Antibody against TIE-2 and method of use

Abstract

To provide anti-Tie2 antibodies with improved properties as well as methods for using the same for therapeutic and diagnostic purposes.SOLUTION: The present invention provides an isolated anti-Tie2 antibody, or an antigen-binding fragment thereof, comprising three heavy chain complementary determining regions (CDR H1-3) and three light chain CDRs (CDR L1-3). The present invention also provides: an immunoconjugate or fusion polypeptide comprising the antibody; a pharmaceutical composition comprising the immunoconjugate or fusion polypeptide; and a method of treating a Tie2 dysregulated disease, the method comprising administering the pharmaceutical composition.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 938,816, filed on November 21, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Field of Invention The present invention relates to anti-Tie2 antibodies and methods for using them. [Background technology]

[0003] Background of the Invention Tie2 is a receptor tyrosine kinase primarily expressed on the surface of endothelial cells, playing a central role in vascular stability, survival, and maturation (Suri, C., et al., 1996. Requisite Role of Angiopoietin-1, a Ligand for the TIE2 Receptor, during Embryonic Angiogenesis. Cell 87(7):1171-80 (Non-Patent Literature 1)), Thurston, G., et al., 1999. Leakage-Resistant Blood Vessels in Mice Transgenically Overexpressing Angiopoietin-1. Science 286(5449):2511-14 (Non-Patent Literature 2), Saharinen, et al., 2010. How Do Angiopoietins Tie with Vascular Endothelial Growth Factors? Current Opinion in Hematology (Non-Patent Literature 3), Augustin, et al., 2009. Control of Vascular Morphogenesis and Homeostasis through the Angiopoietin-Tie System. Nature Reviews. Molecular Cell Biology 10(3):165-77 (Non-Patent Literature 4), Milam, et al., 2015. The Angiopoietin-Tie2 Signaling Axis in the Vascular Leakage of Systemic Inflammation. Tissue Barriers 3(1-2) (Non-Patent Literature 5). Tie2 activity is tightly regulated by at least four soluble protein factors known as angiopoietin 1-4. Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are considered to be major Tie2 function regulators. Under normal physiological conditions, high Ang1 levels and low Ang2 levels maintain constitutive activation of the Tie2 signaling axis.Specifically, Ang1 agonist ligands directly bind to the Tie2 receptor, leading to Tie2 clustering, autophosphorylation, and downstream signaling events, including activation of the PI3 kinase / Akt and MAPK pathways.

[0004] Gene targeting experiments have shown that the Ang / Tie signaling pathway is required for the physiological and pathological remodeling of lymphatic and vascular systems in fetal, postnatal, and adult mice (Eklund L, Kangas J, Saharinen P. Angiopoietin-Tie signaling in the cardiovascular and lymphatic systems. Clin Sci (Lond) 2017;131:87-103 (Non-Patent Literature 6)). In humans, modified expression of angiopoietin is associated with many vascular diseases (Saharinen P, Eklund L, Alitalo K. Therapeutic targeting of the angiopoietin-TIE pathway. Nat Rev Drug Discov 2017;16:635-61 (Non-Patent Literature 7)).

[0005] There is still a need for anti-Tie2 antibodies with improved properties, as well as for their therapeutic and diagnostic applications. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Suri, C., et al., 1996. Requisite Role of Angiopoietin-1, a Ligand for the TIE2 Receptor, during Embryonic Angiogenesis. Cell 87(7):1171-80 [Non-Patent Document 2] Thurston, G., et al., 1999. Leakage-Resistant Blood Vessels in Mice Transgenically Overexpressing Angiopoietin-1. Science 286(5449): 2511-14. [Non-Patent Document 3] Saharinen, et al., 2010. How Do Angiopoietins Tie with Vascular Endothelial Growth Factors? Current Opinion in Hematology [Non-Patent Document 4] Augustin, et al., 2009. Control of Vascular Morphogenesis and Homeostasis through the Angiopoietin-Tie System. Nature Reviews. Molecular Cell Biology 10(3): 165-77 [Non-Patent Document 5] Milam, et al., 2015. The Angiopoietin-Tie2 Signaling Axis in the Vascular Leakage of Systemic Inflammation. Tissue Barriers 3(1-2) [Non-Patent Document 6] Eklund L, Kangas J, Saharinen P. Angiopoietin-Tie signaling in the cardiovascular and lymphatic systems. Clin Sci (Lond) 2017; 131: 87-103 [Non-Patent Document 7] Saharinen P, Eklund L, Alitalo K. Therapeutic targeting of the angiopoietin-TIE pathway. Nat Rev Drug Discov 2017; 16: 635-61 [Summary of the Invention]

[0007] This invention provides anti-Tie2 antibodies and methods for using them for therapeutic and diagnostic purposes. The anti-Tie2 antibodies of this invention exhibit unique properties that make them particularly suitable for therapeutic use.

[0008] The following are specifically intended as part of the disclosed invention: Embodiment 1. An isolated anti-Tie2 antibody or its antigen-binding fragment comprising three heavy chain complementarity-determining regions (CDR H1-3) and three light chain CDRs (CDR L1-3): TIFF0007891318000001.tif109166TIFF0007891318000002.tif179166. Embodiment 2. An isolated anti-Tie2 antibody or its antigen-binding fragment, A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 242, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 243, A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 244, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 245, A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 246, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 247, A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 248, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 249, A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 250, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 251, A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 252, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 253, A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 254, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 255, A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 256, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 257, A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 258, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 259, A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 260, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 261, A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 262, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 263, A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 264, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 265, A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 266, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 267, A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 268, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 269, or A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 270, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 271. An isolated anti-Tie2 antibody or its antigen-binding fragment, containing [the specified substance]. Embodiment 3. An isolated anti-Tie2 antibody, The antibody specifically binds to an epitope within the extracellular domain of human Tie2 cells. The epitope in question is The amino acid residues K312, S316, C332, H358, K387, and T391, as measured by cross-linked mass spectrometry and EU numbering such as Kabat numbering. Isolated anti-Tie2 antibody containing [the specified substance]. Embodiment 4. The antibody according to Embodiments 1 to 3, wherein the antibody is an allosteric activator of Tie2. Embodiment 5. The antibody according to Embodiments 1 to 4, wherein the antibody is a non-ligand competitive binder for Tie2. Embodiment 6. The antibody according to Embodiments 3 to 5, wherein the antibody is cross-reactive to human, mouse, rat, rabbit, and monkey Tie2. Embodiment 7. The antibody according to Embodiments 1 to 6, wherein the antibody is fully human, humanized, monoclonal, or chimeric. Embodiment 8. The antibody according to Embodiments 1 to 7, wherein the antibody is monospecific. Embodiment 9. The antibody according to Embodiments 1 to 7, wherein the antibody is multispecific. Embodiment 10. The antibody according to Embodiment 9, wherein the multispecific antibody is bispecific. Embodiment 11. A bispecific antibody is One binding arm that specifically binds to human Tie-2 as described in Embodiment 8, A second binding arm that specifically binds to VEGF-A, VEGF-B, VEGF-C, VEGF variants, Ang-1, Ang-2, Ang-3, Ang-4, PDGF-β, interleukin-1β, VE-PTP, complement factor C3, integrin α5β1, amyloid beta, PD-1, PD-L1, or CTLA-4. The antibody according to Embodiment 10, including the antibody described above. Embodiment 12. The antibody according to Embodiment 9, wherein the multispecific antibody is a biparatopic antibody. Embodiment 13. A dual paratopic antibody is One binding arm specifically binds to the first epitope on the ECD of human Tie2, The other binding arm specifically binds to a second epitope on the ECD of human Tie2, and The antibody according to Embodiment 12, including the antibody described above. Embodiment 14. The multispecific antibody is a trivalent, tetravalent, pentavalent, or hexavalent antibody. Trivalent, tetravalent, pentavalent, or hexavalent antibodies, The 8th embodiment includes at least one binding arm that specifically binds to human Tie2, Other remaining binding arms that specifically bind to VEGF-A, VEGF-B, VEGF-C, VEGF variants, Ang-1, Ang-2, Ang-3, Ang-4, PDGF-β, interleukin-1β, VE-PTP, complement factor C3, integrin α5β1, amyloid beta, PD-1, PD-L1, or CTLA-4. The antibody according to Embodiment 9, including the antibody described in Embodiment 9. Embodiment 15. The antibody according to Embodiments 1 to 14, wherein the antibody is an antibody fragment that specifically binds to human Tie2. Embodiment 16. The antibody according to Embodiment 15, wherein the antibody fragment is Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. Embodiment 17. The antibody according to Embodiment 16, wherein the multispecific antibody is composed of scFv antibody fragments linked together by a polypeptide linker. Embodiment 18. The antibody according to Embodiments 1 to 17, wherein the antibody possesses reduced effector function. Embodiment 19. The antibody according to Embodiment 18, wherein the antibody contains at least one substitutional mutation at amino acid residues N297, L234, L235, P329, D265, and E430, according to EU numbering such as Kabat numbering. Embodiment 20. The antibody according to Embodiment 19, wherein at least one substitution mutation is selected from the group consisting of amino acid residues N297G, N297A, L234A, L235A, P329G, D265A, and E430G by EU numbering such as Kabat numbering. Embodiment 21. The antibody according to Embodiment 20, wherein the antibody contains a substitution mutation at residue N297A or N297G. Embodiment 22. The antibody according to Embodiment 20, wherein the antibody contains substitutional mutations at residues L234A, L235A, and P329G. Embodiment 23. The antibody according to Embodiment 20, wherein the antibody contains substitutional mutations at residues D265A and N297G. Embodiment 24. The antibody according to Embodiment 21, wherein the antibody further comprises a substitution mutation at residue E430G. Embodiment 25. The antibody according to Embodiment 22, wherein the antibody further comprises a substitution mutation at residue E430G. Embodiment 26. The antibody according to Embodiment 23, wherein the antibody further comprises a substitution mutation at residue E430G. Embodiment 27. The antibody according to Embodiment 25, wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 174 and a light chain containing the amino acid sequence of SEQ ID NO: 175. Embodiment 28. A heavy chain containing the amino acid sequence of SEQ ID NO: 276, and a light chain containing the amino acid sequence of SEQ ID NO: 277, A heavy chain containing the amino acid sequence of SEQ ID NO: 278, and a light chain containing the amino acid sequence of SEQ ID NO: 279. A heavy chain containing the amino acid sequence of SEQ ID NO: 280, and a light chain containing the amino acid sequence of SEQ ID NO: 281. A heavy chain containing the amino acid sequence of SEQ ID NO: 282, and a light chain containing the amino acid sequence of SEQ ID NO: 283, A heavy chain containing the amino acid sequence of SEQ ID NO: 286, and a light chain containing the amino acid sequence of SEQ ID NO: 287, or A heavy chain containing the amino acid sequence of SEQ ID NO: 288, and a light chain containing the amino acid sequence of SEQ ID NO: 289. The antibody according to Embodiment 22, including the antibody described above. Embodiment 29. The amino acid sequence of sequence number 284, or Amino acid sequence of SEQ ID NO: 285 The antibody according to Embodiment 10, including the antibody described above. Embodiment 30. An isolated nucleic acid encoding the antibody described in Embodiments 1 to 29. Embodiment 31. A vector comprising the isolated nucleic acid described in Embodiment 30. Embodiment 32. A host cell containing the vector described in Embodiment 31. Embodiment 33. A method for producing antibodies according to Embodiments 1 to 29, The host cells described in Embodiment 32 are cultured in a culture medium, The obtained antibodies are isolated and Methods that include... Embodiment 34. An immunoconjugate comprising the antibodies described in Embodiments 1 to 29. Embodiment 35. A fusion polypeptide comprising the antibodies described in Embodiments 1 to 29. Embodiment 36. A pharmaceutical composition comprising an antibody described in Embodiments 1 to 29, an immunoconjugate described in Embodiment 34, or a fusion polypeptide described in Embodiment 35. Embodiment 37. The pharmaceutical composition according to Embodiment 36, wherein an antibody, immunoconjugate, or fusion polypeptide is co-formulated with an anti-VEGF antibody or a VEGF extracellular trap protein. Embodiment 38. A method for treating a subject requiring treatment for a Tie2 dysregulation disorder, comprising administering to the subject the pharmaceutical composition described in Embodiment 36. Embodiment 39. A method for treating a subject requiring treatment for a Tie2 dysregulation disorder, comprising administering to the subject the pharmaceutical composition described in Embodiment 37. Embodiment 40. The method according to Embodiment 38, further comprising co-administering a pharmaceutical composition containing an anti-VEGF antibody or a VEGF extracellular trap protein to a target. Embodiment 41. The method according to Embodiments 38-40, wherein the Tie2 dysregulation disorder includes infection, acute respiratory distress syndrome (ARDS), ischemic injury, eye disorder, radiation injury, cancer, systemic sclerosis, traumatic brain injury, neuroinflammation, radiation injury, wound healing, myocardial infarction, blood-brain barrier injury, spongiform malformation, Duchenne muscular dystrophy (DMD), or Clarkson's disease. Embodiment 42. The method according to Embodiments 38-40, wherein the Tie2 dysregulation infection includes sepsis, dengue virus infection, tuberculosis, or influenza. Embodiment 43. The method according to Embodiments 38-40, wherein the Tie2 dysregulation ischemic injury includes diabetic nephropathy, acute kidney injury, chronic kidney disease, organ transplantation, severe lower limb ischemia, traumatic brain injury, or stroke. Embodiment 44. The method according to Embodiments 38-40, wherein the Tie2 accommodative ocular disorder includes diabetic retinopathy, diabetic macular edema (DME), proliferative diabetic retinopathy (PDR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP), or glaucoma. Embodiment 45. An isolated anti-Tie2 antibody according to Embodiments 1 to 29, or an immunoconjugate according to Embodiment 34, or a fusion polypeptide according to Embodiment 35, for use in the treatment of Tie2 dysregulation disorders according to Embodiments 41 to 44. Embodiment 46. Use of an isolated anti-Tie2 antibody described in Embodiments 1-29, or an immunoconjugate described in Embodiment 34, or a fusion polypeptide described in Embodiment 35, for the manufacture of a drug for treating Tie2 dysregulation disorders described in Embodiments 41-44. [Brief explanation of the drawing]

[0009] [Figure 1A] Figures 1A-1C: Anti-Tie2 antibodies increase the levels of phospho-Tie2 (pTie2), as well as phospho-Akt (pAkt) and phospho-ERK (pERK), as determined by Western blotting. HUVEC cells were serum-starved and then treated for 20 minutes with either PBS, Ang1 (12nM), control hIgG1 (12nM), or human anti-Tie2 antibodies #1, #2, and #3, or wild-type (WT) chicken anti-Tie2 antibody (12nM), followed by cell lysis. Figure 1A shows that Tie2 was immunoprecipitated from the lysate using an anti-Tie2 antibody, and the level of pTie2 was determined by Western blotting with an anti-phospho-tyrosine antibody. For pAkt and pERK analysis, Western blotting was performed on pAkt and total Akt in the whole cell lysate (Figure 1B), or pERK and total ERK (Figure 1C). See Example 3. [Figure 1B] Refer to the explanation in Figure 1A. [Figure 1C] Refer to the explanation in Figure 1A. [Figure 2]The functional efficacy of anti-Tie2 antibodies is comparable to that of Ang1 in vitro. HUVEC cells were serum-starved and treated with various concentrations (nM) of Ang1, human anti-Tie2 antibodies #1, #2, and #3, or WT chicken anti-Tie2 antibody, followed by cell lysis. Western blotting was performed using pAkt-specific or total Akt-specific antibodies. pAkt and total Akt signal intensities were measured using a LI-COR scanning fluorometer, and the level of pAkt normalized to total Akt (pAkt / Akt ratio) was plotted on the Y-axis using Graphpad® Prism software, which was then used to determine the EC50 value. See Example 4. [Figure 3] A schematic diagram of a simplified in vitro model of the endothelial barrier is shown. See Example 5. [Figure 4] Anti-Tie2 antibodies can reduce VEGF-induced cell permeability in an in vitro model of endothelial barrier leakage. Results are expressed as a percentage of Ang1 activity (normalized for PBS-treated cells). For statistical analysis (using a one-way ANOVA test), the effect of each antibody was compared to that of a nonspecific IgG1 control antibody. Symbols: **P ≤ 0.01, **** ≤ 0.0001. See Example 5. [Figure 5A]Figures 5A-5F: Anti-Tie2 antibody combined with saturated Ang2 has an additional effect on Tie2 downstream signaling in HUVEC cells. HUVEC cells were serum-starved, treated with reduced Ang2, lysed, and analyzed for pERK and total ERK levels. Normalized values ​​were plotted on the Y-axis, and Ang2 concentration on the X-axis (Figure 5A). Images were quantified using a LI-COR scanning fluorometer, and pERK levels were normalized relative to total ERK. Serum-starved HUVEC cells were treated with 43 nM Ang2 and 12 nM anti-Tie2 antibody for 20 minutes. Lysates were subjected to Western blotting, and images were quantified for pERK and ERK (Figure 5C), and pAKT and AKT (Figure 5E), respectively. pERK (Figure 5D) and pAKT (Figure 5F) levels were normalized relative to total ERK and Akt levels, respectively, to adjust for equivalent protein loading. Please refer to Example 6. [Figure 5B] Refer to the explanation in Figure 5A. [Figure 5C] Refer to the explanation in Figure 5A. [Figure 5D] Refer to the explanation in Figure 5A. [Figure 5E] Refer to the explanation in Figure 5A. [Figure 5F] Refer to the explanation in Figure 5A. [Figure 6] Figures 6A and 6B: Anti-Tie2 antibodies do not compete with either Ang1 (Figure 6A) or Ang2 (Figure 6B) for binding to the Tie2 extracellular domain (ECD). Representative competitive binding data using biolayer interferometry (BLI) is shown. The left panel shows loading of His-tagged Tie2 ECD protein (100 nM) onto a capture biosensor (Ni-NTA) at the start of exposure indicated by arrow (1), followed by exposure to a solution containing individual anti-Tie2 antibodies (100 nM) as indicated by arrow (2), and then exposure to a solution containing Ang1 (Figure 6A) or Ang2 ligand (Figure 6B) (both 100 nM) as indicated by arrow (3). The Y-axis represents the magnitude of binding to the biosensor, and the x-axis represents elapsed time. See Example 7. [Figure 7]Anti-Tie2 antibodies exhibit diversity in epitope recognition as assessed by BLI binding. Recombinant human Tie2 ECD protein was loaded onto each biosensor, then exposed to a solution containing an anti-Tie2 antibody (y-axis), followed by exposure to a second anti-Tie2 antibody (x-axis). The total wavelength shift (nm) after exposure to the second antibody is annotated in white numbers in each box. Black boxes indicate a lack of binding by the second antibody (value < 0.2 nm). The lack of binding was observed only with the same antibody pair. This indicates that all antibodies tested in this panel bind to different epitopes found on Tie2 ECD. See Example 9. [Figure 8] Figures 8A and 8B: Systemic administration of anti-Tie2 antibody did not appear to reduce angiogenesis (NV) (Figure 8A), but it did reduce vaso-obliteration (VO) in an oxygen-induced retinopathy (OIR) model (Figure 8B). Each data point represents an average of two eyes from one pup. Bars represent mean + / - SEM, one-way ANOVA with N=7-8, Tukey posterior |*p<0.05, **p<0.01, ***p<0.001. See Example 10. [Figure 9A] Figures 9A and 9B: Anti-Tie2 antibodies inhibit laser-induced choroidal neovascularization (CNV) in a mouse model. Neovascularization in the lesion and vascular density in the retina were quantified using ImageJ. P-values ​​were evaluated by Student's t-test (significant change, p<0.05). See Example 11. [Figure 9B] See the explanation in Figure 9A. [Figure 10A]Figures 10A and 10B: Schematic diagrams of the Tie2 / VEGF bispecific constructs of the present invention. Figure 10A shows a schematic diagram of an anti-Tie2 antibody linked to two VEGF scFv antibody fragments via a polypeptide linker. Exemplary constructs are embodied in antibody clones #57 and #58. Figure 10B shows a schematic diagram of two anti-Tie2 scFv antibody fragments linked to two VEGF-trap proteins consisting of R1-D2 and R3-D3. Exemplary constructs are embodied in antibody clones #55 and #56. See Example 13. [Figure 10B] Refer to the explanation in Figure 10A. [Figure 11] Tie2 agonism of Tie2 / VEGF bispecific constructs was evaluated by Western blotting. The control used was untreated and anti-Ang1 antibody (Ang1). The positive control was anti-Tie2 antibody clone #3 or nonspecific antibody (NS). The test bispecific antibodies were anti-Tie2 / VEGF bispecific (antibody clone #54), nonspecific antibody / VEGF bispecific (NS / VEGF), VEGF-trap / aTie2 (antibody clone #55), or VEGF-trap alone. See Examples 3 and 14. [Figure 12-1] Figures 12A–12C: Schematic diagrams of the tetravalent and biparatopic anti-Tie2 antibody constructs of the present invention for increasing the valence to Tie2 or crosslinking multiple Tie2 epitopes. Exemplary tetravalent anti-Tie2 construct having heavy and light chain sequences of an anti-Tie2 antibody linked via a polypeptide linker to an anti-Tie2 scFv sequence on the C-terminus of the IgG heavy chain (Figure 12A) or the N-terminus of the heavy chain (Figure 12B). Exemplary biparatopic anti-Tie2 antibody construct (Figure 12C) having two anti-Tie2 scFv sequences from one antibody clone (aTie2 scFv B) linked via a polypeptide linker to the C-terminus of the IgG heavy chain of a second different anti-Tie2 antibody clone (aTie2 Fab A). In all cases, Tie2 binding sites are indicated by hatched boxes. See Example 15. [Figure 12-2]See the explanation in Figure 12-1. [Figure 13] A schematic diagram of the hexamerized anti-Tie2 antibody construct of the present invention for enhancing oligomerization and thereby increasing Tie2 agonism. An exemplary hexamerized anti-Tie2 construct having heavy and light chain sequences of an anti-Tie2 antibody possessing the E430G Fc mutation is shown. See Example 15. [Figure 14] The Tie2 agonism of all the various anti-Tie2 antibody constructs of the present invention was evaluated by pAKT / pERK Western blotting. The controls used were untreated, anti-Ang1 antibody (Ang1), anti-Tie2 antibody clone #3 (aTie2), or nonspecific antibody (NS). The test antibody constructs were a hexamerized anti-Tie2 antibody with the E430G Fc mutation (antibody clone #50) and its control, a nonspecific hexamerized antibody (NS E430G), two tetravalent anti-Tie2 antibody constructs (antibody clones #51 and #52), and a bivalent anti-Tie2 antibody clone #2. See Examples 3, 14, and 15. [Modes for carrying out the invention]

[0010] Detailed description of the invention I. Definition "Tie2" is also known as the angiopoietin-1 receptor, or TEK receptor tyrosine kinase, or CD202B (differentiation cluster 202B), and is a protein encoded by the TEK gene in humans (Partanen Jet al., (April 1992). A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains. Molecular and Cellular Biology. 12(4):1698-707). This receptor possesses a unique extracellular domain containing three immunoglobulin-like loops, three epidermal growth factor-like repeats, and three fibronectin type III-like repeats (see Fiedler et al., 2006. Angiopoietins: A Link between Angiogenesis and Inflammation. Trends in Immunology 27(12):552-58; Barton et al., Crystal structures of the Tie2 receptor ectodomain and the angiopoietin-2-Tie2 complex. Nature Struc.&Mol.Biology, 13, pp524-532 (2006)). The contact residues of angiopoietin-1 and angiopoietin-2 largely overlap on the Tie-2 receptor and are located primarily within the second Ig-like loop, as suggested by analysis of the crystal structure of the Ang2 / Tie2 complex (Barton et al., Nat Str Biol 2006).Other studies support the idea that the binding domains of Ang1 and Ang2 are similar or identical (Fiedler et al., Angiopoietin-1 and angiopoietin-2 share the same binding domains in the Tie-2 receptor involving the first Ig-like loop and the epidermal growth factor-like repeats. JBC. Vol.278(3):1721-7(2003)). An exemplary human Tie2 amino acid sequence can be found in UniProt accession number Q02763 (sequence number 241).

