Antibodies against TIE-2 and methods of using the same

Abstract

The present invention relates to anti-Tie2 antibodies and methods of using the same.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 938,816, filed November 21, 2019, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to anti-Tie2 antibodies and methods of using them. Background Technology

[0004] Tie2 is a receptor tyrosine kinase mainly expressed on the surface of endothelial cells, which plays an important 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); Thurston, G. et al., 1999. Leakage-Resistant Blood Vessels in Mice Transgenically Overexpressing Angiopoietin-1, Science 286(5449):2511–14; Saharinen et al., 2010. How is angiopoietin Tie linked to vascular endothelial growth factor? (How Do Angiopoietins Tie with Vascular Endothelial Growth Factors?) Current Opinion in Hematology; Augustin et al. 2009 Control of Vascular Morphogenesis and Homeostasis through the Angiopoietin-Tie System, Nature Reviews, Molecular Cell Biology 10(3): 165–77; Milam et al. 2015 The Angiopoietin-Tie2 Signaling Axis in the Vascular Leakage of Systemic Inflammation, Tissue Barriers 3(1-2)). Tie2 activity is tightly regulated by at least four soluble protein factors (called angiopoietin 1–4). Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are considered key regulators of Tie2 function. Under normal physiological conditions, high Ang1 levels and low Ang2 levels maintain constitutive activation of the Tie2 signaling axis.Specifically, Ang1 agonist ligands bind directly to the Tie2 receptor, leading to Tie2 aggregation, autophosphorylation, and downstream signaling events, including activation of the PI3-kinase / Akt and MAPK pathways.

[0005] Gene targeting experiments have shown that the Ang / Tie signaling system is essential for the physiological and pathological remodeling of lymphatic vessels and blood vessels in embryonic, neonatal, 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). In humans, altered angiopoietin expression is associated with many vascular diseases (Saharinen P, Eklund L, Alitalo K. Therapeutic targeting of the angiopoietin-TIE pathway), *Nature Reviews Drug Discovery* 2017; 16:635–61.

[0006] There is still a need for anti-Tie2 antibodies with improved properties and their therapeutic and diagnostic applications. Summary of the Invention

[0007] This invention provides an anti-Tie2 antibody and methods of using it for therapeutic and diagnostic purposes. The anti-Tie2 antibody of this invention has properties that make it particularly suitable for therapeutic use.

[0008] As part of the disclosed invention, it specifically covers:

[0009] Example 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), as shown below:

[0010]

[0011]

[0012] Example 2. An isolated anti-Tie2 antibody or its antigen-binding fragment, comprising:

[0013] The heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID No. 242 and the light chain variable (VL) domain containing the amino acid sequence of SEQ ID No. 243;

[0014] The heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID No. 244 and the light chain variable (VL) domain containing the amino acid sequence of SEQ ID No. 245;

[0015] The heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID No. 246 and the light chain variable (VL) domain containing the amino acid sequence of SEQ ID No. 247;

[0016] The heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID No. 248 and the light chain variable (VL) domain containing the amino acid sequence of SEQ ID No. 249;

[0017] The heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID No. 250 and the light chain variable (VL) domain containing the amino acid sequence of SEQ ID No. 251;

[0018] The heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID No. 252 and the light chain variable (VL) domain containing the amino acid sequence of SEQ ID No. 253;

[0019] The heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID No. 254 and the light chain variable (VL) domain containing the amino acid sequence of SEQ ID No. 255;

[0020] The heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID No. 256 and the light chain variable (VL) domain containing the amino acid sequence of SEQ ID No. 257;

[0021] The heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID No. 258 and the light chain variable (VL) domain containing the amino acid sequence of SEQ ID No. 259;

[0022] The heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID No. 260 and the light chain variable (VL) domain containing the amino acid sequence of SEQ ID No. 261;

[0023] The heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID No. 262 and the light chain variable (VL) domain containing the amino acid sequence of SEQ ID No. 263;

[0024] The heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID No. 264 and the light chain variable (VL) domain containing the amino acid sequence of SEQ ID No. 265;

[0025] The heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID No. 266 and the light chain variable (VL) domain containing the amino acid sequence of SEQ ID No. 267;

[0026] The heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID No. 268 and the light chain variable (VL) domain containing the amino acid sequence of SEQ ID No. 269; or

[0027] The heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID No. 270 and the light chain variable (VL) domain containing the amino acid sequence of SEQ ID No. 271.

[0028] Example 3. An isolated anti-Tie2 antibody, wherein the antibody specifically binds to an epitope within the extracellular domain of human Tie2, the epitope comprising amino acid residues K312, S316, C332, H358, K387, and T391 (according to the EU number in Kabat), as measured by crosslinking mass spectrometry.

[0029] Example 4. The antibody according to Examples 1 to 3, wherein the antibody is an allosteric activator of Tie2.

[0030] Example 5. The antibody according to Examples 1 to 4, wherein the antibody is a non-ligand competitive binder of Tie2.

[0031] Example 6. The antibody according to Examples 3 to 5, wherein the antibody is cross-reactive with human, mouse, rat, rabbit and monkey Tie2.

[0032] Example 7. The antibody according to Examples 1 to 6, wherein the antibody is fully human, humanized, monoclonal, or chimeric.

[0033] Example 8. The antibody according to Examples 1 to 7, wherein the antibody is single-specific.

[0034] Example 9. The antibody according to Examples 1 to 7, wherein the antibody is multispecific.

[0035] Example 10. The antibody according to Example 9, wherein the multispecific antibody is bispecific.

[0036] Example 11. The antibody according to Example 10, wherein the bispecific antibody comprises a binding arm that specifically binds to human Tie-2 according to claim 8 and 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 β, PD-1, PD-L1, or CTLA-4.

[0037] Example 12. The antibody according to Example 9, wherein the multispecific antibody is a bispecific antibody.

[0038] Example 13. The antibody according to Example 12, wherein the dual in situ antibody comprises one binding arm of a first epitope on the ECD of human Tie2 specifically binding to and another binding arm of a second epitope on the ECD of human Tie2 specifically binding to.

[0039] Example 14. The antibody according to Example 9, wherein the multispecific antibody is a trivalent, tetravalent, pentavalent, or hexavalent antibody, wherein the trivalent, tetravalent, pentavalent, or hexavalent antibody comprises specifically binding at least one binding arm of human Tie2 according to claim 8 and other remaining binding arms specifically binding 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 β, PD-1, PD-L1, or CTLA-4.

[0040] Example 15. The antibody according to Examples 1 to 14, wherein the antibody is an antibody fragment that specifically binds to human Tie2.

[0041] Example 16. The antibody according to Example 15, wherein the antibody fragment is Fab, Fab'-SH, Fv, scFv or (Fab')2 fragment.

[0042] Example 17. The antibody according to Example 16, wherein the multispecific antibody comprises scFv antibody fragments linked together by linking polypeptides.

[0043] Example 18. An antibody according to Examples 1 to 17, wherein the antibody has reduced effector function.

[0044] Example 19. The antibody according to Example 18, wherein the antibody contains at least one substitution mutation at amino acid residues N297, L234, L235, P329, D265 and E430 (according to the EU number in Kabat).

[0045] Example 20. The antibody according to Example 19, 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 the EU number in Kabat).

[0046] Example 21. The antibody according to Example 20, wherein the antibody comprises a substitution mutation at residue N297A or N297G.

[0047] Example 22. The antibody according to Example 20, wherein the antibody comprises substitution mutations at residues L234A, L235A and P329G.

[0048] Example 23. The antibody according to Example 20, wherein the antibody comprises substitution mutations at residues D265A and N297G.

[0049] Example 24. The antibody according to Example 21, wherein the antibody further comprises a substitution mutation at residue E430G.

[0050] Example 25. The antibody according to Example 22, wherein the antibody further comprises a substitution mutation at residue E430G.

[0051] Example 26. The antibody according to Example 23, wherein the antibody further comprises a substitution mutation at residue E430G.

[0052] Example 27. The antibody according to Example 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.

[0053] Example 28. The antibody according to Example 22, comprising:

[0054] The heavy chain containing the amino acid sequence of SEQ ID No:276 and the light chain containing the amino acid sequence of SEQ ID No:277;

[0055] The heavy chain containing the amino acid sequence of SEQ ID No:278 and the light chain containing the amino acid sequence of SEQ ID No:279;

[0056] Heavy chain containing the amino acid sequence of SEQ ID No:280 and light chain containing the amino acid sequence of SEQ ID No:281;

[0057] The heavy chain containing the amino acid sequence of SEQ ID No:282 and the light chain containing the amino acid sequence of SEQ ID No:283;

[0058] 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

[0059] The heavy chain containing the amino acid sequence of SEQ ID No:288 and the light chain containing the amino acid sequence of SEQ ID No:289.

[0060] Example 29. The antibody according to Example 10, comprising:

[0061] The amino acid sequence of SEQ ID No:284; or

[0062] The amino acid sequence of SEQ ID No:285.

[0063] Example 30. An isolated nucleic acid encoding an antibody as described in Examples 1 to 29.

[0064] Example 31. A vector comprising the isolated nucleic acid as described in Example 30.

[0065] Example 32. A host cell comprising the vector according to Example 31.

[0066] Example 33. A method for producing antibodies according to Examples 1 to 29, the method comprising culturing host cells according to Example 32 in a culture medium and isolating the resulting antibodies.

[0067] Example 34. An immunoconjugate comprising the antibody described in Examples 1 to 29.

[0068] Example 35. A fusion polypeptide comprising the antibody described in Examples 1 to 29.

[0069] Example 36. A pharmaceutical composition comprising an antibody according to Examples 1 to 29, an immunoconjugate according to Example 34, or a fusion polypeptide according to Example 35.

[0070] Example 37. The pharmaceutical composition according to Example 36, wherein the antibody, the immunoconjugate, or the fusion polypeptide is formulated in combination with an anti-VEGF antibody or a VEGF extracellular trap protein.

[0071] Example 38. A method of treating Tie2 disorder in a subject in need, the method comprising administering to the subject the pharmaceutical composition according to Example 36.

[0072] Example 39. A method of treating Tie2 disorder in a subject in need, the method comprising administering to the subject the pharmaceutical composition according to Example 37.

[0073] Example 40. The method according to Example 38, further comprising administering to the subject in combination a pharmaceutical composition comprising an anti-VEGF antibody or a VEGF extracellular trap protein.

[0074] Example 41. The method according to Examples 38 to 40, wherein the Tie2 disorder includes infectious diseases, acute respiratory distress syndrome (ARDS), ischemic injury, eye disease, radiation injury, cancer, systemic sclerosis, traumatic brain injury, neuroinflammation, radiation injury, wound healing, myocardial infarction, blood-brain barrier damage, spongiform malformation, Duchenne muscular dystrophy (DMD), or Clarkson disease.

[0075] Example 42. The method according to Examples 38 to 40, wherein the Tie2 disordered infectious disease includes sepsis, dengue virus infection, tuberculosis, or influenza.

[0076] Example 43. The method according to Examples 38 to 40, wherein the Tie2 dysfunctional ischemic injury includes diabetic nephropathy, acute kidney injury, chronic kidney disease, organ transplantation, severe limb ischemia, traumatic brain injury, or stroke.

[0077] Example 44. The method according to Examples 38 to 40, wherein the Tie2 dysplastic eye disease includes diabetic retinopathy, diabetic macular edema (DME), proliferative diabetic retinopathy (PDR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP), or glaucoma.

[0078] Example 45. The isolated anti-Tie2 antibody according to Examples 1 to 29, or the immunoconjugate according to Example 34, or the fusion polypeptide according to Example 35, is used to treat Tie2 dysregulation diseases according to Examples 41 to 44.

[0079] Example 46. Use of an isolated anti-Tie2 antibody according to Examples 1 to 29, or an immunoconjugate according to Example 34, or a fusion polypeptide according to Example 35 in the manufacture of a medicament for treating Tie2 dysregulation diseases according to Examples 41 to 44. Attached Figure Description

[0080] Figure 1A -C. Anti-Tie2 antibody increased the levels of phosphorylated Tie2 (pTie2), as well as phosphorylated Akt (pAkt) and phosphorylated ERK (pERK), as determined by Western blotting. HUVEC cells were cultured using a serum starvation method and then treated with PBS, Ang1 (12 nM), control hIgG1 (12 nM), or human anti-Tie2 antibodies #1, #2, and #3 or wild-type (WT) chicken anti-Tie2 antibody (12 nM) for 20 minutes, followed by cell lysis. Figure 1A The method demonstrates the immunoprecipitation of Tie2 from lysates using an anti-Tie2 antibody and the determination of pTie2 levels using Western blotting with an anti-phosphorylated tyrosine antibody. For pAkt and pERK analyses, pAkt and total Akt in whole-cell lysates were analyzed. Figure 1B ) or pERK and total ERK ( Figure 1C The protein was determined using Western blotting. See Example 3.

[0081] Figure 2 The functional potency of anti-Tie2 antibodies is comparable to that of Ang1 in vitro. HUVEC cells were cultured using a serum starvation method and treated with different 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 or total Akt-specific antibodies. pAkt and total Akt signal intensities were measured using a LI-COR scanning fluorometer and analyzed using Graphpad. TM The Prism software plots the pAkt level (pAkt / Akt ratio) normalized relative to total Akt on the Y-axis to determine the EC50 value. See Example 4.

[0082] Figure 3 A schematic diagram depicting a simplified in vitro model of the endothelial barrier. See Example 5.

[0083] Figure 4In an in vitro model of endothelial barrier leakage, anti-Tie2 antibodies reduced VEGF-induced cell permeability. Results are expressed as a percentage of Ang1 activity (normalized relative to 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. Meaning of symbols: **P≤0.01; ****≤0.0001. See Example 5.

[0084] Figure 5A -F. The combination of anti-Tie2 antibody and saturated Ang2 had an additive effect on downstream Tie2 signaling in HUVEC cells. HUVEC cells were cultured using a serum starvation method, treated with reduced amounts of Ang2, lysed, and the levels of pERK and total ERK were analyzed. Normalized values ​​were plotted on the Y-axis; Ang2 concentrations were plotted on the X-axis. Figure 5A Images were quantified using a LI-COR scanning fluorometer, and pERK levels were normalized relative to total ERK. HUVEC cells cultured via serum starvation were treated with 43 nM Ang2 and 12 nM anti-Tie2 antibody for 20 min. Lysates were submitted for Western blotting analysis, and pERK and ERK levels were analyzed separately. Figure 5C ) as well as pAKT and AKT ( Figure 5E Quantitative analysis was performed on the images of pERK (). Figure 5D ) and pAKT ( Figure 5F The levels were normalized relative to the total ERK and Akt levels, respectively, to control for equal protein loading. See Example 6.

[0085] Figure 6A -B. Anti-Tie2 antibody does not interact with Ang1 ( Figure 6A ) or Ang2( Figure 6B Competitive binding to the Tie2 extracellular domain (ECD) was demonstrated. Representative competitive binding data using biomembrane layer interference (BLI) were depicted. The left panel shows the loading of his-labeled Tie2 ECD protein (100 nM) onto a capture biosensor (Ni-NTA), followed by exposure as indicated by arrow (1), then exposure to a solution containing a single anti-Tie2 antibody (100 nM) as indicated by arrow (2), and then exposure to a solution containing Ang1 (3). Figure 6A ) or Ang2 ligand ( Figure 6B The solutions were prepared (all at 100 nM). The y-axis represents the degree of binding with the biosensor, and the x-axis represents the elapsed time. See Example 7.

