Patent Publication Number: US-2023134885-A1

Title: HUMANIZED MONOCLONAL ANTIBODIES THAT TARGET VE-PTP (HPTP-Beta)

Description:
CROSS REFERENCE 
     This application is a continuation of U.S. application Ser. No. 16/851,420, filed on Apr. 17, 2020, which is a continuation of U.S. application Ser. No. 16/775,860, filed on Jan. 29, 2020, now U.S. Pat. No. 11,136,389, issued on Oct. 5, 2021, which is a continuation of U.S. application Ser. No. 16/180,850, filed on Nov. 5, 2018, now U.S. Pat. No. 10,604,569, issued on Mar. 31, 2020, which is a continuation of U.S. application Ser. No. 15/654,289, filed on Jul. 19, 2017, now U.S. Pat. No. 10,253,094, issued on Apr. 9, 2019, which claims the benefit of U.S. Provisional Application No. 62/364,381, filed Jul. 20, 2016, and U.S. Provisional Application No. 62/377,072, filed Aug. 19, 2016, each of which is incorporated herein by reference in its entirety. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 20, 2021, is named 61881724305 SL.txt and is 131,666 bytes in size. 
     BACKGROUND OF THE INVENTION 
     Monoclonal antibodies generated from non-human organisms can elicit an immune response when administered to humans. Segments of these non-human monoclonal antibodies can be replaced with humanized sequences through a humanization process to reduce the likelihood of drug immunogenicity while preserving target specificity. 
     INCORPORATION BY REFERENCE 
     Each patent, publication, and non-patent literature cited in the application is hereby incorporated by reference in its entirety as if each was incorporated by reference individually. 
     SUMMARY OF THE INVENTION 
     In some embodiments, the invention provides a compound comprising a sequence that is at least 80% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. 
     In some embodiments, the invention provides a compound comprising a sequence that is at least 80% identical to SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. 
     In some embodiments, the invention provides a compound comprising: a) a heavy chain that comprises a sequence that is at least 80% identical to SEQ ID NO: 30; and b) a light chain that comprises a sequence that is at least 80% identical to SEQ ID NO: 34. 
     In some embodiments, the invention provides a compound comprising: a) a heavy chain that comprises a sequence that is at least 80% identical to SEQ ID NO: 29; and b) a light chain that comprises a sequence that is at least 80% identical to SEQ ID NO: 35. 
     In some embodiments, the invention provides a compound comprising: a) a heavy chain that comprises a sequence that is at least 80% identical to SEQ ID NO: 30; and b) a light chain that comprises a sequence that is at least 80% identical to SEQ ID NO: 37. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG.  1    shows a sequence alignment of the murine V H  sequence (truncated V H0 ; SEQ ID NO: 47) with the four humanized variants SEQ ID NO: 9 (V H1 ), SEQ ID NO: 10 (V H2 ), SEQ ID NO: 11 (V H3 ), and SEQ ID NO: 12 (V H4 ). 
         FIG.  2    shows a sequence alignment of the murine V L , sequence (truncated V L0 ; SEQ ID NO: 48) with the four humanized V L  variants SEQ ID NO: 20 (V L1 ), SEQ ID NO: 21 (V L2 ), SEQ ID NO: 22 (V L3 ), and SEQ ID NO: 23 (V L4 ). 
         FIG.  3    summarizes the hybridoma technology used to generate monoclonal antibodies against the HPTP-β extracellular domain (human VE-PTP/HPTP-β ECD). The 6-HIS tag disclosed in  FIG.  3    is SEQ ID NO: 52. 
         FIG.  4    illustrates an immunoprecipitation western blot (left panel) and flow cytometry results (right panel) of R15E6 bound to endogenous human VE-PTP (HPTP-β) in human endothelial cells. 
         FIG.  5    illustrates the binding specificity of R15E6 to recombinant, 6-His tagged human VE-PTP (HPTP-β) (The “6-His” disclosed in  FIG.  5    is SEQ ID NO: 52), cynomolgus PTP-β, and human PTP-η extracellular domain proteins by western blot. 
         FIG.  6    illustrates a western blot (panel A) and quantification (panel B) of concentration-dependent phosphorylation of Tie2 and enhanced endothelial cell viability (panel C) by R15E6. 
         FIG.  7    illustrates effects of rat anti-mouse VE-PTP monoclonal antibody 109.1 on Tie2 (top left panel), mouse VE-PTP expression (bottom left panel), and Akt (right panel) in mouse endothelial cells as determined by western blot. 
         FIG.  8    illustrates Tie2 activation by polyclonal antibodies against mouse VE-PTP ECD after 1 hour of incubation (panel A) and 3 min of incubation (panel B) in mouse endothelial cells. 
         FIG.  9    illustrates in vitro inhibition of thrombin- and VEGF-induced endothelial cell permeability by mouse VE-PTP ECD polyclonal antibodies. 
         FIG.  10    illustrates in vivo inhibition of VEGF-induced cutaneous vascular permeability by intravenous administration of monoclonal antibody 109.1 (panel A) and mouse VE-PTP ECD polyclonal antibodies (PTP1-8) (panel B), and immunoprecipitation of Tie2 from lung lysates of control IgG (−) or anti-VE-PTP antibody-injected mice (+) (panel C). 
         FIG.  11    depicts effects on retinal neovascularization (panels A and B) and choroidal neovascularization (panel C) after intraocular administration of anti-mouse VE-PTP monoclonal antibody 109.1. 
         FIG.  12    shows quantification of the mean area of retinal neovascularization (panels A and B) and choroidal neovascularization (panel C) after intraocular administration of an anti-mouse VE-PTP monoclonal antibody 109.1. 
         FIG.  13    depicts a reducing (panel A) and a non-reducing (panel B) SDS-PAGE of anti-human VE-PTP (anti-HPTP-β) humanized variants of R15E6. 
         FIG.  14    illustrates binding of HC1 variants of R15E6 and HCOLC0 to HPTP-β. 
         FIG.  15    illustrates binding of HC2 variants of R15E6 and HCOLC0 to HPTP-β 
         FIG.  16    illustrates binding of HC3 variants of R15E6 and HCOLC0 to HPTP-β. 
         FIG.  17    illustrates binding of HC4 variants of R15E6 and HCOLC0 to HPTP-β. 
         FIG.  18    illustrates Tie2 phosphorylation (left panel) and Akt activation (right panel) in the absence of Ang1 by the various humanized antibodies as determined by western blot. 
         FIG.  19    illustrates concentration-dependent Tie2 activation (top panel) and Akt activation (bottom panel) in the absence of ligands by HC2LC4 and HC2LC1 as determined by western blot. 
         FIG.  20    illustrates Tie2 phosphorylation (left panel) and Akt activation (right panel) in the presence of Ang1 and Ang2 by HC2LC4 and HC2LC1 as determined by western blot. 
         FIG.  21    illustrates Tie2 phosphorylation (panel A), human VE-PTP (HPTP-β) expression (panel B), and Akt activation (panel C) in the presence of Ang1 and/or Ang2 by HC2LC1 and HC2LC4 as determined by western blot. 
         FIG.  22    illustrates Akt activation by HC2LC1 alone and with excess recombinant human VE-PTP extracellular domain protein ((3ECD 6His (The “6-His” disclosed in  FIG.  22    is SEQ ID NO: 52) or βECD Fc) as determined by western blot. 
         FIG.  23    illustrates enhanced endothelial cell viability by HC2LC1 and HCOLC0 as compared to the parenteral antibody (R15E6) and a mouse IgG1 control. 
         FIG.  24    illustrates enhanced efficacy of an anti-mouse VE-PTP antibody/aflibercept combination therapy for the treatment of retinal detachment in a mouse model. 
         FIG.  25    illustrates that subcutaneous administration of monoclonal antibody 109.1 reduced ischemic neovascularization in mice compared to vehicle. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present disclosure provides compositions and methods for targeting vascular endothelial protein tyrosine phosphatase (VE-PTP or VEPTP) or human protein tyrosine phosphatase-beta (HPTP-β) for the treatment of ocular disorders that are characterized by, for example, vascular instability, angiogenesis, neovascularization, vascular leakage, and edema. Compositions disclosed herein can activate Tie2 signaling by promoting protein phosphorylation, such as phosphorylation of the Tie2 protein. 
     VE-PTP is a member of the receptor-like family of the protein tyrosine phosphatases (PTPases). VE-PTP is a transmembrane protein found primarily in vascular endothelial cells that displays structural and functional similarity to cell adhesion molecules. VE-PTP is found in various species including, for example, zebrafish, chicken, dog, mouse, marmoset, monkey, and human. The human orthologue of VE-PTP is HPTP-β. 
     Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2) is a membrane receptor tyrosine kinase expressed primarily in vascular endothelial cells throughout development. The principle regulators of Tie2 phosphorylation are angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2). Ang1 is an agonist of Tie2, and binding of Ang1 to Tie2 promotes receptor phosphorylation. Ang2 acts in a context-dependent antagonist or agonist of Tie2. Binding of Ang1 to Tie2 increases the level of endogenous Tie2 receptor phosphorylation and initiates downstream signaling to induce vascular stabilization through highly organized angiogenesis, tightening of the endothelial cell junctions (endothelial cell proximity), enhanced endothelial viability, reduced endothelial inflammation and improved endothelial function. During angiogenesis, Ang2 acts as a negative regulator of Ang1-Tie2 signaling. 
     Under physiological conditions, the duration of Tie2 phosphorylation is regulated by VE-PTP (HPTP-β), which removes the phosphate from the Tie2 receptor. By inhibiting VE-PTP (HPTP-β), the level of Tie2 phosphorylation substantially increases, restoring vascular stability. A VE-PTP (HPTP-β) inhibitor, for example, an antibody that binds VE-PTP (HPTP-β), can activate Tie2 downstream signaling by inhibiting VE-PTP (HPTP-β). Inhibition of VE-PTP (HPTP-β) by the inhibitor can provide vascular stability in subjects with ocular disorders described herein. 
     A VE-PTP (HPTP-β) inhibitor of the disclosure can comprise the murine monoclonal antibody R15E6 produced by hybridoma cell line ATCC No. PTA-7580, which is immunoreactive to the extracellular domain of human VE-PTP (HPTP-β) (SEQ ID NO. 45), is immunoreactive to the first FN3 repeat of human VE-PTP (HPTP-β) (SEQ ID NO. 46). The VE-PTP (HPTP-β) inhibitor can comprise an antibody having the same or substantially the same biological characteristics of R15E6, an antibody fragment of R15E6, wherein the fragment comprises one or both of the heavy and light chain variable regions, a F(ab′)2 of R15E6, dimers or trimers of a Fab, Fv, scFv, and dia-, tria-, or tetrabodies derived from R15E6. In some embodiments, a fragment of a compound described herein can be assayed for any biological activity described herein. In some embodiments, a fragment of a compound described herein can activate Tie2, or provide the biological activity of the corresponding intact antibody at an equipotent, increased, or decreased level. 
     
       
         
           
               
            
               
                 Extracellular domain of human VE-PTP (HPTP-β) 
               
               
                 (SEQ ID NO: 45): 
               
               
                 MLSHGAGLALWITLSLLQTGLAEPERCNFTLAESKASSHSVSIQWRILGS 
               
               
                   
               
               
                 PCNFSLIYSSDTLGAALCPTFRIDNTTYGCNLQDLQAGTIYNFRIISLDE 
               
               
                   
               
               
                 ERTVVLQTDPLPPARFGVSKEKTTSTSLHVWWTPSSGKVTSYEVQLFDEN 
               
               
                   
               
               
                 NQKIQGVQIQESTSWNEYTFFNLTAGSKYNIAITAVSGGKRSFSVYTNGS 
               
               
                   
               
               
                 TVPSPVKDIGISTKANSLLISWSHGSGNVERYRLMLMDKGILVHGGVVDK 
               
               
                   
               
               
                 HATSYAFHGLTPGYLYNLTVMTEAAGLQNYRWKLVRTAPMEVSNLKVTND 
               
               
                   
               
               
                 GSLTSLKVKWQRPPGNVDSYNITLSHKGTIKESRVLAPWITETHFKELVP 
               
               
                   
               
               
                 GRLYQVTVSCVSGELSAQKMAVGRTFPDKVANLEANNNGRMRSLVVSWSP 
               
               
                   
               
               
                 PAGDWEQYRILLFNDSVVLLNITVGKEETQYVMDDTGLVPGRQYEVEVIV 
               
               
                   
               
               
                 ESGNLKNSERCQGRTVPLAVLQLRVKHANETSLSIMWQTPVAEWEKYIIS 
               
               
                   
               
               
                 LADRDLLLIHKSLSKDAKEFTFTDLVPGRKYMATVTSISGDLKNSSSVKG 
               
               
                   
               
               
                 RTVPAQVTDLHVANQGMTSSLFTNWTQAQGDVEFYQVLLIHENVVIKNES 
               
               
                   
               
               
                 ISSETSRYSFHSLKSGSLYSVVVTTVSGGISSRQVVVEGRTVPSSVSGVT 
               
               
                   
               
               
                 VNNSGRNDYLSVSWLLAPGDVDNYEVTLSHDGKVVQSLVIAKSVRECSFS 
               
               
                   
               
               
                 SLTPGRLYTVTITTRSGKYENHSFSQERTVPDKVQGVSVSNSARSDYLRV 
               
               
                   
               
               
                 SWVHATGDFDHYEVTIKNKNNFIQTKSIPKSENECVFVQLVPGRLYSVTV 
               
               
                   
               
               
                 TTKSGQYEANEQGNGRTIPEPVKDLTLRNRSTEDLHVTWSGANGDVDQYE 
               
               
                   
               
               
                 IQLLFNDMKVFPPFHLVNTATEYRFTSLTPGRQYKILVLTISGDVQQSAF 
               
               
                   
               
               
                 IEGFTVPSAVKNIHISPNGATDSLTVNWTPGGGDVDSYTVSAFRHSQKVD 
               
               
                   
               
               
                 SQTIPKHVFEHTFHRLEAGEQYQIMIASVSGSLKNQINVVGRTVPASVQG 
               
               
                   
               
               
                 VIADNAYSSYSLIVSWQKAAGVAERYDILLLTENGILLRNTSEPATTKQH 
               
               
                   
               
               
                 KFEDLTPGKKYKIQILTVSGGLFSKEAQTEGRTVPAAVTDLRITENSTRH 
               
               
                   
               
               
                 LSFRWTASEGELSWYNIFLYNPDGNLQERAQVDPLVQSFSFQNLLQGRMY 
               
               
                   
               
               
                 KMVIVTHSGELSNESFIFGRTVPASVSHLRGSNRNTTDSLWFNWSPASGD 
               
               
                   
               
               
                 FDFYELILYNPNGTKKENWKDKDLTEWRFQGLVPGRKYVLWVVTHSGDLS 
               
               
                   
               
               
                 NKVTAESRTAPSPPSLMSFADIANTSLAITWKGPPDWTDYNDFELQWLPR 
               
               
                   
               
               
                 DALTVFNPYNNRKSEGRIVYGLRPGRSYQFNVKTVSGDSWKTYSKPIFGS 
               
               
                   
               
               
                 VRTKPDKIQNLHCRPQNSTAIACSWIPPDSDFDGYSIECRKMDTQEVEFS 
               
               
                   
               
               
                 RKLEKEKSLLNIMMLVPHKRYLVSIKVQSAGMTSEVVEDSTITMIDRPPP 
               
               
                   
               
               
                 PPPHIRVNEKDVLISKSSINFTVNCSWFSDTNGAVKYFTVVVREADGSDE 
               
               
                   
               
               
                 LKPEQQHPLPSYLEYRHNASIRVYQTNYFASKCAENPNSNSKSFNIKLGA 
               
               
                   
               
               
                 EMESLGGKCDPTQQKFCDGPLKPHTAYRISIRAFTQLFDEDLKEFTKPLY 
               
               
                   
               
               
                 SDTFFSLPITTESEPLFGAIE 
               
               
                   
               
               
                 First FN3 repeat of human VE-PTP (HPTP-β) 
               
               
                 (SEQ ID NO: 46): 
               
               
                 LAEPERCNFTLAESKASSHSVSIQWRILGSPCNFSLIYSSDTLGAALCPT 
               
               
                   
               
               
                 FRIDNTTYGCNLQDLQAGTIYNFRIISLDEERTVVLQTD 
               
            
           
         
       
     
     A VE-PTP (HPTP-β) inhibitor of the disclosure can include an antibody, or an antibody fragment, variant, or derivative thereof, either alone or in combination with other amino acid sequences. The inhibitor can undergo modifications, for example, enzymatic cleavage, and posttranslational modifications. 
     A VE-PTP (HPTP-β) inhibitor of the disclosure can bind a dominant-negative isoform of VE-PTP (HPTP-β). In some embodiments, this dominant-negative isoform can correspond to a form of VE-PTP (HPTP-β) deficient in phosphatase activity that can compete with endogenous VE-PTP (HPTP-β). Functional assessment of dominant-negative VE-PTP (HPTP-β) can occur via delivery of the transgene and determination of the effect on Tie2 phosphorylation. 
     A VE-PTP (HPTP-β) inhibitor of the disclosure can comprise a plurality of VE-PTP (HPTP-β) binding sites. In some embodiments, a VE-PTP (HPTP-β) inhibitor can bind to two VE-PTP (HPTP-β) molecules simultaneously, thereby bringing the two VE-PTP (HPTP-β) molecules into close proximity. A VE-PTP (HPTP-β) inhibitor can bind to three VE-PTP (HPTP-β) molecules simultaneously, thereby bringing the three VE-PTP (HPTP-β) molecules into close proximity. 
     A VE-PTP (HPTP-β) inhibitor of the disclosure can be covalently or non-covalently conjugated to another moiety or vehicle. A moiety or vehicle can, for example, inhibit degradation, increase half-life, increase absorption, reduce toxicity, reduce immunogenicity, and/or increase biological activity of the inhibitor. Non-limiting examples of the moiety include Fc domains of immunoglobulins, polymers such as polyethylene glycol (PEG), polylysine, and dextran, lipids, cholesterol groups such as steroids, carbohydrates, dendrimers, oligosaccharides, and peptides. 
     The compounds of the present invention can be used for targeting VE-PTP (HPTP-β) to restore Tie2 activity and initiate downstream signaling cascades including, for example, Akt/PI3-K signaling, Rac1 signaling, MAPK/Ras signaling. In some embodiments, compounds of the present invention can inhibit NF-κB signaling. The activation of Tie2 can lead to vascular stabilization, which can be beneficial for the treatment of diabetes-related conditions and ocular conditions including, for example, retinopathy, diabetic retinopathy, retinal perfusion, ocular neovascularization, ocular vascular leak, ocular edema, intraocular pressure, ocular hypertension, ocular inflammation, glaucoma, and ocular hemorrhage. The compounds can also be effective for the treatment of peripheral artery disease including, for example, wound healing, destabilized blood flow, cardiac fibrosis, erectile dysfunction, cardiomyopathy, ischemic injury, cardiac hypertrophy, interstitial fibrosis, nephropathy, albuminuria, glomerulosclerosis, renal fibrosis, neuropathy, and neuronal inflammation. 
     Antibodies 
     Antibodies comprise of two identical heavy chain (H) polypeptide sequences and two identical light chain (L) polypeptide sequences. Each of the heavy chains comprises one N-terminal variable (V H ) region and three C-terminal constant (C H 1, C H 2, and C H 3) regions. Each of the light chains comprises one N-terminal variable (V L ) region and one C-terminal constant (C L ) region. The light chain variable region is aligned with the heavy chain variable region and the light chain constant region is aligned with heavy chain constant region C H1 . The pairing of a heavy chain variable region and light chain variable region together forms a single antigen-binding site. Each light chain is linked to a heavy chain by one covalent disulfide bond. The two heavy chains are linked to each other by one or more disulfide bonds depending on the heavy chain isotype. Each heavy and light chain also comprises regularly-spaced intrachain disulfide bridges. 
     The light chain from any vertebrate species can be designated kappa or lambda based on the amino acid sequences of the constant region. Depending on the amino acid sequence of the constant region of the heavy chains, immunoglobulins can be categorized into five classes of immunoglobulins (IgA, IgD, IgE, IgG, and IgM), each having heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively. The alpha and gamma classes are further divided into subclasses on the basis of differences in the sequence and function of the heavy chain constant region. Subclasses of IgA and IgG expressed by humans include IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. 
     A variable (V) region comprises segments that can differ extensively in sequence among antibodies. The variable region mediates antigen-binding and defines specificity of a particular antibody for its antigen. However, the variability is not evenly distributed across the span of the variable regions. Instead, the variable regions consist of relatively invariant stretches called framework regions (FR) of 15-30 amino acids separated by shorter regions of extreme variability called hypervariable regions that are each 9-12 amino acids long. The variable regions of native heavy and light chains each comprise four framework regions, largely adopting a f3-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming a part of, the f3-sheet structure. The hypervariable regions in each chain are held together in close proximity by the framework regions and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC). 
     A hypervariable region can comprise amino acid residues from a complementarity determining region (CDR), for example, around about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and around about 1-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region, and/or residues from a hypervariable loop. 
     A monoclonal antibody can be 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 can be present in minor amounts. In contrast to polyclonal antibody preparations, which include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope. In addition to the specificity, the monoclonal antibodies are advantageous in that each can be synthesized uncontaminated by other antibodies. 
     The monoclonal antibodies used herein can be, for example, chimeric antibodies wherein a portion of the heavy and/or light chain is identical with 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 chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as antigen-binding fragments of such antibodies. 
     An antibody fragment can comprise a portion of a multimeric antibody, for example, the antigen-binding or variable region of the intact antibody. Non-limiting examples of antibody fragments include Fab, Fab′, F(ab′) 2 , dimers, and trimers of Fab conjugates, Fv, scFv, minibodies, dia-, tria-, and tetrabodies, and linear antibodies. 
     Non-limiting examples of epitopes include amino acids, sugars, lipids, phosphoryl, and sulfonyl groups. An epitope can have specific three-dimensional structural characteristics, and/or specific charge characteristics. Epitopes can be conformational or linear. 
     Humanized Monoclonal Antibodies 
     Suitable antibodies that target VE-PTP can be identified using a variety of techniques. For example, candidate agents can be screened for binding to VE-PTP. Agents that bind to VE-PTP can be screened for activity, for example, inhibition of VE-PTP-mediated dephosphorylation of Tie2. In some embodiments, the candidate agents are first screened in vivo for activity. 
     The selection of a suitable assay for use in identification of a specific inhibitor depends on the nature of the candidate agent to be screened. For example, where the candidates are antibodies or peptibodies, which comprise an Fc moiety, fluorescence-activated cell sorting (FACS) analysis allows the candidate agent to be selected based on the ability to bind to a cell that expresses VE-PTP. The cell can endogenously express VE-PTP or can be genetically engineered to express VE-PTP. For other candidate agents such as aptamers, other techniques can be utilized. For example, aptamers that specifically bind to VE-PTP can be selected using systematic evolution of ligands by exponential enrichment (SELEX), which selects specific aptamers through repeated rounds of in vitro selection. 
     VE-PTP inhibitors can be screened for VE-PTP-mediated activity, for example, inhibition of Tie2 dephosphorylation. In one suitable assay based on western blotting, human umbilical vein endothelial cells (HUVEC) are cultured in serum free media in the presence or absence of various concentrations of candidate agent, and lysates of the cells are prepared, immunoprecipitated with a Tie2 antibody, resolved by polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene difluoride (PVDF) membrane. Membrane-bound immunoprecipitated proteins are then serially western blotted with an anti-phosphotyrosine antibody to quantify Tie2 phosphorylation followed by a Tie2 antibody to quantify total Tie2. Tie2 phosphorylation is expressed as the ratio of the anti-phosphotyrosine signal over the total Tie2 signal. Greater levels of the anti-phosphotyrosine signal indicate greater VE-PTP inhibition by the candidate agent. 
     Hybridoma technology is a method for producing large numbers of a monoclonal antibody targeting a specific antigen. Hybridoma development begins by injecting a mammalian host, such as a mouse (murine) or rabbit, with an antigen that provokes an immune response. In response to the antigen, B-lymphocytes (B-cells) in the host produce antibodies that bind to the antigen. The B-cells are then harvested from the host and fused with immortal B cell cancer cells (myeloma cells) to produce a hybrid cell line known as a hybridoma. The hybridoma retains both the antibody-producing ability of the B cell and the exaggerated longevity and reproductivity of the myeloma. The hybridomas can be grown in culture starting with one viable hybridoma cell to produce new cultures consisting of genetically identical hybridomas that, in turn, produce one antibody per culture (monoclonal) or mixtures of different antibodies (polyclonal). The myeloma cell line used in this process is selected for the ability to grow in tissue culture and for the inability to synthesize antibodies. After culturing, a primary screening process is performed to identify and select the hybridomas that produce antibodies with highest specificity. Non-limiting examples of antibody screening techniques include ELISA and immunocytochemical screening. The lead hybridomas identified from screening can then be characterized for reactivity, binding affinity, specificity, and cross-reactivity. 
     In some embodiments, the binding affinity (K D ) to HPTP-β of a compound of the disclosure is from about 70 pM to about 70 nM, 1 nM to about 70 nM, or at least as strong as about 1 nM. In some embodiments, the binding affinity (K D ) to HPTP-β of a compound of the disclosure is from about 4 nM to about 70 nM. 
     Other recombinant antibody engineering techniques involve the use of viruses or yeast rather than mammals. These techniques rely on rapid cloning of immunoglobulin gene segments to create antibody libraries with slightly different amino acid sequences from which antibodies with desired specificities can be selected. Antibody libraries can be used to enhance specificity to target antigens, stability in various environmental conditions, therapeutic efficacy, and detectability in diagnostic applications. 
     For human administration, monoclonal antibodies generated from non-human species can be further optimized by a humanization process to reduce the likelihood of immunogenicity while preserving target specificity. Humanization processes involve the incorporation of human DNA to the genetic sequence of the genes that produce the isolated antibodies. The recombinant DNA is then cloned and expressed in cells for large-scale production of the newly humanized antibodies. 
     An example of a humanized antibody is a modified chimeric antibody. A chimeric antibody is generated as described above. The chimeric antibody is further mutated outside of the CDRs to substitute non-human sequences in the variable regions with the 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. 
     A humanized antibody can be produced in mammalian cells, bioreactors, or transgenic animals, such as mice, chicken, sheep, goat, pig, and marmoset. The transgenic animal can have a substantial portion of the human antibody-producing genome inserted into the genome of the animal. 
     A fully human monoclonal antibody corresponds to an antibody whose antigen-binding residues are fully derived from the human immunoglobulin sequence or fragments thereof undergoing selection. In some embodiments, this selection can occur using phage display techniques in which a series of variable antibody domain is expressed on a filamentous phage coat protein and enriched for binding to a target antigen. In some embodiments, this selection can occur using transgenic animals, for example, mice, rats, or rabbits, in which the entire set of endogenous immunoglobulin genes are replaced with the entire set of human immunoglobulin genes. In some embodiments, the entire set of human immunoglobulin genes can be introduced into the genome of the animal and endogenous antibody production is rendered deficient in the production of antibodies. 
     Humanized Monoclonal Antibodies that Target VE-PTP (HPTP-β) 
     The humanization process of a murine monoclonal antibody that targets VE-PTP (HPTP-β) described herein utilizes a combination of CDR-grafting technologies coupled with antibody structure and a database of mature human IgG sequences. Human framework sequences were used as acceptor frameworks for the CDR sequences. These acceptor sequences all originated from mature Human IgG from a human source and not from phage display. 
     Four humanized variants were designed for the heavy chain and light chain variable domains of a murine monoclonal antibody raised against the recombinant extracellular domain of human VE-PTP (HPTP-β). The designs were based on factors including homology, T-cell epitopes, key residues, and predicted structures. 
     The sequences disclosed herein were identified using antibody numbering systems from IMGT and Kabat. These two numbering systems identified different residues of the murine antibodies as belonging to the CDR, and a combined IMGT/Kabat CDR sequence was used for optimal retention of CDR-loop conformation. 
     Heavy Chain Sequences 
     SEQ ID NO: 1 is the V H  domain of a murine monoclonal antibody R15E6 (V H0 ) that targets human VE-PTP (HPTP-β), which includes the murine signal peptide sequence (underlined). SEQ ID NO: 47 is a truncated sequence of the V H  domain of R15E6 without the murine signal peptide sequence. SEQ ID NO: 2 is the closest human germline gene V-region,  Homo sapiens  IGHV3-73, to the murine V H  domain of R15E6. SEQ ID NO: 3 and SEQ ID NO: 4 are  Homo sapiens  IGHV3-72 and  Homo sapiens  IGHV3-48, respectively, that are additional germline V-regions that are similar to SEQ ID NO: 2. The peptide sequences of SEQ ID NO: 1-4 are presented in TABLE 1. 
     
