Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70517—CD8
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70514—CD4
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/526—CH3 domain
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

• aspects of the invention are drawn to a compositions and methods for treating and preventing disease.
• the disease is an autoimmune disease.
• the reagents include fusion proteins that have a domain including an autoreactive antigen from a diseased animal and a domain that can bind an effector cell.
• the effector-cell binding domain can be an Fc portion of an antibody that can bind to an Fc receptor on an effector cell.
• the effector cell binding domain can be an antibody that can bind an effector cell.
• a fusion protein comprising a desmosomal cadherin or fragment thereof, like a desmoglein (encoded by genes DSG1, 2, 3 or 4) or a desmocollin (encoded by genes DSC1, 2 or 3).
• SEQ ID NOs. 5-28 found in Example 4 and in FIG. 13) include all or parts of these amino acid sequences.
• the fusion protein comprises an extracellular (EC) domain of desmosomal cadherin proteins, like desmoglien 3 (Dsg3) or an EC domain of desmoglein 1 (Dsgl), and an isolated immunoglobulin Fc region or a fragment thereof.
• the fusion protein comprises any one or more of SEQ ID NOs.: 1-4, 29-32 or fragments thereof.
• the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID Nos 1-4 or 29-32.
• the fusion protein targets one or more B cells through its desmosomal cadherin portion.
• the targeted B cell comprises an autoreactive anti-Dsg3 B cell or an autoreactive anti-DSGl B cell.
• the fusion protein also targets effector cells (e.g., NK cells, macrophages) through its Fc region.
• effector cells e.g., NK cells, macrophages
• the Fc region of the fusion protein comprises an amino acid sequence at least 80% identical to the Fc regions of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 or SEQ ID NO 32.
• the Fc region comprises an IgG Fc region.
• the EC domain comprises any one or more of the various sequence IDs provided in FIG. 13.
• the EC domain comprises ECI, EC2, and EC3.
• the fusion protein further comprises a therapeutic moiety, an imaging moiety, a capturing moiety, or a combination thereof.
• the capturing moiety comprises a hydrophilic protein, a bacterial transpeptidase enzyme, a GST tag, a His-Tag, a polyethylene glycol (PEG), or a combination thereof.
• the imaging moiety comprises a fluorophore, a radioisotope, or a combination thereof.
• the therapeutic moiety comprises a cytokine, a toxin, a radiotherapeutic, a T cell-engaging moiety, a natural killer cell engaging moiety, or a combination thereof.
• the fusion protein can comprise a desmosomal cadherin or fragment thereof and an antibody or fragment that can bind an effector cell.
• the effector cell can be a T cell.
• the fusion protein can comprise a desmosomal cadherin or fragment thereof and a radioactive isotope.
• aspects of the invention are drawn towards a method of depleting autoreactive anti-Dsg or anti-Dsc B cells, the method comprising administering to a subject a therapeutically effective amount of a fusion protein described herein, or a pharmaceutical composition described herein.
• the effective amount is positively correlated with the level of anti-Dsg or Dsc antibodies circulating with the subject.
• aspects of the invention are drawn towards a method of treating an autoimmune disease, the method comprising administering to a subject a therapeutically effective amount of a fusion protein described herein, or a pharmaceutical composition described herein, wherein the subject is suffering from the autoimmune disease.
• the disease is selected from the group consisting of an autoimmune blistering disease, lupus, scleroderma, Goodpasture’s disease, Graves’ disease, and immune-mediated vasculitis.
• the autoimmune blistering disease comprises pemphigus vulgaris, bullous pemphigoid, mucous membrane pemphigoid, epidermolysis bullosa acquisita, a linear IgA bullous dermatosis, muscle-specific tyrosine kinase (MuSK) myasthenia gravis (MG), PLA2R Membranous Nephropathy, or Hemophilia A with FVIII Alloantibodies.
• the amount of fusion protein or pharmaceutical composition administered to a subject elicits an immune response against the autoimmune disease.
• FIG. 1 shows images of Pemphigus vulgaris in patients, which can result in painful and severe blistering of the skin and mucosa.
• FIG. 2 shows a representation of desmosome architecture between keratinocytes (Panel A).
• Panel B shows a schematic representation of structure of Dsg3 protein; anti-Dsg3 antibodies result in detachment of keratinocytes (acantholysis).
• the five domains of the extracellular portion of the protein show different levels of immunogenicity. Percentages shown next to each domain is the percentage of PV patients with antibodies specific for that domain. A patient can have antibodies against multiple domains.
• FIG. 3 shows schematic representation of the (panel A) Fc-mediated fusion protein (e.g., Dsg is bound by B cell receptor; NK cells/macrophages are bound by Fc receptor), (panel B) antibody-mediated fusion protein (e.g., Dsg is bound by B cell receptor; T cells are bound by antibody; this approach may be called a “B-cell receptor (BCR)-effector cell engager” approach or, where the effector cell is a T cell, a “BCR-T cell engager approach”), and (panel C) radiotherapy approaches.
• BCR B-cell receptor
• FIG. 4 shows a schematic sortase reaction.
• a protein equipped with an LPETG sortase tag can be labeled with a triglycine containing sortase substrate.
• 'R' can be any biomolecule of interest.
• a His-tag (H6) is used to purify the protein.
• FIG. 5 shows images of a passive transfer disease model.
• Adult B6 mice were injected with AK23 hybridoma cells. Hair loss around Panel A. Mouth Panel B. Eyes Panel C. Back Panel D. Panels H&E of loss of keratinocyte cell adhesion. Active immune model. Immunized Dsg3-/- splenocytes transferred to Rag-2-/- mice. Panel E. Significant size difference 25-35 days post splenocytes transfer in mice that received Dsg3-/- splenocytes (bottom) in contrast to mice that received Dsg3+/- splenocytes (top). Panel F.
• Panel G Intraepithelial blisters in upper esophagus of mice that received Dsg3 -immunized Dsg3-/- splenocytes in contrast to mice that received Dsg3 -immunized Dsg3+/- splenocytes (Panel H). From Amagai M, Tsunoda K, Suzuki H, Nishifuji K, Koyasu S, Nishikawa T. Use of autoantigen-knockout mice in developing an active autoimmune disease model for pemphigus. J Clin Invest. 2000 Mar;105(5):625-631. PMCID: PMC292455.
• FIG. 6 shows (Panel A) mDsg3-IgG2a was labeled with Alexa647 by sortase.
• Panel B SDS-PAGE, in-gel fluorescence and western blot characterization of mDsg3(ECl-3)-IgG2a (expected MW ⁇ 80 kDa).
• Panel C Flow cytometry analysis of anti-Dsg3 F779 and AK23 hybridoma cells with mDsg3-IgG2a-Alexa647, and Nalm6 as a negative control.
• FIG. 7 shows in vitro Fc-mediated cytotoxicity assay for evaluation of mDsg3-mFc chimeric protein.
• Anti-Dsg3 AK23 (Panel A) and F779 (Panel B) cells, and irrelevant hybridoma or Nalm6 control cells were co-cultured with mouse RAW264.7 macrophage cells with 10 nM of mDsg3-IgG2a construct.
• the killing efficacy was measured via standard live-dead assay after the indicated time points. The results were normalized to the 0 nM experiments; *** p ⁇ 0.001.
• FIG. 8 shows schematics of (Panel A) using click reaction to fuse Dsg3 and anti-CD3 scFvs.
• Panel B Using sortase to directly make Dsg3-anti-CD3 fusion protein.
• Panel C SDS- PAGE (Coomassie Staining) characterization of anti-CD19 and anti-CD3 scFv and the fusion protein after direct sortase reaction, (each scFv is ⁇ 35 kDa in size); arrow shows the fusion (expected mass ⁇ 70 kDa).
• FIG. 9 shows data of (Panel A) mDsg3-ECl-3 was labeled with Alexa647 using sortase.
• (Panel C) Flow cytometry analysis of anti-Dsg3 expressing F779, PVB-28 and AK23 hybridoma cells with mDsg3-Alexa647; Nalm6 cells was used as a negative control. Results are representative (n 3-5 for each).
• FIG. 10 shows (Panel A) mDsg3-ECl-3 was site-specifically labeled with DOTA using sortase. It will be radiolabeled using 225-Ac radioisotope as shown.
• FIG. 11 shows Schematic representation of the (Panel A) Fc-mediated, and (Panel B) BCR-T cell engager approaches. Each of the approaches is designed for targeted killing of pathogenic B cells.
• FIG. 12 shows (Panel A) Schematic representation of the Fc-mediated approach to target and kill the autoreactive pathogenic B cells.
• Panel B In vitro characterization of the Dsg3- Fc constructs.
• F779 (Nalm6-leukemia cells engineered with patient-derived anti-Dsg3 antibodies) were incubated with varying concentration of mDsg3-mFc(IgG2a) (top) or hDsg3-hFc(IgGl) (bottom) and analyzed by flow cytometry to calculate the EC50 values.
• Panel C Schematic illustration of the in vivo experimental timeline.
• Panel D Kaplan-Meier survival curve.
• FIG. 13 shows multiple sequence alignment of desmosomal cadherin ectodomains (SEQ ID NOs. 8-28).
• Human, mouse and bovine Dsgl-4 (blue) and Dscl-3 (orange) amino acid sequences were aligned for each EC domain. Residues conserved across both families are highlighted grey; those conserved within Dsgs or Dscs only, are highlighted blue and orange, respectively. Secondary structure corresponding to the human Dsg2 structure and the human Dscl structure are displayed above (blue) and below (orange) the alignment.
• O-linked glycans are shown as violet hexagons; N-linked glycans are shown as wheat hexagons and, where not conserved, individual glycosylated residues are additionally boxed. Disulfide bonds are shown by yellow lines above the alignment for Dsgs and below the alignment for Dscs. Interface residues for which > 10% solvent accessible surface area is buried in the strand-swapped homodimers are indicated by blue (Dsg2), orange (Dscl) and salmon (Dsc2) bars.
• Dsgl which lacks an EC5 domain, is excluded from the EC5 alignment (see Harrison et al., Structural basis of desmosomal cadherin adhesion, Proceedings of the National Academy of Sciences Jun 2016, 113 (26) 7160-7165).
• FIG. 14 shows a non-limiting, exemplary clinical presentation of mucosal-dominant pemphigus vulgaris, which results in painful and severe erosions in the mucosa.
• FIG. 15 shows schematic representation of the (Panel A) Fc-mediated, and (Panel B) BCR-T cell engager approaches. Each of the approaches is designed for targeted killing of pathogenic B cells.
• FIG. 16 shows (Panel A) Site-specific labeling of Dsg3-(ECl-3)-Fc construct via sortase.
• Panel B SDS-PAGE, in-gel fluorescence and western blot characterization of mDsg3- mFc, as indicated on the right side of the gel.
• Panel C Flow cytometric analyses of anti-Dsg3+ F779, PVB28, and AK23 hybridoma cells with murine and human Dsg3-Fc-labeled with Alexa Fluor 647; Nalm6 is used as control.
• FIG. 17 shows in vitro cytotoxicity assay to assess the efficacy of Dsg3-Fc engineered proteins.
• the assays started with 200,000 pre-coated effector cells in 48-well plates. -4-6 hours after the coating, 20,000 target cells, Dsg3-Fc treatment (at different concentrations as indicated), and ImM calcium were added.
• FIG. 18 shows the presence of anti-Dsg3 autoantibody does not neutralize the in vitro efficacy of the treatment.
• FIG. 19 shows the hDsg3-anti-CD3e scFv construct specifically stains F779 and Jurkat cells (human T cells), but not the control Nalm-6 cells.
• FIG. 20 shows the presence of autoantibodies does not inhibit the efficacy of the treatment.
• Mice received 3 doses of IVIG (days -3, -2, -1; 16mg/kg), allowing for a more rapid engraftment of the luciferase + PVB28 cells (1 million cells injected on day 0).
• mDsg3-EC14-mFc is used as the treatment (three times per week for two weeks, and then once per week, starting from day 4).
• Control mice received an isotype IgG2a.
• Auto-Abs group received mix of AK23, AK19 and AK18 (150 ⁇ g total), twice per week for two weeks starting a day prior to initiation of treatment.
• FIG. 21 shows (Panels A-D) Passive transfer model.
• Panel D H&E of loss of keratinocyte cell adhesion.
• Panels E-H Active immune model. Immunized Dsg3-/- splenocytes transferred to Rag-2-/- mice.
• FIG. 22 shows that treatment does not result in cytokine storm and toxicity in immunocompetent mice, even in the presence of autoantibodies.
• Panel A The schedule for injecting mice with treatment (mDsg3-EC14-mFc) and/or the mix of autoantibodies (AK23, AK19, AK18).
• FIG 23 shows that the treatment (mDsg3-ECl-4-mFc) inhibits pathogenic effects of AK23 antibody. All animals received AK23 (12mg/kg; subcutaneous); the AK23+treatment cohort received mDsg3-mFc Ih later (15mg/kg, i.p). Panels A and B show the AK23 group showed significant hair-loss by tape-stripping performed 72h later (Panel A), and showed severe PV phenotype (i.e.
• reagents and methods for treating diseases or conditions in which autoreactive B cells produce autoantibodies in affected animals are designed to target and deplete the autoreactive B cells.
• the reagents and methods are for treating autoimmune blistering diseases, like pemphigus vulgaris, bullous pemphigoid, mucous membrane pemphigoid, epidermolysis bullosa acquisita, linear IgA bullous dermatosis, pemphigus foliaceus, intraepidermal neutrophilic IgA dermatosis, paraneoplastic pemphigus and the like.
