WO2020072937A1 - Anticorps dirigés contre les protéines d'adhérence de la zone de la membrane basale bp180 et bp230 - Google Patents

Anticorps dirigés contre les protéines d'adhérence de la zone de la membrane basale bp180 et bp230

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Publication number
WO2020072937A1
WO2020072937A1 PCT/US2019/054751 US2019054751W WO2020072937A1 WO 2020072937 A1 WO2020072937 A1 WO 2020072937A1 US 2019054751 W US2019054751 W US 2019054751W WO 2020072937 A1 WO2020072937 A1 WO 2020072937A1
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Prior art keywords
antibody
antigen binding
binding fragment
protein
disease
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English (en)
Inventor
Matthias Christoph HAMMERS
Donald L. Siegel
John R. Stanley
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University of Pennsylvania Penn
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University of Pennsylvania Penn
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • BP Bullous pemphigoid
  • BP AG BP antigen
  • BP230 BPAG1, BPAGle
  • BP180 being a critical transmembrane protein mediating adhesion of the basal keratinocytes to dermal adhesion proteins such as laminin 332 (Nishie W et al. Am. J Pathol. 2011; 179: 829-37).
  • NC16A non-collagenous domain 16A
  • the non-collagenous domain 16A (NC16A) of the BP180 ectodomain is the immunodominant epitope in BP patients, and antibody titers against BP 180- NC16A correlate to disease activity, as opposed to anti-BP230 antibody titers (Zillikens D, et al. J. Invest. Dermatol. 1997; 109: 573-9; Dopp R, et al. Journal of the American Academy of Dermatology 2000; 42: 577-83; Sitaru C, et al. Experimental dermatology 2007; 16: 770-7; Holtsche MM, et al. The British journal of dermatology 2018; Daneshpazhooh M, et al.
  • Routine diagnostic confirmation and follow-up of BP relies on direct
  • the present invention provides antibodies against basement membrane zone adhesion proteins BP180 and BP230.
  • the invention provides a fusion protein comprising a first domain and a second domain, wherein the first domain comprises an antigen-binding domain comprising an antibody or antigen binding fragment thereof that binds BP 180 or BP230, and wherein the second domain comprises a therapeutic polypeptide or fragment thereof.
  • the antibody or antigen binding fragment thereof comprises one CDR selected from the group consisting of at least 1, 2, 3, 4, 5, or 6 CDRs as listed in Table 7.
  • the antibody or antigen binding fragment thereof is a scFv.
  • the antibody or antigen binding fragment thereof comprises 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 33-64 or 68-70.
  • the therapeutic polypeptide is a complement-inhibiting peptide.
  • the complement-inhibiting peptide inhibits Cls.
  • the Cls inhibiting peptide is gigastasin.
  • the complement-inhibiting peptide is a factor H-binding peptide.
  • the factor H-binding peptide is 5C6.
  • the fusion protein comprises 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 74-76.
  • the invention provides a recombinant nucleic acid encoding the fusion protein of any one of the above aspects or any aspect of the invention delineated herein.
  • the invention provides a pharmaceutical composition comprising the fusion protein of any one of the above aspects or any aspect of the invention delineated herein.
  • the invention provides a method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of the above aspects or any aspect of the invention delineated herein.
  • the pharmaceutical composition is administered intravenously, intra-articularly, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intravitreously, intra-muscularly, subcutaneously, orally or intranasally.
  • the method further comprises administering a second agent to the subject.
  • the second agent is at least one of carboplatin, cisplatin, paclitaxel, docetaxel, gemcitabine, bevacizumab, olaparib, rucaparib, niraparib,
  • cyclophosphamide FU, abiraterone, flutamide, bicalutamide, leuprolide, goserelin, buserelin, triptorelin, degarelix, Enzalutamide, Apalutamide, Sipuleucel-T, Cabazitaxel, Radium-223, trastuzumab, pertuzumab, lapatinib, tamoxifen, oxaliplatin, capecitabine, leucovorin, Irinotecan, Cetuximab, panitumumab, aflibercept, Regorafenib, Trifluridine-tipiracil, immune checkpoint inhibitors (nivolumab, pembrolizumab), cabozantinib, sunitinib, pazopanib, axitinib, interleukin- 2, interferon alpha, mitomycin C, epirubicin, BCG, bleomycin,
  • the invention provides a method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of the above aspects or any aspect of the invention delineated herein, wherein the fusion protein binds to non-collagenous domain 16A of BP 180, and wherein the disease is bullous pemphigoid, mucous membrane pemphigoid (MMP), linear IgA disease, lichen planus pemphigoides, pemphigoid gestationis, or an autoimmune disease wherein the patient has antibodies against the basement membrane.
  • MMP mucous membrane pemphigoid
  • linear IgA disease lichen planus pemphigoides
  • pemphigoid gestationis or an autoimmune disease wherein the patient has antibodies against the basement membrane.
  • the fusion protein displaces serum IgG against non-collagenous domain 16A of BP 180, and wherein the complement-inhibiting peptide prevents complement deposits at the BMZ.
  • the invention provides an isolated antibody or antigen binding fragment thereof that binds BP180 or BP230, wherein the isolated antibody or antigen binding fragment thereof comprises one CDR selected from the group consisting of at least 1, 2, 3, 4, 5 or 6 CDRs as listed in Table 7.
  • the isolated antibody or antigen binding fragment cross- competes with anti -BP 180 or anti-BP230 antibodies or fragments thereof.
  • said antibody or antigen binding fragment thereof is human, humanized or chimeric.
  • the invention provides a recombinant nucleic acid encoding the antibody or antigen binding fragment thereof of any one of the above aspects or any aspect of the invention delineated herein.
  • the invention provides an antibody-drug conjugate comprising the antibody or antigen binding fragment of any one of the above aspects or any other aspect of the invention delineated herein.
  • the drug is MMAE, ozogamicin, emtansine, amanitin, pyrrolobenzodiazepine (PBD) dimer toxin, a chalichaemicin, a cytotoxic maytansinoid, DM1 , carboplatin, cisplatin, paclitaxel, vedotin, or diphtheria toxin.
  • the invention provides an antibody-protein conjugate.
  • the protein is a therapeutic protein.
  • the therapeutic protein is an enzyme or a pro-apoptotic protein.
  • the invention provides a pharmaceutical composition comprising the isolated antibody or antigen binding fragment of any one of the above aspects or any other aspect of the invention delineated herein.
  • the invention provides a pharmaceutical composition comprising the antibody-drug conjugate of any one of the above aspects or any other aspect of the invention delineated herein.
  • the invention provides a pharmaceutical composition comprising the antibody-protein conjugate of any one of the above aspects or any other aspect of the invention delineated herein.
  • the invention provides a method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of the above aspects or any other aspect of the invention delineated herein.
  • the antibody or antigen binding fragment is administered intravenously, intra-articularly, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intravitreously, intra-muscularly, subcutaneously, orally or intranasally.
  • the method of treating a disease in a subject in need thereof further comprises administering a second agent to the subject.
  • the second agent is at least one of carboplatin, cisplatin, paclitaxel, docetaxel, gemcitabine, bevacizumab, olaparib, rucaparib, niraparib,
  • cyclophosphamide FU, abiraterone, flutamide, bicalutamide, leuprolide, goserelin, buserelin, triptorelin, degarelix, Enzalutamide, Apalutamide, Sipuleucel-T, Cabazitaxel, Radium-223, trastuzumab, pertuzumab, lapatinib, tamoxifen, oxaliplatin, capecitabine, leucovorin, Irinotecan, Cetuximab, panitumumab, aflibercept, Regorafenib, Trifluridine-tipiracil, immune checkpoint inhibitors (nivolumab, pembrolizumab), cabozantinib, sunitinib, pazopanib, axitinib, interleukin- 2, interferon alpha, mitomycin C, epirubicin, BCG, bleomycin,
  • the invention provides a method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of any of the above aspects or any other aspect of the invention delineated herein, wherein the antibody or antigen binding fragment binds to non-collagenous domain 16A of BP180, and wherein the disease is bullous pemphigoid, mucous membrane pemphigoid (MMP), linear IgA disease, lichen planus pemphigoides, pemphigoid gestationis, or an autoimmune disease wherein the patient has antibodies against the basement membrane.