       
         
           
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 Name 
                 Amino acid sequence 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 V H0   
                   MDFGLSWVFFVVFYQGVHC EVQLVETGGGLVQPKGS 
               
               
                   
                   
                 MKLSCAASGFTFNANAMNWIRQAPGKGLEWVARIRTK 
               
               
                   
                   
                 SNNYATYYAGSVKDRFTISRDDAQNMLYLQMNDLKTE 
               
               
                   
                   
                 DTAMYYCVRDYYGSSAWITYWGQGTLVTVSA 
               
               
                   
               
               
                 47 
                 Truncated 
                 EVQLVETGGGLVQPKGSMKLSCAASGFTFNANAMNWI 
               
               
                   
                 V H0   
                 RQAPGKGLEWVARIRTKSNNYATYYAGSVKDRFTISR 
               
               
                   
                   
                 DDAQNMLYLQMNDLKTEDTAMYYCVRDYYGSSAWITY 
               
               
                   
                   
                 WGQGTLVTVSA 
               
               
                   
               
               
                 2 
                 IGHV3-73 
                 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGSAMHWV 
               
               
                   
                   
                 RQASGKGLEWVGRIRSKANSYATAYAASVKGRFTISR 
               
               
                   
                   
                 DDSKNTAYLQMNSLKTEDTAVYYCTR 
               
               
                   
               
               
                 3 
                 IGHV3-72 
                 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWV 
               
               
                   
                   
                 RQAPGKGLEWVGRTRNKANSYTTEYAASVKGRFTISR 
               
               
                   
                   
                 DDSKNSLYLQMNSLKTEDTAVYYCAR 
               
               
                   
               
               
                 4 
                 IGHV3-48 
                 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWV 
               
               
                   
                   
                 RQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDN 
               
               
                   
                   
                 AKNSLYLQMNSLRAEDTAVYYCAR 
               
               
                   
               
            
           
         
       
     
     Online databases of Human IgG sequences were searched for comparison to the murine V H  domain using BLAST search algorithms, and candidate human variable domains were selected from the top 200 BLAST results. The coordinate human variable domains were reduced to four candidates based on a combination of framework homology, maintaining framework residues, and canonical loop structures. TABLE 2 lists the four selected acceptor frameworks, SEQ ID NO: 5-8. SEQ ID NO: 5 is AEX29600, SEQ ID NO: 6 is AAC51024, SEQ ID NO: 7 is AEX29289, and SEQ ID NO: 8 is ABA26204. 
     
       
         
           
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 Name 
                 Amino acid sequence 
               
               
                   
               
             
            
               
                 5 
                 AEX29600 
                 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGSAMHWVR 
               
               
                   
                   
                 QASGKGLEWVGRIRSKANNYATAYAASVKGRFTISRDD 
               
               
                   
                   
                 SKNTAYLQMNSLKTEDTAAYYCIRDYYGATRGFQHWGQ 
               
               
                   
                   
                 GTLVTVSS 
               
               
                   
               
               
                 6 
                 AAC51024 
                 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVR 
               
               
                   
                   
                 QAPGKGLEWVGRTRNKANSYTTEYAASVKGRFTISRDD 
               
               
                   
                   
                 SKNSLYLQMNSLKTEDTAVYYCARYVVGATLDYWGQGT 
               
               
                   
                   
                 LVTVSS 
               
               
                   
               
               
                 7 
                 AEX29289 
                 EVQLVESGGGLVQPGRSLRLSCTASGFSFGDYAMNWVR 
               
               
                   
                   
                 QAPGKGLEWVGFIRSKTYGGTTEYAASVKGRFTISRDD 
               
               
                   
                   
                 SKNIAYLQMNSLKTEDTAVYYCTRDPADFYYYSSGQTG 
               
               
                   
                   
                 WFDPWGQGTLVTVSS 
               
               
                   
               
               
                 8 
                 ABA26204 
                 LVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIR 
               
               
                   
                   
                 QAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAK 
               
               
                   
                   
                 NSLYLQMNSLRAEDTAVHYCARDGYSSSWYVDYWGQGT 
               
               
                   
                   
                 LVTVSS 
               
               
                   
               
            
           
         
       
     
     Grafting the CDRs of the murine V H  into the acceptor frameworks (SEQ ID NO: 5-8) converted these sequences to humanized variants, which are shown in TABLE 3. SEQ ID NO: 9 is V H1 , SEQ ID NO: 10 is V H2 , SEQ ID NO: 11 is V H3 , and SEQ ID NO: 12 is V H4 . 
     
       
         
           
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 Name 
                 Amino acid sequence 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 9 
                 V H1   
                 EVQLVESGGGLVQPGGSLKLSCAASGFTFNANAMNWVRQAS 
               
               
                   
                   
                 GKGLEWVGRIRTKSNNYATYYAGSVKDRFTISRDDSKNTAY 
               
               
                   
                   
                 LQMNSLKTEDTAAYYCVRDYYGSSAWITYWGQGTLVTVSS 
               
               
                   
               
               
                 10 
                 V H2   
                 EVQLVESGGGLVQPGGSLRLSCAASGFTFNANAMNWVRQAP 
               
               
                   
                   
                 GKGLEWVGRIRTKSNNYATYYAGSVKDRFTISRDDSKNSLY 
               
               
                   
                   
                 LQMNSLKTEDTAVYYCVRDYYGSSAWITYWGQGTLVTVSS 
               
               
                   
               
               
                 11 
                 V H3   
                 EVQLVESGGGLVQPGRSLRLSCTASGFTFNANAMNWVRQAP 
               
               
                   
                   
                 GKGLEWVGRIRTKSNNYATYYAGSVKDRFTISRDDSKNIAY 
               
               
                   
                   
                 LQMNSLKTEDTAVYYCVRDYYGSSAWITYWGQGTLVTVSS 
               
               
                   
               
               
                 12 
                 V H4   
                 LVQLVESGGGLVKPGGSLRLSCAASGFTFNANAMNWIRQAP 
               
               
                   
                   
                 GKGLEWVSRIRTKSNNYATYYAGSVKDRFTISRDNAKNSLY 
               
               
                   
                   
                 LQMNSLRAEDTAVHYCVRDYYGSSAWITYWGQGTLVTVSS 
               
               
                   
               
            
           
         
       
     
       FIG.  1    shows a sequence alignment of the murine V H  sequence (truncated V H0 ; SEQ ID NO: 47) with four humanized variants SEQ ID NO: 9 (V H1 ), SEQ ID NO: 10 (V H2 ), SEQ ID NO: 11 (V H3 ), and SEQ ID NO: 12 (V H4 ). Residues important for the V H /V K  interface and canonical loop structure were preserved. 
     The percent homologies of the humanized variants to murine V H  are presented in TABLE 4. The ranked order of homology of the humanized variants is V H2 &gt;V H1 &gt;V H3 &gt;V H4 . 
     
       
         
           
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Humanized variant 
                 Identical amino acids 
                 Consensus amino acids 
               
               
                   
               
             
            
               
                 V H1  (SEQ ID NO: 9) 
                 89.3% 
                 94.3% 
               
               
                 V H2  (SEQ ID NO: 10) 
                 90.2% 
                 96.7% 
               
               
                 V H3  (SEQ ID NO: 11) 
                 87.7% 
                 95.1% 
               
               
                 V H4  (SEQ ID NO: 12) 
                 86.9% 
                 93.4% 
               
               
                   
               
            
           
         
       
     
     Light Chain Sequences 
     In TABLE 5 below, SEQ ID NO: 13 is the murine V L  domain of R15E6 (V L0 ), which includes the murine signal peptide sequence (underlined). SEQ ID NO: 48 is a truncated sequence of the V L  domain of R15E6 without the murine signal peptide sequence. SEQ ID NO: 14 is the closest human germline gene V-region,  Homo sapiens  IGKV1-16, to the murine V L  domain of R15E6. SEQ ID NO: 15 is IGKV4-1 and is very similar to IGKV1-16. 
     
       
         
           
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 Name 
                 Amino acid sequence 
               
               
                   
               
             
            
               
                 13 
                 V L0   
                   MESQTQVFVYMLLWLSGVEG DIVMTQSHKFMSTSVGDR 
               
               
                   
                   
                 VSITCKASQHVGTAVAWYQQKPDQSPKQLIYWASTRHT 
               
               
                   
                   
                 GVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSY 
               
               
                   
                   
                 PFTFGSGTKLEIK 
               
               
                   
               
               
                 48 
                 Truncated 
                 DIVMTQSHKFMSTSVGDRVSITCKASQHVGTAVAWYQQ 
               
               
                   
                 V L0   
                 KPDQSPKQLIYWASTRHTGVPDRFTGSGSGTDFTLTIS 
               
               
                   
                   
                 NVQSEDLADYFCQQYSSYPFTFGSGTKLEIK 
               
               
                   
               
               
                 14 
                 IGKV1-16 
                 DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQ 
               
               
                   
                   
                 KPGKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTIS 
               
               
                   
                   
                 SLQPEDFATYYCQQYNSYP 
               
               
                   
               
               
                 15 
                 IGKV4-1 
                 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNY 
               
               
                   
                   
                 LAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTD 
               
               
                   
                   
                 FTLTISSLQAEDVAVYYCQQYYSTP 
               
               
                   
               
            
           
         
       
     
     Online databases of Human IgK sequences were searched for comparison to the murine V L  domain using BLAST search algorithms, and candidate human variable domains were selected from the top 200 BLAST results. These coordinate human variable domains were reduced to four candidates based on a combination of framework homology, maintaining framework residues and canonical loop structure. TABLE 6 lists the four selected acceptor frameworks. SEQ ID NO: 16 is AF234256_1, SEQ ID NO: 17 is AAD03722, SEQ ID NO: 18 is AAY33352, and SEQ ID NO: 19 is AAZ09113. 
     
       
         
           
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 Name 
                 Amino acid sequence 
               
               
                   
               
             
            
               
                 16 
                 AF234256_1 
                 DVVMTQSPSFLSASVGDRVTITCRASQGISNYLAW 
               
               
                   
                   
                 YQQRPGKAPKLLIYAASTLQTGVPSRFSGSGSGTE 
               
               
                   
                   
                 FTLTISSLQPEDFATYFCQQLGGYPLTFGGGTKLE 
               
               
                   
                   
                 IK 
               
               
                   
               
               
                 17 
                 AAD03722 
                 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNN 
               
               
                   
                   
                 KNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSG 
               
               
                   
                   
                 SGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFG 
               
               
                   
                   
                 QGTKLEIK 
               
               
                   
               
               
                 18 
                 AAY33352 
                 DIQMTQSPFSLSASVGDRVTITCRASQGIGSSLAW 
               
               
                   
                   
                 YQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTD 
               
               
                   
                   
                 FTLTISSLQPEDFATYFCLQHHDYPLTFGGGTKLE 
               
               
                   
                   
                 IK 
               
               
                   
               
               
                 19 
                 AAZ09113 
                 DIVMTQSPDSLAVSLGERATINCKSSQSVFYSSNN 
               
               
                   
                   
                 KNYLAWYQQKPEQPPKLLIYWASTRESGVPDRFSG 
               
               
                   
                   
                 SGSGTDFTLTISSLQAEDVAVYYCQQYYSSPLTFG 
               
               
                   
                   
                 GGTKVEIK 
               
               
                   
               
            
           
         
       
     
     Grafting the CDRs of the murine V L  into these acceptor frameworks converted these sequences to humanized variants, which are shown in TABLE 7. SEQ ID NO: 20 is V L1 , SEQ ID NO: 21 is V L2 , SEQ ID NO: 22 is V L3 , and SEQ ID NO: 23 is V L4 . 
     
       
         
           
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 Name 
                 Amino acid sequence 
               
               
                   
               
             
            
               
                 20 
                 V L1   
                 DVVMTQSPSFLSASVGDRVTITCKASQHVGTAVAWYQQRPGK 
               
               
                   
                   
                 APKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPEDFA 
               
               
                   
                   
                 TYFCQQYSSYPFTFGGGTKLEIK 
               
               
                   
               
               
                 21 
                 V L2   
                 DIVMTQSPDSLAVSLGERATINCKASQHVGTAVAWYQQKPGQ 
               
               
                   
                   
                 PPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDVA 
               
               
                   
                   
                 VYYCQQYSSYPFTFGQGTKLEIK 
               
               
                   
               
               
                 22 
                 V L3   
                 DIQMTQSPFSLSASVGDRVTITCKASQHVGTAVAWYQQKPGK 
               
               
                   
                   
                 APKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFA 
               
               
                   
                   
                 TYFCQQYSSYPFTFGGGTKLEIK 
               
               
                   
               
               
                 23 
                 V L4   
                 DIVMTQSPDSLAVSLGERATINCKASQHVGTAVAWYQQKPEQ 
               
               
                   
                   
                 PPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDVA 
               
               
                   
                   
                 VYYCQQYSSYPFTFGGGTKVEIK 
               
               
                   
               
            
           
         
       
     
       FIG.  2    shows a sequence alignment of the murine V L  sequence (truncated V L0 ; SEQ ID NO: 48) with four humanized V L  variants SEQ ID NO: 20 (V L1 ), SEQ ID NO: 21 (V L2 ), SEQ ID NO: 22 (V L3 ), and SEQ ID NO: 23 (V L4 ). Residues important for the V L /V K  interface and canonical loop structure were preserved. 
     The percent homologies of the humanized variants to murine V L  are presented in TABLE 8. The rank order of homology of the humanized variants is V L3 &gt;V L1 &gt;V L2 &gt;V L4 . 
                             TABLE 8               Humanized variant   Identical amino acids   Consensus amino acids                  V L1  (SEQ ID NO: 20)   81.3%   88.8%       V L2  (SEQ ID NO: 21)   79.4%   88.8%       V L3  (SEQ ID NO: 22)   82.2%   86.9%       V L4  (SEQ ID NO: 23)   78.5%   89.7%                    
Design of Humanized Antibodies: Combination of 4 V H  and 4 V L  Chains
 
     Mouse variable chains were humanized by grafting the murine CDR sequences onto suitable human antibody donor sequences as described above in the context of the IgG4 heavy chain for the expression of intact antibody proteins. A panel of full length humanized antibodies was then codon-optimized for expression in the Chinese hamster ovary (CHO) cell line. The four variants designed for each variable chain using different human donor sequences produced a matrix of 16 human antibodies for expression, the heavy chain and light chain pairings of which are shown in TABLE 9. A chimeric variant with full mouse variable domains (V H0 -V L0 ) grafted to human IgG4 constant domains was created as a positive control in binding and functional assays. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 9 
               
               
                   
               
             
            
               
                 V H1 -V L1   
                 SEQ ID NO: 9-SEQ ID NO: 20 
                 V H3 -V L1   
                 SEQ ID NO: 11-SEQ 
               
               
                   
                   
                   
                 ID NO: 20 
               
               
                 V H1 -V L2   
                 SEQ ID NO: 9-SEQ ID NO: 21 
                 V H3 -V L2   
                 SEQ ID NO: 11-SEQ 
               
               
                   
                   
                   
                 ID NO: 21 
               
               
                 V H1 -V L3   
                 SEQ ID NO: 9-SEQ ID NO: 22 
                 V H3 -V L3   
                 SEQ ID NO: 11-SEQ 
               
               
                   
                   
                   
                 ID NO: 22 
               
               
                 V H1 -V L4   
                 SEQ ID NO: 9-SEQ ID NO: 23 
                 V H3 -V L4   
                 SEQ ID NO: 11-SEQ 
               
               
                   
                   
                   
                 ID NO: 23 
               
               
                 V H2 -V L1   
                 SEQ ID NO: 10-SEQ ID NO: 20 
                 V H4 -V L1   
                 SEQ ID NO: 12-SEQ 
               
               
                   
                   
                   
                 ID NO: 20 
               
               
                 V H2 -V L2   
                 SEQ ID NO: 10-SEQ ID NO: 21 
                 V H4 -V L2   
                 SEQ ID NO: 12-SEQ 
               
               
                   
                   
                   
                 ID NO: 21 
               
               
                 V H2 -V L3   
                 SEQ ID NO: 10-SEQ ID NO: 22 
                 V H4 -V L3   
                 SEQ ID NO: 12-SEQ 
               
               
                   
                   
                   
                 ID NO: 22 
               
               
                 V H2 -V L4   
                 SEQ ID NO: 10-SEQ ID NO: 23 
                 V H4 -V L4   
                 SEQ ID NO: 12-SEQ 
               
               
                   
                   
                   
                 ID NO: 23 
               
               
                   
               
            
           
         
       
     
     T-Cell Epitope Screen 
     Heavy Chain. 
     The presentation of peptide sequences in the groove of MHC Class II molecules leads to activation of CD8+ T-cells and an immunogenic response. To reduce this response, therapeutic proteins can be designed to avoid the incorporation of T-cell epitopes that can activate T-cells by reducing the affinity of binding to the MHC Class II molecules. 
     The murine antibody V H  and V L  and the humanized variant sequences were screened for MHC II binding peptides to determine that the humanization process had removed peptide sequences with high affinity using in silico algorithms. TABLE 10 shows the results of the screen, with high affinity T-cell epitope cores in bold (IC 50 &lt;50 nM). The human germline sequences ICHV3-73, ICHV3-72, and ICHV3-48 were also analyzed for comparison. Any potential T-cell epitopes present in the germline sequence and matched in the humanized variants are italicized. CDRs are underlined. 
     
