Antibodies directed to tie-2 and methods of use

Abstract

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

Description

Technical Field

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

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

Background Art

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

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

[0005] There remains a need for anti-Tie2 antibodies with improved properties, as well as their therapeutic and diagnostic uses.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

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

[0008] The following are specifically contemplated as part of the disclosed invention. Embodiment 1. An isolated anti-Tie2 antibody or antigen-binding fragment thereof comprising the following three heavy-chain complementarity-determining regions (CDR H1-3) and three light-chain CDRs (CDR L1-3): TIFF2025081502000002.tif109166TIFF2025081502000003.tif179166. Embodiment 2. An isolated anti-Tie2 antibody or antigen-binding fragment thereof, a heavy-chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 242, and a light-chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 243, a heavy-chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 244, and a light-chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 245, 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, a heavy-chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 248, and a light-chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 249, a heavy-chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 250, and a light-chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 251, a heavy-chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 252, and a light-chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 253, a heavy-chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 254, and a light-chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 255, A heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 256, and a light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 257 A heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 258, and a light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 259 A heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 260, and a light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 261 A heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 262, and a light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 263 A heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 264, and a light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 265 A heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 266, and a light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 267 A heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 268, and a light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 269, or A heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 270, and a light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 271 An isolated anti-Tie2 antibody or an antigen-binding fragment thereof comprising Embodiment 3. An isolated anti-Tie2 antibody, wherein the antibody specifically binds to an epitope within the extracellular domain of human Tie2, and the epitope comprises amino acid residues K312, S316, C332, H358, K387, and T391 according to EU numbering such as Kabat numbering, as measured by cross-linking mass spectrometry An isolated anti-Tie2 antibody comprising Embodiment 4. The antibody according to any one of Embodiments 1 to 3, wherein the antibody is an allosteric activator of Tie2. Embodiment 5. The antibody according to any one of Embodiments 1 to 4, wherein the antibody is a non-ligand competitive binder of Tie2. Embodiment 6. The antibody according to Embodiments 3 to 5, wherein the antibody is cross-reactive with human, mouse, rat, rabbit, and monkey Tie2. Embodiment 7. The antibody according to Embodiments 1 to 6, wherein the antibody is fully human, humanized, monoclonal, or chimeric. Embodiment 8. The antibody according to Embodiments 1 to 7, wherein the antibody is monospecific. Embodiment 9. The antibody according to Embodiments 1 to 7, wherein the antibody is multispecific. Embodiment 10. The antibody according to Embodiment 9, wherein the multispecific antibody is bispecific. Embodiment 11. The bispecific antibody is one binding arm that specifically binds to human Tie-2 as described in Embodiment 8, and a second binding arm that specifically binds to VEGF-A, VEGF-B, VEGF-C, VEGF variant, Ang-1, Ang-2, Ang-3, Ang-4, PDGF-β, interleukin-1β, VE-PTP, complement factor C3, integrin α5β1, amyloid beta, PD-1, PD-L1, or CTLA-4 The antibody according to Embodiment 10. Embodiment 12. The antibody according to Embodiment 9, wherein the multispecific antibody is a bisparatopic antibody. Embodiment 13. The bisparatopic antibody is one binding arm that specifically binds to a first epitope on the ECD of human Tie2, and the other binding arm that specifically binds to a second epitope on the ECD of human Tie2 The antibody according to Embodiment 12. Embodiment 14. The multispecific antibody is a trivalent, tetravalent, pentavalent, or hexavalent antibody, and the trivalent, tetravalent, pentavalent, or hexavalent antibody is at least one binding arm that specifically binds to human Tie2 as described in Embodiment 8, VEGF-A, VEGF-B, VEGF-C, VEGF variants, Ang-1, Ang-2, Ang-3, Ang-4, PDGF-β, interleukin-1β, VE-PTP, complement factor C3, integrin α5β1, amyloid beta, PD-1, PD-L1, or any other remaining binding arm that specifically binds to CTLA-4 The antibody according to embodiment 9, comprising Embodiment 15. The antibody according to embodiments 1 to 14, wherein the antibody is an antibody fragment that specifically binds to human Tie2. Embodiment 16. The antibody according to embodiment 15, wherein the antibody fragment is Fab, Fab’-SH, Fv, scFv, or (Fab’) 2 fragment. Embodiment 17. The antibody according to embodiment 16, wherein the multispecific antibody is composed of scFv antibody fragments linked together by a polypeptide linker. Embodiment 18. The antibody according to embodiments 1 to 17, wherein the antibody has reduced effector function. Embodiment 19. The antibody according to embodiment 18, wherein the antibody comprises at least one substitution mutation at amino acid residues N297, L234, L235, P329, D265, and E430 according to EU numbering such as Kabat numbering. Embodiment 20. The antibody according to embodiment 19, wherein at least one substitution mutation is selected from the group consisting of N297G, N297A, L234A, L235A, P329G, D265A, and E430G according to EU numbering such as Kabat numbering. Embodiment 21. The antibody according to embodiment 20, wherein the antibody comprises a substitution mutation at residue N297A or N297G. Embodiment 22. The antibody according to embodiment 20, wherein the antibody comprises substitution mutations at residues L234A, L235A, and P329G. Embodiment 23. The antibody according to embodiment 20, wherein the antibody comprises substitution mutations at residues D265A and N297G. Embodiment 24. The antibody according to embodiment 21, wherein the antibody further comprises a substitution mutation at residue E430G. Embodiment 25. The antibody according to embodiment 22, wherein the antibody further comprises a substitution mutation at residue E430G. Embodiment 26. The antibody according to embodiment 23, wherein the antibody further comprises a substitution mutation at residue E430G. Embodiment 27. The antibody according to embodiment 25, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 174 and a light chain comprising the amino acid sequence of SEQ ID NO: 175. Embodiment 28. A heavy chain comprising the amino acid sequence of SEQ ID NO: 276 and a light chain comprising the amino acid sequence of SEQ ID NO: 277, A heavy chain comprising the amino acid sequence of SEQ ID NO: 278 and a light chain comprising the amino acid sequence of SEQ ID NO: 279, A heavy chain comprising the amino acid sequence of SEQ ID NO: 280 and a light chain comprising the amino acid sequence of SEQ ID NO: 281, A heavy chain comprising the amino acid sequence of SEQ ID NO: 282 and a light chain comprising the amino acid sequence of SEQ ID NO: 283, A heavy chain comprising the amino acid sequence of SEQ ID NO: 286 and a light chain comprising the amino acid sequence of SEQ ID NO: 287, or A heavy chain comprising the amino acid sequence of SEQ ID NO: 288 and a light chain comprising the amino acid sequence of SEQ ID NO: 289 The antibody according to embodiment 22, comprising. Embodiment 29. The amino acid sequence of SEQ ID NO: 284, or The amino acid sequence of SEQ ID NO: 285 The antibody according to embodiment 10, comprising. Embodiment 30. An isolated nucleic acid encoding the antibody according to embodiments 1 to 29. Embodiment 31. A vector comprising the isolated nucleic acid according to embodiment 30. Embodiment 32. A host cell comprising the vector according to embodiment 31. Embodiment 33. A method for producing the antibody according to embodiments 1 to 29, comprising: Culturing the host cell according to embodiment 32 in a culture medium, and Isolating the obtained antibody A method comprising. Embodiment 34. An immunoconjugate comprising the antibody described in Embodiments 1 to 29. Embodiment 35. A fusion polypeptide comprising the antibody described in Embodiments 1 to 29. Embodiment 36. A pharmaceutical composition comprising the antibody described in Embodiments 1 to 29, the immunoconjugate described in Embodiment 34, or the fusion polypeptide described in Embodiment 35. Embodiment 37. The pharmaceutical composition according to Embodiment 36, wherein the antibody, immunoconjugate, or fusion polypeptide is co-formulated with an anti-VEGF antibody or a VEGF extracellular trap protein. Embodiment 38. A method of treating a subject in need of treatment for Tie2 dysregulation disorder, the method comprising administering to the subject the pharmaceutical composition according to Embodiment 36. Embodiment 39. A method of treating a subject in need of treatment for Tie2 dysregulation disorder, the method comprising administering to the subject the pharmaceutical composition according to Embodiment 37. Embodiment 40. The method according to Embodiment 38, further comprising co-administering to the subject a pharmaceutical composition comprising an anti-VEGF antibody or a VEGF extracellular trap protein. Embodiment 41. The method according to Embodiments 38 to 40, wherein the Tie2 dysregulation disorder includes an infectious disease, acute respiratory distress syndrome (ARDS), ischemic injury, ocular injury, radiation injury, cancer, systemic sclerosis, traumatic brain injury, neuroinflammation, radiation injury, wound healing, myocardial infarction, blood-brain barrier injury, cerebral cavernous malformation, Duchenne muscular dystrophy (DMD), or Clarkson's disease. Embodiment 42. The method according to Embodiments 38 to 40, wherein the Tie2 dysregulation infectious disease includes sepsis, dengue virus infection, tuberculosis, or influenza. Embodiment 43. The method according to Embodiments 38 to 40, wherein the Tie2 dysregulation ischemic injury includes diabetic nephropathy, acute kidney injury, chronic kidney disease, organ transplantation, critical limb ischemia, traumatic brain injury, or stroke. Embodiment 44. The method according to Embodiments 38 to 40, wherein the Tie2 dysregulation eye disorder includes diabetic retinopathy, diabetic macular edema (DME), proliferative diabetic retinopathy (PDR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP), or glaucoma. Embodiment 45. An isolated anti-Tie2 antibody according to Embodiments 1 to 29, or an immunoconjugate according to Embodiment 34, or a fusion polypeptide according to Embodiment 35, for use in the treatment of Tie2 dysregulation diseases according to Embodiments 41 to 44. Embodiment 46. Use of an isolated anti-Tie2 antibody according to Embodiments 1 to 29, or an immunoconjugate according to Embodiment 34, or a fusion polypeptide according to Embodiment 35, for the manufacture of a medicament for treating Tie2 dysregulation diseases according to Embodiments 41 to 44.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0010] Detailed Description of the Invention I. Definitions "Tie2", also known as angiopoietin-1 receptor, or TEK receptor tyrosine kinase, or CD202B (cluster of differentiation 202B), is a protein encoded by the TEK gene in humans (Partanen J 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 repeats, and three fibronectin type III-like repeats (see Fiedler et al., 2006. Angiopoietins: A Link between Angiogenesis and Inflammation. Trends in Immunology 27(12):552-58, Barton et al., Crystal structures of the Tie2 receptor ectodomain and the angiopoietin-2-Tie2 complex. Nature Struc.&Mol.Biology, 13, pp524-532(2006)). The contact residues of angiopoietin-1 and angiopoietin-2 are mostly overlapping on the Tie-2 receptor, as suggested by the analysis of the crystal structure of the Ang2 / Tie2 complex, and are mainly located within the second Ig-like loop (Barton et al., Nat Str Biol 2006).Other studies 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. JBC. Vol. 278(3):1721-7(2003)). The exemplary amino acid sequence of human Tie2 can be found in UniProt accession number Q02763 (SEQ ID NO: 241).