[0011] As used herein, the term “about” refers to the normal range of error for each value, which is readily known to those skilled in the art. References to values ​​or parameters with “about” herein include (and are described) embodiments that apply to the value or parameter itself.

[0012] For the purposes of this specification, “acceptor human framework” is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or human consensus framework as defined below. An acceptor human framework “derived” from a human immunoglobulin framework or human consensus framework may include the same amino acid sequence or may include amino acid sequence variations. In some embodiments, the number of amino acid variations is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0013] In the context of the antibodies of this invention, "active," "active," or "biologically active" refers to the ability to agonize (partially or completely activate) the biological activity of its target, for example, in vitro and / or in vivo. An example of the biological activity of an antibody is its ability to achieve a measurable improvement in a disorder state related to its target, e.g., in a pathology. For example, with respect to an anti-Tie2 antibody, the disorder could be a Tie2-related disorder such as AMD (e.g., geographic atrophy). The activity of an anti-Tie2 antibody can be determined in vitro or in vivo tests, including binding assays and activity assays (e.g., FRET-based activity assays (e.g., using H2-Opt substrates), or mass spectrometry-based activity assays or signal transduction assays), using relevant animal models or human clinical trials. The activity of the anti-Tie2 antibody of this invention can be determined in vitro or in vivo tests, including binding assays and alternative pathway hemolysis assays (e.g., assays measuring inhibition of alternative pathway complement activity or activation), using relevant animal models or human clinical trials.

[0014] The term "active site of Tie2" is defined as the Ang1 / Ang2 binding domain on Tie2, which is known to be located within the Ig2-like domain of the extracellular domain of Tie2.

[0015] "Affinity" refers to the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by its dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0016] A "affinity-mature" antibody refers to an antibody that possesses one or more modifications in one or more hypervariable regions (CDRs) and / or framework regions (FRs), compared to a parent antibody that does not possess such modifications, and in which case the antibody's affinity for the antigen is improved.

[0017] "Allosteric activation of Tie2" refers to the activation of Tie2 by an agonist anti-Tie2 antibody that specifically interacts with the Tie2 region outside of the described ligand-binding or active site. As a result, binding alters the Tie2 conformation or clustering, enhancing receptor activity.

[0018] The term "antibody" as used herein is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0019] An "antibody fragment" refers to a molecule other than the intact antibody that contains a portion of the intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0020] Papain digestion of the antibody produces two identical antigen-binding fragments called "Fab" fragments and the remaining "Fc" fragment, a name reflecting its ability to readily crystallize. The Fab fragments consist of the entire light (L) chain, along with the variable region domain (VH) of the heavy (H) chain and the first constant domain (CH1) of one heavy chain. Pepsin treatment of the antibody yields a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and is still capable of crosslinking antigens. The Fab' fragments differ from the Fab fragments in that they have several additional residues at the carboxyl terminus of the CH1 domain, which contains one or more cysteines derived from the antibody hinge region. Fab'-SH is the herein designation for Fab' fragments in which the cysteine ​​residue of the constant domain has a free thiol group. The F(ab')2 antibody fragments were originally produced as a pair of Fab' fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.

[0021] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. The term includes both native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0022] "Fv" consists of a dimer of one heavy chain variable domain and one light chain variable domain in a closely coupled, non-covalent association state. The folding of these two domains gives rise to six hypervariable loops (three from the H and L chains respectively) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to an antigen, but often with lower affinity than the entire binding site.

[0023] Single-stranded Fv, also abbreviated as "sFv" or "scFv," is an antibody fragment containing VH and VL antibody domains attached to a single polypeptide chain. The sFv polypeptide may further contain a polypeptide linker between the VH and VL domains, enabling the sFv to form a structure desirable for antigen binding. For an overview of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0024] The term "diabody" refers to a small antibody fragment prepared by constructing an sFv fragment (see previous paragraph) with a short linker (approximately 5-10 residues) between the VH and VL domains so that interchain pairing, rather than intrachain pairing, is achieved in the V domain, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. A bispecific diabody is a heterodimer of two "crossover" sFv fragments, where the VH and VL domains of the two antibodies are located on different polypeptide chains. For more information on diabodies, see, for example, EP404,097, WO93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448, 1993.

[0025] A "blocking" antibody or "antagonist" antibody inhibits or reduces the biological activity of the antigen to which it binds. Certain blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0026] An “agonist” or “activating” antibody is an antibody that activates, stimulates, or increases the biological or signaling activity of the antigen to which it binds. In some situations, an agonist antibody is intended to act in a similar manner to how a ligand engages with and activates its homologous receptor. In other situations, an anti-Tie2 antibody of the present invention is intended to be an agonist if it induces Tie2 signaling, as determined by an increase in the level of one or more of intracellular phosphorylated Tie2 (pTie2), and / or phosphorylated Akt (pAkt), and / or phosphorylated ERK (pERK), as described in Example 3. Furthermore, as described in Examples 6 and 7, an agonist Tie2 antibody of the present invention is also intended to activate downstream signaling of its target antigen in the presence or absence of an endogenously activating (i.e., Ang1) or inhibitory (i.e., Ang2) ligand.

[0027] An antibody that "binds to the same epitope as the reference antibody" refers to an antibody that, compared to the reference antibody, contacts a duplicate set of amino acid residues of the antigen or blocks the binding of the reference antibody to that antigen by 50% or more in a competitive assay. The amino acid residues of the antibody that contact the antigen can be determined, for example, by determining the crystal structure of the antibody in the complex with the antigen or by performing hydrogen / deuterium exchange. In some embodiments, residues of the antibody that are within 5 angstroms from the antigen are considered to be in contact with the antigen. In some embodiments, an antibody that binds to the same epitope as the reference antibody blocks the binding of the reference antibody to that antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to that antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

[0028] As used herein, the term “dual paratopic” refers to a bispecific antibody in which a first antigen-binding moiety and a second antigen-binding moiety bind to different epitopes on the same antigen.

[0029] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0030] The "class" of an antibody refers to the type of constant domain or constant region contained in its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0031] "Complement factors" refer to the various proteins and glycoproteins that make up the complement cascade, a part of the immune system, which enhances (complements) the ability of antibodies and phagocytic cells to eliminate microorganisms and damaged cells from organisms, promote inflammation, and attack the cell membranes of pathogens. They are part of the innate immune system. Examples of complement factors intended herein include C1, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, and C9.

[0032] "Effector function" refers to the biological activity resulting from the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0033] The term "framework," "framework region," or "FR" refers to variable domain residues other than those in the hypervariable region (CDR). The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4.

[0034] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein.

[0035] The term “fusion polypeptide” encompasses, for example, the anti-Tie2 antibody of the present invention fused to an immunoglobulin Fc region. The Fc region may, for example, include the CH3 domain of an immunoglobulin, which may be naturally occurring or modified in some way. Such an Fc fusion polypeptide may exhibit a higher in vivo half-life than its unfused counterpart. Furthermore, fusion to the Fc region allows for dimerization / multimerization of the fusion polypeptide. As intended, the Fc region may be a naturally occurring Fc region or may be modified to improve specific qualities, such as therapeutic quality, circulation time, or reduced aggregation problems. In another embodiment, the fusion polypeptide is intended to be an anti-Tie2 antibody fragment that fuses to a ligand, such as Ang1, to enhance the agonist activity of the fusion polypeptide. In yet another embodiment, the fusion polypeptide is intended to be an anti-Tie2 antibody fragment that fuses to a cytokine, for example, to induce other desired biology.

[0036] As used herein, "hexamerized antibody" refers to an antibody in which the introduction of the E430G mutation in the Fc region facilitates the natural process of antibody hexamer formation by increasing intermolecular Fc-Fc interactions upon binding to membrane-bound antigens on the cell surface (Diebolder et al., Science. 2014, de Jong et al., PLoS Biol. 2016).

[0037] A "human antibody" is one that is produced by a human or human cell, or possesses an amino acid sequence corresponding to the amino acid sequence of an antibody derived from a non-human source that utilizes sequences encoding the human antibody repertoire or other human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0038] The "Human Consensus Framework" is a framework representing the most commonly present amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Generally, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup Kappa I, as described in Kabat et al. In one embodiment, for VH, the subgroup is subgroup III, as described in Kabat et al.

[0039] The "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody that contains a minimal amount of sequences derived from the non-human antibody. In most cases, the humanized antibody is a human immunoglobulin (recipient antibody), and residues from the recipient's hypervariable region are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, that possess the desired antibody specificity, affinity, and capabilities. In some cases, the FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the recipient or donor antibody. These modifications are made to further improve the performance of the antibody. Generally, the humanized antibody will contain substantially all of at least one, typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of the non-human immunoglobulin, and all or substantially all of the FRs being from the human immunoglobulin sequence. Humanized antibodies will also optionally contain at least a portion of the constant region (Fc) of an immunoglobulin, typically that of human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr Op. Struct. Biol. 2:593-596 (1992).

[0040] The term "variable" refers to the fact that a particular segment of the variable domain has a significantly different sequence from antibody to antibody. The variable domain, or "V" domain, mediates antigen binding and defines the specificity of a particular antibody to that particular antigen. However, variability is not uniformly distributed across the span of the variable domain. Instead, the V region consists of relatively invariant extensions called framework regions (FRs) of 15 to 30 amino acids, separated by shorter, highly variable regions called "hypervariable regions," each 9 to 12 amino acids long. As used herein, the term "hypervariable region" or "CDR" refers to the amino acid residues of the antibody responsible for antigen binding. The hypervariable region generally consists of amino acid residues from approximately 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the VL region, and approximately 26-35 (H1), 49-65 (H2), and 95-102 (H3) in the VH region (in one embodiment, H1 is approximately 31-35) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of The variable domains of the natural heavy and light chains each contain four FRs, which primarily employ a beta-sheet configuration and are connected by three hypervariable regions that form loops connecting the beta-sheet structures, and in some cases forming parts thereof.The hypervariable regions of each chain are held together in close proximity by the FR and, together with the hypervariable regions from other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Therefore, the CDR and FR sequences are generally located within the following sequence in the VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. The constant domain does not directly participate in antibody binding to the antigen, but exhibits various effector functions, such as antibody-dependent cytotoxicity (ADCC).

[0041] The terms “Kabat numbering or similar residue numbering,” “Kabat amino acid residues,” or “Kabat numbering or similar amino acid positional numbering,” and variations thereof, refer to the numbering system used in the heavy-chain or light-chain variable domains of antibody compilation in Kabat et al. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion into the FR or CDR of the variable domain. For example, the heavy-chain variable domain may include a single amino acid insert after H2 residue 52 (residue 52a according to Kabat) and an insertion residue after heavy-chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). Kabat residue numbering can be determined for a given antibody by alignment of the antibody sequence in regions of homology with “standard” Kabat numbered sequences.

[0042] The Kabat numbering system is generally used when referring to residues within the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (see, for example, Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues within the constant region of the immunoglobulin heavy chain (for example, the EU index reported by Kabat et al. above). "EU index such as Kabat numbering" refers to the residue numbering of human IgG1 EU antibodies. Unless otherwise specified herein, references to residue numbers in the variable domain of an antibody mean residue numbering by the Kabat numbering system. Unless otherwise specified herein, references to residue numbers in the constant domain of an antibody mean residue numbering by the EU numbering system. Unless otherwise specified, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al. above.

[0043] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules that deliver cell-killing or cell-modifying activity, including but not limited to small molecule drugs (inhibitors or activators), cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes.

[0044] Where used to describe the various antibodies disclosed herein, the term “isolated antibody” means an antibody identified, isolated, and / or recovered from the cells or cell cultures on which it is expressed. Contaminants in its natural environment are typically substances that interfere with the diagnostic or therapeutic use of polypeptides and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, antibodies are purified to a purity of 95% or greater than 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion-exchange or reverse-phase HPLC) approaches. For an overview of antibody purity assessment methods, see, for example, Flatman et al., J.Chromatogr.B 848:79-87 (2007). In certain embodiments, the antibody would be purified to the extent that (1) at least 15 residues of the N-terminal or internal amino acid sequence can be obtained using a spinning cup sequencer, or (2) homogenized by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver staining. The isolated antibody contains the antibody in situ within recombinant cells, as it is devoid of at least one component of the polypeptide's natural environment. However, typically, the isolated polypeptide would be prepared by at least one purification step.

[0045] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, except for possible variant antibodies, such as those containing spontaneous mutations or arising during the production of the monoclonal antibody preparation, and such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain antibodies with different directivity to different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or some of the human immunoglobulin loci, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0046] The term "multispecific antibody" is used in its broadest sense and specifically encompasses antibodies or antibody fragments that possess polyepitope specificity (i.e., those that can bind to two different epitopes on one biological molecule, or to each epitope on different biological molecules). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies having two or more VL and VH domains, antibody fragments, e.g., Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and triabodies, and covalently or non-covalently linked antibody fragments. "Multiepitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. "Dual specificity" or "bispecificity" refers to the ability to specifically bind to two different epitopes on the same or different targets. However, in contrast to bispecific antibodies, dual-specific antibodies have two antigen-binding arms with identical amino acid sequences, and each Fab arm can recognize two antigens. Dual-specificity allows an antibody to interact with two different antigens with high affinity as a single Fab or IgG molecule. According to one embodiment, a multispecific antibody in IgG1 form binds to each epitope with affinities of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM. "Monospecific" refers to an antibody that has the ability to bind to only one epitope on a particular antigen.

[0047] Regarding the binding of an antibody to a target molecule, the term "specific binding" to a particular polypeptide or an epitope on a particular polypeptide target, or "specifically binds to" or "is specific for" a particular polypeptide or an epitope on a particular polypeptide target, means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is shown when the binding of the labeled target to the probe is competitively inhibited by the excess of unlabeled target. As used herein, the term "specific binding" to a particular polypeptide or an epitope on a particular polypeptide target, or "specifically binds to" or "is specific for" a particular polypeptide or an epitope on a particular polypeptide target means, for example, 10 -4 M or less, alternatively 10 -5 M or less, alternatively 10 -6 M or less, alternatively 10 -7 M or less, alternatively 10 -8 M or less, alternatively 10 -9 M or less, alternatively 10 -10 M or less, alternatively 10 -11 M or less, alternatively 10 -12 M or less for the KD for the target, or 10 -4 M to 10 -6 M, or 10 -6 M to 10 -10 M, or 10 -7 M to 10 -9 M and can be shown by a molecule having a KD in the range. As will be understood by those skilled in the art, affinity and KD values are inversely related. High affinity for an antigen is measured by a low KD value. In one embodiment, the term "specific binding" refers to the binding of a molecule, such as an antibody of the present invention, to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0048] The term "non-ligand competitive binder" refers to the anti-Tie2 antibody of the present invention that does not compete with either Ang1 or Ang2 for the active site of Tie2, while still allowing either Ang1 or Ang2 to bind at the active site.

[0049] "Antibody-encoding nucleic acid" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, where such nucleic acid molecules are located at one or more positions within a host cell. In some embodiments, the nucleic acid encodes an anti-Tie2 antibody.

[0050] The term “vector,” as used herein, refers to a nucleic acid molecule capable of increasing the amount of another nucleic acid to be ligated. This term includes vectors as self-replicating nucleic acid structures, and vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors can direct the expression of nucleic acids into which they are operably ligated. Such vectors are referred herein to as “expression vectors.”

[0051] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring regardless of passage number. Offspring may contain mutations, although they may not be completely identical to the parent cells in terms of nucleic acid content. Mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells are included herein.

[0052] The "percentage of amino acid sequence identity (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of amino acid sequence identity can be achieved in various ways within the scope of the art of the art using commonly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A person skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0053] Proteins, including antibodies, are said to be "stable" if they retain essentially intact three-dimensional structure and biological activity. Various analytical techniques for measuring protein stability are available in this field, and are outlined, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones (1993) Adv. Drug Delivery Rev. 10:29-90. Antibody variants with "improved stability" refer to antibody variants that are more stable than the starting reference antibody. Antibody variants with improved stability are variants of the reference (wild-type) antibody in which specific amino acid residues have been modified for the purpose of improving the physical stability, and / or chemical stability, and / or biological activity of the native antibody, and / or reducing immunogenicity.

[0054] In certain embodiments, anti-Tie2 antibodies can be used as therapeutic agents that target and interfere with diseases or conditions involving Tie2 activity. The antibodies may also be subjected to other biological activity assays, for example, to evaluate their efficacy as therapeutic agents. Such assays are known in the art and depend on the target antigen and the intended use of the antibody. Examples include HUVEC inhibition assays, tumor cell growth inhibition assays (e.g., as described in WO89 / 06692), antibody-dependent cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (U.S. Patent No. 5,500,362), and agonist activity or hematopoietic assays (see WO95 / 27062).

[0055] As used herein, the term “triply specific” refers to a type of antibody having three antigen recognition and binding sites, some of which may bind to Tie2. In other cases, at least one binding arm may specifically bind to Tie2, and the other binding sites may bind to either Tie2 or another antigen of interest (listed as “bispecific antibodies”). In one embodiment, such a trispecific antibody comprises an antibody fragment (e.g., Fab, scFv, single-domain antibody). As a non-limiting example, the three antibody-binding fragments of the present invention may be assembled into a trispecific antibody such that at least one antibody-binding fragment binds to Tie2, and the remaining antibody-binding fragments bind to another antigen, such as VEGF. See Runcie et al., Bi-specific and Tri-specific antibodies - the next big thing in solid tumor therapeutics., Mol. Med., 24, (50) (2018).

[0056] As used herein, the term “quadrispecific” refers to a type of antibody having four antigen recognition and binding sites, some of which may bind to Tie2. In other cases, at least one binding arm may specifically bind to Tie2, and the other binding sites may bind to either Tie2 or another antigen of interest (listed as “bispecific antibodies”). In one embodiment, such a quadruplespecific antibody comprises an antibody fragment (e.g., Fab, scFv, single-domain antibody). As a non-limiting example, the four antibody-binding fragments of the present invention may be assembled into a quadruplespecific antibody such that two antibody-binding fragments bind to Tie2 and the other two antibody-binding fragments bind to another antigen, such as VEGF.

[0057] As used herein, the term “quintuple specificity” refers to a type of antibody having five antigen recognition and binding sites, some of which may bind to Tie2. In other cases, at least one binding arm may specifically bind to Tie2, and the other binding sites may bind to either Tie2 or another antigen of interest (listed as “bispecific antibodies”). In one embodiment, such a quintuple specific antibody comprises an antibody fragment (e.g., Fab, scFv, single-domain antibody). As a non-limiting example, the five antibody-binding fragments of the present invention may be assembled into a quintuple specific antibody such that at least one antibody-binding fragment binds to Tie2, and the other antibody-binding fragments bind to another antigen, such as VEGF.

[0058] As used herein, the term “hexaspecific” refers to a type of antibody having six antigen recognition and binding sites, some of which may bind to Tie2. In other cases, at least one binding arm may specifically bind to Tie2, and the other binding sites may bind to either Tie2 or another antigen of interest (listed as “bispecific antibodies”). In one embodiment, such a hexaspecific antibody comprises antibody fragments (e.g., Fab, scFv, single-domain antibodies). As a non-limiting example, the six antibody-binding fragments of the present invention may be assembled into a hexaspecific antibody such that at least one antibody-binding fragment binds to Tie2, and the other antibody-binding fragments bind to another antigen, such as VEGF.

[0059] As used herein, “polypeptide linker” is a polypeptide comprising two or more amino acid residues joined by a peptide bond, used to link two polypeptides (e.g., a VH domain and a VL domain, two scFv antibody fragments, or a variable domain and an extracellular trap protein, or an scFv antibody fragment and an extracellular trap protein). The linker may be flexible or rigid / inflexible. Examples of such linker polypeptides are well known in the art (see, for example, Hollinger P, et al., PNAS USA. 90:6444-6448 (1993), Poljak RJ. Structure 2:1121-1123 (1994)). Non-limiting examples of suitable non-immunogenic linker peptides include flexible peptide linkers of (G4S)n, (SG4)n, or G4(SG4)n, or rigid / inflexible linkers (EAAAK)n or (XP)n (in all cases, "n" is a number from 1 to 10 or from 1 to 4), and oligomers of such linkers.

