WO2024211317A2 - Anticorps monoclonaux dirigés contre la cadhérine 11 et méthodes d'utilisation - Google Patents

Anticorps monoclonaux dirigés contre la cadhérine 11 et méthodes d'utilisation Download PDF

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WO2024211317A2
WO2024211317A2 PCT/US2024/022682 US2024022682W WO2024211317A2 WO 2024211317 A2 WO2024211317 A2 WO 2024211317A2 US 2024022682 W US2024022682 W US 2024022682W WO 2024211317 A2 WO2024211317 A2 WO 2024211317A2
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seq
chain variable
cdh11
variable region
sequence
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WO2024211317A3 (fr
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Percio GULKO
Teresina LARAGIONE
Thomas Moran
James DUTY
Scott Friedman
Dipankar Bhattacharya
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Icahn School of Medicine at Mount Sinai
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Icahn School of Medicine at Mount Sinai
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • Cadherin 11 is part of the cadherin superfamily of membrane proteins involved in homodimeric cell-to-cell interaction.
  • Human CDH11 is a Ca+-dependent, transmembrane protein exhibiting homotypic binding.
  • the 796 amino acid protein ( ⁇ 88 kDa) comprises five extracellular cadherin domains ( ⁇ 110 aa), named EC 1-5, a transmembrane region, and a cytoplasmic tail.
  • CDH11 has a high homology (about 97 %) with mouse CDH11 and shares modest homology with other type 2 cadherins (40-60 %), with increased sequence conservation in the cytoplasmic tails and the EC1-2 domains.
  • CDH11 is expressed on mesenchyme-derived cells, including hepatic stellate cells (HSC), myofibroblasts, and fibroblast-like synoviocytes (FLS). CDH11 engagement increases the production and deposition matrix proteins, including collagens, and fibrosis.
  • HSC hepatic stellate cells
  • FLS fibroblast-like synoviocytes
  • CDH11 has been implicated in a variety of diseases, including fibrotic disorders such as liver fibrosis, pulmonary fibrosis and scleroderma, in rheumatoid arthritis, inflammatory 1 156966279 084284.00294 bowel disease and in cancer. Improved and effective means of addressing these diseases by targeting CDH11 are urgently needed.
  • an anti-cadherin 11 (CDH11) antibody, or CDH11- binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:1, CDR2H comprises SEQ ID NO:11, CDR3H comprises SEQ ID NO:20 or SEQ ID NO:21, CDR1L comprises SEQ ID NO:35, CDR2L comprises SEQ ID NO:43, and CDR3L comprises SEQ ID NO:48 or SEQ ID NO:49; (b) CDR1H comprises SEQ ID NO:1, CDR2H comprises SEQ ID NO:11, CDR3H comprises SEQ ID NO:20 or SEQ ID NO:21, CDR1L comprises SEQ ID NO:35, CDR2L comprises SEQ ID NO:43, and CDR3L comprises SEQ
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:1, CDR2H comprises SEQ ID NO:11, CDR3H comprises SEQ ID NO:20, CDR1L comprises SEQ ID NO:35, CDR2L comprises SEQ ID NO:43, and CDR3L comprises SEQ ID NO:48; (b) CDR1H comprises SEQ ID NO:4, CDR2H comprises SEQ ID NO:13, CDR3H comprises SEQ ID NO:26, CDR1L comprises SEQ ID NO:38, CDR2L comprises SEQ ID NO:45, and CDR3L comprises SEQ ID NO:53; 3 156966279 084284.002
  • an anti-CDH11 antibody, or CDH11-binding fragment wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises: (a) a heavy chain variable region that comprises a sequence that is at least 90% identical to SEQ ID NO:62 and a light chain variable region that comprises a sequence that is at least 90% identical to SEQ ID NO:78; (b) a heavy chain variable region that comprises a sequence that is at least 90% identical 5 156966279 084284.00294 to SEQ ID NO:68 and a light chain variable region that comprises a sequence that is at least 90% identical to SEQ ID NO:83; (c) a heavy chain variable region that comprises a sequence that is at least 90% identical to SEQ ID NO:75 and a light chain variable region that comprises a sequence that is at least 90% identical to SEQ ID NO:90; (d) a heavy chain variable region that comprises a sequence that is at least 90% identical to SEQ ID NO:63 and a light chain variable region
  • an anti-CDH11 antibody, or CDH11-binding fragment wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises: (a) a heavy chain variable region that comprises SEQ ID NO:62 and a light chain variable region that comprises SEQ ID NO:78; (b) a heavy chain variable region that comprises SEQ ID NO:68 and a light chain variable region that comprises SEQ ID NO:83; (c) a heavy chain variable region that comprises SEQ ID NO:75 and a light chain variable region that comprises SEQ ID NO:90; (d) a heavy chain variable region that comprises SEQ ID NO:63 and a light chain variable region that comprises SEQ ID NO:79; (e) a heavy chain variable region that comprises SEQ ID NO:64 and a light chain variable region that comprises SEQ ID NO:80; (f) a heavy chain variable region that comprises SEQ ID NO:65 and a light chain variable region that comprises SEQ ID NO:81; (g) a heavy chain variable region that comprises SEQ ID NO:62 and
  • the CDH11 antibody, or CDH11-binding fragment thereof is a monoclonal antibody. In one embodiment, the CDH11 antibody, or CDH11-binding fragment thereof, is a recombinant antibody. In one embodiment, the CDH11 antibody, or CDH11- binding fragment thereof, comprises a human framework region or a modified human framework region. In one embodiment, the CDH11 antibody, or CDH11-binding fragment thereof, comprises a human constant region or a modified human constant region. In one embodiment, the CDH11-binding fragment is an scFv, Fv, Fab’, Fab, F(ab’)2, or diabody.
  • the anti-CDH11 antibody, or CDH11-binding fragment thereof is deglycosylated. In some embodiments, the anti-CDH11 antibody, or CDH11-binding fragment thereof, is conjugated to one or more of a cytotoxin, a fluorescent label, and an imaging agent. [0014] Provided is a nucleic acid molecule encoding an anti-CDH11 antibody, or CDH11- binding fragment thereof, disclosed herein.
  • the nucleic acid molecule comprises: (a) SEQ ID NO:94 and/or SEQ ID NO:111; (b) SEQ ID NO:95 and/or SEQ ID NO:112; (c) SEQ ID NO:96 and/or SEQ ID NO:113; 8 156966279 084284.00294 (d) SEQ ID NO:97 and/or SEQ ID NO:114; (e) SEQ ID NO:98 and/or SEQ ID NO:115; (f) SEQ ID NO:99 and/or SEQ ID NO:116; (g) SEQ ID NO:100 and/or SEQ ID NO:117; (h) SEQ ID NO:101 and/or SEQ ID NO:118; (i) SEQ ID NO:102 and/or SEQ ID NO:119; (j) SEQ ID NO:103 and/or SEQ ID NO:120; (k) SEQ ID NO:104 and/or SEQ ID NO:121; (l) SEQ ID NO:105 and/or SEQ ID NO:122;
  • a vector or set of vectors comprising a nucleic acid molecule disclosed herein.
  • an isolated host cell comprising a nucleic acid molecule or a vector or set of vectors disclosed herein.
  • a method of producing an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising culturing a host cell disclosed herein under conditions wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, is produced by the host cell.
  • a pharmaceutical composition comprising an anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein, and a pharmaceutically acceptable excipient.
  • a method of inhibiting CDH11-CDH11 interactions between cells comprising contacting the cells with an anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein or a pharmaceutical composition disclosed herein.
  • a method of inhibiting CDH11-CDH11 interactions between cells in a subject in need thereof the method comprising administering to the subject an anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein or a pharmaceutical composition disclosed herein.
  • the method comprises inhibiting CDH11-CDH11 interactions between hepatic stellate cells (HSCs).
  • the fibrosis is liver fibrosis, pulmonary fibrosis, renal fibrosis, cardiac fibrosis, myelofibrosis, pancreatic fibrosis, dermal fibrosis including systemic sclerosis (scleroderma) and keloids, intestinal fibrosis, post-myocardial infarct fibrosis, replacement fibrosis, perivascular fibrosis, or arthrofibrosis.
  • the fibrosis is liver fibrosis.
  • the subject has an infection with hepatitis viruses, nonalcoholic fatty liver disease (NASH), an inherited metabolic disorder, a cholestatic disorder, or an immune disorder.
  • the liver fibrosis is associated with increased alcohol consumption, consumption of excess vitamin A, drug toxicity.
  • the liver fibrosis is associated with an autoimmune disease of the liver or bile ducts, liver transplantation rejection or a genetic disorder of the liver or genetic disorder of the bile ducts.
  • the autoimmune disease of the liver or bile ducts is autoimmune hepatitis, primary biliary cholangitis or primary sclerosing cholangitis.
  • the genetic disorder of the liver is Wilson disease or hemochromatosis or wherein the genetic disorder of the genetic disorder of the bile ducts is biliary atresia.
  • the fibrosis is intestinal fibrosis and intestinal strictures related to colitis.
  • the subject has Inflammatory Bowel Disease (IBD), Crohn’s disease, or colitis.
  • the pulmonary fibrosis is induced by idiopathic pulmonary fibrosis or toxic injury.
  • the subject has scleroderma or systemic sclerosis.
  • the subject has a keloid scar.
  • Figs. 1A and 1B illustrate the immunization strategy for generating anti-CDH11 antibodies.
  • Fig. 1A Strategy for DNA immunization.
  • Fig. 1B Strategy for protein immunization.
  • Figs. 2A and 2B show data for the sera screens that were conducted. Mouse sera were collected after each round of immunizations and tested either by flow cytometry or ELISA.
  • Fig.2A human CDH11 ECD proteins c-terminally tagged with human Fc (IgG1) were coated on ELISA plates at 5 ⁇ g/ml. Sera were added and detected by goat anti-mouse IgG (Fc specific)-horse radish peroxidase (HRP) conjugated secondary antibody (Jackson ImmunoResearch) and developed using ABTS (2,2'-Azinobis [3- 10 156966279 084284.00294 ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) substrate (ThermoFisher). ELISA plates were read for absorbance at OD 405 nm.
  • RGN NMS Normal Mouse Serum (nonimmunized serum).
  • Fig.2B For flow cytometry experiments, Expi293F HEK cells were transfected with either full length human or mouse CDH11, which were tagged C-terminally tagged with GFP.
  • Goat anti-mouse IgG (Fc specific) conjugated with allophycocyanin (APC, Jackson ImmunoResearch) was used as the secondary antibody at a 1:1000 dilution. Normal mouse sera were included as negative controls. Mouse sera were diluted 1:100, 1:1000, and 1:10000. Flow was reported as mean fluorescence intensity (MFI) for APC fluorescence.
  • Protein Mice Bars from left to right: human CDH11 (HuCAD11) 1 st protein boost; human CDH11 2 nd + protein boost; murine CDH11 (MsCAD11) 1 st protein boost; murine CDH112 nd + protein boost. DNA + Protein Mice (see Fig.
  • FIGs.3A and 3B show data for the hybridoma screens that were conducted. Hybridomas were screened against human CDH11 C-terminally tagged with GFP expressed by Expi293F HEK cells (using flow cytometry) or against human CDH11 extracellular domain proteins (tagged with human IgG1 Fc tags) (by ELISA). For flow cytometry, hybridomas were detected by goat anti-mouse IgG Fc specific-APC conjugate (Jackson ImmunoResearch).
  • ELISAs were developed using goat anti-mouse IgG (Fc specific)-HRP conjugate (Jackson ImmunoResearch). Forty-seven clones in total were evaluated. Left bars: flow MFI (right axis). Right bars: ELISA OD 450 nm absorbance (left axis). Hybridoma supernatants were diluted at 1:1000 in FACS buffer, and commercial control anti-huCDH11-antibody 16G5 (BioLegend) was added @ 1 ⁇ g/ml.