  • MMP mucous membrane pemphigoid
  • linear IgA disease lichen planus pemphigoides
  • pemphigoid gestationis or an autoimmune disease wherein the patient has antibodies against the basement membrane.
  • the antibody or antigen binding fragment is an scFv.
  • the antibody or antigen binding fragment displaces serum IgG against non-collagenous domain 16A of BP180.
  • the invention provides a method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of any of the above aspects or any aspect of the invention delineated herein, wherein the disease is bullous pemphigoid, mucous membrane pemphigoid (MMP), linear IgA disease, lichen planus pemphigoides, pemphigoid gestationis, anti-p200 pemphigoid, epidermolysis bullosa acquisita, graft versus host disease (GVHD), toxic epidermolysis bullosa, erythema multiforme, other diseases with interface dermatitis, allergic contact dermatitis, atopic dermatitis, psoriasis, vitiligo or skin cancer.
  • MMP mucous membrane pemphigoid
  • linear IgA disease lichen planus pemphigoides
  • pemphigoid gestationis anti-p200 pemphigoid
  • the antibody portion of the antibody-drug conjugate or the antibody-protein conjugate binds to non-collagenous domain 16A of BP180.
  • the antibody or antigen binding fragment is an scFv.
  • the skin cancer is actinic keratosis, squamous cell carcinoma (SCC), basal cell carcinoma (BCC) or melanoma.
  • Figures 1A-1D illustrate affinity purification (AP) of BP180-NC16A and BP230-C specific antibodies from two representative bullous pemphigoid sera. Eluates from BP180- NCl6A-APs ( Figure 1A, Figure 1C) and BP230-C-APs ( Figure 1B, Figure 1D) demonstrated monospecific reactivity against the BP230-C or BP180-NC16A antigens, showing good effectivity of the AP procedure.
  • AP affinity purification
  • FIGS 2A-2D illustrate indirect immunofluorescence testing of affinity-purified sera on normal human skin and monkey esophagus substrates.
  • IIF indirect immunofluorescence
  • BP230-C-specific polyclonal antibodies Abs
  • BMZ basement membrane zone
  • Figures 3A-3C illustrate that monoclonal antibodies confirm findings from affinity- purified polyclonal antibody testing by indirect immunofluorescence.
  • Antibody phage display- derived scFv mAb clones were confirmed for binding specificity by anti-HA EFISA on BP230-C and BP180-NC16A substrates (Figure 3 A).
  • Deposition of two anti-BP230-C mAbs (BP230-C- mAbl [4-8E, scFv# 28; Table 3] and BP230-C-mAb2 [4-8G, scFv# 14; Table 3]) was observed on NHS and ME substrates (Figure 3B).
  • Figures 4A-4D illustrate double-depletion experiments of two representative BP sera from BP180-NC16A and BP230-C antibodies, each resulting in positive indirect
  • Figures 5A-5B illustrate double indirect immunofluorescence of polyclonal affinity- purified antigen-specific antibodies and a monoclonal antibody. Confirming the binding patterns for anti-BPl 80-NC 16A antibodies seen in Figures 2A-2D and Figures 3A-3C, NC16A expression is present only in normal human skin (Figure 5A), but not in monkey esophagus ( Figure 5B), as shown for two representative BP sera (BP-l, BP-2) and the monoclonal antibody BP180-NC16A- m Ab 1.
  • FIGS 6A-6B illustrate that anti-BPl 80-NC 16A XgE variable heavy chain (VH) gene regions feature significantly lower mutation counts when compared to anti -BP 1 80-NC16A IgG VH gene regions, suggesting direct switch from IgM to IgE.
  • Next generation sequencing detects the majority of BP 180-NC 16A-specific IgE clones in the antigen-specific IgGi 23 4 repertoire of the same patient. Only 2 of 23 anti-BPl 80-NC 16A clones were detected exclusively in IgE (and not IgGi234) repertoire.
  • the majority of BP 180-NC16A IgE mAbs are produced of indirect class- switch recombination, from antigen-specific IgG+ B cells.
  • Figure 7 illustrates that analysis of light chain usage indicates a predominant utilization of the variable light chain gene VL1-47 in anti-BPl 80-NC16A antibodies obtained by genetic cloning.
  • the same variable light chain gene VL1-47 paired with 12 clonal ly different variable heavy chains in both anti-BPl 80-NC 16 A IgE and IgG Abs, suggesting shared structural and/or ammo acid sequence requirements for binding to the antigen.
  • LC-MS/MS was employed on affinity-purified anti-BPl 80-NC 16A serum antibody- light chains from multiple patients (anti-BP230 antibody light chains from same patient sera served as controls). Dominant VL1-47 light chain gene usage may provide a new target for therapy.
  • Figures 8A-8B are two graphs illustrating the binding of two isolated scFv clones to their target antigen.
  • Various dilutions of 3-14G ( Figure 8A) or 3-3E ( Figure 8B) were incubated with BP180 protein as a substrate.
  • ELISA was used as a readout.
  • Separate assays using BP230 were used a negative controls.
  • Figures 9A-9C are a series of micrographs demonstrating the binding of various isolated scFv’s to human skin sections. Normal skin tissue was cryosectioned onto slides and stained with the indicated scFv clones. Indirect immunofluorescence using a FIT C-labe ed, anti-human IgG antibody was then performed to visualize scFv binding. Arrowheads indicate specific staining of the basement membrane.
  • Figure 10 is a pair of micrographs of an in vitro skin blistering assay demonstrating that anti -BP 180 and anti-BP230 scFVs are non-pathogenic.
  • a mixture of scFv antibodies were incubated with a skin organ culture system prior to staining and assessing for nucroblister formation using indirect immunofluorescence.
  • scFv’s alone do not result in microblister formation, and stain the basement membrane (left panel, arrowheads). Toxicity is only observed when bound scFv’s are further crosshnked using rabbit anti-HA antibody, resulting in microblister formation (right panel, asterisks).
  • Figures 11 A-l 1 C are a series of graphs demonstrating that anti-BPl 80 scFv antibodies are capable of displacing activity on polyclonal sera from bullous pemphigoid patients.
  • Polyclonal sera from three BP patients (3419, Figure 11 A; 3389, Figure 1 IB; 3391, Figure 1 1C) was used to coat BP180 substrate in ELISA wells.
  • Increasing concentrations of scFv’s 3-14G (#1), 2-6GL (#29), and 3-3E (#16) were then added to the wells after a brief wash and incubated for 2 hours at 37°C. After two brief washes, new scFv preparations were loaded again to the respective ELISA wells. Displacement of full BP serum IgG antibodies was then detected by developing with anti-human IgG HRP.
  • the anti-BP230 scFv 4-8E.230 (#28) was used as a negative control.
  • Figures 12A-12E are a series of illustrations, graphs, and micrographs illustrating the construction and characterization of the 1005sqv fusion protein.
  • Figure 12A is a diagram showing the sequence of l005sqv with the various features highlighted (bottom) with a linear listing of the features (top).
  • Figure 12B is a protein gel demonstrating the expression and purification of the lOOSsqv fusion protein.
  • Figure 12C is a reactivity assay demonstrating that 1005sqv is able to specifically bind to BP180 substrate as measured by anti-HA-ELISA.
  • the 3- 14G scFv which is used as the antigen- binding domain in 1005sqv was used as a positive control.
  • 1005sqv w3 ⁇ 4s not observed to bind to the basement membrane of human skin sections ( Figure 12D), despite being able to inhibit complement fixation by BP antibodies m in vitro complement-fixation assays ( Figure 12E).
  • Figures 13A-13F are a series of illustrations, graphs, and micrographs demonstrating the design and function of the 1012qxm fusion protein.
  • Figure 13 A is a diagram showing a linear description of the features of 1012qxm (top), with a diagram of the sequence (bottom) with the various features highlighted.
  • Figure 13B is a protein gel demonstrating robust production of the fusion protein m eukaryotic cells. 1012qxm was found to possess significant binding specificity for BP180 protein, as measured by ELISA ( Figure 13C), as well as by binding the BMZ in human skin sections ( Figure 13D) as assessed by indirect immunofluorescence.
  • Figures 14A- ⁇ 4E are a series of illustrations, graphs, and micrographs demonstrating the design and function of the l048nvm fusion protein.
  • Figure 14A is a diagram showing the linear description of the features of l048nvm (top) with a sequence of the fusion protein with the various features highlighted (bottom).
