WO2026030596A1 - Protéines de fusion analogiques env de herv-k (hml-2) pour immunothérapie spécifique à l'antigène et procédés d'utilisation - Google Patents

Protéines de fusion analogiques env de herv-k (hml-2) pour immunothérapie spécifique à l'antigène et procédés d'utilisation

Info

Publication number
WO2026030596A1
WO2026030596A1 PCT/US2025/040127 US2025040127W WO2026030596A1 WO 2026030596 A1 WO2026030596 A1 WO 2026030596A1 US 2025040127 W US2025040127 W US 2025040127W WO 2026030596 A1 WO2026030596 A1 WO 2026030596A1
Authority
WO
WIPO (PCT)
Prior art keywords
herv
hml
env
seq
fusion protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/040127
Other languages
English (en)
Inventor
Marta GARCIA MONTOJO
Ajay Verma
Thomas M. Lancaster
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Twilight Bioscience Inc
Original Assignee
Twilight Bioscience Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Twilight Bioscience Inc filed Critical Twilight Bioscience Inc
Publication of WO2026030596A1 publication Critical patent/WO2026030596A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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

Definitions

  • HERV-K HML-22 envelope (Env) proteins or analogs thereof linked to Fc fragments and their use in the management of HERV-K (HML-2) associated cancers and neurological disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • Fc Fusion Proteins Fc Fusion Proteins
  • Fc fusion proteins are comprised of a species-specific immunoglobin Fc domain that is linked to another peptide such as a protein or peptide with therapeutic potential.
  • fusion protein and “Fc fusion protein” generally mean a protein comprising more than one part, for example from different sources (e.g., different proteins, polypeptides, cells, etc.), that are covalently linked through peptide bonds.
  • Fc fusion proteins are preferably covalently linked by (i) connecting the genes that encode for each part into a single nucleic acid molecule and (ii) expressing in a host cell (e.g., HEK cell or CHO cell) the protein for which TWI-004PCT/60767-PCT3 the nucleic acid molecule encodes.
  • a host cell e.g., HEK cell or CHO cell
  • the fully recombinant synthesis approach is preferred over methods in which the therapeutic protein and Fc fragments are synthesized separately and then chemically conjugated.
  • Fc fragment Fc portion
  • Fc domain Fc polypeptide
  • the Fc fragment, portion, domain, or polypeptide may be a native sequence Fc polypeptide or a variant/mutant Fc polypeptide.
  • the boundaries of the Fc portion of an immunoglobulin heavy chain may vary, they generally comprise some or all of the hinge portion of the heavy chain, the CH2 domain of the heavy chain, and the CH3 domain of the heavy chain.
  • the hinge portion of an Fc fragment comprises amino acid sequences that connect the CH1 domain of the heavy chain to the CH2 domain of the heavy chain and contain one or more cysteines that form one or more inter-heavy chain disulfide bridges to form a homodimer of an Fc fusion protein from two identical but separate monomers of the Fc fusion protein.
  • the hinge portion may comprise all or part of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence.
  • the presence of the Fc domain increases the plasma half-life due to its interaction with the neonatal Fc-receptor (FcRn) in addition to slower renal clearance of the Fc fusion protein due to the large molecule size, resulting in in vivo recycling of the molecule achieving prolonged activity of the linked peptide and improved solubility and stability of the Fc fusion protein molecule.
  • the Fc domain also enables Fc fusion proteins to interact with Fc receptors on immune cells.
  • the therapeutic protein or peptide is linked to the immunoglobin Fc domain via a linker. The therapeutic protein or peptide and linker replace the variable portion of an antibody while keeping the Fc portion intact.
  • An Fc receptor generally means a receptor that binds to an Fc fragment or to the Fc portion of an antibody.
  • the FcR is a native sequence of a mammalian FcR, and the FcR is one which binds an Fc fragment or the Fc portion of an IgG antibody (a gamma receptor) and includes without limitation, receptors of the Fc(gamma) receptor I, Fc(gamma) receptor Ia, Fc(gamma) receptor IIb, and Fc(gamma) receptor III subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • FcR also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG molecules to the fetus and is also responsible for the prolonged in vivo elimination half-lives of antibodies and Fc-fusion proteins in vivo.
  • FcR of human origin are used in vitro (e.g., in an assay) to measure the binding of Fc fusion proteins comprising Fc fragments of any mammalian origin so as to TWI-004PCT/60767-PCT3 assess their FcR binding properties.
  • HERV-K [0009] Human Endogenous Retroviruses (HERVs) are genomic sequence remnants from ancient retroviral infections that occurred during primate evolution and constitute 8% of the human genome.
  • HML-2 which is the most recently acquired HERV, still presents complete ORFs, so it can encode proteins and even form viral-like particles.
  • Expression of HERV-K (HML-2) genes is high during embryonic development but gets mostly silenced in adult tissues.
  • HML-2 aberrant gene expression from HERV-K (HML-2) is rapidly being recognized as a novel disease mechanism in several human cancers and neurological disorders including amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • HERV-K HML-2
  • Env envelope protein
  • transgenic mice expressing the HERV-K (HML-2) Env gene develop ALS-like progressive motor dysfunction and selective motor cortex volume loss. Decreased synaptic activity with dendritic spine abnormalities, nucleolar dysfunction and DNA damage are also seen in the pyramidal neurons of the HERV- K (HML-2) Env transgenic mice. Damage to spinal cord anterior horn cells occurs as well and manifests as an ALS-like atrophy of nerves and muscles.
  • HERV-K HML-2
  • TDP- 43 transactive response DNA binding protein 43
  • HML-2 HERV-K
  • HERV-K HML-2
  • MMTV mouse mammary tumor virus
  • HML-2 human mammary tumor virus
  • BCR B-cell receptor
  • IgG1 is around 65% of the total IgG.
  • IgG2 forms an important host defense against bacteria that are encapsulated.
  • IgG subclasses share more than 90% amino acid sequence homology however they have different antigen binding, immune complex formation, complement activation, effector cell triggering, and half-life. These differences result in significant functional differences.
  • IgG1 and IgG3 are strong mediators of both Fc(gamma) receptors and complement-mediated functions, and are the predominant subclasses involved in the response to protein antigens.
  • IgG2 is a weak mediator of Fc(gamma)R and complement-mediated functions and is involved in the response to polysaccharide antigens.
  • IgG4 has a minimal ability to activate effector cells or fix complement.
  • fusion proteins each comprising a respective HERV-K (HML- 2) Env analog and an Fc fragment, wherein the HERV-K (HML-2) Env analog and the Fc fragment are connected by a peptide linker.
  • the HERV-K (HML- 2) Env analog comprises a HERV-K (HML-2) Env protein comprising a functional fragment, analog, or variant/mutant thereof.
  • the HERV-K (HML-2) Env analog comprises a HERV-K (HML-2) Env analog of SEQ ID NO: 24, or a functional fragment, analog, or variant/mutant thereof.
  • SPVSGPEHPELWRLTVASHHIRIWSGNQTLETRDRKPFYT SEQ ID NO: 24.
  • the HERV-K (HML-2) Env analog comprises a HERV-K (HML-2) Env analog of SEQ ID NO: 25, or a functional fragment, analog, or variant/mutant thereof.
  • the Fc fragment comprises a sequence or functional fragment of SEQ ID NO: 1.
  • the linker comprises the sequence GGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 3).
  • the fusion protein comprises, consists essentially or even consists of a sequence of SEQ ID NO: 46.
  • the fusion protein comprises, consists essentially or even consists of a sequence of SEQ ID NO: 47.
  • the Fc fragment is glycosylated.
  • immunogenic compositions which comprise or consist essentially of a fusion protein(s) according to any embodiments or combinations of embodiments described herein and a pharmaceutically acceptable carrier.
  • the fusion protein is dispersed in the carrier.
  • the compositions further comprise an adjuvant.
  • the adjuvant is Sepivac SWE TM (Seppic, Inc. New Jersey, United States).
  • the fusion protein is emulsified with the adjuvant.
  • the emulsification is prepared onsite before administration.
  • the prepared emulsification is refrigerated (4°C) or room temperature stable for at least 8 hours, preferably up to 24 hours, preferably up to 48 hours or more.
  • the composition is an injectable formulation.
  • the composition is adapted for subcutaneous administration.
  • the composition is adapted for intramuscular administration.
  • the composition is adapted for therapeutic vaccination.
  • HML-2 HERV-K
  • the methods generally comprise administering a therapeutically effective amount of a fusion protein(s) or immunogenic composition(s) according to any embodiments or combinations of embodiments described herein to a patient.
  • the fusion protein or immunogenic composition is administered via injection.
  • the fusion protein or immunogenic composition is administered subcutaneously, intramuscularly, or intranasally.
  • the fusion protein or immunogenic composition is provided as a unit dosage form.
  • the fusion protein or immunogenic composition is co-administered with an adjuvant.
  • the methods further comprise preparing the fusion protein or immunogenic composition for administration, wherein the TWI-004PCT/60767-PCT3 preparation comprises pre-mixing the fusion protein or immunogenic composition with an adjuvant before administration.
  • pre-mixing comprises emulsifying the adjuvant and fusion protein to yield an emulsion and administering the emulsion to the patient.
  • the adjuvant is Sepivac SWE TM (Seppic, Inc. New Jersey, United States).
  • the methods generally comprise transiently transfecting a nucleic acid encoding for the fusion protein into a Chinese Hamster Ovary (CHO) cell, wherein the transfected CHO cell expresses the fusion protein.
  • the fusion protein is secreted by the cells into cell culture media, further comprising purifying or isolating the fusion protein from the media.
  • the yield of the purified or isolated fusion protein is greater than 50 mg/L.
  • Also described herein are cells engineered to express a fusion protein according to any embodiments or combinations of embodiments described herein.
  • the cell is a CHO cell.
  • the fusion protein(s) or immunogenic composition(s) according to any embodiments or combinations of embodiments described herein can be used in therapy and/or as a medicament.
  • the fusion protein(s) or immunogenic composition(s) according to any embodiments or combinations of embodiments described herein can be used for increasing antibody production in a patient.
  • the fusion protein(s) or immunogenic composition(s) according to any embodiments or combinations of embodiments described herein can be used in treatment of HERV-K-associated cancers and neurological disorders including amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • the fusion protein(s) or immunogenic composition(s) can be used as a prophylactic, therapeutic and/or booster vaccine.
  • Particular embodiments concern the fusion protein consisting of SEQ ID NO: 46, the fusion protein consisting of SEQ ID NO: 47, or pharmaceutical compositions of one of these two fusion proteins for use in treatment of HERV-K-associated cancers and neurological disorders including amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • FIG. 1 shows a schematic representation of an HERV-K (HML-2) Env analog-Fc fusion protein homodimer.
  • the glycosylation site shown is the conserved natural glycosylation site on the Fc fragment (glycosylation that may occur on the HERV-K (HML-2) Env analog is not shown).
