Antibodies to betacellulin, their fragments and multispecific binding molecules

Abstract

FIELD: biotechnology.SUBSTANCE: bispecific binding molecule that binds to betacellulin (BTC) and vascular endothelial growth factor (VEGF), as well as to a composition containing it. Also a polynucleotide is disclosed comprising a nucleotide sequence encoding said bispecific binding molecule.EFFECT: treating a patient with an ophthalmological disorder.11 cl, 23 dwg, 18 tbl, 16 ex

Description

CROSS-REFERENCE TO RELATED APPLICATION AND INCLUSION OF SEQUENCE LISTING

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 052,789, filed July 16, 2020, and U.S. Provisional Patent Application No. 63 / 156,709, filed March 4, 2021, which are incorporated herein by reference in their entireties. The sequence listing contained in the file named "PAT058888-WO-PCT SQL_ST25", which is 204,786 bytes (measured on the MS-Windows operating system) and was generated on June 25, 2021, is filed with this document and is incorporated herein by reference. Field of the invention

[0002] The present invention relates to antibodies or antigen-binding fragments thereof, methods for producing them, pharmaceutical compositions containing them, and methods for using them.Background of the invention

[0003] Betacellulin (BTC), a member of the epidermal growth factor (EGF) family, was originally isolated from the conditioned medium of a mouse pancreatic tumor B cell line (Shinget al., Science, 259, 1604-1607, 1993). BTC is a ligand for the ErbB family of receptor tyrosine kinases and mainly activates ErbB1 and ErbB4 homodimers, triggering anti-apoptotic and pre-proliferative signaling pathways such as the Ras / MAPK and PL3K / AKT pathways.

[0004] BTC is initially expressed as a single-span transmembrane protein and is sequentially cleaved (activated) by members of the MMP family to a secreted protein with a molecular mass of 9 kDa. Cleavage of membrane-anchored forms of BTC to release the secreted form occurs predominantly by ADAM-10 (a disintegrin and metalloprotease-10) (Sahin et al., J. Cell Biol. 164, 769–779, 2004; Sahin and Blobel, FEBS Lett 581, 41–44, 2007; and Sanderson et al., J. Biol. Chem. 280, 1826–1837, 2005). Mature, secreted human BTC is a 32-kDa glycoprotein consisting of 80 amino acid residues (Asp1-Tyr80, residues 32-111 of the 178 residue membrane-anchored precursor protein (pro-BTC), described as NP_001720.1 or SEQ ID NO: 156) formed by cleavage of pro-BTC. The carboxyl-terminal 50-residue region of BTC (Arg31-Tyr80) contains a conserved consensus sequence of EGF family proteins.

[0005] Strong BTC mRNA expression has been detected in a number of tissues, including pancreas, liver, kidney, and small intestine, in addition to somewhat lower expression in heart, lung, liver, skeletal muscle, kidney, prostate, testis, ovary, and colon (Sasada et al., Biochem. Biophys. Res. Commun. 190, 1173–1179, 1993; Sasada and Igarashi, Nihon. Rinsho. 51, 3308–3317, 1993; and Seno et al., Growth Factors 13, 181–191, 1996). BTC knockout mice are viable and fertile without exhibiting an obvious phenotype.

[0006] High expression of BTC mRNA suggests that BTC may have a physiological role in pancreatic development and function. BTC levels were found to be elevated 7.5-fold in 9 out of 10 pancreatic cancer cases compared with normal expression levels in the pancreas (Yokoyama et al., 1995). In the pancreas, BTC expression was localized in islet cell populations closely associated with insulin-producing B cells (Miyagawa et al., Endocr. J. 46, 755-764, 1999). BTC may regulate pancreatic islet physiology and may induce fetal pancreatic cell proliferation and stimulate the conversion of non-β-cell cells into insulin-producing β-like cells. Furthermore, BTC overexpression has been reported in endometrial adenocarcinomas (Srinivasan et al., 1999); hepatocellular carcinomas (Moon et al., 2006); head and neck squamous cell carcinomas (O-charoenrat et al., 2000); and gastric carcinomas (Jemal et al., 2011).

[0007] In mammalian eyes, BTC protein is synthesized by retinal pigment epithelium (RPE), endothelial cells, and Müller cells (Anand-Apteet al.,PLoS One5, e13444, 2010) and localized in the outer blood-retinal barrier.

[0008] Although the general role of BTC in vascular endothelial function has been studied, its specific role in the retina is currently unclear. Initial reports of BTC's proliferative effect on RPE cells (Shinget al., 1993) and its proangiogenic functions suggested that it may play a role in the development of proliferative diabetic retinopathy (PDR). Although diabetic mice do not exhibit PDR, they exhibit increased retinal vascular permeability (Poulakiet al., J. Clin. Invest. 109, 805-815, 2002). Furthermore, in a mouse model of diabetes, soluble cleaved BTC was found to be elevated in the retina and contribute to increased retinal vascular permeability (Anand-Apteet al., 2010).

[0009] Subretinal injection of adeno-associated virus expressing soluble BTC leads to a dramatic increase in retinal vascular permeability in mice. Overall, BTC appears to be a potent permeability factor that may play a critical role in the development of increased retinal vascular permeability in diabetic retinopathy and be a potential therapeutic target for this disease.

[0010] Vascular endothelial growth factor (VEGF) has been shown to be a key mediator of neovascularization associated with tumors and intraocular disorders. VEGF is a potent vascular permeability factor and plays a major role in the occurrence of fluid leakage through vessels. In mammalian eyes, VEGF is localized to the inner blood-retinal barrier. VEGF levels are significantly elevated in the vitreous of patients with diabetic macular edema (DME) compared with non-diabetic eyes (Funatsu et al., Ophthalmology 2009 116:73-9).

[0011] Multiple anti-VEGF antibody therapeutics are used to treat ophthalmic disorders such as age-related macular degeneration (AMD) and / or DME, including pegaptanib (anti-VEGF aptamer; Macugen, OSI); ranibizumab (anti-VEGF Fab; Lucentis, Genentech); bevacizumab (full-length humanized antibody; Avastin, Genentech); brolucizumab (anti-VEGF scFv; Beovu, Novartis), and aflibercept (anti-VEGF Fab; Eylea, Regeneron). Other anti-VEGF antibody molecules include soluble VEGF receptor analogs, VEGF decoy (Regeneron), small interfering RNA (siRNA) bevasiranib (Opko Health), and rapamycin (Sirolimus, MacuSight). Anti-VEGF antibody-based drugs are delivered to the eye by injection into the vitreous body under local anesthesia.

[0012] However, there is an unmet need because anti-VEGF antibody-based therapies alone produce suboptimal responses in patients with ophthalmic disorders such as DME. Although anti-VEGF antibody-based agents reduce macular edema, inhibit angiogenesis, and improve vision, not all patients with DME experience significant, long-term improvement. For example, approximately 25% of patients receiving anti-VEGF antibody-based therapies fail to show improvement in visual acuity after 12 months of treatment, and nearly 50% of patients do not achieve the 20 / 40 vision required for legal driving (Mitchellet et al., 2011). Alternative therapies such as laser photocoagulation or intravitreal steroid injection are less effective and have side effects (such as cataracts with intraocular steroid injection (Curr. Ophthalmol. Rep. 2013 Sep 1(3)).

[0013] Therefore, there is a need to identify factors that can further enhance the response to anti-VEGF antibody-based therapies to improve therapy for patients with suboptimal response to anti-VEGF therapy.BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0014] Figure 1 shows the binding structure of Fab to BTC. BTC is shown as a solid surface and Fab is shown as a ribbon. To illustrate the difference in binding modes with Fab, the BTC structure is in the same orientation in all panels: A) BTC / Fab NVS2 complex, B) BTC / Fab NVS3 complex, C) BTC / Fab NVS1 complex, and D) BTC / Fab NVS4 complex.

[0015] Figure 2 shows the structural residues of the Fab epitope binding to BTC. BTC is shown as a ribbon with varying orientations to highlight the epitope residues listed in Tables 12, 13, 14, and 15. The epitope residues are shown in ball-and-stick form and labeled. A) BTC / Fab NVS2 complex, B) BTC / Fab NVS3 complex, C) BTC / Fab NVS1 complex, and D) BTC / Fab NVS4 complex.

[0016] Fig. 3 shows a graphical representation of monospecific and bispecific antibodies.

[0017] Fig. 4 shows the retina images of mice injected with scAAV2-CMV-BTC obtained by fundus recording (left panel) and FFA imaging (right panel).

[0018] Fig. 5 shows scanning laser ophthalmoscopy images of the retina of mice injected with scAAV2-CMV-BTC.

[0019] Fig. 6A-6G show the binding of monospecific and bispecific antibodies to BTC and / or VEGF.

[0020] Fig. 7A-7D show the binding of BTC to ErbB1 in the presence of mono- (NVS1-4) and bispecific (NVS11-14) antibodies.

[0021] Figures 8A-8D show BTC binding to ErbB4 in the presence of mono- (NVS1-4) and bispecific (NVS11-14) antibodies; Figures 8E and 8F show BTC binding to ErB1 or ErbB4 in the presence of NVS1, NVS11, or NVS8; Figure 8G shows BTC-induced EGFR phosphorylation in the presence of NVS1, NVS11, or NVS8.

[0022] Fig. 9A-9C show the binding of VEGF-A to VEGFR2 in the presence of mono-(NVS8) and bispecific (NVS11, NVS12, and NVS14) antibodies.

[0023] Fig. 10A-10D show BTC-induced ERK1 / 2 phosphorylation in the presence of mono- (NVS1-4) and bispecific (NVS11-14) antibodies.

[0024] Fig. 11A-11D show BTC-induced ErbB3 phosphorylation in the presence of mono- (NVS1-4) and bispecific (NVS11-14) antibodies.

[0025] Fig. 12A-12D show BTC-induced HER3 phosphorylation in the presence of mono- (NVS1-2) and bispecific (NVS11-14) antibodies.

[0026] Fig. 13A and 13B show BTC-induced permeabilization of human retinal pigment epithelial (RPE) cells and human retinal microvascular endothelial cells (HRECs) in vitro in the presence of mono- (NVS1 or NVS8) and bispecific (NVS11) antibodies.

[0027] Fig. 14 shows hyperglycemia-induced retinal fluid leakage in diabetic rats in the presence of antibodies to BTC (LZR230) and / or VEGF (4G3).

[0028] Fig. 15A shows optical coherence tomography (OCT) and histological images of rabbit eyes after treatment with VEGF or BTC; Fig. 15B shows illustrative OCT images showing the effect of intravitreal betacellulin injection into the rabbit retina after intravitreal injection of NVS1 or NVS11.

[0029] Fig. 16 shows the change in the retinal thickness of the rabbit eyes after treatment with NVS1 or NVS11.

[0030] Fig. 17 shows the change in the retinal thickness of rabbit eyes after treatment with NVS2 or NVS12.

[0031] Fig. 18A shows the change in retinal thickness of rabbit eyes after treatment with NVS1, NVS11, NVS2, or NVS12; Fig. 18B shows BTC-induced RPE morphological changes in the presence of NVS11 or NVS1; Fig. 18C shows retinal vascular fluid leakage in rabbits (illustrative images) in the presence of NVS11 or NVS8; Fig. 18D shows VEGF-induced retinal vascular fluid leakage in rabbits (quantification by fluorescein angiography) in the presence of NVS11 or NVS8.

[0032] Fig. 19 shows fluorescein angiography images of rabbit eyes after delivery of VEGF or BTC by IVT.

[0033] Fig. 20 shows the vascular fluorescein leakage values ​​from the eyes of individual rabbits after treatment with NVS8, NVS11, or NVS12.

[0034] Fig. 21 shows the vascular fluorescein leakage values ​​from the eyes of individual rabbits after treatment with NVS11, NVS12, or NVS8.

[0035] Fig. 22 shows the change in total retinal thickness values ​​for individual rabbit eyes after treatment with ranibizumab or NVS1.

[0036] Fig. 23 shows the change in retinal thickness in rabbits after treatment with NVS1 or PNVS1. Summary of the invention

[0037] The present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to betacellulin (BTC).

[0038] In one aspect, the antibody or antigen-binding fragment thereof blocks the binding of BTC to ErbB1, ErbB4, or both.

[0039] In one aspect, the antibody or antigen-binding fragment thereof blocks BTC-induced activation of phospho-ERK1 / 2.

[0040] In one aspect, the antibody or antigen-binding fragment thereof blocks BTC-induced activation of phospho-HER3.

[0041] The present invention also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to BTC, wherein the antibody or antigen-binding fragment is characterized by a dissociation constant (KD) of 5 pM or less.

[0042] In one aspect, the antibody or antigen-binding fragment thereof binds to a BTC comprising the amino acid sequence of SEQ ID NO: 157.

[0043] In one aspect, the antibody or antigen-binding fragment thereof binds to at least one residue of SEQ ID NO: 157 selected from the group consisting of G34, H35, F36, S37, R38, C39, P40, K41, Q42, Y43, H45, Y46, R51, R53, F54, V56, A57, E58, Q59, T60, P61, A72, R73, E75 and R76.

[0044] In one aspect, the antibody or antigen-binding fragment thereof binds to R38, C39, P40, K41, Q42, Y43, H45, Y46, F54, Q59, T60, P61 and R73 of SEQ ID NO: 157.

[0045] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain variable region complementarity determining region 1 (HCDR1), a heavy chain variable region complementarity determining region 2 (HCDR2), and a heavy chain variable region complementarity determining region 3 (HCDR3) shown in SEQ ID NOs: 1, 2, and 3, respectively, and a light chain variable region complementarity determining region 1 (LCDR1), a light chain variable region complementarity determining region 2 (LCDR2), and a light chain variable region complementarity determining region 3 (LCDR3) shown in SEQ ID NOs: 14, 15, and 16, respectively.

[0046] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 4, 2 and 3, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 14, 15 and 16, respectively.

[0047] In one aspect, HCDR1 comprises a consensus sequence XYAIS, and / or HCDR2 comprises a consensus sequence GIXPXXGXXXYAQKFQG, and wherein X is any amino acid and may not be the same at different positions.

[0048] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain sequence of SEQ ID NO: 168 and a light chain sequence of SEQ ID NO: 169 or a heavy chain sequence of SEQ ID NO: 170 and a light chain sequence of SEQ ID NO: 171.

[0049] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 5, 6 and 3, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 17, 18 and 19, respectively.

[0050] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 7, 8 and 9, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 20, 18 and 16, respectively.

[0051] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain variable region (VH) and a light chain variable region (VL) comprising an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10 and 21, respectively.

[0052] In one aspect, the amino acid sequence differences are not within the complementarity determining regions.

[0053] In one aspect, the differences in amino acid sequence are conservative substitutions.

[0054] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and VL comprising the amino acid sequence set forth in SEQ ID NO: 10 and 21, respectively.

[0055] In one aspect, VH and VL are encoded by a nucleic acid sequence as set forth in SEQ ID NO: 11 and 22, respectively.

[0056] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain with an amino acid sequence as set forth in SEQ ID NO: 12 and 23, respectively.

[0057] In one aspect, the heavy chain and the light chain are encoded by a nucleic acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13 and 24, respectively.

[0058] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises 1) HCDR1, HCDR2 and HCDR3 contained in a VH with the amino acid sequence of SEQ ID NO: 10, and 2) LCDR1, LCDR2 and LCDR3 contained in a VL with the amino acid sequence of SEQ ID NO: 21.

[0059] In one aspect, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise SEQ ID NO: 1, 2, 3, 14, 15 and 16, respectively; SEQ ID NO: 4, 2, 3, 14, 15 and 16, respectively; SEQ ID NO: 5, 6, 3, 17, 18 and 19, respectively, or SEQ ID NO: 7, 8, 9, 20, 18 and 16, respectively.

[0060] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 5 and 7, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6 and 8, HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 9, and LCDDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 17 and 20, LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 19.

[0061] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and VL comprising the amino acid sequence of SEQ ID NO: 10 and 21, respectively.

[0062] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain with an amino acid sequence as set forth in SEQ ID NO: 12 and 23, respectively.

[0063] The present invention also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to BTCs that comprise VH and VL with the amino acid sequence of SEQ ID NO: 10 and 21, respectively.

[0064] In one aspect, the antibody or antigen-binding fragment thereof binds to P40, K41, Q42, Y43, H45, Y46, E58, Q59, T60, P61, A72, R73, E75 and R76 of SEQ ID NO: 157.

[0065] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 25, 26 and 27, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 38, 39 and 40, respectively.

[0066] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 28, 26 and 27, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 38, 39 and 40, respectively.

[0067] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 29, 30 and 27, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 41, 42 and 43, respectively.

[0068] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 31, 32 and 33, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 44, 42 and 40, respectively.

[0069] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and VL comprising an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 34 and 45, respectively.

[0070] In one aspect, the amino acid sequence differences are not within the complementarity determining regions.

[0071] In one aspect, the differences in amino acid sequence are conservative substitutions.

[0072] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and VL comprising the amino acid sequence set forth in SEQ ID NO: 34 and 45, respectively.

[0073] In one aspect, VH and VL are encoded by a nucleic acid sequence as set forth in SEQ ID NO: 35 and 46, respectively.

[0074] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain with an amino acid sequence as set forth in SEQ ID NO: 36 and 47, respectively.

[0075] In one aspect, the heavy chain and the light chain are encoded by a nucleic acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 37 and 48, respectively.

[0076] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises 1) HCDR1, HCDR2 and HCDR3 contained in a VH with the amino acid sequence of SEQ ID NO: 34, and 2) LCDR1, LCDR2 and LCDR3 contained in a VL with the amino acid sequence of SEQ ID NO: 45.

[0077] In one aspect, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise SEQ ID NO: 25, 26, 27, 38, 39 and 40, respectively; SEQ ID NO: 28, 26, 27, 38, 39 and 40, respectively; SEQ ID NO: 29, 30, 27, 41, 42 and 43, respectively, or SEQ ID NO: 31, 32, 33, 44, 42 and 40, respectively.

[0078] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 28, 29 and 31, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 30 and 32, HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 and 33, and LCDDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 41 and 44, LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 39 and 42, LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 40 and 43.

[0079] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and VL comprising the amino acid sequence of SEQ ID NO: 34 and 45, respectively.

[0080] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain with an amino acid sequence as set forth in SEQ ID NO: 36 and 47, respectively.

[0081] The present invention further provides an isolated antibody or antigen-binding fragment thereof that specifically binds to BTCs that comprise VH and VL with the amino acid sequence of SEQ ID NO: 34 and 45, respectively.

[0082] In one aspect, the antibody or antigen-binding fragment thereof binds to G34, H35, F36, S37, R38, C39, P40, K41, Q42, R51, R53, F54 and V56 of SEQ ID NO: 157.

[0083] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 25, 49 and 50, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 58, 59 and 60, respectively.

[0084] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 28, 49 and 50, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 58, 59 and 60, respectively.

[0085] In one aspect, HCDR1 comprises a consensus sequence XXAMX, and / or HCDR2 comprises a consensus sequence XXXX / -XXXXTXYXDSVКГ, where X is any amino acid and may differ at different positions, and where X / - is any amino acid or a deletion.

[0086] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain sequence of SEQ ID NO: 190 and a light chain sequence of SEQ ID NO: 191.

[0087] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 29, 51 and 50, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 61, 62 and 63, respectively.

[0088] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 31, 52 and 53, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 64, 62 and 60, respectively.

[0089] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and VL comprising an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 54 and 65, respectively.

[0090] In one aspect, the differences in amino acid sequence are not within the complementarity determining regions.

[0091] In one aspect, the differences in amino acid sequence are conservative substitutions.

[0092] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and VL comprising the amino acid sequence set forth in SEQ ID NO: 54 and 65, respectively.

[0093] In one aspect, VH and VL are encoded by a nucleic acid sequence as set forth in SEQ ID NO: 55 and 66, respectively.

[0094] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain with an amino acid sequence presented under SEQ ID NO: 56 and 67, respectively.

[0095] In one aspect, the heavy chain and the light chain are encoded by a nucleic acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 57 and 68, respectively.

[0096] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises 1) HCDR1, HCDR2 and HCDR3 contained in a VH with the amino acid sequence of SEQ ID NO: 54, and 2) LCDR1, LCDR2 and LCDR3 contained in a VL with the amino acid sequence of SEQ ID NO: 65.

[0097] In one aspect, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise SEQ ID NO: 25, 49, 50, 58, 59 and 60, respectively; SEQ ID NO: 28, 49, 50, 58, 59 and 60, respectively; SEQ ID NO: 29, 51, 50, 61, 62 and 63, respectively, or SEQ ID NO: 31, 52, 53, 64, 62 and 60, respectively.

[0098] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 28, 29 and 31, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 51 and 52, HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 50 and 53, and LCDDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 61 and 64, LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 59 and 62, LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 60 and 63.

[0099] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and VL comprising the amino acid sequence of SEQ ID NO: 54 and 65, respectively.

[0100] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain with an amino acid sequence presented under SEQ ID NO: 56 and 67, respectively.

[0101] The present invention also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to BTCs that comprise VH and VL with the amino acid sequence of SEQ ID NO: 54 and 65, respectively.

[0102] In one aspect, the antibody or antigen-binding fragment thereof binds to S37, R38, C39, P40, K41, Q42, Y43, H45, Y46, F54, A57, Q59, T60, P61, A72, R73 and E75 of SEQ ID NO: 157.

[0103] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 69, 70 and 71, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 82, 83 and 84, respectively.

[0104] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 72, 70 and 71, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 82, 83 and 84, respectively.

[0105] In one aspect, HCDR2 comprises the consensus sequence XIXXXXXXXYADSVKG, and / or LCDR3 comprises the consensus sequence QQYDXXXT, and wherein X is any amino acid and may not be the same at different positions.

[0106] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain sequence selected from the group consisting of SEQ ID NO: 194 and 195, respectively; SEQ ID NO: 196 and 197, respectively; SEQ ID NO: 198 and 199, respectively; SEQ ID NO: 200 and 201, respectively; SEQ ID NO: 202 and 203, respectively; SEQ ID NO: 204 and 205, respectively; and SEQ ID NO: 206 and 207, respectively.

[0107] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 73, 74 and 71, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 85, 18 and 86, respectively.

[0108] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 75, 76 and 77, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 87, 18 and 84, respectively.

[0109] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and VL comprising an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 78 and 88, respectively.

[0110] In one aspect, the amino acid sequence differences are not within the complementarity determining regions.

[0111] In one aspect, the differences in amino acid sequence are conservative substitutions.

[0112] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and VL comprising the amino acid sequence set forth in SEQ ID NO: 78 and 88, respectively.

[0113] In one aspect, VH and VL are encoded by a nucleic acid sequence shown as SEQ ID NO: 79 and 89, respectively.

[0114] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain with an amino acid sequence presented under SEQ ID NO: 80 and 90, respectively.

[0115] In one aspect, the heavy chain and the light chain are encoded by a nucleic acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 81 and 91, respectively.

[0116] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises 1) HCDR1, HCDR2 and HCDR3 contained in a VH with the amino acid sequence of SEQ ID NO: 78, and 2) LCDR1, LCDR2 and LCDR3 contained in a VL with the amino acid sequence of SEQ ID NO: 88.

[0117] In one aspect, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise SEQ ID NO: 69, 70, 71, 82, 83 and 84, respectively; SEQ ID NO: 72, 70, 71, 82, 83 and 84, respectively; SEQ ID NO: 73, 74, 71, 85, 18 and 86, respectively, or SEQ ID NO: 75, 76, 77, 87, 18 and 84, respectively.

[0118] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 69, 72, 73 and 75, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 74 and 76, HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71 and 77, and LCDDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 82, 85 and 87, LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 83 and 18, LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 84 and 86.

[0119] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and VL comprising the amino acid sequence of SEQ ID NO: 78 and 88, respectively.

[0120] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain with an amino acid sequence presented under SEQ ID NO: 80 and 90, respectively.

[0121] The present invention also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to BTCs that comprise VH and VL with the amino acid sequence of SEQ ID NO: 78 and 88, respectively.

[0122] In one aspect, the antibody or antigen-binding fragment thereof that specifically binds to BTC is in a format selected from the group consisting of an isolated antibody, Fab, Fab', F(ab')2, Fv and scFv.

[0123] In one aspect, the antibody or antigen-binding fragment thereof that specifically binds to BTC is a Fab.

[0124] In one aspect, the antibody or antigen-binding fragment thereof that specifically binds to BTC is an scFv.

[0125] In one aspect, the antibody or antigen-binding fragment thereof that specifically binds to BTC is an isolated antibody.

[0126] In one aspect, the antibody or antigen-binding fragment thereof that specifically binds to BTC is a human monoclonal antibody.

[0127] In one aspect, the antibody or antigen-binding fragment thereof that specifically binds to BTC is a monoclonal humanized antibody.

[0128] In one aspect, the Fab comprises an Fc region.

[0129] In one aspect, the Fc region is selected from the group consisting of an Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

[0130] In one aspect, the Fc region comprises a human immunoglobulin kappa chain constant region sequence as set forth in SEQ ID NO: 159.

[0131] In one aspect, the Fc region comprises the first constant domain of the Ig heavy chain (CH1 domain) of a human immunoglobulin as shown in SEQ ID NO: 160.

[0132] The present invention provides an isolated antibody or antigen-binding fragment thereof that is capable of competing with an antibody or antigen-binding fragment thereof as described throughout this document for binding to BTC and reducing BTC-mediated signaling.

[0133] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain shown in SEQ ID NOs: 168 and 169, respectively; SEQ ID NOs: 170 and 171, respectively; SEQ ID NOs: 172 and 173, respectively; SEQ ID NOs: 174 and 175, respectively; SEQ ID NOs: 176 and 177, respectively; SEQ ID NOs: 178 and 179, respectively; SEQ ID NOs: 180 and 181, respectively; SEQ ID NOs: 182 and 183, respectively; SEQ ID NOs: 184 and 185, respectively; SEQ ID NOs: 186 and 187, respectively, or SEQ ID NOs: 188 and 189, respectively.

[0134] The present invention provides a polynucleotide comprising nucleotide sequences encoding an antibody or antigen-binding fragment thereof as described throughout this document.

[0135] In one aspect, the expression cassette comprises a polynucleotide as described throughout this document.

[0136] In one aspect, the vector comprises an expression cassette as described throughout this document.

[0137] In one aspect, the host cell comprises a polynucleotide or vector as described throughout this document.

[0138] The present invention also provides a method for producing an antibody or an antigen-binding fragment thereof, comprising culturing a host cell under suitable conditions for expressing the antibody or an antigen-binding fragment thereof.

[0139] In one aspect, the method further comprises purifying the antibody or antigen-binding fragment thereof.

[0140] The present invention further provides a pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof as described throughout this document.

[0141] In one aspect, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, diluent or carrier.

[0142] The present invention provides a method of treating a subject in need thereof, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof or a pharmaceutical composition as described throughout this document.

[0143] In one aspect, the subject has a disease selected from the group consisting of pancreatic carcinoma, breast cancer, endometrial adenocarcinoma, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, and gastric carcinoma.

[0144] In one aspect, the antibody or antigen-binding fragment thereof or pharmaceutical composition is administered by a route selected from the group consisting of intravenous administration, intramuscular administration, subcutaneous administration, parenteral administration, spinal administration and epidermal administration.

[0145] In one aspect, the subject has an ophthalmological disorder.

[0146] In one aspect, the ophthalmological disorder is selected from the group consisting of diabetic macular edema, age-related macular degeneration, neovascular age-related macular degeneration, neovascular glaucoma, diabetic retinopathy, macular edema, pathological myopia, retinal vein occlusions, retinopathy of prematurity, abnormal vascular proliferation associated with phakomatoses, central serous chorioretinopathy, and acute multifocal placoid pigment epitheliopathy.

[0147] In one aspect, the ophthalmological disorder is diabetic macular edema.

[0148] In one aspect, the administration is performed by subretinal injection.

[0149] In one aspect, the administration is performed by intravitreal injection.

[0150] In one aspect, the pharmaceutical composition further comprises an antagonist based on an antibody to VEGF.

[0151] In one aspect, the anti-VEGF antibody antagonist is ranibizumab.

[0152] In one aspect, the anti-VEGF antibody antagonist is bevacizumab.

[0153] In one aspect, the VEGF antibody antagonist is aflibercept.

[0154] ​​In one aspect, the anti-VEGF antibody antagonist is brolucizumab.

[0155] In one aspect, the anti-VEGF antibody antagonist is pegaptanib.

[0156] In one aspect, the VEGF antibody antagonist comprises a heavy chain and a light chain as shown in SEQ ID NO: 103 and 114, respectively.

[0157] In one aspect, the VEGF antibody antagonist is encoded by a nucleic acid sequence as set forth in SEQ ID NOs: 104 and 115.

[0158] In one aspect, the method further comprises administering to the subject an anti-VEGF antibody antagonist.

[0159] In one aspect, the anti-VEGF antibody antagonist is ranibizumab.

[0160] In one aspect, the VEGF antibody antagonist is bevacizumab.

[0161] In one aspect, the anti-VEGF antibody antagonist is aflibercept.

[0162] In one aspect, the anti-VEGF antibody antagonist is brolucizumab.

[0163] In one aspect, the anti-VEGF antibody antagonist is pegaptanib.

[0164] In one aspect, the VEGF antibody antagonist comprises a heavy chain and a light chain as shown in SEQ ID NOs: 103 and 114.

[0165] In one aspect, the VEGF antibody antagonist is encoded by a nucleic acid sequence as set forth in SEQ ID NOs: 104 and 115.

[0166] The present invention provides a kit comprising an antibody or antigen-binding fragment thereof or a pharmaceutical composition as described throughout this document.

[0167] In one aspect, the kit further comprises instructions for use.

[0168] In one aspect, the kit further comprises a syringe.

[0169] The present invention provides a multispecific binding molecule comprising 1) a binding functional unit based on an antibody to BTC and 2) a binding functional unit based on an antibody to VEGF.

[0170] In one aspect, the anti-BTC antibody-based binding functional unit binds to a BTC-containing amino acid sequence of SEQ ID NO: 157.

[0171] In one aspect, the anti-BTC antibody-based binding functional unit binds to at least one residue of SEQ ID NO: 157 selected from the group consisting of G34, H35, F36, S37, R38, C39, P40, K41, Q42, Y43, H45, Y46, R51, R53, F54, V56, A57, E58, Q59, T60, P61, A72, R73, E75 and R76.

