Patent Publication Number: US-2022227881-A1

Title: Humanized Monoclonal Antibody Targeting Pro-N-Cadherin

Description:
TECHNICAL FIELD OF THE INVENTION 
     This invention is related to the area of immunotherapy. In particular, it relates to the use of antibodies for treating diseases associated with misprocessed or mislocated protein. 
     BACKGROUND OF THE INVENTION 
     Fibrosis is the aberrant remodeling of tissue architecture which results in loss of function and inevitably organ failure. It can arise in any organ in the body and manifest from many different disease origins; it is the major and only predictable gross physiological feature of many different diseases that is linearly correlated to organ failure. It is a biologically conserved process regardless of the organ of origin and common endpoint regardless of insult [1]. In the United States, 45% of all deaths can be attributed to some kind of chronic fibrotic related disease [2, 3]. Collectively, fibrotic disease kills more people than cancer. The current scientific theory describes fibrosis as the pathological and constitutive activation state of fibroblasts which result in excessive extracellular matrix turnover and deposition that interferes with normal organ function. Currently, an activated state of these fibroblasts is defined by their acquisition of alpha-smooth muscle actin (α-SMA) protein. Fibroblasts expressing this protein are defined as myofibroblasts, which are an extremely active, synthetic, tissue remodeling cell type. However, a true disease specific marker for these cells does not currently exist. 
     There is a continuing need in the art to detect and eliminate pathological cells involved in fibrosis. 
     SUMMARY OF THE INVENTION 
     According to one aspect of the invention a humanized antibody is provided. It specifically binds to pro-N-cadherin in its pro-domain. The humanized antibody comprises framework portions from a human antibody and six complementarity determining regions (CDRs) from a mouse antibody. The CDRs are SEQ ID NO: 22-27 or SEQ ID NO: 28-33. 
     Another aspect of the invention is a polynucleotide encoding a humanized antibody which binds to pro-N-cadherin in its pro-domain. The polynucleotide comprises segments encoding framework portions of a human antibody and segments encoding six complementarity determining regions (CDRs) from a mouse antibody. The segments encode CDRs having SEQ ID NO: 22-27 or 28-33. The segments may comprise SEQ ID NO: 5-10 or SEQ ID NO: 11-16. 
     Still another aspect of the invention is a method of treating a human with a pathological fibrotic condition. A humanized antibody is administered to the human. The number of pathological fibrotic cells is consequently reduced. The humanized antibody comprises framework portions from a human antibody and six complementarity determining regions (CDRs) from a mouse antibody. The six CDRs are SEQ ID NO: 22-27 or SEQ ID NO: 28-33. 
     Yet another aspect of the invention is a method of treating a human with a tumor. A humanized antibody is administered to the human. The number of tumor cells in the human is consequently reduced. The humanized antibody comprises framework portions from a human antibody and six complementarity determining regions (CDRs) from a mouse antibody. The six CDRs are SEQ ID NO: 22-27 or SEQ ID NO: 28-33. 
     According to another aspect of the invention a chimeric antibody which specifically binds to pro-N-cadherin in its pro-domain is provided. The chimeric antibody comprises framework portions from a non-murine antibody and six complementarity determining regions (CDRs) from a mouse antibody. The CDRs are SEQ ID NO: 22-27 or SEQ ID NO: 28-33. 
     These and other embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with agents and methods for treating fibrotic diseases and tumors. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. 
         FIG. 1  is a schematic of the pro-N-cadherin molecule showing the sequence of nucleotides 92-159 of the pro-domain (SEQ ID NO: 1). The full domain runs from residue 22-159. 
         FIGS. 2A-2F  show that Pro-N-cadherin monoclonal antibody reacts specifically with the pro-domain of pro-N-cadherin from pathological myofibroblasts but not non-pathological.  FIG. 2A . Fibroblasts and myofibroblasts from various origins were stained and analyzed by flow cytometry using pro-N-cadherin mAb (5 ug/mL), excluding dead cells via gating and 7AAD exclusion on Flowjo analysis software. Cell surface pro-N-cadherin positivity was found on myofibroblasts from pathological origins LL97A, CF-DCM, and LX2. Representative data of cardiac myofibroblasts from dilated cardiomyopathy (CF-DCM) were generated from myofibroblasts isolated from two separate explant patient hearts from DUMC. Pro-N-cadherin was not detected on primary normal human lung fibroblasts (NHLF), primary normal human cardiac fibroblasts (NHCF), or immortalized CCD-16Lu myofibroblast cell line from healthy donor.  FIG. 2B . Total protein lysates were analyzed by western blot for each cell line. These corroborate pro-N-cadherin cell surface data.  FIG. 2C . Immunohistochemistry (IHC) performed for pro-N-cadherin was positive in patient tissue from cirrhotic liver and dilated cardiomyopathy but not normal liver.  FIG. 2D . IHC of normal human atrial tissue trimmings from implanted heart (with 10A10 pro-N-cadherin antibody).  FIG. 2E . IHC of fatty liver tissue (with 10A10 pro-N-cadherin antibody).  FIG. 2F . IHC of heart-dilated cardiomyopathy, focus: Interstitial fibroblasts (with 10A10 pro-N-cadherin antibody). 