[0086] Figure 7By BLI binding assessment, anti-Tie2 antibodies exhibited diversity in epitope recognition. Recombinant human Tie2 ECD protein was loaded onto each biosensor and subsequently exposed to a solution containing one anti-Tie2 antibody (y-axis), followed by exposure to a second anti-Tie2 antibody (x-axis). The total wavelength shift (in nm) after exposure to the second antibody is indicated by white numbers in each box. Black boxes indicate a lack of binding to the second antibody (value <0.2 nm). The lack of binding was observed only in the same antibody pairs, indicating that all antibodies tested in this group bound to different epitopes found on the Tie2 ECD. See Example 9.

[0087] Figure 8 AB. Systemic administration of anti-Tie2 antibodies did not appear to reduce neovascularization (NV). Figure 8 A), but reduced vascular occlusion (VO) in the oxygen-induced retinopathy (OIR) model. Figure 8 B). Each data point represents the average of 2 eyes per puppy. Bars represent the mean + / - SEM; N = 7-8 one-way ANOVAs; Tukey post-hoc comparisons | *p < 0.05; **p < 0.01; ***p < 0.001. See Example 10.

[0088] Figure 9A -B. In a mouse model, anti-Tie2 antibody inhibited laser-induced choroidal neovascularization (CNV). Neovascularization in the lesion region and vascular density in the retina were quantified using Image J. P-values ​​were assessed using Student's t-test (significant change, p < 0.05). See Example 11.

[0089] Figure 10A -B. Schematic diagram of the Tie2 / VEGF bispecific construct of the present invention. Figure 10A A schematic diagram of an anti-Tie2 antibody linked to two VEGF scFv antibody fragments via a linker peptide is shown. Exemplary constructs are embodied in antibody clones #57 and #58. Figure 10B A schematic diagram is shown of two anti-Tie2scFv antibody fragments linked to two VEGF-trap proteins containing R1-D2 and R3-D3. Exemplary constructs are embodied in antibody clones #55 and #56. See Example 13.

[0090] Figure 11The Tie2 agonist activity of the Tie2 / VEGF bispecific construct was assessed by Western blotting. The controls used were untreated and anti-Ang1 antibody (Ang1). Positive controls were anti-Tie2 antibody clone #3 or a nonspecific antibody (NS). The assay bispecific antibody was an anti-Tie2 / VEGF bispecific antibody (antibody clone #54), a nonspecific antibody / VEGF bispecific antibody (NS / VEGF), a VEGF-trap / aTie2 (antibody clone #55), or a VEGF-trap alone. See Examples 3 and 14.

[0091] Figure 12 AC. Schematic diagram of the tetravalent and bispecific anti-Tie2 antibody constructs of the present invention for increasing the titer of Tie2 or crosslinking multiple Tie2 epitopes. An exemplary tetravalent anti-Tie2 construct has heavy and light chain sequences of an anti-Tie2 antibody, which are linked to the C-terminus of the IgG heavy chain via a linker polypeptide. Figure 12 A) or the N-terminus of the heavy chain ( Figure 12 The exemplary dual in situ anti-Tie2 antibody construct has two anti-Tie2 scFv sequences from one antibody clone (aTie2scFv B), which are linked by a linker polypeptide to the C-terminus of the IgG heavy chain of a second distinct anti-Tie2 antibody clone (aTie2 Fab A). Figure 12 C). In all cases, the Tie2 binding site is indicated by a shaded box. See Example 15.

[0092] Figure 13 A schematic diagram of the hexameric anti-Tie2 antibody construct of the present invention for enhancing oligomerization and thus enhancing the agonistic effect of Tie2. An exemplary hexameric anti-Tie2 construct having the heavy and light chain sequences of an anti-Tie2 antibody with an E430G Fc mutation is shown. See Example 15.

[0093] Figure 14 The Tie2 agonist activity of the various anti-Tie2 antibody constructs of this invention was evaluated using pAKT / pERK Western blotting. Controls used were untreated anti-Ang1 antibody (Ang1), anti-Tie2 antibody clone #3 (aTie2), or a nonspecific antibody (NS). 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. Detailed Implementation

[0094] I. Definition

[0095] "Tie2," also known as the angiopoietin-1 receptor, or TEK receptor tyrosine kinase, or CD202B (differentiation cluster 202B), is a protein encoded by the TEK gene in the human body (Partanen Jet et 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 has a unique extracellular domain containing three immunoglobulin-like loops, three epidermal growth factor-like repeat sequences, and three type III fibronectin-like repeat sequences (see Fiedler et al., 2006. Angiopoietins: A Link between Angiogenesis and Inflammation, Trends in Immunology, 27(12): 552–58; Crystal structures of the Tie2 receptor ectodomain and the angiopoietin-2–Tie2 complex, Barton et al., Nature Struc. & Mol. Biology, 13, pp. 524–532 (2006). The contact residues of angiopoietin-1 and angiopoietin-2 largely overlap on the Tie-2 receptor and are mainly located in the second Ig-like loop, as indicated by the crystal structure analysis of the Ang2 / Tie2 complex (Barton et al., Nature Structural Biology, 2006).Other works support the concept 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, Journal of Biochemistry (JBC), Vol. 278(3):1721-7(2003)). The amino acid sequence of exemplary human Tie2 can be found according to UniProt accession number Q02763 (SEQ ID No:241).

[0096] As used herein, the term "approximately" refers to the typical range of error for various values ​​that is readily known to those skilled in the art. References to "approximately" values ​​or parameters herein include (and describe) embodiments of that value or parameter itself.

[0097] For the purposes of this document, a “recipient human frame” is a frame comprising the amino acid sequence of a light chain variable domain (VL) frame or a heavy chain variable domain (VH) frame derived from a human immunoglobulin frame or a human common frame, as defined below. A recipient human frame “derived from” a human immunoglobulin frame or a human common frame may contain the same amino acid sequence, or may contain variations in the amino acid sequence. 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 sequence of the VL recipient human frame is identical to the VL human immunoglobulin frame sequence or the human common frame sequence.

[0098] In the context of this invention, “active” or “bioactive” means (e.g., in vitro and / or in vivo) the ability to elicit (partially or fully activate) the bioactivity of its target. An example of antibody bioactivity is the ability to achieve a measurable improvement in the state (e.g., pathology) of a disease associated with its target. For example, for an anti-Tie2 antibody, the disease could be a Tie2-related disease, such as AMD (e.g., geographic atrophy). The activity of anti-Tie2 antibodies can be determined in in vitro or in vivo assays, including binding assays, 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 antibodies of this invention can be determined in in vitro or in vivo assays, including binding assays, alternative pathway hemolysis assays (e.g., assays measuring the activity or inhibition of alternative pathway complement), using relevant animal models, or human clinical trials.

[0099] The term “active site on Tie2” is defined as the Ang 1 / Ang 2 binding domain on Tie2, which is known to be located within the Ig2-like domain of the Tie2 extracellular domain.

[0100] “Affinity” refers to the total strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is typically expressed as a dissociation constant (KD). Affinity can be measured using methods commonly known in the art, including those described herein.

[0101] "Affinity-mature" antibodies are those that have one or more alterations in one or more hypervariable regions (CDRs) and / or frame regions (FRs), which result in increased affinity of the antibody for the antigen compared to parental antibodies that do not have such alterations.

[0102] "Allosteric activation of Tie2" is the activation of Tie2 by an agonistic anti-Tie2 antibody that specifically interacts with the Tie2 region outside the ligand binding or active site, such that the binding leads to a change in the conformation or clustering of Tie2, thereby enhancing the activity of the receptor.

[0103] The term “antibody” is used in the broadest sense herein to include various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity.

[0104] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; dimers; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0105] Papain digestion of the antibody yields two identical antigen-binding fragments, termed the “Fab” fragment, and a residual “Fc” fragment, a name reflecting its tendency to crystallize. The Fab fragment consists of a variable region (VH) domain of both the light (L) and heavy (H) chains, and a first constant domain of one heavy chain (CH1). Pepsin treatment of the antibody yields a single large F(ab’)2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and remains capable of cross-linking the antigen. The Fab’ fragment differs from the Fab fragment in that it has a small number of additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab’-SH is the name given to Fab’ herein, where the cysteine ​​residues in the constant domain have a free thiol group. The F(ab’)2 antibody fragments are initially produced as a pair of Fab’ fragments with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known.

[0106] The term “Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes 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 C-terminus of the heavy chain. However, the C-terminal lysine residue (Lys447) of the Fc region may or may not be present. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th edition, Center for Public Health Services, National Institutes of Health, Bethesda, MD, (1991).

[0107] The “Fv” consists of a dimer composed of a heavy-chain variable region domain and a light-chain variable region domain tightly bound together nonvalently. The folding of these two domains produces six hypervariable rings (three rings each in the H chain and L chain), which provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can recognize and bind antigens, but its affinity is generally lower than that of the entire binding site.

[0108] A single-chain Fv, also known as sFv or scFv, is an antibody fragment containing VH and VL antibody domains linked together to form a single polypeptide chain. sFv polypeptides may further contain linker polypeptides between the VH and VL domains, allowing the sFv to form the structure required for antigen binding. For a review of sFv, see Pluckthun, *The Pharmacology of Monoclonal Antibodies*, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994).

[0109] The term "dimer" 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 to achieve interchain rather than intrachain pairing of the V domains, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. A bispecific dimer is a heterodimer of two "crossed" sFv fragments in which the VH and VL domains of the two antibodies are located on different polypeptide chains. Dimers are described more comprehensively in, for example, EP404,097; WO 93 / 11161; and Hollinger et al., Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 90:6444-6448, 1993.

[0110] "Blocking" or "antagonistic" antibodies are antibodies that inhibit or reduce the biological activity of the antigens they bind to. Some blocking or antagonistic antibodies essentially or completely inhibit the biological activity of the antigen.

[0111] "Agonistic" or "activating" antibodies are antibodies that activate, stimulate, or increase the biological or signaling activity of the antigens they bind to. In some cases, agonist antibodies are expected to function in a manner similar to binding to a ligand and activating its homologous receptor. In other cases, an anti-Tie2 antibody of the present invention is expected to be considered agonist if it induces Tie2 signaling, as determined by an increase in the levels of one or more intracellular phosphorylated Tie2 (pTie2) and / or phosphorylated Akt (pAkt) and / or phosphorylated ERK (pERK), as described in Example 3. Furthermore, it is further expected that the agonist Tie2 antibody of the present invention will also be able to activate downstream signaling of its target antigen in the presence or absence of endogenous activating (i.e., Ang1) or inhibitory (i.e., Ang2) ligands, as described in Examples 6 and 7.

[0112] An antibody “binding to the same epitope as the reference antibody” refers to an antibody that contacts a set of overlapping (compared to the reference antibody) amino acid residues of the antigen, or an antibody that blocks the reference antibody from binding to its antigen by 50% or more in a competitive assay. For example, the amino acid residues of the antibody in contact with the antigen can be determined by determining the crystal structure of the antibody-antigen complex or by performing a hydrogen / deuterium exchange. In some embodiments, antibody residues within 5 angstroms of 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 reference antibody from binding to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the antibody from binding to its antigen by 50% or more in a competitive assay. An exemplary competitive assay is provided herein.

[0113] As used in this article, the term "bispecific" refers to a bispecific antibody in which the first antigen-binding portion and the second antigen-binding portion bind to different epitopes on the same antigen.

[0114] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a specific source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0115] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ.

[0116] "Complement factors" refer to the various proteins and glycoproteins that make up the complement cascade, a part of the immune system that enhances (complement) antibodies and phagocytes' ability to clear inflammatory microbes and damaged cells from the body, as well as attack pathogen cell membranes. It is part of the innate immune system. The complement factors anticipated in this paper include, for example, C1, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, and C9.

[0117] "Effective functions" refer to those biological activities attributed to the Fc region of an antibody, which vary across antibody isotypes. 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.

[0118] The term "framework," "frame region," or "FR" refers to variable domain residues other than the hypervariable region (CDR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4.

[0119] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably in this document and refer to antibodies that have a structure substantially similar to that of natural antibodies or that contain a heavy chain with an Fc region as defined herein.

[0120] The term "fusion peptide" includes the anti-Tie2 antibody of the present invention fused to, for example, the Fc region of an immunoglobulin. The Fc region may contain, for example, the CH3 domain of an immunoglobulin, which may be naturally occurring or modified in some way. Such Fc fusion peptides may exhibit a longer in vivo half-life than their unfused counterparts. Furthermore, fusion with the Fc region allows for dimerization / polymerization of the fusion peptide. As anticipated, the Fc region may be a naturally occurring Fc region or may be modified to improve certain qualities, such as, for example, therapeutic quality, cycle time, or reduction of aggregation problems. In another embodiment, the fusion peptide considers an anti-Tie2 antibody fragment fused with a ligand (e.g., Ang1) to enhance the agonistic activity of the fusion peptide. In another embodiment, the fusion peptide considers an anti-Tie2 antibody fragment fused with, for example, a cytokine to elicit other desired biological properties.

[0121] As used in this article, a “hexamerizing antibody” is an antibody in which the introduction of the E430G mutation in the Fc region promotes the natural process of antibody hexamer formation by increasing intermolecular Fc-Fc interactions when binding to membrane-bound antigens on the cell surface (Diebolder et al., Science 2014; de Jong et al., PLoS Biol. 2016).

[0122] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source using a human antibody library or other human antibody coding sequences. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0123] A “human common framework” is a framework representing the most frequently occurring amino acid residues in the selection of the human immunoglobulin VL or VH framework sequence. Typically, the selection of the human immunoglobulin VL or VH sequence is derived from a subgroup of variable domain sequences. Generally, sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health Publication (NIH Publication) 91-3242, Bethesda, MD, (1991) Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI, as described above by Kabat et al. In one embodiment, for VH, the subgroup is subgroup III, as described above by Kabat et al.

[0124] The “humanized” form of non-human (e.g., rodent) antibodies is a chimeric antibody, containing a minimal sequence derived from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies), where residues from the receptor hypervariable region are replaced by residues from the hypervariable region of a non-human species (donor antibody, such as a mouse, rat, rabbit, or non-human primate possessing the desired antibody specificity, affinity, and capability). In some cases, the FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the receptor or donor antibody. These modifications are intended to further refine the antibody's properties. Generally, humanized antibodies will contain substantially all (at least one, usually two) of the variable domains, where all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. Humanized antibodies may also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically 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).

[0125] The term "variable" refers to the fact that certain segments of the variable domain in an antibody vary considerably in sequence. The variable, or "V," domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the span of the variable domain. Instead, the V region consists of relatively invariant segments of 15–30 amino acids (called frame regions (FRs)), separated by shorter regions of extreme variability (called "hypervariable regions"), each 9–12 amino acids long. As used herein, the term "hypervariable region" or "CDR" refers to the amino acid residues in the antibody responsible for antigen binding. The hypervariable region typically contains amino acid residues from, for example, approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in VL, and approximately residues 26-35 (H1), 49-65 (H2), and 95-102 (H3) in VH (in one embodiment, H1 is approximately residue 31-35); Kabat et al., Sequences of Immunological Interest, 5th ed., NIH Public Health Service, Bethesda, MD, (1991), and / or amino acid residues from the “hypervariable ring” (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in VL, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in VH; Chothia and Lesk, Journal of Molecular Biology, 196:9 01-917 (1987). The variable domains of both the natural heavy and light chains each contain four FRs, primarily employing a β-sheet structure, linked by three hypervariable regions to form loops connecting the β-sheet structures, and in some cases, forming part of the β-sheet structure. The hypervariable regions in each chain are tightly bound to the hypervariable regions of the other chain via FRs, facilitating the formation of antibody antigen-binding sites (see Kabat et al., *Protein Sequences of Immunological Interest*, 5th ed., National Institutes of Health Public Health Service, Bethesda, MD, (1991)). Therefore, CDR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as antibody involvement in antibody-dependent cytotoxicity (ADCC).