  • FIG. 2 is a schematic diagram depicting the organization of the HERV-K (HML-2) provirus.
  • FIG.3A, FIG.3B, FIG.3C, and FIG.3D are a Clustal Omega amino acid sequence comparison showing the alignments of the full-length signal peptide domain (SEQ ID NO: 4), full-length extracellular domain (SEQ ID NO: 5), full-length transmembrane domain (SEQ ID NO: 6), and full-length cytoplasmic domain (SEQ ID NO: 7) of the HERV-K (HML-2) Env consensus sequence with the full-length HERV-K (HML-2) Env consensus sequence (SEQ ID NO: 8).
  • FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, and FIG. 4F show a Clustal Omega amino acid sequence comparison of the full-length HERV-K (HML-2) Env consensus sequence (SEQ ID NO: 8), the 6q14.1 K109 full-length HERV-K Env sequence (SEQ ID NO: 9), the 7p22.1 K109 ERK6 full-length HERV-K Env sequence (SEQ ID NO: 10), the 8p23.1a K115 full-length HERV-K Env sequence (SEQ ID NO: 11), the 11q22.1 K118 full-length HERV-K Env sequence (SEQ ID NO: 12), the 12q14.1 K119 full-length HERV-K Env sequence (SEQ ID NO: 13), the 19p12b K113 full-length HERV-K Env sequence (SEQ ID NO: 14), the 19q11 full-length HERV-K Env sequence (SEQ ID NO: 15), and the 1q23.3 full
  • FIG. 6B are a Clustal Omega amino acid sequence comparison that shows the alignment of the 1q23.3 full-length HERV-K Env sequence (SEQ ID NO: 16) with the HERV-K (HML-2) Env analog of SEQ ID NO: 18.
  • FIG. 7A, FIG. 7B, and FIG. 7C are a Clustal Omega amino acid sequence TWI-004PCT/60767-PCT3 comparison showing the alignment of the full-length HERV-K (HML-2) Env consensus sequence (SEQ ID NO: 8) and the G01 epitope (SEQ ID NO: 17).
  • FIG. 7A, FIG. 7B, and FIG. 7C are a Clustal Omega amino acid sequence TWI-004PCT/60767-PCT3 comparison showing the alignment of the full-length HERV-K (HML-2) Env consensus sequence (SEQ ID NO: 8) and the G01 epitope (SEQ ID NO: 17).
  • FIG. 8 is a Clustal Omega amino acid sequence comparison showing the alignment of a HERV-K (HML-2) Env analog (SEQ ID NO: 18) and its corresponding predicted epitope for binding to B-cell receptors (SEQ ID NO: 26).
  • FIG. 9 is a Clustal Omega amino acid sequence comparison showing the alignment of a HERV-K (HML-2) Env analog (SEQ ID NO: 20) and its corresponding predicted epitope for binding to B-cell receptors (SEQ ID NO: 27).
  • FIG.10 is a Clustal Omega amino acid sequence comparison showing the alignment of a HERV-K (HML-2) Env analog (SEQ ID NO: 21) and its corresponding predicted epitopes for binding to B-cell receptors (SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30).
  • FIG.11 is a Clustal Omega amino acid sequence comparison showing the alignment of a HERV-K (HML-2) Env analog (SEQ ID NO: 22) and its corresponding predicted epitope for binding to B-cell receptors (SEQ ID NO: 31).
  • FIG.12 is a Clustal Omega amino acid sequence comparison showing the alignment of a HERV-K (HML-2) Env analog (SEQ ID NO: 23) and its corresponding predicted epitope for binding to B-cell receptors (SEQ ID NO: 32).
  • FIG.13 is a Clustal Omega amino acid sequence comparison showing the alignment of a HERV-K (HML-2) Env analog (SEQ ID NO: 24) and its corresponding predicted epitopes for binding to B-cell receptors (SEQ ID NO: 33 and SEQ ID NO: 34).
  • FIG.14 is a Clustal Omega amino acid sequence comparison showing the alignment of a HERV-K (HML-2) Env analog (SEQ ID NO: 24) and its corresponding recognized epitope (SEQ ID NO: 35).
  • FIG.15 is a Clustal Omega amino acid sequence comparison showing the alignment of a HERV-K (HML-2) Env analog (SEQ ID NO: 25) and its corresponding predicted epitopes for binding to B-cell receptors (NW EPITOPE and SEQ ID NO: 37).
  • FIG.16 is a Clustal Omega amino acid sequence comparison showing the alignment of a HERV-K (HML-2) Env analog (SEQ ID NO: 25) and its corresponding recognized epitopes (SEQ ID NO: 38 and SEQ ID NO: 39).
  • FIG.17A and FIG.17B show a Clustal Omega amino acid sequence comparison of three HERV-K (HML-2) Env analog-Fc fusion proteins (SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42) that each use a short linker (SEQ ID NO: 2).
  • FIG.18A and FIG.18B show a Clustal Omega amino acid sequence comparison of five HERV-K (HML-2) Env analog-Fc fusion proteins (SEQ ID NO: 43, SEQ ID NO: 44, SEQ TWI-004PCT/60767-PCT3 ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47) that each use a long linker (SEQ ID NO: 3).
  • FIG. 19 is a schematic diagram depicting example modes in which an antigen may interact with an antigen presenting cell, e.g., a dendritic cell.
  • FIG.20 is a graph showing the measured total anti-HERV-K (HML-2) Env IgG titers (individual and median) measured in serum from mice treated with seven different HERV-K (HML-2) Env analog-Fc fusion proteins (SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46).
  • FIG.21 is a graph showing the measured total anti-HERV-K (HML-2) Env IgG titers (individual and median) measured in serum from mice treated with seven different HERV-K (HML-2) Env analog-Fc fusion proteins (SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46) that includes additional data points for SEQ ID NO: 46 from a second immunogenicity study.
  • FIG. 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46 that includes additional data points for SEQ ID NO: 46 from a second immunogenicity study.
  • HML-2 the measured anti-HERV-K (HML-2) Env IgG1/IgG2a ratios (individual and median) measured in serum from mice treated with seven different HERV- K (HML-2) Env analog-Fc fusion proteins (SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46).
  • FIG.23 is a graph showing the measured total anti-HERV-K (HML-2) Env IgG titers (individual and median) measured in serum from mice treated with seven different HERV-K (HML-2) Env analog-Fc fusion proteins (SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 47).
  • FIG.24 is a graph showing the measured total anti-HERV-K (HML-2) Env IgG titers (individual and median) measured in serum from mice treated with seven different HERV-K (HML-2) Env analog-Fc fusion proteins (SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 47) that includes additional data points for SEQ ID NO: 47 from a second immunogenicity study.
  • FIG. 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 47 that includes additional data points for SEQ ID NO: 47 from a second immunogenicity study.
  • FIG. 25 is a graph showing the measured anti-HERV-K (HML-2) Env IgG1/IgG2a ratios (individual and median) measured in serum from mice treated with seven different HERV- K (HML-2) Env analog-Fc fusion proteins (SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 47).
  • HML-2 the measured anti-HERV-K
  • HML-2a ratios measured in serum from mice treated with seven different HERV- K (HML-2) Env analog-Fc fusion proteins
  • HERVs Human Endogenous Retroviruses
  • HERV-K was originally identified by its homology to the mouse mammary tumor virus (MMTV) (US20090297530, “HERV-K Antigens, Antibodies, and methods, Feng Wang- Johanning.)
  • MMTV mouse mammary tumor virus
  • HERV-K (HML-2) is transcriptionally active during embryogenesis but gets mostly silenced in differentiated cells.
  • HML-2 Aberrant expression of HERV-K (HML-2) in adult tissues has been associated with many types of cancer and with neurodegenerative diseases (Garcia-Montojo M, Doucet-O'Hare T, Henderson L, Nath A. Human endogenous retrovirus-K (HML-2): a comprehensive review. Crit Rev Microbiol. 2018 Nov;44(6):715-738. doi: 10.1080/1040841X.2018.1501345. Epub 2018 Oct 14. PMID: 30318978; PMCID: PMC6342650).
  • HML- 2 Overexpression of HERV-K (HML- 2) has been detected in teratocarcinoma, glioblastoma, breast cancer, germ cell tumors, melanoma, ovarian, and prostate cancer (Lower R, Lower J, Frank H, Harzmann R, Kurth R. 1984. Human teratocarcinomas cultured in vitro produce unique retrovirus-like viruses.
  • J Gen Virol 65 (Pt 5):887–898; Shah AH, Rivas SR, Doucet-O'Hare TT, Govindarajan V, DeMarino C, Wang T, Ampie L, Zhang Y, Banasavadi-Siddegowda YK, Walbridge S, Maric D, Garcia- Montojo M, Suter RK, Lee MH, Zaghloul KA, Steiner J, Elkahloun AG, Chandar J, Seetharam D, Desgraves J, Li W, Johnson K, Ivan ME, Komotar RJ, Gilbert MR, Heiss JD, Nath A.
  • Human endogenous retrovirus K contributes to a stem cell niche in glioblastoma. J Clin Invest.2023 Jul 3;133(13):e167929. Johanning GL, Malouf GG, Zheng X, Esteva FJ, Weinstein JN, Wang- Johanning F, Su X. Expression of human endogenous retrovirus-K is strongly associated with the basal-like breast cancer phenotype. Sci Rep. 2017 Feb 6;7:41960. Herbst H, Sauter M, Mueller-Lantzsch N.1996. Expression of human endogenous retrovirus K elements in germ cell and trophoblastic tumors.
  • HERV-K HML-2
  • HML-2 HERV-K
  • HERV-K and specifically HML-2 has been associated with many cancer types such as teratocarcinoma, glioblastoma, breast cancer, germ cell tumors, melanoma, ovarian, and prostate cancer (Lower et al.1984; Herbst et al.1996; Muster et al.2003; Buscher et al.2005; Buscher et al. 2006; Wang-Johanning et al.
  • HML-2 HERV-K
  • RT reverse transcriptase
  • RNA-instructed DNA polymerase activity in a cytoplasmic particulate fraction in brains from Guamanian patients J Exp Med 142(2):483–494.; Steele AJ, Al-Chalabi A, Ferrante K, Cudkowicz ME, Brown RH Jr., Garson JA. 2005. Detection of serum reverse transcriptase activity in patients with ALS and TWI-004PCT/60767-PCT3 unaffected blood relatives. Neurology 64(3):454–458.; McCormick AL, Brown RH Jr., Cudkowicz ME, Al-Chalabi A, Garson JA.2008. Quantification of reverse transcriptase in ALS and elimination of a novel retroviral candidate.
