Anti-beta-cell phosphate antibody, its fragment, and multispecificity binding molecule

Abstract

To improve treatment in patients with an insufficient response to anti-VEGF therapy, this invention provides factors that can further enhance the response to anti-VEGF therapy. [Solution] The present invention provides an isolated antibody or its antigen-binding fragment that specifically binds to beta-cell phosphate (BTC), a method for producing the antibody, a pharmaceutical composition containing the antibody, and a method for using the antibody. The present invention also provides a bispecific antibody comprising a multispecific binding molecule, for example, a BTC-binding moiety and an anti-VEGF-binding moiety.

Description

[Technical Field]

[0001] Cross-referencing of related applications and incorporation of sequence listings This application is a U.S. Provisional Patent Application No. 63 / 052,78, filed on 16 July 2020. Patent No. 9, and U.S. Provisional Patent Application No. 63 / 156,709, filed on March 4, 2021. This application claims priority, and these patent applications, by reference, are entirely incorporated into this application. It will be incorporated. Created on June 25, 2021, 204,786 bytes (operator (Measured using the MS-Windows system) "PAT058888-WO-PCT The sequence listing contained in the file named "SQL_ST25" is submitted with this specification. , incorporated herein by reference.

[0002] The present invention relates to an antibody or an antigen-binding fragment thereof, a method for producing the same, a pharmaceutical composition containing the same, and Regarding its usage. [Background technology]

[0003] Beta-cellulosin (BTC), a member of the epidermal growth factor (EGF) family, is originally These were isolated from the conditional medium of mouse pancreatic B tumor cell lines (Shing et al., Science, 259, 1604-1607, 1993). BTC is the ErbB receptor. It is a ligand for the tyrosine kinase family, mainly Ras / MAPK and PL. E triggers anti-apoptotic and proliferative signaling pathways such as the 3K / AKT pathway. It activates rbB1 and ErbB4 homodimers.

[0004] BTC is initially expressed as a single transmembrane protein, and then subsequently as part of the MMP family. It is cleaved (activated) into a 9kDa secretory protein by the members. It then releases the secretory form. The cleavage of the membrane-immobilized form of BTC is primarily performed by ADAM-10 (disintegrin). (and metalloproteinase-10) is produced by (Sahin et al., JC ell Biol.164,769-779,2004;Sahin and Blob el, FEBS Lett 581, 41-44, 2007; and Sanderson et al., J. Biol. Chem. 280, 1826-1837, 2005). Growth Matured and secreted human BTC is produced by the cleavage of pro-BTC, resulting in 80 amino acids. Acid residues (Asp1-Tyr80, residues 32-111 of 178 residues, NP_001) The membrane-immobilized precursor protein (pro-BT) described as 720.1 or SEQ ID NO: 156 It is a 32kDa glycoprotein composed of C)). The carboxyl terminus of BTC is 5 The zero-residue region (Arg31-Tyr80) is a conserved concept of the EGF family of proteins. It contains a synthus sequence.

[0005] Somewhat lower in the heart, lungs, liver, skeletal muscles, kidneys, prostate, testes, ovaries, and colon. In addition to expression, BTC mRNA is strongly expressed in many tissues, including the pancreas, liver, kidneys, and small intestine. Expression has been detected (Sasada et al., Biochem. Biophy s.Res.Commun.190,1173-1179,1993;Sasada a nd Igarashi, Nihon. Rinsho. 51, 3308-3317, 19 93; and Seno et al., Growth Factors 13, 181-1 91, 1996). BTC knockout mice that do not exhibit a clear phenotype are viable and They are capable of reproduction.

[0006] High expression of BTC mRNA indicates that BTC plays a physiological role in pancreatic development and function. This suggests that it may be present. BTC levels, compared to expression levels in a normal pancreas, It has been found that the risk increases by up to 7.5 times in 9 out of 10 types of pancreatic cancer (Yokoya ma et al., 1995). In the pancreas, BTC expression is closely linked to insulin-producing B cells. It is localized to densely related island cells (Miyagawa et al., Endocr (J.46,755-764,1999). BTC can regulate islet physiology. It induces the proliferation of fetal pancreatic cells and stimulates the conversion of non-β cells into β-like insulin-producing cells. It is possible. Furthermore, overexpression of BTC is associated with endometrial adenocarcinoma (Srinivasan et al. al., 1999), hepatocellular carcinoma (Moon et al., 2006), head and neck suprasplana Skin cancer (O-charoenrat et al., 2000), and gastric cancer (Jemal This has been reported in et al., 2011.

[0007] In mammalian eyes, BTC proteins are found in the retinal pigment epithelium (RPE), endothelium, and Müller's retina. Synthesized by cells (Anand-Apte et al., PLoS One 5, e13444,2010), it is located within the lateral blood-retinal barrier.

[0008] While the general role of BTC in vascular endothelial function has been studied, its specific role in the retina has been investigated. The exact role is currently unknown. (Shing et al.) Al., 1993) and the initial reports on its angiogenesis-promoting function suggest that it is related to the proliferative diabetic network. This suggests that it may play a role in membranous disease (PDR). Diabetic mice demonstrated PDR. Although they do not do so, they certainly indicate an increase in retinal vascular permeability (Poulaki et al., J. Clin. Invest. 109, 805-815, 2002). Canada Furthermore, in a mouse model of diabetes, soluble cleaved BTC increased in the retina, It has been determined that it contributes to increased membrane vascular permeability (Anand-Apte et al. (1., 2010).

[0009] Subretinal injection of adeno-associated virus expressing soluble BTC in mice affects retinal blood vessels. This resulted in a dramatic increase in transparency. Overall, BTC is beneficial for the retina in diabetic retinopathy. It is a potent permeability factor that can play an important role in the manifestation of increased vascular permeability, and in this disease It appears to be a potential therapeutic target.

[0010] Vascular endothelial growth factor (VEGF) is an important component of neovascularization associated with tumors and intraocular disorders. It has been shown to be a dieter. VEGF is a potent vascular permeability factor, and blood vessels It is essential for causing leakage. In mammalian eyes, VEGF is involved in the medial vascular-retinal barrier. It is located in the vitreous humor of patients with diabetic macular edema (DME). VEGF levels are measured in the vitreous humor of patients with diabetic macular edema (DME). In comparison to the eye condition of non-diabetic individuals, the condition is significantly elevated (Funatsu et al.). al., Ophthalmology 2009 116:73-9).

[0011] Pegaptanib (anti-VEGF aptamer; Macugen, OSI); ranibizumab (anti VEGF Fab; Lucentis, Genentech); Bevacizumab (full length) ionized antibodies; Avastin, Genentech); brolucizumab (anti-VEGF sc) FV; Beovu, Novartis); Aflibercept (anti-VEGF Fab; Ey Several anti-VEGF drugs, including lea and Regeneron, are used to treat age-related macular degeneration (AMD) and / or used to treat ophthalmic disorders such as DME. Other anti-VEGF molecules Therefore, soluble VEGF receptor analog, VEGF-Trap (Regeneron), low-molecular-weight Sub-interfering RNA (siRNA) bevacilanib (Opko Health), and rapamycin Examples include (Sirolimus, MacuSight). Anti-VEGF drugs are local anesthesia. It is administered to the eye as an intravitreal injection while the patient is intoxicated.

[0012] However, in patients with ophthalmic disorders such as DME, anti-VEGF therapy alone is insufficient. Unmet needs exist due to insufficient response. Anti-VEGF reagents cause macular effusion. It reduces tumors, inhibits angiogenesis, and improves vision, but not all DME patients experience substantial long-term effects. They do not experience continuous improvement. For example, approximately 25% of patients who received anti-VEGF therapy % showed no improvement in vision after 12 months of treatment, and nearly 50% of patients were 20 / 40 Unable to achieve the legally mandated visual acuity for driving (Mitchell et al., 2011) Alternative therapies such as laser photocoagulation or intravitreal steroid treatment have not been very successful. , has side effects (for example, cataracts caused by intraocular steroid treatment (Curr.Ophtha lmol.Rep.2013 Sep 1(3)).

[0013] Therefore, in order to improve therapy for patients who have an insufficient response to anti-VEGF therapy Therefore, there is a need to identify factors that can further enhance the response to anti-VEGF therapy. . [Brief explanation of the drawing]

[0014] [Figure 1] The structures of Fab binding to BTC are shown. BTC is shown as a solid surface, and Fab is shown as a ribbon. To illustrate the variation in binding modes by Fab, the BTC structures are oriented in the same way 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] The structural epitope residues of Fab that bind to BTC are shown. BTC is shown as ribbons with various orientations to highlight the epitope residues listed in Tables 12, 13, 14, and 15. The epitope residues are shown and labeled as balls and sticks. A) BTC / Fab NVS2 complex, B) BTC / Fab NVS3 complex, C) BTC / Fab NVS1 complex, and D) BTC / Fab NVS4 complex. [Figure 3] This software provides graphical representations of single-specificity antibodies and bispecificity antibodies. [Figure 4] The images show retinal images of mice injected with scAAV2-CMV-BTC, obtained from fundus photography (left panel) and FFA visualization (right panel). [Figure 5] This image shows the retina of mice injected with scAAV2-CMV-BTC using a scanning laser ophthalmoscope. [Figure 6-1] Figure 6: Figures 6A-6G show the binding of monospecific and bispecific antibodies to BTC and / or VEGF. [Figure 6-2] (As stated above.) [Figure 6-3] (As stated above.) [Figure 6-4] (As stated above.) [Figure 7-1]Figure 7: Figures 7A-7D show the binding of BTC to ErbB1 in the presence of monospecific (NVS1-4) antibodies and bispecific (NVS11-14) antibodies. [Figure 7-2] (As stated above.) [Figure 8-1] Figure 8: Figures 8A-8D show the binding of BTC to ErbB4 in the presence of monospecific (NVS1-4) antibodies 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 stated above.) [Figure 8-3] (As stated above.) [Figure 8-4] (As stated above.) [Figure 9-1] Figure 9: Figures 9A-9C show the binding of VEGF-A to VEGFR2 in the presence of monospecific (NVS8) antibodies and bispecific (NVS11, NVS12, and NVS14) antibodies. [Figure 9-2] (As stated above.) [Figure 10-1] Figure 10: Figures 10A-10D show BTC-induced phosphorylation of ERK1 / 2 in the presence of monospecific (NVS1-4) antibodies and bispecific (NVS11-14) antibodies. [Figure 10-2] (As stated above.) [Figure 11-1] Figure 11: Figures 11A-11D show BTC-induced phosphorylation of ErbB3 in the presence of monospecific (NVS1-4) antibodies and bispecific (NVS11-14) antibodies. [Figure 11-2] (As stated above.) [Figure 12-1] Figure 12: Figures 12A-12D show BTC-induced phosphorylation of HER3 in the presence of monospecific (NVS1-2) antibodies and bispecific (NVS11-14) antibodies. [Figure 12-2] (As stated above.) [Figure 13]Figures 13A and 13B show the 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) antibodies and bispecific (NVS11) antibodies. [Figure 14] This 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 pathological images from rabbit eyes after treatment with VEGF or BTC. Figure 15B shows representative OCT images demonstrating the effect of intravitreous beta-cell phosphate on the retina of rabbits after intravitreous injection of NVS1 or NVS11. [Figure 15-2] (As stated above.) [Figure 16] This shows the changes in retinal thickness in rabbit eyes after treatment with NVS1 or NVS11. [Figure 17] This shows the changes 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 morphological changes in BTC-induced RPE in the presence of NVS11 or NVS1. Figure 18C shows VEGF-induced retinal vascular leakage (representative image) in rabbits in the presence of NVS11 or NVS8. Figure 18D shows VEGF-induced retinal vascular leakage (quantified by fluorescein angiography) in rabbits in the presence of NVS11 or NVS8. [Figure 18-2] (As stated above.) [Figure 18-3] (As stated above.) [Figure 19] The images show fluorescein angiography of rabbit eyes after in vitro delivery of VEGF or BTC. [Figure 20] This shows the fluorescein vascular leakage values ​​from the eyes of individual rabbits after treatment with NVS8, NVS11, or NVS12. [Figure 21]This shows the fluorescein vascular leakage values ​​from the eyes of individual rabbits after treatment with NVS11, NVS12, or NVS8. [Figure 22] This shows the change in total retinal thickness from individual rabbit eyes after treatment with ranibizumab or NVS1. [Figure 23] This shows the change in retinal thickness in rabbits after treatment with NVS1 or PNVS1. [Modes for carrying out the invention]

[0015] This disclosure relates to isolated antibodies or antigens that specifically bind to beta-cell phosphate (BTC). Provides a binding fragment.

[0016] In one embodiment, the antibody or its antigen-binding fragment is ErbB1, ErbB4, or so of BTC. Blocks binding to both.

[0017] In one embodiment, an antibody or its antigen-binding fragment is subjected to BTC-induced phosphorylation-ERK1 / 2 activation. Block it.

[0018] In one embodiment, an antibody or its antigen-binding fragment is used to induce BTC-induced phosphorylation-HER3 activation. Lock it.

[0019] This disclosure also provides isolated antibodies or antigen-binding fragments thereof that specifically bind to BTC. Furthermore, this antibody or antigen-binding fragment has a dissociation constant (KD) of 5 pM or less.

[0020] In one embodiment, the antibody or its antigen-binding fragment is a BT containing the amino acid sequence of SEQ ID NO: 157. Combine with C.

[0021] In one embodiment, the antibody or its antigen-binding fragment is 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 R It binds to at least one residue of sequence number 157, selected from a group of 76.

[0022] In one embodiment, the antibody or its antigen-binding fragment is R38, C39, P40 of SEQ ID NO: 157 , K41, Q42, Y43, H45, Y46, F54, Q59, T60, P61, and R Combine with 73.

[0023] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number Heavy chain variable region complementarity determination region 1 (HCDR1), as described in issues 1, 2, and 3, Chain variable region complementarity determination region 2 (HCDR2), and heavy chain variable region complementarity determination region 3 (HC DR3) and light chain variable region phases as described in Sequence IDs 14, 15, and 16, respectively. Complementarity determination region 1 (LCDR1), light chain variable region complementarity determination region 2 (LCDR2), and light This includes the chain variable region complementarity determination region 3 (LCDR3).

[0024] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number HCDR1, HCDR2, and HCDR3 as described in items 4, 2, and 3, and LCDR1, LCDR2, and L as described in sequence numbers 14, 15, and 16, respectively. Includes CDR3.

[0025] In one embodiment, HCDR1 includes the consensus sequence XYAIS and / or HCDR 2 contains the consensus sequence GIXPXXGXXXYAQKFQG, where X is arbitrary. These are amino acids, and they do not have to be the same at different positions.

[0026] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is SEQ ID NO: 168 It includes the heavy chain sequence of SEQ ID NO: 169 and the light chain sequence of SEQ ID NO: 170 and Contains the light chain sequence of sequence number 171.

[0027] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number HCDR1, HCDR2, and HCDR3 as described in items 5, 6, and 3, and LCDR1, LCDR2, and L as described in sequence numbers 17, 18, and 19, respectively. Includes CDR3.

[0028] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number HCDR1, HCDR2, and HCDR3 as described in issues 7, 8, and 9, and LCDR1, LCDR2, and L as described in sequence numbers 20, 18, and 16, respectively. Includes CDR3.

[0029] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number For items 10 and 21, at least 90%, 95%, 96%, 97%, 98%, and 99% or heavy chain variable region (VH) and light chain containing amino acid sequences with 100% sequence identity Includes a variable region (VL).

[0030] In one embodiment, the differences in amino acid sequences are not located within the complementarity-determining region.

[0031] In one embodiment, the difference in amino acid sequences is a conservative substitution.

[0032] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It contains VH and VL having amino acid sequences as described in issues 10 and 21.

[0033] In one embodiment, VH and VL are nucleic acids as described in Sequence ID No. 11 and 22, respectively. It is coded using an array.

[0034] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It includes heavy and light chains having amino acid sequences as described in issues 12 and 23.

[0035] In one embodiment, the heavy chain and the light chain are at least 9 in relation to SEQ ID NOs: 13 and 24, respectively. nuclei with 0%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity It is coded by acid.

[0036] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is 1) SEQ ID NO: 1 HCDR1, HCDR2, and HCDR3 contained in VH having an amino acid sequence of 0 2) LCDR1, LCDR2, contained in VL having the amino acid sequence of SEQ ID NO: 21 and LCDR3, and also include.

[0037] In one embodiment, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and L CDR3 is sequence numbers 1, 2, 3, 14, 15, and 16, respectively; sequence number 4, respectively. , 2, 3, 14, 15, and 16; sequence numbers 5, 6, 3, 17, 18, and 19 respectively ; or including sequence numbers 7, 8, 9, 20, 18, and 16, respectively.

[0038] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is HCDR1, H This includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where a. HCDR1 is an amino acid compound selected from the group consisting of SEQ ID NOs: 1, 4, 5, and 7. The sequence includes an amino acid combination selected from the group consisting of SEQ ID NOs: 2, 6, and 8. The column includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 9. fruit, b. LCDR1 is an amino acid selected from the group consisting of SEQ ID NOs: 14, 17, and 20. The sequence includes an amino acid combination selected from the group consisting of SEQ ID NOs: 15 and 18. The sequence includes a column, and LCDR3 is an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19. Includes.

[0039] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It contains VH and VL, which have amino acid sequences 10 and 21.

[0040] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It includes heavy and light chains having amino acid sequences as described in issues 12 and 23.

[0041] Furthermore, this disclosure includes VH and having the amino acid sequences of SEQ ID NOs. 10 and 21, respectively. An isolated antibody or its antigen-binding fragment containing VL that specifically binds to BTC is provided. ru.

[0042] In one embodiment, the antibody or its antigen-binding fragment is P40, K41, Q42 of SEQ ID NO: 157 , Y43, H45, Y46, E58, Q59, T60, P61, A72, R73, E75 , and R76.

[0043] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number HCDR1, HCDR2, and HCDR3 as described in issues 25, 26, and 27 LCDR1, LCDR2, as described in sequence numbers 38, 39, and 40, respectively. and includes LCDR3.

[0044] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number HCDR1, HCDR2, and HCDR3 as described in issues 28, 26, and 27 LCDR1, LCDR2, as described in sequence numbers 38, 39, and 40, respectively. and includes LCDR3.

[0045] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number HCDR1, HCDR2, and HCDR3 as described in issues 29, 30, and 27 LCDR1, LCDR2, as described in sequence numbers 41, 42, and 43, respectively. and includes LCDR3.

[0046] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number HCDR1, HCDR2, and HCDR3 as described in issues 31, 32, and 33 LCDR1, LCDR2, as described in sequence numbers 44, 42, and 40, respectively. and includes LCDR3.

[0047] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number For items 34 and 45, at least 90%, 95%, 96%, 97%, 98%, and 99% or containing VH and VL containing amino acid sequences having 100% sequence identity.

[0048] In one embodiment, the differences in amino acid sequences are not located within the complementarity-determining region.

[0049] In one embodiment, the difference in amino acid sequences is a conservative substitution.

[0050] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It contains VH and VL with amino acid sequences as described in issues 34 and 45.

[0051] In one embodiment, VH and VL are nucleic acids as described in SEQ ID NOs: 35 and 46, respectively. It is coded using an array.

[0052] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It includes heavy and light chains having amino acid sequences as described in issues 36 and 47.

[0053] In one embodiment, the heavy chain and the light chain are at least 9 in relation to Sequence ID No. 37 and 48, respectively. nuclei with 0%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity It is coded by the acid sequence.

[0054] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is 1) SEQ ID NO: 3 HCDR1, HCDR2, and HCDR3 contained in VH having a 4 amino acid sequence 2) LCDR1, LCDR2, contained in VL having the amino acid sequence of SEQ ID NO: 45 and LCDR3, and also include.

[0055] In one embodiment, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and L CDR3 corresponds to sequence numbers 25, 26, 27, 38, 39, and 40, respectively; each sequence Numbers 28, 26, 27, 38, 39, and 40; sequence numbers 29, 30, 27, and 40 respectively. 1, 42, and 43; or sequence numbers 31, 32, 33, 44, 42, and 40 respectively include.

[0056] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is HCDR1, H This includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where a. HCDR1 is selected from the group consisting of sequence numbers 25, 28, 29, and 31. The HCDR2 contains a mino acid sequence and is selected from the group consisting of SEQ ID NOs: 26, 30, and 32. The amino acid sequence is selected from the group consisting of SEQ ID NOs: 27 and 33, and HCDR3 is selected from the group consisting of SEQ ID NOs: 27 and 33. It contains an amino acid sequence, b. LCDR1 is an amino acid selected from the group consisting of SEQ ID NOs: 38, 41, and 44. The sequence includes an amino acid mixture selected from the group consisting of SEQ ID NOs: 39 and 42. The sequence includes a column, and LCDR3 is an amino acid sequence selected from the group consisting of SEQ ID NOs: 40 and 43. Includes.

[0057] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It contains VH and VL, which have amino acid sequences 34 and 45.

[0058] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It includes heavy and light chains having amino acid sequences as described in issues 36 and 47.

[0059] This disclosure includes VH and VL having the amino acid sequences of SEQ ID NOs. 34 and 45, respectively. The present invention further provides isolated antibodies or antigen-binding fragments thereof that specifically bind to BTC.

[0060] In one embodiment, the antibody or its antigen-binding fragment is G34, H35, F36 of SEQ ID NO: 157 S37, R38, C39, P40, K41, Q42, R51, R53, F54, and V Combine with 56.

[0061] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number HCDR1, HCDR2, and HCDR3 as described in issues 25, 49, and 50 LCDR1, LCDR2, as described in sequence numbers 58, 59, and 60, respectively. and includes LCDR3.

[0062] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number HCDR1, HCDR2, and HCDR3 as described in issues 28, 49, and 50 LCDR1, LCDR2, as described in sequence numbers 58, 59, and 60, respectively. and includes LCDR3.

[0063] In one embodiment, HCDR1 includes the consensus sequence XXAMX and / or HCDR 2 contains the consensus sequence XXXX / -XXXXTXYXDSVKG, where X is Any amino acid, which does not have to be the same at different positions, and X / - is any amino acid or It is missing.

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

[0065] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number HCDR1, HCDR2, and HCDR3 as described in issues 29, 51, and 50 LCDR1, LCDR2, as described in sequence numbers 61, 62, and 63, respectively. and includes LCDR3.

[0066] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number HCDR1, HCDR2, and HCDR3 as described in issues 31, 52, and 53 LCDR1, LCDR2, as described in sequence numbers 64, 62, and 60, respectively. and includes LCDR3.

[0067] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number For numbers 54 and 65, at least 90%, 95%, 96%, 97%, 98%, and 99% or containing VH and VL containing amino acid sequences having 100% sequence identity.

[0068] In one embodiment, the differences in amino acid sequences are not located within the complementarity-determining region.

[0069] In one embodiment, the difference in amino acid sequences is a conservative substitution.

[0070] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It contains VH and VL having amino acid sequences as described in issues 54 and 65.

[0071] In one embodiment, VH and VL are nucleic acids as described in Sequence ID No. 55 and 66, respectively. It is coded using an array.

[0072] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It includes heavy and light chains having amino acid sequences as described in issues 56 and 67.

[0073] In one embodiment, the heavy chain and light chain are at least 9 in relation to Sequence ID No. 57 and 68, respectively. nuclei with 0%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity It is coded by the acid sequence.

[0074] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is 1) SEQ ID NO: 5 HCDR1, HCDR2, and HCDR3 contained in VH having a 4 amino acid sequence 2) LCDR1, LCDR2, contained in VL having the amino acid sequence of SEQ ID NO: 65 and LCDR3, and also include.

[0075] In one embodiment, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and L CDR3 is sequence numbers 25, 49, 50, 58, 59, and 60, respectively; each sequence Numbers 28, 49, 50, 58, 59, and 60; sequence numbers 29, 51, 50, and 60 respectively. 1, 62, and 63; or sequence numbers 31, 52, 53, 64, 62, and 60 respectively include.

[0076] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is HCDR1, H This includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where a. HCDR1 is selected from the group consisting of sequence numbers 25, 28, 29, and 31. The HCDR2 contains a mino acid sequence, and is selected from the group consisting of SEQ ID NOs: 49, 51, and 52. The amino acid sequence is selected from the group consisting of SEQ ID NOs: 50 and 53. It contains an amino acid sequence, b. LCDR1 is an amino acid selected from the group consisting of SEQ ID NOs: 58, 61, and 64. The sequence includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 59 and 62. The sequence includes a column, and LCDR3 is an amino acid sequence selected from the group consisting of SEQ ID NOs: 60 and 63. Includes.

[0077] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It contains VH and VL, which have amino acid sequences 54 and 65.

[0078] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It includes heavy and light chains having amino acid sequences as described in issues 56 and 67.

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

[0080] In one embodiment, the antibody or its antigen-binding fragment is S37, R38, C39 of SEQ ID NO: 157 , P40, K41, Q42, Y43, H45, Y46, F54, A57, Q59, T60 It connects to P61, A72, R73, and E75.

[0081] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number HCDR1, HCDR2, and HCDR3 as described in issues 69, 70, and 71 LCDR1, LCDR2, as described in sequence numbers 82, 83, and 84, respectively. and includes LCDR3.

[0082] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number HCDR1, HCDR2, and HCDR3 as described in issues 72, 70, and 71 LCDR1, LCDR2, as described in sequence numbers 82, 83, and 84, respectively. and includes LCDR3.

[0083] In one embodiment, HCDR2 uses the consensus sequence XIXXXXXXXXYADSVKG Includes and / or LCDR3 includes the consensus sequence QQYDXXXT, where X is These are any amino acids, and they do 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 is, respectively, sequence number Sequence numbers 194 and 195; Sequence numbers 196 and 197 respectively; Sequence number 198 and 199; Sequence IDs 200 and 201 respectively; Sequence IDs 202 and 203 respectively; The group consists of sequence numbers 204 and 205, and sequence numbers 206 and 207, respectively. It includes heavy chain and light chain sequences selected from there.

[0085] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number HCDR1, HCDR2, and HCDR3 as described in issues 73, 74, and 71 LCDR1, LCDR2, as described in sequence numbers 85, 18, and 86, respectively. and includes LCDR3.

[0086] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number HCDR1, HCDR2, and HCDR3 as described in issues 75, 76, and 77 LCDR1, LCDR2, as described in sequence numbers 87, 18, and 84, respectively. and includes LCDR3.

[0087] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number For numbers 78 and 88, at least 90%, 95%, 96%, 97%, 98%, and 99% or containing VH and VL containing amino acid sequences having 100% sequence identity.

[0088] In one embodiment, the differences in amino acid sequences are not located within the complementarity-determining region.

[0089] In one embodiment, the difference in amino acid sequences is a conservative substitution.

[0090] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It contains VH and VL having amino acid sequences as described in issues 78 and 88.

[0091] In one embodiment, VH and VL are nucleic acids as described in Sequence ID No. 79 and 89, respectively. It is coded using an array.

[0092] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It includes heavy and light chains having amino acid sequences as described in issues 80 and 90.

[0093] In one embodiment, the heavy chain and the light chain are, respectively, at least 9 in relation to SEQ ID NOs: 81 and 91. nuclei with 0%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity It is coded by the acid sequence.

[0094] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is 1) SEQ ID NO: 7 HCDR1, HCDR2, and HCDR3 contained in VH having an 8-amino acid sequence 2) LCDR1, LCDR2, contained in VL having the amino acid sequence of SEQ ID NO: 88 and LCDR3, and also include.

[0095] In one embodiment, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and L CDR3 is sequence numbers 69, 70, 71, 82, 83, and 84, respectively; each sequence Numbers 72, 70, 71, 82, 83, and 84; sequence numbers 73, 74, 71, 8 5, 18, and 86; or sequence numbers 75, 76, 77, 87, 18, and 84 respectively include.

[0096] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is HCDR1, H This includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where a. HCDR1 is selected from the group consisting of sequence numbers 69, 72, 73, and 75. The HCDR2 contains a minoic acid sequence, and is selected from the group consisting of SEQ ID NOs: 70, 74, and 76. The amino acid sequence is selected from the group consisting of SEQ ID NOs: 71 and 77. It contains an amino acid sequence, b. LCDR1 is an amino acid selected from the group consisting of SEQ ID NOs: 82, 85, and 87. The sequence includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 83 and 18. The sequence includes a column, and LCDR3 is an amino acid sequence selected from the group consisting of SEQ ID NOs: 84 and 86. Includes.

[0097] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It contains VH and VL, which have amino acid sequences 78 and 88.

[0098] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is, respectively, sequence number It includes heavy and light chains having amino acid sequences as described in issues 80 and 90.

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

[0100] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to BTC is an isolated antibody A form selected from the group consisting of Fab, Fab', F(ab')2, Fv, and scFv. It is an equation.

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

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

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

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

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

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

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

[0108] In one embodiment, the Fc region is a human immunoglobulin molecule as described in Sequence ID No. 159. Includes the P-chain constant region sequence.

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

[0110] Throughout the present disclosure, there is provided an isolated antibody or an antigen-binding fragment thereof that can compete with an antibody or an antigen-binding fragment thereof as described throughout for binding to BTC and reducing BTC-mediated signal transduction.

[0111] In one aspect, an antibody or an antigen-binding fragment thereof that specifically binds to BTC comprises a heavy chain and a light chain 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 a polynucleotide comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment 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 a vector as described throughout.

[0116] In addition, the present disclosure provides a method for producing an antibody or an antigen-binding fragment thereof, which includes culturing a host cell under conditions suitable for the expression of the antibody or the antigen-binding fragment thereof.

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

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

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

[0120] The present disclosure provides a method for treating a subject in need of treatment, which includes administering to the subject an effective amount of an antibody or an antigen-binding fragment thereof, or a 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 the antigen-binding fragment thereof or the pharmaceutical composition is administered via a route selected from the group consisting of intravenous administration, intramuscular administration, subcutaneous administration, parenteral administration, spinal administration, and epithelial administration.

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

[0124] In one aspect, the ophthalmic disorder is diabetic macular edema, age-related macular degeneration, age-related macular degeneration with neovascularization, neovascular glaucoma, diabetic retinopathy, macular edema, pathologic myopia, retinal vein occlusion, retinopathy of prematurity, abnormal vascular proliferation associated with choroiditis, and acute multiple punctate retinopathy, central serous chorioretinopathy, abnormal vascular proliferation associated with nevus flammeus, and acute multiple punctate​​​​​​​​ Selected from the group consisting of pigment epitheliosis.

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

[0126] In one aspect, the administration is carried out via subretinal injection.

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

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

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

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

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

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

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

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

[0135] In one aspect, the anti-VEGF antagonist is as described in SEQ ID NOs: 104 and 115. encoded by such nucleic acid sequences.

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

[0137] In one aspect, 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, anti-VEGF antagonists are described in SEQ ID NOs: 103 and 114. Includes heavy and light eel chains.

[0143] In one embodiment, anti-VEGF antagonists are described in SEQ ID NOs: 104 and 115. It is encoded by nucleic acid sequences.

[0144] This disclosure does not include antibodies or their antigen-binding fragments or pharmaceutical compounds as described throughout. We provide a kit that includes the finished product.

[0145] In one embodiment, the kit further includes an instruction manual.