         FIGS. 3A-3J  show significantly reduced viable cell numbers and proliferation of pathological myofibroblasts as a result of pro-N-cadherin monoclonal antibody treatment at various concentrations.  FIG. 3A . Overnight treatment with pro-N-cadherin monoclonal antibody had no effect on NHLF viable cell numbers but significantly reduced the number of LL97A IPF myofibroblasts.  FIG. 3B . Effects on proliferation from overnight treatment with pro-N-cadherin monoclonal antibody of CF-DCM and LX2 myofibroblasts was analyzed by relative BrdU amount incorporation into newly synthesized DNA.  FIG. 3C . Short duration (1 hr) and time course (0.5-2 hr) treatment of cardiac myofibroblasts from DCM and IPF myofibroblasts, respectively, with pro-N-cadherin monoclonal antibody. LDH activity from the supernatants was used as a marker of cell membrane permeabilization and generalized cell death of myofibroblasts.  FIG. 3D . CF-DCM cells were assessed for cell surface expression of pro-N-cadherin by flow cytometry after overnight pro-N-cadherin monoclonal antibody treatment [0.625 ug/mL] and compared to non-treated control.  FIG. 3E . LX2 hepatic stellate myofibroblasts α-SMA gene expression measured by RT-PCR after overnight pro-N-cadherin monoclonal antibody [2 ug/mL] treatment relative to controls. Representative data from two different pro-N-cadherin monoclonal antibodies are shown.  FIG. 3F . LL97 A a-SMA gene expression measured by rt-PCR after overnight pro-N-cadherin mAb [2 ug/mL] treatment relative to controls and normalized to GAPDH.  FIG. 3G . LX2 cells were plated at 1×10 3  cells/well in 96-well plates and allowed to anchor overnight. Cells were treated with Pro-N-cadherin antibody 10A10 overnight and lifted with trypsin. For each condition, 3 wells were pooled together for each count. Cells were then stained with trypan blue and counted using the Bio-rad TC 20 automated cell counter.  FIG. 3H . Primary cardiac fibroblasts from explant tissue were plated at 1×10 3  cells/well in a 96-well plate. Cells were treated with Pro-N-cadherin antibody 19D8 overnight and lifted with trypsin. Cells were then stained with trypan blue and counted using the Bio-rad TC 20 automated cell counter.  FIG. 3I . Primary cardiac fibroblasts from explant tissue were plated at 1×10 3  cells/well in a 96-well plate. Cells were treated with Pro-N-cadherin antibody 19D8 overnight for 3 nights replacing the media and antibody each day. Cells were then trypsinized and stained with trypan blue and counted using the Bio-rad TC 20 automated cell counter.  FIG. 3J . LL97 A cells were plated at 1×10 3  cells/well in 96-well plates and allowed to anchor overnight. Cells were treated with Pro-N-cadherin antibody 10A10 and BrdU overnight. BrdU incorporation was measured following the manufacturer&#39;s protocol (Millipore Sigma 11647229001). 
         FIGS. 4, 5, and 6 . LL97A Immunostaining. Image showing fibroblast cells expressing pro-N-cadherin (Green—Pro-N-cadherin; Red—Actin Cytoskeleton; Blue—Nuclei; Yellow—Actin/Pro-N-cadherin colocalization). 
         FIG. 7 . Permeabilization of Idiopathic Pulmonary Fibrosis Fibroblast LL97A. LL97A cells were plated at 5×10 4  cells/well in a 6-well plate and allowed to anchor overnight. The following day, the media was replaced with serum free media. Cells were treated with Pro-N-cadherin antibody 10A10 for 4 hours, supernatant was removed and concentrated, and analyzed by western blot. Membrane was probed with LDH antibody. 
         FIGS. 8A-8F  shows immunohistochemistry of 3 patient tissues (some with multiple fields per tissue) stained with an antibody to pro-N-cadherin (PNC). These are representative of 10 patient samples tested and observed, all of which were positive for PNC. PNC is localizing to the alveolar epithelium; this localization indicates that this area of epithelium has undergone an epithelial to mesenchymal transition. 
         FIG. 9 . Pro-N-Cadherin MAb is cytotoxic to fibroblast isolated from idiopathic pulmonary fibrosis. LL97A fibroblasts were plated at 1×10 3  cells/well in 96-well plates and allowed to anchor overnight. Cells were treated with Pro-N-cadherin antibody 10A10 overnight, lifted with trypsin, pooling 6-wells per condition. Cells were then stained with trypan blue and counted using the Bio-Rad TC 20 automated cell counter. 
     