  • mRNA from HERV-K (HML-2) genes, GAG, POL and ENV can be detected in the brains of ALS patients, and RT and ENV proteins are expressed in cortical neurons in the disease (Douville R, Liu J, Rothstein J, Nath A. 2011. Identification of active loci of a human endogenous retrovirus in neurons of patients with amyotrophic lateral sclerosis. Ann Neurol 69(1):141–151.; Li W, Lee MH, Henderson L, Tyagi R, Bachani M, Steiner J, Campanac E, Hoffman DA, von Geldern G, Johnson K et al. 2015. Human endogenous retrovirus-K contributes to motor neuron disease.
  • HML-2 Multiple active HERV-K loci have been identified, although loci in chromosome 7 seem to be differentially expressed in patients and controls (Douville R, Liu J, Rothstein J, Nath A.et al. 2011. Identification of active loci of a human endogenous retrovirus in neurons of patients with amyotrophic lateral sclerosis. Ann Neurol 69(1):141–151.). Forced expression of HERV-K (HML-2) in neurons, either by transfection with the complete consensus sequence or by activation of the endogenous proviruses with a CRISPR/dCAS9 targeting HERV-K (HML-2) LTRs, lead to neuronal injury and cell death.
  • HML-2 HERV-K
  • HML-2 Low levels of antibodies to HERV-K (HML-2) Env in ALS are associated with poor prognosis and decreased survival probability, suggesting a protective role of the antibodies against the disease pathogenesis (Garcia-Montojo, M., Simula, E.R., Fathi, S., McMahan, C., Ghosal, A., Berry, J.D., Cudkowicz, M., Elkahloun, A., Johnson, K., Norato, G., Jensen, P., James, T., Sechi, L.A. and Nath, A. (2022), Antibody Response to HML-2 May Be Protective in Amyotrophic Lateral Sclerosis. Ann Neurol, 92: 782-792).
  • HERV-K provirus There are multiple HERVs that are implicated in Central Nervous System (CNS) diseases.
  • CNS Central Nervous System
  • HML-2 The organization of the HERV-K (HML-2) provirus is shown in FIG.2.
  • GAG The organization of the HERV-K (HML-2) provirus is shown in FIG.2.
  • Splice donor (SD) and SA Splice acceptor sites are shown.
  • LTRs (‘Long TWI-004PCT/60767-PCT3 Terminal Repeat’ sequences found at both ends of a retrovirus) are composed of the U3 and U5 regions separated by the R segment.
  • Transcript 1 has 3 ORFs to encode proteins GAG, protease (PRO) and polymerase (POL).
  • GAG has a start codon (AUG); PRO and POL translation is mediated by 2 ribosomal frame shifts ( ⁇ 1).
  • the figure also shows the ORFs’ organization to encode the different final proteins and domains: matrix (MA), capsid (CA) and nucleocapsid (NC) in GAG, dUTPase in GAG-PRO junction and RT, Rnase H and integrase (IN) in POL).
  • the functional proteins will be formed by proteolysis of the polyproteins GAG, GAG-PRO and GAG-PRO-POL.
  • Transcript 2 encodes the protein ENV, which has 3 different domains: the signal peptide (SP), surface (SU) and transmembrane (TM).
  • Transcript 3 is the product of alternative splicing of the ENV ORF and encodes Rec.
  • This transcript is only produced by type 2 HERV-K (HML-2) proviruses, which do not have the 292 nts deletion. Rec has 87 amino acids in common with ENV, corresponding to its first exon. The second exon starts in a different frame and therefore, the amino acid sequence is not shared with ENV. Transcript 4 is only produced by type 1 proviruses, which present a deletion of 292 nts in the POL-ENV junction. As a result, the SD2 site is lost, and an alternative SD (SDNP9) is used for the splicing. Due to this change only the first 14 amino-acids of NP9 are shared with ENV and Rec.
  • SDNP9 alternative SD
  • HML-2 HERV-K
  • Env Garcia-Montojo, M., Simula, E.R., Fathi, S., McMahan, C., Ghosal, A., Berry, J.D., Cudkowicz, M., Elkahloun, A., Johnson, K., Norato, G., Jensen, P., James, T., Sechi, L.A. and Nath, A. (2022)
  • Antibody Response to HML-2 May Be Protective in Amyotrophic Lateral Sclerosis.
  • Ann Neurol.2022 Oct;92(4):545-561) indicate that triggering or potentiating a humoral response against this neurotoxic protein might be a therapeutic approach to treat ALS. This can be achieved by developing an epitope-specific active immunotherapy against the HERV-K (HML-2) Env protein using an Fc fusion-based vaccine.
  • the vaccine is intended to create Th2 humoral immunity by boosting humoral responses to the HERV-K (HML-2) protein.
  • Antibody against HERV-K (HML-2) Env that targets a conserved region not affected by glycosylation or by native conformation may be useful in diagnostics and/or in therapy (EP3351265A1, “Anti-HERV-K envelope antibody and uses thereof”, US10981977, “Anti- HERV-K envelope protein antibodies and methods for detecting HERV-K envelope protein in patients with sporadic amyotrophic lateral sclerosis”, Geneuro & US Department of Health and Human Services).
  • the murine monoclonal antibody (mAb) named GN_mAb_Env_K01 selectively binds to the SLDKHKHKKLQSFYP (SEQ ID NO: 17) linear epitope on the surface of the HERV-K-ENV protein (also referred to as the G01 epitope)
  • SLDKHKHKKLQSFYP SEQ ID NO: 17 linear epitope on the surface of the HERV-K-ENV protein
  • Step JP Bachani M, Malik N, DeMarino C, Li W, Sampson K, Lee MH, Kowalak J, Bhaskar M, Doucet-O'Hare T, Garcia-Montojo M, Cowen M, Smith B, Reoma LB, Medina J, Brunel J, Pierquin J, Charvet B, Perron H, Nath A.
  • FIG. 7A, FIG. 7B, and FIG. 7C are a Clustal Omega amino acid sequence comparison showing the alignment of the full-length HERV-K (HML-2) Env consensus sequence (SEQ ID NO: 8) and the G01 epitope (SEQ ID NO: 17).
  • GN_mAb_Env_K01 is a full-length antibody of the IgG2b/kappa murine subclass.
  • GN_mAb_Env_01 biological activity has been confirmed by ELISA and Western Blotting immunoassays.
  • GN_MAB_Env_K01 recognized the native and the denatured HERV-K-ENV protein, as well as the non-glycosylated and the glycosylated forms while a commercial anti-HERV-K-ENV antibody failed to detect the glycosylated form.
  • TWI-004PCT/60767-PCT3 The targeted epitope appeared to be highly conserved with a stable amino acid sequence encoded by HERV-K Env genes described in the databases which makes the epitope in the GN_mAb_Env_K01 mAb a key binding site useful for targeting HERV-K envelope proteins.
  • the present disclosure is directed to methods for making and using novel HERV-K (HML-2) Env based Fc fusion proteins (HERV-K (HML-2) Env analog-Fc fusion proteins) which allow for the cost-effective production of vaccines to cause a patient to produce antibodies against HERV-K (HML-2) (e.g., a patient’s endogenously produced HERV-K (HML-2) envelope protein) with the effect of reducing the amount of HERV-K (HML-2) envelope protein present, in particular in the brain, spinal cord, and cerebrospinal fluid of the patient.
  • HML-2 HERV-K
  • HML-2 Env analog-Fc fusion proteins
  • the present disclosure is specifically directed to methods for making and using HERV-K (HML-2) Env analog-Fc fusion proteins for use as a therapeutic vaccine which is efficacious for causing patients to create anti-HERV-K (HML-2) Env antibodies to the HERV-K (HML-2) Env protein, for example to decrease the levels of HERV-K (HML-2) Env protein present, in particular in the brain, spinal cord, and cerebrospinal fluid of the patient or to prevent HERV-K (HML-2) from interacting with the CD98 receptor in order to reduce neurodegeneration symptoms associated with ALS.
  • HERV-K (HML-2) Env analog linked via a linker to an Fc fragment is administered to patients requiring treatment for the pathogenesis of amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • the novel HERV-K (HML-2) Env analog-Fc fusion protein has the effect of stimulating the patient to produce humoral immunity, for example generating increased levels of IgG antibodies compared to an untreated patient, where the antibodies bind HERV-K (HML-2) Env protein in the body.
  • HML- 2 When the immune system develops a robust antibody response to HERV-K (HML- 2), excess levels of HERV-K (HML-2) are removed and may also be blocked from binding to cell-based HERV-K (HML-2) receptors (e.g., CD98 receptor-linked interactions), thereby alleviating neurodegeneration.
  • HML-2 cell-based HERV-K receptors
  • CD98 receptor-linked interactions e.g., CD98 receptor-linked interactions
  • a therapeutic vaccine-based approach to neutralize HERV-K (HML-2) Env protein in the body to reduce levels of HERV-K (HML-2) stimulates the body to generate its own antibodies against the administered HERV-K (HML-2) Env analog-Fc fusion protein, which can then bind and neutralize the endogenously TWI-004PCT/60767-PCT3 produced HERV-K (HML-2) Env protein. Because antibodies are produced endogenously, the frequency of treatment administration is significantly reduced.
  • the immune system also may generate polyclonal antibodies in response to the vaccine, with the potential of providing a more robust response to endogenously-produced HERV-K (HML-2).
  • the articles “a” and “an” refer to one or more than one, e.g., to at least one of the grammatical objects of the article.
  • the use of the words “a” or “an” when used in conjunction with the term “comprising” herein may mean “one”, but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”.
  • the phrase “and/or”, when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed.
  • the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.
  • “about” and “approximately” generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements.
  • an amount of a molecule, compound, conjugate, or substance effective to treat a disorder generally means an amount of the molecule, compound, conjugate, or substance which is effective, upon single or multiple dose administration(s) to a patient, in treating a patient, or in curing, alleviating (e.g., alleviating associated symptoms of ALS), relieving or improving a patient with a condition or disorder (e.g., a disorder described herein) beyond that expected in the absence of such treatment.
  • analog generally means a compound or conjugate (e.g., a compound or conjugate as described herein) having a chemical structure similar to that of another compound or conjugate but differing from it in at least one aspect.
  • antigen or “antigenic agent” generally means any substance that causes a patient’s immune system to produce an immune response (e.g., antibodies and/or T-cell responses) against it.
  • An antigen may be a substance from the environment, such as chemicals, bacteria, viruses, or pollen, or an antigen may also be inside TWI-004PCT/60767-PCT3 the body.
  • the antigen is endogenously-produced (e.g., a self-antigen).
  • a self-antigen is a HERV-K (HML-2) Env protein.
  • An antigen e.g., HERV-K (HML-2) Env
  • an antigen analog e.g., HERV-K (HML-2) Env analog
  • another protein e.g., an Fc fragment
  • antibody or “antibody molecule” generally means an immunoglobulin molecule (Ig), or immunologically active portions of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen binding site that specifically binds, e.g., immunoreacts with, an antigen or a self-antigen.
