Multispecific antibodies targeting HPTP-β (VE-PTP) and VEGF

Abstract

The present disclosure provides compositions comprising multispecific compounds, including compounds that target phosphatases and receptor tyrosine kinase agonists. Also provided are methods of treating conditions associated with angiogenesis, comprising administering a multispecific compound that targets a phosphatase and a receptor tyrosine kinase agonist.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 735,331, filed September 24, 2018, and U.S. Provisional Application No. 62 / 832,461, filed April 11, 2019, each of which is incorporated herein by reference in its entirety. Background Art

[0003] Individual compounds with the ability to modulate different targets can be combined to create multispecific compounds. Such multispecific compounds may have advantages over the parent compounds administered alone. These advantages may include, for example, a simpler dosing regimen, a longer half-life in the subject, or the ability to bind tightly to the target.

[0004] Incorporation by reference

[0005] Each patent, publication, and non-patent document cited in this application is hereby incorporated by reference in its entirety, as if each were individually incorporated by reference. Summary of the Invention

[0006] In some embodiments, the present disclosure provides a compound comprising: (a) a first domain, wherein the first domain regulates a phosphatase, wherein the phosphatase regulates Tie2; and (b) a second domain that specifically binds a receptor tyrosine kinase agonist.

[0007] In some embodiments, the present disclosure provides a method of treating a condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Schematic diagram of a basic four-chain antibody unit. The light chain sequence is represented by "SEQ A." The heavy chain sequence is represented by "SEQ B." N and C indicate the N-terminus and C-terminus, respectively. -SS- indicates a disulfide bond.

[0009] Figure 2 Schematic diagram of a tetravalent bispecific antibody with sequences appended to the C-terminus of the heavy chain. The light chain sequence of the IgG isotype antibody unit is represented by "SEQ A." The heavy chain sequence of the IgG isotype antibody unit is represented by "SEQ B." The linker sequence is represented by "SEQ C." The appended antigen-binding domain sequence is represented by "SEQ D." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0010] Figure 3Schematic diagram of a tetravalent bispecific antibody with sequences appended to the C-terminus of the light chain. The light chain sequence of the IgG isotype antibody unit is represented by "SEQ A." The heavy chain sequence of the IgG isotype antibody unit is represented by "SEQ B." The linker sequence is represented by "SEQ C." The appended antigen-binding domain sequence is represented by "SEQ D." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0011] Figure 4 Schematic diagram of a hexavalent bispecific antibody with sequences appended to the C-termini of the heavy and light chains. The light chain sequence of the IgG isotype antibody unit is represented by "SEQ A." The heavy chain sequence of the IgG isotype antibody unit is represented by "SEQ B." The linker sequences are represented by "SEQ C" and "SEQ E." The appended antigen-binding domain sequence is represented by "SEQ D." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0012] Figure 5 Schematic diagram of a hexavalent trispecific antibody with sequences appended to the C-termini of the heavy and light chains. The light chain sequence of an IgG isotype antibody unit is represented by "SEQ A." The heavy chain sequence of an IgG isotype antibody unit is represented by "SEQ B." The linker sequences are represented by "SEQ C" and "SEQ E." The appended antigen-binding domain sequences are represented by "SEQ D" and "SEQ F." N and C indicate the N-terminus and C-terminus, respectively. -SS- indicates a disulfide bond.

[0013] Figure 6 Schematic diagram of a bivalent bispecific antibody having two different heavy chain sequences and two different light chain sequences. The light chain sequences are represented by "SEQ A" and "SEQ D." The heavy chain sequences are represented by "SEQ B" and "SEQ C." N and C indicate the N-terminus and C-terminus, respectively. -SS- indicates a disulfide bond.

[0014] Figure 7 Schematic diagram of a trivalent, trispecific antibody having two different heavy chain sequences, two different light chain sequences, and a sequence appended to the C-terminus of one light chain. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequence is represented by "SEQ E." The sequence of the appended antigen-binding domain is represented by "SEQ F." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0015] Figure 8Schematic diagram of a trivalent, trispecific antibody having two different heavy chain sequences, two different light chain sequences, and a sequence appended to the C-terminus of one heavy chain. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequence is represented by "SEQ E." The sequence of the appended antigen-binding domain is represented by "SEQ F." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0016] Figure 9 Schematic diagram of a tetravalent antibody having two different heavy chain sequences, two different light chain sequences, and sequences appended to the C-termini of the two light chains. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequences are represented by "SEQ E" and "SEQ F." The sequences of the appended antigen-binding domains are represented by "SEQ G" and "SEQ H." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0017] Figure 10 Schematic diagram of a tetravalent antibody having two different heavy chain sequences, two different light chain sequences, and sequences appended to the C-termini of the two heavy chains. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequences are represented by "SEQ E" and "SEQ F." The sequences of the appended antigen-binding domains are represented by "SEQ G" and "SEQ H." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0018] Figure 11 Schematic diagram of a tetravalent antibody having two different heavy chain sequences, two different light chain sequences, and sequences appended in cis to the C-terminus of one heavy chain and one light chain. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequences are represented by "SEQ E" and "SEQ F." The appended antigen-binding domain sequences are represented by "SEQ G" and "SEQ H." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0019] Figure 12Schematic diagram of a tetravalent antibody having two different heavy chain sequences, two different light chain sequences, and sequences appended in trans to the C-terminus of one heavy chain and one light chain. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequences are represented by "SEQ E" and "SEQ F." The appended antigen-binding domain sequences are represented by "SEQ G" and "SEQ H." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0020] Figure 13 Schematic diagram of a pentavalent antibody having two different heavy chain sequences, two different light chain sequences, and sequences appended to the C-termini of the two heavy chains and one light chain. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequences are represented by "SEQ E," "SEQ F," and "SEQ G." The appended antigen-binding domain sequences are represented by "SEQ H," "SEQ I," and "SEQ J." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0021] Figure 14 Schematic diagram of a pentavalent antibody having two different heavy chain sequences, two different light chain sequences, and sequences appended to the C-termini of one heavy chain and both light chains. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequences are represented by "SEQ E," "SEQ F," and "SEQ G." The appended antigen-binding domain sequences are represented by "SEQ H," "SEQ I," and "SEQ J." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0022] Figure 15 Schematic diagram of a hexavalent antibody having two different heavy chain sequences, two different light chain sequences, and sequences appended to the C-termini of the heavy and light chains. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequences are represented by "SEQ E," "SEQ F," "SEQ G," and "SEQ H." The appended antigen-binding domain sequences are represented by "SEQ I," "SEQ J," "SEQ K," and "SEQ L." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0023] Figure 16 : ELISA data demonstrating binding of the tetravalent bispecific antibody HC2-AFL:LC1 to HPTP-β (upper panel) and VEGF (lower panel).

[0024] Figure 17 : ELISA data demonstrating binding of the tetravalent bispecific antibody HC2-BRO:LC1 to HPTP-β (upper panel) and VEGF (lower panel).

[0025] Figure 18 : ELISA data testing the binding of the tetravalent bispecific antibody HC2-RAN:LC1 to HPTP-β (upper panel) and VEGF (lower panel).

[0026] Figure 19 Tie2 activation in HUVECs treated with tetravalent or hexavalent bispecific antibodies containing brolucizumab-derived or aflibercept-derived VEGF-binding domains. As demonstrated by immunoprecipitation and Western blotting, all bispecific antibodies tested increased basal Tie2 activation (in the absence of exogenous Ang1 or Ang2), while basal VEGFR2 phosphorylation (in the absence of exogenous VEGF) was unaffected.

[0027] Figure 20 Tetravalent and hexavalent bispecific antibodies containing brolucizumab-derived or aflibercept-derived VEGF-binding domains enhanced Tie2 activation and inhibited VEGFR2 activation in HUVECs treated with Ang1 and VEGF, as demonstrated by immunoprecipitation and Western blotting.

[0028] Figure 21A 、 Figure 21B and Figure 21C Results: Tetravalent and hexavalent bispecific antibodies containing abicipar-derived or aflibercept-derived VEGF-binding domains enhanced Tie2 activation and inhibited VEGFR2 activation in HUVECs, including HUVECs treated with Ang1 and VEGF. Figure 21A Western blots are provided. Figure 21B Quantification of Tie2 activation is provided. Figure 21C Quantification of VEGFR2 phosphorylation is provided.

[0029] Figure 22A 、 Figure 22B and Figure 22CHexavalent bispecific antibodies containing brolucizumab-derived, aflibercept-derived, or abicipar-derived VEGF-binding domains enhance Tie2 activation and inhibit VEGFR2 activation in HUVECs, including HUVECs treated with Ang1 and VEGF. Figure 22A Western blots are provided. Figure 22B Quantification of Tie2 activation is provided. Figure 22C Quantification of VEGFR2 phosphorylation is provided.

[0030] Figure 23A and Figure 23B Tetravalent and hexavalent bispecific antibodies containing brolucizumab-derived, aflibercept-derived, or abicipar-derived VEGF-binding domains enhanced Tie2 activation and inhibited VEGFR2 activation in HUVECs treated with Ang1 and VEGF, as demonstrated by quantification of electrochemiluminescence signals. Figure 23A Quantification of Tie2 activation is provided. Figure 23B Quantification of VEGFR2 phosphorylation is provided.

[0031] Figure 24A and Figure 24B Hexavalent bispecific antibodies containing brolucizumab-derived, aflibercept-derived, or abicipar-derived VEGF-binding domains enhanced Tie2 activation and inhibited VEGFR2 activation in HUVECs treated with Ang1 and VEGF, as demonstrated by quantification of electrochemiluminescence signals. Figure 24A Quantification of Tie2 activation is provided. Figure 24B Quantification of VEGFR2 phosphorylation is provided.

[0032] Figure 25A and Figure 25B Tetravalent and hexavalent bispecific antibodies containing a brolucizumab-derived VEGF-binding domain enhanced Tie2 activation and inhibited VEGFR2 activation in HUVECs treated with Angl and VEGF, as demonstrated by quantification of electrochemiluminescence signals. Figure 25A Quantification of Tie2 activation is provided. Figure 25B Quantification of VEGFR2 phosphorylation is provided.

[0033] Figure 26A and Figure 26B Results: Tetravalent and hexavalent bispecific antibodies containing abicipar-derived or aflibercept-derived VEGF-binding domains enhanced Tie2 activation and inhibited VEGFR2 activation in HUVECs treated with Ang1 and VEGF, as demonstrated by quantification of electrochemiluminescence signals. Figure 26A Quantification of Tie2 activation is provided. Figure 26B Quantification of VEGFR2 phosphorylation is provided.

[0034] Figure 27A and Figure 27B Results: Tetravalent and hexavalent bispecific antibodies containing abicipar-derived or aflibercept-derived VEGF-binding domains enhanced Tie2 activation and inhibited VEGFR2 activation in HUVECs treated with Ang1 and VEGF, as demonstrated by quantification of electrochemiluminescence signals. Figure 27A Quantification of Tie2 activation is provided. Figure 27B Quantification of VEGFR2 phosphorylation is provided. DETAILED DESCRIPTION

[0035] The present disclosure provides compositions and methods for regulating phosphatases and kinases, such as receptor tyrosine kinases. Compositions and methods for regulating Tie2, for example, to promote Tie2 phosphorylation, signaling, and / or activation, are provided. In some embodiments, the present disclosure provides compositions and methods for targeting phosphatases that regulate Tie2 signaling. In some embodiments, the phosphatase that regulates Tie2 signaling is human protein tyrosine phosphatase-β (HPTP-β).

[0036] Provided are compositions and methods for modulating receptor tyrosine kinases, e.g., to reduce receptor tyrosine kinase phosphorylation, signaling, and / or activation. In some embodiments, the present disclosure provides compositions and methods for targeting receptor tyrosine kinase agonists, e.g., vascular endothelial growth factor (VEGF). In some embodiments, the present disclosure provides compositions and methods for targeting receptor tyrosine kinases, e.g., VEGF receptors.

[0037] In some embodiments, the present disclosure provides compositions and methods for targeting human protein tyrosine phosphatase-β (HPTP-β) or vascular endothelial protein tyrosine phosphatase (VE-PTP or VEPTP) and vascular endothelial growth factor (VEGF). In some embodiments, the present disclosure provides multispecific compounds, agents, antibodies, fragments or derivatives targeting HPTP-β (VE-PTP) and VEGF.

[0038] The agents disclosed herein can be used to treat disorders characterized by, for example, vascular instability, angiogenesis, neovascularization, vascular leakage, and / or edema. The agents disclosed herein can be used to treat, for example, vascular disorders, ocular disorders, cancer, renal disorders, and diabetic complications.

[0039] HPTP-β / VE-PTP, Tie2 and vascular stability

[0040] HPTP-β is a member of the receptor-like family of protein tyrosine phosphatases (PTPases). HPTP-β is a transmembrane protein found primarily in vascular endothelial cells that exhibits structural and functional similarities to cell adhesion molecules. Orthologs of HPTP-β have been found in various species, including, for example, zebrafish, chicken, dog, mouse, marmoset, and monkey. Orthologs are commonly referred to as vascular endothelial protein tyrosine phosphatases (VE-PTPs). HPTP-β (VE-PTP) can affect vascular stability by acting on Tie2-mediated signaling.

[0041] Tie2 (tyrosine kinase 2 with immunoglobulin and epidermal growth factor homology domains) is a membrane receptor tyrosine kinase expressed primarily in vascular endothelial cells. Upstream factors can regulate Tie2 phosphorylation, influencing downstream signaling and vascular stabilization. Non-limiting examples of such factors include angiopoietin 1 (Ang1 / Angpt1), angiopoietin 2 (Ang2 / Angpt2), and HPTP-β (VE-PTP).

[0042] Ang1 is an agonist of Tie2. Ang1 binding to Tie2 promotes receptor phosphorylation and downstream signaling, inducing vascular stabilization through highly organized angiogenesis, strengthening endothelial cell junctions, enhancing endothelial motility, reducing endothelial inflammation, and improving endothelial function.

[0043] Ang2 acts as a context-dependent antagonist or agonist of Tie2. During angiogenesis, Ang2 acts as a negative regulator of Ang1-Tie2 signaling.

[0044] HPTP-β (VE-PTP) is a phosphatase that can regulate Tie2 signaling. HPTP-β (VE-PTP) can dephosphorylate the Tie2 receptor. Under physiological conditions, HPTP-β (VE-PTP) regulates the duration of Tie2 phosphorylation. Therefore, inhibition of HPTP-β (VE-PTP) can lead to increased Tie2 phosphorylation, increased Tie2-mediated signaling, and enhanced vascular stability. Inhibitors of HPTP-β (VE-PTP) are Tie2 activators. For example, compounds, inhibitors, antibodies, antibody fragments, variants, or derivatives that bind to HPTP-β (VE-PTP) can promote Tie2 phosphorylation, thereby activating Tie2 downstream signaling and promoting vascular stability.

[0045] Through the above process, HPTP-β (VE-PTP) activity can lead to conditions characterized by, for example, vascular instability, angiogenesis, neovascularization, vascular leakage, and / or edema. For example, HPTP-β (VE-PTP) activity can lead to vascular disorders, eye disorders, cancer, kidney disorders, diabetic complications, and other disorders. Inhibition of HPTP-β (VE-PTP) activity can alleviate such disorders.

[0046] VEGF and vascular stability

[0047] Vascular endothelial growth factor (VEGF) is primarily found in endothelial cells and is associated with pathological neovascularization in many diseases. VEGF is a member of the cystine knot growth factor superfamily, the PDGF family, and the VEGF family. VEGF can act as a pro-angiogenic factor. The VEGF family consists of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PGF). There are nine VEGF-A isoforms: VEGF 121 , VEGF 145 , VEGF 148 , VEGF 162 , VEGF 165 , VEGF 165b , VEGF 183 , VEGF 189 and VEGF 206 .

[0048] VEGF is a hypoxia-regulated gene, and VEGF levels are increased in hypoxic or ischemic conditions. VEGF is an agonist of the VEGF receptor (VEGFR). VEGFR is a receptor tyrosine kinase; binding of VEGF to VEGFR can lead to phosphorylation of the receptor, which subsequently leads to phosphorylation of downstream signal transducers. VEGFR-mediated signaling can lead to abnormal angiogenesis, vasculogenesis, and vascular permeability, leading to pathological vascular instability. Therefore, inhibition of VEGF can lead to reduced VEGFR-mediated signaling and enhanced vascular stability. For example, inhibitors, antibodies, antibody fragments, variants, or derivatives thereof that bind to VEGF can reduce VEGFR connections, thereby reducing VEGFR-mediated signaling, and promoting vascular stability. Non-limiting examples of agents that bind to VEGF include aflibercept - a recombinant protein comprising the VEGF-binding portions of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1; brolucizumab - a humanized single-chain antibody fragment (scFv); RTH258 - a humanized single-chain antibody fragment (scFv); ranibizumab —A humanized monoclonal antibody fragment (Fab); bevacizumab - a humanized monoclonal antibody; conbercept - a recombinant fusion protein comprising the extracellular domains from VEGF receptors 1 and 2 fused to the Fc portion of human IgG1; Abicipar - a designed ankyrin repeat protein (DARPin); MP0112 - a DARPin; MP0250 - a DARPin; CT-322 - an adnectin; and PRS-050 - an anticalin.

[0049] Through the above-mentioned process, VEGF can cause diseases characterized by, for example, vascular instability, angiogenesis, neovascularization, vascular leakage and / or edema. For example, VEGF can cause vascular diseases, eye diseases, cancer, kidney diseases, diabetic complications and other diseases. For example, local ischemia of the eye can lead to increased VEGF production, resulting in vascular leakage and pathological neovascularization in the retina. Inhibition of VEGFR-mediated signaling can alleviate such diseases.

[0050] Receptor tyrosine kinases (RTKs) and receptor tyrosine kinase agonists

[0051] Receptor tyrosine kinases (RTKs) are cell surface receptors involved in the regulation of cell growth, differentiation and survival. The combination of agonists and RTKs can cause adjacent RTKs to associate with each other, thereby forming dimers. Dimerization can cause cross-phosphorylation-each RTK in the dimer phosphorylates multiple tyrosine residues on another RTK. Once cross-phosphorylated, the cytoplasmic tail of the RTK can initiate a signal transduction pathway, for example, by serving as a docking platform for various intracellular proteins. RTK signaling can lead to changes in gene transcription and expression in cells.

[0052] Non-limiting examples of RTKs include AATK, AATYK, AATYK1, AATYK2, ACH, ALK, ARK, AXL, BDB, BDB1, BEK, BFGFR, BREK, Brt, CAK, CCK4, CD115, CD117, CD135, CD136, CD140a, CD140b, CD167, CD202b, CD220, CD221, CD246, CD309, CD331, CD332, CD333, CD334, CDHF12, CDHR16, CDw136, CEK, CEK2, CEK3, c-Eyk, CFD1, C-FMS, C-Kit, cprk, c-ros-1, CSF1R, CSFR, D3S3195, DDR1, DDR2, DFNB97, DKFZp761P1010, Dtk, ECT1, EDDR1, EGFR, EphA10, EphA1-8, EphB1, EphB2, EphB3, EphB4, EphB6, ErbB2, ErbB3, ErbB4, Etk-2, FGFR1, FGFR2, FGFR3, FGFR4, FLG, FLK1, FLK2, FLT, FLT1, FLT2, FLT3, FLT4, FMS, GAS9, H2, H3, H4, H5, HGFR, HSCR1, IGF1R, IGFIR, IGFR, INSR, INSRR, IRR, JKT5A, JTK11, JTK12, JTK13, JTK14, JTK2, JTK4, JTK5, JWS, KAL2, KDR, KGFR, KIAA0641, KIAA1079, KIAA1883, KIT, KPI2, K-SAM, LMR1, LMR2, LMR3, LMTK1, LMTK2, LMTK3, LTK, MCF3, MEN2A, MEN2B, Mer, MERTK, MET, MGC18216, MST1R, MTC, MTC1, MTRK1, MuSK, NEP, NOK, N-SAM, NTRK2, NTRK3, NTRK4, NTRKR1, NTRKR2, PBT, PCL, PDGFR, PDGFR1, PDGFR2, PDGFRA, PDGFR-alpha, PDGFRB, PDGFR-beta, PPP1R100, PPP1R101, PPP1R77, PTC, PTK3A, PTK7, PTK8, RCCP2, Rek, RET, RET51, RON, ROR1, ROR2, ROS, ROS1, RP38, RSE, RTK6, RYK, Ryk, RYK1, SCFR, Sky, STK, STYK1, SuRTK106, TEK, TIE1TIE2, Tif, TK14, TK25, TKT, TRK, TrkA, TrkB, TrkC, TYK1, TYKLM3, TYRO10, Tyro12, Tyro3, ​​Tyro7, UFO, VEGFR, VEGFR1, VEGFR2, VEGFR3, VMCM and VMCM1. ,

[0053] Non-limiting examples of RTK agonists include VEGF, Ang1, Ang2, BDNF, EGF, FGF, HGF, IGF, insulin, MSP, NGF, NT-3, and PDGF.

[0054] Antibodies and antigen-binding compounds

[0055] The basic four-chain antibody unit contains two identical heavy chain (H) polypeptide sequences and two identical light chain (L) polypeptide sequences. Each heavy chain may contain an N-terminal variable (V H ) region and three or four C-terminal constant (C H 1. C H 2. C H 3 and C H 4) region. Each light chain may contain an N-terminal variable (V L ) region and a C-terminal constant (C L The light chain variable region is aligned with the heavy chain variable region, and the light chain constant region is aligned with the heavy chain constant region C H1 Aligned. The paired heavy and light chain variable regions together form a single antigen-binding site. Each light chain is linked to a heavy chain by a covalent disulfide bond. Depending on the heavy chain isotype, the two heavy chains are linked to each other by one or more disulfide bonds. Each heavy and light chain also contains regularly spaced intrachain disulfide bonds. The C-terminal constant region of the heavy chain comprises the Fc region of the antibody, which mediates effector functions, for example, by interacting with Fc receptors or complement proteins. Figure 1 A simple representative schematic diagram of the basic four-chain antibody unit is provided; the light chain sequence is represented by "SEQ A". The heavy chain sequence is represented by "SEQ B", -SS- represents a disulfide bond, and N and C represent the N-terminus and C-terminus, respectively.

[0056] Based on the amino acid sequence of the constant region, the light chain can be named κ or λ. Based on the amino acid sequence of the constant region, the heavy chain can be named α, δ, ε, γ or μ. Based on the heavy chain, antibodies are divided into five immunoglobulin classes or isotypes. IgA contains α heavy chains, IgD contains δ heavy chains, IgE contains ε heavy chains, IgG contains γ heavy chains, and IgM contains μ heavy chains. Antibodies of the IgG, IgD and IgE classes contain monomers of the above-mentioned four-chain units (two heavy chains and two light chains), while antibodies of the IgM and IgA classes can contain multimers of the four-chain units. Based on the differences in sequence and function of the heavy chain constant region, the α and γ classes are further divided into subclasses. The subclasses of IgA and IgG expressed by humans include IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

[0057] The constant region is least involved in antigen binding. On the contrary, the constant region can mediate various effector functions. For example, due to the interaction with different Fc receptors and / or complement proteins, different IgG isotypes or subclasses can be associated with different effector functions or therapeutic properties. For example, antibodies comprising an Fc region that engages with activating Fc receptors can participate in antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), induction of signal transduction and cytokine secretion by activation motifs (ITAMs) based on immunoreceptor tyrosine. For example, antibodies comprising an Fc region that engages with inhibitory Fc receptors can induce signal transduction by inhibitory motifs (ITIMs) based on immunoreceptor tyrosine.

[0058] Different antibody subclasses have different abilities to trigger immune effector functions. For example, IgG1 and IgG3 can effectively recruit complement to activate CDC, and IgG2 triggers minimal ADCC. IgG4 has a smaller ability to trigger immune effector functions. The modification of the constant region can also affect antibody properties, for example, the enhancement or reduction of Fc receptor connection, the enhancement or reduction of ADCC, the enhancement or reduction of ADCP, the enhancement or reduction of CDC, the enhancement or reduction of signal transduction by ITAM, the enhancement or reduction of cytokine induction, the enhancement or reduction of signal transduction by ITIM, the extension or shortening of half-life, or the enhancement or reduction of the co-engagement of antigen and Fc receptor. Modification can include, for example, amino acid mutations, changes in post-translational modifications (such as glycosylation), combinations of domains from different isotypes or subclasses, or combinations thereof.

[0059] The compounds or antibodies disclosed herein may comprise a constant region or Fc region, which are selected or modified to provide suitable antibody characteristics, for example, suitable properties for treating a disease or condition disclosed herein. In some embodiments, IgG1 can be used to, for example, promote immune activation effector functions (e.g., ADCC, ADCP, CDC, ITAM signaling, cytokine induction, or a combination thereof, for treating cancer). In some embodiments, for example, IgG4 can be used in the absence of immune effector functions where the antagonistic properties of an antibody are desired (e.g., for treating eye conditions).

[0060] Non-limiting examples of antibody modifications and their effects are provided in Table 1.

[0061]

[0062] The variable (V) region mediates antigen binding and defines the specificity of a particular antibody for the antigen. The variable region comprises relatively invariant sequences known as framework regions and hypervariable regions, the sequences of which vary greatly between antibodies with different binding specificities. The variable region of each antibody heavy or light chain comprises four framework regions separated by three hypervariable regions. The variable regions of the heavy and light chains fold in a manner that tightly binds the hypervariable regions together to form an antigen binding site. The four framework regions primarily adopt a beta-pleated configuration, while the three hypervariable regions form loops that connect, and in some cases form part of, the beta-pleated structure.

[0063] In the hypervariable region, there are amino acid residues that mainly determine the binding specificity of the antibody. The sequence containing these residues is called the complementary determining region (CDR). An antigen-binding site of an antibody contains six CDRs, three in the hypervariable region of the light chain and three in the hypervariable region of the heavy chain. The CDRs in the light chain are named L1, L2 and L3, while the CDRs in the heavy chain are named H1, H2 and H3. CDRs can also be designated as LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 respectively. The contribution of each CDR to antigen binding varies from antibody to antibody. The length of CDR can be different. For example, the length of CDR is generally 5 to 14 residues, but there are CDRs as short as 0 residues or as long as 25 residues or longer.

[0064] There are several methods for predicting or assigning CDR sequences. These methods may use different numbering systems, for example, because sequence insertions and deletions are numbered differently.

[0065] The Kabat method was developed by aligning a limited number of antibody sequences and determining the positions of the most variable residues. Based on the alignment, a numbering scheme was introduced for the residues in the variable region. This numbering scheme can be used to determine the positions that mark the start and end of each CDR. One iteration of the Kabat numbering system uses the following residue positions to determine the CDRs in the light chain variable region: LCDR1 around residues 24-34; LCDR2 around residues 50-56; and LCDR3 around residues 89-97. One iteration of the Kabat numbering system uses the following residue positions to determine the CDRs in the heavy chain variable region: HCDR1 around residues 31-35; HCDR2 around residues 50-65; and HCDR3 around residues 95-102.

[0066] The Chothia method was developed based on analysis of three-dimensional antibody structure. The analysis determined that the hypervariable loops adopt a restricted set of conformations based on the presence of certain residues at key positions in the CDRs and flanking framework regions. This method uses a numbering scheme similar to the Kabat method, but numbers insertions and deletions differently. One iteration of the Chothia numbering system uses the following residue positions to identify the CDRs in the light chain variable region: LCDR1 around residues 24-34; LCDR2 around residues 50-56; and LCDR3 around residues 89-97. One iteration of the Chothia numbering system uses the following residue positions to identify the CDRs in the heavy chain variable region: HCDR1 around residues 26-34; HCDR2 around residues 52-56; and HCDR3 around residues 95-102.