    
    
     A sequence listing and table of sequences forms part of this application.  
     
       
         
           
               
             
               
                 TABLES 
               
             
            
               
                 The patent application contains table(s) that have been included at the end of the specification. 
               
            
           
         
       
     
     DETAILED DESCRIPTION OF THE INVENTION 
     A humanized antibody or a chimeric antibody can be used in the human body with minimal adverse side effects arising from immune reactions to a foreign protein. The humanized or chimeric antibodies directed against pro-N-cadherin surprisingly detect and destroy pathological cells in the body which aberrantly localize pro-N-cadherin to the cell surface without the normal processing to form mature N-cadherin. In some cases, the cytotoxicity of the antibodies is accomplished using an attached cytotoxic moiety, such as a toxin, chemotherapy drug, or radionuclide. 
     Cell-surface located pro-N-cadherin serves as a specific target on a subpopulation of myofibroblasts that only exists in pathological settings. This specific target can distinguish a pathological fibroblast population from surrounding fibroblasts. This specific target of pathological fibroblasts has been found on the cell surface of patient derived myofibroblasts isolated from dilated cardiac myopathy, idiopathic pulmonary fibrosis, and the most well characterized hepatic stellate, myofibroblast cell line LX2, used for studying liver cirrhosis. Importantly, this specific target is not expressed on non-pathologic fibroblasts or myofibroblasts. 
     The specific target is a precursor to N-cadherin (i.e., pro-N-cadherin). Pro-N-cadherin is expressed on the surface of a subpopulation of myofibroblasts derived from several pathological settings of fibrosis, but not on myofibroblasts derived from physiologically normal tissues or on any other normal cell types [4]. It can serve as a disease-specific diagnostic biomarker and as a specific therapeutic target for fibrosis. Pro-N-cadherin is a precursor form of the protein N-cadherin. Pro-N-cadherin is normally processed in the Golgi apparatus of cells by furin proteases to produce the mature form, i.e., N-cadherin, from which the pro-domain has been removed. The processed, mature form is subsequently transported to the cell surface to serve as a cell adhesion molecule [4, 5]. Interestingly, some researchers have reported the presence of pro-N-cadherin on the surface of cancer [6, 7]. This aberrant phenomenon occurs in patient-derived tissues and myofibroblasts from fibrosis of the heart, lung, and liver. Thus, therapeutic targeting of cell-surface expressed pro-N-cadherin is useful for both fibrosis-associated diseases as well as cancers. 
     A murine monoclonal antibody binds to pro-N-cadherin on pathologic myofibroblasts, induces cell death in vitro, and rapidly eliminates this pathologic myofibroblast subpopulation without effecting fibroblasts or myofibroblasts isolated from normal tissue. The specificity of the murine monoclonal antibody is maintained in chimeric and humanized antibodies that share complementarity determining regions and/or variable domains. 
     The term “fibrosis” refers to those diseases/conditions associated with, or characterized by, fibrosis. Examples include, but are not limited to, respiratory conditions such as pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, progressive massive fibrosis, scleroderma, obliterative bronchiolitis, Hermansky-Pudlak syndrome, asbestosis, silicosis, chronic pulmonary hypertension, AIDS associated pulmonary hypertension, sarcoidosis, tumor stroma in lung disease, and asthma; chronic liver disease, primary biliary cirrhosis (PBC), schistosomal liver disease, liver cirrhosis; cardiovascular conditions such as hypertrophic cardiomyopathy, dilated cardiomyopathy (DCM), fibrosis of the atrium, atrial fibrillation, fibrosis of the