Anti-betacellulin antibodies, fragments thereof, and multispecific binding molecules

Abstract

The present disclosure provides anti-BTC antibodies, methods of producing the antibodies, pharmaceutical compositions comprising the antibodies, and methods of using the antibodies. The present disclosure also provides multispecific binding molecules, e.g., bispecific antibodies comprising a BTC-binding portion and an anti-VEGF-binding portion.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCE LISTINGS This application claims priority to 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 by reference in their entireties. The Sequence Listing contained in file entitled "PAT058888-WO-PCT SQL_ST25," created June 25, 2021, and measuring 204,786 bytes (measured on the MS-Windows operating system), is submitted herewith and is incorporated by reference herein.

[0002] The present invention relates to antibodies or antigen-binding fragments thereof, methods for their production, pharmaceutical compositions containing them, and methods of use thereof. [Background technology]

[0003] Betacellulin (BTC), a member of the epidermal growth factor (EGF) family, was originally isolated from the conditioned medium of a murine pancreatic B-tumor cell line (Shing et al., Science, 259, 1604-1607, 1993). BTC is a ligand for the ErbB receptor tyrosine kinase family, primarily activating ErbB1 and ErbB4 homodimers, which trigger anti-apoptotic and pro-growth signaling pathways such as the Ras / MAPK and PL3K / AKT pathways.

[0004] BTC is initially expressed as a single-pass transmembrane protein and then cleaved (activated) into a 9-kDa secreted protein by members of the MMP family. Cleavage of the membrane-anchored form of BTC to release the secreted form occurs primarily 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 composed of 80 amino acid residues (Asp1-Tyr80, residues 32-111 of 178 residues, membrane-anchored precursor protein (pro-BTC) described as NP_001720.1 or SEQ ID NO: 156) generated by cleavage of pro-BTC. The carboxyl-terminal 50-residue region of BTC (Arg31-Tyr80) contains a conserved consensus sequence for the EGF family of proteins.

[0005] Strong expression of BTC mRNA has been detected in many tissues, including the pancreas, liver, kidney, and small intestine, with somewhat lower expression in the 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, which do not display a clear phenotype, are viable and fertile.

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

[0007] In the mammalian eye, BTC proteins are synthesized by the retinal pigment epithelium (RPE), endothelium, and Müller cells (Anand-Apte et al., PLoS One 5, e13444, 2010) and are located 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 unknown. Early reports of BTC's proliferative effect on RPE cells (Shing et al., 1993) and its proangiogenic function suggested that it may play a role in proliferative diabetic retinopathy (PDR). Although diabetic mice do not demonstrate PDR, they do show increased retinal vascular permeability (Poulaki et al., J. Clin. Invest. 109, 805–815, 2002). Additionally, it has been determined that soluble, cleaved BTC is increased in the retina and contributes to increased retinal vascular permeability in a mouse model of diabetes (Anand-Apte et al., 2010).

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

[0010] Vascular endothelial growth factor (VEGF) has been shown to be an important mediator of neovascularization associated with tumors and intraocular disorders. VEGF is a potent vascular permeability factor and is essential for causing vascular leakage. In mammalian eyes, VEGF is located in 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] Several anti-VEGF drugs, 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), have been used to treat ophthalmic disorders such as age-related macular degeneration (AMD) and / or DME. Other anti-VEGF molecules include the soluble VEGF receptor analog, VEGF-Trap (Regeneron), the small interfering RNA (siRNA) bevasiranib (Opko Health), and rapamycin (Sirolimus, MacuSight). Anti-VEGF drugs are delivered to the eye as an intravitreal injection under local anesthesia.

[0012] However, unmet medical needs remain due to inadequate responses to anti-VEGF therapy alone in patients with ophthalmic disorders such as DME. Anti-VEGF agents reduce macular edema, inhibit neovascularization, and improve visual acuity, but not all DME patients experience substantial long-term improvement. For example, approximately 25% of patients receiving anti-VEGF therapy fail to achieve any improvement in visual acuity 12 months after treatment, and nearly 50% of patients are unable to achieve legal driving vision of 20 / 40 (Mitchell et al., 2011). Alternative therapies, such as laser photocoagulation or intravitreal steroid therapy, have been less successful and have side effects (e.g., cataracts caused by intraocular steroid therapy (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 therapy in order to improve therapy for patients who have an inadequate response to anti-VEGF therapy. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows the structure of Fab bound to BTC. BTC is shown as a solid surface, and Fab is shown as a ribbon. To illustrate the variation in binding mode by Fab, the BTC structures are 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. [Figure 2] Structural epitope residues of Fab binding to BTC are shown. BTC is shown as a ribbon with various orientations to highlight the epitope residues listed in Tables 12, 13, 14, and 15. Epitope residues are shown as balls and sticks and labeled. A) BTC / Fab NVS2 complex, B) BTC / Fab NVS3 complex, C) BTC / Fab NVS1 complex, and D) BTC / Fab NVS4 complex. [Figure 3]1 provides a graphical representation of monospecific and bispecific antibodies. [Figure 4] Retinal images of mice injected with scAAV2-CMV-BTC from fundus photography (left panel) and FFA visualization (right panel) are shown. [Figure 5] 1 shows retinal images of mice injected with scAAV2-CMV-BTC from a scanning laser ophthalmoscope. [Figure 6-1] Figure 6: Figures 6A-6G show binding of monospecific and bispecific antibodies to BTC and / or VEGF. [Figure 6-2] (As mentioned above.) [Figure 6-3] (As mentioned above.) [Figure 6-4] (As mentioned above.) [Figure 7-1] Figure 7: Figures 7A-7D show the binding of BTC to ErbB1 in the presence of monospecific (NVS1-4) and bispecific (NVS11-14) antibodies. [Figure 7-2] (As mentioned above.) [Figure 8-1] Figure 8: Figures 8A-8D show the binding of BTC to ErbB4 in the presence of monospecific (NVS1-4) and bispecific (NVS11-14) antibodies. Figures 8E and 8F show the binding of BTC 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. [Figure 8-2] (As mentioned above.) [Figure 8-3] (As mentioned above.) [Figure 8-4] (As mentioned above.) [Figure 9-1] Figure 9: Figures 9A-9C show the binding of VEGF-A to VEGFR2 in the presence of monospecific (NVS8) and bispecific (NVS11, NVS12, and NVS14) antibodies. [Figure 9-2] (As mentioned above.) [Figure 10-1]Figure 10: Figures 10A-10D show BTC-induced phosphorylation of ERK1 / 2 in the presence of monospecific (NVS1-4) and bispecific (NVS11-14) antibodies. [Figure 10-2] (As mentioned above.) [Figure 11-1] Figure 11: Figures 11A-11D show BTC-induced phosphorylation of ErbB3 in the presence of monospecific (NVS1-4) and bispecific (NVS11-14) antibodies. [Figure 11-2] (As mentioned above.) [Figure 12-1] Figure 12: Figures 12A-12D show BTC-induced phosphorylation of HER3 in the presence of monospecific (NVS1-2) and bispecific (NVS11-14) antibodies. [Figure 12-2] (As mentioned above.) [Figure 13] Figures 13A and 13B show BTC-induced permeability of retinal pigment epithelial (RPE) cells and human retinal capillary endothelial (HREC) cells in vitro in the presence of monospecific (NVS1 or NVS8) and bispecific (NVS11) antibodies. [Figure 14] 1 shows hyperglycemia-induced retinal leakage in diabetic rats in the presence of anti-BTC (LZR230) antibody and / or anti-VEGF (4G3) antibody. [Figure 15-1] Figure 15: Figure 15A shows optical coherence tomography (OCT) and pathology images from rabbit eyes after treatment with VEGF or BTC. Figure 15B shows representative OCT images demonstrating the effect of intravitreal betacellulin on rabbit retinas after intravitreal injection of NVS1 or NVS11. [Figure 15-2] (As mentioned above.) [Figure 16] 1 shows the change in retinal thickness in rabbit eyes after treatment with NVS1 or NVS11. [Figure 17] 1 shows the change in retinal thickness in rabbit eyes after treatment with NVS2 or NVS12. [Figure 18-1]Figure 18: Figure 18A shows changes in retinal thickness in rabbit eyes after treatment with NVS1, NVS11, NVS2, or NVS12. Figure 18B shows BTC-induced RPE morphological changes in the presence of NVS11 or NVS1. Figure 18C shows VEGF-induced retinal vascular leakage in rabbits in the presence of NVS11 or NVS8 (representative images). Figure 18D shows VEGF-induced retinal vascular leakage in rabbits in the presence of NVS11 or NVS8 (quantified by fluorescein angiography). [Figure 18-2] (As mentioned above.) [Figure 18-3] (As mentioned above.) [Figure 19] Fluorescein angiography images of rabbit eyes after IVT delivery of VEGF or BTC are shown. [Figure 20] Fluorescein vascular leakage values ​​from individual rabbit eyes after treatment with NVS8, NVS11, or NVS12 are shown. [Figure 21] Fluorescein vascular leakage values ​​from individual rabbit eyes after treatment with NVS11, NVS12, or NVS8 are shown. [Figure 22] 1 shows the change in total retinal thickness values ​​from individual rabbit eyes after treatment with ranibizumab or NVS1. [Figure 23] 1 shows the change in retinal thickness in rabbits after treatment with NVS1 or PNVS1. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present disclosure provides isolated antibodies or antigen-binding fragments thereof that specifically bind to betacellulin (BTC).

[0016] In one embodiment, the antibody or antigen-binding fragment thereof blocks binding of BTC to ErbB1, ErbB4, or both.

[0017] In one aspect, the antibody or antigen-binding fragment thereof blocks BTC-induced phosphorylated-ERK1 / 2 activation.

[0018] In one aspect, the antibody or antigen-binding fragment thereof blocks BTC-induced phosphorylation-HER3 activation.

[0019] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to BTC, wherein the antibody or antigen-binding fragment has a dissociation constant (KD) of 5 pM or less.

[0020] In one embodiment, the antibody or antigen-binding fragment thereof binds to a BTC comprising the amino acid sequence of SEQ ID NO:157.

[0021] 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.

[0022] 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.

[0023] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises heavy chain variable region complementarity determining region 1 (HCDR1), heavy chain variable region complementarity determining region 2 (HCDR2), and heavy chain variable region complementarity determining region 3 (HCDR3) as set forth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable region complementarity determining region 1 (LCDR1), light chain variable region complementarity determining region 2 (LCDR2), and light chain variable region complementarity determining region 3 (LCDR3) as set forth in SEQ ID NOs: 14, 15, and 16, respectively.

[0024] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 4, 2, and 3, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 14, 15, and 16, respectively.

[0025] In one aspect, HCDR1 comprises the consensus sequence XYAIS and / or HCDR2 comprises the consensus sequence GIXPXXGXXXYAQKFQG, where X is any amino acid and does not have to be the same at different positions.

[0026] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises the heavy chain sequence of SEQ ID NO: 168 and the light chain sequence of SEQ ID NO: 169, or comprises the heavy chain sequence of SEQ ID NO: 170 and the light chain sequence of SEQ ID NO: 171.

[0027] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 5, 6, and 3, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 17, 18, and 19, respectively.

[0028] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 7, 8, and 9, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 20, 18, and 16, respectively.

[0029] In one embodiment, 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 amino acid sequences having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 10 and 21, respectively.

[0030] In one embodiment, the difference in amino acid sequence is not within a complementarity determining region.

[0031] In one aspect, the differences in amino acid sequence are conservative substitutions.

[0032] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and a VL comprising the amino acid sequences as set forth in SEQ ID NOs: 10 and 21, respectively.

[0033] In one embodiment, the VH and VL are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 11 and 22, respectively.

[0034] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain having the amino acid sequences as set forth in SEQ ID NOs: 12 and 23, respectively.

[0035] In one aspect, the heavy and light chains are encoded by nucleic acids having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 13 and 24, respectively.

[0036] 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 having the amino acid sequence of SEQ ID NO: 10, and 2) LCDR1, LCDR2, and LCDR3 contained in a VL having the amino acid sequence of SEQ ID NO: 21.

[0037] In one aspect, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise 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.

[0038] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein a. 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, and HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 9; b. LCDR1 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, and LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19.

[0039] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 10 and 21, respectively.

[0040] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain having the amino acid sequences as set forth in SEQ ID NOs: 12 and 23, respectively.

[0041] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to BTC, comprising a VH and a VL having the amino acid sequences of SEQ ID NOs: 10 and 21, respectively.

[0042] 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.

[0043] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 25, 26, and 27, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 38, 39, and 40, respectively.

[0044] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 28, 26, and 27, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 38, 39, and 40, respectively.

[0045] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 29, 30, and 27, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 41, 42, and 43, respectively.

[0046] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 31, 32, and 33, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 44, 42, and 40, respectively.

[0047] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and a VL comprising amino acid sequences having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 34 and 45, respectively.

[0048] In one embodiment, the difference in amino acid sequence is not within a complementarity determining region.

[0049] In one aspect, the differences in amino acid sequence are conservative substitutions.

[0050] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and a VL comprising the amino acid sequences as set forth in SEQ ID NOs: 34 and 45, respectively.

[0051] In one embodiment, the VH and VL are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 35 and 46, respectively.

[0052] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain having the amino acid sequences as set forth in SEQ ID NOs: 36 and 47, respectively.

[0053] In one aspect, the heavy and light chains are encoded by nucleic acid sequences having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 37 and 48, respectively.

[0054] 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 having the amino acid sequence of SEQ ID NO: 34, and 2) LCDR1, LCDR2, and LCDR3 contained in a VL having the amino acid sequence of SEQ ID NO: 45.

[0055] In one aspect, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise 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.

[0056] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein a. 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; and HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 and 33; b. LCDR1 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, and LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 40 and 43.

[0057] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 34 and 45, respectively.

[0058] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain having the amino acid sequences as set forth in SEQ ID NOs: 36 and 47, respectively.

[0059] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof that specifically binds to BTC, comprising a VH and a VL having the amino acid sequences of SEQ ID NOs: 34 and 45, respectively.

[0060] 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.

[0061] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 25, 49, and 50, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 58, 59, and 60, respectively.

[0062] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 28, 49, and 50, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 58, 59, and 60, respectively.

[0063] In one aspect, HCDR1 comprises the consensus sequence XXAMX and / or HCDR2 comprises the consensus sequence XXXX / -XXXXTXYXDSVKG, where X is any amino acid and may not be the same at different positions, and X / - is any amino acid or a deletion.

[0064] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC comprises the heavy chain sequence of SEQ ID NO:190 and the light chain sequence of SEQ ID NO:191.

[0065] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 29, 51, and 50, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 61, 62, and 63, respectively.

[0066] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 31, 52, and 53, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 64, 62, and 60, respectively.

[0067] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and a VL comprising amino acid sequences having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 54 and 65, respectively.

[0068] In one embodiment, the difference in amino acid sequence is not within a complementarity determining region.

[0069] In one aspect, the differences in amino acid sequence are conservative substitutions.

[0070] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and a VL comprising the amino acid sequences as set forth in SEQ ID NOs: 54 and 65, respectively.

[0071] In one embodiment, the VH and VL are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 55 and 66, respectively.

[0072] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain having the amino acid sequences as set forth in SEQ ID NOs: 56 and 67, respectively.

[0073] In one aspect, the heavy and light chains are encoded by nucleic acid sequences having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 57 and 68, respectively.

[0074] 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 having the amino acid sequence of SEQ ID NO: 54, and 2) LCDR1, LCDR2, and LCDR3 contained in a VL having the amino acid sequence of SEQ ID NO: 65.

[0075] In one aspect, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise 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.

[0076] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein a. 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; and HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 50 and 53; b. LCDR1 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, and LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 60 and 63.

[0077] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 54 and 65, respectively.

[0078] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain having the amino acid sequences as set forth in SEQ ID NOs: 56 and 67, respectively.

[0079] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to BTC, comprising a VH and a VL having the amino acid sequences of SEQ ID NOs: 54 and 65, respectively.

[0080] 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.

[0081] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 69, 70, and 71, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 82, 83, and 84, respectively.

[0082] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 72, 70, and 71, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 82, 83, and 84, respectively.

[0083] In one aspect, HCDR2 comprises the consensus sequence XIXXXXXXXXYADSVKG and / or LCDR3 comprises the consensus sequence QQYDXXXT, where X is any amino acid and does not have to be the same at different positions.

[0084] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC comprises heavy and light chain sequences selected from the group consisting of SEQ ID NOs: 194 and 195, respectively; SEQ ID NOs: 196 and 197, respectively; SEQ ID NOs: 198 and 199, respectively; SEQ ID NOs: 200 and 201, respectively; SEQ ID NOs: 202 and 203, respectively; SEQ ID NOs: 204 and 205, respectively; and SEQ ID NOs: 206 and 207, respectively.

[0085] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 73, 74, and 71, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 85, 18, and 86, respectively.

[0086] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 75, 76, and 77, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 87, 18, and 84, respectively.

[0087] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and a VL comprising amino acid sequences having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 78 and 88, respectively.

[0088] In one embodiment, the difference in amino acid sequence is not within a complementarity determining region.

[0089] In one aspect, the differences in amino acid sequence are conservative substitutions.

[0090] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and a VL comprising the amino acid sequences as set forth in SEQ ID NOs: 78 and 88, respectively.

[0091] In one embodiment, the VH and VL are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 79 and 89, respectively.

[0092] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain having the amino acid sequences as set forth in SEQ ID NOs: 80 and 90, respectively.

[0093] In one aspect, the heavy and light chains are encoded by nucleic acid sequences having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 81 and 91, respectively.

[0094] 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 having the amino acid sequence of SEQ ID NO: 78, and 2) LCDR1, LCDR2, and LCDR3 contained in a VL having the amino acid sequence of SEQ ID NO: 88.

[0095] In one aspect, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise 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.

[0096] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein a. 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; and HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71 and 77; b. LCDR1 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, and LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 84 and 86.

[0097] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 78 and 88, respectively.

[0098] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain having the amino acid sequences as set forth in SEQ ID NOs: 80 and 90, respectively.

[0099] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to BTC, comprising a VH and a VL having the amino acid sequences of SEQ ID NOs: 78 and 88, respectively.

[0100] In one embodiment, 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.

[0101] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is a Fab.

[0102] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is an scFV.

[0103] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is an isolated antibody.

[0104] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is a monoclonal human antibody.

[0105] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is a monoclonal humanized antibody.

[0106] In one embodiment, the Fab comprises an Fc region.

[0107] In one embodiment, the Fc region is selected from the group consisting of an Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0108] In one embodiment, the Fc region comprises a human immunoglobulin kappa chain constant region sequence as set forth in SEQ ID NO:159.

[0109] In one embodiment, the Fc region comprises a human immunoglobulin first constant Ig domain (CH1 domain) of the heavy chain as set forth in SEQ ID NO:160.

[0110] The present disclosure provides isolated antibodies or antigen-binding fragments thereof that can compete with antibodies or antigen-binding fragments thereof as described throughout for binding to BTC and reducing BTC-mediated signaling.

[0111] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to BTC comprises heavy and light chains as set forth 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.

[0112] The present disclosure provides polynucleotides comprising nucleotide sequences that encode antibodies or antigen-binding fragments thereof, as described throughout.

[0113] In one aspect, the expression cassette comprises a polynucleotide, as described throughout.

[0114] In one aspect, the vector comprises an expression cassette, as described throughout.

[0115] In one aspect, the host cell comprises a polynucleotide or vector as described throughout.

[0116] The disclosure also provides a method for producing an antibody or antigen-binding fragment thereof, comprising culturing a host cell under conditions suitable for expression of the antibody or antigen-binding fragment thereof.

[0117] In one aspect, the method further comprises purifying the antibody or antigen-binding fragment thereof.

[0118] The disclosure further provides pharmaceutical compositions comprising an effective amount of an antibody or antigen-binding fragment thereof, as described throughout.

[0119] In one aspect, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, diluent, or carrier.

[0120] The present disclosure provides methods of treating a subject in need thereof, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof, or pharmaceutical composition as described throughout.

[0121] In one aspect, the subject has a disease selected from the group consisting of pancreatic cancer, breast cancer, endometrial adenocarcinoma, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, and gastric cancer.

[0122] In one aspect, the antibody or antigen-binding fragment thereof or pharmaceutical composition is administered via a route selected from the group consisting of intravenous administration, intramuscular administration, subcutaneous administration, parenteral administration, spinal administration, and epidermal administration.

[0123] In one aspect, the subject has an ophthalmic disorder.

[0124] In one aspect, the ophthalmologic 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, pathologic myopia, retinal vein occlusion, retinopathy of prematurity, abnormal vascular proliferation associated with central serous chorioretinopathy nevus, and acute multifocal platelet pigment epitheliopathy.

[0125] In one aspect, the ophthalmic disorder is diabetic macular edema.

[0126] In one embodiment, administration is via subretinal injection.

[0127] In one aspect, administration is via intravitreal injection.

[0128] In one embodiment, the pharmaceutical composition further comprises an anti-VEGF antagonist.

[0129] In one embodiment, the anti-VEGF antagonist is ranibizumab.

[0130] In one embodiment, the anti-VEGF antagonist is bevacizumab.

[0131] In one embodiment, the anti-VEGF antagonist is aflibercept.

[0132] In one embodiment, the anti-VEGF antagonist is brolucizumab.

[0133] In one embodiment, the anti-VEGF antagonist is pegaptanib.

[0134] In one embodiment, the anti-VEGF antagonist comprises a heavy chain and a light chain as set forth in SEQ ID NOs: 103 and 114, respectively.

[0135] In one embodiment, the anti-VEGF antagonist is encoded by a nucleic acid sequence as set forth in SEQ ID NOs: 104 and 115.

[0136] In one embodiment, the method further comprises administering to the subject an anti-VEGF antagonist.

[0137] In one embodiment, the anti-VEGF antagonist is ranibizumab.

[0138] In one embodiment, the anti-VEGF antagonist is bevacizumab.

[0139] In one embodiment, the anti-VEGF antagonist is aflibercept.

[0140] In one embodiment, the anti-VEGF antagonist is brolucizumab.

[0141] In one embodiment, the anti-VEGF antagonist is pegaptanib.

[0142] In one embodiment, the anti-VEGF antagonist comprises a heavy chain and a light chain as set forth in SEQ ID NOs: 103 and 114.

[0143] In one embodiment, the anti-VEGF antagonist is encoded by a nucleic acid sequence as set forth in SEQ ID NOs: 104 and 115.