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

[0147] This disclosure relates to a multispecific binding comprising 1) an anti-BTC binding moiety and 2) an anti-VEGF binding moiety. Provides molecules.

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

[0149] In one embodiment, the anti-BTC binding portion is G34, H35, F36, S37, R38, C39 , P40, K41, Q42, Y43, H45, Y46, R51, R53, F54, V56 From A57, E58, Q59, T60, P61, A72, R73, E75, and R76 It binds to at least one residue of sequence number 157 selected from the group.

[0150] In one embodiment, the anti-BTC binding moiety is R38, C39, P40, K41 of Sequence ID No. 157 Connected to Q42, Y43, H45, Y46, F54, Q59, T60, P61, and R73. To combine.

[0151] In one embodiment, the anti-BTC binding moiety is P40, K41, Q42, Y43 of Sequence ID No. 157 H45, Y46, E58, Q59, T60, P61, A72, R73, E75, and R Combine with 76.

[0152] In one embodiment, the anti-BTC binding moiety is G34, H35, F36, S37 of Sequence ID No. 157 Connected to R38, C39, P40, K41, Q42, R51, R53, F54, and V56. To combine.

[0153] In one embodiment, the anti-BTC binding moiety is S37, R38, C39, P40 of SEQ ID NO: 157 , K41, Q42, Y43, H45, Y46, F54, A57, Q59, T60, P61 It connects to A72, R73, and E75.

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

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

[0156] In one embodiment, the anti-BTC binding portion and the anti-VEGF binding portion are isolated antibodies, Fab, The format is selected from a list consisting of Fab', F(ab')2, Fv, and scFv. ru.

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

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

[0159] In one embodiment, HA and HB are connected from the N-terminus to the C-terminus: N-HA-linker The molecules are linked in the form 1-HB-C, with LA and LB connected from the N-terminus to the C-terminus:N They are linked in the form of -LA-Linker 2-LB-C.

[0160] In one embodiment, HA and HB are connected from the N-terminus to the C-terminus: N-HB-linker The elements are linked in the form 1-HA-C, with LA and LB connected from the N-terminus to the C-terminus:N They are linked in the form of -LB-linker2-LA-C.

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

[0162] In one embodiment, linker 1 and linker 2 are coded by the nucleic acid sequence of sequence number 119. It will be transformed.

[0163] In one embodiment, linker 1 and linker 2 are selected from the group consisting of sequence numbers 161 to 167. Includes the selected amino acid sequence.

[0164] In one embodiment, the anti-BTC binding moieties are, respectively, sequence numbers 1, 2, 3, 14, 15, and 1 6; Sequence numbers 4, 2, 3, 14, 15, and 16 respectively; Sequence numbers 5, 6, and 3 respectively , 17, 18, and 19; or as indicated in sequence numbers 7, 8, 9, 20, 18, and 16, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and L are listed. Includes CDR3.

[0165] In one embodiment, the anti-BTC binding moiety has the amino acid sequences of SEQ ID NOs: 10 and 21, respectively. This includes VH and VL.

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

[0167] In one embodiment, the anti-BTC binding moieties are, respectively, sequence numbers 25, 26, 27, 38, and 39. and 40; respective sequence numbers 28, 26, 27, 38, 39, and 40; respective sequence numbers Sequence numbers 29, 30, 27, 41, 42, and 43; or sequence numbers 31, 32, 33 respectively, HCDR1, HCDR2, HCDR3, LCD as described in 44, 42, and 40 Includes R1, LCDR2, and LCDR3.

[0168] In one embodiment, the anti-BTC binding moiety has the amino acid sequences of SEQ ID NOs. 34 and 45, respectively. This includes VH and VL.

[0169] In one embodiment, VH and VL are determined by the nucleic acid sequences of SEQ ID NOs. 127 and 132, respectively. It will be coded.

[0170] In one embodiment, the anti-BTC binding moieties are, respectively, sequence numbers 25, 49, 50, 58, and 59. and 60; respective sequence numbers 28, 49, 50, 58, 59, and 60; respective sequence numbers Sequence numbers 29, 51, 50, 61, 62, and 63; or sequence numbers 31, 52, 53 respectively, HCDR1, HCDR2, HCDR3, LCD as described in 64, 62, and 60 Includes R1, LCDR2, and LCDR3.

[0171] In one embodiment, the anti-BTC binding moiety has the amino acid sequences of SEQ ID NOs. 54 and 65, respectively. This includes VH and VL.

[0172] In one embodiment, VH and VL are determined by the nucleic acid sequences of SEQ ID NOs. 137 and 142, respectively. It will be coded.

[0173] In one embodiment, the anti-BTC binding moieties are, respectively, sequence numbers 69, 70, 71, 82, and 83. and 84; respective sequence numbers 72, 70, 71, 82, 83, and 84; respective sequence numbers Numbers 73, 74, 71, 85, 18, and 86; or sequence numbers 75, 76, 77 respectively, HCDR1, HCDR2, HCDR3, LCD as described in 87, 18, and 84 Includes R1, LCDR2, and LCDR3.

[0174] In one embodiment, the anti-BTC binding moiety has the amino acid sequences of SEQ ID NOs. 78 and 88, respectively. This includes VH and VL.

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

[0176] In one embodiment, the anti-VEGF binding moiety is described in Sequence IDs 92, 93, and 94, respectively. HCDR1, HCDR2, and HCDR3, which are such as, and sequence numbers 105 and 10 This includes LCDR1, LCDR2, and LCDR3 as described in 6 and 107.

[0177] In one embodiment, the anti-VEGF binding moiety is described in SEQ ID NOs. 95, 93, and 94, respectively. HCDR1, HCDR2, and HCDR3, which are such as, and sequence numbers 105 and 10 This includes LCDR1, LCDR2, and LCDR3 as described in 6 and 107.

[0178] In one embodiment, the anti-VEGF binding moiety is described in SEQ ID NOs: 96, 97, and 94, respectively. HCDR1, HCDR2, and HCDR3, which are such as sequence numbers 108 and 10 This includes LCDR1, LCDR2, and LCDR3 as described in 9 and 110.

[0179] In one embodiment, the anti-VEGF binding moieties are described in Sequence IDs 98, 99, and 100, respectively. HCDR1, HCDR2, and HCDR3, which are such as, and sequence numbers 111 and 1, respectively. Includes LCDR1, LCDR2, and LCDR3 as described in 09 and 107. .

[0180] In one embodiment, the anti-VEGF binding moieties are less than those of sequence numbers 101 and 112, respectively. At least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity It contains VH and VL, which have amino acid sequences containing the following:

[0181] In one embodiment, the differences in amino acid sequences are not located within the complementarity-determining region.

[0182] In one embodiment, the difference in amino acid sequences is a conservative substitution.

[0183] In one embodiment, the anti-VEGF binding moieties are described in SEQ ID NOs: 101 and 112, respectively. It contains VH and VL, which have amino acid sequences like the one described.

[0184] In one embodiment, the anti-VEGF binding moieties are described in SEQ ID NOs: 102 and 113, respectively. It contains VH and VL, which have amino acid sequences like the one described.

[0185] In one embodiment, the anti-VEGF binding moieties are described in SEQ ID NOs: 117 and 123, respectively. It contains VH and VL, which have amino acid sequences like the one described.

[0186] In one embodiment, the anti-VEGF binding moieties are described in Sequence ID Nos. 128 and 133, respectively. It contains VH and VL, which have amino acid sequences like the one described.

[0187] In one embodiment, the anti-VEGF binding moieties are described in Sequence ID Nos. 138 and 143, respectively. It contains VH and VL, which have amino acid sequences like the one described.

[0188] In one embodiment, the anti-VEGF binding moieties are described in Sequence ID Nos. 148 and 152, respectively. It contains VH and VL, which have amino acid sequences like the one described.

[0189] In one embodiment, the anti-VEGF binding moieties are described in SEQ ID NOs: 103 and 114, respectively. It includes heavy and light chains having such amino acid sequences.

[0190] In one embodiment, the heavy chain and light chain are, at least, relative to SEQ ID NOs. 104 and 115, respectively. It also has 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. It is encoded by a nucleic acid sequence.

[0191] This disclosure proposes a multispecific binding molecule that includes an anti-BTC binding moiety and an anti-VEGF binding moiety. The anti-BTC binding portion consists of a variable heavy chain domain (VHA) that binds to BTC and a variable light chain domain. It contains a chain domain (VLA), and the anti-VEGF binding moiety is a variable heavy chain domain that binds to VEGF. It includes a yin (VHB) and a variable light chain domain (VLB), where: a. VHA and VLA are amino acids as described in Sequence ID No. 10 and 21, respectively. Includes arrays; b. VHB and VLB are such that they are as described in Sequence ID No. 101 and 112, respectively. Contains an acid sequence.

[0192] In one embodiment, the anti-BTC binding moiety consists of a heavy chain constant domain (CH1A) and a light chain constant domain. It further contains (CKA), and the anti-VEGF binding portion consists of a heavy chain constant domain (CH1B) and a light chain. It further contains chain-constant domains (CKBs).

[0193] In one embodiment, the multispecificity binding molecule is arranged from the N-terminus to the C-terminus:N-VHA- CH1A-Linker-VHB-CH1B-C and N-VLA-CKA-Linker-VLB It is in the format -CKB-C.

[0194] In one embodiment, the multispecific binding molecules are VHA, CH1A, linker, VHB, and CH It contains a heavy chain including 1B, which is as described in Sequence ID No. 120.

[0195] In one embodiment, the heavy chain is encoded by a nucleic acid sequence such as that described in Sequence ID No. 121. It can be done.

[0196] In one embodiment, the multispecific binding molecules are VLA, CKA, linker, VLB, and CKB. It includes a light chain, which is as described in Sequence ID No. 125.

[0197] In one embodiment, the light chain is encoded by a nucleic acid sequence such as that described in Sequence ID No. 126. It can be done.

[0198] Furthermore, this disclosure describes a multispecific binding component including an anti-BTC binding portion and an anti-VEGF binding portion. A child is provided, and the anti-BTC binding moiety is a variable heavy chain domain (VHA) that binds to BTC and It contains a variable light chain domain (VLA), and the anti-VEGF binding moiety is a variable light chain domain that binds to VEGF. It includes a chain domain (VHB) and a variable light chain domain (VLB), where: a. VHA and VLA are amino acids as described in Sequence ID No. 34 and 45, respectively. Includes arrays; b. VHB and VLB are such that they are as described in Sequence ID No. 101 and 112, respectively. Contains an acid sequence.

[0199] In one embodiment, the anti-BTC binding moiety consists of a heavy chain constant domain (CH1A) and a light chain constant domain. It further contains (CKA), and the anti-VEGF binding portion consists of a heavy chain constant domain (CH1B) and a light chain. It further contains chain-constant domains (CKBs).

[0200] In one embodiment, the multispecificity binding molecule is arranged from the N-terminus to the C-terminus:N-VHA- CH1A-Linker-VHB-CH1B-C and N-VLA-CKA-Linker-VLB It is in the format -CKB-C.

[0201] In one embodiment, the multispecific binding molecules are VHA, CH1A, linker, VHB, and CH It contains a heavy chain including 1B, which is as described in Sequence ID No. 130.

[0202] In one embodiment, the heavy chain is encoded by a nucleic acid sequence such as that described in Sequence ID No. 131. It can be done.

[0203] In one embodiment, the multispecific binding molecules are VLA, CKA, linker, VLB, and CKB. It includes a light chain, which is as described in Sequence ID No. 135.

[0204] In one embodiment, the light chain is encoded by a nucleic acid sequence such as that described in Sequence ID No. 136. It can be done.

[0205] This disclosure also describes a multispecific binding molecule comprising an anti-BTC binding moiety and an anti-VEGF binding moiety. The anti-BTC binding portion consists of a variable heavy chain domain (VHA) and a variable light chain domain that binds to BTC. It contains a chain domain (VLA), and the anti-VEGF binding moiety is a variable heavy chain domain that binds to VEGF. It includes a yin (VHB) and a variable light chain domain (VLB), where: a. VHA and VLA are amino acids as described in Sequence ID No. 54 and 65, respectively. Includes arrays; b. VHB and VLB are such that they are as described in Sequence ID No. 101 and 112, respectively. Contains an acid sequence.

[0206] In one embodiment, the anti-BTC binding moiety consists of a heavy chain constant domain (CH1A) and a light chain constant domain. It further contains (CKA), and the anti-VEGF binding portion consists of a heavy chain constant domain (CH1B) and a light chain. It further contains chain-constant domains (CKBs).

[0207] In one embodiment, the multispecificity binding molecule is arranged from the N-terminus to the C-terminus:N-VHA- CH1A-Linker-VHB-CH1B-C and N-VLA-CKA-Linker-VLB It is in the format -CKB-C.

[0208] In one embodiment, the multispecific binding molecules are VHA, CH1A, linker, VHB, and CH It contains a heavy chain including 1B, which is as described in Sequence ID No. 140.

[0209] In one embodiment, the heavy chain is encoded by a nucleic acid sequence such as that described in Sequence ID No. 141. It can be done.

[0210] In one embodiment, the multispecific binding molecules are VLA, CKA, linker, VLB, and CKB. It contains a light chain, which is as described in Sequence ID No. 145.

[0211] In one embodiment, the light chain is encoded by a nucleic acid sequence such as that described in Sequence ID No. 146. It can be done.

[0212] Furthermore, this disclosure describes a multispecific binding component including an anti-BTC binding portion and an anti-VEGF binding portion. A child is provided, and the anti-BTC binding moiety is a variable heavy chain domain (VHA) that binds to BTC and It contains a variable light chain domain (VLA), and the anti-VEGF binding moiety is a variable light chain domain that binds to VEGF. It includes a chain domain (VHB) and a variable light chain domain (VLB), where: a. VHA and VLA are amino acids as described in Sequence ID No. 78 and 88, respectively. Includes arrays; b. VHB and VLB are such that they are as described in Sequence ID No. 101 and 112, respectively. Contains an acid sequence.

[0213] In one embodiment, the anti-BTC binding moiety consists of a heavy chain constant domain (CH1A) and a light chain constant domain. It further contains (CKA), and the anti-VEGF binding portion consists of a heavy chain constant domain (CH1B) and a light chain. It further contains chain-constant domains (CKBs).

[0214] In one embodiment, the multispecificity binding molecule is arranged from the N-terminus to the C-terminus:N-VHA- CH1A-Linker-VHB-CH1B-C and N-VLA-CKA-Linker-VLB It is in the format -CKB-C.

[0215] In one embodiment, the multispecific binding molecules are VHA, CH1A, linker, VHB, and CH It contains a heavy chain including 1B, which is as described in Sequence ID No. 149.

[0216] In one embodiment, the heavy chain is encoded by a nucleic acid sequence such as that described in Sequence ID No. 150. It can be done.

[0217] In one embodiment, the multispecific binding molecules are VLA, CKA, linker, VLB, and CKB. It includes a light chain, which is as described in Sequence ID No. 154.

[0218] In one embodiment, the light chain is encoded by a nucleic acid sequence such as that described in Sequence ID No. 155. It can be done.

[0219] This disclosure relates to a multispecificity binding molecule comprising a first polypeptide chain and a second polypeptide chain. The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 120, and the second poly The peptide chain contains 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 The second polypeptide chain is encoded by the nucleic acid sequence of sequence number 126.

[0221] This disclosure also relates to a multispecific bond comprising a first polypeptide chain and a second polypeptide chain. The molecule provides, the first polypeptide chain contains the amino acid sequence of SEQ ID NO: 130, and the second The polypeptide chain contains 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 The second polypeptide chain is encoded by the nucleic acid sequence of sequence number 136.

[0223] This disclosure relates to a multispecificity binding molecule comprising a first polypeptide chain and a second polypeptide chain. Furthermore, the first polypeptide chain contains the amino acid sequence of SEQ ID NO: 140, and the second The polypeptide chain contains 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 The second polypeptide chain is encoded by the nucleic acid sequence of sequence number 146.

[0225] This disclosure also relates to a multispecific bond comprising a first polypeptide chain and a second polypeptide chain. The molecule provides, the first polypeptide chain contains the amino acid sequence of SEQ ID NO: 149, and the second The polypeptide chain contains 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 The second polypeptide chain is encoded by the nucleic acid sequence of sequence number 155.

[0227] This disclosure codes for nuclei that encode multispecific binding molecules, as described throughout. This provides polynucleotides containing an ocidal sequence.

[0228] This disclosure also describes polynucleotide-containing expression cassettes as described throughout. To provide.

[0229] Furthermore, this disclosure provides vectors containing expression cassettes, as described throughout. To be served.

[0230] Furthermore, this disclosure describes host cells containing polynucleotides, as described throughout. It will be provided.

[0231] This disclosure relates to culturing host cells under conditions suitable for the expression of multispecific binding molecules or their fragments. The present invention provides a method for producing multispecific binding molecules, including the following:

[0232] In one embodiment, the method further includes purifying the multispecific binding molecule.

[0233] This disclosure relates to pharmaceuticals comprising an effective amount of multispecific binding molecules, as described throughout. A composition is provided.

[0234] In one embodiment, 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] Furthermore, this disclosure also includes, in the context of the subject, an effective amount of multiple specificity compounds as described throughout. Treatment of ophthalmic disorders in subjects requiring treatment, including administration of a compound molecule or pharmaceutical composition. A treatment method will be provided.

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

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

[0239] In one aspect, ophthalmic disorders include diabetic macular edema, age-related macular degeneration, and age-related macular degeneration with neovascularization. Degeneration, neovascular glaucoma, diabetic retinopathy, macular edema, pathological myopia, retinal vein occlusion, premature infants The group is selected from those consisting of retinopathy and abnormal vascular proliferation associated with nevus disorders.

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

[0241] In this disclosure, the subject is administered an effective amount of a multispecific binding molecule as described herein. Methods are provided for preventing, treating, or managing ophthalmic disorders, including, multiple specific Sex-binding molecules reduce retinal leakage and / or retinal thickening in the subject compared to the control subject. .

[0242] Furthermore, this disclosure also describes multispecific binding molecules or pharmaceutical compositions as described throughout. A kit including this will be provided.

[0243] In one embodiment, the kit further includes an instruction manual.

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

[0245] In this disclosure, the target is given approximately 0.25 mg / of the multispecific binding molecule described herein. Intravitreal administration, including doses ranging from 7.5 mg / eye, is necessary for prevention or treatment. In the target population, the prevention or treatment of macular edema, DME, AMD, neovascular AMD, or RVO. A method of treatment is provided.

[0246] In one embodiment, the dose is approximately 0.25 mg / eye, 0.75 mg / eye, 2.5 mg / eye, or The dosage is 7.5 mg per 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 embodiment, the doses are 0.25 mg / eye, 0.3 mg / eye, 0.35 mg / eye, and 0.4 mg / eye, 0.45mg / eye, 0.5mg / eye, 0.55mg / eye, 0.6mg / eye, 0 .65mg / eye, 0.7mg / eye, 0.75mg / eye, 0.8mg / eye, 0.85mg / Eye, 0.9mg / eye, 0.95mg / eye, 1.0mg / eye, 1.1mg / eye, 1.2mg / eye, 1.3mg / eye, 1.4mg / eye, 1.5mg / eye, 1.6mg / eye, 1.7mg / eye, 1.8mg / eye, 1.9mg / eye, 2.0mg / eye, 2.1mg / eye, 2.2mg / Eye, 2.3mg / Eye, 2.4mg / Eye, 2.5mg / Eye, 2.6mg / Eye, 2.7mg / eye, 2.8mg / eye, 2.9mg / eye, 3.0mg / eye, 3.1mg / eye, 3.2mg / Eye, 3.3mg / Eye, 3.4mg / Eye, 3.5mg / Eye, 3.6mg / Eye, 3.7mg / eye, 3.8mg / eye, 3.9mg / eye, 4.0mg / eye, 4.1mg / eye, 4.2mg / Eye, 4.3mg / Eye, 4.4mg / Eye, 4.5mg / Eye, 4.6mg / Eye, 4.7mg / eye, 4.8mg / eye, 4.9mg / eye, 5.0mg / eye, 5.1mg / eye, 5.2mg / Eye, 5.3mg / Eye, 5.4mg / Eye, 5.5mg / Eye, 5.6mg / Eye, 5.7mg / eye, 5.8mg / eye, 5.9mg / eye, 6.0mg / eye, 6.1mg / eye, 6.2mg / Eye, 6.3mg / Eye, 6.4mg / Eye, 6.5mg / Eye, 6.6mg / Eye, 6.7mg / eye, 6.8mg / eye, 6.9mg / eye, 7.0mg / eye, 7.1mg / eye, 7.2mg The dosage is / eye, 7.3mg / eye, 7.4mg / eye, or 7.5mg / eye.

[0255] In one embodiment, the multispecificity binding molecule is 1) an amino acid combination of SEQ ID NOs. 10 and 21, respectively. 1) an anti-BTC binding moiety containing a column, and 2) amino acids of sequence numbers 101 and 112, respectively. It contains an anti-VEGF binding moiety.

[0256] In another embodiment, the drug is administered once a month.

[0257] Generally, this disclosure is based in part on the discovery of antibodies specific to beta-cell phosphate (BTC). In particular, the inventors have developed an anti-BTC antibody having properties suitable for therapeutic utility. (i.e., the antibody had sufficient affinity and specificity to achieve the desired therapeutic effect) (Binds to C). Based in part on this discovery, this disclosure relates to another therapeutic part, for example, anti-V Contains an EGF antibody or an antibody specific to BTC bound to an antibody fragment, or a molecule containing an antibody fragment. The therapeutic composition is characterized by an antibody, or a fragment thereof, and BT. Proteins that bind to therapeutic targets in tissues containing C (e.g., the vitreous humor), and their This includes compounds (e.g., low molecular weight compounds) that modulate therapeutic targets in such tissues. If the therapeutic portion contains antibodies, the overall therapeutic composition may contain multispecific antibodies (e.g., (and may be bispecific antibodies). In certain embodiments, this specification also refers to anti-BTC antagonists. By administering necrotic and anti-VEGF antagonists, eye disorders (e.g., AMD) may occur. For example, methods are provided to treat neovascularization (AMD, DME, DR, etc.).

[0258] i.Terms Unless otherwise defined, all technical and scientific terms used herein are defined in accordance with the provisions of this publication. The meaning is the same as that generally understood by those skilled in the art in the relevant technical field. This includes all patents, published patent applications and non-patent publications, as cited herein. Any references are also referenced in their entirety. To facilitate understanding of this disclosure, Some terms and abbreviations used herein are defined below:

[0259] As used herein, the singular forms "a," "an," and "the" refer to the content of Unless explicitly indicated otherwise, it includes multiple referents. For example, a reference to "an antibody" refers to a mixture of two or more such antibodies. Includes.

[0260] Unless otherwise specified or evident from the context, numerical values ​​are used herein. The term "approximately" in relation to this means that it is within the normal acceptable range in the relevant technical field, for example. Therefore, it is understood that it is within two standard deviations of the mean. The values ​​are + / - 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0%. 1%, 0.05%, or 0.01%, preferably within + / - 10% of the indicated value. To obtain. When used before a range or list of numbers, the term "approximately" is used for each number. The following applies: for example, the phrase "approximately 1 to 5" should be interpreted as "approximately 1 to approximately 5," and For example, the phrase "approximately 1, 2, 3, 4" is interpreted as "approximately 1, approximately 2, approximately 3, approximately 4, etc." It should.

[0261] "to include (comprise)", "to include (comprises)", "to include (c Terms such as "comprising" are used in reference to sequences (e.g., amino acid sequences). In all cases, the sequence also "consist", "con It is also limited by terms such as "consists" and "consisting". It should be understood that this is possible. When used in this specification, it means "to be essentially derived from". The terms refer to the genus or species of the active pharmaceutical product contained in the method or composition, as well as the intent of the method or composition. Refers to a genus or species of excipient that is inert for the intended purpose. In some embodiments, it means "from this purpose". The phrase "becomes qualitative" refers to one or more additional activators other than the multispecific binding molecules of this disclosure. Explicitly exclude the inclusion of agents. In some aspects, it means "essentially derived from..." The phrase refers to one or more additional agents other than the multispecific binding molecules and the second concurrently administered agent of this disclosure. Explicitly exclude the inclusion of active ingredients.

[0262] The term "beta-cell phosphate" or "BTC" refers to the increasing number of EGF family members in organisms. This refers to the growth factor. BTC activity is 1) ErbB1; 2) ErbB4; 3) ErbB homozygous. Dimers (e.g., ErbB1 / ErbB1 and ErbB4 / ErB4) and / or 4) ErbB heterodimers (e.g., ErbB1 / ErbB2, ErB1 / ErB3, By binding to ErB1 / ErB4, ErB2 / ErB3, and ErB2 / ErB4) It can be measured. Dunbar and Goddard, Int'l. J. Bi See ochem. & Cell Biol., 2000, 32:805-815. BTC activity can also be measured by the level of phosphorylated ERK1 / 2. In humans, this is a BTC gene located on chromosome 4 at locus 4q13-q21. Encoded by the offspring. Human BTC is a 178-residue protein as pro-BTC. It is expressed as a quality (e.g., NCBI:NP_001720.1 or SEQ ID NO: 156). Mature, secreted human BTC consists of 80 amino acid residues, as described in Sequence ID No. 158. In other words, it is composed of residues 32-111 of pro-BTC. Mouse beta-cell phosphate is 1 It consists of 77 amino acid residues (e.g., NCBI:NP_031594.1). Tobeta-cell phosphate is composed of 177 amino acid residues (e.g., NCBI: GenB ank:BAA96731.1).

[0263] The term "vascular endothelial growth factor" or "VEGF" refers to organisms that possess VEGF activity. For example, it refers to proteins that induce the proliferation and migration of vascular endothelial cells, physiologically and It may be essential for both pathological and angiogenesis. In mammals, the VEGF family consists of five members. Members: VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C, and It contains VEGF-D. Human VEGF is a single gene mRNA alternative splicing At least six isoforms (VEGF) derived from 121 VEGF 145 VE GF 165 VEGF 183 VEGF 189 , and VEGF 206) exists as ( Ferrara N,Davis Smyth T.Endocr Rev 18:1- 22 (1997). The most abundant isoform of VEGF. 165 It is approximately 45,000 It is a basic heparin-binding dimeric glycoprotein with a molecular mass of 0 Daltons. The term "human VEGF" used in this document refers to 165 amino acid human vascular endothelial cell proliferation. Factors, and related 121 amino acids, 189 amino acids, and 206 amino acids, (and others (Isoform) Amino acid vascular endothelial growth factor (Leung et al., Sci ence 246:1306 (1989), and Houck et al., Mol.E Pairs that occur naturally together with ndocrin.5:1806(1991) This refers to the processing methods for protogenetic genes and their growth factors.

[0264] When used herein, the term “anti-BTC binding moiety” refers to a moiety specifically binding to BTC. It means a binding polypeptide (e.g., an antibody, or its antigen-binding fragment). Any misunderstanding To avoid this, non-limiting examples of "anti-BTC binding sites" include full-length antibodies and their anti- Examples of protoconjugated fragments include Fab, scFv, Fv, and single-domain antibodies. In certain embodiments, the anti-BTC antibody is Fab or scFv. In specific embodiments, anti-BTC The binding site specifically binds to human BTC and / or cynomolgus monkey BTC.

[0265] The term “anti-VEGF binding moiety” is used herein, as defined below. Polypeptides that specifically bind to VEGF (for example, as described below) This refers to an antibody, or its antigen-binding fragment. To avoid any misunderstanding, "anti-VEGF" is used. Non-limiting examples of "binding sites" include full-length antibodies and their antigen-binding fragments, for example, as follows: Examples include Fab, scFv, Fv, and single-domain antibodies, as described. In this embodiment, the anti-VEGF antibody is Fab or scFv. In a specific embodiment, anti-VEG The fluorine-binding site specifically binds to human VEGF-A.

[0266] "to bind specifically", "to bind specifically" "Specifically binds" or "Selectively binds" The phrase "actively binds)" refers to the relationship between an antigen (e.g., a protein) and the multiple components of this disclosure. When used to describe interactions between proteins and heavy specificity binding molecules, and other biological products in heterogeneous populations, for example, biological samples, for example, blood, serum, plasma or This refers to the binding reaction that determines the presence of an antigen in a tissue sample. Therefore, a certain designation Under immunoassay conditions, the multispecific binding molecules of this disclosure, which have specific binding specificity, For a given antigen, it binds at least twice the background amount, and also in other samples present in the sample. It does not bind to the antigen in substantially significant amounts. In one embodiment, under specified immunoassay conditions Below, the multispecific binding molecules of this disclosure, which have specific binding specificity, are used against specific antigens. It binds at least 10 times more than the background and is effective against other antigens present in the sample. Specific binding to antibodies or binding agents under such conditions does not occur. The multispecificity binding molecules of this disclosure are selected for their specificity to a particular protein. It may be necessary that it be done. As desired or as needed, this choice may be for other species (for example) can be achieved by subtracting a multispecific binding molecule that cross-reacts with molecules from mice or rats) or other subtypes. This can be achieved by subtracting a multispecific binding molecule that cross-reacts with molecules from mice or rats) or other subtypes.

[0267] In some embodiments, the specific binding of the multispecific binding molecules 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 6 M -1 , at least 5×10 6 M - 1 , at least 10 7 M -1 , at least 5×10 7 M -1 , at least 10 8 M -1 , at least 5×10 8 M -1 , at least 10 9 M -1 , at least 5×10 9 M -1 , at least 10 10 M -1 , at least 5×10 10 M -1 , at least 10 11 M -1 , at least 5×10 11 M -1 , at least 1012 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 1 4 M -1 、 at least 5×10 14 M -1 、 at least 10 15 M -1 、 or at least 5×10 15 M -1 of the equilibrium constant (K A )(k on / k off ). It means a binding accompanied by

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

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

[0270] The term "therapeutic target binding moiety," as used herein, means a molecule that specifically binds to a desired therapeutic target. This molecule can be an antibody or an antigen-binding fragment thereof (such as those described below), an antigen-specific binding moiety, e.g., a DARPin, a Fynomer , an affibody, an adnectin, an affilin, an anticalin, an avimer, a centyrin , or an RNA molecule such as an aptamer. This therapeutic target binding moiety can also be a polypeptide such as a receptor or a part of a receptor that specifically binds to a desired therapeutic target. To avoid any misunderstanding, 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 comprises at least two heavy (H) chains interconnected by disulfide bonds and It is a glycoprotein containing two light (L) chains. Each heavy chain has a heavy chain variable region (as specified herein). It consists of a VH (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains. It is composed of CH1, CH2, and CH3. Each light chain has a light chain variable region (see details below). It consists of the VL (abbreviated as VL in the text) and the light chain steady region. The light chain steady region is one of the CLs. It consists of the following domains. The VH and VL domains are referred to as the Framework Domain (FR). In this hypervariable region, called the Complementarity Determination Region (CDR), a more conservative region is inserted. It can be further subdivided. Each VH and each VL is directed from the amino terminus to the carboxyl terminus. It consists of three CDRs and four FRs arranged in the following order: FR1, CDR 1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains are antigens. It contains a binding domain that interacts with the immunoglobulin and the host group. The constant region of the antibody is the host group of the immunoglobulin. Diverse cells of the tissue or immune system (e.g., effector cells) and the first component of the classical complement system It can mediate binding to factors containing (Clq). The term "antibody" is used for monoclonal antibodies. Human antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, bispecific or multiple specific antibodies Specific antibodies are examples, but are not limited to these. Antibodies can be any isotype / class. S (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass ( For example, those of IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) It is possible.