[0060] "Tie2 dysregulation disorder" is any condition that would benefit from treatment with the anti-Tie2 antibody of the present invention. Non-limiting examples of diseases treated herein include, but are not limited to, any disease or disorder resulting from an imbalance or disruption of the interaction between Ang-1, Ang-2, Ang-3, or Ang-4 and Tie2. Non-limiting examples may include, for example, infections, acute respiratory distress syndrome (ARDS), ischemic injury, eye disorders, radiation injury, cancer, systemic sclerosis, traumatic brain injury, radiation injury, wound healing, myocardial infarction, blood-brain barrier injury (i.e., Alzheimer's disease or other neurodegenerative diseases), neuroinflammation, spongiform malformation, Duchenne muscular dystrophy (DMD), or Clarkson's disease. In one embodiment, Tie2 dysregulation infections include sepsis, dengue virus infection, tuberculosis, or influenza. In another embodiment, Tie2 dysregulation disorders may include, for example, diabetic nephropathy, acute kidney injury, chronic kidney disease, kidney or other organ transplantation, severe limb ischemia, traumatic brain injury, or ischemic injury such as stroke. In another embodiment, Tie2 dysregulation ocular disorders may include, for example, diabetic retinopathy, diabetic macular edema (DME), proliferative diabetic retinopathy (PDR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP), or glaucoma.

[0061] As used herein, “administer” means a method of administering a dosage of a therapeutic agent (e.g., the anti-Tie2 antibody of the present invention, the nucleic acid encoding the anti-Tie2 antibody of the present invention) or a composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising the anti-Tie2 antibody of the present invention) to a subject in need of it. The compositions used in the methods described herein may be administered, for example, intravitreously (e.g., by intravitreous injection), ocularly (e.g., by ocular injection), intraocularly (e.g., by intraocular injection), subcutaneously, or intravenously. The compositions used in the methods described herein may also be administered systemically or topically. The method of administration may vary depending on various factors (e.g., the compound or composition being administered, and the severity of the condition, disease, or disorder being treated).

[0062] As used herein, “co-administration” means administering two or more separate therapeutic agents (e.g., the anti-Tie2 antibody and anti-VEGF antibody therapeutic agents or recombinant VEGF fusion protein therapeutic agents of the present invention) or compositions (e.g., the anti-Tie2 antibody pharmaceutical composition and anti-VEGF antibody composition or recombinant VEGF fusion protein composition of the present invention) concurrently or at the same time to a subject requiring them.

[0063] As used herein, “co-formulation” means two or more distinct therapeutic agents (e.g., the anti-Tie2 antibody and anti-VEGF antibody therapeutic or recombinant VEGF fusion protein therapeutic of the present invention) or compositions (e.g., the anti-Tie2 antibody pharmaceutical composition and anti-VEGF antibody composition or recombinant VEGF fusion protein composition of the present invention) that are combined as a single formulation to be administered to a target requiring it.

[0064] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, livestock (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans, and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human. The “subject” may also be a “patient.”

[0065] As used herein, “treatment” (and “to treat” or “to treat”) refers to a clinical intervention in an attempt to alter the natural course of the individual being treated, which may be performed for preventive purposes or during the course of a clinicopathological condition. Desired effects of treatment include, but are not limited to, prevention of recurrence of disease or disorder, relief of symptoms, reduction of any direct or indirect pathological consequences of disease or disorder, reduction of the rate of disease progression, improvement or mitigation of the state of disease or disorder, and remission or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or disorder, or to slow the progression of disease or disorder.

[0066] Where used herein, the terms “cell,” “cell line,” and “cell culture” are interchangeable, and all such names include offspring. Thus, the terms “transformed” and “transformed cell” include the original target cells and cultures from which they originate, regardless of the number of transfers. It is also understood that, due to intentional or accidental mutations, not all offspring may be exactly identical in DNA content. This includes mutant offspring that possess the same function or biological activity as those screened in the initially transformed cells. Where a separate designation is intended, it will be evident from the context.

[0067] A "mutation" is the deletion, insertion, or substitution of a nucleotide in a reference nucleotide sequence, such as the wild-type sequence.

[0068] A “variant” or “mutant” of a starting polypeptide or reference polypeptide (e.g., a reference antibody or its variable domain / CDR) is a polypeptide that (1) has a different amino acid sequence from that of the starting or reference polypeptide, and (2) is derived from the starting or reference polypeptide through either natural or artificial (artificial) mutagenesis. Such variants include, for example, deletions of residues from within the amino acid sequence of the target polypeptide, and / or insertions of residues, and / or substitutions of residues, which are referred to herein as “amino acid residue modifications.” Thus, a variant CDR refers to a CDR containing a variant sequence with respect to a starting or reference polypeptide sequence (such as that of a source antibody or antigen-binding fragment). Amino acid residue modifications, in this context, refer to amino acids different from the amino acids at the corresponding positions in the starting or reference polypeptide sequence (e.g., the sequence of a reference antibody or fragment thereof). Any combination of deletions, insertions, and substitutions may be performed to arrive at a final variant or mutant construct, provided that the final construct possesses the desired functional characteristics. Amino acid changes can also modify the post-translational processes of the polypeptide, such as changing the number or location of glycosylation sites.

[0069] As used herein, “VEGF extracellular trap protein” or “VEGF-trap” is also known as Aflibercept (Eylea®, Regeneron-Bayer HealthCare, Tarrytown, NY, US). It consists of ligand-binding elements derived from the extracellular components of VEGF receptors 1 and 2, fused to the Fc portion of IgG1. It binds with high affinity to all isoforms of VEGF-A, VEGF-B, and placental growth factor (PlGF), effectively preventing VEGF-A and PlGF ligands from binding to and activating the cell receptor.

[0070] The “wild-type (WT)” or “reference” sequence, i.e., the “wild-type” or “reference” protein / polypeptide sequence, such as the CDR or variable domain of a reference antibody, may be the reference sequence from which the variant polypeptide is derived through the introduction of mutations. Generally, the “wild-type” sequence for a given protein is the most common sequence in nature. Similarly, the “wild-type” gene sequence is the sequence of that gene most commonly found in nature. Mutations can be introduced into a “wild-type” gene (and therefore the protein encoded by that gene) through natural processes or by means induced by humans. The products of such processes are the “variant” or “mutant” form of the original “wild-type” protein or gene.

[0071] When used herein, “reference antibody” refers to an antibody or fragment thereof whose antigen-binding sequence serves as a template sequence on which diversification is performed according to the criteria described herein. The antigen-binding sequence generally includes an antibody variable region, including a framework region, and at least one CDR.

[0072] Compositions and methods The present invention provides novel antibodies that bind to Tie2, as well as methods for producing and using them, for example, for therapeutic and diagnostic applications. The antibodies of the present invention are useful for the diagnosis or treatment of various disorders, including, for example, Tie2 dysregulation disorders described herein.

[0073] The techniques and procedures described or referenced herein are generally well understood, and can be found in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FMAusubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GRTaylor eds. (1995)); Harlow and Lane, eds. (1988); Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1998) Academic Press, Animal Cell Culture (RIFreshney), ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons, Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.), Gene Transfer Vectors for Mammalian Cells (JMMiller and Μ.P.Calos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (JEColigan et al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using Antibodies: A Laboratory Manual (E. Harlow and D.Lane(Cold Spring Harbor Laboratory It is commonly used by those skilled in the art, using conventional methodologies such as those described in *The Antibodies* (M. Zanetti and JDCapra, eds., Harwood Academic Publishers, 1995), which is widely used.

[0074] The anti-Tie2 antibodies of the present invention described herein, and any antibodies for use in the methods described herein, may have any of the characteristics described herein, either alone or in combination.

[0075] In a particular embodiment, the anti-Tie2 antibody provided herein is approximately 1 μM, approximately 100 nM, approximately 10 nM, approximately 1 nM, approximately 0.1 nM, approximately 0.01 nM, or approximately 0.001 nM (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10-9 M~10 -13It has a dissociation constant (KD) of M. For example, in some cases, the antibody provided in this invention binds to human Tie2 (huTie2) with a KD of about 10 nM or less. In some cases, the antibody provided herein binds to huTie2 with a KD of about 5 nM or less. In some cases, the antibody provided herein binds to huTie2 with a KD of about 2 nM or less. For example, in some cases, this antibody is present in concentrations of approximately 25 pM to 2 nM (e.g., approximately 25 pM, 50 pM, 75 pM, 100 pM, 125 pM, 150 pM, 175 pM, 200 pM, 225 pM, 250 pM, 275 pM, 300 pM, 325 pM, 350 pM, 375 pM, 400 pM, 425 pM, 450 pM, 475 pM, 500 pM, 525 pM, 550 pM, 575 pM, and 600 pM). It binds to huTie2 with a KD of approximately 625 pM, 650 pM, 675 pM, 700 pM, 725 pM, 750 pM, 775 pM, 800 pM, 825 pM, 850 pM, 875 pM, 900 pM, 925 pM, 950 pM, 975 pM, 1 nM, 1.1 nM, 1.2 nM, 1.3 nM, 1.4 nM, 1.5 nM, 1.6 nM, 1.7 nM, 1.8 nM, 1.9 nM, or 2 nM. In some cases, this antibody binds to huTie2 with a KD of approximately 75 pM to 600 pM (for example, approximately 75 pM, 100 pM, 125 pM, 150 pM, 175 pM, 200 pM, 225 pM, 250 pM, 275 pM, 300 pM, 325 pM, 350 pM, 375 pM, 400 pM, 425 pM, 450 pM, 475 pM, 500 pM, 525 pM, 550 pM, 575 pM, and 600 pM). In some cases, the antibody binds to huTie2 with a KD of approximately 75 pM to 500 pM. In some cases, the antibody binds to huTie2 at a KD of approximately 75 pM to 400 pM. In some cases, the antibody binds to huTie2 at a KD of approximately 75 pM to 300 pM. In some cases, the antibody binds to huTie2 at a KD of approximately 75 pM to 200 pM. In some cases, the antibody binds to huTie2 at a KD of approximately 75 pM to 150 pM.In some cases, the antibody binds to huTie2 at a KD of approximately 75 pM to 125 pM. In some cases, the antibody binds to huTie2 at a KD of approximately 75 pM to 100 pM. In some cases, the antibody binds to huTie2 at a KD of approximately 80 pM. In some cases, the antibody binds to huTie2 at a KD of approximately 60 pM. In some cases, the antibody binds to huTie2 at a KD of approximately 40 pM.

[0076] In one embodiment, KD is measured by a radiolabeled antigen-binding assay (RIA). In one embodiment, the RIA is performed with the Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of Fab to the antigen is measured in the presence of a step-dose series of unlabeled antigens, with Fab at the lowest concentration ( 125 I) The binding antigen is measured by equilibrating with a labeled antigen and then capturing it with a plate coated with anti-Fab antibody (e.g., Chen et al., J.Mol.Biol.293:865-881 (1999)). To establish conditions for the assay, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS at room temperature (approx. 23°C) for 2–5 hours. In a non-adsorbent plate (Nunc#269620), 100 pM or 26 pM [ 125I) Mix the antigen with a serial dilution of the Fab of interest (see Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although incubation may be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, 150 μl / well of scintillant (MICROSCINT-20®, Packard) is added and the plate is counted on a TOPCOUNT® gamma counter (Packard) for 10 minutes. The concentration of each Fab that gives less than 20% of maximum binding is selected for use in competitive binding assays.

[0077] In another embodiment, KD is measured using a BIACORE® surface plasmon resonance (SPR) assay. For example, assays using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C using an antigen CM5 chip immobilized at approximately 10 response units (RUs). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIAcore, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) in 10 mM sodium acetate at pH 4.8 and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RUs) of the coupling protein. After injecting the antigen, 1M ethanolamine is injected to block unreacted groups. For kinetic analysis, Fab's 2-fold serial dilutions (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μl / min into PBS containing 0.05% polysorbate 20 (TWEEN®-20) surfactant (PBST) at 25°C. The association rate (kon ) and dissociation rate (k off The kD is calculated using a simple one-to-one Langmuir coupled model (BIACORE® evaluation software version 3.2) by simultaneously fitting association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is k off / k on It is calculated as a ratio of . For example, see Chen et al., J.Mol.Biol.293:865-881(1999). The above surface plasmon resonance assay results in an on-rate of 10 6 M -1 s -1 If it exceeds , the on-rate can be determined using fluorescence quenching techniques, which measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of a 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen, measured with a spectrometer such as a stop-flow spectrometer with a stirred cuvette (Aviv Instruments) or an 8000 series SLM-AMINCO® spectrophotometer (ThermoSpectronic). KD may also be measured using the BIACORE® SPR assay, which is known in the art.

[0078] In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For an overview of a particular antibody fragment, see Hudson et al., Nat. Med. 9:129-134 (2003). For an overview of the scFv fragment, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). Also see WO93 / 16185, and U.S. Patents 5,571,894 and 5,587,458. For a discussion of the Fab and F(ab')2 fragments, which contain salvage receptor-binding epitope residues and exhibit increased in vivo half-lives, please refer to U.S. Patent No. 5,869,046.

[0079] Diabodies are antibody fragments having two antigen-binding sites, which may be bivalent or bispecific. See, for example, EP404,097, WO1993 / 01161, Hudson et al. Nat Med.9:129-134(2003), and Hollinger et al. Proc.Natl.Acad.Sci.USA 90:6444-6448(1993). Triabodies and tetrabodies are also described in Hudson et al., Nat.Med.9:129-134(2003).

[0080] A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domains or all or part of the light chain variable domains of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, Mass., e.g., U.S. Patent No. 6,248,516B1).

[0081] Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., Escherichia coli or phages), as described herein.

[0082] In some cases, the anti-Tie2 antibody of the present invention provided herein is Fab.

[0083] In some cases, Fab may include at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 containing any one amino acid sequence from SEQ ID NOs. 1 to 40, (b) CDR-H2 containing any one amino acid sequence from SEQ ID NOs. 41 to 80, (c) CDR-H3 containing one amino acid sequence from SEQ ID NOs. 81 to 120, (d) CDR-L1 containing any one amino acid sequence from SEQ ID NOs. 121 to 160, (e) CDR-L2 containing any one amino acid sequence from SEQ ID NOs. 161 to 200, and (f) CDR-L3 containing any one amino acid sequence from SEQ ID NOs. 201 to 240, or a combination of one or more of the above CDRs and one or more variants thereof having at least approximately 95% sequence identity with any one of SEQ ID NOs. 1 to 240 (e.g., at least 95%, 96%, 97%, 98%, or 99% identity).

[0084] In some cases, Fab binds to Tie2 and contains HCDR1-3 and LCDR1-3, which are antibody clones containing the amino acid sequences of the sequence numbers below. TIFF0007891318000003.tif41166TIFF0007891318000004.tif248166

[0085] In some cases, Fab binds to Tie2 and contains an amino acid sequence that has at least approximately 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity) to any one of the amino acid sequences of SEQ ID NOs. 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, or 272, or an amino acid sequence that has 100% of that sequence. The VH domain and the VL domain comprising (b) an amino acid sequence having at least approximately 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity) to any one of the amino acid sequences of SEQ ID NOs. 242, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, or 273, or having 100% of that sequence.

[0086] In some cases, Fab binds to Tie2 and contains VH and VL domains with amino acid sequences in the following combinations. TIFF0007891318000005.tif128166

[0087] In certain embodiments, the antibodies provided herein are chimeric antibodies. For specific examples of chimeric antibodies, see, for example, U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable domain derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant domain. In further embodiments, a chimeric antibody is a “class-switched” antibody in which the class or subclass is changed from the class or subclass of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0088] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable domains, e.g., a CDR, where the CDR (or a portion thereof) is derived from a non-human antibody and the FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody may optionally also contain at least a portion of the human constant region. In some embodiments, several FR residues in the humanized antibody are replaced with corresponding residues derived from a non-human antibody (e.g., the antibody from which the CDR residues are derived) to restore or improve antibody specificity or affinity, for example.

[0089] Humanized antibodies and methods for their production are outlined, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further, for example, in Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321 and No. 7,087,409, and Kashmiri et al., Methods. This is described in 36:25-34 (2005) (explaining specificity-determining region (SDR) grafting), Padlan, Mol.Immunol.28:489-498 (1991) (explaining "resurfacing"), Dall'Acqua et al., Methods 36:43-60 (2005) (explaining "FR shuffling"), as well as in Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J.Cancer,83:252-260 (2000) (explaining the "guided selection" approach to FR shuffling).

[0090] Human framework regions that can be used for humanization include framework regions selected using the "best fit" method (see, e.g., Sims et.al., J.Immunol. 151:2296 (1993)), framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see, e.g., Carter et.al., Proc.Natl.Acad.Sci.USA, 89:4285 (1992) and Presta et.al. J.Immunol., 151:2623 (1993)), human maturation (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front.Biosci. 13:1619-1633 (2008)), and framework regions obtained from screening of FR libraries (e.g., Baca et. See, al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0091] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk et al., Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0092] Human antibodies can be prepared by administering immunogens to transgenic animals modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci, either replacing endogenous immunoglobulin loci, existing extrachromosomally, or randomly incorporated into the animal's chromosomes. In such transgenic animals, endogenous immunoglobulin loci are generally inactivated. For an overview of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing the XENOMOUSE® technology, U.S. Patent No. 5,770,429 describing the HUANTIBODIES® technology, U.S. Patent No. 7,041,870 describing the KM mouse® technology, and U.S. Patent Application Publication No. 2007 / 0061900 describing the VELOCIMOUSE® technology, as well as U.S. Patent Nos. 9,809,642 and 9,380,769 describing the OmniChicken® technology. The human variable region from intact antibodies produced by such animals may be further modified, for example, by combining it with a different human constant region.

[0093] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human xenomyeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), and Boerner et al., J. Immunol., 147:86 (1991).) For human antibodies produced via human B-cell hybridoma technology, see Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0094] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0095] The antibodies of the present invention can be isolated by screening a combination library for antibodies having the desired activity. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are outlined, for example, in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further, for example, the McCafferty et al., Nature 348:552-554, Clackson et al., Nature 352:624-628 (1991), Marks et al., J.Mol.Biol.222:581-597 (1992), Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003), Sidhu et al., J.Mol.Biol.338(2):299-310 (2004), Lee This is described in et al., J.Mol.Biol.340(5):1073-1093(2004), Fellouse, Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004), and Lee et al., J.Immunol.Methods 284(1-2):119-132(2004).

[0096] In certain phage display methods, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR), randomly recombined within a phage library, and then screened for antigen-binding phages as described in Winter et.al., Ann. Rev. Immunol., 12:433-455 (1994). The phages typically present antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies against immunogens without requiring hybridoma construction. Alternatively, the natural repertoire (e.g., from humans) can be cloned to provide a single antibody source against a wide range of non-self and autoantigens without immunization, as described in Griffiths et.al., EMBO J, 12:725-734 (1993). Finally, natural libraries can also be constructed synthetically by cloning an unrearranged V gene segment from stem cells, encoding a highly variable CDR3 region using PCR primers containing random sequences, and achieving rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Examples of patent publications describing human antibody phage libraries include U.S. Patent No. 5,750,373, and U.S. Patent Publications 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0097] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments in this specification.

[0098] In certain embodiments, the antibodies provided herein are multispecific antibodies, such as, for example, bispecific antibodies, biparatopic antibodies, triplicate antibodies, tetravalent antibodies, pentavalent antibodies, and hexavalent antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites. In certain embodiments, a bispecific antibody may bind to two or more different epitopes of Tie2. In certain embodiments, one of the binding specificities is for Tie2, and the other is for any other antigen, such as VEGF. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. Any of the anti-Tie2 antibodies described herein may be used to manipulate multispecific antibodies.

[0099] In some embodiments, the multispecific anti-Tie2 antibody of the present invention is a bispecific antibody in which one arm binds to Tie2 and the other arm binds to VEGF. In other embodiments, such a bispecific anti-Tie2 antibody of the present invention also has a (abrogate)Fc mutation that blocks ADCC and / or CDC as described herein. Such a bispecific antibody of the present invention comprises a heavy chain and a light chain having amino acid sequences in the following combinations: TIFF0007891318000006.tif53166

[0100] In other instances, the multispecific anti-Tie2 antibody of the present invention is fused with a VEGF extracellular trap protein. In yet another instance, such a multispecific anti-Tie2 antibody fusion protein also has an Fc mutation that blocks ADCC and / or CDC, as described herein. Non-limiting examples of such multispecific antibody fusion proteins include the following amino acid sequences. TIFF0007891318000007.tif23166

[0101] Techniques for producing multispecific antibodies include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO93 / 08829, and Traunecker et al. EMBO, J.10:3655 (1991)), and the "knob-in-hole" engineering (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can also be produced by manipulating the electrostatic steering effect to create antibody Fc-heterodimer molecules (WO2009 / 089004A1), crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)), producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J.Immunol., 148(5):1547-1553 (1992)), using "diabody" techniques to produce bispecific antibody fragments (see, e.g., Hollinger et al., Proc.Natl.Acad.Sci.USA, 90:6444-6448 (1993)), or using single-stranded Fv(sFv) dimers (see, e.g., Gruber They may also be prepared by preparing a triplicate antibody, as described, for example, in Tutt et al., J.Immunol. 147:60 (1991), (see et al., J.Immunol., 152:5368 (1994)).

[0102] Modified antibodies having three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, for example, US2006 / 0025576A1).

[0103] The antibodies or fragments described herein also include “dual-acting FAb” or “DAF” which include an antigen-binding site that binds to Tie2 and another different antigen (see, for example, US2008 / 0069820).