[0011] As used herein, the term "about" refers to the normal error range of each value that is readily known to those of ordinary skill in the art. References to values or parameters with "about" herein include (and describe) embodiments directed to the value or parameter itself.

[0012] For the purposes of this specification, an "acceptor human framework" is a framework that includes the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence or may contain amino acid sequence variations. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework has the same sequence as the VL human immunoglobulin framework sequence or the human consensus framework sequence.

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

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

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

[0016] An "affinity matured" antibody refers to an antibody having one or more modifications in one or more hypervariable regions (CDRs) and / or framework regions (FRs) compared to a parental antibody that does not have such modifications, wherein such modifications result in an improvement in the affinity of the antibody for an antigen.

[0017] "Allosteric activation of Tie2" is activation of Tie2 by an agonist anti-Tie2 antibody that specifically interacts with a region of Tie2 other than the described ligand-binding or active site, such that the binding results in a change in the Tie2 conformation or clustering and an enhancement of the receptor's activity.

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

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

[0020] Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments and a remaining "Fc" fragment, a name reflecting its ability to readily crystallize. The Fab fragment consists of the variable domain of the heavy (H) chain (VH) and the first constant domain of one heavy chain (CH1), along with the entire light (L) chain. Pepsin treatment of an antibody produces a single large F(ab') that generally corresponds to two disulfide-linked Fab fragments having bivalent antigen-binding activity and can still cross-link antigens. 2A fragment is obtained. The Fab’ fragment differs from the Fab fragment by having several additional residues at the carboxy terminus of the CH1 domain that include one or more cysteines from the antibody hinge region. Fab’-SH is the name herein for Fab’ in which the cysteine residue of the constant domain has a free thiol group. F(ab’) 2 Antibody fragments were originally produced as pairs of Fab’ fragments that have hinge cysteines in between. Other chemical couplings of antibody fragments are also known.

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

[0022] “Fv” consists of a dimer of one heavy chain variable domain and one light chain variable domain in a close non-covalent association. From the folding of these two domains, six hypervariable loops (three loops from each of the H and L chains) are generated that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv that contains only the three CDRs specific for the antigen) has the ability to recognize and bind the antigen, but often with lower affinity than the entire binding site.

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

[0024] The term "diabody" refers to a small antibody fragment prepared by constructing an sFv fragment (see preceding paragraph) with a short linker (about 5-10 residues) between the VH and VL domains such that inter-chain pairing, rather than intra-chain pairing of the V domains, is achieved, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. A bispecific diabody is a heterodimer of two "crossover" sFv fragments in which the VH and VL domains of two antibodies are present on different polypeptide chains. See, e.g., EP 404,097, WO 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448, 1993.

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

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

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

[0028] As used herein, the term "biparatopic" refers to a bispecific antibody in which a first antigen-binding portion and a second antigen-binding portion bind to different epitopes on the same antigen.