[0067] The IMGT method (International ImMunoGeneTics database) is developed by integrating existing definitions of framework regions and CDRs, structural data, and data from antibody variable region sequence alignments. This integration results in the identification of conserved residues in the framework regions, which can be used as reference points for identifying CDRs. Examples of conserved residues in the variable region include cysteine ​​at approximately residue 23 (in framework region 1), tryptophan at approximately residue 41 (in framework region 2), hydrophobic amino acids at approximately residue 89 (in framework region 3), cysteine ​​at approximately residue 104 (in framework region 3), and phenylalanine or tryptophan at approximately residue 118 (in framework region 4). By using an algorithm based on computational alignment, CDRs can be identified in the sequence encoding the variable region of an antibody of interest.

[0068] The IMGT numbering method consistently assigns the same number to conserved amino acids, but allows for variation in the length of CDRs and framework regions. Therefore, the IMGT numbering of residues is not necessarily consecutive. The length of CDRs determined by the IMGT method can vary. For example, LCDR1 or HCDR1 can be from about 5 to about 12 amino acids, LCDR2 or HCDR2 can be from about 0 to about 10 amino acids, and LCDR3 or HCDR3 can be from about 5 to about 91 amino acids.

[0069] The Paratome method was developed based on multiple structural alignments of available antibody-antigen complexes. The structural positions at which the antigen is bound were found to be similar between the antibodies examined, and the antibody sequences of the dataset were annotated with antigen-binding regions (ABRs, similar to CDRs). The ABRs in the query sequence can be determined using computational tools that first align the query sequence against antibodies with resolved antibody-antigen structures and then infer the positions of the ABRs based on this alignment. Antibodies with resolved structures can also use structural alignment methods rather than sequence-based alignment methods to determine ABRs.

[0070] The residue subset in CDR contacts the antigen. These residues in contact with the antigen can be referred to as specificity determining residues (SDR). However, other residues except SDR can promote binding activity by helping to maintain the conformation of the binding site. The number of SDR in the antibody can be different according to the size and type of the antigen being recognized. For example, 0-14 SDRs can be found in CDR. SDR can be rich in some residues, such as tyrosine, serine, tryptophan and asparagine.

[0071] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. In contrast to polyclonal antibody preparations that include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope.

[0072] The compounds herein can be monoclonal antibodies, e.g., chimeric antibodies in which portions of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, or antigen-binding fragments of such antibodies.

[0073] Antibody fragments or antigen-binding fragments may comprise a portion of an antibody, such as the antigen-binding region or variable region of an intact antibody. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, dimers and trimers of Fab conjugates, Fv, scFv, minibodies, diabodies, triabodies and tetrabodies, as well as linear antibodies. Fab and Fab' are antigen-binding fragments that may comprise a V domain linked to a light chain by a disulfide bond. L and C L domain-linked heavy chain V H and C H 1 domain. F(ab')2 may comprise two Fab or Fab' linked by a disulfide bond. Fv may comprise a V held together by non-covalent interactions. H and V L scFv (single chain variable fragment) is a fragment that can contain V domains connected by a peptide linker. H and V L Fusion protein of the structural domain. Operator V H and V L The orientation of the domains and the length of the linker can be used to create molecules of different formats, which can be monomeric, dimeric (diabodies), trimeric (tribodies), or tetrameric (tetrabodies). Minibodies are scFv-C antibodies that can assemble into bivalent dimers. H 3 fusion proteins.

[0074] Non-limiting examples of epitopes include amino acids, sugars, lipids, phosphoryl groups, and sulfonyl groups. An epitope can have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope can be conformational or linear.

[0075] For human administration, monoclonal antibodies produced in non-human species can be further optimized through a process called humanization to reduce the potential for immunogenicity while retaining target specificity. The humanization process involves integrating human DNA into the genetic sequence of the gene that produces the isolated antibody. The recombinant DNA is cloned and expressed in cells for large-scale production of the newly humanized antibody.

[0076] An example of a humanized antibody is a modified chimeric antibody. As described above, chimeric antibodies can be produced. Chimeric antibodies are further mutated outside the CDR to replace the non-human sequences in the variable region with homologous human sequences. Another example of a humanized antibody is a CDR-grafted antibody, in which non-human CDR sequences are introduced into the human heavy and light chain variable sequences of a human antibody scaffold to replace the corresponding human CDR sequences.

[0077] Humanized antibodies can be produced in mammalian cells, bioreactors, or transgenic animals such as mice, chickens, sheep, goats, pigs, or marmosets. Transgenic animals have a large portion of the genome that produces human antibodies inserted into the animal's genome.

[0078] In addition to antibodies and antibody fragments, other antigen-binding compounds can also bind to target molecules. Non-limiting examples of non-antibody derived antigen-binding compounds include ankyrin, ankyrin repeat proteins, designed ankyrin repeat proteins (DARPins), affibodies, avimers, adnectins, anticalins, Fynomers, Kunitz domains, knottins, β-hairpin mimetics, and receptors and derivatives thereof, e.g., VEGF receptors, or VEGF binding portions of human VEGF receptors 1 and 2.

[0079] Designed ankyrin repeat proteins (DARPins) can be protein scaffolds based on ankyrin repeat proteins. A DARPin can comprise one or more ankyrin repeats comprising a common sequence and / or structural motif. When compared to one another, individual ankyrin repeats can comprise a common sequence and / or structural motif, despite comprising mutations, substitutions, additions and / or deletions. A DARPin can comprise, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more ankyrin repeats. A DARPin can comprise an N-terminal capping repeat, one or more internal ankyrin repeats and a C-terminal capping repeat. Each ankyrin repeat can comprise framework residues and protein interaction residues. Framework residues can contribute to structural or folding topology, such as the structure of ankyrin repeats or interactions with adjacent ankyrin repeats. Protein interaction residues can promote binding of a target molecule, for example, by direct interaction with the target molecule or by direct interaction residues that are stabilized in a conformation that allows binding.

[0080] The compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure that bind to a target molecule can be, for example, at a concentration of less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM. , less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 9 pM, less than about 8 pM, less than about 7 pM, less than about 6 pM, less than about 5 pM, less than about 4 pM, less than about 3 pM, less than about 2 pM, less than about 1 pM, less than about 900 fM, less than about 800 fM, less than about 700 fM, less than about 600 fM, less than about 500 fM, less than about 400 fM, less than about 300 fM, less than about 200 fM, less than about 100 fM, less than about 90 fM, less than about 80 fM, less than about 70 fM, less than about 60 fM, less than about 50 fM, less than about 40 fM, less than about 30 fM, less than about 20 fM, or less than about 10 fM. D Bind to target.

[0081] In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure that bind to a target molecule can be expressed at, for example, about 10 fM to about 500 nM, about 30 fM to about 500 nM, about 30 fM to about 400 nM, about 30 fM to about 300 nM, about 30 fM to about 200 nM, about 30 fM to about 100 nM, about 30 fM to about 90 nM, about 30 fM to about 80 nM, about 30 fM to about 70 nM, about 30 fM to about 60 nM, about 30 fM to about 50 nM, about 30 fM to about 40 nM, about 30 fM to about 30 nM, about 30 fM to about 20 nM, about 30 fM to about 10 nM, about 30 fM to about 90 nM, about 30 fM to about 80 nM, about 30 fM to about 70 nM, about 30 fM to about 60 nM, about 30 fM to about 50 nM, about 30 fM to about 40 nM, about 30 fM to about 30 nM, about 30 fM to about 20 nM, about 30 fM to about 8nM, about 30fM to about 7nM, about 30fM to about 6nM, about 30fM to about 5nM, about 30fM to about 4nM, about 30fM to about 3nM, about 30fM to about 2nM, about 30fM to about 1nM, about 30fM to about 900pM, about 30fM to about 800pM, about 30fM to about 700pM, about 30fM to about 600pM, about 30fM to about 500pM, about 30fM to about 400pM, about 30fM to about 300pM, about 30fM to about 200pM, about 30fM to about 100pM, about 30fM to about 90pM, about 30fM to about 80pM, about 30fM to about 70pM, about 30fM to about 60pM, about 3 0 fM to about 50 pM, about 30 fM to about 40 pM, about 30 fM to about 30 pM, about 30 fM to about 20 pM, about 30 fM to about 10 pM, about 30 fM to about 1 pM, about 30 fM to about 900 fM, about 30 fM to about 800 fM, about 30 fM to about 700 fM, about 30 fM to about 600 fM, about 30 fM to about 500 fM, about 30 fM to about 400 fM, about 30 fM to about 300 fM, about 30 fM to about 200 fM, about 30 fM to about 100 fM, about 30 fM to about 500 nM, about 30 fM to about 400 nM, about 30 fM to about 300 nM, about 30 fM to about 200 nM, about 30 fM to about 100 nM, about 30 fM to about 90 nM, about 30 fM to about 80 nM, about 30 fM to about 70 nM, about 30 fM to about 60 nM, about 30 fM to about 50 nM, about 30 fM to about 40 nM, about 30 fM to about 30 nM, about 30 fM to about 20 nM, about 30 fM to about 10 nM, about 30 fM to about 9 nM, about 30 fM to about 8 nM, about 30 fM to about 7 nM, about 30 fM to about 6 nM, about 30 fM to about 5 nM, about 30 fM to about 4 nM, about 30 fM to about 3 nM, about 30 fM to about 2 nM, about 30 fM to about 1 nM, about 30 fM to about 900 pM, about 30 fM to about 800 pM, about 30 fM to about 700 pM,about 30 fM to about 600 pM, about 30 fM to about 500 pM, about 30 fM to about 400 pM, about 30 fM to about 300 pM, about 30 fM to about 200 pM, about 30 fM to about 100 pM, about 30 fM to about 90 pM, about 30 fM to about 80 pM, about 30 fM to about 70 pM, about 30 fM to about 60 pM, about 30 fM to about 50 pM, about 30 fM to about 40 pM, about 30 fM to about 30 pM, about 30 fM to about 20 pM, about 30 fM to about 10 pM, about 1 pM to about 500 nM, about 1 pM to about 400 nM, about 1 pM to about 300 nM, about 1 pM to about 200 nM, about 1 pM to about 100 nM, about 1 pM to about 90nM, about 1pM to about 80nM, about 1pM to about 70nM, about 1pM to about 60nM, about 1pM to about 50nM, about 1pM to about 40nM, about 1pM to about 30nM, about 1pM to about 20nM, about 1pM to about 10nM, about 1pM to about 9nM, about 1pM to about 8nM, about 1pM to about 7nM, about 1pM to about 6nM, about 1pM to about 5nM, about 1pM to about 4nM, about 1pM to about 3nM, about 1pM to about 2nM, about 1pM to about 1nM, about 1pM to about 900pM, about 1pM to about 800pM, about 1pM to about 700pM, about 1pM to about 600pM, about 1pM to about 500pM, about 1pM to about 400pM, about 1pM to about 300pM, about 1pM to about 200pM, about 1pM to about 100pM, about 1pM to about 90pM, about 1pM to about 80pM, about 1pM to about 70pM, about 1pM to about 60pM, about 1pM to about 50pM, about 1pM to about 40pM, about 1pM to about 30pM, about 1pM to about 20pM, about 1pM to about 10pM, about 100pM to about 500nM, about 100pM to about 400nM, about 100pM to about 300nM, about 100pM to about 200nM, about 100pM to about 100nM, about 100pM to about 90nM, about 100pM to about 80nM, about 100pM pM to about 5 nM, about 100 pM to about 4 nM, about 100 pM to about 3 nM, about 100 pM to about 2 nM, about 100 pM to about 10 nM, about 100 pM to about 9 nM, about 100 pM to about 8 nM, about 100 pM to about 7 nM, about 100 pM to about 6 nM, about 100 pM to about 5 nM, about 100 pM to about 4 nM, about 100 pM to about 3 nM, about 100 pM to about 2 nM, about 100 pM to about 1 nM, about 100 pM to about 900 pM, about 100 pM to about 800 pM, about 100 pM to about 700 pM, about 100 pM to about 600 pM,a K of about 100 pM to about 500 pM, about 100 pM to about 400 pM, about 100 pM to about 300 pM, or about 100 pM to about 200 pM, D Bind to target.

[0082] In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure can be expressed in a manner such that the K of about 500 fM to about 500 pM is greater than or equal to 1. D Binds to HPTP-β (VE-PTP). In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure can bind to HPTP-β (VE-PTP) with a K of about 1 pM to about 500 pM. D Binds to HPTP-β (VE-PTP). In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure can bind to HPTP-β (VE-PTP) with a K of about 60 pM to about 500 pM. D Binds to HPTP-β (VE-PTP). In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure can bind to HPTP-β (VE-PTP) with a K of about 100 pM to about 500 pM. D Binds to HPTP-β (VE-PTP). In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure can bind to HPTP-β (VE-PTP) with a K of about 1 pM to about 300 pM. D Binds to HPTP-β (VE-PTP). In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure can bind to HPTP-β (VE-PTP) with a K of about 1 pM to about 200 pM. D Binds to HPTP-β (VE-PTP). In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure can bind to HPTP-β (VE-PTP) with a K of about 1 pM to about 120 pM. D Binds to HPTP-β (VE-PTP). In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure can bind to HPTP-β (VE-PTP) with a K of about 1 pM to about 70 pM. D Binds to HPTP-β (VE-PTP).

[0083] In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure can be expressed in a manner such that the K of about 30 fM to about 900 pM is greater than or equal to 1. D In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 30 fM to about 600 pM. D In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 30 fM to about 200 pM. DIn some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 30 fM to about 30 pM. D In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 30 fM to about 40 pM. D In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 30 fM to about 1 pM. D In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 30 fM to about 200 fM. D In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 1 pM to about 900 pM. D In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 1 pM to about 600 pM. D In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 1 pM to about 200 pM. D In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 1 pM to about 30 pM. D In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 1 pM to about 40 pM. D Binds to VEGF.

[0084] In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure can be expressed in a manner such that the K of about 30 fM to about 2 pM is about 0. D In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 35 fM to about 200 fM. D In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 100 fM to about 2 pM. D Binds to VEGF.

[0085] In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure can be expressed in a manner such that the K of about 20 pM to about 1 nM is about 0. DIn some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 20 pM to about 800 pM. D In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 20 pM to about 350 pM. D Binds to VEGF.

[0086] In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure can be expressed in a manner such that the K of about 30 fM to about 700 pM is greater than or equal to 1. D Binds to VEGF.

[0087] In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure can be expressed in a manner such that the K of about 40 fM to about 520 pM is about 0. D In some embodiments, the compounds, antibodies, fragments or derivatives thereof, or other compounds of the present disclosure may bind to VEGF with a K of about 40 fM to about 110 pM. D Binds to VEGF.

[0088] Multivalent and multispecific compounds

[0089] Antigen-binding compounds can be combined to create multivalent and / or multispecific compounds. Such multivalent and / or multispecific compounds may have advantages over the parent compound administered alone. These advantages may include, for example, simpler dosing regimens, longer half-lives in the subject, and the ability to tightly bind to the target antigen.

[0090] Multispecific antibodies can be produced by a variety of methods. In one method, monospecific antibodies or derivatives thereof can be chemically coupled, for example, by chemically coupling two IgG antibody units into a conjugate.

[0091] In another approach, additional antigen binding domains can be appended to conventional IgG antibodies or derivatives thereof using cloning techniques. The additional antigen binding domains can be, for example, single variable domains (sVD), single chain variable fragments (scFv), single chain Fabs, peptides, ankyrin, ankyrin repeat proteins, designed ankyrin repeat proteins (DARPins), affibodies, avimers, adnectins, anticalins, fynomers, Kunitz domains, knottins, beta-hairpin mimetics, tetrameric polyethylene oxide clustered peptides, peptides derived from one or more receptors (e.g., VEGF receptors, or VEGF binding portions of human VEGF receptors 1 and 2), or derivatives thereof.

[0092] Additional antigen binding domains (e.g., sVD, scFv, single-chain Fab, peptide, ankyrin, ankyrin repeat protein, DARPin, affinity body, avimer, adnectin, anticalin, Fynomer, Kunitz domain, knottin, β-hairpin mimetic, tetrameric polyethylene oxide clustered peptide or peptide derived from one or more receptors) can be attached to the N or C terminus of the light chain and / or heavy chain of IgG or Fab, for example, via a peptide connector. In some embodiments, scFv can be attached to the C-terminus of the heavy chain of IgG to provide a tetravalent bispecific antibody. scFv can be attached to the C-terminus of the light chain of IgG to provide a tetravalent bispecific antibody. scFv can be attached to the C-terminus of the light chain of IgG to provide a tetravalent bispecific antibody. scFv can be attached to the C-terminus of the light chain of IgG and the C-terminus of the heavy chain to provide a hexavalent bispecific antibody. In some embodiments, DARPin can be attached to the C-terminus of the heavy chain of IgG to provide a tetravalent bispecific antibody. DARPin can be attached to the C-terminus of the light chain of IgG to provide a tetravalent bispecific antibody. DARPins can be attached to the C-terminus of the light chain and the C-terminus of the heavy chain of IgG to provide a hexavalent bispecific antibody. Other examples of multispecific antibodies produced by cloning techniques include: (i) DVD-Ig TM (Dual variable domain immunoglobulin, the second V H and V L Tandem connection to the N-terminus of HC and LC, respectively), (ii) Tandemab (two V H -C H 1 linked in tandem to a common LC), (iii) DNL (natural association of two antibodies or antibody fragments anchored to the DDD (dimerization and docking domain) from PKA (protein kinase A) and the AD (anchoring domain) from A-kinase anchoring protein (AKAP), respectively), (iv) LUZ-Y (leucine zipper tethered to the C-terminus of HC and subsequently removed by proteolysis), (v) 2-in-1 IgG (identical LC and HC capable of dual recognition), and (vi) mAb 2 (IgG C H 3 domain to obtain a second specificity).

[0093] Another type of multispecific antibody can be characterized by a structure having variable domains or scFv as structural units. Non-limiting examples of such multispecific antibodies include two V domains joined in series. H Domain, diabody (heterodimer containing two polypeptide chains, these polypeptide chains are V H -V L or V L -V H Sequential code V L AV H B and VH AV L B, a linker with 5 amino acids), dsDbs (in the V L With V H interchain disulfide bonds between), DARTs (dual affinity retargeting, 2 V L between interchain disulfide bonds), scDbs (single-chain diabodies), tandAbs (diabody dimers with a flexible linker between them), and two scFvs connected in series via an adjustable linker.

[0094] Another class of multispecific antibodies may comprise different antigen binding fragments while retaining the basic IgG structure. Such antibodies may comprise, for example, two different heavy chains and / or two different light chains. Various techniques may be used to facilitate the pairing of desired light and heavy chain combinations rather than random chain association. Non-limiting examples of such techniques include the use of a common light chain, orthogonal Fab interfaces (introducing complementary mutations at the LC and HC interfaces in one Fab without changes in the other Fab), CrossMab (wherein one Fab V H or C H 1 domain can be used with partner V L or C L domains are switched while the other Fabs are unaffected) and replacing the Fab with a single chain antigen binding domain. Further examples include engineering strategies that can H Mutations can be introduced into the three domains to promote heterodimerization based on steric or electrostatic complementarity. The "knobs in holes" approach can involve creating a "knob" by replacing the threonine at position 366 with a bulky tryptophan residue on one heavy chain, and forming the corresponding "hole" by triple mutations (T366S, L368A, and Y407V) on the partner heavy chain. Another approach can involve adding a C H 3, alternating human IgG and IgA fragments were created to provide so-called SEEDbodies (chain exchange engineered domains) to direct heavy chain heterodimerization.

[0095] A non-limiting schematic diagram of a multispecific antibody is shown in Figure 2-15 Available in.

[0096] Figure 2 A schematic diagram of a tetravalent bispecific antibody with sequences appended to the C-terminus of the heavy chain is provided. The light chain sequence of the IgG isotype antibody unit is represented by "SEQ A." The heavy chain sequence of the IgG isotype antibody unit is represented by "SEQ B." The linker sequence is represented by "SEQ C." The appended antigen-binding domain sequence is represented by "SEQ D." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0097] Figure 3 A schematic diagram of a tetravalent bispecific antibody with sequences appended to the C-terminus of the light chain is provided. The light chain sequence of the IgG isotype antibody unit is represented by "SEQ A." The heavy chain sequence of the IgG isotype antibody unit is represented by "SEQ B." The linker sequence is represented by "SEQ C." The appended antigen-binding domain sequence is represented by "SEQ D." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0098] Figure 4 A schematic diagram of a hexavalent bispecific antibody with sequences appended to the C-termini of the heavy and light chains is provided. The light chain sequence of the IgG isotype antibody unit is represented by "SEQ A." The heavy chain sequence of the IgG isotype antibody unit is represented by "SEQ B." The linker sequences are represented by "SEQ C" and "SEQ E." The appended antigen-binding domain sequence is represented by "SEQ D." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0099] Figure 5 A schematic diagram of a hexavalent trispecific antibody with sequences appended to the C-termini of the heavy and light chains is provided. The light chain sequence of an IgG isotype antibody unit is represented by "SEQ A." The heavy chain sequence of an IgG isotype antibody unit is represented by "SEQ B." The linker sequences are represented by "SEQ C" and "SEQ E." The appended antigen-binding domain sequences are represented by "SEQ D" and "SEQ F." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0100] Figure 6 A schematic diagram of a bivalent bispecific antibody having two different heavy chain sequences and two different light chain sequences is provided. The light chain sequences are represented by "SEQ A" and "SEQ D." The heavy chain sequences are represented by "SEQ B" and "SEQ C." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0101] Figure 7 A schematic diagram of a trivalent, trispecific antibody having two different heavy chain sequences, two different light chain sequences, and a sequence appended to the C-terminus of one light chain is provided. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequence is represented by "SEQ E." The sequence of the appended antigen-binding domain is represented by "SEQ F." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0102] Figure 8A schematic diagram of a trivalent, trispecific antibody having two different heavy chain sequences, two different light chain sequences, and a sequence appended to the C-terminus of one heavy chain is provided. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequence is represented by "SEQ E." The sequence of the appended antigen-binding domain is represented by "SEQ F." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0103] Figure 9 A schematic diagram of a tetravalent antibody having two different heavy chain sequences, two different light chain sequences, and sequences appended to the C-termini of the two light chains is provided. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequences are represented by "SEQ E" and "SEQ F."

[0104] The additional antigen-binding domain sequences are represented by "SEQ G" and "SEQ H." N and C indicate the N-terminus and C-terminus, respectively. -SS- indicates a disulfide bond.

[0105] Figure 10 A schematic diagram of a tetravalent antibody having two different heavy chain sequences, two different light chain sequences, and sequences appended to the C-termini of the two heavy chains is provided. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequences are represented by "SEQ E" and "SEQ F." The additional antigen-binding domain sequences are represented by "SEQ G" and "SEQ H." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0106] Figure 11 A schematic diagram of a tetravalent antibody having two different heavy chain sequences, two different light chain sequences, and sequences appended in cis to the C-terminus of one heavy chain and one light chain is provided. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequences are represented by "SEQ E" and "SEQ F." The appended antigen-binding domain sequences are represented by "SEQ G" and "SEQ H." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0107] Figure 12A schematic diagram of a tetravalent antibody having two different heavy chain sequences, two different light chain sequences, and sequences attached in trans to the C-terminus of one heavy chain and one light chain is provided. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequences are represented by "SEQ E" and "SEQ F." The attached antigen-binding domain sequences are represented by "SEQ G" and "SEQ H." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0108] Figure 13 A schematic diagram of a pentavalent antibody having two different heavy chain sequences, two different light chain sequences, and sequences appended to the C-termini of the two heavy chains and one light chain is provided. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequences are represented by "SEQ E," "SEQ F," and "SEQ G." The appended antigen-binding domain sequences are represented by "SEQ H," "SEQ I," and "SEQ J." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0109] Figure 14 A schematic diagram of a pentavalent antibody having two different heavy chain sequences, two different light chain sequences, and sequences appended to the C-termini of one heavy chain and both light chains is provided. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequences are represented by "SEQ E," "SEQ F," and "SEQ G." The appended antigen-binding domain sequences are represented by "SEQ H," "SEQ I," and "SEQ J." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0110] Figure 15 A schematic diagram of a hexavalent antibody having two different heavy chain sequences, two different light chain sequences, and sequences appended to the C-termini of the heavy and light chains is provided. The light chain sequences of the IgG isotype antibody unit are represented by "SEQ A" and "SEQ D." The heavy chain sequences of the IgG isotype antibody unit are represented by "SEQ B" and "SEQ C." The linker sequences are represented by "SEQ E," "SEQ F," "SEQ G," and "SEQ H." The appended antigen-binding domain sequences are represented by "SEQ I," "SEQ J," "SEQ K," and "SEQ L." N and C represent the N-terminus and C-terminus, respectively. -SS- represents a disulfide bond.

[0111] An antigen-binding compound that is specific for, for example, HPTP-β (VE-PTP) can be combined with an antigen-binding compound that is specific for, for example, an RTK agonist to provide a multivalent, multispecific compound that can bind to both HPTP-β (VE-PTP) and an RTK agonist. For example, an antigen-binding compound that is specific for HPTP-β (VE-PTP) can be combined with an antigen-binding compound that is specific for VEGF, Ang1, Ang2, BDNF, EGF, FGF, HGF, IGF, insulin, MSP, NGF, NT-3, PDGF, or any combination thereof to provide a multivalent, multispecific compound that can bind to both HPTP-β (VE-PTP) and an RTK agonist.

[0112] Antigen-binding compounds that are specific for, for example, HPTP-β (VE-PTP) can be combined with antigen-binding compounds that are specific for, for example, VEGF to provide multivalent, multispecific compounds that bind to both HPTP-β (VE-PTP) and VEGF. Compounds that bind to both HPTP-β (VE-PTP) and VEGF can inhibit HPTP-β (VE-PTP), activate Tie2, inhibit VEGF binding to VEGFR, and inhibit VEGFR signaling.

[0113] Sequences derived from aflibercept (a recombinant protein comprising the VEGF binding portion of human VEGF receptors 1 and 2) can be combined with antigen binding compounds specific for HPTP-β (VE-PTP). For example, sequences derived from aflibercept can be fused to the C-terminus of the heavy chain of an antibody specific for HPTP-β (VE-PTP) to provide a tetravalent bispecific antibody ( Figure 2 A sequence derived from aflibercept can be fused to the C-terminus of the light chain of an HPTP-β (VE-PTP)-specific antibody to provide a tetravalent bispecific antibody ( Figure 3 Sequences derived from aflibercept can be fused to the C-termini of the light and heavy chains of an HPTP-β (VE-PTP)-specific antibody to provide a hexavalent bispecific antibody ( Figure 4 ).

[0114] Sequences derived from brolucizumab (a humanized single-chain antibody fragment (scFv) inhibitor of VEGF that binds to the receptor binding site of VEGF) can be combined with antigen-binding compounds specific for HPTP-β (VE-PTP). For example, sequences from brolucizumab can be fused to the C-terminus of the heavy chain of an antibody specific for HPTP-β (VE-PTP) to provide a tetravalent bispecific antibody ( Figure 2Sequences derived from brolucizumab can be fused to the C-terminus of the light chain of an HPTP-β (VE-PTP)-specific antibody to provide a tetravalent bispecific antibody ( Figure 3 Sequences derived from brolucizumab can be fused to the C-termini of the light and heavy chains of an HPTP-β (VE-PTP)-specific antibody to provide a hexavalent bispecific antibody ( Figure 4 ).

[0115] Sequences derived from ranibizumab, a humanized monoclonal antibody fragment (Fab) that binds to and inhibits the activity of VEGF, can be combined with antigen-binding compounds specific for HPTP-β (VE-PTP). For example, sequences from ranibizumab can be cloned into scFv, and the ranibizumab-derived scFv can be fused to the C-terminus of the heavy chain of an HPTP-β (VE-PTP)-specific antibody to provide a tetravalent bispecific antibody ( Figure 2 Ranibizumab-derived scFv can be fused to the C-terminus of the light chain of an HPTP-β (VE-PTP)-specific antibody to provide a tetravalent bispecific antibody ( Figure 3 Ranibizumab-derived scFv can be fused to the C-termini of the light and heavy chains of an HPTP-β (VE-PTP)-specific antibody to provide a hexavalent bispecific antibody ( Figure 4 ).