[0172] In one aspect, the anti-BTC antibody-based binding functional unit binds to R38, C39, P40, K41, Q42, Y43, H45, Y46, F54, Q59, T60, P61 and R73 of SEQ ID NO: 157.

[0173] In one aspect, the anti-BTC antibody-based binding functional unit binds to P40, K41, Q42, Y43, H45, Y46, E58, Q59, T60, P61, A72, R73, E75 and R76 of SEQ ID NO: 157.

[0174] In one aspect, the anti-BTC antibody-based binding functional unit binds to G34, H35, F36, S37, R38, C39, P40, K41, Q42, R51, R53, F54 and V56 of SEQ ID NO: 157.

[0175] In one aspect, the anti-BTC antibody-based binding functional unit binds to S37, R38, C39, P40, K41, Q42, Y43, H45, Y46, F54, A57, Q59, T60, P61, A72, R73 and E75 of SEQ ID NO: 157.

[0176] In one aspect, the anti-BTC antibody-based binding functional unit is an antibody or antigen-binding fragment thereof as described throughout this document.

[0177] In one aspect, the anti-VEGF antibody-based binding functional unit is an anti-VEGF antibody or an antigen-binding fragment thereof.

[0178] In one aspect, the anti-BTC antibody-based binding functional unit and the anti-VEGF antibody-based binding functional unit are in a format selected from a list consisting of an isolated antibody, Fab, Fab', F(ab')2, Fv and scFv.

[0179] In one aspect, the anti-BTC antibody-based binding functional unit is an anti-BTC Fab, and the anti-VEGF antibody-based binding functional unit is an anti-VEGF Fab.

[0180] In one aspect, the Fab to BTC comprises a heavy chain (HA) and a light chain (LA), and wherein the Fab to VEGF comprises a heavy chain (HB) and a light chain (LB).

[0181] In one aspect, HA and HB are linked in the format from N-terminus to C-terminus: N-HA-linker-1-HB-C, and wherein LA and LB are linked in the format from N-terminus to C-terminus: N-LA-linker-2-LB-C.

[0182] In one aspect, HA and HB are linked in the format from N-terminus to C-terminus: N-HB-linker-1-HA-C, and where LA and LB are linked in the format, from N-terminus to C-terminus: N-LB-linker 2-LA-C.

[0183] In one aspect, linker 1 and linker 2 comprise the amino sequence of SEQ ID NO: 118.

[0184] In one aspect, linker 1 and linker 2 are encoded by the nucleic acid sequence of SEQ ID NO: 119.

[0185] In one aspect, linker 1 and linker 2 comprise an amino sequence selected from the group consisting of SEQ ID NO: 161-167 .

[0186] In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 1, 2, 3, 14, 15 and 16, respectively; SEQ ID NOs: 4, 2, 3, 14, 15 and 16, respectively; SEQ ID NOs: 5, 6, 3, 17, 18 and 19, respectively, or SEQ ID NOs: 7, 8, 9, 20, 18 and 16, respectively.

[0187] In one aspect, the anti-BTC antibody-based binding functional unit comprises VH and VL with the amino acid sequence of SEQ ID NO: 10 and 21, respectively.

[0188] In one aspect, VH and VL are encoded by the nucleic acid sequence of SEQ ID NO: 116 and 122, respectively.

[0189] In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 25, 26, 27, 38, 39 and 40, respectively; SEQ ID NOs: 28, 26, 27, 38, 39 and 40, respectively; SEQ ID NOs: 29, 30, 27, 41, 42 and 43, respectively, or SEQ ID NOs: 31, 32, 33, 44, 42 and 40, respectively.

[0190] In one aspect, the anti-BTC antibody-based binding functional unit comprises VH and VL with the amino acid sequence of SEQ ID NO: 34 and 45, respectively.

[0191] In one aspect, VH and VL are encoded by the nucleic acid sequence of SEQ ID NO: 127 and 132, respectively.

[0192] In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 25, 49, 50, 58, 59 and 60, respectively; SEQ ID NOs: 28, 49, 50, 58, 59 and 60, respectively; SEQ ID NOs: 29, 51, 50, 61, 62 and 63, respectively, or SEQ ID NOs: 31, 52, 53, 64, 62 and 60, respectively.

[0193] In one aspect, the anti-BTC antibody-based binding functional unit comprises VH and VL with the amino acid sequence of SEQ ID NO: 54 and 65, respectively.

[0194] In one aspect, VH and VL are encoded by the nucleic acid sequence of SEQ ID NO: 137 and 142, respectively.

[0195] In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 69, 70, 71, 82, 83 and 84, respectively; SEQ ID NOs: 72, 70, 71, 82, 83 and 84, respectively; SEQ ID NOs: 73, 74, 71, 85, 18 and 86, respectively, or SEQ ID NOs: 75, 76, 77, 87, 18 and 84, respectively.

[0196] In one aspect, the anti-BTC antibody-based binding functional unit comprises VH and VL with the amino acid sequence of SEQ ID NO: 78 and 88, respectively.

[0197] In one aspect, VH and VL are encoded by the nucleic acid sequence of SEQ ID NO: 147 and 151, respectively.

[0198] In one aspect, the anti-VEGF antibody-based binding functional unit comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NOs: 92, 93 and 94, respectively, and LCDR1, LCDR2 and LCDR3 shown as SEQ ID NOs: 105, 106 and 107, respectively.

[0199] In one aspect, the anti-VEGF antibody-based binding functional unit comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NOs: 95, 93 and 94, respectively, and LCDR1, LCDR2 and LCDR3 shown as SEQ ID NOs: 105, 106 and 107, respectively.

[0200] In one aspect, the anti-VEGF antibody-based binding functional unit comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NOs: 96, 97 and 94, respectively, and LCDR1, LCDR2 and LCDR3 shown as SEQ ID NOs: 108, 109 and 110, respectively.

[0201] In one aspect, the anti-VEGF antibody-based binding functional unit comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NOs: 98, 99 and 100, respectively, and LCDR1, LCDR2 and LCDR3 shown as SEQ ID NOs: 111, 109 and 107, respectively.

[0202] In one aspect, the anti-VEGF antibody-based binding functional unit comprises VH and VL comprising an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 101 and 112, respectively.

[0203] In one aspect, the amino acid sequence differences are not within the complementarity determining regions.

[0204] In one aspect, the differences in amino acid sequence are conservative substitutions.

[0205] In one aspect, the anti-VEGF antibody-based binding functional unit comprises VH and VL comprising the amino acid sequence set forth in SEQ ID NO: 101 and 112, respectively.

[0206] In one aspect, the VEGF antibody-based binding functional unit comprises VH and VL encoded by the nucleic acid sequence shown as SEQ ID NO: 102 and 113, respectively.

[0207] In one aspect, the anti-VEGF antibody-based binding functional unit comprises VH and VL encoded by the nucleic acid sequence shown as SEQ ID NO: 117 and 123, respectively.

[0208] In one aspect, the VEGF antibody-based binding functional unit comprises VH and VL encoded by the nucleic acid sequence shown as SEQ ID NO: 128 and 133, respectively.

[0209] In one aspect, the VEGF antibody-based binding functional unit comprises VH and VL encoded by the nucleic acid sequence shown as SEQ ID NO: 138 and 143, respectively.

[0210] In one aspect, the VEGF antibody-based binding functional unit comprises VH and VL encoded by the nucleic acid sequence shown as SEQ ID NO: 148 and 152, respectively.

[0211] In one aspect, the VEGF antibody-based binding functional unit comprises a heavy chain and a light chain with an amino acid sequence shown as SEQ ID NO: 103 and 114, respectively.

[0212] In one aspect, the heavy chain and the light chain are encoded by a nucleic acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 104 and 115, respectively.

[0213] The present invention provides a multispecific binding molecule comprising an anti-BTC antibody-based binding functional unit and an anti-VEGF antibody-based binding functional unit, wherein the anti-BTC antibody-based binding functional unit comprises a heavy chain variable domain (VHA) and a light chain variable domain (VLA) that bind to BTC, and wherein the anti-VEGF antibody-based binding functional unit comprises a heavy chain variable domain (VHB) and a light chain variable domain (VLB) that bind to VEGF, wherein: VHA and VLA comprise the amino acid sequence presented under SEQ ID NO: 10 and 21, respectively, and VHB and VLB comprise the amino acid sequence presented under SEQ ID NO: 101 and 112, respectively.

[0214] In one aspect, the anti-BTC antibody-based binding functional unit further comprises a heavy chain constant domain (CH1A) and a light chain constant domain (CKA), and where the anti-VEGF antibody-based binding functional unit further comprises a heavy chain constant domain (CH1B) and a light chain constant domain (CKB).

[0215] In one aspect, the multispecific binding molecule is in the format from N-terminus to C-terminus as follows: N-VHA-CH1A-linker-VHB-CH1B-C and N-VLA-CKA-linker-VLB-CKB-C.

[0216] In one aspect, the multispecific binding molecule comprises a heavy chain comprising VHA, CH1A, a linker, VHB and CH1B, wherein the heavy chain is shown as SEQ ID NO: 120.

[0217] In one aspect, the heavy chain is encoded by the nucleic acid sequence shown as SEQ ID NO: 121.

[0218] In one aspect, the multispecific binding molecule comprises a light chain comprising a VLA, a CKA, a linker, a VLB, and a CKB, wherein the light chain is shown as SEQ ID NO: 125.

[0219] In one aspect, the light chain is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 126.

[0220] The present invention also provides a multispecific binding molecule comprising an anti-BTC antibody-based binding functional unit and an anti-VEGF antibody-based binding functional unit, wherein the anti-BTC antibody-based binding functional unit comprises a heavy chain variable domain (VHA) and a light chain variable domain (VLA) that bind to BTC, and wherein the anti-VEGF antibody-based binding functional unit comprises a heavy chain variable domain (VHB) and a light chain variable domain (VLB) that bind to VEGF, wherein: VHA and VLA comprise the amino acid sequence presented under SEQ ID NO: 34 and 45, respectively, and VHB and VLB comprise the amino acid sequence presented under SEQ ID NO: 101 and 112, respectively.

[0221] In one aspect, the anti-BTC antibody-based binding functional unit further comprises a heavy chain constant domain (CH1A) and a light chain constant domain (CKA), and where the anti-VEGF antibody-based binding functional unit further comprises a heavy chain constant domain (CH1B) and a light chain constant domain (CKB).

[0222] In one aspect, the multispecific binding molecule is in the format from N-terminus to C-terminus as follows: N-VHA-CH1A-linker-VHB-CH1B-C and N-VLA-CKA-linker-VLB-CKB-C.

[0223] In one aspect, the multispecific binding molecule comprises a heavy chain comprising VHA, CH1A, a linker, VHB and CH1B, wherein the heavy chain is shown as SEQ ID NO: 130.

[0224] In one aspect, the heavy chain is encoded by the nucleic acid sequence shown as SEQ ID NO: 131.

[0225] In one aspect, the multispecific binding molecule comprises a light chain comprising a VLA, a CKA, a linker, a VLB, and a CKB, wherein the light chain is shown as SEQ ID NO: 135.

[0226] In one aspect, the light chain is encoded by the nucleic acid sequence shown as SEQ ID NO: 136.

[0227] The present invention also provides a multispecific binding molecule comprising an anti-BTC antibody-based binding functional unit and an anti-VEGF antibody-based binding functional unit, wherein the anti-BTC antibody-based binding functional unit comprises a heavy chain variable domain (VHA) and a light chain variable domain (VLA) that bind to BTC, and wherein the anti-VEGF antibody-based binding functional unit comprises a heavy chain variable domain (VHB) and a light chain variable domain (VLB) that bind to VEGF, wherein: VHA and VLA comprise the amino acid sequence presented under SEQ ID NO: 54 and 65, respectively, and VHB and VLB comprise the amino acid sequence presented under SEQ ID NO: 101 and 112, respectively.

[0228] In one aspect, the anti-BTC antibody-based binding functional unit further comprises a heavy chain constant domain (CH1A) and a light chain constant domain (CKA), and where the anti-VEGF antibody-based binding functional unit further comprises a heavy chain constant domain (CH1B) and a light chain constant domain (CKB).

[0229] In one aspect, the multispecific binding molecule is in the format from N-terminus to C-terminus as follows: N-VHA-CH1A-linker-VHB-CH1B-C and N-VLA-CKA-linker-VLB-CKB-C.

[0230] In one aspect, the multispecific binding molecule comprises a heavy chain comprising VHA, CH1A, a linker, VHB and CH1B, wherein the heavy chain is shown as SEQ ID NO: 140.

[0231] In one aspect, the heavy chain is encoded by the nucleic acid sequence shown as SEQ ID NO: 141.

[0232] In one aspect, the multispecific binding molecule comprises a light chain comprising a VLA, a CKA, a linker, a VLB, and a CKB, wherein the light chain is shown as SEQ ID NO: 145.

[0233] In one aspect, the light chain is encoded by the nucleic acid sequence shown as SEQ ID NO: 146.

[0234] The present invention further provides a multispecific binding molecule comprising an anti-BTC antibody-based binding functional unit and an anti-VEGF antibody-based binding functional unit, wherein the anti-BTC antibody-based binding functional unit comprises a heavy chain variable domain (VHA) and a light chain variable domain (VLA) that bind to BTC, and wherein the anti-VEGF antibody-based binding functional unit comprises a heavy chain variable domain (VHB) and a light chain variable domain (VLB) that bind to VEGF, wherein: VHA and VLA comprise the amino acid sequence presented under SEQ ID NO: 78 and 88, respectively, and VHB and VLB comprise the amino acid sequence presented under SEQ ID NO: 101 and 112, respectively.

[0235] In one aspect, the anti-BTC antibody-based binding functional unit further comprises a heavy chain constant domain (CH1A) and a light chain constant domain (CKA), and where the anti-VEGF antibody-based binding functional unit further comprises a heavy chain constant domain (CH1B) and a light chain constant domain (CKB).

[0236] In one aspect, the multispecific binding molecule is in the format from N-terminus to C-terminus as follows: N-VHA-CH1A-linker-VHB-CH1B-C and N-VLA-CKA-linker-VLB-CKB-C.

[0237] In one aspect, the multispecific binding molecule comprises a heavy chain comprising VHA, CH1A, a linker, VHB and CH1B, wherein the heavy chain is shown as SEQ ID NO: 149.

[0238] In one aspect, the heavy chain is encoded by the nucleic acid sequence shown as SEQ ID NO: 150.

[0239] In one aspect, the multispecific binding molecule comprises a light chain comprising a VLA, a CKA, a linker, a VLB, and a CKB, wherein the light chain is shown as SEQ ID NO: 154.

[0240] In one aspect, the light chain is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 155.

[0241] The present invention provides a multispecific binding molecule comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 120, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 125.

[0242] In one aspect, the first polypeptide chain is encoded by the nucleic acid sequence of SEQ ID NO: 121, and the second polypeptide chain is encoded by the nucleic acid sequence of SEQ ID NO: 126.

[0243] The present invention also provides a multispecific binding molecule comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 130, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 135.

[0244] In one aspect, the first polypeptide chain is encoded by the nucleic acid sequence of SEQ ID NO: 131, and the second polypeptide chain is encoded by the nucleic acid sequence of SEQ ID NO: 136.

[0245] The present invention further provides a multispecific binding molecule comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 140, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 145.

[0246] In one aspect, the first polypeptide chain is encoded by the nucleic acid sequence of SEQ ID NO: 141, and the second polypeptide chain is encoded by the nucleic acid sequence of SEQ ID NO: 146.

[0247] The present invention also provides a multispecific binding molecule comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154.

[0248] In one aspect, the first polypeptide chain is encoded by the nucleic acid sequence of SEQ ID NO: 150, and the second polypeptide chain is encoded by the nucleic acid sequence of SEQ ID NO: 155.

[0249] The present invention provides a polynucleotide comprising nucleotide sequences encoding a multispecific binding molecule as described throughout this document.

[0250] The present invention also provides an expression cassette comprising a polynucleotide as described throughout this document.

[0251] The present invention also provides a vector comprising an expression cassette as described throughout this document.

[0252] The present invention further provides a host cell comprising a polynucleotide as described throughout this document.

[0253] The present invention provides a method for producing a multispecific binding molecule, comprising culturing a host cell under suitable conditions for expressing the multispecific binding molecule or a fragment thereof.

[0254] In one aspect, the method further comprises purifying the multispecific binding molecule.

[0255] The present invention provides a pharmaceutical composition comprising an effective amount of a multispecific binding molecule as described throughout this document.

[0256] In one aspect, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, diluent or carrier.

[0257] In one aspect, the pharmaceutical composition further comprises one or more therapeutic agents.

[0258] The present invention also provides a method for treating an ophthalmological disorder in a subject in need thereof, comprising administering to the subject an effective amount of a multispecific binding molecule or pharmaceutical composition as described throughout this document.

[0259] In one aspect, the multispecific binding molecule or pharmaceutical composition is administered to the subject intravitreally.

[0260] In one aspect, the multispecific binding molecule or pharmaceutical composition is administered via subretinal injection.

[0261] In one aspect, the ophthalmological disorder is selected from the group consisting of diabetic macular edema, age-related macular degeneration, neovascular age-related macular degeneration, neovascular glaucoma, diabetic retinopathy, macular edema, pathological myopia, retinal vein occlusions, retinopathy of prematurity, abnormal vascular proliferation associated with phakomatoses.

[0262] In one aspect, the ophthalmological disorder is diabetic macular edema.

[0263] The present invention provides a method for preventing, treating or managing an ophthalmological disorder, comprising administering to a subject an effective amount of a multispecific binding molecule described in the present invention, wherein the multispecific binding molecule reduces retinal fluid leakage and / or retinal thickening in the subject relative to a control subject.

[0264] The present invention further provides a kit comprising a multispecific binding molecule or a pharmaceutical composition as described throughout this document.

[0265] In one aspect, the kit further comprises instructions for use.

[0266] In one aspect, the kit further comprises a syringe.

[0267] The present invention provides a method for preventing or treating macular edema, DME, AMD, neovascular AMD, or RVO in a subject in need thereof, comprising intravitreal administration to the subject of a multispecific binding molecule described herein at a dose in the range of about 0.25 mg / eye to 7.5 mg / eye.

[0268] In one aspect, the dose is about 0.25 mg / eye, 0.75 mg / eye, 2.5 mg / eye, or 7.5 mg / eye.

[0269] In one aspect, the dose is 0.25 mg / eye.

[0270] In one aspect, the dose is 0.75 mg / eye.

[0271] In one aspect, the dose is 1 mg / eye.

[0272] In one aspect, the dose is 2.5 mg / eye.

[0273] In one aspect, the dose is 3 mg / eye.

[0274] In one aspect, the dose is 5 mg / eye.

[0275] In one aspect, the dose is 7.5 mg / eye.

[0276] In one aspect, the dose is 0.25 mg / eye, 0.3 mg / eye, 0.35 mg / eye, 0.4 mg / eye, 0.45 mg / eye, 0.5 mg / eye, 0.55 mg / eye, 0.6 mg / eye, 0.65 mg / eye, 0.7 mg / eye, 0.75 mg / eye, 0.8 mg / eye, 0.85 mg / eye, 0.9 mg / eye, 0.95 mg / eye, 1.0 mg / eye, 1.1 mg / eye, 1.2 mg / eye, 1.3 mg / eye, 1.4 mg / eye, 1.5 mg / eye, 1.6 mg / eye, 1.7 mg / eye, 1.8 mg / eye, 1.9 mg / eye, 2.0 mg / eye, 2.1 mg / eye, 2.2 mg / eye, 2.3 mg / eye, 2.4 mg / eye, 2.5 mg / eye, 2.6 mg / eye, 2.7 mg / eye, 2.8 mg / eye, 2.9 mg / eye, 3.0 mg / eye, 3.1 mg / eye, 3.2 mg / eye, 3.3 mg / eye, 3.4 mg / eye, 3.5 mg / eye, 3.6 mg / eye, 3.7 mg / eye, 3.8 mg / eye, 3.9 mg / eye, 4.0 mg / eye, 4.1 mg / eye, 4.2 mg / eye, 4.3 mg / eye, 4.4 mg / eye, 4.5 mg / eye, 4.6 mg / eye, 4.7 mg / eye, 4.8 mg / eye, 4.9 mg / eye, 5.0 mg / eye, 5.1 mg / eye, 5.2 mg / eye, 5.3 mg / eye, 5.4 mg / eye, 5.5 mg / eye, 5.6 mg / eye, 5.7 mg / eye, 5.8 mg / eye, 5.9 mg / eye, 6.0 mg / eye, 6.1 mg / eye, 6.2 mg / eye, 6.3 mg / eye, 6.4 mg / eye, 6.5 mg / eye, 6.6 mg / eye, 6.7 mg / eye, 6,8 mg / eye, 6.9 mg / eye, 7.0 mg / eye, 7.1 mg / eye, 7.2 mg / eye, 7.3 mg / eye, 7.4 mg / eye, or 7.5 mg / eye.

[0277] In one aspect, the multispecific binding molecule comprises 1) an anti-BTC antibody-based binding functional unit comprising the amino acid sequence of SEQ ID NOs: 10 and 21, respectively, and 2) an anti-VEGF antibody-based binding functional unit comprising the amino acid sequence of SEQ ID NOs: 101 and 112, respectively.

[0278] In another aspect, the administration is carried out once a month.DETAILED DESCRIPTION

[0279] In general, the present invention is based in part on the discovery of antibodies specific for betacellulin (BTC). In particular, the inventors of the present invention have discovered antibodies to BTC characterized by properties consistent with therapeutic applicability (i.e., the antibodies bind BTC with affinity and specificity sufficient to achieve the desired therapeutic effect). Based in part on this discovery, the present invention describes therapeutic compositions comprising molecules comprising an antibody or antibody fragment specific for BTC, linked to another therapeutic component, for example, an anti-VEGF antibody or antibody fragment.Such therapeutic components include antibodies or fragments thereof and proteins that bind to therapeutic targets in BTC-containing tissues (e.g., the vitreous humor), as well as compounds (e.g., small molecules) that modulate therapeutic targets in such tissues. In cases where such therapeutic components include antibodies, the therapeutic composition as a whole can be a multispecific antibody (e.g., a bispecific antibody). In specific aspects, this document also provides methods for treating an ocular disorder (e.g., AMD, e.g., nonvascular AMD, DME, DR, etc.) by administering an anti-BTC antibody antagonist and an anti-VEGF antibody antagonist. Terminology.

[0280] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Any literature cited in this document, including, for example, all patents, published patent applications, and non-patent publications, are incorporated by reference in their entirety. To facilitate understanding of the present invention, several terms and abbreviations used in this document are defined below.

[0281] As used herein, the singular forms "a" and "an" include the plural forms unless the content clearly indicates otherwise. For example, a reference to "an antibody" includes a mixture of two or more such antibodies.

[0282] Unless otherwise specifically stated or obvious from the context, the term "about" as used herein with respect to a numerical value is understood to mean being within the range of acceptable error in the art, for example, within two standard deviations from the mean. Thus, "about" may be within + / - 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.05% or 0.01% of the stated value, preferably + / - 10% of the stated value. When used before a numerical range or list of numbers, the term "about" applies to each number in the series, e.g., the phrase "about 1-5" should be interpreted as "about 1 - about 5," or, for example, the phrase "about 1, 2, 3, 4" should be interpreted as "about 1, about 2, about 3, about 4, etc."

[0283] Whenever the term "comprise," "comprises," "comprising," or the like is used in relation to a sequence (e.g., an amino acid sequence), it should be understood that said sequence may also be limited by the term "consist," "consists," "consisting," or the like. As used herein, the phrase "consisting essentially of" refers to the genera or species of active pharmaceutical agents included in the method or composition, as well as any excipients that do not exhibit activity with respect to the intended purpose of using the methods or compositions. In some aspects, the phrase "consisting essentially of" expressly excludes the inclusion of one or more additional active agents other than the multispecific binding molecule of the present invention.In some aspects, the phrase "consisting essentially of" expressly excludes the inclusion of one or more additional active agents other than the multispecific binding molecule of the present invention and a second agent administered together.

[0284] The term "betacellulin" or "BTC" refers to a growth factor of the EGF family in the body. BTC activity can be measured by its binding to 1) ErbB1; 2) ErbB4; 3) ErbB homodimer (e.g., ErbB1 / ErbB1 and ErbB4 / ErB4) and / or 4) ErbB heterodimer (e.g., ErbB1 / ErbB2, ErB1 / ErB3, ErB1 / ErB4, ErB2 / ErB3 and ErB2 / ErB4). See Dunbar and Goddard, Int'l. J. Biochem. & Cell Biol., 2000, 32: 805-815. BTC activity can also be measured by the level of ERK1 / 2 phosphorylation. In humans, it is encoded by the BTC gene located on chromosome 4 at locus 4q13-q21. Human BTC is expressed as a 178-residue protein as pro-BTC (e.g., NCBI: NP_001720.1 or SEQ ID NO: 156). Mature, secreted human BTC consists of 80 amino acid residues, i.e., residues 32-111 of pro-BTC, represented by SEQ ID NO: 158. Mouse betacellulin consists of 177 amino acid residues (e.g., NCBI: NP_031594.1). Rat betacellulin consists of 177 amino acid residues (e.g., NCBI: GenBank: BAA96731.1).

[0285] The term "vascular endothelial growth factor" or "VEGF" refers to a protein characterized by VEGF activity in the body, such as inducing proliferation and migration of vascular endothelial cells, and may be necessary for both physiological and pathological angiogenesis. In mammals, the VEGF family includes five members: VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D. Human VEGF exists in at least six isoforms (VEGF 121 , VEGF 145 , VEGF 165 , VEGF 183 , VEGF 189 and VEGF 206 ), which arise as a result of alternative splicing of mRNA of one gene (Ferrara N, Davis Smyth T. Endocr Rev 18:1-22 (1997)). VEGF 165, the most abundant isoform, is a major heparin-binding dimeric glycoprotein with a molecular weight of approximately 45,000 daltons. The term "human VEGF" as used herein refers to the 165-amino acid human vascular endothelial cell growth factor and related amino acid vascular endothelial cell growth factors of 121, 189, and 206 (and other isoforms) as described by Leung et al., Science 246:1306 (1989), and Houck et al., Mol. Endocrin. 5:1806 (1991), together with naturally occurring allelic and processed forms of these growth factors.

[0286] The term "anti-BTC antibody-based binding functional unit" as used herein means a polypeptide (e.g., an antibody or an antigen-binding fragment thereof) that specifically binds to BTC. For the avoidance of doubt, non-limiting examples of "anti-BTC antibody-based binding functional unit" include full-length antibodies and their antigen-binding fragments, such as Fab, scFv, Fv, single-domain antibodies, and the like. In a particular aspect, the anti-BTC antibody is Fab or scFv. In a particular aspect, the anti-BTC antibody-based binding functional unit specifically binds to human BTC and / or cynomolgus monkey BTC.

[0287] The term "anti-VEGF antibody-based binding functional unit" as used herein means a polypeptide (e.g., an antibody or an antigen-binding fragment thereof as provided below) that specifically binds to VEGF (as defined below). To avoid any doubt, non-limiting examples of the "anti-VEGF antibody-based binding functional unit" include full-length antibodies and their antigen-binding fragments, such as Fab, scFv, Fv, single-domain antibodies, and the like, as provided below. In a particular aspect, the anti-VEGF antibody is Fab or scFv. In a particular aspect, the anti-VEGF antibody-based binding functional unit specifically binds to human VEGF-A.

[0288] The phrase "binds specifically," "specifically binds," or "selectively binds," when used in the context of describing the interaction between an antigen (e.g., a protein) and a multispecific binding molecule of the present invention, refers to a binding reaction that is determinative for establishing the presence of the antigen in a heterogeneous population of proteins and other biological substances, such as in a biological sample, such as blood, blood serum, blood plasma, or a tissue sample. Thus, under certain designated conditions of an immunoassay, the multispecific binding molecule of the present invention, characterized by a specific binding specificity, binds to a specific antigen at least two times more strongly than background levels and does not bind substantially in significant amounts to other antigens present in the sample.In one aspect, under the designated immunoassay conditions, a multispecific binding molecule of the present invention with a specific binding specificity binds to a specific antigen at least ten (10) times stronger than background levels and does not bind substantially to other antigens present in the sample in significant amounts. Specific binding to an antibody or binding agent under such conditions may require that the multispecific binding molecule of the present invention be selected for its specificity for a specific protein. If desired or necessary, this selection can be accomplished by excluding multispecific binding molecules that cross-react with molecules from another species (e.g., mouse or rat) or other subtypes.

[0289] In some aspects, specific binding of a multispecific binding molecule of the present invention means binding with an equilibrium constant (K A ) (k on / k off ), which is at least 10 2 M -1 , at least 5×10 2 M -1 , at least 10 3 M -1 , at least 5×10 3 M -1 , at least 10 4 M - 1 at least 5×10 4 M -1 , at least 10 5 M -1 , at least 5×10 5 M -1 , at least 10 6 M -1 , at least 5×10 6 M -1 , at least 10 7 M -1 , at least 5×10 7 M -1 , at least 10 8 M -1 , at least 5×10 8 M -1 , at least 10 9 M -1 , at least 5×10 9 M -1, at least 10 10 M -1 , at least 5×10 10 M -1 , at least 10 11 M -1 , at least 5×10 11 M -1 , at least 10 12 M -1 , at least 5×10 12 M -1 , at least 10 13 M -1 , at least 5×10 13 M -1 , at least 10 14 M -1 , at least 5×10 14 M -1 , at least 10 15 M -1 or at least 5×10 15 M -1 .

[0290] In some aspects, specific binding of a multispecific binding molecule of the present invention means binding with a rate constant (K D ) (k off / k on ), which is less than 5×10 -2 M, less than 10 -2 M, less than 5×10 -3 M, less than 10 -3 M, less than 5×10 -4 M, less than 10 -4 M, less than 5×10 -5 M, less than 10-5 M, less than 5×10 -6 M, less than 10 -6 M, less than 5×10 -7 M, less than 10 -7 M, less than 5×10 -8 M, less than 10 -8 M, less than 5×10 -9 M, less than 10 -9 M, less than 5×10 -10 M, less than 10 -10 M, less than 5×10 -11 M, less than 10 -11 M, less than 5×10 -12 M, less than 10 -12 M, less than 5×10 -13 M, less than 10 -13 M, less than 5×10 -14 M, less than 10 -14 M, less than 5×10 -15 M or less than 10 -15 M or less, and binds to the target antigen with an affinity that is at least twice its affinity for binding to a non-specific antigen (e.g., HSA).