[0104] In certain embodiments, an amino acid sequence variant of an antibody provided herein (e.g., an antibody variant comprising one or more amino acid residue modifications) is intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variant of the antibody may be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, the deletion of residues in the amino acid sequence of the antibody, and / or the insertion of residues, and / or the substitution of residues. Any combination of deletions, insertions, and substitutions can be performed to arrive at the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

[0105] In a particular embodiment, an antibody variant having one or more amino acid substitutions is provided. Target sites for substitutional mutagenesis include CDRs and FRs. Conservative substitutions are intended, and such substitutions are well known in the art.

[0106] Other amino acid substitutions are described below with reference to amino acid side-chain classes. Amino acid substitutions may be introduced into the antibody of interest, and the product may be screened for desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved or reduced ADCC or CDC. Amino acids can be grouped according to their general side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, lie; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0107] Non-conservative substitution involves swapping one member of one of these classes with one of another.

[0108] One type of substitution variant involves substituting one or more hypervariable region residues and / or FR residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variant, selected for further study, will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, increased stability, increased expression, modified pI, and / or decreased immunogenicity) compared to the parent antibody, and / or substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity-matured antibodies, which can be conveniently generated using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more CDR residues are mutated, the mutant antibody is presented on a phage, and screened for specific biological activity (e.g., binding affinity).

[0109] Modifications (e.g., substitutions) may be made in the CDR, for example, to improve antibody affinity. Such modifications may be made in CDR "hot spots," i.e., residues encoded by codons that are frequently mutated during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol Biol. 207:179-196 (2008)), and / or residues that come into contact with the antigen, and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by secondary library construction and reselection is described, for example, in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, variability is introduced into variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. Next, the library is screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves a CDR-targeted approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0110] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such modifications do not substantially reduce the antibody's antigen-binding ability. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be made in a CDR. Such modifications may be made, for example, outside of antigen-contact residues within a CDR. In certain embodiments of the variant VH and VL sequences provided above, each CDR is either unmodified or contains one, two, or three or fewer amino acid substitutions.

[0111] In certain embodiments, substitutions, insertions, or deletions may occur within one or more FRs, provided that such modifications do not substantially reduce the antibody's antigen-binding ability. Such modifications may, for example, improve antibody affinity and / or stability (e.g., by increasing the melting temperature).

[0112] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) containing amino acid residue modifications (e.g., substitutions) at one or more amino acid positions. In certain embodiments, the present invention aims to provide antibody variants that possess some, but not all, effector functions, which are desirable candidates for applications where the in vivo half-life of the antibody is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxic assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).

[0113] Non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986), and Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), U.S. Patent No. 5,821,337, and Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, for example, ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, Calif.) and CYTOTOX96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be measured in vivo, for example, by Clynes et al., Proc. Nat'l Acad. Sci. USA. The results may be evaluated in animal models, such as those disclosed in 95:652-656 (1998). A C1q binding assay may be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. A CDC assay may also be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al. J. Immunol Methods 202:163 (1996), Cragg et al. Blood 101:1045-1052 (2003), and Cragg et al. Blood 103:2738-2743 (2004)).FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova et al., Int'l.Immunol.18(12):1759-1769(2006)).

[0114] The antibodies of the present invention can be manipulated with reduced effector function (e.g., reduced complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), and antibody-dependent phagocytosis (ADCP)) due to reduced affinity for human FcyRIIIA and / or FcγRIIA and / or FcγRI and / or Clq binding. In some cases, such reduced effector function is achieved by one or more amino acid substitutions of the following Fc region residues, as numbered by EU numbering such as Kabat: N297, L234, L235, D265, and P329. See US6,737,056, US7,332,581, and WO2012 / 130831. In some embodiments, the substitution mutations are one or more of N297G, N297A, L234A, L235A, D265A, and / or P329G. In some embodiments, the substitution mutation is the N297A or N297G substitution mutation. In some embodiments, the substitution mutation includes the so-called "DANA" Fc variant in which residues D265 and N297 are substituted with alanine (U.S. Patent No. 7,332,581). In some embodiments, the substitution mutation includes the so-called "DANG" mutation having residues substituted as D265A and N297G. In some embodiments, the substitution mutation includes the "LALA" Fc variant in which residues L234 and L235 are substituted with alanine (see Lund, J., et al., (1992) Mol.Immunol., 29, 53-59, and Tamm, A. and Schmidt, RE (1997) Int.Rev.Immunol., 16, 57-85). In other embodiments, substitution mutations include the "LALA-PG" Fc mutant, in which residues L234 and L235 are substituted with alanine and P329 is substituted with glycine (see Brunker, P., et al. (2016) Mol. Cancer Ther).

[0115] Certain antibody variants exhibiting improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056, WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001)).

[0116] In some embodiments, the substitution mutation is made within the Fc region resulting in the hexamerized antibody described herein. In some embodiments, such a substitution mutation is E430G. In other embodiments, the E430G mutation may be combined with any of the reduced effector function mutations described above to inhibit CDC function and / or ADCC effector function. In one embodiment, the anti-Tie2 antibody of the present invention comprises a heavy chain and a light chain containing amino acid sequences having the E430G mutation and L234A, L235A, and P329G mutations in the following combinations. TIFF0007891318000008.tif16166

[0117] Antibodies with extended half-lives and improved binding to the neonatal Fc receptor (FcRn), which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J.Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) are described in US2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions that improve binding to FcRn in the Fc region. Examples of such Fc variants include substitutions of one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, substitution of Fc region residue 434 (US Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), US Patent Nos. 5,648,260, 5,624,821, and WO94 / 29351.

[0118] The present invention also provides an immunoconjugate comprising one or more cytotoxic agents, such as chemotherapeutic agents or chemotherapeutic drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes, which are conjugated with the anti-Tie2 antibodies of this specification.

[0119] In one embodiment, the immunoconjugate is an antibody such as mytansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent No. 0425235B1), auristatins such as monomethyl auristatin drug portions DE and DF (MMAE and MMAF) (see U.S. Patents Nos. 5,635,483, 5,780,588 and 7,498,298), drastatin, calicheamycin or its derivatives (see U.S. Patents Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296), Hinman et al., Cancer Res. 53:3336-3342 (1993), and Lode et al., Cancer Res. 58:2925-2928 (1998), anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006), Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006), Torgov et al., Bioconj. Chem. 16:717-721 (2005), Nagy et al. Proc. Natl. Acad. Sci. USA 97:829-834 (2000), Dubowchik et al., Bioorg. & Med. Chem. Letters An antibody-drug conjugate (ADC) conjugated to one or more drugs, including but not limited to methotrexate, vindesine, docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel, taxanes, trichothecenes, and CC1065 (see U.S. Patent No. 6,630,579).

[0120] In another embodiment, the immunoconjugate includes, but is not limited to, antibodies conjugated to enzymatically active toxins or fragments thereof, such as those described herein, including diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modexin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, geronin, mitogenin, restrictoctocin, phenomycin, enomycin, and trichothecenes.

[0121] In another embodiment, the immunoconjugate includes an antibody, as described herein, which is conjugated to a radioactive atom to form a radioconjugate. Various radioisotopes are available for the production of the radioconjugate. For example, At 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 Pb 212 Examples include radioactive isotopes of Lu. When radioconjugates are used for detection, they may contain radioactive atoms for scintigraphy (e.g., Tc99m or 1123) or spin labels for nuclear magnetic resonance (NMR) imaging (also known as MRI) (again, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron, etc.).

[0122] In another embodiment, the immunoconjugate includes the antibody described herein, conjugated to a non-cytotoxic agent, such as an artemisinin such as artusenate, or a cannabinoid, or a metabolite such as naltrexone, or aspirin, or a statin, or metformin, doxycycline, or an anthelmintic.

[0123] Conjugates of antibodies and cytotoxic or non-cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional derivatives of imide esters (dimethyl adipimidoate, HCl, etc.), active esters (disuccinimidyl suberate, etc.), aldehydes (glutaraldehyde, etc.), bis-azide compounds (bis(p-azidobenzoyl)hexanediamine, etc.), bis-diazonium derivatives (bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (toluene 2,6-isocyanate, etc.), and bis-active fluorine compounds (1,5-difluoro-2,4-dinitrobenzene, etc.). For example, see Vitetta et al., Science, 238:1098 (1987) for lysine immunotoxins. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radioactive nucleotides to antibodies. See WO94 / 11026. Linkers may be “cleavable linkers” that facilitate the release of cytotoxic drugs into cells. For example, acid-unstable linkers, peptidase-sensitive linkers, photosensitive linkers, dimethyl linkers, or disulfide-containing linkers may be used (Chari et al., Cancer Res. 52:127-131 (1992), U.S. Patent No. 5,208,020).

[0124] The immunoconjugates or ADCs used herein expressly intend, but are not limited to, such conjugates prepared with a crosslinking reagent. Examples of crosslinking reagents include, but are not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, STAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA) SVSB (succinimidyl-(4-vinylsulfone)benzoate).

[0125] In one embodiment, the anti-Tie2 antibody of the present invention is useful for detecting the presence of Tie2 in a biological sample. As used herein, the term “detection” includes quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissue. In certain embodiments, such tissue includes normal tissue and / or cancerous tissue that express Tie2 at higher levels compared to other tissues.

[0126] In one embodiment, an anti-Tie2 antibody is provided for use in a diagnostic or detection method. In a further embodiment, a method for detecting the presence of Tie2 in a biological sample is provided. In a particular embodiment, the method includes contacting a biological sample with the anti-Tie2 antibody described herein under conditions that allow the binding of the anti-Tie2 antibody to Tie2, and detecting whether a complex is formed between the anti-Tie2 antibody and Tie2. Such a method may be in vitro or in vivo. In one embodiment, the anti-Tie2 antibody is used to select subjects for anti-Tie2 antibody therapy, for example, Tie2 is a biomarker for patient selection.

[0127] Any of the antibodies described herein (e.g., anti-Tie2 antibodies) may be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-Tie2 antibody described herein is provided. Such nucleic acid may encode an amino acid sequence containing VL and / or VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors containing such nucleic acid (e.g., an expression vector) are provided. In a further embodiment, a host cell containing such nucleic acid is provided. In one such embodiment, the host cell comprises (1) a vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and an amino acid sequence containing VH of the antibody, or (2) a first vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and a second vector encoding an amino acid sequence containing VH of the antibody (e.g., transformed with it). In one embodiment, the host cell is a eukaryote, for example, a Chinese hamster ovary (CHO) cell or lymphocyte-like cell (e.g., YO, NSO, Sp20 cells). In one embodiment, a method for producing an anti-Tie2 antibody is provided, which includes culturing host cells containing the nucleic acid encoding the antibody provided above under conditions suitable for antibody expression, and optionally recovering the antibody from the host cells (or host cell culture medium).

[0128] For recombinant production of anti-Tie2 antibodies, for example, as described above, the nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[0129] Suitable host cells for cloning or expressing antibody-coding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. Also, see Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in Escherichia coli (E. coli). After expression, antibodies can be isolated from bacterial cell paste in the soluble fraction and further purified.

[0130] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for antibody-coding vectors, including fungal and yeast strains, where the glycosylation pathway is "humanized," resulting in the production of antibodies with a partially or completely human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0131] Host cells suitable for the expression of glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfection into fall armyworm (Spodoptera frugiperda) cells.

[0132] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0133] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include the SV40(COS-7) transformed monkey kidney CV1 cell line, human embryonic kidney cells (e.g., 293 or 293 cells as described in Graham et al. J. Gen, Virol. 36:59 (1977)), baby hamster kidney cells (BHK), and mouse Sertoli cells (e.g., T cells as described in Mather, Biol. Reprod. 23:243-251 (1980)). M4 Other useful mammalian host cell lines include monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562), TRI cells, MRC 5 cells, and FS4 cells, as described in Mather et al., Annals NYAcad.Sci. 383:44-68 (1982). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad.Sci. USA 77:4216 (1980)), as well as myeloma cell lines such as YO, NSO, and Sp2 / 0. For an overview of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed. Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0134] The anti-Tie2 antibodies provided herein are known in the art and can be identified, screened for their physical / chemical properties and / or biological activity by various assays described herein in the examples and throughout this specification.

[0135] In one embodiment, the antibody of the present invention is tested for its antigen-binding activity by known methods such as ELISA, Western blotting, or surface plasmon resonance assay (e.g., BIACORE®).

[0136] In one embodiment, antigen-binding activity (e.g., as indicated by KD) is measured using a BIACORE® surface plasmon resonance (SPR) assay. For example, assays using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C using an antigen CM5 chip immobilized at approximately 10 response units (RUs). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIAcore, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) in 10 mM sodium acetate at pH 4.8 and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RUs) of the coupling protein. After injecting the antigen, 1M ethanolamine is injected to block unreacted groups. For kinetic analysis, Fab's 2-fold serial dilutions (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μl / min into PBS containing 0.05% polysorbate 20 (TWEEN®-20) surfactant (PBST) at 25°C. The association rate (k on ) and dissociation rate (k offThe kD is calculated using a simple one-to-one Langmuir coupled model (BIACORE® evaluation software version 3.2) by simultaneously fitting association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is k off / k on It is calculated as a ratio of . For example, see Chen et al., J.Mol.Biol.293:865-881(1999). The above surface plasmon resonance assay results in an on-rate of 10 6 M -1 s -1 If it exceeds [value], the on-rate can be determined using fluorescence quenching techniques, which measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing antigen concentrations, measured with a spectrometer such as a stop-flow spectrometer with a stirred cuvette (Aviv Instruments) or an 8000 series SLM-AMINCO® spectrophotometer (ThermoSpectronic). KD may also be measured using the BIACORE® SPR assay.

[0137] In another embodiment, a competitive assay may be used to identify antibodies that compete with antibodies such as those described herein for binding to Tie2. In a particular embodiment, such a competing antibody binds to the same epitope (e.g., a linear or conformational epitope) bound by antibodies such as those described herein. Detailed exemplary methods for mapping the epitopes to which antibodies bind are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0138] In an exemplary competition assay, immobilized Tie2 is incubated in a solution containing a first labeled antibody that binds to Tie2 and a second unlabeled antibody being tested for its ability to compete with the first antibody for binding to Tie2. The second antibody may be present in the hybridoma supernatant. As a control, immobilized Tie2 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to Tie2, excess unbound antibody is removed and the amount of labeling associated with the immobilized Tie2 is measured. If the amount of labeling associated with the immobilized Tie2 is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to Tie2. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0139] In one embodiment, an assay is provided for identifying an anti-Tie2 antibody having biological activity. See the Examples section. Biological activity may include, for example, activating, agonizing, increasing, or enhancing one or more biological activities of Tie2. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0140] In certain embodiments, the antibodies of the present invention are tested for such biological activity. In certain embodiments, an anti-Tie2 antibody binds to Tie2 and reduces or inhibits its serine protease activity against one or more Tie2 substrates, including, for example, H2-Opt substrates, α-casein, β-casein, or BODIPY® FL casein substrates, or any other suitable Tie2 substrates known in the art. In certain embodiments, an anti-Tie2 antibody inhibits Tie2 activity against one or more Tie2 substrates with an IC50 of 50 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 3 nM, 2.5 nM, 2 nM, 1 nM, 800 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, or less.

[0141] In a particular embodiment, a labeled anti-Tie2 antibody is provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromophore labels, high-density electron labels, chemiluminescent labels, and radioactive labels), as well as moieties such as enzymes or ligands that are indirectly detected through enzymatic reactions or molecular interactions. Exemplary labels include radioactive isotopes. 32 P, 14 C, 125 I, 3 H, and 131I, phosphors, such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases, such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases, such as uricase and xanthine oxidase (coupled with enzymes that oxidize the pigment precursor using hydrogen peroxide, such as HRP, lactoperoxidase or microperoxidase), biotin / avidin, spin-labeled, bacteriophage-labeled, stable free radicals, etc., are included but not limited to these. In another embodiment of the present invention, the antibody does not need to be labeled, and the presence of the antibody can be detected using a labeled antibody that binds to an antibody, such as an anti-horseradish peroxidase antibody, which is well known in the art.

[0142] The antibodies of the present invention can be used in any known assay method, including competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. See Zola et al., Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

[0143] Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding to a limited amount of antibody. The amount of antigen in the test sample is inversely proportional to the amount of standard that will bind to the antibody. To facilitate the determination of the amount of standard that will bind, the antibody is generally insolubilized before or after competition, so that the standard and analyte that bind to the antibody can be conveniently separated from the standard and analyte that remain unbound.

[0144] A sandwich assay involves the use of two antibodies that can each bind to different immunogenic moieties or epitopes of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody immobilized on a solid support, and then a second antibody binds to the analyte, thus forming an insoluble ternary complex. See, for example, U.S. Patent No. 4,376,110. The second antibody itself may be labeled at the detectable moiety (direct sandwich assay) or it may be measured using an anti-immunoglobulin antibody that is labeled at the detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is the ELISA assay, in which case the detectable moiety is an enzyme.

[0145] For immunohistochemistry, the sample may be fresh or frozen, or it may be embedded in paraffin and fixed with a preservative such as formalin.

[0146] Pharmaceutical preparations Therapeutic formulations of the fusion polypeptide of the present invention, comprising the anti-Tie2 antibody or antibody fragment or variant thereof, or the immunoconjugate of the present invention, or combinations thereof with an anti-VEGF antibody or recombinant VEGF fusion protein as provided herein, can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the polypeptide of desired purity with an optional "pharmaceutically acceptable" carrier, excipient, or stabilizer (all of which are referred to as "excipients"). Examples include buffers, stabilizers, preservatives, isotonic agents, nonionic surfactants, antioxidants, and various other additives. For example, Remington's Pharmaceutical Sciences, 16 th See edition, A. Osol, Ed. (1980). Such additives must be nontoxic to the recipient at the dosage and concentration used.

[0147] Therapeutic methods and compositions The fusion polypeptides of the present invention, comprising the anti-Tie2 antibody and antibody fragment of the present invention, or the immunoconjugate of the present invention, or combinations thereof with an anti-VEGF antibody or recombinant VEGF fusion protein as provided herein, may be used in therapeutic methods for treating, preventing, and / or alleviating a variety of diseases, including any Tie2 dysregulation disorder as defined herein. [Examples]

[0148] The following are examples of the methods and compositions of the present invention. Considering the above general description, it will be understood that various other embodiments can be implemented.

[0149] Example 1: Immunization of transgenic chickens to isolate fully human anti-Tie2 antibodies Since the extracellular domains (ECDs) of human and chicken Tie2 orthologs are only 62% identical, in contrast to the 90% identity observed between mouse and human Tie2 orthologs, generating Tie2 antibodies using chickens would significantly expand the epitope range accessible by the antibodies.

[0150] The antibodies were produced by immunizing OmniChicken® (Crystal Biosciences, Inc., Emeryville, CA), a proprietary transgenic chicken, with recombinant and purified human Tie2 ECD. OmniChicken can be "humanized" to produce fully human antibodies through deletion of endogenous immunoglobulin-coding genes from the chicken genome and their replacement with human immunoglobulin-coding genes (see U.S. Patents 9,809,642 and 9,380,769).

[0151] Next, millions of B cells were rapidly screened for antibodies specifically binding to Tie2 using a high-throughput single B cell screening and cloning approach, gel-encapsulated microenvironment (GEM) technology (Crystal Bioscience, Inc., Emeryville, CA) (see U.S. Patents 8,415,173 and 8,030,095). GEM involves co-localizing single-antibody-secreting B lymphocytes derived from immunized animals within a gel microdroplet containing one or more particulate reporters (Mettler Izquierdo et al. 2016). The reporters used were polystyrene beads coated with the Tie2 antigen (ECD of human or mouse Tie2 recombinant protein) and / or cells expressing human Tie2. When Tie2 antibody-producing B cells were incorporated into the GEM, binding to the antigen-coated beads or Tie2-expressing cells was detected using a red fluorescent secondary antibody. Through the use of single B-cell cloning techniques, many appropriately paired heavy and light chain variable domains were identified. These sequences were cloned into antibody expression vectors. A total of 236 recombinant antibodies were expressed via transient transfection to confirm specificity and evaluate functional activity in downstream assays.

[0152] Example 2: Primary antibody screening All 236 antibodies generated as described above in Example 1 were tested for binding to recombinant human Tie2 ECD protein using ELISA. The majority of the screened antibodies were found to be potent binders by ELISA, and many had EC50 values ​​in the low pM range.

[0153] Cross-reactivity to mouse Tie2 via binding to recombinant mouse Tie2 ECD protein was used as a primary screening method. These binding studies demonstrated that approximately 90% of the generated anti-Tie2 antibodies were cross-reactive between mouse and human Tie2 proteins, suggesting their potential suitability for in vivo testing in established animal models.

[0154] Next, we screened for the recognition and binding of full-length human Tie2 expressed in its native three-dimensional structure on the plasma membrane of living cells. For these experiments, human umbilical vein endothelial cells (HUVECs) were obtained from donors. Using flow cytometry, we also demonstrated that approximately 70% of antibodies bound to the human Tie2 ECD protein by ELISA bound to native Tie2 expressed on the cells [data not shown].

[0155] Example 3: Agonist antibodies identified by functional screening Next, all Tie2 antibodies conjugated to HUVEC cells, as described in Example 2 above, were tested for agonist properties using a homogeneous immunoassay (AlphaLISA®) screening platform designed to detect intracellular levels of antibody-induced phosphorylated (activated) ERK (pERK or p42 / p44) and phosphorylated (activated) Akt (pAkt). Both are known as downstream signaling effectors of Tie2.

[0156] In this screening method, HUVEC cells were plated in 96-well plates, subjected to serum starvation for 3 hours, and then treated with Ang1 (positive control), hIgG1 (non-specific human IgG1 negative control antibody), or anti-Tie2 antibody at a concentration of 180 nM. After 20 minutes of incubation, the cells were lysed, and the lysates were analyzed by a specific AlphaLISA assay for the presence of pERK and total ERK, as well as pAkt and total Akt. To be designated as an "active agonist anti-Tie2 antibody," the antibody clone needed to induce Tie2 signaling, as determined by an increase in intracellular pAkt and pERK levels (normalized to total Akt and total ERK, respectively). Antibodies that were able to induce an increase in pERK and pAkt levels were ranked based on their activity compared to the pERK and pAkt levels induced by Ang1 treatment. Antibodies that could increase pERK and pAkt levels by more than 75% compared to those seen after Ang1 treatment were considered comparable to Ang1 and were carried over for further testing.