[0144] The present disclosure provides kits comprising an antibody or antigen-binding fragment thereof or a pharmaceutical composition as described throughout.

[0145] In one embodiment, the kit further comprises instructions for use.

[0146] In one embodiment, the kit further comprises a syringe.

[0147] The present disclosure provides multispecific binding molecules comprising 1) an anti-BTC binding moiety and 2) an anti-VEGF binding moiety.

[0148] In one embodiment, the anti-BTC binding moiety binds to a BTC comprising the amino acid sequence of SEQ ID NO:157.

[0149] In one aspect, the anti-BTC binding moiety 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.

[0150] In one aspect, the anti-BTC binding moiety binds to R38, C39, P40, K41, Q42, Y43, H45, Y46, F54, Q59, T60, P61, and R73 of SEQ ID NO: 157.

[0151] In one aspect, the anti-BTC binding moiety binds to P40, K41, Q42, Y43, H45, Y46, E58, Q59, T60, P61, A72, R73, E75, and R76 of SEQ ID NO: 157.

[0152] In one aspect, the anti-BTC binding moiety binds to G34, H35, F36, S37, R38, C39, P40, K41, Q42, R51, R53, F54, and V56 of SEQ ID NO: 157.

[0153] In one aspect, the anti-BTC binding moiety 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.

[0154] In one embodiment, the anti-BTC binding moiety is an antibody or antigen-binding fragment thereof, as described throughout.

[0155] In one embodiment, the anti-VEGF binding moiety is an anti-VEGF antibody or antigen-binding fragment thereof.

[0156] In one embodiment, the anti-BTC binding moiety and the anti-VEGF binding moiety are in a format selected from the list consisting of an isolated antibody, Fab, Fab', F(ab')2, Fv, and scFv.

[0157] In one embodiment, the anti-BTC binding moiety is an anti-BTC Fab and the anti-VEGF binding moiety is an anti-VEGF Fab.

[0158] In one embodiment, the anti-BTC Fab comprises a heavy chain (HA) and a light chain (LA), and the anti-VEGF Fab comprises a heavy chain (HB) and a light chain (LB).

[0159] In one embodiment, HA and HB are linked from the N-terminus to the C-terminus in the following format: N-HA-linker1-HB-C, and LA and LB are linked from the N-terminus to the C-terminus in the following format: N-LA-linker2-LB-C.

[0160] In one embodiment, HA and HB are linked from the N-terminus to the C-terminus in the following format: N-HB-linker1-HA-C, and LA and LB are linked from the N-terminus to the C-terminus in the following format: N-LB-linker2-LA-C.

[0161] In one embodiment, linker 1 and linker 2 comprise the amino acid sequence of SEQ ID NO:118.

[0162] In one embodiment, linker 1 and linker 2 are encoded by the nucleic acid sequence of SEQ ID NO:119.

[0163] In one embodiment, Linker 1 and Linker 2 comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 161-167.

[0164] In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth 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.

[0165] In one embodiment, the anti-BTC binding portion comprises a VH and a VL having the amino acid sequences of SEQ ID NOs: 10 and 21, respectively.

[0166] In one embodiment, the VH and VL are encoded by the nucleic acid sequences of SEQ ID NOs: 116 and 122, respectively.

[0167] In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth 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.

[0168] In one embodiment, the anti-BTC binding portion comprises a VH and a VL having the amino acid sequences of SEQ ID NOs: 34 and 45, respectively.

[0169] In one embodiment, the VH and VL are encoded by the nucleic acid sequences of SEQ ID NOs: 127 and 132, respectively.

[0170] In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth 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.

[0171] In one embodiment, the anti-BTC binding portion comprises a VH and a VL having the amino acid sequences of SEQ ID NOs: 54 and 65, respectively.

[0172] In one embodiment, the VH and VL are encoded by the nucleic acid sequences of SEQ ID NOs: 137 and 142, respectively.

[0173] In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth 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.

[0174] In one embodiment, the anti-BTC binding portion comprises a VH and a VL having the amino acid sequences of SEQ ID NOs: 78 and 88, respectively.

[0175] In one embodiment, the VH and VL are encoded by the nucleic acid sequences of SEQ ID NOs: 147 and 151, respectively.

[0176] In one embodiment, the anti-VEGF binding moiety comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 92, 93, and 94, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 105, 106, and 107, respectively.

[0177] In one embodiment, the anti-VEGF binding moiety comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 95, 93, and 94, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 105, 106, and 107, respectively.

[0178] In one embodiment, the anti-VEGF binding moiety comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 96, 97, and 94, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 108, 109, and 110, respectively.

[0179] In one embodiment, the anti-VEGF binding moiety comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 98, 99, and 100, respectively, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 111, 109, and 107, respectively.

[0180] In one embodiment, the anti-VEGF binding portion comprises a VH and a VL comprising amino acid sequences having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 101 and 112, respectively.

[0181] In one embodiment, the difference in amino acid sequence is not within a complementarity determining region.

[0182] In one aspect, the differences in amino acid sequence are conservative substitutions.

[0183] In one embodiment, the anti-VEGF binding moiety comprises a VH and a VL comprising the amino acid sequences as set forth in SEQ ID NOs: 101 and 112, respectively.

[0184] In one embodiment, the anti-VEGF binding moiety comprises a VH and a VL comprising the amino acid sequences as set forth in SEQ ID NOs: 102 and 113, respectively.

[0185] In one embodiment, the anti-VEGF binding moiety comprises a VH and a VL comprising the amino acid sequences as set forth in SEQ ID NOs: 117 and 123, respectively.

[0186] In one embodiment, the anti-VEGF binding moiety comprises a VH and a VL comprising the amino acid sequences as set forth in SEQ ID NOs: 128 and 133, respectively.

[0187] In one embodiment, the anti-VEGF binding moiety comprises a VH and a VL comprising the amino acid sequences as set forth in SEQ ID NOs: 138 and 143, respectively.

[0188] In one embodiment, the anti-VEGF binding moiety comprises a VH and a VL comprising the amino acid sequences as set forth in SEQ ID NOs: 148 and 152, respectively.

[0189] In one embodiment, the anti-VEGF binding moiety comprises a heavy chain and a light chain having the amino acid sequences as set forth in SEQ ID NOs: 103 and 114, respectively.

[0190] In one aspect, the heavy and light chains are encoded by nucleic acid sequences having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 104 and 115, respectively.

[0191] The present disclosure provides a multispecific binding molecule comprising an anti-BTC binding moiety and an anti-VEGF binding moiety, wherein the anti-BTC binding moiety comprises a variable heavy chain domain (VHA) and a variable light chain domain (VLA) that bind to BTC, and the anti-VEGF binding moiety comprises a variable heavy chain domain (VHB) and a variable light chain domain (VLB) that bind to VEGF, wherein: a. VHA and VLA comprise the amino acid sequences as set forth in SEQ ID NOs: 10 and 21, respectively; b. The VHB and VLB comprise the amino acid sequences as set forth in SEQ ID NOs: 101 and 112, respectively.

[0192] In one embodiment, the anti-BTC binding moiety further comprises a heavy chain constant domain (CH1A) and a light chain constant domain (CKA), and the anti-VEGF binding moiety further comprises a heavy chain constant domain (CH1B) and a light chain constant domain (CKB).

[0193] In one embodiment, the multispecific binding molecule is formatted as follows from N-terminus to C-terminus: N-VHA-CH1A-linker-VHB-CH1B-C and N-VLA-CKA-linker-VLB-CKB-C.

[0194] In one embodiment, the multispecific binding molecule comprises a heavy chain comprising a VHA, a CH1A, a linker, a VHB, and a CH1B, wherein the heavy chain is as set forth in SEQ ID NO: 120.

[0195] In one embodiment, the heavy chain is encoded by a nucleic acid sequence as set forth in SEQ ID NO:121.

[0196] In one embodiment, 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 as set forth in SEQ ID NO: 125.

[0197] In one embodiment, the light chain is encoded by a nucleic acid sequence as set forth in SEQ ID NO:126.

[0198] The disclosure also provides a multispecific binding molecule comprising an anti-BTC binding moiety and an anti-VEGF binding moiety, wherein the anti-BTC binding moiety comprises a variable heavy chain domain (VHA) and a variable light chain domain (VLA) that bind to BTC, and the anti-VEGF binding moiety comprises a variable heavy chain domain (VHB) and a variable light chain domain (VLB) that bind to VEGF, wherein: a. VHA and VLA comprise the amino acid sequences as set forth in SEQ ID NOs: 34 and 45, respectively; b. The VHB and VLB comprise the amino acid sequences as set forth in SEQ ID NOs: 101 and 112, respectively.

[0199] In one embodiment, the anti-BTC binding moiety further comprises a heavy chain constant domain (CH1A) and a light chain constant domain (CKA), and the anti-VEGF binding moiety further comprises a heavy chain constant domain (CH1B) and a light chain constant domain (CKB).

[0200] In one embodiment, the multispecific binding molecule is formatted as follows from N-terminus to C-terminus: N-VHA-CH1A-linker-VHB-CH1B-C and N-VLA-CKA-linker-VLB-CKB-C.

[0201] In one embodiment, the multispecific binding molecule comprises a heavy chain comprising a VHA, a CH1A, a linker, a VHB, and a CH1B, wherein the heavy chain is as set forth in SEQ ID NO: 130.

[0202] In one embodiment, the heavy chain is encoded by a nucleic acid sequence as set forth in SEQ ID NO:131.

[0203] In one embodiment, 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 as set forth in SEQ ID NO: 135.

[0204] In one embodiment, the light chain is encoded by a nucleic acid sequence as set forth in SEQ ID NO:136.

[0205] The present disclosure also provides a multispecific binding molecule comprising an anti-BTC binding moiety and an anti-VEGF binding moiety, wherein the anti-BTC binding moiety comprises a variable heavy chain domain (VHA) and a variable light chain domain (VLA) that binds to BTC, and the anti-VEGF binding moiety comprises a variable heavy chain domain (VHB) and a variable light chain domain (VLB) that binds to VEGF, wherein: a. the VHA and VLA comprise the amino acid sequences as set forth in SEQ ID NOs: 54 and 65, respectively; b. The VHB and VLB comprise the amino acid sequences as set forth in SEQ ID NOs: 101 and 112, respectively.

[0206] In one embodiment, the anti-BTC binding moiety further comprises a heavy chain constant domain (CH1A) and a light chain constant domain (CKA), and the anti-VEGF binding moiety further comprises a heavy chain constant domain (CH1B) and a light chain constant domain (CKB).

[0207] In one embodiment, the multispecific binding molecule is formatted as follows from N-terminus to C-terminus: N-VHA-CH1A-linker-VHB-CH1B-C and N-VLA-CKA-linker-VLB-CKB-C.

[0208] In one embodiment, the multispecific binding molecule comprises a heavy chain comprising a VHA, a CH1A, a linker, a VHB, and a CH1B, wherein the heavy chain is as set forth in SEQ ID NO: 140.

[0209] In one embodiment, the heavy chain is encoded by a nucleic acid sequence as set forth in SEQ ID NO:141.

[0210] In one embodiment, 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 as set forth in SEQ ID NO:145.

[0211] In one embodiment, the light chain is encoded by a nucleic acid sequence as set forth in SEQ ID NO:146.

[0212] Further provided in the present disclosure is a multispecific binding molecule comprising an anti-BTC binding moiety and an anti-VEGF binding moiety, wherein the anti-BTC binding moiety comprises a variable heavy chain domain (VHA) and a variable light chain domain (VLA) that bind to BTC, and the anti-VEGF binding moiety comprises a variable heavy chain domain (VHB) and a variable light chain domain (VLB) that bind to VEGF, wherein: a. VHA and VLA comprise the amino acid sequences as set forth in SEQ ID NOs: 78 and 88, respectively; b. The VHB and VLB comprise the amino acid sequences as set forth in SEQ ID NOs: 101 and 112, respectively.

[0213] In one embodiment, the anti-BTC binding moiety further comprises a heavy chain constant domain (CH1A) and a light chain constant domain (CKA), and the anti-VEGF binding moiety further comprises a heavy chain constant domain (CH1B) and a light chain constant domain (CKB).

[0214] In one embodiment, the multispecific binding molecule is formatted as follows from N-terminus to C-terminus: N-VHA-CH1A-linker-VHB-CH1B-C and N-VLA-CKA-linker-VLB-CKB-C.

[0215] In one embodiment, the multispecific binding molecule comprises a heavy chain comprising a VHA, a CH1A, a linker, a VHB, and a CH1B, wherein the heavy chain is as set forth in SEQ ID NO: 149.

[0216] In one embodiment, the heavy chain is encoded by a nucleic acid sequence as set forth in SEQ ID NO:150.

[0217] In one embodiment, 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 as set forth in SEQ ID NO:154.

[0218] In one embodiment, the light chain is encoded by a nucleic acid sequence as set forth in SEQ ID NO:155.

[0219] The present disclosure 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: 120 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125.

[0220] In one embodiment, 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.

[0221] The present disclosure 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: 130 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135.

[0222] In one embodiment, 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.

[0223] The present disclosure further 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: 140 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145.

[0224] In one embodiment, 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.

[0225] The present disclosure 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.

[0226] In one embodiment, 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.

[0227] The present disclosure provides polynucleotides comprising nucleotide sequences that encode multispecific binding molecules as described throughout.

[0228] The present disclosure also provides expression cassettes comprising the polynucleotides as described throughout.

[0229] Also provided in this disclosure are vectors containing expression cassettes as described throughout.

[0230] Further provided in this disclosure are host cells containing the polynucleotides as described throughout.

[0231] The present disclosure provides a method for producing a multispecific binding molecule comprising culturing a host cell under conditions suitable for expression of the multispecific binding molecule or a fragment thereof.

[0232] In one aspect, the method further comprises purifying the multispecific binding molecule.

[0233] The present disclosure provides pharmaceutical compositions comprising an effective amount of a multispecific binding molecule as described throughout.

[0234] In one aspect, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, diluent, or carrier.

[0235] In one embodiment, the pharmaceutical composition further comprises one or more therapeutic agents.

[0236] Also provided in the present disclosure are methods of treating an ophthalmic 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.

[0237] In one aspect, the multispecific binding molecule or pharmaceutical composition is administered intravitreally to the subject.

[0238] In one aspect, the multispecific binding molecule or pharmaceutical composition is administered via subretinal injection.

[0239] In one aspect, the ophthalmic 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, pathologic myopia, retinal vein occlusion, retinopathy of prematurity, and abnormal vascular proliferation associated with nevus syndrome.

[0240] In one aspect, the ophthalmic disorder is diabetic macular edema.

[0241] The present disclosure provides methods for preventing, treating, or managing an ophthalmic disorder, comprising administering to a subject an effective amount of a multispecific binding molecule as described herein, wherein the multispecific binding molecule reduces retinal leakage and / or retinal thickening in the subject relative to a control subject.

[0242] Further provided in this disclosure are kits comprising the multispecific binding molecules or pharmaceutical compositions as described throughout.

[0243] In one embodiment, the kit further comprises instructions for use.

[0244] In one embodiment, the kit further comprises a syringe.

[0245] The present disclosure provides a method of preventing or treating macular edema, DME, AMD, neovascular AMD, or RVO in a subject in need thereof, comprising intravitreally administering to the subject a multispecific binding molecule described herein at a dose ranging from about 0.25 mg / eye to 7.5 mg / eye.

[0246] In one embodiment, the dose is about 0.25 mg / eye, 0.75 mg / eye, 2.5 mg / eye, or 7.5 mg / eye.

[0247] In one embodiment, the dose is 0.25 mg / eye.

[0248] In one embodiment, the dose is 0.75 mg / eye.

[0249] In one embodiment, the dose is 1 mg / eye.

[0250] In one embodiment, the dose is 2.5 mg / eye.

[0251] In one embodiment, the dose is 3 mg / eye.

[0252] In one embodiment, the dose is 5 mg / eye.

[0253] In one embodiment, the dose is 7.5 mg / eye.

[0254] In one aspect, the dosage 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.

[0255] In one aspect, the multispecific binding molecule comprises 1) an anti-BTC binding portion comprising the amino acid sequences of SEQ ID NOs: 10 and 21 respectively, and 2) an anti-VEGF binding portion comprising the amino acids of SEQ ID NOs: 101 and 112 respectively.

[0256] In another aspect, the administration is once a month.

[0257] In general, the present disclosure is based, in part, on the discovery of antibodies specific for betacellulin (BTC). In particular, the inventors have discovered anti-BTC antibodies with properties compatible with therapeutic utility (i.e., the antibodies bind to BTC with sufficient affinity and specificity to achieve a desired therapeutic effect). Based in part on this discovery, the present disclosure features therapeutic compositions comprising a molecule comprising an antibody specific for BTC, or an antibody fragment, conjugated to another therapeutic moiety, e.g., an anti-VEGF antibody or antibody fragment. Such therapeutic moieties include antibodies, or fragments thereof, and proteins that bind to therapeutic targets in tissues with BTC (e.g., the vitreous), as well as compounds (e.g., low molecular weight compounds) that modulate the therapeutic targets in such tissues. When such therapeutic moieties include antibodies, the overall therapeutic composition can be a multispecific antibody (e.g., a bispecific antibody). In certain aspects, also provided herein are methods for treating ocular disorders (e.g., AMD, e.g., neovascular AMD, DME, DR, etc.) by administering an anti-BTC antagonist and an anti-VEGF antagonist.

[0258] i.Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Any references cited herein, including, for example, all patents, published patent applications, and non-patent publications, are incorporated by reference in their entirety. To facilitate understanding of this disclosure, several terms and abbreviations used herein are defined below as follows:

[0259] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a mixture of two or more such antibodies.

[0260] Unless otherwise specified or clear from the context, as used herein, the term "about" in reference to a numerical value is understood to be within normal tolerances in the art, e.g., within two standard deviations of the mean. Thus, "about" can 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 range or list of numerical values, the term "about" applies to each numerical value in turn, e.g., the phrase "about 1 to 5" should be interpreted as "about 1 to about 5," or the phrase "about 1, 2, 3, 4" should be interpreted as "about 1, about 2, about 3, about 4, etc."

[0261] In all cases where terms such as "comprise," "comprises," "comprising," etc. are used in reference to a sequence (e.g., an amino acid sequence), it is understood that the sequence may also be qualified by terms such as "consist," "consists," "consisting," etc. As used herein, the phrase "consisting essentially of" refers to the genus or species of active pharmaceutical agent included in a method or composition, as well as the genus or species of excipients that are inactive for the intended purpose of the method or composition. In some embodiments, the phrase "consisting essentially of" explicitly excludes the inclusion of one or more additional active agents other than the multispecific binding molecules of the present disclosure. In some embodiments, the phrase "consisting essentially of" explicitly excludes the inclusion of one or more additional active agents other than the multispecific binding molecules of the present disclosure and a second co-administered agent.

[0262] The term "betacellulin" or "BTC" refers to a growth factor of the EGF family in an organism. BTC activity can be measured by its binding to 1) ErbB1; 2) ErbB4; 3) ErbB homodimers (e.g., ErbB1 / ErbB1 and ErbB4 / ErB4) and / or 4) ErbB heterodimers (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 phosphorylated ERK1 / 2. In humans, this is encoded by the BTC gene, which is located on chromosome 4 at locus 4q13-q21. Human BTC is expressed as a 178-residue protein called pro-BTC (e.g., NCBI: NP_001720.1 or SEQ ID NO: 156). Mature, secreted human BTC consists of 80 amino acid residues as set forth in SEQ ID NO: 158, i.e., residues 32 to 111 of pro-BTC. 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).

[0263] The term "vascular endothelial growth factor" or "VEGF" refers to a protein that has VEGF activity in an organism, e.g., induces the proliferation and migration of vascular endothelial cells, and may be essential 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 is expressed in at least six isoforms (VEGF 1, VEGF 2, VEGF 3), which arise from alternative splicing of the mRNA of a single gene. 121 , VEGF 145 , VEGF 165 , VEGF 183 , VEGF 189 , and VEGF 206) (Ferrara N, Davis Smyth T. Endocr Rev 18:1-22 (1997)). The most abundant isoform of VEGF 165 is a basic, heparin-binding, dimeric glycoprotein with a molecular mass of approximately 45,000 daltons. As used herein, the term "human VEGF" refers to the 165-amino acid human vascular endothelial growth factor and the related 121-, 189-, and 206-amino acid (and other isoforms) amino acid vascular endothelial growth factors (as described in Leung et al., Science 246:1306 (1989), and Houck et al., Mol. Endocrin. 5:1806 (1991)), along with naturally occurring alleles and processed forms of those growth factors.

[0264] The term "anti-BTC binding moiety," as used herein, refers to a polypeptide (e.g., an antibody, or antigen-binding fragment thereof) that specifically binds to BTC. For the avoidance of doubt, non-limiting examples of "anti-BTC binding moieties" include full-length antibodies and antigen-binding fragments thereof, such as Fabs, scFvs, Fvs, single-domain antibodies, etc. In certain embodiments, the anti-BTC antibody is a Fab or scFv. In certain embodiments, the anti-BTC binding moiety specifically binds to human BTC and / or cynomolgus BTC.

[0265] The term "anti-VEGF binding moiety," as used herein, means a polypeptide (e.g., an antibody, as described below, or an antigen-binding fragment thereof) that specifically binds to VEGF (as defined below). For the avoidance of doubt, non-limiting examples of "anti-VEGF binding moiety" include full-length antibodies and antigen-binding fragments thereof, such as Fabs, scFvs, Fvs, single-domain antibodies, etc., as described below. In certain embodiments, the anti-VEGF antibody is a Fab or scFv. In certain embodiments, the anti-VEGF binding moiety specifically binds to human VEGF-A.

[0266] The phrases "binds specifically," "specifically binds," or "selectively binds," when used in reference to describing the interaction between an antigen (e.g., a protein) and a multispecific binding molecule of the present disclosure, refer to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologics, for example, in a biological sample, such as a blood, serum, plasma, or tissue sample. Thus, under certain specified immunoassay conditions, a multispecific binding molecule of the present disclosure having a particular binding specificity binds to a particular antigen at least twice above background and does not bind in a substantially significant amount to other antigens present in the sample. In one aspect, under specified immunoassay conditions, a multispecific binding molecule of the present disclosure having a particular binding specificity binds to a particular antigen at least 10 times above background and does not bind in a substantially significant amount to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require that the multispecific binding molecule of the present disclosure be selected for its specificity for a particular protein. As desired or necessary, this selection can be achieved by subtracting out multispecific binding molecules that cross-react with molecules from other species (eg, mouse or rat) or other subtypes.