[0291] The term "K D " or "Kd" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0292] The term "therapeutic target-binding functional unit" as used herein means a molecule that specifically binds a therapeutic target of interest. This molecule may be an antibody or an antigen-binding fragment thereof (as defined below), an antigen-specific binding functional unit such as DARPin, a finomer, an affibody, an adnectin, an affilin, an anticalin, an avimer, a centirin, or an RNA molecule such as an aptamer. This therapeutic target-binding functional unit may also be a polypeptide, such as a receptor or a part of a receptor, that specifically binds the therapeutic target of interest. For the avoidance of doubt, non-limiting examples of "anti-BTC antibody-based binding functional unit" include full-length antibodies and antigen-binding fragments thereof, such as Fab, scFv, Fv, single-domain antibodies, etc., as provided below.

[0293] The term "antibody" as used herein refers to a complete antibody or an antigen-binding fragment thereof. A complete antibody is a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of a single domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs).Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The constant regions of antibodies are capable of mediating the binding of immunoglobulin to tissues or host factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement pathway. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, bispecific, or multispecific antibodies. Antibodies can belong to any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0294] The term "isotype" refers to the class of antibody (e.g., IgM, IgE, IgG such as IgG1 or IgG4) conferred by the heavy chain constant region genes. An isotype also includes modified variants of one of these classes in which modifications have been made to alter Fc function, for example, to enhance or decrease effector functions or binding to Fc receptors. Antibodies can belong to any isotype (e.g., immunoglobulin G (IgG), immunoglobulin E (IgE), immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin A (IgA), and immunoglobulin Y (IgY)), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The term "IgG" or "IgG antibody" as used herein, and unless otherwise specified, means a complete antibody of the G or Ig type.

[0295] The term "light chain" includes a full-length light chain and fragments thereof with sufficient variable region sequence to impart binding specificity. A full-length light chain includes the variable region domain V L and the domain of the constant region C L The variable region domain of the light chain is located at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0296] The term "heavy chain" includes the full-length heavy chain and fragments thereof with sufficient variable region sequence to confer binding specificity. The full-length heavy chain includes the variable region domain V H and three domains of the constant region C H 1, C H 2 and C H 3. Domain V H is located at the amino terminus of the polypeptide, and the C domains H are located at the carboxyl end, with C H3 is located closest to the carboxyl terminus of the polypeptide. Heavy chains can be of any isotype, including IgG (including the subtypes IgG1, IgG2, IgG3, and IgG4), IgA (including the subtypes IgA1 and IgA2), IgM, and IgE.

[0297] The term "variable region" or "variable domain" refers to a portion of the light and / or heavy chains of an antibody, typically comprising approximately 120-130 amino acids at the amino terminal end of the heavy chain and approximately 100-110 amino acids at the amino terminal end of the light chain. In some aspects, the variable regions of different antibodies vary greatly in amino acid sequence, even among antibodies of the same species. The variable region of an antibody typically determines the specificity of a particular antibody for its target.

[0298] The terms "complementarity-determining region" and "CDR" as used herein refer to the amino acid sequences in the variable regions of an antibody that confer antigen specificity and binding affinity. In general, there are three CDRs (HCDR1, HCDR2, and HCDR3) in each variable region of the heavy chain, and three CDRs (LCDR1, LCDR2, and LCDR3) in each variable region of the light chain. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (Kabat numbering scheme); Al-Lazikaniet al., (1997) JMB 273927-948 (numbering scheme according to "Chothia"); and Lefrancet al., (2003) Dev. Comp. Immunol. 27, 55-77 (numbering scheme according to "IMGT").The Kabat definition is the standard for numbering residues in an antibody and is typically used to identify CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28: 214–8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the position of certain regions of the structural loop. See, e.g., Chothia et al., J. Mol. Biol., 196: 901–17 (1986); Chothia et al., Nature, 342: 877–83 (1989).

[0299] Alternatively, other methods for delineating CDR regions can be used, for example, CDR definitions according to both Kabat and Chothia can be combined (a "combined" system). For example, in the case of classical Kabat formats, the amino acid residues of the CDRs in the heavy variable chain (VH) domain are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the amino acid residues of the CDRs in the light variable chain (VL) domain are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). According to Chothia, the amino acids of the CDRs in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). When combining both Kabat and Chothia CDR definitions, the combined CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.As another example, according to IMGT, the CDR amino acid residues in the variable heavy chain (VH) domain are numbered 26-33 (HCDR1), 51-58 (HCDR2), and 97-108 (HCDR3), and the CDR amino acid residues in the variable light chain (VL) domain are numbered 27-36 (LCDR1), 54-56 (LCDR2), and 93-101 (LCDR3).

[0300] As used herein, the term "antibody framework regions" or "FR" refers to the portion of a variable domain, either VL or VH, that serves as the backbone for the antigen-binding loops (CDRs) of that variable domain. Essentially, it represents the variable domain without the CDRs. According to IMGT, antibody CDRs can be determined using the IMGT / DomainGap Align program.

[0301] The term "antigen-binding fragment" of an antibody, as used herein, refers to one or more fragments of an antibody, or one or more polypeptides comprising such a fragment, that retain the ability to specifically bind to a given antigen (e.g., BTC and VEGF). The antigen-binding functions of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed by the term antigen-binding fragment of an antibody include, but are not limited to, a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; an F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked via a disulfide bridge in the hinge region; an Fd fragment, consisting of the VH and CH1 domains; an Fv fragment, consisting of the VL and VH domains; single-chain Fv fragment (scFv), consisting of VL and VH domains linked by a linker sequence, and a single-domain antibody (dAb) fragment (Ward et al., 1989 Nature 341:544–546), which consists of a VH domain or a VL domain. Antigen-binding fragments can also be included in single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126–1136). Antigen-binding portions of antibodies can be grafted onto polypeptide backbones such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide monobodies). Antibody binding fragments can be incorporated into single-chain molecules containing a pair of tandem Fv segments (e.g., V. H -CH1- V H -CH1), which, together with complementary light chain polypeptides (eg, V L -VC-V L -VC) form a pair of antigen-binding regions (Zapata et al., (1995) Protein Eng. 8:1057-1062 and US Patent No. 5,641,870).

[0302] The "Fab" fragment used herein contains one constant and one variable domain from each of the heavy and light chains. The heavy chain of a Fab molecule may not form a disulfide bond with another heavy chain of the molecule.

[0303] "Fab fragment" as used herein comprises one light chain and a portion of one heavy chain that contains a VH domain and a C domain H 1, and also the region between the C domains H 1 and C H 2, so that an interchain disulfide bond can form between the heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0304] "F(ab')2 fragment" as used herein comprises two light chains and two heavy chains containing a portion of the constant region between the C domains H 1 and C H2, so that a disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments, which are held together by a disulfide bond between the two heavy chains.

[0305] The "Fv region" contains the variable regions of both the heavy and light chains, but does not contain the constant regions.

[0306] As used herein, the term "single-chain Fv" or "scFv" refers to antibody fragments that contain V domains H and V L antibodies where these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises an internal polypeptide linker between the V domains. H and V L, which enables the scFv to form the necessary structure for antigen binding. The scFv may also have an engineered internal disulfide bridge, which enhances stability. For a review of scFv, see Plückthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., 1994, Springer-Verlag, New York, pp. 269-315. In preferred aspects, the scFv used in the multispecific binding molecules of the present invention are characterized by the general structures: NH2-V L -linker-V H -COOH or NH2-V H -linker-V L -COOH.

[0307] The term "affinity matured antibody" as used herein is an antibody with one or more alterations in one or more of its CDRs that result in an improvement in the affinity of the antibody for the antigen, compared to the parent antibody that does not possess such alteration(s). Preferred affinity matured antibodies will be characterized by nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are prepared by procedures known in the art. Marks et al. Bio / Technology 10:779-783 (1992) describe affinity maturation by shuffling the VH and VL domains. Random mutagenesis of CDRs and / or framework residues is described in Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al.Gene169:147-155 (1995); Yelton et al.J. Immunol.155:1994-2004 (1995); Jackson et al.,J. Immunol.154(7):3310-9 (1995); and Hawkins et al,J. Mol. Biol.226:889-896 (1992).

[0308] The term "parent" antibody, as used herein, is an antibody encoded by the amino acid sequence used to produce an affinity-matured antibody or variant thereof. Preferably, the parent antibody comprises a human framework region and, if present, comprises a human constant region(s) of a human antibody. For example, the parent antibody may be a humanized or human antibody.

[0309] The term "diabody" as used herein refers to small antibody fragments with two antigen-binding sites, whose fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on one chain, the domains are forced to pair with the complementary domains of another chain and form two antigen-binding sites. Diabodies are described in more detail in, for example, EP 404097; WO 93 / 11161 and Hollinger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0310] The term "monospecific binding molecule" or "monospecific antibody" as used herein refers to a molecule that binds to a single epitope on a target antigen. In one aspect, the monospecific binding molecule or monospecific antibody of the present invention binds to BTC. In another aspect, the monospecific binding molecule or monospecific antibody of the present invention binds to VEGF.

[0311] The term "multispecific binding molecule" or "multispecific antibody" as used herein refers to a molecule that binds to two or more different antigens. Recognition of each antigen is generally accomplished through an "antigen-binding domain" (e.g., "BTC antigen-binding domain," "VEGF antigen-binding domain"). The term "multispecific" includes "bispecific," i.e., a molecule that binds to two different antigens. In some aspects, a multispecific binding molecule comprises one or more polypeptide chains, each of which comprises one antigen-binding domain. In one aspect, a multispecific binding molecule comprises VH or VL. In some aspects, a multispecific binding molecule comprises one or more polypeptide chains, each of which comprises more than one (e.g., two) antigen-binding domains.In some aspects, multispecific binding molecules comprise two, three, four or more polypeptide chains that together comprise a plurality, such as two or more, such as two, three or four antigen-binding domains.

[0312] The term "bispecific binding molecule" or "bispecific antibody" refers to molecules that combine the antigen-binding sites of two antibodies within a single molecule. In one aspect, a bispecific binding molecule or bispecific antibody comprises a single polypeptide. In another aspect, a bispecific binding molecule or bispecific antibody comprises two polypeptides linked by disulfide bridges or any other covalent bonds. Thus, a bispecific antibody is capable of binding two different antigens simultaneously or sequentially. Methods for producing bispecific antibodies are widely known in the art. Various formats for combining two antibodies are also known in the art.Forms of the bispecific antibodies of the present invention include, but are not limited to, diabody, single chain diabody, Fab dimerization product (Fab-Fab), Fab-scFv, and tandem antibody, as known to those skilled in the art.

[0313] The term "bivalent molecule" as used herein refers to a molecule that has two antigen-binding domains. The term "trivalent molecule" as used herein refers to a molecule that has three antigen-binding domains. In some aspects, the trivalent molecule of the present invention is a molecule similar to a trivalent antibody. In some aspects, the trivalent molecule may consist of two antigen-binding domains capable of binding to the same epitope of the same antigen and a third antigen-binding domain that binds to a separate antigen. Such aspects are considered trivalent bispecific molecules.

[0314] The term "multivalent molecule" refers to a molecule that has at least two antigen-binding sites, wherein the antigen-binding sites may have specificity for the same antigen or for different antigens. In some aspects, a multivalent molecule of the present invention is a molecule similar to a multivalent antibody. In some aspects, a multivalent molecule of the present invention is a multivalent antibody. In some aspects, a multivalent molecule is a bivalent molecule, a trivalent molecule, or a tetravalent molecule. The trimerization domain is described, for example, in EP 1012280B1. Pentamerization modules are described, for example, in PCT / EP97 / 05897.

[0315] The term "substantially similar" or "substantially the same" as used herein refers to a sufficiently high level of similarity between two numerical values ​​(typically one associated with an antibody-like molecule of the present invention and the other associated with a reference / comparative antibody or antibody-like molecule) such that a person skilled in the art would consider the difference between the two values ​​to be insignificant or without biological and / or statistical significance in the context of the biological characteristic measured by said values ​​(e.g., Tm values ​​or the amount of antibodies collected). The difference between said two values ​​is preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, preferably less than about 10%, depending on the function of the value for the reference / comparative antibody.

[0316] The term "antigen" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antigen-binding protein (including, for example, an antibody or an immunologically functional fragment thereof). In some aspects, an antigen is capable of being used in an animal to produce antibodies capable of binding to that antigen. An antigen may have one or more epitopes capable of interacting with different antigen-binding proteins, such as antibodies.

[0317] The term "epitope" or "antigenic determinant" as used herein refers to any determinant capable of binding with high affinity to an antibody or antibody-like molecule. An epitope is a region of an antigen that is bound by an antibody (or antibody-like molecule) that specifically targets that antigen, and if the antigen is a protein, the epitope includes specific amino acids that directly contact the antibody or antibody-like molecule. Epitopes are most commonly localized on proteins, but in some cases they can be localized on other types of molecules, such as nucleic acids. Epitopic determinants may include chemically active surface moieties of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0318] In general, multispecific binding molecules specific for a particular target antigen will preferentially recognize an epitope on that target antigen within a complex mixture of proteins and / or macromolecules.

[0319] Regions of a given polypeptide that include an epitope can be identified using a variety of epitope mapping techniques known in the art. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes can be determined, for example, by synthesizing large amounts of peptides in parallel on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and are described in, for example, U.S. Patent No. 4,708,871; Geyse et al., (1984) Proc. Natl. Acad. Sci. USA 8:3998-4002; Geysenet al.,(1985) Proc. Natl. Acad. Sci. USA 82:78-182; Geysenet al.,(1986) Mol. Immunol. 23:709-715.Similarly, conformational epitopes are readily identified by determining the spatial conformation of amino acids, such as by X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols above. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathicity plots, such as those generated by, for example, Omiga version 1.0 software available from the Oxford Molecular Group. This computer program uses the Hoppe / Woods method (Hoppet et al., (1981) Proc. Natl. Acad. Sci USA 78:3824–3828); for determining antigenicity profiles and the Kyte-Doolittle technique (Kyte et al., (1982) J. MoI. Biol. 157:105–132); for hydropathicity plots.

[0320] The term "compete," when used in the context of antigen-binding proteins that compete for a single epitope, means competition between antigen-binding proteins as determined by an assay in which the antigen-binding protein of interest (e.g., an antibody or an immunologically functional fragment thereof) interferes with or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., BTC or a fragment thereof). Many types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, such as solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme-linked immunosorbent assay (EIA), and sandwich competitive assay (see, e.g., Stahliet al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol.137:3614–3619) direct-labeling solid-phase assay, direct-labeling solid-phase sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); direct-labeling solid-phase RIA using I-125 label (see, e.g., Morelet et al., 1988, Molec. Immunol. 25:7–15); direct-labeling solid-phase biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176:546–552), and direct-labeling RIA (Moldenhaueret et al., 1990, Scand. J. Immunol. 32:77–82). Typically, this assay involves the use of purified antigen bound to a solid surface or cells bearing either antigen, unlabeled test antigen-binding protein, and a labeled reference antigen-binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the antigen-binding protein of interest. The antigen-binding protein of interest is typically present in excess.Antigen-binding proteins identified by competitive analysis (competing antigen-binding proteins) include antigen-binding proteins that bind to the same epitope as reference antigen-binding proteins and antigen-binding proteins that bind to an adjacent epitope that is located close enough to the epitope to which the reference antigen-binding protein binds to cause steric hindrance. Additional details regarding methods for determining competitive binding are provided in the examples herein. Typically, when a competing antigen-binding protein is present in excess, it will suppress (e.g., reduce) the specific binding of the reference antigen-binding protein to the common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0321] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The phrases also apply to polymers of amino acids in which one or more amino acid residues are an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as to polymers of naturally occurring amino acids and to a polymer of non-naturally occurring amino acids. Unless otherwise specified, a particular polypeptide sequence also potentially encompasses variants thereof with conservative modifications.

[0322] The term "polypeptide chain" as used herein refers to the entire amino acid chain of the multispecific binding molecule of the present invention, having all constituent regions and domains.

[0323] The terms "constant region" or "constant domain" refer to the carboxy-terminal portion of the light and heavy chains that is not directly involved in antibody-antigen binding but exhibits various effector functions, such as interaction with the Fc receptor. These terms refer to the portion of the immunoglobulin molecule that contains a more conserved amino acid sequence compared to another portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the CH1, CH2, and CH3 domains of the heavy chain and the CL domain of the light chain.

[0324] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code as well as those amino acids that are later modified, such as the cleavage of hydroxyproline, γ-carboxyglutamate, pyroglutamate, C-terminal lysine, and O-phosphoserine, for example, as a result of post-translational modifications. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon bonded to a hydrogen, a carboxylic acid group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium.Such analogues have modified R-groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as the naturally occurring amino acid. Amino acid mimetics are chemical compounds that have a structure different from the general chemical structure of the amino acid but that function similarly to the naturally occurring amino acid.

[0325] For polypeptide sequences, "conservatively modified variants" include single substitutions, deletions, or additions in a polypeptide sequence that result in the replacement of an amino acid with an amino acid of similar chemical properties. Conservative substitution tables listing functionally similar amino acids are well known. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, interspecies homologs, and alleles of the present invention. The following eight groups contain amino acids that are conservative substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T) and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)).In some aspects, the term "conservative sequence modifications" or "conservative modifications" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence.

[0326] In some aspects, the term "dose" refers to an amount of a therapeutic agent, which can be a protein (e.g., an antibody or antigen-binding fragment) or a nucleic acid, or a functional unit that binds a therapeutic target, can be a small molecule (e.g., <900 Daltons) of a therapeutic compound, administered to a subject at one time (single dose) or in two or more administrations over a certain period of time. For example, a dose can refer to an amount of a protein (e.g., an anti-BTC antibody or a functional fragment thereof conjugated to a molecule, e.g., a protein comprising an anti-VEGF antibody or a functional fragment thereof) administered to a subject over a course of three weeks, or one, two, three, four, five, six or more months (e.g., via one administration or two or more administrations).The interval between doses can be any desired amount of time and is called the "dose interval."

[0327] The term "pharmaceutically effective" in relation to a dose means a sufficient amount of a protein (e.g., an antibody or antigen-binding fragment) or other pharmaceutically active agent to provide the desired effect (e.g., improving vision or preventing further vision loss). The amount that is "effective" will vary from subject to subject depending on the age and general condition of the individual, the specific drug or pharmaceutically active agent, and the like. Therefore, it is not always possible to indicate a precise "effective" amount applicable to all patients. However, a suitable "effective" dose in any individual case can be determined by one skilled in the art using routine experimentation.

[0328] The term "human antibody" as used herein is intended to include antibodies comprising variable regions in which both the framework and CDR regions are derived from sequences of human origin. In addition, if the antibody comprises a constant region, the constant region is also derived from such human sequences, such as human germline sequences or mutant versions of human germline sequences, or an antibody comprising consensus framework sequences obtained by analysis of human framework sequences, such as described by Knappik et al. (J. Mol. Biol. 296, 57-86, 2000).The human antibodies of the present invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo, or conservative substitution to ensure stability or manufacture). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mice, have been grafted onto human framework sequences.

[0329] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to polypeptides, including antibodies and antigen-binding fragments, having a substantially identical amino acid sequence or obtained from the same genetic source. This term also includes preparations of antibody molecules of a single-molecule composition. A monoclonal antibody composition exhibits a single specificity and binding affinity for a particular epitope. Methods for producing monoclonal antibodies using phage display technology are known in the art (Proetzel, G., Ebersbach, H. (Eds.) Antibody Methods and Protocols. Humana Press ISBN 978-1-61779-930-3; 2012).

[0330] The term "humanized" forms of non-human (e.g., murine) antibodies refers to chimeric antibodies that contain minimal sequence derived from immunoglobulin of non-human origin. Typically, humanized antibodies are human immunoglobulins (the recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-human species (the donor antibody), such as a mouse, rat, rabbit, or non-human primate, that possess the desired specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding residues of non-human origin. In addition, humanized antibodies may contain residues that are not present in the recipient antibody or the donor antibody. Such modifications are made to further improve the antibody's performance.In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to loops of a non-human immunoglobulin and all or substantially all of the FRs are human immunoglobulin lo sequences. A humanized antibody will optionally also contain at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. For further details, see Jones et al., Nature 321:522–525 (1986); Riechmannet al., Nature 332:323–329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593–596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

[0331] As used herein, the term "identity" refers to the sequence match between two polypeptides, molecules, or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid (e.g., if a position in both polypeptides is occupied by lysine), then the corresponding molecules are identical at that position. The "percent identity" between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that must be introduced to optimally align the two sequences. In general, a comparison is performed when the two sequences are aligned to ensure maximum identity. Such an alignment can be ensured using, for example, the method of Needleman and Wunsch (J. MoI. Biol.(48):444–453 (1970)), the algorithm of which was incorporated into the GAP program in the GCG software package, using either the Blosum 62 matrix or the PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. For sequence comparison, one sequence typically serves as a reference sequence to which the test sequences are compared. When using the sequence comparison algorithm, the test and reference sequences are entered into the computer, subsequence coordinates are set if necessary, and the algorithm's software parameters for sequence analysis are set. The default software parameters can be used, or alternative parameters can be set.The sequence comparison algorithm then calculates percent sequence identity values ​​for the test sequences compared to a reference sequence based on program parameters. A comparison window may be used, where a reference segment is any one of a number of contiguous positions selected from a group consisting of 20 to 600, typically from about 50 to about 200, more typically from about 100 to about 150, in which the sequence can be compared with a reference sequence with the same number of contiguous positions after the two sequences have been optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, using the local homology search algorithm according to Smith and Waterman (1970) Adv. Appl. Math.2:482c, by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970, by the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou ed., 2003)). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschulet al., Nuc. Acids Res. 25:3389–3402, 1977; and Altschulet al., J. Mol. Biol. 215:403–410, 1990, respectively.Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information. This algorithm initially identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy a positively assessed threshold score T when aligned with a word of the same length in a database sequence. T is known as the neighbor word score threshold (Altschulet et al., supra). These initial neighbor word matches act as "seeds" to begin searches to find longer HSPs containing them. Word matches are extended in both directions along each sequence as far as the combined alignment score can be increased.For nucleotide sequences, the overall score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, the scoring matrix is ​​used to calculate the overall score. Extension of word matches in each direction stops when the overall alignment score decreases by X relative to its maximum achieved value; the overall score approaches zero or below due to the accumulation of negatively scoring alignments of one or more residues; or the end of any of the sequences is reached. The W, T, and X parameters of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as default parameters a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as default parameters a wordlength of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989), alignments (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873–5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which indicates the probability with which a match between two nucleotide or amino acid sequences occurred by chance.For example, a nucleic acid is considered to be similar to a reference sequence if the lowest sum probability when comparing the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11–17, 1988), which has been incorporated into the ALIGN program (version 2.0) using the PAM120 residue weight table, a gap extension penalty of 12, and a gap opening penalty of 4. Alternatively, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444–453, 1970), which has been incorporated into the GAP program in the GCG software package (available on the World Wide Web at gcg.com), using either a Blosum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In addition to the percent sequence identity mentioned above, another indicator that two nucleic acid or polypeptide sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies induced by the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, if the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are essentially identical is that the two molecules, or their complementary strands, hybridize to each other under stringent conditions, as described below.Another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the two nucleic acid sequences.

[0332] The term "percent complementarity" or "percent conjugacy" as used herein with respect to two nucleotide sequences is similar to the concept of percent identity, but refers to the percentage of nucleotides of a query sequence that optimally pair or hybridize with nucleotides of a subject sequence when the query and subject sequences are arranged linearly and optimally paired without secondary folding structures such as loops, stems, or hairpins. Such percent complementarity may be between two strands of DNA, two strands of RNA, or a strand of DNA and an RNA strand. "Percent complementarity" is calculated by (i) optimal pairing or hybridization of the two nucleotide sequences in a linear and fully extended arrangement (i.e.,(without folding or secondary structures) in the comparison window, (ii) determining the number of positions that form a base pair between the two sequences in the comparison window to obtain the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the comparison window, and (iv) multiplying this factor by 100% to obtain the percentage complementarity of the two sequences. Optimal base pairing of two sequences can be determined based on known nucleotide base pairs, such as GC, AT, and AU, through hydrogen bonding. If the "percent complementarity" is calculated relative to a reference sequence without specifying a specific comparison window, then the percentage identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence.Thus, for the purposes of the present invention, when two sequences (query and subject) are optimally base-paired (taking into account mismatches or unpaired nucleotides, but without stacking or secondary structures), the "percent complementarity" for the query sequence is equal to the number of base-paired positions between the two sequences divided by the total number of positions in the query sequence over its length (or by the number of positions in the query sequence in the comparison window), which is then multiplied by 100%.

[0333] The term "isolated antibody" refers to an antibody that is substantially free of other antibodies or other proteins with different antigen specificity. Furthermore, an isolated antibody may be substantially free of other cellular materials and / or chemicals, such as an antibody isolated from cell supernatant.

[0334] The term "linked" or "binding" in the context of the anti-BTC antibody-based multispecific binding molecules described herein refers to the attachment of an anti-BTC antibody-based binding functional unit, such as, for example, anti-BTC antibodies or a functional fragment thereof that binds BTCs listed in Table 1, to the molecule. The attachment of the anti-BTC antibody-based binding functional unit to a protein can occur, for example, at the amino- or carboxyl-terminus of a molecule, for example, an anti-VEGF antibody or a functional fragment thereof. The anti-BTC antibody-based binding functional unit can also be attached to both the amino- and carboxyl-termini of a protein. The anti-BTC antibody-based binding functional unit can also be attached to one or more amino acids or nucleic acids within a protein or nucleic acid molecule, respectively.The linking of the anti-BTC antibody-based binding functional unit to the molecule can be achieved by any method known in the art, including, but not limited to, expressing the anti-BTC antibody-based binding functional unit and the molecule as a fusion protein or chemically linking the anti-BTC antibody-based binding functional unit to the molecule after translation either directly to each other or through a linker using disulfide bonds, etc.

[0335] The term "linker" or "linked" in the context of a multispecific binding molecule refers to the attachment of one portion of the multispecific binding molecule directly or indirectly to another portion of the molecule, for example, an anti-BTC antibody-based binding functional unit to an anti-VEGF antibody-based binding functional unit. The linker can be covalently attached to one or both amino or carboxyl termini of the anti-BTC antibody-based binding functional unit and / or a protein or nucleic acid molecule. The peptide linker can also be conjugated to an amino acid or nucleic acid within the protein or nucleic acid molecule sequence, respectively. It is contemplated that in certain aspects, peptide linkers can be, for example, from about 2 to about 25 residues in length.

[0336] The term "nucleic acid" is used herein interchangeably with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers based on them in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified residues or linkages in the backbone, which are synthetic, naturally occurring, and non-naturally occurring, which are characterized by binding properties similar to the reference nucleic acid, and which are metabolized similarly to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, peptide nucleic acids (PNA).Unless otherwise specified, a particular nucleic acid sequence also potentially encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly specified sequence. Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605–2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91–98, 1994).

[0337] The term "operably linked" refers to a functional relationship between two or more segments of a polynucleotide (e.g., DNA). Typically, this term refers to a functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates transcription of the coding sequence in the corresponding host cell or other expression system. In general, transcriptional regulatory promoter sequences that are operably linked to a transcribed sequence are physically adjacent to the transcribed sequence, i.e., they are cis-acting.However, some transcriptional regulatory sequences, such as enhancers, do not necessarily need to be physically adjacent or located in close proximity to the coding sequences whose transcription they enhance.

[0338] As used herein, the term "optimized" or "codon optimization" means that a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the producer cell or organism, typically a eukaryotic cell such as a Pichia cell, a Chinese hamster ovary (CHO) cell, a human cell, or a prokaryotic cell such as an Escherichia coli cell. Codon optimization refers to the discovery that the frequency of synonymous codons (i.e., codons that encode the same amino acid) in the coding DNA varies across species. This codon degeneracy allows for the encoding of an identical polypeptide by different nucleotide sequences. Various methods for codon optimization are known in the art and include, for example, those disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148.An optimized nucleotide sequence is designed to completely, or as closely as possible, preserve the amino acid sequence originally encoded by the original nucleotide sequence, also known as the "original" sequence. Optimized sequences, as used herein, have been designed to have codons that are preferred in mammalian cells. However, optimized expression of these sequences in other eukaryotic or prokaryotic cells is also contemplated within the context of this document. Amino acid sequences encoded by optimized nucleotide sequences are also referred to as optimized.

[0339] As used herein, the term "protein" refers to any organic compound composed of amino acids arranged in one or more linear chains and folded into a three-dimensional conformation. The amino acids in the polymer chain are linked together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The term "protein" further includes, but is not limited to, peptides, single-chain polypeptides, or any complex molecules consisting essentially of two or more amino acid chains. It further includes, but is not limited to, glycoproteins or other known post-translational modifications. It further includes known natural or artificial chemical modifications of natural proteins, such as, but not limited to, glycoengineering, PEGylation, hesylation, etc., the incorporation of unnatural amino acids, and the modification of an amino acid for chemical conjugation with another molecule.

[0340] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The phrases also apply to polymers of amino acids in which one or more amino acid residues are an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as to polymers of naturally occurring amino acids and to a polymer of non-naturally occurring amino acids. Unless otherwise specified, a particular polypeptide sequence also potentially encompasses variants thereof with conservative modifications.

[0341] The term "recombinant host cell" (or simply "host cell") refers to a cell into which one or more recombinant expression vectors have been introduced. It should be understood that such terms are intended to refer not only to the specific host cell but also to the progeny of such a cell. Because certain modifications may arise in subsequent generations either through mutation or environmental influences, such progeny may not actually be identical to the parent cell but are still included within the scope of the term "host cell" as used herein.

[0342] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals), such as non-human primates (e.g., cynomolgus macaques), mice, rats, cats, rabbits, pigs, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where otherwise noted, the terms "patient" and "subject" are used interchangeably in this document. As used herein, the terms "cynomolgus macaque" or "cynomolgus macaque" refer to the cynomolgus macaque (Macaca fascicularis).

[0343] "Warning" or "warn" with respect to the indications described herein such as ophthalmologic conditions or disorders, including conditions or disorders associated with diabetic macular edema, retinal vascular disease, conditions or disorders associated with diabetic retinopathy, and / or conditions or disorders associated with macular edema, means any action that prevents or slows the deterioration of visual function, retinal anatomy, retinal vascular disease index, diabetic retinopathy disease index, and / or macular edema disease index, as described below, in a patient at risk of said deterioration.As used herein, "warning" or "caution" with respect to conditions or disorders other than ophthalmologic, including pancreatic carcinoma, breast cancer, endometrial adenocarcinoma, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, and gastric carcinoma, does not necessarily mean complete prevention of the condition. Partial prevention or alleviation of the severity of a condition or symptom of a condition, or a reduction in the risk of developing the condition, are also encompassed by this term.