  • Fig.3A Fusions 1 and 2.
  • Fig.3B Fusion 3.
  • Fig.4 illustrates the binding of supernatant from selected hybridomas to human and rat primary FLS, as determined by flow cytometry.
  • Fig. 5 illustrates the cross reactivity of isolated anti-CDH11 antibodies with different type 2 cadherins.
  • ANTI-CAD11 mouse anti-human CAD11 antibody, clone 16G5 (BioLegend), positive control. 11 156966279 084284.00294 [0025] Figs.
  • FIG. 7A, 7B, 7C, 7D, 7E, 7F, 7G, and 7H illustrate the effect of monoclonal anti- human CDH11 antibodies on RA (human) FLS invasiveness.
  • Twenty-seven different monoclonal antibodies specific for human CDH11 were tested for invasion-suppressive activity in three to four FLS cell lines derived from patients with RA. Seven different antibodies reduced FLS invasiveness by at least 30% (arrows), some showing a dose-dependent effect.
  • Fig. 7A, 7B, 7C, and 7D Initial batch of monoclonal antibodies.
  • Fig. 7E Selected positive antibodies from the initial batch (from Fig.
  • FIG.7D shows the most significant FLS invasion suppressive effect
  • Figs.7F, 7G, and 7H Second batch of anti-human CDH11 monoclonal antibodies.
  • CADH-11 control antibody.
  • ctl control (cells not treated with antibodies).
  • Figs.8A and 8B illustrate the effect of selected anti-CDH11 antibodies on mouse FLS invasion.
  • Two of the anti-CDH11 antibodies reduced the invasiveness of FLS from a KRN serum induced arthritis (KSIA) mouse (arrow).
  • Fig. 8B illustrate the effect of selected anti-CDH11 antibodies on mouse FLS invasion.
  • Fig.9 illustrates immunofluorescence staining of mouse FLS with selected monoclonal anti-human CDH11 antibodies.
  • FLS derived from a KSIA mouse was used for immunofluorescence with a commercially available anti-CDH11 antibody (16G5, Biolegend, catalog number 368702) or six monoclonal anti-CDH11 antibodies, which had been shown to suppress RA FLS invasion.
  • Four of the six antibodies tested (16A10, 2A10, 3A10 and 5H1) stained positive compared with isotype control, while two stained negative (2F6 and 2A1).
  • Fig.10 illustrates the in vivo testing of selected anti-human CDH11 antibodies in mice with KSIA.
  • Four to five C57BL/6 male mice per treatment group received 100 ⁇ L of the KRN 12 156966279 084284.00294 arthritogenic serum on days 0 and 2. Starting on day 0, mice were started on 10 mg/kg per day of an antibody (IgG1 isotype control, 2A10, 3A10 or 10E1) or PBS, every other day for ten days.
  • Fig. 11 shows that monoclonal anti-CDH11 antibody 3A10 reduced ankle and paw swelling in mice with KSIA (representative images).
  • Figs. 12A, 12B, and 12C illustrate the binding of selected anti-CDH11 antibodies to cellular CDH11. Antibodies 2A10, 3A10, and 10E1 were tested for binding to human CDH11 or murine CDH11 expressed on Expi 293F cells.
  • Fig.12A Antibody binding to human CDH11.
  • Fig.12B Antibody binding to murine CDH11.
  • Fig.12C Antibody binding to human CDH11. Traces at 1 nM from top to bottom: 2A10; 3A10; 16G5; 10E1; mAb2 control antibody; mAb1/isotype control.
  • Fig.13 illustrates the structure and the different domains of human CDH11.
  • Figs. 14A, 14B, and 14C illustrate the results of domain mapping experiments for selected anti-CDH11 antibodies.
  • Five DNA constructs encoding human CDH11 variants (labeled “No Dom 1” to “No Dom 5”) were generated, each CDH11 variant missing one of the five EC domains.
  • the C-termini of the CDH11 variants were fused to GFP.
  • the five constructs or a construct encoding full length human CDH11, respectively, were transfected into Expi293F cells.
  • Figs.14A Bars from left to right: 2A10, 3A10, 10E1, 16G5 (BioLegend, control). 14B. Bars from left to right: 1A1, 2A1, 2D2, 6D5, 16G5 (control).
  • Fig.14C Evaluation of fusion and normal sera from immunized mice. Bars from left to right: 16G5 (control), fusion sera, NMS, isotype control.
  • Figs. 15A, 15B, 15C, 15D, 15E, 15F, 15G, and 15H illustrate the efficacy of anti- CDH11 antibodies in well-established model of fibrosis.
  • Anti-CDH11 antibodies significantly reduced the expression of collagen type I (Col1a1), actin alpha 2, smooth muscle (Acta2), platelet-derived growth factor receptor beta (bPDGFR), matrix metalloproteinase-1 (MMP1), and matrix metalloproteinase-2 (MMP2), both in LX-2 hHSC as well in the primary hHSC line TWNT4. These are genes known to mediate fibrotic processes (* P ⁇ 0.05).
  • Fig.15C and 15D Anti-CDH11 antibodies significantly reduced the production of Col1a1 protein by both LX-2 as well as by the primary hHSC line TWNT4, further demonstrating anti-fibrotic properties (* P ⁇ 0.05).
  • Fig. 15E Anti-CDH11 antibodies significantly reduced the production of Col1a1 protein by both LX-2 as well as by the primary hHSC line TWNT4, further demonstrating anti-fibrotic properties (* P ⁇ 0.05).
  • Anti-CDH11 antibody 3A10 significantly reduced the expression of fibrogenic genes in primary hHSCs.
  • Fig 15F Anti-CDH11 antibody 3A10 significantly reduced the production of Col1a1 in primary hHSCs. * P ⁇ 0.05.
  • Fig.15G Anti- CDH11 antibody 3A10 reduced proliferation of primary hHSCs. * P ⁇ 0.05.
  • Fig. 15H. ⁇ SMA protein expression is significantly downregulated in hPCLS treated with Ab-3A10 for 48 h (Patient 1).
  • Anti-CDH11 antibody 3A10 was not cytotoxic to human precision cut liver slices (hPCLS).
  • CAD11 CDH11
  • CADH-11 CADH-11
  • Fibrotic disorders have been estimated to contribute to 40% of mortality in the population. These conditions include liver fibrosis, pulmonary fibrosis, scleroderma, intestinal strictures in inflammatory bowel disease (IBD) post myocardial infarct fibrosis, and arthrofibrosis.
  • IBD inflammatory bowel disease
  • Liver fibrosis can be caused by several different chronic liver diseases, including common ones such as non-alcoholic fatty liver disease (NASH), increased alcohol consumption, inherited metabolic disorders, drug consumption, consumption of excess vitamin A, cholestatic disorders, and immune disorders. Further, liver fibrosis can progress to liver failure and/or liver cancer and decrease survival.
  • NASH non-alcoholic fatty liver disease
  • CDH11 is expressed predominantly in mesenchyme-derived cells, including HSCs in liver, with increased expression in similar 14 156966279 084284.00294 mesenchymal cells in a range of fibrotic conditions (liver, lung, skin, colitis with strictures).
  • HSCs are mesenchyme-derived cells that play a central role in liver fibrosis and produce type I collagen and other matrix proteins. These cells can be used to study pro- and anti-fibrotic processes in the liver and to screen for new therapeutic agents.
  • CDH11 mRNA levels are increased in the plasma of patients with liver fibrosis. Further, increased levels of CDH11 correlate with collagen type I in cholestatic liver fibrosis.
  • compositions and methods for suppressing HSC fibrogenic phenotypes and functions are also provided herein are compositions and methods for treating fibrotic disorders.
  • Rheumatoid arthritis affects 1% of the population and is associated with increased risk for deformities, disability and with reduced longevity.
  • CDH11 is required for the formation and stability of the synovial tissue lining layer, which is central to the development of synovial hyperplasia and joint erosive changes.
  • CDH11 knock-out mice are protected from arthritis and their FLS have reduced invasiveness (a characteristic has been shown to correlate with reduced disease severity and joint damage in RA and rodent models of RA).
  • CDH11 engagement increases the production of IL-6 by FLS and synergizes with TNF ⁇ and IL-1 ⁇ to activate NF ⁇ B and MAP kinases.
  • compositions and methods for reducing FLS invasiveness and/or joint destruction are also provided herein are compositions and methods for reducing RA disease activity and severity.
  • compositions and methods for preventing or reducing joint damages in patients with RA By targeting the disease site (the synovial membrane), the compositions and methods disclosed herein can achieve disease control by targeting the disease site, thus reducing the risk of systemic immunosuppression.
  • IBD Inflammatory Bowel Disease
  • Treatment typically involves the use of various immunosuppressive agents, yet 40% of the patients do not respond to these treatments.
  • Intestinal fibrosis and strictures are a major 15 156966279 084284.00294 complication of IBD affecting as much as 30% of patients, particularly those with Crohn’s disease.
  • IBD Intestinal mesenchymal cells
  • stromal cells including fibroblasts, myofibroblasts and others
  • IBD intestinal mesenchymal cells
  • Intestinal mesenchymal cells express CDH11 and its levels are increased in the intestines of patients with IBD.
  • Mice deficient for CDH11 (knockout) studied in a model of IBD induced with TNBS developed significantly reduced fibrosis and collagen deposition in the intestinal subepithelial and subserosal areas, compared to wild-type mice that expressed CDH11.
  • Provided herein are compositions and methods for blocking the hemophilic interaction of CDH11 molecules to prevent the CDH11-mediated production of collagen, fibronectin, IL-6 and TGFbeta in the IBD intestines, thus reducing the risk of fibrosis and reducing the progression of established fibrosis.
  • the methods disclosed herein do not immunosuppress patients and therefore can be used in combination with standard of care treatments for IBD, such anti-TNFalpha agents and others.
  • Antibodies are used in the broadest sense and includes monoclonal antibodies (including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies).
  • antibody variable domain refers to the portions of the light and heavy chains of antibody molecules that include amino acid sequences of complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3), and framework regions.
  • V H refers to the variable domain of the heavy chain.
  • V L refers to the variable domain of the light chain.
  • frame regions refers to those variable domain residues other than the CDR residues.
  • the “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct 16 156966279 084284.00294 types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
  • kappa ⁇
  • ⁇ lambda
  • Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.
  • Each CDR can comprise amino acid residues from a CDR as defined by e.g., Kabat (i.e., about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1987, 1991)).
  • Each CDR can also comprise amino acid residues from a “hypervariable loop” (i.e., about residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia & Lesk 196 J. Mol. Biol.901 (1987)).
  • a CDR can include amino acids from both a CDR region defined according to Kabat and a hypervariable loop. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues (primary amino acid sequence).
  • the actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or CDR, of the basic variable domain structure.
  • the correct Kabat numbering of residues may be determined for a given antibody or antigen-binding fragment thereof by alignment of residues of homology in the sequence of the antibody or antigen-binding fragment thereof with a “standard” Kabat numbered sequence.
  • a CDR can be defined according to the ImMunoGeneTics (IMGT) system (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)).
  • constant region refers to a region of an immunoglobulin light chain or heavy chain that is distinct from the variable region.
  • the constant domain of the heavy chain generally comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region), a CH2 domain, and a CH3 domain.
  • an antibody described herein may comprise a polypeptide comprising a CH1 domain; a polypeptide comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide comprising a CH1 domain and a CH3 domain; a polypeptide comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain.
  • a polypeptide comprises a polypeptide chain comprising a CH3 domain.
  • the constant domain of a light chain can be a kappa ( ⁇ ) or lambda ( ⁇ ) constant region.