  • antibody domain generally means a variable or constant region of an immunoglobulin. It is documented in the art that antibodies comprise several classes, for example IgA, IgM, or IgG in the case of mammals (e.g., human).
  • IgG immunoglobulins can be further classified into different isotypes, such as IgG1, IgG2, IgG3 and IgG4 for humans.
  • immunoglobulin isotypes of a given immunoglobulin class will comprise different amino acid sequences, structures, and functional properties from one another (e.g., different binding affinities to Fc(gamma) receptors or CD98 receptors).
  • “Specifically binds” or “immunoreacts with” means that the antibody reacts with one or more antigenic determinants of the desired antigen and has a lower affinity for other polypeptides, e.g., does not react with other polypeptides.
  • HERV-K (HML-2) Env analog generally means a protein comprising a peptide derived from or consisting of all or a portion of a HERV-K (HML-2) Env protein locus, which may have none, one, or more than one amino acid deletions, mutations, or additions.
  • a HERV-K (HML-2) Env analog may be a HERV-K (HML-2) Env protein locus with no changes or mutations, a HERV-K (HML-2) Env protein locus comprising an additional peptide sequence combined with the HERV-K (HML-2) Env protein locus, or a HERV-K (HML-2) Env protein locus with none, one, or more than one amino acid deletions, mutations, or additions from the HERV-K (HML-2) Env protein locus.
  • a HERV-K (HML-2) Env analog may comprise a portion (e.g., a fragment) or truncated section of a HERV-K (HML-2) Env protein locus, which may have none, one, or more than one amino acid deletions, mutations, or additions.
  • a HERV-K (HML-2) Env analog may comprise a portion of an artificial consensus sequence created based on two or more HERV-K (HML-2) Env protein loci, whereby the artificial consensus sequence includes common portions of the two or more HERV-K (HML-2) Env protein loci in addition to none, one, or more than one amino acid deletions, mutations, or additions.
  • the HERV-K (HML-2) Env analog may be linked to an Fc fragment or analog thereof, as illustrated TWI-004PCT/60767-PCT3 in FIG.1.
  • the term “dimer” generally means a protein or a fusion protein comprising two polypeptides linked covalently. In embodiments, two identical polypeptides are linked covalently (e.g., via disulfide bonds) forming a “homodimer” (diagrammatically represented in FIG. 1, which is an illustration of a HERV-K (HML-2) Env analog-Fc fusion protein homodimer).
  • the HERV-K (HML-2) Env analog-Fc fusion protein polypeptide is connected via a linker to an Fc fragment.
  • Disulfide bonds (the total number of disulfide bonds in actuality may be greater or less than the number shown in FIG.1) create a homodimer from two identical Fc fusion proteins.
  • the novel HERV-K (HML-2) Env analog-Fc fusion protein homodimer may be encoded by a single nucleic acid molecule, wherein the homodimer is made recombinantly inside a cell by first forming novel HERV-K (HML-2) Env analog-Fc fusion protein monomers and by then assembling two identical novel HERV-K (HML-2) Env analog-Fc fusion protein monomers into the homodimer upon further processing inside the cell.
  • multimer As used herein, the terms “multimer”, “multimeric”, or “multimeric state” generally mean non-covalent, associated forms of Fc fusion protein dimers that may be in equilibrium with Fc fusion protein dimers or may act as permanently aggregated versions of Fc fusion protein dimers (e.g., dimers of Fc fusion protein homodimers, trimers of Fc fusion protein homodimers, tetramers of Fc fusion protein homodimers, or higher order aggregates containing five or more Fc fusion protein homodimers). It may be expected that multimeric forms of Fc fusion proteins may have different physical, stability, or pharmacologic activities from that of fusion protein homodimers.
  • HERV-K (HML-2) Env analog-Fc fusion protein and a “novel HERV-K (HML-2) Env analog-Fc fusion protein” (which terms may be interchangeably used) generally mean an immunoglobin Fc domain that is linked to a HERV-K (HML-2) Env analog, which is useful in generating antibodies that specifically bind to a HERV-K (HML-2) Env protein.
  • HML-2 Env encompasses protein residues consisting of fragments of one or more native HERV-K (HML-2) Env protein loci.
  • fusion protein and “Fc fusion protein” generally mean a protein comprising more than one part, for example from different sources (e.g., different proteins, polypeptides, cells, etc.), that are covalently linked through peptide bonds.
  • Immunoglobulins (Ig) or antibodies are glycoproteins produced by plasma cells, which have two light chains and two heavy chains in a light-heavy-heavy-light structure arrangement. The heavy chains differ among classes. They have one Fc region that mediates TWI-004PCT/60767-PCT3 biological functions (e.g., the binding capacity to cellular receptors) and a Fab region containing antigen-binding sites.
  • B cells are instructed by specific immunogens to differentiate into plasma cells.
  • Plasma cells are protein-making cells participating in humoral immune responses against bacteria, viruses, fungi, parasites, cellular antigens, chemicals, and synthetic substances.
  • Immunoglobulins constitute about 20% of the protein in plasma.
  • IgG is a type of immunoglobulin in humans that is synthesized mostly in the secondary immune response to pathogens.
  • the IgG antibodies are the main line of acquired defense and the main potentiator of a body’s specific humoral response to pathogens in the host extracellular fluids, including blood, lymph and saliva.
  • IgG is the only immunoglobulin that crosses the placenta as its Fc portion binds to the receptors present on the surface of the placenta.
  • IgG1, IgG2, IgG3, and IgG4 which are differentiated on the position of interchain disulfide bonds, the size of the hinge region and antigenic differences in structure of the heavy chain.
  • IgG2 itself has further subclasses, including IgG2a and IgG2b, which have non-identical molecular forms.
  • the IgG1/IgG2a ratio is calculated as: IgG1 titer / IgG2a titer.
  • a high IgG1/IgG2a ratio ( ⁇ 2) in mice typically means that the immune response will be Th2-biased. Thus, it will be characterized by a predominantly humoral immune response, promoting B cell proliferation and inducing antibody production, and it will have less cellular immune response, which is promoted by Th1 cells. Reporting of IgG1/IgG2a ratios in mice to predict the type of response is common practice.
  • the term “activity”, “biological activity”, “potency”, “bioactive potency”, or “biological potency” generally means the extent to which an Fc fusion protein binds to or activates a cell receptor and/or exerts the production or reduction of native or foreign substances.
  • in vitro activity or “receptor activity” generally means the affinity with which an Fc fusion protein binds to a cell receptor and is typically measured by the concentration of an Fc fusion protein that causes the Fc fusion protein to reach half of its maximum binding (i.e., EC50 value).
  • biosynthesis or “recombinant synthesis”, or “recombinantly made” generally means the process by which an Fc fusion protein is expressed within a host cell by transfecting the cell with a nucleic acid molecule (e.g., vector) encoding the Fc fusion protein (e.g., where the entire Fc fusion protein is encoded by a single nucleic acid molecule).
  • Exemplary host cells include mammalian cells, e.g., CHO cells or HEK293 cells.
  • the cells can be cultured using standard methods in the art and the expressed Fc fusion protein may be TWI-004PCT/60767-PCT3 harvested and purified from the cell culture using standard methods in the art.
  • the term “cell surface receptor” generally means a molecule such as a protein, generally found on the external surface of the membrane of a cell and which interacts with soluble molecules, e.g., molecules that circulate in the blood supply.
  • a cell surface receptor may include a host cell receptor or an Fc receptor which binds to an Fc fragment or the Fc region of an antibody (e.g., an Fc(gamma) receptor, for example Fc(gamma) receptor I or an Fc neonatal receptor, for example FcRn).
  • an Fc(gamma) receptor for example Fc(gamma) receptor I
  • an Fc neonatal receptor for example FcRn
  • in vitro activity or “Fc(gamma) receptor activity” or “Fc(gamma) receptor binding” or “FcRn receptor activity” or “FcRn binding” generally means the affinity with which an Fc fusion protein binds to the Fc receptor (e.g., Fc(gamma) receptor or FcRn receptor) and is typically measured by the concentration of an Fc fusion protein that causes the Fc fusion protein to reach half of its maximum binding (i.e., EC50 value) as measured on an assay (e.g., an enzyme-linked immunosorbent assay (ELISA) assay) using OD 450 nm values as measured on a microplate reader.
  • an assay e.g., an enzyme-linked immunosorbent assay (ELISA) assay
  • the term “immunogenic” or “immunogenicity” generally means the capacity for a given molecule or antigen (e.g., an Fc fusion protein of the present invention) to provoke the immune system of a target patient such that after administration of the molecule, the patient develops antibodies capable of binding to all or specific portions of the molecule (i.e., anti-drug antibodies or ADA).
  • the antibody development may be polyclonal (e.g., a mixture of antibodies capable of binding an Fc fusion protein).
  • neutralizing generally mean the capacity for antibodies developed against an Fc fusion protein (e.g., an antigen or antigen analog-Fc fusion protein) to cross-react, bind and interfere with all or a portion of the self- antigen’s biological activity in the target patient.
  • Fc fusion protein e.g., an antigen or antigen analog-Fc fusion protein
  • the immunogenicity generally means antibodies that bind to the HERV-K (HML-2) Env portion of the HERV-K (HML-2) Env analog-Fc fusion protein but then also cross-react, bind and interfere (e.g., neutralize) with the activity of endogenously produced HERV-K (HML-2) at a cell surface receptor (e.g., a HERV-K (HML-2) receptor, for example a CD98 receptor).
  • a cell surface receptor e.g., a HERV-K (HML-2) receptor, for example a CD98 receptor
  • HML-2 novel HERV-K
  • HML-2 Env analog-Fc fusion protein molecule
  • a pharmaceutical composition thereof are neutralizing when those anti- HERV-K (HML-2) Env antibodies inhibit the binding between an endogenously produced protein (e.g., native HERV-K (HML-2) Env protein) in a patient and a patient’s host cells, which TWI-004PCT/60767-PCT3 is directly related to the bioactivity of endogenously produced HERV-K (HML-2) in the patient.
  • an endogenously produced protein e.g., native HERV-K (HML-2) Env protein
  • an immunogenic composition generally means a pharmaceutical composition or mixture of substances comprising an immunogenic molecule, antigen or agent, that is suitable for administering to a patient.
  • an immunogenic composition may comprise a HERV-K (HML-2) Env analog-Fc fusion protein and a sterile aqueous solution or adjuvant or another carrier.
  • the term “monomer” generally means a protein or a fusion protein comprising a single polypeptide.
  • the “monomer” is a protein or a fusion protein, e.g., a single polypeptide, comprising a HERV-K (HML-2) Env analog polypeptide and an Fc fragment polypeptide, wherein the HERV-K (HML-2) Env fragment and Fc fragment polypeptides are joined by peptide bonds via a linker to form the single polypeptide.
