cattle; MALT, mantle cell, mantle zone, marginal zone, Mediterranean lymphoma mixed lymphocytic-histiocytic, nodular, plasmacytoid, pleomorphic, primary central nervous system, primary effusion, small b-cell, small cleaved cell, small concleaved cell, T-cell lymphomas; convoluted T-cell, cutaneous t-cell, small lymphocytic T-cell, undefined lymphoma, u-cell, undifferentiated, aids-related, central nervous system, cutaneous T-cell, effusion (body cavity based), thymic lymphoma, and cutaneous T cell lymphomas.

[00222] Leukemias and other blood cell malignancies that may be targeted include acute lymphoblastic, acute myeloid, lymphocytic, chronic myelogenous, hairy cell, lymphoblastic, myeloid, lymphocytic, myelogenous, leukemia, hairy cell, T-cell, monocytic, myeloblastic, granulocytic, gross, hand mirror-cell, basophilic, hemoblastic, histiocytic, leukopenic, lymphatic, Schilling's, stem cell, myelomonocyic, prolymphocytic, micromyeloblastic, megakaryoblastic, megakaryoctyic, rieder cell, bovine, aleukemic, mast cell, myelocytic, plamsa cell, subleukemic, multiple myeloma, nonlymphocytic, and chronic myelocytic leukemias.

[00223] Brain and central nervous system (CNS) cancers and tumors that may be targeted include astrocytomas (including cerebellar and cerebral), brain stem glioma, brain tumors, malignant gliomas, ependymoma, glioblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas, primary central nervous system lymphoma, ependymoma, brain stem glioma, visual pathway and hypothalamic glioma, extracranial germ cell tumor, medulloblastoma, myelodysplastic syndromes, oligodendroglioma, myelodysplastic/myeloproliferative diseases, myelogenous leukemia, myeloid leukemia, multiple myeloma, myeloproliferative disorders, neuroblastoma, plasma cell neoplasm/multiple myeloma, central nervous system lymphoma, intrinsic brain tumors, astrocytic brain tumors, gliomas, and metastatic tumor cell invasion in the central nervous system. [00224] Gastrointestimal cancers that may be targeted include extrahepatic bile duct cancer, colon cancer, colon and rectum cancer, colorectal cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastronintestinal carcinoid tumors, gastrointestinal stromal tumors, bladder cancers, islet cell carcinoma (endocrine pancreas), pancreatic cancer, islet cell pancreatic cancer, prostate cancer rectal cancer, salivary gland cancer, small intestine cancer, colon cancer, and polyps associated with colorectal neoplasia.

[00225] Bone cancers that may be targeted include osteosarcoma and malignant fibrous histiocytomas, bone marrow cancers, bone metastases, os- teosarcoma/malignant fibrous histiocytoma of bone, and osteomas and osteosarcomas. Breast cancers that may be targeted include small cell carcinoma and ductal carcinoma.

[00226] Lung and respiratory cancers that may be targeted include bronchial adenomas/carcinoids, esophagus cancer esophageal cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer, lung carcinoid tumor, non-small cell lung cancer, small cell lung cancer, small cell carcinoma of the lungs, mesothelioma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nasopharyngeal cancer, oral cancer, oral cavity and l ip cancer, oropharyngeal cancer; paranasal sinus and nasal cavity cancer, and pleuropulmonary blastoma.

[00227] Urinary tract and reproductive cancers that may be targeted include cervical cancer, endometrial cancer, ovarian epithelial cancer, extragonadal germ cell tumor, extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor, spleen, kidney cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, penile cancer, renal cell cancer (including carcinomas), renal cell cancer, renal pelvis and ureter (transitional cell cancer), transitional cell cancer of the renal pelvis, and ureter, gestational trophoblastic tumor, testicular cancer, ureter and renal pelvis, transitional cell cancer, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vul- var cancer, ovarian carcinoma, primary peritoneal epithelial neoplasms, cervical carcinoma, uterine cancer and solid tumors in the ovarian follicle), superficial bladder tumors, invasive transitional cell carcinoma of the bladder, and muscle-invasive bladder cancer.

[00228] Skin cancers and melanomas (as well as non-melanomas) that may be targeted include cutaneous t-cell lymphoma, intraocular melanoma, tumor progression of human skin keratinocytes, basal cell carcinoma, and squamous cell cancer. Liver cancers that may be targeted include extrahepatic bile duct cancer, and hepatocellular cancers. Eye cancers that may be targeted include intraocular melanoma, retinoblastoma, and intraocular melanoma Hormonal cancers that may be targeted include: parathyroid cancer, pineal and supraten- torial primitive neuroectodermal tumors, pituitary tumor, thymoma and thymic carcinoma, thymoma, thymus cancer, thyroid cancer, cancer of the adrenal cortex, and ACTH-producing tumors.

[00229] Miscellaneous other cancers that may be targeted include advanced cancers, AIDS-related, anal cancer adrenal cortical, aplastic anemia, aniline, betel, buyo cheek, cerebriform, chimney-sweeps, clay pipe, colloid, contact, cystic, dendritic, cancer a deux, duct, dye workers, encephaloid, cancer en cui- rasse, endometrial, endothelial, epithelial, glandular, cancer in situ, kang, kan- gri, latent, medullary, melanotic, mule-spinners', non-small cell lung, occult cancer, paraffin, pitch workers', scar, schistosomal bladder, scirrhous, lymph node, small cell lung, soft, soot, spindle cell, swamp, tar, and tubular cancers.

[00230] Miscellaneous other cancers that may be targeted also include carcinoid (gastrointestinal and bronchal) Castleman's disease chronic myeloproliferative disorders, clear cell sarcoma of tendon sheaths, Ewing's family of tumors, head and neck cancer, l ip and oral cavity cancer, Waldenstrom's macroglobulinemia, metastatic squamous neck cancer with occult primary, multiple endocrine neoplasia syndrome, multiple myeloma/plasma cell neoplasm, Wilms' tumor, mycosis fungoides, pheochromocytoma, sezary syndrome, supratentorial primitive neuroectodermal tumors, unknown primary site, peritoneal effusion, malignant pleural effusion, trophoblastic neo-plasms, and hemangiopericytoma.

Inhibiting VEGFR-3-mediated cellular activities

[00231] In some variations of the invention, the RTK to be targeted by the materials or methods is VEGFR-3. The many biological activities mediated through VEGFR-3 receptor (including but not limited to affecting growth and migration of vascular endothelial cells and blood vessels; promoting growth of lymphatic endothelial cells and lymphatic vessels; increasing vascular permeability; and affecting myelopoiesis) support numerous in vitro and in vivo utilities for the compositions of the invention. For example, the methods of the present disclosures may be a method of inhibiting any one or more of these activities in a cell or in a patient.

[00232] VEGF-C stimulates endothelial cell migration in collagen gel. Thus, inhibitors for use in the invention may be examined to confirm that the inhibitor can reduce or eliminate VEGF-C mediated endothelial cell migration in collagen gel. Exemplary cell migration assays have been described in International Patent Publication No. WO 98/33917, incorporated herein by reference. Briefly, the lymphatic endothelial cells isolated in the invention are seeded on top of a collagen layer in tissue culture plates. VEGF-C is placed in wells made in collagen gel approximately 4 mm away from the location of the attached lymphatic endothelial cells. The number of endothelial cells that have migrated from the original area of attachment in the collagen gel towards the wells containing the VEGF-C is then counted to assess VEGF-C induced cell migration.

[00233] Collagen gels for these assays are prepared by mixing type I collagen stock sol ution (5 mg/m l i n 1 m M HCI ) with a n eq u al vol u m e of 2.times.MEM and 2 volumes of MEM containing 10% newborn calf serum to give a final collagen concentration of 1 .25 mg/ml. Tissue culture plates (5 cm diameter) are coated with about 1 mm thick layer of the solution, which is allowed to polymerize at 37° C. The lymphatic endothelial cells of the invention are seeded atop this layer. [00234] For the migration assays, the cells are allowed to attach inside a plastic ring (1 cm diameter) placed on top of the first collagen layer. After 30 minutes, the ring is removed and unattached cells are rinsed away. A second layer of collagen and a layer of growth medium (5% newborn calf serum (NCS)), solidified by 0.75% low melting point agar (FMC BioProducts, Rockland, Me.), are added. A well (3 mm diameter) is punched through all the layers on both sides of the cell spot at a distance of 4 mm, and media containing a VEGF-C polypeptide is pipetted daily into the wells. Photomicrographs of the cells migrating out from the spot edge are taken, e.g., after six days, through an Olympus CK 2 inverted microscope equipped with phase-contrast optics. The migrating cells are counted after nuclear staining with the fluorescent dye bisbenzimide (1 mg/ml, Hoechst 33258, Sigma).

[00235] The number of cells migrating at different distances from the original area of attachment towards wells containing the VEGF-C, are determined 6 days after addition of the media. The number of cells that migrate out from the original ring of attachment are counted in five adjacent 0.5 mm x 0.5 mm squares using a microscope ocular lens grid and 10x magnification with a fluorescence microscope. Cells migrating further than 0.5 mm are counted in a similar way by moving the grid in 0.5 mm steps.