• the reagents can contain an autoreactive B cell epitope that binds to a B cell receptor on an autoreactive B cell.
• the autoreactive B cell epitope can be from extracellular domains of desmoglein (Dsg) or desmocollin (Dsc) molecules.
• the reagents in addition to the autoreactive B cell epitope, also can contain an effector-cell binding region that can bind to various effector cells, like NK cells, macrophages and T cells. When the autoreactive epitope of the reagent binds to an autoreactive B cell, the effector cells bound to the effector-cell binding region of the reagent are positioned in close proximity to the B cell and can deplete the B cell.
• a reagent having an autoreactive B cell epitope that binds to a B cell receptor on an autoreactive B cell can have an effector-cell binding region that includes an Fc portion of an antibody.
• the Fc portion of an antibody can bind to cells that have an Fc receptor (FcR), including effector cells like macrophages, natural killer (NK) cells, and the like. These effector cells can deplete the autoreactive B cell.
• Fc receptor Fc receptor
• a reagent having an autoreactive B cell epitope that binds to a B cell receptor on an autoreactive B cell can have an effector-cell binding region that includes an antibody that can bind to an effector cell.
• the antibody can be specific for T cells (anti-CD3 antibody).
• the antibody can be specific for cytotoxic T cells.
• the T cells can deplete the autoreactive B cell.
• BCR B cell receptor
• Therapeutics that bind to an effector cell that is a T cell can be called BCR-T cell engagers.
• the reagent molecules as described above may have a spacer between or connecting the autoreactive B cell epitope and the effector-cell binding region.
• the spacer can be an amino acid spacer.
• the spacer can be a flexible spacer.
• the flexible spacer can contain glycine and serine amino acid residues.
• the spacer connecting the autoreactive B cell epitope and the effector-cell binding region can be made using “click” chemistry.
• the spacer can include a C-to-C bond made using copper-mediated azide-alkyne cycloaddition chemistry.
• the spacer can include a N-to-C bond made using a sortase enzyme.
• the term “about” can refer to approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).
• subject or “patient” can refer to any organism to which aspects of the invention can be performed, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes.
• Subjects to which methods as described herein are performed comprise mammals, such as primates, for example humans.
• mammals such as primates, for example humans.
• a wide variety of subjects are suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals and pets such as dogs and cats.
• a wide variety of mammals are suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like.
• the term “living subject” can refer to a subject noted herein or another organism that is alive.
• the term “living subject” can refer to the entire subject or organism and not just a part excised (e.g., a liver or other organ) from the living subject.
• the term “normal subject” can refer to a subject that is not afflicted with a disease or condition, such as a subject that is not afflicted with a cancer.
• the phrase “therapeutic agent” can refer to any agent that elicits a desired pharmacological effect when administered to a subject.
• an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population.
• the appropriate population may be a population of model organisms.
• an appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc.
• a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
• a therapeutically effective amount can refer to an amount of a therapeutic agent whose administration, when viewed in a relevant population, correlates with or is reasonably expected to correlate with achievement of a particular therapeutic effect.
• the therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect).
• a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay and /or alleviate one or more symptoms of the disease, disorder, and/or condition.
• a therapeutically effective amount is administered in a dosing regimen that can comprise multiple unit doses.
• a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) can vary, for example, depending on route of administration, on combination with other pharmaceutical agents.
• the specific therapeutically effective amount (and/or unit dose) for a patient can depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and/or rate of excretion or metabolism of the specific fusion protein employed; the duration of the treatment; and like factors as is well known in the medical arts.
• an effective amount may be administered via a single dose or via multiple doses within a treatment regimen.
• individual doses or compositions are considered to contain a “therapeutically effective amount” when they contain an amount effective as a dose in the context of a treatment regimen.
• a dose or amount may be considered to be effective if it is or has been demonstrated to show statistically significant effectiveness when administered to a population of patients; a particular result need not be achieved in a particular individual patient in order for an amount to be considered to be therapeutically effective as described herein.
• the word “treating” can refer to the medical management of a subject, e.g., an animal, including human, with the intent that a prevention, cure, stabilization, or amelioration of the symptoms or condition will result.
• This term includes active treatment, that is, treatment directed specifically toward improvement of the disorder; palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disorder; preventive treatment, that is, treatment directed to prevention of disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the disorder.
• treatment also includes symptomatic treatment, that is, treatment directed toward constitutional symptoms of the disorder.
• treat or “treatment” can also refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder. “Treatment” can also refer to prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
• Treating” a condition with the compounds of the invention involves administering such a compound, alone or in combination and by any appropriate means, to a patient.
• Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition (e.g., prior to an identifiable disease, disorder, and/or condition), and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
• administration can refer to introducing a pharmaceutical composition or formulation as described herein into a subject.
• One route of administration of the composition is intravenous administration.
• any route of administration such as oral, topical, subcutaneous, peritoneal, intra-arterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into body compartments can be used.
• allergen can refer to a substance that can cause an allergic reaction.
• an allergen can produce an abnormally strong or vigorous immune response (e.g., hypersensitivity of the immune system).
• hypersensitivity can be type-I hypersensitivity.
• the hypersensitivity can be mediated by IgE.
• antibody can refer to a molecule or molecules that binds an antigen.
• antibody can refer to all types of antibodies, fragments and/or derivatives.
• Antibodies include polyclonal and monoclonal antibodies of any suitable isotype or isotype subclass.
• antibody can refer to, but not be limited to Fab, F(ab')2, Fab' single chain antibody, Fv, single chain, mono- specific antibody, bi-specific antibody, tri-specific antibody, multi-valent antibody, chimeric antibody, canine-human chimeric antibody, chimeric antibody, humanized antibody, human antibody, CDR-grafted antibody, shark antibody, nanobody (e.g., antibody consisting of a single monomeric variable domain), camelid antibody (e.g., from the Camelidae family) microbody, intrabody (e.g., intracellular antibody), and/or de-fucosylated antibody and/ or derivative thereof. Mimetics of antibodies are also provided.
• the antibodies disclosed herein are active agents that are part of the compounds disclosed herein that can cross the blood brain barrier.
• Antibody or “antigen-binding polypeptide” can refer to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen.
• An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof.
• antibody can include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen.
• CDR complementarity determining region
• the term “antibody” can refer to an immunoglobulin molecule and immunologically active portions of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen binding site that specifically binds (immunoreacts with) an antigen.
• immunoglobulin immunoglobulin
• immunologically active portions of an immunoglobulin (Ig) molecule i.e., a molecule that contains an antigen binding site that specifically binds (immunoreacts with) an antigen.
• Ig immunoglobulin
• antibody fragment or “antigen-binding fragment”, as used herein, is a portion of an antibody such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody.
• antibody fragment can include aptamers (such as spiegelmers), minibodies, and diabodies.
• antibody fragment can also include any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.
• Antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, dAb (domain antibody), minibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies.
• polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-
• a “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins.
• a single chain Fv (“scFv”) polypeptide molecule is a covalently linked VH:VL heterodimer, which can be expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide-encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883).
• the regions are connected with a short linker peptide of ten to about 25 amino acids.
• the linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N- terminus of the VH with the C-terminus of the VL, or vice versa.
• This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.
• a number of methods have been described to discern chemical structures for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody V region into an scFv molecule, which will fold into a three-dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Patent No. 5,091,5 13; No. 5,892,019; No. 5,132,405; and No. 4,946,778, each of which are incorporated by reference in their entireties.
• Antibody molecules obtained from humans fall into five classes of immunoglobulins: IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.
• immunoglobulins Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (y, p, a, 6, a) with some subclasses among them (e.g., yl-y4).
• Certain classes have subclasses as well, such as IgGi, IgG2, IgGs and IgG4 and others.
• immunoglobulin subclasses e.g., IgGi, IgG2, IgGs, IgG4, IgGs, etc. are well characterized and are known to confer functional specialization.
• IgG a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000.
• the four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “ Y” and continuing through the variable region.
• Immunoglobulin or antibody molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGi, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of an immunoglobulin molecule.
• Light chains are classified as either kappa or lambda (K, ). Each heavy chain class can be bound with either a kappa or lambda light chain.
• the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells, or genetically engineered host cells.
• the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
• Both the light and heavy chains are divided into regions of structural and functional homology.
• variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity.
• the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
• antigen-binding site or "binding portion” can refer to the part of the immunoglobulin molecule that participates in antigen binding.
• the antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light (“L”) chains.
• FR framework regions
• FR can refer to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins.
• the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen-binding surface.
• the antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions,” or "CDRs.” VH and VL regions, which contain the CDRs.
• the term “monoclonal antibody” or “mAb” or “Mab” or “monoclonal antibody composition”, as used herein, can refer to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.
• the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.
• MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
• Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
• a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
• the lymphocytes can be immunized in vitro.
• autoantibody can refer to an antibody produced by an animal that binds to or reacts with an antigen from the animal (e.g., a self-antigen instead of a “foreign” antigen). Generally (e.g., in absence of disease or a condition), animals are tolerant to self-antigens and do not produce autoantibodies.
• autoimmune can refer to an immune response (e.g., antibody production) against a self-antigen.
• Autoimmune disease can refer to a disease or condition resulting from the immune response to self-antigens.
• autoreactive can refer to components of an autoimmune response.
• a B cell that produces an antibody reactive against a self-antigen can be referred to as an autoreactive B cell.
• the antibody can be referred to as an autoreactive antibody.
• the self-antigen to which the autoantibody is reactive can be called an autoreactive antigen.
• B cell can refer to a type of lymphocyte that functions in the humoral immune response of an animal.
• B cells produce antibodies, that are displayed on the surface of the B cell.
• Plasma cells which are a more differentiated form of B cells, can secrete antibodies.
• B cell receptor or “BCR” can refer to the antibody produced by a B cell, which is displayed on the surface, together with a signal transduction moiety.
• depleting can refer removing or diminishing.
• depleting can refer to removing autoreactive B cells.
• Depleting can refer to killing of autoreactive B cells.
• micemoglein and “desmocollin” can refer to families of cadherins that are involved in formation of desmosomes.
• effector cell can refer to cells that can deplete autoreactive B cells.
• effector cells can be macrophages, NK cells, T cells, and the like.
• epitope can refer to the part of an antigen that binds to an antibody (e.g., to a B cell receptor). This epitope can be called a B cell epitope, which can be distinguished from a T cell epitope.
• epitope can include any protein determinant that can specifically bind to an immunoglobulin, a scFv, or a T-cell receptor.
• the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens.
• the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three-dimensional antigen-binding site.
• This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y.
• Epitopic determinants can consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
• antibodies can be raised against N- terminal or C-terminal peptides of a polypeptide.
• the antigen -binding site is defined by three CDRs on each of the VH and VL chains (i.e., CDR-H1, CDR-H2, CDR- H3, CDR-L1, CDR-L2 and CDR-L3).
• extracellular domain can refer to the part of a cellular membrane protein that extends from the exterior surface of the cell membrane of a cell.
• Fc region fragment crystallizable region
• the Fc region can be recovered when an antibody is digested by papain.
• Fc receptor or “FcR” can refer to a receptor that can bind Fc regions of antibodies. Fc receptors are present on the surface of certain cells, including natural killer cells and macrophages.
• immunological binding can refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. In some embodiments, these terms may describe binding of an Fc domain to an Fc receptor.
• the strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity.
• Immunological binding properties of selected polypeptides can be quantified using methods well known in the art.
• One such method entails measuring the rates of antigen- binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions.
• both the "on rate constant” (K O n) and the “off rate constant” (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation.
• K O n the "on rate constant”
• Koff Koff rate constant
• an antibody of the invention can specifically bind to an epitope when the equilibrium binding constant (KD) is ⁇ 1 pM, ⁇ 10 pM, ⁇ 10 nM, ⁇ 10 pM, or ⁇ 100 pM to about 1 pM, as measured by kinetic assays such as radioligand binding assays or similar assays known to those skilled in the art, such as BIAcore or Octet (BLI).
• KD is between about IE-12 M and a KD about IE-11 M. In some embodiments, the KD is between about IE-11
• the KD is between about IE- 10 M and a KD about IE-9 M. In some embodiments, the KD is between about IE-9 M and a KD about IE-8 M. In some embodiments, the KD is between about IE-8 M and a KD about IE-7 M. In some embodiments, the KD is between about IE-7 M and a KD about IE-6 M. For example, in some embodiments, the KD is about IE-12 M while in other embodiments the KD is about IE-11 M. In some embodiments, the KD is about IE-10 M while in other embodiments the KD is about IE-9 M.
• the KD is about IE-8 M while in other embodiments the KD is about IE-7 M. In some embodiments, the KD is about IE-6 M while in other embodiments the KD is about IE-5 M. In some embodiments, for example, the KD is about 3 E-l 1 M, while in other embodiments the KD is about 3E-12 M. In some embodiments, the KD is about 6E-11 M.
• “Specifically binds” or “has specificity to,” can refer to an antibody that binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.
• fusion protein can refer to proteins created by joining genes that originally were separate genes.
• macrophage can refer to a type of phagocytic white blood cell.
• natural killer cell or “NK cell” can refer to a type of cytotoxic lymphocyte.
• polypeptide as used herein can encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
• the term “polypeptide” refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product.
• peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids can refer to “polypeptide” herein, and the term “polypeptide” can be used instead of, or interchangeably with any of these terms.