  • Figure 14B is a protein gel demonstrating robust expression of the fusion protein by eukaryotic cells.
  • “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • conservative sequence modifications is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • A“disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
  • a“disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
  • the animal is a human.
  • Effective amount or“therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • expression as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
  • “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids ( e.g ., naked or contained in liposomes) and viruses (e.g., sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • “Homologous” as used herein refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric
  • immunoglobulins immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human
  • immunoglobulins in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementary-determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Fully human refers to an immunoglobulin, such as an antibody, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody.
  • Identity refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an Arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage.
  • the identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.
  • immune response is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and/or activate lymphocytes to remove the antigen.
  • an“instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention.
  • the instructional material of the kit of the invention may, for example, be affixed to a container which contains the nucleic acid, peptide, and/or composition of the invention or be shipped together with a container which contains the nucleic acid, peptide, and/or composition.
  • the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • A“lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.
  • Modified is meant a changed state or structure of a molecule or cell of the invention.
  • Molecules may be modified in many ways, including chemically, structurally, and functionally.
  • Cells may be modified through the introduction of nucleic acids.
  • modulating is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject.
  • the term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.
  • nucleic acid bases In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine,“C” refers to cytosine,“G” refers to guanosine,“T” refers to thymidine, and“U” refers to uridine.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • tumor antigen or“overexpression” of a tumor antigen is intended to indicate an abnormal level of expression of a tumor antigen in a cell from a disease area like a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ.
  • Patients having solid tumors or a hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art.
  • parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.
  • s.c. subcutaneous
  • i.v. intravenous
  • i.m. intramuscular
  • intrasternal injection or infusion techniques.
  • polynucleotide as used herein is defined as a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and polynucleotides as used herein are interchangeable.
  • nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric“nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • promoter as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
  • promoter/regulatory sequence means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence.
  • this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • A“constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • An“inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • A“tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • A“Sendai virus” refers to a genus of the Paramyxoviridae family. Sendai viruses are negative, single stranded RNA viruses that do not integrate into the host genome or alter the genetic information of the host cell. Sendai viruses have an exceptionally broad host range and are not pathogenic to humans. Used as a recombinant viral vector, Sendai viruses are capable of transient but strong gene expression
  • A“signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
  • the phrase“cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the plasma membrane of a cell.
  • Single chain antibodies refer to antibodies formed by recombinant DNA techniques in which immunoglobulin heavy and light chain fragments are linked to the Fv region via an engineered span of amino acids.
  • Various methods of generating single chain antibodies are known, including those described in U.S. Pat. No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242: 1038-1041.
  • an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample.
  • an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific.
  • an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen.
  • the terms“specific binding” or“specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope“A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled“A” and the antibody, will reduce the amount of labeled A bound to the antibody.
  • a particular structure e.g., an antigenic determinant or epitope
  • A“subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals).
  • A“subject” or“patient,” as used therein, may be a human or non-human mammal.
  • Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals.
  • the subject is human.
  • a“substantially purified” cell is a cell that is essentially free of other cell types.
  • a substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state.
  • a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state.
  • the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.
  • therapeutic means a treatment and/or prophylaxis.
  • a therapeutic effect is obtained by suppression, remission, or eradication of a disease state.
  • transfected or“transformed” or“transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or“transformed” or“transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • To“treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.
  • antibody refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope on an antigen.
  • Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Some antibodies are typically tetramers of immunoglobulin molecules.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, chimeric, hybrid, Fv, Fab, F(ab) 2 , scFv, single chain antibodies, primatized and humanized antibodies.
  • Antibody fragments refer to antigen-binding
  • immunoglobulin peptides which are at least about 5 to about 15 amino acids or more in length, and which retain the capacity to bind to the antigen.
  • an“antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.
  • an“antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.
  • “synthetic antibody or antigen binding fragment thereof’ is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein.
  • the term should also be construed to mean an antibody or antigen binding fragment thereof which has been generated by the synthesis of a DNA molecule encoding the antibody or antigen binding fragment thereof and which DNA molecule expresses an antibody or antigen binding fragment thereof protein, or an amino acid sequence specifying the antibody or antigen binding fragment thereof, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
  • Fab/phage a phage particle which expresses the Fab portion of an antibody.
  • antibody-protein fusion molecule refers to a fusion or chimeric molecule comprising at least two components, including a targeting moiety and peptide or protein.
  • the fusion molecule can also comprise at least two components, including a targeting moiety and a therapeutic moiety. In other embodiments, the fusion molecule can comprise at least two components, including a targeting moiety and a non-protein molecule.
  • non-pathogenic when used in conjuction with the term“antibody” refers to an antibody that does not appreciably alter normal homeostasis of a mammal when introduced into the mammal.
  • the non-pathogenic antibody does not have an appreciable adverse effect on the host mammal when present in the host mammal.
  • scFv/phage a phage particle which expresses the Fv portion of an antibody as a single chain.
  • the nucleic acid encoding the antibody e.g., a plasmid such as, but not limited to, pComb3X, not only comprises a plasmid origin of replication, but also a phage (e.g.
  • Ml 3 origin of replication sequence and an Ml 3 packaging sequence, so that when the nucleic acid is produced, a helper phage can be used to provide the required phage (e.g., Ml 3) proteins in trans to make“phage-like” particles. That is, these particles resemble phage on the outside, but on the inside they contain plasmid (also referred to as“phagemid”) DNA. In other words, the phagemid DNA need not encode any Ml 3 phage proteins, except a piece of Ml 3 gene III fused to the DNA for antibody or peptide.
  • the terms“phage,”“phage particle,”“phage-like particle” and“phagemid” are used interchangeably herein.
  • salt can be washed from a protein by dialyzing a protein
  • an antibody can be removed from the outside of a ce4ll by altering the salt conditions of the cell medium or by removing the salt from the cell medium altogether
  • an unbound phage can be removed from a cell suspension by separating the cell from the phage using a gel filtration technique.
  • therapeutic protein and“therapeutic polypeptide” refer to a protein, a polypeptide, an antibody, a peptide or fragment or variant thereof, having one or more therapeutic and/or biological activity.
  • Therapeutic proteins and polypeptides encompassed by the invention include but are not limited to, proteins, polypeptides, peptides, antibodies, and biologies.
  • the terms peptides, proteins, and polypeptides are used interchangeably herein.
  • a fusion protein of the invention may contain at least a fragment or variant of a therapeutic protein, and/or at least a fragment or variant of an antibody.
  • therapeutic protein may refer to the endogenous or naturally occurring correlate of a therapeutic protein.
  • a polypeptide displaying a“therapeutic activity” or a protein that is“therapeutically active” is meant a polypeptide that possesses one or more known biological and/ or therapeutic activities associated with a therapeutic protein such as one or more of the therapeutic proteins described herein or otherwise known in the art.
  • a“therapeutic protein” is a protein that is useful to treat, prevent or ameliorate a disease, condition or disorder.
  • a non-exhaustive list of“therapeutic protein” portions which may be comprised by a fusion protein of the invention includes, but is not limited to Fas ligand, tumor necrosis factor alpha receptor, CD200, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), transforming growth factor alpha (TGF alpha), programmed death ligand (PD-L1), and epidermal growth factor.
  • TRAIL tumor necrosis factor-related apoptosis-inducing ligand
  • TGF alpha tumor necrosis factor-related apoptosis-inducing ligand
  • TGF alpha transforming growth factor alpha
  • PD-L1 programmed death ligand
  • epidermal growth factor epidermal growth factor.
  • Other useful therapeutic proteins include enzymes, such as those that activate drugs, or those that can act to inhibit an undesirable biological reaction in a mammal. Likewise, other useful therapeutic proteins can bind and thus inhibit undesirable or disease- related biological reactions in
  • a “targeting moiety” or a “cell-specific targeting moiety” is used herein to refer to a molecule comprising an antibody to one or more particular soluble protein, tissue marker, cell surface antigen, cell marker, growth factor, hormone, or cytokine.
  • the targeting moiety of a chimeric composition is an antibody, such as a single chain antibody, an antibody fragment, a Fab, and the likes.
  • pro-apoptotic molecules refers to any molecule that is capable of inducing cell death through apoptotic mechanisms.