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

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

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

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

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

[0034] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to the native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0035] The term "fusion polypeptide" includes, for example, an anti-Tie2 antibody of the present invention fused to an immunoglobulin Fc region. The Fc region can include, for example, the CH3 domain of an immunoglobulin, which can be naturally occurring or modified in some way. Such Fc fusion polypeptides can exhibit a longer half-life in vivo than their unfused counterparts. Also, fusion to the Fc region enables dimerization / multimerization of the fusion polypeptide. As contemplated, the Fc region can be a naturally occurring Fc region or can be modified, for example, to improve certain qualities such as therapeutic quality, circulation time, reduction of aggregation problems. In another embodiment, the fusion polypeptide contemplates an anti-Tie2 antibody fragment fused to a ligand such as Ang1 to enhance the agonist activity of the fusion polypeptide. In another embodiment, the fusion polypeptide contemplates an anti-Tie2 antibody fragment fused to a cytokine, for example, to induce other desired biology.

[0036] As used herein, a "hexamerizing antibody" is one in which the introduction of an E430G mutation in the Fc region promotes the natural process of antibody hexamer formation due to increased intermolecular Fc-Fc interactions upon binding to a membrane-bound antigen on the cell surface (Diebolder et al., Science. 2014, de Jong et al., PLoS Biol. 2016).

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

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

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

[0040] The term "variable" refers to the fact that certain segments of the variable domain have highly variable sequences between antibodies. The variable domain, or "V" domain, mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not uniformly distributed across the span of the variable domain. Instead, the V region consists of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extremely high variability called "hypervariable regions," each 9-12 amino acids in length. As used herein, the term "hypervariable region" or "CDR" refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions generally include, for example, amino acid residues from approximately (around about) residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in VL, and residues 26-35 (H1), 49-65 (H2), and 95-102 (H3) (in one embodiment, H1 is approximately residues 31-35) in VH (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), and / or amino acid residues from "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in VL, and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in VH) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The variable domains of native heavy and light chains each contain four FRs, which mainly adopt a beta-sheet conformation and are connected by three hypervariable regions that form loops connecting the beta-sheet structures and, in some cases, forming part of them.The hypervariable regions of each chain are held together in close proximity by the FRs and, together with the hypervariable regions from other chains, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Thus, the CDR and FR sequences are generally within the following sequences in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. The constant domains do not directly participate in the binding of the antibody to the antigen but exhibit various effector functions such as the antibody's involvement in antibody-dependent cell cytotoxicity (ADCC).

[0041] The terms "residue numbering such as Kabat numbering", "Kabat amino acid residue", or "amino acid position numbering such as Kabat numbering", and variations thereof, refer to the numbering system used in the compilation of the heavy chain variable domain or light chain variable domain of antibodies in the above Kabat et al. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to deletions or insertions in the FR or CDR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues after residue 82 of the heavy chain FR (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat residue numbering can be determined for a given antibody by alignment in the region of homology of the antibody's sequence with the "standard" Kabat numbering sequence.

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

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

[0044] When used to describe the various antibodies disclosed herein, the term "isolated antibody" means an antibody that has been identified and separated and / or recovered from the cells or cell culture in which it was expressed. Contaminant components of its natural environment are substances that typically interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined, for example, by electrophoretic (e.g., SDS-PAGE, isoelectric focusing electrophoresis (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) approaches. For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007). In certain embodiments, the antibody will be purified to the extent that it is (1) sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer or (2) homogeneous by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver staining. Isolated antibodies include antibodies in recombinant cells in situ since at least one component of the natural environment of the polypeptide is absent. However, typically, an isolated polypeptide will be prepared by at least one purification step.

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

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

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

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

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

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

[0051] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cells and progeny derived therefrom regardless of the number of passages. The progeny may not be identical to the parental cell in nucleic acid content and may contain mutations. Mutant progeny having the same function or biological activity as that selected or screened in the originally transformed cell are included herein.

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

[0053] A protein containing an antibody is said to be "stable" if it retains its native conformation and biological activity essentially intact. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones (1993) Adv. Drug Delivery Rev. 10:29-90. An antibody variant having "improved stability" refers to an antibody variant that is more stable compared to a starting reference antibody. An antibody variant having improved stability is a variant of a reference (wild-type) antibody in which specific amino acid residues have been modified for the purpose of improving the physical stability, and / or chemical stability, and / or biological activity of the native antibody and / or reducing immunogenicity.

[0054] In certain embodiments, the anti-Tie2 antibody can be used as a therapeutic agent that targets and interferes with a disease or condition in which Tie2 activity is involved. The antibody may also be subjected to other biological activity assays, for example, to evaluate its effectiveness as a therapeutic agent. Such assays are known in the art and depend on the target antigen and the intended use of the antibody. Examples include HUVEC inhibition assays, tumor cell growth inhibition assays (such as those described in WO89 / 06692), antibody-dependent cell cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (U.S. Patent No. 5,500,362), and agonist activity or hematopoietic assays (see WO95 / 27062).

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

[0056] As used herein, the term "quadruple specificity" refers to a type of antibody that possesses four antigen recognition and binding sites, some of which can bind to Tie2. In another case, at least one binding arm specifically binds to Tie2, and the other binding sites can bind to either Tie2 or another antigen of interest (enumerated in "bispecific antibodies"). In one aspect, such quadruple-specific antibodies include 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 assembled into a quadruple-specific antibody such that two antibody-binding fragments bind to Tie2 and the other two antibody-binding fragments bind to another antigen such as VEGF.

[0057] As used herein, the term "quintuple specificity" refers to a type of antibody that possesses five antigen recognition and binding sites, some of which can bind to Tie2. In another case, at least one binding arm specifically binds to Tie2, and the other binding sites can bind to either Tie2 or another antigen of interest (enumerated in "bispecific antibodies"). In one aspect, such quintuple-specific antibodies include 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 assembled into a quintuple-specific antibody such that at least one antibody-binding fragment binds to Tie2 and the other antibody-binding fragments bind to another antigen such as VEGF.

[0058] As used herein, the term "hexavalent specificity" refers to a type of antibody that possesses six antigen recognition and binding sites, some of which are capable of binding to Tie2. In other cases, at least one binding arm specifically binds to Tie2, and the other binding sites can bind to either Tie2 or another antigen of interest (enumerated in "bispecific antibodies"). In one aspect, such hexavalent specific antibodies include 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 assembled into a hexavalent specific antibody such that at least one antibody binding fragment binds to Tie2 and the other antibody binding fragments bind to another antigen, such as VEGF.

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

[0060] "Tie2 dysregulation diseases" are any conditions that would benefit from treatment with the anti-Tie2 antibodies of the present invention. Non-limiting examples of diseases treated herein include, but are not limited to, any disease or disorder resulting from an imbalance or disruption in the interaction of Ang-1, Ang-2, Ang-3, or Ang-4 with Tie2. Non-limiting examples can include, for example, infectious diseases, acute respiratory distress syndrome (ARDS), ischemic injury, eye disorders, radiation injury, cancer, systemic sclerosis, traumatic brain injury, radiation injury, wound healing, myocardial infarction, blood-brain barrier injury (i.e., Alzheimer's disease or other neurodegenerative diseases), neuroinflammation, cerebral cavernous malformations, 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 can include ischemic injuries such as, for example, diabetic nephropathy, acute kidney injury, chronic kidney disease, kidney or other organ transplantation, critical limb ischemia, traumatic brain injury or stroke. In another embodiment, Tie2 dysregulation eye disorders can include, for example, diabetic retinopathy, diabetic macular edema (DME), proliferative diabetic retinopathy (PDR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP), or glaucoma.