[0267] In some embodiments, the specific binding of a multispecific binding molecule of the present disclosure 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 6M -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 The equilibrium constant (K A )(k on / k off ) means a bond with

[0268] In some embodiments, the specific binding of a multispecific binding molecule of the disclosure is greater than 5×10 -2 Under M, 10 -2 Less than M, 5 x 10 -3 Under M, 10 -3 Less than M, 5 x 10 -4 Under M, 10 -4 Less than M, 5 x 10 -5 Under M, 10 -5 Less than M, 5 x 10-6 Under M, 10 -6 Less than M, 5 x 10 -7 Under M, 10 -7 Less than M, 5 x 10 -8 Under M, 10 -8 Less than M, 5 x 10 -9 Under M, 10 -9 Less than M, 5 x 10 -10 Under M, 10 -10 Less than M, 5 x 10 -11 Under M, 10 -11 Less than M, 5 x 10 -12 Under M, 10 -12 Less than M, 5 x 10 -13 Under M, 10 -13 Less than M, 5 x 10 -14 Under M, 10 -14 Less than M, 5 x 10 -15 Less than M or 10 -15 Dissociation rate constant (K D )(k off / k on ) which binds to a target antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., HSA).

[0269] "K D The term "Kd" or "Kd" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0270] The term "therapeutic target binding moiety," as used herein, refers to a molecule that specifically binds to a therapeutic target of interest. This molecule may be an antibody or antigen-binding fragment thereof (as described below), an antigen-specific binding moiety, e.g., a DARPin, Fynomer, affibody, adnectin, affilin, anticalin, avimer, centilin, or an RNA molecule such as an aptamer. The therapeutic target binding moiety may also be a polypeptide, such as a receptor or portion of a receptor, that specifically binds to a therapeutic target of interest. For the avoidance of doubt, non-limiting examples of "anti-BTC binding moieties" include full-length antibodies and antigen-binding fragments thereof, e.g., Fab, scFv, Fv, single-domain antibodies, etc., as described below.

[0271] The term "antibody," as used herein, refers to a whole antibody or an antigen-binding fragment thereof. A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate binding of the immunoglobulin to various cells of the host tissue or immune system (e.g., effector cells) and factors including the first component (Clq) of the classical complement system. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, bispecific antibodies, or multispecific antibodies. Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0272] The term "isotype" refers to the antibody class (e.g., IgM, IgE, IgG, e.g., IgG1 or IgG4) provided by the heavy chain constant region genes. Isotypes also include modified versions of one of these classes, where the modifications have been made to alter Fc function, for example, to enhance or reduce effector function or binding to an Fc receptor. An antibody can be of 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. As used herein, and unless otherwise specified, the term "IgG" or "IgG antibody" refers to type G whole antibody or Ig.

[0273] The term "light chain" includes full-length light chains and fragments thereof containing sufficient variable region sequence to confer binding specificity. A full-length light chain contains a variable region domain, V L and a constant region domain, C L The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0274] The term "heavy chain" includes full-length heavy chains and fragments thereof containing sufficient variable region sequence to confer binding specificity. A full-length heavy chain contains a variable region domain, V H and three constant region domains, C H 1. C H 2, and C H 3 and V H The domain is located at the amino terminus of the polypeptide and is C H The domain is located at the carboxyl terminus and is C H 3 is closest to the carboxy terminus of the polypeptide. The heavy chain can be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0275] The terms "variable region" or "variable domain" refer to a portion of an antibody's light and / or heavy chain, typically comprising approximately the amino-terminal 120-130 amino acids of the heavy chain and the amino-terminal 100-110 amino acids of the light chain. In certain embodiments, the variable regions of different antibodies vary significantly in amino acid sequence, even among antibodies of the same species. The variable regions of an antibody typically determine the specificity of a particular antibody for its target.

[0276] The terms "complementarity determining region" and "CDR" as used herein refer to amino acid sequences within an antibody variable region that confer antigen specificity and binding affinity. Generally, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). 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 (the "Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (the "Chothia" numbering scheme); and Lefranc et al., (2003) Dev. Comp. Immunol., 27, 55-77 (the "IMGT" numbering scheme). The Kabat definition is the standard for numbering residues in antibodies and is typically used to identify CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28:214-8 (2000). The Chotia definition is similar to the Kabat definition, but takes into account certain structural loop regions (see, e.g., Chothia et al., J. Mol. Biol., 196:901-17 (1986); Chothia et al., Nature, 342:877-83 (1989)).

[0277] Other methods for delineating CDR regions can alternatively be used, for example, combining the CDR definitions of both Kabat and Chotia ("combined" system). For example, for the classical format, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). According to Chothia, the CDR amino acids of the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues of the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the combined CDR consists of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of the human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of the human VL. As another example, according to IMGT, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 26-33 (HCDR1), 51-58 (HCDR2), and 97-108 (HCDR3), and the CDR amino acid residues in the light chain variable domain (VL) are numbered 27-36 (LCDR1), 54-56 (LCDR2), and 93-101 (LCDR3).

[0278] The term "antibody framework" or "FR", as used herein, refers to any portion of a variable domain, VL or VH, that serves as a scaffold for the antigen binding loops (CDRs) of this variable domain. Essentially, it is a variable domain without the CDRs. Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0279] 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 fragments, that retain the ability to specifically bind to a given antigen (e.g., BTC and VEGF). The antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within 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; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains; a single-chain Fv fragment (scFv) consisting of the VL and VH domains connected by a linker sequence; and a single-domain antibody (dAb) fragment consisting of the VH or VL domain (Ward et al., 1989 Nature 341:544-546). Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, 2005 Nature Biotechnology, 23, 9, 1126-1136). Antigen-binding portions of antibodies can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). Antibody-binding fragments can be grafted into scaffolds based on complementary light chain polypeptides (e.g., V L -VC-V L -VC), which together form a pair of antigen-binding regions, H -CH1-V H -CH1) pair (Zapata et al., (1995) Protein Eng. 8:1057-1062; and U.S. Pat. No. 5,641,870).

[0280] As used herein, a "Fab" fragment contains one constant domain and one variable domain from each of a heavy and a light chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0281] As used herein, a "Fab' fragment" refers to one light chain and a VH domain and a C H 1 domain and also C H 1 and C H and a portion of one heavy chain containing the region between the two domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0282] As used herein, a "F(ab')2 fragment" refers to a fragment that contains two light chains and a C H 1 Domain and C H The F(ab')2 fragment contains two heavy chains containing a portion of the constant region between the two domains, which allows the formation of an interchain disulfide bond between the two heavy chains. Thus, the F(ab')2 fragment is composed of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0283] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0284] As used herein, the term "single-chain Fv" or "scFv" refers to the V H and V L Fv refers to an antibody fragment containing V domains, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide contains V domains that enable the scFv to form the desired structure for antigen binding. H Domains and V LThe scFvs further comprise internal polypeptide linkers between the domains. The scFvs can also have engineered internal disulfide bridges that enhance stability. For a discussion of scFvs, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (1994) Springer-Verlag, New York, pp. 269-315. In a preferred embodiment, the scFvs used in the multispecific binding molecules of the present disclosure have the general structure: NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V L Has —COOH.

[0285] As used herein, an "affinity matured" antibody is an antibody that has one or more alterations in one or more of its CDRs that result in an improvement in the antibody's affinity for an antigen compared to a parent antibody that does not have those alterations. Preferred affinity matured antibodies will have nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies can be produced by techniques known in the art. Marks et al. Bio / Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues has been described by: Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (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).

[0286] As used herein, a "parent" or "parental" antibody is an antibody encoded by an amino acid sequence used to prepare an affinity matured antibody or variant thereof. Preferably, the parent antibody has human framework regions and, if present, human antibody constant regions. For example, the parent antibody may be a humanized or human antibody.

[0287] As used herein, the term "diabody" refers to a small antibody fragment having two antigen-binding sites, which fragment comprises a heavy chain variable domain (VH) connected 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 the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies are more fully described, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0288] As used herein, the term "monospecific binding molecule" or "monospecific antibody" refers to a molecule that binds to one epitope on a target antigen. In one embodiment, a monospecific binding molecule or monospecific antibody of the disclosure binds to BTC. In another embodiment, a monospecific binding molecule or monospecific antibody of the disclosure binds to VEGF.

[0289] As used herein, the term "multispecific binding molecule" or "multispecific antibody" refers to a molecule that binds to two or more different antigens. Recognition of each antigen is generally achieved via an "antigen-binding domain" (e.g., a "BTC antigen-binding domain," a "VEGF antigen-binding domain"). The term "multispecific" includes "bispecific," i.e., molecules that bind to two different antigens. In some embodiments, a multispecific binding molecule contains one or more polypeptide chains, each comprising one antigen-binding domain. In one embodiment, a multispecific binding molecule contains a VH or a VL. In some embodiments, a multispecific binding molecule contains one or more polypeptide chains, each comprising two or more (e.g., two) antigen-binding domains. In some embodiments, a multispecific binding molecule comprises two, three, four, or more polypeptide chains that together comprise multiple, e.g., two or more, e.g., two, three, or four antigen-binding domains.

[0290] The term "bispecific binding molecule" or "bispecific antibody" refers to a molecule that combines the antigen-binding sites of two antibodies in a single molecule. In one embodiment, a bispecific binding molecule or bispecific antibody contains a single polypeptide. In another embodiment, a bispecific binding molecule or bispecific antibody contains two polypeptides linked via a disulfide bridge or any other covalent bond. Thus, a bispecific antibody can bind to two different antigens simultaneously or sequentially. Methods for producing bispecific antibodies are well known in the art. Various formats for combining two antibodies are also known in the art. Forms of bispecific antibodies of the present disclosure include, but are not limited to, diabodies, single-chain diabodies, dimerized Fab (Fab-Fab), Fab-scFv, and tandem antibodies, as known to those skilled in the art.

[0291] As used herein, the term "bivalent molecule" refers to a molecule having two antigen-binding domains. As used herein, the term "trivalent molecule" refers to a molecule having three antigen-binding domains. In some embodiments, the trivalent molecules of the present disclosure are trivalent antibody-like molecules. In some embodiments, a trivalent molecule can 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 different antigen. Such embodiments are considered trivalent bispecific molecules.

[0292] The term "multivalent molecule" refers to a molecule having at least two antigen-binding sites, and these antigen-binding molecules may have specificity for the same antigen or different antigens. In some aspects, multivalent molecules of the present disclosure are multivalent antibody-like molecules. In some aspects, multivalent molecules of the present disclosure are multivalent antibodies. In some aspects, multivalent molecules are bivalent, trivalent, or tetravalent molecules. Trimerization domains are described, for example, in EP 1012280 B1. Pentamerization modules are described in PCT / EP 97 / 05897.

[0293] As used herein, the terms "substantially similar" or "substantially identical" refer to a sufficiently high degree of similarity between two numerical values ​​(typically one associated with an antibody-like molecule of the present disclosure and the other associated with a reference / comparator antibody or antibody-like molecule) such that one of skill in the art would consider the difference between the two values ​​to represent little or no biological and / or statistical difference in the context of the biological property measured by the values ​​(e.g., TM value or amount of assembled antibody). The difference between the 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%, as a function of the value for the reference / comparator antibody.

[0294] The term "antigen" refers to a molecule or portion of a molecule that can be 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 embodiments, an antigen can be used to generate antibodies in an animal that can bind to that antigen. An antigen can possess one or more epitopes that can interact with different antigen-binding proteins, e.g., antibodies.

[0295] As used herein, the term "epitope" or "antigenic determinant" refers to any determinant capable of binding with high affinity to an antibody or antibody-like molecule. An epitope is the region of an antigen bound by an antibody (or antibody-like molecule) that specifically targets that antigen, and, if the antigen is a protein, includes specific amino acids that make direct contact with the antibody or antibody-like molecule. In most cases, epitopes reside on proteins, but in some cases, they can reside on other types of molecules, such as nucleic acids. Epitopes can include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups, and can have specific three-dimensional structural and / or charge characteristics.

[0296] In general, a multispecific binding molecule that is specific for a particular target antigen will preferentially recognize an epitope on that target antigen in a complex mixture of proteins and / or macromolecules.

[0297] Regions of a given polypeptide that contain an epitope can be identified using any number of epitope mapping techniques known in the art. See, e.g., "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 by, for example, simultaneously synthesizing a large number of peptides on a solid support (the peptides representing portions of a protein molecule) and reacting the peptides with an antibody while they are still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Pat. No. 4,708,871; Geysen et al., (1984) Proc. Natl. Acad. Sci. USA 8:3998-4002; Geysen et al., (1985) Proc. Natl. Acad. Sci. USA 82:78-182; Geysen et al., (1986) Mol. Immunol. 23:709-715. Similarly, conformational epitopes are readily identified by determining the spatial conformation of amino acids, for example, by X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, the epitope mapping protocol described above. Antigenic regions of proteins can also be identified using standard antigenicity and hydrophobicity plots, such as those calculated using the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program uses the Hopp / Woods method, Hopp et al., (1981) Proc. Natl. Acad. Sci USA 78:3824-3828, for determining antigenic profiles, and the Kyte-Doolittle technique, Kyte et al., (1982) J. MoI. Biol. 157:105-132, for hydrophobicity plots.

[0298] The term "compete," when used in reference to antigen-binding proteins that compete for the same epitope, refers to competition between the antigen-binding proteins as determined by an assay in which the antigen-binding protein being tested (e.g., an antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen-binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., BTC or a fragment thereof). Numerous 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 immunoassay (EIA), sandwich competition assay (see, e.g., Stahl et 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), solid-phase direct label assay, solid-phase direct label sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using I-125 label (see, e.g., Morel et al., 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such assays involve the use of purified antigen or cells bearing either of these bound to a solid surface, an 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 test antigen-binding protein. Typically, the test antigen-binding protein is present in excess.Antigen-binding proteins identified by competitive assays (competing antigen-binding proteins) include antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein and antigen-binding proteins that bind to an adjacent epitope sufficiently close to the epitope bound by the reference antigen-binding protein so that steric hindrance occurs. Further details regarding methods for determining competitive binding are provided in the Examples section of this specification. Typically, when a competing antigen-binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of the reference antigen-binding protein to a 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.

[0299] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The phrase also applies to amino acid polymers that are artificial chemical mimetics of a corresponding naturally occurring amino acid in which one or more amino acid residues correspond, as well as to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0300] As used herein, the term "polypeptide chain" refers to the complete amino acid chain of a multispecific binding molecule of the present disclosure, having all component regions and domains therein.

[0301] The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains that are not directly involved in binding an antibody to an antigen but exhibit various effector functions, such as interaction with Fc receptors. This term refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the other 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.

[0302] 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 amino acids that are later modified, for example, due to post-translational modifications, such as hydroxyproline, γ-carboxyglutamate, pyroglutamate, C-terminal lysine cleavage, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0303] In polypeptide sequences, "conservatively modified variants" include individual substitutions, deletions, or additions to a polypeptide sequence that result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the present disclosure. The following eight groups contain amino acids that are conservative substitutions for one another: 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, e.g., Creighton, Proteins (1984). In some embodiments, the terms "conservative sequence modifications" or "conservative modifications" are used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.

[0304] In certain embodiments, the term "dose" refers to an amount of a therapeutic agent or therapeutic target-binding moiety administered to a subject all at once (a unit dose) or in two or more administrations over a defined time interval, where the therapeutic agent can be a protein (e.g., an antibody or antigen-binding fragment) or a nucleic acid, and the therapeutic target-binding moiety can be a small molecule (e.g., <900 daltons) therapeutic compound. For example, a dose can refer to an amount of a protein (e.g., an anti-BTC antibody or functional fragment thereof conjugated to a molecule, or a protein comprising an anti-VEGF antibody or functional fragment thereof) administered to a subject (e.g., in a single administration or in two or more administrations) over the course of three weeks, or one month, two months, three months, four months, five months, six months, or more. The interval between administrations can be any desired period and is referred to as the "dosing interval."

[0305] The term "pharmaceutically effective" when referring 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., improved vision or prevention of further vision loss). An "effective" amount will vary from subject to subject, depending on the age and general condition of the individual, the particular drug or pharmaceutically active agent, etc. Thus, it is not always possible to specify an exact "effective" amount applicable to all patients. However, in any individual case, an appropriate "effective" dose can be determined by one of ordinary skill in the art using routine experimentation.

[0306] The term "human antibody," as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or variants of human germline sequences, or antibodies containing consensus framework sequences derived from human framework sequence analysis, e.g., as described in Knappik, et al. (J. Mol. Biol. 296, 57-86, 2000). The human antibodies of the present disclosure may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo, or conservative substitutions to facilitate stability or production). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0307] The term "monoclonal antibody" or "monoclonal antibody composition," as used herein, refers to polypeptides, including antibodies and antigen-binding fragments, that have substantially identical amino acid sequences or are derived from the same genetic source. The term also includes preparations of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Methods for generating 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).

[0308] The term "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to further refine antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs correspond to human immunoglobulin Io sequences. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also 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).

[0309] As used herein, "identity" refers to sequence matching between two polypeptides, molecules, or two nucleic acids. If both positions in two compared sequences are occupied by the same base or amino acid (e.g., if a position in each of the two polypeptides is occupied by lysine), the respective 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 that need to be introduced for optimal alignment of the two sequences and the length of each gap. Generally, when two sequences are aligned, they are compared to give the maximum identity. Such alignments can be provided, for example, using the method of Needleman and Wunsch (J. MoI. Biol. (48): 444-453 (1970)) (the algorithm incorporated into the GAP program in the GCG software package, using either a Blosum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6). For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. After optimally sequencing two sequences, a sequence can be compared to a reference sequence by the same number of contiguous positions, and a comparison window can be used, which refers to a segment by any one of the numbers of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, and more usually about 100 to about 150. Methods of aligning sequences for comparison are well known in the art.Optimal sequence alignment for comparison can be achieved, for example, by the local homology algorithm of 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 search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized 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 suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, match or satisfy a certain positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.Word hits are extended in either direction along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is halted when the cumulative alignment score falls by an amount X from its achieved maximum value; when the cumulative score falls below zero due to the accumulation of alignment of one or more negative-scoring residues; or when either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses defaults of a word length (W) of 11, an expectation (E) of 10, M=5, and N=-4, and performs a comparison of both strands. The BLASTP program for amino acid sequences uses a default alignment (B) of 3, an expectation (E) of 10, M=5, and N=-4 with a word length of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) of 50, and performs 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 provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, in a comparison of a test nucleic acid to a reference nucleic acid, if the smallest sum probability is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, then the nucleic acid is considered to be similar to the reference sequence.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), as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can also be determined using the algorithm by Needleman and Wunsch (J. Mol. Biol. 48:444-453, 1970), as incorporated into the GAP program in the GCG software package (available on the Internet at gcg.com), using a Blosum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. In addition to the percentage sequence identity mentioned above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with an antibody to the polypeptide encoded by the second nucleic acid, as described below. Thus, for example, where the two peptides differ only by conservative substitutions, a polypeptide will typically be substantially identical to a second polypeptide. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the two nucleic acid sequences can be amplified using the same primers.

[0310] The terms "percent complementarity" or "percent complementary," as used herein in reference to two nucleotide sequences, are similar to the concept of percent identity, but refer to the percentage of nucleotides in a query sequence that optimally base-pair or hybridize to nucleotides in a subject sequence when the query and subject sequences are linearly arranged and optimally base-paired without secondary fold structures such as loops, stems, or hairpins. Such percent complementarity can be between two DNA strands, two RNA strands, or between a DNA strand and an RNA strand. "Percent complementarity" is calculated by (i) optimally base-pairing or hybridizing two nucleotide sequences in a linear and fully extended configuration (i.e., without folding or secondary structure) over the comparison window, (ii) determining the number of base-pairing positions between the two sequences over 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 quotient by 100% to obtain the percent complementarity of the two sequences. Optimal base pairing of two sequences can be determined based on known pairing of nucleotide bases through hydrogen bonds, such as GC, AT, and AU. When "percent complementarity" is calculated with respect to a reference sequence without specifying a specific comparison window, the percent 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 this disclosure, when two sequences (query and subject) are optimally base-paired (without folding or secondary structure, but allowing for mismatched or non-base-pairing nucleotides), the "percent complementarity" with respect to 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 divided by the number of positions in the query sequence over the comparison window), then multiplied by 100%.

[0311] The term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities or other proteins. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals, e.g., an antibody isolated from a cellular supernatant.

[0312] The terms "linked" or "linkage" with respect to the anti-BTC multispecific binding molecules described herein refer to the attachment of an anti-BTC binding moiety to a molecule, such as, for example, an anti-BTC antibody listed in Table 1 or a functional fragment thereof that binds to BTC. Attachment of the anti-BTC binding moiety to a protein can occur, for example, at the amino or carboxy terminus of the molecule, e.g., an anti-VEGF antibody or functional fragment thereof. The anti-BTC binding moiety can also be attached to both the amino and carboxy termini of the protein. The anti-BTC binding moiety can also be attached to one or more amino acids or nucleic acids within a protein or nucleic acid molecule, respectively. Linking the anti-BTC binding moiety to a molecule can be accomplished by methods known in the art, including, but not limited to, expressing the anti-BTC binding moiety and the molecule as a fusion protein, or post-translationally chemically linking the anti-BTC binding moiety to the molecule, either directly to each other or through a linker, such as a disulfide bond.

[0313] The term "linker" or "linked" with respect to a multispecific binding molecule refers to the attachment, directly or indirectly, of one portion of the multispecific binding molecule to another portion of the molecule, e.g., an anti-BTC binding moiety to an anti-VEGF binding moiety. The linker can be covalently attached to one or both of the amino or carboxy termini of the anti-BTC binding moiety and / or the protein or nucleic acid molecule. Peptide linkers may also be conjugated to amino acids or nucleic acids within the sequence of the protein or nucleic acid molecule, respectively. In certain embodiments, it is contemplated that the peptide linker can be, for example, about 2-25 residues in length.

[0314] As used herein, the term "nucleic acid" is used interchangeably with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. This term encompasses synthetic, natural, and non-natural nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise indicated, a specified nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions can be achieved by generating 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).