  • the pro-apoptotic molecules can induce programmed cell death upon entry into the target cell.
  • Apoptosis, or programmed cell death is a fundamental process controlling normal tissue homeostasis by regulating a balance between cell proliferation and death.
  • Examples of pro-apoptotic molecules include but are not limited to granzymes, Bcl-2 family members (Bax, Bak, Bcl-Xs, Bik, Bok, Bipla, and the like), and caspases.
  • laser absorption molecule is meant a molecule that can absorb laser light radiation. The term is also used to indicate a molecule that can absorb any type of laser energy, including light-based laser energy, but also including any form of electromagnetic or particle-based laser energy.
  • laser absorbing molecule complex refers to a complex comprising at least one laser absorbing molecule and at least one antibody or scFv of the invention.
  • a complex can have the two or molecules associated by way of a non-covalent interaction, or by way of one or more covalent interactions.
  • under transcriptional control or“operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.
  • A“vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term“vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • the term "genetic construct” refers to the DNA or RNA molecules that comprise a nucleotide sequence which encodes protein.
  • the coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered.
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Description
  • the present invention provides a fusion protein comprising two domains.
  • One of the domains is an antigen-binding domain comprising an antibody or antigen binding fragment thereof that binds the human basement membrane zone proteins BP180 or BP230.
  • the second domain comprises a therapeutic polypeptide or fragment thereof.
  • the antibody or antigen binding fragment thereof of the antigen-binding domain comprises one CDR selected from the group consisting of at least 1, 2, 3, 4, 5, or 6 CDRs as listed in Table 7.
  • the antibody or antigen binding fragment thereof of the antigen-binding domain is a scFv.
  • the antibody or antigen binding fragment thereof comprises 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 33-64 or 68-70.
  • the therapeutic polypeptide or fragment thereof inhibits components of the complement system. In some embodiments, the
  • complement-inhibiting polypeptide binds and inhibits complement component Cls.
  • the Cls inhibiting polypeptide is gigastasin.
  • the complement-inhibiting polypeptide binds and inhibits the complement-regulating factor H.
  • the factor H inhibitor is 5C6 peptide.
  • the fusion protein of the invention comprises 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 74-76.
  • the invention further provides a recombinant nucleic acid encoding the fusion protein.
  • composition of the invention comprising the fusion protein of any one of the previous embodiments, as well as a method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition comprising the fusion protein of any one of the previous embodiments.
  • the pharmaceutical composition is administered intravenously, intra-articularly, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intravitreously, intra-muscularly, subcutaneously, orally or intranasally.
  • composition of the invention is further administerd with a second agent to the subject.
  • the second agent is at least one of carboplatin, cisplatin, paclitaxel, docetaxel, gemcitabine, bevacizumab, olaparib, rucaparib, niraparib, cyclophosphamide, FU, abiraterone, flutamide, bicalutamide, leuprolide, goserelin, buserelin, triptorelin, degarelix, Enzalutamide, Apalutamide, Sipuleucel-T, Cabazitaxel, Radium- 223, trastuzumab, pertuzumab, lapatinib, tamoxifen, oxaliplatin, capecitabine, leucovorin, Irinotecan, Cetuximab, panitumumab, aflibercept, Regorafenib, Trifluridine-tipiracil, immune checkpoint inhibitors (nivolumab, pembrab,
  • MMP mucous membrane pemphigoid
  • linear IgA disease lichen planus pemphigoides
  • pemphigoid gestationis or an autoimmune disease wherein the patient has auto antibodies against the basement membrane.
  • the antigen-binding domain of the fusion protein of any one of the previous embodiments displaces auto-reactive, disease-causing serum IgG against non-collagenous domain 16A of BP 180, and the complement-inhibiting therapeutic peptide prevents complement deposits at the BMZ, thereby achieving treatment of the disease.
  • an isolated antibody or antigen binding fragment thereof that binds BP180 or BP230, wherein the isolated antibody or antigen binding fragment thereof comprises at least 1, 2, 3, 4, 5 or 6 CDRs from Table 7.
  • the CDRs are from the same antibody in
  • the CDRs are mixed from different antibodies in Table 7 that bind to the same protein (BP180 or BP230).
  • the antibody or antigen binding fragment thereof binds BP 180.
  • the antibody or antigen binding fragment thereof binds the non-collagenous domain 16A (NC16A) of the BP180 ectodomain.
  • the antibody or antigen binding fragment thereof binds BP230.
  • the isolated antibody or antigen binding fragment thereof comprises 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, and any partial integers therebetween, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 33-64 or 68-70.
  • the antibody or antigen binding fragment thereof is non- pathogenic to its target tissue.
  • the antibody or antigen binding fragment thereof cross-competes with anti-BPl80 or anti-BP230 antibodies or fragments thereof. In further embodiments, the antibody or antigen binding fragment thereof cross-competes with anti-BPl 80-NCl6A antibodies or fragments thereof.
  • the antibody or antigen binding fragment thereof is humanized or chimeric.
  • the antibody or antigen binding fragment thereof is an scFv.
  • nucleic acid encoding the antibody or antigen binding fragment thereof of any one of the previous embodiments.
  • an antibody-drug conjugate comprising the antibody or antigen binding fragment of any one of the previous embodiments.
  • the drug is MMAE, ozogamicin, emtansine, amanitin, pyrrolobenzodiazepine (PBD) dimer toxin, a chalichaemicin, a cytotoxic maytansinoid, DM1 , carboplatin, cisplatin, paclitaxel, vedotin, or diphtheria toxin.
  • an antibody-protein conjugate comprising the antibody or antigen binding fragment of any one of the previous embodiments.
  • the protein is a therapeutic protein.
  • the therapeutic protein is an enzyme or a pro- apoptotic protein.
  • the protein is an inhibitor of complement cascade, for example inhibiting the classical pathway and/or the alternative pathway and/or the lectin pathway.
  • the protein is a cytokine, for example IL-2.
  • the protein is a cytokine-receptor inhibitor such as dupilumab.
  • the protein is a cell surface protein.
  • the protein is PD-l.
  • Provided is a pharmaceutical composition comprising the isolated antibody or antigen binding fragment, or the antibody-drug conjugate or the antibody-protein conjugate of any one of the previous embodiments.
  • the method further comprises administering a second agent to the subject.
  • the second agent is at least one of carboplatin, cisplatin, paclitaxel, docetaxel, gemcitabine, bevacizumab, olaparib, rucaparib, niraparib,
  • cyclophosphamide FU, abiraterone, flutamide, bicalutamide, leuprobde, goserebn, buserebn, triptorebn, degarelix, Enzalutamide, Apalutamide, Sipuleucel-T, Cabazitaxel, Radium-223, trastuzumab, pertuzumab, lapatinib, tamoxifen, oxaliplatin, capecitabine, leucovorin, Irinotecan, Cetuximab, panitumumab, aflibercept, Regorafenib, Trifluridine-tipiracil, immune checkpoint inhibitors (nivolumab, pembrolizumab), cabozantinib, sunitinib, pazopanib, axitinib, interleukin- 2, interferon alpha, mitomycin C, epirubicin, BCG, bleo
  • the second agent is an alkylating agent, antimetabobte, antibiotic, a plant- derived agent, platinum complex, campthotecin derivative, tyrosine kinase inhibitor, monoclonal antibody, interferon, biological response modifier, hormonal anti-tumor agent, anti-tumor viral agent, angiogenesis inhibitor, differentiating agent, PI3K/mTOR/AKT inhibitor, cell cycle inhibitor, apoptosis inhibitor, hsp 90 inhibitor, tubulin inhibitor, DNA repair inhibitor, anti- angiogenic agent, receptor tyrosine kinase inhibitor, topoisomerase inhibitor, taxane, agent targeting Her-2, hormone antagonist, agent targeting a growth factor receptor, or a
  • the anti-tumor agent is citabine, capecitabine, valopicitabine or gemcitabine.
  • the anti-tumor agent is Avastin, Sutent, Nexavar, Recentin, ABT-869, Axitinib, Irinotecan, topotecan, paclitaxel, docetaxel, lapatinib, Herceptin, tamoxifen, progesterone, a steroidal aromatase inhibitor, a non steroidal aromatase inhibitor, Fulvestrant, an inhibitor of epidermal growth factor receptor (EGFR), Cetuximab, Panitumimab, an inhibitor of insulin-like growth factor 1 receptor (IGF1R), and/or CP-751871.