[0061] As used herein, "administering" means a method of administering a dosage of a therapeutic agent (e.g., an 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, e.g., a pharmaceutical composition comprising the anti-Tie2 antibody of the present invention) to a subject in need thereof. The compositions utilized in the methods described herein can be administered, for example, intravitreally (e.g., by intravitreal injection), ocularly (e.g., by ocular injection), intraocularly (e.g., by intraocular injection), subcutaneously, or intravenously. The compositions utilized 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 being administered, and the severity of the condition, disease, or disorder being treated).

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

[0063] As used herein, "co-formulating" means two or more separate therapeutic agents (e.g., an anti-Tie2 antibody of the present invention and an anti-VEGF antibody therapeutic agent or a recombinant VEGF fusion protein therapeutic agent) or compositions (e.g., a pharmaceutical composition of an anti-Tie2 antibody of the present invention and an anti-VEGF antibody composition or a recombinant VEGF fusion protein composition) that are combined as a single formulation for administration to a subject in need thereof.

[0064] The "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic 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 certain embodiments, the individual or subject is a human. The "subject" may be a "patient".

[0065] As used herein, "treatment" (and "treating" or "to treat") refers to a clinical intervention in an attempt to modify the natural course of a subject being treated, which can be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of recurrence of a disease or disorder, alleviation of symptoms, reduction of any direct or indirect pathological consequences of a disease or disorder, decrease in the rate of disease progression, improvement or alleviation of the state of a disease or disorder, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of a disease or disorder or to slow the progression of a disease or disorder.

[0066] As used herein, the expressions "cell", "cell line", and "cell culture" are used interchangeably and all such names include progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or accidental mutations. Mutant progeny having the same function or biological activity as screened in the originally transformed cells are included. Where distinct designations are intended, it will be apparent from the context.

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

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

[0069] As used herein, "VEGF extracellular trap protein" or "VEGF-trap" is known by the alternative name aflibercept (Eylea®, Regeneron - Bayer HealthCare, Tarrytown, NY, US). It consists of ligand-binding elements obtained 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), essentially preventing the VEGF-A and PlGF ligands from binding to and activating cell receptors.

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

[0071] As used herein, a "reference antibody" refers to an antibody or fragment thereof whose antigen-binding sequence functions as a template sequence for which diversification is performed according to the criteria described herein. The antigen-binding sequence generally includes the antibody variable region, which includes framework regions and at least one CDR.

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

[0073] The techniques and procedures described or referenced in this specification are generally well understood. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)), the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)), Oligonucleotide Synthesis (M.J. Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press, Animal Cell Culture (R.I. Freshney), ed., 1987), Introduction to Cell and Tissue Culture (J.P. Mather and P.E.R. Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993 - 8) J.Wiley and Sons, Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.), Gene Transfer Vectors for Mammalian Cells (J.M.Conventional methodologies commonly used by those skilled in the art are employed, such as the widely used methodologies described in Miller and Μ.P.Calos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (J.E.Coligan et al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (C.A.Janeway and P.Travers, 1997), Antibodies (P.Finch, 1997), Antibodies: A Practical Approach (D.Catty., ed., IRL Press, 1988 - 1989), Monoclonal Antibodies: A Practical Approach (P.Shepherd and C.Dean, eds., Oxford University Press, 2000), Using Antibodies: A Laboratory Manual (E.Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M.Zanetti and J.D.Capra, eds,, Harwood Academic Publishers, 1995).

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

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

[0076] In one embodiment, the KD is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed with the Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured by equilibrating the Fab with a minimum concentration of ( 125 I) labeled antigen and then capturing the bound antigen on a plate coated with an anti-Fab antibody (e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the conditions for the assay, a MICROTITER™ multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of the capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23°C). In non-adsorptive plates (Nunc #269620), 100 pM or 26 pM of 125I] Mix the antigen with serial dilutions of the Fab of interest (see Presta et al., Cancer Res. 57:4593 - 4599 (1997)). Then incubate the Fab of interest overnight, although the incubation may be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, transfer the mixture to a capture plate and incubate at room temperature (e.g., for 1 hour). Then remove the solution and wash the plate 8 times with 0.1% polysorbate 20 (TWEEN - 20 (trademark)) in PBS. Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT - 20 (trademark), Packard) and count the plate on a TOPCOUNT (trademark) gamma counter (Packard) for 10 minutes. The concentration of each Fab that gives less than 20% of the maximum binding is selected for use in the competitive binding assay.

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

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

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

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

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

[0082] In some cases, the anti-Tie2 antibodies of the invention provided herein are Fabs.

[0083] In some instances, the Fab binds to Tie2 and comprises at least 1, 2, 3, 4, 5, or 6 CDRs selected from (a) CDR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 1-40, (b) CDR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 41-80, (c) CDR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 81-120, (d) CDR-L1 comprising any one of the amino acid sequences of SEQ ID NOs: 121-160, (e) CDR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 161-200, and (f) CDR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 201-240, or a combination of one or more of the above CDRs and one or more variants thereof having at least about 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 1-240.

[0084] In some instances, the Fab binds to Tie2 and comprises HCDR1-3 and LCDR1-3 comprising the amino acid sequences of SEQ ID NOs. as the following antibody clones. TIFF2025081502000004.tif41166TIFF2025081502000005.tif248166

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

[0086] In some cases, the Fab binds to Tie2 and comprises a VH domain and a VL domain that comprise amino acid sequences in the following combinations. TIFF2025081502000006.tif128166

[0087] In certain embodiments, the antibodies provided herein are chimeric antibodies. For certain chimeric antibodies, see, e.g., U.S. Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody 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 further embodiments, the chimeric antibody is a "class switch" antibody in which the class or subclass has been changed from that of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0088] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the CDRs, e.g., CDR (or a portion thereof), are derived from a non-human antibody and the FRs (or a portion thereof) are derived from human antibody sequences. The humanized antibody will optionally also comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived) to, for example, restore or improve antibody specificity or affinity.

[0089] Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat’l Acad. Sci. USA 86:10029-10033 (1989), U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409, Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting), Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”), Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).

[0090] Human framework regions that can be used for humanization include framework regions selected using the "best fit" method (see, for example, Sims et.al., J.Immunol. 151:2296 (1993)), framework regions derived from the consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions (see, for example, Carter et.al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992), and Presta et.al. J. Immunol., 151:2623 (1993)), human mature (somatic mutated) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions obtained from screening of FR libraries (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997), and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)), but are not limited thereto.

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

[0092] Human antibodies can be prepared by administering an immunogen to a transgenic animal modified to produce intact human antibodies or intact antibodies having human variable regions in response to an antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or is present extrachromosomally, or is randomly integrated into the chromosomes of the animal. In such transgenic animals, the endogenous immunoglobulin locus is generally inactivated. For an overview of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUANTIBODIES™ technology; U.S. Patent No. 7,041,870, which describes K-MOUSE™ technology; U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE™ technology; and U.S. Patent Nos. 9,809,642 and 9,380,769, which describe OmniChicken™ technology). The human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining them with different human constant regions.