• Polypeptide can also refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
• a polypeptide can be derived from a natural biological source or produced by recombinant technology but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. “Recombinant” as it pertains to polypeptides (such as antibodies) or polynucleotides refers to a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be created by combining polynucleotides or polypeptides that would not normally occur together. [0086]
• “sortase” can refer to enzymes from bacteria that recognize and cleave a carboxy-terminal recognition signal, generally in a peptide, polypeptide or protein.
• the recognition signal can include the amino acid motif Leu-Pro-X-Thr-Gly, where “X” can be any amino acid.
• T cell can refer to types of lymphocytes that functions in the cellular immune response of an animal.
• targets when used as a verb, can refer to a cell or molecule that specifically attacks something else (e.g., a fusion protein attaches to a B cell and brings along an effector cell that depletes the B cell; the fusion protein and the effector cell target the B cell).
• Target when used as a noun, can refer for example to the B cell that is depleted.
• the diseases or conditions that the reagents and methods disclosed here are designed to treat can be autoimmune conditions or diseases.
• the diseases or conditions treated can involve those in which autoreactive B cells produce autoreactive antibodies specific for self or autoreactive antigens.
• the diseases can include, for example, autoimmune blistering disease, lupus, scleroderma, Goodpasture’s disease, Graves’ disease, immune-mediated vasculitis, and the like.
• the diseases can include (MuSK) myasthenia gravis (MG), which is a rare, frequently more severe subtype of MG with different pathogenesis and unusual clinical features.
• MoSK myasthenia gravis
• the diseases can include PLA2R membranous nephropathy.
• PLA2R is an autoantigen present in glomerular podocytes.
• MN Membranous nephropathy
• MN can occur when circulating antibodies permeate the glomerular basement membrane and in the subepithelial space, form immune complexes with epitopes on podocyte membranes.
• the diseases can include hemophilia A with FVIII alloantibodies.
• the diseases or conditions can include autoimmune blistering diseases, like pemphigus vulgaris, bullous pemphigoid, mucous membrane pemphigoid, epidermolysis bullosa acquisita, linear IgA bullous dermatosis, pemphigus foliaceus, intraepidermal neutrophilic IgA dermatosis, and paraneoplastic pemphigus.
• the diseases or conditions can be present in animals, including humans, dogs, cats or horses.
• the autoreactive antibodies present in animals with the diseases or conditions can be specific for autoreactive antigens, that include molecules like desmoplakin, envoplakin, periplakin, plectin, bullous pemphigoid antigen 1, corneodesmosin, microtubule actin crossing-linking factor, epiplakin and cadherin.
• the autoreactive antigens can be desmosomal cadherins like desmoglein-1 (Dsgl), desmoglein-2 (Dsg2), desmoglein-3 (Dsg3), desmoglein-4 (Dsg4), desmocollin-1 (Dscl) desmocollin-2 (Dsc2), and desmocollin-3 (Dsc3).
• desmosomal cadherins like desmoglein-1 (Dsgl), desmoglein-2 (Dsg2), desmoglein-3 (Dsg3), desmoglein-4 (Dsg4), desmocollin-1 (Dscl) desmocollin-2 (Dsc2), and desmocollin-3 (Dsc3).
• the regions of the autoreactive antigens of interest in the reagents and methods disclosed herein are parts of the autoreactive antigens that are extracellular (EC) domains, as shown in FIG. 2B and FIG. 13.
• autoreactive antibodies in the diseases and conditions discussed above can be specific for EC domains of autoreactive antigens.
• Specific epitopes within the autoreactive antigens to which the autoreactive antibodies bind can be called autoreactive epitopes.
• the relevant epitopes can be B cell epitopes.
• the B cell autoreactive epitopes of the autoreactive antigens can bind to B cell receptors (BCR) of autoreactive B cells.
• BCR B cell receptors
• the B cells that the reagents and methods disclosed herein are designed to target are autoreactive B cells that have BCRs that can bind to these epitopes, for example from the EC domains of molecules like Dsgl, Dsg2, Dsg3, Dsg4, Dscl, Dsc2, and Dsc3.
• the autoreactive antigens stimulate production of autoreactive antibodies.
• the autoreactive antibodies produced can be “pathogenic.”
• the autoreactive antibodies produced can be “non- pathogenic.”
• Pathogenic antibodies generally produce symptoms and/or pathology of an autoimmune disease, like pemphigus. Without wanting to be held to a mechanism, pathogenic antibodies can inhibit a function of the protein to which they bind.
• therapies described herein can target B cells that produce pathogenic autoreactive antibodies.
• extracellular (EC) domains of autoreactive antigens can be pathogenic or non-pathogenic.
• EC domains of desmogleins and/or desmocollins can be pathogenic or non- pathogenic.
• ECI, EC2 and/or EC4 domains of Dsgl, Dsg2, Dsg3 and/or Dsg4 can be pathogenic.
• ECI, EC2 and/or EC 4 domains ofDsg3 and Dsgl can be pathogenic. While not wishing to be bound by theory, inclusion of non-pathogenic or less pathogenic EC domains in a therapeutic along with pathogenic EC domains is not thought to affect efficacy of the therapeutics disclosed herein.
• levels of immunogenicity of an EC domain may or may not be highly correlative with pathogenicity.
• This disclosure describes multiple approaches for therapy in animals, including humans, having an autoimmune disease or condition.
• the reagents and methods disclosed herein can target and deplete autoreactive B cells in the animals.
• a reagent used to deplete autoreactive B cells can be a fusion protein.
• the fusion proteins can have separate domains or regions.
• a fusion protein can contain one or more autoreactive epitopes that bind to B cell receptors (BCRs) on the surface of B cells reactive with the autoreactive epitope.
• BCRs B cell receptors
• the fusion proteins can also contain a domain that can bind to an effector cell.
• the domain that can bind to an effector cell can be an Fc region of an antibody. These therapeutics can be called Fc-mediated therapeutics.
• the domain that can bind to an effector cell can be an antibody that binds to an effector cell. These therapeutics can be called BCR-effector cell engagers.
• the therapeutics can be called BCR-T cell engagers.
• the fusion proteins can have a domain that includes one or more autoreactive B cell epitopes.
• the autoreactive B cell epitopes can be from any of the autoreactive antigens described above and, in some embodiments, are from EC domains of the autoreactive antigens.
• the autoreactive B cell epitopes are configured such that they can bind to a BCR of a target autoreactive B cell.
• the autoreactive B cell epitope can be located on an end of the fusion protein.
• the B cell epitope can be located at an N-terminal end of the fusion protein.
• “knob-into-hole” strategies can be used to produce Fc-mediated therapeutics that include multiple epitopes, including multiple autoreactive B cell epitopes.
• Knobs-into-holes is an approach that alters separate CH3 domains from, for example, two IgG heavy chains (CH3 domains contain part of the Fc region) to contain “knobs” or complementary “holes”, such that heterodimerization of the separate CH3 domains is favored (Ridgway, John BB, Leonard G. Presta, and Paul Carter. "‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization.” Protein Engineering, Design and Selection 9.7 (1996): 617-621).
• two recombinant polypeptides are designed.
• the first polypeptide can contain a first autoreactive antigen (e.g., one or more EC domains) and one of the “complementary” CH3 domains.
• the second polypeptide can contain a second autoreactive antigen and the other of the “complementary” CH3 domains.
• the two CH3 domains preferentially heterodimerize and disulfide bonds are formed between the polypeptides. Homodimerization is not favored.
• the heterodimerized molecule generally contains a functional Fc region and the two separate autoreactive antigens. In some embodiments, heterodimerized molecules can contain more than two antigens.
• the separate antigens in a “knob-into-hole” molecule can be from different autoreactive antigens.
• the separate antigens can contain different epitopes from the same autoreactive antigen.
• the separate antigens can be from Dsg3 and/or Dsgl.
• the separate antigens can be from different extracellular (EC) domains of a desmoglein.
• the separate antigens can be selected from ECI, EC2, EC3, EC4 and EC5 of a desmoglein molecule.
• the separate antigens can be from ECI and EC2.
• the autoreactive B cell epitopes can come from the ECI, EC2 and/or EC 4 domains of desmosomal cadherins (see Cho, Alice, et al. "Single-cell analysis suggests that ongoing affinity maturation drives the emergence of pemphigus vulgaris autoimmune disease. "Cell reports" 28.4 (2019): 909-922).
• EC3 can be included.
• the autoreactive B cell epitopes come from the ECI, EC2 and/or EC4 domains of Dsg3.
• the autoreactive B cell epitopes come from the ECI and/or EC2 domains of Dsg3.
• a single fusion protein may have multiple EC domains (e.g., two or more of ECI, EC2 and EC3; e.g., EC1-EC4) from one or more desmogleins.
• separate fusion proteins, each containing one ECI, EC2, EC3 or EC4 domains of Dsg3 may be combined into a composition and used for therapy in a patient.
• a therapeutic may be designed to contain EC domains corresponding to those that both bind to autoreactive antibodies in a patient, as well as EC domains corresponding to those for which autoreactive antibodies are not currently detected in the patient.
• epitope spreading a phenomenon known in the art as “epitope spreading,” some patients have autoreactive antibodies reactive with one epitope on an antigen, but not to other epitopes on the antigen. Over time, however, the patients can develop autoreactive antibodies to one or more of the epitopes which did not initially stimulate autoreactive antibodies. Inclusion in the therapeutics disclosed herein, of the epitopes to which autoreactive antibodies later emerge can prevent or lessen symptoms of the disease caused by the later-arising antibodies.
• administration of a therapeutic containing ECI, EC2, EC4, as well as EC5 can be beneficial to the patient.
• the autoreactive B cell epitopes can come from plakins (e.g., envoplakin, periplakin, desmoplakin I, desmoplakin II, epiplakin, plectin, BP230), cadherins ((Dsg3, Dsgl, Dscl, Dsc2, Dsc3, a2-macroglobulin-like molecules), BP 180 (e.g., NCI 6 A, LABD97, LAD-1 domains or C-terminal epitopes), laminin 332, a6p4 integrin, collagen VII, Laminin gammal (p200), Type VII collagen, epidermal transglutaminase, tissue transglutaminase, endomysium or deamidated gliadin.
• plakins e.g., envoplakin, periplakin, desmoplakin I, desmoplakin II, epiplakin, plectin, BP230
• cadherins ((D
• the autoreactive antibodies can be any class of antibody (IgA, IgD, IgE, IgG or IgM). In some instances, the autoreactive antibodies can be IgA or IgG.
• allergens, parts of allergens, or molecules that can mimic allergens can be part of the Fc-mediated therapeutics or BCR-T cell engagers disclosed herein.
• allergens may be from drugs, foods, insects (e.g., insect stings, dust mites), latex, molds, pets/animals (e.g., dander) and pollen.
• Allergens can include urushiol, as found in plants like poison ivy, eastern poison oak, western poison oak, poison sumac, and the like.
• the allergen can be birch pollen allergens, like Bet v 1.
• the allergens/parts of allergens can be proteins, polypeptides or polypeptides.
• the allergies can be type I hypersensitivities.
• the allergies can be mediated by IgE.
• B-cells can produce the IgE.
• the IgE can then bind to IgE- specific receptors found, for example, on mast cells, basophils, and the like, which cells can release mediators of allergies, like cytokines, vasoactive amines (e.g., histamine), proteases, prostaglandins, leukotrienes, and the like.
• allergens that are part of remediated therapeutics or BCR-T cell engagers can bind and deplete B-cells that produce IgE.
• the allergies can be type II, type III or type IV hypersensitivities.
• the fusion proteins can have a domain that binds to an effector cell.
• the fusion protein can be configured such that binding of the autoreactive B cell epitope to a BCR on a B cell, co-localizes effector cells bound to the effector-cell binding domain of the fusion protein in proximity to the B cells such that the effector cells can act on the B cell and deplete the B cell.
• the domain that binds to an effector cell can be a Fc portion of an antibody.
• the domain that binds to an effector cell can be an antibody specific for the effector cell.
• the effector-cell binding domain can be located on an end of the fusion protein. In some examples, the effector-cell binding domain can be located at a C-terminal end of the fusion protein.
• the effector cells to which the effector-cell binding domain of a fusion protein can bind can be a phagocytic cell or phagocyte.
• the phagocyte may include so- called “professional” and “non-professional” phagocytes.
• professional phagocytes can include monocytes, macrophages, neutrophils, dendritic cells and mast cells.
• non-professional phagocytes can include certain T cells (e.g., cytotoxic T cells) and natural killer (NK) cells.
• the domain of the fusion protein that binds to an effector cell can be an Fc portion or domain of an antibody.
• any Fc portion may be used in the reagents and methods disclosed herein.
• any Fc portion that can bind to an effector cell e.g., NK cell, macrophage, and the like
• any Fc portion that can mediate depletion of the target pathogenic B cells e.g., autoreactive B cells
• the Fc portion can bind to a cell that has an Fc receptor (FcR).
• the Fc portion can bind to an FcR on the cell.
• the FcR can be an Fc-gamma receptor (FcyR), an Fc-alpha receptor (FcaR) or an Fc-epsilon receptor (FcsR).
• the FcyR can be an FcyRI, FcyRII or FcyRIII.
• the Fc portion of an antibody is configured such that it can bind to an Fc receptor (FcR) on a cell.
• the Fc portion or domain of an antibody used herein may be modified.
• the modification or modifications can affect binding of the Fc region to Fc receptors on various cells.
• the modifications can affect function of cells to which the Fc regions bind.
• the Fc portion or domain can be heterogeneously modified at a conserved N-glycosylation site on the Fc domain.