[0315] The term "operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, the term refers to the 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 the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, a promoter transcriptional regulatory sequence operably linked to a transcribed sequence is physically contiguous, i.e., cis-acting, with the transcribed sequence. However, some transcriptional regulatory sequences, such as enhancers, need not be physically adjacent or located in close proximity to the coding sequences whose transcription they enhance.

[0316] As used herein, the terms "optimized" or "codon optimization" mean that a nucleotide sequence has been modified to encode an amino acid sequence using codons that are preferred in a production cell or organism, generally a eukaryotic cell, such as a Pichia cell, a Chinese hamster ovary cell (CHO), a human cell, or a prokaryotic cell, such as an Escherichia coli cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that encode the same amino acid) in coding DNA varies across different species. Such codon degeneracy allows identical polypeptides to be encoded by a variety of nucleotide sequences. Various codon optimization methods are known herein, including, for example, at least those disclosed in U.S. Pat. Nos. 5,786,464 and 6,114,148. An optimized nucleotide sequence is engineered to retain, entirely, or as much as possible, the amino acid sequence originally encoded by the starting nucleotide sequence, also known as the "parent" sequence. The optimized sequences herein are engineered to have codons that are preferred in mammalian cells. However, optimized expression of these sequences in other eukaryotic or prokaryotic cells is also contemplated herein. The amino acid sequences encoded by the optimized nucleotide sequences are also referred to as optimized.

[0317] As used herein, the term "protein" refers to any organic compound made from 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" also includes, but is not limited to, peptides, single-chain polypeptides, or any complex molecule composed primarily of two or more chains of amino acids. It also includes, but is not limited to, glycoproteins or other known post-translational modifications. It also includes, but is not limited to, known natural or artificial chemical modifications of native proteins, such as glycoengineering, pegylation, hesylation, incorporation of unnatural amino acids, and amino acid modifications for chemical conjugation to another molecule.

[0318] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The phrase also applies to amino acid polymers that are artificial chemical mimetics of a corresponding naturally occurring amino acid in which one or more amino acid residues correspond, as well as to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0319] 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 particular subject cell but to the progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not be, in fact, identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0320] 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 monkeys), mice, rats, cats, rabbits, pigs, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where noted, the terms "patient" and "subject" are used interchangeably herein. As used herein, the terms "cyno" or "cynomolgus" refer to the cynomolgus monkey (Macaca fascicularis).

[0321] "Prevention" or "preventing," when it relates to an indication described herein, e.g., an ophthalmic condition or disorder, including a condition or disorder associated with diabetic macular edema, a retinal vascular disease, a condition or disorder associated with diabetic retinopathy, and / or a condition or disorder associated with macular edema, refers to any action that prevents or delays a deterioration in visual function, retinal anatomy, retinal vascular disease parameters, diabetic retinopathy disease parameters, and / or macular edema disease parameters, as described below, in a patient at risk of such deterioration. As used herein, "prevention" or "preventing," when it relates to a non-ophthalmic condition or disorder, including pancreatic cancer, breast cancer, endometrial adenocarcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, and gastric cancer, does not necessarily result in complete prevention of the condition. Partial prevention or alleviation of the condition or symptoms of the condition, or reduction of the risk of developing the condition, are also encompassed by the term.

[0322] The term "treating" or "treatment" of a condition or disorder associated with diabetic macular edema, a condition or disorder associated with age-related macular degeneration, e.g., neovascular age-related macular degeneration, a condition or disorder associated with retinal vascular disease, a condition or disorder associated with diabetic retinopathy, and / or a condition or disorder associated with macular edema refers to any action that results in or is intended to result in the improvement or preservation of visual function and / or retinal anatomy. As used herein, "treating" or "treatment" of a non-ophthalmological condition or disorder, including pancreatic cancer, breast cancer, endometrial adenocarcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, and gastric cancer, refers to any action that results in or is intended to result in the improvement or alleviation of the condition or disorder. In another aspect, treatment includes reducing the frequency of repeat administrations and / or reducing doctor / hospital visits. Also included is an aspect / embodiment where treatment involves long-term treatment, e.g., repeated administration over an indefinite period. Methods for assessing disease treatment and / or prevention are known in the art and are described herein below.

[0323] The terms "therapeutically acceptable amount" or "therapeutically effective amount" or "therapeutically effective dose" interchangeably refer to an amount sufficient to produce a desired result (i.e., reduced disease activity, reduced disease progression, reduced disease signs and / or symptoms, etc.). In some embodiments, a therapeutically acceptable amount does not induce or cause undesirable side effects. A therapeutically acceptable amount can be determined by administering an initial low dose and then gradually increasing the dose until the desired effect is achieved. A "prophylactically effective dose" and a "therapeutically effective dose" of a molecule of the present disclosure can prevent the onset of or result in a reduction in the severity of disease symptoms, respectively, including symptoms associated with BTC activity and / or VEGF activity.

[0324] The term "vector" is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. A "plasmid," one type of vector, refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. In another embodiment, polynucleotide sequences can be delivered 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, and additional DNA segments can be ligated into the viral genome. Certain vectors (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced. Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, 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 of utility in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the present disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0325] The term "recombinant," as it refers to a polynucleotide (DNA or RNA) molecule, protein, construct, vector, etc., refers to a polynucleotide or protein molecule or sequence that is man-made, not normally found in nature, and / or exists in a context not normally found in nature, including polynucleotide (DNA or RNA) molecules, proteins, constructs, etc. that contain combinations of two or more polynucleotide or protein sequences that do not naturally occur together in the same way without human intervention, e.g., polynucleotide molecules, proteins, constructs, etc. that contain at least two polynucleotide or protein sequences that are operably linked but heterologous to one another. For example, the term "recombinant" can refer to any combination of two or more DNA or protein sequences in the same molecule (e.g., a plasmid, construct, vector, chromosome, protein, etc.), where such combination is man-made and not normally found in nature. As used in this definition, the phrase "not normally found in nature" means not found in nature without introduction by a human. A recombinant polynucleotide or protein molecule, construct, etc. can include a polynucleotide or protein sequence that is (i) separated from other polynucleotide or protein sequences that occur near each other in nature, and / or (ii) adjacent to (or in close proximity to) other polynucleotide or protein sequences that are not naturally adjacent to each other. Such recombinant polynucleotide molecules, proteins, constructs, etc. can also refer to polynucleotide or protein molecules or sequences that have been genetically engineered and / or assembled outside a cell. For example, a recombinant DNA molecule can include any engineered or artificial plasmid, vector, etc., and can include linear or circular DNA molecules. Such plasmids, vectors, etc. can contain one or more transgenes or expression cassettes in addition to various maintenance elements, including a prokaryotic origin of replication and a selectable marker, and possibly a plant selectable marker gene, etc.

[0326] As used herein, "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, an mRNA, a protein, or a non-coding RNA sequence or molecule).

[0327] As used herein, the term "therapeutic protein" refers to a protein that is useful for treating, preventing, or ameliorating a disease, condition, or disorder.

[0328] As used herein, an "alteration" or "mutation" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, where the change is due to a change in the sequence involving that amino acid residue / position. For example, typical alterations include substitution of the residue (or at that position) with another amino acid (e.g., conservative or non-conservative substitution), insertion of one or more amino acids adjacent to that residue / position, and deletion of that residue / position. An "amino acid substitution" or variation thereof refers to the replacement of an existing amino acid residue in a given (starting) amino acid sequence with a different amino acid residue. Generally and preferably, the alteration results in a change in at least one physico-biochemical activity of the variant polypeptide compared to the polypeptide comprising the starting (or "wild-type") amino acid sequence. For example, in the case of an antibody, the altered physico-biochemical activity can be binding affinity, binding capacity, and / or binding effect on a target molecule.

[0329] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are a species of conservatively modified variants. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. Those of skill in the art will recognize that each codon within a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be altered to result in a functionally identical molecule. Accordingly, within each described sequence, each silent variation of a nucleic acid that encodes a polypeptide is implicit.

[0330] As used herein, "C-terminus" refers to the amino acid at the carboxyl end of a polypeptide chain having a free carboxyl group (-COOH). As used herein, "N-terminus" refers to the amino acid at the amino end of a polypeptide chain having a free amine group (-NH).

[0331] As used herein, phrases such as "patient in need of treatment" or "subject in need of treatment" include subjects, e.g., mammalian subjects, who would benefit from the administration of a molecule or pharmaceutical composition of the present disclosure for use in, e.g., detection, diagnostic procedures, and / or therapy.

[0332] The phrase "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly for use in humans.

[0333] The term "pharmaceutical composition" refers to a mixture of at least one active ingredient (e.g., an antibody or fragment of the present disclosure) with at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0334] "Drug" refers to a substance for use in medical treatment.

[0335] "BTC-mediated disorders" encompass all diseases and medical conditions in which BTC and / or VEGF are implicated, directly or indirectly, in the cause, onset, progression, persistence, or pathology of the disease or condition. BTC-mediated disorders can include, but are not limited to, pancreatic cancer, breast cancer, endometrial adenocarcinoma, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, gastric cancer, diabetic macular edema, age-related macular degeneration, neovascular age-related macular degeneration, neovascular glaucoma, diabetic retinopathy, macular edema, pathological myopia, retinal vein occlusion, retinopathy of prematurity, abnormal vascular proliferation associated with phacomatosis, central serous chorioretinopathy, and acute multifocal platelet pigment epitheliopathy.

[0336] "VEGF-mediated disorders" encompass all diseases and medical conditions in which VEGF is implicated, directly or indirectly, in the cause, onset, progression, persistence, or pathology of the disease or condition. VEGF-mediated disorders can include, but are not limited to, abnormal vascular proliferation associated with central nervous system tumors, capillary hemangioblastoma, meningioma, cerebral edema, pituitary adenoma, nonastrocytic glioma, peritumoral edema, breast cancer, adenocarcinoma, lung cancer, diabetic macular edema, age-related macular degeneration, neovascular age-related macular degeneration, neovascular glaucoma, diabetic retinopathy, macular edema, pathologic myopia, retinal vein occlusion, retinopathy of prematurity, and nevus syndrome.

[0337] ii. Anti-BTC antibody or anti-BTC binding moiety BTC is a member of the EGF family. It is a ligand for the ErbB receptor tyrosine kinase family, primarily activating ErbB1 and ErbB4 homodimers, which trigger anti-apoptotic and pro-proliferative signaling pathways, such as the Ras / MAPK and PL3K / AKT pathways. In the eye, BTC appears to be a potent permeability factor that may play an important role in the development of increased retinal vascular permeability in diabetic retinopathy and is a potential therapeutic target in this disease. An exemplary human pro-BTC amino acid sequence is provided as SEQ ID NO: 156. An exemplary human BTC amino acid sequence is provided as SEQ ID NO: 158. An exemplary human BTC amino acid sequence represented in this disclosure is provided as SEQ ID NO: 157 (remaining amino acid residues are represented in lowercase at the N- and C-termini).

[0338] The structure of human BTC protein bound to four anti-BTC Fab fragments has recently been solved by the applicant via X-ray crystallography (see Example 2). The human BTC structure is an EGF fold with five beta strands in a three-stranded and two-stranded sheet. The structure is stabilized by three disulfide bonds.

[0339] Provided herein are antibodies or antigen-binding fragments thereof that bind to BTC, including human BTC. In some embodiments, the provided antibodies or antigen-binding fragments thereof are polypeptides comprising one or more complementarity-determining regions (CDRs), as described herein. In some embodiments, the CDRs are embedded in "framework" regions, which orient the CDRs such that appropriate antigen-binding properties of the CDRs are achieved. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can interfere with, block, reduce, or modulate the interaction between BTC and ErbB receptors. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can inhibit BTC-mediated activity (including binding). In some embodiments, antigen-binding proteins that bind to these epitopes inhibit, among other things, the interaction between BTC and ErbB receptors and other physiological effects mediated by BTC. In some embodiments, the antigen-binding protein is a human protein, e.g., a fully human antibody or Fab against BTC.

[0340] In some embodiments, the antibody or antigen-binding fragment thereof binds to any one of the epitopes bound by the antibodies discussed herein. In some embodiments, this can be determined by a competition assay between an antibody disclosed herein and other antibodies. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope bound by one of the antibodies listed in Table 1. In some embodiments, the antibody or antigen-binding fragment thereof binds to a specific conformational state of BTC to prevent BTC from interacting with ErbB receptors. In one embodiment, an antibody or antigen-binding fragment thereof of the present disclosure binds to one or more of the five beta chains of human BTC. In one embodiment, the antibody or antigen-binding fragment thereof binds to beta chain 1 of human BTC and prevents BTC from binding to ErbB receptors. In one embodiment, the antibody or antigen-binding fragment thereof binds to beta chain 2 of human BTC and prevents BTC from binding to ErbB receptors. In one embodiment, the antibody or antigen-binding fragment thereof binds to beta chain 3 of human BTC and prevents BTC from binding to ErbB receptors. In one embodiment, the antibody or antigen-binding fragment thereof binds to beta chain 4 of human BTC and prevents BTC from binding to an ErbB receptor. In one embodiment, the antibody or antigen-binding fragment thereof binds to beta chain 5 of human BTC and prevents BTC from binding to an ErbB receptor.

[0341] Disclosed herein are antibodies or antigen-binding fragments thereof that specifically bind to BTC. In some embodiments, the anti-BTC antibody or antigen-binding fragment thereof prevents BTC from functioning in various ways. In some embodiments, 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 embodiments, the anti-BTC antibody or antigen-binding fragment thereof blocks or reduces the ability of BTC to bind to ErbB receptors. In other embodiments, the anti-BTC antibody or antigen-binding fragment thereof blocks BTC-induced phosphorylated ERK1 / 2 activation. In some embodiments, the anti-BTC antibody or antigen-binding fragment thereof blocks BTC-induced phosphorylated HER3 activation.

[0342] Certain antibodies or antigen-binding fragments thereof as provided herein specifically and / or selectively bind to human BTC as set forth in SEQ ID NO: 157 or 158. In some embodiments, the antibodies or antigen-binding fragments thereof selectively bind to a human BTC protein as displayed in Example 2 and Table 1. In some embodiments, the antibodies or antigen-binding fragments thereof specifically and / or selectively bind 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 embodiments, two or more (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 the region bound by the antibody or antigen-binding fragment thereof.

[0343] In some embodiments, the antibody or antigen-binding fragment thereof specifically and / or selectively binds to SEQ ID NO: 157, e.g., R38, C39, P40, K41, Q42, Y43, H45, Y46, F54, Q59, T60, P61, and R73 of NVS1. In some embodiments, the antibody or antigen-binding fragment thereof specifically and / or selectively binds to SEQ ID NO: 157, e.g., P40, K41, Q42, Y43, H45, Y46, E58, Q59, T60, P61, A72, R73, E75, and R76 of NVS2. In some embodiments, the antibody or antigen-binding fragment thereof specifically and / or selectively binds to SEQ ID NO: 157, e.g., G34, H35, F36, S37, R38, C39, P40, K41, Q42, R51, R53, F54, and V56 of NVS3. In some embodiments, the antibody or antigen-binding fragment thereof specifically and / or selectively binds to SEQ ID NO: 157, e.g., S37, R38, C39, P40, K41, Q42, Y43, H45, Y46, F54, A57, Q59, T60, P61, A72, R73, and E75 of NVS4.

[0344] In embodiments in which the antibody or antigen-binding fragment thereof is used for therapeutic purposes, the antibody or antigen-binding fragment thereof can inhibit, interfere with, or modulate one or more biological activities of BTC. In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to human BTC and / or substantially inhibits binding of human BTC to an ErbB receptor by at least about 20%-40%, 40-60%, 60-80%, 80-85%, or more (e.g., by measuring binding in an in vitro competitive binding assay). In some embodiments, the antibody or antigen-binding fragment thereof inhibits binding of human BTC to an ErbB receptor by at least about 20%-40%, 40-60%, 60-80%, 80-85%, or more. -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 K less than M d In some embodiments, the antibody or antigen-binding fragment thereof has an IC50 for blocking binding of an ErbB receptor to a BTC of less than 1 micromolar, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, or 10 pM to 1 pM. 50 It has.

[0345] In some embodiments, the antibody or antigen-binding fragment thereof binds to a variant of BTC that is at least 50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99%, or more percent identical to a form of BTC as set forth in SEQ ID NO: 157 or 158. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope bound by one of the antibodies set forth in Table 1. In some embodiments, the antibody or antigen-binding fragment thereof binds to a specific conformational state of BTC to prevent BTC from interacting with an ErbB receptor.

[0346] The anti-BTC antibody or antigen-binding fragment thereof of the present disclosure comprises heavy chain variable region complementarity determining region 1 (HCDR1), heavy chain variable region complementarity determining region 2 (HCDR2), heavy chain variable region complementarity determining region 3 (HCDR3), light chain variable region complementarity determining region 1 (LCDR1), light chain variable region complementarity determining region 2 (LCDR2), and 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 embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises heavy and light chain CDRs (Kabat, Chothia, IMGT, and / or combined CDRs) as set forth in Table 1 and described below.

[0347] In one embodiment, an anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, according to the Kabat numbering scheme, as set forth in SEQ ID NOs: 4, 2, 3, 14, 15, and 16, respectively. In one embodiment, an anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, according to the Chothia numbering scheme, as set forth in SEQ ID NOs: 5, 6, 3, 17, 18, and 19, respectively. In one embodiment, an anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, according to the combined numbering scheme, as set forth in SEQ ID NOs: 1, 2, 3, 14, 15, and 16, respectively. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, as set forth in SEQ ID NOs: 7, 8, 9, 20, 18, and 16, respectively, according to the IMGT numbering scheme. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof is NVS1, as provided in Table 1. In a particular embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs (e.g., Kabat, Chothia, IMGT, and / or combined CDRs) of antibody NVS1, which comprises the VH and VL of SEQ ID NOs: 10 and 21, respectively.

[0348] In one embodiment, an anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, according to the Kabat numbering scheme, as set forth in SEQ ID NOs: 28, 26, 27, 38, 39, and 40, respectively. In one embodiment, an anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, according to the Chothia numbering scheme, as set forth in SEQ ID NOs: 29, 30, 27, 41, 42, and 43, respectively. In one embodiment, an anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, according to the combined numbering scheme, as set forth in SEQ ID NOs: 25, 26, 27, 38, 39, and 40, respectively. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 31, 32, 33, 44, 42, and 40, respectively, according to the IMGT numbering scheme. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof is NVS2 as provided in Table 1. In a particular embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs (e.g., Kabat, Chothia, IMGT, and / or combined CDRs) of antibody NVS2 comprising the VH and VL of SEQ ID NOs: 34 and 45, respectively.

[0349] In one embodiment, an anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 28, 49, 50, 58, 59, and 60, respectively, according to the Kabat numbering scheme. In one embodiment, an anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 29, 51, 50, 61, 62, and 63, respectively, according to the Chothia numbering scheme. In one embodiment, an anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 25, 49, 50, 58, 59, and 60, respectively, according to the combined numbering scheme. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 31, 52, 53, 64, 62, and 60, respectively, according to the IMGT numbering scheme. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof is NVS3 as provided in Table 1. In a particular embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs (e.g., Kabat, Chothia, IMGT, and / or combined CDRs) of antibody NVS3 comprising the VH and VL of SEQ ID NOs: 54 and 65, respectively.

[0350] In one embodiment, an anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, according to the Kabat numbering scheme, as set forth in SEQ ID NOs: 72, 70, 71, 82, 83, and 84, respectively. In one embodiment, an anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, according to the Chothia numbering scheme, as set forth in SEQ ID NOs: 73, 74, 71, 85, 18, and 86, respectively. In one embodiment, an anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, according to the combined numbering scheme, as set forth in SEQ ID NOs: 69, 70, 71, 82, 83, and 84, respectively. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 75, 76, 77, 87, 18, and 84, respectively, according to the IMGT numbering scheme. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof is NVS4 as provided in Table 1. In a particular embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs (e.g., Kabat, Chothia, IMGT, and / or combined CDRs) of antibody NVS4, which comprises the VH and VL of SEQ ID NOs: 78 and 88, respectively.

[0351] Additionally, the present disclosure also provides anti-BTC antibodies or antigen-binding fragments thereof comprising amino acid sequences homologous to the CDR sequences set forth throughout and in Table 1, wherein the anti-BTC antibodies or antigen-binding fragments thereof bind to BTC and retain the desired functional properties of those described herein. More specifically, the amino acid sequence of the anti-BTC antibodies or antigen-binding fragments thereof can have 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the CDR sequences set forth throughout and in Table 1 and can retain the desired functional properties.

[0352] The present disclosure also provides anti-BTC antibodies or antigen-binding fragments thereof that are homologous to the VH and VL sequences described herein. More specifically, the present disclosure provides proteins comprising amino acid sequences homologous to sequences such as those set forth in Table 1, where the anti-BTC antibodies or antigen-binding fragments bind to a therapeutic target, e.g., an ophthalmic target, and retain the desired functional properties of those described in Table 1 and in the Examples. Antibodies or antigen-binding fragments thereof having VH and VL regions with less than 100% sequence identity to those set forth in Table 1 can be obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of nucleic acid molecules set forth in Table 1, followed by testing the encoded, altered antibodies for retained function using the functional assays described herein and in U.S. Patent Application Publication No. 20120014958. Antibodies or antigen-binding fragments thereof having heavy and light chains with high (i.e., 80% or greater) identity to the heavy and light chains set forth in Table 1 can be obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of nucleic acid molecules encoding such polypeptides, followed by testing the encoded, altered antibodies for retained function, e.g., by using the functional assays described herein.

[0353] The anti-BTC antibodies or antigen-binding fragments thereof of the present disclosure may have a sequence similar to that of SEQ ID NOs: 10 and 21, respectively, of 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 The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) comprising amino acid sequences having 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. It is contemplated that there may be variability within the CDR or framework regions. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises a VH and a VL comprising amino acid sequences as set forth in SEQ ID NOs: 10 and 21, respectively. In another embodiment, the anti-BTC antibody or antigen-binding fragment thereof is NVS1 as provided in Table 1. In another embodiment, the VH and VL are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 11 and 22, respectively. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises a VH and a VL comprising amino acid sequences having at least one, two, or three alterations (e.g., substitutions, e.g., conservative substitutions) but not more than 10 alterations (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequences of SEQ ID NOs: 10 and 21, respectively. In another embodiment, the differences in amino acid sequence are not within the complementarity-determining regions.