[0344] The term "treating" or "treating" conditions or disorders associated with diabetic macular edema, conditions or disorders associated with age-related macular degeneration, such as neovascular age-related macular degeneration, conditions or disorders associated with retinal vascular disease, conditions or disorders associated with diabetic retinopathy, and / or conditions or disorders associated with macular edema means any action that results in, or is expected to result in, improvement or preservation of visual function and / or retinal anatomy.As used herein, "treating" or "treating" conditions or disorders other than ophthalmological, including pancreatic carcinoma, breast cancer, endometrial adenocarcinoma, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, and gastric carcinoma, refers to any action that results in, or is expected to result in, an improvement or reduction in the severity of the conditions or disorders. In another aspect, treating includes reducing the frequency of repeat administrations and / or reducing the number of doctor / hospital visits. Also included is an aspect where treating includes chronic treatment, such as repeated administration for an indefinite period. Methods for assessing treatment and / or preventing disease are known in the art and are described herein below.

[0345] The term "therapeutically acceptable amount" or "therapeutically effective amount" or "therapeutically effective dose" interchangeably refers to an amount sufficient to achieve the desired result (i.e., reducing disease activity, reducing disease progression, reducing signs and / or symptoms of the disease, etc.). In some aspects, a therapeutically acceptable amount does not induce or cause unwanted side effects. A therapeutically acceptable amount can be determined by first administering a low dose and then gradually increasing this dose until the desired effect is achieved. A "prophylactically effective dosage" and a "therapeutically effective dosage" of a molecule of the present invention can prevent the onset or lead to a reduction in the severity, respectively, of disease symptoms, including symptoms associated with BTC activity and / or VEGF activity.

[0346] The term "vector" is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. In another aspect, a polynucleotide sequence can be introduced to a subject using a viral vector, such as an adeno-associated viral vector (AAV, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12), a lentiviral vector, or a retroviral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors that have a bacterial origin of replication and mammalian episomal vectors).Other vectors (e.g., non-episomal mammalian vectors) can integrate into the host cell genome upon introduction into the host cell and thereby replicate along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques often take the form of plasmids. In the present description, "plasmid" and "vector" may be used interchangeably, as the plasmid is the most widely used form of vector. However, the present invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), which perform equivalent functions.

[0347] The term "recombinant" with respect to a polynucleotide molecule (DNA or RNA), protein, construct, vector, etc., refers to a polynucleotide or protein molecule or sequence created by man and not typically found in nature, and / or is present in a context in which it is not typically found in nature, including a polynucleotide molecule (DNA or RNA), protein, construct, etc., containing a combination of two or more polynucleotide or protein sequences that do not occur together in the same way in nature without human intervention, such as a polynucleotide molecule, protein, construct, etc., containing at least two polynucleotide or protein sequences that are operably linked but heterologous with respect to each other. For example, the term "recombinant" may refer to any combination of two or more DNA or protein sequences in a single molecule (e.g., a plasmid, construct, vector, chromosome, protein, etc.), where such combination is man-made and does not normally occur in nature. As used in this definition, the phrase "does not normally occur in nature" means not occurring in nature without the introduction of man. A recombinant polynucleotide or protein molecule, construct, etc., may comprise polynucleotide or protein sequence(s) that are (i) separated from other polynucleotide or protein sequences that exist in close proximity to each other in nature, and / or (ii) adjacent to (or abutting) other polynucleotide or protein sequences that are not in close proximity to each other in nature. Such a recombinant polynucleotide molecule, protein, construct, etc. may also refer to a polynucleotide or protein molecule or sequence that has been genetically created and / or engineered outside a cell.For example, a recombinant DNA molecule may contain any engineered or human-created plasmid, vector, etc., and may comprise a linear or circular DNA molecule. Such plasmids, vectors, etc., may contain various supporting elements, including a prokaryotic origin of replication and a selectable marker, as well as one or more transgenes or expression cassettes, possibly in addition to an introduced plant selectable marker gene, etc.

[0348] As used herein, the term "coding region" or "coding region" refers to a portion of a polynucleotide that encodes a functional unit or molecule (e.g., but not limited to, mRNA, a protein, or a non-coding sequence or RNA molecule).

[0349] As used herein, the term "therapeutic protein" refers to a protein that is used to treat, prevent, or alleviate a disease, condition, or disorder.

[0350] "Modification" or "mutation" of an amino acid residue / position, as used herein, refers to a change in the primary amino acid sequence compared to the original amino acid sequence, where the change results from a change in the sequence including the specified amino acid residue / position. For example, typical modifications include the substitution of a residue (or at the specified position) with another amino acid (e.g., a conservative or non-conservative substitution), the insertion of one or more amino acids adjacent to the specified residue / position, and the deletion of the specified residue / position. "Amino acid substitution" or a variant thereof refers to the replacement of an existing amino acid residue in a predetermined (original) amino acid sequence with another amino acid residue.Generally and preferably, the modification results in an alteration of at least one physicochemical activity of the polypeptide variant compared to a polypeptide containing the original (or "wild-type") amino acid sequence. For example, in the case of an antibody, the altered physicochemical activity may be binding affinity, binding capacity, and / or binding effect with respect to the target molecule.

[0351] The term "conservatively modified variant" applies to both amino acid sequences and nucleic acid sequences. When applied to specific nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode identical or substantially identical amino acid sequences, or, in the case where a nucleic acid does not encode an amino acid sequence, they refer to substantially identical sequences. Due to the degeneracy of the genetic code, any given protein is encoded by a large number of functionally identical nucleic acids. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at each position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide.Such nucleic acid variations are "silent variations," which represent one type of conservatively modified variation. Each nucleic acid sequence herein encoding a polypeptide also describes every possible silent variation of the nucleic acid. One skilled in the art will recognize that every codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Accordingly, every silent variation of the nucleic acid encoding a polypeptide is implicit in each described sequence.

[0352] The term "C-terminus" as used herein refers to the carboxyl-terminal amino acid of a polypeptide chain containing a free carboxyl group (-COOH). The term "N-terminus" as used herein refers to the amino-terminal amino acid of a polypeptide chain containing a free amino group (-NH2).

[0353] As used herein, phrases such as "a patient in need of treatment" or "a subject in need of treatment" include subjects, such as mammals, that would benefit from the administration of a molecule or pharmaceutical composition of the present invention used, for example, for detection, for a diagnostic procedure and / or for treatment.

[0354] The phrase "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.

[0355] The term "pharmaceutical composition" refers to a mixture of at least one active ingredient (e.g., an antibody or fragment of the present invention) and at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0356] "Medicine" refers to a substance used for medical treatment.

[0357] "BTC-mediated disorder" covers all diseases and pathological conditions in which BTC and / or VEGF are directly or indirectly involved in the disease or pathological condition, including the etiology, development, progression, persistence or pathology of the disease or condition. BTC-mediated disorders may include, but are not limited to, pancreatic carcinoma, breast cancer, endometrial adenocarcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, gastric carcinoma, diabetic macular edema, age-related macular degeneration, neovascular age-related macular degeneration, neovascular glaucoma, diabetic retinopathy, macular edema, pathological myopia, retinal vein occlusions, retinopathy of prematurity, phakomatose-associated abnormal vascular proliferation, central serous chorioretinopathy, and acute multifocal placoid pigment epitheliopathy.

[0358] "VEGF-mediated disorder" covers all diseases and pathological conditions in which VEGF is directly or indirectly involved in the disease or pathological condition, including the etiology, development, progression, persistence, or pathology of the disease or condition. VEGF-mediated disorders may include, but are not limited to, central nervous system neoplasm, capillary hemangioblastoma, meningioma, cerebral edema, pituitary adenoma, non-astrocytic glioma, peritumoral edema, breast carcinoma, adenocarcinoma, lung carcinoma, diabetic macular edema, age-related macular degeneration, neovascular age-related macular degeneration, neovascular glaucoma, diabetic retinopathy, macular edema, pathological myopia, retinal vein occlusions, retinopathy of prematurity, and phakomatose-associated abnormal vascular proliferation. Anti-BTC antibody or anti-BTC antibody-based binding functional units

[0359] BTC is a member of the EGF family. It is a ligand for the receptor tyrosine kinase family and primarily activates ErbB1 and ErbB4 homodimers, triggering anti-apoptotic and pre-proliferative signaling pathways, such as the Ras / MAPK and PL3K / AKT pathways. In the eye, BTC is a potent permeability factor that may play a critical role in the development of increased retinal vascular permeability in diabetic retinopathy and be a potential therapeutic target for this disease. An exemplary amino acid sequence of human pro-BTC is provided as SEQ ID NO: 156. An exemplary amino acid sequence of human BTC is provided as SEQ ID NO: 158. An exemplary amino acid sequence of human BTC as described herein is provided as SEQ ID NO: 157 (residual amino acid residues are indicated by lowercase letters at the N- and C-termini).

[0360] The structure of human BTC protein bound to four Fab fragments of BTC was recently determined by the applicant using X-ray crystallography. See Example 2. The structure of human BTC is an EGF fold with five beta chains in a three-chain and two-chain sheet. The structure is stabilized by three disulfide bonds.

[0361] The present document provides an antibody or antigen-binding fragments thereof that bind to BTC, including human BTC. In some aspects, the provided antibody or antigen-binding fragments thereof are polypeptides that comprise one or more complementarity-determining regions (CDRs) as described herein. In some aspects, the CDRs are embedded in a "framework" region that orients the CDRs such that the proper antigen-binding properties of the CDRs are achieved. In some aspects, the antibody or antigen-binding fragments thereof provided herein can interfere with, block, reduce, or modulate the interaction between BTC and the ErbB receptor. In some aspects, the antibody or antigen-binding fragments thereof provided herein are capable of suppressing BTC-mediated activity (including binding).In some aspects, antigen-binding proteins associated with such epitopes inhibit, among other things, interactions between BTC and the ErbB receptor and other BTC-mediated physiological effects. In some aspects, the antigen-binding proteins are human, such as fully human antibodies or Fabs to BTC.

[0362] In some aspects, the antibody or antigen-binding fragments thereof bind to any of the epitopes bound by the antibodies disclosed herein. In some aspects, this can be determined by competition assays between the antibodies disclosed herein and other antibodies. In some aspects, the antibody or antigen-binding fragments thereof bind to one of the epitopes bound by the antibodies described in Table 1. In some aspects, the antibody or antigen-binding fragments thereof bind to a specific conformational state of BTC so as to prevent the interaction of BTC with the ErbB receptor. In one aspect, the antibody or antigen-binding fragments thereof of the present invention bind to one or more of the five beta chains of human BTC. In one aspect, the antibody or antigen-binding fragments thereof bind to the beta chain of human BTC 1 and prevent the binding of BTC to the ErbB receptor.In one aspect, the antibody or antigen-binding fragments thereof bind to the human BTC 2 beta chain and prevent BTC from binding to the ErbB receptor. In one aspect, the antibody or antigen-binding fragments thereof bind to the human BTC 3 beta chain and prevent BTC from binding to the ErbB receptor. In one aspect, the antibody or antigen-binding fragments thereof bind to the human BTC 4 beta chain and prevent BTC from binding to the ErbB receptor. In one aspect, the antibody or antigen-binding fragments thereof bind to the human BTC 5 beta chain and prevent BTC from binding to the ErbB receptor.

[0363] This document discloses an antibody or antigen-binding fragment thereof that specifically binds to BTC. In some aspects, the anti-BTC antibody or antigen-binding fragment thereof prevents BTC from functioning in various ways. In some aspects, the anti-BTC antibody or antigen-binding fragment thereof blocks or reduces the ability of BTC to interact with other substances. For example, in some aspects, the anti-BTC antibody or antigen-binding fragment thereof blocks or reduces the ability of BTC to bind to the ErbB receptor. In other aspects, the anti-BTC antibody or antigen-binding fragment thereof blocks BTC-induced activation of phospho-ERK1 / 2. In some aspects, the anti-BTC antibody or antigen-binding fragment thereof blocks BTC-induced activation of phospho-HER3.

[0364] Certain of the antibodies or antigen-binding fragments thereof provided herein specifically and / or selectively bind to the human BTC shown in SEQ ID NO: 157 or 158. In some aspects, the antibody or antigen-binding fragment thereof selectively binds to the human BTC protein as shown in Example 2 and Table 1. In some aspects, the antibody or antigen-binding fragment thereof specifically and / or selectively binds to at least one residue of SEQ ID NO: 157 selected from the group consisting of G34, H35, F36, S37, R38, C39, P40, K41, Q42, Y43, H45, Y46, R51, R53, F54, V56, A57, E58, Q59, T60, P61, A72, R73, E75 and R76. In some aspects, more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) of the identified BTC residues are part of a region that is bound by the antibody or antigen-binding fragment thereof.

[0365] In some aspects, the antibody or antigen-binding fragment thereof specifically and / or selectively binds to R38, C39, P40, K41, Q42, Y43, H45, Y46, F54, Q59, T60, P61, and R73 of SEQ ID NO: 157, such as NVS1. In some aspects, the antibody or antigen-binding fragment thereof specifically and / or selectively binds to P40, K41, Q42, Y43, H45, Y46, E58, Q59, T60, P61, A72, R73, E75, and R76 of SEQ ID NO: 157, such as NVS2. In some aspects, the antibody or antigen-binding fragment thereof specifically and / or selectively binds to G34, H35, F36, S37, R38, C39, P40, K41, Q42, R51, R53, F54, and V56 of SEQ ID NO: 157, such as NVS3. In some aspects, the antibody or antigen-binding fragment thereof specifically and / or selectively binds to S37, R38, C39, P40, K41, Q42, Y43, H45, Y46, F54, A57, Q59, T60, P61, A72, R73, and E75 of SEQ ID NO: 157, such as NVS4.

[0366] In aspects where the antibody or antigen-binding fragment thereof is used for therapeutic applications, the antibody or antigen-binding fragment thereof can suppress, interfere with, or modulate one or more biological activities of BTC. In one aspect, the antibody or antigen-binding fragment thereof specifically binds to human BTC and / or substantially inhibits the binding of human BTC to the ErbB receptor by at least about 20-40%, 40-60%, 60-80%, 80-85%, or more (e.g., when measuring binding in an in vitro competitive binding assay). In some aspects, the antibody or antigen-binding fragment thereof is characterized by K d , which is less (stronger binding) 10 −7 , 10 −8 , 10, 10 −10 , 10 −11 , 10 −12 , 10 −13 M. In some aspects, the antibody or antigen-binding fragment thereof is characterized by an IC 50to block the binding of the ErbB receptor with BTC of less than 1 microM, from 1000 nM to 100 nM, from 100 nM to 10 nM, from 10 nM to 1 nM, from 1000 pM to 500 pM, from 500 pM to 200 pM, less than 200 pM, from 200 pM to 150 pM, from 200 pM to 100 pM, from 100 pM to 10 pM, from 10 pM to 1 pM.

[0367] In some aspects, the antibody or antigen-binding fragment thereof binds to BTC variants that are at least 50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99% or more percent identical to the BTC form shown under SEQ ID NO: 157 or 158. In some aspects, the antibody or antigen-binding fragment thereof binds to an epitope bound by one of the antibodies described in Table 1. In some aspects, the antibody or antigen-binding fragment thereof binds to a specific conformational state of BTC so as to prevent interaction of BTC with the ErbB receptor.

[0368] The anti-BTC antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region complementarity determining region 1 (HCDR1), a heavy chain variable region complementarity determining region 2 (HCDR2), a heavy chain variable region complementarity determining region 3 (HCDR3), a light chain variable region complementarity determining region 1 (LCDR1), a light chain variable region complementarity determining region 2 (LCDR2), and a light chain variable region complementarity determining region 3 (LCDR3). HCDR1, HCDR2, and HCDR3 are contained in the heavy chain variable region (VH). LCDR1, LCDR2, and LCDR3 are contained in the light chain variable region (VL). In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises the heavy chain and light chain CDRs (e.g., Kabat, Chothia, IMGT, and / or combined CDRs) as shown in Table 1 and described below.

[0369] In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the Kabat numbering scheme. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the Chothia numbering scheme. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the combined numbering scheme. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the IMGT numbering scheme.In one aspect, the anti-BTC antibody or antigen-binding fragment thereof is NVS1 as shown in Table 1. In a particular aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises the heavy chain and light chain CDRs (e.g., Kabat, Chothia, IMGT and / or combined CDRs) of the NVS1 antibody comprising VH and VL, under SEQ ID NO: 10 and 21, respectively.

[0370] In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, according to the Kabat numbering scheme. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, according to the Chothia numbering scheme. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the combined numbering scheme. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the IMGT numbering scheme.In one aspect, the anti-BTC antibody or antigen-binding fragment thereof is NVS2 as shown in Table 1. In a particular aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises the heavy chain and light chain CDRs (e.g., Kabat, Chothia, IMGT and / or combined CDRs) of the NVS2 antibody comprising VH and VL, under SEQ ID NO: 34 and 45, respectively.

[0371] In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, according to the Kabat numbering scheme. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, according to the Chothia numbering scheme. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the combined numbering scheme. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the IMGT numbering scheme.In one aspect, the anti-BTC antibody or antigen-binding fragment thereof is NVS3 as shown in Table 1. In a particular aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises the heavy chain and light chain CDRs (e.g., Kabat, Chothia, IMGT and / or combined CDRs) of the NVS3 antibody comprising VH and VL, under SEQ ID NO: 54 and 65, respectively.

[0372] In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the Kabat numbering scheme. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the Chothia numbering scheme. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the combined numbering scheme. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the IMGT numbering scheme.In one aspect, the anti-BTC antibody or antigen-binding fragment thereof is NVS4 as shown in Table 1. In a particular aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises the heavy chain and light chain CDRs (e.g., Kabat, Chothia, IMGT and / or combined CDRs) of the NVS4 antibody comprising VH and VL, under SEQ ID NO: 78 and 88, respectively.

[0373] Furthermore, the present invention also provides an anti-BTC antibody or antigen-binding fragment thereof comprising amino acid sequences that are homologous to the CDR sequences described throughout and in Table 1, and the anti-BTC antibody or antigen-binding fragment thereof binds to BTC and retains the desired functional properties of the sequences described herein. More specifically, the amino acid sequences of the anti-BTC antibody or antigen-binding fragment thereof may be characterized by greater than or equal to 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with the CDR sequences as described throughout this document and presented in Table 1, and retain their desired functional properties.

[0374] The present invention also provides antibodies to BTC or antigen-binding fragments thereof that are homologous to the VH and VL sequences described herein. More specifically, the present invention provides a protein comprising amino acid sequences that are homologous to sequences such as those described in Table 1, and wherein the antibodies to BTC or antigen-binding fragments bind to a therapeutic target, such as an ophthalmological target, and retain the desired functional properties of those described in Table 1 and the examples.An antibody or antigen-binding fragment thereof comprising VH and VL regions with less than 100% sequence identity to the VH and VL regions described in Table 1 may be produced by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of a nucleic acid molecule described in Table 1, followed by testing the encoded altered antibody for retention of function using the functional assays described herein and in US 20120014958. An antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain with a high (i.e.,80% or greater) identity to the heavy chains and light chains described in Table 1 can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of a nucleic acid molecule encoding such polypeptides, followed by testing the encoded altered antibody for retention of function, e.g., using the functional assays described herein.

[0375] The anti-BTC antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) comprising an amino acid sequence with approximately at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 10 and 21, respectively. It is contemplated that the variability may be in the CDR region or the framework region. In one aspect, an anti-BTC antibody or antigen-binding fragment thereof comprising a VH and VL comprising the amino acid sequence set forth in SEQ ID NOs: 10 and 21, respectively. In another aspect, the anti-BTC antibody or antigen-binding fragment thereof is NVS1, as set forth in Table 1. In another aspect, the VH and VL are encoded by the nucleic acid sequence set forth in SEQ ID NOs: 11 and 22, respectively. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises a VH and VL comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but no more than 10 modifications (e.g., substitutions, e.g.,conservative substitutions) of the amino acid sequence under SEQ ID NO: 10 and 21, respectively. In another aspect, the differences in amino acid sequence are not within the complementarity-determining regions.,

[0376] In one aspect, the anti-BTC antibody or antigen-binding fragment thereof of the present invention comprises a VH and VL comprising an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%,at least 99% or 100% sequence identity to SEQ ID NOs: 34 and 45, respectively. It is contemplated that the variability may be in the CDR region or the framework region. In one aspect, an anti-BTC antibody or antigen-binding fragment thereof comprising a VH and VL comprising the amino acid sequence set forth in SEQ ID NOs: 34 and 45, respectively. In another aspect, the anti-BTC antibody or antigen-binding fragment thereof is NVS2, as set forth in Table 1. In another aspect, the VH and VL are encoded by the nucleic acid sequence set forth in SEQ ID NOs: 35 and 46, respectively. In one aspect, an anti-BTC antibody or antigen-binding fragment thereof comprises a VH and VL comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but no more than 10 modifications (e.g., substitutions, e.g.,conservative substitutions) of the amino acid sequence under SEQ ID NO: 34 and 45, respectively. In another aspect, the differences in amino acid sequence are not within the complementarity-determining regions.,

[0377] In one aspect, the anti-BTC antibody or antigen-binding fragment thereof of the present invention comprises a VH and VL comprising an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%,at least 99% or 100% sequence identity to SEQ ID NOs: 54 and 65, respectively. It is contemplated that the variability may be in the CDR region or the framework region. In one aspect, an anti-BTC antibody or antigen-binding fragment thereof comprising a VH and VL comprising the amino acid sequence set forth in SEQ ID NOs: 54 and 65, respectively. In another aspect, the anti-BTC antibody or antigen-binding fragment thereof is NVS3, as set forth in Table 1. In another aspect, the VH and VL are encoded by the nucleic acid sequence set forth in SEQ ID NOs: 55 and 66, respectively. In one aspect, an anti-BTC antibody or antigen-binding fragment thereof comprises a VH and VL comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but no more than 10 modifications (e.g., substitutions, e.g.,conservative substitutions) of the amino acid sequence under SEQ ID NO: 54 and 65, respectively. In another aspect, the differences in amino acid sequence are not within the complementarity-determining regions.,

[0378] In one aspect, the anti-BTC antibody or antigen-binding fragment thereof of the present invention comprises a VH and VL comprising an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%,at least 99% or 100% sequence identity to SEQ ID NOs: 78 and 88, respectively. It is contemplated that the variability may be in the CDR region or the framework region. In one aspect, an anti-BTC antibody or antigen-binding fragment thereof comprising a VH and VL comprising the amino acid sequence set forth in SEQ ID NOs: 78 and 88, respectively. In another aspect, the anti-BTC antibody or antigen-binding fragment thereof is NVS4, as set forth in Table 1. In another aspect, the VH and VL are encoded by the nucleic acid sequence set forth in SEQ ID NOs: 79 and 89, respectively. In one aspect, an anti-BTC antibody or antigen-binding fragment thereof comprises a VH and VL comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but no more than 10 modifications (e.g., substitutions, e.g.,conservative substitutions) of the amino acid sequence under SEQ ID NO: 78 and 88, respectively. In another aspect, the differences in amino acid sequence are not within the complementarity-determining regions.,

[0379] The anti-BTC antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain comprising an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%,at least 99% or 100% sequence identity to SEQ ID NOs: 12 and 23, respectively. In one aspect, an anti-BTC antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain comprising the amino acid sequence set forth in SEQ ID NOs: 12 and 23, respectively. In one aspect, an anti-BTC antibody can inhibit BTC activity, such as BTC binding to 1) ErbB1; 2) ErbB4; 3) an ErbB homodimer (e.g., ErbB1 / ErbB1 and ErbB4 / ErB4); 4) an ErbB heterodimer (e.g., ErbB1 / ErbB2, ErB1 / ErB3, ErB1 / ErB4, ErB2 / ErB3, and ErB2 / ErB4); and / or 5) can inhibit ERK1 / 2 phosphorylation. In another aspect, the anti-BTC antibody or antigen-binding fragment thereof is NVS1 as shown in Table 1. In another aspect, the heavy chain and the light chain are encoded by a nucleic acid sequence with approximately at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%,at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%,at least 99% or 100% identical or complementary to SEQ ID NO: 13 and 24, respectively. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain encoded by the nucleic acid sequence set forth under SEQ ID NO: 13 and 24, respectively, and is NVS1, as set forth in Table 1.

[0380] In one aspect, the anti-BTC antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain comprising an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 36 and 47, respectively. In one aspect, an anti-BTC antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain comprising the amino acid sequence set forth in SEQ ID NOs: 36 and 47, respectively. In one aspect, an anti-BTC antibody can inhibit BTC activity, such as BTC binding to 1) ErbB1; 2) ErbB4; 3) an ErbB homodimer (e.g., ErbB1 / ErbB1 and ErbB4 / ErB4); 4) an ErbB heterodimer (e.g., ErbB1 / ErbB2, ErB1 / ErB3, ErB1 / ErB4, ErB2 / ErB3, and ErB2 / ErB4); and / or 5) can inhibit ERK1 / 2 phosphorylation. In another aspect, the anti-BTC antibody or antigen-binding fragment thereof is NVS2 as shown in Table 1. In another aspect, the heavy chain and the light chain are encoded by a nucleic acid sequence with approximately at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%,at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%,at least 99% or 100% identical or complementary to SEQ ID NO: 37 and 48, respectively. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain encoded by the nucleic acid sequence set forth under SEQ ID NO: 37 and 48, respectively, and is NVS2, as set forth in Table 1.

[0381] In one aspect, the anti-BTC antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain comprising an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 56 and 67, respectively. In one aspect, an anti-BTC antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain comprising the amino acid sequence set forth in SEQ ID NOs: 56 and 67, respectively. In one aspect, an anti-BTC antibody can inhibit BTC activity, such as BTC binding to 1) ErbB1; 2) ErbB4; 3) an ErbB homodimer (e.g., ErbB1 / ErbB1 and ErbB4 / ErB4); 4) an ErbB heterodimer (e.g., ErbB1 / ErbB2, ErB1 / ErB3, ErB1 / ErB4, ErB2 / ErB3, and ErB2 / ErB4); and / or 5) can inhibit ERK1 / 2 phosphorylation. In another aspect, the anti-BTC antibody or antigen-binding fragment thereof is NVS3 as shown in Table 1. In another aspect, the heavy chain and the light chain are encoded by a nucleic acid sequence with approximately at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%,at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%,at least 99% or 100% identical or complementary to SEQ ID NO: 57 and 68, respectively. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain encoded by the nucleic acid sequence set forth under SEQ ID NO: 57 and 68, respectively, and is NVS3, as set forth in Table 1.

[0382] In one aspect, the anti-BTC antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain comprising an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 80 and 90, respectively. In one aspect, an anti-BTC antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain comprising the amino acid sequence set forth in SEQ ID NOs: 80 and 90, respectively. In one aspect, an anti-BTC antibody can inhibit BTC activity, such as BTC binding to 1) ErbB1; 2) ErbB4; 3) an ErbB homodimer (e.g., ErbB1 / ErbB1 and ErbB4 / ErB4); 4) an ErbB heterodimer (e.g., ErbB1 / ErbB2, ErB1 / ErB3, ErB1 / ErB4, ErB2 / ErB3, and ErB2 / ErB4); and / or 5) can inhibit ERK1 / 2 phosphorylation. In another aspect, the anti-BTC antibody or antigen-binding fragment thereof is NVS4 as shown in Table 1. In another aspect, the heavy chain and the light chain are encoded by a nucleic acid sequence with approximately at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%,at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%,at least 99% or 100% identical or complementary to SEQ ID NO: 81 and 91, respectively. In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain encoded by the nucleic acid sequence set forth under SEQ ID NO: 81 and 91, respectively, and is NVS4, as set forth in Table 1.

[0383] Additionally included within the scope of the present invention are an isolated anti-BTC antibody or an antigen-binding fragment thereof with conservative modifications. More specifically, the present invention relates to anti-BTC antibody-based binding functional units and molecules conjugated to the anti-BTC antibody-based binding functional units with a conservative modification of the anti-BTC antibody-based binding functional units and molecules conjugated to the anti-BTC antibody-based binding functional units from Table 1.In certain aspects, an antibody conjugated to the anti-BTC antibody-based binding functional units of the present invention has a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein one or more of such CDR sequences have the specified amino acid sequences based on the antibodies described herein or conservative modifications thereof, and wherein the antibody retains the desired functional properties of the antibodies of the present invention.

[0384] In a particular aspect, the present document provides polynucleotides whose sequences encode the anti-BTC antibody described herein (e.g., in Table 1) or fragments thereof (e.g., VH or VL). In one aspect, the anti-BTC antibody or antigen-binding fragment thereof is encoded by a polynucleotide whose sequence has been codon-optimized for expression in mammalian cells. In one aspect, the entire construct of the anti-BTC antibody or antigen-binding fragment thereof is encoded by a polynucleotide whose entire sequence has been codon-optimized for expression in mammalian cells, such as human cells.In other aspects, the anti-BTC antibody or antigen-binding fragment thereof is optimized for expression in mammalian cells and has a full-length heavy chain sequence and a full-length light chain sequence, wherein one or more of such sequences have the specified amino acid sequences based on the antibodies described herein or conservative modifications thereof, and wherein the binding functional units based on the anti-BTC antibody retain the desired functional properties of the BTC-binding antibodies of the present invention.Accordingly, the present invention provides an isolated antibody or antigen-binding fragment thereof optimized for expression in mammalian cells, comprising, for example, VH and VL, wherein VH comprises the amino acid sequence of SEQ ID NOs: 10, 34, 54 and 78, and conservative modifications thereof, and VL comprises the amino acid sequence of SEQ ID NOs: 21, 45, 65 and 88, and conservative modifications thereof, that specifically bind to BTC.

[0385] The present invention provides binding functional units based on an antibody to BTC (e.g., antibodies binding to BTC, or fragments thereof) that bind to the same or an overlapping epitope as the antibody to BTC or antigen-binding fragment thereof described in Table 1. Additional antibodies can therefore be identified based on their ability to compete (e.g., competitively inhibit binding in a statistically significant manner) with other antibodies of the present invention in BTC binding assays. The ability of a test antibody to inhibit binding of the molecules of the present invention to BTC demonstrates that the test molecule can compete with that antibody for binding to BTC; such an antibody may, according to a non-limiting theory, bind to the same or a related (e.g., structurally similar or spatially close) epitope on BTC as the antibody with which it competes.In a certain aspect, a molecule that binds to the same epitope on BTC as the antibody of the present invention is a human monoclonal antibody, Fab, or scFv. Such human monoclonal antibodies, Fab, and scFv can be produced and isolated as described herein.