  • the monomer is encoded by a single nucleic acid molecule.
  • the term “carrier” is used herein to include diluents, excipients, vehicles, and the like, in which the Fc fusion protein(s) may be dispersed, emulsified, or encapsulated for administration. Suitable carriers will be pharmaceutically acceptable.
  • pharmaceutically acceptable means not biologically or otherwise undesirable, in that it can be administered to a patient without excessive toxicity, irritation, or allergic response, and does not cause unacceptable biological effects or interact in a deleterious manner with any of the other components of the composition in which it is contained.
  • a pharmaceutically acceptable carrier would naturally be selected to minimize any degradation of the compound or other agents and to minimize any adverse side effects in the patient, as would be well known to one of skill in the art.
  • Pharmaceutically acceptable ingredients include those acceptable for veterinary use as well as human pharmaceutical use and will depend on the route of administration. Any carrier compatible with the excipient(s) and the Fc fusion protein(s) can be used. In example, an adjuvant may be considered one type or subclass of carrier.
  • “pharmacodynamics” or “PD” generally means the biological effects TWI-004PCT/60767-PCT3 of an Fc fusion protein in a patient.
  • the PK generally means the concentration of a novel HERV-K (HML-2) Env analog-Fc fusion protein in the blood or serum of a patient at a given time after the administration of the novel HERV-K (HML-2) Env analog- Fc fusion protein.
  • half-life generally means the time taken for the concentration of Fc fusion protein in the blood or serum of a patient to reach half of its original value as calculated from a first order exponential decay model for drug elimination. Fc fusion proteins with greater “half-life” values demonstrate greater duration of action in the target patient.
  • the contiguous segment of the variant nucleotide sequence may have additional nucleotides or deleted nucleotides with respect to the reference nucleotide sequence.
  • the contiguous segment of the variant amino acid TWI-004PCT/60767-PCT3 sequence may have additional amino acid residues or deleted amino acid residues with respect to the reference amino acid sequence.
  • the percent identity of an amino acid sequence is determined using the Smith-Waterman homology search algorithm using an affine 6 gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix 62.
  • the percent identity of a nucleotide sequence is determined using the Smith-Waterman homology search algorithm using a gap open penalty of 25 and a gap extension penalty of 5.
  • sequence identity can be performed using, for example, the DeCypher Hardware Accelerator from TimeLogic.
  • the term “homology” is used to compare two or more proteins by locating common structural characteristics and common spatial distribution of, for instance, beta strands, helices, and folds. Accordingly, homologous protein structures are defined by spatial analyses.
  • the term “yield” generally means the amount of a fusion protein product (e.g., an Fc fusion protein described herein) resulting from the biosynthesis (e.g., in a mammalian cell, e.g., in a HEK293 cell or CHO cell) per volume of the cell culture.
  • the amount of product may be determined at any step of the production process (e.g., before or after purification), but the yield is always stated per volume of the original cell culture.
  • the term “product yield” or “total protein yield” generally means the total amount of Fc fusion protein expressed by cells and purified via at least one affinity chromatography step (e.g., Protein A or Protein G) and includes monomers of Fc fusion protein, homodimers of Fc fusion protein, and higher-order molecular aggregates of homodimers of Fc fusion protein.
  • percent homodimer or “% homodimer” generally means the proportion of a fusion protein product (e.g., an Fc fusion protein described herein) that is the desired homodimer.
  • the term “homodimer yield” generally means the product of the % homodimer and the total protein yield after Protein A purification step reported per volume of the cell culture.
  • the terms “treat” or “treating” or “treatment” of a patient having a disease or a disorder generally means an intervention performed with the intention of mitigating or preventing symptoms (for example, by preventing motor neuron degeneration and cell death in ALS) associated with the condition or disease (for example but not limited to muscular weakness, paralysis and respiratory failure), and/or reducing the duration of symptoms associated with the condition or disease. Accordingly, “treatment” generally means both therapeutic treatment and prophylactic or preventative measures.
  • Improvement after treatment may be manifested as a decrease or elimination of such symptoms, e.g., by a decrease or elimination of symptoms associated with ALS, and/or by a decrease in the duration of such symptoms, and/or by affecting the biomarkers associated with the pathology [0103]
  • the compositions described herein are useful in treating symptoms associated with ALS.
  • Treating a patient having a condition or disease may include subjecting the patient with the condition or disease to a treatment regimen, for example the administration of a fusion protein such as a HERV-K (HML-2) Env analog-Fc fusion protein described herein, or a pharmaceutical composition of a fusion protein such as a HERV-K (HML-2) Env analog- Fc fusion protein described herein, such that a symptom or biomarker associated with the condition or disease is reduced, alleviated, relieved, altered, remedied, ameliorated, or improved.
  • a treatment regimen for example the administration of a fusion protein such as a HERV-K (HML-2) Env analog-Fc fusion protein described herein, or a pharmaceutical composition of a fusion protein such as a HERV-K (HML-2) Env analog- Fc fusion protein described herein, such that a symptom or biomarker associated with the condition or disease is reduced, alleviated, relieved, altered, remedied,
  • Treating includes administering an amount of a fusion protein such as a HERV-K (HML-2) Env analog-Fc fusion protein described herein, or a pharmaceutical composition of a fusion protein such as a HERV-K (HML-2) Env analog-Fc fusion protein described herein that is effective to reduce, alleviate, relieve, alter, remedy, ameliorate, or improve a symptom associated with the condition or disease.
  • a fusion protein such as a HERV-K (HML-2) Env analog-Fc fusion protein described herein
  • Treating includes administering an amount effective to generate antibodies to an antigen (in this case HERV-K (HML-2)) in a patient that has a condition or disease with the intention of generating antibodies targeted to HERV-K (HML-2) (the antigen) to reduce the antigen and hence the severity or duration of a symptom associated TWI-004PCT/60767-PCT3 with the disease or disorder.
  • an antigen in this case HERV-K (HML-2)
  • HML-2 the antigen
  • a “therapeutic vaccine” generally means a treatment that introduces an antigen or antigen analog into a patient that has a condition or disorder associated with a self- antigen, with the goal that the patient’s immune system will create antibodies for the antigen or antigen analog, enabling the patient’s body to reduce the level of the self-antigen and have an ameliorating effect on symptoms of the condition or disorder that are associated with the self- antigen (for example, symptoms associated with ALS).
  • a “prophylactic vaccine” or “prophylactic immunization” or “preventative vaccine” generally means the artificial establishment of specific immunity through the introduction of antigens into a patient that is not necessarily suffering with the symptoms of a condition or disorder, with the goal that the patient’s immune system will create antibodies against the antigen and thereby prevent any future suffering or increase in suffering with the symptoms of the disorder.
  • a prophylactic vaccine and a therapeutic vaccine, is that a therapeutic vaccine is typically administered to a patient that is already suffering from the symptoms that the immunity targets, and a prophylactic vaccine is typically administered to a patient in anticipation of the patient suffering from the symptoms that the immunity targets.
  • “booster vaccine” generally means an extra administration of a vaccine after the patient has previously received an initial administration of a vaccine, or after a patient has acquired antibodies (i.e., has a measurable antibody titer) through having had a previous treatment which introduced antibodies to reduce the symptoms of the condition or disease, or having had previous exposure to the antigen.
  • an additional dose of a vaccine is beneficial periodically to “boost” the immunity of a patient to an antigen, by increasing the patient’s antigen antibody titer, which in turn ameliorates the symptoms caused by the antigen.
  • an amount of a HERV-K (HML-2) Env analog-Fc fusion protein of the present disclosure or pharmaceutical composition thereof may be TWI-004PCT/60767-PCT3 considered therapeutically effective if symptoms and/or effects of the condition or disease are observably reduced in the patient after the treatment regime.
  • the therapeutically effective dosage of a HERV-K (HML-2) Env analog-Fc fusion protein may vary depending on the size and species of the patient, and/or according to the mode of administration.
  • a linker links or connects a HERV-K (HML-2) Env analog to an IgG1 Fc fragment (SEQ ID NO: 1).
  • Either a short linker (an example of which is GGGSGGGS, SEQ ID NO: 2) or a long linker (an example of which is GGGGGSGGGGSGGGGSGGGGGS, SEQ ID NO: 3) is used.
  • a cited amino acid position is referenced as the position of the amino acid counting from the beginning of the amino acid sequence itself.
  • a mutation of the first serine amino acid of this sequence would be described as a mutation of the 4 th amino acid of the sequence.
  • SEQ ID NO: 8 the first serine amino acid in SEQ ID NO: 8 were mutated to threonine, this could be referred to as an S4T mutation of SEQ ID NO: 8.
  • an “ailment” generally means a HERV-K-(HML-2)-related disease or disorder that is associated with HERV-K (HML-2).
  • HML-2 TWI-004PCT/60767-PCT3 Novel HERV-K (HML-2) Env Analog-Fc Fusion Proteins
  • HML-2 Aberrant expression and/or overexpression of HERV-K (HML-2) in adult tissues has been associated with many types of cancer (including, but not limited to, teratocarcinoma, germ cell tumors, melanoma, breast cancer, glioblastoma, ovarian cancer, and prostate cancer), various features of malignant cells (e.g., tumors), and neurodegenerative or central nervous system (CNS) diseases, especially including, but not limited to, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia.
  • cancer including, but not limited to, teratocarcinoma, germ cell tumors, melanoma, breast cancer, glioblastoma, ovarian cancer, and prostate cancer
  • various features of malignant cells e.g., tumors
  • CNS central nervous system
  • HERV-K (HML-2) envelope (Env) protein has four different domains.
  • the full- length HERV-K (HML-2) Env protein consensus sequence (Dewannieux M, Harper F, Richaud A, Letzelter C, Ribet D, Pierron G, Heidmann T. Identification of an infectious progenitor for the multiple-copy HERV-K human endogenous retroelements. Genome Res. 2006 Dec;16(12):1548-56.) is shown below as SEQ ID NO: 8.
  • the full-length signal peptide domain of the consensus sequence of SEQ ID NO: 8 is shown below as SEQ ID NO: 4.
  • SEQ ID NO: 5 The full-length extracellular domain of the consensus sequence of SEQ ID NO: 8.
  • the full-length transmembrane domain of the consensus sequence of SEQ ID NO: 8 is shown below as SEQ ID NO: 6.
  • IGSTTIINLILILVCLFCLLL SEQ ID NO: 6
  • the full-length cytoplasmic domain of the consensus sequence of SEQ ID NO: 8 is shown below as SEQ ID NO: 7.
  • VCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV SEQ ID NO: 7).
  • FIG.3A, FIG.3B, FIG.3C, and FIG.3D are a Clustal Omega amino acid sequence comparison showing the alignments of the full-length signal peptide domain (SEQ ID NO: 4), full-length extracellular domain (SEQ ID NO: 5), full-length transmembrane domain (SEQ ID NO: 6), and full-length cytoplasmic domain (SEQ ID NO: 7) of the HERV-K (HML-2) Env consensus sequence with the full-length HERV-K (HML-2) Env consensus sequence (SEQ ID NO: 8).