[0126] The terms “residue number as in Kabat,” “Kabat amino acid residue,” or “amino acid position number as in Kabat,” and their variations, refer to the numbering system used by Kabat et al. in their antibody compilations, as described above, for heavy chain or light chain variable domains. Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids, corresponding to shortening or insertion of the variable domain FR or CDR. For example, a heavy chain variable domain could include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 following heavy chain FR residue 82 and after inserted residues (e.g., residues 82a, 82b, and 82c according to Kabat). For a given antibody, the Kabat number of the residue can be determined by comparing the homologous regions of the antibody sequence with a “standard” Kabat numbered sequence.

[0127] When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., Sequences of Interest in Immunology, 5th Edition, National Institutes of Health Public Health Service, Bethesda, MD (1991)). When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is generally used (e.g., the EU index reported in Kabat et al. above). “EU index in Kabat” refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise stated herein, the residue numbers in the variable domain of the antibody referred to are those obtained through the Kabat numbering system. Unless otherwise stated herein, the residue numbers in the constant domain of the antibody referred to are those obtained through the EU numbering system. Unless otherwise stated, CDR residues and other residues (e.g., FR residues) in the variable domain are numbered in this document according to Kabat et al. above.

[0128] "Immunoconjugates" are antibodies conjugated to one or more heterologous molecules that provide cell-killing or cell-altering activity, including but not limited to small molecule drugs (inhibitors or activators), or cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., bacterial, fungal, plant or animal-derived protein toxins, enzyme-active toxins or fragments thereof), or radioactive isotopes.

[0129] The term "isolated antibody," when used to describe the various antibodies disclosed herein, refers to an antibody that has been identified, isolated, and / or recovered from cells or cell cultures expressing the antibody. Its natural environmental contaminants are substances that typically interfere with the diagnostic or therapeutic use of the peptide and may include enzymes, hormones, and other protein or non-protein solutes. In some embodiments, the antibody is purified to a purity greater than 95% or 99% by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., Journal of Chromatography B, 848:79-87 (2007). In some embodiments, the antibody is purified (1) to the extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a rotary cup sequencer, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie Brilliant Blue or silver staining. Isolated antibodies include in situ antibodies from recombinant cells, since at least one component of the peptide in its native environment will not be present. However, isolated peptides are typically prepared via at least one purification step.

[0130] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting this population are identical and / or bind to the same epitopes, except for possible variant antibodies, such as those containing naturally occurring mutations or variants generated during the production of the monoclonal antibody formulation, which are typically present in small quantities. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody's properties are obtained from a substantially homogeneous population of antibodies and is not interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used according to the invention can be prepared 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 part of the human immunoglobulin loci, as described herein and other exemplary methods for preparing monoclonal antibodies.

[0131] The term "multispecific antibody" is used in the broadest sense and specifically covers antibodies or antibody fragments with multi-epitope specificity (i.e., capable of binding to two different epitopes on a single biomolecule or each epitope on different biomolecules). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies with two or more VL and VH domains, antibody fragments (such as Fab, Fv, dsFv, scFv), dimers, bispecific dimers and trimers, and covalently or non-covalently linked antibody fragments. "Multi-epitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. "Bispecific" or "two-specific" refers to the ability to specifically bind to two different epitopes on the same or different targets. However, in contrast to bispecific antibodies, bispecific antibodies have two antigen-binding arms with identical amino acid sequences, and each Fab arm is capable of recognizing two antigens. Bispecificity allows the antibody to interact with two different antigens as a single Fab or IgG molecule with high affinity. According to one embodiment, a multispecific antibody in the form of IgG1 binds to each epitope with an affinity 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. "Single-specific" means that the antibody has the ability to bind to only one epitope on a specific antigen.

[0132] Regarding antibody binding to target molecules, the terms "specific binding," "specifically binding," or "specific to" a specific peptide or an epitope on a specific peptide target refer to binding that is significantly different from nonspecific interactions. For example, specific binding can be measured by comparing the binding of the antibody to that of a control molecule. Specific binding can also be determined by competition with a control molecule similar to the target, such as an excess of unlabeled target. In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, it indicates specific binding. As used herein, the terms "specific binding," "specifically binding," or "specific to" a specific peptide or an epitope on a specific peptide target can be, for example, having a target specificity of 10... -4 M or lower, or 10 -5 M or lower, or 10 -6 M or lower, or 10 -7 M or lower, or 10 -8 M or lower, or 10 -9 M or lower, or 10 -10 M or lower, or 10 -11 M or lower, or 10 -12 M or lower KD, or in the range of 10 -4 M to 10 -6 M or 10 -6 M to 10-10 M or 10 -7 M to 10 -9 Molecules with a KD value within the range of M. Those skilled in the art will understand that affinity is inversely correlated with KD value. High affinity for an antigen is measured by a low KD value. In one embodiment, the term "specific binding" refers to a binding in which a molecule (such as the antibody of the present invention) binds to a specific polypeptide or an epitope on a specific polypeptide and substantially does not bind to any other polypeptide or polypeptide epitope.

[0133] The term "non-ligand competitive binder" refers to the anti-Tie2 antibody of the present invention, which does not compete with Ang 1 or Ang 2 for the active site of Tie2, but still allows Ang 1 or Ang 2 to bind at the active site.

[0134] "Nucleic acid encoding antibody" refers to one or more nucleic acid molecules that encode the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecules in a single vector or in a single vector, and such nucleic acid molecules present at one or more locations in a host cell. In some embodiments, the nucleic acid encodes an anti-Tie2 antibody.

[0135] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors integrated into the host cell's genome, which have been introduced into the host cell. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0136] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny cells of this type. Host cells include “transformers” and “transformed cells,” including primary transformed cells and their derived progeny, regardless of passage number. The nucleic acid content of progeny cells may not be exactly the same as that of parent cells, but may contain mutations. This article includes mutant progeny cells that have the same function or biological activity as those initially transformed cells, screened or selected.

[0137] The "percentage of amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after sequence alignment and cleavage (if necessary) to obtain the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Alignment used to determine the percentage of amino acid sequence identity can be performed in various ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for sequence alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0138] A protein (including antibodies) is said to be “stable” if it retains essentially its complete conformational structure and biological activity. Various analytical techniques exist in the art for measuring protein stability, and reviews can be found, for example, in *Peptide and Protein Drug Delivery*, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones (1993) *Advanced Drug Delivery Rev.* 10:29-90. An antibody variant with “improved stability” refers to an antibody variant that is more stable than the original reference antibody. An antibody variant with improved stability is a variant of the reference (wild-type) antibody in which specific amino acid residues are altered to improve physical stability, and / or chemical stability, and / or biological activity, and / or reduce the immunogenicity of the native antibody.

[0139] In some embodiments, anti-Tie2 antibodies can be used as therapeutic agents that target and interfere with diseases or conditions involving Tie2 activity. Furthermore, other bioactivity assays can be performed on the antibodies, for example, to evaluate their effectiveness as therapeutic agents. Such assays are known in the art and depend on the intended use of the target antigen and the antibody. Examples include HUVEC inhibition assays; tumor cell growth inhibition assays (e.g., as described in WO 89 / 06692); antibody-dependent cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (US Patent No. 5,500,362); and agonist or hematopoietic activity assays (see WO 95 / 27062).

[0140] As used herein, the term “trispecific” refers to a type of antibody having three antigen recognition and binding sites, some of which may bind Tie2. In another case, at least one binding arm specifically binds Tie2, while other binding sites may bind Tie2 or another antigen of interest (listed under “bispecific antibodies”). In one aspect, such trispecific antibodies comprise antibody fragments (e.g., Fab, scFv, single-domain antibodies). As a non-limiting example, the three antibody-binding fragments of the present invention may be combined to form a trispecific antibody such that at least one antibody-binding fragment binds Tie2, while the remaining antibody-binding fragment binds another antigen, such as, for example, VEGF. See Runcie et al., Bi-specific and Tri-specific antibodies – the next big thing in solid tumor therapeutics, *Molecular Medicine*, 24, (50) (2018).

[0141] As used herein, the term "tetravalent" refers to a type of antibody having four antigen recognition and binding sites, some of which may bind Tie2. In another case, at least one binding arm specifically binds Tie2, while other binding sites may bind Tie2 or another antigen of interest (listed under "bispecific antibodies"). In one aspect, such tetravalent antibodies comprise antibody fragments (e.g., Fab, scFv, single-domain antibodies). As a non-limiting example, the four antibody-binding fragments of the present invention can be combined to form a tetravalent antibody such that two antibody-binding fragments bind Tie2, while the other two antibody-binding fragments bind another antigen, such as, for example, VEGF.

[0142] As used herein, the term "pentavalent" refers to a type of antibody having five antigen recognition and binding sites, some of which may bind Tie2. In another case, at least one binding arm specifically binds Tie2, while other binding sites may bind Tie2 or another antigen of interest (listed under "bispecific antibodies"). In one aspect, such pentavalent antibodies comprise antibody fragments (e.g., Fab, scFv, single-domain antibodies). As a non-limiting example, the five antibody-binding fragments of the present invention can be combined to form a pentavalent antibody such that at least one antibody-binding fragment binds Tie2, while the other antibody-binding fragments bind another antigen, such as, for example, VEGF.

[0143] As used herein, the term "hexavalent" refers to a type of antibody having six antigen recognition and binding sites, some of which may bind to Tie2. In another case, at least one binding arm specifically binds to Tie2, while other binding sites may bind to Tie2 or another antigen of interest, listed under "bispecific antibodies". In one aspect, such hexavalent antibodies comprise antibody fragments (e.g., Fab, scFv, single-domain antibodies). As a non-limiting example, the six antibody-binding fragments of the present invention can be combined to form a hexavalent antibody such that at least one antibody-binding fragment binds to Tie2, while the other antibody-binding fragments bind to another antigen, such as, for example, VEGF.

[0144] As used herein, a “linker peptide” is a peptide comprising two or more amino acid residues linked by peptide bonds for linking two peptides (e.g., VH and VL domains, two scFv antibody fragments, or a variable domain with an extracellular trap protein, or an scFv antibody fragment with an extracellular trap protein). The linker can be flexible or rigid / non-flexible. Examples of such linker peptides are well known in the art (see, for example, Hollinger P et al., Proceedings of the National Academy of Sciences (PNAS USA) 90:6444-6448 (1993); Poljak RJ. Structure 2:1121-1123 (1994)). Non-limiting examples of suitable non-immunogenic linker peptides are: (G4S)n, (SG4)n, or G4(SG4)n flexible linker peptides, or rigid / non-flexible linkers (EAAAK)n or (XP)n, in which case “n” is a number between 1 and 10 or 1 and 4, and oligomers of such linkers.

[0145] "Tie2 dysregulation disease" is any condition that would benefit from treatment with the anti-Tie2 antibody of the present invention. Non-limiting examples of diseases to be 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, infectious diseases, acute respiratory distress syndrome (ARDS), ischemic injury, eye disease, radiation injury, cancer, systemic sclerosis, traumatic brain injury, radiation injury, wound healing, myocardial infarction, blood-brain barrier impairment (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 infectious diseases include sepsis, dengue virus infection, tuberculosis, or influenza. In another embodiment, Tie2 dysregulation diseases may include ischemic injuries, such as, for example, diabetic nephropathy, acute kidney injury, chronic kidney disease, kidney or other organ transplantation, severe limb ischemia, traumatic brain injury, or stroke. In another embodiment, Tie2 dysregulation eye disease 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.

[0146] As used herein, “administration” refers to a method of giving a dose of a therapeutic agent (e.g., the anti-Tie2 antibody of the present invention, a nucleic acid encoding the anti-Tie2 antibody of the present invention) or a composition (e.g., a pharmaceutical composition, such as a pharmaceutical composition comprising the anti-Tie2 antibody of the present invention) to a subject in need. The compositions used in the methods described herein can be administered, for example, intravitreal (e.g., by intravitreal injection), ocular (e.g., by ocular injection), intraocular (e.g., by intraocular injection), subcutaneously, or intravenously. The compositions used in the methods described herein can also be administered systemically or locally. The method of administration can vary depending on various factors (e.g., the compound or composition administered, and the severity of the condition, disease, or disorder being treated).

[0147] As used herein, “combined administration” means administering two or more individual 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 combinations thereof (e.g., the anti-Tie2 antibody pharmaceutical composition and anti-VEGF antibody composition or recombinant VEGF fusion protein composition of the present invention) to a subject in need at the same time or simultaneously.

[0148] As used herein, “combination formulation” means combining two or more individual therapeutic agents (e.g., the anti-Tie2 antibody and anti-VEGF antibody or recombinant VEGF fusion protein of the present invention) or combinations (e.g., the anti-Tie2 antibody pharmaceutical composition and anti-VEGF antibody composition or recombinant VEGF fusion protein composition of the present invention) in a single formulation and administering it to a subject in need.

[0149] "Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, 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 some embodiments, the individual or subject is a human. "Subject" may be a "patient".

[0150] As used herein, “treatment” (and treatment or treating) refers to a clinical intervention that attempts to alter the natural course of a disease in an individual receiving treatment, and may be performed for prevention or in the course of clinicopathology. Desired therapeutic effects include, but are not limited to, preventing recurrence of disease or disorder, relieving symptoms, reducing any direct or indirect pathological consequences of disease or disorder, slowing disease progression, improving or alleviating the state of disease or disorder, and alleviating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the development of or slow the progression of disease or disorder.

[0151] As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all these names include progeny. Therefore, the terms “transformant” and “transformed cell” include primary test cells and cultures derived from them, regardless of the number of transformations. It should also be understood that due to intentional or unintentional mutations, the DNA content of all progeny may not be entirely identical. This includes mutant progeny with the same function or biological activity screened from the initially transformed cells. When different names are intended to be used, it will be clear from the context.

[0152] A "mutation" is a deletion, insertion, or substitution of a nucleotide relative to a reference nucleotide sequence, such as a wild-type sequence.

[0153] A “variant” or “mutant” of a starting or reference peptide (e.g., a reference antibody or its variable domain / CDR) is (1) a peptide having an amino acid sequence different from that of the starting or reference peptide, and (2) a peptide derived from the starting or reference peptide through natural or artificial (artificial) mutagenesis. Such variants include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the peptide of interest, referred to herein as “amino acid residue alterations.” Thus, a variant CDR refers to a CDR containing a variant sequence relative to the sequence of the starting or reference peptide (e.g., the sequence of a source antibody or antigen-binding fragment). In this context, an amino acid residue alteration refers to an amino acid that is different from the corresponding amino acid in the sequence of the starting or reference peptide (e.g., the sequence of a reference antibody or its fragment). Any combination of deletions, insertions, and substitutions can be performed to obtain the final variant or mutant construct, provided that the final construct possesses the desired functional characteristics. Amino acid alterations can also alter the post-translational processes of the peptide, such as changing the number or location of glycosylation sites.

[0154] As used in this article, "VEGF extracellular trap protein" or "VEGF-Trap" is also known as aflibercept ( Regeneron-Bayer HealthCare (Tarrington, NY, USA). 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, as well as VEGF-B and placental growth factor (PlGF), and essentially prevents VEGF-A and PlGF ligands from binding to and activating cellular receptors.