[00236] Additionally, the mitogenic activity of VEGF-C and inhibitory effect of binding constructs can be examined using endothelial cell proliferation assays such as that described in Breier et al., Dev 1 14:521 -532 (1992), incorporated herein in its entirety. The cells may be assayed for this effect by adding the VEGF-C to the cells. After three days, the cells are dissociated with trypsin and counted using a cytometer to determine any effects of the peptides on the proliferative activity of the lymphatic endothelial cells.

Patient types

[00237] With regard to the present disclosures, the patient may be any living organism. In some embodiments, the patient is a mammal. As used herein, the term "mammal" refers to any vertebrate animal of the mammalia class, including, but not limited to, any of the monotreme, marsupial, and placental taxas. In some embodiments, the mammal is one of the mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomor- pha, such as rabbits. In certain embodiments, the mammals are from the order Carnivora, including felines (cats) and canines (dogs). In certain embodiments, the mammals are from the order Artiodactyla, including bovines (cows) and swines (pigs) or of the order Perssodactyla, including equines (horses). In some instances, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In particular embodiments, the mammal is a human.

Research and Other Uses

[00238] In addition to the foregoing uses, the compositions of the present disclosures are useful in standard immunochemical procedures (e.g., ELISA, Western blotting, R IA) a n d immunohistochemical procedures (e.g., im- munofluoresence, in situ hybridization, e.g., FISH, tissue staining, FACS), and in other procedures which utilize antibodies specific to VEGFR-3. The binding constructs of the present disclosures are also useful in immunolocalization studies to analyze the distribution of VEGFR-3 during various cellular events, for example, to determine the cellular or tissue-specific distribution of VEGFR- 3 polypeptides under different points in the cell cycle. A particularly useful appl ication of such binding constructs is in purifying native or recombinant VEGFR-3, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.

Kits and Unit Doses

[00239] In some embodiments of the present disclosures, the composition comprising a first binding construct and a second binding construct is packaged in a kit or package or unit dose to, e.g., permit co-administration. Accordingly, provided herein are kits comprising a first binding construct packaged with a second binding construct and optionally also packaged with instructions for use together in a combination therapy. In some aspects, the first binding construct and second binding construct are mixed together e.g., in the same vial. In some aspects, therefore, the two binding constructs are admixed for simultaneous administration to a patient or contacting of cells. In other aspects, the first binding construct is separated from the second binding construct, e.g., the two components are not in admixture, such that their administration to cells or patients can occur simultaneously or separately. In some embodiments, the components to the kit/unit dose are packaged with instructions for administration to a patient, e.g., for treatment of one of the disorders and diseases described herein. In some embodiments, the kit comprises one or more devices for administration to a patient, e.g., a needle and syringe, a dropper, a measuring spoon or cup or like device, an inhaler, and the like. In some aspects, the kit further comprises other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including any of those described herein.

[00240] "Unit dose" is a discrete amount of a therapeutic composition dispersed in a suitable carrier. For example, where polypeptides are being administered parenterally, the polypeptide compositions are generally injected in doses ranging from 1 pg/kg to 100 mg/kg body weight/day, in some embodiments, at doses ranging from 0.1 mg/kg to about 50 mg/kg body weight/day. Parenteral administration in some aspects is carried out with an initial bolus followed by continuous infusion to maintain therapeutic circulating levels of drug product. Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient.

Synergy/Synergism

[00241] Here, two receptor binding constructs are used together in materials or methods to improve efficacy compared to either compound alone, preferably so as to achieve a synergistic effect as judged by one or more of a number of criteria. This synergism may, for example, manifest itself in lower effective doses of one or both inhibitors, which will reduce costs and/or reduce adverse side-effects and toxicity. Achieving therapeutic efficacy (such as inhibition of cell growth or cell migration) for a longer duration also represents synergism. For example, in some embodiments, a combination therapy may be administered for a given time period and then suspended; and cell growth or other indicia of efficacy may be suppressed even after suspension for a greater duration than if only a single compound were employed. In some embodiments, a greater therapeutic window (range between lowest effective dose and toxic dose) is contemplated. In some embodiments, equivalent or better (e.g, synergistic) inhibition is achieved compared to the use of any one compound alone, and with fewer side effects than when a single compound is employed.

[00242] Alternatively, amounts of each inhibitor are considered synergistic by satisfying the following formula:

EeD(InhibitorV) EeD(Inhibitor2) wherein D(lnhibitor1 ) is the dose of the first inhibitor that is administered; and D(lnhibitor2) is the dose of the second inhibitor administered to achieve a particular degree of therapeutic efficacy (e.g., inhibition or prevention of neovascularization or tumor growth); wherein EeD(lnhibitor1 ) is an equi- effective dose of the first inhibitor and EeD(lnhibitor2) is an equi-effective dose of the second inhibitor; wherein the equi-effective dose of first inhibitor and the equi-effective dose of the second inhibitor result in the same quantity of inhibition or prevention of neointimal hyperplasia. See e.g., Berenbaum, "Synergy, Additivism, and Antagonism in Immunosuppression," Clin. Exp. Immunol. 28:1 - 18 (1977); and Berenbaum, J. Antimicrob. Chemother. 19(2):271 -273 (1987); each incorporated herein by reference in its entirety. Such quantity may manifest itself, for example, as fewer neoplastic nuclei, slower neoplastic cell growth, or longer periods of neoplastic growth suppression.

[00243] The following examples are given merely to illustrate the present invention and not in any way to limit its scope.

EXAMPLES [00244] The following materials and methods were used in the studies described in the examples presented herein:

Cell Culture

[00245] Human dermal microvascular endothelial cells (HDMEC) were purchased from Promocell and cultured in endothelial cell medium MV (Promocell, Heidelberg, Germany) according to the manufacturer's instructions. These cells were used between passages 2-7. 293T cells (ATCC) were cultured in DMEM supplemented with 10 % fetal calf serum. Porcine aortic endothelial cells expressing VEGFR-2 were a kind gift from Dr. Lena Claesson-Welsh (University of Uppsala) (Waltenberger J ., J. Biol . Chem. 269, 26988-26995 (1994). The BaF3-VEGFR-3 cell line represents the genetically modified derivative of the murine pro-B cell line BaF3, which stably expresses a chimeric receptor containing the extracellular domain of human VEGFR-3 and the transmembrane and cytoplasmic domains of the mouse erythropoietin receptor. These cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal bovine serum (FBS). For maintenance, the cell cultures were supplemented with 2 ng/mL murine IL-3 (Calbiochem, Gibbs- town, NJ, USA) and 250 g/ml Zeocin (Invitrogen, Karlsruhe, Germany). In the absence of IL-3, BaF3-VEGFR-3 cells grow only in presence of VEGF-C or VEGF-D.

Antibodies

[00246] The following primary antibodies were used in this study: mouse monoclonal against human VEGFR-3: 2E1 1 D1 1 (also called 2E1 1 in the text), 9D9F9 (9D9), rat monoclonal against mouse VEGFR-3: AFL4 (Kubo, H, Involvement of vascular endothelial growth factor receptor-3 in maintenance of integrity of endothelial cell lining during tumor angiogenesis. Blood 96, 546-553 (2000). Antibody 9D9 is available from Chemicon (Millipore, MAB3757) and ReliaTech (101 -M36). The antibody used to block ligand binding to the receptor was 3C5, described in: Persaud K., Involvement of the VEGF receptor 3 in tubular morphogenesis demonstrated with a human anti-human VEGFR-3 monoclonal antibody that antagonizes receptor activation by VEGF-C. J. Cell Sci. 117(Pt 13), 2745-2756 (2004).

FACS analysis

[00247] Cells were detached with the narrow-spectrum proteinase Accutase (PHA, Pasching, Austria) to avoid degradation of VEGFR-3 and incubated at + 4 degrees C with the 2E1 1 or 9D9 antibodies (5 pg/ml) for 30 min. After washing for three times with cold phosphate buffered saline (PBS), the cells were incubated with anti-mouse Alexa488 antibody (Molecular Probes, 1 :400) at + 4 degrees C, washed again three times in cold PBS, fixed in paraformaldehyde for 30 min at room temperature and subjected to FACS analysis in a Becton Dickinson LSR flow cytometer.

Western blotting and immunoprecipitations

[00248] For immunoprecipitation and western blotting, the cells were lysed in 1 ml PLC lysis buffer (PLCLB: 150 mM NaCI, 5 % glycerol, 1 % Triton X-100, 1 .5 M MgCl2, 50 mM Hepes, pH 7.5) supplemented with 1 mM vanadate, 2 mM phenylmethylsulphonyl fluoride (PMSF), 2 g/ml leupeptin and 0.07 U/ml aprotinin. Cleared lysates were incubated with 2 g primary antibody for 2 h. Subseq uently, the immunocomplexes were captured using protein G- sepharose. After three washing steps in the PLCLB buffer, the proteins were separated in 7.5 % polyacrylamide gels under reducing or non-reducing conditions. After blotting of the proteins to nitrocellulose membranes and blocking of the membranes in 5 % BSA, the filters were probed with the monoclonal antibodies (0.5 Mg/ml). After incubation with the second-step HRP-coupled antibodies (Dako, Glostrup, Denmark) the signal was visualized by chemilumines- cence (Pierce, Rockford, IL, USA).