  • these constant domains e.g., the heavy chain or light chain
  • HC refers to the heavy chain, including the V H and the constant region.
  • LC refers to the light chain, including the V L and the constant region.
  • Fc region or “Fc portion” refers to the C terminal region of an immunoglobulin heavy chain.
  • the Fc region can be a native-sequence Fc region or a non- naturally occurring variant Fc region.
  • the Fc region of an immunoglobulin comprises constant domains CH2 and CH3.
  • the human IgG heavy chain Fc region can be defined to extend from an amino acid residue at position C226 or from P230 to the carboxy terminus thereof.
  • the “CH2 domain” of a human IgG Fc region usually extends from about amino acid residue 231 to about amino acid residue 340.
  • N-linked carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule.
  • the CH3 domain” of a human IgG Fc region comprises residues C-terminal to the CH2 domain, e.g., from about amino acid residue 341 to about amino acid residue 447 of the Fc region.
  • the terms “antigen-binding fragment thereof” and “CDH11-binding fragment thereof” are used herein interchangeably.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:1, CDR2H comprises SEQ ID NO:11, CDR3H comprises SEQ ID NO:20 or SEQ ID NO:21, CDR1L comprises SEQ ID NO:35, CDR2L comprises SEQ ID NO:43, and CDR3L comprises SEQ ID NO:48 or SEQ ID NO:49.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:1, CDR2H comprises SEQ ID NO:11, CDR3H comprises SEQ ID 18 156966279 084284.00294 NO:20, CDR1L comprises SEQ ID NO:35, CDR2L comprises SEQ ID NO:43, and CDR3L comprises SEQ ID NO:48.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:1, CDR2H comprises SEQ ID NO:11, CDR3H comprises SEQ ID NO:21, CDR1L comprises SEQ ID NO:35, CDR2L comprises SEQ ID NO:43, and CDR3L comprises SEQ ID NO:49.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:4, CDR2H comprises SEQ ID NO:13, CDR3H comprises SEQ ID NO:25 or SEQ ID NO:26, CDR1L comprises SEQ ID NO:38, CDR2L comprises SEQ ID NO:45, and CDR3L comprises SEQ ID NO:53.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:4, CDR2H comprises SEQ ID NO:13, CDR3H comprises SEQ ID NO:25, CDR1L comprises SEQ ID NO:38, CDR2L comprises SEQ ID NO:45, and CDR3L comprises SEQ ID NO:53.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:4, CDR2H comprises SEQ ID NO:13, CDR3H comprises SEQ ID NO:26, CDR1L comprises SEQ ID NO:38, CDR2L comprises SEQ ID NO:45, and CDR3L comprises SEQ ID NO:53.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and 19 156966279 084284.00294 the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:8, CDR2H comprises SEQ ID NO:17, CDR3H comprises SEQ ID NO:32, CDR1L comprises SEQ ID NO:38, CDR2L comprises SEQ ID NO:45, and CDR3L comprises SEQ ID NO:59.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:2, CDR2H comprises SEQ ID NO:11, CDR3H comprises SEQ ID NO:22, CDR1L comprises SEQ ID NO:36, CDR2L comprises SEQ ID NO:43, and CDR3L comprises SEQ ID NO:50.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:3, CDR2H comprises SEQ ID NO:12, CDR3H comprises SEQ ID NO:23, CDR1L comprises SEQ ID NO:37, CDR2L comprises SEQ ID NO:44, and CDR3L comprises SEQ ID NO:51.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:3, CDR2H comprises SEQ ID NO:12, CDR3H comprises SEQ ID NO:24, CDR1L comprises SEQ ID NO:35, CDR2L comprises SEQ ID NO:43, and CDR3L comprises SEQ ID NO:52.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:5, CDR2H comprises SEQ ID NO:14, CDR3H comprises SEQ ID NO:27, CDR1L comprises SEQ ID NO:39, CDR2L comprises SEQ ID NO:46, and CDR3L comprises SEQ ID NO:54.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:5, CDR2H comprises SEQ ID NO:14, CDR3H comprises SEQ ID NO:28, CDR1L comprises SEQ ID NO:35, CDR2L comprises SEQ ID NO:43, and CDR3L comprises SEQ ID NO:55.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:6, CDR2H comprises SEQ ID NO:15, CDR3H comprises SEQ ID NO:29, CDR1L comprises SEQ ID NO:40, CDR2L comprises SEQ ID NO:45, and CDR3L comprises SEQ ID NO:56.
  • CDR1H comprises SEQ ID NO:6
  • CDR2H comprises SEQ ID NO:15
  • CDR3H comprises SEQ ID NO:29
  • CDR1L comprises SEQ ID NO:40
  • CDR2L comprises SEQ ID NO:45
  • CDR3L comprises SEQ ID NO:56.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:7, CDR2H comprises SEQ ID NO:16, CDR3H comprises SEQ ID NO:30, CDR1L comprises SEQ ID NO:41, CDR2L comprises SEQ ID NO:44, and CDR3L comprises SEQ ID NO:57.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:7, CDR2H comprises SEQ ID NO:16, CDR3H comprises SEQ ID NO:31, CDR1L comprises SEQ ID NO:35, CDR2L comprises SEQ ID NO:43, and CDR3L comprises SEQ ID NO:58.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:9, CDR2H comprises SEQ ID NO:18, CDR3H comprises SEQ ID 21 156966279 084284.00294 NO:33, CDR1L comprises SEQ ID NO:41, CDR2L comprises SEQ ID NO:45, and CDR3L comprises SEQ ID NO:60.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein CDR1H comprises SEQ ID NO:10, CDR2H comprises SEQ ID NO:19, CDR3H comprises SEQ ID NO:34, CDR1L comprises SEQ ID NO:42, CDR2L comprises SEQ ID NO:47, and CDR3L comprises SEQ ID NO:61.
  • an anti-CDH11 antibody or CDH11-binding fragment thereof, comprising at least one, at least two, at least three, at least four, at least five or six of the CDR sequences disclosed in Tables 1 and 2.
  • Table 1 Overview of anti-CDH11 antibody CDR amino acid sequences. Numbers indicate SEQ ID NOs.
  • Table 2 Overview of anti-CDH11 antibody variable chain sequences. Numbers indicate SEQ ID NOs.
  • Antibody VH chain V ⁇ chain VH chain V ⁇ chain Amino acid Nucleic acid B11E1 1 114 , ing fragments thereof disclosed herein also feature humanized frameworks for reduced immunogenicity.
  • the CDRs of the contemplated antibody or antigen- binding fragment thereof are located in frameworks obtained from a human antibody or antigen-binding fragment thereof.
  • surface-exposed framework residues of the contemplated antibody or antigen-binding fragment thereof are replaced with framework residues of a human antibody or antigen-binding fragment thereof.
  • the CDRs may also be located in murine or humanized frameworks linked to human constant regions (i.e., chimeric antibodies).
  • the antibody or antigen-binding fragment thereof comprises a murine variable region and human constant regions.
  • the antibody or antigen-binding fragment thereof comprises a murine variable region, a murine CH1 region and human CH2 and CH3 constant regions.
  • the CDRs of a contemplated antibody or antigen-binding fragment thereof are located in frameworks that are a composite of two or more human antibodies.
  • the contemplated antibodies or antigen-binding fragments thereof comprise two or more sequence segments (“composite”) derived from V-regions of unrelated human antibodies that are selected to maintain monoclonal antibody sequences important for antigen binding of the starting precursor monoclonal antibody, and which have all been filtered for the presence of potential T cell epitopes using “in silico tools” (see, e.g., Holgate & Baker, IDrugs.2009 Apr;12(4):233-7).
  • Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. For example, when a position in the compared nucleotide sequence is occupied by the same base, then the molecules are identical at that position.
  • a degree identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at shared positions.
  • polypeptides having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotides encoding such polypeptides, are contemplated.
  • Methods and computer programs for determining both sequence identity and similarity are publicly available, including, but not limited to, the GCG program package (Devereux et al., Nucleic Acids Research 12: 387, 1984), BLASTP, BLASTN, FASTA (Altschul et al., J. Mol. Biol.
  • BLAST program is publicly available from NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0 at http://www.ncbi.nlm.nih.gov/blast/). In comparing sequences, these methods account for various substitutions, deletions, and other modifications.
  • an anti-CDH11 antibody or CDH11-binding fragment thereof, comprising a heavy chain variable domain comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:62-77.
  • an anti-CDH11 antibody or CDH11-binding fragment thereof, comprising a light chain variable domain comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:78-93.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising (a) a heavy chain variable domain comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:62-77 and (b) a light chain variable domain comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:78-93.
  • an anti-CDH11 antibody or CDH11-binding fragment thereof, comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:62 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:78.
  • an anti-CDH11 antibody or CDH11-binding fragment thereof, comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:68 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:83.
  • an anti-CDH11 antibody or CDH11-binding fragment thereof, comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:75 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:90.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:63 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:79.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% 25 156966279 084284.00294 identical to SEQ ID NO:64 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:80.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:65 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:81.
  • an anti-CDH11 antibody or CDH11-binding fragment thereof, comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:66 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:82.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:67 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:83.
  • an anti-CDH11 antibody or CDH11-binding fragment thereof, comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:69 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:84.
  • an anti-CDH11 antibody or CDH11-binding fragment thereof, comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:70 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:85.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:71 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:86.
  • an anti-CDH11 antibody or CDH11-binding fragment thereof, comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:72 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:87.
  • an anti-CDH11 antibody or CDH11-binding fragment thereof, comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:73 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:88.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:74 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:89.
  • an anti-CDH11 antibody or CDH11-binding fragment thereof, comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:76 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:91.
  • an anti-CDH11 antibody or CDH11-binding fragment thereof, comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% 27 156966279 084284.00294 identical to SEQ ID NO:76 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:92.