[0155] The "wild-type (WT)" or "reference" sequence, or the sequence of a "wild-type" or "reference" protein / peptide, such as the CDR or variable domain of a reference antibody, can be a reference sequence from which a variant peptide is derived by introducing a mutation. Generally, the "wild-type" sequence of a given protein is the most common sequence in nature. Similarly, the "wild-type" gene sequence is the most common gene sequence in nature. Mutations can be introduced into "wild-type" genes (and thus into the proteins they encode) through natural processes or artificial induction. The product of such processes is a "variant" or "mutant" form of the original "wild-type" protein or gene.

[0156] As used herein, a “reference antibody” refers to an antibody or fragment thereof whose antigen-binding sequence serves as a template sequence, and which is diversified on that template sequence according to the criteria described herein. The antigen-binding sequence typically includes an antibody variable region and at least one CDR, including a frame region.

[0157] Ingredients and methods

[0158] This invention provides novel antibodies that bind to Tie2, as well as methods for manufacturing and using these antibodies, for example, for therapeutic and diagnostic purposes. The antibodies of this invention can be used, for example, to diagnose or treat various disorders, including the Tie2 dysregulation disorders described herein.

[0159] The techniques and procedures described or cited herein are generally well understood by those skilled in the art and are frequently used by conventional methods, such as those widely used methods described in the following literature: Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; *Current Protocols in Molecular Biology* (edited by F.M. Usubel et al., (2003)); *Methods in Enzymology* series (Academic Press, Inc.): *PCR 2: A Practical Approach* (edited by M.J. MacPherson, B.D. Hames, and G.G. Taylor, (1995)), edited by Harlow and Lane, (1988); *Antibodies, A Laboratory Manual, and Animal Cell Culture*. * *Culture* (edited by R.Freshney (1987)); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Methods in Molecular Biology*, Humana Press; *Cell Biology: A Laboratory Notebook* (edited by J.E.C. Lis, 1998), Academic Press; *Animal Cell Culture* (edited by R.Freshney, 1987); *Introduction to Cell and Tissue Culture* (edited by J.M. Pather and P.E. Roberts, 1998), Plenum Press; *Cell and Tissue Culture: Laboratory Procedures* (edited by A. Doyle, J.B. Griffiths, and D.G. Newell, 1993-8), John Willie & Sons (J.).Wiley and Sons; *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *PCR: The Polymerase Chain Reaction* (edited by Mullis et al., 1994); *Current Protocols in Immunology* (edited by J.E. Coligan et al., 1991); *Short Protocols in Molecular Biology* (Wiley & Sons, 1999); *Immunobiology* (C.A. Janeway and P. Travers, 1997); *Antibodies* (P. Finch, 1997); *Antibodies: A Practical Approach* * *The Approach to Monoclonal Antibodies* (edited by D. Catty, IRL Press, 1988-1989); *Monoclonal Antibodies: A Practical Approach* (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); *Using Antibodies: A Laboratory Manual* (edited by E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); *Antibodies* (edited by M. Zanetti and JD. Capra, Harwood Academic Publishers, 1995).

[0160] The anti-Tie2 antibody of the present invention described herein, and any antibody used in the methods described herein, may have any of the features described herein (alone or in combination).

[0161] In some embodiments, the dissociation constant (KD) of the anti-Tie2 antibody provided herein is about 1 μM, about 100 nM, about 10 nM, about 1 nM, about 0.1 nM, about 0.01 nM, or about 0.001 nM (e.g., 10 μM). -8M or smaller, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13For example, in some cases, the antibodies provided herein bind human Tie2 (huTie2) with a KD of about 10 nM or less. In some cases, the antibodies provided herein bind huTie2 with a KD of about 5 nM or less. In some cases, the antibodies provided herein bind huTie2 with a KD of about 2 nM or less. For example, in some cases, the antibody is present at a concentration between approximately 25 pM and approximately 2 nM (e.g., approximately 25 pM, approximately 50 pM, approximately 75 pM, approximately 100 pM, approximately 125 pM, approximately 150 pM, approximately 175 pM, approximately 200 pM, approximately 225 pM, approximately 250 pM, approximately 275 pM, approximately 300 pM, approximately 325 pM, approximately 350 pM, approximately 375 pM, approximately 400 pM, approximately 425 pM, approximately 450 pM, approximately 475 pM, approximately 500 pM, approximately 525 pM, approximately 550 pM, approximately 575 pM, approximately 600 pM). KD binding huTie2 (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, approximately 1 nM, approximately 1.1 nM, approximately 1.2 nM, approximately 1.3 nM, approximately 1.4 nM, approximately 1.5 nM, approximately 1.6 nM, approximately 1.7 nM, approximately 1.8 nM, approximately 1.9 nM, approximately 2 nM). In some cases, the antibody binds to huTie2 with a KD ratio between approximately 75 pM and approximately 600 pM (e.g., approximately 75 pM, approximately 100 pM, approximately 125 pM, approximately 150 pM, approximately 175 pM, approximately 200 pM, approximately 225 pM, approximately 250 pM, approximately 275 pM, approximately 300 pM, approximately 325 pM, approximately 350 pM, approximately 375 pM, approximately 400 pM, approximately 425 pM, approximately 450 pM, approximately 475 pM, approximately 500 pM, approximately 525 pM, approximately 550 pM, approximately 575 pM, approximately 600 pM). In some cases, the antibody binds to huTie2 with a KD ratio between approximately 75 pM and approximately 500 pM. In some cases, the antibody binds to huTie2 with a KD ratio between approximately 75 pM and approximately 400 pM. In some cases, the antibody binds to huTie2 with a KD of approximately 75 pM to approximately 300 pM. In some cases, the antibody binds to huTie2 with a KD of approximately 75 pM to approximately 200 pM. In some cases, the antibody binds to huTie2 with a KD of approximately 75 pM to approximately 150 pM. In some cases, the antibody binds to huTie2 with a KD of approximately 75 pM to approximately 125 pM. In some cases, the antibody binds to huTie2 with a KD of approximately 75 pM to approximately 100 pM. In some cases, the antibody binds to huTie2 with a KD of approximately 80 pM.In some cases, the antibody binds to huTie2 with approximately 60 pM of KD. In other cases, the antibody binds to huTie2 with approximately 40 pM of KD.

[0162] In one embodiment, KD is measured by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab to the antigen is determined by measuring the minimum concentration of the unlabeled antigen in the presence of a series of titrations. 125 I) Label the antigen to balance Fab, then use an anti-Fab antibody-coated plate to capture the bound antigen for measurement (see, e.g., Chen et al., Journal of Molecular Biology 293:865-881 (1999)). To establish the assay conditions, a microtester was coated with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) dissolved in 50 mM sodium carbonate (pH 9.6). TM Multi-well plates (Thermo Scientific) were incubated overnight, followed by blocking with 2% (w / v) bovine serum albumin dissolved in PBS at room temperature (approximately 23°C) for 2 to 5 hours. In non-absorbent plates (Nunc#269620), 100 pM or 26 pM [ 125 I] The antigen is mixed with serial dilutions of the Fab of Interest (see Presta et al., Cancer Research 57:4593-4599 (1997)). The Fab of Interest is then incubated overnight; however, incubation may be prolonged (e.g., about 65 hours) to ensure equilibration. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., one hour). The solution is then removed and diluted with 0.1% polysorbate 20 (TWEEN-20) dissolved in PBS. TM Wash the plate eight times. After the plate is dry, add 150 μl / well of scintillation agent (MICROSCINT-20). TM Packard, and TOPCOUNT TM A gamma counter (Packard) was used to measure the plate for several tens of minutes. The concentration of each Fab that produced a maximum binding of less than or equal to 20% was selected for the competitive binding assay.

[0163] According to another embodiment, BIACORE is used. TM Surface plasmon resonance (SPR) measurements are used to measure KD. For example, BIACORE is used. TM -2000 or BIACORE TMThe assay of -3000 (BIAcore, Piscatave, NJ) was performed at 25°C using an immobilized antigen CM5 chip with approximately 10 reaction units (RUs). In one embodiment, the carboxymethylated dextran biosensor chip (CM5, BIAcore) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) before injection at a flow rate of 5 μl / min to obtain approximately 10 reaction units (RUs) of conjugate protein. Following antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, at 25°C, serial dilutions of Fab (0.78 nM to 500 nM) were injected into a solution containing 0.05% polysorbate 20 (TWEEN) at a flow rate of approximately 25 μl / min. TM -20) Surfactant (PBST) in PBS. A simple one-to-one Langmuir binding model (BIACORE) was used by simultaneously fitting association and dissociation sensor maps. TM Evaluation software version 3.2) calculates association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (KD) is calculated as the ratio k. off / k on See, for example, Chen et al., *Journal of Molecular Biology* 293:865-881 (1999). If the onset rate exceeds 10 by the above surface plasmon resonance determination... 6 M -1 s -1 The on / off rate of 20 nM anti-antigen antibody (Fab form) in PBS at pH 7.2 at 25°C with increasing antigen concentration can be determined by using fluorescence quenching techniques (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) that measure the increase or decrease in fluorescence emission intensity. The antigen concentration is measured using a spectrometer, such as an Aviv Instruments 8000 series SLM-AMINCO spectrometer equipped with a stop-flow spectrophotometer or a stirring cuvette. TM A spectrophotometer (ThermoSpectronic) can also be used. Alternatively, it can be measured using a BIACORE spectrophotometer known in the art. TM SPR measurement to measure KD

[0164] In some 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 a review of certain antibody fragments, see Hudson et al., *Nature Medicine*, 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, *The Pharmacology of Monoclonal Antibodies*, Vol. 113, edited by Rosenburg and Moore (Springer, New York), pp. 269-315 (1994); see also WO 93 / 16185; and for a discussion of Fab and F(ab')2 fragments containing rescue receptor-binding epitope residues and having an increased in vivo half-life in U.S. Patent Nos. 5,571,894 and 5,587,458, see U.S. Patent No. 5,869,046.

[0165] Disomy is an antibody fragment with two antigen-binding sites, and can be bivalent or bispecific. See, for example, EP404,097; WO 1993 / 01161; Hudson et al., *Nature Medicine* 9:129-134 (2003); and Hollinger et al., *Proceedings of the National Academy of Sciences* 90:6444-6448 (1993). For trisomy and tetrasomy, see also Hudson et al., *Nature Medicine* 9:129-134 (2003).

[0166] A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Waltham, MA; see, for example, U.S. Patent No. 6,248,516B1).

[0167] Antibody fragments can be manufactured using a variety of techniques, including but not limited to the proteolytic digestion of intact antibodies and production via recombinant host cells (such as E. coli or bacteriophages), as described herein.

[0168] In some cases, the anti-Tie2 antibody of the present invention provided herein is Fab.

[0169] In some cases, Fab binds to Tie2 and may include at least one, two, three, four, five, or six CDRs selected from: (a) CDR-H1 containing the amino acid sequence of any one of SEQ ID No: 1-40; (b) CDR-H2 containing the amino acid sequence of any one of SEQ ID No: 41-80; (c) CDR-H3 containing the amino acid sequence of any one of SEQ ID No: 81-120; (d) CDR-L1 containing the amino acid sequence of any one of SEQ ID No: 121-160; (e) CDR-L2 containing the amino acid sequence of any one of SEQ ID No: 161-200; and (f) CDR-L3 containing the amino acid sequence of any one of SEQ ID No: 201-240, or a combination of one or more of the above CDRs and one or more variants thereof, such variants being associated with SEQ ID No: 1. Any one of the sequences No:1-240 has at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% identity).

[0170] In some cases, Fab binds to Tie2 and contains HCDR 1-3 and LCDR 1-3, which contain the amino acid sequence of SEQ ID No as an antibody clone:

[0171]

[0172] In some cases, Fab binds Tie2 and comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity) or having 100% of the sequence of any one of SEQ ID No: 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, or 272); and (b) a VL ...c) an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, The amino acid sequence of any one of No: 242, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271 or 273 has at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98% or 99% sequence identity) or has 100% of the amino acid sequence described above.

[0173] In some cases, Fab binds to Tie2 and contains VH and VL domains, which contain the following combinations of amino acid sequences:

[0174] Antibody Cloning# VH domain SEQ ID No: VL domain SEQ ID No: 1 242 243 2 244 245 3 246 247 7 248 249 41 250 251 42 252 253 43 254 255 44 256 257 45 258 259 46 260 261 6 262 263 47 264 265 48 266 267 5 268 269 49 270 271 31 272 273

[0175] In some embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proceedings of the National Academy of Sciences, 81:6851-6855 (1984). In one instance, a chimeric antibody comprises 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 another instance, a chimeric antibody is a “class-switching” antibody, wherein the class or subclass has become different from that of the parent antibody. Chimeric antibodies include their antigen-binding fragment.

[0176] In some embodiments, the chimeric antibody is a humanized antibody. Typically, nonhuman antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent nonhuman antibody. Typically, humanized antibodies comprise one or more variable domains, wherein the CDR, for example, a CDR (or a portion thereof) is derived from the nonhuman antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Humanized antibodies may also optionally include at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced by corresponding residues from the nonhuman antibody (e.g., an antibody from which the CDR residues are derived), for example, to restore or enhance antibody specificity or affinity.

[0177] For reviews of humanized antibodies and their manufacturing methods, see, for example, Almagro and Fransson, *Fronts in Bioscience* 13:1619-1633 (2008), and further described in, for example, Riechmann et al., *Nature* 332:323-329 (1988); Queen et al., *Proceedings of the National Academy of Sciences* 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; and Kashmiri et al., *Methods*. Methods, 36:25-34 (2005) (describes specific determinant region (SDR) transplantation); Padlan, Molecular Immunology, 28:489-498 (1991) (describes “surface reconstruction”); Dall'Acqua et al., Methods, 36:43-60 (2005) (describes “FR reorganization”); and Osbourn et al., Methods, 36:61-68 (2005) and Klimka et al., Br.J. Cancer, 83:252-260 (2000) (describes the “guided selection” approach to FR reorganization).

[0178] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a "best fit" method (see, for example, Sims et al., *Journal of Immunology* 151:2296 (1993)); and frame regions derived from specific subgroups of human antibody consortia derived from the light or heavy chain variable regions (see, for example, Carter et al., *Proceedings of the National Academy of Sciences*, 89:4285 (1992); and Presta et al., *Journal of Immunology*, 151:2623 (1993)). ); human maturation (somatic mutation) framework region or human germline framework region (see, for example, Almagro and Fransson Frontiers in Bioscience 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, for example, Baca et al. Journal of Biochemistry 272:10678-10684 (1997) and Rosok et al. Journal of Biochemistry 271:22611-22618 (1996)).

[0179] In some embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using various techniques known in the art. Human antibodies are commonly discussed in van Dijk et al., New Insights in Pharmacology (Curr. Opin. Pharmacol.) 5:368-74 (2001) and Lonberg, New Insights in Immunology (Curr. Opin. Immunol.) 20:450-459 (2008).

[0180] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigen challenges. Such animals typically contain all or part of the human immunoglobulin loci, which replace endogenous immunoglobulin loci, or are present extrachromosomally or randomly integrated into the animal chromosome. In such transgenic animals, endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, *Nature Biotech*, 23:1117-1125 (2005). See also, for example, a description of XENOMOUSE. TM U.S. Patent Nos. 6,075,181 and 6,150,584, describing HUANTIBODIES. TM U.S. Patent No. 5,770,429; describing the KM MOUSE. TM U.S. Patent No. 7,041,870 and the description of VELOCIMOUSE TM The technology described in U.S. Patent Application Publication No. US 2007 / 0061900, and the description of OmniChicken TM(U.S. Patent Nos. 9,809,642 and 9,380,769). The human variable region of the intact antibody produced from such animals can be further modified, for example, by binding to a different human constant region.