  • SEQ ID NO: 8 is equivalent to the concatenation of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
  • HERV-K (HML-2) Env has several loci in the human genome that have open reading frames capable of producing an intact Env protein. The sequences that have been identified in practice are listed below.
  • the 6q14.1 K109 full-length HERV-K Env sequence (UniProt: Q9UKH3) is shown below as SEQ ID NO: 9.
  • the 7p22.1 (ERK6, K108, K(HML.2-HOM), K (C7)) (7p22.1a and 7p22.1b) full- length HERV-K Env sequence (UniProt: Q69384) is shown below as SEQ ID NO: 10.
  • the 8p23.1a (K27, K115) full-length HERV-K Env sequence (UniProt: Q902F8) is shown below as SEQ ID NO: 11.
  • HERV-K Env sequence (UniProt: Q902F9) is shown below as SEQ ID NO: 14.
  • FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, and FIG. 4F show a Clustal Omega amino acid sequence comparison of the full-length HERV-K (HML-2) Env consensus sequence (SEQ ID NO: 8), the 6q14.1 K109 full-length HERV-K Env sequence (SEQ ID NO: 9), the 7p22.1 K109 ERK6 full-length HERV-K Env sequence (SEQ ID NO: 10), the 8p23.1a K115 full-length HERV-K Env sequence (SEQ ID NO: 11), the 11q22.1 K118 full-length HERV-K Env sequence (SEQ ID NO: 12), the 12q14.1 K119 full-length HERV-K Env sequence (SEQ ID NO: 13), the 19p12b K113 full-length HERV-K Env sequence (SEQ ID NO: 14), the 19q11 full-length HERV-K Env sequence (SEQ ID NO: 15
  • the application WO2020006529A1 describes a combination of a different insulin analog, linker and Fc domain that yield a bioactive insulin-Fc fusion protein. Both the application WO2018107117A1 and the application WO2020006529A1 describe combinations of insulin analogs, linkers and Fc domains that have poor TWI-004PCT/60767-PCT3 manufacturability or homogeneity.
  • HERV-K (HML-2) Env analog for the novel HERV-K (HML-2) Env analog-Fc fusion protein it is conceivable that one could choose a HERV-K (HML-2) Env analog that includes some portion of the full-length HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8 or that includes some portion of a full-length HERV-K (HML-2) Env protein locus of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.
  • the HERV-K (HML-2) Env analog for a novel HERV-K (HML-2) Env analog-Fc fusion protein may comprise all or a portion of the signal peptide domain (SEQ ID NO: 4) of the full-length HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8.
  • the HERV-K (HML-2) Env analog for a novel HERV-K (HML-2) Env analog-Fc fusion protein may comprise all or a portion of the extracellular domain (SEQ ID NO: 5) of the full-length HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8.
  • the HERV-K (HML-2) Env analog for a novel HERV- K (HML-2) Env analog-Fc fusion protein may comprise all or a portion of the transmembrane domain (SEQ ID NO: 6) of the full-length HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8.
  • the HERV-K (HML-2) Env analog for a novel HERV-K (HML-2) Env analog-Fc fusion protein may comprise all or a portion of the cytoplasmic domain (SEQ ID NO: 7) of the full-length HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8.
  • the HERV-K (HML-2) Env analog for a novel HERV-K (HML-2) Env analog-Fc fusion protein may comprise all or a portion of the signal peptide domain (SEQ ID NO: 4) and all or a portion of the extracellular domain (SEQ ID NO: 5) of the full-length HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8.
  • the HERV-K (HML-2) Env analog may comprise additional amino acids or polypeptides, for example at the N-terminus of a HERV-K (HML-2) Env analog sequence (collectively then referred to as the HERV-K (HML-2) Env analog).
  • one or more amino acids in the HERV-K (HML-2) Env analog of the novel HERV- K (HML-2) Env analog-Fc fusion protein may be deleted or mutated from their native state.
  • HML-2 HERV-K
  • HML-2 Env analog-Fc fusion protein designs will result in different protein yields (see for example, Azadeh Beygmoradi, Ahmad Homaei, Roohullah Hemmati, Pedro Fernandes, Recombinant protein expression: Challenges in production and folding related matters, International Journal of Biological Macromolecules, Volume 233, 2023, 123407, ISSN 0141-8130, doi.org/10.1016/j.ijbiomac.2023.123407, and Massimo Stefani, Protein misfolding and aggregation: new examples in medicine and biology of the dark side of the protein world, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, Volume 1739,
  • HERV-K (HML-2) Env analog sequences in the HERV-K (HML-2) Env analog, when used to produce the HERV-K (HML-2) Env analog-Fc fusion protein are expected to result in different protein yields.
  • the resulting protein yield when the selected HERV-K (HML-2) Env analog is attached to an Fc fragment can be experimentally determined.
  • the choice of the Fc fragment that is linked to the selected HERV-K (HML-2) Env analog may impact the manufacturability of the HERV-K (HML-2) Env analog-Fc fusion protein.
  • the novel HERV-K (HML-2) Env analog-Fc fusion protein may comprise a peptide linker.
  • the therapeutic protein comprising the HERV-K (HML-2) Env analog is linked to the N-terminal side of the Fc fragment.
  • the novel HERV-K (HML-2) Env analog-Fc fusion protein comprises domains in the following orientation from N- to C-termini: (N-terminus)—antigen—peptide linker—Fc fragment—(C-terminus) (e.g., (N-terminus)— HERV-K (HML-2) Env analog—peptide linker—Fc fragment—(C-terminus)).
  • the length and composition of the linker connecting the HERV-K (HML-2) Env analog to the Fc fragment may impact the protein yield.
  • the design goal is to create a HERV-K (HML-2) Env analog-Fc fusion protein with a protein yield, after production in transiently transfected CHO cells and protein A purification, greater than 50 mg/L (e.g., greater than 75 mg/L, greater than 90 mg/L, greater than 100 mg/L).
  • HML-2 HERV-K
  • HML-2 Env analog-Fc fusion protein
  • the C-terminal lysine that is found in native IgG isotype Fc fragment amino acid sequences is known to result in the accidental production of unwanted amino acid sequence variants during manufacturing (e.g., Fc fragments containing the C-terminal lysine becoming mixed with Fc fragments where the C-terminal lysine is omitted, which can TWI-004PCT/60767-PCT3 occur during production of the desired protein within cells (Dick, LW., (2008) Biotechnol Bioeng. Aug 15;100(6) pp1132-43). Therefore, it was expected that removing the C-terminal lysine would improve manufacturing yield and purity.
  • FIG. 6A and FIG. 6B are a Clustal Omega amino acid sequence comparison that shows the alignment of the 1q23.3 full-length HERV-K Env sequence (SEQ ID NO: 16) with the HERV-K (HML-2) Env analog of SEQ ID NO: 18. As shown in the Clustal Omega amino acid sequence comparison of FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG.
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 18 also partially aligns with amino acids 131- 149 of the HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8 which belong to the extracellular domain of the Env protein.
  • HML-2 endogenous HERV-K
  • HML-2 Env protein
  • the HERV-K (HML-2) Env analog is from a truncated envelope protein encoded by the locus 1q23.3 (SEQ ID NO: 16, Uniprot: O42043). This was the sequence used in a previous study (Arru G et al.
  • HERV-K Modulates the Immune Response in ALS Patients. Microorganisms. 2021 Aug 23;9(8):1784.) to find immunogenic peptides and they found that this HERV-K (HML-2) Env analog elicited an increased humoral response in ALS patients compared to controls. This was confirmed in a posterior study (Garcia-Montojo et al. Antibody TWI-004PCT/60767-PCT3 Response to HML-2 May Be Protective in Amyotrophic Lateral Sclerosis. Ann Neurol.
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 18 contains a peptide DDRCPAKPEEEG (SEQ ID NO: 26) predicted to bind to B-cell receptors, as analyzed by BepiPred Linear Epitope Prediction 2.0 (Department of Health Technology (DTU) Institute, Lyngby Denmark), which is necessary to induce humoral immunity.
  • FIG.8 is a Clustal Omega amino acid sequence comparison showing the alignment of the HERV-K (HML-2) Env analog (SEQ ID NO: 18) and its corresponding predicted epitope for binding to B-cell receptors (SEQ ID NO: 26).
  • HERV-K (HML-2) Env analog of SEQ ID NO: 18 was linked via the short linker GGGSGGGS (SEQ ID NO: 2) to the IgG1 Fc fragment of SEQ ID NO: 1.
  • HERV-K (HML-2) Env analog-Fc fusion protein is given below as SEQ ID NO: 40.
  • the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 40 was manufactured in CHO cells according to Example 4 and was purified according to Example 6.
  • the Fc fusion protein structure of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 40 was confirmed according to Example 7, and sequence identification is performed according to Example 8.
  • the resultant yield of the HERV-K (HML-2) Env analog-Fc fusion protein produced was 140 mg/L.
  • Immunization studies to evaluate the effectiveness of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 40 were performed in 6- to 8-week-old BALB/c mice according to Example 11.
  • mice were administered three doses of 30 ⁇ g of the HERV-K (HML- 2) Env analog-Fc fusion protein of SEQ ID NO: 40 with and without adjuvant 4 weeks apart (on TWI-004PCT/60767-PCT3 Day 0, Day 28 and Day 56) via subcutaneous (s.c.) injection.
  • Blood was obtained from each mouse for antibody titer analysis via ELISA via submandibular venipuncture 21 to 28 days prior to the first injection and then 21 to 28 days after each injection.
  • mice antisera to the HERV-K (HML-2) Env protein and the total levels of IgG anti-HERV-K (HML-2) Env Ab titers in the mice antisera were measured according to Example 12.
  • the resultant total IgG anti-HERV-K (HML-2) Env Ab titers yield in the antisera from mice immunized with the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 40 was 1.1 ⁇ g/mL, which was unacceptably low.
  • mice antisera were measured according to Example 13, and the ratio of IgG1/IgG2a was calculated as 1.13, which was unacceptably low.
  • SEQ ID NO: 40 did meet the design goal of a HERV-K (HML-2) Env analog-Fc fusion protein with a manufacturing yield greater than 50 mg/L.
  • HERV-K (HML-2) Env analog-Fc fusion protein analog-Fc a different portion of the extracellular domain (SEQ ID NO: 5) of the full- length HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8 was selected to create the HERV-K (HML-2) Env analog of SEQ ID NO: 19. It was expected that a short HERV-K (HML- 2) Env analog (17 amino acids in length) would minimize folding which may improve binding and therefore improve immunogenicity.
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 19 is shown below.
  • APGCLMPAVQNWLVEVP SEQ ID NO: 19).