[0116] Sequences derived from bevacizumab (a humanized monoclonal antibody that binds to and inhibits the activity of VEGF) can be combined with antigen-binding compounds specific for HPTP-β (VE-PTP). For example, sequences from bevacizumab can be cloned into scFv, and the bevacizumab-derived scFv can be fused to the C-terminus of the heavy chain of an HPTP-β (VE-PTP)-specific antibody to provide a tetravalent bispecific antibody ( Figure 2 A bevacizumab-derived scFv can be fused to the C-terminus of the light chain of an HPTP-β (VE-PTP)-specific antibody to provide a tetravalent bispecific antibody ( Figure 3 Bevacizumab-derived scFv can be fused to the C-termini of the light and heavy chains of an HPTP-β (VE-PTP)-specific antibody to provide a hexavalent bispecific antibody ( Figure 4 ).

[0117] Sequences derived from Conbercept (a recombinant protein containing the VEGF binding portion of VEGF receptors 1 and 2) can be combined with antigen-binding compounds specific for HPTP-β (VE-PTP). For example, sequences derived from Conbercept can be fused to the C-terminus of the heavy chain of an antibody specific for HPTP-β (VE-PTP) to provide a tetravalent bispecific antibody ( Figure 2 Sequences derived from Conbercept can be fused to the C-terminus of the light chain of an HPTP-β (VE-PTP)-specific antibody to provide a tetravalent bispecific antibody ( Figure 3 Sequences derived from Conbercept can be fused to the C-termini of the light and heavy chains of an HPTP-β (VE-PTP)-specific antibody to provide a hexavalent bispecific antibody ( Figure 4 ).

[0118] Sequences derived from abicipar (a DARPin that binds to VEGF) can be combined with antigen-binding compounds specific for HPTP-β (VE-PTP). For example, sequences derived from abicipar can be fused to the C-terminus of the heavy chain of an antibody specific for HPTP-β (VE-PTP) to provide a tetravalent bispecific antibody ( Figure 2 A sequence derived from abicipar can be fused to the C-terminus of the light chain of an HPTP-β (VE-PTP)-specific antibody to provide a tetravalent bispecific antibody ( Figure 3 Sequences derived from abicipar can be fused to the C-termini of the light and heavy chains of HPTP-β (VE-PTP)-specific antibodies to provide hexavalent bispecific antibodies ( Figure 4 ).

[0119] Antigen binding compounds specific for HPTP-β (VE-PTP) can be combined with DARPin or its amino acid sequence, for example, an amino acid sequence comprising any one of SEQ ID NOs: 158-217. An amino acid sequence comprising any one of SEQ ID NOs: 158-217 can be fused to the C-terminus of the heavy chain of an HPTP-β (VE-PTP)-specific antibody to provide a tetravalent bispecific antibody ( Figure 2 An amino acid sequence comprising any one of SEQ ID NOs: 158-217 can be fused to the C-terminus of the light chain of an HPTP-β (VE-PTP)-specific antibody to provide a tetravalent bispecific antibody ( Figure 3 An amino acid sequence comprising any one of SEQ ID NOs: 158-217 can be fused to the C-termini of the light and heavy chains of an HPTP-β (VE-PTP)-specific antibody to provide a hexavalent bispecific antibody ( Figure 4 ).

[0120] Multi-VEGF specific compounds can be combined with antigen binding compounds specific for HPTP-β (VE-PTP). For example, one binding domain (e.g., a sequence derived from aflibercept) can be fused to the C-terminus of the heavy chain of an anti-HPTP-β (VE-PTP) antibody, while another binding domain (e.g., a sequence derived from brolucizumab) can be fused to the C-terminus of the light chain of an HPTP-β (VE-PTP) specific antibody to provide a hexavalent trispecific antibody ( Figure 5 ).

[0121] If different heavy chains are used in the basic four-chain IgG antibody unit (e.g., using a "knob-in-a-hole" approach), antibodies can be generated that are bivalent, trivalent, tetravalent, pentavalent, or hexavalent, and that are monospecific, bispecific, trispecific, tetraspecific, pentaspecific, or hexaspecific.

[0122] In one non-limiting example, one arm of an antibody unit may comprise CDRs specific for HPTP-β (VE-PTP), while the other arm of the antibody unit may comprise CDRs specific for VEGF ( Figure 6 ).

[0123] In one non-limiting example, two antigen-binding fragments of a basic antibody unit can both be specific for HPTP-β (VE-PTP), and a binding domain specific for VEGF (e.g., an aflibercept-derived sequence, a brolucizumab-derived sequence, a ranibizumab-derived sequence, or a bevacizumab-derived sequence) can be fused to one light chain to provide a trivalent bispecific antibody ( Figure 7 ).

[0124] In one non-limiting example, two antigen-binding fragments of a basic antibody unit can both be specific for HPTP-β (VE-PTP), and a binding domain specific for VEGF (e.g., an aflibercept-derived sequence, a brolucizumab-derived sequence, a ranibizumab-derived sequence, or a bevacizumab-derived sequence) can be fused to one heavy chain to provide a trivalent bispecific antibody ( Figure 8 ).

[0125] In one non-limiting example, two antigen-binding fragments of a basic antibody unit can both be specific for HPTP-β (VE-PTP), and a binding domain specific for VEGF (e.g., an aflibercept-derived sequence, a brolucizumab-derived sequence, a ranibizumab-derived sequence, or a bevacizumab-derived sequence) can be fused to one light chain, and a second, different binding domain specific for VEGF can be fused to the other light chain to provide a tetravalent trispecific antibody ( Figure 9 ).

[0126] In one non-limiting example, two antigen-binding fragments of a basic antibody unit can both be specific for HPTP-β (VE-PTP), a binding domain specific for VEGF (e.g., an aflibercept-derived sequence, a brolucizumab-derived sequence, a ranibizumab-derived sequence, or a bevacizumab-derived sequence) can be fused to one heavy chain, and a second, different binding domain specific for VEGF can be fused to the other heavy chain to provide a tetravalent trispecific antibody ( Figure 10 ).

[0127] In one non-limiting example, two antigen-binding fragments of a basic antibody unit can both be specific for HPTP-β (VE-PTP), and a binding domain specific for VEGF (e.g., an aflibercept-derived sequence, a brolucizumab-derived sequence, a ranibizumab-derived sequence, or a bevacizumab-derived sequence) can be fused to one heavy chain, and a second, different binding domain specific for VEGF can be fused in cis to one light chain to provide a tetravalent, trispecific antibody ( Figure 11 ).

[0128] In one non-limiting example, two antigen-binding fragments of a basic antibody unit can both be specific for HPTP-β (VE-PTP), a binding domain specific for VEGF (e.g., an aflibercept-derived sequence, a brolucizumab-derived sequence, a ranibizumab-derived sequence, or a bevacizumab-derived sequence) can be fused to one heavy chain, and a second, different binding domain specific for VEGF can be fused in trans to one light chain to provide a tetravalent trispecific antibody ( Figure 12 ).

[0129] In one non-limiting example, two antigen-binding fragments of a basic antibody unit can both be specific for HPTP-β (VE-PTP), a binding domain specific for VEGF (e.g., an aflibercept-derived sequence, a brolucizumab-derived sequence, a ranibizumab-derived sequence, or a bevacizumab-derived sequence) can be fused to one heavy chain, a second, distinct binding domain specific for VEGF can be fused to the second heavy chain, and a third, distinct binding domain specific for VEGF can be fused to one light chain to provide a pentavalent, tetraspecific antibody ( Figure 13 ).

[0130] In one non-limiting example, two antigen-binding fragments of a basic antibody unit can both be specific for HPTP-β (VE-PTP), a binding domain specific for VEGF (e.g., an aflibercept-derived sequence, a brolucizumab-derived sequence, a ranibizumab-derived sequence, or a bevacizumab-derived sequence) can be fused to one heavy chain, a second, distinct binding domain specific for VEGF can be fused to one light chain, and a third, distinct binding domain specific for VEGF can be fused to another light chain to provide a pentavalent, tetraspecific antibody ( Figure 14 ).

[0131] In one non-limiting example, two antigen-binding fragments of a basic antibody unit can both be specific for HPTP-β (VE-PTP), a binding domain specific for VEGF (e.g., an aflibercept-derived sequence, a brolucizumab-derived sequence, a ranibizumab-derived sequence, or a bevacizumab-derived sequence) can be fused to one heavy chain, a second, distinct binding domain specific for VEGF can be fused to the other heavy chain, a third, distinct binding domain specific for VEGF can be fused to one light chain, and a fourth binding domain specific for VEGF can be fused to the other light chain, to provide a hexavalent pentaspecific antibody ( Figure 15 ).

[0132] In one non-limiting example, two antigen-binding fragments of a basic antibody unit can both be specific for HPTP-β (VE-PTP), one binding domain (e.g., aflibercept-derived sequence) can be fused to the C-terminus of one heavy chain, and a second binding domain (e.g., a brolucizumab-derived sequence) can be fused to the C-terminus of the other heavy chain to provide a tetravalent trispecific antibody ( Figure 10 In one non-limiting example, the two antigen-binding fragments of the basic antibody unit can both be specific for HPTP-β (VE-PTP), the aflibercept-derived sequence can be fused to the C-terminus of one light chain, and the brolucizumab-derived sequence can be fused to the C-terminus of the other light chain to provide a tetravalent trispecific antibody ( Figure 9 In one non-limiting example, both antigen-binding fragments of a basic antibody unit may be specific for HPTP-β (VE-PTP), the aflibercept-derived sequence may be fused to the C-terminus of one heavy chain, and the brolucizumab-derived sequence to the C-terminus of one light chain, to provide a tetravalent trispecific antibody ( Figure 11 、 Figure 12In one non-limiting example, two antigen-binding fragments of a basic antibody unit can both be specific for HPTP-β (VE-PTP), an aflibercept-derived sequence can be fused to the C-terminus of one heavy chain, a brolucizumab-derived sequence can be fused to the C-terminus of the other heavy chain, and a ranibizumab-derived sequence can be fused to the C-termini of both light chains to provide a hexavalent tetraspecific antibody ( Figure 15 In one non-limiting example, two antigen-binding fragments of a basic antibody unit can both be specific for HPTP-β (VE-PTP), an aflibercept-derived sequence can be fused to the C-terminus of one heavy chain, a brolucizumab-derived sequence can be fused to the C-terminus of the other heavy chain, a ranibizumab-derived sequence can be fused to the C-terminus of one light chain, and a bevacizumab-derived sequence can be fused to the C-terminus of the other light chain, to provide a hexavalent pentaspecific antibody ( Figure 15 ).

[0133] Antigen binding compounds specific for, e.g., HPTP-β (VE-PTP) can be combined with other amino acid sequences to provide multivalent, multispecific compounds that, e.g., enhance Tie2 activation, enhance Tie2 phosphorylation, enhance Tie2 signaling, reduce VEGFR activation, reduce VEGFR phosphorylation, reduce VEGFR signaling, or a combination thereof.

[0134] Antigen binding compounds specific for HPTP-β (VE-PTP) can be combined with collagen IV-derived biomimetic peptides, such as the amino acid sequence comprising SEQ ID NO: 152 or SEQ ID NO: 153. The amino acid sequence comprising SEQ ID NO: 152 or SEQ ID NO: 153 can be fused to the C-terminus of the heavy chain of an HPTP-β (VE-PTP)-specific antibody to provide a tetravalent bispecific antibody ( Figure 2 The amino acid sequence comprising SEQ ID NO: 152 or SEQ ID NO: 153 can be fused to the C-terminus of the light chain of an HPTP-β (VE-PTP)-specific antibody to provide a tetravalent bispecific antibody ( Figure 3 The amino acid sequence comprising SEQ ID NO: 152 or SEQ ID NO: 153 can be fused to the C-termini of the light and heavy chains of an HPTP-β (VE-PTP)-specific antibody to provide a hexavalent bispecific antibody ( Figure 4 ).

[0135] Antigen binding compounds specific for HPTP-β (VE-PTP) can be combined with an Ang1 mimetic, such as vasculotide. Sequences derived from vasculotide can be fused to the C-terminus of the heavy chain of an HPTP-β (VE-PTP)-specific antibody to provide a tetravalent bispecific antibody ( Figure 2 A sequence derived from vasculotide can be fused to the C-terminus of the light chain of an HPTP-β (VE-PTP)-specific antibody to provide a tetravalent bispecific antibody ( Figure 3 Sequences derived from vasculotide can be fused to the C-termini of the light and heavy chains of HPTP-β (VE-PTP)-specific antibodies to provide hexavalent bispecific antibodies ( Figure 4 ).

[0136] An antigen-binding compound that is specific for, for example, HPTP-β (VE-PTP) can be combined with an antigen-binding compound that is specific for, for example, an RTK to provide a multivalent, multispecific compound that can bind to both HPTP-β (VE-PTP) and RTK. For example, an antigen-binding compound that is specific for HPTP-β (VE-PTP) can be combined with an antigen-binding compound that is specific for VEGFR (VEGFR2) to provide a multivalent, multispecific compound that can bind to both HPTP-β (VE-PTP) and VEGFR.

[0137] Compounds that bind to both HPTP-β (VE-PTP) and VEGFR can inhibit HPTP-β (VE-PTP), activate Tie2, inhibit the binding of VEGF to VEGFR, and inhibit VEGFR signaling.

[0138] Sequences derived from ramucirumab (a humanized monoclonal antibody that binds to the extracellular domain of VEGFR2 and inhibits VEGFR2 signaling) can be combined with antigen-binding compounds specific for HPTP-β (VE-PTP). For example, sequences from ramucirumab can be cloned into scFv, and the ramucirumab-derived scFv can be fused to the C-terminus of the heavy chain of an antibody specific for HPTP-β (VE-PTP) to provide a tetravalent bispecific antibody ( Figure 2 Ramucirumab-derived scFv can be fused to the C-terminus of the light chain of an HPTP-β (VE-PTP)-specific antibody to provide a tetravalent bispecific antibody ( Figure 3 Ramucirumab-derived scFv can be fused to the C-termini of the light and heavy chains of an HPTP-β (VE-PTP)-specific antibody to provide a hexavalent bispecific antibody ( Figure 4 ).

[0139] A variety of compounds that enhance Tie2 activation, enhance Tie2 phosphorylation, enhance Tie2 signaling, reduce VEGFR activation, reduce VEGFR phosphorylation, reduce VEGFR signaling, or a combination thereof can be combined with an antigen-binding compound specific for HPTP-β (VE-PTP). For example, one binding domain (e.g., a brolucizumab-derived sequence) can be fused to the C-terminus of an anti-HPTP-β (VE-PTP) antibody heavy chain, while another binding domain (e.g., a vasculotide-derived sequence) can be fused to the C-terminus of an HPTP-β (VE-PTP)-specific antibody light chain to provide a hexavalent trispecific antibody ( Figure 5 ).

[0140] The compounds, antibodies, or derivatives thereof disclosed herein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more domains. Each domain can modulate, bind, antagonize, inhibit, or activate any target disclosed herein, e.g., a phosphatase, a phosphatase that modulates Tie2 signaling, a protein tyrosine phosphatase, a receptor-like protein tyrosine phosphatase, a Tie2 modulator, HPTP-β (VE-PTP), an extracellular domain of HPTP-β (VE-PTP), a first FN3 repeat of an extracellular domain of HPTP-β (VE-PTP), a kinase, a tyrosine kinase, a receptor tyrosine kinase, a receptor tyrosine kinase activator, a receptor tyrosine kinase agonist, a growth factor, a growth factor receptor activator, a growth factor receptor agonist, a cysteine ​​knot growth factor superfamily member, a pro-angiogenic factor, a PDGF family member, a VEGF receptor, a VEGF receptor agonist, a VEGF family member, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PGF, VEGF 121 , VEGF 145 , VEGF 148 , VEGF 162 , VEGF 165 , VEGF 165b , VEGF 183 , VEGF 189 or VEGF 206 .

[0141] Inhibitors, activators, modulators, and binders

[0142] HPTP-β (VE-PTP) inhibitors, modulators or binding agents disclosed herein may include compounds, recombinant proteins, antibodies, antigen-binding fragments, variants or derivatives thereof, Tie2-peptoids, tetrameric polyethylene oxide clustered peptides, collagen IV biomimetic peptides, DARPin or derivatives thereof, affibodies, avimers, adnectins, anticalins, Fynomers, Kunitz domains, knottins, β-hairpin mimetics or peptides derived from one or more receptors, alone or in combination with another amino acid sequence or a plurality of other amino acid sequences. The inhibitors, modulators or binding agents may be modified, for example, by enzymatic cleavage or post-translational modification.

[0143] In some embodiments, the HPTP-β (VE-PTP) inhibitors, modulators, or binding agents of the present disclosure may inhibit HPTP-β (VE-PTP) by interfering with the interaction between HPTP-β (VE-PTP) and Tie2. In some embodiments, the HPTP-β (VE-PTP) inhibitors, modulators, or binding agents of the present disclosure may inhibit HPTP-β (VE-PTP) by stabilizing an inactive conformation of HPTP-β (VE-PTP). In some embodiments, the HPTP-β (VE-PTP) inhibitors, modulators, or binding agents of the present disclosure may inhibit HPTP-β (VE-PTP) by promoting the internalization of HPTP-β (VE-PTP) (e.g., promoting endocytosis and degradation of HPTP-β (VE-PTP)). In some embodiments, the HPTP-β (VE-PTP) inhibitors, modulators, or binding agents of the present disclosure may inhibit HPTP-β (VE-PTP) by blocking the binding of ligands that activate HPTP-β (VE-PTP). In some embodiments, the HPTP-β (VE-PTP) inhibitors, modulators, or binding agents of the present disclosure may inhibit HPTP-β (VE-PTP) by modulating the oligomerization of HPTP-β (VE-PTP).

[0144] In some embodiments, the HPTP-β (VE-PTP) inhibitors, modulators, or binding agents of the present disclosure can bind to a dominant negative isoform of HPTP-β (VE-PTP). The dominant negative isoform can correspond to a form of HPTP-β (VE-PTP) lacking phosphatase activity that can compete with endogenous HPTP-β (VE-PTP).

[0145] The HPTP-β (VE-PTP) inhibitors, modulators, or binding agents of the present disclosure may comprise multiple HPTP-β (VE-PTP) binding sites. In some embodiments, the HPTP-β (VE-PTP) inhibitor, modulator, or binding agent may bind to two HPTP-β (VE-PTP) molecules simultaneously, thereby bringing the two HPTP-β (VE-PTP) molecules into close proximity. In some embodiments, the HPTP-β (VE-PTP) inhibitor, modulator, or binding agent may bind to three HPTP-β (VE-PTP) molecules simultaneously, thereby bringing the three HPTP-β (VE-PTP) molecules into close proximity. In some embodiments, the HPTP-β (VE-PTP) inhibitor, modulator, or binding agent may bind to four HPTP-β (VE-PTP) molecules simultaneously, thereby bringing the four HPTP-β (VE-PTP) molecules into close proximity.

[0146] The HPTP-β (VE-PTP) inhibitors, modulators, or binding agents of the present disclosure can be covalently or non-covalently conjugated to another moiety or vehicle. The moiety or vehicle can, for example, provide binding specificity to another target, inhibit degradation, extend half-life, increase absorption, reduce toxicity, reduce immunogenicity, and / or increase the biological activity of the inhibitor, modulator, or binding agent. Non-limiting examples of moieties that can be conjugated to inhibitors, modulators, or binding agents include Fc domains of immunoglobulins, peptides, lipids, carbohydrates, dendrimers, oligosaccharides, cholesterol groups such as steroids, and polymers such as polyethylene glycol (PEG), polylysine, or dextran.

[0147] The compounds of the present disclosure can be used to target HPTP-β (VE-PTP) to restore Tie2 activity. HPTP-β (VE-PTP) inhibitors, modulators, or binding agents can therefore be Tie2 activators. In some embodiments, the compounds of the present disclosure can initiate or inhibit signaling cascades downstream of HPTP-β (VE-PTP) or Tie2, for example, Akt / PI3-K signaling, Rac1 signaling, MAPK / Ras signaling, or NF-κB signaling.

[0148] Inhibition of HPTP-β (VE-PTP) can lead to vascular stabilization, which may be beneficial for the treatment of conditions characterized by, for example, vascular instability, angiogenesis, neovascularization, vascular leakage and / or edema. For example, inhibition of HPTP-β (VE-PTP) may be beneficial in the treatment of vascular conditions, eye conditions, cancer, kidney conditions, diabetic complications, and other conditions. In some embodiments, the HPTP-β (VE-PTP) inhibitors, modulators, or binding agents of the present disclosure may be used to treat, for example, diabetic retinopathy, non-proliferative diabetic retinopathy (NPDR), glaucoma, intraocular pressure, ocular edema, ocular hemorrhage, ocular hypertension, ocular inflammation, ocular neovascularization, ocular vascular leakage, retinal perfusion, or retinopathy.

[0149] Tie2 activators, modulators, or binders disclosed herein may include, for example, compounds, recombinant proteins, peptides, antibodies, antigen-binding fragments, variants, or derivatives thereof, angiopoietin 1 recombinant proteins, Ang1 mimetics, Tie2 agonists, HPTP-β (VE-PTP) phosphatase inhibitors, Tie2-peptoids, tetrameric polyethylene oxide clustered peptides, collagen IV biomimetic peptides, DARPins or derivatives thereof, affibodies, avimers, adnectins, anticalins, Fynomers, Kunitz domains, knottins, β-hairpin mimetics, or peptides derived from one or more receptors, alone or in combination with another amino acid sequence or multiple other amino acid sequences. The activators, modulators, or binders may be modified, for example, by enzymatic or post-translational modification.

[0150] The Tie2 activators, modulators or binders of the present disclosure can be covalently or non-covalently conjugated to another moiety or vehicle. The moiety or vehicle can, for example, provide binding specificity for another target, inhibit degradation, extend half-life, increase absorption, reduce toxicity, reduce immunogenicity, and / or increase the biological activity of the activator, modulator or binder. Non-limiting examples of moieties that can be conjugated to activators, modulators or binders include Fc domains of immunoglobulins, peptides, lipids, carbohydrates, dendrimers, oligosaccharides, cholesterol groups such as steroids, and polymers such as polyethylene glycol (PEG), polylysine or dextran.

[0151] In some embodiments, compounds of the present disclosure can initiate or inhibit a signaling cascade downstream of Tie2, for example, Akt / PI3-K signaling, Racl signaling, MAPK / Ras signaling, or NF-κB signaling.

[0152] Activation of Tie2 can lead to vascular stabilization, which may be beneficial for the treatment of conditions characterized by, for example, vascular instability, angiogenesis, neovascularization, vascular leakage, and / or edema. For example, activation of Tie2 may be beneficial in the treatment of ocular conditions, cancer, kidney conditions, diabetic complications, and other conditions. In some embodiments, the Tie2 activators, modulators, or binding agents of the present disclosure may be used to treat diabetic retinopathy, non-proliferative diabetic retinopathy (NPDR), glaucoma, intraocular pressure, ocular edema, ocular hemorrhage, ocular hypertension, ocular inflammation, ocular neovascularization, ocular vascular leakage, retinal perfusion, or retinopathy.

[0153] VEGF inhibitors, modulators, or binding agents of the present disclosure may include compounds, recombinant proteins, peptides, antibodies, antigen-binding fragments, variants or derivatives thereof, DARPins or derivatives thereof, affibodies, avimers, adnectins, anticalins, Fynomers, Kunitz domains, knottins, β-hairpin mimetics, tetrameric polyethylene oxide clustered peptides, collagen IV biomimetic peptides, or peptides derived from one or more receptors (e.g., VEGF receptors, or VEGF-binding portions of human VEGF receptors 1 and 2), either alone or in combination with another amino acid sequence or multiple other amino acid sequences. The inhibitors, modulators, or binding agents may be modified, for example, by enzymatic or post-translational modification.

[0154] The VEGF inhibitors, modulators, or binding agents of the present disclosure may comprise multiple VEGF binding sites. In some embodiments, the VEGF inhibitors, modulators, or binding agents may bind to two VEGF molecules simultaneously, thereby bringing the two VEGF molecules into close proximity. In some embodiments, the VEGF inhibitors, modulators, or binding agents may bind to three VEGF molecules simultaneously, thereby bringing the three VEGF molecules into close proximity. In some embodiments, the VEGF inhibitors, modulators, or binding agents may bind to four VEGF molecules simultaneously, thereby bringing the four VEGF molecules into close proximity.

[0155] The VEGF inhibitors, modulators or binding agents of the present disclosure can be covalently or non-covalently conjugated to another moiety or vehicle. The moiety or vehicle can, for example, provide binding specificity to another target, inhibit degradation, extend half-life, increase absorption, reduce toxicity, reduce immunogenicity, and / or increase the biological activity of the inhibitor, modulator or binding agent. Non-limiting examples of moieties that can be conjugated to inhibitors, modulators or binding agents include Fc domains of immunoglobulins, peptides, lipids, carbohydrates, dendrimers, oligosaccharides, cholesterol groups such as steroids, and polymers such as polyethylene glycol (PEG), polylysine or dextran.

[0156] VEGFR inhibitors, modulators or binding agents of the present disclosure may include compounds, recombinant proteins, antibodies, antigen binding fragments, variants or derivatives thereof, tetrameric polyethylene oxide clustered peptides, collagen IV biomimetic peptides, DARPin or derivatives thereof, affibodies, avimers, adnectins, anticalins, Fynomers, Kunitz domains, knottins, β-hairpin mimetics or peptides derived from one or more receptors, alone or in combination with another amino acid sequence or a variety of other amino acid sequences. The inhibitors, modulators or binding agents may be modified, such as by enzymatic cleavage or post-translational modification.

[0157] VEGFR inhibitors, modulators, or binding agents of the present disclosure may comprise multiple VEGFR binding sites. In some embodiments, a VEGFR inhibitor, modulator, or binding agent may bind to two VEGFR molecules simultaneously, thereby bringing the two VEGFR molecules into close proximity. In some embodiments, a VEGFR inhibitor, modulator, or binding agent may bind to three VEGFR molecules simultaneously, thereby bringing the three VEGFR molecules into close proximity. In some embodiments, a VEGFR inhibitor, modulator, or binding agent may bind to four VEGFR molecules simultaneously, thereby bringing the four VEGFR molecules into close proximity.

[0158] VEGFR inhibitors, regulators or binding agents of the present disclosure can be covalently or non-covalently conjugated to another part or vehicle. Part or vehicle can, for example, provide binding specificity to other targets, inhibit degradation, extend half-life, increase absorption, reduce toxicity, reduce immunogenicity, and / or increase the biological activity of inhibitors, regulators or binding agents. Non-limiting examples of parts that can be conjugated to inhibitors, regulators or binding agents include Fc domains of immunoglobulins, peptides, lipids, carbohydrates, dendrimers, oligosaccharides, cholesterol groups such as steroids, and polymers such as polyethylene glycol (PEG), polylysine or dextran.

[0159] In some embodiments, VEGFR inhibitors, regulators or binding agents of the present disclosure can inhibit VEGFR by stabilizing the VEGFR of an inactive conformation. In some embodiments, VEGFR inhibitors, regulators or binding agents of the present disclosure can inhibit VEGFR by promoting the internalization of VEGFR (e.g., promoting the endocytosis and degradation of HVEGFR). In some embodiments, VEGFR inhibitors, regulators or binding agents of the present disclosure can inhibit VEGFR by blocking the binding of the ligand that activates VEGFR (e.g., blocking the VEGF connection of VEGFR). In some embodiments, VEGFR inhibitors, regulators or binding agents of the present disclosure can inhibit VEGFR by regulating the oligomerization of VEGFR (e.g., preventing or reducing the dimerization or oligomerization possibility of VEGFR).