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

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

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

[0096] In certain phage display methods, the repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR), randomly recombined within a phage library, and then screened for antigen-binding phages as described in Winter et.al., Ann.Rev.Immunol., 12:433-455 (1994). Phages typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide antibodies with high affinity for the immunogen without the need to construct hybridomas. Alternatively, natural repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self antigens without immunization, as described in Griffiths et.al., EMBO J, 12:725-734 (1993). Finally, natural libraries can also be synthetically generated by cloning germline V gene segments that have not been rearranged from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro. Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, as well as U.S. Patent Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0097] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

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

[0099] In some cases, the multispecific anti-Tie2 antibodies of the invention are bispecific antibodies in which one arm binds to Tie2 and the other arm binds to VEGF. In other embodiments, such bispecific anti-Tie2 antibodies of the invention also have an Fc mutation that abrogates ADCC and / or CDC as described herein. Such bispecific antibodies of the invention comprise heavy and light chains that contain amino acid sequences in the following combinations. TIFF2025081502000007.tif53166

[0100] In other cases, the multispecific anti-Tie2 antibodies of the invention are fused to a VEGF extracellular trap protein. In yet other examples, such multispecific anti-Tie2 antibody fusion proteins also have an Fc mutation that abrogates ADCC and / or CDC as described herein. Non-limiting examples of such multispecific antibody fusion proteins contain the following amino acid sequences. TIFF2025081502000008.tif23166

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

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

[0103] Antibodies or fragments herein also include "doubly acting FAb" or "DAF" that contain an antigen-binding site that binds to Tie2 and to a different antigen (see, e.g., US2008 / 0069820).

[0104] In certain embodiments, amino acid sequence variants of the antibodies provided herein (e.g., antibody variants that include one or more amino acid residue modifications) are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions of residues within the amino acid sequence of the antibody and / or insertions of residues and / or substitutions of residues. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

[0105] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include the CDRs and FRs. Conservative substitutions are contemplated and are well known in the art.

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

[0107] Non-conservative substitutions involve exchanging one member of one of these classes for another.

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

[0109] Modifications (e.g., substitutions) can be made, for example, in the CDRs to improve antibody affinity. Such modifications can be made at CDR “hot spots,” i.e., residues encoded by codons that mutate at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol Biol. 207:179-196 (2008)), and / or at residues that contact the antigen, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and rescreening of a secondary library is described, for example, in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, N.J., (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, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants having the desired affinity. Another method for introducing diversity involves a CDR-directed approach in which some CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

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

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

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

[0113] Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom et al., Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986), and Hellstrom et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985), U.S. Patent No. 5,821,337, and Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for flow cytometry (CellTechnology, Inc., Mountain View, Calif.), and CYTOTOX96™ Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo, e.g., in an animal model such as those disclosed in Clynes et al., Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may be performed to confirm that the antibody is unable to bind to C1q and, therefore, lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To evaluate complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al. J. Immunol Methods 202:163 (1996), Cragg et al. Blood 101:1045-1052 (2003), and Cragg et al. Blood, 103:2738-2743 (2004)).FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, e.g., Petkova et al., Int’l. Immunol. 18(12):1759-1769 (2006)).

[0114] The antibodies of the present invention can be engineered to have reduced effector function (e.g., reduced complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell phagocytosis (ADCP)) due to reduced affinity for human FcyRIIIA and / or FcγRIIA and / or FcγRI and / or Clq binding. In some cases, such reduced effector function is achieved by amino acid substitution of one or more of the following Fc region residues: N297, L234, L235, D265, and P329, according to EU numbering such as Kabat numbering. See U.S. 6,737,056, U.S. 7,332,581, and WO2012 / 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 an N297A or N297G substitution mutation. In some embodiments, the substitution mutation includes the so-called "DANA" Fc mutant in which residues D265 and N297 are substituted with alanine (U.S. Patent No. 7,332,581). In some embodiments, the substitution mutation includes the so-called "DANG" mutation having residues substituted as D265A and N297G. In some embodiments, the substitution mutation includes the "LALA" Fc mutant in which residues L234 and L235 are substituted with alanine (see Lund, J., et al., (1992) Mol. Immunol., 29, 53-59, and Tamm, A. and Schmidt, R.E. (1997) Int. Rev. Immunol., 16, 57-85). In other embodiments, the substitution mutation includes the "LALA-PG" Fc mutant in which residues L234 and L235 are substituted with alanine and P329 is substituted with glycine (see Brunker, P., et al. (2016) Mol. Cancer Ther).

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

[0116] In some embodiments, the substitution mutations are made within the Fc region that results in the hexamerized antibodies described herein. In some embodiments, such substitution mutation is E430G. In other embodiments, the E430G mutation may be combined with any of the reduced effector function mutations described above to block the CDC function and / or the ADCC effector function. In one embodiment, the anti-Tie2 antibody of the present invention comprises a heavy chain and a light chain comprising an amino acid sequence having the E430G mutation and the L234A, L235A, P329G mutations in the following combination. TIFF2025081502000009.tif16166

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

[0118] The present invention also provides an immunoconjugate comprising an anti-Tie2 antibody of the present specification conjugated to one or more cytotoxic agents such as a chemotherapeutic agent or chemotherapeutic drug, a growth inhibitor, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope.

[0119] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) conjugated to one or more drugs including, but not limited to, maytansinoids (see U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent No. 0425235B1), auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483, 5,780,588, and 7,498,298), dolastatin, calicheamicin or its derivatives (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296, Hinman et al., Cancer Res. 53:3336 - 3342 (1993), and Lode et al., Cancer Res. 58:2925 - 2928 (1998)), anthracyclines such as daunomycin or doxorubicin (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., Bioconj.Chem. 16:717 - 721 (2005), Nagy et al.Proc.Natl.Acad.Sci.USA 97:829 - 834 (2000), Dubowchik et al., Bioorg.&Med.Chem.Letters 12:1529 - 1532 (2002), King et al., J.Med.Chem. 45:4336 - 4343 (2002), and U.S. Patent No. 6,630,579), methotrexate, vindesine, taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel and ortataxel, trichothecene, and CC1065.

[0120] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichothecene.

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

[0122] In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to a non-cytotoxic agent, such as an artemisinin, e.g., artusenate, or a cannabinoid, or naltrexone, or aspirin, or a metabolic agent such as a statin, or metformin, doxocycline, and an anthelmintic.

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

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

[0125] In one aspect, the anti-Tie2 antibody of the invention is useful for detecting the presence of Tie2 in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissues. In certain embodiments, such tissues include normal tissues and / or cancer tissues that express Tie2 at high levels compared to other tissues.

[0126] In one embodiment, an anti-Tie2 antibody for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of Tie2 in a biological sample is provided. In certain embodiments, the method includes contacting a biological sample with an anti-Tie2 antibody described herein under conditions that permit binding of the anti-Tie2 antibody 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 methods. In one embodiment, the anti-Tie2 antibody is used to select a subject for therapy with the anti-Tie2 antibody, e.g., Tie2 is a biomarker for patient selection.

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

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

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

[0130] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors that include fungal and yeast strains that result in the production of antibodies with "humanized" glycosylation pathways and partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

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

[0132] 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, which describe PLANTIBODIES™ technology for producing antibodies in transgenic plants.