[0157] After identifying hits in the primary functional screening, the variable domains from the most active anti-Tie2 antibodies were reformatted onto different human IgG scaffolds more suitable for therapeutic use. Specifically, the variable domains of all candidate leads were cloned into expression plasmids containing human IgG1-derived Fc domains, including an asparagine-to-alanine mutation at amino acid position 297 (N297A). The Tie2 agonist activity of these reformatted antibodies was confirmed by Western blot analysis for antibody-inducible phospho-Tie2 (pTie2) increase (Figure 1A), and by the aforementioned pERK and pAkt (Figures 1B and 1C).

[0158] Therefore, it was found that the anti-Tie2 antibody of the present invention activates both proximal and distal Tie2 signaling events, leading to further research.

[0159] Example 4: Determination of antibody efficacy in vitro To enable the ranking of the anti-Tie2 antibodies of the present invention based on efficacy, we developed a highly accurate assay for determining antibody EC50 values.

[0160] In this assay, HUVEC cells were starved for 3 hours and then treated for 20 minutes with increasing concentrations of Ang1, anti-Tie2 antibody, or negative control hIgG1 before cell lysis. The cell lysates were then subjected to Western blotting and quantitative fluorescence imaging to determine the level of pAkt relative to total Akt, and the respective EC50 values ​​were determined by analysis as follows: Ang1 = 0.54 nM, Ab #1 = 0.91 nM, Ab #2 = 0.48 nM, Ab #3 = 0.45 nM, and WT = 1.33 nM. See Figure 2.

[0161] Analysis of EC50 values ​​revealed that most antibodies exhibited potent Tie2 activation at sub-nanomole concentrations comparable to the potency of Ang1 in the same assay. Overall, the functional assay utilized identified anti-Tie2 antibodies capable of inducing Tie2 signaling to levels comparable to those observed with Ang1.

[0162] Example 5: Anti-Tie2 antibody reduces fluid leakage in an in vitro model of the endothelial barrier. To investigate the potential physiological effects of anti-Tie2 antibodies on the cellular ability to control permeability, a simplified in vitro model of the endothelial barrier was established. This model allowed for further characterization of the antibodies based on their ability to enhance and / or protect physiological permeability induced by increased levels of VEGF in the microenvironment. See Figure 3 for a schematic diagram of the model setting.

[0163] A confluent monolayer of intact HUVEC cells was cultured on a semipermeable membrane to form an adherent structure with tight junctions. Cells were treated with 100 ng / ml VEGF with or without Ang1 or anti-Tie2 antibody. Permeability was assayed at different time points of 6 hours by measuring the amount of fluorescein conjugate dextran that penetrated the cell monolayer into the submembrane receiver well. Leakage rate was measured over time as the number of fluorescein units accumulated in the receiving well. The difference in endothelial barrier leakage between PBS (negative control) and Ang1 (positive control) was determined. The ability of anti-Tie2 antibody to reduce endothelial barrier leakage was normalized against Ang1 treatment in the same assay (%Ang1 activity). See Figure 4.

[0164] Statistical analysis revealed that anti-Tie2 antibodies significantly reduced VEGF-induced fluid leakage through the primary endothelial cell matrix, whereas human IgG1-negative controls did not. Therefore, anti-Tie2 antibodies were able to stimulate downstream Tie2 signaling in biochemical assays and reduce leakage in orthogonal in vitro physiological assays.

[0165] Example 6: Anti-Tie2 agonist antibody activates Tie2 in the presence of high levels of Ang2. Patients with diabetic macular edema (DME) show a marked increase in systemic and intravitreous levels of Ang2 (Loukovaara et al. 2013b, Regula et al. 2017). Considering the possibility that high levels of Ang2 may interfere with anti-Tie2 antibody activity by sharing a similar binding site on Tie2, or whether antibody binding is affected by the allosteric effect of Ang2 on the Tie2 structure, we determined the functional characterization of anti-Tie2 antibodies in the presence of saturated concentrations of Ang2. Ang2 functions as a weak Tie2 agonist that can induce signaling in in vitro assays in the absence of Ang1, but at lower levels than that achievable by Ang1 (Yuan et al. 2009). The in vitro experiments described below supported these findings.

[0166] To determine the saturation level of Ang2 during Tie2 signaling in an in vitro model system, HUVEC cells were starved for 3 hours, treated with increasing concentrations of Ang2, followed by lysis and analysis of pERK / Erk levels by Western blotting. See Figure 4A. The blots were analyzed using quantitative fluorescence imaging, and normalized pERK levels were plotted as a function of Ang2 concentration. The minimum concentration of Ang2 that saturates Tie2 signaling was determined to be 2.5 ug / mL. See Figure 5B. In subsequent experiments, HUVEC cells were co-treated for 20 minutes with Ang2 at a concentration of 2.5 ug / mL (43 nM) and anti-Tie2 antibody at a concentration of 12 nM before cell lysis and analysis of pErk / Erk (Figures 5C and 5D) and pAkt / Akt (Figures 5E and 5F) levels.

[0167] The results of these experiments demonstrated that anti-Tie2 antibodies are potent Tie2 pathway activators, even in the presence of high Ang2 concentrations. Surprisingly, Tie2 activity in cells exposed to both anti-Tie2 antibodies and Ang2 was higher than that observed with either treatment alone.

[0168] Example 7: Anti-Tie2 antibody utilizes a non-ligand competitive binding mechanism. To further investigate the interaction between the anti-Tie2 antibody and angiopoietin at the Tie2 receptor level, a competitive binding assay was established using biolayer interferometry (BLI), a label-free technique for measuring recombinant protein and biomolecular interactions.

[0169] In these experiments, His-tagged recombinant Tie2 ECDs were captured on a Ni-NTA biosensor and subsequently exposed sequentially to a specific anti-Tie2 antibody, and then to Ang1 or Ang2, to evaluate potential binding competition. In these experiments, Ang1 and Ang2 were able to bind to the Tie2 receptor pre-complexed with the anti-Tie2 antibody. See Figures 6A-B. Thus, it was found that the anti-Tie2 agonist antibody of the present invention binds to Tie2 in a non-Ang1 and Ang2 ligand-competitive manner and enhances Tie2 signaling in the presence of Ang2 to a higher level than the anti-Tie2 antibody alone.

[0170] Considering these results in conjunction with the Tie2 pathway signaling results described above, the data characterizing the binding mechanism of the anti-Tie2 antibody of the present invention demonstrate a mechanism of action completely independent of angiopoietin levels.

[0171] Example 8: The anti-Tie2 agonist antibody is cross-reactive to Tie2 variants expressed by other animal species. We investigated the cross-reactivity profiles of anti-Tie2 antibodies against Tie2 orthologs expressed by preclinical animal model species using genetically modified cell lines overexpressing Tie2 orthologs from mouse, rat, rabbit, pig, and cynomolgus monkeys.

[0172] In short, expression plasmids containing various full-length Tie2 coding sequences were transfected into primary human endothelial cells (HUVECs) and human embryonic kidney (HEK293) cells. Stable cell populations were selected by treating transfectants with the antibiotic puromycin for two weeks, and single-cell clones were generated using fluorescence-activated cell sorting. Upon confirmation of cell surface expression of these Tie2 variants, the apparent Kd(EC50) values ​​of anti-Tie2 antibodies on various Tie2 proteins were determined using cell lines. To obtain this data, the cross-reactivity profiles of the anti-Tie2 antibodies of the present invention were determined. Cells were labeled with reduced concentrations of anti-Tie2 antibodies #1, #2, and #3 (10, 5, 2.5, 1.25, 0.63, 0.31, and 0.16 ug / mL) or nonspecific hIgG1 antibody (10 ug / mL), followed by labeling with rabbit anti-human IgG antibody conjugated to Alexa Fluor 488 phosphor (20 ug / mL). EC50 values ​​were generated using ForeCyt® software. See Tables 1 and 2.

[0173] [Table 1]

[0174] [Table 2]

[0175] These experiments demonstrate the interreactive binding of anti-Tie2 antibodies across human and preclinical species.

[0176] Example 9: Anti-Tie2 antibody recognizes distinct epitopes on human Tie2. To understand the diversity of the epitope range of the Tie2 antibody panel, anti-Tie2 antibodies were evaluated for cross-competition using BLI technology.

[0177] Recombinant human Tie2 ECD was loaded onto a biosensor and then exposed to anti-Tie2 antibodies (M1 = mouse anti-Tie2 mAb, Ab #1, Ab #3; M2 = mouse anti-Tie2, Ab #2, WT) to evaluate initial binding. Next, the probe was exposed to a second anti-Tie2 antibody to evaluate the second binding event. A positive wavelength shift indicates that the second antibody can bind to the previously formed Tie2 antibody complex, showing that these antibodies bind to different epitopes and do not compete for binding to the Tie2 protein. Positive wavelength shifts indicating co-binding of two anti-Tie2 antibodies are shown in gray and light gray, with the shift value (nm) embedded in each cell. As expected, the antibody pre-bound to Tie2 prevented the binding of the same antibody to the complex (shaded in black). See Figure 7.

[0178] These experiments demonstrated the absence of cross-competition for binding to Tie2 across a panel of six anti-Tie2 agonist antibodies, showing that each candidate recognizes a different epitope on the receptor.

[0179] Example 10: Oxygen-Induced Retinopathy (OIR) Mouse Model In this study, 40 C57BL / 6J pups were placed in a hyper-oxygen chamber (75% O2) for 5 days starting at 7 days postnatal (P7) (n=10 per cage) to induce central retinal vascular regression. CD-1 rearing mothers were rotated before entering the chamber and 2-3 days after entry. At P12, the pups were returned to room air, where relative hypoxia induced abnormal angiogenesis. They were then intraperitoneally administered either 1×PBS vehicle without endotoxin, 10 mg / kg, HuIgG isotype control, or 10 mg / kg of anti-Tie2 clone #3. At P17, all groups, including naive OIR mice, were euthanized. The eyes were removed and fixed in 4% paraformaldehyde for 1 hour.

[0180] Retinals were dissected and incubated overnight in 1 mM CaCl2 in PBS with rhodamine-labeled lectin (1:100) from Bandeiraea simplicifolia (Griffonia simplicifolia) to visualize areas of angiogenesis (VO) or neovascularization (NV). Stained retinas were mounted flat on slides and imaged on a Zeiss® AxioScan. Images were analyzed on a Visiopharm® to determine the %VO or %NV of the entire retina.

[0181] As shown in Figure 8B, anti-Tie2 mAbs, rather than isotype control mAbs or vehicles, were found to have a significant positive effect on areas of vascular loss (i.e., avascularity), but did not appear to reduce NV (Figure 8A). Promoting the regrowth of healthy vascular systems may be beneficial in mitigating the adverse outcomes of chronic anti-VEGF therapy, such as geographic atrophy or capillary loss, in patients with ocular disease. See Kim J, et al., Science Advances, Vol. 5 Feb 13 (2019), M. Young, et al., Retina 34, 1308-1315 (2014), and T. Kurihara, et al., J. Clin. Invest. 122, 4213-4217 (2012).

[0182] Example 11: Laser-guided choroidal angiogenesis (CNV) mouse model Male C57BL / 6J mice (6-8 weeks old) were anesthetized with a ketamine / xylazine cocktail prior to laser treatment. CNV lesions were induced by laser photocoagulation using a diode laser (IRIDEX®, Oculight® GL) and a slit lamp (Zeiss®), with a spot size of 50 μm, power of 180 mW, and exposure time of 100 ms. Four laser burns were induced around the optic nerve disc of each eye, typically at the 3, 6, 9, and 12 o'clock positions. Tie2-specific antibody (clone #3) or isotype control antibody or anti-mouse VEGF control antibody (B20) was intraperitoneally injected (10 mg / kg) one day prior to laser induction, for a total of three injections every three days. Nine days after laser induction, mice were perfused with FITC-lectin or TRITC-dextran via the tail vein. Five minutes after perfusion, the eyes were removed and fixed with 4% paraformaldehyde (PFA) for 15 minutes.

[0183] The choroid-scleral complex and retina were separated, and anti-CD31 immunofluorescence (IF) was performed to reveal the vascular system by whole-mount staining of both retinal and choroidal tissues. For CD31 IF, rat anti-mouse antibody BD550274 was diluted 1:100 and incubated overnight at 4°C. After 4 hours of incubation with a secondary anti-rat antibody (Life Technologies, A11006), the whole mounts were imaged at 488 nm. See Figure 9A. Neoangiogenesis in the lesion and vascular density in the retina were quantified using Image J. P-values ​​were evaluated by Student's t-test (significant change, p<0.05). See Figure 9B.

[0184] As shown in Figures 9A and 9B, the anti-Tie2 antibody, rather than isotype control mAbs, significantly inhibited the size of choroidal neovascular lesions after laser injury. This result suggests that Tie2 activation after administration of an anti-Tie2 mAb may offer significant clinical benefits to individuals suffering from ophthalmic disorders such as AMD or diabetic retinopathy.

[0185] Example 12: Streptozotocin-induced (STZ) diabetic mouse model Administration of the agonist Tie2 antibody of the present invention may be useful for treating diabetic retinopathy and other pathological conditions associated with diabetes, such as nephropathy. To explore potential therapeutic benefits, the effects of the anti-Tie2 antibody of the present invention will be tested on disease-related endpoints, such as leakage from blood vessels in the eye, visual function as assessed by electroretinography, production of cytokines thought to be involved in human disease, such as IL-1b, and renal function as measured by proteinuria in streptozotocin-induced diabetic mouse models administered mAbs via the intraperitoneal route.

[0186] The weight of 6-7 week old C57BL / 6J mice can be measured, and their baseline blood glucose can be determined (Accu-Chek®, Roche). Mice can be intraperitoneally injected with STZ (Sigma-Aldrich, St. Louis, MO) at a dose of 55 mg / kg for 5 consecutive days. Age-matched controls can be injected with buffer only. Blood glucose can be measured again one week after the last STZ injection, and if non-fasting blood glucose is higher than 17 mM (300 mg / dL), the mouse is considered diabetic. Diabetic C57BL / 6J mice treated with STZ can then be intravitreal (IVT) injected with an appropriate amount of the anti-Tie2 antibody, control antibody, vehicle, or comparative antibody of the present invention, such as an anti-VEGF antibody or recombinant VEGF fusion protein, at least 8 weeks after STZ administration.

[0187] Electroretinography (ERG) assesses the overall function of retinal cells using the UTAS-E visual electrodiagnostic testing system. After overnight dark adaptation, treated mice are anesthetized by subcutaneous injection of a cocktail containing ketamine and xylazine in sterile water. One eye is proptosed, and the pupil is dilated with tropicamide and phenylephrine HCl. The eye is moistened with Genteal® eye drops, and core body temperature is maintained using a heating pad. ERG can be recorded using ultra-low impedance silver / nylon DTL+ electrodes. Needle electrodes can be placed in the middle of the forehead and at the base of the tail. Gold contact lens electrodes are used to record the ERG response. Stimuli may consist, for example, of electronically flashing for 50 milliseconds in a log step. Responses are recorded from stimuli ranging from below threshold to saturation. Analysis may include the maximum amplitude and threshold of the a-wave and b-wave.

[0188] Optokinetic tracking (OKT) can also be recorded as follows: The mouse can be fixed on a turntable surrounded by a set of synchronized monitors displaying a vertical black and white grid (14°). OKT is induced by applying sinusoidal vibrations of the surrounding screen at 1 Hz and 10° / second. Right eye movement is monitored with an infrared-sensing CCD camera under illumination by ANA infrared LED47. Images are sampled at 200 Hz, the center of the pupil is calculated, and the eye position is estimated using Morita's Geteye software program. OKT can be recorded three times for 30 seconds at approximately 30-second intervals.

[0189] Retinal vascular permeability can be measured as follows: Mice are anesthetized and injected into the tail vein with Evans blue dye dissolved in saline. Two hours after tail vein injection, the mice are anesthetized with ketamine and xylazine and can be perfused through the left ventricle using saline. After perfusion, the retina is dissected, weighed, and placed in formamide at 70°C for 18 hours to extract the Evans blue dye. The following day, the retina is centrifuged for 45 minutes and removed from the formamide. Extravasation of Evans blue is measured using a plate reader with an A620. The amount is converted to ng Evans blue / infused tissue weight using a standard curve.

[0190] Tissue collection for targeted transcriptomes can be performed as follows: Mice are sacrificed under anesthesia and both eyes are removed. The retina is then dissected, placed in RNAlater, processed, and analyzed by qRT-PCR.

[0191] Example 13: Dual-specificity biologic construct Simultaneously influencing both the Tie2 and VEGFR-induced signaling pathways may have enhanced benefits compared to regulating either pathway individually. To achieve this, we designed, expressed, and tested for activity bispecific constructs that are designed to affect both Tie2 and VEGFR. Figure 10A shows an exemplary schematic diagram of the bispecific antibody of the present invention having both a Tie2-binding variable domain and a VEGF-binding variable domain. Antibody clone #54 was designed and generated using standard cloning techniques and has the sequences of SEQ ID NOs. 282 and 283. A bispecific construct consisting of VEGFR R1D2 and R3D3 (VEGF trap proteins) and the Tie2-binding domain was also generated (antibody clone #55), which has the sequence of SEQ ID NO. 284. As described in Example 3 above, Tie2 agonism was subsequently evaluated in HUVEC cells using two exemplary aTie2 / VEGF bispecific constructs (antibody clones #54 and #55) at 20 nM each, using the AlphaLISA® screening platform designed to detect intracellular levels of antibody-inducible pERK and pAkt.

[0192] The tested aTie2 / VEGF bispecific constructs were found to possess the ability to induce Tie2 signaling in vitro (Figure 11). These bispecific molecules may enable the administration of a single drug to patients with eye disease, providing substantially higher efficacy than VEGF inhibition or Tie2 activation monotherapy. Promoting healthy vascular regrowth provided by the Tie2 agonist component of the bispecific molecules may also be beneficial in mitigating the potential adverse consequences of the molecule's anti-VEGF component (i.e., geographic atrophy or capillary loss in patients with eye disease).

[0193] Example 14: Increased valence anti-Tie2 antibody and biparatopic anti-Tie2 antibody Antibodies having three or more Tie2 binding moieties, or antibodies capable of binding to multiple epitopes on the Tie2 extracellular domain, may provide a means to enhance in vivo Tie2 pathway activation to a greater extent than is possible with bivalent anti-Tie2 antibodies. Exemplary tetravalent anti-Tie2 constructs having the heavy and light chain sequences of anti-Tie2 antibody clone #3, with a polypeptide linker and B12 scFv sequence added to the C-terminus of the heavy chain (antibody clone #51) or the N-terminus of the heavy chain (antibody clone #52), were generated using standard techniques. See Figures 12A-C for illustrative schematic diagrams.

[0194] As described in Example 3, tetravalent antibody clones #51 and #52 were evaluated for their ability to induce signaling through pAkt measurement. As shown in Figure 14, both clones #51 and #52 show an improved ability to activate Tie2 signaling in HUVEC cells compared to the bivalent anti-Tie2 construct (aTie2) or to angiopoietin 1 (Ang1). Therefore, anti-Tie2 variants with an additional Tie2 binding moiety may provide a means to induce higher-order oligomerization of Tie2 on the cell surface, potentially offering therapeutic benefits in situations where the level of agonism mediated by bivalent anti-Tie2 antibodies or the native agonist ligand Ang1 is insufficient to restore vascular homeostasis in diseased tissue.

[0195] Example 15: Anti-Tie2 antibody having an Fc mutation that promotes the ability to hexamerize on the cell surface Antibodies containing a mutation specific to the heavy chain's Fc domain (i.e., E430G) have been shown to enhance their ability to form hexamers on the cell surface (M. Overdijk, Mol Cancer Ther 2020;19:2126-38). Anti-Tie2 antibodies containing an Fc domain with the E430G mutation were synthesized and compared to the same anti-Tie2 antibody with a comparable Fc containing natural glutamic acid (E) at position 430. See Figure 13 for a schematic diagram of such a hexamerized anti-Tie2 antibody.

[0196] As described in Example 3, Tie2 agonism was evaluated using pAKT / pERK Western blotting. As shown in Figure 14, the anti-Tie2 E430G construct (antibody clone #50) demonstrated an improved ability to induce Tie2 signaling in HUVEC cells compared to the E430 construct. Therefore, B12 variants with mutations that increase the likelihood of hexamerization on the cell surface may be a means of inducing higher-order oligomerization of Tie2 on the cell surface, potentially offering therapeutic benefits in situations where the level of agonism mediated by anti-Tie2 antibodies or the native agonist ligand Ang1 is insufficient to restore vascular homeostasis in diseased tissue.

[0197] Example 16: Determination of Fab binding affinity of anti-Tie2 antibody by SPR BIACORE® (BIAcore, Inc., Piscataway, NJ) was performed at 25°C with human, rat, or mouse Tie2 ECD antigens immobilized on carboxymethylated dextran biosensor chips (CM5) at approximately 10 response units (RUs). CM5 chips were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to supplier instructions. All Tie2 ECD antigens were diluted to 5 μg / ml (approximately 0.2 μM) in 10 mM sodium acetate at pH 4.8 and injected at a flow rate of 5 μl / min to obtain approximately 10 RUs of coupling protein. After each Tie2 ECD injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic analysis, two-fold serial dilutions (0.78 nM to 500 nM) of antibody clone #3 Fab were injected into PBS containing 0.05% polysorbate 20 (TWEEN®-20) surfactant (PBST) at a flow rate of approximately 25 μl / min at 25°C. The association rate (kon) and dissociation rate (koff) were calculated using a simple one-to-one Langmuir binding model (BIACORE® evaluation software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) was calculated as the koff / kon ratio. See, for example, Chen et al., J.Mol.Biol.293:865-881 (1999).