[0160] The compounds of the present disclosure can be used to interfere with the interaction of VEGF and VEGFR, thereby reducing the phosphorylation and downstream signaling of VEGFR. In some embodiments, the inhibition of VEGF can reduce abnormal angiogenesis, angiogenesis or vascular permeability, thereby reducing pathological vascular instability. In some embodiments, the inhibition of VEGF may be beneficial to the treatment of diseases characterized by vascular instability, angiogenesis, new blood vessels, vascular leakage and / or edema. For example, the inhibition of VEGF may be beneficial to the treatment of vascular diseases, eye diseases, cancer, kidney diseases, diabetic complications and other diseases. In some embodiments, VEGF inhibitors, regulators or binding agents of the present disclosure can be used to treat diabetic retinopathy, non-proliferative diabetic retinopathy (NPDR), glaucoma, intraocular pressure, eye edema, eye hemorrhage, ocular hypertension, ocular inflammation, ocular neovascularization, ocular vascular leakage, retinal perfusion or retinopathy. In some embodiments, the inhibition of VEGF can alleviate cancer.

[0161] method

[0162] The promotion of Tie2 signaling and the inhibition of VEGFR signaling can lead to vascular stabilization, which may be beneficial in treating conditions with a vascular component. The compounds disclosed herein can be used to treat, for example, diseases characterized by altered vasculature, diseases characterized by reduced Tie2 activation, or diseases characterized by the involvement of VEGF in pathogenesis, whether progressive or non-progressive, acute or chronic.

[0163] In some embodiments, the compounds disclosed herein can be used to treat eye disorders. The compounds disclosed herein can be used to treat, for example, age-related macular degeneration (dry form), age-related macular degeneration (wet form), atopic keratitis, Best's disease, blepharitis, blurred vision, choroidal neovascularization, chronic retinal detachment, chronic uveitis / vitritis, choroiditis, conjunctivitis, excessive contact lens wear, corneal graft neovascularization, corneal graft rejection, corneal neovascularization, cystoid macular edema, diabetic macular edema, diplopia, diabetic retinopathy, diseases associated with redness (neovascularization of the corner) Diseases caused by abnormal proliferation of fibrovascular or fibrous tissue, including all forms of proliferative vitreoretinopathy, Eales disease, increased intraocular pressure, epidemic keratoconjunctivitis, floaters, glaucoma, hard yellow exudates within the central 500 μm of the fovea with adjacent retinal thickening, hyperviscosity syndrome, infections causing choroiditis, infections causing retinitis, iris neovascularization, ischemic retinopathy, decreased contrast, macular telangiectasia, and mariginolacria. keratolysis), multifocal choroiditis, myopia, neovascular glaucoma, nonproliferative diabetic retinopathy (NPDR), ocular edema, ocular hemorrhage, ocular histoplasmosis, ocular hypertension, ocular inflammation, ocular ischemia, ocular neovascularization, ocular trauma, ocular vascular leakage, optic pit, optic disc edema, pars planitis, phylectenulosis, multipolypoidal choroidal vasculopathy, post-laser complications, proliferative diabetic retinopathy, pterygium keratitis, radial keratotomy, retinal angiomatous proliferation, retinal degeneration, retinal edema (including macular edema), retinal Membrane neovascularization, retinal perfusion, retinal thickening within 1 optic disc diameter of the center of the fovea, retinal thickening within 500 μm of the center of the fovea, retinal vein occlusion (central or branch), retinitis, retinitis pigmentosa, retinopathy, retinopathy of prematurity, scleritis, Stargarts disease, superior limbal keratitis, surgically induced edema, surgically induced neovascularization, Terrien's limbal degeneration, trachoma, trauma, uveitis, vasculitis (e.g., central retinal vein occlusion), or other ophthalmic disease in which the ocular disease or disorder is associated with ocular neovascularization, vascular leakage, or retinal edema, or a combination thereof.

[0164] In some embodiments, the compounds disclosed herein can be used to treat diabetic complications (e.g., comorbidities of diabetes). The compounds disclosed herein can be used to treat, for example, acute glomerulonephritis, acute myocardial infarction, amputation, amyotrophy, aneurysm, angina, aortic aneurysm, aortic dissection, atherosclerosis, atherosclerotic cardiovascular disease, atrial fibrillation, autonomic neuropathy, blindness, cardiovascular complications of diabetes, cerebrovascular complications of diabetes, Charcot arthropathy, chronic glomerulonephritis, chronic renal failure, claudication, clinically significant macular edema, coronary artery disease, cranial nerve palsy, cystoid macular degeneration, cystoid macular edema, diabetic cardiomyopathy, diabetic hand arthropathy, diabetic coma, diabetic encephalopathy, diabetic foot trauma, diabetic hyperglycemia, diabetic hyperlipidemia, diabetic hyperosmolar syndrome, diabetic hypoglycemia, diabetic ketoacidosis, diabetic myonecrosis, diabetic nephropathy, diabetic neuropathy, diabetic eye disease, diabetes peripheral vascular disease, diabetic retinopathy, diffuse idiopathic skeletal hyperostosis, Dupuytren's contracture, embolism, end-stage renal disease, erectile dysfunction, Forestier's disease, gangrene, gas gangrene, gastroparesis / diarrhea, heart failure, hyperglycemic crisis, hypertension, ischemic heart disease, ketoacidosis, lipohypertrophy, metabolic complications of diabetes, mononeuropathy, myocardial infarction, nephritis, nephropathy, nephrotic syndrome, neurogenic bladder, neuropathic arthropathy, neuropathy, orthostatic hypotension, osteoarthritis, osteoporosis, periodontal disease, peripheral vascular disease, polyneuropathy, proliferative retinopathy, renal failure, renal insufficiency, restrictive lung disease, retinal detachment, retinal edema, retinopathy, stroke, thrombosis, transient ischemic attack, ulceration, ventricular fibrillation, vitreous hemorrhage, or a combination thereof.

[0165] In some embodiments, the compounds disclosed herein can be used to treat renal disorders. The compounds disclosed herein can be used to treat, for example, acute kidney injury, acute proliferative glomerulonephritis, adenine phosphoribosyltransferase deficiency, Alport syndrome, indolent nephropathy, autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, Balkan endemic nephropathy, benign nephrosclerosis, Bright disease, cardiorenal syndrome, CFHR5 nephropathy, chronic kidney disease, chronic kidney disease-mineral and bone disorders, congenital nephrotic syndrome, congenital renal syndrome (Conorenal syndrome), contrast-induced nephropathy, cystic kidney disease, Dents disease, diabetic nephropathy, diffuse proliferative nephritis, distal renal tubular acidosis, diuresis, EAST syndrome, end-stage renal disease, epithelial-mesenchymal transition, Epstein syndrome, Fanconi syndrome, Fechtner syndrome, focal proliferative nephritis, focal segmental glomerulosclerosis, Fraley syndrome, Galloway nephropathy, nephrotic syndrome ... Mowat syndrome, Gitelman syndrome, glomerulocystic kidney disease, glomerulopathy, Goodpasture syndrome, high anion gap metabolic acidosis, HIV-associated nephropathy, horseshoe kidney, hydronephrosis, hypertensive nephropathy, IgA nephropathy, interstitial nephritis, juvenile nephropathy, renal cancer, kidney disease, nephrolithiasis, Lightwood–Albright syndrome, lupus nephritis, malarial nephropathy, medullary cystic kidney disease, medullary sponge kidney, membranous glomerulonephritis, Mesoamerican nephropathy, milk-alkali syndrome, minimal membranous glomerulonephritis, polycystic kidney dysplasia, nephritis, nephrocalcinosis, nephrogenic diabetes insipidus, nephrolithiasis large, nephroptosis, nephropathy, nephrotic syndrome, nutcracker syndrome, papillary renal syndrome, phosphate nephropathy, polycystic kidney disease, primary hyperoxaluria, proximal renal tubular acidosis, pyelonephritis, pyonephritis, rapidly progressive glomerulonephritis, renal agenesis, renal colic, renal artery stenosis, renal cyst, renal ischemia, renal osteodystrophy, renal papillary necrosis, renal tubular acidosis, renal vein thrombosis, secondary hypertension, serpentine fibular-polycystic kidney syndrome, shunt nephritis, sickle cell nephropathy, thin basement membrane disease, transplant glomerulopathy, tubulointerstitial nephritis and uveitis, tubular disease, uremia, uremic frost, Wunderlich syndrome, or a combination thereof.

[0166] In some embodiments, the compounds disclosed herein can be used to treat cancer. The compounds disclosed herein can be used to treat, for example, acute leukemia, astrocytoma, biliary tract cancer (cholangiocarcinoma), bone cancer, breast cancer, brain stem glioma, bronchioloalveolar cell lung cancer, adrenal cancer, anal cancer, bladder cancer, endocrine system cancer, esophageal cancer, head or neck cancer, kidney cancer, parathyroid cancer, penile cancer, pleural / peritoneal cancer, salivary gland cancer, small intestine cancer, thyroid cancer, ureteral cancer, urethral cancer, cervical cancer, endometrial cancer, fallopian tube cancer, renal pelvis cancer, renal pelvis cancer, vaginal cancer, vulvar cancer, cervical cancer, chronic leukemia, colon cancer, colorectal cancer, skin melanoma, ependymoma, epidermoid tumor, Ewing's sarcoma, gastric cancer, glioblastoma, multiforme glioma blastoma, glioma, hematologic malignancies, hepatocellular (liver) cancer, hepatoma, Hodgkin's disease, intraocular melanoma, Kaposi's sarcoma, lung cancer, lymphoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, muscle cancer, central nervous system tumors (CNS), neuronal cancers, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pediatric malignancies, pituitary adenoma, prostate cancer, rectal cancer, renal cell carcinoma, soft tissue sarcoma, schwannoma, skin cancer, spinal axis tumors, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, uterine cancer, or tumors and metastases thereof, including refractory forms of any of the foregoing cancers, or a combination thereof.

[0167] In some embodiments, the compounds disclosed herein can be used to treat diseases characterized by altered vasculature, diseases characterized by decreased Tie2 activation, or diseases characterized by the involvement of VEGF in pathogenesis. The compounds disclosed herein can be used to treat, for example, rosacea, acute lung injury, acute respiratory distress syndrome (ARDS), adhesion formation caused by abdominal surgery, obesity, albuminuria, allergic edema, allergic reactions, angina pectoris, angiofibroma, arteriosclerosis, arterial occlusion, ascites, atheroma, atherosclerosis, asthma, avascular necrosis, bacterial ulcers, Bartonella bacilliformis infection, Behcet's disease, Buerger's disease (thromboangiitis obliterans), cardiac fibrosis, cardiac hypertrophy, cardiomyopathy, carotid artery obstructive disease, cerebral infarction, Chemical burns, COPD, Crohn's disease, cytokine-induced vascular leakage, unstable blood flow, diabetes (including non-insulin-dependent diabetes mellitus), dysfunctional uterine bleeding, endometriosis, Epstein-Barr virus infection, erectile dysfunction, excessive hair growth, follicular cysts, foot ulcers (e.g., diabetic foot ulcers), fungal ulcers, giant cell arteritis, glomerulosclerosis, Graves' disease, Hashimoto's autoimmune thyroiditis, hemangiomas, hemangioendotheliomas, hemophilic joints, bleeding, hepatitis C, hereditary hemorrhagic telangiectasia (HHT), herpes simplex infection, herpes zoster infection, hypertension, idiopathic hemorrhage Cytopenic purpura, impaired wound healing, inflammatory and infectious processes (e.g., hepatitis, pneumonia, glomerulonephritis), interstitial fibrosis, ischemia, nephropathy, leishmaniasis, leukomalacia, lipidosis, liver regeneration, lupus nephritis, Lyme disease, lymphoproliferative disorders, malaria (Plasmodium infection), Mooren ulcer, multiple sclerosis, mycobacterial infection, myocardial infarction, nasal polyps, nephropathy, neuronal inflammation, neuropathy, obesity, osteomyelitis, osteophytes, ovarian hyperstimulation, Paget's disease, pannus growth, peripheral arterial disease, peritoneal sclerosis, pemphigoid, polyarteritis, protozoal infection infection, pseudoxanthoma elasticum, psoriasis, pulmonary hypertension, pyogranuloma, renal fibrosis, respiratory distress, rheumatoid arthritis, rickettsial infection, keloid, sepsis, sickle cell anemia, Stevens-Johnson disease, stroke, synovitis, systemic lupus erythematosus, syphilis, goiter, thyroiditis, toxic shock syndrome, toxoplasmosis, trauma, ulcerative colitis, vascular leak, vascular leak syndrome, vascular malformation (e.g., Osler-Weber syndrome), venous occlusion, viral hemorrhagic fever (e.g., dengue fever), vitamin A deficiency, warts, or Wegener's sarcoidosis, or a combination thereof.

[0168] sequence

[0169] As used herein, the abbreviations for the L-enantiomers and D-enantiomers of amino acids are as follows: Alanine (A, Ala); Arginine (R, Arg); Asparagine (N, Asn); Aspartic acid (D, Asp); Cysteine ​​(C, Cys); Glutamic acid (E, Glu); Glutamine (Q, Gln); Glycine (G, Gly); Histidine (H, His); Isoleucine (I, Ile); Leucine (L, Leu); Lysine (K, Lys); Methionine (M, Met); Phenylalanine (F, Phe); Proline (P, Pro); Serine (S, Ser); Threonine (T, Thr); Tryptophan (W, Trp); Tyrosine (Y, Tyr); Valine (V, Val). In some embodiments, the amino acid is the L-enantiomer. In some embodiments, the amino acid is the D-enantiomer.

[0170] Table 2 shows the humanized V binding to HPTP-β (VE-PTP) H The amino acid sequence of the antibody region. SEQ ID NO: 1 is V H1 SEQ ID NO: 2 is V H2 SEQ ID NO: 3 is V H3 , and SEQ ID NO: 4 is V H4 .

[0171]

[0172]

[0173] Table 3 shows the humanized V binding to HPTP-β (VE-PTP) L The amino acid sequence of the antibody region. SEQ ID NO: 5 is V L1 SEQ ID NO: 6 is V L2 SEQ ID NO: 7 is V L3 , and SEQ ID NO: 8 is V L4 .

[0174]

[0175] Each V H The domains can be synthesized in frame with constant domain sequences such as human IgG1, IgG2, IgG3, IgG4, IgE, IgA1, IgA2, IgM or IgD sequences. The DNA sequence encoding the entire heavy chain sequence can be codon optimized and verified.

[0176] Table 4 provides exemplary amino acid sequences of constant domain sequences. In some embodiments, the V HThe V domain is synthesized in frame with a human IgG1 constant domain sequence. The human IgG1 constant domain sequence may comprise SEQ ID NO: 220. In some embodiments, the V domain disclosed herein H The V domain is synthesized in frame with a human IgG2 constant domain sequence. The human IgG2 constant domain sequence may comprise SEQ ID NO: 221. In some embodiments, the V domain disclosed herein H The V domain is synthesized in frame with a human IgG3 constant domain sequence. The human IgG3 constant domain sequence may comprise SEQ ID NO: 222. In some embodiments, the V domain disclosed herein H The V domain is synthesized in frame with the human IgG4 constant domain sequence. The human IgG4 isotype constant domain sequence can be mutated to proline instead of serine at position 228 to reduce Fab-arm exchange (stabilizing S228P mutation). The amino acid sequence of the IgG4 constant domain with the S228P mutation can comprise SEQ ID NO: 9. In some embodiments, the V domains disclosed herein are H The V domain is synthesized in frame with the human IgE constant domain sequence. The human IgE constant domain sequence may comprise SEQ ID NO: 223. In some embodiments, the V domain disclosed herein H The V domain is synthesized in frame with the human IgA1 constant domain sequence. The human IgA1 constant domain sequence may comprise SEQ ID NO: 224. In some embodiments, the V domain disclosed herein H The V domain is synthesized in frame with the human IgA2 constant domain sequence. The human IgA2 constant domain sequence may comprise SEQ ID NO: 225. In some embodiments, the V domain disclosed herein H The V domain is synthesized in frame with a human IgM constant domain sequence. The human IgM constant domain sequence may comprise SEQ ID NO: 226. In some embodiments, the V domain disclosed herein H The domain is synthesized in frame with the human IgD constant domain sequence. The human IgD constant domain sequence may comprise SEQ ID NO: 227.

[0177]

[0178]

[0179] Each V L The domain can be synthesized in frame with a human light chain constant domain sequence, such as a kappa (IgK) or lambda (IgL) chain. The entire light chain sequence can then be codon optimized and the DNA sequence verified. Table 5 provides exemplary light chain constant domain sequences.

[0180] In some embodiments, V disclosed herein LThe V domain is synthesized in frame with the human Igκ constant domain sequence. The human Igκ constant domain sequence may comprise SEQ ID NO: 10. In some embodiments, the V domain disclosed herein L The domain is synthesized in frame with the human IgL constant domain sequence. The human IgL constant domain sequence may comprise SEQ ID NO: 228.

[0181]

[0182] Signal peptides can lead to higher protein expression and / or secretion from cells.The following signal peptides can be appended to all constructs disclosed herein.

[0183] Heavy chain signal peptide (SEQ ID NO: 11): MGWTLVFLFLLSVTAGVHS

[0184] Light chain signal peptide (SEQ ID NO: 12): MVSSAQFLGLLLLCFQGTRC

[0185] Signal peptidases can cleave signal peptides from proteins, for example, during secretion, thereby generating mature proteins that do not contain signal peptide sequences. In some embodiments, signal peptides are cleaved from compounds or antibodies of the present disclosure. In some embodiments, mature compounds or antibodies of the present disclosure do not contain signal peptides.

[0186] Tables 6 and 7 list the complete amino acid sequences of the humanized heavy and light chains that bind to HPTP-β (VE-PTP). H1 、V H2 、V H3 and V H4 A conjugated human IgG4 isotype constant domain with a stabilizing S228P mutation and an appended signal peptide. Amino acids 1-19 of SEQ ID NOs: 13, 14, 15, and 16 are the heavy chain signal peptide (SEQ ID NO: 11). HC1, HC2, HC3, and HC4 are also shown without the appended signal peptide (SEQ ID NOs: 246, 247, 248, and 249). In some embodiments, the mature compounds or antibodies of the present disclosure do not contain a signal peptide.

[0187] LC1, LC2, LC3 and LC4 are respectively connected to V L1 、V L2 、V L3 and V L4The constant domain of the human Igκ isotype is joined and supplemented with a signal peptide. Amino acids 1-20 of SEQ ID NOs: 17, 18, 19, and 20 are the light chain signal peptide (SEQ ID NO: 12). LC1, LC2, LC3, and LC4 are also shown without the signal peptide (SEQ ID NOs: 250, 251, 252, and 253). In some embodiments, the mature compounds or antibodies of the present disclosure do not contain a signal peptide.

[0188]

[0189]

[0190]

[0191]

[0192] Any V disclosed herein H The region may be any V disclosed herein L The signal peptide and constant region sequences can be combined with or without additional sequences to produce compounds that bind to anti-HPTP-β (VE-PTP). For example, the signal peptide and constant region sequences can be appended to the V domains shown in Tables 6 and 7, respectively. H and V L The heavy and light chains can be paired in any combination to form antibodies. Table 8 shows 16 possible pairings of HC1-4 and LC1-4 that bind to HPTP-β (VE-PTP). Transfection and expression of each anti-HPTP-β (VE-PTP) antibody in Table 8 can be explored.

[0193]

[0194] Antibodies or antigen-binding compounds specific for HPTP-β (VE-PTP) can be combined with antibodies or compounds that activate Tie2, inhibit VEGF, or inhibit VEGFR to form multispecific compounds.

[0195] Table 9 provides the sequences of collagen IV-derived biomimetic peptides. SEQ ID NO: 152 is AXT-107, a collagen IV-derived biomimetic peptide targeting integrin that inhibits VEGFR phosphorylation / activation / signaling and promotes Tie2 phosphorylation / activation / signaling. SEQ ID NO: 153 provides a consensus sequence, wherein X is any standard amino acid or a non-genetically encoded amino acid. In some embodiments, X at position 7 is M, A, or G; X at position 9 is F, A, Y, or G; X at position 0 is M, A, G, D-alanine (dA), or norleucine (Nle); X at position 11 is F, A, Y, G, or 4-chlorophenylalanine (4-CiPhe); and X at positions 12 and 18 are independently selected from 2-aminobutyric acid (Abu), G, S, A, V, T, I, L, or allylglycine (AllylGly).

[0196]

[0197] Table 10 provides sequences related to vasculotide, a synthetic Ang1 mimetic peptide that can act as a Tie2 agonist and activate Tie2 signaling. SEQ ID NO: 229 provides the sequence of a synthetic 7-mer that binds to the Tie2 receptor. SEQ ID NO: 230 provides an 8-mer with a cysteine ​​residue added to the N-terminus, allowing covalent tethering to a polyethylene glycol backbone to generate a tetrameric polyethylene oxide clustered form of the peptide.

[0198]

[0199] Antibodies or antigen-binding compounds specific for HPTP-β (VE-PTP) can be combined with antibodies or compounds specific for VEGF or VEGFR to form multispecific compounds that bind to HPTP-β (VE-PTP) and VEGF or VEGFR. Tables 11, 12, 13, 14, 15, and 17 provide example sequences of compounds specific for VEGF. Table 16 provides sequences of antibodies that bind to VEGFR.

[0200] Table 11 provides sequences related to aflibercept, a recombinant protein comprising the VEGF-binding portion of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1. SEQ ID NO: 21 is the complete amino acid sequence of aflibercept. SEQ ID NO: 22 is a shortened sequence comprising the VEGF-binding portion of human VEGF receptors 1 and 2 without the Fc portion of IgG1.

[0201]

[0202] Table 12 provides the sequence of brolucizumab (SEQ ID NO: 23), a humanized single-chain antibody fragment (scFv) inhibitor of VEGF that binds to the receptor binding site of VEGF, thereby interfering with the interaction of VEGF with VEGFR1 and VEGFR2.

[0203]

[0204] Table 13 provides sequences related to ranibizumab, a humanized monoclonal antibody fragment (Fab) that binds to and inhibits VEGF activity. SEQ ID NO: 24 is the heavy chain sequence of ranibizumab. SEQ ID NO: 25 is the light chain sequence of ranibizumab. SEQ ID NO: 26 is a shortened sequence of the ranibizumab heavy chain, which can be used to clone single-chain antibody fragments (scFv). SEQ ID NO: 27 is a shortened sequence of the ranibizumab light chain, which can be used to clone single-chain antibody fragments (scFv). SEQ ID NO: 28 is a single-chain antibody fragment (scFv) comprising SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 33 (connecting peptide, underlined).

[0205]

[0206]

[0207] Table 14 provides sequences related to bevacizumab, a humanized monoclonal antibody that binds to VEGF and inhibits its activity. SEQ ID NO: 29 is the heavy chain sequence of bevacizumab. SEQ ID NO: 30 is the light chain sequence of bevacizumab.

[0208]

[0209] Table 15 provides sequences related to Conbercept, a recombinant protein comprising the extracellular domains of VEGF receptors 1 and 2 fused to the Fc portion of human IgG1. SEQ ID NO: 154 is the complete amino acid sequence of Conbercept. SEQ ID NO: 155 is a shortened version of the VEGF receptors 1 and 2 sequence without the Fc portion of IgG1.

[0210]

[0211]

[0212] Table 16 provides sequences related to ramucirumab, a humanized monoclonal antibody that binds to the extracellular domain of VEGFR2 and inhibits VEGFR2 signaling. SEQ ID NO: 156 is the heavy chain sequence of ramucirumab. SEQ ID NO: 157 is the light chain sequence of ramucirumab.

[0213]

[0214] Table 17 provides amino acid sequences of DARPins and DARPin derivatives that bind to VEGF and inhibit VEGFR signaling. SEQ ID NOs: 158-168 comprise a designed ankyrin repeat with binding specificity for VEGF. SEQ ID NO: 169 comprises a designed ankyrin repeat with binding specificity for VEGF and a designed ankyrin repeat with binding specificity for serum albumin. SEQ ID NO: 170 comprises a designed ankyrin repeat with binding specificity for VEGF, a designed ankyrin repeat with binding specificity for hepatocyte growth factor, and a designed ankyrin repeat with binding specificity for serum albumin. SEQ ID NOs: 171-177 comprise a designed ankyrin repeat with binding specificity for VEGF. SEQ ID NO: 173 provides the sequence of abicipar, which comprises a designed ankyrin repeat with binding specificity for VEGF. SEQ ID NOs: 178-190 provide individual designed ankyrin repeat sequence motifs with binding specificity for VEGF, wherein X represents any amino acid. SEQ ID NOs: 191-217 comprise designed ankyrin repeats with binding specificity for VEGF.

[0215]

[0216]

[0217]

[0218]

[0219]

[0220] An antibody or antigen-binding compound that is specific for VEGF can be combined with an antibody or antigen-binding compound that is specific for HPTP-β (VE-PTP) to form a multispecific compound, such as a bispecific compound that binds to VEGF and HPTP-β (VE-PTP). Any compound disclosed herein that is specific for VEGF can be combined with any compound disclosed herein that is specific for HPTP-β (VE-PTP). Any compound disclosed herein that is specific for VEGF or HPTP-β (VE-PTP) can be modified, if necessary, to generate a multispecific compound. Non-limiting examples of modifications necessary to generate a multispecific compound include the addition of amino acid residues, the removal of amino acid residues, the substitution of amino acid residues, and the use of linkers. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more residues can be added to the N-terminus and / or C-terminus of the sequences disclosed herein, and the resulting sequences can be used to generate multispecific constructs. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more residues can be removed from the N-terminus and / or C-terminus of the sequences disclosed herein, and the remaining sequence can be used to generate multispecific constructs. For example, the N- and / or C-terminal residues can be removed from SEQ ID NO: 173 (e.g., to generate SEQ ID NO: 244), and the truncated sequence can be used in a multispecific construct. In some embodiments, the sequence in any one of SEQ ID NOs: 13-20 or 246-253 can be modified. For example, one or more C-terminal residues can be removed (e.g., the C-terminal lysine can be removed from any one of SEQ ID NOs: 13-16 or 246-249, and the remaining residues (residues 1-467) can be used in a multispecific construct).

[0221] The compounds described herein may include a linker between the different domains of the compound. The linker may be a chemical bond, such as a covalent bond or a non-covalent bond. The linkers described herein may include flexible or rigid linkers.

[0222] The connector of the present disclosure can include a chemical connector. For example, two amino acid sequences of the present disclosure can be linked together by a chemical connector. Each chemical connector of the present disclosure can be an alkylene, alkenylene, alkynylene, sub-assorted alkylene, cycloalkylene, sub-heterocycloalkylene, arylene or sub-heteroaryl, any of which is optionally substituted. In some embodiments, the chemical connector of the present disclosure can be an ester, ether, amide, thioether or polyethylene glycol (PEG). In some embodiments, the connector can, for example, reverse the order of the amino acid sequence in the compound so that the amino acid sequence connected by the connector is head to head, rather than head to tail. The limiting examples of this type of connector include the diester of dicarboxylic acid, such as oxalyl diester, malonyl diester, succinyl diester, glutaryl diester, adipoyl diester, pimetyl diester, fumaryl diester, maleoyl diester, phthaloyl diester, isophthaloyl diester and terephthaloyl diester. Non-limiting examples of such linkers include diamides of dicarboxylic acids such as oxalyl diamide, malonyl diamide, succinyl diamide, glutaryl diamide, adipoyl diamide, pimetyl diamide, fumaryl diamide, maleoyl diamide, phthaloyl diamide, isophthaloyl diamide, and terephthaloyl diamide. Non-limiting examples of such linkers include diamides of diamino linkers such as ethylenediamine, 1,2-bis(methylamino)ethane, 1,3-diaminopropane, 1,3-bis(methylamino)propane, 1,4-bis(methylamino)butane, 1,5-bis(methylamino)pentane, 1,6-bis(methylamino)hexane, and piperazine.