       
         
           
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 Name 
                 Amino acid sequence 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 2 
                 IGHV3-73 
                 EVQLVESGGGLVQPGGSLKLSCAAS GFTFSGSAMH WV 
               
               
                   
                   
                 RQASGKGLEWVGRIRSK ANSYATAYAASV KGRFTISR 
               
               
                   
                   
                 D DSKNTAYLQMNSLKT EDTAVYYC TR   
               
               
                   
               
               
                 3 
                 IGHV3-72 
                 EVQLVESGGGLVQPGGSLRLSCAAS GFTFSDHYMD WV 
               
               
                   
                   
                 RQAPGKGLEWVGRTRNK ANSYTTEYAASV KGRFTISR 
               
               
                   
                   
                 D DSKNSLYLQMNSLKT EDTAVYYC AR   
               
               
                   
               
               
                 4 
                 IGHV3-48 
                 EVQLVESGGGLVQPGGSLRLSCAAS GFTFSSYSMN WV 
               
               
                   
                   
                 RQAPGK GLEWVSYISSSSSTI YYADSVKGRFTISRD N   
               
               
                   
                   
                   AKNSLYLQMNSLR AEDTAVYYC AR   
               
               
                   
               
               
                 47 
                 Trun- 
                 EVQLVETGGGLVQPKGSMKLSCAAS GFTFNANAMN WI 
               
               
                   
                 cated 
                 RQAPGKGLEWVA RIRTKSNNYATYYAGSVKD RFTISR 
               
               
                   
                 V H0   
                 D DAQNMLYLQMNDLKT EDTAMYYC VRDYYGSSAWITY   
               
               
                   
                   
                 WGQGTLVTVSA 
               
               
                   
               
               
                 9 
                 V H1   
                 EVQLVESGGGLVQPGGSLKLSCAAS GFTFNANAMN WV 
               
               
                   
                   
                 RQASGKGLEWVG RIRTKSNNYATYYAGSVKD RFTISR 
               
               
                   
                   
                 D DSKNTAYLQMNSLKT EDTAAYYC VRDYYGSSAWITY   
               
               
                   
                   
                 WGQGTLVTVSS 
               
               
                   
               
               
                 10 
                 V H2   
                 EVQLVESGGGLVQPGGSLRLSCAAS GFTFNANAMN WV 
               
               
                   
                   
                 RQAPGKGLEWVG RIRTKSNNYATYYAGSVKD RFTISR 
               
               
                   
                   
                 D DSKNSLYLQMNSLKT EDTAVYYC VRDYYGSSAWITY   
               
               
                   
                   
                 WGQGTLVTVSS 
               
               
                   
               
               
                 11 
                 V H3   
                 EVQLVESGGGLVQPGRSLRLSCTAS GFTFNANAMN WV 
               
               
                   
                   
                 RQAPGKGLEWVG RIRTKSNNYATYYAGSVKD RFTISR 
               
               
                   
                   
                 D DSKNIAYLQMNSLKT EDTAVYYC VRDYYGSSAWITY   
               
               
                   
                   
                 WGQGTLVTVSS 
               
               
                   
               
               
                 12 
                 V H4   
                 LVQLVESGGGLVKPGGSLRLSCAAS GFTFNANAMN WI 
               
               
                   
                   
                 RQAPGKGLEWVS RIRTKSNNYATYYAGSVKD RFTISR 
               
               
                   
                   
                 D NAKNSLYLQMNSTRA EDTAVHYC VRDYYGSSAWITY   
               
               
                   
                   
                 WGQGTLVTVSS 
               
               
                   
               
               
                 54 
                 Heavy 
                 GFTFNANAMN 
               
               
                   
                 chain 
                   
               
               
                   
                 CDR1 
                   
               
               
                   
               
               
                 55 
                 Heavy 
                 RIRTKSNNYATYYAGSVKD 
               
               
                   
                 chain 
                   
               
               
                   
                 CDR2 
                   
               
               
                   
               
               
                 56 
                 Heavy 
                 VRDYYGSSAWITY 
               
               
                   
                 chain 
                   
               
               
                   
                 CDR3 
               
               
                   
               
            
           
         
       
     
     TABLE 11 shows the results of the screen, with high affinity T-cell epitope cores in bold (IC 50 &lt;50 nM). The human germline sequences IGKV1-16 and IGKV4-1 were also analyzed for comparison, and any potential T-cell epitopes present in the germline sequence and matched in the humanized variants, are italicized. The CDRs are underlined. 
     
       
         
           
               
               
               
             
               
                 TABLE 11 
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 Name 
                 Amino acid sequence 
               
               
                   
               
             
            
               
                 14 
                 IGKV1-16 
                 DIQMTQSPSSLSASVGDRVTITC RASQGISNY   
               
               
                   
                   
                   LA WFQQKPGKA PKSLIYAASSLQ SGVPSRFSG 
               
               
                   
                   
                 SGSGTDFTLTISSLQPEDFATYYC QQYNSYP   
               
               
                   
               
               
                 15 
                 IGKV4-1 
                 DIVMTQSPDSLAVSLGERATINC KSSQSVLYS   
               
               
                   
                   
                   SNNKNYLA WYQQKPGQPPKLLIY WASTRES GV 
               
               
                   
                   
                 PDRFSGSGSGTD FTLTISSLQAEDV AVYYC QQ   
               
               
                   
                   
                 
                   YYSTP 
                 
               
               
                   
               
               
                 48 
                 Truncated 
                 DIVMTQSHKFMSTSVGDRVSITC KASQHVGTA   
               
               
                   
                 V L0   
                   VA WYQQKPDQSPKQLIY WASTRHT GVPDRFTG 
               
               
                   
                   
                 SGSGTDFTLTISNVQSEDLADYFCQ QYSSYPF   
               
               
                   
                   
                   TFGSGTKL EIK 
               
               
                   
               
               
                 20 
                 V L1   
                 DV VMTQSPSFL SASVGDRVTITC KASQHVGTA   
               
               
                   
                   
                   VA WYQQRPGKAPKLLIY WASTRHT GVPSRFSG 
               
               
                   
                   
                 SGSGTEFTLTISSLQPEDFATYFC QQYSSYPF   
               
               
                   
                   
                   T FGGGTKLEIK 
               
               
                   
               
               
                 21 
                 V L2   
                 DIVMTQSPDSLAVSLGERATINC KASQHVGTA   
               
               
                   
                   
                   VA WYQQKPGQPPKLLIY WASTRHT GVPDRFSG 
               
               
                   
                   
                 SGSGTD FTLTISSLQAEDV AVYYCQ QYSSYPF   
               
               
                   
                   
                   TFGQGTKL EIK 
               
               
                   
               
               
                 22 
                 V L3   
                 DIQMTQSPFSLSASVGDRVTITC KASQHVGTA   
               
               
                   
                   
                   VA WYQQKPGKAPKLLIY WASTRHT GVPSRFSG 
               
               
                   
                   
                 SGSGTDFTLTISSLQPEDFATYFC QQYSSYPF   
               
               
                   
                   
                   T FGGGTKLEIK 
               
               
                   
               
               
                 23 
                 V L4   
                 DIVMTQSPDSLAVSLGERATINC KASQHVGTA   
               
               
                   
                   
                   VA WYQQKPEQPPKLLIY WASTRHT GVPDRFSG 
               
               
                   
                   
                 SGSGTD FTLTISSLQAEDV AVYYC QQYSSYPF   
               
               
                   
                   
                   T FGGGTKVEIK 
               
               
                   
               
               
                 57 
                 Light  
                 KASQHVGTAVA 
               
               
                   
                 chain 
                   
               
               
                   
                 CDR1 
                   
               
               
                   
               
               
                 58 
                 Light  
                 WASTRHT 
               
               
                   
                 chain 
                   
               
               
                   
                 CDR2 
                   
               
               
                   
               
               
                 59 
                 Light  
                 QQYSSYPFT 
               
               
                   
                 chain 
                   
               
               
                   
                 CDR3 
               
               
                   
               
            
           
         
       
     
     The murine V L0  contains a T-cell epitope within the CDR3/framework 4 region of the light chain, which is not present in 3 of the humanized variants. 
     Post-Translational Modifications 
     Fv Glycosylation. 
     The N-linked glycosylation motif is NXS/T, where X is any amino acid except proline. This motif was not present in the murine or humanized variants of R15E6 V H  or V L . 
     Deamidation. 
     The amino acid motifs SNG, ENN, LNG, and LNN can be prone to deamidation of asparagine to provide aspartic acid. None of these four motifs were present in the murine or humanized variants of R15E6V H  or V L . 
     Signal Peptides. 
     Murine antibody signal peptides can result in higher levels of expression in CHO cells. The following signal peptides can be incorporated into the humanized variants. 
     
       
         
           
               
               
            
               
                   
                 Heavy chain signal peptide (SEQ ID NO: 24): 
               
               
                   
                 MGWTLVFLFLLSVTAGVHS 
               
               
                   
               
               
                   
                 Light chain signal peptide (SEQ ID NO: 25): 
               
               
                   
                 MVSSAQFLGLLLLCFQGTRC 
               
            
           
         
       
     
     Each of the V H  domains can be synthesized in-frame with a human IgG4 isotype constant domain sequence, with a stabilizing S228P mutation. The entire heavy chain sequence can be codon optimized, and the DNA sequence can be verified. The amino acid sequence of the IgG4 constant domain with S228P mutation (SEQ ID NO: 26) is: 
     
       
         
           
               
            
               
                 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV 
               
               
                   
               
               
                 HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES 
               
               
                   
               
               
                 KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED 
               
               
                   
               
               
                 PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK 
               
               
                   
               
               
                 CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK 
               
               
                   
               
               
                 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSRLTVDKSRWQEG 
               
               
                   
               
               
                 NVFSCSVMHEALHNHYTQKSLSLSLGK 
               
            
           
         
       
     
     Each of the V L  domains can be synthesized in-frame with a human IgK isotype constant domain sequence. The entire light chain sequence can then be codon optimized, and the DNA sequence can be verified. The amino acid sequence of the IgK constant domain (SEQ ID NO: 27) is: 
     
       
         
           
               
            
               
                 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN 
               
               
                   
               
               
                 SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS 
               
               
                   
               
               
                 FNRGEC 
               
            
           
         
       
     
     Each of the variant chains can be verified by DNA sequencing analysis. Then, transient transfection and expression of each of the humanized antibodies can be pursued. One chimeric antibody can be expressed for use as a positive control and can have the murine variable domains, the human Ig constant domains. The 16 humanized variants only have humanized variable domains and human Ig constant domains. TABLE 12 shows the heavy chain and light chain parings that provide the 16 humanized variants with humanized variable domains and human Ig constant domains. 
     
       
         
           
               
               
             
               
                 TABLE 12 
               
               
                   
               
               
                 Chimeric Antibody 
                 HC0-LC0 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Humanized Variants 
                 HC1-LC1 
                 HC1-LC2 
                 HC1-LC3 
                 HC1-LC4 
               
               
                   
                 HC2-LC1 
                 HC2-LC2 
                 HC2-LC3 
                 HC2-LC4 
               
               
                   
                 HC3-LC1 
                 HC3-LC2 
                 HC3-LC3 
                 HC3-LC4 
               
               
                   
                 HC4-LC1 
                 HC4-LC2 
                 HC4-LC3 
                 HC4-LC4 
               
               
                   
               
            
           
         
       
     
     TABLE 13 and TABLE 14 list the full amino acid sequences of each humanized heavy and light chain. 
     
       
         
           
               
               
               
             
               
                 TABLE 13 
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 Name 
                 Amino acid sequence 
               
               
                   
               
             
            
               
                 28 
                 HC0 
                 MGWTLVFLFLLSVTAGVHSEVQLVETGGGLVQPKGSMKL 
               
               
                   
                   
                 SCAASGFTFNANAMNWIRQAPGKGLEWVARIRTKSNNYA 
               
               
                   
                   
                 TYYAGSVKDRFTISRDDAQNMLYLQMNDLKTEDTAMYYC 
               
               
                   
                   
                 VRDYYGSSAWITWGQGTLVTVSAASTKGPSVFPLAPCSR 
               
               
                   
                   
                 STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA 
               
               
                   
                   
                 VLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKV 
               
               
                   
                   
                 DKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI 
               
               
                   
                   
                 SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR 
               
               
                   
                   
                 EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS 
               
               
                   
                   
                 IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK 
               
               
                   
                   
                 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR 
               
               
                   
                   
                 LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                 29 
                 HC1 
                 MGWTLVFLFLLSVTAGVHSEVQLVESGGGLVQPGGSLKL 
               
               
                   
                   
                 SCAASGFTFNANAMNWVRQASGKGLEWVGRIRTKSNNYA 
               
               
                   
                   
                 TYYAGSVKDRFTISRDDSKNTAYLQMNSLKTEDTAAYYC 
               
               
                   
                   
                 VRDYYGSSAWITYWGQGTLVTVSSASTKGPSVFPLAPCS 
               
               
                   
                   
                 RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP 
               
               
                   
                   
                 AVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK 
               
               
                   
                   
                 VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM 
               
               
                   
                   
                 ISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP 
               
               
                   
                   
                 REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS 
               
               
                   
                   
                 SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV 
               
               
                   
                   
                 KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS 
               
               
                   
                   
                 RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                 30 
                 HC2 
                 MGWTLVFLFLLSVTAGVHSEVQLVESGGGLVQPGGSLRL 
               
               
                   
                   
                 SCAASGFTFNANAMNWVRQAPGKGLEWVGRIRTKSNNYA 
               
               
                   
                   
                 TYYAGSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYC 
               
               
                   
                   
                 VRDYYGSSAWITYWGQGTLVTVSSASTKGPSVFPLAPCS 
               
               
                   
                   
                 RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP 
               
               
                   
                   
                 AVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK 
               
               
                   
                   
                 VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM 
               
               
                   
                   
                 ISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP 
               
               
                   
                   
                 REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS 
               
               
                   
                   
                 SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV 
               
               
                   
                   
                 KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS 
               
               
                   
                   
                 RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                 31 
                 HC3 
                 MGWTLVFLFLLSVTAGVHSEVQLVESGGGLVQPGRSLRL 
               
               
                   
                   
                 SCTASGFTFNANAMNWVRQAPGKGLEWVGRIRTKSNNYA 
               
               
                   
                   
                 TYYAGSVKDRFTISRDDSKNIAYLQMNSLKTEDTAVYYC 
               
               
                   
                   
                 VRDYYGSSAWITYWGQGTLVTVSSASTKGPSVFPLAPCS 
               
               
                   
                   
                 RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP 
               
               
                   
                   
                 AVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK 
               
               
                   
                   
                 VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM 
               
               
                   
                   
                 ISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP 
               
               
                   
                   
                 REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS 
               
               
                   
                   
                 SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV 
               
               
                   
                   
                 KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS 
               
               
                   
                   
                 RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                 32 
                 HC4 
                 MGWTLVFLFLLSVTAGVHSLVQLVESGGGLVKPGGSLRL 
               
               
                   
                   
                 SCAASGFTFNANAMNWIRQAPGKGLEWVSRIRTKSNNYA 
               
               
                   
                   
                 TYYAGSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVHYC 
               
               
                   
                   
                 VRDYYGSSAWITYWGQGTLVTVSSASTKGPSVFPLAPCS 
               
               
                   
                   
                 RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP 
               
               
                   
                   
                 AVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK 
               
               
                   
                   
                 VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM 
               
               
                   
                   
                 ISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP 
               
               
                   
                   
                 REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS 
               
               
                   
                   
                 SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV 
               
               
                   
                   
                 KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS 
               
               
                   
                   
                 RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                 TABLE 14 
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 Name 
                 Amino acid sequence 
               
               
                   
               
             
            
               
                 33 
                 LC0 
                 MVSSAQFLGLLLLCFQGTRCDIVMTQSHKFMSTSVGDRV 
               
               
                   
                   
                 SITCKASQHVGTAVAWYQQKPDQSPKQLIYWASTRHTGV 
               
               
                   
                   
                 PDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPFT 
               
               
                   
                   
                 FGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL 
               
               
                   
                   
                 NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL 
               
               
                   
                   
                 SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 34 
                 LC1 
                 MVSSAQFLGLLLLCFQGTRCDVVMTQSPSFLSASVGDRV 
               
               
                   
                   
                 TITCKASQHVGTAVAWYQQRPGKAPKLLIYWASTRHTGV 
               
               
                   
                   
                 PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQYSSYPFT 
               
               
                   
                   
                 FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL 
               
               
                   
                   
                 NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL 
               
               
                   
                   
                 SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 35 
                 LC2 
                 MVSSAQFLGLLLLCFQGTRCDIVMTQSPDSLAVSLGERA 
               
               
                   
                   
                 TINCKASQHVGTAVAWYQQKPGQPPKLLIYWASTRHTGV 
               
               
                   
                   
                 PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSSYPFT 
               
               
                   
                   
                 FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL 
               
               
                   
                   
                 NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL 
               
               
                   
                   
                 SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 36 
                 LC3 
                 MVSSAQFLGLLLLCFQGTRCDIQMTQSPFSLSASVGDRV 
               
               
                   
                   
                 TITCKASQHVGTAVAWYQQKPGKAPKLLIYWASTRHTGV 
               
               
                   
                   
                 PSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYSSYPFT 
               
               
                   
                   
                 FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL 
               
               
                   
                   
                 NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL 
               
               
                   
                   
                 SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 37 
                 LC4 
                 MVSSAQFLGLLLLCFQGTRCDIVMTQSPDSLAVSLGERA 
               
               
                   
                   
                 TINCKASQHVGTAVAWYQQKPEQPPKLLIYWASTRHTGV 
               
               
                   
                   
                 PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSSYPFT 
               
               
                   
                   
                 FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL 
               
               
                   
                   
                 NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL 
               
               
                   
                   
                 SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
            
           
         
       
     
     Target sequences can have 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 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 99.91% 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 to an amino acid sequence provided herein. 
     Various methods and software programs can be used to determine the homology between two or more peptides or nucleic acids, such as NCBI BLAST, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, or another suitable method or algorithm. 
     Pharmaceutical Compositions 
     A pharmaceutical composition of the invention can be a combination of any pharmaceutical compounds described herein with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Pharmaceutical compositions can be administered in therapeutically-effective amounts as pharmaceutical compositions by various forms and routes including, for example, intravenous, intramuscular, oral, parenteral, ophthalmic, and topical administration. 
     A pharmaceutical composition can be administered to the eye via any suitable form or route including, for example, topical, oral, systemic, intravitreal, intracameral, subconjunctival, subtenon, retrobulbar, intraocular, posterior juxtascleral, periocular, subretinal, and suprachoroidal administration. The compositions can be administered by injecting the formulation in any part of the eye including anterior chamber, posterior chamber, vitreous chamber (intravitreal), retina proper, and/or subretinal space. The compositions can be delivered via a non-invasive method. Non-invasive modes of administering the formulation can include using a needleless injection device. Multiple administration routes can be employed for efficient delivery of the pharmaceutical compositions. 
     A pharmaceutical composition can be targeted to any suitable ocular cell including, for example, endothelial cells such as vascular endothelial cells, cells of the retina such as retinal pigment epilthelium (RPE), corneal cells, fibroblasts, astrocytes, glial cells, pericytes, iris epithelial cells, cells of neural origin, 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, and connective tissue cells. 
     A pharmaceutical composition can be administered in a local manner, for example, via injection of the compound directly into an organ, optionally in a depot or sustained release formulation or implant. Pharmaceutical compositions can be provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation. A rapid release form can provide an immediate release. An extended release formulation can provide a controlled release or a sustained delayed release. 
     Pharmaceutical formulations for administration can include aqueous solutions of the active compounds in water-soluble form. Suspensions of the active compounds can be prepared as oily injection suspensions. 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 which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. 
     In practicing the methods of treatment or use provided herein, therapeutically-effective amounts of the compounds described herein are administered in pharmaceutical compositions to a subject having a disease or condition to be treated. In some embodiments, the subject is a mammal such as a human. A 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 compounds used, and other factors. 
     In some embodiments, the compounds described herein can be used singly or in combination with one or more therapeutic agents as components of mixtures. For example, a VE-PTP (HPTP-β) inhibitor of the disclosure can be co-formulated or co-administered with antibodies, for example, anti-VEGF agents. An anti-VEGF agent can be a compound, an antibody, or an antibody fragment, variant, or derivative thereof. Non-limiting examples of an anti-VEGF agent include bevacizumab (Avastin®), ranibizumab (Lucentis®), and aflibercept (Eylea®). In some embodiments, the compounds described herein can be used before, during, or after treatment with an anti-VEGF agent. 
     Pharmaceutical compositions can be formulated using one or more physiologically-acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Formulation can be modified depending upon the route of administration chosen. Pharmaceutical compositions comprising compounds described herein can be manufactured, for example, by mixing, dissolving, emulsifying, encapsulating, entrapping, or compression processes. 
     The pharmaceutical compositions can include at least one pharmaceutically-acceptable carrier, diluent, or excipient and compounds described herein as free-base or pharmaceutically-acceptable salt form. Pharmaceutical compositions can contain solubilizers, stabilizers, tonicity enhancing agents, buffers, and preservatives. 
     Methods for the preparation of compositions comprising the compounds described herein include formulating the compounds with one or more inert, pharmaceutically-acceptable excipients or carriers to form a solid, semi-solid, or liquid composition. Solid compositions include, for example, powders, tablets, dispersible granules, capsules, and cachets. Liquid compositions include, for example, solutions in which a compound is dissolved, emulsions comprising a compound, or a solution containing liposomes, micelles, or nanoparticles comprising a compound as disclosed herein. Semi-solid compositions include, for example, gels, suspensions and creams. The compositions can be in liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or as emulsions. These compositions can also contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically-acceptable additives. 
     Non-limiting examples of dosage forms suitable for use in the invention include liquid, powder, gel, nanosuspension, nanoparticle, microgel, aqueous or oily suspensions, emulsion, and any combination thereof. 
     Non-limiting examples of pharmaceutically-acceptable excipients suitable for use in the invention include binding agents, disintegrating agents, anti-adherents, anti-static agents, surfactants, anti-oxidants, coating agents, coloring agents, plasticizers, preservatives, suspending agents, emulsifying agents, anti-microbial agents, spheronization agents, and any combination thereof. 
     A composition of the invention can be, for example, an immediate release form or a controlled release formulation. An immediate release formulation can be formulated to allow the compounds to act rapidly. Non-limiting examples of immediate release formulations include readily dissolvable formulations. A controlled release formulation can be a pharmaceutical formulation that has been adapted such that release rates and release profiles of the active agent can be matched to physiological and chronotherapeutic requirements or, alternatively, has been formulated to effect release of an active agent at a programmed rate. Non-limiting examples of controlled release formulations include granules, delayed release granules, hydrogels (e.g., of synthetic or natural origin), other gelling agents (e.g., gel-forming dietary fibers), matrix-based formulations (e.g., formulations comprising a polymeric material having at least one active ingredient dispersed through), granules within a matrix, polymeric mixtures, and granular masses. 
     In some, a controlled release formulation is a delayed release form. A delayed release form can be formulated to delay a compound&#39;s action for an extended period of time. A delayed release form can be formulated to delay the release of an effective dose of one or more compounds, for example, for about 4, about 8, about 12, about 16, or about 24 hours. 
     A controlled release formulation can be a sustained release form. A sustained release form can be formulated to sustain, for example, the compound&#39;s action over an extended period of time. A sustained release form can be formulated to provide an effective dose of any compound described herein (e.g., provide a physiologically-effective blood profile) over about 4, about 8, about 12, about 16, or about 24 hours. 
     The disclosed compositions can optionally comprise pharmaceutically-acceptable preservatives. 
     Non-limiting examples of pharmaceutically-acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington&#39;s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &amp; Wilkins 1999), each of which is incorporated by reference in its entirety. 
     A compound described herein can be conveniently formulated into pharmaceutical compositions composed of one or more pharmaceutically-acceptable carriers. See e.g., Remington&#39;s Pharmaceutical Sciences, latest edition, by E. W. Martin Mack Pub. Co., Easton, Pa., incorporated by reference in its entirety, which discloses typical carriers and conventional methods of preparing pharmaceutical compositions. Such carriers can be carriers for administration of compositions to humans and non-humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Pharmaceutical compositions can also include one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents, and anesthetics. 
     Non-limiting examples of pharmaceutically-acceptable carriers include saline, Ringer&#39;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. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the compound. The matrices can be in the form of shaped articles, for example, films, liposomes, microparticles, or microcapsules. 
     The disclosed methods relate to administering an antibody targeting VE-PTP (HPTP-β) as part of a pharmaceutical composition. Compositions suitable for topical administration can be used. In some embodiments, compositions of the invention can comprise a liquid comprising an active agent in solution, in suspension, or both. Liquid compositions can include gels. A liquid composition can be, for example, aqueous. A composition is an in situ gellable aqueous composition. In iteration, the composition is an in situ gellable aqueous solution. Such a composition can comprise a gelling agent in a concentration effective to promote gelling upon contact with the eye or lacrimal fluid in the exterior of the eye. Aqueous compositions can have ophthalmically-compatible pH and osmolality. The composition can comprise an ophthalmic depot formulation comprising an active agent for subconjunctival administration. Microparticles comprising an active agent can be embedded in a biocompatible, pharmaceutically-acceptable polymer or a lipid encapsulating agent. The depot formulations can be adapted to release all or substantially all the active material over an extended period of time. The polymer or lipid matrix, if present, can be adapted to degrade sufficiently to be transported from the site of administration after release of all or substantially all the active agent. The depot formulation can be a liquid formulation, comprising a pharmaceutical acceptable polymer and a dissolved or dispersed active agent. Upon injection, the polymer forms a depot at the injections site, for example, by gelifying or precipitating. The composition can comprise a solid article that can be inserted in a suitable location in the eye, such as between the eye and eyelid or in the conjuctival sac, where the article releases the active agent. Solid articles suitable for implantation in the eye in such fashion can comprise polymers and can be bioerodible or non-bioerodible. 
     Pharmaceutical formulations can include additional carriers, as well as thickeners, diluents, buffers, preservatives, and surface active agents in addition to the agents disclosed herein. 
     The pH of the disclosed composition can range from about 3 to about 12. The pH of the composition can be, for example, from about 3 to about 4, from about 4 to about 5, from about 5 to about 6, from about 6 to about 7, from about 7 to about 8, from about 8 to about 9, from about 9 to about 10, from about 10 to about 11, or from about 11 to about 12 pH units. The pH of the composition can 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 can 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 can 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. If the pH is outside the range desired by the formulator, the pH can be adjusted by using sufficient pharmaceutically-acceptable acids and bases. 
     Depending on the intended mode of administration, the pharmaceutical compositions can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, lotions, creams, or gels, for example, in unit dosage form suitable for single administration of a precise dosage. 
     For solid compositions, nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate. 
     Non-limiting examples of pharmaceutically active agents suitable for combination with compositions of the disclosure include anti-infectives, i.e., aminoglycosides, antiviral agents, antimicrobials, anti-cholinergics/anti-spasmotics, antidiabetic agents, antihypertensive agents, anti-neoplastics, cardiovascular agents, central nervous system agents, coagulation modifiers, hormones, immunologic agents, immunosuppressive agents, and ophthalmic preparations. 
     In some embodiments, the pharmaceutical composition provided herein comprises a therapeutically effective amount of a compound in admixture with a pharmaceutically-acceptable carrier and/or excipient, for example, saline, phosphate buffered saline, phosphate and amino acids, polymers, polyols, sugar, buffers, preservatives, and other proteins. Illustrative agents include octylphenoxy polyethoxy ethanol 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&#39;s and Hank&#39;s solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycol. 
     Methods of Administration and Treatment Methods 
     Pharmaceutical compositions described herein can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, the compositions can be administered to a subject already suffering from a disease or condition, in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition, or to cure, heal, improve, or ameliorate the condition. Compositions can also be administered to lessen a likelihood of developing, contracting, or worsening a condition. Amounts effective for this use can vary based on the severity and course of the disease or condition, previous therapy, the subject&#39;s health status, weight, and response to the drugs, and the judgment of the treating physician. 
     Multiple therapeutic agents can be administered in any order or simultaneously. If simultaneously, the multiple therapeutic agents can be provided in a single, unified form, or in multiple forms, for example, as multiple separate pills. The agents can be packed together or separately, in a single package or in a plurality of packages. One or all of the therapeutic agents can be given in multiple doses. If not simultaneous, the timing between the multiple doses can vary to as much as about a month. 
     Therapeutic agents described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the composition containing a therapeutic agent can vary. For example, the compositions can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition. The compositions can be administered to a subject during or as soon as possible after the onset of the symptoms. The administration of the therapeutic agents can be initiated within the first 48 hours of the onset of the symptoms, within the first 24 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, or within 3 hours of the onset of the symptoms. The initial administration can be via any practical route, such as by any route described herein using any formulation described herein. A therapeutic agent can be administered as soon as is practicable after the onset of a disease or condition is detected or suspected, and for a length of time necessary for the treatment of the disease, such as, for example, from about 1 month to about 3 months. The length of treatment can vary for each subject. 
     Pharmaceutical compositions described herein can be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compounds. The unit dosage can be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged injectables, vials, or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with or without a preservative. Formulations for injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers with a preservative. 
     Pharmaceutical compositions provided herein can be administered in conjunction with other therapies, for example, chemotherapy, radiation, surgery, anti-inflammatory agents, and selected vitamins. The other agents can be administered prior to, after, or concomitantly with the pharmaceutical compositions. 
     Dosing 
     The VE-PTP (HPTP-β) antibody can be administered at a dosage of 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 by weight of the subject. 
     The VE-PTP (HPTP-β) antibody can be administered at any interval desired. The administration of the compound can have irregular dosing schedules to accommodate either the person administering the compound or the subject receiving the compound. For example, the compound can be administered once a week, 2 times a week, 3 times a week, 4 times a week, 6 times a week, 6 times a week, 7 times a week, 8 times a week, 9 times a week or 10 times a week. The interval between daily dosing can be any hourly interval, for example, every hour, every 2 hours, every 3 hours, every 4 hours, every 5 hours, every 6 hours, every 7 hours, every 8 hours, every 9 hours, every 10 hours, every 11 hours, or every 12 hours. The dosing schedules for administration of a VE-PTP (HPTP-β) antibody include, but are not limited to, once daily, three-times weekly, twice weekly, once weekly, three times, monthly, twice monthly, once monthly, once every other month, yearly, two-timed yearly, three-times yearly, or four-times yearly. In some embodiments, the VE-PTP (HPTP-β) antibody can be administered every other week. 
     In addition, the amount of the VE-PTP (HPTP-β) antibody administered can be of the same amount in each dose or the dosage can vary between doses. For example, a first amount dosed in the morning and a second amount administered in the evening. The dosage for administration can vary depending upon the schedule of the anti-VEGF administration. 
     The anti-VE-PTP antibody can be administered in combination with any anti-VEGF agent in any combination, for example, at the beginning of the treatment, at any time during the treatment or at any time after treatment with the anti-VEGF agent has concluded. In addition, the dosage of the VE-PTP (HPTP-β) inhibitors can be adjusted during treatment. Also, the amount of anti-VEGF agent can be adjusted during treatment. 
     Further non-limiting examples of VEGF-modulating agents include non-inflammatory agents, for example, dexamethasone, fluocinolone, and triamcinolone. In addition, the disclosed methods can include implants which deliver an anti-VEGF agent. For example, VE-PTP (HPTP-13) inhibitors can be co-administered either before, during or after an implant is provided to a subject suffering from a disease or condition described herein. 
     Anti-VEGF treatments can be administered, for example, monthly, once every 3 months, once every 6 months, or yearly, wherein the VE-PTP (HPTP-β) inhibitor is administered at any frequency between treatments. 
     Also disclosed herein are methods for treating a disease or condition as disclosed herein. The method comprises administering to a subject:
         a) a therapeutically-effective amount of a VE-PTP (HPTP-β) inhibitor; and   b) a therapeutically-effective amount of an anti-VEGF agent;
 