ventricle, ventricular fibrillation, myocardial fibrosis, Brugada syndrome, myocarditis, endomyocardial fibrosis, myocardial infarction, fibrotic vascular disease, hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC), tubulointerstitial and glomerular fibrosis, atherosclerosis, varicose veins, cerebral infarcts; neurological conditions such as gliosis and Alzheimer&#39;s disease; muscular dystrophy such as Duchenne muscular dystrophy (DMD) or Becker&#39;s muscular dystrophy (BMD); gastrointestinal conditions such as Chron&#39;s disease, microscopic colitis and primary sclerosing cholangitis (PSC); skin conditions such as scleroderma, nephrogenic systemic fibrosis and cutis keloid; arthrofibrosis; Dupuytren&#39;s contracture; mediastinal fibrosis; retroperitoneal fibrosis; myelofibrosis; Peyronie&#39;s disease; adhesive capsulitis; kidney disease (e.g., renal fibrosis, nephritic syndrome, Alport&#39;s syndrome, HIV associated nephropathy, polycystic kidney disease, Fabry&#39;s disease, diabetic nephropathy, chronic glomerulonephritis, nephritis associated with systemic lupus); progressive systemic sclerosis (PSS); chronic graft versus host disease; diseases of the eye such as Grave&#39;s ophthalmopathy, epiretinal fibrosis, retinal fibrosis, subretinal fibrosis (e.g., associated with macular degeneration (e.g., wet age-related macular degeneration (AMD)), diabetic retinopathy, glaucoma, corneal fibrosis, post-surgical fibrosis (e.g., of the posterior capsule following cataract surgery, or of the bleb following trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis; arthritis; fibrotic pre-neoplastic and fibrotic neoplastic disease; and fibrosis induced by chemical or environmental insult (e.g., cancer chemotherapy, pesticides, radiation/cancer radiotherapy). Any of these diseases may be treated with the antibodies to pro-N-cadherin described here. 
     The fibrosis-associated disease/disorder may be one of pulmonary fibrosis, atrial fibrillation, ventricular fibrillation, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), non-alcoholic steatohepatitis (NASH), cirrhosis, chronic kidney disease, scleroderma, systemic sclerosis, keloid, cystic fibrosis, Crohn&#39;s disease, post-surgical fibrosis or retinal fibrosis. Any of these diseases may be treated with the antibodies to pro-N-cadherin described here. 
     Antigen binding fragments of humanized or chimeric antibodies, such as Fab and Fab2 fragments may also be used. The variable heavy (VH) and variable light (VL) domains of the antibody are involved in antigen recognition. Further confirmation was found by “humanization” of rodent antibodies, in which variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parent antibody (see, e.g., Morrison et al (1984) Proc. Natl. Acad. Sd. USA 81, 6851-6855). In some embodiments, complementarity determining regions of the variable domains from a non-human source is substituted into a human antibody framework. Thus, less than the entire variable region is necessary to confer binding specificity. 
     Antibodies may be modified and selected by a process of affinity maturation in which a modified antibody is generated that has an improvement in the affinity of the antibody for antigen, compared to an unmodified parent antibody. Affinity-matured antibodies may be produced by procedures known in the art, e.g., Marks et al., Rio/Technology 10:779-783 (1992); Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):331 0-15 9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992). Affinity may be improved by greater than or equal to 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20-fold by this process. 