[0223] Non-limiting examples of optional substituents include hydroxyl groups, thiol groups, halogens, amino groups, nitro groups, nitroso groups, cyano groups, azide groups, sulfoxide groups, sulfone groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, halo-alkyl groups, alkenyl groups, halo-alkenyl groups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups, acyl groups, acyloxy groups, carbamate groups, amide groups, urea groups, epoxy groups, and ester groups.

[0224] The linker can be a peptide. The linker can comprise a linker sequence, such as a linker peptide sequence. The linker sequence can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 amino acid residues in length.

[0225] Flexible linkers can have sequences containing a stretch of glycine and serine residues. The small size of glycine and serine residues provides flexibility and allows for mobility of the linked functional domains. The incorporation of serine or threonine can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thereby reducing adverse interactions between the linker and the protein moiety.

[0226] The flexible linker may also contain additional amino acids, such as threonine and alanine, to maintain flexibility, and polar amino acids, such as lysine and glutamine, to improve solubility.

[0227] The flexible linker can comprise repeats of SEQ ID NO: 42 (GGGS), for example, SEQ ID NOs: 42-55. The flexible linker can comprise repeats of SEQ ID NO: 31 (GGGGS), for example, SEQ ID NOs: 31-41. Several other types of flexible linkers can also be used, including SEQ ID NO: 59 (KESGSVSSEQLAQFRSLD) and SEQ ID NO: 60 (EGKSSGSGSESKST). SEQ ID NO: 61 (GSAGSAAGSGEF) linkers can also be used, in which large hydrophobic residues are minimized to maintain good solubility in aqueous solution. The length of the flexible linker can be adjusted to allow proper folding or to achieve optimal biological activity of the fusion protein.

[0228] The rigid connector may have, for example, an α-helical structure. α-helical rigid connectors may serve as spacers between protein domains. The rigid connector may comprise repetitions of SEQ ID NO: 62 (EAAAK), for example, SEQ ID NO: 62-66. The rigid connector may comprise repetitions of SEQ ID NO: 67 (EAAAR), for example, SEQ ID NO: 67-72. The rigid connector may have a proline-rich sequence (XP) n, where X represents alanine, lysine, glutamine, or any amino acid. The presence of proline in a non-helical connector may increase rigidity and allow for efficient separation of protein domains.

[0229] The linker may comprise any of the sequences disclosed in Table 18, which may be used to link any portion of a compound disclosed herein to any portion of another compound disclosed herein:

[0230]

[0231]

[0232] The VEGF-binding compounds and VEGFR-binding compounds described in Table 11, Table 12, Table 13, Table 14, Table 15, Table 16, and Table 17 can be combined with the HPTP-β (VE-PTP)-binding compounds described in Table 2, Table 3, Table 6, Table 7, and Table 8.

[0233] Any of the 16 antibodies described in Table 8 can be combined with aflibercept or aflibercept-related sequences to generate bispecific antibodies. Non-limiting illustrative examples are provided in Figure 2 、 Figure 3 and Figure 4 For example, SEQ ID NO: 22 can be appended to SEQ ID NO: 14 or SEQ ID NO: 247 of antibody HC2:LC1 to generate a tetravalent bispecific antibody wherein the heavy chain of HC2:LC1 is appended with SEQ ID NO: 22 ( Figure 2 ). SEQ ID NO: 22 can be appended to SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a tetravalent bispecific antibody in which the light chain of HC2:LC1 has SEQ ID NO: 22 appended ( Figure 3 ). SEQ ID NO:22 can be appended to SEQ ID NO:14 or SEQ ID NO:247, and SEQ ID NO:22 can be appended to SEQ ID NO:17 or SEQ ID NO:250 of antibody HC2:LC1 to generate a hexavalent bispecific antibody in which the heavy and light chains of HC2:LC1 have SEQ ID NO:22 appended ( Figure 4 ).

[0234] Any of the 16 antibodies described in Table 8 can be combined with brolucizumab or brolucizumab-related sequences to generate bispecific antibodies. Non-limiting illustrative examples are provided in Figure 2 、 Figure 3 and Figure 4 For example, SEQ ID NO: 23 can be appended to SEQ ID NO: 14 or SEQ ID NO: 247 of antibody HC2:LC1 to generate a tetravalent bispecific antibody in which the heavy chain of HC2:LC1 is appended with SEQ ID NO: 23 ( Figure 2 ). SEQ ID NO: 23 can be appended to SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a tetravalent bispecific antibody in which the light chain of HC2:LC1 has SEQ ID NO: 23 appended ( Figure 3 ). SEQ ID NO:23 can be appended to SEQ ID NO:14 or SEQ ID NO:247, and SEQ ID NO:23 can be appended to SEQ ID NO:17 or SEQ ID NO:250 of antibody HC2:LC1 to generate a hexavalent bispecific antibody in which the heavy and light chains of HC2:LC1 have SEQ ID NO:23 appended ( Figure 4 ).

[0235] Any of the 16 antibodies described in Table 8 can be combined with ranibizumab or ranibizumab-related sequences to generate bispecific antibodies. Non-limiting illustrative examples are provided in Figure 2 、 Figure 3 and Figure 4 For example, SEQ ID NO: 28 can be appended to SEQ ID NO: 14 or SEQ ID NO: 247 of antibody HC2:LC1 to generate a tetravalent bispecific antibody wherein the heavy chain of HC2:LC1 has SEQ ID NO: 28 appended to it ( Figure 2 ). SEQ ID NO: 28 can be appended to SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a tetravalent bispecific antibody in which the light chain of HC2:LC1 has SEQ ID NO: 28 appended ( Figure 3). SEQ ID NO:28 can be appended to SEQ ID NO:14 or SEQ ID NO:247, and SEQ ID NO:28 can be appended to SEQ ID NO:17 or SEQ ID NO:250 of antibody HC2:LC1 to generate a hexavalent bispecific antibody in which the heavy and light chains of HC2:LC1 have SEQ ID NO:28 appended ( Figure 4 ).

[0236] Any of the 16 antibodies described in Table 8 can be combined with bevacizumab or bevacizumab-related sequences to generate bispecific antibodies. Non-limiting illustrative examples are provided in Figure 2 、 Figure 3 and Figure 4 For example, an antigen-binding scFv for bevacizumab can be generated as shown for ranibizumab in Table 13. A bevacizumab-derived antigen-binding scFv can be appended to SEQ ID NO: 14 or SEQ ID NO: 247 of antibody HC2:LC1 to generate a tetravalent bispecific antibody in which the heavy chain of HC2:LC1 is appended with a bevacizumab-derived antigen-binding scFv ( Figure 2 ). The bevacizumab-derived antigen-binding scFv can be appended to SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a tetravalent bispecific antibody in which the light chain of HC2:LC1 is appended with the bevacizumab-derived antigen-binding scFv ( Figure 3 ). The bevacizumab-derived antigen-binding scFv can be appended to SEQ ID NO: 14 or SEQ ID NO: 247, and the bevacizumab-derived antigen-binding scFv can be appended to SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a hexavalent bispecific antibody in which the heavy and light chains of HC2:LC1 are appended with the bevacizumab-derived antigen-binding scFv ( Figure 4 ).

[0237] Any of the 16 antibodies described in Table 8 can be combined with abicipar or abicipar-related sequences to generate bispecific antibodies. Non-limiting illustrative examples are provided in Figure 2 、 Figure 3 and Figure 4For example, SEQ ID NO: 173 or SEQ ID NO: 244 can be appended to SEQ ID NO: 14, SEQ ID NO: 247, SEQ ID NO: 14 residues 1-467, or SEQ ID NO: 247 residues 1-467 of antibody HC2:LC1 to generate a tetravalent bispecific antibody in which the heavy chain of HC2:LC1 has SEQ ID NO: 173 or SEQ ID NO: 244 appended thereto. Figure 2 ). SEQ ID NO: 173 or SEQ ID NO: 244 can be appended to SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a tetravalent bispecific antibody in which the light chain of HC2:LC1 has SEQ ID NO: 173 or SEQ ID NO: 244 appended ( Figure 3 SEQ ID NO: 173 or SEQ ID NO: 244 can be appended to SEQ ID NO: 14, SEQ ID NO: 247, SEQ ID NO: 14 residues 1-467, or SEQ ID NO: 247 residues 1-467, and SEQ ID NO: 173 or SEQ ID NO: 244 can be appended to SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a hexavalent bispecific antibody in which the heavy and light chains of HC2:LC1 have SEQ ID NO: 173 or SEQ ID NO: 244 appended ( Figure 4 ).

[0238] Any of the 16 antibodies described in Table 8 can be combined with Conbercept or Conbercept-related sequences to generate bispecific antibodies. Non-limiting illustrative examples are provided in Figure 2 、 Figure 3 and Figure 4 For example, SEQ ID NO: 155 can be appended to SEQ ID NO: 14 or SEQ ID NO: 247 of antibody HC2:LC1 to generate a tetravalent bispecific antibody wherein the heavy chain of HC2:LC1 has SEQ ID NO: 155 appended thereto ( Figure 2 ). SEQ ID NO: 155 can be appended to SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a tetravalent bispecific antibody in which the light chain of HC2:LC1 has SEQ ID NO: 155 appended ( Figure 3). SEQ ID NO: 155 can be appended to SEQ ID NO: 14 or SEQ ID NO: 247, and SEQ ID NO: 155 can be appended to SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a hexavalent bispecific antibody in which the heavy and light chains of HC2:LC1 have SEQ ID NO: 155 appended ( Figure 4 ).

[0239] Any of the 16 antibodies described in Table 8 can be combined with ramucirumab or ramucirumab-related sequences to generate bispecific antibodies. Non-limiting illustrative examples are provided in Figure 2 、 Figure 3 and Figure 4 For example, an antigen-binding scFv for ramucirumab can be generated as shown for ranibizumab in Table 13. The ramucirumab-derived antigen-binding scFv can be appended to SEQ ID NO: 14 or SEQ ID NO: 247 of antibody HC2:LC1 to generate a tetravalent bispecific antibody in which the heavy chain of HC2:LC1 is appended with the ramucirumab-derived antigen-binding scFv ( Figure 2 The ramucirumab-derived antigen-binding scFv can be appended to SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a tetravalent bispecific antibody in which the light chain of HC2:LC1 is appended with the ramucirumab-derived antigen-binding scFv ( Figure 3 The ramucirumab-derived antigen-binding scFv can be appended to SEQ ID NO: 14 or SEQ ID NO: 247, and the ramucirumab-derived antigen-binding scFv can be appended to SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a hexavalent bispecific antibody in which the heavy and light chains of HC2:LC1 are appended with the ramucirumab-derived antigen-binding scFv ( Figure 4 ).

[0240] Any of the 16 antibodies described in Table 8 can be combined with a DARPin, a DARPin repeat, or a sequence thereof to generate a multispecific antibody. Non-limiting illustrative examples are provided in Figure 2 、 Figure 3 and Figure 4For example, an amino acid sequence comprising any one of SEQ ID NOs: 158-217 can be appended to SEQ ID NO: 14 or SEQ ID NO: 247 of antibody HC2:LC1 to generate a tetravalent bispecific antibody, wherein the heavy chain of HC2:LC1 is appended with an amino acid sequence comprising any one of SEQ ID NOs: 158-217 ( Figure 2 ). An amino acid sequence comprising any one of SEQ ID NOs: 158-217 can be appended to SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a tetravalent bispecific antibody, wherein the light chain of HC2:LC1 has an amino acid sequence comprising any one of SEQ ID NOs: 158-217 appended thereto ( Figure 3 An amino acid sequence comprising any one of SEQ ID NOs: 158-217 can be appended to SEQ ID NO: 14 or SEQ ID NO: 247 and SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a hexavalent bispecific antibody in which the heavy and light chains of HC2:LC1 are appended with an amino acid sequence comprising any one of SEQ ID NOs: 158-217 ( Figure 4 ).

[0241] The Tie2 activating compounds described in Tables 9 and 10 can be combined with the HPTP-β (VE-PTP) binding compounds described in Tables 2, 3, 6, 7, and 8.

[0242] Any of the 16 antibodies described in Table 8 can be combined with collagen IV derived biomimetic peptide sequences to generate multispecific antibodies. Non-limiting illustrative examples are provided in Figure 2 、 Figure 3 and Figure 4 For example, the amino acid sequence comprising SEQ ID NO: 152 or SEQ ID NO: 153 can be appended to SEQ ID NO: 14 or SEQ ID NO: 247 of antibody HC2:LC1 to generate a tetravalent bispecific antibody, wherein the heavy chain of HC2:LC1 has the amino acid sequence comprising SEQ ID NO: 152 or SEQ ID NO: 153 appended thereto ( Figure 2 ). The amino acid sequence comprising SEQ ID NO: 152 or SEQ ID NO: 153 can be appended to SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a tetravalent bispecific antibody, wherein the light chain of HC2:LC1 has the amino acid sequence comprising SEQ ID NO: 152 or SEQ ID NO: 153 appended thereto ( Figure 3 The amino acid sequence comprising SEQ ID NO: 152 or SEQ ID NO: 153 can be appended to SEQ ID NO: 14 or SEQ ID NO: 247 and SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a hexavalent bispecific antibody in which the heavy and light chains of HC2:LC1 have the amino acid sequence comprising SEQ ID NO: 152 or SEQ ID NO: 153 appended thereto ( Figure 4 ).

[0243] Any of the 16 antibodies described in Table 8 can be combined with an Ang2 mimetic or its sequence to generate a multispecific antibody. Non-limiting illustrative examples are provided in Figure 2 、 Figure 3 and Figure 4 For example, an amino acid sequence comprising any one of SEQ ID NOs: 229-230 can be appended to SEQ ID NO: 14 or SEQ ID NO: 247 of antibody HC2:LC1 to generate a tetravalent bispecific antibody, wherein the heavy chain of HC2:LC1 is appended with an amino acid sequence comprising any one of SEQ ID NOs: 229-230 ( Figure 2 ). An amino acid sequence comprising any one of SEQ ID NOs: 229-230 can be appended to SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a tetravalent bispecific antibody, wherein the light chain of HC2:LC1 has an amino acid sequence comprising any one of SEQ ID NOs: 229-230 appended thereto ( Figure 3 ). An amino acid sequence comprising any one of SEQ ID NOs: 229-230 can be appended to SEQ ID NO: 14 or SEQ ID NO: 247 and SEQ ID NO: 17 or SEQ ID NO: 250 of antibody HC2:LC1 to generate a hexavalent bispecific antibody in which the heavy and light chains of HC2:LC1 are appended with an amino acid sequence comprising any one of SEQ ID NOs: 229-230 ( Figure 4 ).

[0244] The Tie2-activating compounds described in Tables 9 and 10 can be combined with the VEGF-binding and VEGFR-binding compounds described in Tables 11, 12, 13, 14, 15, 16, and 17.

[0245] Any compound of the present disclosure, such as the multispecific fusion constructs described above, can be modified if necessary to promote desired protein folding or biological activity.

[0246] CDR

[0247] Sequence in the present disclosure can comprise complementary determining region (CDR).CDR can be identified by Kabat method, Chothia method, IMGT method or Paratome method.The CDR of this paper sequence can be the length of such as 0 to 91 residues, the length of 0 to 25 residues, the length of 5 to 14 residues, the length of about 0 residue, the length of about 1 residue, the length of about 2 residues, the length of about 3 residues, the length of about 4 residues, the length of about 5 residues, the length of about 6 residues, the length of about 7 residues, the length of about 8 residues, the length of about 9 residues, the length of about 10 residues, the length of about 11 residues, the length of about 12 residues, the length of about 13 residues, the length of about 14 residues, the length of about 15 residues, the length of about 16 residues, the length of about 17 residues, the length of about 18 residues, the length of about 19 residues, the length of about 20 residues, the length of about 21 residues, the length of about 22 residues, the length of about 23 residues, the length of about 24 residues or the length of about 25 residues.

[0248] Compounds of the present disclosure that have binding specificity for or the ability to modulate HPTP-β (VE-PTP) may comprise, for example, any of the CDRs in Table 19, Table 20, Table 21, Table 22, Table 23, or Table 24.

[0249] Table 19 provides non-limiting examples of HCDR1 sequences specific for HPTP-β (VE-PTP).

[0250]

[0251] Table 20 provides non-limiting examples of HCDR2 sequences specific for HPTP-β (VE-PTP).

[0252]

[0253]

[0254] Table 21 provides non-limiting examples of HCDR3 sequences specific for HPTP-β (VE-PTP).

[0255]

[0256] Table 22 provides non-limiting examples of LCDR1 sequences specific for HPTP-β (VE-PTP).

[0257]

[0258] Table 23 provides non-limiting examples of LCDR2 sequences specific for HPTP-β (VE-PTP).

[0259]

[0260] Table 24 provides non-limiting examples of LCDR3 sequences specific for HPTP-β (VE-PTP).

[0261]

[0262] Compounds of the present disclosure that have binding specificity for VEGF or the ability to modulate VEGF may comprise any of the CDRs in Table 25, Table 26, Table 27, Table 28, Table 29, or Table 30.

[0263] Table 25 provides non-limiting examples of HCDR1 sequences specific for VEGF.

[0264]

[0265] Table 26 provides non-limiting examples of HCDR2 sequences specific for VEGF.

[0266]

[0267]

[0268] Table 27 provides non-limiting examples of HCDR3 sequences specific for VEGF.

[0269]

[0270] Table 28 provides non-limiting examples of LCDR1 sequences that are specific for VEGF.

[0271]

[0272] Table 29 provides non-limiting examples of LCDR2 sequences that are specific for VEGF.

[0273]

[0274]

[0275] Table 30 provides non-limiting examples of LCDR3 sequences that are specific for VEGF.

[0276]

[0277] Table 31 provides aflibercept-derived sequences corresponding to the D2 domain of human VEGF receptor 1 (SEQ ID NO: 147) and the D3 domain of human VEGF receptor 2 (SEQ ID NO: 148).

[0278]

[0279] Table 32 provides sequences derived from abicipar. SEQ ID NOs: 233-236 correspond to the ankyrin repeats within abicipar. SEQ ID NOs: 237-242 provide consensus sequences for VEGF-binding ankyrin repeats. In SEQ ID NO: 237, X1 is K, T, or Y; X2 is N or M; X3 is T or F; X4 is S or A; X5 is H or R; X6 is A, Y, H, or N; and X7 is A or T. In SEQ ID NO: 238, X1 is K, M, N, R, or V; X2 is Y, H, M, or V; X3 is F, L, M, or V; X4 is R, H, V, A, K, or N; X5 is F, D, H, T, Y, M, or K; and X6 is A, H, N, or Y. In SEQ ID NO: 239, X1 is L, S, or T; X2 is G, S, or C; X3 is S or A; X4 is Q, S, M, or N; X5 is L, M, or Q; and X6 is A, H, N, Y, or D. In SEQ ID NO: 240, X1 is K, S, I, N, T, or V; X2 is K, N, W, A, H, M, Q, or S; X3 is F, Q, L, H, or V; X4 is F or T; X5 is Q or H; X6 is Y or S; X7 is N, H, Y, or M; and X8 is A, H, N, or Y. In SEQ ID NO: 241, X1 is A, N, R, V, Y, E, H, I, K, L, Q, S, or T; X2 is S, A, N, R, D, F, L, P, T, or Y; X3 is T, V, S, A, L, or F; X4 is W, F, or H; X5 is P, I, A, L, S, T, V, or Y; X6 is W, F, I, L, T, or V; X7 is L or P; and X8 is A, H, N, or Y. In SEQ ID NO: 242, X1 is H, Q, A, K, R, D, I, L, M, N, V, or Y; X2 is Y, F, or H; X3 is Q, F, or T; X4 is W, M, G, H, N, or T; X5 is T, A, M, L, or V; X6 is I, L, V, D, or T; and X7 is A, H, N, or Y. SEQ ID NO: 244 provides a truncated sequence derived from abicipar comprising a designed ankyrin repeat with binding specificity for VEGF.

[0280]

[0281] Homology

[0282] The sequences of the compounds herein can have at least about 70% homology, at least about 71% homology, at least about 72% homology, at least about 73% homology, at least about 74% homology, at least about 75% homology, at least about 76% homology, at least about 77% homology, at least about 78% homology, at least about 79% homology, at least about 80% homology, at least about 81% homology, at least about 82% homology, at least about 83% homology, at least about 84% homology, at least about 85% homology, at least about 86% homology, at least about 87% homology, at least about 88% homology, at least about 89% homology, at least about 90% homology, at least about 91% homology, at least about 92% homology, at least about 93% homology, at least about 94% homology, at least about 95% homology, at least about 96% homology, at least about 97% homology, at least about 98% homology, at least about 99% homology, at least about 100% homology, at least about 101% homology, at least about 102% homology, at least about 103% homology, at least about 104% homology, at least about 105% homology, at least about 106% homology, at least about 107% homology, at least about 108% homology, at least about 109% homology, at least about 110% homology, at least about 111% homology, at least about 112% homology, at least about 113% homology, at least about 114% homology at least about 95% homology, at least about 96% homology, at least about 97% homology, at least about 98% homology, at least about 99% homology, at least about 99.1% homology, at least about 99.2% homology, at least about 99.3% homology, at least about 99.4% homology, at least about 99.5% homology, at least about 99.6% homology, at least about 99.7% homology, at least about 99.8% homology, at least about 99.9% homology, at least about 99.10% homology, at least about 99.11% homology, at least about 99.12% homology, at least about 99.13% homology, at least about 99.14% homology, at least about 99.15% homology, at least about 99.16% homology, at least about 99.17% homology, at least about 99.18% homology, at least about 99.19% homology, at least about 99.20% homology, at least about 99.30% homology, at least about 99.40% homology, at least about 99.50% homology, at least about 99.60% homology, at least about 99.70% homology, at least about 99. At least about 99.8% homology, at least about 99.9% homology, at least about 99.91% homology, at least about 99.92% homology, at least about 99.93% homology, at least about 99.94% homology, at least about 99.95% homology, at least about 99.96% homology, at least about 99.97% homology, at least about 99.98% homology, or at least about 99.99% homology.

[0283] Homology between two or more sequences can be determined using a variety of methods and software programs, such as NCBI BLAST, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, or another suitable method or algorithm.

[0284] Pharmaceutical composition

[0285] The pharmaceutical compositions of the present disclosure can be any combination of the pharmaceutical compounds described herein with other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients. The pharmaceutical compositions facilitate administration of the compound to an organism.

[0286] The pharmaceutical preparation for administration can include an aqueous solution of the active compound in a water-soluble form. The suspension of the active compound can be prepared into an oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. The suspension can also contain suitable stabilizers or reagents that increase the solubility of the compound to allow the preparation of highly concentrated solutions. The active ingredient can be in powder form, for reconstitution with a suitable vehicle such as sterile pyrogen-free water before use.

[0287] In practicing the methods of treatment or use provided herein, a therapeutically effective amount of a compound described herein is administered in the form of a pharmaceutical composition to a subject suffering from the disease or condition to be treated. In some embodiments, the subject is a mammal, such as a human. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors.

[0288] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers comprising excipients and adjuvants that aid in processing the active compound into pharmaceutically acceptable preparations. The formulation can be modified depending on the chosen route of administration. Pharmaceutical compositions comprising the compounds described herein can be prepared, for example, by mixing, dissolving, emulsifying, encapsulating, encapsulating, or compressing processes.

[0289] The pharmaceutical composition may comprise at least one pharmaceutically acceptable carrier, diluent or excipient and a compound described herein in free base or pharmaceutically acceptable salt form. The pharmaceutical composition may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

[0290] Methods for preparing compositions comprising the compounds described herein include formulating the compounds with one or more inert, pharmaceutically acceptable excipients or carriers to form solid, semisolid, or liquid compositions. Solid compositions include, for example, powders, tablets, dispersible granules, capsules, and cachets. Liquid compositions include, for example, solutions in which the compound is dissolved, emulsions containing the compound, or solutions containing liposomes, micelles, or nanoparticles containing the compounds disclosed herein. Semisolid compositions include, for example, gels, suspensions, and creams. The compositions may be liquid solutions or suspensions, solid forms suitable for dissolution or suspension in a liquid prior to use, or as emulsions. These compositions may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, and other pharmaceutically acceptable additives.

[0291] Non-limiting examples of dosage forms suitable for use with the present disclosure include liquids, powders, gels, nanosuspensions, nanoparticles, microgels, aqueous or oily suspensions, emulsions, and any combination thereof.

[0292] Non-limiting examples of pharmaceutically acceptable excipients suitable for use in the present disclosure include binders, disintegrants, anti-adherents, antistatic agents, surfactants, antioxidants, coating agents, colorants, plasticizers, preservatives, suspending agents, emulsifiers, antimicrobial agents, spheronizers, and any combination thereof.

[0293] Compositions of the present disclosure can be, for example, immediate release forms or controlled release formulations. Immediate release formulations can be formulated to allow the compound to act quickly. Non-limiting examples of immediate release formulations include readily soluble formulations. Controlled release formulations can be pharmaceutical formulations that have been adjusted to allow the release rate and release profile of the active agent to match physiological requirements and time treatment requirements, or have been formulated to enable the active agent to release at a programmed rate. Non-limiting examples of controlled release formulations include particles, delayed release particles, (e.g., synthetic or natural origin) hydrogels, other gelling agents (e.g., dietary fibers forming a gel), matrix-based formulations (e.g., formulations comprising polymeric materials having at least one active ingredient dispersed therein), particles within the matrix, polymeric mixtures, and granular masses.

[0294] In some embodiments, the controlled release formulation is a delayed release form. The delayed release form can be formulated to delay the effect of the compound for a period of time that is extended. The delayed release form can be formulated to delay the release of one or more compounds of an effective dose, for example, for about 4 hours, about 8 hours, about 12 hours, about 16 hours, or about 24 hours.

[0295] Controlled release formulations can be sustained release forms. Sustained release forms can be formulated to maintain the effect of, for example, a compound over a period of time that is prolonged. Sustained release forms can be formulated to provide any compound described herein (e.g., providing physiologically effective blood distribution) in an effective dose within about 4 hours, about 8 hours, about 12 hours, about 16 hours, or about 24 hours.

[0296] The disclosed compositions may optionally contain a pharmaceutically acceptable preservative.

[0297] Non-limiting examples of pharmaceutically acceptable excipients can be found in, for example, Remington: The Science and Practice of Pharmacy, 19th ed. (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed. (Lippincott Williams & Wilkins 1999), each of which is herein incorporated by reference in its entirety.

[0298] The compounds described herein can be conveniently formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers. See, for example, Remington's Pharmaceutical Sciences, latest edition, EW Martin Mack Pub. Co., Easton, PA, incorporated by reference in its entirety, for typical carriers and conventional methods for preparing pharmaceutical compositions. Such carriers can be vehicles for administering the compositions to humans and non-humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Pharmaceutical compositions can also contain one or more additional active ingredients, such as antimicrobial agents, anti-inflammatory agents, and anesthetics.

[0299] The limiting examples of pharmaceutically acceptable carriers include saline, Ringer's solution, and dextrose solution. In some embodiments, the pH of the solution can be from about 5 to about 8, and can be from about 7 to about 7.5. Additional carriers include sustained release products, such as semipermeable matrices of solid hydrophobic polymers comprising the compound. The matrix can be a shaped article, for example, in the form of a film, liposome, microparticle, or microcapsule.