Epitope mapping

[00249] The extracellular domains of mouse and human VEGFR-3 were synthesized on a membrane PVDF filter as peptide spots of 20 amino acid residues in length, with a frame shift of three amino acids. Alanine scanning analysis was done for the following peptides (comprising the binding region of 9D9) : E H LRWYRLN LSTLH DAH G N P (SEQ ID NO: 46), RLNLSTLHDAHGNPLLLDCK (SEQ ID NO: 47), LDHAHGNPLLLDCKNVHLFA (SEQ ID NO: 48). For deletion analysis, these peptides were reduced by one amino acid residues from the N- or C-terminus down to the length of six residues. All peptides were immobilized as spots on cellulose membranes and stored at -20° C. After thawing, the membranes were equilibrated in methanol for 10 min at room temperature. The membrane was washed three times in Tris-buffered saline (TBS, pH 8.0) for 10 min and then incubated overnight in blocking buffer (TBS containing 0.05 % Tween-20, 5 % non-fat dry milk and 5% sucrose). On the next day the membrane was washed three times for 10 min in TBST (TBS with 0.05 % Tween-20), followed by incubation with the primary antibody (1 g/ml in blocking buffer) for 1 h. The membrane was then washed three times with TBST and then incubated with the second-step antibody (anti-human IgG-HRP; Dako, Glostrup, Denmark; 1 :10,000) in blocking buffer. Then the membrane was washed two times with TBST and then again two times with PBS. Visualization of the spots was done with the ECL femto- kit (Pierce). Before incubation with another antibody, the membrane was regenerated as follows: incubations (three times for 1 0 min) were done with : TBST, regeneration buffer A (48 % urea, 1 % SDS, 0.1 % - mercaptoethanol), regeneration buffer B (50 % ethanol , 1 0 % acetic acid ), DM F (Ν ' Ν '- dimethylformamide) and methanol.

MTT cell survival assay

[00250] Serial dilutions of AFL4, 9D9 or 2E1 1 antibodies were mixed with predetermined amounts of human full-length VEGF-C (250 ng/ml) in 50 μΙ volumes in 96-well plates. Twenty thousand BaF3-VEGFR-3 cells in 50 μΙ were then added and the plates were incubated at 37° C for 2 days. All assays were in triplicates. At the end of the incubation period, 10 μΙ of MTT substrate (Sigma; 5 mg/ml in PBS) was added and incubated for additional 2 h. One hundred microliters of the lysis solution (1 0 % SDS, 10 mM HCI) was then added to every well, and the plates were incubated overnight at 37° C, fol- lowed by determination of the optical density at 540 nm in a Bichromatic plate reader (Labsystems). All experiments were repeated at least three times and gave similar results.

ELISA

[00251] The freely soluble peptides (at 10 mg/ml) were synthesized by Biosynthesis (Lewisville, TX, USA) and dissolved in water. Soluble extracellular domains of either mouse or human VEGFR-3 fused to alkaline phosphatase (VEGFR-AP) (Pytowski, B., Complete and specific inhibition of adult lymphatic regeneration by a novel VEGFR-3 neutralizing antibody. J. Natl. Cancer Inst. 97, 14-21 (2005) were diluted in PBS to 1 .5 Mg/ml and 100 μΙ were added per well of a 96-well, round-bottom polystyrene plate. After overnight incubation at 5° C, the plate was washed in PBS and blocked for one hour in PBS containing 1 % fat-free milk (PBS-M). AFL4 antibodies were biotinylated and diluted to 1 nM in PBS-M. Peptides were serially diluted in PBS-M to twice the desired concentration, mixed with equal volume of 1 nM AFL4 and incubated for 30 min at room temperature. 100 μΙ of the mixture was added to the VEGFR-3- coated plate for 2 hr at room temperature. The plates were washed three times in PBS with 0.1 % Tween-20 (T-PBS). Binding of AFL4 to the wells was detected by the addition of 100 μΙ of a 1 :4000 dilution of streptavidin coupled to horseradish peroxidase (Strep-HRP; Upstate Biotechnology, Charlottesville, VA, USA). The plates were incubated with Step-HRP for 1 h at room temperature and washed 3 times in T-PBS. Binding was detected by the addition of 50 μΙ of a solution of TMP peroxidase substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MA) and the reaction was stopped with 50 μΙ of H2SO4. Absorption was measured at 450 nm and plotted using SigmaPlot software for Windows V8.

VEGF-C binding assay

[00252] A 96-well plate was pre-coated with 2 μg ml of VEGF-C and nonspecific binding sites were blocked with 1 % BSA. The extracellular domain of VEGFR-3 fused to alkaline phosphatase (AP) was pre-incubated with different concentrations of the antibodies for 20 min and then applied to the VEGF-C pre-coated plates for 20 min. Subsequently, the plates were washed with PBS and binding was detected by the addition of 50 μΙ of an alkaline phosphatase substrate solution (Sigma). Alternatively, the extracellular domain of VEGFR-3 was pre-incubated with the different antibodies and applied to the VEGF-C coated plates for binding. The plates were washed, bound proteins were suspended in 100 μΙ of 1 x Laemmli buffer and analyzed by immunoblotting with anti-VEGFR-3 antibodies.

Migration assays

[00253] Polycarbonate transwells (6.5 mm diameter, 8 μιτι pore diameter) were coated on the underside with 1 0 g/ml gelatin overnight at 4°C. Nonspecific binding sites were blocked with heat-denatured 1 % BSA in PBS for 1 hour at 37°C. Cells were then trypsinized and washed with DMEM containing 0.5 mg/ml trypsin inhibitor. The cells were counted and 10x10⁴ cells were added to each transwell and allowed to attach and migrate for 4h 37°C. Afterwards, the top of each chamber was cleaned with a cotton swab to remove all cells. The cells remaining on the underside were fixed and stained with crystal violet and four randomly chosen fields from triplicate wells were counted at 200x magnification.

3D bead sprouting assay

[00254] Cytodex 3 microcarrier beads (GE Healthcare) were coated with endothelial cells (400 cells per bead) in endothelial growth medium - 2 MV (EGM-2 MV, Lonza), and embedded in 2 mg/ml fibrin gels in 48-well plates by mixing 2 mg/ml fibrinogen (dissolved in Hank's Balanced Salt Solution), 1 .25 U/ml thrombin, and 150 ng/ml aprotinin. Endothelial growth medium (EGM-2, Lonza) containing lung fibroblasts (WI-38, 1 1 000 cells per well) was added to each well in the presence of human VEGF-C (hVEGF-C, 150 ng/ml), HSA conditional medium, anti-VEGFR-3 (7 Mg/ml), anti-VEGFR-2 (7 Mg/ml), or their indicated combinations. The cultures were maintained for 6-9 days by changing the medium every other day before fixation with 4% paraformaldehyde (PFA) for 1 h at room temperature (RT). Bright field images were captured with Axiovert 200 (Zeiss) and sprout lengths were measured with Image J.

Binging affinity measurements by surface plasmon resonance

[00255] The binding of monomeric forms of VEGF-R3D1 -7 to Mabs 9D9, 2E1 1 and Afl4 were analyzed with surface plasmon resonance in the Biacore 2000™ biosensor (GE Helthcare). CM5 biosensor chip flow cells were cova- lently coated either with the VEGF-R3 variants in studying the Mabs as mobile phase analytes, or vice versa to obtain binding affinities of VEGF-R3 analytes to the immobilized Mabs. The coatings were done via standard amine coupling chemistry to 2,000 resonance units (RU) of the receptors or 1 ,000 RU of Mabs. The bindings were analyzed in HBS running buffer (10 mM Hepes, pH 7.4, 150 mM sodium chloride, 1 mM EDTA, 0.005 % surfactant P-20) by varying the analyte concentrations (16-500 nM of VEGFR3 proteins and 63-6000 nM of Mabs). The contact time of analytes to ligands was 5 min and the flow rate 20 L/min. The flow cells were regenerated after every injection with 10 mM glycine, pH 1 .7. The data were evaluated by first subtracting the sensorgram obtained from the empty flow cell from the sensorgrams of the flow cells containing the ligands. The steady-state binding levels (RU) over analyte concentrations were plotted and fitted (SigmaPlot 8.0 software package) assuming 1 :1 binding, for which the dissociation constant, Kd, and standard variations were derived.

Spheroid assay with LEC invected with KSHV

[00256] Wild type (wt) Kaposi sarcoma herpesvirus (KSHV) was produced from the naturally KSHV-infected primary effusion cell line BCBL-1 (NIH AIDS Research and Reference Reagent Program (Cat# 3233 from McGrath and Ganem) induced with 20 ng/ml PMA. The supernatant was collected after three days by ultracentrifugation (Beckman SW28.1 rotor, 21 ,000 rpm at 4oC for 2 h), and resuspended in TNE buffer (150mM NaCI, 1 0mM Tris pH 8, 2mM EDTA, pH 8). For infection, the LECs grown in EC culture medium plus supplement pack (PromoCell) with additional 5% human serum (Sigma) were split at a density of 2.5x105 cells per a 6-well, and spin-infected with KSHV at MOI of ~3 in serum free EC medium supplied with 8 g/ml polybrene.