[0272] The term "isotype" refers to the class of antibodies provided by heavy chain constant region genes (e.g., For example, it refers to IgM, IgE, IgG (e.g., IgG1 or IgG4). The isotype is It also includes a modified version of one of these classes, and that modification alters the Fc functionality. For example, it may be modified to enhance or reduce effector function or binding to Fc receptors. Antibodies can be of any isotype (e.g., immunoglobulin G (IgG), immunoglobulin G Immunoglobulin E (IgE), immunoglobulin M (IgM), immunoglobulin D (IgD), Immunoglobulin A (IgA) and immunoglobulin Y (IgY), class (e.g., Ig IgA1, IgA2, or subclasses of IgA1, IgG2, IgG3, IgG4, IgA1, and IgA2) It may be a body. When used herein, and unless otherwise specified. To the extent that it is used, the terms "IgG" or "IgG antibody" mean G-type total antibody or Ig. do.

[0273] The term "light chain" refers to a full-length light chain and a variable region sufficient to confer binding specificity. It contains a fragment having a column. The full-length light chain has a variable region domain, V L And, steady-state domain Hmm, C L It includes the variable region domain of the light chain, located at the amino terminus of the polypeptide. This includes kappa chains and lambda chains.

[0274] The term "heavy chain" refers to a fully long heavy chain and a sufficient variable region to confer binding specificity. It contains its fragments having columns. The full-length heavy chain has a variable region domain, V H And three steady-state regions Domain, C H 1. C H 2, and C H Includes 3. V H The domain is the polypeptide ami Located at the terminal end, C H The domain is located at the carboxyl terminus, C H 3 is the polypeptide's carbohydrate It is closest to the xy terminus. The heavy chain is IgG(IgG1, IgG2, IgG3, and IgG4). IgA (including IgA1 and IgA2 subtypes), IgM and Ig It could be any isotype, including E.

[0275] The term "variable region" or "variable domain" typically refers to the amino terminus of a heavy chain. Approximately 120-130 amino acids, and about 100-110 amino acids at the amino terminus of the light chain This refers to the light chain and / or heavy chain portion of an antibody, including, in certain embodiments, different antibodies. Variable regions differ significantly in amino acid sequences even among antibodies of the same species. Typical variable regions of antibodies are... The target determines the specificity of a particular antibody against that target.

[0276] The terms “complementarity-determining region” and “CDR” are used herein to mean antigen-specific regions. This refers to the amino acid sequence within the antibody variable region that confers isomerism and binding affinity. Generally, In each heavy chain variable region (HCDR1, HCDR2, and HCDR3), there are three CDRs and There are three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The exact amino acid sequence boundaries of a given CDR are defined in Kabat et al. (1991). “Sequences of Proteins of Immunological Interest,”5th Ed.Public Health Service,N ational Institutes of Health,Bethesda,MD ("Kabat" numbering scheme), Al-Lazikani et al., (19 97) JMB 273,927-948 ("Chothia" numbering scheme); and Lefranc et al.,(2003)Dev.Comp.Immunol.,2 A large number of well-known terms are listed in 7,55-77 ("IMGT" numbering scheme). The determination can be made using one of the schemes. The Kabat definition is antibody This is a criterion for numbering the residues within a molecule, and is typically used to identify the CDR region. It is used. For example, Johnson & Wu, Nucleic Acids Research. See 28:214-8 (2000). The Chotia definition is the Kabat definition. Similar to the above, but the Chotia definition takes into account a specific structural loop region. For example , Chothia et al., J. Mol. Biol., 196:901-17(1 986);Chothia et al.,Nature,342:877-83(19 See 89).

[0277] Other methods can be used as alternatives to depict the CDR area, for example, Kab It is possible to combine CDR definitions from both at and Chotia ("combination"). (system). For example, under Kabat, for the classical form, the heavy chain variable domain ( In VH, the CDR amino acid residues are 31-35 (HCDR1) and 50-65 (HCD). R2) and 95-102 (HCDR3) are numbered; and the light chain variable domain (VL) The CDR amino acid residues in this compound are 24-34 (LCDR1) and 50-56 (LCDR2). They are also numbered 89-97 (LCDR3). Based on Chothia, VH CD The R amino acids are 26-32 (HCDR1), 52-56 (HCDR2), and 95-102. (HCDR3) is numbered as such; and the amino acid residues of VL are 26-32 (LCDR1). They are numbered 50-52 (LCDR2) and 91-96 (LCDR3). Kaba By combining the CDR definitions of both t and Chothia, combined CDR This refers to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) of human VH and 95-102 (HCDR3) and amino acid residues 24-34 (LCDR1) of human VL, It consists of 50-56 (LCDR2) and 89-97 (LCDR3). Another example is IM. According to GT, the number of CDR amino acid residues within the heavy chain variable domain (VH) is 26-33 (HC They are numbered as DR1), 51-58 (HCDR2), and 97-108 (HCDR3). The CDR amino acid residues within the light chain variable domain (VL) are 27-36 (LCDR1 They are numbered as 54-56 (LCDR2) and 93-101 (LCDR3). .

[0278] The terms “antibody framework” or “FR” are used herein to mean “variable” This refers to the domain, VL, or VH portion, and the antigen-binding loop of this variable domain ( It functions as a scaffold for a CD-R. Essentially, it does not have a CD-R. It is a variable domain. Under IMGT, the CDR region of the antibody is programmed as IMGT / D This can be determined using omainGap Align.

[0279] The term "antigen-binding fragment" of an antibody, as used herein, refers to a given antigen (for example) If, then, one or more antibodies that retain the ability to specifically bind to BTC and VEGF Refers to a fragment, or one or more polypeptides containing such fragments. The antigen-binding function of an antibody is This can be performed with intact antibody fragments. This is encompassed within the term "antigen-binding fragment of an antibody." Examples of binding fragments include those consisting of the Fab fragment, VL, VH, CL, and CH1 domain. Monovalent fragment; F(ab)2 fragment, two fragments linked by disulfide bridges in the hinge region A divalent fragment containing a Fab fragment; an Fd fragment consisting of VH and CH1 domains; VL and VH Fv fragment consisting of domains; VL and VH domains joined by a linker sequence A single-stranded Fv fragment (scFv); and a single domain consisting of a VH domain or a VL domain. In antibody (dAb) fragment (Ward et al., 1989 Nature 341: Examples include, but are not limited to, 544-546. Antigen-binding fragments are single domains. Antibodies, maxibody, minibody, intrabody, diabody, triabody, It can also be imported into Trabody, v-NAR, and bisscFv (for example, Hollin ger and Hudson,2005 Nature Biotechnology See 23, 9, 1126-1136). The antigen-binding portion of the antibody is fibrone Graftable within a polypeptide-based scaffold, such as cucin type III (Fn3). It is possible (fibronectin polypeptide monobody is described in U.S. documents 6, 7 (See Specification No. 03,199). The antibody-binding fragment is a complementary light chain polypeptide ( For example, V L -VC-V L -VC) forms a pair of antigen-binding domains, tandem Fv segment (for example, V H -CH1-V H By incorporating the pair of -CH1) into a single-chain molecule This is possible (Zapata et al., (1995) Protein Eng. 8) (1057-1062; and U.S. Patent No. 5,641,870).

[0280] The “Fab” fragment used herein consists of one constant domain each of the heavy chain and the light chain. It contains one variable domain. The heavy chain of the Fab molecule is disulfide bonded to another heavy chain molecule. It cannot be formed.

[0281] As used herein, the “Fab' fragment” consists of one light chain and a VH domain and C H 1 Main and also C H 1 and C H A portion of one heavy chain containing the region between the two domains This includes the formation of interchain disulfide bonds between the two heavy chains of the two Fab' fragments. Thus, it is possible to form an F(ab')2 molecule.

[0282] As used herein, the “F(ab')2 fragment” consists of two light chains and C H 1 domain and C H It contains two heavy chains that include a portion of the constant region between the two domains, thereby 2 An interchain disulfide bond is formed between the two heavy chains. Therefore, the F(ab')2 fragment is Two Fab' fragments are held together by disulfide bonds between the two heavy chains. It consists of.

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

[0284] As used herein, the terms "single-stranded Fv" or "scFv" refer to the V of an antibody. H and V L This refers to antibody fragments containing domains, which exist as a single polypeptide chain. Preferably, the Fv polypeptide has scFv that forms the desired structure for antigen binding. V makes it possible to do H Domain and V L Internal polypeptide linker between the domain Furthermore, scFV also has manipulated internal disulfide bridges to enhance stability. It is possible. For a discussion of scFv, see Plueckthun in The Ph armacology of monoclonal antibodies, vol. 113,Rosenburg and Moore eds.,(1994)Sprin See Ger-Verlag, New York, pp. 269–315. In a more specific embodiment, the scFv used in the multispecific binding molecule of this disclosure has a general structure: NH2 -V L -Linker-V H -COOH or NH2-V H -Linker-V L - Contains COOH ru.

[0285] As used herein, "affinity-matured" antibodies have one or more CDRs. These antibodies have modifications, and compared to parent antibodies that do not have those modifications, they are more effective against the antigen. This results in improved affinity of the antibody. A preferred affinity mature antibody against the target antigen. Affinity-mature antibodies will have nanomolar or even picomolar affinity. It can be manufactured using methods known in the field. Marks et al. Bi o / Technology 10:779-783(1992) describes the VH and VL domains. Affinity maturation through reshuffling is described. CDR and / or framework remaining The original random mutation method is described in: Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813(1994);Schier et al. Gene 169:147-155(1995);Yelton et al.J.Im munol.155:1994-2004(1995);Jackson et al. ,J.Immunol.154(7):3310-9(1995);and Hawkins According to et al, J.Mol.Biol.226:889-896(1992) It is listed.

[0286] The “parent” or “parental” antibodies used herein are Encoded by the amino acid sequence used in the preparation of affinity-mature antibodies or their variants. The antibody is used. Preferably, the parent antibody has a human framework region and is present in the area In combination, it has a constant region of human antibodies. For example, the parent antibody may be a humanized antibody or a human antibody. good.

[0287] As used herein, the term "diabody" refers to a small body having two antigen-binding sites. This refers to a small antibody fragment, which is located in the light chain variable domain (VL) of the same polypeptide chain. Contains a ligated heavy chain variable domain (VH) (VH-VL). Two domains on the same chain. By using linkers that are too short to pair them, the domains are paired with the other chain. It is forced to pair with the complementary domain, creating two antigen-binding sites. For example, see European Patent No. 404,097; International Publication No. 93 / 11161. Nfrett; and Hollinger et al., (1993) Proc. Natl This is fully described in Acad.Sci.USA 90:6444-6448.

[0288] As used herein, the terms "single-specificity binding molecule" or "single-specificity antibody" are defined as follows: This refers to a molecule that binds to a single epitope on a target antigen. In one embodiment, the single specificity of this disclosure The binding molecule or monospecific antibody binds to BTC. In another embodiment, the monospecific antibody of this disclosure The binding molecule or monospecific antibody binds to VEGF.

[0289] As used herein, the terms "multispecificity binding molecule" or "multispecificity antibody" are defined as follows: This refers to molecules that bind to two or more different antigens. The recognition of each antigen is generally called "antigen-binding." via the main domain (e.g., "BTC antigen-binding domain", "VEGF antigen-binding domain") This is achieved by "multispecificity". The term "multispecificity" refers to "dual specificity" of binding to two different antigens. "Specificity," that is, it includes molecules. In some embodiments, the multiple specificity binding molecules are each one It contains one or more polypeptide chains including an antigen-binding domain. In one embodiment, multiple specificity The sex-binding molecule contains VH or VL. In some embodiments, the multispecific binding molecule is Each of the following polypeptide chains contains two or more antigen-binding domains (for example, two) Contains. In some embodiments, multiple specificity binding molecules together, for example, two The above, for example, includes two, three, or four antigen-binding domains, or It contains more than one polypeptide chain.

[0290] The terms "bispecificity conjugation molecule" or "bispecificity antibody" refer to a single molecule containing two antibodies. This refers to molecules that combine antigen-binding sites on the body. In one embodiment, it may refer to a bispecificity binding molecule or a bi The specific antibody contains a single polypeptide. In another embodiment, a bispecificity conjugate molecule or Bispecific antibodies consist of two antibodies linked via disulfide crosslinks or any other covalent bonds. It contains polypeptides. Therefore, the bispecific antibody can simultaneously detect two different antigens. These can be bound sequentially. A method for producing bispecific antibodies is described in the technical field. This is well known in the field. Various forms for combining two antibodies are also known in the field. It is known. The form of the bispecific antibody of this disclosure is known to those skilled in the art, Diabody, single-stranded diabody, dimerized Fab (Fab-Fab), Fab-sc Examples include, but are not limited to, Fv and tandem antibodies.

[0291] As used herein, the term “divalent molecule” refers to a molecule having two antigen-binding domains. Refers to a child. The term “trivalent molecule” as used herein refers to a molecule with three antigen-binding domains. This refers to a molecule possessing a trivalent antibody. In some embodiments, the trivalent molecule of this disclosure is a trivalent antibody-like molecule. In some embodiments, the trivalent molecule can bind to the same epitope of the same antigen. It consists of one antigen-binding domain and a third antigen-binding domain that binds to a different antigen. This is possible. Such a form can be considered a trivalent bispecific molecule.

[0292] The term "polyvalent molecule" refers to a molecule that has at least two antigen-binding sites, These antigen-binding molecules may have specificity for the same antigen or different antigens. In some embodiments, the polyvalent molecules of this disclosure are polyvalent antibody-like molecules. The valent molecule is a polyvalent antibody. In some embodiments, the polyvalent molecule is a divalent molecule, a trivalent molecule, or It is a tetravalent molecule. The trimerized domain is, for example, in European Patent No. 1012280B1. It is described in the book. The pentamerization module is described in PCT / European Patent No. 97 / 05897. It is described in the details.

[0293] As used herein, the terms “substantially similar” or “substantially identical” are: Two numerical values ​​(generally, one relating to the antibody-like molecule of this disclosure and the other to reference / (related to comparator antibodies or antibody-like molecules) Yes, and thereafter a person skilled in the art will know that the difference between two values ​​is the value (for example, the TM value or assembly value) In relation to biological properties measured by the amount of antibodies released, biological and / or This refers to a high degree of similarity where the statistical difference is considered to be little to no. The difference between the two values ​​is: As a function of the value for the reference / comparator antibody, preferably less than about 50%, preferably less than approximately 40%, preferably less than approximately 30%, preferably less than approximately 20%, preferably about 10 It is less than %.

[0294] The term "antigen" refers to an antigen-binding protein (e.g., an antibody or its immunological function). This refers to a molecule or a part of a molecule that can be bound by a selective binder (including a fragment). In some embodiments, an antigen produces antibodies in animals that can bind to that antigen. It can be used to produce antigens, which are different antigen-binding proteins, such as antibodies. It may possess one or more epitopes with which it can interact.

[0295] As used herein, the terms “epitope” or “antigenic determinant” refer to an antibody or antibody. An epitope is an arbitrary determinant that can bind to a molecule with high affinity. , the region of the antigen to which it is bound by an antibody (or antibody-like molecule) that specifically targets that antigen. And, if the antigen is a protein, a specific amino acid comes into direct contact with the antibody or antibody-like molecule. It contains acid. In most cases, epitopes are located on proteins, but in some cases, It can exist on other types of molecules such as nucleic acids. Epitope determinants are amino acids, The molecule contains chemically active surface groups such as sugar side chains, phosphoryl or sulfonyl groups. It can be made possible and may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0296] Generally, multispecificity binding molecules that are specific to a particular target antigen are proteins and / or It will preferentially recognize the epitope on its target antigen within the complex mixture of polymers.

[0297] A given region of polypeptide containing an epitope is one of several known in the art. It can be identified using epitope mapping techniques. For example, Epitope Mapping Protocols in Methods in Molecules ar Biology,Vol.66(Glenn E.Morris,Ed.,199 6) See Humana Press, Totowa, and New Jersey. For example, linear epitopes can simultaneously synthesize a large number of peptides on a solid support ( The peptide is equivalent to a part of a protein molecule, and the peptide is still supported by This can be determined by reacting the peptide with the antibody while it is attached to the antibody. Such techniques are known in the relevant technical field, for example, U.S. Patent No. 4,708 , No. 871; Geysen et al., (1984) Proc.Natl.A cad.Sci.USA 8:3998-4002;Geysen et al.,(1 985)Proc.Natl.Acad.Sci.USA 82:78-182;Gey sen et al., (1986) Mol. Immunol. 23:709-715. It is described. Similarly, the three-dimensional epitope is, for example, determined by X-ray crystallography and two-dimensional analysis. It can be easily identified by determining the spatial three-dimensional structure of amino acids, such as by nuclear magnetic resonance. For example, please refer to the epitope mapping protocol above. (Protein antigen region) The region also has Om, which is available from Oxford Molecular Group, for example. Standard calculations, such as those performed using the IGA version 1.0 software program. This can be identified using antigenicity and hydrophobicity plots. This computer program For determining the antigenic profile, the Hopp / Woods method and the Hopp et al. al.,(1981)Proc.Natl.Acad.Sci USA 78:3824 -3828, and for hydrophobic plots, Kyte-Doolittle technique, Kyt Using e et al., (1982) J.MoI.Biol.157:105-132 ru.

[0298] The term "competing" refers to antigen-binding proteins that compete for the same epitope. When used in this way, the antigen-binding protein being tested (e.g., an antibody or its immune function) A scientifically functional fragment binds to a reference antigen (e.g., BTC or a fragment thereof) To prevent or inhibit the specific binding of a protein (e.g., a ligand or reference antibody) (for example) This means competition among antigen-binding proteins, as determined by assays (which reduce competition). Using a variety of competitive binding assays, one antigen-binding protein binds to another. It can be determined whether there is competition, for example: solid-phase direct or indirect radioimmunotherapy RIA (Rich Induction Activation), Solid-Phase Direct or Indirect Enzyme Immunoassay (EIA), Sandwich Competitive assays (e.g., Stahli et al., 1983, Methods in See Enzymology 9:242-253); Solid-phase direct biotin- Bisin EIA (e.g., Kirkland et al., 1986, J. Immuno) See l.137:3614-3619), solid-phase direct labeling assay, solid-phase direct labeling Recognition sandwich assay (e.g., Harlow and Lane, 1988, Ant) ibodies,A Laboratory Manual,Cold Spring See Harbor Press; Solid-phase direct labeling using I-125 labeling R IA (e.g., Morel et al., 1988, Molec.Immunol.2) See 5:7-15); Solid-phase direct biotin-avidin EIA (e.g., Cheu See ng, et al., 1990, Virology 176:546-552. (Tai); and directly labeled RIA (Moldenhauer et al., 1990, Sc (and.J.Immunol.32:77-82) is an example of this. Typically, The assay involves cells having purified antigen bound to a solid surface or either of these, and non This involves the use of a labeled test antigen-binding protein and a labeled reference antigen-binding protein. The harm is determined by the amount of label bound to a solid surface or cell in the presence of the test antigen-binding protein. It is measured by [method]. Normally, the test antigen-binding protein is present in excess. Competition Antigen-binding proteins (competing antigen-binding proteins) identified by Issey are, When an antigen-binding protein binds to the same epitope as an antigen-binding protein, steric hindrance occurs. In order to do so, the adjacent area is sufficiently close to the epitope bound by the reference antigen-binding protein. It includes an antigen-binding protein that binds to an epitope. A method for determining competitive binding. Further details regarding this are provided in the examples herein. Typically, competing antigen-binding tangs When the protein is present in excess, it leads to the specific binding of the reference antigen-binding protein to the common antigen. at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65% It will inhibit (e.g., reduce) by %, 65-70%, 70-75%, or more than 75%. In some cases, the binding is at least 80-85%, 85-90%, 90-95%, and 95%. ~97%, or more than 97%, of inhibition is achieved.

[0299] The terms "polypeptide" and "protein" are used interchangeably in this specification. This term refers to a polymer of amino acid residues. This phrase also refers to a polymer of one or more amino acid residues. amino acid polymers are artificial chemical mimetic substances of naturally occurring amino acids. - and also, naturally occurring amino acid polymers and naturally occurring amino acid polymers This applies to certain amino acid polymers. Unless otherwise specified, specific polypeptide sequences are used. It implicitly includes the conservatively modified variant.

[0300] As used herein, the term "polypeptide chain" includes all constituent regions and This refers to a complete amino acid chain of the multispecific binding molecule of the present disclosure, which contains a bin domain within it.

[0301] The term "constant region" or "constant domain" directly relates to the binding of an antibody to an antigen. Although not directly involved, the light chain and heavy chain exhibit various effector functions, such as interactions with Fc receptors. This refers to the carboxyl terminus of an immunoglobulin chain. Immunoglobulin components that have a more conserved amino acid sequence compared to other parts, variable domains. This refers to the child portion. The constant domains are the CH1, CH2, and CH3 domains of the heavy chain and the light chain. It contains the CL domain.

[0302] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as naturally occurring Amino acid analogs and amino acid mimetics (imitation substances) that function similarly to existing amino acids. ) refers to naturally occurring amino acids, which are coded by the genetic code, and For example, for post-translational modification, a later modified amino acid, such as hydroxyproline γ-carboxyglutamate, pyroglutamate, C-terminal lysine cleavage, and O-phosphorus It is joselin. Amino acid analogs have the same basic chemical structure as naturally occurring amino acids. In other words, a compound having hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, For example, homoserine, norleucine, methionine sulfoxide, methionine methylsulfon This refers to um. Such analogues are modified R groups (e.g., norleucine) or modified peptides. It has a skeleton, but retains the same basic chemical structure as naturally occurring amino acids. Metics (mimicking substances) have a structure different from the general chemical structure of amino acids, but they are naturally occurring. This refers to chemical compounds that function similarly to amino acids found in the natural world.

[0303] In polypeptide sequences, "conservatively modified variants" are, chemically speaking, amino acids. Individual substitutions in polypeptide sequences, resulting in substitutions by similar amino acids. Includes deletions or additions. Conservative substitution tables that provide functionally similar amino acids are well known. Such conservatively modified variants are considered to be polymorphic variants of the present disclosure, interspecies variants. This adds to homologs and alleles, and does not exclude them. The eight groups are: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamate 1) Asparagine (E); 2) Asparagine (N), Glutamine (Q); 3) Arginine (R), Li Syn(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) are opposite each other. and contain amino acids that are conservative substitutions (e.g., Creighton, Protein See ns(1984). In some aspects, this is called "conservative sequence modification" or "conservative sequence modification". Does the term "existential modification" mean that it does not significantly affect the binding properties of antibodies containing amino acid sequences? Alternatively, it is used to refer to amino acid modifications that do not alter the original amino acid.

[0304] In certain contexts, the term "dose" can mean the entire amount (unit dose) of an object at once, or a fixed amount. The amount of therapeutic agent or therapeutic target binding portion administered in two or more doses over a specified time interval. This refers to a drug whose therapeutic agent is a protein (e.g., an antibody or antigen-binding fragment) or nucleic acid. Even if the therapeutic target binding portion is a small molecule (e.g., <900 Daltons) therapeutic compound, It is also acceptable to do so. For example, the dosage may be for 3 weeks or 1 month, 2 months, 3 months, 4 months, 5 months. The medication is administered to the subject over a period of six months or more (for example, by a single dose, or By administering the protein (e.g., anti-BTC antibody or molecule conjugate) through two or more doses. The functional fragment, or the anti-VEGF antibody or the protein containing the functional fragment. This can refer to the amount of ). The interval between doses may be any desired period, and "dose It is called an "interval."

[0305] When referring to dosage, the term "pharmaceutically effective" means the desired effect (e.g., improvement). Proteins (for example, to provide vision loss or to prevent further vision loss) This refers to a sufficient amount of an antibody or antigen-binding fragment, or other pharmaceutically active agent. The amount of "na" depends on the individual's age and overall health, the specific drug or pharmaceutical active agent, etc. It will vary between patients. Therefore, specifying an exact "effective" dose applicable to all patients is difficult. It is not always possible to do so. However, in any individual case, appropriate The "effective" dose can be determined by those skilled in the art using routine experiments.

[0306] The term "human antibody" is used herein in the framework area and CDR. The intention is to include antibodies in which both regions have variable regions derived from human-derived sequences. Furthermore, if the antibody contains a constant region, the constant region is also such a human sequence, for example, human biological Reproductive line sequences, or variants of human germline sequences, or, for example, Knappik, et al. As described in al. (J.Mol.Biol.296,57-86,2000), Antibodies containing consensus framework sequences derived from human framework sequence analysis The human antibodies of this disclosure are derived from amino acid residues not encoded by the human sequence (e.g., , random or site-directed mutagenesis in vitro or somatic mutation in vivo This may include mutations introduced or conservative substitutions to enhance stability or production. However, as used herein, the term “human antibody” refers to a different mammalian species such as a mouse. This antibody contains a germline-derived CDR sequence graphed onto a human framework sequence. That was not the intention.

[0307] The terms "monoclonal antibody" or "monoclonal antibody composition" used herein refer to... The term refers to antibodies and This term refers to polypeptides containing antigen-binding fragments. This term also includes the preparation of antibody molecules as single-molecule compositions. It also includes agents. Monoclonal antibody compositions have a single binding specificity to a particular epitope. It exhibits sex and affinity. Monoclonal antibodies are generated using phage display technology. The method for doing so is known in the art (Proetzel, G., Eber). sbach, H. (Eds.) Antibody Methods and Proto cols.Humana Press ISBN 978-1-61779-930-3 (2012).

[0308] The term "humanized" form of non-human (e.g., mouse) antibodies refers to non-human immunoglobulins. It is a chimeric antibody containing the minimum sequence derived from [the original source]. In most cases, humanized antibodies are, The residues from the recipient's hypervariable region possess the desired specificity, affinity, and ability. Hypervariable regions (donor antibodies) of non-human species such as mice, rats, rabbits, or non-human primates. Human immunoglobulin (recipient antibody) that has been replaced by residues from In some cases, the framework region (FR) residues of human immunoglobulins correspond It is replaced by non-human residues. Furthermore, humanized antibodies are either recipient antibodies or donors. These modifications may include residues that cannot be found in the antibody. These modifications further refine antibody performance. This may be done for that purpose. Generally, humanized antibodies have at least one and typically two possible This includes virtually all of the variable domains, and within them all or substantially all of the hypervariable loops. All correspond to non-human immunoglobulins, and all or virtually all of FR are human immunoglobulins. It corresponds to the robulin lo sequence. Humanized antibodies can also optionally target the immunoglobulin constant region. This would include at least some of Fc), typically those of human immunoglobulins. For further details, see Jones et al., Nature 321:522-525. (1986);Riechmann et al.,Nature 332:323-3 29 (1988); and Presta, Curr Op. Struct. Biol. 2:5 See 93-596 (1992). Also, see the review article and the references therein. See also the bibliography: Vaswani and Hamilton, Ann. All ergy, Asthma & Immunol.1:105-115(1998);Ha rris,Biochem.Soc.Transactions 23:1035-10 38(1995);Hurle and Gross,Curr.Op.Biotech .5:428-433 (1994).

[0309] As used herein, “identity” refers to two polypeptides, intermolecular or nuclei. This refers to sequence matching between acids. It involves matching the positions of the same base or amino acid in both sequences being compared. When occupied by an acid (for example, the position in each of the two polypeptides is lig (When occupied by n), each molecule is identical at that position. Between the two sequences The "identity percentage" is a value that needs to be introduced for the optimal alignment of the two sequences. Considering the number of gaps and the length of each gap, the same position shared by these sequences It is a function of the number of. In general, when two arrays are sorted, to give the greatest identity A comparison is made. Such alignments include, for example, Needleman and Wuns Ch's method (J.MoI.Biol.(48):444-453(1970))(Blo Either a sum62 matrix or a PAM250 matrix, and 16, 14, 1 Gap weighting of 2, 10, 8, 6, or 4 and length weighting of 1, 2, 3, 4, 5, or 6 Using the search function, the GAP program of the GCG software package is incorporated. It can be provided using an algorithm. In the case of array comparison, typically one The sequence acts as a reference sequence, which is then compared to the test sequence. When using rhythms, enter the test sequence and reference sequence into the computer, and if necessary Specify the sub-array coordinates and the array algorithm program parameters. Initial settings The program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm tests the reference sequence based on the program parameters. The percentage sequence identity of the sequences is calculated. After optimally determining the sequences of the two sequences, the sequences are... , comparable to a reference array with the same number of consecutive positions, 20-600, typically about 50-2 00, more typically, the number of consecutive positions selected from a group consisting of approximately 100 to 150. If a segment is mentioned using any one of these, use the comparison window. This is possible. For comparison, the sequence alignment method is well known in the art. The optimal sequence alignment for comparison is, for example, Smith and Waterm. Local homology algorithm by an(1970)Adv.Appl.Math.2:482c According to Needleman and Wunsch, J.Mol.Biol.48 According to the homology alignment algorithm by :443,1970, Pearson a nd Lipman,Proc.Nat'l.Acad.Sci.USA 85:244 The similarity search method 4,1988 allows for the computerized implementation of these algorithms. (Wisconsin Genetics Software Package, Ge netics Computer Group,575 Science Dr.,Ma (Dison, WI's GAP, BESTFIT, FASTA, and TFASTA) Or manual alignment and visual inspection (e.g., Brent et al., Cur rent Protocols in Molecular Biology,John See Wiley & Sons, Inc. (Ringbou ed., 2003). This can also be done by (the desired method). Determine the sequence identity percentage and sequence similarity percentage. Two examples of algorithms suitable for determining this are, respectively, Altschul et al. ., Nuc. Acids Res. 25:3389-3402, 1977; and Alts chul et al., J. Mol. Biol. 215:403-410, 1990. The BLAST and BLAST 2.0 algorithms described are presented. BLAST solution The software used for the analysis is from the National Center for Biotechnology Information (NCCI). Officially from the Inter for Biotechnology Information It is available. This algorithm performs array resolution using words of the same length in the database array. When set, the query distribution matches or satisfies a certain positive threshold score T. First, identify high-scoring sequence pairs (HSPs) by identifying short word lengths W within the sequence. This involves setting T to the adjacent word score threshold (Altschul et al., previously mentioned) These initial adjacent word hits find longer HSPs that contain them. It acts as a seed to initiate the search. As long as the cumulative alignment score can increase... Then, word hits are extended in any direction along each sequence. The cumulative score is Nuku In rheotide sequences, the parameter M (reward score for matching residue pairs; constant) Using >0) and N (penalty score for mismatch residues; always <0) Calculate. For amino acid sequences, use a scoring matrix to calculate the cumulative score. If the cumulative alignment score decreases by an amount X from its maximum value achieved; 1 Due to the accumulation of alignment of more than one negative score residue, the cumulative score decreases to zero or less. If; or if the end of any of the arrays is reached, stop the expansion of word hits in each direction. To stop. Alignment is determined by the BLAST algorithm parameters W, T, and X. The sensitivity and speed of the test are determined using the BLASTN program (for nucleotide sequences). This uses default settings of 11 word length (W), 10 expected value (E), M=5, and N=-4. Use and compare both chains. In the BLASTP program for amino acid sequences, 3 The word length, the expected value (E) of 10, and the BLOSUM62 scoring matrix of 50. Henikoff and Henikoff, Proc. Natl. Academia See Sci.USA 89:10915, 1989) Alignment (B), We will use the expected value (E) of 10, M=5, and N=-4 as defaults and compare both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (for example) Karlin and Altschul, Proc. Natl. Acad. Sci. See .USA 90:5873-5787, 1993. BLAST algorithm One measure of similarity provided by the sm is the minimum sum probability (P(N)), and this This provides an index of the probability that compatibility between two nucleotide or amino acid sequences occurs by chance. For example, in the comparison of a test nucleic acid to a reference nucleic acid, the minimum sum probability is less than approximately 0.2. If, more preferably, less than about 0.01, and most preferably less than about 0.001, then the nuclear The acid is considered similar to the reference sequence. The percentage of identity between the two amino acid sequences. The PAM120 weighted residue table, 12 gap length penalties, and 4 gaps It was incorporated into the ALIGN program (version 2.0) that uses a penalty. Furthermore, the algorithms of E. Meyers and W. Miller (Comput.Appl. This can be determined using Biosci., 4:11-17, 1988. The percentage of identity between two amino acid sequences is determined by the Blosum62 matrix or P AM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 GCG software uses length weighting of 1, 2, 3, 4, 5, or 6. The GAP program is included in the package (available on the internet at gcg.com). Needleman and Wunsch (J.Mol, Biol.4) were incorporated into the project. The decision can also be made using the algorithm described in 8:444-453, 1970. Other than the sequence identity percentages mentioned above, two nucleic acid sequences or polypeptides are substantially Another indicator of identity is the coding of the first nucleic acid, as described below. The lipeptide immunologically interacts with the antibody against the polypeptide encoded by the second nucleic acid. It is differential reactivity. Therefore, for example, two peptides can be substituted only by conservative substitutions. When they differ, the polypeptide is typically substantially identical to the second polypeptide. Yes. Another indicator that two nucleic acid sequences are substantially identical is described below: Two molecules, or their complements, hybridize with each other under strict conditions. Another indicator that two nucleic acid sequences are substantially identical is that they use the same primers. This allows for the amplification of two nucleic acid sequences.