[0181] Human antibodies can also be prepared using hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines used to produce human monoclonal antibodies have been described. (See, for example, Kozbor, *Journal of Immunology*, 133:3001 (1984); Brodeur et al., *Monoclonal Antibody Production Techniques and Applications*, pp. 51–63 (Marcel Decker, New York, 1987); and Boerner et al., *Journal of Immunology*, 147:86 (1991).) Human antibodies produced via human B-cell hybridoma technology have also been described by Li et al., *Proceedings of the National Academy of Sciences*, 103:3557–3562 (2006). Other methods include, for example, those described in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni Modern Immunology 26(4):265-268 (2006) (describing human-human hybridoma). Human hybridoma technology (tri-source hybridoma 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).

[0182] Human antibodies can also be generated by isolating variable domain sequences of Fv clones selected from a human phage display library. These variable domain sequences can then bind to the desired human constant domain. Techniques for selecting human antibodies from an antibody library are described below.

[0183] The antibodies of the present invention can be isolated by screening a library of antibodies with the desired activity. For example, various methods are known in the art for generating phage display libraries and screening antibodies with the desired binding characteristics from such libraries. Such methods are reviewed, for example, in Hoogenboom et al., *Methods in Molecular Biology* 178:1-37 (edited by O'Brien et al., Human Press, Totoa, NJ, 2001), and in McCafferty et al., *Nature* 348:552-554; Clackson et al., *Nature* 352:624-628 (1991); Marks et al., *Journal of Molecular Biology* 222:581-597 (1992); Marks and Bradbury, *Methods in Molecular Biology* 248:161-175 (edited by Lo, Human Press, Totoa, NJ, 2003); S Such methods are further described in Idhu et al., Journal of Molecular Biology 338(2):299-310 (2004); Lee et al., Journal of Molecular Biology 340(5):1073-1093 (2004); Fellouse, Proceedings of the National Academy of Sciences 101(34):12467-12472 (2004); and Lee et al., Journal of Immunology Methods 284(1-2):119-132 (2004).

[0184] In some phage display methods, VH and VL gene libraries are cloned separately by polymerase chain reaction (PCR), and random recombination is performed in the phage library, followed by screening for antigen-binding phages, as described by Winter et al. in *Annalen Pharmacology Review*, 12:433-455 (1994). Phages typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries derived from immune sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, proto-libraries (e.g., cloned from humans) can be cloned to provide monogenic antibodies against a variety of non-autoantigens and autoantigens without any immunization, as described by Griffiths et al. in *European Organization for Molecular Biology Journal*, 12:725-734 (1993). Finally, the original library can also be prepared by cloning an unrearranged V-gene fragment from stem cells, encoding a highly variable CDR3 region using PCR primers containing random sequences, and performing rearrangement in vitro, as described by Hoogenboom and Winter, *Journal of Molecular Biology* 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0185] In this paper, antibodies or antibody fragments isolated from human antibody libraries can be considered as human antibodies or human antibody fragments.

[0186] In some embodiments, the antibodies provided herein are multispecific antibodies, such as, for example, bispecific antibodies, bispecific in situ antibodies, trispecific antibodies, tetravalent antibodies, pentavalent antibodies, hexavalent antibodies, to name just a few. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites. In some embodiments, a bispecific antibody may bind to two or more different epitopes of Tie2. In some embodiments, one binding specificity is against Tie2, while the other is against any other antigen, such as, for example, VEGF. Bispecific antibodies may be prepared as full-length antibodies or antibody fragments. Any anti-Tie2 antibody described herein can be used to construct multispecific antibodies.

[0187] In some cases, the multispecific anti-Tie2 antibody of the present invention is a bispecific antibody, with one arm binding to Tie2 and the other arm binding to VEGF. In other embodiments, such bispecific anti-Tie2 antibodies of the present invention also have an Fc mutation that eliminates ADCC and / or CDC, as described herein. Such bispecific antibodies of the present invention comprise a heavy chain and a light chain, the heavy chain and the light chain comprising the following amino acid sequences:

[0188] Antibody Cloning# Heavy chain SEQ ID No: Light chain SEQ ID No: 51 276 277 52 278 279 53 280 281 54 282 283 57 286 287 58 288 289

[0189] In other cases, the multispecific anti-Tie2 antibody of the present invention is fused with a VEGF extracellular trap protein. In still other cases, such multispecific anti-Tie2 antibody fusion proteins also have an Fc mutation that eliminates ADCC and / or CDC, as described herein. Non-limiting examples of such multispecific antibody fusion proteins comprise the following amino acid sequences:

[0190] Antibody Cloning# SEQ ID No: 55 284 56 285

[0191] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829 and Traunecker et al., CERN 10:3655 (1991) and "knob-in-hole" engineering (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared through engineered electrostatic manipulation effects for the preparation of antibody Fc-heterodimer molecules (WO). 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); generating bispecific antibodies using leucine zippers (see, for example, Kostelny et al., Journal of Immunology, 148(5):1547-1553 (1992)); preparing bispecific antibody fragments using “dimer” techniques (see, for example, Hollinger et al., Proceedings of the National Academy of Sciences, 90:6444-6448 (1993)); and using single-chain Fv(sFv) dimers (see, for example, Gruber et al., Journal of Immunology, 152:5368 (1994)); and preparing trispecific antibodies as described in, for example, Tutt et al., Journal of Immunology, 147 / 60 (1991).

[0192] This article also includes engineered antibodies with three or more functional antigen-binding sites, including “octopus antibodies” (see, for example, US 2006 / 0025576A1).

[0193] The antibodies or fragments described herein also include “dual-acting FAbs” or “DAFs”, which contain an antigen-binding site that binds to Tie2 and another different antigen (see, for example, US 2008 / 0069820).

[0194] In some embodiments, consideration is given to amino acid sequence variants of the antibodies provided herein (e.g., antibody variants comprising one or more altered amino acid residues). For example, it may be necessary to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

[0195] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include CDR and FR. Conservative substitutions are considered, and this is well known in the art.

[0196] Other amino acid substitutions are described below with reference to amino acid side chain categories. Amino acid substitutions can be introduced into antibodies of interest, and products can be screened for desired activities, such as preserved / improved antigen binding, reduced immunogenicity, or improved or reduced ADCC or CDC. Amino acids can be grouped according to common side chain characteristics:

[0197] (1) Hydrophobic: Leucine, methionine, alanine, valine, leucine, isoleucine:

[0198] (2) Neutral hydrophilic: cysteine, serine, threonine, asparagine, glutamine;

[0199] (3) Acids: Aspartic acid, glutamic acid;

[0200] (4) Alkaline: Histidine, Lysine, Arginine;

[0201] (5) Residues that affect chain orientation: glycine, proline;

[0202] (6) Aromatic: tryptophan, tyrosine, phenylalanine.

[0203] Non-conservative substitution requires replacing a member of one of these classes with a member of another class.

[0204] A substitution variant involves replacing one or more hypervariable region residues and / or FR residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further research will be modified (e.g., improved) relative to the parent antibody in terms of certain biological properties (e.g., increased affinity, increased stability, increased expression, altered pI, and / or decreased immunogenicity), and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be readily generated, for example, using phage display-based affinity maturation techniques, as described herein. In short, one or more CDR residues are mutated, and a variant antibody is displayed on a phage and screened for its specific biological activity (e.g., binding affinity).

[0205] For example, alterations (e.g., substitutions) can be made in the CDR to improve antibody affinity. Such alterations can be made in CDR “hotspots,” i.e., residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, for example, Chowdhury, Methods in Molecular Biology 207:179-196 (2008)), and / or residues that contact the antigen, where the binding affinity of the resulting variant VH or VL is tested. Affinity maturation has been described, for example, by constructing and reselecting from a secondary library, in, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Publishing, Totoa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. This library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves CDR-directed methods, in which several CDR residues (e.g., 4 to 6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. Specifically, CDR-H3 and CDR-L3 are frequently targeted.

[0206] In some embodiments, substitutions, insertions, or deletions may occur in one or more CDRs, as long as such changes do not significantly reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions, as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. For example, such changes may be made outside the antigen-contacting residues in the CDRs. In some embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0207] In some embodiments, substitution, insertion, or deletion may occur in one or more FRs, as long as such changes do not significantly reduce the antibody's ability to bind to the antigen. For example, such changes can improve antibody affinity and / or stability (e.g., as assessed by elevated melting temperatures).

[0208] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby creating Fc region variants. Fc region variants may comprise a human Fc region sequence (e.g., the human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid residue alterations (e.g., substitutions) at one or more amino acid positions. In some embodiments, the invention envisions antibody variants having some, but not all, effector functions, making them ideal candidates for applications where the in vivo antibody half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays may be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay may be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding capacity. Primary cells used to mediate ADCC, NK cells, 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's Annual Review of Immunology 9:457-492 (1991).

[0209] Non-limiting examples of in vitro assays for assessing ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom et al., Proceedings of the National Academy of Sciences 83:7059-7063 (1986) and Hellstrom et al., Proceedings of the National Academy of Sciences 82:1499-1502 (1985); U.S. Patent No. 5,821,337; and Bruggemann et al., Journal of Experimental Medicine 166:1351-1361 (1987). Alternatively, non-radioactive assays may be used (see, for example, ACTI for flow cytometry). TM Non-radioactive cytotoxicity assays (Cell Technology, Inc., Mountain View, California); and CYTOTOX 96 TMNon-radioactive cytotoxicity assays (Promega, Madison, Wisconsin). Effective effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example in animal models, such as those disclosed in Clynes et al., Proceedings of the National Academy of Sciences 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., *Journal of Immunological 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 assays can also be performed using methods known in the art (see, for example, Petkova et al., *International Journal of Immunology* 18(12):1759-1769 (2006)).

[0210] The antibodies of this invention can be engineered to reduce effector function, such as by reducing affinity for binding to human FcyRIIIA and / or FcyRIIA and / or FcyRI and / or Clq, thereby reducing complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), and antibody-dependent phagocytosis (ADCP). In some cases, such reduction in effector function is achieved by amino acid substitution of one or more of the following Fc region residues: N297, L234, L235, D265, and P329 according to EU numbers in Kabat. See US6,737,056, US7,332,581, and WO / 2012 / 130831. In some embodiments, the substitution mutation is one or more of N297G, N297A, L234A, L235A, D265A, and / or P329G. In some embodiments, the substitution mutation is either N297A or N297G. In some embodiments, the substitution mutation includes the so-called “DANA” Fc mutation, in which residues D265 and N297 are replaced with alanine (US Patent No. 7,332,581). In some embodiments, the substitution mutation includes the so-called “DANG” mutation, in which residues are replaced with D265A and N297G. In some embodiments, the substitution mutation includes the “LALA” Fc mutation, in which residues L234 and L235 are replaced with alanine (see Lund, J. et al. (1992), *Molecular Immunology*, 29:53–59; and Tamm, A. and Schmidt, RE (1997), *International Review of Immunology*, 16:57–85). In other embodiments, the substitution mutation includes the “LALA-PG” Fc mutation, in which residues L234 and L235 are replaced with alanine and P329 is replaced with glycine (see Brünker, P. et al. (2016), *Molecular Cancer Therapy*).

[0211] Certain antibody variants that bind to FcR with enhanced or diminished binding are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., Journal of Biochemistry 9(2):6591-6604(2001)).

[0212] In some embodiments, a substitution mutation is performed in the Fc region to produce a hexamerized antibody, as described herein. In some embodiments, such a substitution mutation is E430G. In other embodiments, the E430G mutation may be combined with any of the aforementioned reduced effector function mutations to eliminate 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, the heavy chain and the light chain comprising an amino acid sequence having the E430G mutation and the L234A, L235A, P329G mutations in combination of the following:

[0213] Antibody Cloning# Heavy chain SEQ ID No: Light chain SEQ ID No: 50 174 175

[0214] Antibodies with prolonged half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for transferring maternal IgG to the fetus (Guyer et al., *Journal of Immunology* 117:587 (1976) and Kim et al., *Journal of Immunology* 24:249 (1994)), are further described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions that improve the binding of the Fc region to the FcRn. Such Fc variants include those with substitutions at one or more 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, the substitution of Fc region residue 434 (U.S. Patent No. 7,371,826). See also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent Nos. 5,648,260 and 5,624,821; and WO 94 / 29351 concerning other examples of Fc region variants.

[0215] The present invention also provides immunoconjugates comprising anti-Tie2 antibodies conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, bacterial, fungal, plant or animal-derived enzyme-active toxins or fragments thereof) or radioisotopes.

[0216] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to maytansine (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); auristatin, such as the monomethylauristatin drug portions DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483, 5,780,588 and 7,498,298); dolastatin; chachiin or derivatives thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5...). Nos. 767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Research 53:3336-3342 (1993); and Lode et al., Cancer Research 58:2925-2928 (1998); anthracycline antibiotics, such as doxorubicin or doxycycline (see Kratz et al., Current Med). .Chem.) 13:477-523 (2006); Jeffrey et al. Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al. Bioconjugate Chemistry 16:717-721 (2005); Nagy et al. Proceedings of the National Academy of Sciences 97:829-83 4 (2000); Dubowchik et al. Bioorganic & Medicinal Chemistry Letters 12:1529-1532 (2002); King et al. Journal of Medicinal Chemistry 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vinorelbine; taxanes such as docetaxel, paclitaxel, lalotaxel, testataxyl, and oxadecane; trichothecene; and CC1065.

[0217] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or a fragment thereof, including but not limited to diphtheria toxin A chain, a non-conjugated active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin toxin A chain, modizol A chain, α-salicylic acid, tung oil protein, carnation toxin protein, American pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, jatropha toxin protein, crotonin protein, soapwort inhibitor, white tree toxin, mitogellin, localized aspergillin, phenolmycin, enoxamin, and trichothecene compounds.

[0218] In another embodiment, the immunoconjugate comprises an antibody, as described herein, coupled with a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to generate the radioconjugate. Examples include At... 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 Pb 212 And radioactive isotopes of Lu. When radiocoupled materials are used for detection, they may contain radioactive atoms for scintillation studies, such as TC99M or TC1123, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0219] In another embodiment, the immunoconjugate comprises an antibody conjugated to a non-cytotoxic agent as described herein, such as, for example, artemisinin, artesunate, or cannabis, or naltrexone, or aspirin, or statins, or a metabolite such as metformin, doxycycline, and an anti-helmintic.

[0220] A variety of bifunctional protein conjugates may be used to prepare conjugates of antibodies and cytotoxic or non-cytotoxic agents, such as N-succinimide-3-(2-pyridyl dithio)propionate (SPDP), succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), iminothiacyclopentane (IT), bifunctional derivatives of imide esters (such as dimethyl adipate hydrochloride), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), diazid derivatives (such as bis(p-diazidobenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and diactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared, as described by Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radioactive nucleotides to antibodies. See WO94 / 11026. The adapter can be a “cleavable adapter” that promotes the release of cytotoxic drugs from cells. For example, acid-instable adapters, peptidase-sensitive adapters, light-instable adapters, dimethyl adapters, or disulfide-containing adapters can be used (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0221] This article explicitly envisions, but is not limited to, such conjugates prepared with cross-linking agents, including but not limited to BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, STAB, SMCC, SMPB, SMPH, thioEMCS, thioGMBS, thioKMUS, thioMBS, thioSIAB, thioSMCC and thioSMPB, and SVSB (succinimide-(4-vinyl sulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Illinois, USA).