[00257] Confluent monolayers of LECs infected with KSHV for 6-8 days (K- LECs), and grown in basal EC culture medium plus supplement pack (Promo- Cell) for 1 2 to 24 hours were seeded into 0.5% agarose pre-coated, nonadherent round-bottom 96-well plates at 4000 cells per well with or without VEGF-C (100ng/ml). During the formation of spheroids the medium was also supplemented with either 10pg/nnl control IgG, 10pg/nnl 2E1 1 or 10pg/nnl 3C5 antibodies or with the mixture of 2E1 1 and 3C5 (5 g/ml+5 g/ml). After 16 to 24 h incubation at 37°C, the formed spheroids were harvested and embedded into fibrin gel consisting of plasminogen-free human fibrinogen (final concentration 3mg/ml; Calbiochem) and human thrombin (final concentration 2 U/ml; Sigma) in 50 μΙ Hank's Balanced Salt Solution supplemented with 400 g/ml aprotinin (Sigma). The gels were cast onto the bottom of 24-well plates and incubated for 1 -2 h at 37°C to allow complete gelling followed by addition of the EC culture medium supplemented with the same combinations and concentrations of antibodies as described above. The sprouting was followed by phase-contrast microscopy for 3 days.To quantify the sprouting of the spheroids in duplicate wells the number of sprouts per spheroid was determined from phase contrast images acquired with a Zeiss Axiovert 200 epifluorescence microscope (eight to 16 spheroids were quantified per condition). Next the average length of the sprouts was determined using Zeiss AxioVision 3.1 software from the same phase contrast images. Relative sprouting was obtained by multiplying the number of sprouts with the average length of the sprout.

In vivo Matricge pluc assay

[00258] 50 000 of B ECs or L ECs transfected with lentivi ral Cherry- fluorescence vector were diluted in growth factor reduced Matrigel (BD Biosciences) containing 200 ng/ml of VEGF-C and 5 g/ml of blocking antibody as single treatment or 2.5 g/ml of each blocking antibody as combination, and injected intradermal^ to the mouse ear of NOD SCID gamma mice (Jackson Laboratories) in a volume of 30 μΙ . Mice were injected intravenously with 1 mg/kg of blocking antibodies every day. The mice were sacrificed 10 days following plug implantation by intracardiac perfusion with 1 % paraformaldehyde (PFA). The plugs were dissected and processed for frozen sectioning. Samples were mounted with Vectashield (VectorLabs) and analyzed with a confocal microscope (Zeiss LSM 510 DUO, 10x objective with a numerical aperture of 0.4) by using multichannel scanning in frame mode. Three-dimensional projections were digitally constructed from confocal z-stacks. The color images were converted to 8-bit grayscale using Adobe PhotoShop software (San Jose, CA). The images were then exported to ImageJ software for quantification of the area covered by BECs or LECs (Cherry positive area), which was divided by the number of individual BEC or LECs clusters to yield the median cluster size in pixels. Statistical analysis was carried out using one-way ANOVA; a p- value of less than 0.05 was considered to be statistically significant.

EXAMPLE 1

Identification of a new VEGFR-3 target epitope for blocking VEGFR-3 activity

[00259] Monoclonal antibodies against the extracellular domain of VEGFR-3 were tested for blocking of VEGFR-3 activation and survival/proliferation of BaF3 cells expressing a VEGFR-3/ only in the presence of a VEGFR-3 ligand in the culture medium ((Makinen, T., Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3. Nat. Med. 7, 199-205 (2001 ). Figure 1 A shows comparison of four antibodies in erythropoietin (Epo) receptor chimera. In the absence of IL-3 these cells survive this assay. As can be seen from the Figure, the addition of increasing amounts of the 2E1 1 antibody, but not of 9D9 or AFL4 antibody to the medium containing 25 ng/ml human recombinant VEGF-C inhibited the survival of the cells. The previously published human antihuman VEGFR-3 antibody 3C5 (Persaud, K., Involvement of the VEGF receptor 3 in tubular morphogenesis demonstrated with a human anti-human VEGFR-3 monoclonal antibody that antagonizes receptor activation by VEGF-C. J. Cell. Sci. 117, 2745-2756 (2004) and the previously published VEGFR-3-lg soluble receptor (Makinen, T., Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3. Nat. Med. 7, 199-205 (2001 ) were used as positive controls for VEGFR-3 inhibition. The inhibition of VEGFR-3 activation by the 2E1 1 and 3C5 antibodies was confirmed by using VEGF-C induced VEGFR-3 phosphorylation in endothelial cells (Figure 1 B). These data indicated that 2E1 1 is a VEGFR-3 blocking antibody and suggested that it differs from the previously described VEGFR-3 blocking antibody 3C5. They also suggested that 2E1 1 does not inhibit VEGFR-3 by inducing receptor internalization followed by downregulation.

[00260] A common mechanism for antibody inhibition of receptor activation is to block ligand binding to the receptor. It has been shown that the 3C5 antibody strongly inhibits the binding of VEGF-C to VEGFR-3 and the VEGF-C- induced mitogenic response in cells that express a chimeric human VEGFR-3- FMS receptor (Persaud, K., Involvement of the VEGF receptor 3 in tubular morphogenesis demonstrated with a human anti-human VEGFR-3 monoclonal antibody that antagonizes receptor activation by VEGF-C. J . Cell . Sci. 777, 2745-2756 (2004). However, unlike the 3C5 antibody, the 2E1 1 antibody did not block the binding of VEGFR-3 extracellular domain to immobilized VEGF-C (Figure 1 C). A similar result was obtained by using the soluble VEGFR-3 extracellular domain fused to alkaline phosphatase (Fig. 1 D). These data show that although 2E1 1 and 3C5 both inhibit VEGFR-3 activation, their inhibition mechanisms are different.

[00261] The binding epitopes of AFL4 and 9D9 were mapped to linear peptide sequences in VEGFR-3 Ig-like domain (D) 5 and D6, respectively, of the extracellular domain (Figures 6A and B). In contrast, the 2E1 1 binding site could not be mapped to a linear epitope. The 2E1 1 epitope was not in the ligand binding region (D1 -3), as this antibody recognized a VEGFR-3 mutant in which this region had been deleted (Figure 6C). This antibody bound to non- reduced, but not to reduced VEGFR-3 polypeptides in western blotting analysis (Figure 2B), suggesting that the epitope is conformational, and sensitive to de- naturation of the VEGFR-3 protein. Figure 13 shows the Kd values for 2E1 1 , 9D9 and AFL4 obtained from surface plasmon resonance analysis using monomeric VEGF-R3D1 -7. Because the antibody bound better to the nonre- duced receptor, we searched for the binding epitope in D5 that undergoes a proteolytic cleavage after receptor biosynthesis, while the fragments remain bound by a disulfide bridge.

[00262] (Figure 6A) Peptide scan (SPOT) analysis of the whole extracellular domain of VEGFR-3 to detect the exact binding sites of antibodies 9D9, 2E1 1 and AFL4. Peptides of 20 aa length with a transition frame of +3 were spotted on cellulose membranes and binding of the antibodies to the membranes was assessed by immunoblotting. The 2E1 1 antibody did not display reactivity to a linear epitope (data not shown). For 9D9, strong binding (+) was found within a linear epitope comprising peptides covering the region from E586 to A617, which is located in D6. To find out the exact residues involved in the binding of 9D9, alanine scan and staggered end deletion analysis were employed (described in detail in the "materials and methods"). According to these analyses L598HDAHGNP605 turned out to be the minimal peptide epitope. The residues that lost 9D9 binding when mutated to alanine are marked in red. The crucial H602 corresponds to Q602 in the corresponding mouse sequence, thus providing an explanation for the species specificity of this antibody. Indeed, in our hands, 9D9 did not recognize murine VEGFR-3 in immunohistochemical stainings or in peptide scan analysis (data not shown). (Figur 6B) Probing of a SPOTS membrane containing the mouse VEGFR-3 peptides with the AFL4 antibodies. Binding (+) was found within to the region from E491 to D525, which is located in D5. This was identical in the human VEGFR-3 sequence except for S506 was T506 in the human sequence. SPOTS analysis of the extracellular domain of human VEGFR-3 indeed showed that the AFL4 antibody binds to the corresponding human sequence (data not shown). (Figure 6C) Mapping the 2E1 1 binding domain. Since it was impossible to locate the 2E1 1 epitope, we made VEGFR-3 receptor with deletion of first three extracellular domains (D1 -D3) named VEGFR-3 Δ1 -3. This construct was expressed in 293T cells along with WT VEGFR-3, both containing the Streptag l l l at C- terminus. The proteins precipitated with streptactin beads, run on SDS-PAGE under non-reducing conditions and blotted with the 2E1 1 and 3C5 antibodies. (Figure 13) Surface plasmon resonance analysis of the binding of monomeric VEGF-R3D1 -7 to Mabs 9D9, 2E1 1 and AFL4.

EXAMPLE 2

A polypeptide loop extending from the VEGFR-3 D5 is critical for 2E11 antibody binding and receptor activation.