wherein the administration of the VE-PTP (HPTP-β) inhibitor and the anti-VEGF agent can be conducted in any manner desired by the administrator, for example, as further described herein.
       

     A VE-PTP (HPTP-β) binding agent or an anti-VEGF agent can be administered in any amount necessary or convenient. For example, a compound described herein can be administered in an amount from 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, 0.1 mg to about 1.5 mg, about 0.05 mg to about 1 mg, or about 0.1 mg to about 1 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.11 mg, about 0.12 mg, about 0.13 mg, about 0.14 mg, about 0.15 mg, about 0.16 mg, about 0.17, mg, about 0.18 mg, about 0.19 mg, about 0.2 mg, about 0.21 mg, about 0.22 mg, about 0.23 mg, about 0.24 mg, about 0.25 mg, about 0.26 mg, about 0.27, mg, about 0.28 mg, about 0.29 mg, about 0.3 mg, about 0.31 mg, about 0.32 mg, about 0.33 mg, about 0.34 mg, about 0.35 mg, about 0.36 mg, about 0.37, mg, about 0.38 mg, about 0.39 mg, about 0.4 mg, about 0.41 mg, about 0.42 mg, about 0.43 mg, about 0.44 mg, about 0.45 mg, about 0.46 mg, about 0.47, mg, about 0.48 mg, about 0.49 mg, about 0.5 mg, about 0.51 mg, about 0.52 mg, about 0.53 mg, about 0.54 mg, about 0.55 mg, about 0.56 mg, about 0.57, mg, about 0.58 mg, about 0.59 mg, about 0.6 mg, about 0.61 mg, about 0.62 mg, about 0.63 mg, about 0.64 mg, about 0.65 mg, about 0.66 mg, about 0.67, mg, about 0.68 mg, about 0.69 mg, about 0.7 mg, about 0.71 mg, about 0.72 mg, about 0.73 mg, about 0.74 mg, about 0.75 mg, about 0.76 mg, about 0.77, mg, about 0.78 mg, about 0.79 mg, about 0.8 mg, about 0.81 mg, about 0.82 mg, about 0.83 mg, about 0.84 mg, about 0.85 mg, about 0.86 mg, about 0.87, mg, about 0.88 mg, about 0.89 mg, about 0.9 mg, about 0.91 mg, about 0.92 mg, about 0.93 mg, about 0.94 mg, about 0.95 mg, about 0.96 mg, 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, 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 per dose for a subject by any route of administration. 
     Intraocular Delivery 
     Disclosed herein are methods for intraocular delivery of compositions of the invention to a subject having a disease or condition as disclosed herein. The delivery method can include an invasive method for direct delivery of the composition to ocular cells. In some embodiments, a liquid pharmaceutical composition comprising the antibody is delivered via a subretinal injection. In some embodiments, a liquid pharmaceutical composition comprising the antibody is delivered via intravitreal or subcutaneous injection. In some embodiments, a liquid pharmaceutical composition comprising the antibody is delivered via intracameral injection. In some embodiments, the composition is delivered via multiple administration routes, for example, subretinal and/or intravitreous, to increase efficiency of the antibody delivery. In some embodiments, the subretinal and/or intravitreal injection is preceded by a vitrectomy. 
     The intraocular injection can be performed over any interval of time to improve efficiency of delivery and/or to minimize or avoid damage to surrounding tissue. The interval of time for the intraocular injection can be from, for example, about 1 minute to about 60 minutes, about 1 minute to about 5 minutes, about 5 minutes to about 10 minutes, about 10 minutes to about 15 minutes, about 15 minutes to about 20 minutes, about 20 minutes to about 25 minutes, about 25 minutes to about 30 minutes, about 30 minutes to about 35 minutes, about 35 minutes to about 40 minutes, about 40 minutes to about 45 minutes, about 45 minutes to about 50 minutes, about 50 minutes to about 55 minutes, or about 55 minutes to about 60 minutes. 
     The intraocular injection can be performed at any rate. The rate of intraocular injection can be from, for example, about 1 μL/min to about 200 μL/min, about 1 μL/min to about 10 μL/min, about 10 μL/min to about 20 μL/min, about 20 μL/min to about 30 μL/min, about 30 μL/min to about 40 μL/min, about 40 μL/min to about 50 μL/min, about 50 μL/min to about 60 μL/min, about 60 μL/min to about 70 μL/min, about 70 μL/min to about 80 μL/min, about 80 μL/min to about 90 μL/min, about 90 μL/min to about 100 μL/min, about 100 μL/min to about 110 μL/min, about 110 μL/min to about 120 μL/min, about 120 μL/min to about 130 μL/min, about 130 μL/min to about 140 μL/min, about 140 μL/min to about 150 μL/min, about 150 μL/min to about 160 μL/min, about 160 μL/min to about 170 μL/min, about 170 μL/min to about 180 μL/min, about 180 μL/min to about 190 μL/min, or about 190 μL/min to about 200 μL/min. 
     Kits 
     The present disclosure further relates to kits containing the composition of the disclosure. A kit can comprise:
         A) a composition comprising an antibody targeting VE-PTP (HPTP-β); and   B) a carrier for delivering the composition to a subject.       

     The kits can be modified to fit the dosing regimen prescribed for the subject being treated. The following is a non-limiting example of a kit for use with a subject receiving a composition of the disclosure by an intraocular injection:
         A) an aqueous composition containing:
           a) an antibody targeting the VE-PTP (HPTP-β) extracellular domain; and   b) a carrier system, comprising:
               i) a tonicity agent; and   ii) water   wherein the tonicity agent is present in an amount such that the such that the re-constituted formula comprises from about 0.5% to about 10% mass to volume of the tonicity agent; and   
               
           B) a component for delivering the aqueous composition.       

     In some embodiments, a kit of the invention comprises:
         A) a composition for delivering an anti-VE-PTP antibody; and   B) a composition for delivering an anti-VEGF agent.       

     The kits can be modified to fit the dosing regimen prescribed for the subject being treated. The following is a non-limiting example of a kit for use with a patient receiving an intravenously-delivered composition comprising the disclosed compounds and an intravitreally-administered anti-VEGF agent. This example provides dosing of the disclosed compounds twice daily for 3 months and for an injection of ranibizumab at week 12.
         A. 3 packages, each package containing 4 vials. Each vial comprising a sufficient amount of a VE-PTP (HPTP-β) inhibitor to provide 2 daily injections of 5 mg of the disclosed compounds for 7 days; and   B. a vial of ranibizumab for injection at the end of week 12 which provides 0.5 mg of ranibizumab.       