[0222] In one aspect, the anti-Tie2 antibody of the present invention can be used to detect the presence of Tie2 in biological samples. As used herein, the term "detection" includes quantitative or qualitative detection. In some embodiments, the biological sample comprises cells or tissues. In some embodiments, such tissues include normal and / or cancerous tissues that express Tie2 at higher levels relative to other tissues.

[0223] In one embodiment, an anti-Tie2 antibody is provided for use in a diagnostic or detection method. In another embodiment, a method is provided for detecting the presence of Tie2 in a biological sample. In some embodiments, the method includes contacting a biological sample with an anti-Tie2 antibody as described herein, under conditions allowing the anti-Tie2 antibody to bind to Tie2, and detecting whether a complex is formed between the anti-Tie2 antibody and Tie2. Such methods can be in vitro or in vivo. In one embodiment, the anti-Tie2 antibody is used to select subjects who meet the criteria for anti-Tie2 antibody treatment, for example, where Tie2 is a biomarker used for patient selection.

[0224] Recombinant methods and compositions may be used to generate any of the antibodies described herein (e.g., anti-Tie2 antibodies), such as those described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding the anti-Tie2 antibody described herein is provided. Such nucleic acid may encode an amino acid sequence containing VL and / or an amino acid sequence containing VH (e.g., the light chain and / or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. In another embodiment, a host cell containing such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., having been transformed by): (1) a vector containing a nucleic acid encoding an amino acid sequence containing VL and an amino acid sequence containing VH of the antibody, or (2) a first vector containing a nucleic acid encoding an amino acid sequence containing VL and a second vector containing a nucleic acid encoding an amino acid sequence containing VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as Chinese hamster ovary (CHO) cells or lymphocytes (e.g., YO, NSO, Sp20 cells). In one embodiment, a method for preparing an anti-Tie2 antibody is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding an antibody as described above under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0225] For the recombinant production of anti-Tie2 antibodies, for example, the nucleic acid encoding the antibody as described above 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 routine procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody.

[0226] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be generated in bacteria, particularly when glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and peptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. See also Charlton's *Methods in Molecular Biology*, Vol. 248 (edited by BKCLo, Humanities Press, Totoa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in *E. coli*. Following expression, the antibody can be isolated from the soluble fraction of the bacterial cell body and can be further purified.

[0227] Besides prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains with "humanized" glycosylation pathways, thereby producing antibodies with partial or complete human glycosylation patterns. See Gerngross, Nature Biotechnology, 22:1409-1414 (2004), and Li et al., Nature Biotechnology, 24:210-215 (2006).

[0228] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be conjugated to insect cells, particularly for transfecting fall armyworm cells.

[0229] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (description of PLATNIBODIES for generating antibodies in transgenic plants). TM technology).

[0230] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for suspension growth may be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed with SV40 (COS-7); the human embryonic kidney cell line (293 or 293 cells, as described by Graham et al. in the Journal of General Virology 36:59 (1977)); juvenile hamster kidney cells (BHK); and mouse supporting cells (T... M4Cells, such as those described by Mather in *Biol. Reprod.* 23:243-251 (1980); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells, such as those described by Mather et al. in *Annals of the New York Academy of Sciences* 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proceedings of the National Academy of Sciences 77:4216 (1980)); and myeloma cell lines such as YO, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, Humana Press, Totoa, NJ), pp. 255–268 (2003).

[0231] The anti-Tie2 antibodies provided herein can be identified, screened, or characterized by their physical / chemical properties and / or biological activity using a variety of assays known in the art and described herein in examples and throughout the specification.

[0232] On one hand, the antigen-binding activity of the antibodies of the present invention was tested, for example, by known methods such as ELISA, Western blotting, and surface plasmon resonance assays (e.g., BIACORE). TM )wait.

[0233] On the one hand, using BIACORE TM Surface plasmon resonance (SPR) assays are used to measure antigen-binding activity (e.g., represented by KD). For example, BIACORE is used. TM -2000 or BIACORE TMThe assay of -3000 (BIAcore, Piscatave, NJ) was performed at 25°C using an immobilized antigen CM5 chip with approximately 10 reaction units (RUs). In one embodiment, the carboxymethylated dextran biosensor chip (CM5, BIAcore) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) before injection at a flow rate of 5 μl / min to obtain approximately 10 reaction units (RUs) of conjugate protein. Following antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, at 25°C, serial dilutions of Fab (0.78 nM to 500 nM) were injected into a solution containing 0.05% polysorbate 20 (TWEEN) at a flow rate of approximately 25 μl / min. TM -20) Surfactant (PBST) in PBS. A simple one-to-one Langmuir binding model (BIACORE) was used by simultaneously fitting association and dissociation sensor maps. TM Evaluation software version 3.2) calculates association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (KD) is calculated as the ratio k. off / k on See, for example, Chen et al., *Journal of Molecular Biology* 293:865-881 (1999). If the onset rate exceeds 10 by the above surface plasmon resonance determination... 6 M -1 s -1 The on / off rate of 20 nM anti-antigen antibody (Fab form) in PBS at pH 7.2 at 25°C with increasing antigen concentration can be determined by using fluorescence quenching techniques (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) that measure the increase or decrease in fluorescence emission intensity. The antigen concentration is measured using a spectrometer, such as an 8000 series SLM-AMINCO spectrophotometer equipped with a stop-flow spectrophotometer (Aviv Instruments) or a stirring cuvette. TM A spectrophotometer (ThermoSpectronic) can also be used. BIACORE can also be used. TM SPR measurement is used to measure KD.

[0234] On the other hand, competitive assays can be used to identify antibodies that compete with the antibodies described herein for binding to Tie2. In some embodiments, such competitive antibodies bind to the same epitopes (e.g., linear or conformational epitopes) that the antibodies described herein bind to. Detailed exemplary methods for mapping antibody-binding epitopes are provided in Morris (1996), “Epitope Mapping Protocols,” *Methods in Molecular Biology*, Vol. 66 (Humana Press, Totoa, NJ).

[0235] In an exemplary competitive assay, immobilized Tie2 is incubated in a solution containing a first labeled antibody that binds Tie2 and a second unlabeled antibody that tests its ability to compete with the first antibody for Tie2 binding. 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 in the test sample is significantly reduced relative to the control sample, it indicates that the second antibody competes with the first antibody for Tie2 binding. See Harlow and Lane (1988), *Antibodies: A Laboratory Handbook*, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).

[0236] In one aspect, an assay is provided for identifying anti-Tie2 antibodies as having biological activity. See the Examples section. Biological activity may include, for example, one or more biological activities of Tie2, such as activation, agonism, amplification, or enhancement. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0237] In some embodiments, such biological activities of the antibodies of the present invention were tested. In some embodiments, the anti-Tie2 antibody binds to Tie2 and reduces or inhibits its serine protease activity against one or more Tie2 substrates, said substrates including, for example, H2-Opt substrates, α-casein, β-casein, or BODIPY known in the art. TM FL casein substrate, or any other suitable Tie2 substrate. In some embodiments, for one or more Tie2 substrates, the anti-Tie2 antibody inhibits Tie2 activity with an IC50 of less than 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.

[0238] In some embodiments, labeled anti-Tie2 antibodies are provided. Labeling includes, but is not limited to, labels or portions that are directly detectable (e.g., fluorescence, chromophores, electron-dense, chemiluminescence, and radioactive labels), and portions (e.g., those detected indirectly (e.g., by enzyme reactions or molecular interactions) such as enzymes or ligands. Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C 125 I, 3 H and 131 I. Fluoresceins such as rare earth chelates or luciferin and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases (e.g., firefly luciferase and bacterial luciferase) (US Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, β-galactosidase, glucosylamylase, 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 use hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labeling, bacterial phage labeling, stable free radicals, etc. In another embodiment of the invention, labeled antibodies are not required, and the presence of antibodies can be detected using labeled antibodies bound to antibodies, such as, for example, anti-horseradish peroxidase antibodies well known in the art.

[0239] The antibodies of this invention can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. (Zola et al., Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).)

[0240] Competitive binding assays rely on the ability of labeled standards and test sample analytes to compete for binding with a limited amount of antibody. The amount of antigen in the test sample is inversely proportional to the amount of standard bound to the antibody. To facilitate determination of the amount of bound standard, the antibody is typically insoluble before or after the competition, thus allowing for easy separation of antibody-bound standards and analytes from those remaining unbound.

[0241] Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic moiety or epitope of the protein to be detected. In a sandwich assay, a first antibody immobilized on a solid support binds to the test sample analyte, and then a second antibody binds to the analyte, forming an insoluble three-part complex. See, for example, U.S. Patent No. 4,376,110. The second antibody itself can be labeled with the detectable moiety (direct sandwich assay), or it can be measured using an anti-immunoglobulin antibody labeled with the detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which the detectable moiety is an enzyme.

[0242] For immunohistochemistry, samples can be fresh or frozen, or they can be embedded in paraffin and fixed with preservatives such as formalin.

[0243] pharmaceutical preparations

[0244] Therapeutic formulations of the anti-Tie2 antibody or antibody fragment or variant thereof, or the immunoconjugates or fusion peptides of the present invention (including combinations thereof with the anti-VEGF antibody or recombinant VEGF fusion protein provided herein), may be prepared as lyophilized formulations or stored in aqueous solutions by mixing the peptide, having the desired purity, with optional "pharmaceutically acceptable" carriers, excipients, or stabilizers (all of which are referred to as "excipients") commonly used in the art. Examples include buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and a variety of other additives. See, for example, Remington's Pharmaceutical Sciences, 16th edition, edited by A. Osol (1980). Such additives must be non-toxic to the recipient at the dosage and concentration used.

[0245] Treatment methods and compositions

[0246] Any anti-Tie2 antibody and antibody fragment of the present invention, or the immunoconjugate of the present invention, or the fusion polypeptide of the present invention (including its combination with the anti-VEGF antibody or recombinant VEGF fusion protein provided herein), may be used in the treatment of, prevention of, and / or relief of various diseases, including any Tie2 dysregulation disease as defined herein.

[0247] Example

[0248] The following are examples of the methods and compositions of the present invention. It should be understood that various other embodiments can be practiced in light of the general description provided above.

[0249] Example 1: Immunizing transgenic chickens to isolate intact human anti-Tie2 antibodies

[0250] Because the extracellular domain (ECD) of the direct homolog of Tie2 in humans and chickens is only 62% identical, while 90% identity has been observed between the direct homologs of Tie2 in mice and humans, using chickens to generate Tie2 antibodies will greatly increase the epitope coverage that antibodies can obtain.

[0251] Using a recombinantly generated and purified human Tie2 ECD antibody against proprietary transgenic chickens (Crystal Biosciences Inc., Emeryville, California) Immunization is used to generate antibodies. By deleting endogenous immunoglobulin-coding genes from the chicken genome and replacing them with human immunoglobulin-coding genes, OmniChickens is "humanized" and can produce complete human antibodies (see U.S. Patents 9,809,642 and 9,380,769).

[0252] Next, a high-throughput single B-cell screening and cloning method—a gel encapsulation microenvironment (GEM) technology (Crystal Biosciences, Emeryville, CA)—was used to rapidly screen millions of B cells for antibodies that specifically bind to Tie2 (see U.S. Patents 8,415,173 and 8,030,095). GEM involves co-localizing single antibody-secreting B lymphocytes from immunized animals within gel droplets containing one or more particle reporter molecules (Mettler Izquierdo et al. 2016). The reporter molecules used were polystyrene beads coated with 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. Using single B-cell cloning technology, numerous 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, which were used to confirm specificity and assess functional activity in downstream assays.

[0253] Example 2: First Antibody Screening

[0254] The binding of all 236 antibodies generated as described in Example 1 above to the recombinant human Tie2 ECD protein was tested using an ELISA method. The ELISA revealed that most of the screened antibodies were strong binders, and many had EC50 values ​​in the low pM range.

[0255] Cross-reactivity with mouse Tie2 was used as a preliminary screening criterion by binding to recombinant mouse Tie2 ECD protein. These binding studies showed that approximately 90% of the generated anti-Tie2 antibodies were cross-reactive between mouse and human Tie2 proteins, making them potentially suitable for in vivo testing in established animal models.

[0256] Next, screening was performed to identify and bind to full-length human Tie2 expressed in its native conformation on the cell membrane of living cells. For these experiments, human umbilical vein endothelial cells (HUVECs) were obtained from donors. Using flow cytometry, approximately 70% of the antibodies that bound to the human Tie2 ECD protein by ELISA also demonstrated the binding of the antibodies to native Tie2 expressed on the cells [data not shown].

[0257] Example 3: Agonistous antibodies identified through functional screening

[0258] Then, homogeneous immunoassay (AlphaLISA) was used. TM The screening platform tests the agonistic properties of all Tie2 antibodies that bind to HUVEC cells as described in Example 2 above. This platform is designed to detect the intracellular levels of antibody-induced phosphorylated (activated) ERK (pERK or p42 / p44) and phosphorylated (activated) Akt (pAkt), both of which are known downstream signal transduction effectors of Tie2.

[0259] In this screening method, HUVEC cells were plated in 96-well plates, starved of serum 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 incubation for 20 minutes, cells were lysed, and the presence of pERK and total ERK, as well as pAkt and total Akt, in the lysates was analyzed by a specific AlphaLISA assay. To be designated as an "active agonist anti-Tie2 antibody," the antibody clone needed to induce Tie2 signaling, which was determined by an increase in intracellular pAkt and pERK levels (normalized relative to total Akt and total ERK, respectively). Antibodies capable of inducing an increase in pERK and pAkt levels were graded according to their activity relative to the pERK and pAkt levels induced by Ang1 treatment. Antibodies that increased pERK and pAkt levels by ≥75% after Ang1 treatment were considered equivalent to Ang1 and were further tested.

[0260] Following initial functional screening and identification of the target, the variable domains from the most active anti-Tie2 antibody were reformatted onto different human IgG scaffolds more suitable for therapeutic use. Specifically, all candidate variable domains were cloned into expression plasmids containing an Fc domain (from human IgG1) with an asparagine-to-alanine mutation at amino acid position 297 (N297A). In antibody-induced phosphate-Tie2 (pTie2) increase Western blot assays… Figure 1A ), and through the previously described pERK and pAkt ( Figure 1B and 1C This confirmed the Tie2 agonist activity of these recombinant antibodies.

[0261] Therefore, it was found that the anti-Tie2 antibody of the present invention activates proximal and distal Tie2 signaling events and was used for further research.

[0262] Example 4: In vitro determination of antibody titer

[0263] In order to rank the anti-Tie2 antibodies of the present invention based on their titers, a high-resolution assay for determining the EC50 value of the antibodies has been developed.

[0264] In this assay, HUVEC cells were serum-starved for 3 hours, then treated for 20 minutes with increased 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 EC50 values ​​were determined as follows: Ang1 = 0.54 nM, Ab#1 = 0.91 nM, Ab#2 = 0.48 nM, Ab#3 = 0.45 nM, WT = 1.33 nM. See also... Figure 2 .

[0265] Analysis of EC50 values ​​showed that most antibodies exhibited effective Tie2 activation at sub-nanomolar concentrations, comparable to Ang1 titers in the same assay. In summary, the functional assay utilized an identified anti-Tie2 antibody capable of inducing Tie2 signaling to levels comparable to Ang1.