[0198] The table below shows the Fab binding affinity of antibody clone #3 against each Tie2 ECD antigen tested. TIFF0007891318000011.tif26166

[0199] Although the present invention described herein is described in some detail by examples and embodiments for the purpose of clarifying understanding, the description and embodiments of the invention should not be construed as limiting the scope of the invention. All patent and scientific literature disclosures cited herein are expressly incorporated in their entirety by reference.

[0200] Array information SEQUENCE LISTING <110> Unity Biotechnology, Inc. <120> Antibodies Directed to TIE-2 and Methods of Use <150> US 62 / 938,816 <151> 2019-11-21 <160> 289 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.3 <400> 1 Asp Ser Tyr Gly Met 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.8 <400> 2 Asp Ser Tyr Gly Met 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.9 <400> 3 Ser Val Tyr Gly Met 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.10 <400> 4 Ser Val Tyr Ala Met 1 5 <210> 5 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.4 <400> 5 Ser Asn Tyr Val Met 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.11 <400> 6 Gly Val Tyr Gly Met 1 5 <210> 7 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.12 <400> 7 Ser Ile Tyr Ala Met 1 5 <210> 8 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.13 <400> 8 Ser Val Tyr Gly Met 1 5 <210> 9 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.14 <400> 9 Asp Ile Tyr Gly Met 1 5 <210> 10 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.15 <400> 10 Asp Ile Tyr Gly Met 1 5 <210> 11 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.16 <400> 11 Ser Asn Tyr Val Met 1 5 <210> 12 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.17 <400> 12 Asn Val Tyr Ala Met 1 5 <210> 13 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.18 <400> 13 Asp Ile Tyr Gly Met 1 5 <210> 14 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.19 <400> 14 Ser Ile Tyr Ala Met 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.20 <400> 15 Asp Ile Tyr Gly Met 1 5 <210> 16 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p2.21 <400> 16 Ser Val Tyr Gly Met 1 5 <210> 17 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.22 <400> 17 Ile Asn Phe Ala Met 1 5 <210> 18 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.23 <400> 18 Ser Ser Tyr Ala Met 1 5 <210> 19 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.24 <400> 19 Ser Ser Tyr Ala Met 1 5 <210> 20 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.25 <400> 20 Arg Asn Tyr Gly Met 1 5 <210> 21 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.1 <400> 21 Ser Ser Tyr Ala Met 1 5 <210> 22 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.26 <400> 22 Ser Ser Tyr Ala Met 1 5 <210> 23 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.27 <400> 23 Asp Val Tyr Ala Met 1 5 <210> 24 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.28 <400> 24 Ser Ser Tyr Ala Met 1 5 <210> 25 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.29 <400> 25 Ser Ser Tyr Ala Met 1 5 <210> 26 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.30 <400> 26 Asp Val Tyr Ala Met 1 5 <210> 27 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.6 <400> 27 Asp Val Tyr Ala Met 1 5 <210> 28 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.2 <400> 28 Ile Asn Phe Ala Met 1 5 <210> 29 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.31 <400> 29 Ile Asn Phe Ala Met 1 5 <210> 30 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.5 <400> 30 Asp Val Tyr Ala Met 1 5 <210> 31 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.32 <400> 31 Asp Val Tyr Ala Met 1 5 <210> 32 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.33 <400> 32 Arg Asn Tyr Gly Met 1 5 <210> 33 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.39 <400> 33 Ile Asn Phe Ala Met 1 5 <210> 34 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p1.22 <400> 34 Ile Asn Phe Ala Met 1 5 <210> 35 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p3.1.34 <400> 35 Ser Asn Tyr Ala Met 1 5 <210> 36 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p3.1.35 <400> 36 Ser Ser Tyr Gly Met 1 5 <210> 37 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p3.1.36 <400> 37 Ser Tyr Tyr Ala Met 1 5 <210> 38 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p3.1.37 <400> 38 Arg Ser Tyr Ala Met 1 5 <210> 39 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p3.2.38 <400> 39 Ser Ser Tyr Gly Met 1 5 <210> 40 <211> 5 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC1; clone: p3.2.40 <400> 40 Ser Tyr Tyr Ala Met 1 5 <210> 41 <211> 15 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.3 <400> 41 Ser Ile Asn Val Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val Arg 1 5 10 15 <210> 42 <211> 15 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.8 <400> 42 Ser Ile Asn Val Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val Arg 1 5 10 15 <210> 43 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.9 <400> 43 Arg Ile Ser Gly Ser Gly Asp Ser Thr Phe Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 44 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.10 <400> 44 Arg Ile Ser Gly Asn Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 45 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.4 <400> 45 Ala Ile Ser His Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Glu 1 5 10 15 <210> 46 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.11 <400> 46 Arg Ile Ser Gly Ser Gly Asp Ser Thr Phe Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 47 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.12 <400> 47 Gly Ile Ser Gly Ser Gly Ala Leu Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 48 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.13 <400> 48 Arg Ile Ser Gly Ser Gly Asp Ser Thr Phe Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 49 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.14 <400> 49 Arg Ile Ser Val Arg Gly Arg Gly Ala Glu Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 50 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.15 <400> 50 Arg Ile Ser Gly Asn Gly Gly Ser Thr Phe Tyr Ser Glu Ser Val Lys 1 5 10 15 <210> 51 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.16 <400> 51 Ala Ile Ser His Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 52 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.17 <400> 52 Arg Ile Ser Gly Asn Gly Gly Ser Thr Tyr Tyr Ala Glu Ser Val Lys 1 5 10 15 <210> 53 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.18 <400> 53 Arg Ile Ser Gly Ser Gly Gly Thr Thr Phe Tyr Ala Glu Ser Val Lys 1 5 10 15 <210> 54 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.19 <400> 54 Gly Ile Ser Gly Ser Gly Ala Leu Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 55 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.20 <400> 55 Arg Ile Ser Val Arg Gly Arg Gly Ala Glu Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 56 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p2.21 <400> 56 Arg Ile Ser Gly Ser Gly Asp Ser Thr Phe Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 57 <211> 18 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.22 <400> 57 Leu Ile Ser Asp Asp Gly Arg Gly Asn Arg Pro Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys <210> 58 <211> 14 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.23 <400> 58 Arg Ile Ser Asp Tyr Thr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 <210> 59 <211> 14 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.24 <400> 59 Arg Ile Ser Asp Tyr Thr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 <210> 60 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.25 <400> 60 Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ile Asp Ser Val Lys 1 5 10 15 <210> 61 <211> 14 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.1 <400> 61 Arg Ile Ser Asp Tyr Thr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 <210> 62 <211> 14 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.26 <400> 62 Arg Ile Ser Asp Tyr Thr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 <210> 63 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.27 <400> 63 Leu Ile Ser Gly Thr Gly Arg Met Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 64 <211> 14 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.28 <400> 64 Arg Ile Ser Asp Tyr Thr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 <210> 65 <211> 14 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.29 <400> 65 Arg Ile Ser Asp Tyr Thr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 <210> 66 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.30 <400> 66 Leu Ile Ser Gly Thr Gly Arg Met Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 67 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.6 <400> 67 Leu Ile Ser Gly Thr Gly Arg Met Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 68 <211> 18 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.2 <400> 68 Leu Ile Ser Asp Asp Gly Arg Gly Asn Arg Pro Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys <210> 69 <211> 18 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.31 <400> 69 Leu Ile Ser Asp Asp Gly Arg Gly Asn Arg Pro Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys <210> 70 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.5 <400> 70 Leu Ile Ser Gly Thr Gly Arg Met Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 71 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.32 <400> 71 Leu Ile Ser Gly Thr Gly Arg Met Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 72 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.33 <400> 72 Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ile Asp Ser Val Lys 1 5 10 15 <210> 73 <211> 18 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.39 <400> 73 Leu Ile Ser Asp Asp Gly Arg Gly Asn Arg Pro Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys <210> 74 <211> 18 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p1.22 <400> 74 Leu Ile Ser Asp Asp Gly Arg Gly Asn Arg Pro Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys <210> 75 <211> 21 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p3.1.34 <400> 75 Ser Ile Ser Gly Asn Gly Ile Ser His Ser Gly Gly Ser Thr Tyr Tyr 1 5 10 15 Ala Asp Ser Val Lys 20 <210> 76 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p3.1.35 <400> 76 Ile Ile Ser Gly Asp Gly Val Ile Thr Ser Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 77 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p3.1.36 <400> 77 Arg Ile Ser Gly Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 78 <211> 21 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p3.1.37 <400> 78 Ser Ile Ser Gly Asn Gly Ile Ser His Ser Gly Gly Ser Thr Tyr Tyr 1 5 10 15 Ala Asp Ser Val Lys 20 <210> 79 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p3.2.38 <400> 79 Ile Ile Ser Gly Asp Gly Val Ile Thr Ser Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 80 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC2; clone: p3.2.40 <400> 80 Arg Ile Ser Gly Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys 1 5 10 15 <210> 81 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.3 <400> 81 Trp Asn Ser Phe Phe Asp Tyr Trp 1 5 <210> 82 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.8 <400> 82 Trp Asn Ser Phe Phe Asp Tyr Trp 1 5 <210> 83 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.9 <400> 83 Thr Trp Asn Asn Phe Phe Asp Tyr Trp 1 5 <210> 84 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.10 <400> 84 Thr Leu Asn Asn Phe Phe Asp Tyr Trp 1 5 <210> 85 <211> 12 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.4 <400> 85 Asp Leu Gly Tyr Trp Asn Asn Phe Phe Asp Tyr Trp 1 5 10 <210> 86 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.11 <400> 86 Thr Trp Asn Asn Phe Phe Asp Tyr Trp 1 5 <210> 87 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.12 <400> 87 Pro Leu Asn Asn Phe Phe Asp Tyr Trp 1 5 <210> 88 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.13 <400> 88 Thr Trp Asn Asn Phe Phe Asp Tyr Trp 1 5 <210> 89 <211> 11 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.14 <400> 89 Glu Asn Asn Trp Asn Ser Phe Phe Asp Tyr Trp 1 5 10 <210> 90 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.15 <400> 90 Thr Leu Asn Asn Phe Phe Asp Tyr Trp 1 5 <210> 91 <211> 12 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.16 <400> 91 Asp Leu Gly Tyr Trp Asn Asn Phe Phe Asp Tyr Trp 1 5 10 <210> 92 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.17 <400> 92 Thr Trp Asn Asn Phe Phe Asp Tyr Trp 1 5 <210> 93 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.18 <400> 93 Thr Leu Asn Asn Phe Phe Asp Tyr Trp 1 5 <210> 94 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.19 <400> 94 Pro Leu Asn Asn Phe Phe Asp Tyr Trp 1 5 <210> 95 <211> 11 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.20 <400> 95 Glu Asn Asn Trp Asn Ser Phe Phe Asp Tyr Trp 1 5 10 <210> 96 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p2.21 <400> 96 Thr Trp Asn Asn Phe Phe Asp Tyr Trp 1 5 <210> 97 <211> 10 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.22 <400> 97 Val Ser Trp Asp Val Phe Phe Asp Tyr Trp 1 5 10 <210> 98 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.23 <400> 98 Ser Gly Pro Lys Ser Gly Lys Tyr Trp Asn Asn Phe Phe Asp Ser Trp 1 5 10 15 <210> 99 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.24 <400> 99 Ser Gly Pro Lys Ser Gly Lys Tyr Trp Asn Asn Phe Phe Asp Ser Trp 1 5 10 15 <210> 100 <211> 12 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.25 <400> 100 Asp Arg Gly Asn Ser Tyr Gly Phe Tyr Asp Tyr Trp 1 5 10 <210> 101 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.1 <400> 101 Ser Gly Pro Lys Ser Gly Lys Tyr Trp Asn Asn Phe Phe Asp Ser Trp 1 5 10 15 <210> 102 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.26 <400> 102 Ser Gly Pro Lys Ser Gly Lys Tyr Trp Asn Asn Phe Phe Asp Ser Trp 1 5 10 15 <210> 103 <211> 11 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.27 <400> 103 Asp Pro Phe Gly His Gly Phe Phe Asp Tyr Trp 1 5 10 <210> 104 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.28 <400> 104 Ser Gly Pro Lys Ser Gly Lys Tyr Trp Asn Asn Phe Phe Asp Ser Trp 1 5 10 15 <210> 105 <211> 16 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.29 <400> 105 Ser Gly Pro Lys Ser Gly Lys Tyr Trp Asn Asn Phe Phe Asp Ser Trp 1 5 10 15 <210> 106 <211> 11 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.30 <400> 106 Asp Pro Phe Gly His Gly Phe Phe Asp Tyr Trp 1 5 10 <210> 107 <211> 11 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.6 <400> 107 Asp Pro Phe Gly His Gly Phe Phe Asp Tyr Trp 1 5 10 <210> 108 <211> 10 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.2 <400> 108 Val Ser Trp Asp Val Phe Phe Asp Tyr Trp 1 5 10 <210> 109 <211> 10 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.31 <400> 109 Val Ser Trp Asp Val Phe Phe Asp Tyr Trp 1 5 10 <210> 110 <211> 11 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.5 <400> 110 Asp Pro Phe Gly His Gly Phe Phe Asp Tyr Trp 1 5 10 <210> 111 <211> 11 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.32 <400> 111 Asp Pro Phe Gly His Gly Phe Phe Asp Tyr Trp 1 5 10 <210> 112 <211> 12 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.33 <400> 112 Asp Arg Gly Asn Ser Tyr Gly Phe Tyr Asp Tyr Trp 1 5 10 <210> 113 <211> 10 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.39 <400> 113 Val Ser Trp Asp Val Phe Phe Asp Tyr Trp 1 5 10 <210> 114 <211> 10 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p1.22 <400> 114 Val Ser Trp Asp Val Phe Phe Asp Tyr Trp 1 5 10 <210> 115 <211> 13 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p3.1.34 <400> 115 Asp Leu Gly Thr Trp Asn Ser Tyr Gly Phe Asp Tyr Trp 1 5 10 <210> 116 <211> 11 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p3.1.35 <400> 116 Arg Asp His Arg Ser Thr Phe Phe Asp Tyr Trp 1 5 10 <210> 117 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p3.1.36 <400> 117 Thr Leu Asn Asn Phe Phe Asp Tyr Trp 1 5 <210> 118 <211> 13 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p3.1.37 <400> 118 Asp Leu Gly Thr Trp Asn Ser Tyr Gly Phe Asp Tyr Trp 1 5 10 <210> 119 <211> 11 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p3.2.38 <400> 119 Arg Asp His Arg Ser Thr Phe Phe Asp Tyr Trp 1 5 10 <210> 120 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:HC3; clone: p3.2.40 <400> 120 Thr Leu Asn Asn Phe Phe Asp Tyr Trp 1 5 <210> 121 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.3 <400> 121 Ser Gln Asn Val Arg Ser Asp Leu Ala 1 5 <210> 122 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.8 <400> 122 Ser Gln Ser Val Asn Ser Lys Leu Ala 1 5 <210> 123 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.9 <400> 123 Ser Gln Thr Val Lys Thr Tyr Leu Ala 1 5 <210> 124 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.10 <400> 124 Ser Gln Gly Ile Val Gly Asn Leu Ala 1 5 <210> 125 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.4 <400> 125 Ser Gln Asn Val Arg Ser Asp Leu Ala 1 5 <210> 126 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.11 <400> 126 Ser Gln Thr Val Lys Thr Tyr Leu Ala 1 5 <210> 127 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.12 <400> 127 Ser Gln Ser Val Asn Ser Asn Leu Ala 1 5 <210> 128 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.13 <400> 128 Ser Gln Thr Val Lys Thr Tyr Leu Ala 1 5 <210> 129 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.14 <400> 129 Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 <210> 130 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.15 <400> 130 Ser Gln Thr Val Gly Ser Lys Leu Ala 1 5 <210> 131 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.16 <400> 131 Ser Gln Ser Val His Ser Tyr Leu Ala 1 5 <210> 132 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.17 <400> 132 Ser Gln Ser Val Lys Thr Tyr Leu Ala 1 5 <210> 133 <211> 10 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.18 <400> 133 Ser His Ser Val Ser Ser Thr Tyr Leu Ala 1 5 10 <210> 134 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.19 <400> 134 Ser Gln Asn Val Arg Ser Asp Leu Ala 1 5 <210> 135 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.20 <400> 135 Ser Gln Ser Val Gly Ser Asn Leu Ala 1 5 <210> 136 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p2.21 <400> 136 Ser Gln Thr Val Lys Thr Tyr Leu Ala 1 5 <210> 137 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.22 <400> 137 Ser Gln Pro Ile Asp Val Tyr Leu Ala 1 5 <210> 138 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.23 <400> 138 Ser Gln Thr Val Gly Ser Lys Leu Ala 1 5 <210> 139 <211> 10 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.24 <400> 139 Ser Gln Ser Val Gly Ser Tyr Tyr Leu Ala 1 5 10 <210> 140 <211> 10 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.25 <400> 140 Ser Gln Ser Val Ser Ala Ser Gln Leu Ala 1 5 10 <210> 141 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.1 <400> 141 Ser Gln Ser Val Lys Thr Asp Leu Ala 1 5 <210> 142 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.26 <400> 142 Ser Gln Ser Val Lys Thr Asp Leu Ala 1 5 <210> 143 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.27 <400> 143 Ser Gln Ser Val Asn Arg Asn Leu Ala 1 5 <210> 144 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.28 <400> 144 Ser Gln Pro Ile Asn Thr Tyr Leu Ala 1 5 <210> 145 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.29 <400> 145 Ser Gln Thr Val Gly Ser Lys Leu Ala 1 5 <210> 146 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.30 <400> 146 Ser Gln Ser Val Asn Arg Asn Leu Ala 1 5 <210> 147 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.6 <400> 147 Ser Gln Ser Val Asn Arg Asn Leu Ala 1 5 <210> 148 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.2 <400> 148 Ser Gln Pro Ile Asp Val Tyr Leu Ala 1 5 <210> 149 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.31 <400> 149 Ser Gln Pro Ile Asp Val Tyr Leu Ala 1 5 <210> 150 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.5 <400> 150 Ser Gln Thr Val Gly Ser Lys Leu Ala 1 5 <210> 151 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.32 <400> 151 Ser Gln Ser Val Asn His Asn Leu Ala 1 5 <210> 152 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.33 <400> 152 Asn Arg Ser Ile Leu Thr Ser Leu Ala 1 5 <210> 153 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.39 <400> 153 Ser Gln Pro Ile Asp Val Tyr Leu Ala 1 5 <210> 154 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p1.22 <400> 154 Ser Gln Pro Ile Asp Val Tyr Leu Ala 1 5 <210> 155 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p3.1.34 <400> 155 Ser Gln Thr Val Asn Thr Lys Leu Ala 1 5 <210> 156 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p3.1.35 <400> 156 Ser Gln Ser Val Ser Ser Asn Leu Ala 1 5 <210> 157 <211> 10 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p3.1.36 <400> 157 Ser Gln Ser Val Ser Asp Thr Tyr Leu Ala 1 5 10 <210> 158 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p3.1.37 <400> 158 Ser Gln Thr Val Thr Thr Lys Leu Ala 1 5 <210> 159 <211> 9 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p3.2.38 <400> 159 Ser Gln Ser Val Ser Ser Asn Leu Ala 1 5 <210> 160 <211> 10 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC1; clone: p3.2.40 <400> 160 Ser Gln Ser Val Ser Asp Thr Tyr Leu Ala 1 5 10 <210> 161 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.3 <400> 161 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 162 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.8 <400> 162 Asp Ala Ser Ser Arg Ala Thr 1 5 <210> 163 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.9 <400> 163 Ala Ala Ser Ser Arg Ala Ala 1 5 <210> 164 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.10 <400> 164 Asp Ala Ser Ser Arg Ala Thr 1 5 <210> 165 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.4 <400> 165 Gly Ala Ser Thr Arg Ala Thr 1 5 <210> 166 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.11 <400> 166 Asp Ala Ser Thr Arg Ala Thr 1 5 <210> 167 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.12 <400> 167 Asp Ala Ser Ser Arg Ala Thr 1 5 <210> 168 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.13 <400> 168 Asp Ala Ser Thr Arg Ala Thr 1 5 <210> 169 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.14 <400> 169 Gly Ala Ser Thr Arg Ala Thr 1 5 <210> 170 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.15 <400> 170 Asp Ala Ser Thr Arg Ala Thr 1 5 <210> 171 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.16 <400> 171 Asp Ala Ser Ser Arg Ala Thr 1 5 <210> 172 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.17 <400> 172 Asp Ala Ser Asp Arg Ala Thr 1 5 <210> 173 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.18 <400> 173 Asp Ala Ser Thr Arg Ala Thr 1 5 <210> 174 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.19 <400> 174 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 175 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.20 <400> 175 Ala Ala Ser Ser Arg Ala Thr 1 5 <210> 176 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p2.21 <400> 176 Asp Ala Ser Thr Lys Ala Thr 1 5 <210> 177 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.22 <400> 177 Gly Ala Ser Arg Arg Ala Thr 1 5 <210> 178 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.23 <400> 178 Ala Ala Ser Ser Arg Asp Thr 1 5 <210> 179 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.24 <400> 179 Gly Ala Ser Arg Arg Ala Thr 1 5 <210> 180 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.25 <400> 180 Asp Ala Thr Thr Arg Ala Thr 1 5 <210> 181 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.1 <400> 181 Gly Ala Thr Thr Arg Ala Thr 1 5 <210> 182 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.26 <400> 182 Gly Ala Thr Thr Arg Ala Thr 1 5 <210> 183 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.27 <400> 183 Asp Ala Arg Thr Arg Ala Thr 1 5 <210> 184 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.28 <400> 184 Asp Ala Ser Thr Arg Ala Thr 1 5 <210> 185 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.29 <400> 185 Ser Ala Ser Ser Arg Ala Thr 1 5 <210> 186 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.30 <400> 186 Asp Ala Arg Thr Arg Ala Thr 1 5 <210> 187 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.6 <400> 187 Asp Ala Arg Thr Arg Ala Thr 1 5 <210> 188 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.2 <400> 188 Gly Ala Asn Arg Arg Ala Ile 1 5 <210> 189 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.31 <400> 189 Gly Ala Asn Arg Arg Ala Ile 1 5 <210> 190 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.5 <400> 190 Asp Ala Ser Thr Arg Ala Thr 1 5 <210> 191 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.32 <400> 191 Asp Ala Arg Thr Arg Ala Thr 1 5 <210> 192 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.33 <400> 192 Asp Ala Ser Thr Arg Ala Thr 1 5 <210> 193 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.39 <400> 193 Gly Ala Asn Arg Arg Ala Ile 1 5 <210> 194 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p1.22 <400> 194 Gly Ala Ser Arg Arg Ala Thr 1 5 <210> 195 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p3.1.34 <400> 195 Asp Ala Ser Thr Arg Ala Thr 1 5 <210> 196 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p3.1.35 <400> 196 Gly Ala Asn Thr Arg Ala Thr 1 5 <210> 197 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p3.1.36 <400> 197 Asp Thr Ser Thr Arg Ala Thr 1 5 <210> 198 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p3.1.37 <400> 198 Asp Ala Ser Thr Arg Ala Thr 1 5 <210> 199 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p3.2.38 <400> 199 Gly Ala Asn Thr Arg Ala Thr 1 5 <210> 200 <211> 7 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC2; clone: p3.2.40 <400> 200 Asp Thr Ser Thr Arg Ala Thr 1 5 <210> 201 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.3 <400> 201 Gln Gln Tyr Ser Asn Trp Pro Pro 1 5 <210> 202 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.8 <400> 202 Gln Gln Tyr Asn Thr Trp Pro Pro 1 5 <210> 203 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.9 <400> 203 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 204 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.10 <400> 204 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 205 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.4 <400> 205 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 206 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.11 <400> 206 Gln Gln Ser Tyr Asp Trp Pro Pro 1 5 <210> 207 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.12 <400> 207 Gln Gln Tyr Asn Asn Trp Pro Pro 1 5 <210> 208 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.13 <400> 208 Gln Gln Ser Tyr Asp Trp Pro Pro 1 5 <210> 209 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.14 <400> 209 Gln Gln Tyr Asn Asn Trp Pro Pro 1 5 <210> 210 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.15 <400> 210 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 211 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.16 <400> 211 Gln Gln Tyr Ser Asn Trp Pro Pro 1 5 <210> 212 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.17 <400> 212 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 213 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.18 <400> 213 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 214 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.19 <400> 214 Gln Gln Tyr Asn Asn Trp Pro Pro 1 5 <210> 215 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.20 <400> 215 Gln Gln Tyr Asn Asn Trp Pro Pro 1 5 <210> 216 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p2.21 <400> 216 Gln Gln Ser Tyr Asp Trp Pro Pro 1 5 <210> 217 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.22 <400> 217 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 218 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.23 <400> 218 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 219 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.24 <400> 219 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 220 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.25 <400> 220 Gln Gln Tyr Tyr Asp Trp Arg Pro 1 5 <210> 221 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.1 <400> 221 Gln Gln Tyr Tyr Val Trp Pro Pro 1 5 <210> 222 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.26 <400> 222 Gln Gln Tyr Tyr Val Trp Pro Pro 1 5 <210> 223 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.27 <400> 223 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 224 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.28 <400> 224 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 225 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.29 <400> 225 Gln Gln Tyr Asn Asn Trp Pro Pro 1 5 <210> 226 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.30 <400> 226 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 227 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.6 <400> 227 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 228 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.2 <400> 228 Gln Gln Tyr Met Thr Trp Pro Pro 1 5 <210> 229 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.31 <400> 229 Gln Gln Tyr Met Thr Trp Pro Pro 1 5 <210> 230 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.5 <400> 230 Gln Gln Tyr Asn Asn Trp Pro Pro 1 5 <210> 231 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.32 <400> 231 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 232 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.33 <400> 232 Gln Gln Tyr Tyr Asp Trp Arg Pro 1 5 <210> 233 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.39 <400> 233 Gln Gln Tyr Met Thr Trp Pro Pro 1 5 <210> 234 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p1.22 <400> 234 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 235 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p3.1.34 <400> 235 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 236 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p3.1.35 <400> 236 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 237 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p3.1.36 <400> 237 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 238 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p3.1.37 <400> 238 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 239 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p3.2.38 <400> 239 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 240 <211> 8 <212> PRT <213> Artificial sequence <220> <223> human mAb CDR:LC3; clone: p3.2.40 <400> 240 Gln Gln Tyr Tyr Asp Trp Pro Pro 1 5 <210> 241 <211> 1124 <212> PRT <213> Homo sapiens <400> 241 Met Asp Ser Leu Ala Ser Leu Val Leu Cys Gly Val Ser Leu Leu Leu 1 5 10 15 Ser Gly Thr Val Glu Gly Ala Met Asp Leu Ile Leu Ile Asn Ser Leu 20 25 30 Pro Leu Val Ser Asp Ala Glu Thr Ser Leu Thr Cys Ile Ala Ser Gly 35 40 45 Trp Arg Pro His Glu Pro Ile Thr Ile Gly Arg Asp Phe Glu Ala Leu 50 55 60 Met Asn Gln His Gln Asp Pro Leu Glu Val Thr Gln Asp Val Thr Arg 65 70 75 80 Glu Trp Ala Lys Lys Val Val Trp Lys Arg Glu Lys Ala Ser Lys Ile 85 90 95 Asn Gly Ala Tyr Phe Cys Glu Gly Arg Val Arg Gly Glu Ala Ile Arg 100 105 110 Ile Arg Thr Met Lys Met Arg Gln Gln Ala Ser Phe Leu Pro Ala Thr 115 120 125 Leu Thr Met Thr Val Asp Lys Gly Asp Asn Val Asn Ile Ser Phe Lys 130 135 140 Lys Val Leu Ile Lys Glu Glu Asp Ala Val Ile Tyr Lys Asn Gly Ser 145 150 155 160 Phe Ile His Ser Val Pro Arg His Glu Val Pro Asp Ile Leu Glu Val 165 170 175 His Leu Pro His Ala Gln Pro Gln Asp Ala Gly Val Tyr Ser Ala Arg 180 185 190 Tyr Ile Gly Gly Asn Leu Phe Thr Ser Ala Phe Thr Arg Leu Ile Val 195 200 205 Arg Arg Cys Glu Ala Gln Lys Trp Gly Pro Glu Cys Asn His Leu Cys 210 215 220 Thr Ala Cys Met Asn Asn Gly Val Cys His Glu Asp Thr Gly Glu Cys 225 230 235 240 Ile Cys Pro Pro Gly Phe Met Gly Arg Thr Cys Glu Lys Ala Cys Glu 245 250 255 Leu His Thr Phe Gly Arg Thr Cys Lys Glu Arg Cys Ser Gly Gln Glu 260 265 270 Gly Cys Lys Ser Tyr Val Phe Cys Leu Pro Asp Pro Tyr Gly Cys Ser 275 280 285 Cys Ala Thr Gly Trp Lys Gly Leu Gln Cys Asn Glu Ala Cys His Pro 290 295 300 Gly Phe Tyr Gly Pro Asp Cys Lys Leu Arg Cys Ser Cys Asn Asn Gly 305 310 315 320 Glu Met Cys Asp Arg Phe Gln Gly Cys Leu Cys Ser Pro Gly Trp Gln 325 330 335 Gly Leu Gln Cys Glu Arg Glu Gly Ile Gln Arg Met Thr Pro Lys Ile 340 345 350 Val Asp Leu Pro Asp His Ile Glu Val Asn Ser Gly Lys Phe Asn Pro 355 360 365 Ile Cys Lys Ala Ser Gly Trp Pro Leu Pro Thr Asn Glu Glu Met Thr 370 375 380 Leu Val Lys Pro Asp Gly Thr Val Leu His Pro Lys Asp Phe Asn His 385 390 395 400 Thr Asp His Phe Ser Val Ala Ile Phe Thr Ile His Arg Ile Leu Pro 405 410 415 Pro Asp Ser Gly Val Trp Val Cys Ser Val Asn Thr Val Ala Gly Met 420 425 430 Val Glu Lys Pro Phe Asn Ile Ser Val Lys Val Leu Pro Lys Pro Leu 435 440 445 Asn Ala Pro Asn Val Ile Asp Thr Gly His Asn Phe Ala Val Ile Asn 450 455 460 Ile Ser Ser Glu Pro Tyr Phe Gly Asp Gly Pro Ile Lys Ser Lys Lys 465 470 475 480 Leu Leu Tyr Lys Pro Val Asn His Tyr Glu Ala Trp Gln His Ile Gln 485 490 495 Val Thr Asn Glu Ile Val Thr Leu Asn Tyr Leu Glu Pro Arg Thr Glu 500 505 510 Tyr Glu Leu Cys Val Gln Leu Val Arg Arg Gly Glu Gly Gly Glu Gly 515 520 525 His Pro Gly Pro Val Arg Arg Phe Thr Thr Ala Ser Ile Gly Leu Pro 530 535 540 Pro Pro Arg Gly Leu Asn Leu Leu Pro Lys Ser Gln Thr Thr Leu Asn 545 550 555 560 Leu Thr Trp Gln Pro Ile Phe Pro Ser Ser Glu Asp Asp Phe Tyr Val 565 570 575 Glu Val Glu Arg Arg Ser Val Gln Lys Ser Asp Gln Gln Asn Ile Lys 580 585 590 Val Pro Gly Asn Leu Thr Ser Val Leu Leu Asn Asn Leu His Pro Arg 595 600 605 Glu Gln Tyr Val Val Arg Ala Arg Val Asn Thr Lys Ala Gln Gly Glu 610 615 620 Trp Ser Glu Asp Leu Thr Ala Trp Thr Leu Ser Asp Ile Leu Pro Pro 625 630 635 640 Gln Pro Glu Asn Ile Lys Ile Ser Asn Ile Thr His Ser Ser Ala Val 645 650 655 Ile Ser Trp Thr Ile Leu Asp Gly Tyr Ser Ile Ser Ser Ile Thr Ile 660 665 670 Arg Tyr Lys Val Gln Gly Lys Asn Glu Asp Gln His Val Asp Val Lys 675 680 685 Ile Lys Asn Ala Thr Ile Thr Gln Tyr Gln Leu Lys Gly Leu Glu Pro 690 695 700 Glu Thr Ala Tyr Gln Val Asp Ile Phe Ala Glu Asn Asn Ile Gly Ser 705 710 715 720 Ser Asn Pro Ala Phe Ser His Glu Leu Val Thr Leu Pro Glu Ser Gln 725 730 735 Ala Pro Ala Asp Leu Gly Gly Gly Lys Met Leu Leu Ile Ala Ile Leu 740 745 750 Gly Ser Ala Gly Met Thr Cys Leu Thr Val Leu Leu Ala Phe Leu Ile 755 760 765 Ile Leu Gln Leu Lys Arg Ala Asn Val Gln Arg Arg Met Ala Gln Ala 770 775 780 Phe Gln Asn Val Arg Glu Glu Pro Ala Val Gln Phe Asn Ser Gly Thr 785 790 795 800 Leu Ala Leu Asn Arg Lys Val Lys Asn Asn Pro Asp Pro Thr Ile Tyr 805 810 815 Pro Val Leu Asp Trp Asn Asp Ile Lys Phe Gln Asp Val Ile Gly Glu 820 825 830 Gly Asn Phe Gly Gln Val Leu Lys Ala Arg Ile Lys Lys Asp Gly Leu 835 840 845 Arg Met Asp Ala Ala Ile Lys Arg Met Lys Glu Tyr Ala Ser Lys Asp 850 855 860 Asp His Arg Asp Phe Ala Gly Glu Leu Glu Val Leu Cys Lys Leu Gly 865 870 875 880 His His Pro Asn Ile Ile Asn Leu Leu Gly Ala Cys Glu His Arg Gly 885 890 895 Tyr Leu Tyr Leu Ala Ile Glu Tyr Ala Pro His Gly Asn Leu Leu Asp 900 905 910 Phe Leu Arg Lys Ser Arg Val Leu Glu Thr Asp Pro Ala Phe Ala Ile 915 920 925 Ala Asn Ser Thr Ala Ser Thr Leu Ser Ser Gln Gln Leu Leu His Phe 930 935 940 Ala Ala Asp Val Ala Arg Gly Met Asp Tyr Leu Ser Gln Lys Gln Phe 945 950 955 960 Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Gly Glu Asn Tyr 965 970 975 Val Ala Lys Ile Ala Asp Phe Gly Leu Ser Arg Gly Gln