     Kits can comprise any combination of elements. In addition, when an anti-VE-PTP antibody disclosed herein is provided orally, a single container with sufficient doses of the disclosed compounds can be supplied with the kit. 
     A kit can also provide written instructions for use and disposal of the compositions to be delivered. The instructions can be modified from kit to kit to reflect the dosing regimen prescribed. The instructions can describe any therapy, compounds, excipients, or method of administration described herein. 
     The disclosed compositions can comprise, for example, from about 1.5% to about 90% mass by volume of a carrier system. Non-limiting examples of tonicity agents include dextrose, mannitol, and glycerin. The formulator can utilize more than one tonicity agent. 
     The kit can further comprise devices for administration, such as a syringe, filter needle, extension tubing, cannula, and subretinal injector. Non-limiting examples of routes of administration include intraocular, parenteral, and topical. Intraocular routes of administration can include, for example, intravitreal, intracameral, subconjunctival, subtenon, retrobulbar, intraocular, posterior juxtascleral, periocular, subretinal, and suprachoroidal. Delivery can be by, for example, syringe, needle, infusion pump, or injector. Syringes and injectors can be, for example, single-dose, multi-dose, fixed-dose, or variable-dose. Non-limiting examples of injectors include, pen injectors, auto-injectors, and electronic patch injector systems. 
     The kits can comprise suitable components for the administration of a composition of the invention to a subject. In some embodiments a composition of the invention is present in the kit as a unit dosage form. As such, the formulator can provide delivery devices having a higher concentration of compound and adjust the delivered volume to provide an amount of compound that is less than the amount in the entire solution. 
     A set of instructions can be included in any kit described herein. The instructions can relate to the dosing amount, timing of dosing, reconstitution of the composition when the kit contains a dry composition, and methods of disposal of delivery vehicles and unused composition. The instructions can describe any therapy, compounds, excipients, or method of administration described herein. 
     Methods 
     The invention provides compositions and methods for the treatment or prevention of diseases or conditions of the eye, for example, ocular edema, diabetic macular edema, vascular leak, age-related macular degeneration (wet form), age-related macular degeneration (dry form), choroidal neovascularization, diabetic retinopathy, ocular ischemia, uveitis, retinal vein occlusion (central or branch), ocular trauma, surgery induced edema, surgery induced neovascularization, cystoid macular edema, ocular ischemia, and uveitis. These diseases or conditions can be characterized by changes in the ocular vasculature, whether progressive or non-progressive, whether a result of an acute disease or condition, or a chronic disease or condition. These diseases can be characterized by an increased level of plasma vascular endothelial growth factor (VEGF). 
     The disclosure provides a method of treating ocular neovascularization in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of an antibody targeting VE-PTP (HPTP-β). 
     In some embodiments, the VE-PTP (HPTP-β) inhibitor stabilizes the vasculature against leakage and neovascularization. 
     Improvement of clinical symptoms can be monitored, for example, by indirect ophthalmoscopy, fundus photography, fluorescein angiopathy, electroretinography, external eye examination, slit lamp biomicroscopy, applanation tonometry, pachymetry, optical coherence tomography, or autorefraction. In some embodiments, the disclosed methods relate to the administration of the VE-PTP (HPTP-β) antibody, including compositions comprising an anti-VEGF agent. 
     In some embodiments, the methods of the disclosure include co-administration of a VE-PTP (HPTP-β) antibody with one or more anti-VEGF agents, which can stabilize the vasculature against leakage. 
     In some embodiments, the methods of the disclosure are drawn towards co-administration of an anti-VE-PTP (HPTP-β) antibody with one or more anti-VEGF agents, which can stabilize the vasculature against neovascularization. 
     In some embodiments, the anti-VE-PTP (HPTP-β) antibody can stabilize the vasculature against leakage and neovascularization. 
     In some embodiments, a human subject with at least one visually-impaired eye is treated with from about 0.1 mg to about 100 mg of a VE-PTP (HPTP-β) antibody. 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 and autorefraction. As described herein, the dosing can occur at any frequency determined by the administrator. After cessation of the anti-VEGF agent treatment, subsequent doses can be administered, for example, weekly or monthly, e.g., with a frequency of about 2-8 weeks or about 1-12 months apart depending upon the response. 
     Diseases that are a direct or indirect result of diabetes include, inter alia, diabetic macular edema and diabetic retinopathy. The ocular vasculature of the diabetic becomes unstable over time and leads to conditions such as non-proliferative retinopathy, macular edema, and proliferative retinopathy. As fluid leaks into the center of the macula, the part of the eye where sharp, straight-ahead vision occurs, the buildup of fluid and the associated protein begin to deposit on or under the macula. This deposit results in swelling that causes the subject&#39;s central vision gradually to become distorted. This condition is macular edema. Another condition that can occur is non-proliferative retinopathy in which vascular changes, such as microaneurysms, outside the macular region of the eye can be observed. 
     These conditions can be associated with diabetic proliferative retinopathy, which is characterized by increased neovascularization. The new blood vessels are fragile and are susceptible to bleeding. The result is scarring of the retina and occlusion or total blockage of the light pathway through the eye due to the overformation of new blood vessels. Subjects having diabetic macular edema often suffer from the non-proliferative stage of diabetic retinopathy; however, subjects often only begin to manifest macular edema at the onset of the proliferative stage. 
     Diabetic retinopathy is the most common cause of vision loss in working-aged Americans. Severe vision loss occurs due to tractional retinal detachments that complicate retinal neovascularization, but the most common cause of moderate vision loss is diabetic macular edema (DME). 
     VEGF is a hypoxia-regulated gene, and VEGF levels are increased in hypoxic or ischemic retina. Under most circumstances, Ang2 binds Tie2 but does not stimulate phosphorylation, and acts as a Tie2 antagonist. In the eye, Ang2 is upregulated at sites of neovascularization and acts as a permissive factor for VEGF. Increased expression of VEGF in the retina does not stimulate sprouting of neovascularization from the superficial or intermediate capillary beds of the retina or the choriocapillaris, but does stimulate sprouting from the deep capillary bed where constitutive expression of Ang2 occurs. Co-expression of VEGF and Ang2 at the surface of the retina causes sprouting of neovascularization from the superficial retinal capillaries. 
     Angiogenesis, the process of creating new blood vessels from pre-existing vessels, is essential to a wide range of physiological and pathological events, including embryological development, menstruation, wound healing, and tumor growth. Most, if not all, tumors require angiogenesis to grow and proliferate. VEGF can be a major factor in angiogenesis by increasing vessel permeability and capillary number. 
     VEGF is a protein that is primarily found in endothelial cells and has functions in vasculogenesis, angiogenesis, and permeabilization of blood vessels. The expression of VEGF is induced by hypoxia, activated oncogenes, and cytokines. VEGF activation can lead to angiogenesis in normal human cells and tissues, but also angiogenesis in tumors and allows for tumor progression and growth. Inhibition of VEGF can inhibit tumor growth leading to tumor regression. A variety of retinopathies are associated with increased levels of VEGF; ischemia in the eye leads to an induction of VEGF production due to lack of oxygen. This increase in VEGF can cause hyperproliferation of blood vessels in the retina and can lead to blindness. The disclosed anti-VE-PTP (HPTP-β) antibodies can act to stabilize ocular vasculature and can counteract the stimulation caused by VEGF and other inflammatory agents present in the diseased retina. In some embodiments, administration of an anti-VE-PTP (HPTP-β) antibody to a subject can maintain the level of disease reversal after administration of anti-VEGF drugs to the subject has been withdrawn. 
     Macular degeneration is characterized by a gradual loss or impairment of eyesight due to cell and tissue degeneration of the yellow macular region in the center of the retina. Macular degeneration is often characterized as one of two types, non-exudative (dry form) or exudative (wet form). Although both types are bilateral and progressive, each type can reflect different pathological processes. The wet form of age-related macular degeneration (AMD) is the most common form of choroidal neovascularization and a leading cause of blindness in the elderly. AMD affects millions of Americans over the age of 60, and is the leading cause of new blindness among the elderly. 
     Choroidal neovascular membrane (CNVM) is a problem that is related to a wide variety of retinal diseases, but is most commonly linked to age-related macular degeneration. With CNVM, abnormal blood vessels stemming from the choroid (the blood vessel-rich tissue layer just beneath the retina) grow up through the retinal layers. These new vessels are very fragile and break easily, and cause blood and fluid to pool within the layers of the retina. 
     Diabetes (diabetes mellitus) is a metabolic disease caused by the inability of the pancreas to produce or use insulin. The most common types of diabetes are type 1 diabetes (often referred to as Juvenile Onset Diabetes Mellitus) and type 2 diabetes (often referred to as Adult Onset Diabetes Mellitus). Type 1 diabetes results from the body&#39;s failure to produce insulin due to loss of insulin producing cells, and presently requires the person to inject insulin. Type 2 diabetes generally results from insulin resistance, a condition in which cells fail to use insulin properly. Diabetes can be correlated to a large number of other conditions, including conditions or diseases of the eye including diabetic retinopathy and diabetic macular edema (DME). 
     Diabetic retinopathy is a complication of diabetes that results from damage to the blood vessels of the light-sensitive tissue at the back of the eye (retina). At first, diabetic retinopathy can cause no symptoms or only mild vision problems. Eventually diabetic retinopathy can result in blindness. Diabetic retinopathy can develop in anyone who has type 1 diabetes or type 2 diabetes. 
     At the earliest stage of non-proliferative retinopathy, microaneurysms occur in the retina&#39;s tiny blood vessels. As the disease progresses, more of these blood vessels become damaged or blocked and these areas of the retina send signals into the regional tissue to grow new blood vessels for nourishment. This stage is called proliferative retinopathy. The new blood vessels grow along the retina and along the surface of the clear, vitreous gel that fills the inside of the eye. The vessels have thin, fragile walls and without timely treatment, the new blood vessels can leak blood, for example, whole blood or some constituents thereof, and can result in severe vision loss and even blindness. Also, fluid can leak into the center of the macula, the part of the eye where sharp, straight-ahead vision occurs. The fluid and the associated protein begin to deposit on or under the macula swell the subject&#39;s central vision becomes distorted. This condition is called macular edema and can occur at any stage of diabetic retinopathy, but is more likely to occur as the disease progresses. 
     Uveitis is a condition in which the uvea becomes inflamed. The eye is hollow on the inside with three different layers of tissue surrounding a central cavity. The outermost is the sclera (white coat of the eye) and the innermost is the retina. The middle layer between the sclera and the retina is called the uvea. The uvea contains many of the blood vessels that nourish the eye. Complications of uveitis include glaucoma, cataracts, and new blood vessel formation (neovascularization). 
     Ocular trauma is any sort of physical or chemical injury to the eye. Ocular trauma can affect anyone and major symptoms include redness or pain in the affected eye. The symptoms tend not to occur if tiny projectiles cause the trauma. 
     Surgery-induced edema is the development of swelling in the eye tissues following surgery on the retina or other part of the eye. Cystoid macular edema (CME) is an example of this phenomenon. CME can occur not only in people who have had cataract surgery, but also those with diabetes, retinitis pigmentosa, AMD, or conditions that cause chronic inflammation in the eye. The major symptoms of CME are blurred or decreased central vision. 
     Ocular ischemic syndrome (OIS) encompasses the signs and symptoms that result from chronic vascular insufficiency. The condition is caused by ocular hypoperfusion due to occlusion or stenosis of the common or internal carotid arteries. OIS generally affects subjects of age 50-80, who often exhibit systemic diseases, such as hypertension or diabetes. The major symptoms of OIS are orbital pain, vision loss, changes of the visual field, asymmetric cataract, and sluggish reaction to light. 
     Retinal vein occlusion (RVO) is the most common retinal vascular disease after diabetic retinopathy. Depending on the area of retinal venous drainage effectively occluded, the condition is broadly classified as central retinal vein occlusion (CRVO), hemispheric retinal vein occlusion (HRVO), or branch retinal vein occlusion (BRVO). Presentation of RVO is with variable painless visual loss with any combination of fundal findings consisting of retinal vascular tortuosity, retinal hemorrhages (blot and flame shaped), cotton wool spots, optic disc swelling and macular edema. In a CRVO, retinal hemorrhages can be found in all four quadrants of the fundus, while the hemorrhages are restricted to either the superior or inferior fundal hemisphere in a HRVO. In a BRVO, hemorrhages are largely localized to the area drained by the occluded branch retinal vein. Vision loss occurs secondary to macular edema or ischemia. 
     Compositions of the disclosure act to stabilize ocular vasculature and, in some embodiments, an agent of the disclosure can counteract the stimulation caused by VEGF and other inflammatory agents that can be present in the diseased retina. In some embodiments, administration of an antibody of the disclosure to a subject can be used to maintain the level of disease reversal after administration of anti-VEGF drugs to the subject has been withdrawn. 
     Treatment of Subjects 
     In some embodiments, a single administration of the composition of the disclosure in a subject having a disease or condition as disclosed herein results in sustained intraocular expression of a VE-PTP (HPTP-β) inhibitor at a level sufficient for long-term suppression of ocular neovascularization. 
     For example, the level of VE-PTP (HPTP-β) inhibitor produced in a host ocular cell can be at least 100 pg/mL, at least 200 pg/mL, at least 300 pg/mL, at least 400 pg/mL, at least 500 pg/mL, at least 600 pg/mL, at least 00 pg/mL, at least 800 pg/mL, at least 900 pg/mL, at least 1000 pg/mL, at least 2000 pg/mL, at least 3000 pg/mL, at least 4000 pg/mL, at least 5000 pg/mL, at least 6000 pg/mL, at least 7000 pg/mL, at least 8000 pg/mL, at least 9000 pg/mL or at least 10,000 pg/mL. The level of VE-PTP (HPTP-β) inhibitor produced in host ocular cell can be at most 100 pg/mL, at most 200 pg/mL, at most 300 pg/mL, at most 400 pg/mL, at most 500 pg/mL, at most 600 pg/mL, at most 700 pg/mL, at most 800 pg/mL, at most 900 pg/mL, at most 1000 pg/mL, at most 2000 pg/mL, at most 3000 pg/mL, at most 4000 pg/mL, at most 5000 pg/mL, at most 6000 pg/mL, at most 7000 pg/mL, at most 8000 pg/mL, at most 9000 pg/mL, or at most 10,000 pg/mL. 
     Protein levels can be measured at least about 0.1, at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 14, at least about 21, at least about 30, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, or at least about 365 days after administering a pharmaceutical composition of the disclosure. Protein levels can be measured at most about 0.1, at most about 0.2, at most about 0.3, at most about 0.4, at most about 0.5, at most about 0.6, at most about 0.7, at most about 0.8, at most about 0.9, at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 6, at most about 7, at most about 14, at most about 21, at most about 30, at most about 50, at most about 75, at most about 100, at most about 125, at most about 150, at most about 175, at most about 200, at most about 225, at most about 250, at most about 275, at most about 300, at most about 325, at most about 350, or at most about 365 days after administering a pharmaceutical composition of the disclosure. 
     Central Foveal Thickness 
     Also disclosed herein are methods for decreasing the Central Foveal Thickness (CFT) in a subject having a disease or condition as disclosed herein. The method comprises administering to an eye an antibody targeting VE-PTP (HPTP-β), wherein the administration of the antibody can be conducted in any manner. 
     The level of decrease in Central Foveal Thickness can be for example, from about 50 μm to about 1000 μm. The level of decrease in Central Foveal Thickness can be for example, from about 50 μm to about 500 μm, from about 50 μm to about 750 μm, from about 150 μm to about 500 μm, from about 200 μm to about 500 μm, from about 200 μm to about 1000 μm, from about 250 μm to about 650 μm, or from about 400 μm to about 700 μm. 
     Visual Acuity 
     Further disclosed herein are methods for increasing the visual acuity of a subject having a disease or condition as disclosed herein. 
     Visual acuity is acuteness or clearness of vision, which depends on the sharpness of the retinal focus within the eye and the sensitivity of the interpretative faculty of the brain. Visual acuity is a measure of the spatial resolution of the visual processing system. Visual acuity is tested by requiring the subject to identify characters, typically numbers or letters, on a chart from a set distance. Chart characters are represented as black symbols against a white background. The distance between the subject&#39;s eyes and the testing chart is set at a sufficient distance to approximate infinity in the way the lens attempts to focus. Twenty feet, or six meters, is essentially infinity from an optical perspective. In the present disclosure, an improvement in visual acuity was assessed by an increase in the number of letters that can be read from the chart. 
     One non-limiting test for measuring visual acuity is the use of the ESV-3000 ETDRS testing device and self-calibrated test lighting. The ESV-3000 device incorporates LED light source technology. The auto-calibration circuitry constantly monitors the LED light source and calibrates the test luminance to 85 cd/m 2  or 3 cd/m 2 . 
     Although designed for clinical trials, where large-format ETDRS testing (up to 20/200) is performed at 4 meters, the device can be used in a non-research setting, i.e., hospital or clinic where ocular disease monitoring is conducted. To evaluate ETDRS properly, the test should be conducted under standardized lighting conditions, for example, photopic test level of 85 cd/m 2 . Scoring of visual acuity can be accomplished in any manner chosen by the monitor. After providing a baseline evaluation, the increase or decrease in the number of letters that can be identified by the test subject provides a measure of sight increase or decrease during treatment. 
     Disclosed herein is a method for increasing visual acuity in a subject having a disease or condition of the eye disclosed herein. This method comprises administering to a subject having the disease or condition of the eye, an antibody targeting VE-PTP (HPTP-β), wherein the administration of the antibody can be conducted in any manner described herein. The increase in the number of letters recognized by a treated eye can be, for example, from about 1 to about 30 letters, from about 5 to about 25 letters, from about 5 to about 20 letters, from about 5 to about 15 letters, from about 5 to about 10 letters, from about 10 to about 25 letters, from about 15 to about 25 letters, or from about 20 to about 25 letters. The increase in visual acuity can be about 1 letter, about 5 letters, about 10 letters, about 15 letters, about 20 letters, or about 25 letters. 
     EXAMPLES 
     Example 1: Generation of Monoclonal Antibodies to the VE-PTP (HPTP-β) Extracellular Domain Using Hybridoma Technology 
     Hybridoma technology was used to generate monoclonal antibodies against the N-terminal extracellular domain (ECD) of human VE-PTP (HPTP-β) or mouse VE-PTP as shown in the schematic of  FIG.  3   . Mice were challenged with human VE-PTP (HPTP-β) ECD protein to generate antibodies against the target protein. B-cells were then harvested from the spleen and lymph nodes and fused with myeloma cells to produce the hybridoma cell line. The fused cells were cultured in a medium where only myeloma cells that have genes derived from the antigen exposed mouse can grow. Colonies that were derived from a single myeloma cell were generated by limiting dilution. Antibodies produced by the hybridoma colonies were then screened for binding to human VE-PTP (HPTP-β) ECD by ELISA. One hybridoma, R15E6, was selected for further analysis. R15E6 antibody bound to endogenous human VE-PTP (HPTP-β) from human umbilical vein endothelial cells (HUVECs) as shown by immunoprecipitation (IP) western blot (left panel) and flow cytometry (right panel) in  FIG.  4   . No binding of R15E6 to the bead control (protein A/G beads without antibody; BC) or VEGFR2 or Tie2 (R2/T2) was observed. 
     Example 2: Tie2 Activation in HUVECS with R15E6 
     R15E6 activated Tie2 in a concentration-dependent manner as demonstrated by IP experiments shown in panels A and B of  FIG.  6   . The antibody also enhanced the viability of serum starved endothelial cells in a concentration-dependent manner in HUVECs as shown in panel C. 
     Example 3: Tie2 Activation and Cellular Permeability Experiments with Murine Monoclonal and Polyclonal Anti-Mouse VE-PTP ECD Antibodies 
     To characterize therapeutic effects of a VE-PTP antibody in vitro, Tie2 activation and cellular permeability experiments were performed using anti-mouse VE-PTP antibodies. Monoclonal and polyclonal antibodies against mouse VE-PTP ECD were generated by immunizing rats with a VE-PTP-Fc fusion protein of the N-terminal 8 FN3 repeats of the extracellular domain. Immunization, hybridoma-fusion, and screening were performed as described above. In Example 3, monoclonal antibody 109.1 (mAb 109.1) and polyclonal antibodies (pAbs) against the extracellular fibronectin type III (FN3)-like domains 1-8 of mouse VE-PTP were selected for further analysis. 
       FIG.  7    illustrates the effect of mAb 109.1 on Tie2 activation (top left panel), mouse VE-PTP protein expression (bottom left panel), and Akt (right panel) in mouse endothelial cells (bEnd) as determined by western blot. Treatment with mAb 109.1 resulted in the activation of Tie2 as indicated by Tie2 phosphorylation (pTyr; top left panel). Akt was constitutively activated in the tested mouse endothelial cells as confirmed by the absence of increased phosphorylation with increasing concentrations of antibody (right panel). 
     In vitro experiments with mouse pAb 1-8 also enhanced Tie2 (Tie-2) activation and downstream signaling in endothelial cells. Cultured mouse endothelial (bEnd.5) cells were treated with pAb 1-8 or preimmune (control) antibodies for 1 hr and subsequently immunoprecipitated for Tie2, followed by immunoblotting with anti-phosphotyrosine antibodies (pTyr) and antibodies against Tie2. Aliquots of cell lysates with identical protein content were directly immunoblotted for VE-PTP and Tie2 (bottom panels).  FIG.  8    illustrates the immunoprecipitation experiments in which pAbs rapidly induced Tie2 activation in bEnd.5 cells treated with polyclonal antibodies against mouse VE-PTP or preimmune antibodies for 1 h (panel A) or 3 min (panel B). 
     Consistent with the vascular-stabilizing effects of Tie2 activation, VE-PTP ECD antibodies reduced thrombin- and VEGF-induced permeability of endothelial monolayers in vitro as shown in  FIG.  9   . Paracellular permeability for 250 kD FITC-dextran was determined for cultured HUVEC monolayers grown in transwell filters. Permeability was induced either with thrombin (panel A) or VEGF (panel B). Permeability of PBS-treated cells was set to 100%. For VE-PTP targeting, cells were treated with anti-VE-PTP pAbs. As a control, cells were treated with preimmune antibodies (control Ab). The data represent two independent experiments. 
     Consistent with the vascular-stabilizing effects of Tie2 activation, VE-PTP ECD antibodies also blocked VEGF-induced cutaneous vascular permeability in vivo as shown in  FIG.  10   . Both monoclonal and polyclonal VE-PTP ECD antibodies, mAb 109.1 (panel A) and pAb PTP 1-8 (panel B), inhibited VEGF-induced cutaneous vascular permeability as demonstrated by the Miles assay. The Miles assay can be used as an in vivo model of vascular permeability, which is characteristic of vascular leakage and neovascularization. Mice were injected intravenously with 100 μg of control IgG or anti-mouse VE-PTP antibodies 30 min before starting the Miles assay. Evan&#39;s blue was then injected to the mice intravenously, followed by intradermal injection of VEGF (striped bars) or PBS (solid black bars) after 10 min. After 30 min, the mice were sacrificed and the dye was extracted from the skin samples and quantified. Data sets were checked for normality and equal variance. Values are presented as means±standard error of mean (SEM). As shown in panel C, the blockade of permeability correlates with Tie2 phosphorylation as demonstrated in lung tissue of mice treated with pAb PTP 1-8. 
     Example 4: Effects of a Mouse VE-PTP ECD Monoclonal Antibody on Retinal and Choroidal Neovascularization in Mice 
     In an ischemic retinopathy model that mimics the aspects of proliferative diabetic retinopathy, a VE-PTP ECD monoclonal antibody (mAb 109.1) significantly reduced retinal neovascularization after administration of the antibody as shown in  FIG.  11    (panel A) and quantified in  FIG.  12    (panel A). At P12, mice with ischemic retinopathy were administered an intravitreous injection of 0.1, 0.5, or 2 μg of mAb 109.1 or 2 μg IgG isotype control (n≥12 for each). At P17, extensive GSA-stained retinal neovascularization was observed in control IgG-injected eyes and significantly less retinal neovascularization was observed in eyes injected with 2 μg anti-VE-PTP. *P&lt;0.001. 
     Similarly, in two models that mimic different aspects of wet age-related macular edema (retinal angiomatous proliferation and choroidal neovascularization), a single 2 μg intravitreal administration of the VE-PTP ECD monoclonal antibody (mAb 109.1) significantly reduced both retinal neovascularization (panel B of  FIG.  11    and quantified in panel B of  FIG.  12   ). At P15, six Rho-VEGF transgenic mice were given an intravitreous injection of 0.5 or 2 of mAb 109.1 in one eye and a corresponding dose of control IgG in the fellow eye. At P21, significantly less GSA-stained subretinal neovascularization was observed in the eyes treated with 0.5 or 2 μg of mAb 109.1 than in the control IgG-treated eyes. *P=0.01 by unpaired t-test for comparison with IgG control fellow eyes. Scale bar: 100 μm. 
     Intravitreous injection of 2 μg of mAb 109.1 significantly reduced the area of choroidal neovascularization at Bruch&#39;s membrane rupture sites compared with control IgG (panel C of  FIG.  11    and quantified in panel C of  FIG.  12   ). *P&lt;0.001 by 1-way ANOVA with Bonferroni&#39;s correction. Scale bar: 100 μm. 
     Subcutaneous administration of mAb 109.1 also reduced ischemic neovascularization in an ischemic retinopathy mouse model. The mean area of retinal neovascularization was reduced by about 30% compared to vehicle, as shown in  FIG.  25   . Mice with ischemic retinopathy were administered a subcutaneous injection of 2 mg/kg q.o.d. (every other day)×3. 
     Example 5: Effects of a Mouse VE-PTP ECD Monoclonal Antibody in Combination with an Anti-VEGF Agent on Retinal Detachment in Mice 
     To evaluate the effects of combination therapies using an anti-VE-PTP antibody disclosed herein, a Tet/opsin/VEGF mouse model was used. Tet/opsin/VEGF transgenic mice develop severe subretinal neovascularization and vascular leakage, and leads to exudative retinal detachment mediated by inducible retinal overexpression of human VEGF.  FIG.  24    shows enhanced efficacy of the combination of an anti-mouse VE-PTP antibody (mAb 109.1) and aflibercept compared to either therapy alone to prevent retinal detachment in the mice, as denoted by the double asterisks. Rat IgG was used as the control. 
     Example 6: Generation and Purification of Humanized Antibodies from R15E6 Against Human VE-PTP (HPTP-β) 
     R15E6 is a 150 kDa antibody containing heavy chains and light chains. Sixteen different humanized variations of the antibody were synthesized using different combinations of heavy chain sequence to light chain sequences as shown in TABLE 12. 
     R15E6 humanized variants, as described in Example 1, were cloned into the pVitro DHFR3 mammalian expression vector. Medium-scale transient transfection expression analysis was carried out to determine expression yield from CHO cells. Suspension-adapted CHO cells were cultivated at 2.0-3.0×10 5  cells/mL with 85% CO 2  at 37° C. at 150 rpm in Pro CHO4 serum-free medium supplemented with 8 mM L-glutamine and 10 mL/L hypoxanthine/thymidine in 500 mL vented flasks. Maxi-preps of each construct were prepared using a Nucleobond pc500 kit according to manufacturer instructions. Vector DNA was quantified using a NanoDrop Lite™ spectrophotometer. 
     100 mL of cells at a final density of 1.0×10 6  cells/mL were transiently transfected with 1.25 μg/mL of plasmid DNA in Pro CHO5 serum-free medium supplemented with 8 mM L-glutamine and 10 mL/L hypoxanthine/thymidine in 500 mL vented flasks. Transfected cultures were incubated for 8 to 11 days with 85% CO 2  at 37° C. at 150 rpm before harvesting by centrifugation at 4,000 rpm at 4° C. for 40 min. 
     Following centrifugation, the media were filtered through a 0.8 μm cellulose acetate filter. Each batch was purified using an Amersham Biosciences AKTA chromatography system. Expressed humanized antibodies were purified via Protein A affinity chromatography. A 1-mL HiTrap Protein A column was used for all humanized antibody purifications. All purifications were carried out using Fusion Antibodies in-house wash and elution buffers. After loading of the protein into the column, any bound antibody was eluted using a glycine/Tris buffer (pH 3). All eluted 1 mL fractions were neutralized with 100 μL Tris buffer (pH 8.5). Eluted fractions corresponding to the elution peak were selected for overnight dialysis in PBS. Following dialysis, the concentration of the antibody was measured with a NanoDrop Lite™ spectrophotometer. 
     Example 7: Evaluation of Humanized Antibodies Against Human VE-PTP (HPTP-β) 
     Samples of the humanized antibodies were analyzed by reducing and non-reducing SDS-PAGE ( FIG.  13   ) to determine the presence of heavy and light chains and the purity of the antibodies. The mouse/human chimeric antibody, HC0LC0, was derived from R15E6 by fusion of the murine heavy and light chain variable regions to the human Fc region. In both SDS-PAGE gels, lane 1: SeeBlue Plus2 pre-stained protein standard; lane 2: HCOLC0 #1; lane 3: HC1LC1 #2; lane 4: HC1LC2 #1; lane 5: HC1LC3 #1; lane 6: HC1LC3 #2; lane 7: HC1LC4 #1; lane 8: HC2LC1 #1; lane 9: HC2LC2 #1; lane 10: HC2LC3 #1; lane 11: HC2LC3 #2; lane 12: HC2LC4 #1; lane 13: HC3LC1 #1; lane 14: HC3LC2 #1; lane 15: HC3LC3 #1; lane 16: HC3LC4 #1; lane 17: HC4LC1 #1; lane 18: HC4LC1 #2; lane 19: HC4LC2 #1; lane 20: HC4LC3 #1; lane 21: HC4LC3 #2; lane 22: HC4LC4 #1; lane 23: HC4LC4 #2; and lane 24: SeeBlue Plus2 pre-stained protein standard. The SDS-PAGE gel analysis showed high purity of all analyzed samples. In the reducing SDS-PAGE, the heavy and light chains are visible and are at the expected molecular weight of 50 kDa for the heavy chain and 25 kDa for the light chain. In the non-reducing SDS-PAGE, no free heavy or light chains were observed at around 50 kDa or 25 kDa, respectively. The gels show a low amount of protein for all HC4 variants and HC1LC3, HC2LC3, and HC3LC3 variants. 
     Purified R15E6 was quantified using the extinction coefficient of 13.7 as the standard reference for IgG at 280 nm using a NanoDrop Lite™ spectrophotometer. TABLE 16 summarizes the results of the SDS-PAGE analysis. In agreement with the SDS-PAGE analysis, all of the heavy chain 4 variants expressed little to no antibody. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 16 
               
               
                   
               
               
                   
                   
                   
                 Total 
                   
               
               
                 R15E6 
                   
                 Concentration 
                 yield 
                   
               
               
                 Variant 
                 Volume 
                 (mg/ml) 
                 (mg) 
                 SDS PAGE 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 HC0 LC0 #1 
                 100 ml 
                 0.403 
                 0.85 
                 HC and LC present 
               
               
                 HC1 LC1 #1 
                 100 ml 
                 0 
                 0 
                 No antibody purified 
               
               
                 HC1 LC1 #2 
                 160 ml 
                 0.141 
                 0.28 
                 HC and LC present 
               
               
                 HC1 LC2 #1 
                 100 ml 
                 0.127 
                 0.23 
                 HC and LC present 
               
               
                 HC1 LC3 #1 
                 100 ml 
                 0.045 
                 0.086 
                 HC and LC present faint 
               
               
                 HC1 LC3 #2 
                 160 ml 
                 0.175 
                 0.39 
                 HC and LC present 
               
               
                 HC1 LC4 #1 
                 100 ml 
                 0.1 
                 0.18 
                 HC and LC present 
               
               
                 HC2 LC1 #1 
                 100 ml 
                 0.147 
                 0.28 
                 HC and LC present 
               
               
                 HC2 LC2 #1 
                 100 ml 
                 0.538 
                 1.08 
                 HC and LC present 
               
               
                 HC2 LC3 #1 
                 100 ml 
                 0.034 
                 0.068 
                 HC and LC present faint 
               
               
                 HC2 LC3 #2 
                 160 ml 
                 0.591 
                 1.18 
                 HC and LC present 
               
               
                 HC2 LC4 #1 
                 100 ml 
                 0.165 
                 0.39 
                 HC and LC present 
               
               
                 HC3 LC1 #1 
                 100 ml 
                 0.078 
                 0.16 
                 HC and LC present 
               
               
                 HC3 LC2 #1 
                 100 ml 
                 0.264 
                 0.53 
                 HC and LC present 
               
               
                 HC3 LC3 #1 
                 100 ml 
                 0.043 
                 0.07 
                 HC and LC very faint 
               
               
                 HC3 LC4 #1 
                 100 ml 
                 0.406 
                 0.812 
                 HC and LC present 
               
               
                 HC4 LC1 #1 
                 100 ml 
                 0.025 
                 0.06 
                 No bands 
               
               
                 HC4 LC1 #2 
                 160 ml 
                 0.051 
                 0.11 
                 No bands 
               
               
                 HC4 LC2 #1 
                 100 ml 
                 0.051 
                 0.11 
                 No bands 
               
               
                 HC4 LC3 #1 
                 100 ml 
                 0.053 
                 0.13 
                 No bands 
               
               
                 HC4 LC3 #2 
                 160 ml 
                 0.035 
                 0.07 
                 No bands 
               
               
                 HC4 LC4 #1 
                 100 ml 
                 0.041 
                 0.09 
                 No bands 
               
               
                 HC4 LC4 #2 
                 160 ml 
                 0.031 
                 0.068 
                 No bands 
               
               
                   
               
            
           
         
       
     
     Example 8: Selectivity of R15E6 and Humanized Variants Against Human VE-PTP (HPTP-β), Cynomologus VE-PTP (Cyno PTP-β), and Human HPTP-η 
     Tissue culture supernatants containing the antibodies from hybridomas were tested against targets human VE-PTP (Human Beta ½ ECD), cyno VE-PTP (Cyno Beta), and human HPTP-η (Human Eta) to determine binding affinity by ELISA. 
     Human HPTP-β, cyno PTPβ, and human HPTP-η were independently produced and purified using HEK293 cells. The HEK293 cells were seeded in a shaker flask for 24 hrs before transfection with plasmids expressing species-specific VE-PTP or HPTP-η. The HEK293 cells were grown using serum-free chemically defined media. The DNA for the species-specific VE-PTP or HPTP-η construct was transiently transfected into a 30 mL suspension of HEK293. After 24 hrs, the cells were counted to assess the viability of the cells and determine the viable cell count. Additional readings were taken throughout the transient transfection process. The cell culture was harvested at day 5 of the transient transfection. The conditioned media supernatant harvested from the transient transfection was clarified by centrifugation. The supernatant was filtered using a 0.2 μm membrane filter. The protein was then purified by anti-His tag affinity chromatography. After purification, buffer exchange of the protein to PBS (pH 7.4) was performed. Then, SDS-Gel Capillary Electrophoresis (CE-SDS) analysis was performed and the protein was purified. The amino acid sequences of the DNA plasmid inserts, the DNA plasmid inserts, and corresponding wildtype sequences are shown in TABLE 15 below. The signal peptide of the protein sequences is underlined. 
     