[0266] Example 5: Anti-Tie2 antibody reduces fluid leakage in an in vitro model of the endothelial barrier.

[0267] To investigate the potential physiological role of anti-Tie2 antibodies in controlling cellular permeability, a simplified in vitro model of the endothelial barrier was established. Based on the antibody's ability to enhance and / or protect physiological permeability induced by increased VEGF levels in the microenvironment, this model allows for further characterization of the antibody. A schematic diagram of the model setup is shown below. Figure 3 .

[0268] Complete confluent monolayers of HUVEC cells were cultured on a semipermeable membrane to form a tightly connected, adhesive structure. Cells were treated with 100 ng / ml VEGF (with or without Ang1 or anti-Tie2 antibody). Permeability was measured at different time points over 6 hours by measuring the amount of fluorescein-conjugated dextran that permeates the cell monolayer into the submembrane receiver pores. Leakage rate was measured over time by the number of fluorescein units accumulated in the receiver pores. 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 relative to the effect of Ang1 treatment in the same assay (Ang1 activity %). See also Figure 4 .

[0269] Statistical analysis showed that anti-Tie2 antibodies significantly reduced VEGF-induced fluid leakage through the primary endothelial cell matrix, while the human IgG1 negative control did not. Therefore, anti-Tie2 antibodies can stimulate downstream Tie2 signaling in biochemical assays and reduce leakage in in vitro physiological orthogonal assays.

[0270] Example 6: Anti-Tie2 agonist antibodies activate Tie2 in the presence of high levels of Ang2.

[0271] Patients with diabetic macular edema (DME) have shown significantly increased systemic and intravitreal levels of Ang2 (Loukovaara et al. 2013b; Regula et al. 2017). Considering the possibility that high levels of Ang2 might interfere with the activity of anti-Tie2 antibodies due to similar binding sites on Tie2, or if antibody binding is affected by the allosteric effect of Ang2 on the Tie2 structure, the functional properties of anti-Tie2 antibodies in the presence of saturated concentrations of Ang2 were determined. Ang2 functions as a weak Tie2 agonist, capable of inducing signal transduction in in vitro assays in the absence of Ang1, although at levels lower than those achievable with Ang1 (Yuan et al. 2009). The following in vitro experiments confirm these findings.

[0272] To determine the saturation level of Ang2 for Tie2 signaling in an in vitro model system, HUVEC cells were starved of serum for 3 hours, treated with increased concentrations of Ang2, and then lysed and analyzed for pERK / Erk levels using Western blotting. See also Figure 4 A. Quantitative fluorescence imaging was used to analyze the blot, and normalized pERK levels were plotted as a function of Ang2 concentration. The minimum concentration of Ang2 required to saturate Tie2 signal transduction was determined to be 2.5 μg / mL. See also Figure 5B In subsequent experiments, cell lysis and analysis of pErk / Erk ( Figure 5C and 5D) and pAkt / Akt( Figure 5E and 5F Before reaching the target level, HUVEC cells were simultaneously treated for 20 minutes with Ang2 at a concentration of 2.5 μg / mL (43 nM) and anti-Tie2 antibody at a concentration of 12 nM.

[0273] The results of these experiments demonstrate that anti-Tie2 antibodies are effective Tie2 pathway activators, even in the presence of high concentrations of Ang2. Surprisingly, Tie2 activity in cells exposed to both anti-Tie2 antibodies and Ang2 was higher than that observed with either treatment alone.

[0274] Example 7: Anti-Tie2 antibody utilizes a non-ligand competitive binding mechanism

[0275] To further investigate the interaction between anti-Tie2 antibody and angiopoietin at the Tie2 receptor level, a competitive binding assay was established using recombinant protein and biomembrane interference (BLI), a label-free technique for measuring biomolecular interactions.

[0276] In these experiments, recombinant his-labeled Tie2 ECDs were captured onto a Ni-NTA biosensor and subsequently exposed sequentially to a specific anti-Tie2 antibody, followed by exposure to Ang1 or Ang2, to assess potential binding competition. In these experiments, Ang1 and Ang2 were able to bind to the Tie2 receptor pre-conjugated with the anti-Tie2 antibody. See also Figure 6A -B. Therefore, it has been 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 to a higher level in the presence of Ang2 than the anti-Tie2 antibody alone.

[0277] When combined with the above-mentioned Tie2 pathway signal transduction results, the data characterizing the binding mechanism of the anti-Tie2 antibody of the present invention demonstrate a mode of action that is completely independent of angiopoietin levels.

[0278] Example 8: Cross-reactivity of anti-Tie2 agonist antibodies with Tie2 variants expressed in other animal species

[0279] We investigated the cross-reactivity profile of anti-Tie2 antibodies with Tie2 direct homologs expressed in preclinical animal model species using engineered cell lines that overexpressed direct homologs of Tie2 in mice, rats, rabbits, pigs, and cynomolgus monkeys.

[0280] In summary, expression plasmids containing the full-length Tie2 coding sequence for each species were used to transfect primary human endothelial cells (HUVECs) and human embryonic kidney (HEK293) cells. Stable cell populations were selected by treating transfectants with puromycin antibiotic over 2 weeks, and single-cell clones were generated using fluorescence-activated cell sorting. After confirming cell surface expression of these Tie2 variants, cell lines were used to determine the epigenetic Kd (EC50) values ​​of anti-Tie2 antibodies on various Tie2 proteins. To obtain this data, the cross-reactivity profile of the anti-Tie2 antibodies of this invention was determined. Cells were labeled with reduced concentrations of anti-Tie2 antibodies #1, #2, and #3 (10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1 μg / mL, 25 μg / mL, 0.63 μg / mL, 0.31 μg / mL, and 0.16 μg / mL) or nonspecific hIgG1 antibody (10 μg / mL), followed by labeling with rabbit anti-human IgG antibody (20 μg / mL) conjugated to the Alexa Fluor 488 fluorophore. ForeCyt was used. TM The software generates EC50 values. See Tables 1 and 2.

[0281] Table 1

[0282]

[0283] Table 2

[0284]

[0285] These experiments demonstrate cross-reactive binding of anti-Tie2 antibodies across humans and preclinical species.

[0286] Example 9: Anti-Tie2 antibodies recognize different epitopes on human Tie2.

[0287] To understand the diversity of epitope coverage in the Tie2 antibody population, cross-competition of anti-Tie2 antibodies was assessed using BLI technology.

[0288] Recombinant human Tie2 ECD was loaded onto a biosensor and then exposed to anti-Tie2 antibodies (M1 = mouse anti-Tie2 mAb; antibody #1; antibody #3; M2 = mouse anti-Tie2; antibody #2; WT) to assess initial binding. The probe was then exposed to a second anti-Tie2 antibody to assess a second binding event. Positive wavelength shifts indicate that the second antibody can bind to the previously formed Tie2-antibody complex, suggesting that these antibodies bind to different epitopes and do not compete for binding to the Tie2 protein. Positive wavelength shifts indicating co-binding of the two anti-Tie2 antibodies are shown in gray and light gray, with shift values ​​(nm) embedded in each cell. As expected, the antibody pre-bound to Tie2 prevented the same antibody from binding to the complex (black shaded area). See also Figure 7 .

[0289] These experiments demonstrate that among a group of six anti-Tie2 agonist antibodies, there is no cross-competition for binding to Tie2, indicating that each candidate antibody recognizes a different epitope on the receptor.

[0290] Example 10: Oxygen-Induced Retinopathy (OIR) Mouse Model

[0291] In this study, 40 C57BL / 6J pups (n=10 per cage) on day 7 postpartum (P7) were placed in a hyperoxia chamber (75% O2) for 5 days, resulting in vascular degeneration in the central retina. CD1 surrogate mothers were rotated 2–3 days before and after entering the chamber. At P12, the pups were returned to the room air, where relative hypoxia induced abnormal new angiogenesis, and then placed in 10 mg / kg 1X PBS carrier without endotoxin; intraperitoneal administration of HuIgG isotype control or 10 mg / kg anti-Tie2 clone #3 was also administered. At P17, all groups, including uninfected OIR mice, were euthanized. Eyes were removed and the mice were fixed in 4% paraformaldehyde for 1 hour.

[0292] The retina was dissected and incubated overnight in 1 mm CaCl2 dissolved in PBS with a rhodamine-labeled lectin from *Bandeiraea simplicifolia* (Griffonia simplicifolia) (1:100) to observe areas of vascular occlusion (VO) or neovascularization (NV). The stained retina was laid flat on a glass slide and... Imaging on AxioScan. The images were analyzed to determine the total retinal VO% or NV%.

[0293] like Figure 8As shown in Figure B, anti-Tie2 mAb, rather than the allotype control mAb or the carrier, was found to have a significant positive effect on vascular occlusion (i.e., avascular) areas, but did not appear to reduce NV (vascular occlusion). Figure 8 A). Promoting the regeneration of healthy vascular systems may help mitigate the adverse consequences of chronic anti-VEGF therapy, such as geographic atrophy or capillary detachment in patients with eye disease. See Kim J et al., Science Advances, Vol. 5, Feb. 13 (2019); M. Young et al., Retina, 34, 1308–1315 (2014); T. Kurihara et al., Journal of Clinical Research, 122, 4213–4217 (2012).

[0294] Example 11: Laser-induced choroidal neovascularization (CNV) mouse model

[0295] Male C57BL / 6J mice were anesthetized (6-8 weeks) with a ketamine / toluidine mixture prior to laser treatment. A diode laser was then used. GL) and slit lamp CNV lesions were induced by laser photocoagulation under the following conditions: a spot size of 50 μm, a power of 180 mW, and an exposure duration of 100 ms. Typically, four laser burns were induced at the 3 o'clock, 6 o'clock, 9 o'clock, and 12 o'clock positions around the optic disc of each eye. One day prior to laser induction, mice were intraperitoneally injected with a Tie2-specific antibody (clone #3), an allotype control antibody, or an anti-mouse VEGF control antibody (B20) (10 mg / kg), for a total of three injections every three days. Nine days after laser induction, mice were perfused via the tail vein with FITC-phytohemagglutinin or TRITC-glucan. Five minutes after perfusion, the eyes were enucleated and fixed in 4% paraformaldehyde (PFA) for 15 minutes.

[0296] The choroid-scleral complex and retina were isolated and subjected to anti-CD31 immunofluorescence (IF) to demonstrate the vascular system through whole-body staining of the retinal and choroidal tissues. For CD31 IF, rat anti-mouse antibody BD 550274 was diluted 1:100 and incubated overnight at 4°C. After incubation for 4 hours with an anti-rat secondary antibody (Life Technologies, A11006), the whole slide was imaged at 488 nm. See [link to documentation]. Figure 9A Image J was used to quantify neovascularization in the lesion region and retinal vessel density. Student's t-test was used to assess p-values ​​(significant change, p < 0.05). See also Figure 9B .

[0297] like Figure 9A and 9BAs shown, the anti-Tie2 antibody, rather than the isotype control mAb, significantly suppressed the size of choroidal neovascularization lesions following laser injury. This result suggests that activation of Tie2 following administration of the anti-Tie2 mAb has the potential to provide significant clinical benefit for individuals with ophthalmic conditions such as AMD or diabetic retinopathy.

[0298] Example 12: Streptozotocin-induced (STZ) diabetic mouse model

[0299] Administration of the anti-Tie2 antibody of the present invention can be used to treat diabetic retinopathy and other diabetes-related diseases, such as nephropathy. To explore potential therapeutic benefits, the effects of the anti-Tie2 antibody of the present invention on disease-related endpoints will be tested, such as vascular leakage in the eye, visual function assessed by electroretinography, production of cytokines believed to be associated with human disease (e.g., IL-1β), and renal function measured by proteinuria in a streptozotocin-induced diabetic mouse model. The mAb is administered via an intraperitoneal route.

[0300] Six- to seven-week-old C57BL / 6J mice can be weighed and their baseline blood glucose levels measured. Roche (the company) can administer STZ to mice via intraperitoneal injection at a rate of 55 mg / kg for 5 consecutive days (Sigma-Aldrich, St. Louis, Missouri). Age-matched control mice can only be injected with buffer. Blood glucose levels can be measured again one week after the last STZ injection; mice are considered to have diabetes if their non-fasting blood glucose level is higher than 17 mM (300 mg / dL). Then, at least 8 weeks after STZ administration, an appropriate amount of the present invention's anti-Tie2 antibody, control antibody, vector, or comparative antibody, such as, for example, anti-VEGF antibody or recombinant VEGF fusion protein, can be injected intravitreally (IVT) into STZ-treated diabetic C57BL / 6J mice.

[0301] Electroretinography (ERG) was performed using the UTAS-E visual electrodiagnostic testing system to assess overall retinal cell function. After overnight dark adaptation, treated mice were anesthetized by subcutaneous injection of a mixture of ketamine and toluenethiazide dissolved in sterile water (BW). One eye was tilted forward and the pupil dilated using tropicamide and phenformin hydrochloride. Eye drops are used to keep the eyes moist, and a heating pad is used to maintain core body temperature. ERG can be recorded using ultra-low impedance silver / nylon DTL Plus electrodes. Needle electrodes can be placed in the middle of the forehead and at the base of the tailbone. Gold contact lens electrodes are used to record ERG responses. Stimulation may include, for example, 50 ms of electrophoresis in a logging step. Stimulation responses are recorded from below the threshold to the saturation range. Analysis may include the maximum amplitude and threshold of the a and b waves.

[0302] Optical-motor tracking (OKT) can also be recorded as follows: The mouse is fixed on a turntable surrounded by a set of synchronized monitors displaying a vertical black-and-white grating (14°). A surrounding screen vibrates sinusoidally at a peak velocity of 1 Hz and 10° / sec to induce OKT. Right eye movement is monitored using an infrared-sensitive CCD camera under illumination from an ana infrared LED47. Images are sampled at a sampling rate of 200 Hz, and the center of the pupil is calculated using the Morita Geteye software program to estimate the eye position. OKT can be recorded for 30 seconds, with three recordings approximately 30 seconds apart.

[0303] Retinal vascular permeability can be measured as follows: Mice are anesthetized and injected via the tail vein with Evans blue staining solution dissolved in saline. Two hours after the tail vein injection, mice are anesthetized with ketamine and toluenethiazide, and can be perfused through the left ventricle with saline. After perfusion, the retina is dissected, weighed, and then placed in formamide at 70°C for 18 hours to extract the Evans blue staining solution. The next day, the retina is centrifuged for 45 minutes and removed from the formamide. Extravasation of the Evans blue staining solution is measured using an ELISA reader at A620. A standard curve is used to convert to units of ng Evans blue / wet tissue weight.

[0304] Tissue collection for targeted transcriptome analysis can be performed as follows: Mice are euthanized under anesthesia, and both eyes are removed. The retina is then dissected, placed in an RNAlater, and processed for analysis using qRT-PCR.