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

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

[0135] In one aspect, the antibodies of the invention are tested for their antigen-binding activity by known methods such as ELISA, Western blot, surface plasmon resonance assays (e.g., BIACORE™), etc.

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

[0137] In another aspect, a competition assay can be used to identify antibodies that compete with the antibodies as described herein with respect to binding to Tie2. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) that is bound by an antibody as described herein. Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.).

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

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

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

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

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

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

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

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

[0146] Pharmaceutical formulations The therapeutic formulations of the fusion polypeptides of the present invention, which comprise an anti-Tie2 antibody or antibody fragment or variant thereof of the present invention, or an immunoconjugate of the present invention, or combinations thereof with an anti-VEGF antibody or recombinant VEGF fusion protein as provided herein, can be prepared for storage as a lyophilized formulation or an aqueous solution by mixing a polypeptide having the desired purity with an optional "pharmaceutically acceptable" carrier, excipient, or stabilizer (all of which are referred to herein as "excipients"). For example, buffering agents, stabilizing agents, preservatives, isotonicity agents, nonionic surfactants, antioxidants, and various other additives. See, for example, Remington’s Pharmaceutical Sciences, 16 th edition, A. Osol, Ed. (1980). Such additives must be non-toxic to the recipient at the dosages and concentrations employed.

[0147] Therapeutic Methods and Compositions The fusion polypeptides of the present invention, which comprise an anti-Tie2 antibody and antibody fragment of the present invention, or an immunoconjugate of the present invention, or combinations thereof with an anti-VEGF antibody or recombinant VEGF fusion protein as provided herein, can be used in therapeutic methods for treating, preventing, and / or alleviating various diseases, including any Tie2 dysregulation disease as defined herein.

Examples

[0148] The following are examples of the methods and compositions of the present invention. It is understood that various other embodiments can be practiced in view of the general description above.

[0149] Example 1: Immunization of Transgenic Chickens to Isolate a Fully Human Anti-Tie2 Antibody The extracellular domains (ECDs) of the human and chicken Tie2 orthologs are only 62% identical, in contrast to the 90% identity observed between the mouse and human Tie2 orthologs. Therefore, using chickens to generate Tie2 antibodies will greatly expand the range of epitopes accessible to the antibodies.

[0150] Antibodies were generated by immunizing OmniChicken® (Crystal Biosciences, Inc, Emeryville, CA), a unique transgenic chicken, with recombinantly produced and purified human Tie2 ECD. By deletion of the endogenous immunoglobulin-encoding genes from the chicken genome and replacement of them with human immunoglobulin-encoding genes, OmniChicken can be "humanized" to produce fully human antibodies (see US Patent Nos. 9,809,642 and 9,380,769).

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

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

[0153] Cross-reactivity to mouse Tie2 by binding to recombinant mouse Tie2 ECD protein was used as the primary screen. These binding studies demonstrated that approximately 90% of the anti-Tie2 antibodies generated were cross-reactive between mouse and human Tie2 proteins, suggesting that they may be suitable for in vivo testing in established animal models.

[0154] Next, screening was performed for the recognition and binding of full-length human Tie2 expressed in its native conformation on the plasma membrane of live cells. For these experiments, human umbilical vein endothelial cells (HUVECs) were obtained from donors. Using flow cytometry, it was also demonstrated [data not shown] that approximately 70% of the antibodies that bind to the human Tie2 ECD protein bind to native Tie2 expressed on the cells.

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

[0156] In this screening method, HUVEC cells were plated in 96-well plates, serum-starved 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 a 20-minute incubation, the cells were lysed and the lysates were analyzed by a specific AlphaLISA assay for the presence of pERK and total ERK, as well as pAkt and total Akt. To be designated as an "active agonist anti-Tie2 antibody", the antibody clone had to induce Tie2 signaling such that it was determined by an increase in the levels of intracellular pAkt and pERK (normalized to total Akt and total ERK, respectively). Antibodies that were able to induce an increase in the levels of pERK and pAkt were ranked based on their activity compared to the levels of pERK and pAkt induced by Ang1 treatment. Antibodies that could increase the pERK and pAkt levels by more than 75% compared to those seen after Ang1 treatment were considered comparable to Ang1 and carried forward for further testing.

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

[0158] Thus, the anti-Tie2 antibodies of the present invention were found to activate both proximal and distal Tie2 signaling events and were advanced for further study.

[0159] Example 4: Determination of Antibody Potency In Vitro To enable ranking of the anti-Tie2 antibodies of the present invention based on efficacy, an assay with high precision for determining antibody EC50 values was developed.

[0160] In this assay, HUVEC cells were starved for 3 hours and then treated with increasing concentrations of Ang1, anti-Tie2 antibody, or negative control hIgG1 for 20 minutes 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 analyzed as follows to determine the respective EC50 values: Ang1 = 0.54 nM, Ab #1 = 0.91 nM, Ab #2 = 0.48 nM, Ab #3 = 0.45 nM, and WT = 1.33 nM. See Figure 2.

[0161] Analysis of the EC50 values revealed that most antibodies showed potent Tie2 activation at sub-nanomolar concentrations comparable to the efficacy of Ang1 in the same assay. Collectively, in the functional assay, identified anti-Tie2 antibodies with the ability to induce Tie2 signaling up to levels comparable to those seen with Ang1 were utilized.

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

[0163] An intact confluent monolayer of HUVEC cells was cultured on a semi-permeable membrane to form an adhesion structure with tight junctions. Cells were treated with 100 ng / ml VEGF regardless of the presence of Ang1 or anti-Tie2 antibody. Permeability was assayed at different time points over 6 hours by measuring the amount of fluorescein-conjugated dextran that penetrated through the cell monolayer into the receiver well beneath the membrane. The leakage rate was measured over time as the number of fluorescein units accumulated in the receiving well. The difference in endothelial barrier leakage between PBS (negative control) and Ang1 (positive control) was determined. The ability of the anti-Tie2 antibody to reduce endothelial barrier leakage was normalized to the effect of Ang1 treatment in the same assay (% Ang1 activity). See Figure 4.

[0164] Statistical analysis revealed that the anti-Tie2 antibody significantly reduced VEGF-induced fluid leakage through the matrix of these primary endothelial cells, while the human IgG1 negative control did not. Thus, the anti-Tie2 antibody was able to stimulate downstream Tie2 signaling in the biochemical assay and also reduce leakage in an orthogonal in vitro physiological assay.

[0165] Example 6: Anti-Tie2 agonist antibody activates Tie2 in the presence of high levels of Ang2 Patients suffering from diabetic macular edema (DME) show a marked increase in systemic and intravitreal levels of Ang2 (Loukovaara et.al. 2013b, Regula et.al. 2017). Considering that high levels of Ang2 may interfere with anti-Tie2 antibody activity by sharing similar binding sites on Tie2, or whether 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 saturating concentrations of Ang2 were determined. Ang2 functions as a weak Tie2 agonist that can induce signaling in in vitro assays in the absence of Ang1, but at lower levels than can be achieved by Ang1 (Yuan et al. 2009). The in vitro experiments described below supported these findings.