       
         
           
               
               
               
             
               
                 TABLE 15 
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 Name 
                 Sequence 
               
               
                   
               
             
            
               
                 38 
                 Protein 
                   MEWSWVFLFFLSVTTGVHS EPERCNFTL 
               
               
                   
                 sequence of 
                 AESKASSHSVSIQWRILGSPCNFSLIYS 
               
               
                   
                 Human VE-PTP 
                 SDTLGAALCPTFRIDNTTYGCNLQDLQA 
               
               
                   
                 (HPTP-β) 
                 GTIYNFKIISLDEERTVVLQTDPLPPAR 
               
               
                   
                 containing 
                 FGVSKEKTTSTGLHVWWTPSSGKVTSYE 
               
               
                   
                 first eight FN3 
                 VQLFDENNQKIQGVQIQESTSWNEYTFF 
               
               
                   
                 repeats with  
                 NLTAGSKYNIAITAVSGGKRSFSVYTNG 
               
               
                   
                 C-terminal 
                 STVPSPVKDIGISTKANSLLISWSHGSG 
               
               
                   
                 His 6  tag (SEQ 
                 NVERYRLMLMDKGILVHGGVVDKHATSY 
               
               
                   
                 ID NO: 52) 
                 AFHGLTPGYLYNLTVMTEAAGLQNYRWK 
               
               
                   
                 (Human ½  
                 LVRTAPMEVSNLKVTNDGSLTSLKVKWQ 
               
               
                   
                 VE-PTP ECD) 
                 RPPGNVDSYNITLSHKGTIKESRVLAPW 
               
               
                   
                   
                 ITETHFKELVPGRLYQVTVSCVSGELSA 
               
               
                   
                   
                 QKMAVGRTFPLAVLQLRVKHANETSLSI 
               
               
                   
                   
                 MWQTPVAEWEKYIISLADRDLLLIHKSL 
               
               
                   
                   
                 SKDAKEFTFTDLVPGRKYMATVTSISGD 
               
               
                   
                   
                 LKNSSSVKGRTVPAQVTDLHVANQGMTS 
               
               
                   
                   
                 SLFTNWTQAQGDVEFYQVLLIHENVVIK 
               
               
                   
                   
                 NESISSETSRYSFHSLKSGSLYSVVVTT 
               
               
                   
                   
                 VSGGISSRQVVVEGRTVPSSVSGVTVNN 
               
               
                   
                   
                 SGRNDYLSVSWLLAPGDVDNYEVTLSHD 
               
               
                   
                   
                 GKVVQSLVIAKSVRECSFSSLTPGRLYT 
               
               
                   
                   
                 VTITTRSGKYENHSFSQERTVPDKVQGV 
               
               
                   
                   
                 SVSNSARSDYLRVSWVHATGDFDHYEVT 
               
               
                   
                   
                 IKNKNNFIQTKSIPKSENECVFVQLVPG 
               
               
                   
                   
                 RLYSVTVTTKSGQYEANEQGNGRTIPEK 
               
               
                   
                   
                 GNSADIQHSGGRSSLEGPRFERTGGGHH 
               
               
                   
                   
                 HHHH 
               
               
                   
               
               
                 39 
                 Wild-type 
                 MLSHGAGLALWITLSLLQTGLAEPERCN 
               
               
                   
                 protein 
                 FTLAESKASSHSVSIQWRILGSPCNFSL 
               
               
                   
                 sequence of 
                 IYSSDTLGAALCPTFRIDNTTYGCNLQD 
               
               
                   
                 Human VE-PTP 
                 LQAGTIYNFRIISLDEERTVVLQTDPLP 
               
               
                   
                 (HPTP-β) 
                 PARFGVSKEKTTSTSLHVWWTPSSGKVT 
               
               
                   
                   
                 SYEVQLFDENNQKIQGVQIQESTSWNEY 
               
               
                   
                   
                 TFFNLTAGSKYNIAITAVSGGKRSFSVY 
               
               
                   
                   
                 TNGSTVPSPVKDIGISTKANSLLISWSH 
               
               
                   
                   
                 GSGNVERYRLMLMDKGILVHGGVVDKHA 
               
               
                   
                   
                 TSYAFHGLTPGYLYNLTVMTEAAGLQNY 
               
               
                   
                   
                 RWKLVRTAPMEVSNLKVTNDGSLTSLKV 
               
               
                   
                   
                 KWQRPPGNVDSYNITLSHKGTIKESRVL 
               
               
                   
                   
                 APWITETHFKELVPGRLYQVTVSCVSGE 
               
               
                   
                   
                 LSAQKMAVGRTFPDKVANLEANNNGRMR 
               
               
                   
                   
                 SLVVSWSPPAGDWEQYRILLFNDSVVLL 
               
               
                   
                   
                 NITVGKEETQYVMDDTGLVPGRQYEVEV 
               
               
                   
                   
                 IVESGNLKNSERCQGRTVPLAVLQLRVK 
               
               
                   
                   
                 HANETSLSIMWQTPVAEWEKYIISLADR 
               
               
                   
                   
                 DLLLIHKSLSKDAKEFTFTDLVPGRKYM 
               
               
                   
                   
                 ATVTSISGDLKNSSSVKGRTVPAQVTDL 
               
               
                   
                   
                 HVANQGMTSSLFTNWTQAQGDVEFYQVL 
               
               
                   
                   
                 LIHENVVIKNESISSETSRYSFHSLKSG 
               
               
                   
                   
                 SLYSVVVTTVSGGISSRQVVVEGRTVPS 
               
               
                   
                   
                 SVSGVTVNNSGRNDYLSVSWLLAPGDVD 
               
               
                   
                   
                 NYEVTLSHDGKVVQSLVIAKSVRECSFS 
               
               
                   
                   
                 SLTPGRLYTVTITTRSGKYENHSFSQER 
               
               
                   
                   
                 TVPDKVQGVSVSNSARSDYLRVSWVHAT 
               
               
                   
                   
                 GDFDHYEVTIKNKNNFIQTKSIPKSENE 
               
               
                   
                   
                 CVFVQLVPGRLYSVTVTTKSGQYEANEQ 
               
               
                   
                   
                 GNGRTIPEPVKDLTLRNRSTEDLHVTWS 
               
               
                   
                   
                 GANGDVDQYEIQLLFNDMKVFPPFHLVN 
               
               
                   
                   
                 TATEYRFTSLTPGRQYKILVLTISGDVQ 
               
               
                   
                   
                 QSAFIEGFTVPSAVKNIHISPNGATDSL 
               
               
                   
                   
                 TVNWTPGGGDVDSYTVSAFRHSQKVDSQ 
               
               
                   
                   
                 TIPKHVFEHTFHRLEAGEQYQIMIASVS 
               
               
                   
                   
                 GSLKNQINVVGRTVPASVQGVIADNAYS 
               
               
                   
                   
                 SYSLIVSWQKAAGVAERYDILLLTENGI 
               
               
                   
                   
                 LLRNTSEPATTKQHKFEDLTPGKKYKIQ 
               
               
                   
                   
                 ILTVSGGLFSKEAQTEGRTVPAAVTDLR 
               
               
                   
                   
                 ITENSTRHLSFRWTASEGELSWYNIFLY 
               
               
                   
                   
                 NPDGNLQERAQVDPLVQSFSFQNLLQGR 
               
               
                   
                   
                 MYKMVIVTHSGELSNESFIFGRTVPASV 
               
               
                   
                   
                 SHLRGSNRNTTDSLWFNWSPASGDFDFY 
               
               
                   
                   
                 ELILYNPNGTKKENWKDKDLTEWRFQGL 
               
               
                   
                   
                 VPGRKYVLWVVTHSGDLSNKVTAESRTA 
               
               
                   
                   
                 PSPPSLMSFADIANTSLAITWKGPPDWT 
               
               
                   
                   
                 DYNDFELQWLPRDALTVFNPYNNRKSEG 
               
               
                   
                   
                 RIVYGLRPGRSYQFNVKTVSGDSWKTYS 
               
               
                   
                   
                 KPIFGSVRTKPDKIQNLHCRPQNSTAIA 
               
               
                   
                   
                 CSWIPPDSDFDGYSIECRKMDTQEVEFS 
               
               
                   
                   
                 RKLEKEKSLLNIMMLVPHKRYLVSIKVQ 
               
               
                   
                   
                 SAGMTSEVVEDSTITMIDRPPPPPPHIR 
               
               
                   
                   
                 VNEKDVLISKSSINFTVNCSWFSDTNGA 
               
               
                   
                   
                 VKYFTVVVREADGSDELKPEQQHPLPSY 
               
               
                   
                   
                 LEYRHNASIRVYQTNYFASKCAENPNSN 
               
               
                   
                   
                 SKSFNIKLGAEMESLGGKCDPTQQKFCD 
               
               
                   
                   
                 GPLKPHTAYRISIRAFTQLFDEDLKEFT 
               
               
                   
                   
                 KPLYSDTFFSLPITTESEPLFGAIEGVS 
               
               
                   
                   
                 AGLFLIGMLVAVVALLICRQKVSHGRER 
               
               
                   
                   
                 PSARLSIRRDRPLSVHLNLGQKGNRKTS 
               
               
                   
                   
                 CPIKINQFEGHFMKLQADSNYLLSKEYE 
               
               
                   
                   
                 ELKDVGRNQSCDIALLPENRGKNRYNNI 
               
               
                   
                   
                 LPYDATRVKLSNVDDDPCSDYINASYIP 
               
               
                   
                   
                 GNNFRREYIVTQGPLPGTKDDFWKMVWE 
               
               
                   
                   
                 QNVHNIVMVTQCVEKGRVKCDHYWPADQ 
               
               
                   
                   
                 DSLYYGDLILQMLSESVLPEWTIREFKI 
               
               
                   
                   
                 CGEEQLDAHRLIRHFHYTVWPDHGVPET 
               
               
                   
                   
                 TQSLIQFVRTVRDYINRSPGAGPTVVHC 
               
               
                   
                   
                 SAGVGRTGTFIALDRILQQLDSKDSVDI 
               
               
                   
                   
                 YGAVHDLRLHRVHMVQTECQYVYLHQCV 
               
               
                   
                   
                 RDVLRARKLRSEQENPLFPIYENVNPEY 
               
               
                   
                   
                 HRDPVYSRH 
               
               
                   
               
               
                 40 
                 DNA sequence 
                 ATGGAATGGAGCTGGGTCTTTCTCTTCT 
               
               
                   
                 of Human ½ 
                 TCCTGTCAGTAACGACTGGTGTCCACTC 
               
               
                   
                 VE-PTP ECD 
                 CGAGCCCGAGAGATGCAACTTCACCCTG 
               
               
                   
                 containing  
                 GCCGAGTCCAAGGCCTCCTCCCACTCCG 
               
               
                   
                 first eight  
                 TGTCTATCCAGTGGCGGATCCTGGGCTC 
               
               
                   
                 FN3 repeats 
                 CCCCTGCAACTTCTCTCTGATCTACTCC 
               
               
                   
                 (SEQ ID NO: 38) 
                 TCCGACACCCTGGGCGCTGCCCTGTGCC 
               
               
                   
                   
                 CTACCTTCAGAATCGACAACACCACCTA 
               
               
                   
                   
                 CGGCTGCAACCTGCAGGATCTGCAGGCC 
               
               
                   
                   
                 GGCACCATCTACAACTTCAAGATCATCT 
               
               
                   
                   
                 CCCTGGACGAGGAACGGACCGTGGTGCT 
               
               
                   
                   
                 GCAGACCGATCCTCTGCCTCCTGCCAGA 
               
               
                   
                   
                 TTCGGCGTGTCCAAAGAAAAGACCACCT 
               
               
                   
                   
                 CCACCGGACTGCACGTGTGGTGGACCCC 
               
               
                   
                   
                 TTCCAGCGGCAAAGTGACCTCCTACGAG 
               
               
                   
                   
                 GTGCAGCTGTTCGACGAGAACAACCAGA 
               
               
                   
                   
                 AAATCCAGGGCGTGCAGATCCAGGAATC 
               
               
                   
                   
                 CACCTCCTGGAACGAGTACACCTTCTTC 
               
               
                   
                   
                 AACCTGACCGCCGGCTCCAAGTACAATA 
               
               
                   
                   
                 TCGCCATCACCGCCGTGTCCGGCGGCAA 
               
               
                   
                   
                 GAGATCCTTCTCCGTGTACACCAACGGC 
               
               
                   
                   
                 TCCACCGTGCCCAGCCCCGTGAAGGACA 
               
               
                   
                   
                 TCGGCATCTCCACC 
               
               
                   
                   
                 AAGGCCAACTCCCTGCTGATCTCCTGGT 
               
               
                   
                   
                 CCCACGGCTCCGGCAACGTGGAACGGTA 
               
               
                   
                   
                 CAGACTGATGCTGATGGACAAGGGCATC 
               
               
                   
                   
                 CTGGTGCACGGCGGCGTGGTGGATAAGC 
               
               
                   
                   
                 ACGCCACCTCTTACGCCTTCCACGGCCT 
               
               
                   
                   
                 GACCCCTGGCTACCTGTACAATCTGACC 
               
               
                   
                   
                 GTGATGACCGAGGCCGCTGGACTGCAGA 
               
               
                   
                   
                 ACTACCGGTGGAAGCTCGTGCGGACCGC 
               
               
                   
                   
                 CCCCATGGAAGTGTCCAACCTGAAAGTG 
               
               
                   
                   
                 ACCAACGACGGCTCCCTGACCTCTCTGA 
               
               
                   
                   
                 AAGTGAAGTGGCAGAGGCCCCCTGGCAA 
               
               
                   
                   
                 TGTGGACTCCTACAACATCACCCTGTCC 
               
               
                   
                   
                 CACAAGGGCACCATCAAAGAATCCCGGG 
               
               
                   
                   
                 TGCTGGCCCCTTGGATCACCGAGACACA 
               
               
                   
                   
                 CTTCAAAGAACTGGTGCCTGGCCGGCTG 
               
               
                   
                   
                 TACCAAGTGACCGTGTCCTGTGTGTCTG 
               
               
                   
                   
                 GCGAGCTGTCCGCCCAGAAAATGGCCGT 
               
               
                   
                   
                 GGGCAGAACCTTCCCTCTGGCCGTGCTG 
               
               
                   
                   
                 CAGCTGAGAGTGAAGCACGCTAACGAGA 
               
               
                   
                   
                 CATCCCTGTCCATCATGTGGCAGACCCC 
               
               
                   
                   
                 CGTGGCCGAGTGGGAGAAGTACATCATC 
               
               
                   
                   
                 AGCCTGGCCGACCGGGACCTGCTGCTGA 
               
               
                   
                   
                 TCCACAAGTCCCTGAGCAAGGACGCCAA 
               
               
                   
                   
                 AGAGTTCACCTTCACCGACCTGGTGCCC 
               
               
                   
                   
                 GGCAGAAAGTACATGGCCACCGTGACCT 
               
               
                   
                   
                 CCATCTCCGGCGACCTGAAGAACTCCTC 
               
               
                   
                   
                 CAGCGTGAAGGGCAGGACCGTGCCTGCC 
               
               
                   
                   
                 CAAGTGACAGACCTGCACGTGGCCAACC 
               
               
                   
                   
                 AGGGCATGACCTCCTCCCTGTTCACCAA 
               
               
                   
                   
                 CTGGACCCAGGCTCAGGGCGACGTGGAA 
               
               
                   
                   
                 TTCTACCAGGTGCTGCTGATTCATGAGA 
               
               
                   
                   
                 ACGTCGTGATCAAGAACGAGTCCATCTC 
               
               
                   
                   
                 CTCCGAGACAAGCCGGTACTCCTTCCAC 
               
               
                   
                   
                 TCCCTGAAGTCCGGCAGCCTGTACTCCG 
               
               
                   
                   
                 TGGTCGTGACCACAGTGTCCGGGGGCAT 
               
               
                   
                   
                 CTCCTCTAGACAGGTGGTGGTGGAAGGC 
               
               
                   
                   
                 CGCACCGTGCCTAGTTCAGTGTCAGGCG 
               
               
                   
                   
                 TGACCGTGAACAACAGCGGCCGGAACGA 
               
               
                   
                   
                 CTACCTGTCCGTGTCTTGGCTGCTGGCT 
               
               
                   
                   
                 CCTGGGGACGTGGACAACTACGAAGTGA 
               
               
                   
                   
                 CCCTGAGCCACGACGGCAAGGTGGTGCA 
               
               
                   
                   
                 GTCTCTCGTGATCGCCAAGTCCGTGCGC 
               
               
                   
                   
                 GAGTGCTCCTTCAGCTCTCTGACACCTG 
               
               
                   
                   
                 GCAGACTGTATACCGTGACCATCACCAC 
               
               
                   
                   
                 CAGATCCGGGAAGTACGAGAACCACAGC 
               
               
                   
                   
                 TTCTCCCAGGAACGCACAGTGCCCGACA 
               
               
                   
                   
                 AGGTGCAGGGCGTGTCAGTGTCTAACTC 
               
               
                   
                   
                 CGCCAGATCTGACTACCTGCGGGTGTCC 
               
               
                   
                   
                 TGGGTGCACGCTACCGGCGACTTCGACC 
               
               
                   
                   
                 ATTATGAAGTGACAATCAAGAACAAGAA 
               
               
                   
                   
                 CAACTTCATCCAGACCAAGTCCATCCCC 
               
               
                   
                   
                 AAGTCCGAGAACGAGTGCGTGTTCGTGC 
               
               
                   
                   
                 AGCTGGTGCCAGGCAGACTGTACTCTGT 
               
               
                   
                   
                 GACAGTGACCACCAAGTCCGGCCAGTAC 
               
               
                   
                   
                 GAGGCCAACGAGCAGGGCAACGGCAGGA 
               
               
                   
                   
                 CCATCCCTGAGAAGGGCAACTCCGCCGA 
               
               
                   
                   
                 CATCCAGCACTCTGGCGGCAGATCCTCT 
               
               
                   
                   
                 CTGGAAGGCCCCAGATTCGAGAGAACCG 
               
               
                   
                   
                 GCGGAGGCCACCACCATCATCACCATTG 
               
               
                   
                   
                 A 
               
               
                   
               
               
                 41 
                 Protein 
                   MEWSWVFLFFLSVTTGVHS ERCNFTLAE 
               
               
                   
                 sequence of 
                 SKASSHSVSIRWRIWGSPCNFNLTYSSD 
               
               
                   
                 Cynomolgus 
                 TLGAASCPPFRLDNTTYGCNLQDLQAGT 
               
               
                   
                 VE-PTP (Cyno 
                 IYNFRIVSLDGEERTVVLQTDPLPPARF 
               
               
                   
                 PTP-β) 
                 GVSKEKTTSTSLHVWWTPSPGKVTSYEV 
               
               
                   
                 containing first 
                 QLFDENNQKIQGVQIQESTSWNKYTFFN 
               
               
                   
                 eight FN3 
                 LTAGSKYNITITAVSGGKRSSSVYTNGS 
               
               
                   
                 repeats with C- 
                 TVPSPVKDIGISTKANSLLVSWSHGSGN 
               
               
                   
                 terminal His 10   
                 VERYRLMLMDKGILVHGSVVDRQATSYT 
               
               
                   
                 tag (SEQ ID 
                 FNGLTPGYLYNLTVVTEAAGLQNYKWKL 
               
               
                   
                 NO: 53)(Cyno 
                 VRTAPMEVSNLKVTNDGSLTSLKVKWQR 
               
               
                   
                 Beta ½ VE-PTP 
                 PPGNVDSYNITLSHKGTIKESRVLAPRV 
               
               
                   
                 ECD) 
                 TETHFKELTPGRLYQVTVSCVSGELSAQ 
               
               
                   
                   
                 RMAVGRTFPLPVLQLRVKHANETSLSII 
               
               
                   
                   
                 WQPPVAEWEEYIISLADRDLRLIHKSLS 
               
               
                   
                   
                 KDAKEFTFTDLVPGRKYMATVTSISGDL 
               
               
                   
                   
                 KNSSSVKGRTVPAQVTDLHVANQGMTSS 
               
               
                   
                   
                 LFTNWTQAQGDVEFYQVLLIHENVVIKN 
               
               
                   
                   
                 ESIPSETSGYNFHFLKSGSLYSVVVTTV 
               
               
                   
                   
                 SGGISSRQVVVEGRTVPSSVSGVTVNNS 
               
               
                   
                   
                 GRNDYLSVSWLPAPGDVDNYEVTLSHDG 
               
               
                   
                   
                 RVVQSLVIAKSVRECSFSSLTPGRLYTV 
               
               
                   
                   
                 TITTRSGKYENHSFSQERTVPDKVQGVS 
               
               
                   
                   
                 VSNSARSDYLRVSWVHATGDFDHYEVTI 
               
               
                   
                   
                 KNKNNFIETKSIPKSENECVFVQLVPGR 
               
               
                   
                   
                 LYSVTVTTKSGQYEASEQGNGRTGGGHH 
               
               
                   
                   
                 HHHHHHHH 
               
               
                   
               
               
                 42 
                 DNA sequence 
                 ATGGAATGGAGCTGGGTCTTTCTCTTCT 
               
               
                   
                 of Cyno Beta ½ 
                 TCCTGTCAGTAACGACTGGTGTCCACTC 
               
               
                   
                 VE-PTP ECD 
                 CGAGCGGTGCAACTTTACCCTGGCCGAG 
               
               
                   
                 containing first 
                 TCCAAGGCCTCCTCCCACTCCGTGTCTA 
               
               
                   
                 eight FN3 
                 TCCGGTGGCGGATCTGGGGCTCCCCCTG 
               
               
                   
                 repeats (SEQ 
                 CAACTTCAACCTGACCTACTCCTCCGAT 
               
               
                   
                 ID NO: 41) 
                 ACCCTGGGCGCTGCCTCCTGTCCTCCTT 
               
               
                   
                   
                 TCCGGCTGGACAACACCACCTACGGCTG 
               
               
                   
                   
                 CAACCTGCAGGATCTGCAGGCCGGCACC 
               
               
                   
                   
                 ATCTACAACTTCCGGATCGTGTCCCTGG 
               
               
                   
                   
                 ACGGCGAGGAACGGACAGTGGTGCTGCA 
               
               
                   
                   
                 GACCGATCCTCTGCCCCCTGCCAGATTC 
               
               
                   
                   
                 GGCGTGTCCAAAGAAAAGACCACCTCCA 
               
               
                   
                   
                 CCTCCCTGCACGTGTGGTGGACCCCTAG 
               
               
                   
                   
                 CCCTGGCAAAGTGACCTCCTACGAGGTG 
               
               
                   
                   
                 CAGCTGTTCGACGAGAACAACCAGAAAA 
               
               
                   
                   
                 TCCAGGGCGTGCAGATCCAGGAATCCAC 
               
               
                   
                   
                 CTCCTGGAACAAGTACACCTTCTTCAAT 
               
               
                   
                   
                 CTGACCGCCGGCTCCAAGTACAACATCA 
               
               
                   
                   
                 CCATCACCGCCGTGTCCGGCGGCAAGAG 
               
               
                   
                   
                 ATCCTCCTCCGTGTACACCAACGGCTCC 
               
               
                   
                   
                 ACCGTGCCCAGCCCCGTGAAGGACATCG 
               
               
                   
                   
                 GCATCTCCACCAAGGCCAACTCCCTGCT 
               
               
                   
                   
                 GGTGTCCTGGTCCCACGGCTCCGGCAAC 
               
               
                   
                   
                 GTGGAACGGTACAGACTGATGCTGATGG 
               
               
                   
                   
                 ACAAGGGCATCCTGGTGCACGGCAGCGT 
               
               
                   
                   
                 GGTGGATAGACAGGCCACCTCCTACACC 
               
               
                   
                   
                 TTCAACGGCCTGACCCCCGGCTACCTGT 
               
               
                   
                   
                 ATAACCTGACCGTCGTGACCGAGGCCGC 
               
               
                   
                   
                 TGGACTGCAGAACTACAAGTGGAAGCTC 
               
               
                   
                   
                 GTGCGGACCGCCCCCATGGAAGTGTCCA 
               
               
                   