[0386] In one aspect, a molecule that competes with an anti-BTC antibody or antigen-binding fragment thereof of the present invention binds to at least one residue of SEQ ID NO: 157 selected from the group consisting of G34, H35, F36, S37, R38, C39, P40, K41, Q42, Y43, H45, Y46, R51, R53, F54, V56, A57, E58, Q59, T60, P61, A72, R73, E75, and R76. In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that is capable of competing with those described in Table 1, for example, NVS1, NVS2, NVS3, and NVS4, to bind to BTC and reduce BTC-mediated signaling. In another aspect, the competing antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain as set forth in SEQ ID NOs: 168-189 in Table 5.

[0387] The anti-BTC antibody or antigen-binding fragment thereof of the present invention is in a format selected from the group consisting of an isolated antibody, Fab, Fab', F(ab')2, Fv and scFv. In a preferred aspect, the anti-BTC antibody or antigen-binding fragment thereof is a Fab, including a Fab comprising an Fc region. In another aspect, the Fc region is selected from the group consisting of an Fc region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD. In one aspect, the Fc region comprises a human immunoglobulin kappa chain constant region sequence as set forth in SEQ ID NO: 159. In another aspect, the Fc region comprises the first constant domain of a human immunoglobulin heavy chain (CH1 domain) as set forth in SEQ ID NO: 160.

[0388] In one aspect, the anti-BTC antibody or antigen-binding fragment thereof is an isolated antibody, such as a human monoclonal antibody or a humanized monoclonal antibody. In certain aspects, the anti-BTC antibodies may be in scFv or Fab format. In certain aspects, the anti-BTC antibodies may be in scFv or Fab format. Table 1. Exemplary anti-BTC Fabs NVS1 SEQ ID NO: 1 (combined) HCDR1 GGTFSSYAIS SEQ ID NO: 2 (combined) HCDR2 GIVPWMGEAVYAQKFQG SEQ ID NO: 3 (combined) HCDR3 SSSTYGIHAFDY SEQ ID NO: 4 (Related) HCDR1 SIX SEQ ID NO: 2 (Related) HCDR2 GIVPWMGEAVYAQKFQG SEQ ID NO: 3 (Related) HCDR3 SSSTYGIHAFDY SEQ ID NO: 5 (Chothia) HCDR1 GGTFSSY SEQ ID NO: 6 (Chothia) HCDR2 VPWMGE SEQ ID NO: 3 (Chothia) HCDR3 SSSTYGIHAFDY SEQ ID NO: 7 (IMGT) HCDR1 GGTFSSYA SEQ ID NO: 8 (IMGT) HCDR2 IVPWMGEA SEQ ID NO: 9 (IMGT) HCDR3 ARSSSTYGIHAFDY SEQ ID NO: 10 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIVPWMGEAVYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSSSTYGIHAFDYWGQGTLVTVSS SEQ ID NO: 11 DNA encoding VH CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAAAACCGGGCAGGCGTGAAAGTTAGCTGCAAAGCATCCGGAGGGACGTTTAGCAGCTATGCGATTAGCTGGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTGGGGGGCGTATCGTTCCGTGGATGGGGAAGCTGTTGTTCG CCCAGAAATTTCAGGGCCGGGTGACCATTACCGCCGATGAAAGCACCAGCACCGCCTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACGGCCGTGTTATTGCGCGCGTTCTTTCTACTTACGGTATCCATGCTTTCGATTACTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCA SEQ ID NO: 12 Heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGGTTFSSYAISWVRQAPGQGLEWMGGIVPWMGEAVYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSSSTYGIHAFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTCGVHTFPAVLQSSGLYSLSSVVTVPSSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID NO: 13 DNA encoding the heavy chain CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAAAACCGGGCAGGCGTGAAAGTTAGCTGCAAAGCATCCGGAGGGACGTTTAGCAGCTATGCGATTAGCTGGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTGGGGGCGGTATCGTTCCGTGGATGGGGC GAAGCTGTTTACGCCCAGAAATTTCAGGGCCGGGTGACCATTACCGCCGATGAAAGCACCAGCACCGCCTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACGGCCGTGTTATTGCGCGCGTTCTTCTCTTACTTACGGTATCCATGCTTTCGATTACTGGGGC CAAGGCACCCTGGTGACTGTTAGCTCAGCCTCCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGGCGGCACAGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACC TCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGTGT SEQ ID NO: 14 (combined) LCDR1 RASQSISNFLN SEQ ID NO: 15 (combined) LCDR2 AASNLQS SEQ ID NO: 16 (combined) LCDR3 QQYDDFPMT SEQ ID NO: 14 (Related) LCDR1 RASQSISNFLN SEQ ID NO: 15 (Related) LCDR2 AASNLQS SEQ ID NO: 16 (Related) LCDR3 QQYDDFPMT SEQ ID NO: 17 (Chothia) LCDR1 SQSISNF SEQ ID NO: 18 (Chothia) LCDR2 AAS SEQ ID NO: 19 (Chothia) LCDR3 YDDFPM SEQ ID NO: 20 (IMGT) LCDR1 QSISNF SEQ ID NO: 18 (IMGT) LCDR2 AAS SEQ ID NO: 16 (IMGT) LCDR3 QQYDDFPMT SEQ ID NO: 21 VL DIQMTQSPSSLSASVGDRVTITCRASQSISNFLNWYQQKPGKAPKLLIAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDDFPMTFGQGTKVEIK SEQ ID NO: 22 DNA encoding VL gatatccagatgacccagagcccgagcagcctgagcgccagcgtgggcgcgtgaccattacctgcagagccagccagcttatttctaacttcctgaactggtaccagcagaaccgggcaaagcgcgaacttattaatctacgctgctctaacc tgcaaagcggcgtgccgagccgctttagcggcagcggatccggcaccgatttcacctgaccattagctctctgcaaccggaagactttgcgacctattattgccagcagtacgacgacttcccgatgaccttggccaggcacgaaagttgaaattaaa SEQ ID NO: 23 Light chain DIQMTQSPSSLSASVGDRVTITCRASQSISNFLNWYQQKPGKAPKLLIAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDDFPMTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 24 DNA encoding the light chain gatatccagatgacccagagcccgagcagcctgagcgccagcgtgggcgcgtgaccattacctgcagagccagccagcttatttctaacttcctgaactggtaccagcagaaccgggcaaagcgcgaacttattaatctacgctgctctaacc tgcaaagcggcgtgccgagccgctttagcggcagcggatccggcaccgatttcacctgaccattagctctctgcaaccggaagactttgcgacctattattgccagcagtacgacgacttcccgatgaccttggccaggcacgaaagttgaaattaaa cgtacggtggccgctcccagcgtgttcatcttcccccccgacgagcagctgaagagcggcaccgcgtggtgcctgctgaaacctctccccggggccaaggtgcagtggaaggtggacaacgccctgcagcggcaaccagg aaagcgtcaccgagcaggacagcagactcacctacagcctgagcagcctgaccctgagcgaggtgacccagggcctgtccagccccgtgaccagagcttcaacggggcgagtgt NVS2 SEQ ID NO: 25 (combined) HCDR1 GFTFSSYAMS SEQ ID NO: 26 (combined) HCDR2 AISGSGGSTYYADSVKG SEQ ID NO: 27 (combined) HCDR3 QRYYFGEFDL SEQ ID NO: 28 (Related) HCDR1 SYAMS SEQ ID NO: 26 (Related) HCDR2 AISGSGGSTYYADSVKG SEQ ID NO: 27 (Related) HCDR3 QRYYFGEFDL SEQ ID NO: 29 (Chothia) HCDR1 GFTFSSY SEQ ID NO: 30 (Chothia) HCDR2 SGSGGS SEQ ID NO: 27 (Chothia) HCDR3 QRYYFGEFDL SEQ ID NO: 31 (IMGT) HCDR1 GFTFSSYA SEQ ID NO: 32 (IMGT) HCDR2 ISGSGGST SEQ ID NO: 33 (IMGT) HCDR3 ARQRYYFGEFDL SEQ ID NO: 34 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQRYYFGEFDLWGQGTLVTVSS SEQ ID NO: 35 DNA encoding VH GAAGTGCAGCTGCTGGAAAGCGGTGGCGGTCTGGTGCAGCCAGGTGGTAGCCTGCGCCTGAGCTGTGCCGCAAGCGGCTTTACCTTTAGCAGCTATGCCATGAGCTGGGTGCGCCAAGCACCAGGCAAAGGCCTGGAATGGGTGAGCGCTTAGCGGCAGCGGTGGCAGCACCTATT ATGCCGATAGCGTGAAAGGTCGCTTTACCATTAGTCGCGATAACAGCAAAAACACCCTGTATCTGCAAATGAACAGCCCTGCGGGCAGAAGATACCGCAGTTTTATTGCGCGCGACAACGTTACTACTTCGGTGAGTTCGACCTGTGGGGCCAGGGCACCCTGGTTACTGTCTCGAGC SEQ ID NO: 36 Heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQRYYFGEFDLWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTCGVHTFPAVLQSSGLYSLSSVVTVPSSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID NO: 37 DNA encoding the heavy chain GAAGTGCAGCTGCTGGAAAGCGGTGGCGGTCTGGTGCAGCCAGGTGGTAGCCTGCGCCTGAGCTGTGCCGCAAGCGGCTTTACCAGCTATGCCATGAGCTGGGTGCGCCAAGCACCAGGCAAAGGCCTGGAATGGGTGAGCGCCATTAGCGGCAGCGGTG GCAGCACCTATTATGCCGATAGCGTGAAAGGTCGCTTTACCATTAGTCGCGATAACAGCAAAAACACCCTGTATCTGCAAATGAACAGCCCTGCGGGCAGAAGATACCGCAGTTTTATTGCGCGCGACAACGTTACTACTTCGGTGAGTTCGACCTGTGGGGCCAG GGCACCCTGGTTACTGTCTCGAGCGCCAGCACAAAGGGACCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGTCTACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCTCTGACAA GCGGCGTGCACACCTTTCCAGCCGTGCTCCAGAGCAGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTATCTGCAACGTGAACCACAAGCCCAAGCAACACAAAGGTGGACAAGCGGTGGAACCCAAGTCCTGC SEQ ID NO: 38 (combined) LCDR1 SGDKLGDKYAY SEQ ID NO: 39 (combined) LCDR2 QDSKRPS SEQ ID NO: 40 (combined) LCDR3 QAFDYLYSLGV SEQ ID NO: 38 (Related) LCDR1 SGDKLGDKYAY SEQ ID NO: 39 (Related) LCDR2 QDSKRPS SEQ ID NO: 40 (Related) LCDR3 QAFDYLYSLGV SEQ ID NO: 41 (Chothia) LCDR1 DKLGDKY SEQ ID NO: 42 (Chothia) LCDR2 QDS SEQ ID NO: 43 (Chothia) LCDR3 FDYLYSLG SEQ ID NO: 44 (IMGT) LCDR1 KLGDKY SEQ ID NO: 42 (IMGT) LCDR2 QDS SEQ ID NO: 40 (IMGT) LCDR3 QAFDYLYSLGV SEQ ID NO: 45 VL SYELTQPPSVSVSPGQTASITCSGDKLGDKYAYWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQAFDYLYSLGVFGGGTKLTVL SEQ ID NO: 46 DNA encoding VL agctatgaactgacccagccgcgaggttagcgttagcccaggccagacgccagcattacctgtagcggcgaaactgggcgaaatacgcctactggtatcagcagaaaccgggccagagcccggtgctggttatcttatcagatagcaaacgccg agcggcattccagaacgctttagcggcagcaacagcggcaaccgccaccctgaccattagcggcacccaggcgaagacgaagccgattactgtcaggctttcgactacctgtattccctgggtgtgtttggcggcggtaccaagctgaccgtgctg SEQ ID NO: 47 Light chain SYELTQPPSVSVSPGQTASITCSGDKLGDKYAYWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQAFDYLYSLGVFGGGTKLTV LGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 48 DNA encoding the light chain agctatgaactgacccagccgccgaggttagcgttagcccaggccagacgccagcattacctgtagcggcgaaactgggcgaaatacgcctactggtatcagcagaaaccgggccagagcccggtgctggttatctatcaggatagcaaacgcc cgagcggcattccagaacgctttagcggcagcaacagcggcaaccgccaccctgaccattagcggcacccaggccgaagacgaagccgattactgtcaggctttcgactacctgtattccctgggtgtgtttggcggcggtaccaagctgaccgtg ctgggccagcccaaagccgccctagcgtgaccctgttccccccaagcagcgaggaactccaggccaaaggccaccctcgtgtgcctgatcagcgacttctaccctggcgccgtgaccgtggcctggaaggccgatagcagccctgtgaaggcggg tggaaccaccacccccagcagcagcaacaaatacgccgccagctacctgaccccgagcagtggaagtcccacagatctacagctgccaggtcacacacgagggcagcaccgtggaaagaccgtggcccaccgagtgcagc NVS3 SEQ ID NO: 25 (combined) HCDR1 GFTFSSYAMS SEQ ID NO: 49 (combined) HCDR2 GLGHVGYTTYTDSVKG SEQ ID NO: 50 (combined) HCDR3 DYLDFGYYFDV SEQ ID NO: 28 (Related) HCDR1 SYAMS SEQ ID NO: 49 (Related) HCDR2 GLGHVGYTTYTDSVKG SEQ ID NO: 50 (Related) HCDR3 DYLDFGYYFDV SEQ ID NO: 29 (Chothia) HCDR1 GFTFSSY SEQ ID NO: 51 (Chothia) HCDR2 GHVGY SEQ ID NO: 50 (Chothia) HCDR3 DYLDFGYYFDV SEQ ID NO: 31 (IMGT) HCDR1 GFTFSSYA SEQ ID NO: 52 (IMGT) HCDR2 LGHVGYT SEQ ID NO: 53 (IMGT) HCDR3 ARDYLDFGYYFDV SEQ ID NO: 54 VH QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGLGHVGYTTYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYLDFGYYFDVWWGQGTLVTVSS SEQ ID NO: 55 DNA encoding VH caggtgcagctgctggaatcaggcggcggactggtgcagcctgcggtagcctgactgactgcgctgctgcttcaccttctagctacgctatgagctgggtcggcaggcccctggcaaaggcctgggctcggctggctcgcgtgggctactactaca ccgatagcgtgaagggccggttcactatctctagggataactctaagaacaccctgtacctgcagatgaatagcctgagagccgaggacaccgctctactactggctagactacctggacttcggctactacttcgacgtgtggggggggcacctggtcacgtgtctagc SEQ ID NO: 56 Heavy chain QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGLGHVGYTTYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYLDFGYYFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTCGVHTFPAVLQSSGLYSLSSVVTVPSSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID NO: 57 DNA encoding the heavy chain caggtgcagctgctggaatcaggcggcggactggtgcagcctgcggtagcctgactgactgcgctgctgcttcaccttctagctacgctatgagctgggtccggcaggcccctggcaaaggcctgggctcggactgggtcacgtgggct acactacctacaccgatagcgtgaagggccggttcactatctctctagggataactctaagaacaccctgtacctgcagatgaatagcctgagagcgaggacaccgctctactactgcgctagactacctggacttcggctactactcgacgtgtggggccag ggcaccctggtcaccgtgtctagcgctagcactaagggcccctcgtgttcctctgcccttccagcaagtctacctggcggcaccgctgctgggctggtgaaggactctccctgagcctgtgacagtcctggaactctggcgcctgcctc ccggcgtgcacaccttccctgccgtgctgcagtcctcggcctgtactccctgtcctccgtggtgcaacgtgaaccacaagccttccacaccaaggtggacaagcgggtggagcctaagtcatgc SEQ ID NO: 58 (combined) LCDR1 SGDKIGKKYVH SEQ ID NO: 59 (combined) LCDR2 DDSDRPS SEQ ID NO: 60 (combined) LCDR3 QAWDMQSVV SEQ ID NO: 58 (Related) LCDR1 SGDKIGKKYVH SEQ ID NO: 59 (Related) LCDR2 DDSDRPS SEQ ID NO: 60 (Related) LCDR3 QAWDMQSVV SEQ ID NO: 61 (Chothia) LCDR1 DKIGKKY SEQ ID NO: 62 (Chothia) LCDR2 DDS SEQ ID NO: 63 (Chothia) LCDR3 WDMQSV SEQ ID NO: 64 (IMGT) LCDR1 KIGKKY SEQ ID NO: 62 (IMGT) LCDR2 DDS SEQ ID NO: 60 (IMGT) LCDR3 QAWDMQSVV SEQ ID NO: 65 VL SYELTQPLSVSVALGQTARITCSGDKIGKKYVHWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISRAQAGDEADYYCQAWDMQSVVFGGGTKLTVL SEQ ID NO: 66 DNA encoding VL agctacgagctgactcagcccctgagcgtcagcgtggccctgggccagaccgctagaatcacctgtagcggcgataagatcggcaagaatacgtgcactggtatcagcccggccaggccccgtgctggtcatctacgacgatagcgataga cctagcggaatccccgagcggtttagcggctaatatagcggcaaccgcctgactatctctctagggctcaggccggcgacgacgactactactgtcaggcctggatatgcagtcagtggtcggcggcactaagctgaccgtgctg SEQ ID NO: 67 Light chain SYELTQPLSVSVALGQTARITCSGDKIGKKYVHWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISRAQAGDEADYYCQAWDMQSVVFGGGTKLTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 68 DNA encoding the light chain agctacgagctgactcagcccctgagcgtcagcgtggccctgggccagaccgctagaatcacctgtagcggcgataagatcggcaagaatacgtgcactggtatcagcccggccaggccccgtgctggtcatctacgacgatagcgataga cctagcggaatccccgagcggtttagcggctaatatagcggcaaccgcctgactatctctctagggctcaggccggcgacgacgactactactgtcaggcctggatatgcagtcagtggtcggcggcactaagctgaccgtgctg ggccagcctaaggctgcccccagcgtgaccctgttccccccgcagcgaggagctgcaggccaaaggccaccctgtgtgcctgatcgcgactctacccaggcgccgtgaccgtggcctggaaggccgacagcccgtgaaggcggcgtg gagaccaccacccccagcaagcagagcaacaagtacgccgccagctacctgaccccgagcagtggaagagccaggctacagctgcaggtgacccacgagggcagcgtggaaagaccgtggcccacgagcagcagc NVS4 SEQ ID NO: 69 (combined) HCDR1 GFTFSRYWIS SEQ ID NO: 70 (combined) HCDR2 YIDSTGTFINYADSVKG SEQ ID NO: 71 (combined) HCDR3 GGSLFDY SEQ ID NO: 72 (Related) HCDR1 RYWIS SEQ ID NO: 70 (Related) HCDR2 YIDSTGTFINYADSVKG SEQ ID NO: 71 (Related) HCDR3 GGSLFDY SEQ ID NO: 73 (Chothia) HCDR1 GFTFSRY SEQ ID NO: 74 (Chothia) HCDR2 DSTGTF SEQ ID NO: 71 (Chothia) HCDR3 GGSLFDY SEQ ID NO: 75 (IMGT) HCDR1 GFTFSRYW SEQ ID NO: 76 (IMGT) HCDR2 IDSTGTFI SEQ ID NO: 77 (IMGT) HCDR3 ARGGSLFDY SEQ ID NO: 78 VH QVQLLESGGGLVQPGGSLRLSCAASGFTFSRYWISWVRQAPGKGLEWVSYIDSTGTFINYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSLFDYWGQGTLVTVSS SEQ ID NO: 79 DNA encoding VH caggtgcagctgctggaatcaggcggcggactggtgcagcctgcggtagcctgactgactgcgctgtagtgcttcaccttctctaggtactgattagctgggtcggcaggcccctggcaaaggcctggggctctctatatcgactctacggcggcttatt aactacgccgataggtcgaagggccggttcactatctctctagggataactctaagaacaccctgtacctgcagatgaatagcctgagagccgaggacaccgctctactactgcgctagaggcggtagtctgttcgactactggggccagggcaccctggtcaccgtgctagc SEQ ID NO: 80 Heavy chain QVQLLESGGGLVQPGGSLRLSCAASGFTFSRYWISWVRQAPGKGLEWVSYIDSTGTFINYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSLFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID NO: 81 DNA encoding the heavy chain caggtgcagctgctggaatcaggcggcggactggtgcagcctgcggtagcctgactgactgcgctgctgcttcaccttctaggtactgattagctgggtcggcaggcccctggcaaaggcctggggctctatatatcgactctaccgg caccttattaactacgccgatagcgtgaagggccggttcactatctctagggataactctaagaacaccctgtacctgcagatgaatagcctgagagcgaggacaccgctctactactgcgctagaggcggtagtctgttcgactactggggccaggc ccctggtcaccgtgtctagcgctagcactaagggcccctccgtgttcctctggcccttccagcaagtctacctggcggcacctgctctgggctgtgaaggactctccctgagcctgtgacagtcgtcgcttggcgcctgacctc ggcgtgcacaccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtggtgcaacgtgaaccacaagccttccacaccaaggtggacaagcgggtggagcctaagtcatgc SEQ ID NO: 82 (combined) LCDR1 RASQGIISYLG SEQ ID NO: 83 (combined) LCDR2 AASSLQS SEQ ID NO: 84 (combined) LCDR3 QQYDALNT SEQ ID NO: 82 (Related) LCDR1 RASQGIISYLG SEQ ID NO: 83 (Related) LCDR2 AASSLQS SEQ ID NO: 84 (Related) LCDR3 QQYDALNT SEQ ID NO: 85 (Chothia) LCDR1 SQGIISY SEQ ID NO: 18 (Chothia) LCDR2 AAS SEQ ID NO: 86 (Chothia) LCDR3 YDALN SEQ ID NO: 87 (IMGT) LCDR1 QGIISY SEQ ID NO: 18 (IMGT) LCDR2 AAS SEQ ID NO: 84 (IMGT) LCDR3 QQYDALNT SEQ ID NO: 88 VL DIQMTQSPSSLSASVGDRVTITCRASQGIISYLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDALNTFGQGTKVEIK SEQ ID NO: 89 DNA encoding VL gatattcagatgactcagtcacctagtagctgagcgctagtgtgggcgatagagtgactatcacctgtagacctctcaggggattattagctacctgggctggtatcagcccggcaaagcccctaagctgctgatctacgccgcctagc ctgcagtcaggcgtgccctctaggtttagcggtagggtagtggcaccgacttcaccctgacttagtagcctgcagccgaggactcgctacctactgtcagcagtacgacgccctgaacaccttcggccaggcactaaggtcgagattaag SEQ ID NO: 90 Light chain DIQMTQSPSSLSASVGDRVTITCRASQGIISYLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDALNTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 91 DNA encoding the light chain gatattcagatgactcagtcacctagtagctgagcgctagtgtgggcgatagagtgactatcacctgtagacctctcaggggattattagctacctgggctggtatcagcccggcaaagcccctaagctgctgatctacgccgcctagc ctgcagtcaggcgtgccctctaggtttagcggtaggtagtagtgcaccgacttcaccctgacttagtagcctgcagccgaggactcgctacctactgtcagcagtacgacgccctgaacaccttcggccaggcactaaggtcgagattaagc gtacggtggccgctcccagcgtgttcatcttcccccccgacgacgctgaagagcggcaccgcgtggtgcctgctgaaacttctccccggggaggccaaggtgcagtggaaggtggacaacgccctgcagcggcaaccagg gagcgtcaccgagcaggacagcaaggactccacctacagcctgagcagcaccctgacctgagcgactacgagagaagcataaggtgtacgccgaggtgacccagggcctgtccagccccgtgaccagagcttcacaggggcgagtgc Binding partner of the antibody to BTC

[0389] As mentioned above, the present invention includes compositions comprising one or more antibodies to BTC or antigen-binding fragments thereof conjugated (e.g., covalently linked, non-covalently linked, or fused) to a functional unit that binds a therapeutic target specific for a target located in or near a tissue containing BTC (e.g., significant levels of BTC, such as the eye, pancreas, etc.). For example, the present invention includes compositions with one or more antibodies to BTC or antigen-binding fragments thereof attached to a functional unit that binds a therapeutic target suitable for the treatment of BTC-associated conditions or diseases (e.g., DR, DME, AMD, such as neovascular AMD, and / or RVO).In another aspect, the present invention includes a pharmaceutical composition comprising an anti-BTC antibody or an antigen-binding fragment thereof and a functional unit binding a therapeutic target, and the pharmaceutical composition can be used in the treatment of BTC-mediated conditions or diseases. In yet another aspect, the present invention includes a method of treating a subject in need thereof, comprising administering to the subject an effective amount of an anti-BTC antibody or an antigen-binding fragment thereof according to the present invention, followed by administering to the subject a functional unit binding a therapeutic target (e.g., a VEGF inhibitor), wherein the subject has BTC-mediated conditions or diseases.

[0390] In certain preferred aspects, the therapeutic target-binding functional unit is an antibody or antigen-binding fragment thereof that binds the therapeutic target (e.g., in the format of an scFv, Fab, single-domain antibody, or diabody), or a polypeptide that binds the therapeutic target (e.g., a soluble receptor). Such therapeutic targets may be, for example, those associated with an ophthalmological disorder, such as VEGF, PDGF, PDGF-BB, angiopoietin, angiopoietin-2, S1P, integrins αvβ3, αvβ5, α5β1, apelin / APJ, erythropoietin, complement factor D, TNFα, C2, factor B, factor H, factor P, CFHR3, C1q, C3, C3b, C5, C5a, C3a, HtrA1, ARMS2, EPO, EPOR, TIMP3, HLA, IL8, CX3CR1, TLR3, TLR4, CETP, LIPC, COL10A1, IL-1β, IL-17A, FGFR2 and TNFRSF10A. Additional therapeutic targets include factor P, factor D, IL-6, IL-12, IL-18, bFGF, MCP-1, CD132, IL-6R, CD20, and IGF-1.

[0391] In one aspect, the present invention includes multispecific binding molecules comprising at least one of a binding functional unit based on an antibody to BTC and one or more functional units that bind a therapeutic target.Accordingly, in one aspect, the present invention includes, for example, bispecific molecules (e.g., bispecific antibodies) characterized by combinations of binding selectivities selected from the following: BTC and VEGF, BTC and PDGF, BTC and PDGF-BB, BTC and angiopoietin, BTC and angiopoietin-2, BTC and S1P, BTC and itegrin αvβ3, BTC and αvβ5, BTC and α5β1, BTC and apelin / APJ, BTC and erythropoietin, BTC and complement factor D, BTC and TNFα, BTC and C2, BTC and factor B, BTC and factor H, BTC and CFHR3, BTC and C1q, BTC and C3, BTC and C3b, BTC and C5, BTC and C5a, BTC and C3a, BTC and HtrA1, BTC and ARMS2, BTC and EPO, BTC and EPOR, BTC and TIMP3, BTC and HLA, BTC and IL8, BTC and CX3CR1, BTC and TLR3, BTC and TLR4, BTC and CETP, BTC and LIPC, BTC and COL10A1, BTC and IL-1β, BTC and IL-17A, BTC and FGFR2, BTC and TNFRSF10A, BTC and factor P, BTC and factor D, BTC and IL-6, BTC and IL-12, BTC and IL-18, BTC and bFG, BTC and MCP-1, BTC and CD132, BTC and IL-6R, BTC and CD20, or BTC and IGF-1.

[0392] In one aspect, the functional unit that binds the therapeutic target may be an anti-VEGF antibody antagonist. In one aspect, the anti-VEGF antibody antagonist is ranibizumab (Lucentis®; WO 98 / 45331; WO 98 / 45331; US ​​6884879; US 6407213; US 7060269; US 7365166). In one aspect, the anti-VEGF antibody antagonist is bevacizumab (Avastin®; US 6054297; US 7169901; US ​​7375193; US 7297334). In one aspect, the anti-VEGF antibody antagonist is aflibercept (Eylea®; US 7,279,159). In one aspect, the anti-VEGF antibody antagonist is brolucizumab (Beovu®; WO 2009 / 155724; US 8,349,322; US 9,090,684; US 9,873,737; WO 03 / 097697; WO 2016 / 073915; WO / 2016 / 073918). In one aspect, the anti-VEGF antibody antagonist is pegaptanib (Macugen®). In one aspect, the anti-VEGF antibody antagonist is KH902 (WO 2005 / 121176; US 7,750,138).In one aspect, the anti-VEGF antibody antagonist comprises a heavy chain and a light chain as set forth in SEQ ID NOs: 103 and 114, respectively. In one aspect, the anti-VEGF antibody antagonist is encoded by a nucleic acid sequence as set forth in SEQ ID NOs: 104 and 115. Multispecific binding molecule.

[0393] The present invention also provides a multispecific binding molecule comprising 1) a binding functional unit based on an antibody to BTC and 2) a binding functional unit based on an antibody to VEGF.The binding functional unit based on an antibody to BTC

[0394] In one aspect, the anti-BTC antibody binding functional unit binds to human BTC as shown in SEQ ID NO: 157 or 158. In some aspects, the anti-BTC antibody binding functional unit selectively binds to human BTC protein as illustrated in Example 2 and Table 4. In some aspects, the anti-BTC antibody binding functional unit specifically and / or selectively binds to at least one residue of SEQ ID NO: 157 selected from the group consisting of G34, H35, F36, S37, R38, C39, P40, K41, Q42, Y43, H45, Y46, R51, R53, F54, V56, A57, E58, Q59, T60, P61, A72, R73, E75 and R76. In some aspects, more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) of the identified BTC residues are part of a region that is bound by a BTC antibody-based binding functional unit.In specific aspects, such an anti-BTC antibody-based binding functional unit selectively binds to a human BTC protein and inhibits (e.g., partially inhibits) BTC activity.