  • an anti-CDH11 antibody or CDH11-binding fragment thereof, comprising a heavy chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:77 and a light chain variable region that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:93.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:62 and a light chain variable region that comprises SEQ ID NO:78.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:68 and a light chain variable region that comprises SEQ ID NO:83.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:75 and a light chain variable region that comprises SEQ ID NO:90.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:63 and a light chain variable region that comprises SEQ ID NO:79.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:64 and a light chain variable region that comprises SEQ ID NO:80.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:65 and a light chain variable region that comprises SEQ ID NO:81.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:66 and a light chain variable region that comprises SEQ ID NO:82.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:67 and a light chain variable region that comprises SEQ ID NO:83. 28 156966279 084284.00294
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:69 and a light chain variable region that comprises SEQ ID NO:84.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:70 and a light chain variable region that comprises SEQ ID NO:85.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:71 and a light chain variable region that comprises SEQ ID NO:86.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:72 and a light chain variable region that comprises SEQ ID NO:87.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:73 and a light chain variable region that comprises SEQ ID NO:88.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:74 and a light chain variable region that comprises SEQ ID NO:89.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:76 and a light chain variable region that comprises SEQ ID NO:91.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:76 and a light chain variable region that comprises SEQ ID NO:92.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof comprising a heavy chain variable region that comprises SEQ ID NO:77 and a light chain variable region that comprises SEQ ID NO:93.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:1, CDR2H comprises SEQ ID NO:11, CDR3H comprises SEQ ID NO:20, CDR1L comprises SEQ ID NO:35, CDR2L comprises SEQ ID NO:43, and CDR3L 29 156966279 084284.00294 comprises SEQ ID NO:48; (b) the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:62 and (c) the light chain variable region comprises a sequence that is
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:1, CDR2H comprises SEQ ID NO:11, CDR3H comprises SEQ ID NO:21, CDR1L comprises SEQ ID NO:35, CDR2L comprises SEQ ID NO:43, and CDR3L comprises SEQ ID NO:49; (b) the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:63; and (c) the light chain variable region comprises a sequence that is at least 80%, at least 85%, at
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:4, CDR2H comprises SEQ ID NO:13, CDR3H comprises SEQ ID NO:25, CDR1L comprises SEQ ID NO:38, CDR2L comprises SEQ ID NO:45, and CDR3L comprises SEQ ID NO:53; (b) the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:67; and (c) the light chain variable region comprises a sequence that is at least 80%, at least 85%, at
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:4, CDR2H comprises SEQ ID NO:13, CDR3H comprises SEQ ID NO:26, CDR1L comprises SEQ ID NO:38, CDR2L comprises SEQ ID NO:45, and CDR3L 30 156966279 084284.00294 comprises SEQ ID NO:53; (b) the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:68; and the (c) light chain variable region comprises a sequence that
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:8, CDR2H comprises SEQ ID NO:17, CDR3H comprises SEQ ID NO:32, CDR1L comprises SEQ ID NO:38, CDR2L comprises SEQ ID NO:45, and CDR3L comprises SEQ ID NO:59; (b) the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:75; and (c) the light chain variable region comprises a sequence that is at least 80%, at least 85%, at
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:8, CDR2H comprises SEQ ID NO:17, CDR3H comprises SEQ ID NO:32, CDR1L comprises SEQ ID NO:38, CDR2L comprises SEQ ID NO:45, and CDR3L comprises SEQ ID NO:59; (b) the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:74; and (c) the light chain variable region comprises a sequence that is at least 80%, at least 85%, at
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:2, CDR2H comprises SEQ ID NO:11, CDR3H comprises SEQ ID NO:22, CDR1L comprises SEQ ID NO:36, CDR2L comprises SEQ ID NO:43, and CDR3L 31 156966279 084284.00294 comprises SEQ ID NO:50; (b) heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:64; and (c) the light chain variable region comprises a sequence that is
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:3, CDR2H comprises SEQ ID NO:12, CDR3H comprises SEQ ID NO:23, CDR1L comprises SEQ ID NO:37, CDR2L comprises SEQ ID NO:44, and CDR3L comprises SEQ ID NO:51; (b) heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:65; and (c) the light chain variable region comprises a sequence that is at least 80%, at least 85%, at least
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:3, CDR2H comprises SEQ ID NO:12, CDR3H comprises SEQ ID NO:24, CDR1L comprises SEQ ID NO:35, CDR2L comprises SEQ ID NO:43, and CDR3L comprises SEQ ID NO:52; (b) the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:66; and (c) the light chain variable region comprises a sequence that is at least 80%, at least 85%, at
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:5, CDR2H comprises SEQ ID NO:14, CDR3H comprises SEQ ID NO:27, CDR1L comprises SEQ ID NO:39, CDR2L comprises SEQ ID NO:46, and CDR3L 32 156966279 084284.00294 comprises SEQ ID NO:54; (b) the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:69; and (c) the light chain variable region comprises a sequence that
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:5, CDR2H comprises SEQ ID NO:14, CDR3H comprises SEQ ID NO:28, CDR1L comprises SEQ ID NO:35, CDR2L comprises SEQ ID NO:43, and CDR3L comprises SEQ ID NO:55; (b) the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:70; and (c) the light chain variable region comprises a sequence that is at least 80%, at least 85%, at
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:6, CDR2H comprises SEQ ID NO:15, CDR3H comprises SEQ ID NO:29, CDR1L comprises SEQ ID NO:40, CDR2L comprises SEQ ID NO:45, and CDR3L comprises SEQ ID NO:56; (b) the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:71; and (c)the light chain variable region comprises a sequence that is at least 80%, at least 85%, at
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:7, CDR2H comprises SEQ ID NO:16, CDR3H comprises SEQ ID NO:30, CDR1L comprises SEQ ID NO:41, CDR2L comprises SEQ ID NO:44, and CDR3L 33 156966279 084284.00294 comprises SEQ ID NO:57; (b) the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:72; and (c) the light chain variable region comprises a sequence
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:7, CDR2H comprises SEQ ID NO:16, CDR3H comprises SEQ ID NO:31, CDR1L comprises SEQ ID NO:35, CDR2L comprises SEQ ID NO:43, and CDR3L comprises SEQ ID NO:58; (b) the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:73; and (c) the light chain variable region comprises a sequence that is at least 80%, at least 85%,
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:9, CDR2H comprises SEQ ID NO:18, CDR3H comprises SEQ ID NO:33, CDR1L comprises SEQ ID NO:41, CDR2L comprises SEQ ID NO:45, and CDR3L comprises SEQ ID NO:60; (b) the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:76; and (c) the light chain variable region comprises a sequence that is at least 80%, at least 85%,
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:9, CDR2H comprises SEQ ID NO:18, CDR3H comprises SEQ ID NO:33, CDR1L comprises SEQ ID NO:41, CDR2L comprises SEQ ID NO:45, and CDR3L 34 156966279 084284.00294 comprises SEQ ID NO:60; (b) the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:76; and (c) the light chain variable region comprises a sequence
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof wherein the anti-CDH11 antibody, or CDH11-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein: (a) CDR1H comprises SEQ ID NO:10, CDR2H comprises SEQ ID NO:19, CDR3H comprises SEQ ID NO:34, CDR1L comprises SEQ ID NO:42, CDR2L comprises SEQ ID NO:47, and CDR3L comprises SEQ ID NO:61; (b) the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:77; and (c) the light chain variable region comprises a sequence that is at least 80%, at least 85%,
  • the antibodies disclosed herein may comprises modifications in their variable and their constant regions.
  • provides herein are antibodies comprising modified, but functionally equivalent variable regions and/or CDRs.
  • the modification does not significantly affect the properties of the antibody or antigen-binding fragment thereof.
  • the modification leads to enhanced or decreased activity and/or affinity.
  • the amino acid sequence of an anti-CDH11 antibody, or CDH11-binding fragment thereof, thereof may be mutated to obtain an antibody with the desired binding affinity to CDH11. Modification of polypeptides is routine practice in the art and need not be described in detail herein.
  • modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids which do not significantly deleteriously change the functional activity, or which mature (enhance) the affinity of the polypeptide for its ligand, or use of chemical analogs.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag.
  • substitution variants of the antibody molecule include the fusion 35 156966279 084284.00294 to the N- or C-terminus of the antibody or antigen-binding fragment thereof with an enzyme or a polypeptide which increases the half-life of the antibody in the blood circulation.
  • Substitution variants of the antibodies and antigen-binding fragments thereof have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. Substitutional mutagenesis may be directed to the hypervariable regions, but framework alterations are also contemplated. Examples of conservative substitutions are shown in Table 3. Table 3. Conservative amino acid substitutions.
  • Naturally occurring residues are divided into groups based on common side-chain properties: (1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile; (2) Polar without charge: Cys, Ser, Thr, Asn, Gln; (3) Acidic (negatively charged): Asp, Glu; (4) Basic (positively charged): Lys, Arg; (5) Residues that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe, His. 36 156966279 084284.00294 [0125] Conservative substitutions can also be made by exchanging a member of one of these classes for another member of the class.
  • substitution for example, that may be made is to change one or more cysteines in the antibody, which may be chemically reactive, to another residue, such as, without limitation, alanine or serine.
  • alanine or serine for example, there can be a substitution of a non-canonical cysteine.
  • the substitution can be made in a CDR or framework region of a variable domain or in the constant region of an antibody.
  • the cysteine is canonical. Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking.
  • the antibody disclosed is a monoclonal antibody or the antigen- binding fragment thereof is a fragment of a monoclonal antibody.
  • the term “monoclonal antibody” as used herein refers to an antibody member of a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
  • such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target on CDH11, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences.
  • the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones.
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
  • an identified monoclonal antibody can be produced by non-hybridoma techniques, e.g., by appropriate recombinant means once the sequence thereof is identified.
  • the antibody disclosed herein is a human antibody and/or the antigen-binding fragment thereof is a fragment of a human antibody.
  • a “human 37 156966279 084284.00294 antibody” is one whose sequences correspond to (i.e., are identical in sequence to) an antibody that could be produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein, but not one which has been made in a human.
  • a “human antibody” as used herein can be produced using various techniques known in the art, including phage-display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol.
  • the antigen e.g., CDH11 or an entity comprising such
  • mice See also KM Mouse® system, described in PCT Publication WO 02/43478 by Ishida et al., in which the mouse carries a human heavy chain transchromosome and a human light chain transgene, and the TC mouse system, described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727, in which the mouse carries both a human heavy chain transchromosome and a human light chain transchromosome, both of which are hereby incorporated by reference in their entireties.
  • the transgenes and/or transchromosomes carried by the mice comprise human immunoglobulin variable and constant region sequences.
  • human antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are sequences of human origin or identical thereto other than antibodies naturally occurring in a human or made in a human. Furthermore, if the antibody (e.g., an intact antibody rather than, for example, a Fab fragment) contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences.
  • the human antibodies disclosed herein may include amino acid residues not encoded by human sequences (e.g., mutations 38 156966279 084284.00294 introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the human antibodies are human monoclonal antibodies
  • such antibodies can be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • the antibody, or antigen-binding fragment thereof is a recombinant antibody.
  • recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created, or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. [0130] In some embodiments, the antibody, or antigen-binding fragment thereof, is isolated.
  • the term “isolated antibody” refers to an antibody that by virtue of its origin or source of derivation meets one, two, three or four of the following criteria: (1) is not substantially associated with naturally associated components that accompany it in its native state, (2) is substantially free of other proteins from the same species, (3) is expressed by a cell from a different species, and (4) does not occur in nature.
  • the antibody, or antigen-binding fragment thereof comprises a human constant region or modified constant region.
  • the antibody, or antigen- binding fragment thereof has a non-human constant region or a modified non-human constant region.
  • antibody, or antigen-binding fragment thereof has murine constant region or modified murine constant region.
  • the antibody, or antigen- binding fragment thereof has a non-human primate constant region or modified non-human 39 156966279 084284.00294 primate constant region.
  • the antibody, or antigen-binding fragment thereof has a non-human constant region or a modified non-human constant region.
  • constant region is from a non-human primate, a mouse, a rat, a sheep, a goat, or a rabbit. [0132] Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes.
  • the antibody or antigen- binding fragment can be, e.g., any of an IgG, IgD, IgE, IgA or IgM antibody or fragment thereof, respectively.
  • the antibody is an immunoglobulin G.
  • the antibody fragment is a fragment of an immunoglobulin G.
  • the antibody is an IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4.
  • the antibody comprises sequences from a mammalian IgG1, mammalian IgG2, mammalian IgG3 or mammalian IgG4.
  • the antibody comprises sequences from a human IgG1, human IgG2, human IgG3 or human IgG4.
  • an IgG generally has a serum half-life of 23 days, IgA 6 days, IgM 5 days, IgD 3 days, and IgE 2 days.
  • the antibody, or antigen-binding fragment thereof may comprise sequences from more than one class or isotype.
  • the antibody, or antigen-binding fragment thereof comprises an Fc domain that has the same sequence or 99% or greater sequence similarity with a human IgG1 Fc domain. In an embodiment, the antibody, or antigen-binding fragment thereof, comprises an Fc domain that has the same sequence or 99% or greater sequence similarity with a human IgG2 Fc domain. In an embodiment, the antibody, or antigen-binding fragment thereof, comprises an Fc domain that has the same sequence or 99% or greater sequence similarity with a human IgG3 Fc domain. In an embodiment, the antibody, or antigen-binding fragment thereof, comprises an Fc domain that has the same sequence or 99% or greater sequence similarity with a human IgG4 Fc domain.
  • the Fc domain is not mutated.
  • the Fc domain is mutated at the CH2–CH3 domain interface to increase the affinity of IgG for FcRn at acidic but not neutral pH.