                   
                 ACCTGAAAGTGACCAACGACGGCTCCCT 
               
               
                   
                   
                 GACCTCTCTGAAAGTGAAGTGGCAGAGG 
               
               
                   
                   
                 CCCCCTGGCAATGTGGACAGCTACAATA 
               
               
                   
                   
                 TCACCCTGTCCCACAAGGGCACCATCAA 
               
               
                   
                   
                 AGAATCCCGGGTGCTGGCCCCCAGAGTG 
               
               
                   
                   
                 ACCGAGACACACTTCAAAGAGCTGACCC 
               
               
                   
                   
                 CTGGCCGGCTGTACCAAGTGACCGTGTC 
               
               
                   
                   
                 CTGTGTGTCTGGCGAGCTGTCTGCCCAG 
               
               
                   
                   
                 AGAATGGCCGTGGGCAGAACCTTCCCTC 
               
               
                   
                   
                 TGCCCGTGCTGCAGCTGAGAGTGAAGCA 
               
               
                   
                   
                 CGCCAACGAGACATCCCTGTCCATCATC 
               
               
                   
                   
                 TGGCAGCCCCCTGTGGCCGAGTGGGAAG 
               
               
                   
                   
                 AGTACATCATCAGCCTGGCCGACCGGGA 
               
               
                   
                   
                 CCTGCGGCTGATCCACAAGTCCCTGAGC 
               
               
                   
                   
                 AAGGACGCCAAAGAGTTCACCTTCACCG 
               
               
                   
                   
                 ACCTGGTGCCTGGCCGGAAGTACATGGC 
               
               
                   
                   
                 CACCGTGACCTCCATCTCCGGCGACCTG 
               
               
                   
                   
                 AAGAACTCCTCCAGCGTGAAGGGCAGGA 
               
               
                   
                   
                 CCGTGCCTGCCCAAGTGACAGACCTGCA 
               
               
                   
                   
                 TGTGGCCAACCAGGGCATGACCTCCAGC 
               
               
                   
                   
                 CTGTTCACCAACTGGACCCAGGCTCAGG 
               
               
                   
                   
                 GCGACGTGGAATTCTACCAGGTGCTGCT 
               
               
                   
                   
                 GATCCATGAGAACGTCGTGATCAAGAAC 
               
               
                   
                   
                 GAGTCCATCCCCTCCGAGACAAGCGGCT 
               
               
                   
                   
                 ACAACTTTCACTTCCTGAAGTCCGGCAG 
               
               
                   
                   
                 CCTGTACTCCGTGGTCGTGACCACAGTG 
               
               
                   
                   
                 TCCGGGGGCATCTCCTCTAGAAGGTGGT 
               
               
                   
                   
                 GGTGGAAGGCCGCACCGTGCCTAGTTCA 
               
               
                   
                   
                 GTGTCAGGCGTGACCGTGAACAACAGCG 
               
               
                   
                   
                 GCCGGAACGACTACCTGTCCGTGTCTTG 
               
               
                   
                   
                 GCTGCCTGCCCCTGGGGACGTGGACAAC 
               
               
                   
                   
                 TACGAAGTGACCCTGTCTCACGACGGCC 
               
               
                   
                   
                 GGGTGGTGCAGTCTCTCGTGATCGCTAA 
               
               
                   
                   
                 GTCCGTGCGCGAGTGCTCCTTCAGCAGC 
               
               
                   
                   
                 CTGACACCTGGCAGACTGTATACCGTGA 
               
               
                   
                   
                 CCATCACCACCAGATCCGGGAAGTACGA 
               
               
                   
                   
                 GAACCACAGCTTCTCCCAGGAACGAACC 
               
               
                   
                   
                 GTGCCCGACAAGGTGCAGGGCGTGTCAG 
               
               
                   
                   
                 TGTCTAACTCCGCCAGATCTGACTACCT 
               
               
                   
                   
                 GAGAGTGTCCTGGGTGCACGCCACCGGC 
               
               
                   
                   
                 GACTTCGACCATTATGAAGTGACAATCA 
               
               
                   
                   
                 AGAACAAGAACAACTTCATCGAGACAAA 
               
               
                   
                   
                 GAGCATCCCCAAGTCCGAGAACGAGTGC 
               
               
                   
                   
                 GTGTTCGTGCAGCTGGTGCCAGGCAGGC 
               
               
                   
                   
                 TGTATTCTGTGACAGTGACCACCAAGTC 
               
               
                   
                   
                 CGGCCAGTACGAGGCCTCTGAGCAGGGC 
               
               
                   
                   
                 AATGGCAGAACCGGCGGTGGACACCACC 
               
               
                   
                   
                 ATCATCACCATCACCACCATCACTAG 
               
               
                   
               
               
                 43 
                 Protein 
                   MEWSWVFLFFLSVTTGVHS AGGTPSPIP 
               
               
                   
                 sequence of 
                 DPSVATVATGENGITQISSTAESFHKQN 
               
               
                   
                 Human HPTP-η 
                 GTGTPQVETNTSEDGESSGANDSLRTPE 
               
               
                   
                 with C-terminal 
                 QGSNGTDGASQKTPSSTGPSPVFDIKAV 
               
               
                   
                 His 10  tag (SEQ 
                 SISPTNVILTWKSNDTAASEYKYVVKHK 
               
               
                   
                 ID NO: 53) 
                 MENEKTITVVHQPWCNITGLRPATSYVF 
               
               
                   
                 (Human HPTP- 
                 SITPGIGNETWGDPRVIKVITEPIPVSD 
               
               
                   
                 η ECD) 
                 LRVALTGVRKAALSWSNGNGTASCRVLL 
               
               
                   
                   
                 ESIGSHEELTQDSRLQVNISGLKPGVQY 
               
               
                   
                   
                 NINPYLLQSNKTKGDPLGTEGGLDASNT 
               
               
                   
                   
                 ERSRAGSPTAPVHDESLVGPVDPSSGQQ 
               
               
                   
                   
                 SRDTEVLLVGLEPGTRYNATVYSQAANG 
               
               
                   
                   
                 TEGQPQAIEFRTNAIQVFDVTAVNISAT 
               
               
                   
                   
                 SLTLIWKVSDNESSSNYTYKIHVAGETD 
               
               
                   
                   
                 SSNLNVSEPRAVIPGLRSSTFYNITVCP 
               
               
                   
                   
                 VLGDIEGTPGFLQVHTPPVPVSDFRVTV 
               
               
                   
                   
                 VSTTEIGLAWSSHDAESFQMHITQEGAG 
               
               
                   
                   
                 NSRVEITTNQSIIIGGLFPGTKYCFEIV 
               
               
                   
                   
                 PKGPNGTEGASRTVCNRTVPSAVFDIHV 
               
               
                   
                   
                 VYVTTTEMWLDWKSPDGASEYVYHLVIE 
               
               
                   
                   
                 SKHGSNHTSTYDKAITLQGLIPGTLYNI 
               
               
                   
                   
                 TISPEVDHVWGDPNSTAQYTRPSNVSNI 
               
               
                   
                   
                 DVSTNTTAATLSWQNFDDASPTYSYCLL 
               
               
                   
                   
                 IEKAGNSSNATQVVTDIGITDATVTELI 
               
               
                   
                   
                 PGSSYTVEIFAQVGDGIKSLEPGRKSFC 
               
               
                   
                   
                 TDPASMASFDCEVVPKEPALVLKWTCPP 
               
               
                   
                   
                 GANAGFELEVSSGAWNNATHLESCSSEN 
               
               
                   
                   
                 GTEYRTEVTYLNFSTSYNISITTVSCGK 
               
               
                   
                   
                 MAAPTRNTCTTGITDPPPPDGSPNITSV 
               
               
                   
                   
                 SHNSVKVKFSGFEASHGPIKAYAVILTT 
               
               
                   
                   
                 GEAGHPSADVLKYTYEDFKKGASDTYVT 
               
               
                   
                   
                 YLIRTEEKGRSQSLSEVLKYEIDVGNES 
               
               
                   
                   
                 TTLGYYNGKLEPLGSYRACVAGFTNITF 
               
               
                   
                   
                 HPQNKGLIDGAESYVSFSRYSDAVSLPQ 
               
               
                   
                   
                 DPGVICGGGHHHHHHHHHH 
               
               
                   
               
               
                 44 
                 DNA sequence 
                 ATGGAATGGAGCTGGGTCTTTCTCTTCT 
               
               
                   
                 of Human 
                 TCCTGTCAGTAACGACTGGTGTCCACTC 
               
               
                   
                 HPTP-η with C- 
                 CGCAGGTGGCACCCCTAGTCCAATTCCT 
               
               
                   
                 terminal His 10   
                 GACCCTTCAGTAGCAACTGTTGCCACAG 
               
               
                   
                 tag (SEQ ID 
                 GGGAAAATGGCATAACGCAGATCAGCAG 
               
               
                   
                 NO: 43) 
                 TACAGCAGAATCCTTTCATAAACAGAAT 
               
               
                   
                 (“His 10 ” 
                 GGAACTGGAACACCTCAGGTGGAAACAA 
               
               
                   
                 disclosed as 
                 ACACCAGTGAGGATGGTGAAAGCTCTGG 
               
               
                   
                 SEQ ID NO: 53) 
                 AGCCAACGATAGTTTAAGAACACCTGAA 
               
               
                   
                   
                 CAAGGATCTAATGGGACTGATGGGGCAT 
               
               
                   
                   
                 CTCAAAAAACTCCCAGTAGCACTGGGCC 
               
               
                   
                   
                 CAGTCCTGTGTTTGACATTAAAGCTGTT 
               
               
                   
                   
                 TCCATCAGTCCAACCAATGTGATCTTAA 
               
               
                   
                   
                 CTTGGAAAAGTAATGACACAGCTGCTTC 
               
               
                   
                   
                 TGAGTACAAGTATGTAGTAAAGCATAAG 
               
               
                   
                   
                 ATGGAAAATGAGAAGACAATTACTGTTG 
               
               
                   
                   
                 TGCATCAACCATGGTGTAACATCACAGG 
               
               
                   
                   
                 CTTACGTCCAGCGACTTCATATGTATTC 
               
               
                   
                   
                 TCCATCACTCCAGGAATAGGCAATGAGA 
               
               
                   
                   
                 CTTGGGGAGATCCCAGAGTCATAAAAGT 
               
               
                   
                   
                 CATCACAGAGCCGATCCCAGTTTCTGAT 
               
               
                   
                   
                 CTCCGTGTTGCCCTCACGGGTGTGAGGA 
               
               
                   
                   
                 AGGCTGCTCTCTCCTGGAGCAATGGCAA 
               
               
                   
                   
                 TGGCACTGCCTCCTGCCGGGTTCTTCTT 
               
               
                   
                   
                 GAAAGCATTGGAAGCCATGAGGAGTTGA 
               
               
                   
                   
                 CTCAAGACTCAAGACTTCAGGTCAATAT 
               
               
                   
                   
                 CTCGGGCCTGAAGCCAGGGGTTCAATAC 
               
               
                   
                   
                 AACATCAACCCGTATCTTCTACAATCAA 
               
               
                   
                   
                 ATAAGACAAAGGGAGACCCCTTGGGCAC 
               
               
                   
                   
                 AGAAGGTGGCTTGGATGCCAGCAATACA 
               
               
                   
                   
                 GAGAGAAGCCGGGCAGGGAGCCCCACCG 
               
               
                   
                   
                 CCCCTGTGCATGATGAGTCCCTCGTGGG 
               
               
                   
                   
                 ACCTGTGGACCCATCCTCCGGCCAGCAG 
               
               
                   
                   
                 TCCCGAGACACGGAAGTCCTGCTTGTCG 
               
               
                   
                   
                 GGTTAGAGCCTGGCACCCGATACAATGC 
               
               
                   
                   
                 CACCGTTTATTCCCAAGCAGCGAATGGC 
               
               
                   
                   
                 ACAGAAGGACAGCCCCAGGCCATAGAGT 
               
               
                   
                   
                 TCAGGACAAATGCTATTCAGGTTTTTGA 
               
               
                   
                   
                 CGTCACCGCTGTGAACATCAGTGCCACA 
               
               
                   
                   
                 AGCCTGACCCTGATCTGGAAAGTCAGCG 
               
               
                   
                   
                 ATAACGAGTCGTCATCTAACTATACCTA 
               
               
                   
                   
                 CAAGATACATGTGGCGGGGGAGACAGAT 
               
               
                   
                   
                 TCTTCCAATCTCAACGTCAGTGAGCCTC 
               
               
                   
                   
                 GCGCTGTCATCCCCGGACTCCGCTCCAG 
               
               
                   
                   
                 CACCTTCTACAACATCACAGTGTGTCCT 
               
               
                   
                   
                 GTCCTAGGTGACATCGAGGGCACGCCGG 
               
               
                   
                   
                 GCTTCCTCCAAGTGCACACCCCCCCTGT 
               
               
                   
                   
                 TCCAGTTTCTGACTTCCGAGTGACAGTG 
               
               
                   
                   
                 GTCAGCACGACGGAGATCGGCTTAGCAT 
               
               
                   
                   
                 GGAGCAGCCATGATGCAGAATCATTTCA 
               
               
                   
                   
                 GATGCATATCACACAGGAGGGAGCTGGC 
               
               
                   
                   
                 AATTCTCGGGTAGAAATAACCACCAACC 
               
               
                   
                   
                 AAAGTATTATCATTGGTGGCTTGTTCCC 
               
               
                   
                   
                 TGGAACCAAGTATTGCTTTGAAATAGTT 
               
               
                   
                   
                 CCAAAAGGACCAAATGGGACTGAAGGGG 
               
               
                   
                   
                 CATCTCGGACAGTTTGCAATAGAACTGT 
               
               
                   
                   
                 TCCCAGTGCAGTGTTTGACATCCACGTG 
               
               
                   
                   
                 GTCTACGTCACCACCACGGAGATGTGGC 
               
               
                   
                   
                 TGGACTGGAAGAGCCCTGACGGTGCTTC 
               
               
                   
                   
                 CGAGTATGTCTACCATTTAGTCATAGAG 
               
               
                   
                   
                 TCCAAGCATGGCTCTAACCACACAAGCA 
               
               
                   
                   
                 CGTATGACAAAGCGATTACTCTCCAGGG 
               
               
                   
                   
                 CCTGATTCCGGGCACCTTATATAACATC 
               
               
                   
                   
                 ACCATCTCTCCAGAAGTGGACCACGTCT 
               
               
                   
                   
                 GGGGGGACCCCAACTCCACTGCACAGTA 
               
               
                   
                   
                 CACACGGCCCAGCAATGTGTCCAACATT 
               
               
                   
                   
                 GATGTAAGTACCAACACCACAGCAGCAA 
               
               
                   
                   
                 CTTTAAGTTGGCAGAACTTTGATGACGC 
               
               
                   
                   
                 CTCTCCCACGTACTCCTACTGCCTTCTT 
               
               
                   
                   
                 ATTGAGAAGGCTGGAAATTCCAGCAACG 
               
               
                   
                   
                 CAACACAAGTAGTCACGGACATTGGAAT 
               
               
                   
                   
                 TACTGACGCTACAGTCACTGAATTAATA 
               
               
                   
                   
                 CCTGGCTCATCATACACAGTGGAGATCT 
               
               
                   
                   
                 TTGCACAAGTAGGGGATGGGATCAAGTC 
               
               
                   
                   
                 ACTGGAACCTGGCCGGAAGTCATTCTGT 
               
               
                   
                   
                 ACAGATCCTGCGTCCATGGCCTCCTTCG 
               
               
                   
                   
                 ACTGCGAAGTGGTCCCCAAAGAGCCAGC 
               
               
                   
                   
                 CCTGGTTCTCAAATGGACCTGCCCTCCT 
               
               
                   
                   
                 GGCGCCAATGCAGGCTTTGAGCTGGAGG 
               
               
                   
                   
                 TCAGCAGTGGAGCCTGGAACAATGCGAC 
               
               
                   
                   
                 CCACCTGGAGAGCTGCTCCTCTGAGAAT 
               
               
                   
                   
                 GGCACTGAGTATAGAACGGAAGTCACGT 
               
               
                   
                   
                 ATTTGAATTTTTCTACCTCGTACAACAT 
               
               
                   
                   
                 CAGCATCACCACTGTGTCCTGTGGAAAG 
               
               
                   
                   
                 ATGGCAGCCCCCACCCGGAACACCTGCA 
               
               
                   
                   
                 CTACTGGCATCACAGATCCCCCTCCTCC 
               
               
                   
                   
                 AAGATGGATCCCCTATATTACATCTGTC 
               
               
                   
                   
                 AGTCACAATTCAGTAAAGGTCAAGTTCA 
               
               
                   
                   
                 GTGGATTTGAAGCCAGCCACGGACCCAT 
               
               
                   
                   
                 CAAAGCCTATGCTGTCATTCTCACCACC 
               
               
                   
                   
                 GGGGAAGCTGGTCACCCTTCTGCAGATG 
               
               
                   
                   
                 TCCTGAAATACACGTATGAGGATTTCAA 
               
               
                   
                   
                 AAAGGGAGCCTCAGATACTTATGTGACA 
               
               
                   
                   
                 TACCTCATAAGAACAGAAGAAAAGGGAC 
               
               
                   
                   
                 GTTCTCAGAGCTTGTCTGAAGTTTTGAA 
               
               
                   
                   
                 ATATGAAATTGACGTTGGGAATGAGTCA 
               
               
                   
                   
                 ACCACACTTGGTTATTACAATGGGAAGC 
               
               
                   
                   
                 TGGAACCTCTGGGCTCCTACCGGGCTTG 
               
               
                   
                   
                 TGTGGCTGGCTTCACCAACATTACCTTC 
               
               
                   
                   
                 CACCCTCAAAACAAGGGGCTCATTGATG 
               
               
                   
                   
                 GGGCTGAGAGCTATGTGTCCTTCAGTCG 
               
               
                   
                   
                 CTACTCAGATGCTGTTTCCTTGCCCCAG 
               
               
                   
                   
                 GATCCAGGTGTCATCTGTGGCGGTGGAC 
               
               
                   
                   
                 ACCACCATCATCACCATCACCACCATCA 
               
               
                   
                   
                 CTAG 
               
               
                   
               
            
           
         
       
     
     HPTP-β, cyno PTP-β, and HPTP-η proteins were coated onto ELISA plates in coating buffer (pH 9.4). Each protein was prepared as a 1 pg/mL protein stock in 50 mM carbonate-bicarbonate. The coated proteins were aliquoted at a volume of 100 μL/well into sterile, clear, polystyrene, flat bottom 96-well plates. The plates were incubated overnight at 4° C. After overnight incubation, the plates were washed 3× with 200 μL/well of 1×PBS-T (PBS with 0.05% Tween 20). The immunizing protein was human VE-PTP (HPTP-β). The plates were then blocked with 200 μL/well of 5% blotting grade blocker non-fat dry milk in PBS-T and incubated for 1 hr at room temperature. 
     Primary antibody samples were prepared for the humanized variants and the negative control, anti-hCD20-hIgG4S228P, using 1 pg/well in 5% blotting grade blocker non-fat dry milk in PBS. Samples were tested in triplicates. The Fab antibody fragments were treated as the IgG antibodies. The plates were then washed 3× with PBS-T and 100 μL of primary antibody preparation was added in each well. The plates were then incubated for 1 hr at room temperature. 
     Secondary antibody samples were prepared by diluting peroxidase-conjugated affinity purified donkey anti-human IgG specific secondary antibody at 1:2,000 dilution in blotting grade blocker non-fat dry milk in PBS. The plates were then washed 3× with PBS-T and 100 μL of the secondary antibody preparation was added in each well. The plates were then incubated for 1 hr at room temperature. After incubation, the plates were washed 3× with PBS-T. 
     Then, 75 pg/1-Step™ Ultra TMB-ELISA was added in each well, and the plate was allowed to develop for 5 min at room temperature. After 5 min, 50 μL of 2 M sulfuric acid was added to each well to stop the reaction. The plates were then analyzed on a plate reader at an absorbance wavelength of 450 nm. 
       FIG.  5    illustrates western blots of R15E6 screened against species-specific VE-PTP and HPTP-η. R15E6 was highly selective for human VE-PTP (HPTP-β; lanes 1 and 4). No significant binding to human PTP-η or cyno VE-PTP (cyno PTP-β) was observed. The indicated recombinant hexahistidine-tagged proteins (6-His) (SEQ ID NO: 52) were probed sequentially with R15E6 and a commercially-available 6-His antibody (“6-His” is SEQ ID NO: 52). The commercially-available 6-His antibody (“6-His” is SEQ ID NO: 52) exhibited binding to all protein targets, and showed poor selectivity for human VE-PTP (HPTP-β). 
     Each humanized variant was measured for affinity to the antigen, human VE-PTP (HPTP-β), using ELISA. 100 mg/well of the VE-PTP (HPTP-β) antigen were immobilized onto 96-well Maxisorp plates in coating buffer (0.5 mM NaHCO 3  brought to pH 9.6 by the addition of 0.5 mM Na 2 CO 3 ) overnight at 4° C. The coating buffer was then removed and 200 μL/well of block solution (3% w/v semi-skimmed milk powder, PBS) was added and agitated for 2 hrs at room temperature. The plate was washed four times with PBS-T (1% v/v Tween 20). 100 μL/well was added of purified R15E6 variants serially diluted from 10,000 ng/mL to 9.765 ng/mL in PBS-TB (0.05% v/v Tween 20, 0.5% w/v BSA). Following agitation for 2 hrs at room temperature, the plate was washed 4× with PBS-T. 100 μL/well goat anti-human HRP (Fc specific) at a ratio of 1:60,000 PBS was then added and the plates were incubated for an additional hour with agitation at room temperature. The plate was then washed 6× with PBS-T and once in PBS. Next, 100 μL/well of TMB substrate solution was added and incubated at 37° C. for 10 min. 50 μL 1 M HCl was added per well and the plate immediately read at 450 nm on a Tecan Sunrise plate reader. The ELISA results are shown in  FIGS.  14 - 17   . The HC1, HC2 and HC3 variants generally displayed similar binding characteristics compared to the chimeric HCOLC0 antibody. The binding of the HC4 variants was much lower than with the other heavy chains variants compared to HCOLC0 ( FIG.  17   ). 
     Samples of each variant were further assessed by Biacore analysis to determine the binding affinity. Antibody binding experiments were performed on Biacore 3000 at 25° C. Assay buffer: 10 mM HEPES buffer (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.05% P20 (polyoxyethylenesorbitan). Regeneration buffer: 10 mM glycine HCl (pH 1.75). Conjugation buffer: 10 mM sodium acetate buffer (pH 5). The flow rate used for capturing the ligand was 5 μL/min. The flow rate for the kinetic analysis was 50 μL/min. Flow cell 1 and 2 of the CMS chip were coated with maximum amounts of goat anti-human IgG. Approximately 150 RU of the test antibody was captured on flow cell 2, 3, and 4 as indicated. The antigen was flowed over the chip. Binding of the antigen to the antibodies was monitored in real time. The K D  was determined from the observed k on  and k off  values. 
     A scouting analysis was performed using single analyte concentrations as indicated. At these concentrations, binding can be observed even if the ligand binding is weak. Flow cell 1 response was used for reference subtraction. 
     A full kinetic analysis was performed for the control antibody with a 2-fold serial dilution with the range of concentrations the analyte as indicated: 50, 25, 12.5, 6.25, 3.125, and 0 nM. 
     TABLE 17 shows the binding kinetic parameters of the binding of each humanized antibody to VE-PTP (HPTP-β) ECD, including the association rate (k a ), dissociation rate (k d ), maximum binding capacity (R max ), the association constant (K A ), and the dissociation constant (K D ) as determined by Biacore binding assays. R max  was measured as the relative response (RU). All of the humanized candidates exhibited similar binding affinities as the mouse/human chimeric antibody, HC0LC0. 
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                 TABLE 17 
               
               
                   
               
               
                   
                   
                   
                   
                   
                 Conc. Of 
                   
                   
               