     Similarly, antibodies may be modified to remove features that may be problematic in the human body or problematic in a cell line used for production or manufacture. These modifications can be accomplished by site directed mutagenesis, for example. Examples of features that may be liabilities include T-cell epitopes, glycosylation sites, and deamidation. Codon optimization may be performed on a nucleic acid construct to use the most efficient codons for a particular amino acid sequence for expression in a particular species. This process may be done to adapt a sequence to a particular producer cell. For example, is a rodent cell is to be used, codons that are efficiently used and recognized in that rodent species may be substituted for the codons that were present in the donor or acceptor species of the antibody construct. This process typically enhances manufacturing rather than altering the product antibody itself. 
     To achieve a suitable therapeutic index, it is important that antibodies have and retain specificity for the pro-domain sequence and do not bind to other portions of the N-cadherin molecule. Other portions of the N-cadherin molecule are typically found on the cell surface, even in the non-diseased state. Recognition of other portions could lead to cytotoxicity to non-diseased cells that express non-pathological, mature N-cadherin on their cell surfaces. 
     Cytoxic agents or moieties that may be coupled to an antibody, preferably to a constant region of an antibody, but also possible to a variable domain, include bacterial toxins such as  Pseudomonas  exotoxin, diphtheria toxin, ricin A chain toxin, and saporin toxin. Chemotherapy drugs such as 2-(Hydroxymethyl)anthraquinone, Doxorubicin, methotrexate, and cyclopropanecarbonyl (CPC) chloridemay be used. Radionuclides emitting α-particles, β-particles or Auger electrons may be used as cytotoxic moieties. 
     Any type of human antibody can be used as a framework for humanization, including but not limited to IgA, IgD. IgE, IgG, and IgM. Antibodies may be, for example, IgG1, IgG2, IgG3, or IgG4. Chimeric antibodies and humanized antibodies may be seen as overlapping categories. The former may be used to denote a construct with entire variable regions from a non-human source, for example. The latter may be used to denote antibodies in which only the complementarity determining regions of an antibody are non-human. Both are non-naturally occurring constructed entities which aim to capitalize on and combine the beneficial properties of different species&#39; antibodies. Variant humanized heavy and light chains of 10A10 and 19D8 antibodies are shown in SEQ ID NO: 35-39, 41-45, 47-51, 52-57. These retain the CDR sequences of the parent murine antibodies. A heavy chain variant from 10A10 may be used in combination with a light chain variant from 10A10. The two chains may derive from the same or a different variant. Similarly, a heavy chain variant from 19D8 can be used in combination with a light chain variant from 19D8. The two chains may derive from the same or a different variant. 
     Vectors for expression of antibody sequences may be, for example, episomes, viral, phage, artificial chromosomes, without limitation. Antibody sequences may be expressed in recombinant production systems, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. 
     The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention. 
     Example 1—a Monoclonal Antibody Specific for the Precursor (Pro) Domain of Pro-N-Cadherin Binds to Pathological Myofibroblasts from Lung, Heart and Liver but not Non-Pathological Fibroblasts and Myofibroblasts 
     We investigated a murine monoclonal antibody (mAb) highly specific for the precursor (pro) domain of pro-N-cadherin[5]. Our studies show that this mAb recognizes pro-N-cadherin protein from human pathological myofibroblasts from lung (LL97A), heart (CF-DCM) and liver (LX2) ( FIG. 2B ). In addition, this antibody reacts with pro-N-cadherin on the cell surface of human pathological myofibroblasts from lung, heart and liver ( FIG. 2A ). Immunohistochemistry confirmed pro-N-cadherin protein expression in patient tissue derived from cirrhotic liver and dilated cardiomyopathy but not normal liver ( FIG. 2C ). 
     Example 2—the Effects of Pro-N-Cadherin mAb on Proliferation and Viability of Pathological Myofibroblasts 