  • the antibody, or antigen-binding fragment thereof comprises an Fc domain that has the same sequence as a human IgG1 Fc domain. 40 156966279 084284.00294 [0135]
  • the antibody, or antigen-binding fragment thereof has a modified IgG Fc region. Modified IgG Fc regions are well known in the art. For example, see any of the mutations listed in Table 1 of Wang et al. Protein Cell (2018), 9(1):63–73.
  • the modified Fc region relative to the unmodified Fc region, has enhanced complement-based effector function, increased or decreased Fc ⁇ R-based effector function, reduced effector function, enhanced coengagement of antigen and Fc ⁇ Rs, and/or increased serum half-life.
  • Fc modifications to modulate antibody effector function for IgG1 see Wang et al.
  • Protein Cell (2018), 9(1):63–73 include are: (a) Increased Fc ⁇ RIIIa binding: F243L/ R292P/ Y300L/ V305I/ P396L (b) Increased Fc ⁇ RIIIa binding: S239D/ I332E (c) Increased Fc ⁇ RIIIa binding: decreased Fc ⁇ RIIb binding S239D/ I332E/ A330L (d) Increased Fc ⁇ RIIIa binding: S298A/ E333A/ K334A. i. For example, in one heavy chain: L234Y/ L235Q/ G236W/ S239M/ H268D/ D270E/ S298A ii.
  • IgG1 L234A/ L235A ii.
  • IgG4 F234A/ L235A
  • m Reduced Fc ⁇ R and C1q binding
  • m Reduced Fc ⁇ R and C1q binding
  • IgG2 H268Q/ V309L/ A330S /P331S
  • n Reduced Fc ⁇ R and C1q binding
  • IgG2 V234A/ G237A/ P238S/ H268A/ V309L/ A330S/ P331S
  • p Increased FcRn binding at pH 6.0: M428L/ N434S
  • q Increased Fc ⁇ RIIb binding: S267E /L328F 41 156966279 084284.00294
  • r Increased Fc ⁇ RIIa binding, decreased Fc ⁇ RIIIa binding
  • Antibody Binding [0138] Provided herein are anti-CDH11 antibodies, or antigen-binding fragments thereof, that bind to human CDH11. Provided herein are anti-CDH11 antibodies, or antigen-binding fragments thereof, that bind to murine CDH11. Provided herein are anti-CDH11 antibodies, or antigen-binding fragments thereof, that bind to human CDH11 and to murine CDH11. Provided herein are anti-CDH11 antibodies, or antigen-binding fragments thereof, that bind to ectodomain 3 (extracellular cadherin domain, EC3) of CDH11. Provided herein are anti- CDH11 antibodies, or antigen-binding fragments thereof, that bind to EC4 of CDH11.
  • anti-CDH11 antibodies or antigen-binding fragments thereof, that bind to EC5 of CDH11.
  • anti-CDH11 antibodies, or antigen-binding fragments thereof that bind to EC3 of human CDH11.
  • anti-CDH11 antibodies, or antigen-binding fragments thereof that bind to EC3 of murine CDH11.
  • anti-CDH11 antibodies or antigen-binding fragments thereof, that bind to EC4 of murine CDH11.
  • anti-CDH11 antibodies, or antigen-binding fragments thereof that bind to EC5 of murine CDH11.
  • the anti-CDH11 antibody, or CDH11-binding fragment thereof binds CDH11 with a binding affinity (K D ) of from about 1 x 10 -6 M to about 1 x 10 -10 M, from about 1 x 10 -7 M to about 1 x 10 -10 M, from about 1 x 10 -8 M to about 1 x 10 -10 M, from about 1 x 10- 8 M to about 1 x 10 -19 M.
  • K D binding affinity
  • the anti-CDH11 antibody, or CDH11-binding fragment thereof binds CDH11 with a binding affinity (KD) of about 1 x 10 -7 M, about 1 x 10- 8 M, about 1 x 10 -9 M, or about 1 x 10 -10 M.
  • KD binding affinity
  • the anti-CDH11 antibody, or CDH11-binding fragment thereof, described herein is binds to CDH11 with a dissociation constant that is ⁇ 1 ⁇ M, ⁇ 1 nM, or ⁇ 10 pM.
  • the K D of the anti-CDH11 antibody, or CDH11-binding fragment thereof, for CDH11 is less than 100 nM.
  • the K D of the anti-CDH11 antibody, or CDH11-binding fragment thereof, for CDH11 is less than 10 nM. In an embodiment, the KD of the anti-CDH11 antibody, or CDH11- binding fragment thereof, CDH11 is less than 1.0 nM.
  • K D is intended to refer to the dissociation constant of an antibody-antigen interaction.
  • One way of determining the K D or binding affinity of antibodies to their antigen is by measuring binding 42 156966279 084284.00294 affinity Using a Dip and Read assay using an immobilized antigen and monoclonal antibodies (Octet Red96 ForteBio, Fremont, CA). (The affinity constant is the inverted dissociation constant).
  • Biotinylated antigen can be diluted into PBS + 0.1 % BSA, 0.02 % Tween 20 and 0.05 % sodium azide (Kinetics Buffer, ForteBio) and dipped into wells containing serial diluted mAbs starting from 37.75 nM.
  • concentrations of the Fab proteins are determined by ELISA and/or SDS-PAGE electrophoresis using an IgG1 standard monoclonal antibody of known concentration as a standard.
  • Kinetic association rates (kon) and dissociation rates (koff) are obtained simultaneously by fitting the data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994).
  • a molecular entity is said to exhibit “specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances.
  • An antibody “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target.
  • anti-CDH11 antibodies or antigen-binding fragments thereof, that specifically bind to human CDH11.
  • anti-CDH11 antibodies, or antigen-binding fragments thereof that specifically bind to murine CDH11.
  • an anti- CDH11 antibody, or CDH11-binding fragment thereof does not cross-react with murine CDH11.
  • an anti-CDH11 antibody, or antigen-binding fragment thereof that does not significantly bind to one or more of CDH5, CDH6, CDH7, CDH8, CDH9, CDH18, CDH20, or CDH24.
  • the anti-CDH11 antibody, or CDH11-binding fragment thereof binds to a linear epitope. In embodiments, the anti-CDH11 antibody, or CDH11-binding fragment 43 156966279 084284.00294 thereof, binds to a non-linear epitope. In one embodiment, anti-CDH11 antibody, or CDH11- binding fragment thereof, binds to its antigen with one, two, three, four, five, or six CDRs.
  • the term “compete”, as used herein with regard to an antibody means that a first antibody, or an antigen-binding portion thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody, or an antigen-binding portion thereof, such that the result of binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody.
  • the alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope.
  • each antibody detectably inhibits the binding of the other antibody with its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope(s).
  • competing and cross-competing antibodies are disclosed herein. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope, or portion thereof), the skilled artisan would appreciate, based upon the teachings provided herein, that such competing and/or cross-competing antibodies are encompassed and can be useful for the methods disclosed herein.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof that competes with any of the anti-CDH11 antibodies, or CDH11-binding fragments thereof, disclosed herein.
  • bispecific antibodies comprising any of the CDR sequences, variable heavy chain sequences, or variable light chain sequences disclosed herein.
  • a bispecific antibody refers to an antibody that is capable of binding two antigens simultaneously.
  • antibody Fragments may be a fragment comprising a Fab, Fab', F(ab')2, Fd, Fv, domain antibodies (dAbs such as shark and camel antibodies), ScFv, a maxibody, a minibody, a nanobody, an intrabody, a diabody, a triabody, a tetrabody, a v-NAR and a bis-scFv, or a polypeptide that contain at least 44 156966279 084284.00294 certain portions of an immunoglobulin sufficient to confer specific antigen-binding to the polypeptide.
  • Antibody fragments can be prepared, for example, by cleaving an intact antibody or by recombinant means. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989), hereby incorporated by reference in its entirety). Antigen-binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies or by molecular biology techniques.
  • the antibody disclosed herein is a Fab fragment, which comprises or consist essentially of a variable (VL) and constant (CL) domain of the light chain and a variable domain (VH) and the first constant domain (CH1) of the heavy chain.
  • the antibody fragment is a Fab' fragment, which refers to a Fab fragment having one or more cysteine residues at the C- terminus of the CH1 domain.
  • the antibody fragment is an Fd fragment comprising or consisting essentially of VH and CH1 domains.
  • the antibody fragment is an Fd' fragment comprising VH and CH1 domains and one or more cysteine residues at the C- terminus of the CH1 domain.
  • Single-chain Fv or scFv antibody fragments comprise or consist essentially of the VH and VL domains of an antibody, such that these domains are present in a single polypeptide chain.
  • an Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen-binding.
  • the antibody fragment is a Fv fragment comprising or consisting essentially of the VL and VH domains of a single arm of an antibody.
  • the antibody fragment is a diabody comprising two antigen-binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the antibody fragment is a dAb fragment comprising or consisting essentially of a VH domain.
  • the antibody fragment is a F(ab')2 fragment, which comprises a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge at the hinge region.
  • the antibody fragment is a linear antibody comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding regions.
  • F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
  • the antibody fragment of choice is a single chain Fv fragment (scFv). See, for example, WO 93/16185.
  • scFv single chain Fv fragment
  • these fragments can also be produced directly by recombinant host cells.
  • antibody fragments can be isolated from the antibody phage libraries discussed herein.
  • Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992).
  • an antibody, or antigen-binding fragment thereof, disclosed herein may include a modification, including, but not limited to glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
  • the process of making chemical modifications is known in the art, and may include, but are not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the molecules may contain one or more non-classical amino acids.
  • an antibody, or antigen-binding fragment thereof is conjugated to a functional moiety.
  • useful functional moieties include, but are not limited to, a blocking moiety, a detectable moiety, a diagnostic moiety, a targeting moiety, and a therapeutic moiety.
  • Exemplary blocking moieties include moieties of sufficient steric bulk and/or charge such that reduced glycosylation occurs, for example, by blocking the ability of a glycosidase to glycosylate the antibody or antigen-binding fragment thereof.
  • the blocking moiety may, 46 156966279 084284.00294 additionally or alternatively, reduce effector function, for example, by inhibiting the ability of the Fc region to bind a receptor or complement protein.
  • Preferred blocking moieties include cysteine adducts and PEG moieties.
  • the blocking moiety is a cysteine, preferably a cysteine that has associated with a free cysteine, e.g., during or subsequent to the translation of the Fc containing polypeptide, e.g., in cell culture.
  • Other blocking cysteine adducts include cystine, mixed disulfide adducts, or disulfide linkages.
  • the blocking moiety is a polyalkylene glycol moiety, for example, a PEG moiety and preferably a PEG-maleimide moiety.
  • Preferred pegylation moieties can be, for example, polyethylene glycol (“PEG”), polypropylene glycol (“PPG”), polyoxyethylated glycerol (“POG”) and other polyoxyethylated polyols, polyvinyl alcohol (“PVA”) and other polyalkylene oxides, polyoxyethylated sorbitol, or polyoxyethylated glu-cose.
  • the polymer can be a homopolymer, a random or block copolymer, a terpolymer based on the monomers listed above, straight chain or branched, substituted or unsubstituted as long as it has at least one active sulfone moiety.
  • the polymeric portion can be of any length or molecular weight but these characteristics can affect the biological properties. Polymer average molecular weights particularly useful for decreasing clearance rates in pharmaceutical applications are in the range of 2,000 to 35,000 Daltons.
  • the length of the polymer can impact upon the effective distance, and other spatial relationships, between the two groups. Thus, one skilled in the art can vary the length of the polymer to optimize or confer the desired biological activity.
  • PEG is useful in biological applications for several reasons.
  • PEG typically is clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyze, and is nontoxic.