               
                 Ligand 
                 Analyte 
                 k a  (1/Ms) 
                 k d  (1/s) 
                 R max  (RU) 
                 Analyte 
                 K A  (1/M) 
                 K D  (M) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 HC0:LC0 
                 ½ VE-PTP 
                 2.09 × 10 5   
                 6.56 × 10 −4   
                 27.2 
                 50-0 
                 nM 
                 3.18 × 10 8   
                 3.14 × 10 −9   
               
               
                 (150RU) 
                 ECD 
                 6.71 × 10 4   
                 4.17 × 10 −4   
                 69.6 
                 100 
                 nM 
                 1.61 × 10 8   
                 6.22 × 10 −9   
               
               
                 HC2:LC4 
                 ½ VE-PTP 
                 9.86 × 10 5   
                 4.29 × 10 −4   
                 13.9 
                 50-0 
                 nM 
                 2.30 × 10 9   
                     4.36 × 10 −10   
               
               
                 (145RU) 
                 ECD 
                 5.15 × 10 4   
                 6.68 × 10 −4   
                 67.3 
                 100 
                 nM 
                 7.72 × 10 7   
                 1.30 × 10 −8   
               
               
                 HC2:LC1 
                 ½ VE-PTP 
                 1.08 × 10 6   
                 7.23 × 10 −5   
                 8.38 
                 50-0 
                 nM 
                     1.5 × 10 10   
                     6.68 × 10 −11   
               
               
                 (140RU) 
                 ECD 
                 4.98 × 10 4   
                 6.88 × 10 −4   
                 54.2 
                 100 
                 nM 
                 7.24 × 10 7   
                 1.38 × 10 −8   
               
               
                 HC1:LC2 
                 ½ VE-PTP 
                 4.10 × 10 5   
                 9.47 × 10 −4   
                 29.6 
                 50-0 
                 nM 
                 4.33 × 10 8   
                 2.31 × 10 −9   
               
               
                 (150RU) 
                 ECD 
                 2.51 × 10 5   
                 7.69 × 10 −4   
                 52.4 
                 100 
                 nM 
                 3.27 × 10 8   
                 3.06 × 10 −9   
               
               
                 HC2:LC3 
                 ½ VE-PTP 
                 8.90 × 10 5   
                 1.23 × 10 −3   
                 22.6 
                 25-0 
                 nM 
                 6.75 × 10 8   
                 1.48 × 10 −9   
               
               
                 (145RU) 
                 ECD 
                 2.22 × 10 5   
                 6.62 × 10 −4   
                 38.8 
                 100 
                 nM 
                 3.36 × 10 8   
                 2.98 × 10 −9   
               
               
                 HC3:LC2 
                 ½ VE-PTP 
                 5.39 × 10 4   
                 1.23 × 10 −3   
                 104 
                 25-0 
                 nM 
                 4.30 × 10 7   
                 2.32 × 10 −8   
               
               
                 (140RU) 
                 ECD 
                 2.05 × 10 5   
                 9.93 × 10 −4   
                 36.8 
                 100 
                 nM 
                 2.06 × 10 8   
                 4.84 × 10 −9   
               
               
                 HC1:LC4 
                 ½ VE-PTP 
                 5.96 × 10 5   
                 1.34 × 10 −3   
                 24.6 
                 50-0 
                 nM 
                 4.44 × 10 8   
                 2.25 × 10 −9   
               
               
                 (150RU) 
                 ECD 
                 1.79 × 10 5   
                 7.92 × 10 −4   
                 49 
                 100 
                 nM 
                 2.26 × 10 8   
                 4.42 × 10 −9   
               
               
                 HC2:LC2 
                 ½ VE-PTP 
                 4.25 × 10 4   
                 2.15 × 10 −3   
                 313 
                 25-0 
                 nM 
                 1.98 × 10 7   
                 5.06 × 10 −8   
               
               
                 (145RU) 
                 ECD 
                 1.49 × 10 5   
                 5.24 × 10 −4   
                 40.8 
                 100 
                 nM 
                 2.85 × 10 8   
                 3.51 × 10 −9   
               
               
                 HC3:LC4 
                 ½ VE-PTP 
                 1.34 × 10 5   
                 1.09 × 10 −3   
                 42.8 
                 25-0 
                 nM 
                 1.23 × 10 8   
                 8.14 × 10 −9   
               
               
                 (140RU) 
                 ECD 
                 1.39 × 10 5   
                 8.87 × 10 −4   
                 44.7 
                 100 
                 nM 
                 1.59 × 10 8   
                 6.29 × 10 −9   
               
               
                   
               
            
           
         
       
     
     TABLE 18 shows the binding selectivity of the humanized antibodies compared to the parental antibody (R15E6) and the inactive control (IgG4) as determined by ELISA. A value that is less than or equal to 0.2 signifies no binding. The humanized antibody candidates, the parental R15E6, and the mouse/human chimeric antibody (HC0:LC0) all exhibited high selectivity for human VE-PTP (HPTP-β) as indicated by no significant binding to human PTP-η and cyno PTP-β. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 18 
               
               
                   
               
               
                   
                 Clone 
                 Human 
                 Human 
                 Cyno 
               
               
                   
                 (1 μg/well) 
                 VE-PTP 
                 PTPη 
                 VE-PTP 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 HC0:LC0 
                 1.8 
                 0.16 
                 0.12 
               
               
                   
                 HC1:LC2 
                 1.75 
                 0.12 
                 0.13 
               
               
                   
                 HC1:LC4 
                 1.74 
                 0.12 
                 0.19 
               
               
                   
                 HC2:LC1 
                 1.81 
                 0.13 
                 0.18 
               
               
                   
                 HC2:LC2 
                 1.92 
                 0.12 
                 0.12 
               
               
                   
                 HC2:LC3 
                 1.55 
                 0.12 
                 0.13 
               
               
                   
                 HC2:LC4 
                 1.65 
                 0.11 
                 0.13 
               
               
                   
                 HC3:LC4 
                 2.00 
                 0.11 
                 0.14 
               
               
                   
                 Parental 
                 2.43 
                 0.14 
                 0.17 
               
               
                   
                 IgG4 
                 0.20 
                 0.13 
                 0.16 
               
               
                   
               
            
           
         
       
     
     The humanized antibodies used for targeting human VE-PTP (HPTP-β) can restore Tie2 activity by autophosphorylation and initiate downstream effectors including, for example, proteins in the PI3K-Akt pathway. In addition to assessing binding of the antibodies to human VE-PTP (HPTP-β), the ability of the antibodies to activate these effector proteins was measured.  FIG.  18    illustrates the activation of Tie2 (left panel) and Akt (right panel) in the absence of Tie2 ligands (e.g., Ang1 or Ang2) by the humanized variants as determined by western blot. The activities of the humanized variants were similar to that of the parental murine antibody R15E6 and the chimeric antibody HC0LC0. Human IgG4 was used as a control. All antibodies were tested at 50 nM. The top panel of each blot shows phosphorylation of either Tie2 or Akt, indicating Tie2 or Akt activation. Tie2 and Akt exhibited greater phosphorylation in samples treated with the humanized antibodies than those samples treated with the control antibodies, and indicated that the humanized antibodies were effective in activating Tie2 and Akt even in the absence of ligands. 
       FIG.  19    illustrates concentration-dependent activation of Tie2 (top panel) and Akt (bottom panel) in the absence of Tie2 ligands (e.g., Ang1 or Ang2) by humanized antibodies, HC2LC4 and HC1LC1 as determined by western blot. Tie2 and Akt signaling activity was observed at low nanomolar concentrations of the humanized variants. The upper panel of each blot shows phosphorylation of either Tie2 or Akt, indicating Tie2 or Akt activation. Tie2 and Akt exhibited greater phosphorylation in the samples treated with the humanized antibodies than exhibited in samples treated with the control antibodies. This result indicates that the humanized antibodies were effective in activating Tie2 and Akt even in the absence of Ang1. 
       FIG.  20    illustrates the activation of Tie2 (left panel) and Akt (right panel) in the presence of Ang1 and Ang2 by the humanized antibody candidates, HC2LC4 and HC1LC1, as determined by western blot. HC2LC4 and HC1LC1 activated both Tie2 and Akt in the presence of Ang2, and enhanced Ang1-mediated Tie2 and Akt activation, as indicated by the increase in Tie2 and Akt phosphorylation, respectively. All antibodies were tested at 10 nM. 
       FIG.  21    illustrates the activation of Tie2 (panel A), human VE-PTP (HPTP-β) expression (panel B), and Akt (panel C) in the presence of Ang1 or Ang2 by the humanized antibody candidates, HC2LC1 and HC2LC4, as determined by western blot. HC2LC1 and HC2LC4 activated Tie2 in the presence of Ang1 alone, Ang2 alone, and in the presence of both Ang1 and Ang2. The antibodies were pre-incubated for 15 min and then stimulated by Ang1 or Ang2 for 10 min at a concentration of 600 ng/mL. All antibodies were tested at 10 nM. 
       FIG.  22    illustrates the activation of Akt in HUVECs using the humanized antibody candidate, HC2LC1. The upper panel of the first 3 blots shows phosphorylation of Akt, indicating Akt activation, at increasing concentrations of HC2LC1. Consistent with the binding affinity study, HC2LC1 activated Akt at nanomolar concentrations of antibody. Akt activation by HC2LC1 was blocked by pre-incubation of the antibody with the human VE-PTP (HPTP-β) extracellular domain (βECD 6His (“6-His” is SEQ ID NO: 52) and βECD Fc). This result indicates that HC2LC1-mediated Akt activation occurs through HC2LC1 binding to the human VE-PTP (HPTP-β) extracellular domain. 
       FIG.  23    illustrates the enhanced viability in serum-starved HUVECs using humanized antibody candidates HC2LC1 and HC0LC0, as compared to the parental antibody, R15E6, and the mIgG1 control. 
     EMBODIMENTS 
     Embodiment 1. A compound comprising a sequence that is at least 80% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. 
     Embodiment 2. The compound of embodiment 1, wherein the sequence is at least 85% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. 
     Embodiment 3. The compound of any one of embodiments 1-2, wherein the sequence is at least 90% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. 
     Embodiment 4. The compound of any one of embodiments 1-3, wherein the sequence is at least 95% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. 
     Embodiment 5. The compound of embodiment 1, wherein the sequence is at least 80% identical to SEQ ID NO: 9. 
     Embodiment 6. The compound of any one of embodiments 1-5, wherein the sequence is SEQ ID NO: 9. 
     Embodiment 7. The compound of embodiment 1, wherein the sequence is at least 80% identical to SEQ ID NO: 10. 
     Embodiment 8. The compound of any one of embodiments 1˜4 and 7, wherein the sequence is SEQ ID NO: 10. 
     Embodiment 9. The compound of embodiment 1, wherein the sequence is at least 80% identical to SEQ ID NO: 11. 
     Embodiment 10. The compound of any one of embodiments 1˜4 and 9, wherein the sequence is SEQ ID NO: 11. 
     Embodiment 11. The compound of embodiment 1, wherein the sequence is at least 80% identical to SEQ ID NO: 12. 
     Embodiment 12. The compound of any one of embodiments 1˜4 and 11, wherein the sequence is SEQ ID NO: 12. 
     Embodiment 13. The compound of embodiment 1, wherein the sequence is at least 80% identical to SEQ ID NO: 29. 
     Embodiment 14. The compound of embodiment 1, wherein the sequence is SEQ ID NO: 
     29. 
     Embodiment 15. The compound of embodiment 1, wherein the sequence is at least 80% identical to SEQ ID NO: 30. 
     Embodiment 16. The compound of embodiment 1, wherein the sequence is SEQ ID NO: 30. 
     Embodiment 17. The compound of embodiment 1, wherein the sequence is at least 80% identical to SEQ ID NO: 31. 
     Embodiment 18. The compound of embodiment 1, wherein the sequence is SEQ ID NO: 31. 
     Embodiment 19. The compound of embodiment 1, wherein the sequence is at least 80% identical to SEQ ID NO: 32. 
     Embodiment 20. The compound of embodiment 1, wherein the sequence is SEQ ID NO: 32. 
     Embodiment 21. The compound of any one of embodiments 1-20, wherein the compound inhibits a tyrosine phosphatase. 
     Embodiment 22. The compound of any one of embodiments 1-21, wherein the compound inhibits HPTP-β. 
     Embodiment 23. The compound of any one of embodiments 1-22, wherein the compound inhibits VE-PTP. 
     Embodiment 24. The compound of any one of embodiments 1-23, wherein the compound activates Tie2. 
     Embodiment 25. The compound of any one of embodiments 1-24, wherein the compound activates Akt. 
     Embodiment 26. The compound of any one of embodiments 1-25, wherein the compound binds an extracellular domain of HPTP-β. 
     Embodiment 27. The compound of any one of embodiments 1-26, wherein the compound binds the first FN3 repeat of an extracellular domain of HPTP-β. 
     Embodiment 28. The compound of any one of embodiments 1-27, wherein a binding affinity (K D ) of the compound to the extracellular domain of HPTP-β is about 70 μM to about 70 nM. 
     Embodiment 29. A compound comprising a sequence that is at least 80% identical to SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. 
     Embodiment 30. The compound of embodiment 29, wherein the sequence is at least 85% identical to SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. 
     Embodiment 31. The compound of any one of embodiments 29-30, wherein the sequence is at least 90% identical to SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. 
     Embodiment 32. The compound of any one of embodiments 29-31, wherein the sequence is at least 95% identical to SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. 
     Embodiment 33. The compound of embodiment 29, wherein the sequence is at least 80% identical to SEQ ID NO: 20. 
     Embodiment 34. The compound of any one of embodiments 29-33, wherein the sequence is SEQ ID NO: 20. 
     Embodiment 35. The compound of embodiment 29, wherein the sequence is at least 80% identical to SEQ ID NO: 21. 
     Embodiment 36. The compound of any one of embodiments 29-32 and 35, wherein the sequence is SEQ ID NO: 21. 
     Embodiment 37. The compound of embodiment 29, wherein the sequence is at least 80% identical to SEQ ID NO: 22. 
     Embodiment 38. The compound of any one of embodiments 29-32 and 37, wherein the sequence is SEQ ID NO: 22. 
     Embodiment 39. The compound of embodiment 29, wherein the sequence is at least 80% identical to SEQ ID NO: 23. 
     Embodiment 40. The compound of any one of embodiments 29-32 and 39, wherein the sequence is SEQ ID NO: 23. 
     Embodiment 41. The compound of embodiment 29, wherein the sequence is at least 80% identical to SEQ ID NO: 34. 
     Embodiment 42. The compound of embodiment 29, wherein the sequence is SEQ ID NO: 34. 
     Embodiment 43. The compound of embodiment 29, wherein the sequence is at least 80% identical to SEQ ID NO: 35. 
     Embodiment 44. The compound of embodiment 29, wherein the sequence is SEQ ID NO: 35. 
     Embodiment 45. The compound of embodiment 29, wherein the sequence is at least 80% identical to SEQ ID NO: 36. 
     Embodiment 46. The compound of embodiment 29, wherein the sequence is SEQ ID NO: 36. 
     Embodiment 47. The compound of embodiment 29, wherein the sequence is at least 80% identical to SEQ ID NO: 37. 
     Embodiment 48. The compound of embodiment 29, wherein the sequence is SEQ ID NO: 37. 
     Embodiment 49. The compound of any one of embodiments 29-48, wherein the compound inhibits a tyrosine phosphatase. 
     Embodiment 50. The compound of any one of embodiments 29-49, wherein the compound inhibits HPTP-β. 
     Embodiment 51. The compound of any one of embodiments 29-50, wherein the compound inhibits VE-PTP. 
     Embodiment 52. The compound of any one of embodiments 29-51, wherein the compound activates Tie2. 
     Embodiment 53. The compound of any one of embodiments 29-52, wherein the compound activates Akt. 
     Embodiment 54. The compound of any one of embodiments 29-53, wherein the compound binds an extracellular domain of HPTP-β. 
     Embodiment 55. The compound of any one of embodiments 29-54, wherein the compound binds the first FN3 repeat of an extracellular domain of HPTP-β. 
     Embodiment 56. The compound of any one of embodiments 29-55, wherein a binding affinity (K D ) of the compound to the extracellular domain of HPTP-β is about 70 pM to about 70 nM. 
     Embodiment 57. A compound comprising: a) a heavy chain that comprises a sequence that is at least 80% identical to SEQ ID NO: 30; and b) a light chain that comprises a sequence that is at least 80% identical to SEQ ID NO: 34. 
     Embodiment 58. The compound of embodiment 57, wherein the heavy chain is at least 85% identical to SEQ ID NO: 30; and the light chain is at least 85% identical to SEQ ID NO: 34. 
     Embodiment 59. The compound of any one of embodiments 57-58, wherein the heavy chain is at least 90% identical to SEQ ID NO: 30; and the light chain is at least 90% identical to SEQ ID NO: 34. 
     Embodiment 60. The compound of any one of embodiments 57-59, wherein the heavy chain is at least 95% identical to SEQ ID NO: 30; and the light chain is at least 95% identical to SEQ ID NO: 34. 
     Embodiment 61. The compound of any one of embodiments 57-60, wherein the heavy chain is SEQ ID NO: 30 and the light chain is SEQ ID NO: 34. 
     Embodiment 62. The compound of any one of embodiments 57-61, wherein the compound inhibits a tyrosine phosphatase. 
     Embodiment 63. The compound of any one of embodiments 57-62, wherein the compound inhibits HPTP-β. 
     Embodiment 64. The compound of any one of embodiments 57-63, wherein the compound inhibits VE-PTP. 
     Embodiment 65. The compound of any one of embodiments 57-64, wherein the compound activates Tie2. 
     Embodiment 66. The compound of any one of embodiments 57-65, wherein the compound activates Akt. 
     Embodiment 67. The compound of any one of embodiments 57-66, wherein the compound binds an extracellular domain of HPTP-β. 
     Embodiment 68. The compound of any one of embodiments 57-67, wherein the compound binds the first FN3 repeat of an extracellular domain of HPTP-β. 
     Embodiment 69. The compound of any one of embodiments 57-68, wherein a binding affinity (K D ) of the compound to the extracellular domain of HPTP-β is about 70 pM to about 70 nM. 
     Embodiment 70. A compound comprising: a) a heavy chain that comprises a sequence that is at least 80% identical to SEQ ID NO: 29; and b) a light chain that comprises a sequence that is at least 80% identical to SEQ ID NO: 35. 
     Embodiment 71. The compound of embodiment 70, wherein the heavy chain is at least 85% identical to SEQ ID NO: 29; and the light chain is at least 85% identical to SEQ ID NO: 35. 
     Embodiment 72. The compound of any one of embodiments 70-71, wherein the heavy chain is at least 90% identical to SEQ ID NO: 29; and the light chain is at least 90% identical to SEQ ID NO: 35. 
     Embodiment 73. The compound of any one of embodiments 70-72, wherein the heavy chain is at least 95% identical to SEQ ID NO: 29; and the light chain is at least 95% identical to SEQ ID NO: 35. 
     Embodiment 74. The compound of any one of embodiments 70-73, wherein the heavy chain is SEQ ID NO: 29 and the light chain is SEQ ID NO: 35. 
     Embodiment 75. The compound of any one of embodiments 70-74, wherein the compound inhibits a tyrosine phosphatase. 
     Embodiment 76. The compound of any one of embodiments 70-75, wherein the compound inhibits HPTP-β. 
     Embodiment 77. The compound of any one of embodiments 70-76, wherein the compound inhibits VE-PTP. 
     Embodiment 78. The compound of any one of embodiments 70-77, wherein the compound activates Tie2. 
     Embodiment 79. The compound of any one of embodiments 70-78, wherein the compound activates Akt. 
     Embodiment 80. The compound of any one of embodiments 70-79, wherein the compound binds an extracellular domain of HPTP-β. 
     Embodiment 81. The compound of any one of embodiments 70-80, wherein the compound binds the first FN3 repeat of an extracellular domain of HPTP-β. 
     Embodiment 82. The compound of any one of embodiments 70-81, wherein a binding affinity (K D ) of the compound to the extracellular domain of HPTP-β is about 70 pM to about 70 nM. 
     Embodiment 83. A compound comprising: a) a heavy chain that comprises a sequence that is at least 80% identical to SEQ ID NO: 30; and b) a light chain that comprises a sequence that is at least 80% identical to SEQ ID NO: 37. 
     Embodiment 84. The compound of embodiment 83, wherein the heavy chain is at least 85% identical to SEQ ID NO: 30; and the light chain is at least 85% identical to SEQ ID NO: 37. 
     Embodiment 85. The compound of any one of embodiments 83-84, wherein the heavy chain is at least 90% identical to SEQ ID NO: 30; and the light chain is at least 90% identical to SEQ ID NO: 37. 
     Embodiment 86. The compound of any one of embodiments 83-85, wherein the heavy chain is at least 95% identical to SEQ ID NO: 30; and the light chain is at least 95% identical to SEQ ID NO: 37. 
     Embodiment 87. The compound of any one of embodiments 83-86, wherein the heavy chain is SEQ ID NO: 30 and the light chain is SEQ ID NO: 37. 
     Embodiment 88. The compound of any one of embodiments 83-87, wherein the compound inhibits a tyrosine phosphatase. 
     Embodiment 89. The compound of any one of embodiments 83-88, wherein the compound inhibits HPTP-β. 
     Embodiment 90. The compound of any one of embodiments 83-89, wherein the compound inhibits VE-PTP. 
     Embodiment 91. The compound of any one of embodiments 83-90, wherein the compound activates Tie2. 
     Embodiment 92. The compound of any one of embodiments 83-91, wherein the compound activates Akt. 
     Embodiment 93. The compound of any one of embodiments 83-92, wherein the compound binds an extracellular domain of HPTP-β. 
     Embodiment 94. The compound of any one of embodiments 83-93, wherein the compound binds the first FN3 repeat of an extracellular domain of HPTP-β. 
     Embodiment 95. The compound of any one of embodiments 83-94, wherein a binding affinity (K D ) of the compound to the extracellular domain of HPTP-β is about 70 pM to about 70 nM. 
     Embodiment 96. A method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of the compound of any one of embodiments 1-95. 
     Embodiment 97. The method of embodiment 96, wherein the condition is an ocular condition. 
     Embodiment 98. The method of embodiment 96 or 97, wherein the condition is diabetic retinopathy. 
     Embodiment 99. The method of any one of embodiments 96-97, wherein the condition is neovascularization. 
     Embodiment 100. The method of any one of embodiments 96-97, wherein the condition is vascular leak. 
     Embodiment 101. The method of any one of embodiments 96-97, wherein the condition is increased intraocular pressure. 
     Embodiment 102. The method of any one of embodiments 96-97, wherein the condition is ocular edema. 
     Embodiment 103. The method of embodiment 96 or 97, wherein the condition is diabetic macular edema. 
     Embodiment 104. The method of any one of embodiment 96 or 97, wherein the condition is ocular hypertension. 
     Embodiment 105. The method of embodiment 96 or 97, wherein the condition is ocular inflammation. 
     Embodiment 106. The method of any one of embodiments 96-105, wherein the administration is to an eye of the subject. 
     Embodiment 107. The method of any one of embodiments 96-106, wherein the administration is intravitreal. 
     Embodiment 108. The method of any one of embodiments 96-105, wherein the administration is subcutaneous. 
     Embodiment 109. The method of any one of embodiments 96-106, wherein the administration is topical. 
     Embodiment 110. The method of any one of embodiments 96-109, wherein the subject is a human. 
     Embodiment 111. The method of any one of embodiments 96-110, wherein the therapeutically-effective amount of the compound is from about 0.25 mg to about 200 mg. 
     Embodiment 112. The method of any one of embodiments 96-110, wherein the therapeutically-effective amount of the compound is from about 1 mg/kg to about 10 mg/kg. 
     Embodiment 113. The method of any one of embodiments 96-110, wherein the therapeutically-effective amount of the compound is from about 1 mg to about 50 mg. 
     Embodiment 114. The method of any one of embodiments 96-110, wherein the therapeutically-effective amount of the compound is from about 50 mg to about 200 mg.