  • EGFR epidermal growth factor receptor
  • Cetuximab Cetuximab
  • Panitumimab an inhibitor of insulin-like growth factor 1 receptor (IGF1R)
  • CP-751871 insulin-like growth factor 1 receptor
  • a method of treating a disease in a subject in need thereof comprising administering to the subject an effective amount of a pharmaceutical composition comprising the isolated antibody or antibody fragment of any one of the previous embodiments.
  • the antibody or antibody fragment binds to non-collagenous domain 16A of BP 180.
  • the disease is bullous pemphigoid, mucous membrane
  • the antibody or antigen binding fragment is an scFv. In further embodiments, the antibody or antigen binding fragment displaces serum IgG against non- collagenous domain 16A of BP 180.
  • a method of treating a disease in a subject in need thereof comprising administering to the subject an effective amount of a pharmaceutical composition comprising the antibody-protein conjugate or the antibody-drug conjugate of any one of the previous
  • the antibody or antibody fragment binds to non- collagenous domain 16A of BP180. In further embodiments, the antibody or antibody fragment that binds to non-collagenous domain 16A of BP180 is listed in Table 3. In yet further embodiments, the antibody or antigen binding fragment thereof comprises 91%, 92%, 93%,
  • the disease is bullous pemphigoid, mucous membrane pemphigoid (MMP), linear IgA disease, lichen planus pemphigoides, pemphigoid gestationis, anti-p200 pemphigoid, epidermolysis bullosa acquisita, graft versus host disease (GVHD), toxic epidermolysis bullosa, erythema multiforme, other diseases with interface dermatitis, allergic contact dermatitis, atopic dermatitis, psoriasis, vitiligo, other diseases in which activated lymphocytes cause damage in the epidermis, skin cancer, and diseases affecting any other tissue known to express the antigens BP180 and/or BP230.
  • MMP mucous membrane pemphigoid
  • linear IgA disease lichen planus pemphigoides
  • pemphigoid gestationis anti-p200 pemphigoid
  • epidermolysis bullosa acquisit
  • the skin cancer is actinic keratosis, squamous cell carcinoma (SCC), basal cell carcinoma (BCC) or melanoma.
  • tissues different from skin and mucous membranes that express BP180/BP230 are the retina of the eye (Claudepierre, T et al. J Comp Neurol 2005; 487(2): 190-203) and brain tissue (Seppanen, A et al. Brain Res 2007; 1158:50-6).
  • the antibody or antigen binding fragment is an scFv.
  • Monoclonal antibodies directed against full length or peptide fragments of a protein or peptide may be prepared using any well-known monoclonal antibody preparation procedures, such as those described for example in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.) and in Tuszynski et al. (1988, Blood, 72: 109-115).
  • Quantities of the desired peptide may also be synthesized using chemical synthesis technology.
  • DNA encoding the desired peptide may be cloned and expressed from an appropriate promoter sequence in cells suitable for the generation of large quantities of peptide.
  • Monoclonal antibodies directed against the peptide are generated from mice immunized with the peptide using standard procedures as referenced herein.
  • Monoclonal antibodies may also be obtained using antibody phage display libraries as previously described (Hammers CM et al. The Journal of investigative dermatology 2014; 134: 1-5; Payne AS et al. J. Clin. Invest. 2005; 115: 888-99).
  • Nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using technology which is readily available in the art, and is described, for example, in Wright et al. (1992, Ciritical Rev. Immunol. 12: 125-168), and references cited therein. Further, the antibody of the invention may be“humanized” using the technology described in, for example, Wright et al., and in the references cited therein, and in Gu et al. (1997, Thrombosis and Hematocyst 77:755-759), and other methods of humanizing antibodies well-known in the art or to be developed.
  • a human antibody or antigen binding fragment thereof that binds BP180.
  • the antibody or antigen binding fragment thereof binds the non-collagenous domain 16A (NC16A) of the BP 180 ectodomain.
  • the antibody or antibody fragment that binds to non-collagenous domain 16A of BP 180 is listed in Table 3.
  • the antibody or antigen binding fragment thereof comprises 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, and any partial integers therebetween, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 61, 62, 63, 64, and 70.
  • a human antibody or antigen binding fragment thereof that binds BP230.
  • the antibody or antibody fragment that binds to BP230 is listed in Table 3.
  • a human antibody or antigen binding fragment thereof that binds BP180 or BP230, wherein the isolated antibody or antigen binding fragment thereof comprises at least 1, 2, 3, 4, 5 or 6 CDRs from Table 7.
  • a humanized antibody or antigen binding fragment thereof that binds BP180.
  • the antibody or antigen binding fragment thereof binds the non- collagenous domain 16A (NC16A) of the BP180 ectodomain.
  • a humanized antibody or antigen binding fragment thereof that binds BP230 binds BP230.
  • a humanized antibody or antigen binding fragment thereof that binds BP180 or BP230, wherein the isolated antibody or antigen binding fragment thereof comprises at least 1 , 2, 3, 4, 5 or 6 CDRs from Table 7.
  • Humanized forms of non-human (e g. murine) antibodies are genetically engineered chimeric antibodies or antigen binding fragments thereof having preferably minimal portions derived from non-human antibodies.
  • Humanized antibodies include antibodies in which CDRs of a human antibody (recipient antibody) are replaced by residues from a CDR region of a non human species (donor antibody) such as mouse, rat or rabbit having the desired functionality.
  • donor antibody such as mouse, rat or rabbit having the desired functionality.
  • Fv framework residues of the human antibody are replaced by
  • Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework (FR) sequences.
  • the humanized antibody may comprise substantially all of at least one, typically two, variable domains domains in which all or substantially ail of the framework regions correspond to those of a relevant human consensus sequence.
  • Humanized antibodies may also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, for example, Jones et al, 1986. Nature 321 :522-525;
  • ammo acids in the human acceptor sequence may be replaced by the corresponding amino acids from the donor sequence, for example where: (1) the amino acid is in a CDR; (2) the amino acid is in a human framework region (e.g., the amino acid is immediately adjacent to one of the CDRs). See, U.8. Patent No. 5,530,101 and 5,585,089, incorporated herein by reference, which provide detailed instructions for construction of humanized antibodies.
  • humanized antibodies often incorporate all six CDRs (e.g, as defined by Rabat, but often also including hypervariable loop HI as defined by Chotiua) from a mouse antibody, they can also be made with fewer mouse CDRs and/or less than complete mouse CDR sequence(s) (e.g., a functional fragment of a CDR) (e.g., Pascaiis et al. J Immunol. 169:3076, 2002: Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al, Mol. Immunol. 36: 1079-1091, 1999; Tamura et al., Journal of Immunology!, 164: 1432-1441, 2000).
  • CDRs e.g., as defined by Rabat, but often also including hypervariable loop HI as defined by Chotiua
  • humanized antibody has one or more amino acid residues introduced into it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as “import” residues, which are typically taken from an“import” variable domain. Thus, humanized antibodies comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions from human.
  • humanized chimeric antibodies substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • variable domains both light and heavy
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity.
  • sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151 :2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference herein in their entirety).
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • the same framework may be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al, J. Immunol., 151 :2623 (1993), the contents of which are incorporated herein by reference herein in their entirety).
  • Antibodies can be humanized with retention of high affinity for the target antigen and other favorable biological properties.
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three- dimensional conformational structures of selected candidate immunoglobulin sequences.
  • Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved.
  • the CDR residues are directly and most substantially involved in influencing antigen binding.
  • A“humanized” antibody retains a similar antigenic specificity as the original antibody.
  • the affinity and/or specificity of binding of the antibody for human CD3 antigen may be increased using methods of“directed evolution,” as described by Wu et al., J. Mol. Biol., 294: 151 (1999), the contents of which are incorporated herein by reference herein in their entirety.
  • the antibody is a synthetic antibody, human antibody, a humanized antibody, single chain variable fragment, single domain antibody, an antigen binding fragment thereof, and any combination thereof.
  • a humanized antibody including a light chain comprising at least one CDR from Table 7 and a human variable region framework; and a heavy chain comprising at least one CDR from Table 7 and a human variable region framework.
  • the humanized antibody includes said light chain and said heavy chain together with a light chain constant region and a heavy chain constant region.