  • HERV-K (HML-2) Env analog comprising amino acids 168-184 of the HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8 belongs to the extracellular domain of the Env protein it was anticipated that endogenous HERV-K (HML-2) Env protein might be accessible to the circulating antibodies generated by the immunization with the TWI-004PCT/60767-PCT3 compound, facilitating the binding.
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 19 was linked via the short linker GGGSGGGS (SEQ ID NO: 2) to the IgG1 Fc fragment of SEQ ID NO: 1.
  • HERV-K (HML-2) Env analog-Fc fusion protein is given below as SEQ ID NO: 41.
  • the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 41 was manufactured in CHO cells according to Example 4 and was purified according to Example 6.
  • the Fc fusion protein structure of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 41 was confirmed according to Example 7, and sequence identification is performed according to Example 8.
  • the resultant yield of the HERV-K (HML-2) Env analog-Fc fusion protein produced was 81 mg/L.
  • Immunization studies to evaluate the effectiveness of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 41 were performed in 6- to 8-week-old BALB/c mice according to Example 11.
  • mice were administered three doses of 30 ⁇ g of the HERV-K (HML- 2) Env analog-Fc fusion protein of SEQ ID NO: 41 with and without adjuvant 4 weeks apart (on Day 0, Day 28 and Day 56) via subcutaneous (s.c.) injection.
  • Blood was obtained from each mouse for antibody titer analysis via ELISA via submandibular venipuncture 21 to 28 days prior to the first injection and then 21 to 28 days after each injection.
  • the binding capacity of the mice antisera to the HERV-K (HML-2) Env protein and the total levels of IgG anti-HERV-K (HML-2) Env Ab titers in the mice antisera were measured according to Example 12.
  • the resultant total IgG anti-HERV-K (HML-2) Env Ab titers yield in the antisera from mice immunized with the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 41 was 1.078 ⁇ g/mL, which was unacceptably low.
  • the IgG1 and IgG2a anti- TWI-004PCT/60767-PCT3 HERV-K (HML-2) Env Ab titers in the mice antisera were measured according to Example 13, and the ratio of IgG1/IgG2a was calculated as 1.27, which was unacceptably low.
  • SEQ ID NO: 41 did meet the design goal of a HERV-K (HML-2) Env analog-Fc fusion protein with a manufacturing yield greater than 50 mg/L. Manufacturability was good, but total IgG titers and ratio were very low indicating that the HERV-K (HML-2) Env analog selection was not successful. This may have been because it does not contain any peptide able to bind to B-cell receptors, as per analysis with BepiPred Linear Epitope Prediction 2.0 (Department of Health Technology (DTU) Institute, Lyngby Denmark).
  • DTU Department of Health Technology
  • HML-2 HERV-K
  • HML-2 HERV-K
  • HML-2 HERV-K
  • HML-2 HERV-K
  • SEQ ID NO: 5 a portion of the extracellular domain of the full-length HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8 was selected to create the HERV-K (HML- 2) Env analog of SEQ ID NO: 20. It was expected that a short HERV-K (HML-2) Env analog (18 amino acids in length) would minimize folding which may improve binding and therefore improve immunogenicity.
  • FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F are a Clustal Omega amino acid sequence comparison that includes the alignment of the full-length HERV-K (HML- 2) Env consensus sequence (SEQ ID NO: 8) with the HERV-K (HML-2) Env analog of SEQ ID NO: 20.
  • HERV-K (HML-2) Env analog comprising amino acids 141-158 of the HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8 belongs to the extracellular domain of the Env protein it was anticipated that endogenous HERV-K (HML-2) Env protein might be accessible to the circulating antibodies generated by the immunization with the compound, facilitating the binding. It contains a peptide CPAKPEEEGM (SEQ ID NO: 27) predicted to bind to B-cell receptors, as analyzed by BepiPred Linear Epitope Prediction 2.0 (Department of Health Technology (DTU) Institute, Lyngby Denmark), which is necessary to induce humoral immunity.
  • DTU Department of Health Technology
  • FIG.9 is a Clustal Omega amino acid sequence comparison showing the alignment of the HERV-K (HML-2) Env analog (SEQ ID NO: 20) and its corresponding predicted epitope for binding to B-cell receptors (SEQ ID NO: 27).
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 20 was linked via the short linker GGGSGGGS (SEQ ID NO: 2) to the IgG1 Fc fragment of SEQ ID NO: 1.
  • HERV-K (HML-2) Env analog-Fc fusion protein is given below as SEQ ID NO: 42.
  • the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 42 was manufactured in CHO cells according to Example 4 and was purified according to Example 6.
  • the Fc fusion protein structure of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 42 was confirmed according to Example 7, and sequence identification is performed according to Example 8.
  • the resultant yield of the HERV-K (HML-2) Env analog-Fc fusion protein produced was 112 mg/L.
  • Immunization studies to evaluate the effectiveness of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 42 were performed in 6- to 8-week-old BALB/c mice according to Example 11.
  • mice were administered three doses of 30 ⁇ g of the HERV-K (HML- 2) Env analog-Fc fusion protein of SEQ ID NO: 42 with and without adjuvant 4 weeks apart (on Day 0, Day 28 and Day 56) via subcutaneous (s.c.) injection.
  • Blood was obtained from each mouse for antibody titer analysis via ELISA via submandibular venipuncture 21 to 28 days prior to the first injection and then 21 to 28 days after each injection.
  • the binding capacity of the mice antisera to the HERV-K (HML-2) Env protein and the total levels of IgG anti-HERV-K (HML-2) Env Ab titers in the mice antisera were measured according to Example 12.
  • the resultant total IgG anti-HERV-K (HML-2) Env Ab titers yield in the antisera from mice immunized with the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 42 was 11.2 ⁇ g/mL, which was unacceptably low.
  • the IgG1 and IgG2a anti- HERV-K (HML-2) Env Ab titers in the mice antisera were measured according to Example 13, and the ratio of IgG1/IgG2a was calculated as 24.03, which was acceptable.
  • SEQ ID NO: 42 did meet the design goal of a HERV-K (HML-2) Env analog-Fc TWI-004PCT/60767-PCT3 fusion protein with a manufacturing yield greater than 50 mg/L. Manufacturability was good, and total IgG titers and ratio were higher than previously-evaluated sequences (SEQ ID NO: 40 and SEQ ID NO: 41) but overall, the immunogenicity was not sufficient, indicating that the HERV-K (HML-2) Env analog selection was not successful. In consequence a combination of a different linker and a different HERV-K (HML-2) Env analog from the extracellular domain was selected. A longer linker could also potentially improve immunogenicity.
  • HERV-K (HML-2) Env analog-Fc fusion protein As a fourth attempt in creating a novel HERV-K (HML-2) Env analog-Fc fusion protein, a longer portion (63 amino acids) of the full-length HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8 was selected to create the HERV-K (HML-2) Env analog of SEQ ID NO: 21. This longer HERV-K (HML-2) Env analog may potentially incorporate more epitopes which may improve binding and therefore increase immunogenicity.
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 21 is shown below.
  • FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F are a Clustal Omega amino acid sequence comparison that includes the alignment of the full-length HERV-K (HML- 2) Env consensus sequence (SEQ ID NO: 8) with the HERV-K (HML-2) Env analog of SEQ ID NO: 21.
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 21 was created by selecting a section of the signal peptide of SEQ ID NO: 4 and a section of the extracellular domain of SEQ ID NO: 5.
  • This HERV-K (HML-2) Env analog comprises amino acids 35-97 of the full-length HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8.
  • the signal peptide is highly conserved across all HERV-K Env loci.
  • the signal peptide of HERV-K Env has shown to translocate to the nucleoli (Ruggieri A, Maldener E, Sauter M, Mueller-Lantzsch N, Meese E, Fackler OT, Mayer J.
  • Human endogenous retrovirus HERV-K(HML-2) encodes a stable signal peptide with biological properties distinct from Rec. Retrovirology. 2009 Feb 16;6:17) and nucleolar dysfunction has been described in the neurons of HERV-K Env transgenic mice (Li W, Lee MH, Henderson L, Tyagi R, Bachani M, Steiner J, Campanac E, Hoffman DA, von Geldern G, Johnson K, Maric D, Morris HD, Lentz M, Pak K, Mammen A, Ostrow L, Rothstein J, Nath A. Human endogenous retrovirus-K contributes to motor neuron disease. Sci Transl Med.
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 21 contains 3 different peptides TWI-004PCT/60767-PCT3 predicted to bind to B-cell receptors, as analyzed by BepiPred Linear Epitope Prediction 2.0 (Department of Health Technology (DTU) Institute, Lyngby Denmark), which is necessary to induce humoral immunity: 1) EQMKLPSTKKAEPPTWAQLKK (SEQ ID NO: 28); 2) TQLATKYLENTKVTQT (SEQ ID NO: 29); and 3) PMPAGAA (SEQ ID NO: 30).
  • FIG.10 is a Clustal Omega amino acid sequence comparison showing the alignment of the HERV-K (HML-2) Env analog (SEQ ID NO: 21) and its corresponding predicted epitopes for binding to B-cell receptors (SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30).
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 21 was linked via the long linker GGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 3) to the IgG1 Fc fragment of SEQ ID NO: 1.
  • HERV-K (HML-2) A long linker was used in an attempt to facilitate presentation of the HERV-K (HML- 2) analog and/or modify potential enzymatic processing of the analog-Fc fusion protein within immune cells, thereby increasing immunogenicity.
  • HML-2 The resultant HERV-K (HML-2) Env analog-Fc fusion protein is given below as SEQ ID NO: 43.
  • the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 43 was manufactured in CHO cells according to Example 4 and was purified according to Example 6.
  • the Fc fusion protein structure of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 43 was confirmed according to Example 7, and sequence identification is performed according to Example 8.
  • the resultant yield of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 43 produced was 65 mg/L.
  • SEQ ID NO: 43 did meet the design goal of a HERV-K (HML-2) Env analog-Fc fusion protein with a manufacturing yield greater than 50 TWI-004PCT/60767-PCT3 mg/L.
  • Immunization studies to evaluate the effectiveness of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 43 were performed in 6- to 8-week-old BALB/c mice according to Example 11. Mice were administered three doses of 23 ⁇ g of the HERV-K (HML- 2) Env analog-Fc fusion protein of SEQ ID NO: 43 with and without adjuvant 4 weeks apart (on Day 0, Day 28 and Day 56) via subcutaneous (s.c.) injection.
  • the resultant total IgG anti-HERV-K (HML-2) Env Ab titers yield in the antisera from mice immunized with the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 43 was 5.34 ⁇ g/mL, which was unacceptably low.
  • the IgG1 and IgG2a anti- HERV-K (HML-2) Env Ab titers in the mice antisera were measured according to Example 13, and the ratio of IgG1/IgG2a was calculated as 2.60, which was unacceptably low.