• a complex, biantennary glycan can be attached at the conserved N-glycosylation site on the Fc domain (see Li, Tiezheng, et al.
• any type of Fc domain modification can be used, as long as the modified Fc domain can bind to an effector cell (e.g., NK cell, macrophage, and the like).
• an effector cell e.g., NK cell, macrophage, and the like.
• any type of Fc domain modification can be used, as long as the modified Fc domain can mediate depletion of the target pathogenic B cells (e.g., autoreactive B cells).
• the Fc portion can also be configured such that an effector cell to which the Fc portion binds is co-localized in proximity to the autoreactive B cell to which the autoreactive B cell epitope of the fusion protein binds.
• Effector cells that have an FcR, and to which the Fc portion of the fusion protein can bind can include for example, neutrophils, monocytes, macrophages, mast cells and dendritic cells.
• the domain of the fusion protein that binds to an effector cell can be an antibody that binds to an effector cell.
• fusion proteins that contain an autoreactive antigen and an antibody that binds to an effector cell can be called BCR-effector cell engagers.
• the antibody can be an antibody that binds T cells (e.g., an anti-CD3 antibody) or specific types of T cells.
• the antibody can be an antibody that binds cytotoxic T cells (e.g., an anti-CD8 antibody) or an antibody that binds helper T cells (e.g., an anti- CD4 antibody).
• the antibody can be an antibody that binds, for example, macrophages, natural killer (NK) cells or other phagocytes.
• an antibody or combination of antibodies that bind NK cells can be used.
• an anti-CD56 antibody may be used.
• the antibody can be a single-chain variable fragment (scFv).
• the antibody that binds to an effector cell is configured such that it can bind to the specific effector cell. In some embodiments, the antibody can bind to one or more desired effectors cells, but not bind or bind less efficiently to other cells.
• the antibody that binds to an effector cell is also configured such that an effector cell to which the antibody binds is co-localized in proximity to the autoreactive B cell to which the autoreactive B cell epitope of the fusion protein binds. This co-localization facilitates the ability of the effector cell to interact with/act on the autoreactive B cell.
• the fusion proteins disclosed herein can additionally have a spacer or linker.
• a spacer can be located in the fusion protein between the autoreactive B cell epitope domain and the domain that binds to an effector cell.
• the spacer can facilitate the separate domains within a fusion protein to each perform their desired function.
• a spacer located between a domain of a fusion protein that includes an autoreactive B cell epitope and a domain that binds to an effector cell can provide for the autoreactive B cell epitope to bind to a BCR on an autoreactive B cell while, simultaneously, the effector-cell binding domain can bind to and co-localize the effector cell in proximity to the autoreactive B cell.
• the spacer can provide for the co-localized effector cell to deplete the autoreactive B cell.
• the spacer can be an amino acid spacer. In some embodiments, the spacer can be between about 2 and 50 amino acids in length. In some embodiments, the spacer can be between about 5 and 45, 6 and 40, 7 and 35, 8 and 30, 9 and 25, or 10 and 20 amino acids in length. In some embodiments, the spacer can be a flexible spacer that can increase flexibility of the fusion protein.
• flexible peptide spacers can include small, polar (e.g., Ser, Thr) or non-polar (e.g., Gly) amino acids. The flexible peptide spacers can have sequences of Gly and Ser residues (e.g., a “GS” linker).
• An example GS linker amino acid sequence can include (Gly- Gly-Gly-Gly-Ser)n.
• An example peptide spacer can be (Gly-Gly-Gly-Gly-Ser)3.
• Other types of flexible spacers can include KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, (Gly)s, GSAGSAAGSGEF and (GGGGS)4 (Chen, Xiaoying, Jennica L. Zaro, and Wei-Chiang Shen. "Fusion protein linkers: property, design and functionality." Advanced drug delivery reviews 65.10 (2013): 1357-1369.).
• Other example types of spacers can be rigid spacers or cleavable spacers.
• the domains that include an autoreactive B cell epitope and the effector-cell binding domain can be connected to one another using various chemistries.
• so-called “click” chemistry can be used.
• sortase enzymes can be used.
• the domains can be connected using C-to-C fusion chemistry.
• sortase can be used to install click “handles” at C-termini of the autoreactive C cell epitope domain and the effector-cell binding domains.
• the two modified domains can be “clicked” together using, for example, using azide-alkyne click chemistry, producing the C-to-C fusion (FIG. 8A).
• the domains can be connected using N-to-C fusion chemistry.
• a tri gly cine molecule (Gly-Gly-Gly) can be used to catalyze formation N-to-C fusion of the two domains.
• One of the domains (first domain) can be modified to have a Leu-Pro-Glu-Thr-Gly at its C terminus.
• the other domain (second domain) can be modified to have a Leu-Pro-Glu-Thr-Gly-Gly-Gly at its N terminus.
• the Leu-Pro-Glu-Thr segment is removed from the second modified domain and converted to Gly3.
• the Gly3-second domain can then react with the modified first domain to form the fusion protein (FIG. 8B).
• the fusion proteins disclosed here can include additional moieties/domains, like a therapeutic moiety, an imaging moiety, a capturing moiety, and combinations thereof. In some embodiments, these moieties can be added to the fusion proteins using click chemistry and/or sortase enzymes.
• a therapeutic moiety can include a cytotoxin, a toxin, a radiotherapeutic agent, a T cell-engaging moiety, a natural killer (NK) cell engaging moiety, or a combination thereof.
• a therapeutic moiety can include IL-2 or a molecule having IL-1 activity.
• an imaging moiety can include a fluorophore, a radioisotope, or a combination thereof.
• a capturing moiety can include a hydrophilic protein, a bacterial transpeptidase enzyme, a GST tag, a His-Tag, polyethylene glycol (PEG), or a combination thereof.
• fusion proteins disclosed herein and individual domains of those proteins (e.g., autoreactive epitopes, Fc regions, antibodies that bind effector cells)
• individual substitutions, deletions or additions of amino acids to peptide, polypeptide, or protein sequence, or to nucleotides of a nucleic acid sequence, which alters, adds, deletes, or substitutes a single amino acid or a small percentage of amino acids in the encoded sequence is collectively referred to herein as a "conservatively modified variant".
• the alteration results in the substitution of an amino acid with a chemically similar amino acid.
• Conservative substitution tables providing functionally similar amino acids are well known in the art.
• Such conservatively modified variants of the Fc domains or antibodies disclosed herein can exhibit increased effector-cell binding in comparison to unmodified sequences.
• a “conservative amino acid substitution” is one 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, including 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
• basic side chains e.g., lysine, arginine, histidine
• acidic side chains e.g., aspartic acid
• a nonessential amino acid residue in an immunoglobulin polypeptide is replaced with another amino acid residue from the same side chain family.
• a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
• the autoreactive epitopes, Fc regions, or antibodies that bind effector cells can have a specified percentage identity or similarity to the amino acid or nucleotide sequences of the autoreactive epitopes, Fc regions, or effector-cell-binding antibodies described herein.
• “homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position.
• a degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
• the molecules described herein can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
• the molecules described herein can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
• sequence identity or similarity to the nucleic acids and proteins of the present invention can be determined by sequence comparison and/or alignment by methods known in the art, for example, using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. For example, sequence comparison algorithms (i.e. BLAST or BLAST 2.0), manual alignment or visual inspection can be utilized to determine percent sequence identity or similarity for the nucleic acids and proteins of the present invention.
• sequence comparison algorithms i.e. BLAST or BLAST 2.0
• manual alignment or visual inspection can be utilized to determine percent sequence identity or similarity for the nucleic acids and proteins of the present invention.
• therapeutic preparation can refer to any compound or composition that can be used or administered for therapeutic effects.
• therapeutic effects can refer to effects sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
• Embodiments as described herein can be administered to a subject in the form of a pharmaceutical composition or therapeutic preparation prepared for the intended route of administration.
• Such compositions and preparations can comprise, for example, the active ingredient(s) and a pharmaceutically acceptable carrier.
• Such compositions and preparations can be in a form adapted to oral, subcutaneous, parenteral (such as, intravenous, intraperitoneal), intramuscular, rectal, epidural, intratracheal, intranasal, dermal, vaginal, buccal, ocularly, or pulmonary administration, such as in a form adapted for administration by a peripheral route or is suitable for oral administration or suitable for parenteral administration.
• compositions can be prepared in a manner well-known to the person skilled in the art, e.g., as generally described in “Remington's Pharmaceutical Sciences”, 17. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., U.S.A., 1985 and more recent editions and in the monographs in the “Drugs and the Pharmaceutical Sciences” series, Marcel Dekker.
• the compositions and preparations can appear in conventional forms, for example, solutions and suspensions for injection, capsules and tablets, in the form of enteric formulations, e.g., as disclosed in U.S. Pat. No. 5,350,741, and for oral administration.
• Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
• the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• suitable carriers include physiological saline, bacteriostatic water, Cremophor EMTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
• the composition can be sterile and can be fluid to the extent that easy syringeability exists. In embodiments, it can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal.
• isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein.
• examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• Oral compositions can include an inert diluent or an edible carrier.
• Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
• Oral formula of the drug can be administered once a day, twice a day, three times a day, or four times a day, for example, depending on the half-life of the drug.
• compositions administered to a subject can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel ® (sodium starch glycolate) , or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
• a binder such as microcrystalline cellulose, gum tragacanth or gelatin
• an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel ® (sodium starch glycolate) , or corn starch
• a lubricant such as magnesium stearate or sterotes
• Systemic administration can also be by transmucosal or transdermal means.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
• Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
• the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
• administering can comprise the placement of a pharmaceutical composition into a subject by a method or route which results in at least partial localization of the composition at a desired site such that desired effect is produced.
• the pharmaceutical composition can be administered by bolus injection or by infusion.
• a bolus injection can refer to a route of administration in which a syringe is connected to the IV access device and the medication is injected directly into the subject.
• the term “infusion” can refer to an intravascular injection.
• Embodiments as described herein can be administered to a subject one time (e.g., as a single injection, bolus, or deposition).
• administration can be once or twice daily to a subject for a period of time, such as from about 2 weeks to about 28 days. Administration can continue for up to one year. In embodiments, administration can continue for the life of the subject. It can also be administered once or twice daily to a subject for period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof.
• compositions as described herein can be administered to a subject chronically.
• Chronic administration can refer to administration in a continuous manner, such as to maintain the therapeutic effect (activity) over a prolonged period of time.
• a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the particular antibodies, variant or derivative thereof used, the patient's age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art.
• the amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.
• a therapeutically effective amount of a reagent or therapeutic composition of the invention can be the amount needed to achieve a therapeutic objective. As noted herein, this can be a binding interaction between the reagent or therapeutic composition and its target that, in certain cases, interferes with the functioning of the target.
• the amount required to be administered will furthermore depend on the binding affinity of the reagent or therapeutic composition for its specific target and will also depend on the rate at which an administered reagent or therapeutic composition is depleted from the free volume other subject to which it is administered.
• the dosage administered to a subject (e.g., a patient) of the binding polypeptides described herein is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight, between 0.1 mg/kg and 20 mg/kg of the patient's body weight, or 1 mg/kg to 10 mg/kg of the patient's body weight.
• Human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible.
• the dosage and frequency of administration of reagent or therapeutic composition of the disclosure may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
• Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention can be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight.
• Common dosing frequencies can range, for example, from twice daily to once a week.
• fragments e.g., antibody fragments
• the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred.
• peptide molecules can be designed that retain the ability to bind the target protein sequence.
• Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. (See, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)).
• the formulation can also contain more than one active compound as necessary for the particular indication being treated, for example, those with complementary activities that do not adversely affect each other.
• the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine (e.g., IL- 15), chemotherapeutic agent, or growth-inhibitory agent.
• a cytotoxic agent e.g., IL- 15
• chemotherapeutic agent e.g., IL- 15
• growth-inhibitory agent e.g., IL- 15
• Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
• the active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in_macroemulsions. Sustained-released preparations can be prepared.
• the pharmaceutical or therapeutic carrier or diluent employed can be a conventional solid or liquid carrier.
• solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose.
• liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water.
• the carrier or diluent can include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
• the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or it can be in the form of a troche or lozenge.
• the amount of solid carrier will vary widely but will usually be from about 25 mg to about 1 g.
• the preparation can be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.
• composition and/or preparation can also be in a form suited for local or systemic injection or infusion and can, as such, be formulated with sterile water or an isotonic saline or glucose solution.
• the compositions can be in a form adapted for peripheral administration only, with the exception of centrally administrable forms.
• compositions and/or preparations can be in a form adapted for central administration.
• compositions and/or preparations can be sterilized by conventional sterilization techniques which are well known in the art.
• the resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution prior to administration.
• the compositions and/or preparations can contain pharmaceutically and/or therapeutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
• autoimmune diseases including an autoimmune blistering disease like pemphigus
• the therapeutics described herein contain autoreactive epitopes to which the autoreactive antibodies in the patient could possibly bind.
• a potential concern can be that the autoreactive antibodies could bind to and/or inactivate the therapeutic after it has been administered to a patient.
• data presented herein e.g., see Example 7, FIGs. 17 & 18
• effectiveness of the therapeutics disclosed herein are not decreased or are minimally decreased by the presence of autoreactive antibodies in the patient to which the therapeutic is administered.
• nucleic acids encoding all or part of the fusion proteins described herein.
• vectors e.g., plasmids, viral, and the like
• cells e.g., prokaryotic, eukaryotic
• nucleic acids or vectors can express the fusion proteins.