[0354] In one embodiment, the anti-BTC antibodies or antigen-binding fragments thereof of the present disclosure have a binding affinity of 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 The anti-BTC antibody or antigen-binding fragment thereof comprises a VH and a VL comprising amino acid sequences having 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. It is contemplated that there may be variability within the CDR or framework regions. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises a VH and a VL comprising the amino acid sequences as set forth in SEQ ID NOs: 34 and 45, respectively. In another embodiment, the anti-BTC antibody or antigen-binding fragment thereof is NVS2 as provided in Table 1. In another embodiment, the VH and VL are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 35 and 46, respectively. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises a VH and a VL comprising amino acid sequences having at least one, two, or three alterations (e.g., substitutions, e.g., conservative substitutions) but not more than 10 alterations (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequences of SEQ ID NOs: 34 and 45, respectively. In another embodiment, the differences in amino acid sequence are not within the complementarity-determining regions.

[0355] In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof of the present disclosure has a binding affinity of 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 The anti-BTC antibody or antigen-binding fragment thereof comprises a VH and a VL comprising amino acid sequences having 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. It is contemplated that there may be variability within the CDR or framework regions. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises a VH and a VL comprising the amino acid sequences as set forth in SEQ ID NOs: 54 and 65, respectively. In another embodiment, the anti-BTC antibody or antigen-binding fragment thereof is NVS3 as provided in Table 1. In another embodiment, the VH and VL are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 55 and 66, respectively. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises a VH and a VL comprising amino acid sequences having at least one, two, or three alterations (e.g., substitutions, e.g., conservative substitutions) but not more than 10 alterations (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequences of SEQ ID NOs: 54 and 65, respectively. In another embodiment, the differences in amino acid sequence are not within the complementarity-determining regions.

[0356] In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof of the present disclosure has a binding affinity of 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 The anti-BTC antibody or antigen-binding fragment thereof comprises a VH and a VL comprising amino acid sequences having 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. It is contemplated that there may be variability within the CDR or framework regions. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises a VH and a VL comprising amino acid sequences as set forth in SEQ ID NOs: 78 and 88, respectively. In another embodiment, the anti-BTC antibody or antigen-binding fragment thereof is NVS4 as provided in Table 1. In another embodiment, the VH and VL are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 79 and 89, respectively. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises a VH and a VL comprising amino acid sequences having at least one, two, or three alterations (e.g., substitutions, e.g., conservative substitutions) but not more than 10 alterations (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequences of SEQ ID NOs: 78 and 88, respectively. In another embodiment, the differences in amino acid sequence are not within the complementarity-determining regions.

[0357] The anti-BTC antibodies or antigen-binding fragments thereof of the present disclosure comprise heavy and light chains comprising amino acid sequences having 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 embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises heavy and light chains comprising the amino acid sequences as set forth in SEQ ID NOs: 12 and 23, respectively. In one embodiment, the anti-BTC antibody can inhibit BTC activity, e.g., binding of BTC to 1) ErbB1; 2) ErbB4; 3) ErbB homodimers (e.g., ErbB1 / ErbB1 and ErbB4 / ErB4); 4) ErbB heterodimers (e.g., ErbB1 / ErbB2, ErB1 / ErB3, ErB1 / ErB4, ErB2 / ErB3, and ErB2 / ErB4); and / or 5) can inhibit ERK1 / 2 phosphorylation. In another embodiment, the anti-BTC antibody or antigen-binding fragment thereof is NVS1 as provided in Table 1.In another embodiment, the heavy and light chains have a sequence similar to or similar to 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%, or 100% identity or complementarity to the nucleic acid sequence as set forth in SEQ ID NOs: 13 and 24, respectively.

[0358] In one embodiment, the anti-BTC antibodies or antigen-binding fragments thereof of the present disclosure have a binding affinity of 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 The anti-BTC antibody or antigen-binding fragment thereof comprises heavy and light chains comprising amino acid sequences that have 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 the heavy and light chains. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises heavy and light chains comprising the amino acid sequences as set forth in SEQ ID NOs: 36 and 47, respectively. In one embodiment, the anti-BTC antibody can inhibit BTC activity, e.g., binding of BTC to 1) ErbB1; 2) ErbB4; 3) ErbB homodimers (e.g., ErbB1 / ErbB1 and ErbB4 / ErB4); 4) ErbB heterodimers (e.g., ErbB1 / ErbB2, ErB1 / ErB3, ErB1 / ErB4, ErB2 / ErB3, and ErB2 / ErB4); and / or 5) inhibit ERK1 / 2 phosphorylation. In another embodiment, the anti-BTC antibody or antigen-binding fragment thereof is NVS2 as provided in Table 1.In another embodiment, the heavy and light chains have a sequence similar to or similar to 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%, or 100% identity or complementarity to the nucleic acid sequence as set forth in SEQ ID NOs: 37 and 48, respectively.

[0359] In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof of the present disclosure has a binding affinity of 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 The anti-BTC antibody or antigen-binding fragment thereof comprises heavy and light chains comprising amino acid sequences that have 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 the heavy and light chains. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises heavy and light chains comprising the amino acid sequences as set forth in SEQ ID NOs: 56 and 67, respectively. In one embodiment, the anti-BTC antibody can inhibit BTC activity, e.g., binding of BTC to 1) ErbB1; 2) ErbB4; 3) ErbB homodimers (e.g., ErbB1 / ErbB1 and ErbB4 / ErB4); 4) ErbB heterodimers (e.g., ErbB1 / ErbB2, ErB1 / ErB3, ErB1 / ErB4, ErB2 / ErB3, and ErB2 / ErB4); and / or 5) inhibit ERK1 / 2 phosphorylation. In another embodiment, the anti-BTC antibody or antigen-binding fragment thereof is NVS3 as provided in Table 1.In another embodiment, the heavy and light chains have a sequence similar to or similar to 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%, or 100% identity or complementarity to the nucleic acid sequence as set forth in SEQ ID NOs: 57 and 68, respectively.

[0360] In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof of the present disclosure has a nucleotide sequence similar to or at least about 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 In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain comprising amino acid sequences having 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 the heavy chain. In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain comprising the amino acid sequences as set forth in SEQ ID NOs: 80 and 90, respectively. In one embodiment, the anti-BTC antibody can inhibit BTC activity, e.g., binding of BTC to 1) ErbB1; 2) ErbB4; 3) ErbB homodimers (e.g., ErbB1 / ErbB1 and ErbB4 / ErB4); 4) ErbB heterodimers (e.g., ErbB1 / ErbB2, ErB1 / ErB3, ErB1 / ErB4, ErB2 / ErB3, and ErB2 / ErB4); and / or 5) inhibit ERK1 / 2 phosphorylation. In another embodiment, the anti-BTC antibody or antigen-binding fragment thereof is NVS4 as provided in Table 1.In another embodiment, the heavy and light chains have a sequence similar to or identical to 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%, or 100% identity or complementarity to the nucleic acid sequence as set forth in SEQ ID NOs: 81 and 91, respectively.

[0361] Additionally, isolated anti-BTC antibodies or antigen-binding fragments thereof with conservative modifications are included within the scope of this disclosure. More specifically, this disclosure relates to anti-BTC binding moieties and molecules conjugated to the anti-BTC binding moieties that have conservative modifications relative to the anti-BTC binding moieties and molecules conjugated to the anti-BTC binding moieties of Table 1. In certain embodiments, antibodies conjugated to anti-BTC binding moieties of this disclosure have 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 these CDR sequences have a specific amino acid sequence based on an antibody or conservative variant thereof described herein, and the antibody retains the desired functional properties of the antibodies of this disclosure.

[0362] In certain aspects, provided herein are polynucleotides whose sequences encode anti-BTC antibodies or fragments thereof (e.g., VH or VL) described herein (e.g., Table 1). 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, e.g., 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, one or more of which sequences have a specific amino acid sequence based on an antibody or conservative variant thereof described herein, and the anti-BTC binding portion retains the desired functional properties of the anti-BTC binding antibodies of the present disclosure. Thus, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to BTC, optimized for expression in mammalian cells, for example, comprising a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NOs: 10, 34, 54, and 78, and conservative variants thereof; and the VL comprises the amino acid sequence of SEQ ID NOs: 21, 45, 65, and 88, and conservative variants thereof.

[0363] The present disclosure provides anti-BTC binding moieties (e.g., BTC-binding antibodies or fragments thereof) that bind to the same or overlapping epitopes as the anti-BTC antibodies or antigen-binding fragments thereof listed in Table 1. Accordingly, additional antibodies can be identified based on their ability to compete (e.g., competitively inhibit binding in a statistically significant manner) with other antibodies of the present disclosure in BTC binding assays. The ability of a test antibody to inhibit binding of a molecule of the present disclosure to BTC demonstrates that the test molecule can compete with that antibody for binding to BTC; such an antibody may, according to non-limiting theory, bind to the same or related (e.g., structurally similar or spatially proximal) epitope on BTC as the competing antibody. In certain embodiments, a molecule that binds to the same epitope on BTC as an antibody of the present disclosure is a human monoclonal antibody, Fab, or scFv. Such human monoclonal antibodies, Fabs, and scFvs can be prepared and isolated as described herein.

[0364] In one embodiment, a molecule that competes with an anti-BTC antibody or antigen-binding fragment thereof of the present disclosure 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 embodiment, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that can compete with those listed in Table 1, e.g., NVS1, NVS2, NVS3, and NVS4, for binding to BTC and reducing BTC-mediated signaling. In another embodiment, 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 of Table 5.

[0365] The anti-BTC antibody or antigen-binding fragment thereof of the present disclosure is in a format selected from the group consisting of isolated antibody, Fab, Fab', F(ab')2, Fv, and scFv. In a preferred embodiment, the anti-BTC antibody or antigen-binding fragment thereof is a Fab, including a Fab comprising an Fc region. In another embodiment, the Fc region is selected from the group consisting of Fc regions from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. In one embodiment, the Fc region comprises a human immunoglobulin kappa chain constant region sequence as set forth in SEQ ID NO: 159. In another embodiment, the Fc region comprises the first constant Ig domain (CH1 domain) of a human immunoglobulin heavy chain as set forth in SEQ ID NO: 160.

[0366] In one embodiment, the anti-BTC antibody or antigen-binding fragment thereof is an isolated antibody, e.g., a monoclonal human antibody or a monoclonal humanized antibody. In certain embodiments, the anti-BTC antibody can be in scFv or Fab format. In certain embodiments, the anti-BTC antibody can be in scFv or Fab format.

[0367] [Table 1]

[0368] [Table 2]

[0369] [Table 3]

[0370] [Table 4]

[0371] [Table 5]

[0372] [Table 6]

[0373] [Table 7]

[0374] [Table 8]

[0375] [Table 9]

[0376] iii. Binding partners of anti-BTC antibodies As noted above, the present disclosure features compositions comprising one or more anti-BTC antibodies or antigen-binding fragments thereof conjugated (e.g., covalently linked, non-covalently linked, or fused) to a therapeutic target-binding moiety specific for a target located within or near a tissue bearing BTC (e.g., substantially at the level of BTC, e.g., the eye, pancreas). For example, the present disclosure features compositions having one or more anti-BTC antibodies or antigen-binding fragments thereof attached to a therapeutic target-binding moiety relevant to the treatment of a BTC-associated condition or disease (e.g., DR, DME, AMD, e.g., neovascular AMD, and / or RVO). In another aspect, the present disclosure features pharmaceutical compositions comprising anti-BTC antibodies or antigen-binding fragments thereof and therapeutic target-binding moieties, which pharmaceutical compositions can be used to treat a BTC-mediated condition or disease. In yet another aspect, the disclosure features a method of treating a subject in need of treatment, wherein the subject has a BTC-mediated condition or disease, comprising administering to the subject an effective amount of an anti-BTC antibody, or antigen-binding fragment thereof, of the disclosure, and subsequently administering to the subject a therapeutic target-binding moiety (e.g., a VEGF inhibitor).

[0377] In certain preferred embodiments, the therapeutic target binding moiety is an antibody or antigen-binding fragment thereof (e.g., in the form of an scFv, Fab, single domain antibody, or diabody) that binds to the therapeutic target, or is a polypeptide that binds to the therapeutic target (e.g., a soluble receptor). Such therapeutic targets can be, for example, those associated with ophthalmic disorders, such as VEGF, PDGF, PDGF-BB, angiopoietin, angiopoietin-2, S1P, integrin 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.

[0378] In one aspect, the disclosure features multispecific binding molecules having at least one each of anti-BTC binding moieties and one or more therapeutic target binding moieties. Thus, in one aspect, the disclosure features bispecific molecules (e.g., bispecific antibodies) having a combination of binding selectivities selected from, for example, the following: BTC and VEGF, BTC and PDGF, BTC and PDGF-BB, BTC and angiopoietin, BTC and angiopoietin-2, BTC and S1P, BTC and integrin 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 CFHR3. C 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.

[0379] In one embodiment, the therapeutic target binding moiety can be an anti-VEGF antagonist. In one embodiment, the anti-VEGF antagonist is ranibizumab (Lucentis®; WO 98 / 45331; WO 98 / 45331; U.S. Pat. No. 6,884,879; U.S. Pat. No. 6,407,213; U.S. Pat. No. 7,060,269; U.S. Pat. No. 7,365,166). In one embodiment, the anti-VEGF antagonist is bevacizumab (Avastin®; U.S. Pat. No. 6,054,297; U.S. Pat. No. 7,169,901; U.S. Pat. No. 7,375,193; U.S. Pat. No. 7,297,334). In one embodiment, the anti-VEGF antagonist is aflibercept (Eylea®; U.S. Pat. No. 7,279,159). In one embodiment, the anti-VEGF antagonist is brolucizumab (Beovu®; WO 2009 / 155724; U.S. Pat. No. 8,349,322; U.S. Pat. No. 9,090,684; U.S. Pat. No. 9,873,737; WO 03 / 097697; WO 2016 / 073915; WO 2016 / 073918). In one embodiment, the anti-VEGF antagonist is pegaptanib (Macugen®). In one embodiment, the anti-VEGF antagonist is KH902 (WO 2005 / 121176; U.S. Pat. No. 7,750,138). In one embodiment, the anti-VEGF antagonist comprises a heavy chain and a light chain as set forth in SEQ ID NOs: 103 and 114, respectively. In one embodiment, the anti-VEGF antagonist is encoded by a nucleic acid sequence as set forth in SEQ ID NOs: 104 and 115, respectively.

[0380] iv. Multispecific binding molecules Also provided in this disclosure are multispecific binding molecules comprising 1) an anti-BTC binding moiety and 2) an anti-VEGF binding moiety.

[0381] a) Anti-BTC binding part In one embodiment, the anti-BTC binding moiety binds to human BTC, as set forth in SEQ ID NO: 157 or 158. In some embodiments, the anti-BTC binding moiety selectively binds to a human BTC protein displayed in Example 2 and Table 4. In some embodiments, the anti-BTC binding moiety 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 embodiments, two or more of the identified BTC residues (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) are part of the region bound by an anti-BTC binding moiety. In certain embodiments, such anti-BTC binding moieties selectively bind to human BTC protein and inhibit (e.g., partially inhibit) BTC activity.

[0382] In some embodiments, the anti-BTC binding portion specifically and / or selectively binds to SEQ ID NO: 157, e.g., R38, C39, P40, K41, Q42, Y43, H45, Y46, F54, Q59, T60, P61, and R73 of NVS11. In some embodiments, the antibody or antigen-binding fragment thereof specifically and / or selectively binds to SEQ ID NO: 157, e.g., P40, K41, Q42, Y43, H45, Y46, E58, Q59, T60, P61, A72, R73, E75, and R76 of NVS12. In some embodiments, the antibody or antigen-binding fragment thereof specifically and / or selectively binds to SEQ ID NO: 157, e.g., G34, H35, F36, S37, R38, C39, P40, K41, Q42, R51, R53, F54, and V56 of NVS13. In some embodiments, the antibody or antigen-binding fragment thereof specifically and / or selectively binds to SEQ ID NO: 157, e.g., S37, R38, C39, P40, K41, Q42, Y43, H45, Y46, F54, A57, Q59, T60, P61, A72, R73, and E75 of NVS14. In one embodiment, the anti-BTC binding moiety is an anti-BTC antibody or antigen-binding fragment thereof, as described throughout. In certain embodiments, such anti-BTC binding moieties selectively bind to human BTC protein and inhibit (eg, partially inhibit) BTC activity.

[0383] In embodiments where the anti-BTC binding moiety is used for therapeutic purposes, the anti-BTC binding moiety can inhibit, interfere with, or modulate one or more biological activities of BTC. In one embodiment, the anti-BTC binding moiety specifically binds to human BTC and / or substantially inhibits binding of human BTC to an ErbB receptor by at least about 20%-40%, 40-60%, 60-80%, 80-85%, or more (e.g., by measuring binding in an in vitro competitive binding assay). In some embodiments, the anti-BTC binding moiety is capable of inhibiting, interfering with, or modulating one or more biological activities of BTC. -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 K less than M dIn some embodiments, the anti-BTC binding moiety has an IC50 for blocking binding of an ErbB receptor to BTC of 1 micromolar, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, or 10 pM to 1 pM. 50 In certain embodiments, such anti-BTC binding moieties selectively bind to human BTC protein and inhibit (e.g., partially inhibit) BTC activity.

[0384] In some embodiments, the anti-BTC binding moiety binds to a variant of BTC that is about at least 50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99%, or more percent identical to a form of BTC such as set forth in SEQ ID NO: 157 or 158. In some embodiments, the anti-BTC binding moiety binds to an epitope bound by one of the antibodies set forth in Table 3. In some embodiments, the anti-BTC binding moiety binds to a specific conformational state of BTC to prevent BTC from interacting with an ErbB receptor.

[0385] The anti-BTC binding portion of the multispecific binding molecule of the present disclosure comprises heavy chain variable region complementarity determining region 1 (HCDR1), heavy chain variable region complementarity determining region 2 (HCDR2), heavy chain variable region complementarity determining region 3 (HCDR3), light chain variable region complementarity determining region 1 (LCDR1), light chain variable region complementarity determining region 2 (LCDR2), and 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 embodiment, the anti-BTC antibody or antigen-binding fragment thereof comprises heavy and light chain CDRs (e.g., Kabat, Chothia, IMGT, and / or combined CDRs) as set forth in Table 3 and described below.

[0386] In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 4, 2, 3, 14, 15, and 16, respectively, according to the Kabat numbering scheme. In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 5, 6, 3, 17, 18, and 19, respectively, according to the Chothia numbering scheme. In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 1, 2, 3, 14, 15, and 16, respectively, according to the combined numbering scheme. In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 7, 8, 9, 20, 18, and 16, respectively, according to the IMGT numbering scheme. In one embodiment, the anti-BTC binding moiety is comprised in NVS11, as provided in Table 3.

[0387] In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 28, 26, 27, 38, 39, and 40, respectively, according to the Kabat numbering scheme. In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 29, 30, 27, 41, 42, and 43, respectively, according to the Chothia numbering scheme. In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 25, 26, 27, 38, 39, and 40, respectively, according to the combined numbering scheme. In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 31, 32, 33, 44, 42, and 40, respectively, according to the IMGT numbering scheme. In one embodiment, the anti-BTC binding moiety is comprised in NVS12, as provided in Table 3.

[0388] In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 28, 49, 50, 58, 59, and 60, respectively, according to the Kabat numbering scheme. In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 29, 51, 50, 61, 62, and 63, respectively, according to the Chothia numbering scheme. In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 25, 49, 50, 58, 59, and 60, respectively, according to the combined numbering scheme. In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 31, 52, 53, 64, 62, and 60, respectively, according to the IMGT numbering scheme. In one embodiment, the anti-BTC binding moiety is comprised in NVS13, as provided in Table 3.

[0389] In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 72, 70, 71, 82, 83, and 84, respectively, according to the Kabat numbering scheme. In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 73, 74, 71, 85, 18, and 86, respectively, according to the Chothia numbering scheme. In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 69, 70, 71, 82, 83, and 84, respectively, according to the combined numbering scheme. In one embodiment, the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 75, 76, 77, 87, 18, and 84, respectively, according to the IMGT numbering scheme. In one embodiment, the anti-BTC binding moiety is comprised in NVS14, as provided in Table 3.

[0390] Additionally, the present disclosure also provides anti-BTC binding moieties comprising amino acid sequences homologous to the CDR sequences set forth throughout and in Table 3, wherein the anti-BTC binding moieties bind to BTC and retain the desired functional properties of those described herein. More specifically, the amino acid sequence of the anti-BTC binding moiety can have 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to the CDR sequences as set forth throughout and in Table 3 and retain the desired functional properties thereof.

[0391] The present disclosure also provides anti-BTC binding moieties that are homologous to the VH and VL sequences described herein. More specifically, the present disclosure provides proteins comprising amino acid sequences homologous to sequences such as those set forth in Table 3, where the anti-BTC binding moieties bind to an ophthalmic target and retain the desired functional properties of those described in Table 3 and in the Examples. Anti-BTC binding moieties having VH and VL regions with less than 100% sequence identity to those set forth in Table 3 can be obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of nucleic acid molecules set forth in Table 3, followed by testing the encoded, altered antibodies for retained function using the functional assays described herein and in U.S. Patent Application Publication No. 20120014958. Anti-BTC binding moieties having heavy and light chains with high (i.e., 80% or greater) identity to the heavy and light chains set forth in Table 3 can be obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of nucleic acid molecules encoding such polypeptides, followed by testing the encoded, altered antibodies for retained function using the functional assays described herein.

[0392] Anti-BTC binding moieties of the present disclosure have a binding affinity of 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%. , 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. It is contemplated that there may be variability within the CDR or framework regions. In one embodiment, the anti-BTC binding moiety comprises a VH and a VL comprising amino acid sequences as set forth in SEQ ID NOs: 10 and 21, respectively. In another embodiment, the anti-BTC binding moiety is NVS11 as provided in Table 3. In another embodiment, the VH and VL are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 116 and 122, respectively. In one embodiment, the anti-BTC binding moiety comprises a VH and a VL comprising amino acid sequences having at least one, two, or three alterations (e.g., substitutions, e.g., conservative substitutions) but not more than ten alterations (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequences of SEQ ID NOs: 10 and 21, respectively. In another embodiment, the differences in amino acid sequence are not within the complementarity determining regions.