[0395] In some aspects, the anti-BTC antibody-based binding functional unit specifically and / or selectively binds to R38, C39, P40, K41, Q42, Y43, H45, Y46, F54, Q59, T60, P61, and R73 of SEQ ID NO: 157, for example, NVS11. In some aspects, the antibody or antigen-binding fragment thereof specifically and / or selectively binds to P40, K41, Q42, Y43, H45, Y46, E58, Q59, T60, P61, A72, R73, E75, and R76 of SEQ ID NO: 157, for example, NVS12. In some aspects, the antibody or antigen-binding fragment thereof specifically and / or selectively binds to G34, H35, F36, S37, R38, C39, P40, K41, Q42, R51, R53, F54, and V56 of SEQ ID NO: 157, such as NVS13. In some aspects, the antibody or antigen-binding fragment thereof specifically and / or selectively binds to S37, R38, C39, P40, K41, Q42, Y43, H45, Y46, F54, A57, Q59, T60, P61, A72, R73, and E75 of SEQ ID NO: 157, such as NVS14.In one aspect, the anti-BTC antibody-based binding functional unit is an anti-BTC antibody or antigen-binding fragment thereof, as described throughout this document. In particular aspects, such an anti-BTC antibody-based binding functional unit selectively binds to the human BTC protein and inhibits (e.g., partially inhibits) BTC activity.

[0396] In aspects where the anti-BTC antibody-based binding functional unit is used for therapeutic applications, the anti-BTC antibody-based binding functional unit can suppress, interfere with, or modulate one or more biological activities of BTC. In one aspect, the anti-BTC antibody-based binding functional unit specifically binds to human BTC and / or significantly suppresses the binding of human BTC to the ErbB receptor by at least about 20-40%, 40-60%, 60-80%, 80-85%, or more (e.g., when measuring binding in an in vitro competitive binding assay). In some aspects, the above-mentioned anti-BTC antibody-based binding functional unit is characterized by K d , which is less (stronger binding) 10 −7 , 10 −8 , 10, 10 −10 , 10 −11 , 10 −12 , 10 −13M. In some aspects, the anti-BTC antibody-based binding functional unit is characterized by an IC 50 for blocking the binding of the ErbB receptor to BTC of less than 1 microM, from 1000 nM to 100 nM, from 100 nM to 10 nM, from 10 nM to 1 nM, from 1000 pM to 500 pM, from 500 pM to 200 pM, less than 200 pM, from 200 pM to 150 pM, from 200 pM to 100 pM, from 100 pM to 10 pM, from 10 pM to 1 pM. In particular aspects, such an anti-BTC antibody-based binding functional unit selectively binds to the human BTC protein and inhibits BTC activity (e.g., partially inhibits).

[0397] In some aspects, the anti-BTC antibody-based binding functional unit binds to BTC variants that are at least about 50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99% or more percent identical to the BTC form provided under SEQ ID NO: 157 or 158. In some aspects, the anti-BTC antibody-based binding functional unit binds to an epitope bound by one of the antibodies described in Table 3. In some aspects, the anti-BTC antibody-based binding functional unit binds to a specific conformational state of BTC so as to prevent interaction of BTC with the ErbB receptor.

[0398] The anti-BTC antibody-based binding functional unit of the multispecific binding molecule of the present invention comprises a heavy chain variable region complementarity determining region 1 (HCDR1), a heavy chain variable region complementarity determining region 2 (HCDR2), a heavy chain variable region complementarity determining region 3 (HCDR3), a light chain variable region complementarity determining region 1 (LCDR1), a light chain variable region complementarity determining region 2 (LCDR2), and a light chain variable region complementarity determining region 3 (LCDR3). HCDR1, HCDR2, and HCDR3 are contained in the heavy chain variable region (VH). LCDR1, LCDR2, and LCDR3 are contained in the light chain variable region (VL).In one aspect, the anti-BTC antibody or antigen-binding fragment thereof comprises the heavy chain and light chain CDRs (e.g., Kabat, Chothia, IMGT, and / or combined CDRs) shown in Table 3 and described below.

[0399] In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the Kabat numbering scheme. In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the Chothia numbering scheme. In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the combined numbering scheme. In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the IMGT numbering scheme.In one aspect, the anti-BTC antibody-based binding functional unit is contained in NVS11 as shown in Table 3.

[0400] In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the Kabat numbering scheme. In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the Chothia numbering scheme. In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the combined numbering scheme. In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the IMGT numbering scheme.In one aspect, the anti-BTC antibody-based binding functional unit is contained in NVS12 as shown in Table 3.

[0401] In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the Kabat numbering scheme. In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the Chothia numbering scheme. In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the combined numbering scheme. In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the IMGT numbering scheme.In one aspect, the anti-BTC antibody-based binding functional unit is contained in NVS13 as shown in Table 3.

[0402] In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the Kabat numbering scheme. In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the Chothia numbering scheme. In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the combined numbering scheme. In one aspect, the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, according to the IMGT numbering scheme.In one aspect, the anti-BTC antibody-based binding functional unit is contained in NVS14 as shown in Table 3.

[0403] Furthermore, the present invention also provides an anti-BTC antibody-based binding functional unit comprising amino acid sequences homologous to the CDR sequences described throughout and in Table 3, and wherein the anti-BTC antibody-based binding functional unit binds to BTC and retains the desired functional properties of the sequences described herein. More specifically, the amino acid sequences of the anti-BTC antibody-based binding functional unit may have greater than or equal to 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with the CDR sequences as described throughout and shown in Table 3, and retain their desired functional properties.

[0404] The present invention also provides binding functional units based on an antibody to BTC that are homologous to the VH and VL sequences described herein. More specifically, the present invention provides a protein comprising amino acid sequences that are homologous to sequences such as those described in Table 3, and wherein the binding functional units based on an antibody to BTC bind to an ophthalmological target and retain the desired functional properties described in Table 3 and the examples.An anti-BTC antibody-based binding functional unit comprising VH and VL regions with less than 100% sequence identity to the VH and VL regions described in Table 3 can be produced by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of the nucleic acid molecules described in Table 3, followed by testing the encoded altered antibody for retention of function using the functional assays described herein and in US 20120014958. An anti-BTC antibody-based binding functional unit comprising a heavy chain and a light chain with a high (i.e.,80% or greater) identity to the heavy chains and light chains described in Table 3 can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding such polypeptides, followed by testing the encoded altered antibody for retention of function using the functional assays described herein.

[0405] The anti-BTC antibody binding functional unit of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) comprising an amino acid sequence with approximately at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NOs: 10 and 21, respectively. It is contemplated that the variability may be in the CDR region or the framework region. In one aspect, the anti-BTC antibody-based binding functional unit comprising VH and VL comprises the amino acid sequence presented under SEQ ID NOs: 10 and 21, respectively. In another aspect, the anti-BTC antibody-based binding functional unit is NVS11, as presented in Table 3. In another aspect, VH and VL are encoded by the nucleic acid sequence presented under SEQ ID NOs: 116 and 122, respectively. In one aspect, the anti-BTC antibody-based binding functional unit comprises a VH and a VL comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but no more than 10 modifications (e.g., substitutions,(e.g., conservative substitutions) of the amino acid sequence under SEQ ID NO: 10 and 21, respectively. In another aspect, the differences in amino acid sequence are not within the complementarity-determining regions.,

[0406] In one aspect, the anti-BTC antibody binding functional unit of the present invention comprises a VH and a VL comprising an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs: 34 and 45, respectively. It is contemplated that the variability may be in the CDR region or the framework region. In one aspect, the anti-BTC antibody-based binding functional unit comprising VH and VL comprises the amino acid sequence presented under SEQ ID NOs: 34 and 45, respectively. In another aspect, the anti-BTC antibody-based binding functional unit is NVS12, as presented in Table 3. In another aspect, VH and VL are encoded by the nucleic acid sequence presented under SEQ ID NOs: 127 and 132, respectively. In one aspect, the anti-BTC antibody-based binding functional unit comprises a VH and a VL comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but no more than 10 modifications (e.g., substitutions, e.g.,conservative substitutions) of the amino acid sequence under SEQ ID NO: 34 and 45, respectively. In another aspect, the differences in amino acid sequence are not within the complementarity-determining regions.,

[0407] In one aspect, the anti-BTC antibody binding functional unit of the present invention comprises a VH and a VL comprising an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs: 54 and 65, respectively. It is contemplated that the variability may be in the CDR region or the framework region. In one aspect, the anti-BTC antibody-based binding functional unit comprising VH and VL comprises the amino acid sequence presented under SEQ ID NOs: 54 and 65, respectively. In another aspect, the anti-BTC antibody-based binding functional unit is NVS13, as presented in Table 3. In another aspect, VH and VL are encoded by the nucleic acid sequence presented under SEQ ID NOs: 137 and 142, respectively. In one aspect, the anti-BTC antibody-based binding functional unit comprises a VH and a VL comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but no more than 10 modifications (e.g., substitutions, e.g.,conservative substitutions) of the amino acid sequence under SEQ ID NO: 54 and 65, respectively. In another aspect, the differences in amino acid sequence are not within the complementarity-determining regions.,

[0408] In one aspect, the anti-BTC antibody binding functional unit of the present invention comprises a VH and a VL comprising an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs: 78 and 88, respectively. It is contemplated that the variability may be in the CDR region or the framework region. In one aspect, the anti-BTC antibody-based binding functional unit comprising VH and VL comprises the amino acid sequence presented under SEQ ID NOs: 78 and 88, respectively. In another aspect, the anti-BTC antibody-based binding functional unit is NVS14, as presented in Table 3. In another aspect, VH and VL are encoded by the nucleic acid sequence presented under SEQ ID NOs: 147 and 151, respectively. In one aspect, the anti-BTC antibody-based binding functional unit comprises a VH and a VL comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but no more than 10 modifications (e.g., substitutions, e.g.,conservative substitutions) of the amino acid sequence under SEQ ID NO: 78 and 88, respectively. In another aspect, the differences in amino acid sequence are not within the complementarity-determining regions.,

[0409] In one aspect, the anti-BTC antibody binding functional unit of the present invention comprises 1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising SEQ ID NOs: 1, 2, 3, 14, 15 and 16, respectively (combined numbering scheme); SEQ ID NOs: 4, 2, 3, 14, 15 and 16, respectively (Kabat numbering scheme); SEQ ID NOs: 5, 6, 3, 17, 18 and 19, respectively (Chothia numbering scheme) or SEQ ID NOs: 7, 8, 9, 20, 18 and 16, respectively (IMGT numbering scheme), and 2) VH and VL comprising the amino acid sequence shown as SEQ ID NOs: 10 and 21, respectively. In another aspect, the anti-BTC antibody-based binding functional unit is contained in NVS11, as shown in Table 3.

[0410] In one aspect, the anti-BTC antibody binding functional unit of the present invention comprises 1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising SEQ ID NOs: 25, 26, 27, 38, 39 and 40, respectively (combined numbering scheme); SEQ ID NOs: 28, 26, 27, 38, 39 and 40, respectively (Kabat numbering scheme); SEQ ID NOs: 29, 30, 27, 41, 42 and 43, respectively (Chothia numbering scheme) or SEQ ID NOs: 31, 32, 33, 44, 42 and 40, respectively (IMGT numbering scheme), and 2) VH and VL comprising the amino acid sequence set forth under SEQ ID NOs: 34 and 45, respectively. In another aspect, the anti-BTC antibody-based binding functional unit is comprised in NVS12, as shown in Table 3.

[0411] In one aspect, the anti-BTC antibody binding functional unit of the present invention comprises 1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising SEQ ID NOs: 25, 49, 50, 58, 59 and 60, respectively (combined numbering scheme); SEQ ID NOs: 28, 49, 50, 58, 59 and 60, respectively (Kabat numbering scheme); SEQ ID NOs: 29, 51, 50, 61, 62 and 63, respectively (Chothia numbering scheme) or SEQ ID NOs: 31, 52, 53, 64, 62 and 60, respectively (IMGT numbering scheme), and 2) VH and VL comprising the amino acid sequence set forth under SEQ ID NOs: 54 and 65, respectively. In another aspect, the anti-BTC antibody-based binding functional unit is comprised in NVS13, as shown in Table 3.

[0412] In one aspect, the anti-BTC antibody binding functional unit of the present invention comprises 1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising SEQ ID NOs: 69, 70, 71, 82, 83 and 84, respectively (combined numbering scheme); SEQ ID NOs: 72, 70, 71, 82, 83 and 84, respectively (Kabat numbering scheme); SEQ ID NOs: 73, 74, 71, 85, 18, and 86, respectively (Chothia numbering scheme) or SEQ ID NOs: 75, 76, 77, 87, 18, and 84, respectively (IMGT numbering scheme) and 2) VH and VL comprising the amino acid sequence set forth under SEQ ID NOs: 78 and 88, respectively. In another aspect, the anti-BTC antibody-based binding functional unit is comprised in NVS14, as shown in Table 3. The anti-VEGF antibody-based binding functional unit

[0413] The present invention also provides an anti-VEGF antibody-based binding functional unit contained in a multispecific binding molecule. In specific aspects, the anti-VEGF antibody-based binding functional unit comprises a heavy chain variable region complementarity determining region 1 (HCDR1), a heavy chain variable region complementarity determining region 2 (HCDR2), a heavy chain variable region complementarity determining region 3 (HCDR3), a light chain variable region complementarity determining region 1 (LCDR1), a light chain variable region complementarity determining region 2 (LCDR2), and a light chain variable region complementarity determining region 3 (LCDR3). HCDR1, HCDR2, and HCDR3 are contained in a heavy chain variable region (VH). LCDR1, LCDR2 and LCDR3 are contained in the variable region of the light chain (VL).In one aspect, the anti-VEGF antibody-based binding functional unit comprises heavy chain and light chain CDRs (e.g., Kabat, Chothia, IMGT, and / or combined CDRs), as shown in Table 2 or 3 and described below. In one aspect, the anti-VEGF antibody-based binding functional unit is an anti-VEGF antibody or an antigen-binding fragment thereof. In one aspect, the anti-VEGF antibody-based binding functional unit is an anti-VEGF antibody or an antigen-binding fragment thereof that inhibits (e.g., partially inhibits) VEGF activity.

[0414] In one aspect, the anti-VEGF antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, according to the Kabat numbering scheme. In one aspect, the anti-VEGF antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, according to the Chothia numbering scheme. In one aspect, the anti-VEGF antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, according to the combined numbering scheme. In one aspect, the anti-VEGF antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, according to the IMGT numbering scheme.In one aspect, the anti-VEGF antibody-based binding functional unit is NVS8 as shown in Table 2, or is contained in any of NVS11-NVS14 as shown in Table 3. In another aspect, the anti-VEGF antibody-based binding functional unit is brolucizumab (Beovu®).

[0415] Furthermore, the present invention also provides an anti-VEGF antibody-based binding functional unit comprising amino acid sequences that are homologous to the CDR sequences described throughout and in Table 2 or 3, and wherein the anti-VEGF antibody-based binding functional unit binds to VEGF and retains the desired functional properties of the sequences described herein. More specifically, the amino acid sequences of the anti-VEGF antibody-based binding functional unit may have greater than or equal to 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with the CDR sequences as described throughout this document and presented in Table 2 or 3, and retain their desired functional properties.

[0416] The present invention also provides binding functional units based on an antibody to VEGF that are homologous to the VH and VL sequences described herein. More specifically, the present invention provides a protein comprising amino acid sequences that are homologous to sequences such as those described in Table 2 or 3, and wherein the binding functional units based on an antibody to VEGF bind an ophthalmological target and retain the desired functional properties of the sequences described in Table 2 or 3 and in the examples.An anti-VEGF antibody-based binding functional unit comprising VH and VL regions with less than 100% sequence identity to the VH and VL regions described in Table 2 or 3 may be produced by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of the nucleic acid molecules described in Table 2 or 3, followed by testing the encoded altered antibody for retention of function using the functional assays described herein and in US 20120014958. An anti-VEGF antibody-based binding functional unit comprising a heavy chain and a light chain with a high (i.e.,80% or greater) identity to the heavy chains and light chains described in Table 2 or 3 can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding such polypeptides, followed by testing the encoded altered antibody for retention of function using the functional assays described herein.

[0417] The anti-VEGF antibody binding functional unit of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) comprising an amino acid sequence with approximately at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs: 101 and 112, respectively. It is contemplated that the variability may be in the CDR region or the framework region. In one aspect, the VH and VL-containing antibody-based binding functional unit comprises the amino acid sequence set forth in SEQ ID NOs: 101 and 112, respectively. In another aspect, the anti-VEGF antibody-based binding functional unit is NVS8 as presented in Table 2, or is contained in any of NVS11-NVS14 as presented in Table 3. In one aspect, the anti-VEGF antibody-based binding functional unit comprises a VH and a VL comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but no more than 10 modifications (e.g., substitutions, e.g.,conservative substitutions) of the amino acid sequence under SEQ ID NO: 101 and 112, respectively. In another aspect, the differences in amino acid sequence are not within the complementarity-determining regions.,

[0418] In one aspect, the VH and VL of the anti-VEGF antibody-based binding functional unit are encoded by the nucleic acid sequence shown in SEQ ID NO: 102 and 113, respectively. In one aspect, the anti-VEGF antibody-based binding functional unit is NVS8, as shown in Table 2.

[0419] In one aspect, the VH and VL of the anti-VEGF antibody-based binding functional unit are encoded by the nucleic acid sequence shown as SEQ ID NO: 117 and 123, respectively. In one aspect, the anti-VEGF antibody-based binding functional unit is contained in NVS11, as shown in Table 3.

[0420] In one aspect, the VH and VL of the anti-VEGF antibody-based binding functional unit are encoded by the nucleic acid sequence shown as SEQ ID NO: 128 and 133, respectively. In one aspect, the anti-VEGF antibody-based binding functional unit is contained in NVS12, as shown in Table 3.

[0421] In one aspect, the VH and VL of the anti-VEGF antibody-based binding functional unit are encoded by the nucleic acid sequence shown as SEQ ID NO: 138 and 143, respectively. In one aspect, the anti-VEGF antibody-based binding functional unit is contained in NVS13, as shown in Table 3.

[0422] In one aspect, the VH and VL of the anti-VEGF antibody-based binding functional unit are encoded by the nucleic acid sequence shown as SEQ ID NO: 148 and 152, respectively. In one aspect, the anti-VEGF antibody-based binding functional unit is contained in NVS14, as shown in Table 3.

[0423] In one aspect, the anti-VEGF antibody binding functional unit of the present invention comprises 1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising SEQ ID NOs: 92, 93, 94, 105, 106 and 107, respectively (combined numbering scheme); SEQ ID NOs: 95, 93, 94, 105, 106 and 107, respectively (Kabat numbering scheme); SEQ ID NOs: 96, 97, 94, 108, 109 and 110, respectively (Chothia numbering scheme) or SEQ ID NOs: 98, 99, 100, 111, 109 and 107, respectively (IMGT numbering scheme), and 2) VH and VL comprising the amino acid sequence set forth under SEQ ID NOs: 101 and 112, respectively. In another aspect, the anti-VEGF antibody-based binding functional unit is NVS8, as set forth in Table 2, or is contained in any of NVS11-NVS14, as set forth in Table 3. In one aspect, the anti-VEGF antibody-based binding functional unit suppresses (e.g., partially suppresses) VEGF activity.

[0424] In one aspect, the anti-VEGF antibody binding functional unit of the present invention (e.g., an anti-VEGF antibody binding functional unit that suppresses VEGF activity) comprises a heavy chain and a light chain comprising an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 103 and 114, respectively. In one aspect, the anti-VEGF antibody-based binding functional unit comprises a heavy chain and a light chain comprising the amino acid sequence set forth in SEQ ID NOs: 103 and 114, respectively. In another aspect, the anti-VEGF antibody-based binding functional unit is NVS8, as shown in Table 2. In another aspect, the heavy chain and the light chain are encoded by a nucleic acid sequence with about at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%,at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical or complementary to SEQ ID NO: 104 and 115, respectively. In one aspect, the anti-VEGF antibody-based binding functional unit comprises a heavy chain and a light chain,encoded by the nucleic acid sequence shown as SEQ ID NO: 104 and 115, respectively, and is NVS8, as shown in Table 2. The bispecific binding molecule,

[0425] In specific aspects, the present invention is provided by a multispecific binding molecule comprising an anti-BTC antibody-based binding functional unit and an anti-VEGF antibody-based binding functional unit. The present invention provides a multispecific binding molecule comprising an anti-BTC antibody-based binding functional unit and an anti-VEGF antibody-based binding functional unit, 1) wherein the anti-BTC antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the anti-BTC antibody shown in Table 1 (e.g., NVS1, NVS2, NVS3 or NVS4); and 2) wherein the anti-VEGF antibody-based binding functional unit comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the anti-VEGF antibody presented in Table 2 or 3. In one aspect, the multispecific binding molecule is NVS11 presented in Table 3.

[0426] The present invention provides a multispecific binding molecule comprising an anti-BTC antibody-based binding functional unit and an anti-VEGF antibody-based binding functional unit, 1) wherein the anti-BTC antibody-based binding functional unit comprises 1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising SEQ ID NOs: 1, 2, 3, 14, 15 and 16, respectively (combined numbering scheme); SEQ ID NOs: 4, 2, 3, 14, 15 and 16, respectively (Kabat numbering scheme); SEQ ID NOs: 5, 6, 3, 17, 18, and 19, respectively (Chothia numbering scheme) or SEQ ID NOs: 7, 8, 9, 20, 18, and 16, respectively (IMGT numbering scheme); and 2) wherein the anti-VEGF antibody binding functional unit comprises SEQ ID NOs: 92, 93, 94, 105, 106, and 107, respectively (combined numbering scheme); SEQ ID NOs: 95, 93, 94, 105, 106, and 107, respectively (Kabat numbering scheme);SEQ ID NOs: 96, 97, 94, 108, 109, and 110, respectively (Chothia numbering scheme) or SEQ ID NOs: 98, 99, 100, 111, 109, and 107, respectively (IMGT numbering scheme). In one aspect, the multispecific binding molecule is NVS11 as shown in Table 3. In one aspect, such a multispecific binding molecule 1) selectively binds to human BTC protein and inhibits BTC activity (e.g., partially inhibits) and 2) selectively binds to human VEGF protein and inhibits VEGF activity (e.g., partially inhibits).;

[0427] The present invention also provides a multispecific binding molecule comprising an anti-BTC antibody-based binding functional unit and an anti-VEGF antibody-based binding functional unit, 1) wherein the anti-BTC antibody-based binding functional unit comprises 1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising SEQ ID NOs: 25, 26, 27, 38, 39 and 40, respectively (combined numbering scheme); SEQ ID NOs: 28, 26, 27, 38, 39 and 40, respectively (Kabat numbering scheme); SEQ ID NOs: 29, 30, 27, 41, 42, and 43, respectively (Chothia numbering scheme) or SEQ ID NOs: 31, 32, 33, 44, 42, and 40, respectively (IMGT numbering scheme); and 2) wherein the anti-VEGF antibody binding functional unit comprises SEQ ID NOs: 92, 93, 94, 105, 106, and 107, respectively (combined numbering scheme); SEQ ID NOs: 95, 93, 94, 105, 106, and 107, respectively (Kabat numbering scheme);SEQ ID NOs: 96, 97, 94, 108, 109, and 110, respectively (Chothia numbering scheme) or SEQ ID NOs: 98, 99, 100, 111, 109, and 107, respectively (IMGT numbering scheme). In one aspect, the multispecific binding molecule is NVS12, as shown in Table 3.;

[0428] The present invention also provides a multispecific binding molecule comprising an anti-BTC antibody-based binding functional unit and an anti-VEGF antibody-based binding functional unit, 1) wherein the anti-BTC antibody-based binding functional unit comprises 1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising SEQ ID NOs: 25, 49, 50, 58, 59 and 60, respectively (combined numbering scheme); SEQ ID NOs: 28, 49, 50, 58, 59 and 60, respectively (Kabat numbering scheme); SEQ ID NOs: 29, 51, 50, 61, 62, and 63, respectively (Chothia numbering scheme) or SEQ ID NOs: 31, 52, 53, 64, 62, and 60, respectively (IMGT numbering scheme); and 2) wherein the anti-VEGF antibody binding functional unit comprises SEQ ID NOs: 92, 93, 94, 105, 106, and 107, respectively (combined numbering scheme); SEQ ID NOs: 95, 93, 94, 105, 106, and 107, respectively (Kabat numbering scheme);SEQ ID NOs: 96, 97, 94, 108, 109, and 110, respectively (Chothia numbering scheme) or SEQ ID NOs: 98, 99, 100, 111, 109, and 107, respectively (IMGT numbering scheme). In one aspect, the multispecific binding molecule is NVS13, as shown in Table 3.;

[0429] The present invention also provides a multispecific binding molecule comprising an anti-BTC antibody-based binding functional unit and an anti-VEGF antibody-based binding functional unit, 1) wherein the anti-BTC antibody-based binding functional unit comprises 1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising SEQ ID NOs: 69, 70, 71, 82, 83 and 84, respectively (combined numbering scheme); SEQ ID NOs: 72, 70, 71, 82, 83 and 84, respectively (Kabat numbering scheme); SEQ ID NOs: 73, 74, 71, 85, 18, and 86, respectively (Chothia numbering scheme) or SEQ ID NOs: 75, 76, 77, 87, 18, and 84, respectively (IMGT numbering scheme); and 2) wherein the anti-VEGF antibody binding functional unit comprises SEQ ID NOs: 92, 93, 94, 105, 106, and 107, respectively (combined numbering scheme); SEQ ID NOs: 95, 93, 94, 105, 106, and 107, respectively (Kabat numbering scheme);SEQ ID NOs: 96, 97, 94, 108, 109, and 110, respectively (Chothia numbering scheme) or SEQ ID NOs: 98, 99, 100, 111, 109, and 107, respectively (IMGT numbering scheme). In one aspect, the multispecific binding molecule is NVS14, as shown in Table 3.;

[0430] In one aspect, the multispecific binding molecule of the present invention comprises 1) VHA and VLA that bind to BTC, wherein the VHA and VLA comprise an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 10 and 21, respectively; and 2) VHB and VLB that bind to VEGF, wherein the VHB and VLB comprise an amino acid sequence with approximately at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 101 and 112, respectively. In another aspect, a multispecific binding molecule of the present invention comprises 1) VHA and VLA that bind to BTC, comprising the amino acid sequence of SEQ ID NOs: 10 and 21, respectively; and 2) VHB and VLB that bind to VEGF, comprising the amino acid sequence of SEQ ID NOs: 101 and 112, respectively. In another aspect, VHA and VLA are encoded by a nucleic acid sequence with approximately at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 116 and 122, respectively. In another aspect, VHB and VLB are encoded by a nucleic acid sequence with approximately at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 117 and 123, respectively. In one aspect, the multispecific binding molecule is NVS11 as shown in Table 3. In one aspect, such a multispecific binding molecule 1) selectively binds to a human BTC protein and inhibits BTC activity (e.g.,partially suppresses) and 2) selectively binds to the human VEGF protein and suppresses VEGF activity (e.g., partially suppresses).,

[0431] In one aspect, the multispecific binding molecule of the present invention comprises 1) VHA and VLA that bind to BTC, wherein the VHA and VLA comprise an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 34 and 45, respectively; and 2) VHB and VLB that bind to VEGF, wherein the VHB and VLB comprise an amino acid sequence with approximately at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 101 and 112, respectively. In another aspect, a multispecific binding molecule of the present invention comprises 1) VHA and VLA that bind to BTC, comprising the amino acid sequence of SEQ ID NOs: 34 and 45, respectively; and 2) VHB and VLB that bind to VEGF, comprising the amino acid sequence of SEQ ID NOs: 101 and 112, respectively. In another aspect, VHA and VLA are encoded by a nucleic acid sequence with approximately at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 127 and 132, respectively. In another aspect, VHB and VLB are encoded by a nucleic acid sequence with approximately at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 128 and 133, respectively. In one aspect, the multispecific binding molecule is NVS12 as shown in Table 3. In one aspect, such a multispecific binding molecule 1) selectively binds to a human BTC protein and inhibits BTC activity (e.g.,partially suppresses) and 2) selectively binds to the human VEGF protein and suppresses VEGF activity (e.g., partially suppresses).,

[0432] In one aspect, the multispecific binding molecule of the present invention comprises 1) VHA and VLA that bind to BTC, wherein the VHA and VLA comprise an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 54 and 65, respectively; and 2) VHB and VLB that bind to VEGF, wherein the VHB and VLB comprise an amino acid sequence with approximately at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 101 and 112, respectively. In another aspect, a multispecific binding molecule of the present invention comprises 1) VHA and VLA that bind to BTC, comprising the amino acid sequence of SEQ ID NOs: 54 and 65, respectively; and 2) VHB and VLB that bind to VEGF, comprising the amino acid sequence of SEQ ID NOs: 101 and 112, respectively. In another aspect, VHA and VLA are encoded by a nucleic acid sequence with approximately at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 137 and 142, respectively. In another aspect, VHB and VLB are encoded by a nucleic acid sequence with approximately at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 138 and 143, respectively. In one aspect, the multispecific binding molecule is NVS13 as shown in Table 3. In one aspect, such a multispecific binding molecule 1) selectively binds to a human BTC protein and inhibits BTC activity (e.g.,partially suppresses) and 2) selectively binds to the human VEGF protein and suppresses VEGF activity (e.g., partially suppresses).,

[0433] In one aspect, the multispecific binding molecule of the present invention comprises 1) VHA and VLA that bind to BTC, wherein the VHA and VLA comprise an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 78 and 88, respectively; and 2) VHB and VLB that bind to VEGF, wherein the VHB and VLB comprise an amino acid sequence with approximately at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 101 and 112, respectively. In another aspect, a multispecific binding molecule of the present invention comprises 1) VHA and VLA that bind to BTC, comprising the amino acid sequence of SEQ ID NOs: 78 and 88, respectively; and 2) VHB and VLB that bind to VEGF, comprising the amino acid sequence of SEQ ID NOs: 101 and 112, respectively. In another aspect, VHA and VLA are encoded by a nucleic acid sequence with approximately at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 147 and 151, respectively. In another aspect, VHB and VLB are encoded by a nucleic acid sequence with approximately at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 and 152, respectively. In one aspect, the multispecific binding molecule is NVS14 as shown in Table 3. In one aspect, such a multispecific binding molecule 1) selectively binds to a human BTC protein and inhibits BTC activity (e.g.,partially suppresses) and 2) selectively binds to the human VEGF protein and suppresses VEGF activity (e.g., partially suppresses).,

[0434] The multispecific binding molecule of the present invention comprises 1) a heavy chain comprising VHA, CH1A, a linker, VHB, and CH1B, wherein the heavy chain comprises an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, by at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 120; and 2) a light chain comprising a VLA, a CKA, a linker, a VLB, and a CKB, wherein the light chain comprises an amino acid sequence with approximately at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 125. In one aspect, the heavy chain and the light chain are encoded by a nucleic acid sequence with about at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 121 and 126, respectively. In one aspect, the multispecific binding molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120,and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125, as set forth in relation to NVS11, as presented in Table 3. In one aspect, such a multispecific binding molecule 1) selectively binds to the human BTC protein and inhibits BTC activity (e.g., partially inhibits) and 2) selectively binds to the human VEGF protein and inhibits VEGF activity (e.g., partially inhibits).,