  • Pegylation can improve pharmacokinetic performance of a molecule by increasing the molecule's apparent molecular weight. The increased apparent molecular weight reduces the rate of clearance from the body following subcutaneous or systemic administration. In many cases, pegylation can decrease antigenicity and immunogenicity. In addition, pegylation can increase the solubility of a biologically-active molecule.
  • detectable moieties for the detection of the antibodies and antigen-binding fragments thereof disclosed herein include fluorescent moieties or labels, imaging agents, radioisotopic moieties, radiopaque moieties, and the like, e.g. detectable labels such as biotin, fluorophores, chromophores, spin resonance probes, or radiolabels.
  • detectable labels such as biotin, fluorophores, chromophores, spin resonance probes, or radiolabels.
  • Exemplary fluorophores include fluorescent dyes (e.g. fluorescein, rhodamine, and the like) and other luminescent 47 156966279 084284.00294 molecules (e.g. luminol).
  • a fluorophore may be environmentally-sensitive such that its fluorescence changes if it is located close to one or more residues in the modified protein that undergo structural changes upon binding a substrate (e.g. dansyl probes).
  • exemplary radiolabels include small molecules containing atoms with one or more low sensitivity nuclei ( 13 C, 15 N, 2 H, 125 I, 123 I, 99 Tc, 43 K, 52 Fe, 67 Ga, 68 Ga, 111 In and the like). Other useful moieties are known in the art.
  • therapeutic moieties include, but are not limited to, anti-inflammatory agents, non-steroidal anti-inflammatory drugs, steroids, disease-modifying antirheumatic drugs (DMARDs), or recombinant proteins, including any such anti-inflammatory agents, non- steroidal anti-inflammatory drugs, steroids, disease-modifying antirheumatic drugs (DMARDs), or recombinant proteins disclosed herein.
  • the functional moiety may also have one or more of the above-mentioned functions.
  • a salvage receptor binding epitope may refer to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for in-creasing the in vivo serum half-life of the IgG molecule (e.g., Ghetie et al., 18 Ann. Rev. Immunol.
  • a nucleic acid molecule encoding the salvage receptor binding epitope can be linked in frame to a nucleic acid encoding a polypeptide sequence described herein so that the fusion protein expressed by the engineered nucleic acid molecule comprises the salvage receptor binding epitope and a polypeptide sequence described herein.
  • the serum half-life can also be in-creased, for example, by attaching other polypeptide sequences.
  • Other types of functional moieties are known in the art and can be readily used in the methods and compositions of the present disclosure based on the teachings contained herein.
  • Nucleic Acids and Vectors [0170] Also provided herein are nucleic acids encoding the antibodies, or antigen-binding fragments thereof, disclosed herein, as well as vectors, host cells, and expression systems.
  • nucleic acid refers to a polymeric form of nucleotides of any length, 48 156966279 084284.00294 either ribonucleotides or desoxyribonucleotides.
  • this term includes, but is not limited to, single-, double- or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • a nucleic acid that encodes a CDR or a variable chain sequence disclosed herein.
  • nucleic acid that encodes a variable chain sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:62-77.
  • nucleic acid that encodes a variable chain sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:78-93.
  • nucleic comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:94-127.
  • nucleic acid or set of nucleic acids that comprises two or more sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:94- 127.
  • nucleic acid sequence encoding a heavy chain variable region comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 94-110.
  • nucleic acid sequence encoding a light chain variable region the nucleic acid sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 111-127.
  • nucleic acid sequence encoding a heavy chain variable region the nucleic acid sequence comprising any one of SEQ ID NOs: 94-110.
  • nucleic acid sequence encoding a light chain variable region the nucleic acid sequence comprising any one of SEQ ID NOs: 111-127.
  • nucleic acids encoding any of the pairs of variable heavy and light chains disclosed in Table 2 (or variants thereof).
  • the nucleic acids encoding the antibody, or antigen-binding fragment thereof, disclosed herein may be, e.g., DNA, cDNA, RNA, synthetically produced DNA or RNA, or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination.
  • an expression vector or set of expression vectors comprising a polynucleotide sequence encoding an antibody, or antigen-binding fragment thereof, described herein operably linked to expression control sequences suitable for expression in a eukaryotic and/or prokaryotic host cell.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • a “vector” includes, but is not limited to, a viral vector, a plasmid, an RNA vector or a linear or circular DNA or RNA molecule which may consists of a chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids.
  • the employed vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available.
  • Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno associated viruses, AAV), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.
  • rabies and vesicular stomatitis virus paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).
  • Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, and spumavirus.
  • vectors comprising nucleic acids encoding the antibodies, or antigen-binding fragments thereof, disclosed herein.
  • vectors comprising a nucleic acid that encodes a variable chain sequence disclosed herein.
  • nucleic acid that encodes a variable chain sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:62- 77.
  • a vector comprising a nucleic acid that encodes a variable chain sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:78-93.
  • a vector comprising a nucleic comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:94-127.
  • a vector comprising a nucleic acid or set of nucleic acids that comprises two or more sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:94-127.
  • a vector comprising a nucleic acid sequence encoding a heavy chain variable region, the nucleic acid sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 94-110.
  • a vector comprising a nucleic acid sequence encoding a light chain variable region, the nucleic acid sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 111-127.
  • a vector comprising a nucleic acid sequence encoding a heavy chain variable region, the nucleic acid sequence comprising any one of SEQ ID NOs: 94-110.
  • a vector comprising a nucleic acid sequence encoding a light chain variable region, the nucleic acid sequence comprising any one of SEQ ID NOs: 111-127.
  • a vector comprising a nucleic acid encoding one or more of any of the amino acid sequences in Tables 1 or 2 (or variants thereof).
  • a set of vectors comprising nucleic acids encoding any of the pairs of variable heavy and light chains disclosed in Table 2 (or variants thereof).
  • a vector or a set of vectors comprising (1) a nucleic acid sequence comprising any one of SEQ ID NOs:94-110 and (2) a nucleic acid sequence comprising any one of SEQ ID NOs:111-127.
  • a vector or a set of vectors comprising: (a) SEQ ID NO:94 and/or SEQ ID NO:111; (b) SEQ ID NO:95 and/or SEQ ID NO:112; (c) SEQ ID NO:96 and/or SEQ ID NO:113; (d) SEQ ID NO:97 and/or SEQ ID NO:114; (e) SEQ ID NO:98 and/or SEQ ID NO:115; (f) SEQ ID NO:99 and/or SEQ ID NO:116; (g) SEQ ID NO:100 and/or SEQ ID NO:117; 51 156966279 084284.00294 (h) SEQ ID NO:101 and/or SEQ ID NO:118; (i) SEQ ID NO:102 and/or SEQ ID NO:119; (j) SEQ ID NO:103 and/or SEQ ID NO:120; (k) SEQ ID NO:104 and/or SEQ ID NO:121; (l) SEQ ID NO:105 and
  • a variety of expression vectors have been developed for the efficient synthesis of an antibody, or antigen-binding fragment thereof, in prokaryotic cells such as bacteria and in eukaryotic systems, including but not limited to yeast and mammalian cell culture systems have been developed.
  • the vectors can comprise segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • cells comprising expression vectors for the expression of the disclosed the antibody, or antigen-binding fragment thereof.
  • a host cell comprising a nucleic acid molecule described herein, or a vector described herein, is provided. The host cell can be isolated.
  • the cell is a mammalian, an insect cell, a fungal cell, or a bacterial cell.
  • Antibody Production comprising culturing a cell comprising one or more nucleic acid molecules encoding for an antibody, or antigen-binding fragment thereof, disclosed herein under conditions whereby the antibody, or antigen-binding fragment thereof, is produced by the host cell.
  • Nucleic acids encoding light and heavy chain variable regions, optionally linked to constant regions may be inserted into the same expression vectors.
  • the nucleic acids encoding light and heavy chain variable regions, optionally linked to constant regions are inserted into different expression vectors.
  • the expression vector may further comprise one or more expression control sequences, which include, but are not limited to, promoters (e.g., homologous or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • promoters e.g., homologous or heterologous promoters
  • signal sequences e.g., enhancer elements, and transcription termination sequences.
  • the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells.
  • the host is maintained under conditions suitable for high-level expression of the nucleotide sequences, and the collection and purification of the cross-reacting antibodies after the vector is incorporated into the appropriate host.
  • expression vectors contain selection markers (e.g., ampicillin- resistance, hygromycin-resistance, tetracycline resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequences.
  • selection markers e.g., ampicillin- resistance, hygromycin-resistance, tetracycline resistance or neomycin resistance
  • the host used to express the antibodies, or antigen-binding fragments thereof, disclosed herein can be a prokaryotic or eukaryotic host.
  • suitable hosts include bacterial or eukaryotic hosts, including yeast, insects, fungi, bird and mammalian cells either in vivo, or in situ, or host cells of mammalian, insect, bird, or yeast origin.
  • the mammalian cell or tissue can be of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog, or cat origin, but any other mammalian cell may be used.
  • Examples of bacterial hosts that can be used to express the antibodies, antigen- binding fragments disclosed herein can be E. coli, bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • Yeasts may also be used as hosts for expressing the express the antibodies, antigen- binding fragments or the fusion protein disclosed herein.
  • Saccharomyces and Pichia are exemplary yeast hosts, with suitable vectors having expression control sequences (e.g., promoters), an origin of replication, termination sequences, and the like as desired.
  • Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes.
  • Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for methanol, maltose, and galactose utilization.
  • Mammalian cells in culture may also be used as host cells for expressing the antibodies, antigen-binding fragments or the fusion proteins disclosed herein.
  • suitable host cell lines capable of secreting heterologous proteins include CHO cell lines, various COS cell lines, HeLa cells, 293 cells, myeloma cell lines, transformed B-cells, and hybridomas.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer and necessary processing information sites such as ribosome binding site, RNA splice site and/or transcriptional terminator sequences.
  • expression control sequences examples include SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like.
  • the antibodies, or antigen-binding fragments thereof, disclosed herein can be expressed using a single expression construct or vector or multiple expression constructs or vectors (e.g., two or three expression constructs). When the antibody heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins.
  • the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms disclosed herein can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel electrophoresis, and the like (see generally Scopes, Protein Purification (Springer-Verlag, N.Y., (1982)). Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses. [0196]
  • the antibodies, or antigen-binding fragments thereof, disclosed herein can be made by any method known in the art.
  • compositions comprising an anti- CDH11 antibody, or CDH11-binding fragment thereof as described herein.
  • a pharmaceutical composition comprising an antibody, or antigen- binding fragment thereof, described herein, and a pharmaceutically acceptable excipient, is provided.
  • the pharmaceutically acceptable excipient can be a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), solvent or encapsulating material, involved in carrying or transporting the therapeutic compound for 54 156966279 084284.00294 administration to the subject, bulking agent, salt, surfactant and/or a preservative.
  • a pharmaceutically-acceptable material, composition or vehicle such as a liquid or solid filler, diluent, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), solvent or encapsulating material, involved in carrying or transporting the therapeutic compound for 54 156966279 084284.00294 administration to the subject, bulking agent, salt, surfactant and/or a preservative.
  • materials which can serve as pharmaceutically-acceptable excipients include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gelatin; talc; waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as ethylene glycol and propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents; water; isotonic saline; pH buffered solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
  • sugars such as lactose, glucose and sucrose
  • starches such as corn starch and potato star
  • compositions or pharmaceutical compositions comprising the antibody, or antigen- binding fragment thereof, disclosed herein comprise stabilizers to prevent loss of activity or structural integrity of the protein due to the effects of denaturation, oxidation, or aggregation over a period of time during storage and transportation prior to use.
  • the compositions or pharmaceutical compositions can comprise one or more of any combination of salts, surfactants, pH and tonicity agents such as sugars that contribute to overcoming aggregation problems.