[00263] Figure 2A shows the sequence comparison of human and mouse VEGFR-3 and VEGFR-2 D5. The VEGFR-3 proteolytic cleavage site is marked with a red arrowhead and the cysteine residues are marked bold. Fig. 2C and D show a computer model of VEGFR-3 D5 structure based on myelin basic protein-C immunoglobulin-homology domain. In VEGFR-3 D5, the extended loop (underlined in Fig. 2A; including the SLRRRQQQ sequence) contains the cleavage site betweenR472 and S473 (red arrowhead in Fig. 2 A and C). In Figure 2D the surface of D5 is colored red for negative charge and blue for positive charge. This model suggested that the positively charged residues of the D5 elongated loop "arm" could contact the negatively charged surface of the D5 "armpit", thus contributing to dimer stabilization and activation of the receptor.

[00264] Figure 3A schematically outlines the mutagenesis strategy used to interrogate the importance of D5 and its elongated, cleaved loop structure for 2E1 1 antibody binding and receptor function. The disulfide bonds in the figure are hypothetical and based on deductions from the D5 model. The effect of D5 cysteine to serine residue replacements on VEGFR-3 expression, cleavage and autophosphorylation in transfected 293T cells in the absence and presence of VEGF-C are shown in Figure 3B (left panel). The transfected cells were analysed by VEGFR-3 immunoprecipitation and western blotting using the anti-phosphotyrosine (pY) or VEGFR-3 antibodies. As can be seen from the results, the C445S and C534S mutations and their combination prevented receptor autophosphorylation and processing. The C466S mutation decreased VEGFR-3 expression levels while retaining at least some phosphorylation, and blocked cleavage of the receptor, while C486S allowed both processing and ligand-induced phosphorylation. [00265] (Figure 2A) Sequence alignment of D5 of human and mouse VEGFR-3 and VEGFR-2. The predicted extra loop and proteolytic processing site have been marked, as are the deleted and swapped sequences, plus the AFL4 binding peptide. The cysteine residues are marked in red and the two N- linked glycosylation sites have been underlined. (Figure 2B) Sensitivity of the antibody epitopes to reduction of disulfide bonds. VEGFR-3-streptag III was stably expressed in 293T cells, precipitated with streptactin sepharose and analyzed by blotting with 2E1 1 and 9D9 antibodies under reducing and non- reducing conditions. (Figure 2C) A th ree-dimensional VEGFR-3 D5 model (Phyre), based on the MyBP-C structure (PDB code 1 GXE), with the cysteine residues highlighted in yellow. Note that in VEGFR3 D5, C445 and C534 make a disulfide bridge typical for immunoglobulin (Ig) homology domains. C466 and C486 are far apart in the model but probably interact in VEGFR-3 D5. In MyBP-C, there are no counterparts for residues S473-Q480 (dotted line). R472-S473 is the identified protease cleavage site. In (Figure 2D), the surface is colored according to the electrostatic potential (red = negative, blue = positive charge). Note that the acidic residues in the AFL4 antibody-binding site center around F510 (see also Figure 6B). Notably, the loop area, including the residues missing from MyBP-C (SLRRRQQQ, dotted line) is positively charged. (Figure 14) Immunofluorescent staining of 293T cells transfected with WT, LD and LS VEGFR-3. 293T cells were transfected with different VEGFR-3 constructs and stained for VEGFR-3 with 2E1 1 antibodies as in Figure 3C. Scale bar 20 μιτι.

[00266] A similar analysis was carried out with a chimeric VEGFR-3 receptor where the loop region was substituted with the corresponding amino acid sequence of VEGFR-2, or where the loop was deleted. As shown in Figure 3B, the loop swap (LS) from VEGFR-2 to VEGFR-3 leads to slightly decreased VEGFR-3 phosphorylation and loss of both the chimeric VEGFR-3 cleavage and VEGF-C-inducible activation. In contrast, loop deletion (LD) leads to significant decrease of receptor phosphorylation even in the presence of VEGF-C. These results suggest that D5 plays acrucial role in VEGFR-3 activation. [00267] Since point mutations and deletions in the D5 loop area of VEGFR-3 had a significant effect on VEGFR-3 activation, the 2E1 1 antibodies were tested for binding to the different VEGFR-3 constructs in transient transfection experiments. As shown in Figure 3C and other cell-based experiments, the 2E1 1 antibodies do not recognize VEGFR-3 LD, but recognize VEGFR-3 LS, although they failed to detect VEGFR-2 expressed in 293T cells (data not shown). When the other VEGFR-3 mutants were expressed in 293T cells and precipitated with 2E1 1 or 9D9 followed by western blotting with 9D9 antibodies, 2E1 1 failed to precipitate those mutants that had lost VEGF-C inducible activation (Figure 3D). These data indicate that the 2E1 1 epitope is at least partially located in D5 and sensitive to conformational changes in the loop region. Furthermore, the 2E1 1 antibodies recognize an epitope that correlates with the ability of the receptor to be activated.

EXAMPLE 3

2E11 antibodies bind to VEGFR-3 D5 and provide synergistic inhibition in combination with 3C5 antibodies.

[00268] Although the mutagenesis data strongly suggested that the 2E1 1 epitope is located VEGFR-3 D5, it was not possible to map this epitope using linear peptides. To further prove that 2E1 1 recognizes D5, this domain was cloned to pSecTag vector and expressed in 293T cells. As shown in Figure 3E, the 2E1 1 (and AFL4) antibodies readily precipitated the D5 domain form the conditioned medium, whereas the 9D9 antibodies did not, because their epitope maps to D6 (Figure 6A). The above experiments showed that the 3C5 and 2E1 1 antibodies bind to different regions of VEGFR-3 and inhibit receptor activity by different mechanisms. This fact raised a question of possible synergistic inhibition of VEGFR-3 activity by 2E1 1 and 3C5. As shown in Figure 4A, the 2E1 1 and 3C5 antibodies in combination blocked VEGFR-3 activity better than either antibody alone when VEGF-C was used at 10 ng/ml . At 100 ng/ml, 3C5 had provided very little inhibition, while 2E1 1 retained some activity (Figure 4B). Strikingly, at this ligand concentration, the two antibodies together provided synergistic inhibition of VEGFR-3 activation. Thus these data show that antibodies with different mechanisms of inhibition provide higher efficacy when used in combination. These data also suggest that at high doses of VEGF-C, the 3C5 antibodies cannot efficiently block VEGFR-3 activation because they compete with ligand binding, whereas the 2E1 1 antibodies retain activity because their mechanism is based on blocking receptor dimerization rather than ligand binding. Consistent with this hypothesis, the 3C5 antibodies were not able to inhibit Erk1 ,2 activation at high concentrations of VEGF-C in the BaF cells expressing the VEGFR-3/EpoR chimera, although 2E1 1 showed efficient inhibition at all tested VEGF-C concentrations (Figure 4C,D). Interestingly, as shown in Figure 4E, a similar difference between the 2E1 1 and 3C5 antibodies was detected in HDMEC cells when VEGFR-3 was analyzed for phosphorylation after different VEGF-C concentrations. Interestingly, only the 2E1 1 antibody was able to inhibit some of the Erk1 ,2 phosphorylation induced by 25 ng/ml of VEGF-C, whereas no effect was not observed when using the 3C5 antibodies (Figure 4F).

EXAMPLE 4

The 2E1 1 antibody inhibits VEGF-C induced VE GFR-2A/E GFR-3 het- erodimerization and VEGFR-2 activation.

[00269] Previous studies have shown that VEGF-C can induce the formation of VEGFR-2A/EGFR-3 heterodimers that show distinct phophorylation patterns in comparison with receptor homodimers (Dixelius, J., Ligand-induced vascular endothelial growth factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2 in primary lymphatic endothelial cells regulates tyrosine phosphorylation sites. J . Biol . Chem. 278, 40973-40979 (2003). We investigated the possibility that the VEGFR-3 D5 binding antibody 2E1 1 that inhibited VEGFR-3 activation could act in trans to inhibit also the formation of the heterodimers. HDMECs that express both receptors were stimulated with VEGF-C in the presence of the VEGFR-3 antibodies. VEGFR-3 was immunoprecipitated and the immune complexes were subjected to western blotting using VEGFR-2 specific antibodies. As can be seen from the results shown in Fig. 5A, the two VEGFR-3 blocking antibod ies (2E1 1 and 3C5) inhibited the formation of VEGFR-2A/EGFR-3 heterodimers, whereas the AFL4 antibodies did not significantly decrease VEGFR-2 coprecipitation. Interestingly, inhibition of het- erodimer formation was associated with decreased VEGFR-2 activation by VEGF-C, particularly when the 2E1 1 antibodies were used (Fig. 5B), correlating with the decreased downstream signaling via the MAP kinase Erk1 ,2 (Fig. 4F). In contrast, VEGFR-2 signaling was not affected by the 2E1 1 or 3C5 antibodies in transfected porcine aortic endothelial (PAE) cells expressing only VEGFR-2 (Fig. 5C). Furthermore, preincubation with the 2E1 1 or 3C5 antibodies did not affect VEGF-induced VEGFR-2 and none of the antibodies induced receptor downregulation phosphorylation. These results indicate that the 2E1 1 antibod ies inhibit signal ing of both VEGFR-3 homodimers and VEGFR- 3A/EGFR-2 heterodimers.