• PV Pemphigus vulgaris
• Dsg3 desmoglein 3
• Dsgl epithelial detachment
• anti-CD20 antibodies e.g., rituximab
• anti-CD20 antibodies e.g., rituximab
• Relapse in PV can be due to expansion of the same autoreactive cells, not depleted during initial treatment; thus, complete depletion of autoreactive B cells can be a cure.
• Neonatal Fc receptor and Bruton’s Tyrosine Kinase inhibitors currently in clinical trials, cannot circumvent the off-target immunosuppressive effects.
• Fc-mediated approach this approach is analogous to the use of rituximab in PV, with major advantage of targeting anti-Dsg3 B cells.
• BCR-T cell engager approach The Dsg3-anti-CD3e construct can establish the molecular clustering and formation of the immunologic synapses between the target anti-Dsg3 B cells and the T cells, which will consequently result in the proliferation and activation of T cells to specifically kill the targeted anti-Dsg3 B cells, leaving other B cells intact.
• cytokine release syndrome is not expected in treatment of autoimmune diseases, given the burden of autoreactive B cell is lower than for malignant clones.
• Dsg3-Fc can be safe and can replace the use of Rituximab.
• the other approach provides an attractive alternative option for patients who fail to respond to available treatments. Therefore, this project has the potential to transform the treatment of not only PV, but open an avenue for developing treatments for several autoantibody/immune-complex mediated diseases to known antigenic targets, ranging from other autoimmune blistering diseases (e.g., bullous pemphigoid, mucous membrane pemphigoid, epidermolysis bullosa acquisita, linear IgA bullous dermatosis), subtypes of lupus, scleroderma, Goodpasture’s disease, Graves’ disease, to immune-mediated vasculitis. Therefore, without wishing to be bound by theory, the therapeutics disclosed herein can be considered a ground-breaking addition to the therapeutic toolbox for autoimmune diseases.
• PV Pemphigus vulgaris
• Dsg3 desmoglein 3
• acantholysis epithelial detachment
• anti-CD20 antibodies against B cells e.g., rituximab
• off-target immunosuppressive effects including increased risk of infection albeit lower than traditional immunosuppressive agents and a high risk of relapse after treatment.
• Neonatal Fc receptor and Bruton’s Tyrosine Kinase inhibitors currently in clinical trials, cannot circumvent the off-target immunosuppressive effects.
• Early-stage cell-based therapies such as autologous polyclonal regulatory T cells 14 and chimeric autoantigen receptor (CAAR) T cells 16, if proven effective, cannot be globally immunosuppressive.
• CAAR chimeric autoantigen receptor
• Fc-mediated this approach is analogous to the use of rituximab in PV, in the sense that B cells are targeted, with a major advantage of targeting anti-Dsg3 B cells-only.
• BCR-T cell engager The Dsg3-anti-CD3e construct can establish the molecular clustering and formation of the immunological synapses between the target anti-Dsg3 B cells and the T cells, which can consequently result in the proliferation and activation of T cells to specifically kill the targeted anti-Dsg3 B cells, leaving other B cells intact.
• cytokine release syndrome is not expected in treatment of autoimmune diseases, given the burden of autoreactive B cell is lower than for malignant clones.
• Radiotherapy In clinical practice, the short range of alpha particles in tissue ( ⁇ 0.1 mm), allows for selective killing of the targeted cells without damaging neighboring tissue; the killing is independent of oxygenation status, cell cycle, cell internalization and prior treatments. Radiotherapy can therefore offer an attractive alternative for patients who are resistant to traditional therapy, Rituximab, or our Dsg3-Fc and Dsg3-anti-CD3 approaches.
• Sortase is a bacterial transpeptidase enzyme that recognizes an “LPETG” motif and cleaves the bond between threonine and glycine to form a thio-acyl intermediate, which is substituted with a triglycine containing probe (GGG-Probe).
• GGG-Probe triglycine containing probe
• sortase can convert a “protein-LPETG” to “protein-LPET-GGG-probe” product, where the probe can be any biomolecule of interest (FIG. 4), rapidly, with near complete yield.
• sortase to install fluorophores, polymers, radioisotopes or a secondary protein into biomolecules.
• Plasmid and cell lines We used three cell lines: 1) AK23 hybridoma cells, which produces anti-mouse Dsg3 antibodies, which induces PV in mice.
• the AK23 antibody recognizes the ECI domain and cross-reacts with human Dsg3-ECl; 2 and 3) Nalm-6 F779 and Nalm-6 PVB- 28 cancer cell lines.
• Nalm-6 is a B cell precursor leukemia cell line
• F779 and PVB-28 are the cells engineered to express patient-derived anti-Dsg3 antibodies that recognize Dsg3-ECl and Dsg3-EC2 domains, respectively.
• the P3F3 antibody is an IgGl antibody that binds specifically to human Dsg3-ECl domain, inducing strong acantholysis when added to human keratinocytes.
• the AK23 model has advantages, however, treatment can overcome the fast-proliferating hybridoma cells. Therefore, as a second model, we used an active PV immune model that closely mimics the clinical phenotype of human disease, has lower frequency of autoreactive Dsg3-specific B cells that resembles what happens clinically, and finally has a high serum polyclonal autoantibody level. In this model, we developed an active polyclonal pemphigus mouse disease model by first immunizing Dsg3-/- mice (JAX - 002911) with recombinant extracellular domain of mouse Dsg3, followed by the transfer of splenocytes into RAG2-/- mice.
• mice can be infected with active LCMV-C113. Animals can be injected with AK23 hybridoma 21 days post-infection followed by our treatment strategies according to experimental design described herein. Viral load for infected and control mice can be compared 30 days post-injection. Alternatively, at day 7 post our PV therapy, mice can be challenged orally with Listeria monocytogenes 40 or intravenously with LCMV infection. Microbial load in liver and spleen will be checked 10 days post-infection and compared with infection-only mice. In both experiments, and without wishing to be bound by theory, there will not be any difference in the viral load.
• Non-limiting, exemplary results We have made both mouse and human Fc-fusion proteins: mDsg3-ECl-3-IgG2a (mFc) and hDsg3-ECl-3-IgGl (hFc). Fc regions from human IgGl and mouse IgG2a were chosen for their high Fc-mediated killing activities.
• the proteins were engineered to have a FLAG tag, a sortase recognition motif and a His6 tag at the C-terminus.
• the FLAG, an 8 amino acid sequence (DYKDDDDK) can be used for Western Blot and flow cytometry analyses, and subsequently for analysis of the construct metabolic fate when used in vivo.
• the His6 tag was used for purification and the sortase tag was used to site-specifically introduce modifications.
• a Gly3-Alexa647 sortase substrate was synthesized and used to site-specifically label the fusion proteins with Alexa Fluor 647. Sortase reaction was performed by mixing the construct (50 pM), the Gly3- Alexa647 substrate (500 pM), and sortase (5 pM) for 1 h, at 4°C.
• Alexa647-labeled constructs were purified via size-exclusion column chromatography and the labeling were further confirmed by ingel fluorescence scan of the constructs (FIG. 6 panel B).
• Alexa647-labeled mDsg3-ECl-3-mFc to stain the anti-mDsg3 AK23 hybridoma and anti-hDsg3 F779 cells.
• the cells were stained with affinity and specificity (at low nM concentration) (FIG. 6 panel C), indicating that the patient derived BCRs on the F779 cells cross-react with mouse Dsg3 protein. Nalm-6 cells, used as control, did not show any binding (FIG. 6 panel C).
• fusion protein can be intravenously injected at 0, 2 or 5 days after AK23 injection (model 1). These time points can help us identify the relationship between disease burden and the effectiveness of the treatment, that is, to determine whether the treatments are preventative and/or can they be able to rescue severe disease phenotype.
• the treatment proteins can be used at varying concentrations (5 mg/kg - 15 mg/kg 46,47 in 100 pl final volume via tail-vein IV injection) to find the optimum condition. The range can be determined based upon the antibody concentrations used for B cell depletion in autoimmune mouse models.
• the treatments will be performed at Day 0, 14, or 21 post adoptive cell transfer in Rag2-/- mice, as previous studies have reported circulating anti-Dsg3 IgG was in the sera of recipient Rag2-/- mice as early as day 4 followed by a rapid increase and a plateau around day 21.
• the readout for model 1 experiments will be bioluminescence imaging (IVIS) of AK23 hybridoma cells (as they are luciferase positive) that allows sensitive longitudinal measurements for the AK23 cells.
• IVIS bioluminescence imaging
• serum anti- Dsg3 titers measured at days 5 and 14 post-initiation of treatments can be performed.
• B cell counts can be performed as well, to ensure they remain within normal levels during the treatment. Without wishing to be bound by theory, the treatments result in clinical and histological resolution of the disease.
• the construct was designed as such to have the Dsg3 on the N-terminus and anti-CD3e on the C-terminus, similar to other bi-specific T cell engagers; furthermore, this structure can ensure that the EC1-EC3 domains will be available for recognition by the Dsg3-reactive B-cells.
• a flexible (G4S)3 spacer was included between Dsg3 and scFv proteins, similar to other bi-specific T cell engagers. The protein was expressed using the Expi293F cells.
• C-to-C fusion using click chemistry Without wishing to be bound by theory, through using sortase, we can site-specifically install click handles at the C-termini of Dsg3 and anti-CD3e scFv proteins, which then can allow us to “click” them together using azide-alkyne click chemistry. This will result in the production of a C-to-C fusion, which is impossible to make through traditional genetic approaches. Such an approach is site-specific, thereby the product can retain its functionality.
• the anti-CD3escFv was installed with the alkyne substrate, and the anti -CD 19 scFv was installed with the azide substrate, both at their C-termini via sortase.
• the click-functionalized scFvs were purified via FPLC and subsequently clicked together via the copper-mediated azide-alkyne cycloaddition chemistry (FIG. 8 panel A).
• the product was purified via FPLC and characterized using SDS-PAGE and flow cytometry analyses, which confirmed that the clicked C-to-C fusion stains both Nalm6 cells and human T cells with a strong affinity. We will use the approach to make C-to-C fusion of Dsg3-anti-CD3e as well. 2.
• Sortase-mediated synthesis of the N-to-C fusion The click chemistry approach is an alternative to the traditional genetic fusion when and if the expression of a genetically fused heterodimer protein proves to be difficult.
• N- to-C fusion of Dsg3-anti-CD3e without any prior modification on either of the proteins, as follows:
• a triglycine molecule (GGG) can be used to catalyze the formation of a C-to-N protein fusion: protein A is engineered to have an “LPETG” motif at the C-terminus, and protein B will be engineered to have an “LPETGGG” at the N-terminus.
• sortase and the triglycine molecule can remove the “LPET” part from the protein B and convert it into "Gly3-B".
• the Gly3- B protein, produced in situ, can then react with protein A to form the fusion product, “A-LPET- GGG-B” with no click chemistry involved (FIG. 8 panel B).
• the reaction can be done in a dialysis cassette allowing for the excess GGG catalyst to leave the reaction mixture, which would further move the reaction toward the formation of the fusion product.
• anti-CD19-anti-CD3 scFv fusion The anti- CD 19 scFv has “LPETG” sortase tag at its C-terminus.
• anti-CD3 scFv protein was engineered to have the following sequence: “MHis6- LPETGGG-anti-CD3 scFv” (proteins always start with an M, as the methionine codon (ATG) serves as the start codon; His6 was used to facilitate purification).
• ATG methionine codon
• His6 was used to facilitate purification.
• the product formation was confirmed via SDS-PAGE analysis and the fusion protein was purified via FPLC size-exclusion chromatography (FIG. 8 panel C); the fusion was used to stain Nalm6 cells (CD 19+) and Jurkat cells (CD3+).
• Standard live-dead analysis of cells will be performed using flow cytometric analysis. Production of IFN-y, as an indicator of T cell activation, will be assessed using ELISpot assay. Delineating the efficacy of the approach on patients PBMCs in in vitro assays: Similar to aim 1, hDsg3-anti-hCD3e fusion protein can be added to human PBMC culture obtained from PV patients; healthy PBMCs can be used as control. No additional T cells will be added to the mixture, as (without wishing to be bound by theory) the patients’ own T cells to be directed and activated to kill the targeted anti-Dsg3 B cells.
• Varying concentrations of the fusion can be used to find the optimum condition (10 pg/mL to 1 ng/mL).
• Recombinant protein can be intravenously injected at 0, 2 or 5 days after AK23 cell injection (model 1) at varying concentrations (0.5 mg/kg - 1 mg/kg), similar to those used in oncological treatment settings in 100 pl final volume to find the optimum condition.
• recombinant protein will be injected at Day 0, 14, or 21 post adoptive cell transfer in Rag2-/- mice (as previously described).
• Dsgl-anti CD3e complex can be used at the same concentration as the treatment protein.
• readout along with luciferase (for model 1), immunofluorescence and histological imaging, and anti-Dsg3 ELISA titers, cells can be isolated from the skin biopsies of mice with and without treatment. Normalized count of CD3+ cells will be analyzed through flow cytometry analysis at Day 5, 14 and 20 post treatment. There can be an increase in T cell counts in mice injected with Dsg3-anti-CD3e treatment confirming that the recombinant protein can recruit T cells. Long term monitoring and any potential side-effects will be performed as explained herein.
• the sortase reaction can be monitored via SDS-PAGE and/or mass spectrometry.
• the product can be purified via size-exclusion chromatography.
• the azide-alkyne cycloaddition is a well characterized reaction extensively used for bioconjugation of proteins, with near complete yield.