Glu Val Tyr 980 985 990 Val Lys Lys Thr Met Gly Arg Leu Pro Val Arg Trp Met Ala Ile Glu 995 1000 1005 Ser Leu Asn Tyr Ser Val Tyr Thr Thr Asn Ser Asp Val Trp Ser 1010 1015 1020 Tyr Gly Val Leu Leu Trp Glu Ile Val Ser Leu Gly Gly Thr Pro 1025 1030 1035 Tyr Cys Gly Met Thr Cys Ala Glu Leu Tyr Glu Lys Leu Pro Gln 1040 1045 1050 Gly Tyr Arg Leu Glu Lys Pro Leu Asn Cys Asp Asp Glu Val Tyr 1055 1060 1065 Asp Leu Met Arg Gln Cys Trp Arg Glu Lys Pro Tyr Glu Arg Pro 1070 1075 1080 Ser Phe Ala Gln Ile Leu Val Ser Leu Asn Arg Met Leu Glu Glu 1085 1090 1095 Arg Lys Thr Tyr Val Asn Thr Thr Leu Tyr Glu Lys Phe Thr Tyr 1100 1105 1110 Ala Gly Ile Asp Cys Ser Ala Glu Glu Ala Ala 1115 1120 <210> 242 <211> 122 <212> PRT <213> Artificial sequence <220> <223> human mab p1.1- VH <400> 242 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Ser Asp Tyr Thr Ile Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys 85 90 95 Ser Gly Pro Lys Ser Gly Lys Tyr Trp Asn Asn Phe Phe Asp Ser Trp 100 105 110 Gly Leu Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 243 <211> 108 <212> PRT <213> Artificial sequence <220> <223> human mab p1.1 - VL <400> 243 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Thr Cys Arg Ala Ser Gln Ser Val Lys Thr Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Thr Asn Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Val Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 244 <211> 120 <212> PRT <213> Artificial sequence <220> <223> human Mab p1.2 - VH <400> 244 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ile Asn Phe 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Glu Gly Pro Glu Trp Val 35 40 45 Ser Leu Ile Ser Asp Asp Gly Arg Gly Asn Arg Pro Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Thr Tyr 85 90 95 Tyr Cys Ala Lys Val Ser Trp Asp Val Phe Phe Asp Tyr Trp Gly Leu 100 105 110 Gly Thr Val Val Thr Val Ser Ser 115 120 <210> 245 <211> 108 <212> PRT <213> Artificial sequence <220> <223> human Mab p1.2 VL <400> 245 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Val Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Leu Ser Cys Arg Ala Ser Gln Pro Ile Asp Val Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Asn Arg Arg Ala Ile Asp Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Asn Ser Leu Gln Asn 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr Met Thr Trp Pro Pro 85 90 95 Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 246 <211> 115 <212> PRT <213> Artificial sequence <220> <223> human Mab p2.3 - VH <400> 246 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asn Val Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Ile Ile Ser Arg Asp Ser Ser Arg Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Asn Trp Asn Ser Phe Phe Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 247 <211> 108 <212> PRT <213> Artificial sequence <220> <223> human Mab p2.3 - VL <400> 247 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Ile Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val Arg Ser Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 248 <211> 115 <212> PRT <213> Artificial sequence <220> <223> human mAb p4.1.7 - VH <400> 248 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asn Val Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Ile Ile Ser Arg Asp Ser Ser Arg Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Asn Trp Asn Ser Phe Phe Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 249 <211> 108 <212> PRT <213> Artificial sequence <220> <223> human mAb p4.1.7 - VL <400> 249 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Leu Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 250 <211> 117 <212> PRT <213> Artificial sequence <220> <223> human mAb p4.3.41 - VH <400> 250 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Ile 35 40 45 Ser Gly Ile Ser Gly Ser Gly Ala Leu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Leu Asn Asn Phe Phe Asp Tyr Trp Gly Leu Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 251 <211> 108 <212> PRT <213> Artificial sequence <220> <223> human mAb p4.3.41 - VL <400> 251 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Lys 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Phe Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Asp Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 252 <211> 115 <212> PRT <213> Artificial sequence <220> <223> human mAb p4.3.42 - VH <400> 252 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asn Val Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Ile Ile Ser Arg Asp Ser Ser Arg Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Asn Trp Asn Ser Phe Phe Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 253 <211> 108 <212> PRT <213> Artificial sequence <220> <223> human mAb p4.3.42 - VL <400> 253 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Ser Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Val Gly Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Lys Asp Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 254 <211> 118 <212> PRT <213> Artificial sequence <220> <223> human mAb p5.43 - VH <400> 254 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Pro Thr Tyr 20 25 30 Ala Leu Ser Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val 35 40 45 Ser Ser Thr Ile Trp Gly Gly Asp Thr Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Ser Ser Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gln Gly Thr Trp Asn Ile Phe Phe Asp Tyr Trp Gly Leu Gly Val 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 255 <211> 108 <212> PRT <213> Artificial sequence <220> <223> human mAb p5.43 - VL <400> 255 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ala Gly Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 256 <211> 117 <212> PRT <213> Artificial sequence <220> <223> human mAb p4.2.44 - VH <400> 256 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Leu Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Ser Gly Arg Asp Gly Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Leu Asn Asn Phe Phe Asp Tyr Trp Gly Leu Gly Thr Leu 100 105 110 Val Thr Val Ser Leu 115 <210> 257 <211> 109 <212> PRT <213> Artificial sequence <220> <223> human mAb p4.2.44 - VL <400> 257 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ile Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Thr 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Asp Ala Ser Asn Arg Ala Ala Asp Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Asp Trp Pro 85 90 95 Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 258 <211> 114 <212> PRT <213> Artificial sequence <220> <223> human mAb p5.45 VH <400> 258 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ile Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Asn 85 90 95 Trp Asn Ser Phe Phe Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> 259 <211> 108 <212> PRT <213> Artificial sequence <220> <223> human mAb p5.45 VL <400> 259 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Thr Cys Arg Ala Ser Gln His Val Asn Thr His 20 25 30 Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Ile Ile 35 40 45 Tyr Asp Ala Thr Asn Arg Ala Asn Asp Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Ile Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 260 <211> 114 <212> PRT <213> Artificial sequence <220> <223> human mAb p5.46 - VH <400> 260 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Thr Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ile Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Asn 85 90 95 Trp Asn Ser Phe Phe Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> 261 <211> 108 <212> PRT <213> Artificial sequence <220> <223> human mAb p5.46 - VL <400> 261 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Thr Cys Arg Ala Ser Gln His Val Asn Thr His 20 25 30 Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Ile Ile 35 40 45 Tyr Asp Ala Thr Asn Arg Ala Asn Asp Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Ile Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 262 <211> 119 <212> PRT <213> Artificial sequence <220> <223> human mAb p1.6 - VH <400> 262 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Phe Asp Val Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Thr Leu Ile Ser Gly Thr Gly Arg Met Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Pro Phe Gly His Gly Phe Phe Asp Tyr Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 263 <211> 108 <212> PRT <213> Artificial sequence <220> <223> human mAb p1.6 - VL <400> 263 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Thr Leu Ser Pro Gly 1 5 10 15 Glu Thr Ala Thr Leu Thr Cys Arg Ala Ser Gln Ser Val Asn Arg Asn 20 25 30 Leu Ala Trp Tyr Gln Glu Lys Pro Asp Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Arg Thr Arg Ala Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Val Cys Gln Gln Tyr Tyr Asp Trp Pro Pro 85 90 95 Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 264 <211> 122 <212> PRT <213> Artificial sequence <220> <223> human mAb p4.2.47 - VH <400> 264 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Arg Pro Gly Glu 1 5 10 15 Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr His Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Gly Ser Trp Thr Ser Ser Gly Phe Phe Asp Tyr Trp 100 105 110 Gly Leu Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 265 <211> 108 <212> PRT <213> Artificial sequence <220> <223> human mAb p4.2.47 - VL <400> 265 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Lys Ala Thr Leu Asn Cys Arg Ala Ser Gln Asn Ile Asn Ser Lys 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Gly Asn Arg Ala Ala Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Asp Trp Arg Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 266 <211> 117 <212> PRT <213> Artificial sequence <220> <223> human mAb p5.48 - VH <400> 266 Asp Val Gln Leu Val Glu Ser Gly Gly Ala Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asp Ile Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Ser Gly Asn Gly Gly Ser Thr Phe Tyr Ser Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Thr Leu Asn Asn Phe Phe Asp Tyr Trp Gly Leu Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 267 <211> 108 <212> PRT <213> Artificial sequence <220> <223> human mAb p5.48 - VL <400> 267 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Gly Gly Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Asp Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 268 <211> 119 <212> PRT <213> Artificial sequence <220> <223> human mAb p1.5 - VH <400> 268 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Phe Asp Val Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Thr Leu Ile Ser Gly Thr Gly Arg Met Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Pro Phe Gly His Gly Phe Phe Asp Tyr Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 269 <211> 108 <212> PRT <213> Artificial sequence <220> <223> human mAb p1.5 - VL <400> 269 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Val Gly Ser Lys 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 270 <211> 120 <212> PRT <213> Artificial sequence <220> <223> human mAb p3.2.49 - VH <400> 270 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Asp Ile Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val 35 40 45 Ser Thr Leu Thr Gly Ile Ser Gly Arg Gly Gly Ser Thr Phe Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Asp Asn 65 70 75 80 Ile Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile 85 90 95 Tyr Tyr Cys Ala Gly Gly Thr Asp Ile Phe Phe Asp Tyr Trp Gly Leu 100 105 110 Gly Ile Leu Val Thr Val Ser Ser 115 120 <210> 271 <211> 108 <212> PRT <213> Artificial sequence <220> <223> human mAb p3.2.49 - VL <400> 271 Glu Met Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 272 <211> 120 <212> PRT <213> Artificial sequence <220> <223> human mAb p1.31 - VH <400> 272 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Pro Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ile Asn Phe 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Glu Gly Pro Glu Trp Val 35 40 45 Ser Leu Ile Ser Asp Asp Gly Arg Gly Asn Arg Pro Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Thr Tyr 85 90 95 Tyr Cys Ala Lys Val Ser Trp Asp Val Phe Phe Asp Tyr Trp Gly Leu 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 273 <211> 108 <212> PRT <213> Artificial sequence <220> <223> human mAb p1.31 - VL <400> 273 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Val Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Leu Ser Cys Arg Ala Ser Gln Pro Ile Asp Val Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Asn Arg Arg Ala Ile Asp Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Asn Ser Leu Gln Asn 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr Met Thr Trp Pro Pro 85 90 95 Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 274 <211> 445 <212> PRT <213> Artificial sequence <220> <223> Artificial p2.3 with pro-hexamerizing and ADCC / CDC abrogating Fc mutations (heavy chain) <400> 274 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asn Val Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Ile Ile Ser Arg Asp Ser Ser Arg Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Asn Trp Asn Ser Phe Phe Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Gly Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 275 <211> 215 <212> PRT <213> Artificial sequence <220> <223> Artificial p2.3 with pro-hexamerizing and ADCC / CDC abrogating Fc mutations (light chain (kappa)) <400> 275 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Ile Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val Arg Ser Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 276 <211> 696 <212> PRT <213> Artificial sequence <220> <223> Artificial p2.3 IgG-scFv fusion tetravalent mAb with HC C-terminal p2.3-scFv and ADCC / CDC abrogating Fc mutations (heavy chain) <400> 276 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asn Val Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Ile Ile Ser Arg Asp Ser Ser Arg Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Asn Trp Asn Ser Phe Phe Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly 435 440 445 Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly 450 455 460 Thr Leu Ser Leu Ser Pro Gly Glu Ile Ala Thr Leu Ser Cys Arg Ala 465 470 475 480 Ser Gln Asn Val Arg Ser Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly 485 490 495 Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly 500 505 510 Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 515 520 525 Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln 530 535 540 Gln Tyr Ser Asn Trp Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Val 545 550 555 560 Glu Ile Lys Gly Gln Ser Ser Arg Ser Ser Gly Gly Gly Gly Ser Ser 565 570 575 Gly Gly Gly Gly Ser Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val 580 585 590 Val Arg Pro Gly Glu Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe 595 600 605 Thr Phe Asp Ser Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys 610 615 620 Gly Leu Glu Trp Val Ser Ser Ile Asn Val Gly Asp Asn Thr Tyr Tyr 625 630 635 640 Ala Asp Ser Val Arg Gly Arg Phe Ile Ile Ser Arg Asp Ser Ser Arg 645 650 655 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala 660 665 670 Val Tyr Tyr Cys Ala Asn Trp Asn Ser Phe Phe Asp Tyr Trp Gly Leu 675 680 685 Gly Thr Leu Val Thr Val Ser Ser 690 695 <210> 277 <211> 215 <212> PRT <213> Artificial sequence <220> <223> Artificial p2.3 IgG-scFv fusion tetravalent mAb with HC C-terminal p2.3-scFv and ADCC / CDC abrogating Fc mutations (light chain (kappa)) <400> 277 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Ile Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val Arg Ser Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 278 <211> 696 <212> PRT <213> Artificial sequence <220> <223> Artificial p2.3 IgG-scFv fusion tetravalent mAb with HC N-terminal p2.3-scFv and ADCC / CDC abrogating Fc mutations (heavy chain) <400> 278 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Ile Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val Arg Ser Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gln Ser Ser 100 105 110 Arg Ser Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Ser Asp Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu Ser Leu 130 135 140 Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Ser Tyr Gly Met 145 150 155 160 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser 165 170 175 Ile Asn Val Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val Arg Gly Arg 180 185 190 Phe Ile Ile Ser Arg Asp Ser Ser Arg Asn Thr Leu Tyr Leu Gln Met 195 200 205 Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Asn Trp 210 215 220 Asn Ser Phe Phe Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr Val Ser 225 230 235 240 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val 245 250 255 Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu Ser Leu Arg Leu Ser 260 265 270 Cys Thr Ala Ser Gly Phe Thr Phe Asp Ser Tyr Gly Met Ser Trp Val 275 280 285 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Asn Val 290 295 300 Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val Arg Gly Arg Phe Ile Ile 305 310 315 320 Ser Arg Asp Ser Ser Arg Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 325 330 335 Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Asn Trp Asn Ser Phe 340 345 350 Phe Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr Val Ser Ser Ala Ser 355 360 365 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 370 375 380 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 385 390 395 400 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 405 410 415 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 420 425 430 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 435 440 445 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 450 455 460 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 465 470 475 480 Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 485 490 495 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 500 505 510 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 515 520 525 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 530 535 540 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 545 550 555 560 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 565 570 575 Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 580 585 590 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 595 600 605 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 610 615 620 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 625 630 635 640 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 645 650 655 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 660 665 670 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 675 680 685 Ser Leu Ser Leu Ser Pro Gly Lys 690 695 <210> 279 <211> 215 <212> PRT <213> Artificial sequence <220> <223> Artificial p2.3 IgG-scFv fusion tetravalent mAb with HC N-terminal p2.3-scFv and ADCC / CDC abrogating Fc mutations (light chain (kappa)) <400> 279 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Ile Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val Arg Ser Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 280 <211> 701 <212> PRT <213> Artificial sequence <220> <223> Artificial p2.3 IgG-scFv fusion biparatopic mAb with HC C-terminal p1.2-scFv and ADCC / CDC abrogating Fc mutations (heavy chain) <400> 280 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asn Val Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Ile Ile Ser Arg Asp Ser Ser Arg Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Asn Trp Asn Ser Phe Phe Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly 435 440 445 Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly 450 455 460 Thr Val Ser Leu Ser Pro Gly Glu Arg Val Thr Leu Ser Cys Arg Ala 465 470 475 480 Ser Gln Pro Ile Asp Val Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 485 490 495 Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Asn Arg Arg Ala Ile Asp 500 505 510 Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu 515 520 525 Thr Ile Asn Ser Leu Gln Asn Glu Asp Phe Ala Val Tyr Phe Cys Gln 530 535 540 Gln Tyr Met Thr Trp Pro Pro Val Thr Phe Gly Gly Gly Thr Lys Val 545 550 555 560 Glu Ile Lys Gly Gln Ser Ser Arg Ser Ser Gly Gly Gly Gly Ser Ser 565 570 575 Gly Gly Gly Gly Ser Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val 580 585 590 Val Arg Pro Gly Glu Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 595 600 605 Ser Phe Ile Asn Phe Ala Met Thr Trp Val Arg Gln Ala Pro Gly Glu 610 615 620 Gly Pro Glu Trp Val Ser Leu Ile Ser Asp Asp Gly Arg Gly Asn Arg 625 630 635 640 Pro Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 645 650 655 Asn Ser Lys Asn Ile Leu Tyr Leu Gln Met Asn Ser Leu Arg Val Glu 660 665 670 Asp Thr Ala Thr Tyr Tyr Cys Ala Lys Val Ser Trp Asp Val Phe Phe 675 680 685 Asp Tyr Trp Gly Leu Gly Thr Val Val Thr Val Ser Ser 690 695 700 <210> 281 <211> 215 <212> PRT <213> Artificial sequence <220> <223> Artificial p2.3 IgG-scFv fusion biparatopic mAb with HC C-terminal p1.2-scFv and ADCC / CDC abrogating Fc mutations (light chain (kappa)) <400> 281 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Ile Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val Arg Ser Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 282 <211> 701 <212> PRT <213> Artificial sequence <220> <223> Artificial p2.3 IgG-scFv fusion Tie2 / VEGF bispecific mAb with HC C-terminal VEGF-scFv and ADCC / CDC abrogating Fc mutations (heavy chain) <400> 282 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asn Val Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Ile Ile Ser Arg Asp Ser Ser Arg Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Asn Trp Asn Ser Phe Phe Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly 435 440 445 Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 450 455 460 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala 465 470 475 480 Ser Gln Val Ile Arg Arg Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly 485 490 495 Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Ala Ser Gly 500 505 510 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 515 520 525 Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln 530 535 540 Gln Ser Asn Thr Ser Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu 545 550 555 560 Ile Lys Gly Gln Ser Ser Arg Ser Ser Gly Gly Gly Gly Ser Ser Gly 565 570 575 Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 580 585 590 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser 595 600 605 Ile Asn Gly Ser Trp Ile Phe Trp Val Arg Gln Ala Pro Gly Lys Gly 610 615 620 Leu Glu Trp Val Gly Ala Ile Trp Pro Phe Gly Gly Tyr Thr His Tyr 625 630 635 640 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys 645 650 655 Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 660 665 670 Val Tyr Tyr Cys Ala Arg Trp Gly His Ser Thr Ser Pro Trp Ala Met 675 680 685 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 690 695 700 <210> 283 <211> 215 <212> PRT <213> Artificial sequence <220> <223> Artificial p2.3 IgG-scFv fusion Tie2 / VEGF bispecific mAb with HC C-terminal VEGF-scFv and ADCC / CDC abrogating Fc mutations (light chain (kappa)) <400> 283 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Ile Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val Arg Ser Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 284 <211> 682 <212> PRT <213> Artificial sequence <220> <223> Aflibercept-p2.3 scFv fusion Tie2 / VEGF bispecific mAb with C-terminal p2.3-scFv <400> 284 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 1 5 10 15 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25 30 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40 45 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50 55 60 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 65 70 75 80 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85 90 95 Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile 100 105 110 Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120 125 Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130 135 140 His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly 145 150 155 160 Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr 165 170 175 Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 180 185 190 Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr 195 200 205 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 210 215 220 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 225 230 235 240 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 245 250 255 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 260 265 270 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 275 280 285 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 290 295 300 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 305 310 315 320 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 325 330 335 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 340 345 350 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 355 360 365 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 370 375 380 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 385 390 395 400 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 405 410 415 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 420 425 430 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser 435 440 445 Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Ile Ala Thr Leu Ser Cys 450 455 460 Arg Ala Ser Gln Asn Val Arg Ser Asp Leu Ala Trp Tyr Gln Gln Lys 465 470 475 480 Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala 485 490 495 Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 500 505 510 Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr 515 520 525 Cys Gln Gln Tyr Ser Asn Trp Pro Pro Leu Thr Phe Gly Gly Gly Thr 530 535 540 Lys Val Glu Ile Lys Gly Gln Ser Ser Arg Ser Ser Gly Gly Gly Gly 545 550 555 560 Ser Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val Glu Ser Gly Gly 565 570 575 Gly Val Val Arg Pro Gly Glu Ser Leu Arg Leu Ser Cys Thr Ala Ser 580 585 590 Gly Phe Thr Phe Asp Ser Tyr Gly Met Ser Trp Val Arg Gln Ala Pro 595 600 605 Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Asn Val Gly Asp Asn Thr 610 615 620 Tyr Tyr Ala Asp Ser Val Arg Gly Arg Phe Ile Ile Ser Arg Asp Ser 625 630 635 640 Ser Arg Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Thr Ala Glu Asp 645 650 655 Thr Ala Val Tyr Tyr Cys Ala Asn Trp Asn Ser Phe Phe Asp Tyr Trp 660 665 670 Gly Leu Gly Thr Leu Val Thr Val Ser Ser 675 680 <210> 285 <211> 682 <212> PRT <213> Artificial sequence <220> <223> Aflibercept-p2.3 scFv fusion Tie2 / VEGF bispecific mAb with C-terminal p2.3-scFv and ADCC / CDC abrogating Fc mutations <400> 285 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 1 5 10 15 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25 30 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40 45 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50 55 60 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 65 70 75 80 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85 90 95 Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile 100 105 110 Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120 125 Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130 135 140 His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly 145 150 155 160 Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr 165 170 175 Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 180 185 190 Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr 195 200 205 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 210 215 220 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 225 230 235 240 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 245 250 255 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 260 265 270 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 275 280 285 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 290 295 300 Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr 305 310 315 320 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 325 330 335 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 340 345 350 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 355 360 365 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 370 375 380 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 385 390 395 400 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 405 410 415 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 420 425 430 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser 435 440 445 Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Ile Ala Thr Leu Ser Cys 450 455 460 Arg Ala Ser Gln Asn Val Arg Ser Asp Leu Ala Trp Tyr Gln Gln Lys 465 470 475 480 Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala 485 490 495 Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 500 505 510 Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr 515 520 525 Cys Gln Gln Tyr Ser Asn Trp Pro Pro Leu Thr Phe Gly Gly Gly Thr 530 535 540 Lys Val Glu Ile Lys Gly Gln Ser Ser Arg Ser Ser Gly Gly Gly Gly 545 550 555 560 Ser Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val Glu Ser Gly Gly 565 570 575 Gly Val Val Arg Pro Gly Glu Ser Leu Arg Leu Ser Cys Thr Ala Ser 580 585 590 Gly Phe Thr Phe Asp Ser Tyr Gly Met Ser Trp Val Arg Gln Ala Pro 595 600 605 Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Asn Val Gly Asp Asn Thr 610 615 620 Tyr Tyr Ala Asp Ser Val Arg Gly Arg Phe Ile Ile Ser Arg Asp Ser 625 630 635 640 Ser Arg Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Thr Ala Glu Asp 645 650 655 Thr Ala Val Tyr Tyr Cys Ala Asn Trp Asn Ser Phe Phe Asp Tyr Trp 660 665 670 Gly Leu Gly Thr Leu Val Thr Val Ser Ser 675 680 <210> 286 <211> 696 <212> PRT <213> Artificial sequence <220> <223> p2.3-ranibizumab Tie2 / VEGF bispecific mAb with HC C-terminal ranibizumab-scFv and ADCC / CDC abrogating Fc mutations (heavy chain) <400> 286 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asn Val Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Ile Ile Ser Arg Asp Ser Ser Arg Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Asn Trp Asn Ser Phe Phe Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly 435 440 445 Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly 450 455 460 Thr Leu Ser Leu Ser Pro Gly Glu Ile Ala Thr Leu Ser Cys Arg Ala 465 470 475 480 Ser Gln Asn Val Arg Ser Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly 485 490 495 Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly 500 505 510 Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 515 520 525 Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln 530 535 540 Gln Tyr Ser Asn Trp Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Val 545 550 555 560 Glu Ile Lys Gly Gln Ser Ser Arg Ser Ser Gly Gly Gly Gly Ser Ser 565 570 575 Gly Gly Gly Gly Ser Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val 580 585 590 Val Arg Pro Gly Glu Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe 595 600 605 Thr Phe Asp Ser Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys 610 615 620 Gly Leu Glu Trp Val Ser Ser Ile Asn Val Gly Asp Asn Thr Tyr Tyr 625 630 635 640 Ala Asp Ser Val Arg Gly Arg Phe Ile Ile Ser Arg Asp Ser Ser Arg 645 650 655 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala 660 665 670 Val Tyr Tyr Cys Ala Asn Trp Asn Ser Phe Phe Asp Tyr Trp Gly Leu 675 680 685 Gly Thr Leu Val Thr Val Ser Ser 690 695 <210> 287 <211> 215 <212> PRT <213> Artificial sequence <220> <223> p2.3-ranibizumab Tie2 / VEGF bispecific mAb with HC C-terminal ranibizumab-scFv and ADCC / CDC abrogating Fc mutations (light chain (kappa)) <400> 287 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Ile Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val Arg Ser Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 288 <211> 696 <212> PRT <213> Artificial sequence <220> <223> p2.3-ranibizumab Tie2 / VEGF bispecific mAb with HC N-terminal ranibizumab-scFv and ADCC / CDC abrogating Fc mutations (heavy chain) <400> 288 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Ile Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val Arg Ser Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gln Ser Ser 100 105 110 Arg Ser Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Ser Asp Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu Ser Leu 130 135 140 Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Ser Tyr Gly Met 145 150 155 160 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser 165 170 175 Ile Asn Val Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val Arg Gly Arg 180 185 190 Phe Ile Ile Ser Arg Asp Ser Ser Arg Asn Thr Leu Tyr Leu Gln Met 195 200 205 Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Asn Trp 210 215 220 Asn Ser Phe Phe Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr Val Ser 225 230 235 240 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val 245 250 255 Glu Ser Gly Gly Gly Val Val Arg Pro Gly Glu Ser Leu Arg Leu Ser 260 265 270 Cys Thr Ala Ser Gly Phe Thr Phe Asp Ser Tyr Gly Met Ser Trp Val 275 280 285 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Asn Val 290 295 300 Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val Arg Gly Arg Phe Ile Ile 305 310 315 320 Ser Arg Asp Ser Ser Arg Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 325 330 335 Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Asn Trp Asn Ser Phe 340 345 350 Phe Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr Val Ser Ser Ala Ser 355 360 365 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 370 375 380 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 385 390 395 400 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 405 410 415 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 420 425 430 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 435 440 445 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 450 455 460 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 465 470 475 480 Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 485 490 495 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 500 505 510 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 515 520 525 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 530 535 540 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 545 550 555 560 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 565 570 575 Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 580 585 590 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 595 600 605 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 610 615 620 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 625 630 635 640 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 645 650 655 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 660 665 670 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 675 680 685 Ser Leu Ser Leu Ser Pro Gly Lys 690 695 <210> 289 <211> 215 <212> PRT <213> Artificial sequence <220> <223> p2.3-ranibizumab Tie2 / VEGF bispecific mAb with HC N-terminal ranibizumab-scFv and ADCC / CDC abrogating Fc mutations (light chain (kappa)) <400> 289 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Ile Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val Arg Ser Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Asn Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215