[00270] The 2E1 1 antibody does not inhibit VEGF-A induced VEGFR-2 phosphorylation or induce VEGFR-2 or VEGFR-3 downregulation. (Figure 16A) VEGF-A-induced VEGFR-2 phosphorylation and intracellular signaling in HDME cells in presence of 2E1 1 and IMC1 121 B (positive control) antibodies. HDME cells were pre-incubated with different antibodies for 15 min and then stimulated with VEGF-A or VEGF-C for 5 min for VEGFR-2 phosphorylation or 30 min for Erk1 ,2 and Akt phosphorylation. Lysates were either precipitated with polyclonal VEGFR-2 antibodies and blotted with pY antibodies or analysed for Erk1 ,2 and Akt phosphorylation . (Figure 1 6B) Western blotting analysis of cellular VEGFR-2 and VEGFR-3 after ligand and antibody treatment. HDME cells were incubated with indicated antibodies for different time periods and the cell lysates were analyzed by blotting with VEGFR-2 and VEGFR-3 antibodies. Note downregulation by VEGF-C, used as a positive control. (Figure 9) Analysis of the HDME cells by anti-podoplanin immunofluorescence staining followed by flow cytometry. The percentages of positive (LEC) and negative (BEC) cells are indicated above the graph.

EXAMPLE 5

The 2E11 antibody inhibits VEGF-C induced migration and sprouting of normal as well as transformed endothelial cells. [00271] The effects of the VEGFR-3 blocking antibodies were next analyzed in sprouting and migration assays in cultured HDME cells (Fig. 8). The 2E1 1 , 3C5 and IMC1 121 B antibodies inhibited HDME cell migration and sprouting with comparable efficacy. As shown by flow cytometric analysis in Figure 9, the HDME cells used consisted of almost equal proportions of blood vascular and lymphatic endothelial cells (BECs and LECs, respectively). Figure 10A and B summarize the results of migration assays performed with isolated LECs and BECs. As can be seen from the figure, 2E1 1 effectively inhibited the migration of both cell types, while 3C5 inhibited only LEC migration. Similar results were obtained in the LEC sprouting assay (Fig. 10C), while BECs did not sprout in this assay (data not shown).

[00272] As shown in Fig. 10D, in LECs, both Erk1 ,2 and Akt phosphorylation were inhibited by blocking VEGFR-3 activation using 3C5 or 2E1 1 antibodies, while in BECs only the VEGFR-2 blocking antibodies (IMC1 121 B) inhibited Erk1 ,2 activation . Furthermore, the very slight Akt activation seen in the VEGF-C treated BECs was inhibited only by the IMC1 121 antibodies.

EXAMPLE 6

Stronger inhibition of sprout outgrowth by combination treatment

[00273] Kaposi sarcoma herpesvirus (KSHV)-infected ECs represent a biologically relevant model of KSHV-induced Kaposi sarcoma (KS); these cells have been demonstrated to robustly express VEGFR-3 (Jussila et al., Cancer Res. 58, 1599-1604 1998) Moreover, the KS tumors show inhanced levels of VEGFR-3 and VEGF-C, which may play a key role in KSHV biology as LECs are considered to be the favored target of KSHV infection. We therefore tested the effect of the VEGFR-3 blocking antibodies on the capillary outgrowth of KSHV-infected LEC (K-LEC) spheroids grown in a crosslinked 3D fibrin matrix. Extensive sprouting in response to VEGF-C was observed in the K-LEC spheroids (Figure 1 1 A), whereas the control LECs sprouted to a lesser extent (data not shown). In the absence of VEGF-C the sprouting was greatly reduced. To assess the effect of the 2E1 1 and 3C5 antibodies on the sprouting of K-LEC spheroids, the cultures were treated with 10 g/ml of 2E1 1 or 3C5 antibodies or with 5 pg/irnl of both 2E1 1 and 3C5. The IgG control did not influence the sprouting over the untreated K-LEC spheroids in the presence of VEGF-C (data not shown), whereas incubation with either 2E1 1 or 3C5 antibodies reduced sprout outgrowth significantly. The combination treatment with 2E1 1 and 3C5 antibodies led to a stronger inhibition of sprout outgrowth (Figure 1 1 A,B).

[00274] (Figure 15A) Expression of VEGFRs and neuropilins in the endothelial cells. VEGFR2, VEGFR3, Nrp1 , Nrp2 and Proxl levels were determined by western blot analysis in BECs, HDMECs and LECs. (Figure 15B) VEGFR-3 phosphorylation in BECs stimulated with 25 ng/ml of VEGF-C. The cells were immunoprecipitated with VEGFR-3 antibodies followed by western blot with pTyr and VEGFR-3 antibodies. (Figure 15C) Antibody inhibition of LEC sprouting. Shown are PECAM-1 stained immunofluorescent images of LECs sprouting from microbeads in the presence of 3C5, 2E1 1 or the combination, and statistical evaluation of sprout length and number. Note that the antibody combination decreases sprout length significantly more than either antibody alone, and a similar trend is seen for the number of sprouts. ^*p<0.05, ^**p<0.01 , ^***p<0.001 compared to hlgG. Error bars represent +/- SEM.

EXAMPLE 6

Strong inhibition of vascular morphogenesis in vivo by the 2E11 and 3C5 antibody combination.

[00275] To test if the antibodies blocking ligand binding and receptor dimeri- zation would be effective in vivo, we implanted human BECs or LECs in VEGF- C contain ing Matrigel plugs into the ears of immunodeficient NOD-SCID- gamma mice. We found that the 2E1 1 and 3C5 antibodies suppressed LEC tube formation with similar efficiency (Figure 12A-C). Importantly, combining 2E1 1 to 3C5 provided a stronger inhibition of tube formation than either blocking antibody alone at the same antibody dose (Figure 12B). Intriguingly, the combination of 2E1 1 and 3C5 also dramatically suppressed the survival of the transplanted LECs (Figure 12A, C and data not shown). The 2E1 1 and 3C5 combination also inhibited the ability of transplanted BECs to form vascular networks in vivo more efficiently than either antibody alone (Figure 12D-F). However, unlike for the LECs, further suppression of BEC survival was not observed when comparing the combination treatment to the single treatments (Figure 12F). These results indicate that VEGF-C driven tube formation and survival of LECs as well as the vascular networks formation of BECs are inhibited by the antibodies, and most efficiently by the combination of the two antibodies.

Discussion of the Foregoing Examples

[00276] Here we describe how a VEGFR-3 antibody, called 2E1 1 , displays inhibitory activity toward human VEGFR-3. We show that 2E1 1 antibodies block VEGFR-3 phosphorylation and mitogenicactivity. Detailed analysis revealed that their mechanism of inhibition strikingly differs from other inhibitory antibodies against VEGFRs. First, the 2E1 1 antibodies did not block VEGF-C binding to VEGFR-3, yet they effectively inhibited the VEGFR-3 phosphorylation and mitogenic signal transduction, even at high concentrations of VEGF-C, when the 3C5 antibodies that occupy the ligand binding site in VEGFR-3 displayed only moderate inhibition. These features indicated that the 2E1 1 inhibition is based on a new mechanism that is not related to the blocking of ligand binding. Even more strikingly, this new mechanism of inhibition seems to syn- ergize with the inhibition of ligand binding to VEGFR-3.

[00277] The VEGFR-3 D5 is the only site of proteolytic processing found in the VEGFR-3 family, but the reason for processing is not known. Our previous studies have shown that processing occurs only after the receptor is glycosylated and appears on cell surface (Pajusola Oncogene). One question is whether the VEGFR-3 cleavage is required for receptor activation. The tyrosyl phosphorylation of the uncleaved VEGFR-3 polypeptide band could depend on heterodimerization with the cleaved form of the receptor. Previous attempts to mutate the cleavage site to answer this question led to intracellular accumulation of the mutant receptor. In the present study, mutagenesis of select cysteine resides inhibited VEGFR-3 cleavage, and yet we could detect phosphorylation of the uncleaved form of the VEGFR-3 upon VEGF-C stimulation. This makes it unlikely that the cleavage is essential for receptor activation. Our pre- sent data also indicate that a receptor where the extended loop of D5, including the cleavage site has been deleted or replaced by a corresponding shorter, nonhomologous loop of VEGFR-2 D5 cannot be cleaved, although VEGFR-3 LS shows a small increase of tyrosyl phosphorylation upon ligand stimulation. Thus, proteolytic cleavage of VEGFR-3 is not required for receptor activity, and the extended loop of D5 where the cleavage occurs can be exchanged with a nonhomologous loop without complete loss of ligand stimulated activity. On the other hand, the presence of a loop stucture and its internal Cys bonds seem to be important for maintaining a conformation in D5 that supports receptor activity.

[00278] So far, published blocking antibodies towards the VEGFRs have been isolated from a screen of ligand binding inhibitors by using a receptor binding immunoassay Krupitskaya Y., Curr. Opin. Investig. Drugs. 10, 597-605 (2009). However, our primary screen for anti-VEGFR-3 employed immunization with the full-length receptor, followed by FACS analysis of VEGFR-3 expressing and nonexpressing cells as the primary screen. The 9D9 antibody obtained from this screen has been shown to work very well in different experimental settings, like immunoprecipitation, immunoblotting, immunohistochem- istry and FACS analysis Paavonen K., Am. J. Pathol. 156, 1499-1504 (2000). The secondary screen of the generated antibodies revealed different VEGFR-3 binding epitopes, some of which were associated with inhibition of receptor activation, including the 2E1 1 epitope.