[0305] Example 13: Bispecific Biologic Construct

[0306] Simultaneous influence on Tie2 and VEGFR-induced signaling pathways may have enhanced benefits compared to regulating either pathway alone. To achieve this, a bispecific construct influencing Tie2 and VEGFR was designed, and its activity was expressed and tested. Figure 10A An exemplary schematic diagram of the bispecific antibody of the present invention, possessing both a Tie2-binding variable domain and a VEGF-binding variable domain, is shown. Antibody clone #54, having the sequences of SEQ ID Nos. 282 and 283, was designed and generated using standard cloning techniques. A bispecific construct (antibody clone #55), having the sequence of SEQ ID No. 284, comprising VEGFR R1D2 and R3D3 (VEGF trap proteins) and a Tie2-binding domain, was also generated. AlphaLISA was subsequently used. TM The screening platform evaluated the Tie2 agonistic activity of two exemplary aTie2 / VEGF bispecific constructs (antibody clones #54 and #55) in HUVEC cells using 20 nm per molecule, as described by the AlphaLISA.TM The screening platform is designed to detect antibody-induced intracellular levels of pERK and pAkt, as described in Example 3 above.

[0307] The tested aTie2 / VEGF bispecific construct was found to have the ability to induce Tie2 signaling in vitro. Figure 11 These bispecific molecules allow patients with eye diseases to receive monotherapy, which is significantly more effective than monotherapy that inhibits VEGF or activates Tie2. The Tie2-activating component of the bispecific molecules can promote the regeneration of healthy vascular systems, which may also help mitigate the potential adverse effects of the anti-VEGF component of the molecule (i.e., geographic atrophy or capillary loss in patients with eye diseases).

[0308] Example 14: Anti-Tie2 antibodies with increased titers and double in situ anti-Tie2 antibodies

[0309] Antibodies having two or more Tie2-binding moieties, or those capable of binding to multiple epitopes on the Tie2 extracellular domain, can provide a way to enhance activation of the Tie2 in vivo pathway to a degree greater than that possible with bivalent anti-Tie2 antibodies. An exemplary tetravalent anti-Tie2 construct was produced using standard techniques, having the heavy and light chain sequences of anti-Tie2 antibody clone #3, with a linker polypeptide and a B12 scFv sequence added at the C-terminus of the heavy chain (antibody clone #51) or the N-terminus of the heavy chain (antibody clone #52). An exemplary schematic diagram is shown below. Figure 12 AC.

[0310] As described in Example 3, the ability of the tetravalent antibody clones #51 and #52 to induce signal transduction was assessed by measuring pAkt. Figure 14 As shown, clones #51 and #52 demonstrated enhanced ability to activate Tie2 signaling in HUVEC cells compared to the bivalent anti-Tie2 construct (aTie2) or compared to angiopoietin 1 (Ang1). Therefore, anti-Tie2 variants with an additional Tie2-binding moiety could provide a way to induce higher-order oligomerization of Tie2 on the cell surface and potentially offer therapeutic benefits when the level of agonistic activity mediated by the bivalent anti-Tie2 antibody or the natural agonist Ang1 is insufficient to restore vascular homeostasis in diseased tissues.

[0311] Example 15: Anti-Tie2 antibody with Fc mutation that promotes cell surface hexamerization

[0312] Antibodies containing a specific mutation in the heavy chain Fc domain (i.e., E430G) have been shown to enhance their ability to form hexamers on the cell surface (M. Overdijk, Molecular Cancer Therapy 2020; 19:2126–38). Anti-Tie2 antibodies containing an Fc domain with the E430G mutation were synthesized and compared with the same anti-Tie2 antibody containing a matching Fc domain containing native glutamic acid (E) at position 430. A schematic diagram of such hexamerized anti-Tie2 antibodies is shown below. Figure 13 .

[0313] As described in Example 3, the pAKT / pERK Western blotting method was used to assess Tie2 agonism. Figure 14 As shown, the anti-Tie2 E430G construct (antibody clone #50) demonstrated an enhanced ability to induce Tie2 signaling in HUVEC cells compared to the E430 construct. Therefore, the B12 variant with mutated potential for increased cell surface hexamerization could provide a pathway to induce higher-order oligomerization of Tie2 on the cell surface and may offer therapeutic benefits when the level of agonistic activity mediated by anti-Tie2 antibodies or the natural agonist Ang1 is insufficient to restore vascular homeostasis in diseased tissues.

[0314] Example 16: Determination of Fab binding affinity of anti-Tie2 antibody by SPR

[0315] BIACORE TM (BIAcore, Piscavenge, NJ) Immobilized human, rat, or mouse Tie2 ECD antigens were analyzed at approximately 10 reaction units (RUs) on a carboxymethylated dextran biosensor chip (CM5) at 25°C. The CM5 chip was activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. All Tie2 ECD antigens were diluted to 5 μg / ml (approximately 0.2 M) with 10 mM sodium acetate (pH 4.8) before injection at a flow rate of 5 μl / min to obtain approximately 10 RU of conjugated protein. After injection of each Tie2 ECD, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, at 25°C and a flow rate of approximately 25 μl / min, serial dilutions (0.78 nM to 500 nM) of antibody clone #3Fab were injected into a solution containing 0.05% polysorbate 20 (TWEEN). TM -20) Surfactant (PBST) in PBS. A simple one-to-one Langmuir binding model (BIACORE) was used by simultaneously fitting association and dissociation sensor maps. TMThe evaluation software (version 3.2) was used to calculate the association rate (kon) and dissociation rate (koff). The equilibrium dissociation constant (KD) was calculated as the ratio koff / kon. See, for example, Chen et al., Journal of Molecular Biology 293:865-881 (1999).

[0316] The table below shows the binding affinity of antibody clone #3Fab for each Tie2 ECD antigen tested.

[0317]

[0318] Although the foregoing invention has been described in detail with reference to illustrations and examples for purposes of clarity, these descriptions and examples should not be construed as limiting the scope of the invention. All disclosures of patents and scientific literature cited herein are expressly incorporated herein by reference in their entirety. sequence list <110> Uniti Biotechnology Company <120> Antibodies against TIE-2 and their usage <130> UNITY-022WO <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; Cloner: 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; Cloner: 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; Clones: 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; Cloner: 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; Cloner: 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; Cloning: 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; Cloner: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloner: 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; Cloning: 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; Cloner: 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; Cloner: p1.25 <400> 20 Arg Asn Tyr Gly Met 1 5 <210> twenty one <211> 5 <212> PRT <213> Artificial sequence <220> <223> Human mAb CDR:HC1; Cloner: p1.1 <400> twenty one Ser Ser Tyr Ala Met 1 5 <210> twenty two <211> 5 <212> PRT <213> Artificial sequence <220> <223> Human mAb CDR:HC1; Cloner: p1.26 <400> twenty two Ser Ser Tyr Ala Met 1 5 <210> twenty three <211> 5 <212> PRT <213> Artificial sequence <220> <223> Human mAb CDR:HC1; Cloning: p1.27 <400> twenty three Asp Val Tyr Ala Met 1 5 <210> twenty four <211> 5 <212> PRT <213> Artificial sequence <220> <223> Human mAb CDR:HC1; Cloning: p1.28 <400> twenty four Ser Ser Tyr Ala Met 1 5 <210> 25 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Human mAb CDR:HC1; Cloning: 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; Cloner: 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; Cloner: 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; Cloner: 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; Cloning: 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; Cloner: 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; Cloner: 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; Clones: 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; Cloning: 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; Cloner: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloner: p2.3 <400> 41 Ser Ile Asn Val Gly Asp Asn Thr Tyr 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; Cloner: p2.8 <400> 42 Ser Ile Asn Val Gly Asp Asn Thr Tyr 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; Cloner: 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; Cloner: p2.10 <400> 44 Arg Ile Ser Gly Asn Gly Gly Ser Thr Tyr 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; Cloner: 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; Cloning: 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; Cloner: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: p2.17 <400> 52 Arg Ile Ser Gly Asn Gly Gly Ser Thr Tyr 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: p1.25 <400> 60 Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr 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; Cloner: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloner: 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; Cloner: 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; Cloning: 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; Cloner: 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; Cloning: 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; Cloning: p1.33 <400> 72 Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr 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; Cloning: 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; Cloning: 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> twenty one <212> PRT <213> Artificial sequence <220> <223> Human mAb CDR:HC2; Cloning: 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; Cloning: 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; Cloning: 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> twenty one <212> PRT <213> Artificial sequence <220> <223> Human mAb CDR:HC2; Cloning: 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; Cloning: 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; Cloning: 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; Cloner: 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; Cloner: 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; Clones: 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; Cloning: 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; Cloner: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloner: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloner: 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; Cloner: 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; Cloning: 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; Cloner: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloning: 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; Cloner: 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; Cloner: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Cloner: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Cloning: 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; Cloning: 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; Cloning: 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; Clones: 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; Clones: 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; Clones: 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; Clones: p2.8 <400> 162 Asp Ala Ser Ser Ser Arg Ala Thr 1 5 <210> 163 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Human mAb CDR:LC2; Clones: p2.9 <400> 163 Ala Ala Ser Ser Ser Arg Ala Ala 1 5 <210> 164 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Human mAb CDR:LC2; Clones: p2.10 <400> 164 Asp Ala Ser Ser Ser Arg Ala Thr 1 5 <210> 165 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Human mAb CDR:LC2; Clones: 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; Clones: 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; Clones: p2.12 <400> 167 Asp Ala Ser Ser Ser Arg Ala Thr 1 5 <210> 168 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Human mAb CDR:LC2; Clones: 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; Clones: 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; Clones: 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; Clones: p2.16 <400> 171 Asp Ala Ser Ser Ser Arg Ala Thr 1 5 <210> 172 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Human mAb CDR:LC2; Clones: 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; Clones: 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; Clones: 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; Clones: p2.20 <400> 175 Ala Ala Ser Ser Ser Arg Ala Thr 1 5 <210> 176 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Human mAb CDR:LC2; Cloning: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Cloning: 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; Clones: 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; Cloning: 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; Cloning: 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; Clones: 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; Cloning: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Cloning: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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; Clones: 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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 mutation (heavy chain) with pre-hexamerization and Fc mutation removing ADCC / CDC. <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 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 pre-hexamerization and Fc mutations that remove ADCC / CDC. Mutation (light chain (K)) <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 C-terminal p2.3-scFv with removal of ADCC / CDC Fc mutation p2.3 IgG-scFv fusion tetravalent mAb (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 C-terminal p2.3-scFv with removal of ADCC / CDC Fc mutation p2.3 IgG-scFv fusion tetravalent mAb (light chain (K)) <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 Fc mutation with HC N-terminal p2.3-scFv and removal of ADCC / CDC p2.3 IgG-scFv fusion tetravalent mAb (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 Fc mutation with HC N-terminal p2.3-scFv and removal of ADCC / CDC p2.3 IgG-scFv fusion tetravalent mAb (light chain (K)) <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 C-terminal p1.2-scFv with removal of ADCC / CDC Fc mutation p2.3 IgG-scFv fusion dual in situ mAb (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 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 C-terminal p1.2-scFv with removal of ADCC / CDC Fc mutation p2.3 IgG-scFv fusion dual in situ mAb (light chain (K)) <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 VEGF-scFv with HC C-terminus and Fc mutations that remove ADCC / CDC p2.3 IgG-scFv fusion Tie2 / VEGF bispecific mAb (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 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 VEGF-scFv with HC C-terminus and Fc mutations that remove ADCC / CDC p2.3 IgG-scFv fusion Tie2 / VEGF bispecific mAb (light chain (K)) <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 with Tie2 / VEGF with C-terminal p2.3-scFv Bispecific mAb <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 with C-terminal p2.3-scFv and Fc mutations that remove ADCC / CDC p2.3 scFv fusion Tie2 / VEGF bispecific mAb <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> With HC C-terminal ranibizumab-scFv and Fc mutations that remove ADCC / CDC p2.3-Ranibizumab Tie2 / VEGF bispecific mAb (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> With HC C-terminal ranibizumab-scFv and Fc mutations that remove ADCC / CDC p2.3-Ranibizumab Tie2 / VEGF Bispecific mAb (Light Chain (K)) <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> With HC N-terminal ranibizumab-scFv and Fc mutations that remove ADCC / CDC p2.3-Ranibizumab Tie2 / VEGF bispecific mAb (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> With HC N-terminal ranibizumab-scFv and Fc mutations that remove ADCC / CDC p2.3-Ranibizumab Tie2 / VEGF Bispecific mAb (Light Chain (K)) <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 antigen-binding fragment thereof, wherein the anti-Tie2 antibody binds to Tie2 in a non-Ang1 and Ang2 ligand competitive manner, wherein the anti-Tie2 antibody or antigen-binding fragment thereof comprises three heavy chain complementary determining regions (CDR H1-3) and three light chain CDRs (CDR L1-3), as shown below:

2. An isolated anti-Tie2 antibody or antigen-binding fragment thereof, comprising: A heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID No.246 and a light chain variable (VL) domain comprising the amino acid sequence of SEQ ID No.

247.

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

4. The antibody according to any one of claims 1 to 3, wherein the antibody is a non-ligand competitive binder of Tie2.

5. The antibody according to any one of claims 3 to 4, wherein the antibody has cross-reactivity with human, mouse, rat, rabbit and monkey Tie2.

6. The antibody of any one of claims 1 to 5, wherein the antibody is fully human, humanized, monoclonal or chimeric.

7. The antibody according to any one of claims 1 to 6, wherein the antibody is monospecific. 8 . The antibody according to any one of claims 1 to 7 , wherein the antibody is an antibody fragment that specifically binds to human Tie2.

9. The antibody according to claim 8, wherein the antibody fragment is a Fab, Fab'-SH, Fv, scFv or (Fab')2 fragment.

10. The antibody of any one of claims 1 to 9, wherein the antibody has reduced effector function.

11. The antibody of claim 10, wherein the antibody comprises at least one substitution mutation at amino acid residues N297, L234, L235, P329, D265 and E430 according to EU numbering in Kabat.

12. The antibody of claim 11, 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 in Kabat.

13. The antibody of claim 12, wherein the antibody comprises a substitution mutation at residue N297A or N297G.

14. The antibody of claim 12, wherein the antibody comprises substitution mutations at residues L234A, L235A and P329G.

15. The antibody of claim 12, wherein the antibody comprises substitution mutations at residues D265A and N297G.

16. The antibody of claim 13, wherein the antibody further comprises a substitution mutation at residue E430G.

17. The antibody of claim 14, wherein the antibody further comprises a substitution mutation at residue E430G.

18. The antibody of claim 15, wherein the antibody further comprises a substitution mutation at residue E430G.

19. An isolated nucleic acid encoding the antibody of any one of claims 1 to 18.

20. A vector comprising the isolated nucleic acid according to claim 19.

21. A host cell comprising the vector according to claim 20.

22. A method of producing an antibody according to any one of claims 1 to 18, the method comprising culturing the host cell according to claim 21 in a culture medium and isolating the resulting antibody.

23. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 18.

24. The pharmaceutical composition of claim 23, wherein the antibody is formulated in combination with an anti-VEGF antibody or a VEGF extracellular trap protein.

25. Use of the pharmaceutical composition of claim 23 or 24 in the preparation of a medicament for treating a Tie2 disorder in a subject in need thereof, wherein the Tie2 disorder is selected from proliferative diabetic retinopathy and age-related macular degeneration (AMD).

26. The use according to claim 25, wherein the Tie2 disorder disease is proliferative diabetic retinopathy (PDR).

27. Use of the anti-Tie2 antibody of any one of claims 1-18 in the preparation of a medicament for treating a Tie2 disorder in a subject, wherein the Tie2 disorder is selected from proliferative diabetic retinopathy and age-related macular degeneration (AMD).

28. The use according to claim 27, wherein the Tie2 disorder disease is proliferative diabetic retinopathy (PDR).

29. The use according to claim 25 or 26, wherein the medicament further comprises a pharmaceutical composition comprising an anti-VEGF antibody or a VEGF extracellular trap protein.

Images