Claims

1. An isolated anti-Tie2 antibody or its antigen-binding fragment comprising three heavy chain complementarity-determining regions (CDR H1-3) and three light chain CDRs (CDR L1-3), CDR H1-3 and CDR L1-3 are CDR H1 containing the amino acid sequence of SEQ ID NO: 28, CDR H2 containing the amino acid sequence of SEQ ID NO: 68, CDR H3 containing the amino acid sequence of SEQ ID NO: 108, CDR L1 containing the amino acid sequence of SEQ ID NO: 148, CDR L2 containing the amino acid sequence of SEQ ID NO: 188, and CDR L3 containing the amino acid sequence of SEQ ID NO: 228, or CDR H1-3 and CDR L1-3 are CDR H1 containing the amino acid sequence of SEQ ID NO: 29, CDR H2 containing the amino acid sequence of SEQ ID NO: 69, CDR H3 containing the amino acid sequence of SEQ ID NO: 109, CDR L1 containing the amino acid sequence of SEQ ID NO: 149, CDR L2 containing the amino acid sequence of SEQ ID NO: 189, and CDR L3 containing the amino acid sequence of SEQ ID NO:

229. Isolated anti-Tie2 antibody or its antigen-binding fragment.

2. It includes a heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 244, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 245, or A heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 272, and a light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 273, Isolated anti-Tie2 antibody or its antigen-binding fragment.

3. The antibody according to claim 1 or 2, which is an allosteric activator of Tie2.

4. The antibody according to claim 1 or 2, which is a non-ligand competitive binder for Tie2.

5. The antibody according to claim 1 or 2, which is cross-reactive to human, mouse, rat, rabbit, and monkey Tie2.

6. The antibody according to claim 1 or 2, which is a fully human antibody, a humanized antibody, a monoclonal antibody, or a chimeric antibody.

7. The antibody according to claim 1 or 2, which is an antibody fragment that specifically binds to human Tie2.

8. The antibody fragment is Fab, Fab'-SH, Fv, scFv, or (Fab') ２ A fragment of the antibody according to claim 7.

9. The antibody according to claim 1 or 2, having reduced effector function.

10. The antibody according to claim 9, comprising at least one substitution mutation at amino acid residues N297, L234, L235, P329, D265, and E430, as assigned by EU numbering such as Kabat numbering.

11. The antibody according to claim 10, wherein the at least one substitution mutation is selected from the group consisting of amino acid residues N297G, N297A, L234A, L235A, P329G, D265A, and E430G, according to EU numbering such as Kabat numbering.

12. The antibody according to claim 11, comprising the substitution mutation in residue N297A or N297G.

13. The antibody according to claim 11, comprising the substitution mutations in residues L234A, L235A, and P329G.

14. The antibody according to claim 11, comprising the substitution mutations in residues D265A and N297G.

15. An isolated nucleic acid encoding the antibody according to claim 1 or 2.

16. A vector comprising the isolated nucleic acid described in claim 15.

17. A host cell comprising the vector according to claim 16.

18. A method for producing the antibody according to claim 1 or 2, The host cells described in claim 17 are cultured in a culture medium, The obtained antibodies are isolated and Methods that include...

19. An immunoconjugate or fusion polypeptide comprising the antibody described in claim 1 or 2.

20. A pharmaceutical composition comprising the antibody according to claim 1 or 2, or the immunoconjugate or fusion polypeptide according to claim 19.

21. A pharmaceutical composition according to claim 20 for treating Tie2 dysregulation disorder.

22. The pharmaceutical composition according to claim 21, wherein the Tie2 dysregulation disorder includes infectious diseases, acute respiratory distress syndrome (ARDS), ischemic injury, eye disorders, radiation injury, cancer, systemic sclerosis, traumatic brain injury, neuroinflammation, radiation injury, wound healing, myocardial infarction, blood-brain barrier injury, spongiform malformation, Duchenne muscular dystrophy (DMD), or Clarkson's disease.

23. The pharmaceutical composition according to claim 22, wherein the Tie2 dysregulation infection includes sepsis, dengue virus infection, tuberculosis, or influenza.

24. The pharmaceutical composition according to claim 22, wherein Tie2 dysregulation ischemic injury includes diabetic nephropathy, acute kidney injury, chronic kidney disease, organ transplantation, severe lower limb ischemia, traumatic brain injury, or stroke.

25. The pharmaceutical composition according to claim 22, wherein the Tie2 accommodative ocular disorder includes diabetic retinopathy, diabetic macular edema (DME), proliferative diabetic retinopathy (PDR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP), or glaucoma.

26. Use of an isolated anti-Tie2 antibody according to any one of claims 1 to 14, or an immunoconjugate or fusion polypeptide according to claim 19, for the manufacture of a pharmacopoeia for treating a Tie2 dysregulation disorder according to any one of claims 22 to 25.

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