[00279] We used peptides covering the whole extracellular domains of human and mouse VEGFR-3 to determine the binding epitopes of the antibodies. The AFL4 and 9D9 binding epitopes could be readily determined, since they comprise linear sequences in D5 and D6, respectively. However, 2E1 1 did not react with synthetic VEGFR-3 peptides. This is in line with the poor performance of this antibody in western blotting in reducing conditions, suggesting a conformational epitope suitable for applications, where the native antigen is detected, such as FACS analysis or immunoprecipitation, implying a conforma- tional/discontinous epitope for this antibody. The fact that the replacement of the VEGFR-3 D5 loop with the essentially non-homologous loop ofVEGFR-2 rescued 2E1 1 binding activity, suggests that the loop affects the 3D structure of VEGFR-3 D5.

[00280] We found a linear binding region in the D5 of mouse as well as human VEGFR-3 that bound the rat anti-mouse antibody AFL4, which has been shown to inhibit neoangiogenesis by inducing micro-hemorrhages in mouse tumor xenografts (Kubo,H., Blood 96, 546-553 (2000)). However, another study showed that the AFL4 antibody does not inhibit ligand binding or phosphorylation of VEGFR-3 nor does it block VEGF-C-stimulated cell proliferation. Despite the fact that AFL4 antibodies bind very close to the epitope of the 2E1 1 function-blocking antibody, we could confirm the lack of AFL4 inhibition of ligand binding. However, we also obtained no evidence that AFL4 inhibits cellular VEGFR-3 activation. Several studies have used AFL4 antibodies to inhibit angiogenesis in the cornea and in the ischemic hindlimb after femoral artery ligation. Although we do not understand how the AFL4 antibody inhibits angiogenesis, the possibility remains that it works for example by accelerating receptor downregulation or by immune opsonization of cells expressing VEGFR-3, as has been described for some of the anti-ErbB2 antibodies that inhibit tumor growth (Valabrega, G., Ann. Oncol. 18, 977-984 (2007)).

[00281] The VEGFRs transduce their effects according to the consensus scheme for receptor tyrosine kinases. Binding of ligand leads to dimerization of tyrosine kinase receptors by close apposition of the receptor intracellular domains and exposure of the kinase active site (Hubbard, S.R., Prog. Biophys. Mol. Biol. 71, 343-358 (1999). Tyrosine phosphorylation then initiates signal transduction cascades, which ultimatelylead to cellular responses such as proliferation, motility and survival. Crystal structures of complexes of VEGF-A and PIGF have been determined, providing the first structural insights into ligand binding within the VEGF family. Analysis mutants of the extracellular parts of VEGFR-1 and VEGFR-2 showed that both D2 and D3 are needed for high affinity VEGF-A binding. Similarly, the recently published analysis of the VEGF- AA/EGFR-2 complex by electron microscopy suggested that VEGF-A binds to D2 and D3 (Ruch, C, Nat. Struct. Mol. Biol. 14, 249-250 (2007). Interestingly, in this study it was found that the VEGFR-2 dinners are further stabilized by receptor-receptor contacts mediated by D4 and D7. The related Kit and PDGF receptors similarly interact in the membrane proximal D5 (Yuzawa, S. 2007) (Yang, Y., Proc. Natl. Acad. Sci. U. S. A. 107, 1906-191 1 (2008). One could thus envision that antibodies binding to the domains involved in dimeric recep- torreceptor contacts could interfere with the close apposition of the downstream tyrosine kinase domains, thus blocking receptor activity. However, according to our search of the literature, no such antibodis have been previously characterized for the VEGFRs.

[00282] It should be mentioned that the ErbB2 antibody trastuzumab, one of the first monoclonal antibodies used in clinical practice, acts through a mechanism not involving inhibition of ligand binding since a soluble ligand for ErbB2 has not been found. The exact mechanism of ErbB2 inhibition by trastuzumab is not completely understood, but these antibodies have little effect on ErbB2- ErbB3 heterodimerization (Agus et al., Cancer Cell. 2, 127-137 (2002). Rather they are thought to act through antibody-dependent cellular cytotoxicity or inhibition of ErbB2 shedding (Valabrega et al., Ann. Oncol. 18, 977-984 (2007). Interestingly, another ErbB2 blocking antibody, pertuzumab, acts through blocking heterodimerization of ErbB2 with other members of the ErbB family by binding to domain II and sterically masking a binding pocket necessary for receptor-receptor interaction (Franklin et al ., Cancer Cell. 5, 31 7-328 (2004). Thus in addition to antibody-dependent cellular cytotoxicicity, pertuzumab binding directly inhibits ErbB2 heterodimerization, which blocks the ErbB2 signaling cascade. This difference between trastuzumab and pertuzumab explains why pertuzumab is effective in carcinomas that express low levels of ErbB2, whereas trastuzumab is not.

[00283] Antibodies that block ligand binding to receptor need to compete with the ligand for receptor binding, i.e. the outcome of therapeutic targeting is dependent on the stoicheometry between ligand and antibody. At high ligand concentrations such antibodies are less effective than antibodies blocking re- ceptor dimerization, as seen in our analysis in the BaF3/VEGFR-3 cultures. In the cultured microvascular endothelial cells, only VEGFR-2A/EGFR-2 h et- erodimers, but not VEGFR-2 homodimers were inhibited by the 2E1 1 antibodies. Importantly however, our data indicated that a combination of antibodies blocking ligand binding and receptor dimerization is more effective in inhibiting both blood vascular and lymphatic endothelial cell sprouting, in particular sprout elongation, than either antibody alone. This was also the case in the analysis of blood and lymphatic endothelial vascular network formation in vivo in matrigel plugs, where the antibody combination furthermore compromised the survival of lymphatic endothelial cells. Similarly, stronger inhibition of sprouting was observed with LECs transformed with the KSHV human tumor virus when the combination of antibodies was used. Depending on the assay, the combination of blocking antibodies thus provided an additive or a synergistic inhibition. It would be interesting to know if such effects could be further improved by inclusion of the recently published antibodies against neuropilin-2 that block VEGF-C binding and LEC sprout elongation (Xu et al., J. Cell Biol. 188, 1 15-130 (2010).

[00284] Three ongoing clinical trials are addressing if a combination of tras- tuzumab and pertuzumab results in a better therapeutic outcome than either of t h e t w o a n t i b o d i e s a l o n e (http://clinicaltrials.gov/ct2/results?term=trastuzumab+pertuzumab). Although such studies have not yet been carried out with VEGFR targeting antibodies, our data on vascular network formation of BECs and LECs suggest that employing a combination of ligand binding and dimerization inhibitors would provide more effective blocking of VEGFRs and enhanced inhibition of tumor an- giogenesis and lymphangiogenesis in vivo. The combination could also form a potential treatment modality for Kaposi sarcoma tumor cells that are known to expresses VEGFR-3 (Jussila et al., Cancer Res. 58, 1599-1604 (1998).

[00285] In conclusion, our results define a novel class of VEGFR blocking antibodies, which provide interesting mechanistic insight into receptor structure and activation. Importantly, the dimerization inhibitor unveils a biologically meaningful rationale for suppressing endothelial activation and angiogenesis in tumors. The use of a combination of antibodies inhibiting ligand binding and receptor dimerization should translate into improved anti-angiogenic and anti- lymphangiogenic therapies in the future.

[00286] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[00287] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open- ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.

[00288] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[00289] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.

[00290] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inven- tors intend for the invention to be practiced otherwise than as specifically described herein . Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as perm itted by appl icable law. Moreover, any combination of the above- described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1 . A composition comprising a first binding construct that binds to a first epitope of a Receptor Tyrosine Kinase (RTK) extracellular domain (ECD) and a second binding construct that binds to a second epitope of the RTK ECD that differs from the first epitope, wherein each of the first binding construct and second binding construct reduces ligand-induced activation of the RTK.

2. The composition of claim 1 , further comprising a pharmaceutically acceptable diluent or carrier.

3. A kit comprising a first binding construct that binds to a first epitope of a Receptor Tyrosine Kinase (RTK) extracellular domain (ECD) and a second binding construct that binds to a second epitope of the RTK ECD that differs from the first epitope, wherein each of the first binding construct and second binding construct reduces ligand-induced activation of the RTK, wherein the first and second binding constructs are packaged together but not in admixture.

4. A method of inhibiting ligand-induced activiation of a receptor tyrosine kinase (RTK), the method comprising contacting the RTK with a first binding construct that binds to a first epitope of a Receptor Tyrosine Kinase (RTK) extracellular domain (ECD) and with a second binding construct that binds to a second epitope of the RTK ECD that differs from the first epitope, wherein each of the first binding construct and second binding construct reduces ligand-induced activation of the RTK.

5. The method of claim 4, wherein the RTK is expressed on the cells of a mammalian subject, and the contacting comprises administering the first and second binding constructs to the mammalian subject in amounts effective to inhibit ligand-induced activation of the RTK.