  • an isolated human or humanized single chain antibody that binds specifically to BP180 or BP230.
  • a human or humanized single chain antibody specific for binding to BP180 or BP230 referred to herein as a“human anti-BP180 single chain antibody,” “humanized anti-BP180 single chain antibody,”“human anti-BP230 single chain antibody” or “humanized anti-BP230 single chain antibody” is fused to an Fc polypeptide.
  • the Fc polypeptide is an Fc region of an IgG immunoglobulin, such as an IgG immunogl obulin selected from the group consisting of IgG 1 isotype, IgG2 isotype, IgG3 isotype, XgG4 isotype, IgE isotype, IgAl isotype, XgA2 isotype and IgM isotype.
  • an IgG immunoglobulin such as an IgG immunogl obulin selected from the group consisting of IgG 1 isotype, IgG2 isotype, IgG3 isotype, XgG4 isotype, IgE isotype, IgAl isotype, XgA2 isotype and IgM isotype.
  • the human or humanized single chain antibody is fused to the carboxy terminus of the Fc polypeptide. In some embodiments, the human or humanized single chain antibody is fused to the amino terminus of the Fc polypeptide.
  • the fusions are constructed as a single genetic construct and are expressed in cells in culture.
  • the engineered cytokines of the invention were codon optimized so as to enhance their ability to modulate the immune response in a mammal into which they are introduced.
  • the invention includes sequences that are homologous to the sequences disclosed herein. Sequence homology for nucleotides and amino acids may be determined using FASTA, BLAST and Gapped BLAST (Altschul et al., Nuc. Acids Res., 1997, 25, 3389, which is incorporated herein by reference in its entirety) and PAUP* 4.0b 10 software (D. L. Swofford, Sinauer Associates, Massachusetts). "Percentage of similarity" is calculated using PAUP*
  • the BLAST algorithm which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity (Altschul et al, J. Mol. Biol., 1990, 215, 403-410, which is incorporated herein by reference in its entirety).
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached.
  • the Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al, Proc. Natl. Acad.
  • a nucleic acid is considered similar to another if the smallest sum probability in comparison of the test nucleic acid to the other nucleic acid is less than about 1 , preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid encoding any of the engineered antibodies of the invention into a mammal or introduction of a nucleic acid encoding the antibodies or antibody fragments into the cells of a mammal for the purpose of generating antibodies or antibody fragments can be accomplished using technology available in the art.
  • the genetic construct(s) When taken up by a cell, the genetic construct(s) may remain present in the cell as a functioning extrachromosomal molecule and/or integrate into the cell's chromosomal DNA.
  • DNA may be introduced into cells where it remains as separate genetic material in the form of a plasmid or plasmids.
  • linear DNA that can integrate into the chromosome may be introduced into the cell.
  • reagents that promote DNA integration into chromosomes may be added.
  • DNA sequences that are useful to promote integration may also be included in the DNA molecule.
  • RNA may be administered to the cell. It is also contemplated to provide the genetic construct as a linear minichromosome including a centromere, telomeres and an origin of replication. Gene constructs may remain part of the genetic material in attenuated live microorganisms or recombinant microbial vectors which live in cells. Gene constructs may be part of genomes of recombinant viral vectors where the genetic material either integrates into the chromosome of the cell or remains
  • Genetic constructs include regulatory elements necessary for gene expression of a nucleic acid molecule.
  • the elements include: a promoter, an initiation codon, a stop codon, and a polyadenylation signal.
  • enhancers are often required for gene expression of the sequence that encodes the antibody or the immunomodulating protein. It is necessary that these elements be operable linked to the sequence that encodes the desired proteins and that the regulatory elements are operably in the individual to whom they are administered.
  • Initiation codons and stop codon are generally considered to be part of a nucleotide sequence that encodes the desired protein. However, it is necessary that these elements are functional in the individual to whom the gene construct is administered. The initiation and termination codons must be in frame with the coding sequence. Promoters and polyadenylation signals used must be functional within the cells of the individual.
  • promoters useful to practice the present invention include but are not limited to promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (MV) such as the BIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human Actin, human Myosin, human Hemoglobin, human muscle creatine and human metalothionein.
  • SV40 Simian Virus 40
  • MMTV Mouse Mammary Tumor Virus
  • MV Human Immunodeficiency Virus
  • LTR Long Terminal Repeat
  • ALV Moloney virus
  • CMV Cytomegalovirus
  • EBV Epstein Barr Virus
  • RSV Rous Sarcoma Virus
  • polyadenylation signals useful to practice the present invention, include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals.
  • the SV40 polyadenylation signal that is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to as the SV40 polyadenylation signal, is used.
  • enhancers may be selected from the group including but not limited to: human Actin, human Myosin, human Hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV.
  • Genetic constructs can be provided with mammalian origin of replication in order to maintain the construct extrachromosomally and produce multiple copies of the construct in the cell.
  • Plasmids pVAXl, pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr virus origin of replication and nuclear antigen EBNA-l coding region which produces high copy episomal replication without integration.
  • regulatory sequences may be selected which are well suited for gene expression in the cells the construct is administered into.
  • codons may be selected which are most efficiently transcribed in the cell.
  • One having ordinary skill in the art can produce DNA constructs that are functional in the cells.
  • the engineered antibodies of the invention i.e., the engineered antibodies of the invention
  • one having ordinary skill in the art can, using well known techniques, produce and isolate proteins of the invention using well known techniques.
  • the protein for example, one having ordinary skill in the art can, using well known techniques, inserts DNA molecules that encode a protein of the invention into a commercially available expression vector for use in well-known expression systems.
  • the commercially available plasmid pSE420 Invitrogen, San Diego, Calif.
  • the commercially available plasmid pYES2 Invitrogen, San Diego, Calif.
  • S S.
  • the commercially available MAXBAC.TM. complete baculovirus expression system may, for example, be used for production in insect cells.
  • the commercially available plasmid pcDNA I or pcDNA3 may, for example, be used for production in mammalian cells such as Chinese Hamster Ovary cells.
  • compounds may also be synthesized as proteins in the patient by introducing DNA expression vectors into that subject (Medi, BM et al. Methods Mol Biol 2008; 423:225-32;
  • Expression systems containing the requisite control sequences such as promoters and polyadenylation signals, and preferably enhancers are readily available and known in the art for a variety of hosts. See e.g., Sambrook et al, Molecular Cloning Third Ed. Cold Spring Harbor Press (2001).
  • Genetic constructs include the protein coding sequence operably linked to a promoter that is functional in the cell line into which the constructs are transfected. Examples of constitutive promoters include promoters from cytomegalovirus or SV40. Examples of inducible promoters include mouse mammary leukemia virus or metallothionein promoters.
  • the expression vector including the DNA that encodes the protein is used to transform the compatible host which is then cultured and maintained under conditions wherein expression of the foreign DNA takes place.
  • the protein produced is recovered from the culture, either by lysing the cells or from the culture medium as appropriate and known to those in the art.
  • One having ordinary skill in the art can, using well known techniques, isolate protein that is produced using such expression systems.
  • the methods of purifying protein from natural sources using antibodies which specifically bind to a specific protein as described above may be equally applied to purifying protein produced by recombinant DNA methodology.
  • automated peptide synthesizers may also be employed to produce isolated, essentially pure protein. Such techniques are well known to those having ordinary skill in the art and are useful if derivatives which have substitutions not provided for in DNA-encoded protein production.
  • polynucleotides encoding the engineered antibodies of the invention may be delivered using any of several well-known technologies including DNA injection, recombinant vectors such as recombinant adenovirus, recombinant adenovirus associated virus and recombinant vaccinia virus.
  • Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intra-arterial, intraocular
  • Preferred routes of administration include intravenous, intramuscular, intraperitoneal, intradermal and subcutaneous injection.
  • Genetic constructs may be administered by means including, but not limited to, electroporation methods and devices, traditional syringes, microneedling devices, needleless injection devices, or microprojectile bombardment.
  • the invention includes a method for treating a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody- drug conjugate (ADC), wherein the antibody or antigen binding fragment portion of the ADC binds to BP180 or BP230.
  • ADC antibody- drug conjugate
  • the drug used to make the ADC is MMAE (monomethyl auristatin E), ozogamicin, emtansine, amantin, pyrrolobenzodiazepine (PBD) dimer toxin, a chalichaemicin, a cytotoxic maytansinoid, for example DM1.