• Final fusion constructs can be further characterized by mass spectroscopy, SDS-PAGE, flow cytometry and immunoblot analysis.
• the synthesis of the various modified peptides is routine in the laboratory, and (without wishing to be bound by theory) any difficulty in the production and scaling up of the synthesis of the modified peptides.
• the PEG spacer can lead to a higher stabilization of the construct; this PEG spacer size can be changed if a given fusion has low stability or has low solubility. Furthermore, we can analyze whether the size of the spacer affects the killing abilities of the constructs. We have shown that our mouse and human Dsg3-ECl-3 constructs, bacterially expressed and then refolded, binds with great affinity (low nanomolar) to all the anti-Dsg3 cells and antibodies that we have in hand. Furthermore, glycosylation of Dsg3 was shown not to play a significant role in its binding to patient anti-Dsg3 antibodies. Therefore, we will continue to use our established approach to make Dsg3 constructs.
• a side-effect for the anti-CD3 treatment can be the engagement of CD4 T cells, for example T follicular helper cells (Tfh), with autoreactive B cells, which can result in their expansion and thus exacerbation of the disease. Without wishing to be bound by theory, this will not happen, as the bi-specific T cell engagers used in the clinic are effective in recruiting CD8 cytotoxic T cells to kill the target cells.
• CD4 T cells for example T follicular helper cells (Tfh)
• autoreactive B cells autoreactive B cells
• the frequency of CD8 T cell recruitment can be higher than the frequency of recruited Tfh cells; this is because most, if not all, of CD8 T cells, with any TCR, can be recruited to kill the autoreactive Dsg3 cells, whereas engagement of CD4 T cells (expressing unrelated TCR) with the autoreactive B cells is not expected to result in B cell expansion; perhaps engagement of only a few CD4 T cells, with reactivities against Dsg3 -derived peptide-MHC complexes, can result in an expansion of autoreactive B cells.
• the DOTA-labeled Dsg3 will be installed with a 225 Ac radioisotope. Formation of product and radiochemistry yield will be determined using radio-HPLC analysis. Stability of the purified 225 Ac -Dsg3 constructs will be assessed by incubation in PBS and in human serum at 37 °C. To ensure radiolabeling will not affect functionality of the desmoglein protein, positive anti-Dsg3 cells and negative cells will be incubated with 225 Ac-Dsg3 for 30 min. Cells will be washed, 3 times, and radioactivity will be measured. We expect the anti- Dsg3 cells to be radioactive, while the negative cells can or cannot have any radioactivity.
• Incubation with radiolabeled Dsg3 or the control isotope will be performed for 20 min, followed by washing out (3x) any unbound reagent/isotope to prevent non-specific cell death from irradiation.
• Analyses will be performed at 4, 16, 24 and 48h post-incubation with radiolabeled Dsg3, via an ELISpot assay for antigen-specific and total IgG B-cell depletion.
• We will measure the percentage decrease of the autoreactive anti-Dsg3 IgGs and total IgGs level.
• analysis will be performed via flow cytometry, and standard live- dead analyses on the nonspecific cells, including B and T cells, which will act as an internal standard for the assay. Controls, inclusion and exclusion criteria will be same as mentioned in aims 1 and 2. Expected results will confirm targeted killing of Dsg3 autoreactive B cells.
• Radiolabeled recombinant protein will be intravenously injected at 0, 2 or 5 days after AK23 cell injection (model 1), with the 225 Ac- labeled Dsg3 constructs ( ⁇ 30 kBq activity and 50 pg Dsg3 per animal 72 ).
• 225 Ac-DOTA the chelator molecule
• control the amount of activity ( ⁇ 30 kBq), ⁇ 3.0 pg DOTA per animal).
• Radiolabeling proteins can affect binding affinity of autoreactive antibodies on Dsg3 proteins. This can be due to the high positive charge of actinium cation (3+). If the radiolabeling affects the binding capability of the Dsg3 protein, we will synthesize a sortase substrate with a PEG spacer (for example Gly3-PEG5-DOTA). This should allow proper distance between the Ac cation (3+) and the Dsg3 protein. Syntheses of the peptides are routine in our laboratory.
• 225 -Ac isotope We chose to use 225 -Ac isotope, not only because it has been used in the clinic, but because it has unique benefits over other existing isotopes, such as limited range in tissue, good linear energy transfer, relatively short half-life (-10 days), and multiple (4 net) alpha particles emitted with each decay. If using 225 - Actinium revealed to have other unexpected issues, we can use 213 -Bismuth, also used in the clinical setting, as an alternative. In vivo studies: The issue of radiotherapeutics is renal toxicity. We will monitor for renal toxicity and use approaches such as attachment of polyethylene glycol (PEG), a biologically inert and hydrophilic polymer to decrease the kidney uptake.
• PEG polyethylene glycol
• PEGylation decreases kidney uptake of radiolabeled antibody fragments in preclinical studies (by >10 times). Furthermore, PEGylation has been shown to be safe in the clinic, as there are several approved PEGylated protein therapeutics. We will use different size PEG (5, 10 and 20 kDa) to evaluate the effect of PEGylation on the kidney clearance. Alternatively, we can co-inject mice with 150 mg/kg Gelofusine, which have been shown to reduce kidney uptake and renal toxicities.
• this approach can be an option for patients who fail existing treatments, with potential future applications to be expanded to other autoimmune blistering diseases, for example, in paraneoplastic or immunotherapy induced autoimmune blistering diseases when the use of immunosuppression is associated with worse outcome 83 .
• the radiotherapy approach may be used concurrently with other drugs that lower anti-Dsg3 titer and/or saturate FcRns, such as IVIG.
• the Dsg3 -containing constructs will not incorporate themselves into desmosomes in the skin, as it requires the full structure to be on the cell-surface including the transmembrane domain for a potentially long-term stability.
• extensive studies on Dsg3-CAAR T cells in animal models did not show any accumulation of the Dsg3-CAAR in the animal skin and, similarly, when human skin was transplanted into mice skin, the hDsg3-CAAR T cells were not detected in the transplanted skin, indicating the Dsg3 constructs are not expected to incorporate themselves into the desmosome. This will be studied through histology analyses.
• PBMCs will be collected from mice that received our proposed therapy and percentages of Thl, Th2, Thl7, Tregs, and CD8 T cells will be analyzed by flow cytometry analysis. Age and sex matched unmanipulated WT B6 mice will be used as control. Without wishing to be bound by theory, there will be no difference in the percentages of immune cell population suggesting no adverse long-term effect of the treatments. [00199]
• the approaches herein to target autoreactive B cells as well as our strategies to develop these therapeutics site-specific installation of the radioisotope, synthesis of C-to-C fusions and N-to-C sortase mediated fusions) are unique.
• compositions and methods described herein can transform the treatment of not only PV, but open an avenue for developing treatments for several autoantibody/immune-complex mediated diseases to known antigenic targets, ranging from other autoimmune blistering diseases (e.g., bullous pemphigoid, mucous membrane pemphigoid, epidermolysis bullosa acquisita, linear IgA bullous dermatosis), subtypes of lupus, scleroderma, Goodpasture’s disease, Graves’ disease, to immune-mediated vasculitis.
• autoimmune blistering diseases e.g., bullous pemphigoid, mucous membrane pemphigoid, epidermolysis bullosa acquisita, linear IgA bullous dermatosis
• subtypes of lupus, scleroderma, Goodpasture’s disease, Graves’ disease to immune-mediated vasculitis.
• Pemphigus vulgaris is a devastating B-cell mediated autoimmune disease.
• Autoantibodies targeting desmoglein 3 (Dsg3) a desmosomal protein critical for intercellular adhesion of skin and mucous membrane, is responsible for the disease phenotype.
• Standard treatments include immunosuppressive drugs that systemically suppress the immune system.
• Our in vitro and in vivo experiments indicate the approach results in efficient and specific depletion of the autoreactive B cells.
• PBMCs peripheral blood mononuclear cells
• PV Pemphigus vulgaris
• Dsg3 desmoglein 3
• acantholysis epithelial detachment
• anti- CD20 antibodies e.g., rituximab 3
• off-target immunosuppressive effects including obtunding humoral response to vaccines with current, real lifetime implications, and increased risk of infection albeit lower than traditional immunosuppressive agents 10 11 and a high risk of relapse after treatment 10 “ 12 .
• Neonatal Fc receptor and Bruton’s Tyrosine Kinase inhibitors currently in clinical trials, cannot circumvent the off-target immunosuppressive effects.
• Early-stage cell-based therapies such as autologous polyclonal regulatory T cells 13 and chimeric autoantigen receptor (CAAR) T cells 14 , if proven effective, cannot be globally immunosuppressive.
• CAAR chimeric autoantigen receptor
• Pemphigus vulgaris affects a young, healthy group of patients (i.e. young/middle aged-adults, average 40-60 years of age) compared to other autoimmune blistering diseases.
• Current existing treatment strategies is limited to immunosuppressive medications - for example, systemic steroid, mycophenolate mofetil, azathioprine, and rituximab, with risk of infection, higher risk of malignancy in the long run and obtunding response to new vaccination with current, real-life implications.
• the disease can be brought into remission with intense period of immunosuppression, but the risk of recurrence is as high as 80% 10 .
• the clinical effect of the therapeutic is visible to the human eye and there is commercially available serum test for monitoring recurrence.
• the disease prevalence in the US is estimated to be 5.2 cases per 100,000 adults 15 . Patients can have relapses throughout their life, requiring repeat treatments. There is a higher incidence in those of Jewish ancestry ( ⁇ 4- and 10- fold higher than the average) 15 17 .
• Existing treatments i.e. prednisone, mycophenolate mofetil, azathioprine, rituximab
• up-and-coming treatment e.g., neonatal Fc receptor antagonist, Burton’s Tyrosine Kinase inhibitors
• the method herein is based on the common shared autoantigen which can be manufactured without the need for personalization.
• the treatment described herein can be used in an outpatient setting in a manner analogous to Rituximab (which can be given in an outpatient infusion center).
• This treatment will be used both in the initial stage of treatment and with relapses.
• the disease population will be patients with mucosal-dominant pemphigus vulgaris, with a clear goal to expand to other autoimmune blistering diseases in the future, such as pemphigus foliaceus (where the main autoantigen is Dsgl) in the next step.
• This treatment can replace current standard of treatment, Rituximab (anti-CD20), as it is selective and does not have off-target effect/global immunosuppression from complete depletion of B cell pool by Rituximab.
• the antigen-Fc (Dsg3- Fc) fusion protein will effectively and specifically kill Dsg3 -recognizing cells.
• the Dsg3-portion of the construct will bind to the anti-Dsg3 BCR of autoreactive pathogenic B cells and the Fc domain will induce Fc-mediated killing of the Dsg3 -recognizing cells by recruitment of phagocytic and natural killer (NK) cells (FIG. 12 panel A).
• NK phagocytic and natural killer
• the FLAG an 8 amino acid sequence (DYKDDDDK)
• the His6-tag was used for purification and the sortase tag was used to site-specifically introduce modifications, such as attachment of fluorophores.
• Alexa647- mDsg3-mFc was used to stain the anti-mDsg3 AK23 hybridoma 24 and anti-hDsg3 F779 cells 14 , a B lymphoma cell line (Nalm6) engineered with a patient-derived pathogenic anti-Dsg3 antibody.
• the cells were stained with great affinity and specificity (at low nM concentrations) (FIG. 12 panel B), suggesting that the patient-derived BCRs on the F779 cells cross-react with mouse Dsg3 protein. Nalm-6 cells, used as control, did not show any binding. Similar experiments were performed using the Alexa647-labeled hDsg3-hFc, which confirmed that the construct binds strongly to the F779 cells (FIG. 12 panel B) and stained the mouse AK23 cells too (similar to
• mDsg3-mFc was injected 5 times a week to ensure enough drug is available in the circulation, for the duration of the study.
• the disease progression was monitored by bioluminescence imaging and mice body scoring.
• PBMC was collected via submandibular bleeding and animals were euthanized. The result showed the control group had a mean survival of 26 days, whereas the treatment group had a survival of 57 days.
• FIG. 12 panel D The flow cytometric analyses of PBMC from the two groups revealed that the treatment group had near 100% antigen-loss (FIG. 12 panel E), indicating that the treatment was effective in mediating the depletion of antigenpositive F779 cells. Of note, the F779 cells are malignant and thus rapidly proliferate.
• time point for the tissue harvest will be determined to observe acantholysis (by H&E staining).
• the peak disease time is expected to be around Day 7 24 ; we expect the mice to exhibit hair loss 24 , weight loss, and have blistering in hair follicles and oral mucosa along with AK23 autoantibodies produced by AK23 cells binding to epidermal keratinocytes with physiologic anti-Dsg3 IgG levels (300-400 RU/ml) 26 .
• AK23 model has advantages such as easy read out, however, treatment can overcome the fast-proliferating hybridoma cells. Therefore, as a second model, we will use an active PV immune model that mimics the clinical phenotype of human disease, has lower frequency of autoreactive Dsg3-specific B cells that resembles what happens clinically, and finally has a high serum polyclonal autoantibody level. In this model, we can develop an active polyclonal pemphigus mouse disease model by first immunizing Dsg3-/- mice (J AX - 002911) with recombinant extracellular domain of mouse Dsg3, followed by the transfer of splenocytes into RAG2-/- mice 27,28 .
• Pemphigus is the most common autoimmune skin disease diagnosed in veterinary medicine, for which there is no cure, and the prognosis remains poor 31 “ 37 .