[0435] The multispecific binding molecule of the present invention comprises 1) a heavy chain comprising VHA, CH1A, a linker, VHB, and CH1B, wherein the heavy chain comprises an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, by at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 130, and 2) a light chain comprising a VLA, a CKA, a linker, a VLB, and a CKB, wherein the light chain comprises an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 135. In one aspect, the heavy chain and the light chain are encoded by a nucleic acid sequence with about at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 131 and 136, respectively. In one aspect, the multispecific binding molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130,and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135, as set forth in relation to NVS12, as presented in Table 3. In one aspect, such a multispecific binding molecule 1) selectively binds to the human BTC protein and inhibits BTC activity (e.g., partially inhibits) and 2) selectively binds to the human VEGF protein and inhibits VEGF activity (e.g., partially inhibits).,

[0436] The multispecific binding molecule of the present invention comprises 1) a heavy chain comprising VHA, CH1A, a linker, VHB, and CH1B, wherein the heavy chain comprises an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, by at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 140; and 2) a light chain comprising a VLA, a CKA, a linker, a VLB, and a CKB, wherein the light chain comprises an amino acid sequence with approximately at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 145. In one aspect, the heavy chain and the light chain are encoded by a nucleic acid sequence with about at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 141 and 146, respectively. In one aspect, the multispecific binding molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140,and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145, as set forth in relation to NVS13, as presented in Table 3. In one aspect, such a multispecific binding molecule 1) selectively binds to the human BTC protein and inhibits BTC activity (e.g., partially inhibits) and 2) selectively binds to the human VEGF protein and inhibits VEGF activity (e.g., partially inhibits).,

[0437] The multispecific binding molecule of the present invention comprises 1) a heavy chain comprising VHA, CH1A, a linker, VHB, and CH1B, wherein the heavy chain comprises an amino acid sequence with about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, by at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 149; and 2) a light chain comprising a VLA, a CKA, a linker, a VLB, and a CKB, wherein the light chain comprises an amino acid sequence with approximately at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 154. In one aspect, the heavy chain and the light chain are encoded by a nucleic acid sequence with about at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 150 and 155, respectively. In one aspect, the multispecific binding molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149,and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154, as set forth in relation to NVS14, as presented in Table 3. In one aspect, such a multispecific binding molecule 1) selectively binds to the human BTC protein and inhibits BTC activity (e.g., partially inhibits) and 2) selectively binds to the human VEGF protein and inhibits VEGF activity (e.g., partially inhibits).,

[0438] The multispecific binding molecule of the present invention (e.g., NVS11-NVS14) can: simultaneously bind to BTC and VEGF; suppress the binding of soluble BTC to ErbB1 or ErB4 and the subsequent phosphorylation of Erb receptors; suppress the phosphorylation of ERK1 / 2 induced by soluble BTC; bind to membrane-bound BTC and suppress the juxtacrine activation of ErbB1 phosphorylation induced by membrane-bound BTC; suppress the binding of soluble VEGF-A165 to soluble VEGFR2; limit the permeability of human iPSC-derived RPE induced by BTC in an external BRBin vitro model; limit the permeability of human retinal endothelial cells (HREC) induced by VEGF in an internal BRBin vitro model; suppress BTC-induced retinal thickening; and / or inhibit VEGF-induced retinal vascular fluid leakage. Table 2. Illustrative Fab to VEGF NVS8 SEQ ID NO: 92 (combined) HCDR1 GFSLTDYYMT SEQ ID NO: 93 (combined) HCDR2 FIDPDDDPYATWAKG SEQ ID NO: 94 HCDR3 GDHNSGWGLDI SEQ ID NO: 95 (Kabat) HCDR1 DYYYMT SEQ ID NO: 93 (Kabat) HCDR2 FIDPDDDPYYATWAKG SEQ ID NO: 94 (Kabat) HCDR3 GDHNSGWGLDI SEQ ID NO: 96 (Chothia) HCDR1 GFSLTDYY SEQ ID NO: 97 (Chothia) HCDR2 DPDDD SEQ ID NO: 94 (Chothia) HCDR3 GDHNSGWGLDI SEQ ID NO: 98 (IMGT) HCDR1 GFSLTDYYY SEQ ID NO: 99 (IMGT) HCDR2 IDPDDDP SEQ ID NO: 100 (IMGT) HCDR3 AGGDHNSGWGLDI SEQ ID NO: 101 VH EVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDHNSGWGLDIWGQGTLVTVSS SEQ ID NO: 102 ДНК, кодирующая VH GAGGGTGCAATTGGTTGAATCTGGGGGCGGACTGGTGCAGCCCGGTGGATCTTTGCGCCTGTCCTGTACAGCTTCTGGCTTCTCCTTGACCGACTATTACATGACTTGGGTTTCGCCAAGCCCCAGGCAAAGGGCTTGAATGGGTGGGGTTCATTGACCCCGACGATGATCCTTACTAC GCCACATGGGCAAAGGGCCGGTTTACTATCAGCCGGGATAATTCCAAAAACACATTGTATTGCAAATGAACTCACTGAGAGCAGAAGATACGGCTGTGTACTATTGCGCAGGCGGCGATCATAACTCCGGCTGGGGCCTGGACATCTGGGGGCAGGGGACCCTGGTGACAGTCAGCTCA SEQ ID NO: 103 Тяжелая цепь EVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDHNSGWGLDIWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTCGVHTFPAVLQSSGLYSLSSVVTVPSSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID NO: 104 DNA encoding the heavy chain GAGGTGCAATTGGTTGAATCTGGGGGCGGACTGGTGCAGCCCGGTGGATCTTTGCGCCTGTCCTGTACAGCTTCTGGCTTCTCCTTGACCGACTACTATTACTTGGGTTCGCCAAGCCCCAGGCAAAGGGCTTGAATGGGTGGGGTTCATTGACCCCGACGA TGATCCTTACTACGCCACATGGGCAAAGGGCCGGTTTACTATCAGCCGGGATAATTCCAAAAACATTGTATTTGCAAATGAACTCACTGAGAGCAGAAGATACGGCTGTGTACTATTGCGCAGGCGGCGATCATAACTCCGGCTGGGGCCTGGACATCTGGGGGC AGGGGACCCTGGTGACAGTCAGCTCAGCCTCAACGAAGGGGCCCAGCGTGTTTCCTTTGGCCCCAAGCAGCAAGTCCACGTCCGGTGGGACTGCAGCTCTTGGTTGTCTGGTCAAGGATTATTTCCCAGAACCCGTGACCGTGTCTTGGAACAGGTGGTGCATTGACA TCAGGAGTGCATACATTCCCAGCTGTGCTGCAGAGCTCTGGCCTGTATAGCCTTTCCTCTGTTGTCACGGTGCCCAGCTCCAGCCCTGGGGACGCAGACCTATTTGTAACGTGAACCATAAACCCTCCAACACCAAGGTTGATAAAAGAGTGGAGCCCAAGTCTTGT SEQ ID NO: 105 (combined) LCDR1 QASEIIHSWLA SEQ ID NO: 106 (combined) LCDR2 LASTLAS SEQ ID NO: 107 (combined) LCDR3 QNVYLASTNGAN SEQ ID NO: 105 (Related) LCDR1 QASEIIHSWLA SEQ ID NO: 106 (Related) LCDR2 LASTLAS SEQ ID NO: 107 (Related) LCDR3 QNVYLASTNGAN SEQ ID NO: 108 (Chothia) LCDR1 SEIIHSW SEQ ID NO: 109 (Chothia) LCDR2 LAS SEQ ID NO: 110 (Chothia) LCDR3 VYLASTNGA SEQ ID NO: 111 (IMGT) LCDR1 EIIHSW SEQ ID NO: 109 (IMGT) LCDR2 LAS SEQ ID NO: 107 (IMGT) LCDR3 QNVYLASTNGAN SEQ ID NO: 112 VL EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLK SEQ ID NO: 113 DNA encoding VL gagattgtgatgactcagagcccttcaacgctgtctgcatccgtaggtcgcgtcattattacctgcaagcctcagagatcattcactcttgctcgcctggtatcagcccggtaaggcccccaagctgctgatctcttgcttcaaccctcgcga gcggggtgccctcccgcttcgggctccggctctgccgaatttaccctgacaatcagctctctccaacccgatttcggacttactgtcagaatgtctacttggcctcaaccaacggagccaacttcggccaggccaaactgaccgtcttaag SEQ ID NO: 114 Light chain EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTV LKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 115 DNA encoding the light chain gagattgtgatgactcagagcccttcaacgctgtctgcatccgtaggtcgcgtcattattaccgtcaagcctcagagatcattcactcttgctcgcctggtatcagcccggtaaggcccccaagctgctgatctcttgcttcaacctcg cgagcggggtgccctcccgcttcgggctccggctctgccgaatttaccctgacaatcagctctctccaacccgatttcgcgacttactgtcagaatgtctacttggcctcaaccaacggagccaacttcggccaggccaaactgaccgtc cttaagcgtacggtggcagctccgtctttttcatcttccacctagcgacgagcactcaaaagtggtacagcatccgtggttgtctgctgaacaatttttaccccagggaggctaaggtccagtggaaagtcgataacgctcttcagtctggcaacgtc aggagagcgtcacagagcaggactctaaggatagcacttatagtctgtcctccacgctgacactgtctaaagcggattatgagaagcaaggtttacgcctgtgaggtaacgcaccaggactctcctccccagttaccaaatctttcaacagagagagatgt Table 3. Illustrative bispecific Fabs to BTC / to VEGF NVS11 SEQ ID NO: 1 (combined) HCDR1 1 GGTFSSYAIS SEQ ID NO: 2 (combined) HCDR2 1 GIVPWMGEAVYAQKFQG SEQ ID NO: 3 (number) HCDR3 1 SSSTYGIHAFDY SEQ ID NO: 4 (Kabat) HCDR1 1 SYAIS SEQ ID NO: 2 (Kabat) HCDR2 1 GIVPWMGEAVYAQKFQG SEQ ID NO: 3 (Kabat) HCDR3 1 SSSTYGIHAFDY SEQ ID NO: 5 (Chothia) HCDR1 1 GGTFSSY SEQ ID NO: 6 (Chothia) HCDR2 1 VPWMGE SEQ ID NO: 3 (Chothia) HCDR3 1 SSSTYGIHAFDY SEQ ID NO: 7 (IMGT) HCDR1 1 GGTFSSYA SEQ ID NO: 8 (IMGT) HCDR2 1 IVPWMGEA SEQ ID NO: 9 (IMGT) HCDR3 1 ARSSSTYGIHAFDY SEQ ID NO: 10 VH 1 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIVPWMGEAVYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSSSTYGIHAFDYWGQGTLVTVSS SEQ ID NO: 116 Video, video, VH 1 CAAGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCCGGCTCCTCCGTGAAAGTGTCCTGCAAGGCCTCCGGCGGCACCTTCTCCAGCTACGCCATCTCCTGGGTCGACAGGCCCAGGCCAGGGCCTGGAGTGGATGGGCGGCATCGTGCCTTGGATGGGCGAGGCCGTTGTACG CCCAGAAATTCCAGGGCAGAGTGACCATCACCGCCGACGAGTCCACCTCCACCGCCTACATGGAACTGTCCTCCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCCGGTCCTCCTCCACCTACGGCATCCACGCCTTCGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCC SEQ ID NO: 92 HCDR1 2 GFSLTDYYYMT SEQ ID NO: 93 (number) HCDR2 2 FIDPDDDPYYATWAKG SEQ ID NO: 94 HCDR3 2 GDHNSGWGLDI SEQ ID NO: 95 (Kabat) HCDR1 2 DYYYMT SEQ ID NO: 93 (Kabat) HCDR2 2 FIDPDDDPYYATWAKG SEQ ID NO: 94 (Kabat) HCDR3 2 GDHNSGWGLDI SEQ ID NO: 96 (Chothia) HCDR1 2 GFSLTDYY SEQ ID NO: 97 (Chothia) HCDR2 2 DPDDD SEQ ID NO: 94 (Chothia) HCDR3 2 GDHNSGWGLDI SEQ ID NO: 98 (IMGT) HCDR1 2 GFSLTDYYY SEQ ID NO: 99 (IMGT) HCDR2 2 IDPDDDP SEQ ID NO: 100 (IMGT) HCDR3 2 AGGDHNSGWGLDI SEQ ID NO: 101 VH 2 EVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDHNSGWGLDIWGQGTLVTVSS SEQ ID NO: 117 Video, video, VH 2 GAAGTCCAGCTGGTGGAATCCGGCGGAGGCCTGGTGCAGCCAGGCGGATCCCTGAGGCTGTCTTGCACCGCCTCCGGCTTCCCTGACCGACTACTACTGACTTGGGTCCGCCAGGCTCCCGGAAAAGGACTGGAGTGGGTCGGATTCATCGACCCCGACGACGACCCCTACTAC GCCACCTGGGCCAAGGGCCGGTTCAACCATCCTCCCGGGACAACTCCAAGAACACCCTGTAACCTGCAGATGAACTCCCTGAGGCCGAAGATACAGCTGTGTACTATTGCGCTGGCGGCGACCACAACTCCGGCTGGGGGCCTGGATATCTGGGGACAGGGAACACTCGTGACAGTGTCCAGC SEQ ID NO: 118 HC-LINKER GSGGGGSGGGGSGGG SEQ ID NO: 119 DNA encoding the HC linker ggctctggcggcggaggatggcggaggcggtagcggaggcgga SEQ ID NO: 120 Heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIVPWMGEAVYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSSSTYGIHAFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTCGVHTFPAVLQSSGLYSLSSVVTVPSSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGSGGGS GGGGSGGGEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDDPYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDHNSGWGL DIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID NO: 121 DNA encoding the heavy chain CAAGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCCGGCTCCTCCGTGAAAGTGTCCTGCAAGGCCTCCGGCGGCACCTTCTCCAGCTACGCCATCTCCTGGGTCCGACAGGCCCCAGGCCAGGGCCTGGAGTGGATGGGCGGCATCGTGCCTTGGATGGGCGAGGCCGTGTACGCCCAGAAATTCCAGGGCAGAGTGACCATCACCGCCGACGAGTCCACCTCCACCGCCTACATGGAACTGTCCTCCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCCGGTCCTCCTCCACCTACGGCATCCACGCCTTCGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCCGCCTCCACCAAGGGACCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGCACCGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAAGTGGACAAGCGGGTGGAACCCAAGTCCTGCGGCTCTGGCGGCGGAGGATCTGGCGGAGGCGGTAGCGGAGGCGGAGAAGTCCAGCTGGTGGAATCCGGCGGAGGCCTGGTGCAGCCAGGCGGATCCCTGAGGCTGTCTTGCACCGCCTCCGGCTTCTCCCTGACCGACTACTACTACATGACTTGGGTCCGCCAGGCTCCCGGAAAAGGACTGGAGTGGGTCGGATTCATCGACCCCGACGACGACCCCTACTACGCCACCTGGGCCAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAAGATACAGCTGTGTACTATTGCGCTGGCGGCGACCACAACTCCGGCTGGGGCCTGGATATCTGGGGACAGGGAACACTCGTGACAGTGTCCAGCGCCAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACCGCTGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACAGTGCCTTCCTCCAGCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCATGC SEQ ID NO: 14 (combined) LCDR1 1 RASQSISNFLN SEQ ID NO: 15 (combined) LCDR2 1 AASNLQS SEQ ID NO: 16 (combined) LCDR3 1 QQYDDFPMT SEQ ID NO: 14 (Related) LCDR1 1 RASQSISNFLN SEQ ID NO: 15 (Related) LCDR2 1 AASNLQS SEQ ID NO: 16 (Related) LCDR3 1 QQYDDFPMT SEQ ID NO: 17 (Chothia) LCDR1 1 SQSISNF SEQ ID NO: 18 (Chothia) LCDR2 1 AAS SEQ ID NO: 19 (Chothia) LCDR3 1 YDDFPM SEQ ID NO: 20 (IMGT) LCDR1 1 QSISNF SEQ ID NO: 18 (IMGT) LCDR2 1 AAS SEQ ID NO: 16 (IMGT) LCDR3 1 QQYDDFPMT SEQ ID NO: 21 VL 1 DIQMTQSPSSLSASVGDRVTITCRASQSISNFLNWYQQKPGKAPKLLIAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDDFPMTFGQGTKVEIK SEQ ID NO: 122 DNA encoding VL GACATCCAGATGACCCAGAGCCCCTCCAGCCTGTCCGCCTCCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGTCTATCCAACTTCCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCTCCAACC TGCAGTCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCAGCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACGACGACTTCCCCATGACCTTCGGCCAGGGCACCAAAGTGGAAATCAAG SEQ ID NO: 105 (combined) LCDR1 2 QASEIIHSWLA SEQ ID NO: 106 (combined) LCDR2 2 LASTLAS SEQ ID NO: 107 (combined) LCDR3 2 QNVYLASTNGAN SEQ ID NO: 105 (Related) LCDR1 2 QASEIIHSWLA SEQ ID NO: 106 (Related) LCDR2 2 LASTLAS SEQ ID NO: 107 (Related) LCDR3 2 QNVYLASTNGAN SEQ ID NO: 108 (Chothia) LCDR1 2 SEIIHSW SEQ ID NO: 109 (Chothia) LCDR2 2 LAS SEQ ID NO: 110 (Chothia) LCDR3 2 VYLASTNGA SEQ ID NO: 111 (IMGT) LCDR1 2 EIIHSW SEQ ID NO: 109 (IMGT) LCDR2 2 LAS SEQ ID NO: 107 (IMGT) LCDR3 2 QNVYLASTNGAN SEQ ID NO: 112 VL 2 EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLK SEQ ID NO: 123 The DNA encoding VL GAGATCGTGATGACCCAGTCCCCTAGCACCCTGAGCGCCAGCGTGGGAGATCGCGTGATCATCACATGCCAGGCCTCCGAGATCATCCACAGCTGGCTGGCTTGGTCAGCAGAAACCTGGAAAAGCTCCCAAGCTCCTGATCTATCTGGCCAGCACCCTGGCCT CTGGCGTGCCCAGCAGATTCAGCGGCTCCGGCAGCGGCGCTGAGTTTACCCTGACAATCAGCTCCCTGCAGCCTGACGATTTTGCTACCTACTATTTGTCAGAACGTGTACCTGGCCTCCACCAACGGCGCCAACTTTGGCCAGGAACAAAGCTGACCGTGCTGAAG SEQ ID NO: 118 LC-linker GSGGGGSGGGGSGGG SEQ ID NO: 124 DNA encoding the LC linker ggctccggcggaggcggatctggtggcggaggctggcggtggc SEQ ID NO: 125 Light chain DIQMTQSPSSLSASVGDRVTITCRASQSISNFLNWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDDFPMTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGSGGGGSGG GGSGGGEIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTK LTVLKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 126 Yes, it's worth it. GACATCCAGATGACCCAGAGCCCCTCCAGCCTGTCCGCCTCCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGTCTATCTCCAACTTCCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCTCCAACCTGCAGTCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACGACGACTTCCCCATGACCTTCGGCCAGGGCACCAAAGTGGAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTCGTGTGCCTGCTGAACAACTTCTACCCTCGCGAGGCCAAAGTGCAGTGGAAAGTGGACAACGCCCTGCAGAGCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGCGAGTGTGGCTCCGGCGGAGGCGGATCTGGTGGCGGAGGATCTGGCGGTGGCGAGATCGTGATGACCCAGTCCCCTAGCACCCTGAGCGCCAGCGTGGGAGATCGCGTGATCATCACATGCCAGGCCTCCGAGATCATCCACAGCTGGCTGGCTTGGTATCAGCAGAAACCTGGAAAAGCTCCCAAGCTCCTGATCTATCTGGCCAGCACCCTGGCCTCTGGCGTGCCCAGCAGATTCAGCGGCTCCGGCAGCGGCGCTGAGTTTACCCTGACAATCAGCTCCCTGCAGCCTGACGATTTTGCTACCTACTATTGTCAGAACGTGTACCTGGCCTCCACCAACGGCGCCAACTTTGGCCAGGGAACAAAGCTGACCGTGCTGAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC NVS12 SEQ ID NO: 25 (number) HCDR1 1 GFTFSSYAMS SEQ ID NO: 26 (number) HCDR2 1 AISGSGGSTYYADSVKG SEQ ID NO: 27 (number) HCDR3 1 QRYYFGEFDL SEQ ID NO: 28 (Kabat) HCDR1 1 SYAMS SEQ ID NO: 26 (Kabat) HCDR2 1 AISGSGGSTYYADSVKG SEQ ID NO: 27 (Kabat) HCDR3 1 QRYYFGEFDL SEQ ID NO: 29 (Chothia) HCDR1 1 GFTFSSY SEQ ID NO: 30 (Chothia) HCDR2 1 SGSGGS SEQ ID NO: 27 (Chothia) HCDR3 1 QRYYFGEFDL SEQ ID NO: 31 (IMGT) HCDR1 1 GFTFSSYA SEQ ID NO: 32 (IMGT) HCDR2 1 ISGSGGST SEQ ID NO: 33 (IMGT) HCDR3 1 ARQRYYFGEFDL SEQ ID NO: 34 VH 1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQRYYFGEFDLWGQGTLVTVSS SEQ ID NO: 127 DNA encoding VH GAAGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGCTCCCTGAGGCTGTCTTGTGCCGCCTCCGGCTTCACCTTCCAGCTACGCCATGTCCTGGGTCCGAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCTCCGGCTCCGTCCCTAGTCCCCT ACGCCGACTCCGTGAAGGGCCGGTCCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACCGCCGTGTACTACTGCGCCAGACAGCGGTACTACTCGGCGAGTTCGACCTGTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCC SEQ ID NO: 92 (combined) HCDR1 2 GFSLTDYYMT SEQ ID NO: 93 (combined) HCDR2 2 FIDPDDDPYATWAKG SEQ ID NO: 94 (combined) HCDR3 2 GDHNSGWGLDI SEQ ID NO: 95 (Related) HCDR1 2 INCHES SEQ ID NO: 93 (Related) HCDR2 2 FIDPDDDPYATWAKG SEQ ID NO: 94 (Related) HCDR3 2 GDHNSGWGLDI SEQ ID NO: 96 (Chothia) HCDR1 2 GFSLTDYY SEQ ID NO: 97 (Chothia) HCDR2 2 DPDDD SEQ ID NO: 94 (Chothia) HCDR3 2 GDHNSGWGLDI SEQ ID NO: 98 (IMGT) HCDR1 2 GFSLTDYYY SEQ ID NO: 99 (IMGT) HCDR2 2 IDPDDDP SEQ ID NO: 100 (IMGT) HCDR3 2 AGGDHNSGWGLDI SEQ ID NO: 101 VH 2 EVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDDPYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGDHNSGWGLDIWGQGTLVTVSS SEQ ID NO: 128 DNA encoding VH GAAGTCCAGCTGGTGGAAAGCGGCGGAGGCCTGGTCCAGCCAGGCGGATCCCTGAGGCTCAGCTGCACCGCCTCTGGCTTCTCCCTGACCGACTACTACTATGACTTGGGTCCGCCAGGCTCCCGGAAAAGGACTCGAATGGGTCGGATTCATCGACCCCGACGGACCGACCCTTACTAC GCCACCTGGGCCAAGGGCAGATTCACCATCAGCAGAGACAACAGCAAGAACACTCTATCTCCAGATGAACTCCCTGAGGGCTGAAGATACCGCTGTCTATTACTGCGCTGGCGGCGACCAACTCCGGCTGGGGCCTGGATATCTGGGGACAAGGGCAGATTCACCATCAGGGGGGGACAAGGGCAGATTCGGGGATCTGGGGACAAGGGCAGATCACTCCAGTC SEQ ID NO: 118 HC linker GSGGGGSGGGGSGGG SEQ ID NO: 129 DNA encoding the HC linker ggctctggcggaggcggaagtggtggcggatcaggcggcgga SEQ ID NO: 130 Heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQRYYFGEFDLWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTCGVHTFPAVLQSSGLYSLSSVVTVPSSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGSGGGGSGSG GGGSGGGEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDDPYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGDHNSGWGLD IWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID NO: 131 DNA encoding the heavy chain GAAGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGCTCCCTGAGGCTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCCAGCTACGCCATGTCCTGGGTCCGACAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCTCCGGCTCCGGCGGCTCTACCTACTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGACAGCGGTACTACTTCGGCGAGTTCGACCTGTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCCGCCTCCACCAAGGGACCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGCACCGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCCGGCGCTCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAAGTGGACAAGCGGGTGGAACCCAAGTCCTGCGGCTCTGGCGGAGGCGGAAGTGGTGGCGGAGGATCAGGCGGCGGAGAAGTCCAGCTGGTGGAAAGCGGCGGAGGCCTGGTCCAGCCAGGCGGATCCCTGAGGCTCAGCTGCACCGCCTCTGGCTTCTCCCTGACCGACTACTACTATATGACTTGGGTCCGCCAGGCTCCCGGAAAAGGACTCGAATGGGTCGGATTCATCGACCCCGACGACGACCCTTACTACGCCACCTGGGCCAAGGGCAGATTCACCATCAGCAGAGACAACAGCAAGAACACACTCTATCTCCAGATGAACTCCCTGAGGGCTGAAGATACCGCTGTCTATTACTGCGCTGGCGGCGACCACAACTCCGGCTGGGGCCTGGATATCTGGGGACAGGGCACACTCGTGACAGTGTCCAGCGCCAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACCGCTGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACAGTGCCTTCCTCCAGCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCATGC SEQ ID NO: 38 (combined) LCDR1 1 SGDKLGDKYAY SEQ ID NO: 39 (combined) LCDR2 1 QDSKRPS SEQ ID NO: 40 (combined) LCDR3 1 QAFDYLYSLGV SEQ ID NO: 38 (Related) LCDR1 1 SGDKLGDKYAY SEQ ID NO: 39 (Related) LCDR2 1 QDSKRPS SEQ ID NO: 40 (Related) LCDR3 1 QAFDYLYSLGV SEQ ID NO: 41 (Chothia) LCDR1 1 DKLGDKY SEQ ID NO: 42 (Chothia) LCDR2 1 QDS SEQ ID NO: 43 (Chothia) LCDR3 1 FDYLYSLG SEQ ID NO: 44 (IMGT) LCDR1 1 KLGDKY SEQ ID NO: 42 (IMGT) LCDR2 1 QDS SEQ ID NO: 40 (IMGT) LCDR3 1 QAFDYLYSLGV SEQ ID NO: 45 VL 1 SYELTQPPSVSVSPGQTASITCSGDKLGDKYAYWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQAFDYLYSLGVFGGGTKLTVL SEQ ID NO: 132 DNA encoding VL TCCTACGAGCTGACCCAGCCTCCCTCCGTGTCCGTGTCTCCTGGCCAGACCGCCTCCACCTGTTCCGGCGACAAGCTGGGCGATAAGTACGCCTACTGGTATCAGCAGAAGCCCGGCCAGTCCCCTGTGCTGGTCATCTACCAGGACTCCAAGCGGCCC TCCGGCATCCCTGAGCGGTTCTCCGGCTCCAACTCCGGCAACACCGCCACCCTGACCATCTCCGGCACCCAGGCCGAGGACGAGGCCGACTACTACTGCCAGGCCTTCGACTACCTGTACTCCCTGGGCGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTG SEQ ID NO: 105 (combined) LCDR1 2 QASEIIHSWLA SEQ ID NO: 106 (combined) LCDR2 2 LASTLAS SEQ ID NO: 107 (combined) LCDR3 2 QNVYLASTNGAN SEQ ID NO: 105 (Related) LCDR1 2 QASEIIHSWLA SEQ ID NO: 106 (Related) LCDR2 2 LASTLAS SEQ ID NO: 107 (Related) LCDR3 2 QNVYLASTNGAN SEQ ID NO: 108 (Chothia) LCDR1 2 SEIIHSW SEQ ID NO: 109 (Chothia) LCDR2 2 LAS SEQ ID NO: 110 (Chothia) LCDR3 2 VYLASTNGA SEQ ID NO: 111 (IMGT) LCDR1 2 EIIHSW SEQ ID NO: 109 (IMGT) LCDR2 2 LAS SEQ ID NO: 107 (IMGT) LCDR3 2 QNVYLASTNGAN SEQ ID NO: 112 VL 2 EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLK SEQ ID NO: 133 The DNA encoding VL GAGATCGTGATGACCCAGTCCCCTTCCACCCTGTCCGCCTCCGTGGGCGACAGAGTGATCATCACCTGTCAGGCCTCCGAGATCATCCACAGCTGGCTGGCTTGGTCAGCAGAAACCTGGCAAGGCCCCTAAGCTGCTGATCTACCTGGCCTCCCACCCTGGCCT CCGGCGTGCCCTCCAGATTCTCCGGATCTGGCTCTGGCGCCGAGTTCACCCTGACAATCAGCTCCCTGCAGCCCGACGACTTCGCCACCTACTACTGTCAGAACGTGTACCTGGCCAGCACCAACGGCGCCAACTTCGGCCAGGGCACAACTGACAGTGCTGAAG SEQ ID NO: 118 LC-linker GSGGGGSGGGGSGGG SEQ ID NO: 134 DNA encoding the LC linker ggctctggtggcggaggatggcggaggcggttctggcggcgga SEQ ID NO: 135 Light chain SYELTQPPSVSVSPGQTASITCSGDKLGDKYAYWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNATLTTISGTQAEDEADYYCQAFDYLYSLGVFGGGTKLTVLGQP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSGSGGGGGSGG GGSGGGEIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTK LTVLKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 136 Yes, it's worth it. TCCTACGAGCTGACCCAGCCTCCCTCCGTGTCCGTGTCTCCTGGCCAGACCGCCTCCATCACCTGTTCCGGCGACAAGCTGGGCGATAAGTACGCCTACTGGTATCAGCAGAAGCCCGGCCAGTCCCCTGTGCTGGTCATCTACCAGGACTCCAAGCGGCCCTCCGGCATCCCTGAGCGGTTCTCCGGCTCCAACTCCGGCAACACCGCCACCCTGACCATCTCCGGCACCCAGGCCGAGGACGAGGCCGACTACTACTGCCAGGCCTTCGACTACCTGTACTCCCTGGGCGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTGGGCCAGCCCAAGGCCGCTCCTTCCGTGACCCTGTTCCCTCCATCCTCCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCTCCGACTTCTACCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTTCCAAGCAGTCCAACAACAAATACGCCGCCTCCTCCTACCTGTCCCTGACCCCTGAGCAGTGGAAGTCCCACCGGTCCTACAGCTGCCAAGTCACACACGAGGGCTCCACCGTGGAAAAGACCGTGGCCCCTACCGAGTGCTCCGGCTCTGGTGGCGGAGGATCTGGCGGAGGCGGTTCTGGCGGCGGAGAGATCGTGATGACCCAGTCCCCTTCCACCCTGTCCGCCTCCGTGGGCGACAGAGTGATCATCACCTGTCAGGCCTCCGAGATCATCCACAGCTGGCTGGCTTGGTATCAGCAGAAACCTGGCAAGGCCCCTAAGCTGCTGATCTACCTGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGATTCTCCGGATCTGGCTCTGGCGCCGAGTTCACCCTGACAATCAGCTCCCTGCAGCCCGACGACTTCGCCACCTACTACTGTCAGAACGTGTACCTGGCCAGCACCAACGGCGCCAACTTCGGCCAGGGCACAAAACTGACAGTGCTGAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC NVS13 SEQ ID NO: 25 (number) HCDR1 1 GFTFSSYAMS SEQ ID NO: 49 (number) HCDR2 1 GLGHVGYTTYTDSVKG SEQ ID NO: 50 HCDR3 1 DYLDFGYYFDV SEQ ID NO: 28 (Kabat) HCDR1 1 SYAMS SEQ ID NO: 49 (Kabat) HCDR2 1 GLGHVGYTTYTDSVKG SEQ ID NO: 50 (Kabat) HCDR3 1 DYLDFGYYFDV SEQ ID NO: 29 (Chothia) HCDR1 1 GFTFSSY SEQ ID NO: 51 (Chothia) HCDR2 1 GHVGY SEQ ID NO: 50 (Chothia) HCDR3 1 DYLDFGYYFDV SEQ ID NO: 31 (IMGT) HCDR1 1 GFTFSSYA SEQ ID NO: 52 (IMGT) HCDR2 1 LGHVGYT SEQ ID NO: 53 (IMGT) HCDR3 1 ARDYLDFGYYFDV SEQ ID NO: 54 VH 1 QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGLGHVGYTTYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYLDFGYYFDVWGQGTLVTVSS SEQ ID NO: 137 Video, video, VH 1 CAAGTGCAAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGCTCCCTGAGGCTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCCAGCTACGCCATGTCCTGGGTCCGACAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGGCCTGGGCCACGTGGGCTACACCACCTACA CCGACTCCGTGAAGGGCCGGTTCACCATCCTCCCGGGACAACTCCAAGAACACCCTGTAACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCGTGTACTACTGCGCCAGAGACTACCTGGACTTCGGCTACTACTTCGACGTGTGTGGGCCAGGGCACCCTGGTCACCGTGTCCTCC SEQ ID NO: 92 HCDR1 2 GFSLTDYYYMT SEQ ID NO: 93 (number) HCDR2 2 FIDPDDDPYYATWAKG SEQ ID NO: 94 HCDR3 2 GDHNSGWGLDI SEQ ID NO: 95 (Kabat) HCDR1 2 DYYYMT SEQ ID NO: 93 (Kabat) HCDR2 2 FIDPDDDPYYATWAKG SEQ ID NO: 94 (Kabat) HCDR3 2 GDHNSGWGLDI SEQ ID NO: 96 (Chothia) HCDR1 2 GFSLTDYY SEQ ID NO: 97 (Chothia) HCDR2 2 DPDDD SEQ ID NO: 94 (Chothia) HCDR3 2 GDHNSGWGLDI SEQ ID NO: 98 (IMGT) HCDR1 2 GFSLTDYYY SEQ ID NO: 99 (IMGT) HCDR2 2 IDPDDDP SEQ ID NO: 100 (IMGT) HCDR3 2 AGGDHNSGWGLDI SEQ ID NO: 101 VH 2 EVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDDPYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGDHNSGWGLDIWGQGTLVTVSS SEQ ID NO: 138 DNA encoding VH GAAGTGCAGCTGGTCGAGAGTGGCGGAGGCCTCGTCCAGCCAGGCGGATCCCTGAGGCTCAGCTGCACCGCCTCTGGCTTCTCCCTGACCGACTACTACTATGACTTGGGTCCGCCAGGCTCCCGGAAAAGGACTCGAATGGGTCGGATTCATCGACCCCGACGACGACCCCTACTAC GCCACCTGGGCCAAGGGCAGATTCACCATCAGCAGAGACAACAGCAAGAACACTCTATCTCCAGATGAACTCCCTGAGGGCTGAAGATACCGCTGTCTATTACTGCGCTGGCGGCGACCAACTCCGGCTGGGGCCTGGATATCTGGGGACAAGGGCAGATTCACCATCAGGGGGGGACAAGGGCAGATTCGGGGATCTGGGGACAAGGGCAGATCACTCCAGTC SEQ ID NO: 118 HC linker GSGGGGSGGGGSGGG SEQ ID NO: 139 DNA encoding the HC linker GGCTCTGGCGGAGGCGGAAGTGGTGGCGGAGGATCAGGCGGCGGA SEQ ID NO: 140 Heavy chain QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGLGHVGYTTYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYLDFGYYFDVWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTCGVHTFPAVLQSSGLYSLSSVVTVPSSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGSGGGGSGSG GGGSGGGEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDDPYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGDHNSGWGLD IWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID NO: 141 DNA encoding the heavy chain CAAGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGCTCCCTGAGGCTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCCAGCTACGCCATGTCCTGGGTCCGACAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGGCCTGGGCCACGTGGGCTACACCACCTACACCGACTCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGACTACCTGGACTTCGGCTACTACTTCGACGTGTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCCGCCTCCACCAAGGGACCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGCACCGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAAGTGGACAAGCGGGTGGAACCCAAGTCCTGCGGCTCTGGCGGAGGCGGAAGTGGTGGCGGAGGATCAGGCGGCGGAGAAGTGCAGCTGGTCGAGAGTGGCGGAGGCCTCGTCCAGCCAGGCGGATCCCTGAGGCTCAGCTGCACCGCCTCTGGCTTCTCCCTGACCGACTACTACTATATGACTTGGGTCCGCCAGGCTCCCGGAAAAGGACTCGAATGGGTCGGATTCATCGACCCCGACGACGACCCCTACTACGCCACCTGGGCCAAGGGCAGATTCACCATCAGCAGAGACAACAGCAAGAACACACTCTATCTCCAGATGAACTCCCTGAGGGCTGAAGATACCGCTGTCTATTACTGCGCTGGCGGCGACCACAACTCCGGCTGGGGCCTGGATATCTGGGGACAGGGCACACTCGTGACAGTGTCCAGCGCCAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACCGCTGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACAGTGCCTTCCTCCAGCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCATGC SEQ ID NO: 58 (combined) LCDR1 1 SGDKIGKKYVH SEQ ID NO: 59 (combined) LCDR2 1 DDSDRPS SEQ ID NO: 60 (combined) LCDR3 1 QAWDMQSVV SEQ ID NO: 58 (Related) LCDR1 1 SGDKIGKKYVH SEQ ID NO: 59 (Related) LCDR2 1 DDSDRPS SEQ ID NO: 60 (Related) LCDR3 1 QAWDMQSVV SEQ ID NO: 61 (Chothia) LCDR1 1 DKIGKKY SEQ ID NO: 62 (Chothia) LCDR2 1 DDS SEQ ID NO: 63 (Chothia) LCDR3 1 WDMQSV SEQ ID NO: 64 (IMGT) LCDR1 1 KIGKKY SEQ ID NO: 62 (IMGT) LCDR2 1 DDS SEQ ID NO: 60 (IMGT) LCDR3 1 QAWDMQSVV SEQ ID NO: 65 VL 1 SYELTQPLSVSVALGQTARITCSGDKIGKKYVHWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISRAQAGDEADYYCQAWDMQSVVFGGGTKLTVL SEQ ID NO: 142 DNA encoding VL TCCTACGAGCTGACCCAGCCCCTGTCCGTGTCTGTGGCTCTGGGCCAGACCCCGGATCACCTGTTCCGGCGAAGATCGGCAAGAAATACGTGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCTGTGCTGGTCATCTACGACGACTCCGACCGG CCCTCCGGCATCCCTGAGCGGTTCTCCGGCTCCAACTCCGGCAACACCGCCACCCTGACCATCCCAGAGCCCAGGCCGGCGACGAGGCCGACTACTACTGCCAGGCCTGGGACATGCAGTCCGTGGTGTTCCGGCGGAGGCACCAAGCTGACCGTGCTG SEQ ID NO: 105 (combined) LCDR1 2 QASEIIHSWLA SEQ ID NO: 106 (combined) LCDR2 2 LASTLAS SEQ ID NO: 107 (combined) LCDR3 2 QNVYLASTNGAN SEQ ID NO: 105 (Related) LCDR1 2 QASEIIHSWLA SEQ ID NO: 106 (Related) LCDR2 2 LASTLAS SEQ ID NO: 107 (Related) LCDR3 2 QNVYLASTNGAN SEQ ID NO: 108 (Chothia) LCDR1 2 SEIIHSW SEQ ID NO: 109 (Chothia) LCDR2 2 LAS SEQ ID NO: 110 (Chothia) LCDR3 2 VYLASTNGA SEQ ID NO: 111 (IMGT) LCDR1 2 EIIHSW SEQ ID NO: 109 (IMGT) LCDR2 2 LAS SEQ ID NO: 107 (IMGT) LCDR3 2 QNVYLASTNGAN SEQ ID NO: 112 VL 2 EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLK SEQ ID NO: 143 The DNA encoding VL GAGATCGTGATGACCCAGTCCCCTTCCACCCTGTCCGCCTCCGTGGGCGACAGAGTGATCATCACCTGTCAGGCCTCCGAGATCATCCACAGCTGGCTGGCTTGGTATCAGCAGAAACCTGGCAAGGCTCCCAAGCTGCTGATCTACCTGGCCTCCCACCCTGGCCT CCGGCGTGCCCTCCAGATTCTCCGGATCTGGCTCTGGCGCCGAGTTCACCCTGACAATCAGCTCCCTGCAGCCCGACGACTTCGCCACCTACTACTGTCAGAACGTGTACCTGGCCAGCACCAACGGCGCCAACTTCGGCCAGGGCACAACTGACAGTGCTGAAG SEQ ID NO: 118 LC-linker GSGGGGSGGGGSGGG SEQ ID NO: 144 DNA encoding the LC linker GGCTCTGGTGGCGGAGGATCTGGCGGAGGCGGTTCTGGCGGCGGA SEQ ID NO: 145 Light chain SYELTQPLSVSVALGQTARITCSGDKIGKKYVHWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISRAQAGDEADYYCQAWDMQSVVFGGGTKLTVLGQPKA APSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSGSGGGGSGGG GSGGGEIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTK LTVLKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 146 Yes, it's worth it. TCCTACGAGCTGACCCAGCCCCTGTCCGTGTCTGTGGCTCTGGGCCAGACCGCCCGGATCACCTGTTCCGGCGACAAGATCGGCAAGAAATACGTGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCTGTGCTGGTCATCTACGACGACTCCGACCGGCCCTCCGGCATCCCTGAGCGGTTCTCCGGCTCCAACTCCGGCAACACCGCCACCCTGACCATCTCCAGAGCCCAGGCCGGCGACGAGGCCGACTACTACTGCCAGGCCTGGGACATGCAGTCCGTGGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTGGGCCAGCCCAAGGCCGCTCCCTCTGTGACCCTGTTCCCTCCATCCTCCGAGGAACTGCAGGCCAACAAGGCCACCCTCGTGTGCCTGATCTCCGACTTCTACCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCTCTCCTGTGAAGGCCGGCGTGGAAACCACCACCCCTTCCAAGCAGTCCAACAACAAATACGCCGCCTCCTCCTACCTGTCCCTGACCCCTGAGCAGTGGAAGTCCCACCGGTCCTACAGCTGCCAAGTCACACACGAGGGCTCCACCGTGGAAAAGACCGTGGCCCCTACCGAGTGCTCCGGCTCTGGTGGCGGAGGATCTGGCGGAGGCGGTTCTGGCGGCGGAGAGATCGTGATGACCCAGTCCCCTTCCACCCTGTCCGCCTCCGTGGGCGACAGAGTGATCATCACCTGTCAGGCCTCCGAGATCATCCACAGCTGGCTGGCTTGGTATCAGCAGAAACCTGGCAAGGCTCCCAAGCTGCTGATCTACCTGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGATTCTCCGGATCTGGCTCTGGCGCCGAGTTCACCCTGACAATCAGCTCCCTGCAGCCCGACGACTTCGCCACCTACTACTGTCAGAACGTGTACCTGGCCAGCACCAACGGCGCCAACTTCGGCCAGGGCACAAAACTGACAGTGCTGAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC NVS14 SEQ ID NO: 69 (number) HCDR1 1 GFTFSRYWIS SEQ ID NO: 70 (number) HCDR2 1 YIDSTGTFINYADSVKG SEQ ID NO: 71 HCDR3 1 GGSLFDY SEQ ID NO: 72 (Kabat) HCDR1 1 RYWIS SEQ ID NO: 70 (Kabat) HCDR2 1 YIDSTGTFINYADSVKG SEQ ID NO: 71 (Kabat) HCDR3 1 GGSLFDY SEQ ID NO: 73 (Chothia) HCDR1 1 GFTFSRY SEQ ID NO: 74 (Chothia) HCDR2 1 DSTGTF SEQ ID NO: 71 (Chothia) HCDR3 1 GGSLFDY SEQ ID NO: 75 (IMGT) HCDR1 1 GFTFSRYW SEQ ID NO: 76 (IMGT) HCDR2 1 IDSTGTFI SEQ ID NO: 77 (IMGT) HCDR3 1 ARGGSLFDY SEQ ID NO: 78 VH 1 QVQLLESGGGLVQPGGSLRLSCAASGFTFSRYWISWVRQAPGKGLEWVSYIDSTGTFINYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSLFDYWGQGTLVTVSS SEQ ID NO: 147 Video, video, VH 1 CAAGTGCAAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGCTCCCTGAGGCTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCCCGGTACTGGATCTCCTGGGTCCGACAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCTACATCGACTCCACCGGCACCTTCATC AACTACGCCGACTCCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACTCCAAGAACACCCTGTAACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGGCGGCAGCCTGTTCGACTACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCC SEQ ID NO: 92 HCDR1 2 GFSLTDYYYMT SEQ ID NO: 93 (number) HCDR2 2 FIDPDDDPYYATWAKG SEQ ID NO: 94 HCDR3 2 GDHNSGWGLDI SEQ ID NO: 95 (Kabat) HCDR1 2 DYYYMT SEQ ID NO: 93 (Kabat) HCDR2 2 FIDPDDDPYYATWAKG SEQ ID NO: 94 (Kabat) HCDR3 2 GDHNSGWGLDI SEQ ID NO: 96 (Chothia) HCDR1 2 GFSLTDYY SEQ ID NO: 97 (Chothia) HCDR2 2 DPDDD SEQ ID NO: 94 (Chothia) HCDR3 2 GDHNSGWGLDI SEQ ID NO: 98 (IMGT) HCDR1 2 GFSLTDYYY SEQ ID NO: 99 (IMGT) HCDR2 2 IDPDDDP SEQ ID NO: 100 (IMGT) HCDR3 2 AGGDHNSGWGLDI SEQ ID NO: 101 VH 2 EVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDDPYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGDHNSGWGLDIWGQGTLVTVSS SEQ ID NO: 148 DNA encoding VH GAAGTGCAGCTGGTCGAGAGTGGCGGAGGCCTCGTCCAGCCAGGCGGATCCCTGAGGCTCAGCTGCACCGCCTCTGGCTTCTCCCTGACCGACTACTACTACATGGGTCCGCCAGGCTCCCGGAAAAGGACTCGAATGGGTCGGATTCATCGACCCCGACGACGACCCCTACCTACC GCCACCTGGGCCAAGGGCAGATTCCACCATCCAGAGATAACAGCAAGAACACTCTATCTCCAGATGAACTCCCTGAGGGCTGAAGATACCGCTGTCTATTACTGCGCTGGCGGCGACCAACTCCGGCTGGGGCCTGGATATCTGGGGACAAGGGAACAGGGGGGGATCTGGGGACAAGGGAACAGATCCAGGGGGGGGGGACAAGTC SEQ ID NO: 118 HC linker GSGGGGSGGGGSGGG SEQ ID NO: 139 DNA encoding the HC linker GGCTCTGGCGGAGGCGGAAGTGGTGGCGGAGGATCAGGCGGCGGA SEQ ID NO: 149 Heavy chain QVQLLESGGGLVQPGGSLRLSCAASGFTFSRYWISWVRQAPGKGLEWVSYIDSTGTFINYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSSLFDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGSGGGGGSGG GGSGGGEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDHNSGWGLD IWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID NO: 150 Yes, it's worth it. CAAGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGCTCCCTGAGGCTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCCCGGTACTGGATCTCCTGGGTCCGACAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCTACATCGACTCCACCGGCACCTTCATCAACTACGCCGACTCCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGGCGGCAGCCTGTTCGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCCGCCTCCACCAAGGGACCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGCACCGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAAGTGGACAAGCGGGTGGAACCCAAGTCCTGCGGCTCTGGCGGAGGCGGAAGTGGTGGCGGAGGATCAGGCGGCGGAGAAGTGCAGCTGGTCGAGAGTGGCGGAGGCCTCGTCCAGCCAGGCGGATCCCTGAGGCTCAGCTGCACCGCCTCTGGCTTCTCCCTGACCGACTACTACTACATGACATGGGTCCGCCAGGCTCCCGGAAAAGGACTCGAATGGGTCGGATTCATCGACCCCGACGACGACCCCTACTACGCCACCTGGGCCAAGGGCAGATTCACCATCTCCAGAGATAACAGCAAGAACACACTCTATCTCCAGATGAACTCCCTGAGGGCTGAAGATACCGCTGTCTATTACTGCGCTGGCGGCGACCACAACTCCGGCTGGGGCCTGGATATCTGGGGACAGGGAACACTCGTGACAGTGTCCAGCGCCAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACCGCTGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACAGTGCCTTCCTCCAGCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCATGC SEQ ID NO: 82 (number) LCDR1 1 RASQGIISYLG SEQ ID NO: 83 LCDR2 1 AASSLQS SEQ ID NO: 84 LCDR3 1 QQYDALNT SEQ ID NO: 82 (Kabat) LCDR1 1 RASQGIISYLG SEQ ID NO: 83 (Kabat) LCDR2 1 AASSLQS SEQ ID NO: 84 (Kabat) LCDR3 1 QQYDALNT SEQ ID NO: 85 (Chothia) LCDR1 1 SQGIISY SEQ ID NO: 18 (Chothia) LCDR2 1 AAS SEQ ID NO: 86 (Chothia) LCDR3 1 YDALN SEQ ID NO: 87 (IMGT) LCDR1 1 QGIISY SEQ ID NO: 18 (IMGT) LCDR2 1 AAS SEQ ID NO: 84 (IMGT) LCDR3 1 QQYDALNT SEQ ID NO: 88 VL 1 DIQMTQSPSSLSASVGDRVTITCRASQGIISYLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDALNTFGQGTKVEIK SEQ ID NO: 151 DNA encoding VL GACATCCAGATGACCCAGAGCCCCTCCAGCCCTGTCCGCCTCCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGGGCATCATCTCCTACCTGGGCTGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCTCC CTGCAGTCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCCAGCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACGACGCCCTGAACACCTTCGGCCAGGGCACCAAAGTGGAAATCAAG SEQ ID NO: 105 (combined) LCDR1 2 QASEIIHSWLA SEQ ID NO: 106 (combined) LCDR2 2 LASTLAS SEQ ID NO: 107 (combined) LCDR3 2 QNVYLASTNGAN SEQ ID NO: 105 (Related) LCDR1 2 QASEIIHSWLA SEQ ID NO: 106 (Related) LCDR2 2 LASTLAS SEQ ID NO: 107 (Related) LCDR3 2 QNVYLASTNGAN SEQ ID NO: 108 (Chothia) LCDR1 2 SEIIHSW SEQ ID NO: 109 (Chothia) LCDR2 2 LAS SEQ ID NO: 110 (Chothia) LCDR3 2 VYLASTNGA SEQ ID NO: 111 (IMGT) LCDR1 2 EIIHSW SEQ ID NO: 109 (IMGT) LCDR2 2 LAS SEQ ID NO: 107 (IMGT) LCDR3 2 QNVYLASTNGAN SEQ ID NO: 112 VL 2 EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLK SEQ ID NO: 152 The DNA encoding VL GAGATCGTGATGACCCAGTCCCCTAGCACCCTGAGCGCCAGCGTGGGAGATCGCGTGATCATCACATGCCAGGCCTCCGAGATCATCCACAGCTGGCTGGCTTGGTCAGCAGAAACCTGGAAAAGCTCCCAAGCTCCTGATCTATCTGGCCAGCACCCTGGCCT CTGGCGTGCCCAGCAGATTCAGCGGCTCCGGCAGCGGCGCTGAGTTTACCCTGACAATCAAGCTCTCTGCAGCCTGACGATTTTGCTACCTACTATTTGTCAGAACGTGTACCTGGCCTCCACCAACGGCGCCAACTTTGGCCAGGAACAAAGCTGACCGTGCTGAAG SEQ ID NO: 118 LC-linker GSGGGGSGGGGSGGG SEQ ID NO: 153 DNA encoding the LC linker GGCTCCGGCGGAGGCGGATCTGGTGGCGGAGGATCTGGCGGTGGC SEQ ID NO: 154 Light chain DIQMTQSPSSLSSASVGDRVTITCRASQGIISYLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDALNTFGQGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGSGGGGSGGG GSGGGEIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTK LTVLKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 155 DNA encoding the light chain GACATCCAGATGACCCAGAGCCCCTCCAGCCTGTCCGCCTCCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGGGCATCATCTCCTACCTGGGCTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCTCCCTGCAGTCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACGACGCCCTGAACACCTTCGGCCAGGGCACCAAAGTGGAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTCGTGTGCCTGCTGAACAACTTCTACCCTCGCGAGGCCAAAGTGCAGTGGAAAGTGGACAACGCCCTGCAGAGCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGCGAGTGTGGCTCCGGCGGAGGCGGATCTGGTGGCGGAGGATCTGGCGGTGGCGAGATCGTGATGACCCAGTCCCCTAGCACCCTGAGCGCCAGCGTGGGAGATCGCGTGATCATCACATGCCAGGCCTCCGAGATCATCCACAGCTGGCTGGCTTGGTATCAGCAGAAACCTGGAAAAGCTCCCAAGCTCCTGATCTATCTGGCCAGCACCCTGGCCTCTGGCGTGCCCAGCAGATTCAGCGGCTCCGGCAGCGGCGCTGAGTTTACCCTGACAATCAGCTCTCTGCAGCCTGACGATTTTGCTACCTACTATTGTCAGAACGTGTACCTGGCCTCCACCAACGGCGCCAACTTTGGCCAGGGAACAAAGCTGACCGTGCTGAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC Linkers