[0300] Compositions suitable for topical administration can be used. In some embodiments, the compositions of the present disclosure may include a liquid containing an active agent in the form of a solution, a suspension, or both. The liquid composition may include a gel. The liquid composition may be, for example, aqueous. The composition is an aqueous composition that can be gelled in situ. In an alternative, the composition is an aqueous solution that can be gelled in situ. Such a composition may contain a gelling agent whose concentration effectively promotes gelation when in contact with the eye or with tear fluid outside the eye. The aqueous composition may have an ophthalmically compatible pH and osmotic pressure. The composition may include an ophthalmic depot formulation containing an active agent for subconjunctival administration. Microparticles containing the active agent may be embedded in a biocompatible, pharmaceutically acceptable polymer or lipid encapsulating agent. The depot formulation may be suitable for releasing all or substantially all of the active agent over an extended period of time. The polymer or lipid matrix (if present) may be suitable for sufficient degradation after releasing all or substantially all of the active agent for delivery from the site of administration. The depot formulation may be a liquid formulation containing a pharmaceutically acceptable polymer and a dissolved or dispersed active agent. Upon injection, the polymer forms a reservoir at the injection site, for example by gelling or precipitation. The composition may comprise a solid product that can be inserted into a suitable location in the eye, such as between the eye and the eyelid or in the conjunctival sac, where the product releases the active agent. Solid products suitable for implantation in the eye in this manner may comprise a polymer and may be bioerodible or non-bioerodible.

[0301] In addition to the agents disclosed herein, pharmaceutical formulations may contain additional carriers, as well as thickeners, diluents, buffers, preservatives, and surfactants.

[0302] The pH of the disclosed compositions may be in the range of about 3 to about 12. The pH of the composition may be, for example, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 10 to about 11, or about 11 to about 12 pH units. The pH of the composition may be, for example, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 pH units. The pH of the composition may be, for example, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 pH units. The pH of the composition may be, for example, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, or at most 12 pH units. The pH of the pharmaceutical formulations disclosed herein may be about 5.5 to about 6.5. For example, the pH of the formulations of the present disclosure can be about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5. In some embodiments, the pH is 6.2 ± 0.3, 6.2 ± 0.2, 6.2 ± 0.1, about 6.2, or 6.2.

[0303] If the pH is outside the range desired by the formulator, sufficient pharmaceutically acceptable acids and bases can be used to adjust the pH.

[0304] Depending on the intended mode of administration, the pharmaceutical composition may be in the form of a solid, semisolid or liquid dosage form, such as a tablet, suppository, pill, capsule, powder, liquid, suspension, lotion, cream or gel, e.g., in a unit dosage form suitable for single administration of a precise dose.

[0305] For solid compositions, non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, sucrose, and magnesium carbonate.

[0306] Non-limiting examples of pharmaceutically active agents suitable for combination with the compositions of the present disclosure include anti-infectives (i.e., aminoglycosides), antivirals, antimicrobials, anticholinergics / anticospasmodics, antidiabetics, antihypertensives, antineoplastic agents, cardiovascular agents, central nervous system agents, coagulation regulators, hormones, immunosuppressants, and ophthalmic products.

[0307] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a compound in admixture with a pharmaceutically acceptable carrier and / or excipient, such as saline, phosphate buffered saline, phosphate and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary agents include octylphenoxypolyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's solution and Hank's solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinyl pyrrolidone, polyethylene, and glycols.

[0308] In some embodiments, the pharmaceutical formulations disclosed herein may comprise: (i) a compound or antibody disclosed herein; (ii) a buffered solvate; (iii) a non-ionic detergent; (iv) a tonicity agent; and (v) a stabilizer. In some embodiments, the pharmaceutical formulations disclosed herein are stable liquid pharmaceutical formulations.

[0309] In some embodiments, the ophthalmic formulations disclosed herein may comprise: (i) a compound or antibody disclosed herein; (ii) a buffered solvate; (iii) a nonionic detergent; (iv) a tonicity agent; and (v) a stabilizer. In some embodiments, the ophthalmic formulations disclosed herein are stable liquid pharmaceutical formulations or stable liquid ophthalmic formulations.

[0310] In some embodiments, the pharmaceutical or ophthalmic formulations disclosed herein are liquid formulations that may contain about 5 mg / mL to about 150 mg / mL of an antibody or compound, about 7.5 mg / mL to about 140 mg / mL of an antibody or compound, about 10 mg / mL to about 130 mg / mL of an antibody or compound, about 10 mg / mL to about 100 mg / mL of an antibody or compound, about 20 mg / mL to about 80 mg / mL of an antibody or compound, or about 30 mg / mL to about 70 mg / mL of an antibody or compound. For example, the formulations of the present disclosure may contain about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg. / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 120 mg / mL, about 140 mg / mL or about 150 mg / mL of a compound, antibody or antigen-binding fragment thereof described herein.

[0311] In some embodiments, the pharmaceutical preparations or ophthalmic preparations disclosed herein may include a buffer. In some embodiments, the buffer is used to maintain a stable pH and help stabilize the compounds or antibodies disclosed herein. In some embodiments, the buffer or buffer system includes at least one buffer whose buffer range completely or partially overlaps with the range of pH 5.5-7.4. In some embodiments, the pKa of the buffer is about 6.2 ± 0.5. In some embodiments, the buffer includes a sodium phosphate buffer. In some embodiments, sodium phosphate is present at a concentration of about 5mM to about 15mM, about 6mM to about 14mM, about 7mM to about 13mM, about 8mM to about 12mM, about 9mM to about 11mM, or about 10mM. In certain embodiments, the buffer system includes 10mM sodium phosphate at a pH of 6.2 ± 0.3 or 6.1 ± 0.3.

[0312] In some embodiments, the pharmaceutical or ophthalmic formulations disclosed herein may include a nonionic detergent. In some embodiments, the nonionic detergent is a nonionic polymer containing a polyoxyethylene moiety. In some embodiments, the nonionic detergent is any one or more of polysorbate 20, poloxamer 188, or polyethylene glycol 3350. In some embodiments, the nonionic detergent is polysorbate 20. In some embodiments, the nonionic detergent is polysorbate 80. In some embodiments, the pharmaceutical or ophthalmic formulations disclosed herein may include from about 0.01% to about 1% of a nonionic detergent. For example, the formulations of the present disclosure may comprise about 0.0085%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.20%, about 0.21%, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.30%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.36%, about 0.37%, about 0.38%, about 0.39%, about 0.40%, about 0.41%, about 0.42%, about 0.43%, about 0.44%, about 0.45%, about 0.46%, about 0.47%, about 0.48%, about 0.49%, about 0.50%, about 0.51%, about 0.52%, about 0.53%, about 0.54%, about 0.55%, about 0.56%, about 0.57%, about 0.58%, about 0.59%, about 0.60%, about 0.61%, about 0.62%, about 0.63%, about 0.64%, about 0.65%, about 0.66%, about 0.67%, about 0.68%, about 0.69%, about 0.70%, about About 25%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.15%, about 1.2%, about 1.25%, about 1.3%, about 1.35%, about 1.4%, about 1.45%, about 1.5%, about 1.55%, about 1.6%, about 1.65%, about 1.7%, about 1.75%, about 1.8%, about 1.85%, about 1.9%, about 1.95% or about 2% of polysorbate 20, polysorbate 80 or poloxamer 188.

[0313] In some embodiments, the pharmaceutical or ophthalmic formulations disclosed herein may include a tonicity agent. In some embodiments, the tonicity agent is sodium chloride or potassium chloride. In some embodiments, the tonicity agent is sodium chloride. In some embodiments, sodium chloride is present at a concentration of about 5 mM to about 100 mM, about 10 mM to about 50 mM, or about 40 mM.

[0314] In some embodiments, the pharmaceutical preparations or ophthalmic preparations disclosed herein may include a stabilizer. In some embodiments, the stabilizer is a thermal stabilizer that can stabilize the antibodies or compounds disclosed herein under heat stress conditions. In some embodiments, when a solution comprising a compound or antibody and a thermal stabilizer is kept at about 45°C for up to about 28 days, the stabilizer maintains a compound or antibody of greater than about 93% of its native conformation. In some embodiments, when a solution comprising a compound or antibody and a thermal stabilizer is kept at about 45°C for up to about 28 days, the stabilizer prevents aggregation of the compound or antibody and less than 4% of the compound or antibody aggregates. In some embodiments, when a solution comprising a compound or antibody and a thermal stabilizer is kept at about 37°C for up to about 28 days, the stabilizer maintains more than about 96% of the compound or antibody in its native conformation. In some embodiments, when a solution comprising a compound or antibody and a thermal stabilizer is kept at about 37°C for up to about 28°C, the stabilizer prevents aggregation of the compound or antibody and less than about 2% of the compound or antibody aggregates.

[0315] In some embodiments, the heat stabilizer is a sugar or sugar alcohol, such as sucrose, sorbitol, glycerol, trehalose or mannitol, or any combination thereof. In some embodiments, the stabilizer is a sugar. In some embodiments, the sugar is sucrose, mannitol or trehalose. In some embodiments, the stabilizer is sucrose. In some embodiments, pharmaceutical preparations disclosed herein or ophthalmic preparations may include about 1% to about 20% sugar or sugar alcohol, about 2% to about 18% sugar or sugar alcohol, about 3% to about 15% sugar or sugar alcohol, about 4% to about 10% sugar or sugar alcohol or about 5% sugar or sugar alcohol. For example, pharmaceutical preparations of the present disclosure or ophthalmic preparations may include about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13% or about 14% sugar or sugar alcohol (such as sucrose, trehalose or mannitol). In some embodiments, the stabilizer is present at a concentration of about 1% w / v to about 20% w / v. In some embodiments, the stabilizer is sucrose at a concentration of about 1% w / v to about 15% w / v or about 1% w / v to about 10% w / v. In some embodiments, the stabilizer is sucrose at a concentration of 5% w / v or about 5% w / v. In some embodiments, the stabilizer is sucrose at a concentration of 7.5% w / v or about 7.5% w / v. In some embodiments, the stabilizer is sucrose at a concentration of 10% w / v or about 10% w / v. In some embodiments, the stabilizer is sucrose at a concentration of 12.5% ​​w / v or about 12.5% ​​w / v. In some embodiments, the stabilizer is sucrose at a concentration of 15% w / v or about 15% w / v. In some embodiments, the stabilizer is sucrose at a concentration of 20% w / v or about 20% w / v.

[0316] Administration of pharmaceutical compositions

[0317] The pharmaceutical compositions disclosed herein can be administered in a therapeutically effective amount by various forms and routes, including, for example, oral, topical, parenteral, intravenous injection, intravenous infusion, subcutaneous injection, subcutaneous infusion, intramuscular injection, intramuscular infusion, intradermal injection, intradermal infusion, intraperitoneal injection, intraperitoneal infusion, intracerebral injection, intracerebral infusion, subarachnoid injection, subarachnoid infusion, intraocular injection, intraspinal injection, intrasternal injection, ophthalmic administration, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, intraarterial administration, intrathecal administration, inhalation, intralesional administration, intradermal administration, epidural administration, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa), intracapsular administration, subcapsular administration, intracardiac administration, transtracheal administration, subcutaneous administration, subarachnoid administration, subcapsular administration, intraspinal administration, or intrasternal administration.

[0318] The pharmaceutical composition can be used in a local manner, for example, by direct injection of the compound into an organ, optionally in the form of a depot preparation or a sustained release formulation or an implant. The pharmaceutical composition can be provided in the form of a quick-release formulation, in the form of an extended-release formulation or in the form of an intermediate-release formulation. The quick-release form can provide immediate release. Extended-release formulations can provide controlled release or sustained delayed release.

[0319] In some embodiments, a pump can be used to deliver a pharmaceutical composition. In some embodiments, a pen delivery device can be used, for example, to deliver a composition of the present disclosure subcutaneously. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device can use a replaceable cartridge containing a pharmaceutical composition disclosed herein. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. A disposable pen does not have a replaceable cartridge. Instead, a disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0320] Pharmaceutical compositions disclosed herein can be for example administered to eyes by any suitable form or approach, including for example topical, oral, systemic, intravitreal, intracameral, intracranial, subconjunctival, subbulbar, posterior, intraocular, intrastromal, intracorneal, near sclera, periocular, subretinal or suprachoroidal administration. Delivery method can include an invasive method for delivering compositions directly to eye cells. In some embodiments, the liquid pharmaceutical composition comprising antibody or compound can be by subretinal injection, intravitreal injection (for example, before, during or after vitreous injection), intravitreal implant, intraorbital injection, intraorbital administration, subcutaneous injection, intracameral injection, intracranial injection, subconjunctival injection, subconjunctival implant, through temporal side to intracameral injection, intrastromal injection, intracorneal injection, aqueous humor injection, subbulbar injection or subbulbar implant. Compositions can be administered by injecting preparation into any part of eyes including anterior chamber, posterior chamber, vitreous chamber (vitreal), intrinsic retina and / or subretinal space.

[0321] The pharmaceutical compositions disclosed herein can be delivered by non-invasive methods. Examples of non-invasive modes of administration of the formulation may include the use of a needle-free injection device, as well as topical administration, such as eye drops to the cornea. A variety of routes of administration can be used to effectively deliver the pharmaceutical composition. In some embodiments, the composition is delivered by a variety of routes of administration, such as subretinal and intravitreal, to increase the efficiency of antibody delivery. In some embodiments, vitrectomy is performed prior to subretinal and / or intravitreal injection.

[0322] In some embodiments, a liquid formulation comprising 10 mg / mL to 120 mg / mL of an antibody or compound is in a pre-filled syringe and is administered intravitreally in a volume of up to about 500 μL. In some embodiments, a liquid formulation comprising 10 mg / mL to 120 mg / mL of an antibody or compound is in a pre-filled syringe and is administered intravitreally in a volume of up to about 100 μL. In some embodiments, a liquid formulation comprising 10 mg / mL to 120 mg / mL of an antibody or compound is in a pre-filled syringe and is administered intravitreally in a volume of about 50 μL.

[0323] The pharmaceutical compositions disclosed herein can be targeted to any suitable ocular cell, including, for example, endothelial cells such as vascular endothelial cells, retinal cells such as retinal pigment epithelium (RPE), corneal cells, fibroblasts, astrocytes, glial cells, pericytes, iris epithelial cells, neurogenic cells, ciliary epithelial cells, Müller cells, muscle cells surrounding and attached to the eye, such as cells of the lateral rectus muscle, orbital fat cells, cells of the sclera and episclera, cells of the trabecular meshwork, or connective tissue cells.

[0324] Drug administration

[0325] The compounds, antibodies, or therapeutic agents described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administering a composition containing the compound, antibody, or therapeutic agent can be varied. For example, the composition can be used as a prophylactic and can be continuously administered to a subject susceptible to a condition or disease to reduce the likelihood of the disease or condition occurring. The composition can be administered to a subject already suffering from the disease or condition in an amount sufficient to cure or at least partially prevent the symptoms of the disease or condition or to cure, treat, improve, or alleviate the condition. The composition can be administered to the subject as soon as possible during or after the onset of symptoms. Administration of the compound, antibody, or therapeutic agent can be started within 48 hours before the onset of symptoms, within 24 hours before the onset of symptoms, within 6 hours before the onset of symptoms, or within 3 hours of the onset of symptoms. Any formulation described herein can be used, via any practical route, such as initial administration by any route described herein. The compound, antibody, or therapeutic agent can be administered as soon as feasible after the onset of a disease or condition is detected or suspected, and continues to treat the disease for the necessary length of time, for example, from about 1 month to about 3 months. The length of treatment can vary from subject to subject. Amounts effective for this use may vary based on the severity and course of the disease or condition, previous therapy, the subject's health status, weight, and response to the drugs, and the judgment of the attending physician. Improvement of clinical symptoms can be monitored by, for example, indirect ophthalmoscopy, fundus photography, fluorescein angiopathy, electroretinography, external eye examination, slit-lamp biomicroscopy, applanation tonometry, pachymetry, optical coherence tomography, or autorefaction.

[0326] The pharmaceutical compositions described herein can be in the form of unit dosage forms suitable for single administration of precise doses. In unit dosage forms, the formulations can be divided into unit doses containing an appropriate amount of one or more compounds, antibodies, or therapeutic agents. The unit doses can be in the form of packages containing discrete amounts of the formulation. Non-limiting examples are packaged injections, vials, and ampoules. The aqueous suspension compositions disclosed herein can be packaged in non-reclosable single-dose containers. Reclosable multi-dose containers can be used, for example, in combination with a preservative or without a preservative. The injectable formulations disclosed herein can be present in unit dosage form, for example, in ampoules or multi-dose containers with a preservative.

[0327] The various compounds, antibodies, or therapeutic agents disclosed herein can be administered in any order or simultaneously. If administered simultaneously, the various compounds, antibodies, or therapeutic agents can be provided in a single, unified form or in multiple forms, such as as multiple independent injections or infusions. The compounds, antibodies, or therapeutic agents can be packaged together in a single package or individually in multiple packages. One or all of the compounds, antibodies, or therapeutic agents can be administered in multiple doses. If not administered simultaneously, the timing between multiple administrations can vary by up to about a month.

[0328] Intraocular injections can be performed at any time intervals to improve delivery efficiency and / or minimize or avoid damage to surrounding tissues. The time interval between two or more intraocular injections can be, for example, from about 1 minute to about 60 minutes, from about 1 minute to about 5 minutes, from about 5 minutes to about 10 minutes, from about 10 minutes to about 15 minutes, from about 15 minutes to about 20 minutes, from about 20 minutes to about 25 minutes, from about 25 minutes to about 30 minutes, from about 30 minutes to about 35 minutes, from about 35 minutes to about 40 minutes, from about 40 minutes to about 45 minutes, from about 45 minutes to about 50 minutes, from about 50 minutes to about 55 minutes, or from about 55 minutes to about 60 minutes.

[0329] Intraocular injection can be performed at any rate. The rate of intraocular injection can be, for example, from about 1 μL / sec to about 500 μL / sec, from about 1 μL / sec to about 10 μL / sec, from about 10 μL / sec to about 20 μL / sec, from about 20 μL / sec to about 30 μL / sec, from about 30 μL / sec to about 40 μL / sec, from about 40 μL / sec to about 50 μL / sec, from about 50 μL / sec to about 60 μL / sec, from about 60 μL / sec to about 70 μL / sec, from about 70 μL / sec to about 80 μL / sec, from about 80 μL / sec to about 90 μL / sec, from about 90 μL / sec to about 100 μL / sec, from about 100 μL / sec to about 110 μL / sec, from about 110 μL / sec to about 120 μL / sec, or from about 130 μL / sec to about 140 μL / sec. L / sec to about 120 μL / sec, about 120 μL / sec to about 130 μL / sec, about 130 μL / sec to about 140 μL / sec, about 140 μL / sec to about 150 μL / sec, about 150 μL / sec to about 160 μL / sec, about 160 μL / sec to about 170 μL / sec, about 170 μL / sec to about 180 μL / sec, about 180 μL / sec to about 190 μL / sec, about 190 μL / sec to about 200 μL / sec, about 200 μL / sec to about 300 μL / sec, about 300 μL / sec to about 400 μL / sec, or about 400 μL / sec to about 500 μL / sec.

[0330] The compounds, antibodies, or therapeutic agents disclosed herein can be administered at a dosage of 0.0001 mg / kg to about 1000 mg / kg, about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 20 mg / kg, about 0.02 mg / kg to about 7 mg / kg, about 0.03 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 3 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 0.5 mg / kg, or about 0.01 mg / kg to about 20 mg / kg.

[0014] In some embodiments, the present invention provides a dosage of about 1 mg / kg to about 2 mg / kg, about 0.2 mg / kg to about 0.6 mg / kg, about 0.3 mg / kg to about 0.7 mg / kg, about 0.4 mg / kg to about 0.8 mg / kg, about 0.1 mg / kg to about 0.9 mg / kg, about 0.01 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 1 mg / kg to about 5 mg / kg, or about 3 mg / kg to about 7 mg / kg of subject mass.

[0331] The compounds, antibodies or therapeutic agents described herein can be administered at any desired interval. The administration of the compound, antibody or therapeutic agent can have a regular or irregular dosing schedule, suitable for the person administering the compound, antibody or therapeutic agent or the subject receiving the compound, antibody or therapeutic agent. For example, the compound, antibody or therapeutic agent can be administered twice a day, once a day, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every five weeks, once every six weeks, once every eight weeks, once every two months, once every twelve weeks, once every three months, once every four months, once every six months, once a year or with less frequency. In some embodiments, administration is once every other week.

[0332] The amount administered can be the same amount in each dose, or the dose can vary between doses. For example, a first amount can be administered in the morning and a second amount can be administered in the evening.

[0333] The compounds, antibodies, or therapeutic agents described herein can be administered in any amount necessary or convenient. For example, the compounds described herein can be administered by any route of administration in an amount of about 0.05 mg to about 300 mg, about 0.1 mg to about 300 mg, about 0.1 mg to about 200 mg, about 0.1 mg to about 100 mg, about 0.05 mg to about 1.5 mg, about 0.1 mg to about 1.5 mg, about 0.05 mg to about 1 mg, about 1 mg to about 1.5 mg, about 0.5 mg to about 6 mg, about 1 mg to about 4 mg, about 2 mg to about 10 mg, about 10 mg to about 30 mg, about 30 mg to about 50 mg, about 50 mg to about 70 mg, about 70 mg to about 100 mg, or about 0.1 mg to about 1 mg, about 0.05 ...0.05 mg, about 0.05 mg, about 0.05 mg, about 0.05 mg, about 0.05 mg, about 0.05 mg, about 0.05 mg, about 0.05 mg, about 0.05 mg, about 0.05 mg, about 0.05 mg, about 0.05 mg, about 0.05 mg, about 0.05 mg 6mg, about 0.07mg, about 0.08mg, about 0.09mg, about 0.1mg, about 0.11mg, about 0.12mg, about 0.13mg, about 0.14mg, about 0.15mg, about 0.16mg, about 0.17mg, about 0.18mg, about 0.19mg, about 0.2mg, about 0.21mg, about 0.22mg, about 0.23mg, about 0.24mg, about 0.25mg, about 0.26mg, about 0.27mg, about 0.28mg, about 0.29mg, about 0.3mg, about 0.31mg, about 0.32mg, about 0.33mg, about 0.34mg, about 0.35mg, about 0.36mg , about 0.37,mg, about 0.38mg, about 0.39mg, about 0.4mg, about 0.41mg, about 0.42mg, about 0.43mg, about 0.44mg, about 0.45mg, about 0.46mg, about 0.47,mg, about 0.48mg, about 0.49mg, about 0.5mg, about 0.51mg, about 0.52mg, about 0.53mg, about 0.54mg, about 0.55mg, about 0.56mg, about 0.57,mg, about 0.58mg, about 0.59mg, about 0.6mg, about 0.61mg, about 0.62mg, about 0.63mg, about 0.64mg, about 0.65mg, about 0.66mg, about 0.67,mg, about 0.68mg, about 0.69mg, about 0.7mg, about 0.71mg, about 0.72mg, about 0.73mg, about 0.74mg, about 0.75mg, about 0.76mg, about 0.77,mg, about 0.78mg, about 0.79mg, about 0.8mg, about 0.81mg, about 0.82mg, about 0.83mg, about 0.84mg, about 0.85mg, about 0.86mg, about 0.87,mg, about 0.88mg, about 0.89mg, about 0.9mg, about 0.91mg, about 0.92mg, about 0.93mg, about 0.94mg, about 0.95mg, about 0.96mg, about 0.97, mg, about 0.98 mg, about 0.99 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, An amount of about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, or about 300 mg is administered to a subject.

[0334] Combination therapy

[0335] The pharmaceutical compositions provided herein can be administered with other therapies, such as chemotherapy, radiotherapy, surgery, anti-inflammatory agents or vitamins. Other agents can be administered before, after or concomitantly with the pharmaceutical composition.

[0336] In some embodiments, the compounds or antibodies described herein may be used alone or in combination with one or more therapeutic agents as components of a mixture.

[0337] In some embodiments, the present disclosure provides for the co-administration of multispecific compounds or antibodies that target, for example, HPTP-β (VE-PTP) and VEGF, with one or more additional anti-VEGF agents, which can stabilize the vasculature to prevent neovascularization. In some embodiments, the co-administration of multispecific compounds or antibodies with one or more additional anti-VEGF agents can stabilize the vasculature to prevent leakage. The anti-VEGF agent can be a compound, a recombinant protein, an antibody, an antigen-binding fragment, a variant or derivative thereof (e.g., scFv), a protein comprising one or more designed ankyrin repeats, a designed ankyrin repeat protein (DARPin), an ankyrin, ankyrin repeat protein, an affibody, an avimer, an adnectin, anticalin, a fynomer, a Kunitz domain, a knottin, a β-hairpin mimetic, or a peptide derived from one or more receptors, such as a VEGF receptor, or a VEGF-binding portion of human VEGF receptors 1 and 2.

[0338] Non-limiting examples of anti-VEGF agents include bevacizumab Ranibizumab Aflibercept Conbercept, brolucizumab, RTH258, VEGF receptor tyrosine kinase inhibitors such as sorafenib, sunitinib, axitinib, pazopanib, vandetinib, cabozantinib, regorafenib and 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), VEGF variants, soluble VEGF receptor fragments or traps, aptamers capable of blocking VEGF or VEGFR (e.g. 培加尼布 ), neutralizing anti-VEGFR antibodies or fragments thereof (e.g., ramucirumab, p1C11, 1121, 1121B), anti-KDR antibodies, anti-flt1 antibodies, low molecular weight inhibitors of VEGFR tyrosine kinase, DARPins that bind to VEGF (e.g., abicipar, MP0112, MP0250), proteins comprising one or more designed ankyrin repeats that bind to VEGF, adnectins (e.g., CT-322), anticalins (e.g., PRS-050), collagen IV-derived biomimetic peptides (e.g., AXT-107).

[0339] Other non-limiting examples of VEGF modulators include anti-inflammatory agents such as dexamethasone, fluocinolone, and triamcinolone. The multispecific compound or antibody that targets, for example, HPTP-β (VE-PTP) and VEGF can be administered in combination with any additional anti-VEGF agent in any combination, for example, at the start of treatment, at any time during treatment, or at any time after treatment with the additional anti-VEGF agent has ended. In addition, the dose of the multispecific compound or antibody can be adjusted during treatment. In addition, the dose of the additional anti-VEGF agent can be adjusted during treatment. The multispecific compound or antibody can be administered, for example, once a month, once every three months, once every six months, or once a year, with the additional anti-VEGF agent being administered at any frequency between treatments. Also disclosed herein are methods of treating a disease or condition disclosed herein. The method comprises administering to a subject:

[0340] a) a therapeutically effective amount of a multispecific compound or antibody targeting, for example, HPTP-β (VE-PTP) and VEGF; and

[0341] b) a therapeutically effective amount of an additional anti-VEGF agent;

[0342] Administration of the multispecific compound or antibody and the additional anti-VEGF agent can be performed as described herein.

[0343] In some embodiments, the present disclosure provides for the co-administration of multispecific compounds or antibodies targeting, for example, HPTP-β (VE-PTP) and VEGF with one or more additional anti-HPTP-β (VE-PTP) agents, which can stabilize the vasculature to prevent neovascularization. In some embodiments, the co-administration of multispecific compounds or antibodies with one or more additional anti-HPTP-β (VE-PTP) agents can stabilize the vasculature to prevent leakage. The anti-HPTP-β (VE-PTP) agent can be a compound, a recombinant protein, an antibody, an antigen-binding fragment, a variant or derivative thereof (e.g., scFv), a protein comprising one or more designed ankyrin repeats, a designed ankyrin repeat protein (DARPin), an ankyrin, ankyrin repeat protein, an affibody, an avimer, an adnectin, anticalin, a Fynomer, a Kunitz domain, a knottin, a β-hairpin mimetic, or a peptide derived from one or more receptors. In some embodiments, the additional anti-HPTP-β (VE-PTP) agent can activate Tie2 signaling by promoting protein phosphorylation, such as phosphorylation of the Tie2 protein. In some embodiments, additional anti-HPTP-β (VE-PTP) agents may be combined with HPTP-β (VE-PTP),

[0344] A multispecific compound or antibody targeting, for example, HPTP-β (VE-PTP) and VEGF can be co-administered with any additional anti-HPTP-β (VE-PTP) agent in any combination, e.g., at the start of treatment, at any time during treatment, or at any time after treatment with the additional anti-HPTP-β (VE-PTP) agent has ended. The dosage of the multispecific compound or antibody can be adjusted during treatment. The dosage of the additional anti-HPTP-β (VE-PTP) agent can be adjusted during treatment. For example, the multispecific compound or antibody can be administered monthly, once every three months, once every six months, or once a year, with the additional anti-HPTP-β (VE-PTP) agent administered at any frequency between treatments. Also disclosed herein are methods of treating a disease or condition disclosed herein. The method comprises administering to a subject:

[0345] a) a therapeutically effective amount of a multispecific compound or antibody targeting, for example, HPTP-β (VE-PTP) and VEGF; and

[0346] b) a therapeutically effective amount of an additional anti-HPTP-β (VE-PTP) agent;

[0347] Administration of the multispecific compound or antibody and the additional anti-HPTP-β (VE-PTP) agent can be performed as described herein.