[0310] "Percent complementarity" or "Complementary percentage" The term "percent complementary" has two meanings. When used herein with reference to the nucleotide sequence, the concept of identity percentage and Similarly, the query array and the target array are arranged linearly, and loops, stems, or heaps are used. When optimal base pairing occurs without secondary folding structures such as apins, the target sequence One hundred nucleotides of query sequences that optimally form base pairs or hybridize with nucleotides. This refers to a fraction. Such percentage complementarity is between two DNA strands, between two RNA strands. , or it may be between a DNA strand and an RNA strand. "Percent complementarity" is (i)2 Two nucleotide sequences are compared in linear and fully extended configurations across a comparison window (that is, (ii) optimally base-pair or hybridize without folding or secondary structure, Across the comparison window, the number of base-pairing positions between the two sequences is determined, and complementary positions are identified. (iii) Obtain the number of positions, and divide the number of complementary positions by the total number of positions in the comparison window. (iv) Multiply this quotient by 100% to obtain the percentage complementarity of the two sequences. It is calculated by the following: The optimal base pairing of two sequences is GC, AT, and AU. This can be determined based on known pairings of nucleotide bases through hydrogen bonds, etc. "Percent complementarity" refers to the reference sequence without specifying a particular comparison window. When calculated, the percentage identity is the number of complementary positions between two linear arrays, compared to the reference array. It is determined by dividing by the total length of . Therefore, for the purposes of this disclosure, two sequences When (query and target) are optimally base-paired (mismatch or non-base-pairing nuclei) Otid is acceptable, but without folding or secondary structure, for query arrays, "parse "Base pair complementarity" is the number of base pairing positions between two sequences over the length of the query sequence. Divide by the total number of positions within (or by the number of positions in the query array across the comparison window) It is then equal to multiplying it by 100%.

[0311] The term "isolated antibody" refers to other antibodies or proteins that have different antigen specificity. This refers to antibodies that are substantially free of cellulose. Furthermore, isolated antibodies can be used with other cell materials and / or It may contain virtually no chemical substances, such as antibodies isolated from cell supernatant.

[0312] With respect to the anti-BTC multi-characteristic binding molecules described herein, the terms "bound" or "linked" The term refers, for example, to the anti-BTC antibodies listed in Table 1 or their functional decomposition to bind to BTC. This refers to the attachment of anti-BTC binding sites to molecules such as ligatures. The attachment is, for example, an amino acid of a molecule, such as an anti-VEGF antibody or its functional fragment. Alternatively, it can occur at the carboxyl terminus. The anti-BTC binding site is also at the amino end of the protein. It can be attached to both the terminal and carboxyl terminus. The anti-BTC binding portion is also These can be attached to one or more amino acids or nucleic acids within a protein or nucleic acid molecule. The linkage of the anti-BTC binding portion to the molecule is performed as a fusion protein between the anti-BTC binding portion and the molecule. After expression or translation, the anti-BTC binding site is directly attached to the molecule, either to each other or as a disulfide. This can be achieved by chemical bonding, such as through a linker. However, this is not limited to methods that can be achieved by known methods in the relevant art. It is possible.

[0313] The terms "linker" or "linked" in relation to multispecific binding molecules are used to describe multispecific A part of an isomerized molecule is directly or indirectly attached to another part of the molecule, for example. This refers to the anti-BTC binding portion being attached to the anti-VEGF binding portion. The linker is anti BTC binding site and / or amino or carboxyl terminus of protein or nucleic acid molecule It can be covalently attached to one or both of them. The peptide linker can also be attached to each These may be conjugated to amino acids or nucleic acids within the sequence of a protein or nucleic acid molecule. In certain embodiments, the peptide linker can be, for example, about 2 to 25 residues in length. This is the intended outcome.

[0314] In this specification, the term "nucleic acid" is interchangeable with the term "polynucleotide." Used in either single-stranded or double-stranded form, deoxyribonucleotides or This term refers to ribonucleotides and their polymers. This term also refers to known nucleotide analogs or This is a nucleic acid containing modified main chain residues or bonds, having similar binding properties to a reference nucleic acid. It includes synthetic, natural, and unnatural nucleic acids that are metabolized in a similar manner to the reference nucleotide. Examples of such analogues include phosphorothioates, phosphoramidates, and methylphosphonates. Phosphorate, chiral methylphosphonate, 2-O-methylribonucleotide, peptide nucleus Acids (PNA) are examples, but are not limited to these. Unless otherwise specified, some details Nucleic acid sequences are also implicitly represented by their conservatively modified variants (e.g., degenerate codon substitutions). ), including alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. In particular, degenerate codon substitution involves the third position of one or more (or all) selected codons. However, by creating sequences substituted with mixed bases and / or deoxyinosine residues This is achievable (Batzer et al., Nucleic Acid Res. 19: 5081,1991;Ohtsuka et al.,J.Biol.Chem.260 :2605-2608, 1985; and Rossolini et al., Mol.C (ell. Probes 8:91-98, 1994).

[0315] The term "operably linked" refers to two or more polynucleotides (e.g., DN A) Refers to the functional relationship between segments. Typically, this term refers to the relationship between transcriptional regulatory sequences. This refers to the functional relationship to the sequence being mapped. For example, promoter sequences or enhancer sequences. The column stimulates or modulates the transcription of coding sequences within appropriate host cells or other expression systems. When rated, it is operably linked to the code sequence. Generally, it is linked to the sequence being transcribed. The movably linked promoter transcription regulatory sequence is physically adjacent to the sequence being transcribed. In other words, it is cis-acting. However, some transcriptional regulatory sequences, such as enhancers, Transcriptions need to be physically adjacent to the coding sequences they enhance, or they need to be located in close proximity. There isn't any.

[0316] As used herein, the terms “optimized” or “codon optimized” are used in a non-verbal sense. The creotide sequence is produced by a cell or organism that produces it, generally a eukaryotic cell, for example, Pichia (Pic hia) cells, Chinese hamster ovary cells (CHO), human cells, or prokaryotes In cells, for example, Escherichia coli cells, preferred cod This means that it has been modified to encode an amino acid sequence using codons. Optimization refers to synonymous codons in coding DNA (i.e., those encoding the same amino acid). This refers to the discovery that the frequency of codon occurrences is biased across different species. Such codon degeneracy is This makes it possible for the same polypeptide to be encoded by various nucleotide sequences. Various codon optimization methods are known herein, for example, at least U.S. 5, This includes the methods disclosed in Specification No. 786,464 and Specification No. 6,114,148. The optimized nucleotide sequence is also known as the “parent” sequence, or the starting nucleotide sequence. The process is designed to preserve, or as much as possible, the amino acid sequence that is originally encoded. The optimized sequences described herein have preferred codons in mammalian cells. It is operated in such a way. However, this specification also refers to other eukaryotic or prokaryotic cells. The optimized expression of these sequences is also intended. The amino acid sequence being processed is also said to be optimized.

[0317] As used herein, the term "protein" refers to a chain of one or more linear units. This refers to any organic compound made from amino acids folded into a three-dimensional structure. The amino acids in the mer chain have a gap between the carboxyl group and the amino group of adjacent amino acid residues. They are linked together by butyl bonds. The term "protein" is further limited However, peptides, single-chain polypeptides, or chains of two or more amino acids are not formed. This includes any complex molecule, which may also include, but is not limited to, glycoproteins or other This includes known post-translation modifications. It also includes, but is not limited to, glycotechnology, pegylation, and hemodynamics. Known natural or artificial chemical modifications of natural proteins, such as sylation, and non-natural amino acids This includes amino acid modifications for incorporation and chemical bonding with other molecules.

[0318] The terms "polypeptide" and "protein" are used interchangeably in this specification. This term refers to a polymer of amino acid residues. This term also refers to a polymer in which one or more amino acid residues correspond. Amino acid polymers are artificial chemical mimetic substances of naturally occurring amino acids. Furthermore, suitable for naturally occurring amino acid polymers and amino acid polymers that do not exist in nature. It is used. Unless otherwise specified, specific polypeptide sequences are used in their conservatively modified form. It implicitly includes riant as well.

[0319] The term “recombinant host cell” (or simply “host cell”) refers to one or more recombinant expression cells. This refers to cells into which a receptor has been introduced. Such terminology can refer to not only specific target cells, but also... It should be understood that this also refers to the offspring of cells like those mentioned above. Because certain modifications can occur in later generations due to either of the following influences, such later generations are Although they may not be identical to the parent cell, the term "host cell" is used herein. It remains within the scope of [the specified range].

[0320] The term "subject" includes humans and non-human animals. Non-human animals include non-human primates. For example, crab-eating macaques, mice, rats, cats, rabbits, pigs, sheep, dogs, cows, All vertebrates, including chickens, amphibians, and reptiles (e.g., mammals and non-mammals) Included. Unless otherwise noted, the terms “patient” and “subject” are used interchangeably in this specification. Used herein. The term "cynomolgus macaque" or "cynomolgus macaque" as used herein refers to a cynomolgus macaque. The term "cynomolgus" refers to the crab-eating macaque (cynomolgus monk). ey) (Macaca fascicularis) vinegar.

[0321] "Prevention" or "prevention" means that it is for an indication described herein, such as diabetes. Conditions or disorders related to pathological macular edema, retinal vascular disease, and conditions related to diabetic retinopathy. Ophthalmic conditions including conditions or disorders, and / or conditions or disorders related to macular edema When related to the disorder, the following may be considered: visual function, retinal anatomical morphology, retinal blood Tube disease parameters, diabetic retinopathy disease parameters, and / or macular edema disease parameters Any action to prevent or delay the worsening of the condition in patients at risk of such worsening It means for the purpose of preventing pancreatic Non-ophthalmic conditions including visceral cancer, breast cancer, endometrial adenocarcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, and gastric cancer. Or, when relating to a disorder, it is not necessary to bring about complete prevention of the condition. Partial prevention or mitigation of the condition, or reduction of the risk of developing the condition, are also included in this term. It will be done.

[0322] Conditions or disorders related to diabetic macular edema, conditions related to age-related macular degeneration or Disorders, such as neovascular age-related macular degeneration, conditions or disorders related to retinal vascular disease, diabetes Conditions or disorders associated with pathological retinopathy, and / or conditions or disorders associated with macular edema The terms "to treat" or "treatment" refer to the modification of visual function and / or retinal anatomical form. This means any action that brings about or intends to bring about good or preservation. When used in the specification, pancreatic cancer, breast cancer, endometrial adenocarcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, And "treating" or "treating" non-ophthalmic conditions or disorders, including gastric cancer, any action that brings about or is intended to bring about improvement or reduction of a disability This refers to the purpose of treatment. In another aspect, treatment involves reducing the frequency of repeated administrations and / or visits to a doctor / hospital. This includes reducing the number of questions. Also, the treatment involves long-term treatment, for example, repeated administration indefinitely. This includes one aspect / appearance of the case. Methods for evaluating the treatment and / or prevention of a disease are in the art. It is known in this context and is described below in this specification.

[0323] The terms "tolerable therapeutic dose," "effective therapeutic dose," or "effective therapeutic dose" are interchangeable. Desired outcome (i.e., reduction of disease activity, reduction of disease progression, reduction of disease signs and / or symptoms) This refers to an amount sufficient to bring about a reduction (or similar effect). In some embodiments, the therapeutic dose is desirable. It does not induce or cause any side effects. The therapeutic dose is administered initially at a low dose. Next, it is determined by gradually increasing the dose until the desired effect is achieved. Yes, it is possible. The “prophylactically effective dose” and “therapeutically effective dose” of the molecules in this disclosure are B To prevent the onset of disease symptoms, including those related to TC activity and / or VEGF activity. It can either reduce the severity of the condition or lead to a reduction in its severity.

[0324] The term "vector" refers to another polynucleotide molecule linked together. This term is intended to refer to polynucleotide molecules capable of transporting nucleotides. It is a type of vector. A "plasmid" refers to a circular double-stranded DNA loop, to which additional DNA segments may be linked. It can be concluded. In another embodiment, the polynucleotide sequence is an adeno-associated virus vector (A AV, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV 7. AAV8, AAV9, AAV10, AAV11, and AAV12), lentiviruses The virus is delivered to the target using a vector, or a viral vector such as a retroviral vector. This allows additional DNA segments to be ligated into the viral genome. Vectors (e.g., bacterial vectors with bacterial origins of replication and mammalian episomal vectors) The vectors can self-replicate in the host cells into which they are introduced. Other vectors ( For example, when a non-episome mammalian vector is introduced into a host cell, it introduces the host cell's genotype. It can be incorporated into the genome, thereby replicating together with the host genome. Furthermore, specific vectors These devices can direct the expression of genes that are operably linked. Vectors such as the above are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). It is called a "creator". Generally, useful expression vectors in recombinant DNA technology are plus It is often in the form of a plasma. In this specification, "plasmid" and "vector" are used interchangeably. It can be used interchangeably when Sumid is the most commonly used form of vector. However, this disclosure does not describe a viral vector that performs an equivalent function (e.g., a replication-deficient viral vector). This includes other forms of expression vectors such as adenoviruses and adeno-associated viruses. That is the intention.

[0325] Polynucleotides (DNA or RNA) molecules, proteins, constructs, vectors, etc. The term "recombinant" refers to something artificial, not normally found in nature, and / or normally. These are polynucleotides or protein molecules that exist in a situation not found in nature. These refer to sequences, and these contain two elements that do not naturally exist together in the same way without human intervention. Polynucleotides (DN) containing the above combinations of polynucleotides or protein sequences. This includes molecules (A or RNA), proteins, constructs, etc., and for example, are functionally linked. However, it contains at least two polynucleotides or protein sequences that are heterogeneous. These include polynucleotide molecules, proteins, and constructs. For example, the term "recombination." This refers to the same molecule (e.g., plasmid, construct, vector, chromosome, protein, etc.) This can refer to any combination of two or more DNA or protein sequences, and here, Such combinations are artificial and not typically found in nature. In this case, the phrase "not normally found in nature" means that it is not found in nature without human introduction. This means that it cannot be done. Recombinant polynucleotides or protein molecules, constructs, etc., ( i) In nature, they are separated from other polynucleotides or protein sequences that are located nearby. (ii) other polynucleotides that are not naturally close to each other or Polynucleotides or protein sequences adjacent to (or close to) a protein sequence This may include recombinant polynucleotide molecules, proteins, constructs, etc. Also, genetically engineered and / or polynucleotides constructed outside the cell This can refer to a DNA molecule or protein molecule or sequence. For example, a recombinant DNA molecule is It may contain any manipulated or artificial plasmids, vectors, etc., and may be linear or Some may contain circular DNA molecules. Such plasmids, vectors, etc., are prokaryotic. Various maintenance elements, including the origin of replication and selectable markers for cellular organisms, as well as Probably in addition to plant-selectable marker genes, one or more transgenes or expression cases It can contain .

[0326] As used herein, “encoded area” or “code area” means a functional unit or fraction This refers to a portion of a polynucleotide that codes for a child (for example, mRNA, but not limited to this). (proteins, or non-coding RNA sequences or molecules).

[0327] As used herein, the term “therapeutic protein” means a disease, condition, or disorder. This refers to proteins that are useful for treating, preventing, or improving a condition.

[0328] "Modification" or "mutation" of an amino acid residue / position is, when used herein, the start amino acid This refers to a change in the primary amino acid sequence compared to the no-acid sequence, and in this case, the change is the remaining amino acid sequence. This is due to a change in the sequence involved in the base / position. For example, a typical modification involves another anechoic. Substitution of residues by no acids (or at that position) (e.g., conservative or non-conservative substitution), This includes the insertion of one or more amino acids adjacent to a residue / position, and the deletion of such residue / position. "Amino acid substitution" or its variations is an existing amino acid in a given (start) amino acid sequence. This refers to the substitution of an amino acid residue with a different amino acid residue. Generally and preferably, modified Compared to polypeptides containing the start (or "wild-type") amino acid sequence, variant proteins This results in a change in at least one physicobiochemical activity of the lipeptide. For example, an antibody In this case, the altered physicochemical activity is the binding affinity, binding ability, and / or the target molecule. It could be a binding effect.

[0329] The term "conservatively modified variant" refers to either the amino acid sequence or the nucleic acid sequence. This also applies to the following: With respect to a specific nucleic acid sequence, a conservatively modified variant is considered identical to the same variant. A nucleic acid that codes for an amino acid sequence or an amino acid sequence that is essentially the same as an amino acid sequence, or a nucleic acid that is an amino acid If it does not code for an acid sequence, it refers to essentially the same sequence. This is due to the degenerate nature of the gene code. In this case, a large number of functionally identical nucleic acids encode any given protein. For example, GCA, GCC, GCG, and GCU, all of which are derived from the amino acid alanine, are all composed of Therefore, alanine is coded at every position specified by the codon. Without altering the polypeptide, the codon is changed to one of the corresponding codons listed. Such nucleic acid variations can be modified into any of the following. These nucleic acid variations are conservatively modified. This is a type of variant called a "silent variation." It codes for polypeptides. Therefore, all nucleic acid sequences in this specification are all possible silent variations of nucleic acids. This will also be described. Those skilled in the art will know each codon in nucleic acids (usually only the methionine codon). By modifying AUG and (excluding TGG, which is usually a codon containing only tryptophan), functionally It will be recognized that this can result in the same molecule. Therefore, within each sequence described This implies that each silent variation of the nucleic acid encoding the polypeptide is involved.

[0330] As used herein, "C-terminus" has a free carboxyl group (-COOH). This refers to the amino acid at the carboxyl terminus of a polypeptide chain. When used herein, The "N-terminus" is the amino terminus of a polypeptide chain that has a free amine group (-NH2). It refers to anoacids.

[0331] As used herein, “patients requiring treatment” or “subjects requiring treatment” Such phrases are, for example, used in detection, diagnostic procedures, and / or treatment of the part of this disclosure. This includes subjects who benefit from the administration of the child or pharmaceutical composition, such as mammals.

[0332] The phrase "pharmaceutically acceptable" is approved by a federal or state regulatory body. Approved or approved for use in the U.S. Pharmacopoeia or in animals, especially for use in humans This means that it is listed in other generally recognized pharmacopoeias.

[0333] The term "pharmaceutical composition" means at least one active ingredient (e.g., the antibody or A mixture of a fragment and at least one pharmaceutically acceptable excipient, diluent, or carrier. To point.

[0334] "Pharmaceuticals" refer to substances used in medical procedures.

[0335] "Disruptions mediated by BTC" refers to disruptions caused by BTC and / or VEGF, whether directly or indirectly. Whether it is related, the cause, onset, progression, persistence, or pathology of a disease or condition, including the disease or It encompasses all diseases and medical conditions related to medical status. It is mediated by BTC. The diseases that can occur include pancreatic cancer, breast cancer, endometrial adenocarcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, gastric cancer, and diabetes. Uropathic macular edema, age-related macular degeneration, neovascular age-related macular degeneration, neovascular glaucoma, diabetic retina Abnormal vascularization associated with conditions such as macular edema, pathological myopia, retinal vein occlusion, retinopathy of prematurity, and nevus disorders. Examples include bipolar disorder, central serous chorioretinopathy, and acute multiple plaque epithelial disease. , but not limited to these.

[0336] "VEGF-mediated disorders" refers to diseases in which VEGF is directly or indirectly involved. Or relating to a disease or medical condition, including the cause, onset, progression, duration, or pathology of the condition. It includes all diseases and medical conditions that are affected, as disorders mediated by VEGF. This includes central nervous system tumors, hemangioblastomas, meningiomas, cerebral edema, pituitary adenomas, and non-astrocytic tumors (non-astrocytic tumors). strocytic) glioma, peritumoral edema, breast cancer, adenocarcinoma, lung cancer, diabetic macular floater Macular edema, age-related macular degeneration, neovascular age-related macular degeneration, neovascular glaucoma, diabetic retinopathy, macular edema , among others, abnormal vascular proliferation associated with pathological myopia, retinal vein occlusion, retinopathy of prematurity, and nevus disorders. These are possible, but not limited to them.

[0337] ii. Anti-BTC antibody or anti-BTC binding moiety BTC is a member of the EGF family. It is an ErbB receptor tyrosine receptor. It is a ligand for the -ase family, primarily Ras / MAPK and PL3K / AK ErbB1 triggers anti-apoptotic and proliferative signaling pathways such as the T pathway. It activates the ErbB4 homodimer. In the eye, BTC is used in diabetic retinopathy. It is a powerful permeability factor that can play an important role in the manifestation of increased retinal vascular permeability. This is a potential therapeutic target in this disease. Exemplary human pro-BTC amino acids. The sequence is presented as sequence number 156. An example human BTC amino acid sequence is given by sequence number 156. Presented as No. 158. The exemplary human BTC amino acid sequences represented in this disclosure are sequences Presented as number 157 (at the N-terminus and C-terminus, the residual amino acid residues are shown in lowercase) (This represents)

[0338] The structure of the human BTC protein bound to the four anti-BTC Fab fragments is shown in the X-ray crystal structure. The present applicant has recently elucidated this through structural analysis. Please refer to Example 2. Human BTC structure The structure is an EGF fold with 3-strand and 2-strand sheets and 5 beta strands. It is stabilized by three disulfide bonds.

[0339] An antibody that binds to BTC, including human BTC, or an antigen-binding fragment thereof, is provided herein. In some embodiments, the antibody or antigen-binding fragment provided is as described herein. Thus, it is a polypeptide containing one or more complementarity-determining regions (CDRs). In this embodiment, the CDR is embedded within the "framework" area, and the framework area The region directs the CDR so that the appropriate antigen-binding properties of the CDR are achieved. In this embodiment, the antibody or antigen-binding fragment provided herein is used for BTC and ErbB receptor It can interfere with, block, reduce, or modulate interactions between the body and other parts of the body. In that embodiment, the antibody or antigen-binding fragment provided herein has BTC-mediated activity (binding It can inhibit (including binding). In some embodiments, binding to these epitopes Antigen-binding proteins, in particular, are BTC and ErbB receptors and are mediated by BTC. It inhibits interactions with other physiological effects. In some embodiments, antigen binding The protein is a human protein, for example, a fully human antibody against BTC or Fab That is the case.

[0340] In some embodiments, the antibody or its antigen-binding fragment is provided by the antibody discussed herein. It binds to any one of the combined epitopes. In some embodiments, this is true This can be determined by a competitive assay between the antibody disclosed in the document and other antibodies. In some embodiments, the antibody or its antigen-binding fragment is one of the antibodies listed in Table 1. It binds to an epitope conjugated by one of the following: In some embodiments, an antibody or its anti The primordial binding fragment prevents BTC from interacting with the ErbB receptor. It binds to a specific three-dimensional structural state. In one embodiment, the antibody or antigen-binding fragment of the present disclosure is a It binds to one or more of the five beta chains of BTC. In one embodiment, it binds to an antibody or its antigen. The binding fragment binds to the beta chain 1 of human BTC, causing BTC to bind to the ErbB receptor. It interferes with the following. In one embodiment, the antibody or its antigen-binding fragment binds to the beta chain 2 of human BTC. This prevents BTC from binding to the ErbB receptor. In one embodiment, an antibody or its antigen The binding fragment binds to the β-chain 3 of human BTC, causing BTC to bind to the ErbB receptor. It interferes with the following. In one embodiment, the antibody or its antigen-binding fragment binds to the beta chain 4 of human BTC. This prevents BTC from binding to the ErbB receptor. In one embodiment, an antibody or its antigen The binding fragment binds to the beta chain 5 of human BTC, causing BTC to bind to the ErbB receptor. To obstruct.

[0341] This specification discloses antibodies or antigen-binding fragments thereof that specifically bind to BTC. In some embodiments, an anti-BTC antibody or its antigen-binding fragment enables BTC to function in various ways. To prevent BTC from To block or reduce the ability to interact with other substances. For example, in some embodiments Anti-BTC antibodies or their antigen-binding fragments block the ability of BTC to bind to the ErbB receptor. To reduce or decrease. In other embodiments, an anti-BTC antibody or its antigen-binding fragment is used to reduce BTC-induced It blocks the activation of oxidized ERK1 / 2. In some embodiments, an anti-BTC antibody or the The antigen-binding fragment blocks BTC-induced phosphorylation of HER3.

[0342] Certain antibodies or antigen-binding fragments thereof, as provided herein, are related to SEQ ID NO: 157 Or it binds specifically and / or selectively to human BTC as described in 158. In several embodiments, the antibody or its antigen-binding fragment is as shown in Example 2 and Table 1. It selectively binds to the BTC protein. In some embodiments, it breaks the binding of the antibody or its antigen. The models are G34, H35, F36, S37, R38, C39, P40, K41, Q42, Y 43, H45, Y46, R51, R53, F54, V56, A57, E58, Q59, T Sequence ID numbers selected from the group consisting of 60, P61, A72, R73, E75, and R76. It specifically and / or selectively binds to at least one residue of 157. Several embodiments So, two or more of the identified BTC residues (for example, 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 one of the regions to which the antibody or its antigen-binding fragment is bound. It is a department.

[0343] In some embodiments, the antibody or its antigen-binding fragment is, for example, SEQ ID NO: 157, NVS 1 R38, C39, P40, K41, Q42, Y43, H45, Y46, F54, Q5 9, T60, P61, and R73 bind specifically and / or selectively. In this case, the antibody or its antigen-binding fragment is, for example, SEQ ID NO: 157, P40, K of NVS2. 41, Q42, Y43, H45, Y46, E58, Q59, T60, P61, A72, R It binds specifically and / or selectively to 73, E75, and R76. In some embodiments, The antibody or its antigen-binding fragment is, for example, SEQ ID NO: 157, NVS3 G34, H35, F36, S37, R38, C39, P40, K41, Q42, R51, R53, F54, And it binds specifically and / or selectively to V56. In some embodiments, the antibody or its The antigen-binding fragment is SEQ ID NO: 157, e.g., S37, R38, C39, P40 of NVS4. , K41, Q42, Y43, H45, Y46, F54, A57, Q59, T60, P61 It binds specifically and / or selectively to A72, R73, and E75.

[0344] In embodiments in which antibodies or their antigen-binding fragments are used for therapeutic purposes, the antibody or its antigen-binding fragments are cleaved. The compound can inhibit, interfere with, or modulate one or more biological activities of BTC. In one embodiment, the antibody or its antigen-binding fragment specifically binds to human BTC and / or The binding of BTC to the ErbB receptor was at least approximately 20%-40%, 40-60%, and 6%. Substantially inhibiting 0-80%, 80-85%, or more (e.g., in vitro competitive inhibition) (By measuring binding in a binding assay). In some embodiments, the antibody or its The antigen-binding fragment is 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10-13 Less than M K d It has (better binding). In some embodiments, the antibody Or the antigen-binding fragment is less than 1 microM, 1000 nM to 100 nM, 100 nM to 10nM, 10nM~1nM, 1000pM~500pM, 500pM~200pM, 2 Less than 00 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM IC regarding the blocking of ErbB receptor binding to BTC at M, 10 pM to 1 pM 50 of To possess.

[0345] In some embodiments, the antibody or its antigen-binding fragment is as described in SEQ ID NO: 157 or 158. For forms of BTC that are used, at least 50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99%, or higher percentages It binds to a variant of BTC that is identical. In some embodiments, the antibody or its antigen binds. The composite fragment binds to an epitope that is conjugated by one of the antibodies listed in Table 1. In some embodiments, an antibody or its antigen-binding fragment interacts with the ErbB receptor. To prevent its use, it binds to a specific three-dimensional structural state of BTC.

[0346] The anti-BTC antibody or its antigen-binding fragment is a heavy chain variable region complementarity determining region 1 (HC DR1), Heavy Chain Variable Region Complementarity Determination Region 2 (HCDR2), Heavy Chain Variable Region Complementarity Determination Region 3 (HCDR3), Light chain variable region complementarity determination region 1 (LCDR1), Light chain variable region complementarity It includes decision region 2 (LCDR2) and light chain variable region complementarity decision region 3 (LCDR3). HCDR1, HCDR2, and HCDR3 are included in the heavy-chain variable region (VH). LC DR1, LCDR2, and LCDR3 are included in the light chain variable region (VL). In one embodiment The anti-BTC antibody or its antigen-binding fragment is listed in Table 1 and described below. heavy chain and light chain CDR (Kabat, Chothia, IMGT, and / or combination) Includes CD-R.

[0347] In one embodiment, an anti-BTC antibody or its antigen-binding fragment is used according to the Kabat numbering scheme. These are HCDs, as described in sequence numbers 4, 2, 3, 14, 15, and 16, respectively. Includes R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. In this case, anti-BTC antibodies or their antigen-binding fragments are classified according to the Chothia numbering scheme. And, as described in sequence numbers 5, 6, 3, 17, 18, and 19 respectively, HCDR 1. Includes HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. One embodiment Then, the anti-BTC antibody or its antigen-binding fragment is assigned according to the combination numbering scheme. HCDR1, H Includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3. In one embodiment, The anti-BTC antibody or its antigen-binding fragment is distributed according to the IMGT numbering scheme. HCDR1, HCDR2, as described in columns 7, 8, 9, 20, 18, and 16. , including HCDR3, LCDR1, LCDR2, and LCDR3. In one embodiment, anti-BTC The antibody or its antigen-binding fragment is an NVS1 as provided in Table 1. In certain embodiments, The anti-BTC antibody or its antigen-binding fragment is VH and VL, respectively, of SEQ ID NOs. 10 and 21. The heavy and light chain CDRs of antibody NVS1 (e.g., Kabat, Chothia, IM) Includes GT and / or combined CDR.