[0393] In one embodiment, an anti-BTC binding portion of the disclosure comprises a VH and a VL comprising amino acid sequences having 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 there may be variability within the CDR or framework regions. In one embodiment, the anti-BTC binding moiety comprises a VH and a VL comprising the amino acid sequences as set forth in SEQ ID NOs: 34 and 45, respectively. In another embodiment, the anti-BTC binding moiety is NVS12 as provided in Table 3. In another embodiment, the VH and VL are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 127 and 132, respectively. In one embodiment, the anti-BTC binding moiety comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 34 and 45, respectively, having at least one, two, or three alterations (e.g., substitutions, e.g., conservative substitutions) but not more than ten alterations (e.g., substitutions, e.g., conservative substitutions). In another embodiment, the differences in amino acid sequence are not within the complementarity determining regions.

[0394] In one embodiment, an anti-BTC binding portion of the disclosure comprises a VH and a VL comprising amino acid sequences having 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 there may be variability within the CDR or framework regions. In one embodiment, the anti-BTC binding moiety comprises a VH and a VL comprising the amino acid sequences as set forth in SEQ ID NOs: 54 and 65, respectively. In another embodiment, the anti-BTC binding moiety is NVS13 as provided in Table 3. In another embodiment, the VH and VL are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 137 and 142, respectively. In one embodiment, the anti-BTC binding moiety comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 54 and 65, respectively, having at least one, two, or three alterations (e.g., substitutions, e.g., conservative substitutions) but not more than 10 alterations (e.g., substitutions, e.g., conservative substitutions). In another embodiment, the differences in amino acid sequence are not within the complementarity determining regions.

[0395] In one embodiment, an anti-BTC binding portion of the disclosure comprises a VH and a VL comprising amino acid sequences having 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 there may be variability within the CDR or framework regions. In one embodiment, the anti-BTC binding moiety comprises a VH and a VL comprising the amino acid sequences as set forth in SEQ ID NOs: 78 and 88, respectively. In another embodiment, the anti-BTC binding moiety is NVS14 as provided in Table 3. In another embodiment, the VH and VL are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 147 and 151, respectively. In one embodiment, the anti-BTC binding moiety comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 78 and 88, respectively, having at least one, two, or three alterations (e.g., substitutions, e.g., conservative substitutions) but not more than ten alterations (e.g., substitutions, e.g., conservative substitutions). In another embodiment, the differences in amino acid sequence are not within the complementarity determining regions.

[0396] In one embodiment, an anti-BTC binding moiety of the present disclosure 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) a VH and VL comprising the amino acid sequences as set forth in SEQ ID NOs: 10 and 21, respectively. In another embodiment, the anti-BTC binding moiety is comprised in NVS11 as provided in Table 3.

[0397] In one embodiment, an anti-BTC binding moiety of the present disclosure 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) a VH and VL comprising the amino acid sequences as set forth in SEQ ID NOs: 34 and 45, respectively. In another embodiment, the anti-BTC binding moiety is comprised in NVS12 as provided in Table 3.

[0398] In one embodiment, an anti-BTC binding moiety of the present disclosure 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) a VH and VL comprising the amino acid sequences as set forth in SEQ ID NOs: 54 and 65, respectively. In another embodiment, the anti-BTC binding moiety is comprised in NVS13 as provided in Table 3.

[0399] In one embodiment, an anti-BTC binding moiety of the present disclosure 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) a VH and VL comprising the amino acid sequences as set forth in SEQ ID NOs: 78 and 88, respectively. In another embodiment, the anti-BTC binding moiety is comprised in NVS14 as provided in Table 3.

[0400] b) Anti-VEGF binding part The present disclosure also provides anti-VEGF binding moieties contained in multispecific binding molecules. In certain embodiments, the anti-VEGF binding moiety comprises heavy chain variable region complementarity determining region 1 (HCDR1), heavy chain variable region complementarity determining region 2 (HCDR2), heavy chain variable region complementarity determining region 3 (HCDR3), light chain variable region complementarity determining region 1 (LCDR1), light chain variable region complementarity determining region 2 (LCDR2), and 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 embodiment, the anti-VEGF binding moiety comprises heavy and light chain CDRs (e.g., Kabat, Chothia, IMGT, and / or combined CDRs) as set forth in Table 2 or 3 and described below. In one aspect, the anti-VEGF binding moiety is an anti-VEGF antibody or antigen-binding fragment thereof. In one aspect, the anti-VEGF binding moiety is an anti-VEGF antibody or antigen-binding fragment thereof that inhibits (e.g., partially inhibits) VEGF activity.

[0401] In one embodiment, the anti-VEGF binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 95, 93, 94, 105, 106, and 107, respectively, according to the Kabat numbering scheme. In one embodiment, the anti-VEGF binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 96, 97, 94, 108, 109, and 110, respectively, according to the Chothia numbering scheme. In one embodiment, the anti-VEGF binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 92, 93, 94, 105, 106, and 107, respectively, according to the combined numbering scheme. In one embodiment, the anti-VEGF binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 98, 99, 100, 111, 109, and 107, respectively, according to the IMGT numbering scheme. In one embodiment, the anti-VEGF binding moiety is NVS8 as provided in Table 2, or included in any one of NVS11-NVS14 as provided in Table 3. In another embodiment, the anti-VEGF binding moiety is brolucizumab (Beovu®).

[0402] In addition, the present disclosure also provides anti-VEGF binding moieties comprising amino acid sequences homologous to the CDR sequences described throughout and set forth in Tables 2 or 3, wherein the anti-VEGF binding moieties bind to VEGF and retain the desired functional properties of those described herein. More specifically, the amino acid sequence of the anti-VEGF binding moiety can have 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the CDR sequences as described throughout and set forth in Tables 2 or 3 and can retain the desired functional properties thereof.

[0403] The present disclosure also provides anti-VEGF binding moieties that are homologous to the VH and VL sequences described herein. More specifically, the present disclosure provides proteins comprising amino acid sequences homologous to sequences such as those set forth in Table 2 or 3, where the anti-VEGF binding moieties bind to an ophthalmic target and retain the desired functional properties of those described in Table 2 or 3 and in the Examples. Anti-VEGF binding moieties having VH and VL regions with less than 100% sequence identity to the VH and VL regions of those set forth in Table 2 or 3 can be obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of nucleic acid molecules set forth in Table 2 or 3, followed by testing the encoded, altered antibodies for retained function using the functional assays described herein and in U.S. Patent Application Publication No. 20120014958. Anti-VEGF binding moieties having heavy and light chains with high (i.e., 80% or greater) identity to the heavy and light chains set forth in Tables 2 or 3 can be obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of nucleic acid molecules encoding such polypeptides, followed by testing the encoded, altered antibodies for retained function using the functional assays described herein.

[0404] Anti-VEGF binding moieties of the present disclosure may have a binding affinity of 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%. The anti-VEGF binding moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL) comprising amino acid sequences that have 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. It is contemplated that there may be variability within the CDR or framework regions. In one embodiment, the anti-VEGF binding moiety comprises a VH and a VL comprising amino acid sequences as set forth in SEQ ID NOs: 101 and 112, respectively. In another embodiment, the anti-VEGF binding moiety is NVS8 as provided in Table 2 or is included in any one of NVS11-NVS14 as provided in Table 3. In one embodiment, the anti-VEGF binding moiety comprises a VH and a VL comprising amino acid sequences having at least one, two, or three alterations (e.g., substitutions, e.g., conservative substitutions) but not more than ten alterations (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequences of SEQ ID NOs: 101 and 112, respectively. In another embodiment, the differences in amino acid sequence are not within the complementarity determining regions.

[0405] In one embodiment, the VH and VL of the anti-VEGF binding moiety are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 102 and 113, respectively. In one embodiment, the anti-VEGF binding moiety is NVS8 as provided in Table 2.

[0406] In one embodiment, the VH and VL of the anti-VEGF binding moiety are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 117 and 123, respectively. In one embodiment, the anti-VEGF binding moiety is comprised in NVS11, as provided in Table 3.

[0407] In one embodiment, the VH and VL of the anti-VEGF binding moiety are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 128 and 133, respectively. In one embodiment, the anti-VEGF binding moiety is comprised in NVS12, as provided in Table 3.

[0408] In one embodiment, the VH and VL of the anti-VEGF binding moiety are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 138 and 143, respectively. In one embodiment, the anti-VEGF binding moiety is comprised in NVS13, as provided in Table 3.

[0409] In one embodiment, the VH and VL of the anti-VEGF binding moiety are encoded by nucleic acid sequences as set forth in SEQ ID NOs: 148 and 152, respectively. In one embodiment, the anti-VEGF binding moiety is comprised in NVS14, as provided in Table 3.

[0410] In one aspect, an anti-VEGF binding moiety of the present disclosure 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) a VH and VL comprising the amino acid sequences as set forth in SEQ ID NOs: 101 and 112, respectively. In another aspect, the anti-VEGF binding moiety is NVS8 as provided in Table 2 or is included in any one of NVS11-NVS14 as provided in Table 3. In one embodiment, the anti-VEGF binding moiety inhibits (eg, partially inhibits) VEGF activity.

[0411] In one aspect, an anti-VEGF binding moiety of the disclosure (e.g., an anti-VEGF binding moiety that inhibits VEGF activity) has a VEGF activity that is 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 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%. The anti-VEGF binding moiety comprises a heavy chain and a light chain comprising amino acid sequences having 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 the heavy chain. In one embodiment, the anti-VEGF binding moiety comprises a heavy chain and a light chain comprising amino acid sequences as set forth in SEQ ID NOs: 103 and 114, respectively. In another embodiment, the anti-VEGF binding moiety is NVS8 as provided in Table 2.In another embodiment, the heavy and light chains have a sequence similar to or different from 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% of SEQ ID NOs: 104 and 115, respectively. , 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% identity or complementarity to the anti-VEGF binding moiety. In one embodiment, the anti-VEGF binding moiety comprises a heavy chain and a light chain encoded by a nucleic acid sequence as set forth in SEQ ID NOs: 104 and 115, respectively, and is NVS8 as provided in Table 2.

[0412] c) Bispecific binding molecules In certain embodiments, the present disclosure provides a multispecific binding molecule comprising an anti-BTC binding moiety and an anti-VEGF binding moiety. The present disclosure provides a multispecific binding molecule comprising an anti-BTC binding moiety and an anti-VEGF binding moiety, wherein 1) the anti-BTC binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an anti-BTC antibody (e.g., NVS1, NVS2, NVS3, or NVS4) as described in Table 1, and 2) the anti-VEGF binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an anti-VEGF antibody as described in Table 2 or 3. In one embodiment, the multispecific binding molecule is NVS11 as provided in Table 3.

[0413] In the present disclosure, multispecific binding molecules are provided that include an anti-BTC binding portion and an anti-VEGF binding portion, wherein 1) the anti-BTC binding portion comprises 1) HCDR1, HCDR2, and HCDR3 that comprise 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) the anti-VEGF binding moiety 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 embodiment, the multispecific binding molecule is NVS11 as provided in Table 3. In one embodiment, such a multispecific binding molecule 1) selectively binds to human BTC protein and inhibits (e.g., partially inhibits) BTC activity, and 2) selectively binds to human VEGF protein and inhibits (e.g., partially inhibits) VEGF activity.

[0414] Also provided in the present disclosure are multispecific binding molecules comprising an anti-BTC binding portion and an anti-VEGF binding portion, wherein 1) the anti-BTC binding portion comprises 1) an HCDR1 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). , HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and 2) the anti-VEGF binding moiety 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 embodiment, the multispecific binding molecule is NVS12 as provided in Table 3.

[0415] Also provided in the present disclosure are multispecific binding molecules comprising an anti-BTC binding portion and an anti-VEGF binding portion, wherein 1) the anti-BTC binding portion comprises 1) an HCDR1 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). , HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and 2) the anti-VEGF binding moiety 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 embodiment, the multispecific binding molecule is NVS13 as provided in Table 3.

[0416] Also provided in the present disclosure are multispecific binding molecules comprising an anti-BTC binding portion and an anti-VEGF binding portion, wherein 1) the anti-BTC binding portion comprises 1) an HCDR1 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). , HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and 2) the anti-VEGF binding moiety 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 embodiment, the multispecific binding molecule is NVS14 as provided in Table 3.

[0417] In one aspect, a multispecific binding molecule of the disclosure comprises: 1) a VHA and a VLA that bind to BTC, wherein the VHA and VLA have a binding affinity to SEQ ID NOs: 10 and 21, respectively, of 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 and a VHA comprising an amino acid sequence having 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 a VHA. and VLA, and 2) VHB and VLB that bind VEGF, wherein the VHB and VLB have a binding affinity of 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 200%, at least 210%, at least 211%, at least 212%, at least 213%, at least 214%, at least 215%, at least 216%, at least 217%, at least 218%, at least 219%, at least 220%, at least 221%, at least 222%, at least 223%, at least 224%, at least 225%, at least 226%, at least 227%, at least 228%, at least 229%, at least 230%, at least 231%, at least 232%, at and VHBs and VLBs comprising amino acid sequences having 8%, 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.In another embodiment, a multispecific binding molecule of the disclosure comprises 1) a VHA and a VLA that bind to BTC comprising the amino acid sequences of SEQ ID NOs: 10 and 21, respectively, and 2) a VHB and a VLB that bind to VEGF comprising the amino acid sequences of SEQ ID NOs: 101 and 112, respectively. In another embodiment, the VHA and VLA are encoded by nucleic acid sequences of 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 to SEQ ID NOs: 116 and 122, respectively. In another embodiment, the VHB and VLB are encoded by a nucleic acid sequence having 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 to SEQ ID NOs: 117 and 123, respectively. In one embodiment, the multispecific binding molecule is NVS11 as provided in Table 3. In one embodiment, such a multispecific binding molecule 1) selectively binds to human BTC protein and inhibits (e.g., partially inhibits) BTC activity, and 2) selectively binds to human VEGF protein and inhibits (e.g., partially inhibits) VEGF activity.

[0418] In one aspect, a multispecific binding molecule of the disclosure comprises: 1) a VHA and a VLA that bind to BTC, wherein the VHA and VLA have a binding affinity to SEQ ID NOs: 34 and 45, respectively, of 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 and a VHA comprising an amino acid sequence having 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 a VHA. and VLA, and 2) VHB and VLB that bind VEGF, wherein the VHB and VLB have a binding affinity of 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 200%, at least 210%, at least 211%, at least 212%, at least 213%, at least 214%, at least 215%, at least 216%, at least 217%, at least 218%, at least 219%, at least 220%, at least 221%, at least 222%, at least 223%, at least 224%, at least 225%, at least 226%, at least 227%, at least 228%, at least 229%, at least 230%, at least 231%, at least 232%, at and VHBs and VLBs comprising amino acid sequences having 8%, 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.In another embodiment, a multispecific binding molecule of the disclosure comprises 1) a VHA and a VLA that bind to BTC comprising the amino acid sequences of SEQ ID NOs: 34 and 45, respectively, and 2) a VHB and a VLB that bind to VEGF comprising the amino acid sequences of SEQ ID NOs: 101 and 112, respectively. In another embodiment, the VHA and VLA are encoded by nucleic acid sequences of 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 to SEQ ID NOs: 127 and 132, respectively. In another embodiment, the VHB and VLB are encoded by a nucleic acid sequence having 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 to SEQ ID NOs: 128 and 133, respectively. In one embodiment, the multispecific binding molecule is NVS12 as provided in Table 3. In one embodiment, such a multispecific binding molecule 1) selectively binds to human BTC protein and inhibits (e.g., partially inhibits) BTC activity, and 2) selectively binds to human VEGF protein and inhibits (e.g., partially inhibits) VEGF activity.

[0419] In one aspect, a multispecific binding molecule of the disclosure comprises: 1) a VHA and a VLA that bind to BTC, wherein the VHA and VLA have a binding affinity to SEQ ID NOs: 54 and 65, respectively, of 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 and a VHA comprising an amino acid sequence having 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 a VHA. and VLA, and 2) VHB and VLB that bind VEGF, wherein the VHB and VLB have a binding affinity of 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 200%, at least 210%, at least 211%, at least 212%, at least 213%, at least 214%, at least 215%, at least 216%, at least 217%, at least 218%, at least 219%, at least 220%, at least 221%, at least 222%, at least 223%, at least 224%, at least 225%, at least 226%, at least 227%, at least 228%, at least 229%, at least 230%, at least 231%, at least 232%, at and VHBs and VLBs comprising amino acid sequences having 8%, 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.In another embodiment, a multispecific binding molecule of the disclosure comprises 1) a VHA and a VLA that bind to BTC comprising the amino acid sequences of SEQ ID NOs: 54 and 65, respectively, and 2) a VHB and a VLB that bind to VEGF comprising the amino acid sequences of SEQ ID NOs: 101 and 112, respectively. In another embodiment, the VHA and VLA are encoded by nucleic acid sequences of 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 to SEQ ID NOs: 137 and 142, respectively. In another embodiment, the VHB and VLB are encoded by a nucleic acid sequence having 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 to SEQ ID NOs: 138 and 143, respectively. In one embodiment, the multispecific binding molecule is NVS13 as provided in Table 3. In one embodiment, such a multispecific binding molecule 1) selectively binds to human BTC protein and inhibits (e.g., partially inhibits) BTC activity, and 2) selectively binds to human VEGF protein and inhibits (e.g., partially inhibits) VEGF activity.

[0420] In one aspect, a multispecific binding molecule of the disclosure comprises: 1) a VHA and a VLA that bind to BTC, wherein the VHA and VLA have a binding affinity to SEQ ID NOs: 78 and 88, respectively, of 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 and a VHA comprising an amino acid sequence having 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 a VHA. and VLA, and 2) VHB and VLB that bind VEGF, wherein the VHB and VLB have a binding affinity of 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 200%, at least 210%, at least 211%, at least 212%, at least 213%, at least 214%, at least 215%, at least 216%, at least 217%, at least 218%, at least 219%, at least 220%, at least 221%, at least 222%, at least 223%, at least 224%, at least 225%, at least 226%, at least 227%, at least 228%, at least 229%, at least 230%, at least 231%, at least 232%, at and VHBs and VLBs comprising amino acid sequences having 8%, 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.In another embodiment, a multispecific binding molecule of the disclosure comprises 1) a VHA and a VLA that bind to BTC comprising the amino acid sequences of SEQ ID NOs: 78 and 88, respectively, and 2) a VHB and a VLB that bind to VEGF comprising the amino acid sequences of SEQ ID NOs: 101 and 112, respectively. In another embodiment, the VHA and VLA are encoded by nucleic acid sequences of 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 to SEQ ID NOs: 147 and 151, respectively. In another embodiment, the VHB and VLB are encoded by a nucleic acid sequence having 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 to SEQ ID NOs: 148 and 152, respectively. In one embodiment, the multispecific binding molecule is NVS14 as provided in Table 3. In one embodiment, such a multispecific binding molecule 1) selectively binds to human BTC protein and inhibits (e.g., partially inhibits) BTC activity, and 2) selectively binds to human VEGF protein and inhibits (e.g., partially inhibits) VEGF activity.

[0421] Multispecific binding molecules of the disclosure include: 1) a heavy chain comprising a VHA, a CH1A, a linker, a VHB, and a CH1B, wherein the heavy chain has a sequence similar to or different from 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 and amino acid sequences having 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. and 2) a light chain comprising a VLA, a CKA, a linker, a VLB, and a CKB, wherein the light chain has a sequence similar to or different from SEQ ID NO: 125 by 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 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125. and a light chain comprising an amino acid sequence having 8%, 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 the antibody.In one embodiment, the heavy and light chains are encoded by nucleic acid sequences of 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 to SEQ ID NOs: 121 and 126, respectively. In one embodiment, 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 NVS11 provided in Table 3. In one embodiment, such a multispecific binding molecule 1) selectively binds to human BTC protein and inhibits (e.g., partially inhibits) BTC activity, and 2) selectively binds to human VEGF protein and inhibits (e.g., partially inhibits) VEGF activity.

[0422] Multispecific binding molecules of the disclosure include: 1) a heavy chain comprising a VHA, a CH1A, a linker, a VHB, and a CH1B, wherein the heavy chain has a sequence identity of 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 and amino acid sequences having 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. and 2) a light chain comprising a VLA, a CKA, a linker, a VLB, and a CKB, wherein the light chain has a sequence similar to or different from SEQ ID NO: 135 by 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 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at and a light chain comprising an amino acid sequence having 8%, 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 the antibody.In one embodiment, the heavy and light chains are encoded by nucleic acid sequences of 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 to SEQ ID NOs: 131 and 136, respectively. In one embodiment, 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 NVS12 provided in Table 3. In one embodiment, such a multispecific binding molecule 1) selectively binds to human BTC protein and inhibits (e.g., partially inhibits) BTC activity, and 2) selectively binds to human VEGF protein and inhibits (e.g., partially inhibits) VEGF activity.

[0423] Multispecific binding molecules of the disclosure include: 1) a heavy chain comprising a VHA, a CH1A, a linker, a VHB, and a CH1B, wherein the heavy chain has a sequence similar to or different from 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 and amino acid sequences having 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. and 2) a light chain comprising a VLA, a CKA, a linker, a VLB, and a CKB, wherein the light chain has a sequence similar to or different from SEQ ID NO: 145 by 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 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at and a light chain comprising an amino acid sequence having 8%, 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 the antibody.In one embodiment, the heavy and light chains are encoded by nucleic acid sequences of 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 to SEQ ID NOs: 141 and 146, respectively. In one embodiment, 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 NVS13 provided in Table 3. In one embodiment, such a multispecific binding molecule 1) selectively binds to human BTC protein and inhibits (e.g., partially inhibits) BTC activity, and 2) selectively binds to human VEGF protein and inhibits (e.g., partially inhibits) VEGF activity.

[0424] Multispecific binding molecules of the disclosure include: 1) a heavy chain comprising a VHA, a CH1A, a linker, a VHB, and a CH1B, wherein the heavy chain has a sequence identity of 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 and amino acid sequences having 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. and 2) a light chain comprising a VLA, a CKA, a linker, a VLB, and a CKB, wherein the light chain has a sequence similar to or different from SEQ ID NO: 154 by 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 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at and a light chain comprising an amino acid sequence having 8%, 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 the antibody.In one embodiment, the heavy and light chains are encoded by nucleic acid sequences of 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 to SEQ ID NOs: 150 and 155, respectively. In one embodiment, 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 NVS14 provided in Table 3. In one embodiment, such a multispecific binding molecule 1) selectively binds to human BTC protein and inhibits (e.g., partially inhibits) BTC activity, and 2) selectively binds to human VEGF protein and inhibits (e.g., partially inhibits) VEGF activity.