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

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

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

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

[0170] When considered in conjunction with the above Tie2 pathway signaling results, the data characterizing the binding mechanism of the anti-Tie2 antibody of the present invention indicates a mechanism of action that is completely independent of angiopoietin levels.

[0171] Example 8: The anti-Tie2 agonist antibody is cross-reactive with Tie2 variants expressed by other animal species Genetically engineered cell lines overexpressing Tie2 orthologs of mouse, rat, rabbit, pig, and cynomolgus monkey were used to investigate the cross-reactivity profile of the anti-Tie2 antibody against Tie2 orthologs expressed by preclinical animal model species.

[0172] Briefly stated, primary human endothelial cells (HUVEC) as well as human embryonic kidney (HEK293) cells were transfected using expression plasmids containing various full-length Tie2 coding sequences. Stable cell populations were selected by treating the transfectants with puromycin antibiotic over a two-week period, and single cell clones were generated using fluorescence-activated cell sorting. When confirming the cell surface expression of these Tie2 variants, cell lines were used to determine the apparent 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 the present invention was determined. Cells were labeled with decreasing concentrations of anti-Tie2 antibodies #1, #2, and #3 (10, 5, 2.5, 1.25, 0.63, 0.31, and 0.16 ug / mL) or a non-specific hIgG1 antibody (10 ug / mL), and subsequently labeled with a rabbit anti-human IgG antibody conjugated to Alexa Fluor 488 fluorophore (20 ug / mL). EC50 values were generated using ForeCyt™ software. See Tables 1 and 2.

[0173]

Table 1

[0174]

Table 2

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

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

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

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

[0179] Example 10: Oxygen-Induced Retinopathy (OIR) Mouse Model In this study, 40 C57BL / 6J pups were housed in a hyperoxic chamber (75% O 2 ) for 5 days starting at postnatal day 7 (P7) (n = 10 per cage), resulting in vascular regression in the central part of the retina. CD-1 foster mothers were changed before and 2 - 3 days after entering the chamber. At P12, the pups were returned to room air, where abnormal angiogenesis was induced by relative hypoxia. Then, endotoxin-free 1×PBS vehicle, 10 mg / kg, HuIgG isotype control, or 10 mg / kg of anti-Tie2 clone #3 was administered intraperitoneally. At P17, all groups including naive OIR mice were euthanized. The eyes were removed and fixed in 4% paraformaldehyde for 1 hour.

[0180] The retina was dissected and incubated overnight in PBS with 1 mM CaCl with rhodamine-labeled lectin (1:100) from Bandeiraea simplicifolia (Griffonia simplicifolia) to visualize the vascular obliteration (VO) or neovascularization (NV) areas. The stained retina was mounted flat on slides and imaged on a Zeiss® AxioScan. Images were analyzed on Visiopharm® to determine the %VO or %NV of the whole retina. 2 As shown in Figure 8B, anti-Tie2 mAb, but not isotype control mAb or vehicle, was found to have a significant positive effect on the vascular obliteration area (i.e., avascular), but did not appear to reduce NV (Figure 8A). Promotion of the regrowth of a healthy vasculature could be beneficial to mitigate the detrimental consequences of chronic anti-VEGF therapies such as geographic atrophy or capillary dropout in patients with eye diseases. 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., J. Clin. Invest. 122, 4213-4217 (2012).

[0181]

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

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

[0184] As shown in Figures 9A and 9B, the anti-Tie2 antibody, but not the isotype control mAb, significantly inhibited the size of choroidal neovascularization lesions after laser injury. This result indicates that activation of Tie2 after administration of anti-Tie2 mAb may have significant clinical benefits for humans suffering from ophthalmic disorders such as AMD or diabetic retinopathy.

[0185] Example 12: Streptozotocin-induced (STZ) diabetic mouse model Administration of the agonist Tie2 antibody of the present invention may have utility for the treatment of diabetic retinopathy and other pathological conditions associated with diabetes such as nephropathy. To explore potential therapeutic advantages, the effects of the anti-Tie2 antibody of the present invention were determined on disease-related endpoints such as leakage from blood vessels in the eye, visual function as assessed by electroretinogram, production of cytokines such as IL-1b thought to be involved in human disease, and kidney function as measured by proteinuria in a streptozotocin-induced diabetic mouse model in which the mAb was administered via the intraperitoneal route.

[0186] The weights of 6- to 7-week-old C57BL / 6J mice can be measured, and their baseline blood glucose can be measured (Accu-Chek®, Roche). The mice can be injected intraperitoneally with STZ (Sigma-Aldrich, St. Louis, MO) at 55 mg / kg for 5 consecutive days. The age-matched controls can be injected with buffer only. Blood glucose can be measured again 1 week after the last STZ injection, and the mice are considered diabetic if their non-fasting blood glucose is higher than 17 mM (300 mg / dL). Next, the STZ-treated diabetic C57BL / 6J mice can be injected intravitreally (IVT) with an appropriate amount of the anti-Tie2 antibody, control antibody, vehicle, or comparative antibody of the present invention, such as an anti-VEGF antibody or recombinant VEGF fusion protein, at least 8 weeks after STZ administration.

[0187] The electroretinogram (ERG) evaluates the overall function of retinal cells using a UTAS-E visual electrodiagnostic test system. After overnight dark adaptation, the treated mice are anesthetized by subcutaneous injection of BW of a cocktail containing ketamine and xylazine in sterile water. One eye is proptosed, and the pupil is dilated with tropicamide and phenylephrine HCL. The eye is moistened with Genteal® eye drops, and the deep body temperature is maintained using a heating pad. The ERG can be recorded using an ultra-low impedance silver / nylon DTL plus electrode. The needle electrodes can be placed at the center of the forehead and the base of the tail. A gold contact lens electrode is used to record the ERG response. The stimulus can be composed, for example, of electronically flickering at 50 milliseconds in log steps. The response is recorded from stimuli ranging from below threshold to saturation. The analysis can include the maximum amplitude and threshold of the a-wave and b-wave.

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

[0189] Retinal vascular permeability can be measured as follows. Anesthetize the mouse and inject Evans blue dye dissolved in physiological saline into the tail vein. Two hours after the tail vein injection, the mouse can be anesthetized with ketamine and xylazine and perfused through the left ventricle using physiological saline. After perfusion, dissect the retina, measure the weight, place it in formamide at 70 °C for 18 hours, and extract the Evans blue dye. The next day, centrifuge the retina for 45 minutes and remove it from the formamide. Measure the extravascular leakage of Evans blue using a plate reader at A620. Use a standard curve to convert to units of ng Evans blue / wet tissue weight.

[0190] Tissue collection for the targeted transcriptome can be performed as follows. Sacrifice the mouse under anesthesia and remove both eyes. Then, dissect the retina, place it in RNAlater, process it, and analyze it by qRT-PCR.