[0348] In one embodiment, an anti-BTC antibody or its antigen-binding fragment is used according to the Kabat numbering scheme. These are described in sequence numbers 28, 26, 27, 38, 39, and 40, respectively. Includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 In one embodiment, an anti-BTC antibody or its antigen-binding fragment is used in the Chothia numbering scheme. Accordingly, as described in sequence numbers 29, 30, 27, 41, 42, and 43, respectively. , HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 Includes. In one embodiment, the anti-BTC antibody or its antigen-binding fragment is a combination numbering scheme. Accordingly, as described in sequence numbers 25, 26, 27, 38, 39, and 40, respectively. , HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 Includes. In one embodiment, an anti-BTC antibody or its antigen-binding fragment is included in the IMGT numbering scheme. Therefore, as described in Sequence IDs 31, 32, 33, 44, 42, and 40 respectively , including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 In one embodiment, the anti-BTC antibody or its antigen-binding fragment is an NVS as provided in Table 1. 2. In certain embodiments, the anti-BTC antibody or its antigen-binding fragment is, respectively, SEQ ID NO: 3 Heavy and light chain CDRs of antibody NVS2 containing VH and VL of 4 and 45 (e.g., Kaba Includes t, Chothia, IMGT, and / or combined CDRs.

[0349] In one embodiment, an anti-BTC antibody or its antigen-binding fragment is used according to the Kabat numbering scheme. These are described in sequence numbers 28, 49, 50, 58, 59, and 60, respectively. Includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 In one embodiment, an anti-BTC antibody or its antigen-binding fragment is used in the Chothia numbering scheme. Accordingly, they are described in sequence numbers 29, 51, 50, 61, 62, and 63, respectively. , HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 Includes. In one embodiment, the anti-BTC antibody or its antigen-binding fragment is a combination numbering scheme. Accordingly, they are described in sequence numbers 25, 49, 50, 58, 59, and 60, respectively. , HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 Includes. In one embodiment, an anti-BTC antibody or its antigen-binding fragment is included in the IMGT numbering scheme. Therefore, as described in Sequence IDs 31, 52, 53, 64, 62, and 60 respectively , including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 In one embodiment, the anti-BTC antibody or its antigen-binding fragment is an NVS as provided in Table 1. 3. In certain embodiments, the anti-BTC antibody or its antigen-binding fragment is, respectively, SEQ ID NO: 5 Heavy and light chain CDRs of antibody NVS3 containing VH and VL of 4 and 65 (e.g., Kaba Includes t, Chothia, IMGT, and / or combined CDRs.

[0350] In one embodiment, an anti-BTC antibody or its antigen-binding fragment is used according to the Kabat numbering scheme. These are described in sequence numbers 72, 70, 71, 82, 83, and 84, respectively. Includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 In one embodiment, an anti-BTC antibody or its antigen-binding fragment is used in the Chothia numbering scheme. Accordingly, as described in sequence numbers 73, 74, 71, 85, 18, and 86, respectively. , HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 Includes. In one embodiment, the anti-BTC antibody or its antigen-binding fragment is a combination numbering scheme. Accordingly, as described in sequence numbers 69, 70, 71, 82, 83, and 84, respectively. , HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 Includes. In one embodiment, an anti-BTC antibody or its antigen-binding fragment is included in the IMGT numbering scheme. Therefore, as described in Sequence IDs 75, 76, 77, 87, 18, and 84 respectively , including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 In one embodiment, the anti-BTC antibody or its antigen-binding fragment is an NVS as provided in Table 1. 4. In certain embodiments, the anti-BTC antibody or its antigen-binding fragment is, respectively, SEQ ID NO: 7 Heavy and light chain CDRs of antibody NVS4 containing VH and VL of 8 and 88 (e.g., Kaba Includes t, Chothia, IMGT, and / or combined CDRs.

[0351] In addition, this disclosure also relates to the CDR sequences throughout and listed in Table 1. The present invention provides an anti-BTC antibody or its antigen-binding fragment containing a mino acid sequence, and the anti-BTC antibody or its anti The original binding fragment binds to BTC and retains the desired functional properties of the one described herein. More specifically, the amino acid sequence of the anti-BTC antibody or its antigen-binding fragment is, throughout For CDR sequences listed and shown in Table 1, 80% or more, 90% or more, 95% In addition, if they have an identity of 96% or more, 97% or more, 98% or more, or 99% or more And it can maintain its desired functional characteristics.

[0352] This disclosure also relates to anti-BTC antibodies homologous to the VH and VL sequences described herein or the This disclosure provides antigen-binding fragments, such as those listed in Table 1. The protein contains an amino acid sequence homologous to the anti-BTC antibody or antigen-binding fragment. , binding to therapeutic targets, for example, ophthalmic targets, and desired mechanisms as described in Table 1 and the examples. It retains functional properties. Less than 100% of the sequence is present in the VH and VL regions of those listed in Table 1. Antibodies or antigen-binding fragments having unified VH and VL regions are listed in Table 1. Mutagenesis of nucleic acid molecules (e.g., site-directed mutagenesis or PCR-mediated mutagenesis), followed by Described herein and as described in U.S. Patent Application Publication No. 20120014958 Functional assays are used to test modified antibodies that encode the retained function. This can be obtained by experimentation. High (s) compared to the heavy chain and light chain listed in Table 1. In other words, antibodies or their antigen-binding fragments having heavy and light chains that are identical (80% or more) , mutagenesis of nucleic acid molecules encoding such polypeptides (e.g., site-directed mutation) Mutagenesis (induced by induction or PCR), followed by, for example, a functional assay as described herein. By using (I), the retained function is coded, and modified antibodies are tested. It can be obtained through experience.

[0353] The anti-BTC antibody or its antigen-binding fragment described herein corresponds to SEQ ID NOs. 10 and 21, respectively. And, approximately at least 60%, at least 61%, at least 62%, at least 63%, At least 64%, at least 65%, at least 66%, at least 67%, and 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%, less 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%, less At least 86%, at least 87%, at least 88%, at least 89%, at least 9 0%, at least 91%, at least 92%, at least 93%, at least 94%, small At least 95%, at least 96%, at least 97%, at least 98%, at least Heavy chain variable region (VH) containing amino acid sequences with 99% or 100% sequence identity and This includes the light chain variable region (VL). Variability may exist within the CDR or framework region. This is intended. In one embodiment, the anti-BTC antibody or its antigen-binding fragment is, respectively, SEQ ID NO: It includes VH and VL having amino acid sequences as described in 10 and 21. In another embodiment, The anti-BTC antibody or its antigen-binding fragment is an NVS1 as provided in Table 1. In this embodiment, VH and VL are nucleic acid sequences such as those described in Sequence ID No. 11 and 22, respectively. Encoded by: In one embodiment, the anti-BTC antibody or its antigen-binding fragment is, At least one, two, or three modifications to the amino acid sequences of SEQ ID NOs. 10 and 21 (for example, It has substitutions (e.g., conservative substitutions), but more than 10 modifications (e.g., substitutions, e.g., conservative substitutions) It includes VH and VL containing amino acid sequences that do not have (storage substitutions). In another embodiment, amino acids The differences in the sequences are not within the complementarity determination region.

[0354] In one embodiment, the anti-BTC antibody or its antigen-binding fragment of the present disclosure is, respectively, SEQ ID NO: 34 and For 45, approximately at least 60%, at least 61%, at least 62%, and 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%, less 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%, less At least 81%, at least 82%, at least 83%, at least 84%, at least 8 5%, at least 86%, at least 87%, at least 88%, at least 89%, small 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%, VH and VL containing amino acid sequences with at least 99% or 100% sequence identity. This includes. It is intended that there may be variability within the CDR or framework area. One embodiment Therefore, the anti-BTC antibody or its antigen-binding fragment is described in SEQ ID NOs. 34 and 45, respectively. It includes VH and VL having such amino acid sequences. In another embodiment, an anti-BTC antibody or the The antigen-binding fragment is NVS2 as provided in Table 1. In another embodiment, VH and VL These are encoded by nucleic acid sequences such as those described in SEQ ID NOs. 35 and 46, respectively. In one embodiment, the anti-BTC antibody or its antigen-binding fragment is the same as SEQ ID NOs. 34 and 45, respectively. At least one, two, or three modifications of the amino acid sequence (e.g., substitution, e.g., conservative substitution) A has substitutions but does not have more than 10 modifications (e.g., substitutions, e.g., conservative substitutions). It includes VH and VL containing amino acid sequences. In another embodiment, the difference in amino acid sequences is phase It is not within the complementarity determination region.

[0355] In one embodiment, the anti-BTC antibody or its antigen-binding fragment of the present disclosure is, respectively, SEQ ID NO: 54 and For 65, approximately at least 60%, at least 61%, at least 62%, and 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%, less 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%, less At least 81%, at least 82%, at least 83%, at least 84%, at least 8 5%, at least 86%, at least 87%, at least 88%, at least 89%, small 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%, VH and VL containing amino acid sequences with at least 99% or 100% sequence identity. This includes. It is intended that there may be variability within the CDR or framework area. One embodiment Therefore, the anti-BTC antibody or its antigen-binding fragment is described in SEQ ID NOs. 54 and 65, respectively. It includes VH and VL having such amino acid sequences. In another embodiment, an anti-BTC antibody or the The antigen-binding fragment is NVS3 as provided in Table 1. In another embodiment, VH and VL These are encoded by nucleic acid sequences such as those described in SEQ ID NOs. 55 and 66, respectively. In one embodiment, the anti-BTC antibody or its antigen-binding fragment is the same as SEQ ID NOs. 54 and 65, respectively. At least one, two, or three modifications of the amino acid sequence (e.g., substitution, e.g., conservative substitution) A has substitutions but does not have more than 10 modifications (e.g., substitutions, e.g., conservative substitutions). It includes VH and VL containing amino acid sequences. In another embodiment, the difference in amino acid sequences is phase It is not within the complementarity determination region.

[0356] In one embodiment, the anti-BTC antibody or its antigen-binding fragment of the present disclosure is, respectively, SEQ ID NO: 78 and For 88, approximately at least 60%, at least 61%, at least 62%, and 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%, less 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%, less At least 81%, at least 82%, at least 83%, at least 84%, at least 8 5%, at least 86%, at least 87%, at least 88%, at least 89%, small 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%, VH and VL containing amino acid sequences with at least 99% or 100% sequence identity. This includes. It is intended that there may be variability within the CDR or framework area. One embodiment Therefore, the anti-BTC antibody or its antigen-binding fragment is described in SEQ ID NOs. 78 and 88, respectively. It includes VH and VL having such amino acid sequences. In another embodiment, an anti-BTC antibody or the The antigen-binding fragment is NVS4 as provided in Table 1. In another embodiment, VH and VL These are encoded by nucleic acid sequences such as those described in SEQ ID NOs. 79 and 89, respectively. In one embodiment, the anti-BTC antibody or its antigen-binding fragment is the same as SEQ ID NOs. 78 and 88, respectively. At least one, two, or three modifications of the amino acid sequence (e.g., substitution, e.g., conservative substitution) A has substitutions but does not have more than 10 modifications (e.g., substitutions, e.g., conservative substitutions). It includes VH and VL containing amino acid sequences. In another embodiment, the difference in amino acid sequences is phase It is not within the complementarity determination region.

[0357] The anti-BTC antibody or its antigen-binding fragment described herein corresponds to SEQ ID NOs. 12 and 23, respectively. And, approximately at least 60%, at least 61%, at least 62%, at least 63%, At least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, At least 73%, at least 74%, at least 75%, at least 76%, and 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%, less 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%, less At least 95%, at least 96%, at least 97%, at least 98%, at least 9 It contains heavy and light chains with amino acid sequences having 9% or 100% sequence identity. In this case, the anti-BTC antibody or its antigen-binding fragment is described in SEQ ID NOs. 12 and 23, respectively. It comprises a heavy chain and a light chain having an amino acid sequence such that it can be used. In one embodiment, the anti-BTC antibody is BT C activity, for example, of BTC 1) ErbB1; 2) ErbB4; 3) ErbB homodimer ( For example, ErbB1 / ErbB1 and ErbB4 / ErbB4); 4) ErbB heterodimer Body (for example, ErbB1 / ErbB2, ErB1 / ErB3, ErB1 / ErB4, Er It can inhibit binding to B2 / ErB3 and ErB2 / ErB4); and / or 5) ERK1 / 2 phosphorylation can be inhibited. In another embodiment, an anti-BTC antibody or The antigen-binding fragment is NVS1 as provided in Table 1. In another embodiment, the heavy chain and light chain are used. The chains are approximately at least 50% and at least 51% of the sequence numbers 13 and 24, respectively. , at least 52%, at least 53%, at least 54%, at least 55%, less 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%, less At least 65%, at least 66%, at least 67%, at least 68%, at least 6 9%, at least 70%, at least 71%, at least 72%, at least 73%, small 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%, and 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%, less 96%, at least 97%, at least 98%, at least 99%, or 100% Encoded by nucleic acid sequences that are identical or complementary. In one embodiment, an anti-BTC antibody Alternatively, the antigen-binding fragment may be a nucleic acid sequence as described in SEQ ID NOs: 13 and 24, respectively. Therefore, it is an NVS1 that includes coded heavy and light chains, as provided in Table 1.

[0358] In one embodiment, the anti-BTC antibody or its antigen-binding fragment of the present disclosure is, respectively, SEQ ID NO: 36 and For 47, approximately at least 60%, at least 61%, at least 62%, and 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%, less 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%, less At least 81%, at least 82%, at least 83%, at least 84%, at least 8 5%, at least 86%, at least 87%, at least 88%, at least 89%, small 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%, Heavy and light chains containing amino acid sequences with at least 99% or 100% sequence identity. Includes. In one embodiment, the anti-BTC antibody or its antigen-binding fragment is SEQ ID NO: 36 and It comprises a heavy chain and a light chain having an amino acid sequence as described in 47. In one embodiment, it is an anti-BTC Antibodies are used to test for BTC activity, for example, 1) ErbB1; 2) ErbB4; 3) ErbB Homodimers (e.g., ErbB1 / ErbB1 and ErbB4 / ErB4); 4) Erb B heterodimers (e.g., ErbB1 / ErbB2, ErbB1 / ErB3, ErbB1 / ErB3) It can inhibit binding to rB4, ErB2 / ErB3, and ErB2 / ErB4. ; and / or 5) ERK1 / 2 phosphorylation can be inhibited. In another embodiment, anti-BT The C antibody or its antigen-binding fragment is an NVS2 as provided in Table 1. In another embodiment, The heavy chain and light chain are approximately 50% less than those in SEQ ID NOs. 37 and 48, respectively. At least 51%, at least 52%, at least 53%, at least 54%, at least 5 5%, at least 56%, at least 57%, at least 58%, at least 59%, small 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%, and 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%, less 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%, less At least 91%, at least 92%, at least 93%, at least 94%, at least 9 5%, at least 96%, at least 97%, at least 98%, at least 99%, and It is encoded by a nucleic acid sequence having 100% identity or complementarity. In one embodiment, The anti-BTC antibody or its antigen-binding fragment is as described in SEQ ID NOs. 37 and 48, respectively. NVS comprising heavy and light chains encoded by nucleic acid sequences, such as those provided in Table 1. The answer is 2.

[0359] In one embodiment, the anti-BTC antibody or its antigen-binding fragment of the present disclosure is, respectively, SEQ ID NO: 56 and For 67, approximately at least 60%, at least 61%, at least 62%, and 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%, less 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%, less At least 81%, at least 82%, at least 83%, at least 84%, at least 8 5%, at least 86%, at least 87%, at least 88%, at least 89%, small 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%, Heavy and light chains containing amino acid sequences with at least 99% or 100% sequence identity. Includes. In one embodiment, the anti-BTC antibody or its antigen-binding fragment is SEQ ID NO: 56 and It comprises a heavy chain and a light chain having an amino acid sequence as described in 67. In one embodiment, it contains an anti-BTC Antibodies are used to test for BTC activity, for example, 1) ErbB1; 2) ErbB4; 3) ErbB Homodimers (e.g., ErbB1 / ErbB1 and ErbB4 / ErB4); 4) Erb B heterodimers (e.g., ErbB1 / ErbB2, ErbB1 / ErB3, ErbB1 / ErB3) It can inhibit binding to rB4, ErB2 / ErB3, and ErB2 / ErB4. ; and / or 5) ERK1 / 2 phosphorylation can be inhibited. In another embodiment, anti-BT The C antibody or its antigen-binding fragment is an NVS3 as provided in Table 1. In another embodiment, The heavy chain and light chain are approximately 50% less than those in SEQ ID NOs. 57 and 68, respectively. At least 51%, at least 52%, at least 53%, at least 54%, at least 5 5%, at least 56%, at least 57%, at least 58%, at least 59%, small 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%, and 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%, less 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%, less At least 91%, at least 92%, at least 93%, at least 94%, at least 9 5%, at least 96%, at least 97%, at least 98%, at least 99%, and It is encoded by a nucleic acid sequence having 100% identity or complementarity. In one embodiment, The anti-BTC antibody or its antigen-binding fragment is as described in SEQ ID NOs. 57 and 68, respectively. NVS comprising heavy and light chains encoded by nucleic acid sequences, such as those provided in Table 1. The answer is 3.

[0360] In one embodiment, the anti-BTC antibody or its antigen-binding fragment of the present disclosure is, respectively, SEQ ID NO: 80 and For 90, approximately at least 60%, at least 61%, at least 62%, and 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%, less 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%, less At least 81%, at least 82%, at least 83%, at least 84%, at least 8 5%, at least 86%, at least 87%, at least 88%, at least 89%, small 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%, Heavy and light chains containing amino acid sequences with at least 99% or 100% sequence identity. Includes. In one embodiment, the anti-BTC antibody or its antigen-binding fragment is SEQ ID NO: 80 and It comprises a heavy chain and a light chain having an amino acid sequence as described in 90. In one embodiment, it is an anti-BTC Antibodies are used to test for BTC activity, for example, 1) ErbB1; 2) ErbB4; 3) ErbB Homodimers (e.g., ErbB1 / ErbB1 and ErbB4 / ErB4); 4) Erb B heterodimers (e.g., ErbB1 / ErbB2, ErbB1 / ErB3, ErbB1 / ErB3) It can inhibit binding to rB4, ErB2 / ErB3, and ErB2 / ErB4. ; and / or 5) ERK1 / 2 phosphorylation can be inhibited. In another embodiment, anti-BT The C antibody or its antigen-binding fragment is an NVS4 as provided in Table 1. In another embodiment, The heavy chain and light chain are approximately at least 50% less than those in SEQ ID NOs. 81 and 91, respectively. At least 51%, at least 52%, at least 53%, at least 54%, at least 5 5%, at least 56%, at least 57%, at least 58%, at least 59%, small 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%, and 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%, less 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%, less At least 91%, at least 92%, at least 93%, at least 94%, at least 9 5%, at least 96%, at least 97%, at least 98%, at least 99%, and It is encoded by a nucleic acid sequence having 100% identity or complementarity. In one embodiment, The anti-BTC antibody or its antigen-binding fragment is as described in SEQ ID NOs. 81 and 91, respectively. NVS comprising heavy and light chains encoded by nucleic acid sequences, such as those provided in Table 1. The answer is 4.

[0361] Furthermore, isolated anti-BTC antibodies or their antigen-binding fragments having conservative modifications are provided for in this disclosure. It is included within the scope. More specifically, this disclosure includes the anti-BTC binding portion and anti-BTC binding portion of Table 1. The anti-BTC binding portion and the molecule conjugated to the binding portion have a conservative modification. This relates to a molecule conjugated to the anti-BTC portion of the present disclosure. In certain embodiments, the anti-B The antibody conjugated to the TC binding site contains CDR1, CDR2, and CDR3 sequences. It has a heavy chain variable region and a light chain variable region containing CDR1, CDR2, and CDR3 sequences. One or more of these CDR sequences are antibodies or their conservative variants as described herein. Having a specific amino acid sequence based on [a certain characteristic], this antibody retains the desired functional properties of the antibody of this disclosure. To hold.

[0362] In certain embodiments, the sequence is an anti-BTC antibody as described herein (e.g., Table 1) or Polynucleotides encoding fragments (e.g., VH or VL) are provided herein. In one embodiment, an anti-BTC antibody or its antigen-binding fragment is such that its sequence is in mammalian cells Encoded by polynucleotides that are codon-optimized for expression. One aspect So, the entire construct of an anti-BTC antibody or its antigen-binding fragment, the entire sequence of which is found in mammalian cells For example, polynucleotides that have been codon-optimized for expression in human cells This is coded as follows. In other embodiments, the anti-BTC antibody or its antigen-binding fragment is a mammalian antibody. It is optimized for expression within cells and has a full-length heavy chain sequence and a full-length light chain sequence, One or more of these sequences are based on the antibodies or their conservative variants described herein. Having a specific amino acid sequence, the anti-BTC binding moiety is the desired function of the anti-BTC binding antibody of this disclosure. It retains the characteristic. Therefore, this disclosure includes, for example, VH and VL, where VH is sequence number It includes amino acid sequences 10, 34, 54, and 78, and their conservative variants; VL is sequence BTC includes amino acid sequences numbered 21, 45, 65, and 88, and their conserved variants. Isolated antibodies optimized for expression in mammalian cells that specifically bind or It provides antigen-binding fragments.

[0363] This disclosure is the same as or overlaps with the anti-BTC antibodies or their antigen-binding fragments listed in Table 1. Provides an anti-BTC binding moiety that binds to an epitope (e.g., a BTC-binding antibody or a fragment thereof). Therefore, the additional antibody will compete with the other antibodies in the BTC binding assay. For example, they can be identified based on their ability to competitively inhibit binding in a statistically significant manner. Yes. The ability of the test antibody to inhibit the binding of the molecule of this disclosure to BTC is such that the test molecule is B To demonstrate that it can compete with that antibody for binding to TC; such an antibody According to a non-limiting theory, the body is the same as or related to competing antibodies (for example, structurally similar). It can bind to epitopes on BTC that are similar or spatially proximal. In certain embodiments, Molecules that bind to the same epitope as the antibodies disclosed herein are human monoclonal The antibody, Fab, or scFv. Such human monoclonal antibody, Fab, and sc Fv can be prepared and isolated as described herein.

[0364] In one embodiment, a molecule that competes with the anti-BTC antibody or its antigen-binding fragment of the present disclosure is G34. H35, F36, S37, R38, C39, P40, K41, Q42, Y43, H45, Y46, R51, R53, F54, V56, A57, E58, Q59, T60, P61, Among the sequence numbers 157 selected from the group consisting of A72, R73, E75, and R76 It binds to at least one residue. In one embodiment, the present disclosure is for binding to BTC and To reduce BTC-mediated signaling, use the methods listed in Table 1, for example, NVS1 Isolated antibodies or their anti-NVS2, NVS3, and NVS4 that can compete with NVS2, NVS3, and NVS4 The proto-binding fragment is provided. In another embodiment, the competing antibody or its antigen-binding fragment is given by the sequence number in Table 5. This includes heavy and light chains as described in issues 168-189.

[0365] The anti-BTC antibody or its antigen-binding fragments described herein include isolated antibodies, Fab, Fab', This form is selected from the group consisting of F(ab')2, Fv, and scFv. Preferred form In this case, the anti-BTC antibody or its antigen-binding fragment is Fab, and Fab containing the Fc region is included. In another embodiment, the Fc region contains IgG1, IgG2, IgG3, IgG4, and IgA Selected from the group consisting of Fc regions from IgM, IgE, and IgD. In one embodiment, The Fc region is a constant region of human immunoglobulin kappa chain, as described in Sequence ID No. 159. Includes a column. In another embodiment, the Fc region is a human immunoglobulin as described in Sequence ID No. 160. It contains the first constant Ig domain (CH1 domain) of the brin heavy chain.

[0366] In one embodiment, the anti-BTC antibody or its antigen-binding fragment is an isolated antibody, for example, monoclonal It is a ronal human antibody or a monoclonal humanized antibody. In certain embodiments, it is an anti-BTC antibody. The antibody may be in scFv or Fab form. In certain embodiments, the anti-BTC antibody is s It can be in cFv 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 for anti-BTC antibodies As stated above, this disclosure is not valid for organizations that possess BTC (e.g., a substantial amount of BTC, e.g. For example, therapeutic targets specific to targets located within or near the eyes, pancreas, etc. Conjugate to the joint (e.g., covalently, non-covalently, or fuse). (For example) A composition comprising one or more anti-BTC antibodies or their antigen-binding fragments. This disclosure does not affect BTC-related conditions or diseases (e.g., DR, DME, AMD, etc.). Attached to therapeutic target binding sites related to the treatment of vascular AMD and / or RVO The composition is characterized by having one or more anti-BTC antibodies or antigen-binding fragments thereof. This disclosure relates to pharmaceutical compositions and therapeutic target binding sites comprising an anti-BTC antibody or its antigen-binding fragment. This pharmaceutical composition is characterized by its use in the treatment of BTC-mediated conditions or diseases. Yes, it is possible. In yet another embodiment, the Disclosure may provide an effective amount of the anti-BTC antibody or its antigenic compound of the Disclosure. The combined fragment is administered to the target, followed by the therapeutic target binding site (e.g., VEGF inhibitory linkage) being targeted to the target. The method of treating a subject in need of treatment includes administering a harmful agent, and the subject is Having a BTC-mediated condition or disease

[0377] In a particular preferred embodiment, the therapeutic target binding portion is an antibody that binds to the therapeutic target, or Antigen-binding fragments (e.g., scFv, Fab, single-domain antibodies, or diabolic bodies) It is in the form of, or a polypeptide that binds to a therapeutic target (e.g., a soluble receptor). Such therapeutic targets may be related to ophthalmic disorders, for example, VEG. F, PDGF, PDGF-BB, Angiopoietin, Angiopoietin-2, S1P Indegrin 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, TIMP 3, HLA, IL8, CX3CR1, TLR3, TLR4, CETP, LIPC, COL 10A1, IL-1β, IL-17A, FGFR2, and TNFRSF10A may be Additional therapeutic targets include factor P, factor D, IL-6, IL-12, IL-18, and bF. Examples include GF, MCP-1, CD132, IL-6R, CD20, and IGF-1.

[0378] In one embodiment, the present disclosure includes at least one anti-BTC binding portion and one or more It is characterized by a multispecific binding molecule having a therapeutic target binding site. Therefore, in one embodiment, This disclosure provides, for example, a bispecific component having a combination of binding selectivity selected from the following: Characterized by offspring (e.g., bispecific antibodies): BTC and VEGF, BTC and PDGF, B TC and PDGF-BB, BTC and angiopoietin, BTC and angiopoietin-2 BTC and S1P, BTC and Indigrin αvβ3, BTC and αvβ5, BTC and α5β 1. BTC and apelin / APJ, BTC and erythropoietin, BTC and complement factors D and B TC and TNFα, BTC and C2, BTC and factor B, BTC and factor H, BTC and CFHR3 BTC and C1q, BTC and C3, BTC and C3b, BTC and C5, BTC and C5a, B TC and C3a, BTC and HtrA1, BTC and ARMS2, BTC and EPO, BTC and E POR, 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, B TC 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 site may be an anti-VEGF antagonist. Anti-VEGF antagonists include ranibizumab (Lucentis®); international public Pamphlet No. 98 / 45331; International release pamphlet No. 98 / 45331; USA Japanese Patent No. 6884879; U.S. Patent No. 6407213; U.S. Patent No. 70 U.S. Patent No. 60269; U.S. Patent No. 7365166). In one embodiment, anti-VE GF antagonists include bevacizumab (Avastin®); U.S. 605 U.S. Patent No. 4297; U.S. Patent No. 7169901; U.S. Patent No. 7375193 (Detailed description; U.S. Patent No. 7,297,334). In one embodiment, anti-VEGF antagonist The strike is Aflibercept (Eylea®; U.S. Patent No. 7279159). (Book) In one aspect, the anti-VEGF antagonist is brolucizumab (Beovu( Registered trademark; International Publication No. 2009 / 155724; U.S. Patent No. 83493 U.S. Patent No. 22; U.S. Patent No. 9090684; U.S. Patent No. 9873737 International Publication No. 03 / 097697; International Publication No. 2016 / 073915 This is a pamphlet (International Publication No. / 2016 / 073918). In one aspect... The anti-VEGF antagonist is pegaptanib (Macugen®). In one aspect, the anti-VEGF antagonist is KH902 (International Publication No. 2005 / 121). This is Pamphlet No. 176; U.S. Patent No. 7750138. In one embodiment, anti-V EGF antagonists are heavy chains, as described in SEQ ID NOs. 103 and 114, respectively. and light chains are included. In one embodiment, the anti-VEGF antagonist is sequence number 104 and It is encoded by a nucleic acid sequence, as described in bi115.

[0380] iv. Multispecific binding molecules Furthermore, in this disclosure, a multi-part compound comprising 1) an anti-BTC binding portion and 2) an anti-VEGF binding portion is also disclosed. A highly specific binding molecule is provided.

[0381] a) Anti-BTC binding part In one embodiment, the anti-BTC binding moiety is such as that described in Sequence ID No. 157 or 158. It binds to BTC. In some embodiments, the anti-BTC binding portion is shown in Example 2 and Table 4. It selectively binds to the indicated human BTC protein. In some embodiments, it is anti-BTC binding. The parts are G34, H35, F36, S37, R38, C39, P40, K41, Q42, Y43, H45, Y46, R51, R53, F54, V56, A57, E58, Q59, A sequence selected from the group consisting of T60, P61, A72, R73, E75, and R76. It specifically and / or selectively binds to at least one residue of number 157. In this embodiment, two or more of the identified BTC residues (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 2 2, 23, 24, or 25) are parts of the region bound by the anti-BTC binding moiety. In certain embodiments, such anti-BTC binding moieties selectively bind to human BTC proteins. Combined, it inhibits (for example, partially inhibits) BTC activity.

[0382] In some embodiments, the anti-BTC binding moiety is R of SEQ ID NO: 157, for example, NVS11. 38, C39, P40, K41, Q42, Y43, H45, Y46, F54, Q59, T It binds specifically and / or selectively to 60, P61, and R73. In some embodiments, The antibody or its antigen-binding fragment is, for example, SEQ ID NO: 157, P40, K41 of NVS12. , Q42, Y43, H45, Y46, E58, Q59, T60, P61, A72, R73 It binds specifically and / or selectively to E75 and R76. In some embodiments, it binds to anti The body or its antigen-binding fragments are, for example, G34, H35, F of SEQ ID NO: 157, NVS13. 36, S37, R38, C39, P40, K41, Q42, R51, R53, F54, and It binds specifically and / or selectively to V56. In some embodiments, the antibody or its anti The original binding fragment is S37, R38, C39, P40 of NVS14, for example, SEQ ID NO: 157. , K41, Q42, Y43, H45, Y46, F54, A57, Q59, T60, P61 , specifically and / or selectively binds to A72, R73, and E75. In one embodiment, anti The BTC binding portion is an anti-BTC antibody or its antigen-binding fragment, as described throughout. In certain embodiments, such anti-BTC binding moieties are selectively targeted to human BTC protein. It binds to BTC and inhibits (e.g., partially inhibits) BTC activity.