[0425] Multispecific binding molecules of the disclosure (e.g., NVS11-NVS14) include: a. capable of binding to BTC and VEGF simultaneously; b. capable of inhibiting the binding of soluble BTC to ErbB1 or Erb4 and the subsequent phosphorylation of the Erb receptor; c. It can inhibit soluble BTC-induced phosphorylation of ERK1 / 2; d. Can bind to membrane-bound BTC and inhibit juxtacrine activation of membrane-bound BTC-induced phosphorylation of ErbB1; e. inhibiting the binding of soluble VEGF-A165 to soluble VEGFR2; f. It can inhibit BTC-induced human iPSC-induced RPE permeability in an in vitro outer BRB model; g. It can inhibit VEGF-induced human retinal endothelial cell (HREC) permeability in an in vitro inner BRB model; h. capable of inhibiting BTC-induced retinal thickening; and / or i. It can inhibit VEGF-induced retinal vascular leakage.

[0426] [Table 10]

[0427] [Table 11]

[0428] [Table 12]

[0429] [Table 13]

[0430] [Table 14]

[0431] [Table 15]

[0432] [Table 16]

[0433] [Table 17]

[0434] [Table 18]

[0435] [Table 19]

[0436] Table 20

[0437] Table 21

[0438] Table 22

[0439] Table 23

[0440] Table 24

[0441] Table 25

[0442] Table 26

[0443] Table 27

[0444] Table 28

[0445] Table 29

[0446] v. Linker In certain aspects of the present disclosure, the anti-BTC binding moiety can be linked to a molecule, e.g., an anti-VEGF binding moiety, by a linker. More specifically, the anti-BTC binding moiety can be linked to a peptide linker (e.g., (Gly)) having an optimized length and / or amino acid composition. n -Ser n ) n or (Ser n -Gly n ) n The proteins or nucleic acids may be linked by peptide linkers (peptide linkers). It is known that the length of the peptide linker can greatly affect 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, U.S. 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.

[0447] The peptide linker sequence can 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 can be composed of naturally occurring or non-naturally occurring amino acids. In some embodiments, the linker is a glycine polymer. In some embodiments, the amino acids glycine and serine comprise the amino acids within the linker sequence. In certain embodiments, the linker region is a set of glycine repeats (GlySerGly3). nwherein n is a positive integer equal to or greater than 1, e.g., 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 linker region orientation comprises a set of glycine repeats (SerGly3) n wherein n is a positive integer greater than or equal to 1, for example, n=3 (SEQ ID NO: 167).

[0448] Peptide linkers can also include, but are not limited to, (Gly4Ser)4 (SEQ ID NO: 161) or (Gly4Ser)3 (SEQ ID NO: 162). The amino acid residues Glu and Lys can be interspersed within the Gly-Ser peptide linker for better solubility. In certain embodiments, the peptide linker can comprise multiple repeats of (Gly3Ser), (Gly2Ser), or (GlySer). In certain embodiments, the peptide linker can comprise multiple repeats of (SerGly3), (SerGly2), or (SerGly). In other embodiments, the peptide linker can comprise a multiple set of (Gly3Ser) + (Gly4Ser) + (GlySer) (SEQ ID NO: 163). In still other embodiments, Ser can be replaced with Ala, for example, (Gly4Ala) or (Gly3Ala). In yet another embodiment, the linker comprises the motif (GluAlaAlaAlaLys) n (SEQ ID NO: 164), where n is an integer greater than or equal to 1. In certain embodiments, the peptide linker may also include a cleavable linker.

[0449] Peptide linkers can be of various lengths. In particular, peptide linkers are about 5 to about 50 amino acids in length, about 10 to about 40 amino acids in length, about 15 to about 30 amino acids in length, or about 15 to about 20 amino acids in length. Varying the length of the peptide linker can maintain or enhance activity, resulting in superior efficacy in activity tests. Peptide linkers can be introduced into polypeptide and protein sequences using techniques known in the art. For example, PCR mutagenesis can be used. Modifications can be confirmed by DNA sequence analysis. Plasmid DNA can be used to transform host cells for stable production of the resulting polypeptide.

[0450] The peptide linker, anti-BTC binding moiety, and protein (e.g., anti-VEGF binding moiety) can be encoded in the same vector and expressed and assembled in the same host cell. Alternatively, each peptide linker, anti-BTC binding moiety, anti-VEGF binding moiety, and protein or nucleic acid can be produced separately and then conjugated to each other. The peptide linker, anti-BTC binding moiety, and protein or nucleic acid can be prepared by conjugating the components using methods known in the art. Site-specific conjugation can be achieved using sortase-mediated enzymatic conjugation (Mao H, et al., J. Am. Chem. Soc. 2004 Mar. 10;126(9):2670-1). Various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686; Liu, MA et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus, 1985 Behring Ins. Mitt. No. 78, 118-132; Brennan et al., 1985 Science 229:81-83; and Glennie et al., 1987 J. Immunol. 139:2367-2375. Conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0451] vi. Forms and types of multi-property binding molecules In some embodiments, the multispecific binding molecule is a bispecific antibody or bispecific antibody-like molecule. In some embodiments, the bispecific antibody or antibody-like molecule can be multivalent, e.g., bivalent for one antigen and monovalent for the other antigen. An exemplary bispecific antibody molecule or bispecific antibody-like molecule is characterized by a first antigen-binding domain (e.g., comprising a first heavy chain and a first light chain) that has binding specificity for a first antigen or epitope (e.g., BTC) and a second antigen-binding domain (e.g., comprising a second heavy chain and a second light chain) that has binding specificity for a second antigen or epitope (e.g., VEGF).

[0452] In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In certain embodiments, a bispecific antibody molecule or bispecific antibody-like molecule comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope or antigen, and heavy and light chain variable domain sequences that have binding specificity for a second epitope or antigen.

[0453] In some embodiments, a bispecific antibody molecule or antibody-like molecule comprises a half antibody that has binding specificity for a first epitope or antigen and a half antibody that has binding specificity for a second epitope or antigen. In certain embodiments, a bispecific antibody molecule or antibody-like molecule comprises a half antibody or fragment thereof that has binding specificity for a first epitope or antigen and a half antibody or fragment thereof that has binding specificity for a second epitope or antigen.

[0454] In certain aspects, a bispecific antibody molecule or bispecific antibody-like molecule comprises an scFv or Fab or fragment thereof that has binding specificity for a first epitope or antigen and an antibody or fragment thereof that has binding specificity for a second epitope or antigen. In certain aspects, a bispecific antibody molecule or bispecific antibody-like molecule comprises two scFvs or Fabs or fragments thereof that have binding specificity for a first epitope or antigen and an antibody or fragment thereof that has binding specificity for a second epitope or antigen. In certain aspects, a bispecific antibody molecule or bispecific antibody-like molecule comprises an scFv or fragment thereof that has binding specificity for a first epitope or antigen and a Fab or fragment thereof that has binding specificity for a second epitope or antigen.

[0455] In certain embodiments, the antibody or antibody-like molecule is a multispecific (e.g., bispecific or trispecific) antibody or antibody-like molecule. Protocols for making bispecific or heterodimeric antibodies or antibody-like molecules are known in the art, such as the "knobs-in-holes" method described in U.S. Pat. No. 5,731,168; electrostatic steering Fc pairing, as described in WO 09 / 089004, WO 06 / 106905, and WO 2010 / 129304; strand exchange engineering domains (SEs), as described in WO 07 / 110205, and WO 2010 / 129304. ED) heterodimer formation; e.g., Fab arm exchange as described in WO 08 / 119353, WO 2011 / 131746 and WO 2013 / 060867; dual antibody conjugates by antibody cross-linking using, for example, heterobifunctional reagents with amine-reactive and sulfhydryl-reactive groups to create bispecific structures, as described, for example, in U.S. Pat. No. 4,433,059; bispecific antibodies or antibody-like molecule determinants made by combining half antibodies (heavy-light chain pairs or Fab) from different antibodies or antibody-like molecules by cycles of reduction and oxidation of the disulfide bond between the two heavy chains, as described in, for example, U.S. Pat. No. 5,273,743; trispecific antibodies, e.g., three Fab' fragments cross-linked via sulfhydryl-reactive groups, as described in, for example, U.S. Pat. No. 5,534,254; biosynthetic binding proteins, e.g., dimeric antibodies ... Pairs of scFvs cross-linked via sulfide or amine reactive chemical cross-linking; bifunctional antibodies, e.g., Fab fragments with different binding specificities dimerized via leucine zippers (e.g., c-fos and c-jun) replacing the constant domains, as described, for example, in U.S. Pat. No. 5,582,996; bispecific and oligospecific monovalent and oligovalent receptors, e.g., combining the CH1 region of one antibody with the V domain of the other antibody, normally associated with a light chain, as described, for example, in U.S. Pat. No. 5,591,828. HV of two antibodies (two Fab fragments) linked via a polypeptide spacer between the V domains H -CH1 region (Fd region); bispecific DNA antibody conjugates, e.g., as described in U.S. Pat. No. 5,635,602, for example, cross-linking of antibodies or Fab fragments via double-stranded DNA fragments; bispecific fusion proteins, e.g., as described in U.S. Pat. No. 5,637,481, for example, an expression construct containing two scFvs and a complete constant region with a hydrophilic helical peptide linker between them; multivalent and multispecific binding proteins, e.g., as described in U.S. Pat. No. 5,837,242, for example, a dimer of a polypeptide having a first domain with a binding region of an Ig heavy chain variable region and a second domain with a binding region of an Ig light chain variable region, commonly referred to as a diabody (including higher order structures that generate bispecific, trispecific or tetraspecific molecules); linked V domains further linked to antibody hinge and CH3 regions by a peptide spacer, which can be dimerized to form bispecific / multivalent molecules, e.g., as described in U.S. Pat. No. 5,837,821. L and V H V, V, VIII, VIV, VIVIII ... H and V L domains; for example, trimers and tetramers as described in U.S. Pat. No. 5,844,094; V domains forming a series of FVs (or scFVs), for example, as described in U.S. Pat. No. 5,864,019; L V linked by peptide bonds with a crosslinkable group further associated with the domain at the C-terminus H domain (or V in family members) L a V domain) to which one of the antigens is monovalently bound and one of the antigens is bivalently bound, optionally including a heterodimeric Fc region, for example as described in WO 2011 / 028952; L and V Hdomains, scFv, or Fab; and V linked via peptide linkers that are conjugated into multivalent structures via non-covalent or chemical crosslinking to form homobivalent, heterobivalent, trivalent, and tetravalent structures, using both scFv or diabody-type formats, e.g., as described in U.S. Pat. No. 5,869,620. L and V H Examples of suitable binding polypeptides include, but are not limited to, single-chain binding polypeptides having both the nucleotide and nucleotide domains.

[0456] Further exemplary multispecific and bispecific molecules and methods of making the same are described in, e.g., U.S. Pat. Nos. 5,910,573, 5,932,448, 5,959,083, 5,989,830, 6,005,079, 6,239,259, 6,294,353, 6,333,396, 6,476,198, 6,511,663, 6,670,453, 6,743,896, 6,809,101, 6,910,573, 6,932,448, 6,959,083, 6,989,830, 6,005,079, 6,239,259, 6,294,353, 6,333,396, 6,476,198, 6,511,663, 6,670,453, 6,743,896, 6,809,101, 6,910,573, 6,920,574, 6,930,575, 6,940,576, 6,950,577, 6,960,578, 6,970,579, 6,980,579, 6,9 ... 85, U.S. Patent No. 6,833,441, U.S. Patent No. 7,129,330, U.S. Patent No. 7,183,076, U.S. Patent No. 7,521,056, U.S. Patent No. 7,527,787, U.S. Patent No. 7,534,866, U.S. Patent No. 7,612,181, U.S. Patent Application Publication No. 2002004587A1, U.S. Patent Application Publication No. 2002076406A1, U.S. Patent Application Publication No. 2002103345A1, U.S. Patent Application Publication No. 2003207346A1, U.S. Patent Application Publication No. 2003211078A 1, U.S. Patent Application Publication No. 2004219643A1, U.S. Patent Application Publication No. 2004220388A1, U.S. Patent Application Publication No. 2004242847A1, U.S. Patent Application Publication No. 2005003403A1, U.S. Patent Application Publication No. 2005004352A1, U.S. Patent Application Publication No. 2005069552A1, U.S. Patent Application Publication No. 2005079170A1, U.S. Patent Application Publication No. 2005100543A1, U.S. Patent Application Publication No. 2005136049 ...100543A1, U.S. Patent Application Publication No. 2005136049A1, U.S. Patent Application Publication No. 2005100543A1, U. 005136051A1, U.S. Patent Application Publication No. 2005163782A1, U.S. Patent Application Publication No. 2005266425A1, U.S. Patent Application Publication No. 2006083747A1, U.S. Patent Application Publication No. 2006120960A1, U.S. Patent Application Publication No. 2006204493A1, U.S. Patent Application Publication No. 2006263367A1, U.S. Patent Application Publication No. 2007004909A1, U.S. Patent Application Publication No. 2007087381A1, U.S. Patent Application Publication No. 2007128150A1,US Patent Application Publication No. 2007141049A1, US Patent Application Publication No. 2007154901A1, US Patent Application Publication No. 2007274985A1, US Patent Application Publication No. 2008050370A1, US Patent Application Publication No. 2008069820A1, US Patent Application Publication No. 2008152645A1, US Patent Application Publication No. 2008171855A1, US Patent Application Publication No. 2008241884A1, US Patent Application Publication No. 2008254512A1, US Patent Application Publication No. US Patent Application Publication No. 2008260738A1, US Patent Application Publication No. 2009130106A1, US Patent Application Publication No. 2009148905A1, US Patent Application Publication No. 2009155275A1, US Patent Application Publication No. 2009162359A1, US Patent Application Publication No. 2009162360A1, US Patent Application Publication No. 2009175851A1, US Patent Application Publication No. 2009175867A1, US Patent Application Publication No. 2009232811A1, US Patent Application Publication No. 200923410 5A1, U.S. Patent Application Publication No. 2009263392A1, U.S. Patent Application Publication No. 2009274649A1, European Patent Application Publication No. 346087A2, International Publication No. 0006605A2, International Publication No. 02072635A2, International Publication No. 04081051A1, International Publication No. 06020258A2, International Publication No. 2007044887A2, International Publication No. 2007095338A2, International Publication No. 2007137760A2 FRET, WO 2008119353A1, WO 2009021754A2, WO 2009068630A1, WO 9103493A1, WO 9323537A1, WO 9409131A1, WO 9412625A2, WO 9509917A1, WO 9637621A2, WO 9964460A1. The contents of the above-referenced applications are found inThe entirety of which is incorporated herein by reference.

[0457] Thus, in some embodiments, the BTC / VEGF multispecific binding molecules of the present disclosure comprise a BTC-binding domain and a VEGF-binding domain in any one of the multispecific or bispecific formats known in the art and described throughout. Preferred formats for the multispecific binding molecules of the present disclosure are described in more detail below.

[0458] The multispecific binding molecules of the present disclosure comprise an anti-BTC binding portion and an anti-VEGF binding portion, wherein the anti-BTC binding portion comprises a variable heavy chain domain (VHA) and a variable light chain domain (VLA) that bind to BTC, and the anti-VEGF binding portion comprises a variable heavy chain domain (VHB) and a variable light chain domain (VLB) that bind to VEGF. In one embodiment, the VHA and VLA are covalently linked, e.g., via a disulfide bond. In one embodiment, the VHB and VLB are covalently linked, e.g., via a disulfide bond. In one embodiment, the multispecific binding molecule is in the format, from N- to C-terminus: N-VHA-linker1-VHB-C and N-VLA-linker2-VLB-C. In another embodiment, the multispecific binding molecule is in the format, from N- to C-terminus: N-VHB-linker1-VHA-C and N-VLB-linker2-VLA-C. Linker1 and linker2 can be the same or different.

[0459] In one embodiment, the anti-BTC binding moiety further comprises a heavy chain constant domain (CH1A) and a light chain constant domain (CKA), and the anti-VEGF binding moiety further comprises a heavy chain constant domain (CH1B) and a light chain constant domain (CKB). In one embodiment, the multispecific binding molecule is in the format, from N- to C-terminus: N-VHA-CH1A-linker-VHB-CH1B-C and N-VLA-CKA-linker-VLB-CKB-C, e.g., NVS11, NVS12, NVS13, and NVS14, as provided in Table 3. In another embodiment, the multispecific binding molecule is in the format, from N- to C-terminus: N-VHB-CH1B-linker-VHA-CH1A-C and N-VLB-CKB-linker-VLA-CKA-C. In one embodiment, the two linkers are the same. In another embodiment, the two linkers are different.

[0460] In certain embodiments, the CH1 constant region is present with the VH and the Cκ constant region is present with the VL, such that a Fab fragment is formed by dimerization of the respective light and heavy chains (i.e., VHA-CH1 will form a Fab fragment with VLA-Cκ, and VHB-CH1 will form a Fab fragment with VLB-Cκ). In certain embodiments, the multispecific binding molecules of the disclosure are in a Fab-Fab format, in which both the anti-BTC binding moiety and the anti-VEGF binding moiety form Fab fragments. In other embodiments, the CH1 constant region is present with the VH and the Cλ constant region is present with the VL, such that a Fab fragment is formed by dimerization of the respective light and heavy chains (i.e., VHA-CH1 will form a Fab fragment with VLA-Cλ, and VHB-CH1 will form a Fab fragment with VLB-Cλ).

[0461] The multispecific binding molecule of the present disclosure comprises an anti-BTC binding portion and an anti-VEGF binding portion, wherein the anti-BTC binding portion is a Fab and comprises a heavy chain (HA) and a light chain (LA), and the anti-VEGF binding portion is a Fab and comprises a heavy chain (HB) and a light chain (LB). In one embodiment, HA and HB are linked N- to C-terminally as follows: N-HA-Linker1-HB-C, and LA and LB are linked N- to C-terminally as follows: N-LA-Linker2-LB-C. In another embodiment, HA and HB are linked N- to C-terminally as follows: N-HB-Linker1-HA-C, and LA and LB are linked N- to C-terminally as follows: N-LB-Linker2-LA-C. Linker1 and Linker2 can be the same or different. In one embodiment, Linker1 and Linker2 comprise the amino acid sequence of SEQ ID NO: 118 or are encoded by the nucleic acid sequence of SEQ ID NO: 119. In another embodiment, Linker 1 and Linker 2 comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 161-167.

[0462] In one aspect, a Fab multispecific binding molecule of the disclosure has the structure depicted in Figures 1-2.

[0463] In one embodiment, a bispecific antibody of the present disclosure comprises two polypeptide chains, one chain comprising an anti-VEGF scFv and a light chain variable domain (VLB) of an anti-BTC antibody also having a light chain constant region CL (VLB-CL), which are linked to each other by a linker peptide, and the other chain comprising a heavy chain variable domain (VHB) of an anti-BTC antibody also having a heavy chain constant region CHI (VHB-CH1). In another embodiment, a bispecific antibody of the present disclosure comprises two polypeptide chains, one chain comprising an anti-VEGF scFv and a heavy chain variable domain (VHB) of an anti-BTC antibody also having a heavy chain constant region CHI (VHB-CH1), which are linked to each other by a linker peptide, and the other chain comprising a light chain variable domain (VLB) of an anti-BTC antibody also having a light chain constant region CL (VLB-CL).

[0464] In certain aspects, the orientation of the scFv on one polypeptide chain of a bispecific antibody of the present disclosure can be NH2-VLB-CL-linker2-scFv-COOH or NH2-VHB-CH1-linker2-scFv-COOH. In one aspect, the linker sequence between the VL and VH domains on the scFv has the sequence (GGGGS)4 (SEQ ID NO: 63), and the VL and VH are in the format NH2-VLA-linker2-VHA-COOH.

[0465] In another embodiment, one of the binding domains of the bispecific antibodies of the present disclosure forms a Fab and the other binding domain forms a single-chain (scFv) antibody fragment. Those skilled in the art will recognize that other orientations are possible in addition to those shown. For example, an scFv-Fab format is possible, or the binding specificity can be rearranged.

[0466] In some embodiments, the multispecific binding molecule is a bispecific antibody or bispecific antibody-like molecule. In other embodiments, the present disclosure features multispecific molecules that include a domain with specificity for BTC (i.e., an anti-BTC binding moiety) and another domain (therapeutic target binding moiety) with specificity for another therapeutic target, e.g., VEGF. For example, a multispecific molecule can include an anti-BTC binding moiety, antibody, or antigen-binding fragment thereof, and a nucleic acid moiety of the present disclosure. Anti-BTC antibodies are known in the art, e.g., as described in U.S. Pat. No. 6,183,971 and WO 2004 / 083241 A2.

[0467] An antibody of the present disclosure, or an antigen-binding fragment thereof, can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., a ligand for another antibody or receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. An antibody of the present disclosure can, in fact, be derivatized or linked to two or more other functional molecules to generate a multispecific molecule that binds to three or more different binding sites and / or target molecules (such multispecific molecules are intended to be encompassed by the term "bispecific molecule" as used herein). To create a bispecific molecule of the present disclosure, an antibody of the present disclosure can be operatively linked (e.g., by chemical bond, genetic fusion, noncovalent bond, or otherwise) to one or more other binding molecules, such as another antibody, antigen-binding fragment, peptide, or binding mimetic, thereby resulting in a bispecific molecule.

[0468] Thus, the present disclosure includes bispecific molecules comprising a first binding specificity for at least one BTC and a second target epitope, e.g., a second binding specificity for another therapeutic target. For example, the second target epitope is an epitope of VEGF.

[0469] In one embodiment, the anti-BTC binding moiety and the anti-VEGF binding moiety comprised within the multispecific binding molecule are in a format comprising, for example, Fab, Fab', F(ab')2, Fv, or single-chain Fv (scFv). In another embodiment, the anti-BTC binding moiety is an anti-BTC Fab and the anti-VEGF binding moiety is an anti-VEGF Fab. In another embodiment, the anti-BTC binding moiety is an scFv and the anti-VEGF binding moiety is an scFv. The anti-BTC binding moiety and the anti-VEGF binding moiety comprised within the multispecific binding molecule can also be a light or heavy chain dimer, or any minimal fragment thereof, such as an Fv or single-chain construct as described in U.S. Patent No. 4,946,778 to Ladner et al.