6. Use of a first binding construct that binds to a first epitope of a Receptor Tyrosine Kinase (RTK) extracellular domain (ECD) and a second bind- ing construct that binds to a second epitope of the RTK ECD that differs from the first epitope, wherein each of the first binding construct and second binding construct reduces ligand-induced activation of the RTK, in a combination therapy for inhibiting ligand-induced activation of a receptor tyrosine kinase (RTK) in cells of a mammalian subject.

7. A composition comprising a first binding construct that binds to a first epitope of a Receptor Tyrosine Kinase (RTK) extracellular domain (ECD) and a second binding construct that binds to a second epitope of the RTK ECD that differs from the first epitope, wherein each of the first binding construct and second binding construct reduces ligand-induced activation of the RTK, for use in a combination therapy for inhibiting ligand-induced activation of a receptor tyrosine kinase (RTK) in cells of a mammalian subject.

8. The composition, kit, method, or use of any one of claims 1 to 7, wherein the first binding construct comprises an antibody that binds the first epitope, or an antigen binding fragment thereof.

9. The composition, kit, method, or use of any one of claims 1 to 8, wherein the second binding construct comprises an antibody that binds the second epitope, or antigen binding fragment thereof.

10. The composition, kit, method, or use of claim 9, wherein the antibodies are monoclonal antibodies.

1 1 . The composition, kit, method, or use of claim 9 or 10, wherein the antibodies are humanized or human antibodies.

12. The composition, kit, method, or use of any one of claims 1 to 1 1 , wherein each of the first and second binding constructs is an antigen binding fragment of an antibody, said antigen binding fragment selected from the group consisting of: fab, f(ab)₂', fab3, scFv, diabody, triabody, tetrabody, minibody, and single-domain antibody.

13. The composition, kit, method, or use of any one of claims 1 to 12, wherein the first binding construct reduces activation of the RTK by inhibiting binding between the RTK and a ligand that binds to the RTK.

14. The composition, kit, method, or use of claim 13, wherein the first epitope is a portion of a ligand binding domain of the RTK.

15. The composition, kit, method, or use of any one of claims 13 to 14, wherein the second binding construct does not inhibit binding between the RTK and the ligand.

16. The composition, kit, method, or use of any one of claims 13 to 15, wherein the second epitope is an extracellular epitope that does not participate in ligand binding.

17. The composition, kit, method, or use of any one of claims 1 to 16, wherein the second binding construct inhibits RTK dimerization.

18. The composition, kit, method, or use of any one of claims 1 to 17, wherein the RTK is selected from the group consisting of Vascular Endothelial Growth Factor Receptors -1 , -2, and -3 (VEGFR-1 , VEGFR-2, and VEGFR-3) and Platelet-Derived Growth Factor Receptors -alpha and -beta (PDGFR-a, and PDGFR-β).

19. The composition, kit, method, or use of any one of claims 1 to 17, wherein the RTK is human VEGFR-3.

20. The composition, kit, method, or use of claim 19, wherein the first epitope comprises at least a portion of the Immunoglobulin (Ig) homology domain D1 , the Ig homology domain D2, the Ig homology domain D3, or a combination thereof, of the VEGFR-3 ECD.

21 .The composition, kit, method, or use of claim 20, wherein the first epitope is the epitope of antibody 3C5 (Imclone).

22. The composition, kit, method, or use of claim 21 , wherein the first binding construct comprises antibody 3C5 (Imclone), or an antigen binding fragment thereof.

23. The composition, kit, method, or use of any one of claims 19 to 22, wherein the second epitope is not located within any of D1 to D3 of VEGFR-3.

24. The composition, kit, method, or use of any one of claims 19 to 23, wherein the second epitope is a conformational epitope and wherein high affinity binding between the RTK and the ligand requires a disulfide bond between the Cys445 and Cys534 of VEGFR-3 (SEQ ID NO: 6).

25. The composition, kit, method, or use of any of any one of claims 19 to 24, wherein the second epitope comprises at least a portion of the Ig homology domain D5 of the VEGFR-3 ECD.

26. The composition, kit, method, or use of any of claims 19 to 25, wherein the second epitope is the epitope of antibody 2E1 1 (Accession No. 01083129).

27. The composition, kit, method, or use of claim 26, wherein the second binding construct comprises antibody 2E1 1 , or an antigen binding fragment thereof.

28. The composition, kit, method, or use of any one of claims 1 to 27, wherein at least one of the first binding construct and the second binding construct is conjugated to a heterologous moiety selected from the group consisting of: a polymer, a cytokine and a cytotoxic agent.

29. The method of any one of claims 5 and 8-28, wherein the mammalian subject has a neoplastic disorder, and the binding constructs are administered in an amount effective to inhibit neoplastic cell growth.

30. The use according to any one of claims 6 to 28, wherein the therapy is for a neoplastic disorder.

31 .A method of inhibiting ligand-induced activation of a RTK in a cell, the method comprising contacting the cell with a composition of any one of claims 1 -2 and 7-28, in an amount effective to inhibit ligand-induced activation of the RTK.

32. The method of claim 31 , wherein the cell is a lymphatic endothelial cell, a blood endothelial cell, or a hematopoietic progenitor cell.

33. The method of any one of claims 5 to 29 and 31 -32, wherein the first and second binding constructs are administered separately to the mammalian subject.

34. The method or use of any preceding method or use claim, wherein the mammal is a human.

35. Use of the composition of any of claims 1 -2 and 6-28 in the preparation of a medicament for treating a neoplastic disorder in a mammal.

36. The use according to claim 35, wherein said neoplastic disorder is Kaposis sarcoma.

37. An isolated monoclonal antibody, or antigen bind ing fragment thereof, that binds to the extracellular domain (ECD) of a receptor tyrosine kinase (RTK) selected from the group consisting of VEGFR-1 , VEGFR-2, PDGFR-alpha, and PDGFR-beta, wherein the antibody or fragment permits a ligand of the RTK to bind the RTK but inhibits ligand-mediated phosphorylation of the RTK.

38. The antibody or antibody fragment according to claim 37 that is human or humanized.

39. A composition comprising the antibody of claim 37 or 38 and a pharmaceutically acceptable carrier.

40. A method for inhibiting cell growth in a mammalian organism, the method compring: administering to a mammalian organism the composition of claim 39, wherein the organism has cells that express the RTK, and the antibody or fragment is present in the composition in an amount effective to inhibit ligand-mediated phosphorylation of the RTK, thereby inhibiting growth of the cells that express the RTK.

41 . The composition of claim 39, further comprising a second monoclonal antibody, or fragment thereof, that binds to the RTK and inhibits the ligand from binding to the RTK.

42. A method for inhibiting cell growth in a mammalian organism, the method comprising: administering to a mammalian organism the composition of claim 41 , wherein the organism has cells that express the RTK, and the antibodies or fragments are present in the composition in amounts effective to inhibit ligand-mediated phosphorylation of the RTK, thereby inhibiting growth of the cells that express the RTK.

43. The composition of claim 39 or 41 , further comprising a third monoclonal antibody, or fragment thereof, that binds to the ligand and inhibits the ligand from binding to the RTK.

44. In a method of treatment that includes administering to a mammalian subject a first antibody, or antigen binding fragment thereof, that inhibits a ligand from the PDGF or VEGF family of ligands from binding to a receptor tyrosine kinase for the ligand, an improvement comprising administering to the mammalian subject a second antibody, or antigen binding fragment thereof, that binds to the extracellular domain (ECD) of the RTK, wherein the second antibody or fragment inhibits dimerization of the RTK and inhibits ligand- mediated phosphorylation of the RTK.

45. The improvement of claim 44, wherein the first antibody binds to the ligand.

46. The improvement of claim 44, wherein the first antibody binds to the

RTK.

47. A method of making a binding construct comprising: (a) screening a library of compounds to identify a candidate compound that binds the extracellular domain (ECD) of a receptor tyrosine kinase (RTK), permits a ligand of the RTK to bind to the RTK, and inhibits the RTK from dimerizing; and (b) making a binding compound containing the candidate compound identified in (a), or a fragment thereof that retains the binding and inhibition properties.

48. The method of claim 47, wherein the library contains antibodies or antigen binding fragments of antibodies.

49. The method of claim 47 or 48, wherein the RTK is selected from the group consisting of VEGFR-1 , VEGFR-2, PDGFR-alpha, and PDGFR-beta.

50. The method of claim 47 or 48, wherein the RTK is VEGFR-3, and wherein the antibody binds to a different epitope of VEGFR-3 than the epitope recoginzied by antibody 2E1 1 .

51 . A binding construct made by a method according to any one of claims 47 to 50.

52. An antibody or antigen binding fragment thereof that comprises the antigen binding domain of an antibody made according to the method of any one of claims 48 to 50.

53. The composition of any preceding composition claim, further including a standard of care cancer therapeutic.

54. A method for inhibiting ligand-induced sprouting of microvascular endothelial cells in a mammalian organism, the method comprising: administering to a mammalian organism the composition of claim 41 , wherein the organism has cells that express the RTK, and the antibodies or fragments are present in the composition in amounts effective to inh ibit ligand-induced sprouting of microvascular endothelial cells in a mammalian organism.