[0383] In embodiments where the anti-BTC binding portion is used for therapeutic purposes, the anti-BTC binding portion is 1 of BTC It can inhibit, interfere with, or modulate one or more biological activities. In one embodiment, anti-B The TC binding moiety specifically binds to human BTC and / or to the ErbB receptor of human BTC. The binding to at least approximately 20%-40%, 40-60%, 60-80%, and 80-85% or substantially inhibits (for example, in an in vitro competitive binding assay, binding) (by measuring) In some embodiments, its anti-BTC binding portion is 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 Less than M K d of It has (better binding). In some embodiments, the anti-BTC binding portion is 1 micron M, 1000nM~100nM, 100nM~10nM, 10nM~1nM, 1000p M~500pM, 500pM~200pM, less than 200pM, 200pM~150pM, ErbB receptors at concentrations of 200 pM to 100 pM, 100 pM to 10 pM, and 10 pM to 1 pM IC to block binding to BTC 50 It has. In certain embodiments, such resistance The BTC binding site selectively binds to human BTC protein and inhibits BTC activity (e.g., For example, partially inhibiting it.

[0384] In some embodiments, the anti-BTC binding moiety is described in Sequence ID No. 157 or 158. Regarding the BTC form, approximately 50%, 50-60%, 60-70%, 70% ~80%, 80-90%, 90-95%, 95-99%, or higher percentages. It binds to a variant of BTC that is of the opposite sex. In some embodiments, the anti-BTC binding portion is table It binds to an epitope that is conjugated by one of the antibodies described in 3. In this embodiment, the anti-BTC binding moiety prevents BTC from interacting with the ErbB receptor. It binds to a specific three-dimensional structural state of BTC.

[0385] The anti-BTC binding portion of the multispecific binding molecule of this disclosure is the heavy chain variable region complementarity determination region 1( HCDR1), Heavy Chain Variable Region Complementarity Determination Region 2 (HCDR2), Heavy Chain Variable Region Complementarity Determination Region 3 (HCDR3), Light Chain Variable Region Complementarity Determination Region 1 (LCDR1), Light Chain Variable Region Phase Includes complementarity determination region 2 (LCDR2) and light chain variable region complementarity determination region 3 (LCDR3). HCDR1, HCDR2, and HCDR3 are included in the heavy chain variable region (VH). LCDR1, LCDR2, and LCDR3 are included in the light chain variable region (VL). In this case, the anti-BTC antibody or its antigen-binding fragment is listed in Table 3 and is described below. heavy and light chain CDRs (e.g., Kabat, Chothia, IMGT, and / or combinations) Includes (combined CD-Rs).

[0386] In one embodiment, the anti-BTC binding moieties are sequenced according to the Kabat numbering scheme. HCDR1, HCDR2, H as described in numbers 4, 2, 3, 14, 15, and 16. Includes CDR3, LCDR1, LCDR2, and LCDR3. In one embodiment, anti-BTC binding The parts follow the Chothia numbering scheme, with sequence numbers 5, 6, 3, and 17 respectively. HCDR1, HCDR2, HCDR3, LCDR1, as described in 18 and 19 Includes LCDR2 and LCDR3. In one embodiment, the anti-BTC binding portion is a combination number The following are the corresponding sequences, as described in sequence numbers 1, 2, 3, 14, 15, and 16, respectively, according to the attachment scheme. Examples include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 This includes. In one embodiment, the anti-BTC binding portion follows the IMGT numbering scheme, respectively HCDR1, HCDR2 as described in sequence numbers 7, 8, 9, 20, 18, and 16. , including HCDR3, LCDR1, LCDR2, and LCDR3. In one embodiment, anti-BTC The joint portion is included in NVS11, as provided in Table 3.

[0387] In one embodiment, the anti-BTC binding moieties are sequenced according to the Kabat numbering scheme. HCDR1, HCDR as described in numbers 28, 26, 27, 38, 39, and 40. 2, including HCDR3, LCDR1, LCDR2, and LCDR3. In one embodiment, anti-BT The C-bonded portion follows the Chothia numbering scheme, corresponding to sequence numbers 29 and 30, respectively. HCDR1, HCDR2, HCDR3, as described in 27, 41, 42, and 43, Includes LCDR1, LCDR2, and LCDR3. In one embodiment, the anti-BTC bonding portion is a combination According to the matching numbering scheme, sequence numbers 25, 26, 27, 38, 39, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR 2, and LCDR3 are included. In one embodiment, the anti-BTC binding portion is an IMGT numbering ski As per the instructions, see sequence numbers 31, 32, 33, 44, 42, and 40 respectively. Includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 In one embodiment, the anti-BTC binding portion is included in NVS12 as provided in Table 3. ru.

[0388] In one embodiment, the anti-BTC binding moieties are sequenced according to the Kabat numbering scheme. HCDR1, HCDR as described in numbers 28, 49, 50, 58, 59, and 60. 2, including HCDR3, LCDR1, LCDR2, and LCDR3. In one embodiment, anti-BT The C-bonded parts follow the Chothia numbering scheme, corresponding to sequence numbers 29 and 51, respectively. HCDR1, HCDR2, HCDR3, as described in 50, 61, 62, and 63, Includes LCDR1, LCDR2, and LCDR3. In one embodiment, the anti-BTC bonding portion is a combination According to the matching numbering scheme, sequences 25, 49, 50, 58, 59, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR 2, and LCDR3 are included. In one embodiment, the anti-BTC binding portion is an IMGT numbering ski As per the instructions, see sequence numbers 31, 52, 53, 64, 62, and 60 respectively. Includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 In one embodiment, the anti-BTC binding portion is included in NVS13 as provided in Table 3. ru.

[0389] In one embodiment, the anti-BTC binding moieties are sequenced according to the Kabat numbering scheme. HCDR1, HCDR as described in numbers 72, 70, 71, 82, 83, and 84. 2, including HCDR3, LCDR1, LCDR2, and LCDR3. In one embodiment, anti-BT The C-bonded portions follow the Chothia numbering scheme, corresponding to sequence numbers 73 and 74, respectively. HCDR1, HCDR2, HCDR3, as described in 71, 85, 18, and 86 Includes LCDR1, LCDR2, and LCDR3. In one embodiment, the anti-BTC bonding portion is a combination According to the matching numbering scheme, sequence numbers 69, 70, 71, 82, 83, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR 2, and LCDR3 are included. In one embodiment, the anti-BTC binding portion is an IMGT numbering ski As per the instructions, see sequence numbers 75, 76, 77, 87, 18, and 84 respectively. Includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 In one embodiment, the anti-BTC binding portion is included in NVS14 as provided in Table 3. ru.

[0390] In addition, this disclosure also relates to the CDR sequences throughout and in Table 3, which are homologous to the CDR sequences described in Table 3. It provides an anti-BTC binding moiety containing a mino acid sequence, and the anti-BTC binding moiety binds to BTC, and the present invention It retains the desired functional characteristics of those described in the details. More specifically, the anti-BTC binding part. The amino acid sequence of the minute is described throughout, and is compared to the CDR sequence described in Table 3. and have 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more of identity. This allows for the maintenance of desired functional characteristics.

[0391] This disclosure also provides anti-BTC binding moieties homologous to the VH and VL sequences described herein. Provides. More specifically, this disclosure provides an ami with sequences homologous to those listed in Table 3, etc. The protein contains an acid sequence, and the anti-BTC binding moiety binds to an ophthalmic target, as shown in Table 3 and The desired functional characteristics of the items described in the examples are maintained. VH and Anti-B cells have VH and VL regions with less than 100% sequence identity to the VL region. The TC binding site is responsible for mutagenesis of nucleic acid molecules as listed in Table 3 (for example, site-directed mutagenesis or (This refers to mutagenesis by PCR), as described herein, U.S. Patent Application Publication No. 20120 Regarding the retained function, use the functional assay described in Specification No. 014958. This can be obtained by testing coded and modified antibodies. (See Table 3) Heavy and light chains that have high (i.e., 80% or more) identity with respect to heavy and light chains The anti-BTC binding moiety having such a polypeptide induces mutation in nucleic acid molecules encoding such polypeptides. Mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis), followed by the method described herein. Using functional assays, modified antibodies were used to encode the retained function. This can be obtained by testing.

[0392] The anti-BTC binding moieties of this disclosure are approximately at least as shown for Sequence ID Nos. 10 and 21, respectively. 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%, and 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%, less 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%, less At least 87%, at least 88%, at least 89%, at least 90%, at least 9 1%, at least 92%, at least 93%, at least 94%, at least 95%, small At least 96%, at least 97%, at least 98%, at least 99%, or 100% Heavy chain variable region (VH) and light chain variable region (VH) containing amino acid sequences with % sequence identity Includes L). It is intended that there may be variability within the CDR or framework area. In this embodiment, the anti-BTC binding portion is as described in Sequence ID Nos. 10 and 21, respectively. It includes VH and VL containing a minoic acid sequence. In another embodiment, the anti-BTC binding moiety is provided in Table 3. In another embodiment, VH and VL are such that SEQ ID NO: 11. Encoded by nucleic acid sequences such as those described in 6 and 122. In one embodiment, anti-BT The C-bonding portion consists of at least one and two amino acid sequences from sequence numbers 10 and 21, respectively. or having three modifications (e.g., substitution, e.g., conservative substitution), but more than 10 modifications ( For example, VH and VL include amino acid sequences that do not have substitutions (e.g., conservative substitutions). In another aspect, the differences in amino acid sequences are not located within the complementarity-determining region.

[0393] In one embodiment, the anti-BTC binding moieties of the present disclosure correspond to Sequence ID Nos. 34 and 45, respectively. Approximately at least 60%, at least 61%, at least 62%, at least 63%, less 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%, less At least 73%, at least 74%, at least 75%, at least 76%, at least 7 7%, at least 78%, at least 79%, at least 80%, at least 81%, small 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%, and 95%, at least 96%, at least 97%, at least 98%, at least 99% or containing VH and VL containing amino acid sequences with 100% sequence identity. CDR or It is intended that there may be variability within the framework domain. In one embodiment, anti-BTC bonds The combined portion contains a VH containing an amino acid sequence, as described in SEQ ID NOs. 34 and 45, respectively. and VL. In another embodiment, the anti-BTC binding moiety is NVS1 as provided in Table 3. 2. In another embodiment, VH and VL are described in Sequence ID No. 127 and 132, respectively. It is encoded by nucleic acid sequences such as the following. In one embodiment, the anti-BTC binding moieties are each The amino acid sequences of sequence numbers 34 and 45 have been modified by at least one, two, or three modifications (for example) If it has substitutions (e.g., conservative substitutions), but has more than 10 modifications (e.g., substitutions, e.g., It includes VH and VL containing amino acid sequences that do not have (conservative substitutions). In another embodiment, The differences in the no-acid sequence are not within the complementarity-determining region.

[0394] In one embodiment, the anti-BTC binding portion of the present disclosure corresponds to Sequence ID No. 54 and 65, respectively. Approximately at least 60%, at least 61%, at least 62%, at least 63%, less 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%, less At least 73%, at least 74%, at least 75%, at least 76%, at least 7 7%, at least 78%, at least 79%, at least 80%, at least 81%, small 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%, and 95%, at least 96%, at least 97%, at least 98%, at least 99% or containing VH and VL containing amino acid sequences with 100% sequence identity. CDR or It is intended that there may be variability within the framework domain. In one embodiment, anti-BTC bonds The combined portion contains a VH containing an amino acid sequence, as described in SEQ ID NOs. 54 and 65, respectively. and VL. In another embodiment, the anti-BTC binding moiety is NVS1 as provided in Table 3. 3. In another embodiment, VH and VL are described in Sequence ID No. 137 and 142, respectively. It is encoded by nucleic acid sequences such as the following. In one embodiment, the anti-BTC binding moieties are each The amino acid sequences of sequence numbers 54 and 65 have been modified by at least one, two, or three modifications (for example) If it has substitutions (e.g., conservative substitutions), but has more than 10 modifications (e.g., substitutions, e.g., It includes VH and VL containing amino acid sequences that do not have (conservative substitutions). In another embodiment, The differences in the no-acid sequence are not within the complementarity-determining region.

[0395] In one embodiment, the anti-BTC binding moieties of the present disclosure correspond to Sequence ID Nos. 78 and 88, respectively. Approximately at least 60%, at least 61%, at least 62%, at least 63%, less 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%, less At least 73%, at least 74%, at least 75%, at least 76%, at least 7 7%, at least 78%, at least 79%, at least 80%, at least 81%, small 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%, and 95%, at least 96%, at least 97%, at least 98%, at least 99% or containing VH and VL containing amino acid sequences with 100% sequence identity. CDR or It is intended that there may be variability within the framework domain. In one embodiment, anti-BTC bonds The combined portion contains a VH with an amino acid sequence as described in SEQ ID NOs. 78 and 88, respectively. and VL. In another embodiment, the anti-BTC binding moiety is NVS1 as provided in Table 3. 4. In another embodiment, VH and VL are described in Sequence ID No. 147 and 151, respectively. It is encoded by nucleic acid sequences such as the following. In one embodiment, the anti-BTC binding moieties are each The amino acid sequences of sequence numbers 78 and 88 have been modified by at least one, two, or three modifications (for example) If it has substitutions (e.g., conservative substitutions), but has more than 10 modifications (e.g., substitutions, e.g., It includes VH and VL containing amino acid sequences that do not have (conservative substitutions). In another embodiment, The differences in the no-acid sequence are not within the complementarity-determining region.

[0396] In one embodiment, the anti-BTC binding portion of the present disclosure is 1) Sequence IDs 1, 2, 3, and 14, respectively. 15 and 16 (combination numbering scheme); array numbers 4, 2, 3, and 14 respectively, 15 and 16 (Kabat numbering scheme); array indices 5, 6, 3, and 17 respectively. 18 and 19 (Chothia numbering scheme); or sequence numbers 7, 8, and 9 respectively. HCDR1, HCDR2, including 20, 18, and 16 (IMGT numbering scheme) HCDR3, LCDR1, LCDR2, and LCDR3, and 2) Sequence ID 10 and VH and VL having amino acid sequences as described in 21, and in another embodiment, The anti-BTC binding moiety is contained within NVS11 as provided in Table 3.

[0397] In one embodiment, the anti-BTC binding portion of the present disclosure is 1) Sequence IDs 25, 26, and 27, respectively. 38, 39, and 40 (combination numbering scheme); sequence numbers 28, 26, respectively. 27, 38, 39, and 40 (Kabat numbering scheme); array index 29, respectively. 30, 27, 41, 42, and 43 (Chothia numbering scheme); or each Includes sequence numbers 31, 32, 33, 44, 42, and 40 (IMGT numbering scheme) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and 2 ) VH and VL, respectively, containing amino acid sequences as described in SEQ ID NOs. 34 and 45. , including. In another embodiment, the anti-BTC binding moiety is in NVS12 as provided in Table 3. It is included.

[0398] In one embodiment, the anti-BTC binding portion of the present disclosure is 1) Sequence IDs 25, 49, and 50, respectively. 58, 59, and 60 (combination numbering scheme); sequence numbers 28, 49, respectively. 50, 58, 59, and 60 (Kabat numbering scheme); each with sequence number 29, 51, 50, 61, 62, and 63 (Chothia numbering scheme); or each Includes sequence numbers 31, 52, 53, 64, 62, and 60 (IMGT numbering scheme) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and 2 ) VH and VL, respectively, containing amino acid sequences as described in SEQ ID NOs. 54 and 65. , including. In another embodiment, the anti-BTC binding moiety is in NVS13 as provided in Table 3. It is included.

[0399] In one embodiment, the anti-BTC binding portion of the present disclosure is 1) Sequence IDs 69, 70, and 71, respectively. 82, 83, and 84 (combination numbering scheme); sequence numbers 72, 70, respectively. 71, 82, 83, and 84 (Kabat numbering scheme); respectively, sequence number 73, 74, 71, 85, 18, and 86 (Chothia numbering scheme); or each Includes sequence numbers 75, 76, 77, 87, 18, and 84 (IMGT numbering scheme). HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and 2 ) VH and VL containing amino acid sequences as described in SEQ ID NOs. 78 and 88 respectively. , including. In another embodiment, the anti-BTC binding moiety is in NVS14 as provided in Table 3. It is included.

[0400] b) Anti-VEGF binding part Furthermore, this disclosure also discloses the anti-VEGF binding moiety contained within the multispecific binding molecule. In certain embodiments, the anti-VEGF binding moiety is the heavy chain variable region complementarity determination region 1 (HCDR1 ), Heavy Chain Variable Region Complementarity Determination Region 2 (HCDR2), Heavy Chain Variable Region Complementarity Determination Region 3 (H CDR3), Light Chain Variable Region Complementarity Determination Region 1 (LCDR1), Light Chain Variable Region Complementarity Determination Region Includes region 2 (LCDR2) and light chain variable region complementarity determination region 3 (LCDR3). HCD R1, HCDR2, and HCDR3 are located within the heavy chain variable region (VH). LCDR1 LCDR2 and LCDR3 are included in the light chain variable region (VL). In one embodiment, The VEGF binding sites are listed in Table 2 or 3, and the heavy chain and light chain CD are as described below. R (e.g., Kabat, Chothia, IMGT, and / or combined CDR) Includes. In one embodiment, the anti-VEGF binding portion is an anti-VEGF antibody or its antigen-binding fragment. In one embodiment, the anti-VEGF binding moiety inhibits VEGF activity (for example, partially inhibits it). It is an anti-VEGF antibody or its antigen-binding fragment.

[0401] In one embodiment, the anti-VEGF binding moieties are arranged according to the Kabat numbering scheme. HCDR1 as described in columns 95, 93, 94, 105, 106, and 107, Includes HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. In one embodiment, The anti-VEGF binding moieties follow the Chothia numbering scheme, with each corresponding sequence number 9. HCDR1, HCDR as described in 6, 97, 94, 108, 109, and 110 2, including HCDR3, LCDR1, LCDR2, and LCDR3. In one embodiment, anti-VE The GF bond portions follow the combination numbering scheme, with sequence numbers 92, 93, and 9 respectively. HCDR1, HCDR2, HCDR 3, including LCDR1, LCDR2, and LCDR3. In one embodiment, the anti-VEGF binding portion These follow the IMGT numbering scheme, with sequence numbers 98, 99, 100, and 111 respectively. HCDR1, HCDR2, HCDR3, LCDR as described in 109 and 107 1. Includes LCDR2 and LCDR3. In one embodiment, the anti-VEGF binding moieties are shown in Table 2. Either the provided NVS8 or one of the NVS11 to NVS14 provided in Table 3. It is included in one. In another embodiment, the anti-VEGF binding moiety is brolucizumab (Beovu( It is a registered trademark.

[0402] In addition, this disclosure also relates to the CDR sequences described throughout and listed in Table 2 or 3. It provides an anti-VEGF binding moiety containing homologous amino acid sequences, and the anti-VEGF binding moiety is VE It binds to GF and retains the desired functional properties of those described herein. More specifically The amino acid sequence of the anti-VEGF binding portion is described throughout and is shown in Table 2 or 3. For CDR sequences that are such that 80% or more, 90% or more, 95% or more, 96% or more, 9 It can have an identity of 7% or more, 98% or more, or 99% or more, and its desired functional It can retain its characteristics.

[0403] This disclosure also relates to anti-VEGF binding moieties homologous to the VH and VL sequences described herein. Provides. More specifically, this disclosure provides sequences homologous to those described in Table 2 or 3. It provides a protein containing a certain amino acid sequence, and the anti-VEGF binding moiety binds to ophthalmic targets. The desired functional characteristics of those described in Table 2 or 3 and the examples are maintained. For the VH and VL regions of the described material, VH and VL regions with less than 100% sequence identity The anti-VEGF binding moiety having a VL region induces mutagenesis of nucleic acid molecules as described in Table 2 or 3 (e.g.) For example, site-directed mutagenesis or PCR-induced mutagenesis, followed by the Specified and U.S. Patent Publications Using the functional assay described in Patent Publication No. 20120014958, retention The modified function can be obtained by testing the modified antibody, which is coded for the modified function. It is possible. A high (i.e., 80% or more) ratio of the heavy and light chains listed in Table 2 or 3. The anti-VEGF binding moiety having a unisexual heavy chain and a light chain is such a polypeptide Mutagenesis of nucleic acid molecules (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) ), and then, using the functional assay described herein, the retained function is assessed. This can be obtained by testing modified antibodies.

[0404] The anti-VEGF binding moieties of this disclosure are approximately less than those of SEQ ID NOs: 101 and 112, respectively. At least 60%, at least 61%, at least 62%, at least 63%, at least 6 4%, at least 65%, at least 66%, at least 67%, at least 68%, small 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%, and 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%, less 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 Heavy chain variable region (VH) and light chain variable region containing amino acid sequences with 100% sequence identity Includes the region (VL). It is intended that there may be variability within the CDR or framework region. In one embodiment, the anti-VEGF binding moieties are described in Sequence IDs 101 and 112, respectively. It includes VH and VL containing amino acid sequences such as the above. In another embodiment, the anti-VEGF binding site This is either NVS8 as provided in Table 2, or NVS11 as provided in Table 3. It is included in any one of ~NVS14. In one embodiment, the anti-VEGF binding moieties are each At least one, two, or three modifications to the amino acid sequences of sequence numbers 101 and 112 (e.g.) For example, it has substitutions (e.g., conservative substitutions), but more than 10 modifications (e.g., substitutions, for example) In another embodiment, VH and VL include amino acid sequences that do not have conservative substitutions. The differences in the mino acid sequence are not located within the complementarity-determining region.

[0405] In one embodiment, the anti-VEGF binding moieties VH and VL are, respectively, sequence numbers 102 and 11 Encoded by nucleic acid sequences, as described in 3. In one embodiment, anti-VEGF binding The portion is NVS8 as provided in Table 2.

[0406] In one embodiment, the anti-VEGF binding moieties VH and VL are, respectively, sequence numbers 117 and 12 It is encoded by a nucleic acid sequence, as described in 3. In one embodiment, anti-VEGF binding The portion is included in NVS11, as provided in Table 3.

[0407] In one embodiment, the anti-VEGF binding moieties VH and VL are, respectively, sequence numbers 128 and 13 Encoded by nucleic acid sequences, as described in 3. In one embodiment, anti-VEGF binding The portion is included in NVS12, as provided in Table 3.

[0408] In one embodiment, the anti-VEGF binding moieties VH and VL are, respectively, sequence numbers 138 and 14. It is encoded by a nucleic acid sequence, as described in 3. In one embodiment, anti-VEGF binding The portion is included in NVS13, as provided in Table 3.

[0409] In one embodiment, the anti-VEGF binding moieties VH and VL are, respectively, sequence numbers 148 and 15 Encoded by a nucleic acid sequence, as described in 2. In one embodiment, anti-VEGF binding The portion is included in NVS14, as provided in Table 3.

[0410] In one embodiment, the anti-VEGF binding moieties of the present disclosure are 1) Sequence IDs 92, 93, and 94, respectively. , 105, 106, and 107 (combination numbering scheme); each with sequence number 95 , 93, 94, 105, 106, and 107 (Kabat numbering scheme); respectively Sequence numbers 96, 97, 94, 108, 109, and 110 (Chothia numbering scheme) ; or sequence numbers 98, 99, 100, 111, 109, and 107 respectively (IM HCDR1, HCDR2, HCDR3, LCDR1, LC (including GT numbering scheme) DR2 and LCDR3, and 2) as described in sequence numbers 101 and 112 respectively. The present invention includes VH and VL containing amino acid sequences. In another embodiment, the anti-VEGF binding moiety is table It is either an NVS8 as provided in 2, or an NVS11-NV as provided in Table 3. It is included in any one of S14. In one embodiment, the anti-VEGF binding moiety controls VEGF activity. To inhibit (for example, to partially inhibit).

[0411] In one embodiment, the anti-VEGF binding moiety of the present disclosure (for example, an anti-VEGF binding moiety that inhibits VEGF activity) The F-bond portion is approximately at least 60% of the portion of sequence numbers 103 and 114, respectively. 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%, and 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%, less 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%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 9 6%, at least 97%, at least 98%, at least 99%, or 100% of the sequence It includes a heavy chain and a light chain containing a monogeneous amino acid sequence. In one embodiment, the anti-VEGF binding portion These include heavy chains containing amino acid sequences, as described in SEQ ID NOs. 103 and 114, respectively. It includes a light chain. In another embodiment, the anti-VEGF binding moiety is NVS8 as provided in Table 2. In another embodiment, the heavy chain and light chain are, respectively, for SEQ ID NOs. 104 and 115. Approximately at least 50%, at least 51%, at least 52%, at least 53%, less 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%, less At least 63%, at least 64%, at least 65%, at least 66%, at least 6 7%, at least 68%, at least 69%, at least 70%, at least 71%, small 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%, and 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%, less 94%, at least 95%, at least 96%, at least 97%, at least 98% Nucleic acid sequences having %, at least 99%, or 100% identity or complementarity are used. It is converted. In one embodiment, the anti-VEGF binding moiety is located in sequence numbers 104 and 115, respectively. Includes heavy and light chains encoded by nucleic acid sequences, as described, as provided in Table 2. It is an NVS8 that can be used in this way.

[0412] c) Bispecific binding molecules In certain embodiments, the present invention comprises multiple specific moieties including an anti-BTC binding moiety and an anti-VEGF binding moiety. It is a sex-binding molecule. In this disclosure, it includes an anti-BTC binding moiety and an anti-VEGF binding moiety. A hemispecific binding molecule is provided, and 1) the anti-BTC binding moiety is an anti-BT molecule as described in Table 1. HCDR1, HCD C antibodies (e.g., NVS1, NVS2, NVS3, or NVS4) Includes R2, HCDR3, LCDR1, LCDR2, and LCDR3, 2) Anti-VEGF binding The combined portion is HCDR1, HCDR2, an anti-VEGF antibody as described in Table 2 or 3. , including HCDR3, LCDR1, LCDR2, and LCDR3. In one embodiment, multiple singular The sex-binding molecule is NVS11, as provided in Table 3.

[0413] In this disclosure, a multispecific binding molecule comprising an anti-BTC binding moiety and an anti-VEGF binding moiety is described. Provided are 1) anti-BTC binding moieties, 1) sequence numbers 1, 2, 3, 14, 15 respectively. and 16 (combination numbering scheme); respectively, sequence numbers 4, 2, 3, 14, 15, and 16 (Kabat numbering scheme); array indices 5, 6, 3, 17, 18 respectively, and 19 (Chothia numbering scheme); or sequence numbers 7, 8, 9, and 20 respectively HCDR1, HCDR2, HCD, including 18 and 16 (IMGT numbering scheme) 1) comprising R3, LCDR1, LCDR2, and LCDR3, and 2) anti-VEGF binding portion However, these correspond to sequence numbers 92, 93, 94, 105, 106, and 107 respectively (combination numbers) Attachment scheme); respective sequence numbers 95, 93, 94, 105, 106, and 107 (K abat numbering scheme); respective sequence numbers 96, 97, 94, 108, 109, and 110 (Chothia numbering scheme); or sequence numbers 98, 99, and 10 respectively Includes 0, 111, 109, and 107 (IMGT numbering scheme). In one embodiment, multiple The highly specific binding molecule is NVS11, as provided in Table 3. In one embodiment, These multispecific binding molecules 1) selectively bind to human BTC protein and also activate BTC activity 1) inhibits (e.g., partially inhibits), 2) selectively binds to human VEGF protein, Furthermore, it inhibits VEGF activity (for example, partially inhibits it).

[0414] Furthermore, in this disclosure, a multispecific binding including an anti-BTC binding portion and an anti-VEGF binding portion is also described. The molecules provided are 1) anti-BTC binding moieties, and 1) SEQ ID NOs. 25, 26, 27, 3 respectively. 8, 39, and 40 (combination numbering scheme); sequence numbers 28, 26, and 2 respectively. 7, 38, 39, and 40 (Kabat numbering scheme); sequence numbers 29 and 3, respectively. 0, 27, 41, 42, and 43 (Chothia numbering scheme); or each H including column numbers 31, 32, 33, 44, 42, and 40 (IMGT numbering scheme) Includes CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, 2) The anti-VEGF binding moieties are, respectively, sequence numbers 92, 93, 94, 105, and 106. , and 107 (combination numbering scheme); array numbers 95, 93, 94, and 1 respectively. 05, 106, and 107 (Kabat numbering scheme); array indices 96 and 9 respectively. 7, 94, 108, 109, and 110 (Chothia numbering scheme); or that These are sequence numbers 98, 99, 100, 111, 109, and 107 (IMGT numbering scheme). It includes (a). In one embodiment, the multispecific binding molecule is NVS12 as provided in Table 3. That is the case.

[0415] Furthermore, in this disclosure, a multispecific binding including an anti-BTC binding portion and an anti-VEGF binding portion is also described. The molecules provided are 1) anti-BTC binding moieties, and 1) SEQ ID NOs: 25, 49, 50, 5 8, 59, and 60 (combination numbering scheme); sequence numbers 28, 49, and 5 respectively. 0, 58, 59, and 60 (Kabat numbering scheme); array indices 29 and 5 respectively. 1, 50, 61, 62, and 63 (Chothia numbering scheme); or each H including column numbers 31, 52, 53, 64, 62, and 60 (IMGT numbering scheme) Includes CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, 2) The anti-VEGF binding moieties are, respectively, sequence numbers 92, 93, 94, 105, and 106. , and 107 (combination numbering scheme); array numbers 95, 93, 94, and 1 respectively. 05, 106, and 107 (Kabat numbering scheme); array indices 96 and 9 respectively. 7, 94, 108, 109, and 110 (Chothia numbering scheme); or that These are sequence numbers 98, 99, 100, 111, 109, and 107 (IMGT numbering scheme). It includes (a). In one embodiment, the multispecific binding molecule is NVS13 as provided in Table 3. That is the case.

[0416] Furthermore, in this disclosure, a multispecific binding including an anti-BTC binding portion and an anti-VEGF binding portion is also described. The molecules provided are 1) anti-BTC binding moieties, and 1) SEQ ID NOs: 69, 70, 71, 8 respectively. 2, 83, and 84 (combination numbering scheme); sequence numbers 72, 70, and 7 respectively. 1, 82, 83, and 84 (Kabat numbering scheme); sequence numbers 73 and 7, respectively. 4, 71, 85, 18, and 86 (Chothia numbering scheme); or each H including column numbers 75, 76, 77, 87, 18, and 84 (IMGT numbering scheme) Includes CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, 2) The anti-VEGF binding moieties are, respectively, sequence numbers 92, 93, 94, 105, and 106. , and 107 (combination numbering scheme); array numbers 95, 93, 94, and 1 respectively. 05, 106, and 107 (Kabat numbering scheme); array indices 96 and 9 respectively. 7, 94, 108, 109, and 110 (Chothia numbering scheme); or that These are sequence numbers 98, 99, 100, 111, 109, and 107 (IMGT numbering scheme). It includes (a). In one embodiment, the multispecific binding molecule is NVS14 as provided in Table 3. That is the case.