     Pathological myofibroblasts from heart, lung and liver were challenged with pro-N-cadherin mAb and effects were measured by several proliferation assays, flow cytometry, cytotoxicity assays, dose titrations and time course in vitro. Dose titrations of the monoclonal antibodies revealed the hook effect on each pathological myofibroblast culture tested by proliferation and cytotoxicity assays ( FIGS. 3A, 3B and 3C ).  FIGS. 3A-3C  demonstrate an example of the hook effect, in which the epitope is saturated by competitive binding of the mAb at high monoclonal antibody concentrations. The hook effect is a well characterized phenomenon, exclusively indicative of monoclonal antibody activity[8, 9]. In this case, when the epitope is saturated, steric hindrance limits antibody-antigen interactions to monovalent binding that limits crosslinking of the pro-N-cadherin antigen and decreases efficacy. At optimal concentrations, the antibody binds bivalently and optimal efficacy of cytotoxicity and reduced proliferation is observed. Furthermore, after overnight treatment of CF-DCM with optimal mAb concentration, the pathological subpopulation of myofibroblasts can no longer be detected by measuring cell surface pro-N-cadherin of the remaining myofibroblast culture via flow cytometry ( FIG. 3D ). Significantly reduced α-SMA gene expression was also observed after LX2 hepatic stellate myofibroblasts were treated with pro-N-cadherin mAb overnight ( FIG. 3E ). 
     REFERENCES 
     The disclosure of each reference cited is expressly incorporated herein.
     1. Rockey, D. C., P. D. Bell, and J. A. Hill,  Fibrosis—a common pathway to organ injury and failure . New England Journal of Medicine, 2015. 372(12): p. 1138-1149.   2. Wynn, T.,  Cellular and molecular mechanisms of fibrosis . The Journal of Pathology: A Journal of the Pathological Society of Great Britain and Ireland, 2008. 214(2): p. 199-210.   3. Wynn, T. A.,  Fibrotic disease and the T H  1 /T H  2  paradigm . Nature Reviews Immunology, 2004. 4(8): p. 583.   4. Ozawa, M. and R. Kemler,  Correct proteolytic cleavage is required for the cell adhesive function of uvomorulin . The Journal of Cell Biology, 1990. 111(4): p. 1645-1650.   5. Wahl, J. K., et al.,  N - cadherin - catenin complexes form prior to cleavage of the proregion and transport to the plasma membrane . Journal of Biological Chemistry, 2003. 278(19): p. 17269-17276.   6. Maret, D., et al.,  Surface expression of precursor N - cadherin promotes tumor cell invasion . Neoplasia, 2010. 12(12): p. 1066-1080.   7. Nelson, E. R., et al.,  Chemotherapy enriches for an invasive triple - negative breast tumor cell subpopulation expressing a precursor form of N - cadherin on the cell surface . Oncotarget, 2016. 7(51): p. 84030.   8. Caron, P. C., et al.,  Murine and humanized constructs of monoclonal antibody M 195 ( anti - CD 33)  for the therapy of acute myelogenous leukemia . Cancer, 1994. 73(S3): p. 1049-1056.   9. Taborda, C. P., et al.,  More is not necessarily better: prozone - like effects in passive immunization with IgG . The Journal of Immunology, 2003. 170(7): p. 3621-3630.   10. Habiel, D. M., et al.,  Modeling idiopathic pulmonary fibrosis in humanized severe combined immunodeficient mice . The American journal of pathology, 2018. 188(4): p. 891-903.   11. Elrick, L. J., et al.,  Generation of a monoclonal human single chain antibody fragment to hepatic stellate cells—a potential mechanism for targeting liver anti - fibrotic therapeutics . Journal of hepatology, 2005. 42(6): p. 888-896.   12. Poelstra, K. and D. Schuppan,  Targeted therapy of liver fibrosis/cirrhosis and its complications . Journal of hepatology, 2011. 55(3): p. 726-728.   13. Hmiel, L. K., K. A. Brorson, and M. T. Boyne,  Post - translational structural modifications of immunoglobulin G and their effect on biological activity . Analytical and bioanalytical chemistry, 2015. 407(1): p. 79-94.   14. Asmani, M., et al.,  Fibrotic microtissue array to predict anti - fibrosis drug efficacy . Nature communications, 2018. 9(1): p. 2066.   