[0439] In certain aspects of the present invention, an anti-BTC antibody-based binding functional unit may be linked to a molecule, such as an anti-VEGF antibody-based binding functional unit, via a linker. More specifically, the anti-BTC antibody-based binding functional units may be linked to a protein or nucleic acid via a peptide linker (e.g., a Gly linker) n -Sir n ) n or (Ser n -Gly n ) n) with optimized length and / or amino acid composition. It is known that the length of a peptide linker can greatly influence how the linked proteins fold and interact. For examples of linker orientation and size, see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, US Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT Publication Nos. WO 2006 / 020258 and WO 2007 / 024715, which are incorporated herein by reference.

[0440] The peptide linker sequence may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more amino acid residues in length. The peptide linker sequence may be composed of naturally occurring or non-naturally occurring amino acids. In some aspects, the linker is a glycine polymer. In some aspects, the amino acids in the linker sequence are glycine and serine amino acids. In certain aspects, the linker region comprises glycine repeat groups (GlySerGly3) n, where n is a positive integer that is equal to or greater than 1, such as n = 3 (SEQ ID NO: 118). More specifically, the linker sequence can be GlySerGlyGlyGly (SEQ ID NO: 165). Alternatively, the linker sequence can be GlySerGlyGly (SEQ ID NO: 166). In certain other aspects, the orientation of the linker region provides for groups of glycine repeats (SerGly3) n , where n is a positive integer that is equal to or greater than 1, for example n=3 (SEQ ID NO: 167).

[0441] Peptide linkers may also include, but are not limited to, (Gly4Ser)4(SEQ ID NO: 161) or (Gly4Ser)3(SEQ ID NO: 162). Glu and Lys amino acid residues may be distributed within the Gly-Ser peptide linkers to provide improved solubility. In certain aspects, peptide linkers may comprise multiple repeats of (Gly3Ser), (Gly2Ser), or (GlySer). In certain aspects, peptide linkers may comprise multiple repeats of (SerGly3), (SerGly2), or (SerGly). In other aspects, peptide linkers may include combinations and multiples of (Gly3Ser) + (Gly4Ser) + (GlySer) (SEQ ID NO: 163). In other aspects, Ser can be replaced by Ala, for example (Gly4Ala) or (Gly3Ala). In st...

Claims

1. A bispecific binding molecule that binds to betacellulin (BTC) and vascular endothelial growth factor (VEGF), wherein the bispecific binding molecule comprises: 1) a fragment that binds the BTC, and 2) VEGF-binding fragment, where the BTC-binding fragment contains the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3: a) SEQ ID NO: 1, 2, 3, 14, 15 and 16, respectively; SEQ ID NO: 4, 2, 3, 14, 15 and 16 respectively; SEQ ID NO: 5, 6, 3, 17, 18 and 19 respectively; or SEQ ID NO: 7, 8, 9, 20, 18 and 16 respectively; b) SEQ ID NO: 25, 26, 27, 38, 39 and 40, respectively; SEQ ID NO: 28, 26, 27, 38, 39 and 40 respectively; SEQ ID NO: 29, 30, 27, 41, 42 and 43 respectively; or SEQ ID NO: 31, 32, 33, 44, 42 and 40 respectively; c) SEQ ID NO: 25, 49, 50, 58, 59 and 60, respectively; SEQ ID NO: 28, 49, 50, 58, 59 and 60 respectively; SEQ ID NO: 29, 51, 50, 61, 62, and 63, respectively; or SEQ ID NO: 31, 52, 53, 64, 62, and 60, respectively; or d) SEQ ID NO: 69, 70, 71, 82, 83 and 84, respectively; SEQ ID NO: 72, 70, 71, 82, 83 and 84 respectively; SEQ ID NO: 73, 74, 71, 85, 18 and 86, respectively; or SEQ ID NO: 75, 76, 77, 87, 18 and 84 respectively, wherein the BTC-binding fragment and the VEGF-binding fragment are an antibody or an antigen-binding fragment thereof.

2. The bispecific binding molecule of claim 1, wherein the VEGF-binding moiety and / or the BTC-binding moiety are in a format selected from the group consisting of an isolated antibody, CrossMab, Fab, Fab', F(ab')2, Fv, and scFv.

3. The bispecific binding molecule of claim 2, wherein the BTC-binding moiety is an anti-BTC Fab, and the VEGF-binding moiety is an anti-VEGF Fab, wherein the anti-BTC Fab comprises a heavy chain (HA) and a light chain (LA), and wherein the anti-VEGF Fab comprises a heavy chain (HB) and a light chain (LB).

4. The bispecific binding molecule of claim 3, wherein HA and HB are linked in the format from N-terminus to C-terminus: N-HA-linker-1-HB-C, and where LA and LB are connected in the format from N-terminus to C-terminus: N-LA-linker-2-LB-C.

5. The bispecific binding molecule of claim 1, wherein the BTC-binding moiety comprises VH and VL comprising an amino acid sequence that is at least 90% identical to: a) SEQ ID NO: 10 and 21, respectively; b) SEQ ID NO: 34 and 45, respectively; c) SEQ ID NO: 54 and 65, respectively; or d) SEQ ID NO: 78 and 88, respectively.

6. The bispecific binding molecule of claim 5, wherein the BTC-binding moiety comprises a heavy chain and a light chain with an amino acid sequence: a) SEQ ID NO:12 and 23, respectively; b) SEQ ID NO:36 and 47, respectively; c) SEQ ID NO:56 and 67, respectively; or d) SEQ ID NO:80 and 90, respectively.

7. The bispecific binding molecule of claim 1, wherein the VEGF binding moiety comprises VH and VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 101 and 112, respectively.

8. The bispecific binding molecule of claim 7, wherein the VEGF binding moiety comprises a heavy chain and a light chain with the amino acid sequence of SEQ ID NO: 103 and 114, respectively, or wherein the heavy chain and light chain are encoded by a nucleotide sequence that is 100% identical to SEQ ID NO: 104 and 115, respectively.

9. The bispecific binding molecule of claim 1, wherein the BTC-binding fragment comprises a variable heavy chain domain (VHA) and a variable light chain domain (VLA) that bind to BTC, and wherein the VEGF-binding fragment comprises a variable heavy chain domain (VHB) and a variable light chain domain (VLB) that bind to VEGF, wherein the bispecific binding molecule is in the format from N-terminus to C-terminus as follows: N-VHA-heavy chain constant domain (CH1A)-linker-1-VHB-heavy chain constant domain (CH1B)-C and N-VLA-light chain constant domain (CKA)-linker-2-VLB-light chain constant domain (CKB)-C, which comprises a heavy chain comprising VHA, CH1A, linker 1, VHB and CH1B, and which comprises a light chain comprising VLA, CKA, linker 2, VLB and CKB, where the heavy chain and light chain are as follows: a) SEQ ID NO: 120 and 125, respectively; b) SEQ ID NO: 130 and 135, respectively; c) SEQ ID NO: 140 and 145, respectively; or d) SEQ ID NO: 149 and 154, respectively.

10. A polynucleotide for expressing a bispecific binding molecule according to claim 1, wherein the polynucleotide comprises a nucleotide sequence encoding the bispecific binding molecule according to claim 1.

11. A pharmaceutical composition comprising an effective amount of a bispecific binding molecule according to claim 1 for treating a patient with an ophthalmological disorder, where the ophthalmological disorder is diabetic macular edema (DME) or neovascular age-related macular degeneration (nAMD).

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