[0191] Example 13: Bispecific Biologic Construct Simultaneously affecting both the Tie2 and VEGFR - induced signaling pathways may have enhanced benefits compared to modulating either pathway alone. To achieve this, bispecific constructs designed to affect both Tie2 and VEGFR were designed, expressed, and tested for activity. Figure 10A shows an exemplary schematic of a bispecific antibody of the invention having both a Tie2 - binding variable domain and a VEGF - binding variable domain. Antibody clone #54 was designed and generated using standard cloning techniques and has the sequences of SEQ ID NOs: 282 and 283. A bispecific construct consisting of VEGFR R1D2 and R3D3 (VEGF trap protein) and the Tie2 - binding domain was also generated (antibody clone #55), and this construct has the sequence of SEQ ID NO: 284. As described in Example 3 above, subsequently, an AlphaLISA™ screening platform designed to detect the intracellular levels of antibody - induced pERK and pAkt was used to evaluate Tie2 agonism for two exemplary aTie2 / VEGF bispecific constructs (antibody clones #54 and #55) in HUVEC cells, each using 20 nM.

[0192] The aTie2 / VEGF bispecific constructs tested were found to have the ability to induce Tie2 signaling in vitro (Figure 11). These bispecific molecules may make it possible to administer a single drug to patients with eye diseases and provide substantially higher efficacy than monotherapy with VEGF inhibition or Tie2 activation alone. Promoting the regrowth of a healthy vasculature provided by the Tie2 agonist component of the bispecific molecule may also be beneficial in mitigating potential adverse effects of the anti - VEGF component of the molecule (i.e., geographic atrophy or capillary dropout in patients with eye diseases).

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

[0194] As described in Example 3, tetravalent antibody clones #51 and #52 were evaluated for their ability to induce signaling through measurement of pAkt. As shown in Figure 14, both clones #51 and #52 demonstrate improved ability to activate Tie2 signaling in HUVEC cells compared to the bivalent anti-Tie2 construct (aTie2) or compared to angiopoietin 1 (Ang1). Thus, anti-Tie2 variants having additional Tie2 binding portions may provide a means to induce higher-order oligomerization of Tie2 on the cell surface and potentially provide a therapeutic benefit in situations where the level of agonism mediated by bivalent anti-Tie2 antibodies or the natural agonist ligand Ang1 is not appropriate to restore vascular homeostasis in diseased tissue.

[0195] Example 15: Anti-Tie2 Antibodies with Fc Mutations that Promote the Ability to Hexamerize on the Cell Surface Antibodies containing mutations specific to the Fc domain of the heavy chain (i.e., E430G) have been demonstrated to enhance their ability to form hexamers on the cell surface (M. Overdijk, Mol Cancer Ther 2020;19:2126-38). Anti-Tie2 antibodies containing an Fc domain with the E430G mutation were synthesized and compared to the same anti-Tie2 antibodies with a comparable Fc containing the native glutamic acid (E) at position 430. See Figure 13 for a schematic of such hexamerizing anti-Tie2 antibodies.

[0196] As described in Example 3, Tie2 agonism was evaluated using pAKT / pERK Western blot. As shown in Figure 14, the anti-Tie2 E430G construct (antibody clone #50) demonstrated an improved ability to induce Tie2 signaling in HUVEC cells compared to the E430 construct. Thus, the B12 variant with mutations that increase the likelihood of hexamerization at the cell surface could represent a means to induce higher-order oligomerization of Tie2 on the cell surface and potentially provide a therapeutic benefit in situations where the level of agonism mediated by anti-Tie2 antibodies or the natural agonist ligand Ang1 is not appropriate to restore vascular homeostasis in diseased tissue.

[0197] Example 16: Determination of Fab Binding Affinity of Anti-Tie2 Antibodies by SPR BIACORE (trademark) (BIAcore, Inc., Piscataway, N.J.) was performed at 25°C with human, rat or mouse Tie2 ECD antigen immobilized on a carboxymethylated dextran biosensor chip (CM5) at approximately 10 response units (RU). 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 μM) with 10 mM sodium acetate at pH 4.8 and then injected at a flow rate of 5 μl / min to obtain approximately 10 RU of coupled protein. After injecting each Tie2 ECD, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of the Fab of antibody clone #3 (0.78 nM to 500 nM) were injected at a flow rate of approximately 25 μl / min at 25°C into PBS containing 0.05% polysorbate 20 (TWEEN (trademark)-20) surfactant (PBST). The association rate (kon) and dissociation rate (koff) were calculated using a simple 1:1 Langmuir binding model (BIACORE (trademark) evaluation software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) was calculated as the ratio of koff / kon. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999).

[0198] The following table shows the Fab binding affinity of antibody clone #3 for each Tie2 ECD antigen tested. TIFF2025081502000012.tif26166

[0199] The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, but the description and examples of the invention should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are hereby expressly incorporated by reference in their entirety.

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

Claims

1. 1. An isolated anti-Tie2 antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions (CDRs H1-3) and three light chain CDRs (CDRs L1-3), wherein CDR H1 comprises the amino acid sequence of SEQ ID NO:1, CDR H2 comprises the amino acid sequence of SEQ ID NO:41, CDR H3 comprises the amino acid sequence of SEQ ID NO:81, CDR L1 comprises the amino acid sequence of SEQ ID NO:121, CDR L2 comprises the amino acid sequence of SEQ ID NO:161, and CDR L3 comprises the amino acid sequence of SEQ ID NO:

201.

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 of claim 1 which is an allosteric activator of Tie2.

4. The antibody of claim 1 which is a non-ligand competitive binder of Tie2.

5. The antibody of claim 1 , which is cross-reactive with human, mouse, rat, rabbit and monkey Tie2.

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

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

8. The antibody fragment is Fab, Fab'-SH, Fv, scFv, or (Fab') ２ The antibody of claim 7 which is a fragment.

9. The antibody of claim 1, which possesses reduced effector function.

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

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

12. 12. The antibody of claim 11, comprising the substitution mutation at residue N297A or N297G.

13. The antibody of claim 11, comprising the substitution mutations at residues L234A, L235A, and P329G.

14. The antibody of claim 11, comprising the substitution mutations at residues D265A and N297G.

15. An isolated nucleic acid encoding the antibody of claim 1.

16. A vector comprising the isolated nucleic acid of claim 15.

17. A host cell comprising the vector of claim 16.

18. A method for producing the antibody of claim 1, comprising: Culturing the host cell of claim 17 in a culture medium; Isolating the resulting antibody. A method comprising:

19. An immunoconjugate or fusion polypeptide comprising the antibody of claim 1.

20. 20. A pharmaceutical composition comprising the antibody of claim 1 or the immunoconjugate or fusion polypeptide of claim 19.

21. 21. The pharmaceutical composition of claim 20 for treating a Tie2 dysregulated disease.

22. 22. The pharmaceutical composition of claim 21, wherein the Tie2 dysregulated disease comprises infectious disease, acute respiratory distress syndrome (ARDS), ischemic injury, eye damage, radiation injury, cancer, systemic sclerosis, traumatic brain injury, neuroinflammation, radiation injury, wound healing, myocardial infarction, blood-brain barrier damage, cerebral cavernous malformation, Duchenne muscular dystrophy (DMD) or Clarkson's disease.

23. 23. The pharmaceutical composition of claim 22, wherein the Tie2 dysregulated infection comprises sepsis, dengue virus infection, tuberculosis, or influenza.

24. 23. The pharmaceutical composition of claim 22, wherein the Tie2 dysregulated ischemic injury comprises diabetic nephropathy, acute kidney injury, chronic kidney disease, organ transplantation, critical limb ischemia, traumatic brain injury or stroke.

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

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

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