     
       
         
           
               
            
               
                   
               
               
                 Table of Sequences 
               
            
           
           
               
               
               
               
               
            
               
                   
                 SEQ ID NO  
                 Clone Name  
                 Length  
                 Type 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 1  
                 A10A HEAVY  
                 402  
                 DNA 
               
               
                   
                 2  
                 A10A LIGHT  
                 384  
                 DNA 
               
               
                   
                 3  
                 19D8 HEAVY  
                 417  
                 DNA 
               
               
                   
                 4  
                 19D8 LIGHT  
                 381  
                 DNA 
               
               
                   
                 5  
                 A10A CDR1 (H)  
                 15  
                 DNA 
               
               
                   
                 6  
                 A10A CDR2 (H)  
                 51  
                 DNA 
               
               
                   
                 7  
                 A10A CDR3 (H)  
                 18  
                 DNA 
               
               
                   
                 8  
                 A10A CDR1 (L)  
                 30  
                 DNA 
               
               
                   
                 9  
                 A10A CDR2(L)  
                 21  
                 DNA 
               
               
                   
                 10  
                 A10A CDR3(L)  
                 27  
                 DNA 
               
               
                   
                 11  
                 19D8 CDR1 (H)  
                 15  
                 DNA 
               
               
                   
                 12  
                 19D8 CDR2 (H)  
                 48  
                 DNA 
               
               
                   
                 13  
                 19D8 CDR3 (H)  
                 36  
                 DNA 
               
               
                   
                 14  
                 19D8 CDR1(L)  
                 33  
                 DNA 
               
               
                   
                 15  
                 19D8 CDR2(L)  
                 21  
                 DNA 
               
               
                   
                 16  
                 19D8 CDR2(L)  
                 27  
                 DNA 
               
               
                   
                 17  
                 PRODOMAIN  
                 68  
                 Protein 
               
               
                   
                 18  
                 A10A HEAVY  
                 134  
                 Protein 
               
               
                   
                 19  
                 A10A LIGHT  
                 128  
                 Protein 
               
               
                   
                 20  
                 19D8 HEAVY  
                 139  
                 Protein 
               
               
                   
                 21  
                 19D8 LIGHT  
                 127  
                 Protein 
               
               
                   
                 22  
                 A10A CDR1 (H)  
                 5  
                 Protein 
               
               
                   
                 23  
                 A10A CDR2(H)  
                 17  
                 Protein 
               
               
                   
                 24  
                 A10A CDR3(H)  
                 6  
                 Protein 
               
               
                   
                 25  
                 A10A CDR1 (L)  
                 10  
                 Protein 
               
               
                   
                 26  
                 A10A CDR2(L)  
                 7  
                 Protein 
               
               
                   
                 27  
                 A10A CDR3(L)  
                 9  
                 Protein 
               
               
                   
                 28  
                 19D8 CDR1(H)  
                 5  
                 Protein 
               
               
                   
                 29  
                 19D8 CDR2(H)  
                 16  
                 Protein 
               
               
                   
                 30  
                 19D8 CDR3(H)  
                 12  
                 Protein 
               
               
                   
                 31  
                 19D8 CDR1(L)  
                 11  
                 Protein 
               
               
                   
                 32  
                 19D8 CDR2(L)  
                 7  
                 Protein 
               
               
                   
                 33  
                 19D8 CDR3(L)  
                 9  
                 Protein 
               
               
                   
                 34  
                 A10A HC0  
                 461  
                 Protein 
               
               
                   
                 35  
                 A10A HC1  
                 461  
                 Protein 
               
               
                   
                 36  
                 A10A HC2  
                 461  
                 Protein 
               
               
                   
                 37  
                 A10A HC3  
                 461  
                 Protein 
               
               
                   
                 38  
                 A10A HC4  
                 461  
                 Protein 
               
               
                   
                 39  
                 A10A HC5  
                 461  
                 Protein 
               
               
                   
                 40  
                 A10A LC0  
                 233  
                 Protein 
               
               
                   
                 41  
                 A10A LC1  
                 233  
                 Protein 
               
               
                   
                 42  
                 A10A LC2  
                 233  
                 Protein 
               
               
                   
                 43  
                 A10A LC3  
                 233  
                 Protein 
               
               
                   
                 44  
                 A10A LC4  
                 233  
                 Protein 
               
               
                   
                 45  
                 A10A LC5  
                 233  
                 Protein 
               
               
                   
                 46  
                 19D8 HC0  
                 469  
                 Protein 
               
               
                   
                 47  
                 19D8 HC1  
                 469  
                 Protein 
               
               
                   
                 48  
                 19D8 HC2  
                 469  
                 Protein 
               
               
                   
                 49  
                 19D8 HC3  
                 468  
                 Protein 
               
               
                   
                 50  
                 19D8 HC4  
                 469  
                 Protein 
               
               
                   
                 51  
                 19D8 HC5  
                 469  
                 Protein 
               
               
                   
                 52  
                 19D8 LC0  
                 234  
                 Protein 
               
               
                   
                 53  
                 19D8 LC1  
                 234  
                 Protein 
               
               
                   
                 54  
                 19D8 LC2  
                 234  
                 Protein 
               
               
                   
                 55  
                 19D8 LC3  
                 234  
                 Protein 
               
               
                   
                 56  
                 19D8 LC4  
                 234  
                 Protein 
               
               
                   
                 57  
                 19D8LC5  
                 234  
                 Protein