[0348] In some embodiments, the present disclosure provides for the co-administration of a multispecific compound or antibody targeting, for example, HPTP-β (VE-PTP) and VEGF with one or more additional Tie2 receptor activating compounds. The additional Tie2 receptor activating compound can be, for example, angiopoietin 1 recombinant protein, Ang1 mimetic, Tie2 agonist, peptide, HPTP-β (VE-PTP) phosphatase inhibitor, Tie2-peptide, tetrameric polyethylene oxide clustered peptide, collagen IV biomimetic peptide, compound, recombinant protein, antibody, antigen binding fragment, variant or derivative thereof (e.g., scFv), affibody, avimer, adnectin, protein comprising one or more designed ankyrin repeats, designed ankyrin repeat protein (DARPin), ankyrin, ankyrin repeat protein, affibody, avimer, adnectin, anticalin, Fynomer, Kunitz domain, knottin, β-hairpin mimetic, or peptide derived from one or more receptors. In some embodiments, the one or more additional Tie2 receptor activating compounds are small molecules. In some embodiments, the one or more additional Tie2 receptor activating compounds improve drainage through the ocular lymphatic vessels, Schlemm's canal, or limbal lymphatic vessels. In some embodiments, the one or more additional Tie2 receptor activating compounds are administered in the form of eye drops. In some embodiments, the one or more additional Tie2 receptor activating compounds are administered to treat primary open-angle glaucoma, age-related macular degeneration, cardiovascular disease, or cystic kidney disease. In some embodiments, the one or more additional Tie2 receptor activating compounds can be, for example, MAN-01, AXT-107, or vasculotide. In some embodiments, the compounds disclosed herein can be co-administered with, for example, MAN-01, AXT-107, or vasculotide.

[0349] The multispecific compound or antibody targeting HPTP-β (VE-PTP) and VEGF can be co-administered with any additional Tie2 receptor activating compound in any combination, for example, at the start of treatment, at any time during treatment, or at any time after treatment with the additional Tie2 receptor activating compound has ended. The dosage of the multispecific compound or antibody can be adjusted during treatment. The dosage of the additional Tie2 receptor activating compound can be adjusted during treatment. For example, the multispecific compound or antibody can be administered monthly, once every three months, once every six months, or once a year, wherein the additional Tie2 receptor activating compound is administered at any frequency between treatments with the multispecific compound or antibody. Also disclosed herein are methods of treating a disease or condition disclosed herein. The method comprises administering to a subject:

[0350] a) a therapeutically effective amount of a multispecific compound or antibody targeting, for example, HPTP-β (VE-PTP) and VEGF; and

[0351] b) a therapeutically effective amount of a Tie2 activator;

[0352] Administration of the multispecific compound or antibody and the additional Tie2 receptor activating compound can be performed as described herein.

[0353] Mouse model

[0354] Oxygen-induced ischemic retinopathy model

[0355] The oxygen-induced ischemic retinopathy model can be considered to mimic aspects of proliferative retinal neovascularization and proliferative diabetic retinopathy. One-week-old mice can be placed in a sealed chamber and exposed to hyperoxia (75±3% oxygen) for five days, resulting in hyperoxia-induced neovascularization between postnatal days 17 and 21, for example, at the junction between vascularized and avascular retina. The compound of interest, such as an antibody or compound of the present disclosure, can be administered to the mice to determine the effect on neovascularization and / or vascular leakage. Neovascularization and / or vascular leakage can be assessed as follows,

[0356] Rho / VEGF mouse model

[0357] The Rho / VEGF mouse model can simulate various aspects of neovascular age-related macular degeneration. Transgenic mice with vascular endothelial growth factor (VEGF) expression driven by the rhodopsin promoter (rho / VEGF mice) can develop retinal neovascularization, retinal angiomatous proliferation, and retinal vascular leakage. In rho / VEGF mice, VEGF expression in photoreceptors can begin between the 5th and 10th days after birth, during which time the deep capillary bed develops. Neovascularization can originate from the deep capillary bed of the retina and grow into the subretinal space. The mice can be administered a compound of interest, such as an antibody or compound of the present disclosure, to determine the effect on neovascularization and / or vascular leakage. Neovascularization and / or vascular leakage can be assessed as described below.

[0358] Tet / opsin / VEGF mouse model

[0359] Mice with VEGF under the control of a reverse tetracycline transactivator (rtTA) inducible promoter coupled to the rhodopsin promoter (Tet / opsin / VEGF mice) can be used as an inducible model for neovascularization, retinal vascular leakage, and retinal detachment. In these mice, VEGF transgenic expression in the retina can be induced by administering doxycycline. Three to four days after VEGF induction, neovascularization may become apparent. Neovascularization may be more extensive and may lead to outer retinal wrinkling, and then to complete retinal detachment within about five days. The mice can be administered with a compound of interest, such as an antibody or compound of the present disclosure, to determine the effect on neovascularization, vascular leakage, and / or retinal detachment. Neovascularization and / or vascular leakage can be assessed as described below. To assess retinal detachment, the eyes can be frozen in cutting temperature embedding solution, ten-micrometer sections cut through the entire eye, and the sections stained with Hoechst. The sections can be examined by light microscopy, the average length of retinal detachment for each section measured by image analysis, and the percentage of retinal detachment calculated.

[0360] Tet / opsin / Ang2 mouse model

[0361] Tet / opsin / Ang2 mice have inducible Ang2 expression in the retina. These mice can be used to study the effects of Ang2 expression under various experimental conditions. For example, Tet / opsin / Ang2 mice can be used to determine the effect of Ang2 expression on neovascularization when VEGF levels are high versus low, or to determine the effect of Ang2 expression in an oxygen-induced ischemic retinopathy model. Compounds of interest, such as antibodies or compounds of the present disclosure, can be administered to mice to determine the effect of Ang2 expression on therapeutic efficacy.

[0362] Laser-induced choroidal neovascularization model

[0363] It is believed that the laser-induced choroidal neovascularization model can simulate various aspects of neovascular age-related macular degeneration. Anesthetized mice can be dilated and burns can be applied to the retina using a krypton laser, for example, using a slit lamp system and a cover glass as a contact lens. Multiple burns can be produced in one eye, for example, three burns in each eye. The burns can cause Bruch's membrane to rupture. Choroidal neovascularization can be assessed at multiple time points after laser treatment, for example, one, two, or four weeks after laser treatment. The mice can be administered with a compound of interest, such as an antibody or compound of the present disclosure, to determine the effect on neovascularization. The eye cup can be stained with FITC-labeled GSA, the choroid can be flat-mounted, and the area of ​​choroidal neovascularization at each Bruch's membrane rupture site can be measured by fluorescence microscopy and image analysis. Neovascularization and / or vascular leakage can also be assessed as described below.

[0364] Assessment of neovascularization

[0365] A variety of techniques can be used to assess neovascularization.

[0366] Fluorescein angiography can be performed by taking serial fundus photographs after injection of a dye to visualize the blood vessels, allowing the presence, location, and size of neovascular complexes to be determined. For example, 0.3 mL of 1% sodium fluorescein can be injected intraperitoneally, serial fundus photographs can be taken, and the area of ​​choroidal neovascularization, total lesion area, and leakage area can be measured.

[0367] The eyes can be processed for evaluation by fluorescence, light, or electron microscopy. For example, mice can be perfused with fluorescently labeled dextran, or the eyes can be injected with GSA or antibodies targeting PECAM. The eyes can be processed for observation under a fluorescence microscope, and the extent of neovascularization can be quantified, for example, by quantifying the area of ​​neovascularization per retina, by quantifying the area of ​​neovascularization per site of Bruch's membrane rupture, or by quantifying the number of nuclei of new vessels extending from the retina to the vitreous.

[0368] The area of ​​retinal neovascularization can be determined, for example, using FITC-labeled GSA lectin and fluorescence microscopy. The eyes can be fixed in 10% formalin, completely dissected, washed with PBS, blocked in 8% porcine serum, and stained with FITC-labeled GSA lectin for a period of time (e.g., 40-50 minutes) suitable for staining retinal neovascularization and vitreous blood vessels, but not staining normal retina. The retina can be tiled and sealed, and digital images can be obtained using a fluorescence microscope and merged into a single image of the entire retina. Software can be used to measure the area of ​​neovascularization of each retina.

[0369] The area of ​​subretinal neovascularization can be determined, for example, using FITC-labeled GSA lectin and fluorescence microscopy. Eyes can be fixed in 10% formalin, the retinas can be dissected, blocked with 5% porcine serum, stained with FITC-conjugated GSA for 2 hours to stain vascular cells, and flat-mounted with the photoreceptor side facing up. The area of ​​subretinal neovascularization can be measured by fluorescence microscopy and image analysis.

[0370] Assessment of retinal vascular leakage

[0371] Retinal vascular leakage can be assessed by measuring extravasated serum albumin using immunofluorescence techniques. For example, eyes can be fixed in 10% formalin, and the retinas can be dissected, washed, blocked, and stained using an anti-albumin antibody and a fluorescently conjugated secondary antibody. Blood vessels can be labeled by counterstaining with GSA lectin, and the retinas can be flat-mounted. The retinas can be examined by fluorescence microscopy, and the area of ​​albumin staining determined by image analysis. Retinal vascular leakage is associated with, for example, diabetic macular edema and macular edema due to retinal vein occlusion.

[0372] Miles assay for vascular leakage

[0373] The Miles assay can be used to assess vascular leakage in the subcutaneous vessels of the dermis. Evans blue is a dye that binds to albumin. Under physiological conditions, the endothelium is impermeable to albumin, so the albumin bound by Evans blue is confined to the blood vessels. Under pathological conditions that promote increased vascular permeability, the endothelial cells partially lose their tight contacts. The endothelium becomes permeable to small proteins such as albumin. Mice can be injected intravenously with 1% Evans blue dye in PBS and injected intradermally with VEGF. Thirty minutes after the intradermal injection, the tissue at the intradermal injection site can be excised and extracted with formamide to assess the extravasation of Evans blue dye. Vascular leakage can be quantified by measuring the dye incorporated per milligram of tissue, for example, using optical density measurements and a standard curve. The compound of interest, such as the antibody or compound of the present disclosure, can be administered to mice to determine the effect on vascular leakage.

[0374] Cancer models

[0375] The compounds or antibodies of the present disclosure can be tested for their efficacy as cancer treatments in a variety of cancer models. In some cancer models, cancer cells from a cell line can be implanted into a recipient animal, such as a mouse. Non-limiting examples of suitable mouse strains include C57BL6, BALB / C, C3H, FVB / N, and FVB / N-Tg(MMTV-PyVT)634Mul. Non-limiting examples of suitable cancer cell lines include 4T1, E0771, and P0008. For example, cells from the 4T1 or E0771 cell lines can be implanted into the mammary pad as a model of breast cancer. 4T1 or E0771 cells can be implanted, for example, into the third mammary fat pad of a female nude mouse.

[0376] The size of solid tumors can be measured with calipers and tumor volume calculated. Tumors can be processed for histopathological evaluation or fluorescence microscopy to assess, for example, tumor area, tumor grade, metastasis count, metastatic area, number of nuclei per tumor focus, intratumoral vessel diameter, intratumoral vessel density, tumor vessel maturity, perivascular cell coverage, proximity between perivascular cells and endothelial cells, or to grade tumor foci as intravascular or extravascular.

[0377] The spontaneous metastasis model can be used to study the effects of compounds or antibodies of the present disclosure on metastasis. After tumor implantation, the primary tumor can be excised (e.g., when it reaches 5 mm in size) and the animal then evaluated for metastasis by naked eye or histopathology, e.g., to determine the number and size of metastases in the lungs, liver, lymph nodes, and bones.

[0378] To evaluate the effect of vascular stability on metastasis, a model can be used in which tumor cells can be injected intravenously and the animals can then be evaluated for intravascular versus extravasation metastasis. For example, 4T-1 cells can be injected intravenously and the lungs processed for histopathological evaluation to quantify intravascular versus extravascular metastasis.

[0379] Metastases can be counted and measured visually, for example, by immersing the lungs in Bouin's solution and examining them with a stereomicroscope.

[0380] To measure tumor vascular permeability in vivo, in vivo multiphoton microscopy can be used. For example, animals can be injected with fluorescently labeled bovine serum albumin at pre- and post-treatment time points. At each time point, two different regions within the tumor can be selected, and a multiphoton laser scanning microscope can be used to capture 200-micron image stacks of BSA fluorescence every ten minutes over an hour through each region. This analysis method can involve three-dimensional vessel tracking to create vascular metrics and a three-dimensional plot of voxel intensity versus distance to the nearest vessel over time. Using three-dimensional image registration, images can be corrected for sample movement over time. Normalized transvascular flux can then be calculated.

[0381] Illustrative CDR Combinations

[0382] In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise LCDR1. In some embodiments, the antibodies or compounds of the present disclosure comprise LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise LCDR3.

[0383] In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1 and HCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1 and HCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1 and LCDR1. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1 and LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1 and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2 and HCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2 and LCDR1. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2 and LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2 and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR3 and LCDR1. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR3 and LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR3 and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise LCDR1 and LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise LCDR1 and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise LCDR2 and LCDR3.

[0384] In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR2, and HCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR2, and LCDR1. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR2, and LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR2, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR3, and LCDR1. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR3, and LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR3, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, LCDR1, and LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, LCDR1, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, LCDR2, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2, HCDR3, and LCDR1. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2, HCDR3, and LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2, HCDR3, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2, LCDR1, and LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2, LCDR1, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2, LCDR2, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR3, LCDR1, and LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR3, LCDR1, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR3, LCDR2, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise LCDR1, LCDR2, and LCDR3.

[0385] In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR2, HCDR3, and LCDR1. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR2, HCDR3, and LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR2, HCDR3, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR2, LCDR1, and LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR2, LCDR1, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR2, LCDR2, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR3, LCDR1, and LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR3, LCDR1, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR3, LCDR2, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, LCDR1, LCDR2, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2, HCDR3, LCDR1, and LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2, HCDR3, LCDR1, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2, HCDR3, LCDR2, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2, LCDR1, LCDR2, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR3, LCDR1, LCDR2, and LCDR3.

[0386] In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR2, HCDR3, LCDR1, and LCDR2. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR2, HCDR3, LCDR1, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR2, HCDR3, LCDR2, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR2, LCDR1, LCDR2, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3. In some embodiments, the antibodies or compounds of the present disclosure comprise HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0387] In some embodiments, an antibody or compound of the present disclosure comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0388] In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 88-90. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 91-93. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 97-98.

[0389] In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80 and any one of SEQ ID NOs: 81-87. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80 and any one of SEQ ID NOs: 88-90. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80 and any one of SEQ ID NOs: 91-93. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80 and any one of SEQ ID NOs: 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80 and any one of SEQ ID NOs: 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87 and any one of SEQ ID NOs: 88-90. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87 and any one of SEQ ID NOs: 91-93. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87 and any one of SEQ ID NOs: 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87 and any one of SEQ ID NOs: 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 88-90 and any one of SEQ ID NOs: 91-93. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 88-90 and any one of SEQ ID NOs: 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 88-90 and any one of SEQ ID NOs: 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 91-93 and any one of SEQ ID NOs: 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 91-93 and any one of SEQ ID NOs: 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 94-96 and any one of SEQ ID NOs: 97-98.

[0390] In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 81-87, and any one of SEQ ID NOs: 88-90. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 81-87, and any one of SEQ ID NOs: 91-93. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 81-87, and any one of SEQ ID NOs: 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 81-87, and any one of SEQ ID NOs: 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 88-90, and any one of SEQ ID NOs: 91-93. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 88-90, and any one of SEQ ID NOs: 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 88-90, and any one of SEQ ID NOs: 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 91-93, and any one of SEQ ID NOs: 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 91-93, and any one of SEQ ID NOs: 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 94-96, and any one of SEQ ID NOs: 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87, any one of SEQ ID NOs: 88-90, and any one of SEQ ID NOs: 91-93.In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87, 88-90, and 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87, 88-90, and 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87, 91-93, and 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87, 91-93, and 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87, 94-96, and 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 88-90, 91-93, and 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 88-90, 91-93, and 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 88-90, 91-93, and 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 88-90, 94-96, and 97-98. In some embodiments, an antibody or compound of the present disclosure comprises any one of SEQ ID NOs: 91-93, any one of SEQ ID NOs: 94-96, and any one of SEQ ID NOs: 97-98.

[0391] In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 81-87, any one of SEQ ID NOs: 88-90, and any one of SEQ ID NOs: 91-93. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 81-87, any one of SEQ ID NOs: 88-90, and any one of SEQ ID NOs: 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 81-87, any one of SEQ ID NOs: 88-90, and any one of SEQ ID NOs: 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 81-87, any one of SEQ ID NOs: 91-93, and any one of SEQ ID NOs: 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 81-87, any one of SEQ ID NOs: 91-93, and any one of SEQ ID NOs: 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 81-87, any one of SEQ ID NOs: 94-96, and any one of SEQ ID NOs: 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 88-90, any one of SEQ ID NOs: 91-93, and any one of SEQ ID NOs: 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 88-90, any one of SEQ ID NOs: 91-93, and any one of SEQ ID NOs: 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 88-90, any one of SEQ ID NOs: 94-96, and any one of SEQ ID NOs: 97-98.In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 91-93, any one of SEQ ID NOs: 94-96, and any one of SEQ ID NOs: 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87, any one of SEQ ID NOs: 88-90, any one of SEQ ID NOs: 91-93, and any one of SEQ ID NOs: 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87, any one of SEQ ID NOs: 88-90, any one of SEQ ID NOs: 91-93, and any one of SEQ ID NOs: 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87, 88-90, 94-96, and 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87, 91-93, 94-96, and 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 88-90, 91-93, 94-96, and 97-98.

[0392] In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, 81-87, 88-90, 91-93, and 94-96. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, 81-87, 88-90, 91-93, and 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, 81-87, 88-90, 91-93, and 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, 81-87, 91-93, 94-96, and 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 76-80, 88-90, 91-93, 94-96, and 97-98. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 81-87, 88-90, 91-93, 94-96, and 97-98.

[0393] In some embodiments, an antibody or compound of the present disclosure comprises any one of SEQ ID NOs: 76-80, any one of SEQ ID NOs: 81-87, any one of SEQ ID NOs: 88-90, any one of SEQ ID NOs: 91-93, any one of SEQ ID NOs: 94-96, and any one of SEQ ID NOs: 97-98.

[0394] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 81. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 88. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 82.

[0395] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76 and SEQ ID NO: 81. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76 and SEQ ID NO: 88. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76 and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76 and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76 and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 81 and SEQ ID NO: 88. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 81 and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 81 and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 81 and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 88 and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 88 and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 88 and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 91 and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 91 and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 94 and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77 and SEQ ID NO: 82. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77 and SEQ ID NO: 88. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77 and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77 and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77 and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 82 and SEQ ID NO: 88.In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 82 and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 82 and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 82 and SEQ ID NO: 97.

[0396] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, and SEQ ID NO: 88. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 88, and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 88, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 88, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 91, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 81, SEQ ID NO: 88, and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 81, SEQ ID NO: 88, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 81, SEQ ID NO: 88, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 81, SEQ ID NO: 91, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 81, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 81, SEQ ID NO: 94, and SEQ ID NO: 97.In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 88, SEQ ID NO: 91, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 88, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 88, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 91, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 82, and SEQ ID NO: 88. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 82, and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 82, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 82, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 88, and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 88, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 88, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 91, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO: 82, SEQ ID NO: 88, and SEQ ID NO: 91. In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO: 82, SEQ ID NO: 88, and SEQ ID NO: 94.In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 82, SEQ ID NO: 88, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 82, SEQ ID NO: 91, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 82, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 82, SEQ ID NO: 94, and SEQ ID NO: 97.

[0397] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 88, and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 88, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 88, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 91, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 88, SEQ ID NO: 91, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 88, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 88, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 91, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 81, SEQ ID NO: 88, SEQ ID NO: 91, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 81, SEQ ID NO: 88, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO:81, SEQ ID NO:88, SEQ ID NO:94, and SEQ ID NO:97.In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 81, SEQ ID NO: 91, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 88, and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 88, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 88, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 91, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 88, SEQ ID NO: 91, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 88, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 88, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 91, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO:82, SEQ ID NO:88, SEQ ID NO:91, and SEQ ID NO:94.In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 82, SEQ ID NO: 88, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 82, SEQ ID NO: 88, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 94, and SEQ ID NO: 97.

[0398] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 88, SEQ ID NO: 91, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 88, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 88, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 91, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 91, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 81, SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 88, SEQ ID NO: 91, and SEQ ID NO: 94. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 88, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 88, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO:82, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:94, and SEQ ID NO:97.

[0399] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 94, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 94, and SEQ ID NO: 97.

[0400] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 83. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 92. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 97.

[0401] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78 and SEQ ID NO: 83. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78 and SEQ ID NO: 89. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78 and SEQ ID NO: 92. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78 and SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78 and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 83 and SEQ ID NO: 89. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 83 and SEQ ID NO: 92. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 83 and SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 83 and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89 and SEQ ID NO: 92. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89 and SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89 and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 92 and SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 92 and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 95 and SEQ ID NO: 97.

[0402] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 83, and SEQ ID NO: 89. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 83, and SEQ ID NO: 92. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 83, and SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 83, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 89, and SEQ ID NO: 92. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 89, and SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 89, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 92, and SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 92, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 95, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 83, SEQ ID NO: 89, and SEQ ID NO: 92. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 83, SEQ ID NO: 89, and SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 83, SEQ ID NO: 89, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 83, SEQ ID NO: 92, and SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 83, SEQ ID NO: 92, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 83, SEQ ID NO: 95, and SEQ ID NO: 97.In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89, SEQ ID NO: 92, and SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89, SEQ ID NO: 92, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89, SEQ ID NO: 95, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 92, SEQ ID NO: 95, and SEQ ID NO: 97.

[0403] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 83, SEQ ID NO: 89, and SEQ ID NO: 92. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 83, SEQ ID NO: 89, and SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 83, SEQ ID NO: 89, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 83, SEQ ID NO: 92, and SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 83, SEQ ID NO: 92, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 83, SEQ ID NO: 95, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 89, SEQ ID NO: 92, and SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 89, SEQ ID NO: 92, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 89, SEQ ID NO: 95, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 92, SEQ ID NO: 95, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 83, SEQ ID NO: 89, SEQ ID NO: 92, and SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 83, SEQ ID NO: 89, SEQ ID NO: 92, and SEQ ID NO: 97. In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO:83, SEQ ID NO:89, SEQ ID NO:95, and SEQ ID NO:97.In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 83, SEQ ID NO: 92, SEQ ID NO: 95, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89, SEQ ID NO: 92, SEQ ID NO: 95, and SEQ ID NO: 97.

[0404] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 83, SEQ ID NO: 89, SEQ ID NO: 92, and SEQ ID NO: 95. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 83, SEQ ID NO: 89, SEQ ID NO: 92, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 83, SEQ ID NO: 89, SEQ ID NO: 95, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 83, SEQ ID NO: 92, SEQ ID NO: 95, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 78, SEQ ID NO: 83, SEQ ID NO: 92, SEQ ID NO: 95, and SEQ ID NO: 97. In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO:83, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:97.

[0405] In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO:78, SEQ ID NO:83, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:97.

[0406] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 84. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 90. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 93. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 86.

[0407] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79 and SEQ ID NO: 84. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79 and SEQ ID NO: 90. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79 and SEQ ID NO: 93. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79 and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79 and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 84 and SEQ ID NO: 90. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 84 and SEQ ID NO: 93. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 84 and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 84 and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 90 and SEQ ID NO: 93. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 90 and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 90 and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 93 and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 93 and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 96 and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79 and SEQ ID NO: 86. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 86 and SEQ ID NO: 90. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 86 and SEQ ID NO: 93. In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO: 86 and SEQ ID NO: 96. In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO: 86 and SEQ ID NO: 98.

[0408] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, and SEQ ID NO: 90. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, and SEQ ID NO: 93. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 90, and SEQ ID NO: 93. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 90, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 90, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 93, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 93, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 84, SEQ ID NO: 90, and SEQ ID NO: 93. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 84, SEQ ID NO: 90, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 84, SEQ ID NO: 90, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 84, SEQ ID NO: 93, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 84, SEQ ID NO: 93, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 84, SEQ ID NO: 96, and SEQ ID NO: 98.In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 90, SEQ ID NO: 93, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 90, SEQ ID NO: 93, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 90, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 93, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 86, and SEQ ID NO: 90. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 86, and SEQ ID NO: 93. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 86, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 86, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 86, SEQ ID NO: 90, and SEQ ID NO: 93. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 86, SEQ ID NO: 90, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 86, SEQ ID NO: 90, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 86, SEQ ID NO: 93, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 86, SEQ ID NO: 93, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 86, SEQ ID NO: 96, and SEQ ID NO: 98.

[0409] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 90, and SEQ ID NO: 93. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 90, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 90, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 93, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 93, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 90, SEQ ID NO: 93, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 90, SEQ ID NO: 93, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 90, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 90, SEQ ID NO: 93, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 84, SEQ ID NO: 90, SEQ ID NO: 93, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 84, SEQ ID NO: 90, SEQ ID NO: 93, and SEQ ID NO: 98. In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO:84, SEQ ID NO:90, SEQ ID NO:96, and SEQ ID NO:98.In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 84, SEQ ID NO: 93, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 90, SEQ ID NO: 93, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 86, SEQ ID NO: 90, and SEQ ID NO: 93. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 86, SEQ ID NO: 90, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 86, SEQ ID NO: 90, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 86, SEQ ID NO: 93, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 86, SEQ ID NO: 93, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 86, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 93, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 93, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 86, SEQ ID NO: 93, SEQ ID NO: 96, and SEQ ID NO: 98.

[0410] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 90, SEQ ID NO: 93, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 90, SEQ ID NO: 93, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 90, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 93, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 93, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 84, SEQ ID NO: 90, SEQ ID NO: 93, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 93, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 93, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 86, SEQ ID NO: 93, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO:86, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:96, and SEQ ID NO:98.

[0411] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 90, SEQ ID NO: 93, SEQ ID NO: 96, and SEQ ID NO: 98. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 79, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 93, SEQ ID NO: 96, and SEQ ID NO: 98.

[0412] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 85. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 87.

[0413] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80 and SEQ ID NO: 85. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80 and SEQ ID NO: 89. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80 and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80 and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80 and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 85 and SEQ ID NO: 89. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 85 and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 85 and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 85 and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89 and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89 and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89 and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 91 and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 91 and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 96 and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80 and SEQ ID NO: 87. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 87 and SEQ ID NO: 89. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 87 and SEQ ID NO: 91. In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO: 87 and SEQ ID NO: 96. In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO: 87 and SEQ ID NO: 97.