  • the composition may have a pH value in an approximately neutral pH range.
  • surfactant levels are minimized to avoid bubbles in the formulation which are detrimental for injection into subjects.
  • the composition or pharmaceutical composition is in liquid form and stably supports high concentrations of bioactive antibody in solution and is suitable for inhalational or parenteral administration.
  • the composition or pharmaceutical composition is suitable for intravenous, intramuscular, intraperitoneal, intradermal and/or subcutaneous injection.
  • the composition or pharmaceutical composition is in liquid form and has minimized risk of bubble formation and anaphylactoid side effects.
  • the composition or pharmaceutical composition is isotonic.
  • the composition or pharmaceutical composition has a pH or 6.8 to 7.4.
  • the antibodies, or fragments of antibodies, or compositions, or pharmaceutical compositions described herein can also be lyophilized or provided in any suitable forms 55 156966279 084284.00294 including, but not limited to, injectable solutions or inhalable solutions, gel forms, and tablet forms.
  • Methods of Using the Antibodies Described Herein [0204] In embodiments, the anti-CDH11 antibodies, or CDH11-binding fragments thereof, disclosed herein interfere with cellular CDH11:CDH11 binding. In embodiments, the anti- CDH11 antibodies, or CDH11-binding fragments thereof, disclosed herein interfere with CDH11:CDH11 binding between HSCs.
  • the anti-CDH11 antibodies, or CDH11-binding fragments thereof, disclosed herein interfere with CDH11:CDH11 binding between HSCs other mesenchyme derived CDH11 + pro-fibrotic cells.
  • the anti-CDH11 antibodies, or CDH11-binding fragments thereof suppress a CDH11 + cell’s pro-fibrotic phenotype.
  • Provided herein is a method of inhibiting CDH11-CDH11 interactions between cells, the method comprising contacting the cells with an anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein, or a pharmaceutical composition comprising anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein.
  • a method of inhibiting CDH11-CDH11 interactions in a subject in need thereof comprising administering to the subject an anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein, or a pharmaceutical composition comprising an anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein.
  • the subject is an animal.
  • the subject is a mammal.
  • the subject is a non-human mammal.
  • the subject is a non- primate (e.g., cows, buffalos, pigs, horses, cats, dogs, rats, mouse, guinea pigs, sheep, goats etc.) or a primate (e.g., monkey and human).
  • a primate e.g., monkey and human
  • the subject is human.
  • Individuals and patients are also subjects herein. 56 156966279 084284.00294
  • the terms “treat,” “treated,” “treating,” or “treatment” as used herein refer to therapeutic treatment, wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease.
  • Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
  • prevention refers to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or to minimize the extent of the disease or disorder or slow its course of development.
  • a method of treating fibrosis in a subject in need thereof comprising administering to the subject an anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein, or a pharmaceutical composition comprising an anti- CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein for use in treating fibrosis in a subject in need thereof.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein in the manufacture of a medicament or in the manufacture of a pharmaceutical composition for treating fibrosis in a subject in need thereof.
  • the anti-CDH11 antibody, or CDH11-binding fragment thereof reverses or prevents disease progression in fibrosis.
  • the fibrosis is liver fibrosis, pulmonary fibrosis, renal fibrosis, cardiac fibrosis, myelofibrosis, pancreatic fibrosis, dermal fibrosis, intestinal fibrosis, post-myocardial infarct fibrosis, replacement fibrosis, perivascular fibrosis, or arthrofibrosis.
  • the fibrosis is liver fibrosis.
  • the subject has an infection with hepatitis viruses, nonalcoholic fatty liver disease (NASH), an inherited metabolic disorder, a cholestatic disorder, or an immune disorder.
  • the liver fibrosis is associated with increased alcohol consumption or consumption of excess vitamin A.
  • the liver fibrosis is associated with autoimmune diseases of the liver or bile ducts (including, but not limited to, autoimmune hepatitis, primary biliary cholangitis or primary sclerosing cholangitis), drug toxicity, liver 57 156966279 084284.00294 transplantation rejection or genetic disorders of the liver (including, but not limited to, Wilson disease or hemochromatosis) or bile ducts (including, but not limited to, biliary atresia).
  • the fibrosis is intestinal fibrosis.
  • the subject has Inflammatory Bowel Disease (IBD), Crohn’s disease, or colitis.
  • the pulmonary fibrosis is induced as idiopathic pulmonary fibrosis, toxic injury (including, but not limited to, bleomycin, silicosis).
  • the subject has scleroderma or systemic sclerosis.
  • the subject has a keloid scar.
  • a method of treating rheumatoid arthritis in a subject in need thereof comprising administering to the subject an anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein, or a pharmaceutical composition comprising an anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein for use in treating rheumatoid arthritis in a subject in need thereof.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein in the manufacture of a medicament or in the manufacture of a pharmaceutical composition for treating rheumatoid arthritis in a subject in need thereof.
  • the antibodies, or antigen-binding fragments thereof, disclosed herein can be administered with to one or more additional therapeutic agents to induce disease remission/control and to maximize a therapeutic response without further impairing systemic immune responses to microbes.
  • Suitable additional therapeutic agents in this regard include, but are not limited to, anti-TNF alpha agents.
  • an anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed herein, or a pharmaceutical composition comprising an anti-CDH11 antibody, or CDH11-binding fragment thereof, disclosed is administered alone, or in combination with one or more other therapeutic agents (e.g., anti-inflammatory or metabolic agents) or agents that inhibit mesenchymal cell activation through growth factor receptor inhibition or fibrogenic cell depletion.
  • Suitable anti-inflammatory agents that are useful for treating inflammatory joint disorders, particularly RA, which can be administered in combination with anti-CDH11 antibodies, or CDH11-binding fragments thereof, disclosed herein, include, but are not limited to, (i) non-steroidal anti-inflammatory drugs (NSAIDs; e.g., detoprofen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenameate, mefenamic acid, meloxicam, nabumeone, naproxen sodium, oxaprozin, piroxicam, sulindac, tolmetin, celecoxib, rofecoxib, aspirin, choline salicylate, salsalte, and sodium and magnesium 58 156966279 084284.00294 salicylate); (ii) steroids (e.g., cor
  • the devices provided herein comprise one or more of the anti-CDH11 antibodies, or CDh11- binding fragments, disclosed herein. In embodiments, the devices provided herein comprise one or more of the antibodies disclosed herein. [0217] In embodiments, the antigen to be detected is immobilized directly onto a surface of a multi-well microtiter and is then contacted with an anti-CDH11 antibody or antigen-binding fragment thereof, disclosed herein, wherein the anti-CDH11 antibody or antigen-binding fragment thereof is equipped with a detection moiety.
  • the antigen to be detected is immobilized directly onto a surface of a multi-well microtiter and is then contacted with an anti-CDH11 antibody or antigen-binding fragment thereof, disclosed herein.
  • the anti-CDH11 antibody or antigen-binding fragment thereof is detected by a secondary antibody that binds to the anti-CDH11 antibody or antigen- binding fragment thereof and that is equipped with a detection moiety.
  • the anti-CDH11 antibodies or antigen-binding fragments disclosed herein are used in a sandwich ELISA or variation thereof. In a sandwich ELISA, the analyte to be measured is bound between two primary antibodies, each detecting a different epitope of the antigen.
  • the first primary antibody, the capture antibody, is often immobilized on a surface.
  • the second primary antibody is often equipped with a detection moiety.
  • the second 59 156966279 084284.00294 primary antibody may also be unlabeled, and may in turn be detected by a secondary antibody that binds to the second primary antibody and that is equipped with a detection moiety.
  • the anti-CDH11 antibodies, or antigen-binding fragments, disclosed herein are used as a first primary antibody.
  • the anti-CDH11 antibodies, or antigen- binding fragments, disclosed herein are used as a second primary antibody.
  • CDH11 Provided herein is a method of detecting CDH11, the method comprising contacting a biological sample with an anti-CDH11 antibody, or CDH11-binding fragment thereof, as described herein and detecting binding of the anti-CDH11 antibody, or CDH11-binding fragment thereof, to CDH11.
  • Binding of the anti-CDH11 antibody, or CDH11-binding fragment thereof, to its antigen can be detected in numerous ways, including, but not limited to (1) attaching a reporting moiety to the anti-CDH11 antibody, or CDH11-binding fragment thereof, or (2) contacting the CDH11 bound by a first anti-CDH11 antibody, or CDH11-binding fragment thereof, with a second anti-CDH11 antibody, or CDH11-binding fragment thereof (which in turn may be attached to a reporting moiety), or (3) contacting the second anti-CDH11 antibody, or CDH11-binding fragment thereof, with a third antibody (which in turn may be attached to a reporting moiety) that recognizes an antigen located on the second anti-CDH11 antibody, or CDH11-binding fragment thereof, such as the constant region of the second anti anti-CDH11 antibody, or CDH11-binding fragment thereof.
  • the reporting entity comprises an enzyme.
  • the enzyme is horseradish peroxidase (HRP) or alkaline phosphatase (AP).
  • the reporting entity comprises a gold nanoparticle.
  • the anti-CDH11 antibody, or CDH11-binding fragment thereof may be affixed to a solid support of a device.
  • the solid support comprises nitrocellulose.
  • the device further comprises a fluid sample pad prior in sequential order to the first and second portions.
  • a kit containing one or more of the antibodies or antigen- binding fragments disclosed herein and one or more buffers.
  • the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes those possibilities).
  • All other referenced patents and applications are incorporated herein by reference in their entirety.
  • Example 1 Generation of Anti-CDH11 Antibodies [0230] CDH11 Constructs [0231] Human (ENST00000268603.8) and mouse (ENSMUST00000075190.4) CDH11 full length and extracellular domains (ECD) CDS DNA were synthesized (GeneArt, ThermoFisher) and cloned into a variety of vectors with and without various c-terminal tags. For immunizations, full length human CDH11 was cloned into the pcDNA3.1 mammalian expression vector (ThermoFisher), untagged.
  • CDH11 For flow cytometry-based screening, human and mouse full length CDH11 were cloned into pSelect-cGFP vectors (InVivoGen) producing full length CDH11 membrane-tethered proteins fused with a c-terminal, intracellular GFP tag. These constructs were transfected into Expi293 cells (ThermoFisher) and analyzed by flow cytometry.
  • human CDH11 ECD DNA was 61 156966279 084284.00294 cloned into pFuse_Fc tag vectors which recombinantly fuses a human G1 Fc or mouse G2a Fc tag to the c-terminal end of the ECD protein domain of CDH11. These constructs were used to transfect Expi293 cells for the production of recombinantly Fc tagged protein secreted into the supernatant. Protein was purified from supernatants using Protein A/G.
  • CDS DNA for eight different full length human cadherins (CAD 5, 6, 7, 8, 18, 20 & 24) were also synthesized and cloned in pSelect_cGFP which produced c-terminal, tagged GFP recombinant membrane- tethered proteins used for flow cytometry as described above.
  • Mouse Fc tagged proteins were used primarily as boosting immunogens and human Fc tagged proteins were used as the final boost to encourage a CDH11 specific response.
  • Ms# 135-138 were given protein immunizations only (Fig.1B).
  • Mouse sera from various points of time after immunizations were screened by ELISA on human CDH11 ECD protein tagged with human IgG1-Fc coated at 5 ⁇ g/ml (Fig.2A).
  • Sera 62 156966279 084284.00294 were also screened by flow on transfected Expi293F cells (Invitrogen) with the GFP tagged human and mouse full length CDH11 constructs (Fig.2B).