  • antibody-drug conjugates comprising an antibody or antigen binding fragment thereof that bind BP180.
  • antibody-drug conjugates comprising an antibody or antigen binding fragment thereof that binds BP230.
  • the drug is MMAE (monomethyl auristatin E), ozogamicin, emtansine, amanitin, pyrrolobenzodiazepine (PBD) dimer toxin, a chalichaemicin, a cytotoxic maytansinoid, for example DM1.
  • the drug is connected to the antibody or antigen binding fragment thereof via a linker.
  • Suitable linkers are known in the art. See for example U/S. Patent No.
  • the antibody portion of the antibody-drug conjugate or the antibody-protein conjugate binds to non-collagenous domain 16A of BP 180 or binds to BP230, and the disease that is treated is bullous pemphigoid, mucous membrane pemphigoid (MMP), linear IgA disease, lichen planus pemphigoides, pemphigoid gestationis, anti-p200 pemphigoid, epidermolysis bullosa acquisita, graft versus host disease (GVHD), toxic epidermolysis bullosa, erythema multiforme, other diseases with interface dermatitis, allergic contact dermatitis, atopic dermatitis, psoriasis, vitiligo, other diseases in which activated lymphocytes cause damage in the epidermis, skin cancer, and diseases affecting any other tissue known to express BP180 and/or BP230 (e.g., ocular diseases or brain diseases).
  • MMP mucous membrane
  • the skin cancer is actinic keratosis, squamous cell carcinoma (SCC), basal cell carcinoma (BCC) or melanoma.
  • tissues different from skin and mucous membranes that express BP180/BP230 are the retina of the eye (Claudepierre, T et al. J Comp Neurol 2005; 487(2): 190-203) and brain tissue (Seppanen, A et al. Brain Res 2007; 1158:50-6).
  • compositions according to the present invention are formulated according to the mode of administration to be used.
  • pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen free and particulate free.
  • An isotonic formulation is preferably used.
  • additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose.
  • isotonic solutions such as phosphate buffered saline are preferred.
  • Stabilizers include gelatin and albumin.
  • a vasoconstriction agent is added to the formulation.
  • the antibody or antigen binding fragment may be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • Remington The Science and Practice of Pharmacy, l9th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA.
  • the antibody or antigen binding fragment may be administered orally, bucally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate, delayed or controlled-release applications.
  • the antibody or antigen binding fragment may also be administered via intracavernosal injection.
  • the antibody or antigen binding fragment may also be administered parenterally. In some embodiments, the antibody or antigen binding fragment may be administered
  • the antibody or antigen binding fragment is administered by infusion techniques.
  • the antibody or antigen binding fragment is used in the form of a sterile aqueous solution that may contain other substances, for example, sufficient salts or glucose (or other sugars) to make the solution isotonic with blood.
  • a sterile aqueous solution should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • suitable parenteral formulations under sterile conditions is readily accomplished by standard
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with blood.
  • Suitable formulations for parenteral administration also include aqueous and non-aqueous suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers.
  • the daily dosage level of the antibody or antigen binding fragment that binds BP180 or BP230 will usually be from 10 to 200 g per adult (i.e., from about 0.1 to 2 g/kg), administered in single or multiple or divided doses.
  • the dosage level may be from about 1 mg/kg to about 10 mg/kg, depending on the site and exact route of application, e. g., intraocular injection or topical administration.
  • the antibody or antigen binding fragment is administered intranasally or by inhalation.
  • the antibody or antigen binding fragment may be delivered in the form of a dry powder inhaler or an aerosol spray from a pressurized container, pump, spray or nebulizer with the use of a propellant.
  • the antibody or antigen binding fragment is administered by DNA injection and electroporation of the DNA encoded antibody into muscle or skin.
  • a method of treating a disease in a subject in need thereof comprising administering to the subject an effective amount of any one of the antibody or antigen binding fragments described herein.
  • the subject is human.
  • the antibody or antigen binding fragment is provided in a pharmaceutical composition.
  • the pharmaceutical composition may be delivered orally, parenterally, for example as a parenteral injection, intravenously, for example as an intravenous infusion, or by inhalation.
  • a sample is a tissue or a bodily fluid sample.
  • the sample is a tumor sample, a blood sample, a blood plasma sample, a peritoneal fluid sample, an exudate or an effusion.
  • a second agent is administered to the subject.
  • the second agent is at least one of carboplatin, cisplatin, paclitaxel, docetaxel, gemcitabine, bevacizumab, olaparib, rucaparib, niraparib, cyclophosphamide, FU, abiraterone, flutamide, bicalutamide, leuprolide, goserelin, buserelin, triptorelin, degarelix, Enzalutamide, Apalutamide, Sipuleucel-T, Cabazitaxel, Radium-223, trastuzumab, pertuzumab, lapatinib, tamoxifen, oxaliplatin, capecitabine, leucovorin, Irinotecan, Cetuximab, panitumumab, aflibercept, Regorafenib, Trifluridine-tipiracil, immune
  • the second agent is an alkylating agent, antimetabolite, antibiotic, a plant-derived agent, platinum complex, campthotecin derivative, tyrosine kinase inhibitor, monoclonal antibody, interferon, biological response modifier, hormonal anti-tumor agent, anti-tumor viral agent, angiogenesis inhibitor, differentiating agent, PI3K/mTOR/AKT inhibitor, cell cycle inhibitor, apoptosis inhibitor, hsp 90 inhibitor, tubulin inhibitor, DNA repair inhibitor, anti- angiogenic agent, receptor tyrosine kinase inhibitor, topoisomerase inhibitor, taxane, agent targeting Her-2, hormone antagonist, agent targeting a growth factor receptor, or a
  • the anti-tumor agent is citabine, capecitabine, valopicitabine or gemcitabine.
  • the anti-tumor agent is Avastin, Sutent, Nexavar, Recentin, ABT-869, Axitinib, Irinotecan, topotecan, paclitaxel, docetaxel, lapatinib, Herceptin, tamoxifen, progesterone, a steroidal aromatase inhibitor, a non steroidal aromatase inhibitor, Fulvestrant, an inhibitor of epidermal growth factor receptor (EGFR), Cetuximab, Panitumimab, an inhibitor of insulin-like growth factor 1 receptor (IGF1R), and/or CP-751871.
  • EGFR epidermal growth factor receptor
  • Cetuximab Cetuximab
  • Panitumimab an inhibitor of insulin-like growth factor 1 receptor (IGF1R)
  • CP-751871 insulin-like growth factor 1 receptor
  • the disease is cancer.
  • the cancer is skin cancer.
  • the skin cancer is actinic keratosis, squamous cell carcinoma (SCC), basal cell carcinoma (BCC) or melanoma.
  • the antibody or antigen binding fragment binds to non- collagenous domain 16A of BP180, and the disease is bullous pemphigoid, mucous membrane pemphigoid (MMP), linear IgA disease, lichen planus pemphigoides or pemphigoid gestationis, or any other disease affecting tissues that express BP180/BP230.
  • MMP mucous membrane pemphigoid
  • linear IgA disease lichen planus pemphigoides or pemphigoid gestationis, or any other disease affecting tissues that express BP180/BP230.
  • ADCC Antibody- Cell-Mediated Cytotoxicity
  • FcRs Fc receptors
  • NK cells nonspecific cytotoxic cells that express Fc receptors
  • NK cells express FcgRIII only, whereas monocytes express FcgRI, FcgRII and FcgRIII.
  • FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).
  • an in vitro ADCC assay such as that described in U.S. Patent Nos. 5,500,362 or 5,821,337 may be performed.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model.
  • administering to the subject an effective amount of any one of the antibody or antigen binding fragments described herein results in ADCC.
  • IIF Indirect immunofluorescence
  • Binding of bound antibodies was detected with anti-human IgG F(ab’)2 FITC conjugate (dilution, 1 : 100; Bio-Rad). Finally, slides were washed as above and mounted with DAPI Fluoromo20unt-G mounting medium (SouthernBiotech).