[0417] In one embodiment, the multispecific binding molecules of this disclosure are 1) VHA and VLA that bind to BTC. Therefore, VHA and VLA are approximately at least for Sequence ID No. 10 and 21, respectively. 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%, and 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%, less 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%, less At least 87%, at least 88%, at least 89%, at least 90%, at least 9 1%, at least 92%, at least 93%, at least 94%, at least 95%, small At least 96%, at least 97%, at least 98%, at least 99%, or 100% 1) VHA and VLA containing amino acid sequences with 1% sequence identity, and 2) bound to VEGF VHB and VLB, wherein VHB and VLB are sequence numbers 101 and 11, respectively. For 2, approximately at least 60%, at least 61%, at least 62%, and at least 6 3%, at least 64%, at least 65%, at least 66%, at least 67%, small 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%, and 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%, less 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%, less VHB and VL contain amino acid sequences with at least 99% or 100% sequence identity. B and, including. In another embodiment, the multispecific binding molecules of the present disclosure are 1) SEQ ID NO: 1 VHA and VLA that bind to BTC containing amino acid sequences 0 and 21, and 2) each VHB and VLB, which bind to VEGF containing amino acid sequences in columns 101 and 112, This includes. In another embodiment, VHA and VLA correspond to sequence numbers 116 and 122, respectively. And, approximately at least 80%, at least 81%, at least 82%, at least 83%, At least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, At least 93%, at least 94%, at least 95%, at least 96%, and at least Nucleic acid sequences with 97%, at least 98%, at least 99%, or 100% sequence identity. It is coded by. In another embodiment, VHB and VLB are coded by sequence number 117, respectively. And for 123, approximately at least 80%, at least 81%, at least 82%, and less At least 83%, at least 84%, at least 85%, at least 86%, at least 8 7%, at least 88%, at least 89%, at least 90%, at least 91%, small 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% array It is encoded by a nucleic acid sequence of identity. In one embodiment, the multispecific binding molecules are shown in Table 3. The provided NVS11 is one such example. In one embodiment, such a multispecific binding molecule is 1 ) Selectively binds to human BTC protein and inhibits BTC activity (for example, partially) 1) inhibits, and 2) selectively binds to human VEGF protein and inhibits VEGF activity. (For example, partially inhibit it).

[0418] In one embodiment, the multispecific binding molecules of this disclosure are 1) VHA and VLA that bind to BTC. Therefore, VHA and VLA are approximately at least for SEQ ID NOs. 34 and 45, respectively. 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%, and 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%, less 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%, less At least 87%, at least 88%, at least 89%, at least 90%, at least 9 1%, at least 92%, at least 93%, at least 94%, at least 95%, small At least 96%, at least 97%, at least 98%, at least 99%, or 100% 1) VHA and VLA containing amino acid sequences with 1% sequence identity, and 2) bound to VEGF VHB and VLB, wherein VHB and VLB are sequence numbers 101 and 11, respectively. For 2, approximately at least 60%, at least 61%, at least 62%, and at least 6 3%, at least 64%, at least 65%, at least 66%, at least 67%, small 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%, and 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%, less 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%, less VHB and VL contain amino acid sequences with at least 99% or 100% sequence identity. B and, including. In another embodiment, the multispecific binding molecules of the present disclosure are 1) SEQ ID NO: 3 VHA and VLA that bind to BTC containing amino acid sequences 4 and 45, and 2) each VHB and VLB, which bind to VEGF containing amino acid sequences in columns 101 and 112, This includes. In another embodiment, VHA and VLA correspond to sequence numbers 127 and 132, respectively. And, approximately at least 80%, at least 81%, at least 82%, at least 83%, At least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, At least 93%, at least 94%, at least 95%, at least 96%, and at least Nucleic acid sequences with 97%, at least 98%, at least 99%, or 100% sequence identity. It is coded by. In another embodiment, VHB and VLB are each sequence number 128. And for 133, approximately at least 80%, at least 81%, at least 82%, and less At least 83%, at least 84%, at least 85%, at least 86%, at least 8 7%, at least 88%, at least 89%, at least 90%, at least 91%, small 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% array It is encoded by a nucleic acid sequence of identity. In one embodiment, the multispecific binding molecules are shown in Table 3. The provided NVS12 is one such example. In one embodiment, such a multispecific binding molecule is 1 ) Selectively binds to human BTC protein and inhibits BTC activity (for example, partially) 1) inhibits, and 2) selectively binds to human VEGF protein and inhibits VEGF activity. (For example, partially inhibit it).

[0419] In one embodiment, the multispecific binding molecules of this disclosure are 1) VHA and VLA that bind to BTC. Therefore, VHA and VLA are approximately at least for SEQ ID NOs. 54 and 65, respectively. 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%, and 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%, less 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%, less At least 87%, at least 88%, at least 89%, at least 90%, at least 9 1%, at least 92%, at least 93%, at least 94%, at least 95%, small At least 96%, at least 97%, at least 98%, at least 99%, or 100% 1) VHA and VLA containing amino acid sequences with 1% sequence identity, and 2) bound to VEGF VHB and VLB, wherein VHB and VLB are sequence numbers 101 and 11, respectively. For 2, approximately at least 60%, at least 61%, at least 62%, and at least 6 3%, at least 64%, at least 65%, at least 66%, at least 67%, small 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%, and 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%, less 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%, less VHB and VL contain amino acid sequences with at least 99% or 100% sequence identity. B and, including. In another embodiment, the multispecific binding molecules of the present disclosure are 1) SEQ ID NO: 5 VHA and VLA that bind to BTC containing amino acid sequences 4 and 65, and 2) each VHB and VLB, which bind to VEGF containing amino acid sequences in columns 101 and 112, This includes. In another embodiment, VHA and VLA correspond to sequence numbers 137 and 142, respectively. And, approximately at least 80%, at least 81%, at least 82%, at least 83%, At least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, At least 93%, at least 94%, at least 95%, at least 96%, and at least Nucleic acid sequences with 97%, at least 98%, at least 99%, or 100% sequence identity. It is coded by. In another embodiment, VHB and VLB are each sequence number 138. And for 143, approximately at least 80%, at least 81%, at least 82%, and less At least 83%, at least 84%, at least 85%, at least 86%, at least 8 7%, at least 88%, at least 89%, at least 90%, at least 91%, small 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% array It is encoded by a nucleic acid sequence of identity. In one embodiment, the multispecific binding molecules are shown in Table 3. The provided NVS13 is one such example. In one embodiment, such a multispecific binding molecule is 1 ) Selectively binds to human BTC protein and inhibits BTC activity (for example, partially) 1) inhibits, and 2) selectively binds to human VEGF protein and inhibits VEGF activity. (For example, partially inhibit it).

[0420] In one embodiment, the multispecific binding molecules of this disclosure are 1) VHA and VLA that bind to BTC. Therefore, VHA and VLA are approximately at least for sequence numbers 78 and 88, respectively. 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%, and 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%, less 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%, less At least 87%, at least 88%, at least 89%, at least 90%, at least 9 1%, at least 92%, at least 93%, at least 94%, at least 95%, small At least 96%, at least 97%, at least 98%, at least 99%, or 100% 1) VHA and VLA containing amino acid sequences with 1% sequence identity, and 2) bound to VEGF VHB and VLB, wherein VHB and VLB are sequence numbers 101 and 11, respectively. For 2, approximately at least 60%, at least 61%, at least 62%, and at least 6 3%, at least 64%, at least 65%, at least 66%, at least 67%, small 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%, and 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%, less 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%, less VHB and VL contain amino acid sequences with at least 99% or 100% sequence identity. B and, including. In another embodiment, the multispecific binding molecules of this disclosure are 1) SEQ ID NO: 7 VHA and VLA that bind to BTC containing amino acid sequences 8 and 88, and 2) each VHB and VLB, which bind to VEGF containing amino acid sequences in columns 101 and 112, This includes. In another embodiment, VHA and VLA correspond to sequence numbers 147 and 151, respectively. And, approximately at least 80%, at least 81%, at least 82%, at least 83%, At least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, At least 93%, at least 94%, at least 95%, at least 96%, and at least Nucleic acid sequences with 97%, at least 98%, at least 99%, or 100% sequence identity. It is coded by. In another embodiment, VHB and VLB are each sequence number 148. And for 152, approximately at least 80%, at least 81%, at least 82%, and less At least 83%, at least 84%, at least 85%, at least 86%, at least 8 7%, at least 88%, at least 89%, at least 90%, at least 91%, small 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% array It is encoded by a nucleic acid sequence of identity. In one embodiment, the multispecific binding molecules are shown in Table 3. The provided NVS14 is one such example. In one embodiment, such a multispecific binding molecule is 1 ) Selectively binds to human BTC protein and inhibits BTC activity (for example, partially) 1) inhibits, and 2) selectively binds to human VEGF protein and inhibits VEGF activity. (For example, partially inhibit it).

[0421] The multispecific binding molecules of this disclosure are 1) VHA, CH1A, linker, VHB, and CH A heavy chain containing 1B, wherein the heavy chain is approximately 60% less than that of SEQ ID NO: 120. At least 61%, at least 62%, at least 63%, at least 64%, at least 6 5%, at least 66%, at least 67%, at least 68%, at least 69%, small 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%, and 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%, less 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 A heavy chain containing an amino acid sequence having properties, and 2) VLA, CKA, linker, VLB, A light chain containing CKB, wherein the light chain is approximately at least 60% of the amount of SEQ ID NO: 125. 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%, and 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%, less 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%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 9 6%, at least 97%, at least 98%, at least 99%, or 100% of the sequence A light chain comprising a monochromatic amino acid sequence is included. In one embodiment, the heavy chain and the light chain are For sequence numbers 121 and 126, the percentages were approximately at least 80%, at least 81%, and less than 80%. At least 82%, at least 83%, at least 84%, at least 85%, at least 8 6%, at least 87%, at least 88%, at least 89%, at least 90%, small 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%, Alternatively, it is encoded by 100 sequence-identical nucleic acid sequences. In one embodiment, multiple specificity sequences The combined molecule comprises a first polypeptide chain and a second polypeptide chain, and is provided in Table 3. As described in VS11, the first polypeptide chain is the amino acid sequence of SEQ ID NO: 120. The second polypeptide chain includes the amino acid sequence of SEQ ID NO: 125. In one embodiment, Such multispecific binding molecules 1) selectively bind to human BTC protein, and B 1) Inhibit TC activity (e.g., partially inhibit), and 2) selectively target human VEGF protein It binds and inhibits (e.g., partially inhibits) VEGF activity.

[0422] The multispecific binding molecules of this disclosure are 1) VHA, CH1A, linker, VHB, and CH A heavy chain containing 1B, wherein the heavy chain is approximately 60% less than that of SEQ ID NO: 130. At least 61%, at least 62%, at least 63%, at least 64%, at least 6 5%, at least 66%, at least 67%, at least 68%, at least 69%, small 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%, and 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%, less 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 A heavy chain containing an amino acid sequence having properties, and 2) VLA, CKA, linker, VLB, A light chain containing CKB, wherein the light chain is approximately at least 60% of the amount of SEQ ID NO: 135. 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%, and 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%, less 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%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 9 6%, at least 97%, at least 98%, at least 99%, or 100% of the sequence A light chain comprising a monochromatic amino acid sequence is included. In one embodiment, the heavy chain and the light chain are For sequence numbers 131 and 136, the percentages were approximately at least 80%, at least 81%, and less than 80% respectively. At least 82%, at least 83%, at least 84%, at least 85%, at least 8 6%, at least 87%, at least 88%, at least 89%, at least 90%, small 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%, Alternatively, it is encoded by 100 sequence-identical nucleic acid sequences. In one embodiment, multiple specificity sequences The combined molecule comprises a first polypeptide chain and a second polypeptide chain, and is provided in Table 3. As described in VS12, the first polypeptide chain is the amino acid sequence of SEQ ID NO: 130. The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 135. In one embodiment, Such multispecific binding molecules 1) selectively bind to human BTC protein, and B 1) Inhibit TC activity (e.g., partially inhibit), and 2) selectively target human VEGF protein It binds and inhibits (e.g., partially inhibits) VEGF activity.

[0423] The multispecific binding molecules of this disclosure are 1) VHA, CH1A, linker, VHB, and CH A heavy chain containing 1B, wherein the heavy chain is approximately 60% less than that of SEQ ID NO: 140. At least 61%, at least 62%, at least 63%, at least 64%, at least 6 5%, at least 66%, at least 67%, at least 68%, at least 69%, small 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%, and 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%, less 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 A heavy chain containing an amino acid sequence having properties, and 2) VLA, CKA, linker, VLB, A light chain containing CKB, wherein the light chain is approximately at least 60% of the amount of SEQ ID NO: 145. 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%, and 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%, less 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%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 9 6%, at least 97%, at least 98%, at least 99%, or 100% of the sequence A light chain comprising a monochromatic amino acid sequence is included. In one embodiment, the heavy chain and the light chain are For sequence numbers 141 and 146, the percentages were approximately at least 80%, at least 81%, and less than 80% respectively. At least 82%, at least 83%, at least 84%, at least 85%, at least 8 6%, at least 87%, at least 88%, at least 89%, at least 90%, small 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%, Alternatively, it is encoded by 100 sequence-identical nucleic acid sequences. In one embodiment, multiple specificity sequences The combined molecule comprises a first polypeptide chain and a second polypeptide chain, and is provided in Table 3. As described in VS13, the first polypeptide chain is the amino acid sequence of SEQ ID NO: 140 The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 145. In one embodiment, Such multispecific binding molecules 1) selectively bind to human BTC protein, and B 1) Inhibit TC activity (e.g., partially inhibit), and 2) selectively target human VEGF protein It binds and inhibits (e.g., partially inhibits) VEGF activity.

[0424] The multispecific binding molecules of this disclosure are 1) VHA, CH1A, linker, VHB, and CH A heavy chain containing 1B, wherein the heavy chain is approximately 60% less than that of SEQ ID NO: 149. At least 61%, at least 62%, at least 63%, at least 64%, at least 6 5%, at least 66%, at least 67%, at least 68%, at least 69%, small 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%, and 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%, less 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 A heavy chain containing an amino acid sequence having properties, and 2) VLA, CKA, linker, VLB, A light chain containing CKB, wherein the light chain is approximately at least 60% of the amount of SEQ ID NO: 154. 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%, and 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%, less 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%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 9 6%, at least 97%, at least 98%, at least 99%, or 100% of the sequence A light chain comprising a monochromatic amino acid sequence is included. In one embodiment, the heavy chain and the light chain are For SEQ ID NOs. 150 and 155, the percentages were approximately at least 80%, at least 81%, and less than 80% respectively. At least 82%, at least 83%, at least 84%, at least 85%, at least 8 6%, at least 87%, at least 88%, at least 89%, at least 90%, small 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%, Alternatively, it is encoded by 100 sequence-identical nucleic acid sequences. In one embodiment, multiple specificity sequences The combined molecule comprises a first polypeptide chain and a second polypeptide chain, and is provided in Table 3. As described in VS14, the first polypeptide chain is the amino acid sequence of SEQ ID NO: 149 The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 154. In one embodiment, Such multispecific binding molecules 1) selectively bind to human BTC protein, and B 1) Inhibit TC activity (e.g., partially inhibit), and 2) selectively target human VEGF protein It binds and inhibits (e.g., partially inhibits) VEGF activity.

[0425] The multispecific binding molecules of this disclosure (e.g., NVS11-NVS14) are: a. Can be bound to BTC and VEGF simultaneously; b. Binding of soluble BTC to ErbB1 or ErB4, and subsequent binding of Erb receptors It can inhibit phosphorylation; c. It can inhibit the soluble BTC-induced phosphorylation of ERK1 / 2; d. Juxtaclin binds to membrane-bound BTC and induces membrane-bound BTC-induced phosphorylation of ErbB1. It can inhibit activation; e. The binding of soluble VEGF-A165 to soluble VEGFR2 can be inhibited; f. In the in vitro external BRB model, BTC-induced human iPSC-induced RPE permeability It can be inhibited; g. In an in vitro medial BRB model, VEGF-induced human retinal endothelial cells (HRECs) It can inhibit permeability; h. It can inhibit 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 embodiments of this disclosure, the anti-BTC binding moiety is linked to a molecule by a linker, for example, It can be linked to the anti-VEGF binding site. More specifically, the anti-BTC binding site is optimized Peptide linkers having a length and / or amino acid composition (e.g., (Gly n -Ser n ) n or (Ser n -Gly n ) n By linkers, by proteins or nucleic acids They may be linked. The peptide linker length is determined by how the linked proteins fold. Linker orientation is known to have a significant impact on how they fold and interact. For examples of sizes, see, for example, Holling, incorporated herein by reference. er et al.1993 Proc Natl Acad.Sci.USA9 0:6444-6448, U.S. Patent Application Publication No. 2005 / 0100543, the same Specification No. 2005 / 0175606, Specification No. 2007 / 0014794, and P CT Public International Publication No. 2006 / 020258 and International Publication No. 2007 / 0 Please refer to pamphlet number 24715.

[0447] The peptide linker sequence is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 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 It is possible. The peptide linker sequence may consist of naturally occurring amino acids, or amino acids that do not exist naturally. It can be composed of anoacids. In some embodiments, the linker is a glycine polymer. In some embodiments, the amino acids glycine and serine include amino acids in the linker sequence. In certain embodiments, the linker region is a set of glycine repeats (GlySerGl y3) n The formula includes n, where n is a positive integer greater than or equal to 1, for example, n=3 (Sequence ID 118). ) More specifically, the linker sequence is GlySerGlyGlyGly(sequence number) It could be code 165). Alternatively, the linker sequence could be GlySerGlyGly(sequence number 165). 166) It may be that in certain other embodiments, the linker region orientation is glycine repeat Set (SerGly3) n The expression includes n, where n is a positive integer greater than or equal to 1, for example, n =3 (sequence number 167).

[0448] As a peptide linker, (Gly4Ser)4 (SEQ ID NO: 161) or (Gl One example is y4Ser)3 (sequence number 162), but it is not limited to these. The amino acid residues Glu and Lys are used in the Gly-Ser peptide for better solubility. It can be dispersed within the linker. In a particular embodiment, the peptide linker is (Gl It may include multiple repeats of (y3Ser), (Gly2Ser), or (GlySer). Yes, it is possible. In certain embodiments, the peptide linker is (SerGly3), (SerGl It may include multiple repeats of y2), or (SerGly). In other embodiments, pep Chidlinker is (Gly3Ser)+(Gly4Ser)+(GlySer)(arrangement number) It may include multiples of (163). In yet another embodiment, Ser is replaced with Ala. It can be, for example, (Gly4Ala) or (Gly3Ala). Furthermore, other states In this case, the linker is the motif (GluAlaAlaAlaLys) n (Sequence ID 164) The formula includes, where n is an integer of 1 or more. In certain embodiments, the peptide linker also , may include a severable linker.

[0449] Peptide linkers can be of various lengths. In particular, peptide linkers are approximately 5 ~Length of approximately 50 amino acids, length of approximately 10 to 40 amino acids, length of approximately 15 to 30 amino acids The length, or the length of approximately 15 to 20 amino acids. Variations in the length of the peptide linker. This modification can maintain or enhance activity, resulting in superior efficacy in activity tests. Peptide linkers use techniques known in the art to link polypeptides and It can be introduced into protein sequences. For example, PCR mutagenesis can be used. Modifications can be confirmed by DNA sequence analysis. Plasmid DNA is used. Therefore, in order to ensure stable production of the polypeptides produced, the host cells can be transformed. ru.

[0450] Peptide linker, anti-BTC binding site and protein, for example, anti-VEGF binding site It can be encoded in the same vector and expressed and assembled in the same host cell. Alternatively, each peptide linker, anti-BTC binding site, anti-VEGF binding site, and protein Nucleic acids or nucleic acids can be produced separately and then conjugated together. Peptide linkers, anti-BTC binding sites, and proteins or nucleic acids are used in the art. It can be prepared by conjugating the constituent components using known methods. Yes, it is possible. Site-specific conjugation uses saltase-mediated enzyme conjugation. It can be achieved using (Mao H, et al., J. Am. Chem. So c.2004 Mar 10;126(9):2670-1). Various coupling agents or Crosslinking agents can be used in covalent conjugation. Examples of crosslinking agents include Protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-fe Niredienmaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio) Propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl )Examples include cyclohexane-l-carboxylate (sulfo-SMCC) (for example, Karpovsky et al.,1984 J.Exp.Med.160:1686 ;Liu,MA et al.,1985 Proc.Natl.Acad.Sci.U See SA 82:8648). Another method is Paulus, 1985. Behring Ins.Mitt.No.78,118-132;Brennan e t al., 1985 Science 229:81-83), and Glennie (as described in et al., 1987 J.Immunol.139:2367-2375) Examples include the following: The conjugate agents are SATA and sulfo-SMCC, and both Both are available from Pierce Chemical Co. (Rockford, IL). It is Noh.

[0451] vi. Forms and types of multi-characteristic coupled molecules In some embodiments, the multispecificity binding molecule is a bispecificity antibody or a bispecificity antibody-like molecule. It is a child. In some embodiments, the bispecific antibody or antibody-like molecule is polyvalent, for example, one It can be bivalent against one antigen and monovalent against other antigens. Exemplary bispecific antibody fraction The child or bispecific antibody-like molecule reacts to the first antigen or epitope (e.g., BTC) A first antigen-binding domain having binding specificity (for example, comprising a first heavy chain and a first light chain) ), and the second antigen or epitope (e.g., VEGF) having binding specificity. Characterized by two antigen-binding domains (e.g., including a second heavy chain and a second light chain) It can be done.

[0452] In some embodiments, the first and second epitopes are located on the same antigen, for example, the same protein. It is located on the quality (or a subunit of a certain multimeric protein). In one embodiment, bispecific antibodies The molecule or bispecific antibody-like molecule has binding specificity to the first epitope or antigen. The heavy chain variable domain sequence and light chain variable domain sequence, and the second epitope or antigen It includes a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity.

[0453] In some embodiments, the bispecific antibody molecule or antibody-like molecule targets a first epitope or antibody. A semi-antibody that has binding specificity to the protozoan, and a semi-antibody that has binding specificity to a second epitope or antigen. It includes a semi-antibody having a bispecific antibody molecule or antibody-like molecule. A semi-antibody or fragment thereof having binding specificity to an epitope or antigen, and a second epi It comprises a semi-antibody or fragment thereof having binding specificity to a tope or antigen.

[0454] In one embodiment, a bispecific antibody molecule or a bispecific antibody-like molecule is a first epitope or This comprises scFv or Fab or a fragment thereof that has binding specificity to the antigen, and a second epi A certain embodiment includes an antibody or fragment thereof having binding specificity to a tope or antigen. So, a bispecific antibody molecule or bispecific antibody-like molecule targets a first epitope or antigen. Two scFv or Fab or fragment thereof having binding specificity, and a second epito It comprises an antibody or fragment thereof having binding specificity to a p or antigen. In one embodiment, The bispecific antibody molecule or bispecific antibody-like molecule reacts to the first epitope or antigen. A binding-specific scFv or fragment thereof binds to a second epitope or antigen. Includes a Fab or fragment thereof having specificity.

[0455] In certain embodiments, an antibody or antibody-like molecule exhibits multispecificity (e.g., bispecificity or It is a triple-specific antibody or antibody-like molecule. It is a bispecific or heterodimer antibody or an anti Protocols for producing body-like molecules are known in the art; for example, U.S. patents. The “knob-in-hole” method described in Specification No. 5731168; for example, International Publication No. 09 Pamphlet No. / 089004, International Publication No. 06 / 106905 and International Electrostatic steering F as described in the brochure No. 2010 / 129304 c-pair formation; for example, as described in International Publication No. 07 / 110205 Strand exchange domain (SEED) heterodimer formation; e.g., International Publication No. 08 / 1193 Pamphlet No. 53, International Publication No. 2011 / 131746 Pamphlet and International Publication No. Fab arm replacement as described in the 2013 / 060867 pamphlet; for example As described in U.S. Patent No. 4,433,059, for example, amine-reactive groups and A bispecific structure is prepared using a heterobifunctional reagent containing a sulfhydryl reactive group. Double antibody conjugates by antibody crosslinking; for example, U.S. Patent No. 4444878 As described in the book, the reduction and oxidation cycle of the disulfide bond between the two heavy chains By combining half-antibodies (heavy-light chain pairs or Fab) from different antibodies or antibody-like molecules... Bispecific antibodies or antibody-like molecular determinants produced thereby; for example, U.S. Patent No. 52737 A trifunctional antibody as described in Specification No. 43, for example, via a sulfhydryl-reactive group Three Fab' fragments that are cross-linked; for example, as described in U.S. Patent No. 5,534,254. Biosynthetic binding proteins, for example, via the C-terminal chain, preferably disulfide. Pairs of scFv crosslinked via filo- or amine-reactive chemical crosslinking; for example, US Special As described in Specification No. 5582996, a bifunctional antibody, for example, a constant domain Dimerization occurs via leucine zippers that replace the original leucine (e.g., c-fos and c-jun). Fab fragments having different binding specificities; for example, U.S. Patent No. 5591828 As described in the book, bispecific and oligospecific monovalent and oligovalent receptors, for example, The CH1 region of one antibody and the V region of the other antibody, which is usually associated with the light chain. H Poly between the region V of two antibodies (two Fab fragments) linked via a peptide spacer H -CH1 Area (Fd area); for example, two as described in U.S. Patent No. 5635602. Heavily specific DNA antibody conjugates, for example, antibodies or F2 conjugates mediated by fragments of DNA double helix. Crosslinking of ab fragments; for example, double as described in U.S. Patent No. 5,637,481 Specific fusion proteins, for example, two proteins having a hydrophilic helical peptide linker between them. An expression construct containing one scFv and a complete constant region; for example, U.S. 5 Polyvalent and multispecific binding proteins as described in Specification No. 837242, for example The first domain has a binding region for the Ig heavy chain variable region, which is generally called the diabody. A polypeptide dimer having a second domain that has a binding region for the Ig light chain variable region ( This also includes higher-order structures that generate bispecific, triplicate, or quadruplicate molecules; for example. , as described in U.S. Patent No. 5,837,821, dimerized and bispecific / Polyvalent molecules can be formed, and the peptide spacer further enhances the antibody hinge region and CH3 region. A connected V L and V H A minibody construct having a dimer; A short peptide linker (e.g.,) can be formed in either direction to create a bispecific diabody. For example, linked by 5 or 10 amino acids, or linked without any linkers at all. V H and V L Domain; for example, as described in U.S. Patent No. 5844094 Trimers and tetramers of; for example, as described in U.S. Patent No. 5,864,019. V that forms a series of FV (or scFv) L The domain and the C-terminus are further linked and can be crosslinked. V linked by peptide bonds with a group H Domain (or Family Members) Keru V L A sequence of domains; for example, International Publication No. 2011 / 028952 pamphlet One of the antigens, which optionally contains a heterodimer Fc region as described in the text, is monovalent. Combined, one of the antigens is bivalently bound to V L and V H Domain, scFv, or Fab; and, for example, as described in U.S. Patent No. 5,869,620. For example, using both scFv or diabody type forms, homobivalent, heterobivalent, trivalent It is compounded into a polyvalent structure via non-covalent or chemical crosslinking that forms a tetravalent structure. V linked via peptide linker L and V H Single-chain binding polyp Petit de la Muscat is one example, but it is not limited to these.

[0456] Further exemplary multispecific and bispecific molecules and methods for producing them are, for example, from the United States Patent Office. U.S. Patent No. 5910573, U.S. Patent No. 5932448, U.S. Patent No. 5959 U.S. Patent No. 083, U.S. Patent No. 5989830, U.S. Patent No. 6005079 Book, U.S. Patent No. 6,239,259, U.S. Patent No. 6,294,353, U.S. Patent U.S. Patent No. 6333396, U.S. Patent No. 6476198, U.S. Patent No. 65116 U.S. Patent No. 63, U.S. Patent No. 6670453, U.S. Patent No. 6743896 U.S. Patent No. 6809185, U.S. Patent No. 6833441, U.S. U.S. Patent No. 7129330, U.S. Patent No. 7183076, U.S. Patent No. 752105 U.S. Patent No. 6, U.S. Patent No. 7527787, U.S. Patent No. 7534866, U.S. Patent No. 7612181, U.S. Patent Application Publication No. 2002004587A1 Detailed description, U.S. Patent Application Publication No. 2002076406A1 specification, U.S. Patent Application Publication No. 20 Specification No. 02103345A1, U.S. Patent Application Publication No. 2003207346A1 , U.S. Patent Application Publication No. 2003211078A1, U.S. Patent Application Publication No. 2004 Specification No. 219643A1, U.S. Patent Application Publication No. 2004220388A1, U.S. National Patent Application Publication No. 2004242847A1, U.S. Patent Application Publication No. 200500 Specification No. 3403A1, U.S. Patent Application Publication No. 2005004352A1, U.S. Patent Patent Application Publication No. 2005069552A1, U.S. Patent Application Publication No. 20050791 Specifications No. 70A1, U.S. Patent Application Publication No. 2005100543A1, U.S. Patent Application Publication Specification of Patent Application Publication No. 2005136049A1, U.S. Patent Application Publication No. 2005136051 Specification A1, U.S. Patent Application Publication No. 2005163782A1 Specification, U.S. Patent Application Publication Specification No. 2005266425A1, U.S. Patent Application Publication No. 2006083747A1 Specification No., U.S. Patent Application Publication No. 2006120960A1, U.S. Patent Application Publication No. Specification No. 2006204493A1, U.S. Patent Application Publication No. 2006263367A1 Detailed description, U.S. Patent Application Publication No. 2007004909A1 specification, U.S. Patent Application Publication No. 20 Specification No. 07087381A1, U.S. Patent Application Publication No. 2007128150A1 , U.S. Patent Application Publication No. 2007141049A1 specification, U.S. Patent Application Publication No. 2007 Specification No. 154901A1, U.S. Patent Application Publication No. 2007274985A1, U.S. National Patent Application Publication No. 2008050370A1, U.S. Patent Application Publication No. 200806 Specifications No. 9820A1, U.S. Patent Application Publication No. 2008152645A1, U.S. Patent Patent Application Publication No. 2008171855A1, U.S. Patent Application Publication No. 20082418 Specifications 84A1, U.S. Patent Application Publication No. 2008254512A1, U.S. Patent Publication Patent Application Publication No. 2008260738A1, U.S. Patent Application Publication No. 2009130106 Specification A1, U.S. Patent Application Publication No. 2009148905A1 Specification, U.S. Patent Application Publication Specification No. 2009155275A1, U.S. Patent Application Publication No. 2009162359A1 Specification No., U.S. Patent Application Publication No. 2009162360A1, U.S. Patent Application Publication No. Specification No. 2009175851A1, U.S. Patent Application Publication No. 2009175867A1 Detailed description, U.S. Patent Application Publication No. 2009232811A1 specification, U.S. Patent Application Publication No. 20 Specification No. 09234105A1, U.S. Patent Application Publication No. 2009263392A1 , U.S. Patent Application Publication No. 2009274649A1, European Patent Application Publication No. 3460 Specification No. 87A2, Brochure No. 0006605A2, International Publication No. 0207 Pamphlet No. 2635A2, International Publication No. 04081051A1, International Publication Pamphlet No. 06020258A2, International Publication No. 2007044887A2 Fret, International Publication No. 2007095338A2 pamphlet, International Publication No. 20071 Pamphlet No. 37760A2, International Publication No. 2008119353A1, International Publication No. 2009021754A2, International Publication No. 2009068630 Pamphlet A1, International Publication No. 9103493; Pamphlet A1, International Publication No. 932 Pamphlet No. 3537A1, International Publication No. 9409131A1, International Publication Pamphlet No. 9412625A2, International Publication Pamphlet No. 9509917A1, International Publication No. 9637621A2 pamphlet, International Publication No. 9964460A1 pamphlet The contents of the application referenced above are incorporated herein by reference in their entirety. To be absorbed.