[0414] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, and SEQ ID NO: 89. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 89, and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 89, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 89, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 91, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 85, SEQ ID NO: 89, and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 85, SEQ ID NO: 89, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 85, SEQ ID NO: 89, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 85, SEQ ID NO: 91, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 85, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 85, SEQ ID NO: 96, and SEQ ID NO: 97.In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 91, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, and SEQ ID NO: 89. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 87, SEQ ID NO: 89, and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 87, SEQ ID NO: 89, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 87, SEQ ID NO: 89, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 87, SEQ ID NO: 91, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 87, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 87, SEQ ID NO: 96, and SEQ ID NO: 97.

[0415] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 89, and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 89, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 89, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 91, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 89, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 91, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO:85, SEQ ID NO:89, SEQ ID NO:96, and SEQ ID NO:97.In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 85, SEQ ID NO: 91, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, SEQ ID NO: 89, and SEQ ID NO: 91. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, SEQ ID NO: 89, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, SEQ ID NO: 89, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, SEQ ID NO: 91, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 96, and SEQ ID NO: 97.

[0416] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 91, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 96. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, an antibody or compound of the present disclosure comprises SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:96, and SEQ ID NO:97.

[0417] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 96, and SEQ ID NO: 97. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 96, and SEQ ID NO: 97.

[0418] In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 124-131. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 132-137. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 138-143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 144-146.

[0419] In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113 and any one of SEQ ID NOs: 114-123. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113 and any one of SEQ ID NOs: 124-131. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113 and any one of SEQ ID NOs: 132-137. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113 and any one of SEQ ID NOs: 138-143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113 and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123 and any one of SEQ ID NOs: 124-131. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123 and any one of SEQ ID NOs: 132-137. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123 and any one of SEQ ID NOs: 138-143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123 and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 124-131 and any one of SEQ ID NOs: 132-137. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 124-131 and any one of SEQ ID NOs: 138-143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 124-131 and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 132-137 and any one of SEQ ID NOs: 138-143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 132-137 and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 138-143 and any one of SEQ ID NOs: 144-146.

[0420] In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 114-123, and any one of SEQ ID NOs: 124-131. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 114-123, and any one of SEQ ID NOs: 132-137. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 114-123, and any one of SEQ ID NOs: 138-143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 114-123, and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 124-131, and any one of SEQ ID NOs: 132-137. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 124-131, and any one of SEQ ID NOs: 138-143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 124-131, and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 132-137, and any one of SEQ ID NOs: 138-143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 132-137, and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 138-143, and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 124-131, and any one of SEQ ID NOs: 132-137.In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 124-131, and any one of SEQ ID NOs: 138-143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 124-131, and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 132-137, and any one of SEQ ID NOs: 138-143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 132-137, and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 138-143, and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 124-131, any one of SEQ ID NOs: 132-137, and any one of SEQ ID NOs: 138-143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 124-131, any one of SEQ ID NOs: 132-137, and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 124-131, any one of SEQ ID NOs: 138-143, and any one of SEQ ID NOs: 144-146. In some embodiments, an antibody or compound of the present disclosure comprises any one of SEQ ID NOs: 132-137, any one of SEQ ID NOs: 138-143, and any one of SEQ ID NOs: 144-146.

[0421] In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, 114-123, 124-131, and 132-137. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, 114-123, 124-131, and 138-143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, 114-123, 124-131, and 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 132-137, and any one of SEQ ID NOs: 138-143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 132-137, and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 138-143, and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 124-131, any one of SEQ ID NOs: 132-137, and any one of SEQ ID NOs: 138- 143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 124-131, any one of SEQ ID NOs: 132-137, and any one of SEQ ID NOs: 144-146.In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, 124-131, 138-143, and 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, 132-137, 138-143, and 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123, 124-131, 132-137, and 138-143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 124-131, any one of SEQ ID NOs: 132-137, and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 124-131, any one of SEQ ID NOs: 138-143, and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 132-137, any one of SEQ ID NOs: 138-143, and any one of SEQ ID NOs: 144-146. In some embodiments, an antibody or compound of the present disclosure comprises any one of SEQ ID NOs: 124-131, any one of SEQ ID NOs: 132-137, any one of SEQ ID NOs: 138-143, and any one of SEQ ID NOs: 144-146.

[0422] In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 124-131, any one of SEQ ID NOs: 132-137, and any one of SEQ ID NOs: 138- 143. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 124-131, any one of SEQ ID NOs: 132-137, and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 124-131, any one of SEQ ID NOs: 138-143, and any one of SEQ ID NOs: 144- 146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 132-137, any one of SEQ ID NOs: 138-143, and any one of SEQ ID NOs: 144-146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 124-131, any one of SEQ ID NOs: 132-137, any one of SEQ ID NOs: 138-143, and any one of SEQ ID NOs: 144- 146. In some embodiments, the antibodies or compounds of the present disclosure comprise any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 124-131, any one of SEQ ID NOs: 132-137, any one of SEQ ID NOs: 138-143, and any one of SEQ ID NOs: 144-146.

[0423] In some embodiments, an antibody or compound of the present disclosure comprises any one of SEQ ID NOs: 99-113, any one of SEQ ID NOs: 114-123, any one of SEQ ID NOs: 124-131, any one of SEQ ID NOs: 132-137, any one of SEQ ID NOs: 138-143, and any one of SEQ ID NOs: 144-146.

[0424] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 103, SEQ ID NO: 118, SEQ ID NO: 125, SEQ ID NO: 132, SEQ ID NO: 140, and SEQ ID NO: 144. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 108, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 135, SEQ ID NO: 143, and SEQ ID NO: 145. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 113, SEQ ID NO: 123, SEQ ID NO: 130, SEQ ID NO: 135, SEQ ID NO: 143, and SEQ ID NO: 145.

[0425] In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO 103, SEQ ID NO: 118, SEQ ID NO: 125, SEQ ID NO: 132, SEQ ID NO: 140, and SEQ ID NO: 144. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO 103, SEQ ID NO: 118, SEQ ID NO: 125, SEQ ID NO: 132, SEQ ID NO: 140, and SEQ ID NO: 144. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO 108, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 135, SEQ ID NO: 143, and SEQ ID NO: 145. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO: 80, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO 108, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 135, SEQ ID NO: 143, and SEQ ID NO: 145. In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO:80, SEQ ID NO:85, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:113, SEQ ID NO:123, SEQ ID NO:130, SEQ ID NO:135, SEQ ID NO:143, and SEQ ID NO:145.In some embodiments, the antibodies or compounds of the present disclosure comprise SEQ ID NO:80, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO 113, SEQ ID NO:123, SEQ ID NO:130, SEQ ID NO:135, SEQ ID NO:143, and SEQ ID NO:145.

[0426] Example

[0427] Example 1: Tetravalent bispecific antibody comprising antibody HC2:LC1 and aflibercept-derived sequence.

[0428] To generate a tetravalent bispecific antibody fused to the heavy chain of antibody HC2:LC1 to the aflibercept-derived VEGF-binding domain, an amino acid sequence was generated that contained the following additional amino acid sequence from the N-terminus to the C-terminus:

[0429] 1) SEQ ID NO: 14 (heavy chain of antibody HC2:LC1);

[0430] 2) SEQ ID NO: 31 (connector peptide, underlined); and

[0431] 3) SEQ ID NO: 22 (Aficept-derived sequence).

[0432] The resulting polypeptide (SEQ ID NO: 149) was co-expressed with SEQ ID NO: 17 to provide a tetravalent bispecific antibody HC2-AFL: LC1 comprising the sequences shown in Table 33. Amino acids 1-19 of SEQ ID NO: 149 are the heavy chain signal peptide (SEQ ID NO: 11). Amino acids 1-20 of SEQ ID NO: 17 are the light chain signal peptide (SEQ ID NO: 12). In some embodiments, the mature HC2-AFL: LC1 does not comprise a signal peptide. For example, a mature HC2-AFL: LC1 of the present disclosure may comprise SEQ ID NO: 254 and SEQ ID NO: 250.

[0433]

[0434]

[0435] Example 2: Tetravalent bispecific antibodies comprising antibody HC2:LC1 and brolucizumab-derived sequences.

[0436] To generate a tetravalent bispecific antibody with the heavy chain of antibody HC2:LC1 fused to a brolucizumab-derived VEGF-binding domain, an amino acid sequence was generated that contained the following additional amino acid sequence from N-terminus to C-terminus:

[0437] 1) SEQ ID NO: 14 (heavy chain of antibody HC2:LC1);

[0438] 2) SEQ ID NO: 31 (connector peptide, underlined); and

[0439] 3) SEQ ID NO: 23 (brolucizumab-derived sequence).

[0440] The resulting polypeptide (SEQ ID NO: 150) was co-expressed with SEQ ID NO: 17 to provide a tetravalent bispecific antibody HC2-BRO: LC1 comprising the sequences shown in Table 34. Amino acids 1-19 of SEQ ID NO: 150 are the heavy chain signal peptide (SEQ ID NO: 11). Amino acids 1-20 of SEQ ID NO: 17 are the light chain signal peptide (SEQ ID NO: 12). In some embodiments, the mature HC2-BRO: LC1 does not comprise a signal peptide. For example, a mature HC2-BRO: LC1 of the present disclosure may comprise SEQ ID NO: 255 and SEQ ID NO: 250.

[0441]

[0442] Example 3: Tetravalent bispecific antibodies comprising antibody HC2:LC1 and ranibizumab-derived sequences.

[0443] To generate a tetravalent bispecific antibody with the heavy chain of antibody HC2:LC1 fused to a ranibizumab-derived VEGF-binding domain, an amino acid sequence was generated that contained the following additional amino acid sequence from N-terminus to C-terminus:

[0444] 1) SEQ ID NO: 14 (heavy chain of antibody HC2:LC1);

[0445] 2) SEQ ID NO: 31 (connector peptide, underlined); and

[0446] 3) SEQ ID NO: 28 (ranibizumab-derived sequence).

[0447] The resulting polypeptide (SEQ ID NO: 151) was co-expressed with SEQ ID NO: 17 to provide a tetravalent bispecific antibody HC2-RAN: LC1 comprising the sequences shown in Table 35. Amino acids 1-19 of SEQ ID NO: 151 are the heavy chain signal peptide (SEQ ID NO: 11). Amino acids 1-20 of SEQ ID NO: 17 are the light chain signal peptide (SEQ ID NO: 12). In some embodiments, the mature HC2-RAN: LC1 does not comprise a signal peptide. For example, a mature HC2-RAN: LC1 of the present disclosure may comprise SEQ ID NO: 256 and SEQ ID NO: 250.

[0448]

[0449]

[0450] Example 4: Characterization of bispecific compounds.

[0451] The following multispecific antibodies of the present disclosure were generated: (i) HC2-RAN:LC1, (ii) HC2-AFL:LC1, (iii) HC2-BRO:LC1, (iv) HC2-ABI:LC1, (v) HC2:LC1-AFL, (vi) HC2:LC1-BRO, (vii) HC2:LC1-ABI, (viii) HC2-AFL:LC1-AFL, (ix) HC2-BRO:LC1-BRO, and (x) HC2-ABI:LC1-ABI. Enzyme-linked immunosorbent assays (ELISAs) were performed to determine the binding of these antibodies to VEGF and HPTP-β (VE-PTP). Binding to HPTP-β (VE-PTP) was confirmed for all constructs, and binding to VEGF was confirmed for all constructs except HC2-RAN:LC1.

[0452] As shown in Table 36, Figure 16 、 Figure 17 and Figure 18 As described, the tetravalent bispecific antibodies described in Examples 1-3 were generated and characterized.

[0453] Table 36 provides the results of small-scale production and characterization of bispecific candidates.

[0454]

[0455] **m / c=measured / calculated

[0456] Figure 16ELISA data demonstrating binding of the tetravalent bispecific antibody HC2-AFL:LC1 to HPTP-β (upper panel) and VEGF (lower panel) are provided. Binding was compared to controls (HC2:LC1 in the upper panel, VEGF-R2-Fc chimera in the lower panel).

[0457] Figure 17 ELISA data demonstrating binding of the tetravalent bispecific antibody HC2-BRO:LC1 to HPTP-β (upper panel) and VEGF (lower panel) are provided. Binding was compared to controls (HC2:LC1 in the upper panel, VEGF-R2-Fc chimera in the lower panel).

[0458] Figure 18 ELISA data demonstrating binding of the tetravalent bispecific antibody HC2-RAN:LC1 to HPTP-β (upper panel) and VEGF (lower panel) are provided. Binding was compared to controls (HC2:LC1 in the upper panel, VEGF-R2-Fc chimera in the lower panel).

[0459] Example 5: Tetravalent bispecific antibodies comprising antibody HC2:LC1 and abicipar-derived sequences.

[0460] To generate a tetravalent bispecific antibody fused to the heavy chain of antibody HC2:LC1 to the abicipar-derived VEGF-binding domain, an amino acid sequence was generated that contained the following additional amino acid sequence from N-terminus to C-terminus:

[0461] 1) residues 1-467 of SEQ ID NO: 14 (heavy chain of antibody HC2:LC1);

[0462] 2) SEQ ID NO: 31 (connector peptide, underlined); and

[0463] 3) SEQ ID NO: 244 (abicipar-derived sequence).

[0464] The resulting polypeptide (SEQ ID NO: 231) was co-expressed with SEQ ID NO: 17 to provide a tetravalent bispecific antibody HC2-ABI: LC1 comprising the sequences shown in Table 37. Amino acids 1-19 of SEQ ID NO: 231 are the heavy chain signal peptide (SEQ ID NO: 11). Amino acids 1-20 of SEQ ID NO: 17 are the light chain signal peptide (SEQ ID NO: 12). In some embodiments, the mature HC2-ABI: LC1 does not comprise a signal peptide. For example, a mature HC2-ABI: LC1 of the present disclosure may comprise SEQ ID NO: 257 and SEQ ID NO: 250.

[0465]

[0466]

[0467] Example 6: Hexavalent bispecific antibody comprising sequences derived from antibody HC2:LC1 and brolucizumab.

[0468] To generate a heavy chain with a brolucizumab-derived VEGF-binding domain added to the C-terminus, an amino acid sequence was generated that contained the following additional amino acid sequence from the N-terminus to the C-terminus:

[0469] 1) SEQ ID NO: 14 (heavy chain of antibody HC2:LC1);

[0470] 2) SEQ ID NO: 31 (connector peptide, underlined); and

[0471] 3) SEQ ID NO: 23 (brolucizumab-derived sequence).

[0472] To generate a light chain with a brolucizumab-derived VEGF-binding domain added to the C-terminus, an amino acid sequence was generated that contained the following additional amino acid sequence from the N-terminus to the C-terminus:

[0473] 1) SEQ ID NO: 17 (light chain of antibody HC2:LC1);

[0474] 2) SEQ ID NO: 31 (connector peptide, underlined); and

[0475] 3) SEQ ID NO: 23 (brolucizumab-derived sequence).

[0476] The resulting polypeptides SEQ ID NO: 150 and SEQ ID NO: 218 were co-expressed to provide the hexavalent bispecific antibody HC2-BRO: LC1-BRO shown i...

Claims

1. An antibody comprising: (a) a first domain that binds to HPTP-β, wherein the first domain comprises: (i) a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 76-80, an amino acid sequence of any one of SEQ ID NOs: 81-87, and an amino acid sequence of any one of SEQ ID NOs: 88-90; and (ii) a light chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 91-93, an amino acid sequence of any one of SEQ ID NOs: 94-96, and an amino acid sequence of any one of SEQ ID NOs: 97-98; and (b) a second domain that binds to VEGF, wherein the second domain comprises: (i) a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 99-113, an amino acid sequence of any one of SEQ ID NOs: 114-123, and an amino acid sequence of any one of SEQ ID NOs: 124-131; and (ii) a light chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 132-137, an amino acid sequence of any one of SEQ ID NOs: 138-143, and an amino acid sequence of any one of SEQ ID NOs: 144-146.

2. The antibody according to claim 1, wherein the antibody is a multispecific antibody.

3. The antibody of claim 1, wherein the antibody is a tetravalent antibody. The antibody according to claim 1 , wherein the antibody is a hexavalent antibody. The antibody according to claim 1 , wherein the antibody is a bispecific antibody. The antibody of claim 1 , wherein the antibody is a tetravalent bispecific antibody.

7. The antibody of claim 1, wherein the antibody is a hexavalent bispecific antibody.

8. The antibody according to any one of claims 1 to 7, wherein the first domain binds to the extracellular domain of HPTP-β.

9. The antibody according to any one of claims 1 to 7, wherein the first domain binds to the first FN3 repeat sequence of the extracellular domain of HPTP-β.

10. The antibody of any one of claims 1-7, wherein the second domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:28, or SEQ ID NO:

244.

11. The antibody of any one of claims 1-7, wherein the second domain comprises the amino acid sequence of SEQ ID NO:

22.

12. The antibody according to any one of claims 1 to 7, wherein: The second domain comprises the amino acid sequence of SEQ ID NO:

23.

13. The antibody according to any one of claims 1 to 7, wherein: The second domain comprises the amino acid sequence of SEQ ID NO:

244.

14. The antibody according to any one of claims 1 to 7, wherein: The second domain comprises the amino acid sequence of SEQ ID NO:

28.

15. The antibody of claim 1, wherein the antibody comprises: (a) a heavy chain amino acid sequence comprising the heavy chain variable region of the first domain, and its sequence is at least 80% identical to SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256 or SEQ ID NO: 257; and (b) a light chain amino acid sequence comprising the light chain variable region of the first domain, and its sequence is at least 80% identical to SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252 or SEQ ID NO:

253.

16. The antibody of claim 1, wherein the antibody comprises: (a) a heavy chain amino acid sequence comprising the heavy chain variable region of the first domain, and its sequence is SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256 or SEQ ID NO: 257; and (b) a light chain amino acid sequence comprising the light chain variable region of the first domain, and its sequence is SEQ ID NO:

250.

17. The antibody of claim 1, wherein the antibody comprises: (a) a heavy chain amino acid sequence comprising the heavy chain variable region of the first domain, and its sequence is SEQ ID NO: 254; and (b) a light chain amino acid sequence comprising the light chain variable region of the first domain, and its sequence is SEQ ID NO:

250.

18. The antibody of claim 1, wherein the antibody comprises: (a) a heavy chain amino acid sequence comprising the heavy chain variable region of the first domain, and whose sequence is at least 80% identical to any one of SEQ ID NO: 262 or SEQ ID NO: 246-249; and (b) a light chain amino acid sequence comprising the light chain variable region of the first domain, and its sequence is at least 80% identical to SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:261 or SEQ ID NO:

260.

19. The antibody of claim 1, wherein the antibody comprises: (a) a heavy chain amino acid sequence comprising the heavy chain variable region of the first domain, and its sequence is SEQ ID NO: 262 or SEQ ID NO: 247; and (b) a light chain amino acid sequence comprising the light chain variable region of the first domain, and its sequence is SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260 or SEQ ID NO:

261.

20. The antibody of claim 1, wherein the antibody comprises: (a) a heavy chain amino acid sequence comprising the heavy chain variable region of the first domain, and its sequence is SEQ ID NO: 247; and (b) a light chain amino acid sequence comprising the light chain variable region of the first domain, and its sequence is SEQ ID NO:

259.

21. The antibody of claim 1, wherein the antibody comprises: (a) a heavy chain amino acid sequence comprising the heavy chain variable region of the first domain, and its sequence is at least 80% identical to SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256 or SEQ ID NO: 257; and (b) a light chain amino acid sequence comprising the light chain variable region of the first domain, and its sequence is at least 80% identical to SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260 or SEQ ID NO:

261.

22. The antibody of claim 1, wherein the antibody comprises: (a) a heavy chain amino acid sequence comprising the heavy chain variable region of the first domain, and its sequence is SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256 or SEQ ID NO: 257; and (b) a light chain amino acid sequence comprising the light chain variable region of the first domain, and its sequence is SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260 or SEQ ID NO:

261.

23. The antibody of claim 1, wherein the antibody comprises: (a) a heavy chain amino acid sequence comprising the heavy chain variable region of the first domain, and its sequence is SEQ ID NO: 254; and (b) a light chain amino acid sequence comprising the light chain variable region of the first domain, and its sequence is SEQ ID NO:

259.

24. The antibody of claim 1, wherein the antibody is a tetravalent bispecific antibody, wherein: (a) the second domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:28 or SEQ ID NO:244; (b) the antibody comprises a heavy chain amino acid sequence comprising the heavy chain variable region of the first domain, and its sequence is at least 80% identical to SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248 or SEQ ID NO:249; (c) the antibody comprises a light chain amino acid sequence comprising a light chain variable region of the first domain, and its amino acid sequence is at least 80% identical to SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253; and (d) the antibody comprises a linker, wherein the linker connects the second domain to the heavy chain amino acid sequence, wherein the linker comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 31-75.

25. The antibody of claim 1, wherein the antibody is a tetravalent bispecific antibody, wherein: (a) the second domain comprises the amino acid sequence of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:28 or SEQ ID NO:244; (b) the antibody comprises a heavy chain amino acid sequence, the heavy chain amino acid sequence comprises the heavy chain variable region of the first domain, and its sequence is SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248 or SEQ ID NO: 249; (c) the antibody comprises a light chain amino acid sequence, the light chain amino acid sequence comprises the light chain variable region of the first domain, and its sequence is SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253; and (d) the antibody comprises a linker, wherein the linker connects the second domain to the light chain amino acid sequence, wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs: 31-75.

26. The antibody of claim 1, wherein the antibody is a tetravalent bispecific antibody, wherein: (a) the second domain comprises the amino acid sequence of SEQ ID NO: 22; (b) the antibody comprises a heavy chain amino acid sequence, the heavy chain amino acid sequence comprises the heavy chain variable region of the first domain, and the sequence thereof is SEQ ID NO: 247; (c) the antibody comprises a light chain amino acid sequence, the light chain amino acid sequence comprises the light chain variable region of the first domain, and the sequence thereof is SEQ ID NO: 250; and (d) the antibody comprises a linker, wherein the linker connects the second domain to the heavy chain amino acid sequence, wherein the linker comprises the amino acid sequence of SEQ ID NO:

31.

27. The antibody of claim 1, wherein the antibody is a hexavalent bispecific antibody, wherein: (a) the second domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:28 or SEQ ID NO:244; (b) the antibody comprises a heavy chain amino acid sequence comprising the heavy chain variable region of the first domain, and its sequence is at least 80% identical to SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248 or SEQ ID NO:249; (c) the antibody comprises a first linker, wherein the first linker connects the second domain to the heavy chain amino acid sequence, wherein the first linker comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 31-75; (d) the antibody comprises a light chain amino acid sequence comprising the light chain variable region of the first domain, and the sequence thereof is at least 80% identical to SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252 or SEQ ID NO:253; (e) the antibody comprises a third domain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:28 or SEQ ID NO:244; and (f) the antibody comprises a second linker, wherein the second linker connects the third domain to the light chain amino acid sequence, wherein the second linker comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 31-75.

28. The antibody of claim 1, wherein the antibody is a hexavalent bispecific antibody, wherein: (a) the second domain comprises the amino acid sequence of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:28 or SEQ ID NO:244; (b) the antibody comprises a heavy chain amino acid sequence, the heavy chain amino acid sequence comprises the heavy chain variable region of the first domain, and its sequence is SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248 or SEQ ID NO: 249; (c) the antibody comprises a first linker, wherein the first linker connects the second domain to the heavy chain amino acid sequence, wherein the first linker comprises an amino acid sequence of any one of SEQ ID NOs: 31-75; (d) the antibody comprises a light chain amino acid sequence, the light chain amino acid sequence comprises the light chain variable region of the first domain, and its sequence is SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253; (e) the antibody comprises a third domain, wherein the third domain comprises the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 28 or SEQ ID NO: 244; and (f) the antibody comprises a second linker, wherein the second linker connects the third domain to the light chain amino acid sequence, wherein the second linker comprises the amino acid sequence of any one of SEQ ID NOs: 31-75.

29. The antibody of claim 1, wherein the antibody is a hexavalent bispecific antibody, wherein: (a) the second domain comprises the amino acid sequence of SEQ ID NO: 22; (b) the antibody comprises a heavy chain amino acid sequence, the heavy chain amino acid sequence comprises the heavy chain variable region of the first domain, and the sequence thereof is SEQ ID NO: 247; (c) the antibody comprises a first linker, wherein the first linker connects the second domain to the heavy chain amino acid sequence, wherein the first linker comprises the amino acid sequence of SEQ ID NO: 31; (d) the antibody comprises a light chain amino acid sequence, the light chain amino acid sequence comprises the light chain variable region of the first domain, and its sequence is SEQ ID NO: 250; (e) the antibody comprises a third domain, wherein the third domain comprises the amino acid sequence of SEQ ID NO: 22; and (f) the antibody comprises a second linker, wherein the second linker connects the third domain to the light chain amino acid sequence, wherein the second linker comprises the amino acid sequence of SEQ ID NO:

31.

30. The antibody of any one of claims 1-7, wherein the heavy chain variable region of the first domain comprises the amino acid sequence of SEQ ID NO: 2, and the light chain variable region of the first domain comprises the amino acid sequence of SEQ ID NO:

5.

31. An antibody according to any one of claims 1-7, wherein the antibody comprises a light chain amino acid sequence, the light chain amino acid sequence comprises the light chain variable region of the first domain, and its sequence is SEQ ID NO: 250; and a heavy chain amino acid sequence, the heavy chain amino acid sequence comprises the heavy chain variable region of the first domain, and its sequence is SEQ ID NO: 247 or SEQ ID NO:

262.

32. The antibody of any one of claims 1-7, wherein the heavy chain variable region of the first domain comprises the amino acid sequence of SEQ ID NO: 76, the amino acid sequence of SEQ ID NO: 81, and the amino acid sequence of SEQ ID NO: 88, and the light chain variable region of the first domain comprises the amino acid sequence of SEQ ID NO: 91, the amino acid sequence of SEQ ID NO: 94, and the amino acid sequence of SEQ ID NO:

97.

33. The antibody according to any one of claims 1-7 and 15-29, wherein the binding affinity (K) of the antibody to HPTP-β is D ) is 30fM to 70nM.

34. The antibody according to any one of claims 1-7 and 15-29, wherein the binding affinity (K) of the antibody to VEGF is D ) is 30fM to 70nM.

35. The antibody according to any one of claims 1-7 and 15-29, wherein the binding affinity (K) of the antibody to HPTP-β is D ) is 30 fM to 70 nM, and the binding affinity (K D ) is 30fM to 70nM.

36. Use of an antibody according to any one of claims 1-35 in the preparation of a medicament for treating a condition in a subject in need thereof.

37. The use according to claim 36, wherein the condition is an eye condition.

38. The use according to claim 36, wherein the condition is diabetic retinopathy.

39. The use according to claim 36, wherein the condition is neovascularization.

40. The use according to claim 36, wherein the condition is vascular leakage.

41. The use according to claim 36, wherein the condition is increased intraocular pressure.

42. The use according to claim 36, wherein the condition is ocular edema.

43. The use according to claim 36, wherein the condition is diabetic macular edema.

44. The use according to claim 36, wherein the condition is ocular hypertension.

45. The use according to claim 36, wherein the condition is ocular inflammation.

46. ​​The use according to claim 36, wherein the condition is glaucoma.

47. The use of claim 36, wherein the medicament is formulated for administration to the eye of the subject.

48. The use according to claim 36, wherein the medicament is formulated for intravitreal administration.

49. The use according to claim 36, wherein the medicament is formulated for subcutaneous administration.

50. The use according to claim 36, wherein the medicament is formulated for topical administration.

51. The use according to claim 36, wherein the subject is a human.

52. The use according to claim 36, wherein the therapeutically effective amount is 0.25 mg to 200 mg.

53. The use according to claim 36, wherein the therapeutically effective amount is 1 mg / kg to 10 mg / kg.

54. The use according to claim 36, wherein the therapeutically effective amount is 1 mg to 50 mg.

55. The use according to claim 36, wherein the therapeutically effective amount is 50 mg to 200 mg.

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