• the common autoantigens in canine pemphigus are, Dsg3 (pemphigus vulgaris) and Dscl (desmocollin-1; pemphigus foliaceus (PF)) 31,38-41 .
• canine models can serve as a bridge between preclinical and human trials.
• Production of such proteins is now straightforward in our lab and we anticipate no obstacles in making a large amount of these proteins. Improvement of lesions and autoantibody titers in circulation during the first three months of treatment will be carefully assessed.
• compositions and methods described herein can transform the treatment of not only pemphigus, but also open an avenue for developing treatments for several autoantibody/immune- complex mediated diseases to known antigenic targets, ranging from other autoimmune blistering diseases (e.g., pemphigus foliaceus, bullous pemphigoid, mucous membrane pemphigoid, epidermolysis bullosa acquisita, linear IgA bullous dermatosis), subtypes of lupus, psoriasis, scleroderma, Goodpasture’s disease, Graves’ disease, to immune-mediated vasculitis.
• autoimmune blistering diseases e.g., pemphigus foliaceus, bullous pemphigoid, mucous membrane pemphigoid, epidermolysis bullosa acquisita, linear IgA bullous dermatosis), subtypes of lupus, psoriasis, sclero
• compositions and methods described herein can be to the therapeutic toolbox for autoimmune diseases.
• Ploegh HL Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions. Nat Protoc. 2013 Sep;8(9): 1787-1799. PMID: 23989673
• Nishikawa T A mouse model of pemphigus vulgaris by adoptive transfer of naive splenocytes from desmoglein 3 knockout mice. Br J Dermatol. 2004 Aug;151(2):346-354. PMID: 15327541 [00247] 29. Amagai M, Tsunoda K, Suzuki H, Nishifuji K, Koyasu S, Nishikawa T. Use of autoantigen-knockout mice in developing an active autoimmune disease model for pemphigus. J Clin Invest. 2000 Mar;105(5):625-631. PMCID: PMC292455
• Dsg3 is a calcium-binding transmembrane glycoprotein component of desmosomes in vertebrate epithelial cells
• -Desmosomes are cell-cell junctions between epithelial, myocardial, and certain other cell types
• -Anti-CD20 antibodies e.g., rituximab
• rituximab are first line option (targets all B cells)
• PBMCs peripheral blood mononuclear cells
• sortase technology can be used to site-specifically attach any biomolecule of interest at the C-terminus of a protein.
• biomolecules of interest can be attached non-specifically as well, for example, via maleimide reaction with free cysteines, or NHS-active groups with free amines of lysine side-chains.
• Certain embodiments can comprise multiple antigens on single fusion protein.
• An embodiment comprises a knob-into-hole Fc. Under one embodiment, an antigen is placed on the hole, and a second antigen on the knob. Such embodiments permit inclusion of two major antigens in one drug.
• embodiments can comprise an Dsg3-knob, and an Dsgl-hole or vice versa.
• Embodiments can comprise specific extracellular (EC) domains of Dsgl or Dsg3, for example, as antigens included in these therapeutics. Such embodiments can be useful in the treatment for pemphigus patients.
• autoimmune disorders are provided in US Patent No. 7,332,168, which is hereby incorporated by reference in its entirety.
• Non-limiting examples of autoimmune disorders are provided below:
• Paraneoplastic pemphigus IgG Abs against predominantly plakins (envoplakin, periplakin, desmoplakin I, desmoplakin II, epiplakin, plectin, BP230), cadherins (Dsg3, Dsgl, Dscl, Dsc2, Dsc3), a2-macroglobulin-like molecules)
• IgA pemphigus IgA against Dsgl, Dsg3, Dscl, Dsc2, Dsc3
• Pemphigus herpetiformis IgG Dsgl, Dsg3, Dscl, Dsc3
• Lichen planus pemphigoides IgG against BP180 (NC16A) domain, BP230 [00317] Pemphigoid gestation: Anti IgG against BP180 (NC16A domain; 90% of cases), BP230 (less prevalent)
• Mucous membrane pemphigoid IgG/IgA against BP 180 [NC16A domain / C- terminal epitopes], laminin 332, BP230, a6p4 integrin, and collagen VII
• Anti-laminin gamma l/p200 pemphigoid IgG against Laminin gammal (p200)
• Pemphigus vulgaris is a devastating autoimmune disease, which can be characterized by painful blistering of the skin and mucosa 1-4 .
• Desmogleins a part of the cadherin family, are the primary cellular glue between keratinocytes in stratified epithelium. Autoantibodies against desmoglein molecules inhibit their adhesive function resulting in loss of cell-to-cell adhesion, known as acantholysis 5,6 . Additionally, the autoantibodies’ interference in cell-to-cell adhesion leads to cell signaling pathways that augment the pathological autoimmune response and contribute to acantholysis 2,3 ’ 7 .
• Mucosal-dominant PV is caused by the production of autoantibodies against desmoglein-3 (Dsg3), resulting in painful mucosal erosions 8,2, 9 (FIG. 14) Oral mucosal erosions are particularly painful and troubling for patients causing difficulty in eating and drinking. Mucosal erosions can occur in other mucosal services as well including nasal, vaginal, perianal, laryngeal/esophageal, urogenital, and conjunctival mucosa (FIG. 14). There is no cure for PV; treatment strategies typically include systemic steroids and immunosuppressive drugs 10-22 . Anti-Dsg3 antibodies from autoreactive B cells are the primary drivers of mucosal-dominant-PV 23,24 .
• anti-CD20 antibodies e.g., rituximab
• anti-CD20 antibodies have become the first-line option for moderate-to-severe disease 11,4,27-30
• off-target immunosuppressive effects including an increased risk of infection (albeit lower than traditional immunosuppressive agents), depletion of B cells, obtunding the humoral response to vaccination or new infections for 6 or more months, and a high risk of relapse after treatment 29,31-34 .
• Treatment options including neonatal Fc receptor antagonists 35,36 , blocking antibodies against B cellactivating factor receptor (BAFF-R) 37 , and Bruton’s Tyrosine Kinase inhibitors (BTKi) 38,39 , some currently in clinical trials, would not circumvent the off-target immunosuppressive effects of obtunding the global humoral immunity and carry a high risk of severe hypogammaglobulinemia, particularly in the case of neonatal Fc receptor antagonists 40 .
• BAFF-R B cellactivating factor receptor
• BTKi Tyrosine Kinase inhibitors
• cell-based therapies such as autologous polyclonal regulatory T cells 44 and chimeric autoantigen receptor (CAAR) T cells 45 , if proven effective, cannot be globally immunosuppressive.
• cell-based therapies can be personalized to an individual patient, and have specific requirements for manufacturing, storage, and transport, which cannot be feasible in all hospitals and clinics treating this disease group.
• IVS Inducible Co-Stimulator
• T cells which influences the activity of T follicular helper cells that play critical roles in development of anti-Dsg3 pathogenic B cells
• anti-ICOS antibody has shown results in delaying the progression of PV in animal models 46 ; however, this is a less direct way of targeting anti-Dsg3 antibody production compared to targeting anti-Dsg3 B cells themselves, without wishing to be bound by theory, can only delay the progression of the disease, and may have negative impacts on the production of other protective antibodies.
• the development of a targeted treatment that precisely depletes autoreactive cells while preserving the protective immunity is an unmet need and will be lifesaving for many patients.
• Dsg3 and Dsgl have different expression patterns in skin and mucosa 1 .
• Dsg3 can be expressed through the mucosa and in the deeper layers of the adult cutaneous skin; Dsgl and cadherin are unable to compensate for the loss of Dsg3 function in the mucosa in mucosal- dominant PV.
• Anti-desmoglein autoantibodies play a pathogenic role in inducing loss of adhesion between keratinocytes and formation of blisters and erosions. Experimental models have shown that Dsg3 and Dsgl autoantibodies are directly pathogenic 24,25 ’ 47 . Anti -Dsgl and anti-Dsg3 autoantibody levels can correlate with disease severity 48 .
• Perilesional autoreactive B and T lymphocytes that work together to produce autoreactive antibodies are the cellular players in disease pathogenesis 49 ; relapse can be correlated with the return of autoreactive T and B cells, which work together to cause disease phenotype. Remission is induced by lowering or depletion of autoreactive antibodies targeting desmoglein. Relapse is higher in patients who have incomplete depletion of autoreactive B-cells 50 .
• the Fc portion then induces Fc-mediated killing of the pathogenic B cells recognizing Dsg3 by recruitment of phagocytic and natural killer (NK) cells (FIG. 15 panel A).
• NK natural killer
• compositions and methods described herein can provide a platform for the treatment of not only for mucosal-dominant PV, other variant of pemphigus, but also antibody-driven autoimmune diseases with known antigens.
• compositions and methods described herein can transform the treatment of not only mucosal-dominant PV, but also opens avenues for the development of treatments for autoantibody/immune-complex mediated diseases with known antigenic targets, ranging from other autoimmune blistering diseases (e.g., mucocutaneous pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, mucous membrane pemphigoid, epidermolysis bullosa acquisita, linear IgA bullous dermatosis), subtypes of lupus, scleroderma, Goodpasture’s disease, Graves’ disease, to immune-mediated vasculitis.
• autoimmune blistering diseases e.g., mucocutaneous pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, mucous membrane pemphigoid, epidermolysis bullosa acquisita, linear IgA bullous dermatosis), subtype
• Sortase a bacterial transpeptidase
• the enzyme has a short recognition sequence (LPETG), cleaves between the T and G to form an intermediate thioester, allowing resolution by a nucleophilic reaction with a Gly 3 -containing substrate.
• the reaction can provide near-quantitative yield and can be rapid, robust, and reproducible.
• sortase to install functionalities onto proteins, such as fluorophores, polymers, and radioisotopes 56-61 .
• the sortase-mediated site-specific labeling ensures the binding capacity of proteins will not be compromised after labeling, and therefore, here, we will use the sortase technology to modify proteins.
• Anti-Dsg3 cell lines and antibodies We can use three cell lines and four anti-Dsg3 antibodies: 1) AK23 hybridoma cells, which produces anti-mouse Dsg3 antibodies that induces PV in mice63. The AK23 antibody recognizes ECI domain and cross-react with human Dsg3- EC1; 2 and 3) Nalm-6 F779 and Nalm-6 PVB28 cancer cell lines. Nalm-6 is a B cell precursor leukemia cell line, and F779 and PVB28 are the Nalm-6 cells engineered to express patient-derived anti-Dsg3 antibodies that recognize Dsg3-ECl and Dsg3-EC2 domains, respectively 45 .
• the P3F3 antibody is an IgGl antibody that binds specifically to human Dsg3-ECl domain, inducing strong acantholysis when added to human keratinocytes 64 .
• P1F5, P5D4, and P5G6 antibodies bind to the Dsg3-EC2, Dsg3-EC4 and Dsg3- EC1 domains respectively and together induce strong acantholysis 64 .
• the combined monoclonal antibodies cause higher dissociation of keratinocytes than any single one of them.
• the proteins were engineered to have a FLAG tag, a sortase recognition motif and a His6 tag at the C-terminus (FIG. 16 panel A).
• the FLAG an 8 amino acid sequence (DYKDDDDK)
• DYKDDDDK 8 amino acid sequence
• the His6 tag was used for purification and the sortase tag (LPETG) was used to site-specifically introduce modifications.
• LETG sortase tag
• constructs using the mammalian HEK293 cells, purified via Ni-NTA affinity column and characterized using SDS-PAGE and western blotting analyses, which confirmed the formation of the constructs (FIG. 16 panels A-B).
• a Gly3-Alexa647 sortase substrate was synthesized and used to site-specifically label the fusion proteins with AlexaFluor647. Sortase reaction was performed by mixing the construct (50 pM), the Gly3- Alexa647 substrate (500 pM), and sortase (5 pM) for 1 h, at 4°C.
• Alexa647-labeled constructs were purified via size-exclusion column chromatography and the installation of the fluorophore was further confirmed by in-gel fluorescence scan of the constructs (FIG. 16 panel B).
• Alexa647-labeled mDsg3-mFc to stain the anti-mDsg3 AK23 hybridoma, and the anti-hDsg3 F779 and PVB28 cells.
• the cells were stained with great affinity and specificity (at 10 nM concentration) (FIG. 16 panel C), suggesting that the patient-derived BCRs on the F779 and PVB28 cells cross-react with mouse Dsg3 protein.
• Results showed a high rate of the killing of targeted cells (-90% killing), with no non-specific killing of anti -Dsg3 -negative Nalm6 cells, at 10 and 100 nM, suggesting the treatment remains highly effective and specific at higher concentration ranges (FIG. 17).
• Results showed highly effective and specific killing, similar to that of mDsg3-mFc experiment discussed above (FIG. 17).
• THP1 cells 70 (a widely used and established human monocyte cell line derived from an AML patient) as effector cells and used hDsg3-hFc construct as the treatment.
• Results showed an effective killing of target cells with no killing of the control anti -Dsg3 -negative Nalm-6 cells (FIG. 17).
• We performed the killing assay using a range of doses for the treatment (0, 1, 10, and 100 nM of the hDsg3-hFc construct (FIG. 17) and observed an effective killing efficacy for all tested concentrations, suggesting the treatment is effective at low concentrations and remains highly specific at high concentrations.
• a potential concern is whether the presence of anti-Dsg3 autoantibodies (as occurs in PV patients) can block the treatment from binding to the target cells, and thus decrease or neutralize the treatment efficacy.