  • Fusion Screens [0239] Three fusions were performed on responding mice over a six-month period with all mice being fused except mouse #138 and 139, which gave very low titers. Screens were performed with hybridoma supernatants on Expi293F cells transfected with human CDH11 fused to GFP. ELISAs were also performed using the human Fc (IgG1) tagged human CDH11 ECD protein at 5 ⁇ g/ml (Fig. 3).
  • clones Forty-seven clones were identified that produced a positive signal by flow or ELISA after two rounds of screening (Fig. 3 and Table 6).
  • fusion sera shown at 1:1000 dilution
  • the commercial anti-human CDH11, clone 16G5, (BioLegend) at 1 ⁇ g/ml
  • Normal mouse sera at 1:1000 dilution
  • Isotyping was performed using standard mouse isotyping kits. Clones that showed flow reactivity were considered higher in priority over clones that were just ELISA positive, due to a better expectation of conformational binding.
  • Example 2 Binding of Hybridoma Supernatants to Primary Human and Rat Fibroblast-Like Synoviocytes (FLS) [0241] Primary human and rat FLS were used to assess binding of selected hybridoma supernatants from Fusion 1 and 2 by flow cytometry, based on the availability of the cells.
  • Commercial cadherin control antibody 16G5 was used as a positive control (1 ⁇ g/ml) as was fusion sera (1:1000 dilution). Normal mouse sera (1:1000 dilution) and irrelevant isotype control antibody (1 ⁇ g/ml).
  • APC conjugated goat anti-mouse IgG (Fc specific) was used as secondary antibody.
  • Clones 2D2, 10E1, 10F1, 11D2, and 11D3 showed positive staining to both human and rat FLS (Fig.4).
  • Clone 5A2 showed small positivity to human synoviocytes.
  • Human FLS were not available during the screening of the Fusion 3 hybridomas, but fusion 3 hybridomas were assessed for binding to primary rat FLS.
  • Two clones showed reactivity to rat FLS, namely 2A1 and 2B9.
  • Example 3 Cross-reactivity of Hybridoma Supernatants to Type 2 Cadherins and Mouse CDH11
  • hybridomas were screened by flow cytometry on Expi293F cells expressing one of 63 156966279 084284.00294 eight different human type 2 cadherins (Cadherin 5, 6, 8, 9, 18, 20, and 24) fused to GFP (Table 5).
  • Hybridoma supernatants from CDH11-binding clones were screened by flow cytometry for binding to Expi293F HEK cells expressing different cadherins C-terminally tagged with GFP.
  • Results are shown as percentage graphs, relative for each clone.
  • Commercial anti-huCDH11 antibody 16G5, fusion sera, and normal mouse sera were used as controls.
  • Cross reactivity was also assessed using with purified antibody.
  • Lead antibodies were purified from VelcoImmune-derived hybridoma supernatants and tested on transiently transfected Expi293 cells with either human or mouse CAD11 tagged with GFP.
  • Antibodies were stained at a concentration of 2.5 ⁇ g/ml and compared to a mouse anti-Human CAD11 control (clone 16G5) or isotope control (mouse G1) or cells alone (mock transfected) by flow cytometry.
  • the clones that showed cross-reactivity to other constructs were predominantly ELISA positive clones (for example 3F8, 3G8, 5C4, and 1G11 in fusion 1; 2F6, 3G6 and 17C1 in fusion 2; 2B9 and 5E4 in fusion 3) and showed very low binding to all cadherins by flow (these mostly were ELISA positive clones) or they were broadly reactive to all human cells (3F8, 3G8, 5C4 and 5E4) as shown in the pie diagrams. Clone 2D2 from fusion 1 showed a slight positivity to cadherin 8 but was overwhelmingly positive for human and mouse CDH11.
  • Table 7 provides a summary of the tested clones. 64 156966279 Table 5. Cross reactivity of hybridoma supernatants with different type 2 cadherins.
  • HuCAD human cadherin.
  • MoCAD murine cadherin.
  • Hybridoma supernatants from CDH11-binding clones were screened by flow cytometry for binding to Expi293F HEK cells expressing different cadherins C-terminally tagged with GFP.
  • Commercial anti-huCDH11 antibody 16G5, fusion sera, and normal mouse sera were used as controls.
  • Anti- HuCA HuCA HuCA HuCA HuCA HuCA HuCA MoC body D 5 D 6 D 7 D 8 D 9 D 18 D 20 D 24 AD 11 2 3 4 1 0 1 8 2 4 6 8 3 8 8 5
  • Table 7 Summary of clones. Summary of binding analysis of 47 generated clones. Binding MFI or absorbance OD measurements for ELISA were normalized to commercial control antibody (16G5) and ranked. Binding was considered “low” for any events lower than 25% of the control, positive for any events greater than 25% of the control, and double positive for events within the control range or higher ( ⁇ 10%). Clones that were within range of the negative control (normal mouse sera and mock cells) ( ⁇ 5%) were considered negative. Summaries are also included for interpretations of cross reactivity against the other type 2 cadherins, as well as results of the binding to primary FLS cells. Human FLS cells were only available to a select group of clones from fusion 1 and 2. Rat FLS were available for testing hybridomas from all three fusions. Antibody ELISA hCDH11 MsCDH11 Human Rat Positive (Expi 293) (Expi 293) Primary Primary Primary Primary
  • the GTPase Rac Regulates the Proliferation and Invasion of Fibroblast-Like Synoviocytes from Rheumatoid Arthritis Patients. Mol Med. 2007;13(5-6):297-304, incorporated by reference in its entirety herein.
  • matrigel FLS invasion correlates with joint damage. Briefly, FLS invasion was studied in a trans well system using Matrigel-coated inserts (BD Biosciences). Cells that were 70-80 % confluent were harvested by trypsin-EDTA digestion and washed with serum-free DMEM.
  • Example 6 Immunofluorescence Staining of Mouse FLS with Anti-CDH11 Antibodies
  • KRN serum induced arthritis (KSIA)-derived FLS cell line was used for immunofluorescence staining.
  • Four of the six antibodies tested (16A10, 2A10, 3A10 and 5H1) stained positive compared with isotype control, while two stained negative (2F6 and 2A1) (Fig.9).
  • Example 7 Efficacy of Anti-CDH11 Antibodies In Vivo in a KRN Serum Induced Arthritis (KSIA) Model
  • KSIA KRN Serum Induced Arthritis
  • Example 8 Binding Affinity of Selected Anti-CDH11 Antibodies
  • Antibodies 2A10, 3A10, and 10E1 were evaluated for affinity (KD) based on binding to transfected human CDH11 Expi293 cells (Fig. 12A, Table 10) and mouse CDH11 transfected Expi293 cells (Fig.12B, Table 11). KD values were calculated based on nonlinear fit regression analysis of the binding data (GraphPad Prism). In a third experiment (Fig.
  • Example 10 Efficacy of Anti-CDH11 Antibodies in a Well-Established Model of Fibrosis
  • human hepatic stellate cells hHSC
  • Cells were cultured for 24 hours or 48 hours in the presence of different concentrations of anti-CDH11 antibody 3A10, and compared with a control isotype.
  • Anti-CD11 antibody 3A10 had no effect on the hHSC lines’ viability (Fig. 15A) or proliferation (not shown).
  • Cells were harvested at 24 hours and 48 hours and run in triplicate for qPCR and for collagen type I (Col1a1) protein quantification in the supernatant.
  • An Alk5 inhibitor (Alk5i) was used as a positive control.
  • Anti-CDH11 antibody 3A10 significantly reduced the expression of collagen type I (Col1a1), actin alpha 2, smooth muscle (Acta2), platelet-derived growth factor receptor beta (bPDGFR), matrix metalloproteinase-1 (MMP1), and matrix metalloproteinase-2 (MMP2), which are genes implicated in fibrosis and in the fibrotic phenotype of fibroblasts, hHSC and myofibroblasts (Fig.15B, results shown for LX2, TWNT4, and JS1 cells).
  • Anti-CDH11 antibody 3A10 also significantly reduced the production of Col1a1 protein by LX-2 and TWNT4, further demonstrating its anti-fibrotic properties (Figs.15C and 15D).
  • Anti CDH113A10 significantly reduced the expression of fibrogenic genes (Fig.15E), reduced the production of Col1a1 (Fig. 15F), reduced proliferation (Fig. 15G), and reduced ⁇ SMA protein expression in primary hHSCs isolated from patients (Fig.15H).
  • Example 11 Anti-CDH113A10 was not Cytotoxic to Human Precision Cut Liver Slices (hPCLS)
  • An LDH-assay was used to assess to assess cytotoxicity in hPCLS treated with anti- CDH11 3A10.
  • Human precision-cut liver slices (hPCLS) were generated from discarded remnants of surgically resected human livers. Two different male patient backgrounds for liver samples were selected for the hPCLS study.
  • Normal-appearing resection margins surrounding HCC or metastatic regions were selected for hPCLS generation.
  • the ischemic time between post-hepatectomy and generated PCLS was 3 - 4 hours.
  • the resected liver pieces were transported to the lab in ice-cold Krebs-Henseleit buffer. Cores of 8 mm diameter were generated by using a stainless-steel coring tool, and the liver core was attached to the specimen holder by solvent- free cyanoacrylate adhesive.
  • 74 156966279 was mounted in the buffer tray, which was submerged in carbogen saturated ice-cold Krebs- Henseleit buffer supplemented with 25 mM glucose, and the liver slices were created using VT1200S tissue slicer (Leica Biosystem, IL). During slicing Krebs-Henseleit buffer was continuously supplied with carbogen. Intact liver slices were collected from the buffer tray and transferred to six well tissue culture plate (3-4 slices/well) filled with 37 °C warm William’s E GlutaMAX media (Thermo Fisher Scientific, MA) supplemented with 15% Hepatocyte Media (Takara Bio Inc.), 25 mM glucose and 50 ⁇ g/ml gentamycin (Thermo Fisher Scientific, MA).
  • the slices were pre-incubated in a 37 °C incubator supplemented with 5% CO 2 and 95% O 2 on a gentle rocker (10 rpm) for 24 hours to equilibrate the tissues. After 24 hours pre-incubation for equilibration, fresh medium was added containing either IgG1 (1000 ng/ml) control or two different concentration of 3A10 (100 ng/ml or 1000 ng/ml) for additional 48 hours. At 24 hours of antibody treatment the old media was replace by new culture media containing respective doses of IgG1 or 3A10 antibody. At the end of drug treatment (at 24 and 48 hours) culture media from each condition were collected.
  • Example 12 Anti-CDH11 Antibodies Result in Downregulation of Fibrotic Genes
  • LX-2 cells were treated separately for 24 and 48 hours with either 100 ng/ml or 10,000 ng/ml of these hybridoma supernatants comprising selected antibodies, respectively.
  • PY102 10,000 ng/ml
  • Alk5i (10 uM) were used in parallel as IgG and positive treatment controls.
  • SIN SEQ ID NO.
  • SIN V ⁇ chain 10E1 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLGWYQQK Y Y P T Y L L L L L G Y L L S P Y

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Abstract

L'invention concerne des anticorps et des fragments de liaison à l'antigène de ceux-ci se liant à la cadhérine 11 (CDH11) ainsi que des acides nucléiques codant de tels anticorps et des fragments de liaison à l'antigène de ceux-ci et des vecteurs comprenant lesdits acides nucléiques. L'invention concerne en outre des méthodes d'utilisation et des dispositifs utilisant de tels anticorps et/ou fragments de liaison à l'antigène, comprenant, mais sans s'y limiter, des méthodes d'inhibition d'interactions CDH11-CDH11 entre des cellules et des méthodes de traitement d'une maladie chez un sujet en ayant besoin.
PCT/US2024/022682 2023-04-07 2024-04-02 Anticorps monoclonaux dirigés contre la cadhérine 11 et méthodes d'utilisation Ceased WO2024211317A2 (fr)

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