[0457] Therefore, in some embodiments, the BTC / VEGF multispecific binding molecule of this disclosure is The BTC binding domain and the VEGF binding domain are known in the art and all It includes either a multispecificity or a bispecificity form described throughout the body. The preferred form for the multispecific binding molecule shown is described in more detail below.

[0458] The multispecific binding molecule of this disclosure comprises an anti-BTC binding moiety and an anti-VEGF binding moiety, and The BTC binding portion consists of a variable heavy chain domain (VHA) and a variable light chain domain that bind to BTC. (VLA) is included, and the anti-VEGF binding moiety is a variable heavy chain domain (VH) that binds to VEGF. B) and variable light chain domains (VLBs). In one embodiment, VHA and VLA are shared. For example, they are linked via disulfide bonds. In one embodiment, VHB and VLB and They are linked via covalent bonds, such as disulfide bonds. In one embodiment, multiple features The isomerized molecule is arranged from the N-terminus to the C-terminus: N-VHA-linker 1-VHB- The forms are C and N-VLA-linker 2-VLB-C. In another embodiment, multiple specificity bonds are used. The combined molecule is arranged from the N-terminus to the C-terminus as follows: N-VHB-linker 1-VHA-C and The format is N-VLB-linker2-VLA-C. Linker 1 and linker 2 are the same. It may be different, or it may be different.

[0459] In one embodiment, the anti-BTC binding moiety consists of a heavy chain constant domain (CH1A) and a light chain constant domain. It further contains (CKA), and the anti-VEGF binding portion consists of a heavy chain constant domain (CH1B) and a light chain. It further contains a chain-constant domain (CKB). In one embodiment, the multispecific binding molecule is N-terminal or Towards the C-terminus: N-VHA-CH1A-linker-VHB-CH1B-C and N The format is -VLA-CKA-linker-VLB-CKB-C, for example, as shown in Table 3. These are NVS11, NVS12, NVS13, and NVS14. In another embodiment, The multi-specific binding molecule is arranged from the N-terminus to the C-terminus as follows: N-VHB-CH1B- Linker-VHA-CH1A-C and N-VLB-CKB-Linker-VLA-CKA-C This is the form. In one embodiment, the two linkers are the same. In another embodiment, the two linkers — is different.

[0460] In certain embodiments, the CH1 steady-state region exists together with VH, and the Cκ steady-state region exists together with VL. It exists, and as a result, the Fab fragment is formed by the dimerization of the respective light and heavy chains. (That is, VHA-CH1 forms a Fab fragment containing VLA-Cκ) Therefore, VHB-CH1 will form a Fab fragment containing VLB-Cκ. In some embodiments, the multispecific binding molecule of the present disclosure is in a Fab-Fab configuration, and among these, the anti-B Both the TC binding portion and the anti-VEGF binding portion form a Fab fragment. In another embodiment, The CH1 steady-state region exists together with VH, and the Cλ steady-state region exists together with VL, and thus, F ab fragments are formed by the dimerization of their respective light and heavy chains (i.e., VHA) -CH1 will form a Fab fragment containing VLA-Cλ, and VHB-CH1 will be V This will result in the formation of a Fab fragment containing LB-Cλ.

[0461] The multispecific binding molecule of this disclosure comprises an anti-BTC binding moiety and an anti-VEGF binding moiety, and The BTC binding region is Fab, and it contains a heavy chain (HA) and a light chain (LA), and the anti-VEGF is F ab, and includes a heavy chain (HB) and a light chain (LB). In one embodiment, HA and HB are N- Linked from terminal to C-terminal in the form of N-HA-linker1-HB-C, LA And LB is in the form N-LA-linker2-LB-C, from the N-terminus to the C-terminus. They are connected by: In another embodiment, HA and HB are connected from the N-terminus to the C-terminus: Linked in the form N-HB-linker1-HA-C, LA and LB are connected from the N-terminus to the C- The links are connected towards the end in the form: N-LB-linker2-LA-C. Linker 1 Linker 1 and Linker 2 may be the same or different. In one embodiment, Linker 1 and Linker 2 Nucleic acid 2 contains the amino acid sequence of SEQ ID NO: 118 or the nucleic acid sequence of SEQ ID NO: 119 Therefore, it is coded. In another embodiment, linker 1 and linker 2 are sequence numbers 161~ It contains an amino acid sequence selected from a group of 167.

[0462] In one embodiment, the Fab multispecific binding molecule of this disclosure has the structure shown in Figures 1-2. ru.

[0463] In one embodiment, the bispecific antibody of the present disclosure comprises two polypeptide chains, one of which is: Anti-VEGF scFv and light chain constant region C linked together by a linker peptide. It contains the light chain variable domain (VLB) (VLB-CL) of an anti-BTC antibody that also has L, and the other The chain is the heavy chain variable domain (VHB) of an anti-BTC antibody, which also has a heavy chain constant region CH1. -CH1) is included. In another embodiment, the bispecific antibody of this disclosure comprises two polypeptide chains It contains, and one chain is linked to the other by a linker peptide, anti-VEGF scFv, The heavy chain variable domain (VHB) of the anti-BTC antibody also has a heavy chain constant region CH1 (VHB- The other chain contains CH1), and the light chain variable domain of an anti-BTC antibody also has a light chain constant region CL. Includes (VLB)(VLB-CL).

[0464] In certain embodiments, the scFv on one polypeptide chain of the bispecific antibody of this disclosure The orientation is NH2-VLB-CL-linker2-scFv-COOH or NH2-VHB- It could be CH1-linker2-scFv-COOH. In one embodiment, VL on scFv The linker sequence between the main and VH domains is the sequence of (GGGGS)4 (sequence number 63). ) has, and VL and VH are in the form of NH2-VLA-linker2-VHA-COOH ru.

[0465] In another embodiment, one of the binding domains of the bispecific antibody of this disclosure forms a Fab. The other binding domain forms a single-chain (scFv) antibody fragment. Those skilled in the art will know that You will notice that other orientations are possible in addition to those shown. For example, scF The v-Fab format is possible, or the binding specificity can be reorganized.

[0466] In some embodiments, the multispecificity binding molecule is a bispecificity antibody or a bispecificity antibody-like molecule. It is a child. In another aspect, the present disclosure has a domain specific to BTC (i.e. (anti-BTC binding moiety), and another therapeutic target, for example, another specific to VEGF It is characterized by a multispecific molecule containing a domain (therapeutic target binding portion). For example, multispecific The sex molecule comprises an anti-BTC binding moiety, an antibody, or its antigen-binding fragment, and the nucleic acid moiety of this disclosure. It is possible. Anti-BTC antibodies are described, for example, in U.S. Patent No. 6,183,971 and international publications. As described in brochure No. 2004 / 083241A2, in the technical field It is known.

[0467] The antibodies or antigen-binding fragments thereof of this disclosure have at least two different binding sites or target regions. To generate a bispecific molecule that binds to its offspring, another functional molecule, such as another peptide, is required. Alternatively, it may be derivatized into a protein (for example, a ligand for another antibody or receptor), or can be bound thereto. The antibodies of this disclosure actually have three or more different binding sites and / or multispecific molecules that bind to the target molecule (such multispecific molecules are also used herein) (intended to be encompassed by the term "bispecific molecule") Therefore, it may be derivatized or linked to two or more other functional molecules. To create specific molecules, the antibodies of this disclosure may be combined with other antibodies, antigen-binding fragments, peptides, or It can functionally link to one or more other binding molecules, such as binding mimetic molecules (for example) (by chemical bonding, gene fusion, non-covalent bonding, or other means), thereby achieving bispecificity. It brings forth molecules.

[0468] Therefore, this disclosure provides a first binding specificity for at least one BTC, and a second A bispecific component that includes a target epitope, for example, a second binding specificity to another therapeutic target. This includes offspring. For example, the second target epitope is the VEGF epitope.

[0469] In one embodiment, the anti-BTC binding portion and the anti-VEGF binding portion are contained within the multispecific binding molecule. The terms are, for example, Fab, Fab', F(ab')2, Fv, or single-stranded Fv(scFv). This is a form that includes. In another embodiment, the anti-BTC binding portion is anti-BTC Fab and anti-V The EGF binding moiety is anti-VEGF Fab. In another embodiment, the anti-BTC binding moiety is s It is cFv, and the anti-VEGF binding portion is scFv. It is contained within the multispecific binding molecule. The anti-BTC binding portion and the anti-VEGF binding portion are also a light chain or heavy chain dimer, or Fv Alternatively, as described in Ladner et al.'s U.S. Patent No. 4,946,778 This could be any smallest fragment of the main chain structure, etc.

[0470] In one embodiment, the multispecific binding molecule of this disclosure may be a diabody. The links are connected by linkers that are too short to allow pairing between two domains on the same chain. The VH and VL domains within it are represented as a bivalent double on a single-stranded polypeptide. It is a specific molecule. The VH and VL domains pair with the complementary domain of another chain, and Therefore, it creates two antigen-binding sites (for example, Holliger et al., 19 93 Proc.Natl.Acad.Sci.USA 90:6444-6448;P oljak et al., 1994 Structure 2:1121-1123 (See reference). The diamond body has structures VHA-VLB and VHB-VLA (VH-VL (Configuration), or having either VLA-VHB and VLB-VHA (VL-VH configuration) It can be produced by expressing two polypeptide chains within the same cell. Most of these can be expressed in bacteria in a soluble form. Single-stranded diabolic bodies (scDb) ) links two diabody-forming polypeptide chains with a linker of approximately 15 amino acid residues. It is produced by (Holliger and Winter, 1997 C ancer Immunol. Immunother.,45(3-4):128-30 ;Wu et al.,1996 Immunotechnology,2(1):21 (See -36). scDb can be expressed in bacteria in a soluble, active monomeric form. (Holliger and Winter, 1997 Cancer Immunology) l.Immunother.,45(34):128-30;Wu et al.,19 96 Immunotechnology,2(1):21-36;Pluckthun and Pack,1997 Immunotechnology,3(2):83- 105;Ridgway et al.,1996 Protein Eng.,9(7 (See 617-21). The Diabody is fused with Fc and called "Diabody". It is possible to generate "ィ" (Lu et al., 2004 J. Biol. Chem See .,279(4):2856-65. Other antibodies that can be used include mouse, chimeric, and humanized monoclonal antibodies.

[0471] The protocol for producing bispecific or heterodimer antibodies or antibody-like molecules is: Known in the art; for example, as described in U.S. Patent No. 5,731,168 "Nobu in Hole" Law; for example, International Publication No. 09 / 089004, International Publication Pamphlet No. 06 / 106905 and International Publication No. 2010 / 129304 Electrostatic steering Fc pair formation as described in the book; for example, International Publication No. 07 / 11 Strand exchange operation domain (SEED) heterogeneity as described in pamphlet No. 0205 Merger formation; e.g., International Publication No. 08 / 119353, International Publication No. 2011 In pamphlet No. / 131746 and international publication No. 2013 / 060867 Fab arm replacement as described; for example, as described in U.S. Patent No. 4433059 As described, for example, heterodiplex groups having amine-reactive groups and sulfhydryl-reactive groups Dual antibody conjugates created by antibody crosslinking using functionalized reagents to produce a bispecific structure. For example, as described in U.S. Patent No. 4444878, the gap between two heavy chains The reduction and oxidation cycle of sulfide bonds leads to the formation of semi-antibodies from different antibodies or antibody-like molecules. Bispecific antibodies or antibodies produced by combining heavy-light chain pairs or Fab Molecular determinants; for example, three functions as described in U.S. Patent No. 5,273,743. Sex antibodies, for example, three Fab' fragments crosslinked via sulfhydryl reactive groups; for example For example, biosynthetic binding proteins as described in U.S. Patent No. 5,534,254, e.g. For example, via the C-terminal chain, preferably via a disulfide or amine-reactive chemical crosslink. A pair of cross-linked scFv; for example, as described in U.S. Patent No. 5,582,996. As such, a bifunctional antibody, for example, a leucine zipper with a replaced constant domain (for example, Fab cleavage with different binding specificities, dimerized via c-fos and c-jun. For example, dual specificity and O as described in U.S. Patent No. 5591828. Ligo-specific monovalent and oligovalent receptors, for example, the CH1 region of one antibody and typically the light chain Two polypeptide spacers are linked between the VH region of the other antibody that has associated with it. VH-CH1 region (Fd region) of two antibodies (two Fab fragments); for example, U.S. 5 A bispecific DNA antibody conjugate as described in Specification No. 635602, for example For example, crosslinking of antibodies or Fab fragments via DNA double-stranded fragments; for example, U.S. Patent No. 56 A bispecific fusion protein as described in Specification No. 37481, for example, between It contains two scFvs having hydrophilic helical peptide linkers and a complete constant region. Expression constructs; for example, as described in U.S. Patent No. 5,837,242 Polyvalent and multispecific binding proteins, such as the Ig heavy chain commonly called a diabody. A first domain having a binding region for the variable region and a second domain having a binding region for the Ig light chain variable region Dimers of polypeptides having two domains (bispecific, tripspecific, or quadrispecific components) This also includes higher-order structures that generate offspring; for example, as described in U.S. Patent No. 5,837,821. As described, the peptide space can be dimerized to form a bispecific / polyvalent molecule. The linked VL and VH, which are further conjugated to the antibody hinge region and CH3 region by the sir, A minibody construct that can form a dimer to create a bispecific diabody. They are linked by a short peptide linker (e.g., 5 or 10 amino acids) in either direction. VH and VL domains linked together or without any linker; for example, U.S. patent Trimers and tetramers as described in Patent No. 5844094; for example, in the United States V forms a series of FVs (or scFvs) as described in Specification No. 5864019 The L-domain is further linked to a crosslinkable group at the C-terminus via a peptide bond. A sequence of VH domains (or VL domains in family members); for example, a country As described in the brochure published in 2011 / 028952, arbitrarily heterodiliter One of the antigens is monovalently bound, and the other antigen is bivalently bound, including the body's Fc region. VL and VH domains, scFv, or Fab; and, for example, U.S. Article 58 As described in Specification No. 69620, for example, scFv or diamond body type Non-covalent bonding that forms homodivalent, heterodivalent, trivalent, and tetravalent structures using both forms. Alternatively, VL and linked via peptide linkers that are compounded into a multivalent structure via chemical crosslinking. Examples include, but are not limited to, single-chain binding polypeptides having both a VH domain and a VH domain. do not have.

[0472] Further exemplary multispecific and bispecific molecules and methods for producing them are, for example, from the United States Patent Office. U.S. Patent No. 5910573, U.S. Patent No. 5932448, U.S. Patent No. 5959 U.S. Patent No. 083, U.S. Patent No. 5989830, U.S. Patent No. 6005079 Book, U.S. Patent No. 6,239,259, U.S. Patent No. 6,294,353, U.S. Patent U.S. Patent No. 6333396, U.S. Patent No. 6476198, U.S. Patent No. 65116 U.S. Patent No. 63, U.S. Patent No. 6670453, U.S. Patent No. 6743896 U.S. Patent No. 6809185, U.S. Patent No. 6833441, U.S. U.S. Patent No. 7129330, U.S. Patent No. 7183076, U.S. Patent No. 752105 U.S. Patent No. 6, U.S. Patent No. 7527787, U.S. Patent No. 7534866, U.S. Patent No. 7612181, U.S. Patent Application Publication No. 2002004587A1 Detailed description, U.S. Patent Application Publication No. 2002076406A1 specification, U.S. Patent Application Publication No. 20 Specification No. 02103345A1, U.S. Patent Application Publication No. 2003207346A1 , U.S. Patent Application Publication No. 2003211078A1, U.S. Pat...

Claims

1. An isolated antibody or its antigen-binding fragment that specifically binds to beta-cell phosphate (BTC). The antibody or its antigen-binding fragment has a dissociation constant (KD) of 5 pM or less. The antibody or its antigen-binding fragment.

2. a. G34, H35, F36, S37, R38, C39, P40, K41, Q42, Y 43, H45, Y46, R51, R53, F54, V56, A57, E58, Q59, T Sequence ID selected from the group consisting of 60, P61, A72, R73, E75, and R76. Binds to at least one of the 157 residues, b. Sequence ID 157: R38, C39, P40, K41, Q42, Y43, H45, Y 46, F54, Q59, T60, P61, and R73 are coupled, c. P40, K41, Q42, Y43, H45, Y46, E58, Q of Sequence ID No. 157 59, T60, P61, A72, R73, E75, and R76 are coupled, d. Sequence ID 157: G34, H35, F36, S37, R38, C39, P40, K 41, Q42, R51, R53, F54, and V56 are coupled, or e. S37, R38, C39, P40, K41, Q42, Y43, H of Sequence ID No. 157 45, Y46, F54, A57, Q59, T60, P61, A72, R73, and E75 The antibody or antigen-binding fragment thereof according to claim 1, which binds to the antibody.

3. Heavy chain variable region complementarity determination region 1 (HCDR1), heavy chain variable region complementarity determination region 2 (HC DR2), and heavy chain variable region complementarity determination region 3 (HCDR3), and light chain variable region complementarity determination Fixed region 1 (LCDR1), light chain variable region complementarity determination region 2 (LCDR2), and light chain variable The region complementarity determination region 3 (LCDR3) includes the HCDR1, HCDR2, and HCD R3, LCDR1, LCDR2, and LCDR3 are a. Sequence numbers 1, 2, 3, 14, 15, and 16 respectively, and sequence numbers 4, 2 respectively, 3, 14, 15, and 16, respectively, sequence numbers 5, 6, 3, 17, 18, and 19, Whether they are as described in sequence numbers 7, 8, 9, 20, 18, and 16, b. Sequence IDs 25, 26, 27, 38, 39, and 40, respectively, and sequence ID 2, respectively. Sequence numbers 8, 26, 27, 38, 39, and 40 correspond to sequences 29, 30, 27, 41, and 4, respectively. 2, and 43, or as indicated in sequence numbers 31, 32, 33, 44, 42, and 40, respectively. Is it as it is listed? c. Sequence IDs 25, 49, 50, 58, 59, and 60, respectively, and sequence ID 2, respectively. Sequence numbers 8, 49, 50, 58, 59, and 60 correspond to sequence numbers 29, 51, 50, 61, and 6, respectively. 2, and 63, or as indicated in sequence numbers 31, 52, 53, 64, 62, and 60, respectively. Is it as stated, or d. Sequence IDs 69, 70, 71, 82, 83, and 84, respectively, and sequence ID 7, respectively. Sequence numbers 2, 70, 71, 82, 83, and 84 correspond to sequence numbers 73, 74, 71, 85, and 1, respectively. As indicated in 8 and 86, or sequence numbers 75, 76, 77, 87, 18, and 84, respectively. The antibody or antigen-binding fragment thereof according to claim 1 or 2, as described above.

4. a. For sequence numbers 10 and 21 respectively, b. For sequence numbers 34 and 45 respectively, c. For sequence numbers 54 and 65 respectively, or d. For SEQ ID NOs. 78 and 88 respectively, at least 90%, 95%, 96%, and 9% Heavy chain variable containing amino acid sequences with 7%, 98%, 99%, or 100% sequence identity. The antibody according to any one of claims 1 to 3, comprising a region (VH) and a light chain variable region (VL). A body or its antigen-binding fragment.

5. a. As described in Sequence IDs 12 and 23, b. As described in Sequence IDs 36 and 47, respectively, c. As described in Sequence IDs 56 and 67, respectively, or d. Heavy chains having amino acid sequences as described in SEQ ID NOs. 80 and 90, respectively, and An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising a light chain.

6. Nuclei encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 A polynucleotide containing an ocidal sequence.

7. An expression cassette or vector comprising the polynucleotide described in claim 6.

8. The polynucleotide described in claim 6 or the expression cassette or vector described in claim 7. — a host cell containing

9. A medical device comprising an effective amount of the antibody or antigen-binding fragment thereof described in any one of claims 1 to 8. A pharmaceutical composition.

10. A method of providing treatment for ophthalmic disorders to those who require it, The effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 or claim The procedure includes administering the pharmaceutical composition described in 9 to the subject, wherein the ophthalmic disorder is diabetic jaundice. Macular edema, age-related macular degeneration, neovascular glaucoma, diabetic retinopathy, macular edema, pathological myopia, retinal stabilization Cardiac occlusion, retinopathy of prematurity, abnormal vascular proliferation associated with nevus disease, central serous chorioretinopathy, and The method is selected from the group consisting of and acute multiple plaque pigment epitheliopathy.

11. The pharmaceutical composition further comprises an anti-vascular epidermal growth factor (VEGF) antagonist, or The method further comprises administering an anti-VEGF antagonist to the subject, EGF antagonists include ranibizumab, bevacizumab, aflibercept, and brolucid. Selected from the group consisting of mab and pegaptanib, or the anti-VEGF antagonist is Claim 1, comprising a heavy chain and a light chain as described in Sequence ID No. 103 and 114, respectively. The method described in 0.

12. A multispecific binding molecule comprising 1) an anti-BTC binding moiety and 2) an anti-VEGF binding moiety.

13. The anti-VEGF binding moiety is the isolated antibody, Fab, Fab', F(ab') ２ F Anti-VEGF antibodies or their antigen-binding fragments in a form selected from the group consisting of v and scFv. The multispecific binding molecule according to claim 12.

14. The anti-BTC binding portion is an anti-BTC Fab, and the anti-VEGF binding portion is an anti-VEG Fab. F Fab, wherein the anti-BTC Fab comprises a heavy chain (HA) and a light chain (LA), The polymorphism according to claim 13, wherein the anti-VEGF Fab comprises a heavy chain (HB) and a light chain (LB). Heavy specificity binding molecules.

15. The HA and HB are arranged from the N-terminus to the C-terminus: N-HA-linker 1- They are connected in the form of HB-C, with LA and LB extending from the N-end towards C- The multiplexing according to claim 14, linked in the form of N-LA-linker2-LB-C. Specificity binding molecules.

16. The anti-BTC binding portion is a. Sequence numbers 1, 2, 3, 14, 15, and 16 respectively, and sequence numbers 4, 2 respectively, 3, 14, 15, and 16, respectively, sequence numbers 5, 6, 3, 17, 18, and 19, As described in sequence numbers 7, 8, 9, 20, 18, and 16, b. Sequence IDs 25, 26, 27, 38, 39, and 40, respectively, and sequence ID 2, respectively. Sequence numbers 8, 26, 27, 38, 39, and 40 correspond to sequences 29, 30, 27, 41, and 4, respectively. 2, and 43, or as indicated in sequence numbers 31, 32, 33, 44, 42, and 40, respectively. Like the kind that will be published, c. Sequence IDs 25, 49, 50, 58, 59, and 60, respectively, and sequence ID 2, respectively. Sequence numbers 8, 49, 50, 58, 59, and 60 correspond to sequence numbers 29, 51, 50, 61, and 6, respectively. 2, and 63, or as indicated in sequence numbers 31, 52, 53, 64, 62, and 60, respectively. It will be published, or d. Sequence IDs 69, 70, 71, 82, 83, and 84, respectively, and sequence ID 7, respectively. Sequence numbers 2, 70, 71, 82, 83, and 84 correspond to sequence numbers 73, 74, 71, 85, and 1, respectively. As indicated in 8 and 86, or sequence numbers 75, 76, 77, 87, 18, and 84, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and A multispecific binding molecule according to any one of claims 12 to 15, comprising LCDR3.

17. The anti-BTC binding portion is a. For sequence numbers 10 and 21 respectively, b. For sequence numbers 34 and 45 respectively, c. For sequence numbers 54 and 65 respectively, or d. For SEQ ID NOs. 78 and 88 respectively, at least 90%, 95%, 96%, and 9% VH and containing amino acid sequences having 7%, 98%, 99%, or 100% sequence identity A multispecific binding molecule according to any one of claims 12 to 16, comprising VL.

18. The anti-BTC binding portion is a. As described in Sequence IDs 12 and 23, b. As described in Sequence IDs 36 and 47, respectively, c. As described in Sequence IDs 56 and 67, respectively, or d. Heavy chains having amino acid sequences as described in SEQ ID NOs. 80 and 90, respectively, and A multispecific binding molecule according to any one of claims 12 to 17, comprising a light chain.

19. The anti-VEGF binding portion is a. These are described in sequence numbers 92, 93, 94, 105, 106, and 107, respectively. eel, b. These are described in sequence numbers 95, 93, 94, 105, 106, and 107, respectively. eel, c. These are described in sequence numbers 96, 97, 94, 108, 109, and 110, respectively. eel, or d. As described in sequence numbers 98, 99, 100, 111, 109, and 107, respectively. such as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 A multispecific binding molecule according to any one of claims 12 to 18, comprising:

20. The anti-VEGF binding moieties, respectively, are at least as follows with respect to SEQ ID NOs. 101 and 112. Having sequence identity of 90%, 95%, 96%, 97%, 98%, 99%, or 100% Multiplex according to any one of claims 12 to 19, comprising VH and VL containing amino acid sequences. Specificity binding molecules.

21. The anti-VEGF binding portion is a. As described in sequence numbers 102 and 113, b. As described in Sequence IDs 117 and 123, respectively, c. As described in Sequence IDs 128 and 133, respectively, d. As described in Sequence IDs 138 and 143, respectively, or e. Encoded by nucleic acid sequences such as those described in SEQ ID NOs. 148 and 152, respectively. The multispecific binding component according to any one of claims 12 to 20, comprising VH and VL. child.

22. The anti-VEGF binding portion is such that it is as described in Sequence ID No. 103 and 114, respectively. It comprises a heavy chain and a light chain having a mino acid sequence, or the heavy chain and light chain each have a sequence number 1 For 04 and 115, at least 90%, 95%, 96%, 97%, 98%, and 99% or encoded by a nucleic acid sequence having 100% sequence identity, claims 12-2 A multispecific binding molecule as described in any one of item 1.

23. The anti-BTC binding moiety consists of a variable heavy chain domain (VHA) and a variable light chain that bind to BTC. It includes a domain (VLA), and the anti-VEGF binding moiety is a variable heavy chain that binds to VEGF. The multispecific binding molecule comprises a main (VHB) and a variable light chain domain (VLB), From the N-terminus to the C-terminus: N-VHA-CH1A-linker-VHB-CH1B -C and N-VLA-CKA-linker-VLB-CKB-C, and this This includes a heavy chain comprising VHA, CH1A, linker, VHB, and CH1B, and this is The light chain includes VLA, CKA, linker, VLB, and CKB, and the heavy chain and the Light chain, a. Whether they are as described in Sequence IDs 120 and 125, respectively. b. Whether they are as described in Sequence IDs 130 and 135, respectively. c. As described in Sequence IDs 140 and 145, respectively, or d. Claims 12 to 22, as described in Sequence IDs 149 and 154, respectively. A multispecific binding molecule as described in any one of the items.

24. Nucleo encoding a multispecific binding molecule according to any one of claims 12 to 23 A polynucleotide containing a cytonucleotide sequence.

25. An expression cassette or vector comprising the polynucleotide described in claim 24.

26. A pharmaceutical product comprising an effective amount of the multispecific binding molecule described in any one of claims 12 to 23. composition.

27. A method for treating ophthalmic disorders in a person requiring treatment for ophthalmic disorders, the aforementioned The target is an effective amount of the multispecific binding molecule described in any one of claims 12 to 23 or The present invention includes administering the pharmaceutical composition described in item 26, wherein the ophthalmic disorder is diabetic macular edema. Macular tumor, age-related macular degeneration, neovascular glaucoma, diabetic retinopathy, macular edema, pathological myopia, retinal vein occlusion A group selected from the group consisting of embolus, retinopathy of prematurity, and abnormal vascular proliferation associated with nevus disease, How to write.

28. The multispecific binding molecule according to any one of claims 12 to 23 or the molecule according to claim 26 A kit containing a pharmaceutical composition.

29. Prophylaxis or treatment of macular edema, DME, AMD, neovascular AMD, or RVO is required. A method for preventing or treating them in a subject, wherein the subject is as described in claims 12 to 23. The multispecific binding molecule described in any one of the items is administered at approximately 0.25 mg / eye, 0.75 mg / eye, A method comprising intravitreal administration at a dose of 2.5 mg / eye or 7.5 mg / eye.

30. The aforementioned doses are 0.25 mg / eye, 0.3 mg / eye, 0.35 mg / eye, and 0.4 mg / eye. , 0.45mg / eye, 0.5mg / eye, 0.55mg / eye, 0.6mg / eye, 0.65m g / eye, 0.7mg / eye, 0.75mg / eye, 0.8mg / eye, 0.85mg / eye, 0. 9mg / eye, 0.95mg / eye, 1.0mg / eye, 1.1mg / eye, 1.2mg / eye, 1 .. 3mg / eye, 1.4mg / eye, 1.5mg / eye, 1.6mg / eye, 1.7mg / eye, 1 .. 8mg / eye, 1.9mg / eye, 2.0mg / eye, 2.1mg / eye, 2.2mg / eye, 2 .. 3mg / eye, 2.4mg / eye, 2.5mg / eye, 2.6mg / eye, 2.7mg / eye, 2 .. 8mg / eye, 2.9mg / eye, 3.0mg / eye, 3.1mg / eye, 3.2mg / eye, 3 .. 3mg / eye, 3.4mg / eye, 3.5mg / eye, 3.6mg / eye, 3.7mg / eye, 3 .. 8mg / eye, 3.9mg / eye, 4.0mg / eye, 4.1mg / eye, 4.2mg / eye, 4 .. 3mg / eye, 4.4mg / eye, 4.5mg / eye, 4.6mg / eye, 4.7mg / eye, 4 .. 8mg / eye, 4.9mg / eye, 5.0mg / eye, 5.1mg / eye, 5.2mg / eye, 5 .. 3mg / eye, 5.4mg / eye, 5.5mg / eye, 5.6mg / eye, 5.7mg / eye, 5 .. 8mg / eye, 5.9mg / eye, 6.0mg / eye, 6.1mg / eye, 6.2mg / eye, 6 .. 3mg / eye, 6.4mg / eye, 6.5mg / eye, 6.6mg / eye, 6.7mg / eye, 6 .. 8mg / eye, 6.9mg / eye, 7.0mg / eye, 7.1mg / eye, 7.2mg / eye, 7 The method according to claim 29, wherein the dose is 3 mg / eye, 7.4 mg / eye, or 7.5 mg / eye.

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