• anti-Dsg3 autoantibodies as occurs in PV patients
• the AK23 antibody recognizes ECI domain on Dsg3, as discussed above, and thus can potentially neutralize the treatment. Experiment was performed following identical setting as above.
• Results showed a very effective killing of the AK23 cells (either with Raw cells or THP1 cells as effector cells, and mDsg3-mFc or hDsg3-hFc as treatments), similar to what we observed for the F779 and PVB28 cells (FIG. 17).
• the hDsg3-hFc fusion protein (1 nM and 10 nM) will be added to the cells following incubation at 37 °C in a standard cell incubator. No additional phagocytic cells will be added to the mixture, as we expect the patients’ own phagocytic cells and NK cells, present in the PBMCs, to be able to kill the targeted anti-Dsg3+ pathogenic B cells.
• an ELISpot assay will be performed to evaluate for antigen-specific and total IgG B-cell depletion.
• Results will reveal percentage decrease of the autoreactive anti-Dsg3 IgGs compared to total IgG level (which is expected to be unaffected; therefore, used as control). Analysis will be performed via flow cytometry, and standard live-dead analyses, including on B and T cells, which will act as an internal standard for the assay.
• the hDsg3 protein (with no Fc) is already in hand.
• hDsg3 protein (EC 1-3) following a similar protocol as we used to make the described proteins (standard cloning, following expression, purification, and site-specific labeling via sortase to install Alexa647). This will allow gating on anti-Dsg3+ B cells present in the PV patients’ PBMCs. Flow cytometry analysis before and after the assay, which includes staining with Alexa647-labeled Dsg3 protein, will reveal how effectively anti-Dsg3 cells were targeted and killed. As a second control, healthy PBMCs will be used with the same settings to further confirm the observed result (here, we expect no change to occur).
• PBMCs will be collected from patients meeting the following inclusion/exclusion criteria: Inclusion criteria: 1) biopsy proven PV; 2) circulating anti-Dsg3 IgG autoantibodies by standard ELISA; 3) normal B lymphocyte counts demonstrated by CD 19 and CD20 at the time of PBMC collection. Exclusion criteria: 1) Patients with known blood-born malignancies including B and T cell lymphomas/leukemias. Treatment in the 12-months preceding and at the time of PBMC collection will be documented including any treatment that may affect PBMC level or function. Without wishing to be bound by theory, pathogenic B cells in patients’ PBMCs can be efficiently and specifically killed, without affecting the normal B cells.
• the protein expression yield is low for a particular construct, we can explore different routes to address the issue, including, but not limited to: 1) re-optimize the nucleotide codons used for the expression, 2) use a different signal peptide sequence, and/or 3) use other systems for protein expression instead of HEK293 cells, such as CHO, Expi293F or insect cells.
• antibodies can bind to different epitopes on Dsg3 protein; many antibodies will therefore have non-overlapping epitopes.
• the complex (antibody bound to Dsg3-Fc) can still bind to anti-Dsg3 BCRs (on the surface of pathogenic B cells) with nonoverlapping epitopes and, as such, bridge the effector to the target cells, help to form the cluster between the two cells, and thus mediate the killing of the target cells.
• Dsg3-Fc construct reaching to all pathogenic clones of anti-Dsg3 BCRs remains statistically high.
• the dosing of these constructs can be patient-dependent, potentially based on the level of circulating anti-Dsg3 titer, similar to, but not directly analogous to, drugs like omalizumab (an IgGlk antihuman IgE, used based on the IgE level).
• the therapeutic can be used concurrently with other drugs that lower anti-Dsg3 titer such as IVIG 72-76 .
• BCMA B-cell maturation antigen
• Dsg3-Fc fused with IL-2 which can engage more cells and enhance the killing. If this increases the efficacy, we can explore other cytokines (e.g., IL-15) as well.
• cytokines e.g., IL-15
• Dsgl and Dsg3 are expressed throughout the squamous layer of mucosa, however, Dsg3 is expressed at a higher level than Dsgl 1,3,23 .
• Dsgl is expressed throughout the epidermis, it is more intensely present in the superficial layers.
• Dsg3 is more intensely expressed in the lower portion of the epidermis, including in the basal and parabasal layers and on a lesser extent in the superficial layers.
• Dsg3 behaves as the major pathogenic antigen in patients with the mucosal-dominant type of pemphigus vulgaris, who develop deep erosions in their mucous with no or minimal involvement of the skin (as Dsgl can compensate for it in the skin); all of the mucosal-dominant PV patients have high levels of anti-Dsg3 IgG autoantibodies 3,23 ; ii) patients with high-levels of anti-Dsg3 and anti-Dsgl IgG autoantibodies develop mucocutaneous type of pemphigus vulgaris and show deep erosions in mucous and skin; and iii) patients with pemphigus foliaceus (PF) have only high-levels of anti- Dsgl autoreactive antibodies and only show superficial skin blisters with no mucosal involvement, suggesting the Dsgl being the major pathogenic autoantigen in PF.
• PF pemphigus foliaceus
• Dsg3-Fc fusion proteins which can be used to treat mucosal -dominant type of PV.
• patients with mucocutaneous PV can be treated with a mix of Dsg3-Fc and Dsgl-Fc, and patients with PF can be treated with Dsgl-Fc.
• Dsg3-Dsgl-Fc knobs-into-holes structure 82 (Dsg3-knobs + Dsgl-holes), which allows heterodimerization of the CH3 in the Fc part, and thus can target both Dsgl and Dsg3 autoreactive pathogenic B cells.
• the Dsg3-anti-CD3e construct will, without wishing to be bound by theory, establish the molecular clustering and formation of the immunological synapses between the target anti-Dsg3 B cells and the T cells, which will consequently result in the activation of T cells 52,53 and to specifically kill the targeted anti-Dsg3 B cells, leaving other B cells intact 52,53,86-88 .
• cytokine release syndrome is not expected in treatment of autoimmune diseases, given the burden of autoreactive B cells is much lower than for malignant clones.
• the N-to-C Dsg3-anti-CD3e fusion plasmid was designed to contain cDNA for the hDsg3-ECl-3 and the OKT3 anti-CD3e scFv.
• OKT3 clone has been used in the clinic as the anti-CD3e portion of the bi-specific T cell engagers 89 , and as such we chose to use this clone; of note, CD3e is found on all mature T lymphocytes.
• the construct was designed to have the Dsg3 on the N-terminus and anti-CD3e scFv on the C-terminus, similar to other bispecific T cell engagers; furthermore, this structure will ensure that the Dsg3-ECl and EC2 domains, that are most pathogenic, will be available for recognition by the Dsg3-reactive B-cells.
• a flexible (G4S)3 spacer/linker was included between Dsg3 and scFv proteins, similar to other bispecific T cell engagers.
• the protein was expressed using the HEK293 cells, purified via Ni-NTA beads, and size-exclusion chromatography, and further characterized by SDS-PAGE, anti-FLAG western blot, and flow cytometric analysis.
• a Dsg3-Fc construct can mediate targeted depletion of the circulating anti-Dsg3 + autoreactive B cells.
• the presence of autoantibodies does not affect the targeted depletion.
• NSG mice lack mature T cells, B cells, and NK cells, show reduced dendritic cell function, and defective macrophage activity, and as such are widely used for the engraftment of a wide range of human cells for preclinical studies. Accordingly, the NSG mice were intravenously injected via tail vein (i.v.) with luciferase-positive PVB28 cells (IxlO 6 ) on day 0.
• mice were pretreated with 16 mg/kg IVIG (intraperitoneal (i.p.) injection) on days -3, -2 and -1 prior to PBV28 cell administration, to block nonspecific killing via FcyR (i.e., to enhance engraftment of the PVB28 cells).
• mDsg3-mFc treatment was administered to the treatment group, injected i.p. 3 times a week for two weeks and then one per week, starting on day 4.
• the control group received an isotype IgG2a.
• the autoantibodies group received a mix of AK23, AK19 and AK18 (150 pg; anti-Dsg antibodies from hybridomas) twice per week for two weeks, starting a day prior to initiation of treatment (i.e., on day 3) all mice (treatment and control). Disease progression was monitored by bioluminescence imaging (BLI).
• FIG. 20 panel A shows BLI imaging showing a slower signal enhancement in treatment-recipient mice, even in the presence of antibodies.
• a purpose of this experiment is to delineate the effect of the presence of pathogenic anti-Dsg3 autoantibodies in the circulation on the efficacy of the treatment.
• the read-out will be the BLI analyses and survival, same as above.
• polyclonal pathogenic B cells mixture of F779 and PVB28
• polyclonal anti-Dsg3 autoantibodies mixture of AK23, P1F5, P3F3, P5G6, and P5D4 antibodies; 200 pg total antibody per injection, 40 pg of each. This can show that the treatment will remain effective even in the presence of polyclonal autoantibodies and polyclonal pathogenic Dsg3-reactive B cells.
• mice can exhibit hair loss, weight loss, and have blistering in hair follicles and oral mucosa along with autoantibodies binding to epidermal keratinocytes (FIG. 21 panels A-D) 91,48 .
• BLI imaging can be used to monitor the killing efficacy of the treatment, similar to above.
• Two sets of control can be used: mice that will not receive the treatment (negative control), and mice that will receive the treatment but not the autoantibodies (positive control; we have already showed treatment results in near full response in this setting).
• the passive disease model has advantages; however, treatment should overcome the fast-proliferating malignant cells, which may not be the case in PV patients. Therefore, as a second model, we can use an active PV immune model that closely mimics the clinical phenotype of human disease, has lower frequency of autoreactive Dsg3-specific B cells that resembles what happens clinically, and finally has a high serum polyclonal anti-Dsg3 autoantibody level.
• Serum anti-Dsg3 titers measured at days 5 and 14 post-initiation of treatments can be performed to further assess the efficacy of the approach.
• B cell counts can be performed as well to ensure they remain within normal levels during the treatment. Without wishing to be bound by theory, the treatments can result in clinical and histological resolution of the disease, the intended outcome.
• Dsg3-recognizing autoreactive B cells the target of our therapeutic
• Dsg3-Fc inherently target our therapeutics
• anti-Dsg3 levels can be measured by ELISA once per week to test long-lasting remission. Mice can be tested by a blinded investigator to check for skin lesions to ensure no disease relapse.
• the Fc-mediated approach can show relapse, as with rituximab, but without wishing to be bound by theory, they can respond with retreatment.
• some studies indicate that majority of patients treated with multiple cycles of rituximab over several years show long-term remission 100 .
• rituximab depletes the patients’ whole B cell pool, and repeated infusion can result in delayed repopulation of B cells, it carries risk of infection and would obliterate the humoral response to new infections or vaccinations.
• the targeted nature of the Dsg3-Fc approach developed here allows it to be used as a continuous treatment in multiple cycles without the fear of any major side-effects.
• mice with active PV model 2
• Dsg3-Fc can be infected with active LCMV-C113 (2* 10 6 PFU, i.v.).
• C57BL/6 will be used as a positive control.
• Viral load in liver and blood of infected mice can be compared 30 days post-injection using plaque assay 107-109 .
• mice can be challenged orally with Listeria monocytogenes (10 4 — 10 5 CFU) or intraperitoneally with LCMV Armstrong (2* 10 5 PFU) 110-112 .
• Microbial load in liver and spleen can be checked 10 days post-infection and compared with infection-only mice. In both experiments, without wishing to be bound by theory, there will not be any difference in the viral load.
• the BCR-T cell engager approach (Dsg3-fusion to anti-CD3) can be an alternative approach, as discussed herein.
• Dsg3-fusion to anti-CD3 can be an alternative approach, as discussed herein.
• PMCID PMC5932349 [00394] 8. Mascaro JM, Espana A, Liu Z, Ding X, Swartz SJ, Fairley JA, Diaz LA.
• PD-L1 is an activation-independent marker of brown adipocytes. Nat Commun.
• PMCID PMC6587433 [00458] 72. Amagai M, Ikeda S, Shimizu H, lizuka H, Hanada K, Aiba S, Kaneko F,
• EXAMPLE 8 Animal models of pemphigus disease in mice are known (discussed herein). In experiments, the therapeutics disclosed herein have been administered to these mice to determine their effects on disease in the mice (e.g., Example 7). The data from these experiments show that the therapeutics are effective. Additional studies like this are described in this example.
• FIG. 22 Panels A-D demonstrate efficacy, as well as that treatment does not result in a cytokine storm nor toxicity, in immunocompetent mice, even in presence of autoantibodies.
• the schedule for injecting mice with treatment (mDsg3-EC14-mFc) and/or the mix of autoantibodies (AK23, AK19, AK18) is shown in Panel A.
• Panel D shows ELISA analyses that demonstrated no cytokine storm in any of the three groups that received either the treatment alone, autoantibodies, or both autoantibodies and the treatment (using ELISA MAXTM Standard Set Mouse (BioLegend)).
• Sera from 6 mice that did not receive anything were used for healthy controls in each gender.
• Samples from 3 mice that received 2mg/kg of lipopolysaccharide (LPS) were used to mimic a cytokine storm in each gender.
• Unpaired t-test was used to test for significant differences between groups, (ns, P> 0.05; *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001).
• FIG. 23 Panels A-E shows that the treatment (mDsg3-ECl-4-mFc) inhibits pathogenic effects of AK23 antibody. All animals received AK23 (12mg/kg; subcutaneous); the AK23+treatment cohort received mDsg3-mFc Ih later (15mg/kg, i.p). Panels A and B show the AK23 group demonstrated significant hair-loss by tape-stripping performed 72h later (Panel A), and showed severe PV phenotype (i.e.

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