[0470] In one embodiment, the multispecific binding molecule of the present disclosure can be a diabody. Diabodies are bivalent, bispecific molecules in which the VH and VL domains are represented on a single polypeptide chain, linked by a linker that is too short to allow pairing between the two domains on the same chain. The VH and VL domains pair with complementary domains on another chain, thereby creating two antigen-binding sites (see, e.g., Holliger et al., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al., 1994 Structure 2:1121-1123). Diabodies can be produced by expressing two polypeptide chains with either the VHA-VLB and VHB-VLA (VH-VL configuration) or VLA-VHB and VLB-VHA (VL-VH configuration) structures in the same cell. Most of them can be expressed in soluble form in bacteria. Single-chain diabodies (scDbs) are produced by linking two diabody-forming polypeptide chains with a linker of approximately 15 amino acid residues (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45(3-4):128-30; Wu et al., 1996 Immunotechnology, 2(1):21-36). scDbs can be expressed in bacteria in a soluble, active monomeric form (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45(34):128-30; Wu et al., 1996 Immunotechnology, 2(1):21-36; Pluckthun and Pack, 1997 Immunotechnology, 3(2):83-105; Ridgway et al., 1996 Protein Eng., 9(7):617-21). Diabodies can be fused to Fc to generate "didiabodies" (see Lu et al., 2004 J. Biol. Chem., 279(4):2856-65). Other antibodies that can be used in the bispecific molecules of the present disclosure are murine, chimeric, and humanized monoclonal antibodies.

[0471] Protocols for making bispecific or heterodimeric antibodies or antibody-like molecules are known in the art; for example, the "knobs-in-holes" method described in U.S. Pat. No. 5,731,168; electrostatic steering Fc pairing as described in, for example, WO 09 / 089004, WO 06 / 106905, and WO 2010 / 129304; strand exchange engineering domain (SEED) heterodimerization as described in, for example, WO 07 / 110205; Fab arm exchange as described in WO 08 / 119353, WO 2011 / 131746 and WO 2013 / 060867; dual antibody conjugates by antibody cross-linking using, for example, heterobifunctional reagents with amine-reactive groups and sulfhydryl-reactive groups to create bispecific structures, as described, for example, in U.S. Pat. No. 4,433,059; reduction and oxidation of disulfide bonds between two heavy chains, as described, for example, in U.S. Pat. No. 4,444,878. Bispecific antibody or antibody-like molecule determinants made by combining half antibodies (heavy-light chain pairs or Fab) from different antibodies or antibody-like molecules by cycling; trifunctional antibodies, e.g., as described in U.S. Pat. No. 5,273,743, e.g., three Fab' fragments cross-linked via sulfhydryl-reactive groups; biosynthetic binding proteins, e.g., as described in U.S. Pat. No. 5,534,254, e.g., pairs of scFvs cross-linked via C-terminal chains, preferably via disulfide or amine-reactive chemical bridges; Bifunctional antibodies, such as Fab fragments with different binding specificities dimerized via leucine zippers (e.g., c-fos and c-jun) replacing the constant domains, as described in U.S. Pat. No. 5,582,996; bispecific and oligospecific monovalent and oligovalent receptors, such as those described in U.S. Pat. No. 5,591,828, such as the VH-CH1 regions (Fd regions) of two antibodies (two Fab fragments) linked via a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody, usually associated with a light chain;Bispecific DNA antibody conjugates, e.g., cross-linking of antibodies or Fab fragments via double-stranded fragments of DNA, as described, for example, in U.S. Pat. No. 5,635,602; bispecific fusion proteins, e.g., expression constructs containing two scFvs and complete constant regions with a hydrophilic helical peptide linker between them, as described, for example, in U.S. Pat. No. 5,637,481; multivalent and multispecific binding proteins, e.g., dimers of polypeptides having a first domain with a binding region of an Ig heavy chain variable region and a second domain with a binding region of an Ig light chain variable region, commonly referred to as diabodies (including higher order structures that generate bispecific, trispecific or tetraspecific molecules), as described, for example, in U.S. Pat. No. 5,837,242; antibodies linked by peptide spacers that can dimerize to form bispecific / multivalent molecules, as described, for example, in U.S. Pat. No. 5,837,821. minibody constructs having linked VL and VH domains further joined to hinge and CH3 regions; VH and VL domains linked in either orientation by a short peptide linker (e.g., 5 or 10 amino acids) or without any linker at all, which can dimerize to form bispecific diabodies; trimers and tetramers, for example, as described in U.S. Pat. No. 5,844,094; strings of VH domains (or VL domains in family members) linked by peptide bonds with a crosslinkable group further associated at the C-terminus with the VL domain forming a series of FVs (or scFvs), for example, as described in U.S. Pat. No. 5,864,019; VL and VH domains, scFvs, or Fabs, optionally comprising a heterodimeric Fc region, to which one of the antigens is monovalently bound and one of the antigens is bivalently bound, for example, as described in WO 2011 / 028952;and single-chain binding polypeptides having both VL and VH domains linked via peptide linkers that are conjugated into multivalent structures via non-covalent or chemical crosslinking to form homobivalent, heterobivalent, trivalent, and tetravalent structures, e.g., using both scFv or diabody-type formats, e.g., as described in U.S. Pat. No. 5,869,620;

[0472] Further exemplary multispecific and bispecific molecules and methods of making the same are described in, e.g., U.S. Pat. Nos. 5,910,573, 5,932,448, 5,959,083, 5,989,830, 6,005,079, 6,239,259, 6,294,353, 6,333,396, 6,476,198, 6,511,663, 6,670,453, 6,743,896, 6,809,101, 6,910,573, 6,932,448, 6,959,083, 6,989,830, 6,005,079, 6,239,259, 6,294,353, 6,333,396, 6,476,198, 6,511,663, 6,670,453, 6,743,896, 6,809,101, 6,910,573, 6,920,574, 6,930,575, 6,940,576, 6,950,577, 6,960,578, 6,970,579, 6,980,579, 6,9 ... 85, U.S. Patent No. 6,833,441, U.S. Patent No. 7,129,330, U.S. Patent No. 7,183,076, U.S. Patent No. 7,521,056, U.S. Patent No. 7,527,787, U.S. Patent No. 7,534,866, U.S. Patent No. 7,612,181, U.S. Patent Application Publication No. 2002004587A1, U.S. Patent Application Publication No. 2002076406A1, U.S. Patent Application Publication No. 2002103345A1, U.S. Patent Application Publication No. 2003207346A1, U.S. Patent Application Publication No. 2003211078A 1, U.S. Patent Application Publication No. 2004219643A1, U.S. Patent Application Publication No. 2004220388A1, U.S. Patent Application Publication No. 2004242847A1, U.S. Patent Application Publication No. 2005003403A1, U.S. Patent Application Publication No. 2005004352A1, U.S. Patent Application Publication No. 2005069552A1, U.S. Patent Application Publication No. 2005079170A1, U.S. Patent Application Publication No. 2005100543A1, U.S. Patent Application Publication No. 2005136049 ...100543A1, U.S. Patent Application Publication No. 2005136049A1, U.S. Patent Application Publication No. 2005100543A1, U. 005136051A1, U.S. Patent Application Publication No. 2005163782A1, U.S. Patent Application Publication No. 2005266425A1, U.S. Patent Application Publication No. 2006083747A1, U.S. Patent Application Publication No. 2006120960A1, U.S. Patent Application Publication No. 2006204493A1, U.S. Patent Application Publication No. 2006263367A1, U.S. Patent Application Publication No. 2007004909A1, U.S. Patent Application Publication No. 2007087381A1, U.S. Patent Application Publication No. 2007128150A1,US Patent Application Publication No. 2007141049A1, US Patent Application Publication No. 2007154901A1, US Patent Application Publication No. 2007274985A1, US Patent Application Publication No. 2008050370A1, US Patent Application Publication No. 2008069820A1, US Patent Application Publication No. 2008152645A1, US Patent Application Publication No. 2008171855A1, US Patent Application Publication No. 2008241884A1, US Patent Application Publication No. 2008254512A1, US Patent Application Publication No. US Patent Application Publication No. 2008260738A1, US Patent Application Publication No. 2009130106A1, US Patent Application Publication No. 2009148905A1, US Patent Application Publication No. 2009155275A1, US Patent Application Publication No. 2009162359A1, US Patent Application Publication No. 2009162360A1, US Patent Application Publication No. 2009175851A1, US Patent Application Publication No. 2009175867A1, US Patent Application Publication No. 2009232811A1, US Patent Application Publication No. 200923410 5A1, U.S. Patent Application Publication No. 2009263392A1, U.S. Patent Application Publication No. 2009274649A1, European Patent Application Publication No. 346087A2, International Publication No. 0006605A2, International Publication No. 02072635A2, International Publication No. 04081051A1, International Publication No. 06020258A2, International Publication No. 2007044887A2, International Publication No. 2007095338A2, International Publication No. 2007137760A2 FRET, WO 2008119353A1, WO 2009021754A2, WO 2009068630A1, WO 9103493A1, WO 9323537A1, WO 9409131A1, WO 9412625A2, WO 9509917A1, WO 9637621A2, WO 9964460A1. The contents of the above-referenced applications are found inThe entirety of which is incorporated herein by reference.

[0473] Bispecific molecules can be prepared by conjugating the binding specificities using methods known in the art, for example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one another.

[0474] When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonds at the C-terminal hinge regions of the two heavy chains. In certain embodiments, the hinge region is modified to contain an odd number of sulfhydryl residues, e.g., one sulfhydryl residue, prior to conjugation. Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2, ligand x Fab, anti-BTC antibody or functional fragment thereof x mAb, crossmab format, BITE format, or anti-BTC antibody or functional fragment thereof x Fab fusion protein. The bispecific molecules of the present disclosure can be single-chain molecules comprising one single-chain antibody and a binding determinant, or single-chain bispecific molecules comprising two binding determinants. The bispecific molecule can comprise at least two single-chain molecules. Methods for preparing bispecific molecules are described, for example, in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

[0475] Binding of multispecific binding or multivalent molecules to their specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0476] In another aspect, the present disclosure provides multivalent molecules comprising at least two identical or different antigen-binding portions of antibodies of the present disclosure that bind to a target, such as BTC. In a further aspect, the present disclosure provides multivalent compounds comprising at least two identical or different antigen-binding portions of BTC-binding portions and / or therapeutic target-binding portions. The antigen-binding portions can be linked to each other via protein fusion or covalent or non-covalent bonds. Alternatively, methods of linking are described for multispecific molecules. Tetravalent compounds can be obtained, for example, by cross-linking an antibody of the present disclosure with an antibody that binds to the constant region, e.g., the Fc or hinge region, of an antibody of the present disclosure.

[0477] vii. Modification of the molecules of the present disclosure The present application encompasses variants of the molecules and / or fragments thereof described herein, as well as fusions and conjugates of the disclosed molecules, with various modifications in the variable and / or constant regions. For example, the Fc region of the disclosed multispecific binding molecules, e.g., CH1 and / or Cκ, can be wild-type, or it can be modified to achieve various results. Preferred modifications to the Fc include "LS" mutations (M428L, N434S, (EU numbering)) and "YTE" mutations (M252Y, S254T, T256E (EU numbering)) for half-life extension, "DAPA" mutations (D265A, P329A (EU numbering)) for effector silencing, and knob-in-hole mutations (e.g., knob S354C, T366W; hole Y349C, T366S, L368A, Y407V (EU numbering)) that promote proper chain pairing.

[0478] The Fc region can also be modified to "silence" effector function, for example, to reduce or eliminate the ability of the BTC-binding molecule to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP). This can be achieved, for example, by introducing mutations into the Fc region. Such mutations have been described in the art: LALA and N297A (Strohl, 2009, Curr. Opin. Biotechnol. 20(6):685-691); and D265A (Baudino et al., 2008, J. Immunol. 181:6664-69; Strohl, supra). An example of a silent Fc IgG1 antibody includes the so-called LALA mutant, which contains the L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of a silent IgG1 antibody contains the D265A mutation. Another silent IgG1 antibody contains the so-called DAPA mutant, which contains D265A and P329A in the IgG1 Fc amino acid sequence. Another silent IgG1 antibody contains the N297A mutation, which results in an aglycosylated / aglycosylated antibody.

[0479] Each of the VH and VL domains of the anti-BTC antibodies or antigen-binding fragments thereof and / or multispecific binding molecules of the present disclosure comprises hypervariable region CDR1, CDR2, and CDR3 sequences. In certain aspects, one or more of these CDR sequences have conservative amino acid sequence modifications, such that the modified molecule retains or has enhanced binding properties compared to the parent antibody.

[0480] In addition, it has been found that in certain cases it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen-binding ability of an antibody (see, e.g., Queen et al., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370). The molecules of the present disclosure can be modified by introducing such mutations into their variable region frameworks to improve binding characteristics.

[0481] Another type of variable region modification, known as "affinity maturation," involves mutating amino acid residues within the VH CDR1, VH CDR2, and / or VH CDR3 domains and / or the VL CDR1, VL CDR2, and / or VL CDR3 domains to thereby improve one or more binding characteristics (e.g., affinity) of a molecule of interest (e.g., an antibody or antibody-like molecule). Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations, and the effect on binding or other functional properties of the antibody of interest can be assessed in in vitro or in vivo assays as described herein. Conservative modifications (discussed above) can be introduced. Mutations can be amino acid substitutions, additions, or deletions. In addition, typically no more than one, two, three, four, or five residues within the CDR regions are also modified.

[0482] Amino acid sequence variants of the molecules of the present invention can be prepared by introducing appropriate nucleotide changes into the encoding DNA or by synthesizing the desired variant. Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence of the molecules of the present invention. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired antigen-binding properties. Amino acid changes can also alter post-translational processes of the molecule, such as changing the number or position of glycosylation sites.

[0483] The present application includes variants of the molecules described herein, and / or fragments thereof, having conservative amino acid modifications in the variable and / or constant regions.

[0484] viii. Nucleic Acids, Expression Vectors, and Host Cells The present disclosure provides purified nucleic acid molecules (e.g., substantially purified nucleic acid molecules) encoding multispecific binding molecules comprising an anti-BTC antibody or antigen-binding fragment thereof, and / or a BTC-binding portion, and another binding portion (e.g., an anti-VEGF binding portion) described herein. In certain aspects, the present disclosure provides purified nucleic acid molecules (e.g., substantially purified nucleic acid molecules) encoding an anti-BTC binding portion described in Table 1. In another aspect, the present disclosure provides purified nucleic acid molecules (e.g., substantially purified nucleic acid molecules) encoding multispecific binding molecules comprising a BTC-binding portion and an anti-VEGF binding portion described in Table 3.

[0485] The nucleic acid molecules of the present disclosure can encode both the variable and constant regions of an antibody. Some nucleic acid sequences of the present disclosure contain nucleotides that encode a modified heavy chain sequence that has substantial identity (e.g., at least 80%, 90%, 95%, or 99%) to the original heavy chain sequence (e.g., substantial identity to the heavy chain of NVS1, NVS2, NVS3, NVS4, NVS11, NVS12, NVS13, or NVS14). Some other nucleic acid sequences contain nucleotides that encode a modified light chain sequence that has substantial identity (e.g., at least 80%, 90%, 95%, or 99%) to the original light chain sequence (e.g., substantial identity to the light chain of NVS1, NVS2, NVS3, NVS4, NVS11, NVS12, NVS13, or NVS14).

[0486] Polynucleotide sequences encoding anti-BTC antibodies or binding fragments thereof can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences (e.g., sequences as described in the Examples below). Direct chemical synthesis of nucleic acids can be achieved by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859, 1981; and the solid support method of U.S. Pat. No. 4,458,066. Introduction of mutations into polynucleotide sequences by PCR can be carried out, for example, as described in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0487] The present disclosure also provides expression cassettes, vectors, and host cells for producing multispecific binding molecules comprising an anti-BTC binding moiety and / or a BTC binding moiety and an anti-VEGF binding moiety. More specifically, the present disclosure provides expression cassettes and / or vectors comprising a nucleic acid encoding an anti-BTC binding moiety having a sequence as set forth in Table 1, or alternatively, an expression cassette and / or vector comprising a nucleic acid encoding an anti-BTC binding moiety conjugated to a molecule as described herein. In certain aspects, the expression cassette and / or vector comprises a nucleic acid encoding any one of the molecules conjugated to its anti-BTC binding moiety set forth in Table 1. In another aspect, the present disclosure provides an expression cassette and / or vector comprising a nucleic acid encoding a multispecific binding molecule comprising a BTC binding moiety and an anti-VEGF binding moiety as set forth in Table 3.

[0488] In one aspect, the disclosure provides an expression cassette and / or vector comprising a nucleic acid molecule encoding a VH and VL of an anti-BTC antibody or antigen-binding fragment thereof, the nucleic acid molecule having a VH and VL sequence similar to or 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 80%, 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 119%, at least 119%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 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.

[0489] In one aspect, the disclosure provides expression cassettes and / or vectors comprising nucleic acid molecules encoding the heavy and light chains of an anti-BTC antibody or antigen-binding fragment thereof, wherein the nucleic acid molecules have a sequence similar to or different from 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 80%, 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 1 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.

[0490] In one aspect, the disclosure provides: 1) a VHA and a VLA that bind to BTC, wherein the VHA and VLA have a binding affinity of 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 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% 1) a VHA and a VLA encoded by a nucleic acid sequence that is at least 98%, at least 99%, or 100% identical or complementary to SEQ ID NOs: 117 and 123, respectively; and 2) a VHB and a VLB that bind to VEGF, wherein the VHB and VLB have a nucleic acid sequence that is 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%, or at least 64% identical or complementary to SEQ ID NOs: 117 and 123, respectively. 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%,Expression cassettes and / or vectors are provided comprising nucleic acid molecules encoding multispecific binding molecules, including VHBs and VLBs, encoded by nucleic acid sequences that are 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 each other.

[0491] In one aspect, the disclosure provides: 1) a VHA and a VLA that bind to BTC, wherein the VHA and VLA have a binding affinity of 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 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% 1) a VHA and a VLA encoded by a nucleic acid sequence that is at least 98%, at least 99%, or 100% identical or complementary to SEQ ID NOs: 128 and 133, respectively; and 2) a VHB and a VLB that bind to VEGF, wherein the VHB and VLB have a nucleic acid s...

Claims

1. 1. A multispecific binding molecule comprising: 1) an anti-BTC binding portion; and 2) an anti-VEGF binding portion, The anti-BTC binding portion is a. Set forth 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; b. Set forth 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; c. Set forth 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; or d. Set forth 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; comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; the anti-BTC binding moiety and the anti-VEGF binding moiety are antibodies or antigen-binding fragments thereof; The multispecific binding molecule.

2. The anti-VEGF binding moiety and / or the anti-VEGF binding moiety may be an isolated antibody, Fab arm exchange, Crossmab, Fab, Fab', F(ab') ２ 2. The multispecific binding molecule of claim 1, which is in a format selected from the group consisting of Fv, Fv, and scFv.

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

4. The multispecific binding molecule of claim 3, wherein the HA and the HB are linked from the N-terminus to the C-terminus in the following format: N-HA-linker1-HB-C, and the LA and the LB are linked from the N-terminus to the C-terminus in the following format: N-LA-linker2-LB-C.

5. The anti-BTC binding portion is a. SEQ ID NOs: 10 and 21, respectively: b. to SEQ ID NOs: 34 and 45, respectively: c. to SEQ ID NOs: 54 and 65, respectively; or d. to SEQ ID NOs: 78 and 88, respectively:

2. The multispecific binding molecule of claim 1, comprising a VH and a VL comprising amino acid sequences with at least 90% sequence identity.

6. The anti-BTC binding portion is a. Set forth in SEQ ID NOs: 12 and 23, respectively; b. Set forth in SEQ ID NOs: 36 and 47, respectively; c. Set forth in SEQ ID NOs: 56 and 67, respectively; or d. Set forth in SEQ ID NOs: 80 and 90, respectively 6. The multispecific binding molecule of claim 5, comprising a heavy chain and a light chain having the amino acid sequence

7. 2. The multispecific binding molecule of claim 1, wherein the anti-VEGF binding portion comprises a VH and a VL comprising the amino acid sequences set forth in SEQ ID NOs: 101 and 112, respectively.

8. 8. The multispecific binding molecule of claim 7, wherein the anti-VEGF binding portion comprises a heavy chain and a light chain having the amino acid sequences set forth in SEQ ID NOs: 103 and 114, respectively, or the heavy chain and light chain are encoded by nucleic acid sequences having 100% sequence identity to SEQ ID NOs: 104 and 115, respectively.

9. the anti-BTC binding moiety comprises a variable heavy chain domain (VHA) and a variable light chain domain (VLA) that bind to BTC, the anti-VEGF binding moiety comprises a variable heavy chain domain (VHB) and a variable light chain domain (VLB) that bind to VEGF, and the multispecific binding molecule is in the format from N-terminus to C-terminus: N-VHA-heavy chain constant domain (CH1A)-linker1-VHB-heavy chain constant domain (CH1B)-C and N-VLA-light chain constant domain (CKA)-linker2-VLB-light chain constant domain (CKB)-C, wherein the multispecific binding molecule comprises a heavy chain comprising the VHA, CH1A, linker1, VHB, and CH1B, and a light chain comprising the VLA, CKA, linker2, VLB, and CKB, and wherein the heavy chain and the light chain are a. As set forth in SEQ ID NOs: 120 and 125, respectively; b. As set forth in SEQ ID NOs: 130 and 135, respectively; c. As set forth in SEQ ID NOs: 140 and 145, respectively; or d. As set forth in SEQ ID NOs: 149 and 154, respectively; The multispecific binding molecule of claim 1 .

10. A polynucleotide comprising a nucleotide sequence encoding the anti-BTC binding moiety in the multispecific binding molecule of claim 1.

11. An expression cassette or vector comprising the polynucleotide of claim 10.

12. A pharmaceutical composition comprising an effective amount of the multispecific binding molecule of claim 1.

13. 13. A method for treating an ophthalmic disorder in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 12, wherein the ophthalmic disorder is selected from the group consisting of abnormal vascular proliferation associated with the macula (DME), age-related macular degeneration (AMD), neovascular AMD, neovascular glaucoma, diabetic retinopathy, macular edema, pathological myopia, retinal vein occlusion, retinopathy of prematurity, and nevus syndrome. The pharmaceutical composition of claim 12.

14. 14. The pharmaceutical composition of claim 13, wherein the ophthalmological disorder is DME.

15. 14. The pharmaceutical composition of claim 13, wherein the ophthalmic disorder is neovascular AMD.

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