  • His-tagged BP180-NC16A and BP230-C fragments were obtained from EUROIMMUN and coupled to magnetic beads (DynabeadsTM His-Tag Isolation & Pulldown; ThermoFisher Scientific), following the instructions of the manufacturer. Serum was diluted 1 : 10 diluted in DPBS/0.05% Tween 20, pH 7.1, and incubated with antigen-loaded beads for 90 minutes. After extensive washing with DPBS/0.05% Tween 20, pH 7.1, antigen- specific antibodies were eluted with 76 mM citric acid, pH 2.2, and immediately neutralized with 2M TRIS buffer, pH 11.1.
  • antibody phage display (APD) libraries were constructed from peripheral mononuclear cell-RNA, obtained from a patient with active BP (confirmed by clinical features, DIF, IIF, ELISA).
  • the PCR- amplified variable heavy and light chains were assembled via overlap-PCR, digested with Sfi-I (Roche), and ligated into the pComb3X vector (Scripps Institute, La Jolla).
  • phage libraries were panned on BP180/230-ELISA wells (MBL, Nagoya, Japan). Bound polyclonal phages were eluted with 76 mM citric acid, amplified, and re panned for up to four rounds.
  • both BP230 clones used herein reacted on both the MBL BP230-C/N and the EUROIMMUN BP230-CF-ELISA systems, we concluded that both clones react against the BP230-C domain (clones termed BP230-C-mAbl [4-8E, scFv# 28; Table 3] and BP230-C-mAb2 [4-8G, scFv# 14; Table 3]).
  • Monoclonals against BP180- NC16A used herein were termed BPl80-NCl6A-mAbl (3-4E scFv# 17; Table 3) and BP180- NCl6A-mAb2 (3-14G, scFv# 1; Table 3).
  • Displacement of serum IgG by scFv was studied using the following protocol.
  • Patient serum was incubated with NCl6A-wells (5 wells) plus one well without serum (here: blocking buffer) for 30 minutes at 37 degrees Celsius.
  • the patient serum was titered before, for an OD of about 0.5/linear range.
  • scFvs #1, 29, 16 and 28 in different dilutions stock at about 1 ug/uL): 1 : 10, 1 : 100, 1 : 1,000, 1 : 10,000.
  • 2 wells were without scFv (positive control with serum IgG only; negative control).
  • the wells were incubated for 2 hours at 37 degrees Celsius.
  • Optical density (OD) values are given in Tables 4-6.
  • GGC AGAGT GGTT GGGT GTTCGGCGGAGGC ACC AAGCT GACCGT CCT AGGCGGT GGT
  • ELTLTQSPGTLSLSPGERATLSCRASQSVNNKYLAWYQQKPGQAPTLLIYGGSMRATGIP DRF S GS GSETDFTLTITRLEPEDF AVYYCQQ Y GRSPITFGQGTRLEIKGGS SRS S S S GGGG S GGGGQ V QF V Q S GAEVKKPGS S VKV S CKAS GGTF SNS AIS WVRQ APGQGFEWMGAIIPI FGTPNYAQRFQGRVTITADESTRTAYMEFGSFRSEDTAVYYCARDQPSWGSGWSPDHY YGFD VWGQGTTVTVS S
  • ELVLTQPPSASGTPGQRVTISCSGSGSNIGRNYVYWYQQLPGTAPKLFIFRNDQRPSGVP DRF S GSKS GTS ASL AIS GLRSEDE AD YY C AS WDDRQ S GWVFGGGTKLTVLGGGS SRS S S S GGGGS GGGGE V QLVES GAEVKKPGS S VKV S CKAS GGTF S S YDI S WVRQ APGQGLEW MGGIIPIF GTASYAQKFQGRVTITADESTSTAYMELS SLRSEDT AVYY C ARAPIIVLV SRG MD VWGQGTTVTVS S
  • ELVMTQ SPLSLP VTPGEP ASI S CRS S Q SLLHSDGYNYLD WYLQKPGQ SPQLLI YLGS YRA S GVPDRF S GS GS GTDFTLKISRVE AED V GVYY CMQ ALQTP YTFGQGTKLEIKGGS SRS S S S GGGGS GGGGE V QL V Q S GAEVKKPGS S VKV S CK AS GGTF S S S YAIS WVRQ APGQGLEW
  • ELVLTQPPS V SE APRQRVTIPCSGGS SNIGNNAV S WYQQLPGKAPKLLIYYDDLLPSGV S DRFSGSKSGASASLAISGLQSEDEADYYCAAWDDSLNAWVFGGGTKVTVLGGGSSRSS S S GGGGS GGGGQ V QLV Q S GAEVKKPGAS VKV S CKAS GYTFTGYYLHWLRQ APGQGPE WMGWINPNSGDTNFAQKFQGSVTLTRDTSSNTAYMELSSLTSGDTAVYYCARGGSGLG AFDVWGQGTVVTVSS
  • ELVMTQ SP ATLS AS V GDRVTIT CRAS Q SISRWLAWY Q QKPGK APKLLI Y AAS SLQ S GVP SRF S GS GS GTDFTLTIS SLQPEDF AT YY CQQ AN SFPITF GQGTRLEIKGGS SRS S S S GGGGS GGGGE V QLLES GGGWRPGGSLRLS C AAS GFTFDD Y GMS WVRQ APGKGLEWV S GINW N GGS T GY AD S VKGRFTISRDNAKN SL YLQMN SLRAEDT AVYY C ARVY SRWLRYFEYW GQGTLVTVSS
  • ACCCCC AATTTT GT ATTT ATTT ATTTTA ATT ATTTT GT GC AGCGATGGGGGCGGGG
  • GGGGAAC A AAGGCTGCGT GCGGGGT GT GT GT GCGT GGGGGGGTGAGC AGGGGGT GT G
  • ACCCCC AATTTT GT ATTT ATTT ATTTTA ATT ATTTT GT GC AGCGATGGGGGCGGGG
  • GGGGAAC A AAGGCTGCGT GCGGGGT GT GT GT GCGT GGGGGGGTGAGC AGGGGGT GT G

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Abstract

La présente invention concerne des anticorps ou des fragments de liaison à un antigène associés dirigés contre les protéines d'adhérence de la zone de la membrane basale BP180 et BP230. L'invention concerne également des procédés de fabrication d'anticorps ou de fragments de liaison associés dirigés contre des protéines d'adhérence de la zone basale et leurs méthodes d'utilisation. Ces anticorps ou leurs fragments de liaison peuvent être utilisés seuls ou en tant que protéines de fusion pour le diagnostic et le traitement de maladies affectant les tissus qui expriment BP180/BP230, pour le traitement de maladies dans lesquelles des lymphocytes activés provoquent une détérioration de l'épiderme, pour le traitement avec une dermatite d'interface et pour le traitement d'un cancer de la peau.
PCT/US2019/054751 2018-10-04 2019-10-04 Anticorps dirigés contre les protéines d'adhérence de la zone de la membrane basale bp180 et bp230 Ceased WO2020072937A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
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CN114252607A (zh) * 2020-09-24 2022-03-29 首都医科大学附属北京世纪坛医院 尿液v-atpase亚基s1及其多肽片段在过敏性疾病中的应用
US20220195014A1 (en) * 2020-12-23 2022-06-23 Regeneron Pharmaceuticals, Inc. Nucleic acids encoding anchor modified antibodies and uses thereof
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EP4703371A1 (fr) 2024-09-03 2026-03-04 SeraDiaLogistics Propriétés diagnostiques et thérapeutiques d'épitopes peptidiques dérivés de protéines d'adhésion matricielle extracellulaires de pemphigoïde bulleuses

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CN114252607A (zh) * 2020-09-24 2022-03-29 首都医科大学附属北京世纪坛医院 尿液v-atpase亚基s1及其多肽片段在过敏性疾病中的应用
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WO2024155750A1 (fr) 2023-01-18 2024-07-25 Lycia Therapeutics, Inc. Molécules bifonctionnelles de ciblage lysosomal pour la dégradation d'auto-anticorps
EP4703371A1 (fr) 2024-09-03 2026-03-04 SeraDiaLogistics Propriétés diagnostiques et thérapeutiques d'épitopes peptidiques dérivés de protéines d'adhésion matricielle extracellulaires de pemphigoïde bulleuses
WO2026052631A1 (fr) 2024-09-03 2026-03-12 SeraDiaLogistics Propriétés diagnostiques et thérapeutiques d'épitopes peptidiques dérivés de protéines d'adhésion à une matrice extracellulaire de pemphigoïde bulleuse

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