  • the recombinant HERV-K Env used in the ELISA assay to detect antibodies in the mouse sera after immunization with the compounds does not include the signal peptide (which comprises amino acids 0 through 89, i.e., the recombinant HERV-K Env used in the ELISA assay comprises amino acids 90 through 632).
  • the signal peptide which comprises amino acids 0 through 89, i.e., the recombinant HERV-K Env used in the ELISA assay comprises amino acids 90 through 632.
  • HERV-K (HML-2) Env analog-Fc fusion protein As a fifth attempt in creating a novel HERV-K (HML-2) Env analog-Fc fusion protein a different portion of the extracellular domain (SEQ ID NO: 5) of the full-length HERV- K (HML-2) Env consensus sequence of SEQ ID NO: 8 was selected to create the HERV-K (HML-2) Env analog of SEQ ID NO: 22. It was expected that a short HERV-K (HML-2) Env analog would minimize folding which may improve binding and therefore improve immunogenicity.
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 22 is shown below. MVSGMSLRPRVNYLQDFSYQRS (SEQ ID NO: 22). [0179] FIG.
  • FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F are a Clustal Omega amino acid sequence comparison that includes the alignment of the full-length HERV-K (HML- 2) Env consensus sequence (SEQ ID NO: 8) with the HERV-K (HML-2) Env analog of SEQ ID NO: 22.
  • HERV-K (HML-2) Env analog comprising amino acids 196-217 of the HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8 belongs to the extracellular domain of the Env protein, it was anticipated that endogenous HERV-K (HML-2) Env protein might be accessible to the circulating antibodies generated by the immunization with HERV-K (HML-2) Env analog-Fc fusion protein using the HERV-K (HML-2) Env analog of SEQ ID NO: 22, facilitating the binding.
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 22 contains a peptide LRPRVNYLQDFSYQRS (SEQ ID NO: 31) predicted to bind to B-cell receptors, as analyzed by BepiPred Linear Epitope Prediction 2.0 (Department of Health Technology (DTU) Institute, Lyngby Denmark), which is necessary to induce humoral immunity.
  • FIG. 11 is a Clustal Omega amino acid sequence comparison showing the alignment of the HERV-K (HML- 2) Env analog (SEQ ID NO: 22) and its corresponding predicted epitope for binding to B-cell receptors (SEQ ID NO: 31).
  • HERV-K (HML-2) Env analog of SEQ ID NO: 22 was linked via the long linker GGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 3) to the IgG1 Fc fragment of SEQ ID NO: 1.
  • HML-2 HERV-K
  • HML-2 HERV-K
  • SEQ ID NO: 44 SEQ ID NO: 44.
  • MVSGMSLRPRVNYLQDFSYQRSGGGGGSGGGGGGSGGGGSGGGGGSDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG (SEQ ID NO: 44).
  • the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 44 was manufactured in CHO cells according to Example 4 and was purified according to Example 6.
  • the Fc fusion protein structure of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ TWI-004PCT/60767-PCT3 ID NO: 44 was confirmed according to Example 7, and sequence identification is performed according to Example 8.
  • the resultant yield of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 44 produced was 46 mg/L.
  • SEQ ID NO: 44 did not meet the design goal of a HERV-K (HML-2) Env analog-Fc fusion protein with a manufacturing yield greater than 50 mg/L.
  • Immunization studies to evaluate the effectiveness of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 44 were performed in 6- to 8-week-old BALB/c mice according to Example 11. Mice were administered three doses of 20 ⁇ g of the HERV-K (HML- 2) Env analog-Fc fusion protein of SEQ ID NO: 44 with and without adjuvant 4 weeks apart (on Day 0, Day 28 and Day 56) via subcutaneous (s.c.) injection.
  • the resultant total IgG anti-HERV-K (HML-2) Env Ab titers yield in the antisera from mice immunized with the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 44 was 4.673 ⁇ g/mL, which was unacceptably low.
  • the IgG1 and IgG2a anti- HERV-K (HML-2) Env Ab titers in the mice antisera were measured according to Example 13, and the ratio of IgG1/IgG2a was calculated as 2.27, which was unacceptably low. It was reasoned that a different HERV-K (HML-2) Env analog from the extracellular domain might improve the results.
  • HERV-K (HML-2) Env analog-Fc fusion protein As a sixth attempt in creating a novel HERV-K (HML-2) Env analog-Fc fusion protein, a portion of the extracellular domain (SEQ ID NO: 5) of the full-length HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8 was selected to create the HERV-K (HML- 2) Env analog of SEQ ID NO: 23. It was expected that the short HERV-K (HML-2) Env analog would minimize folding which may result in good binding and therefore good immunogenicity.
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 23 is shown below.
  • WAYVPFPPLIRAVTWMDNP SEQ ID NO: 23.
  • FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F are a Clustal Omega amino acid sequence comparison that includes the alignment of the full-length HERV-K (HML- 2) Env consensus sequence (SEQ ID NO: 8) with the HERV-K (HML-2) Env analog of SEQ ID NO: 23.
  • HERV-K (HML-2) Env analog comprising amino acids 104-122 of the HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8 belongs to the extracellular TWI-004PCT/60767-PCT3 domain of the Env protein, it was anticipated that endogenous HERV-K (HML-2) Env protein might be accessible to the circulating antibodies generated by the immunization with a HERV- K (HML-2) Env analog-Fc fusion protein using the HERV-K (HML-2) Env analog of SEQ ID NO: 23, facilitating the binding.
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 23 contains a peptide LIRAVTWMDNP (SEQ ID NO: 32) predicted to bind to B-cell receptors, as analyzed by BepiPred Linear Epitope Prediction 2.0 (Department of Health Technology (DTU) Institute, Lyngby Denmark), which is necessary to induce humoral immunity.
  • FIG.12 is a Clustal Omega amino acid sequence comparison showing the alignment of the HERV-K (HML-2) Env analog (SEQ ID NO: 23) and its corresponding predicted epitope for binding to B-cell receptors (SEQ ID NO: 32).
  • HERV-K (HML-2) Env analog of SEQ ID NO: 23 was linked via the long linker GGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 3) to the IgG1 Fc fragment of SEQ ID NO: 1.
  • HML-2 HERV-K
  • HML-2 HERV-K
  • SEQ ID NO: 45 The resultant HERV-K (HML-2) Env analog-Fc fusion protein is given below as SEQ ID NO: 45.
  • the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 45 was manufactured in CHO cells according to Example 4 and was purified according to Example 6.
  • the Fc fusion protein structure of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 45 was confirmed according to Example 7, and sequence identification is performed according to Example 8.
  • the resultant yield of the HERV-K (HML-2) Env analog-Fc fusion TWI-004PCT/60767-PCT3 protein of SEQ ID NO: 45 produced was 37 mg/L.
  • SEQ ID NO: 45 did not meet the design goal of a HERV-K (HML-2) Env analog-Fc fusion protein with a manufacturing yield greater than 50 mg/L.
  • Immunization studies to evaluate the effectiveness of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 45 were performed in 6- to 8-week-old BALB/c mice according to Example 11. Mice were administered three doses of 17 ⁇ g of the HERV-K (HML- 2) Env analog-Fc fusion protein of SEQ ID NO: 45 with and without adjuvant 4 weeks apart (on Day 0, Day 28 and Day 56) via subcutaneous (s.c.) injection.
  • the resultant total IgG anti-HERV-K (HML-2) Env Ab titers yield in the antisera from mice immunized with the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 45 was 11.893 ⁇ g/mL, which was unacceptably low.
  • the IgG1 and IgG2a anti- HERV-K (HML-2) Env Ab titers in the mice antisera were measured according to Example 13, and the ratio of IgG1/IgG2a was calculated as 28.30, which was acceptable. Immunogenicity improved slightly over SEQ ID NO: 44 but was still insufficient.
  • HML-2 HERV-K
  • HML-2 HERV-K
  • SEQ ID NO: 5 a different portion of the extracellular domain of the full-length HERV- K (HML-2) Env consensus sequence of SEQ ID NO: 8 was selected to create the HERV-K (HML-2) Env analog of SEQ ID NO: 24. It was expected that a longer HERV-K (HML-2) Env analog (40 amino acids in length) may potentially incorporate more epitopes which may improve binding and therefore increase immunogenicity.
  • FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F are a Clustal Omega amino acid sequence comparison that includes the alignment of the full-length HERV-K (HML- 2) Env consensus sequence (SEQ ID NO: 8) with the HERV-K (HML-2) Env analog of SEQ ID NO: 24.
  • HERV-K (HML-2) Env analog comprising amino acids 329-368 of the HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8 belongs to the extracellular domain of the Env protein it was anticipated that endogenous HERV-K (HML-2) Env protein TWI-004PCT/60767-PCT3 might be accessible to the circulating antibodies generated by the immunization with the compound, facilitating the binding.
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 24 contains 2 different peptides predicted to bind to B-cell receptors, as analyzed by BepiPred Linear Epitope Prediction 2.0 (Department of Health Technology (DTU) Institute, Lyngby Denmark), which is necessary to induce humoral immunity: 1) SPVSGPEHPE (SEQ ID NO: 33); and 2) IRIWSGNQTLETRDRKPFYT (SEQ ID NO: 34).
  • FIG. 13 is a Clustal Omega amino acid sequence comparison showing the alignment of the HERV-K (HML-2) Env analog (SEQ ID NO: 24) and its corresponding predicted epitopes for binding to B-cell receptors (SEQ ID NO: 33 and SEQ ID NO: 34).
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 24 was linked via the long linker GGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 3) to the IgG1 Fc fragment of SEQ ID NO: 1.
  • HERV-K (HML-2) A long linker was used in an attempt to facilitate presentation of the HERV-K (HML-2) analog and/or modify potential enzymatic processing of the analog-Fc fusion protein within immune cells, thereby increasing immunogenicity.
  • HML-2 HERV-K
  • SEQ ID NO: 46 The resultant HERV-K (HML-2) Env analog-Fc fusion protein is given below as SEQ ID NO: 46.
  • the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 46 was manufactured in CHO cells according to Example 4 and was purified according to Example 6.
  • the Fc fusion protein structure of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 46 was confirmed according to Example 7, and sequence identification is performed according to Example 8.
  • the resultant yield of the HERV-K (HML-2) Env analog-Fc fusion TWI-004PCT/60767-PCT3 protein produced was 127 mg/L.
  • mice antisera to the HERV-K (HML-2) Env protein and the total levels of IgG anti-HERV-K (HML-2) Env Ab titers in the mice antisera were measured according to Example 12.
  • the resultant total IgG anti-HERV-K (HML-2) Env Ab titers yield in the antisera from mice immunized with the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 46 was 99.5 ⁇ g/mL, which was unexpectedly high.
  • the IgG1 and IgG2a anti- HERV-K (HML-2) Env Ab titers in the mice antisera were measured according to Example 13, and the ratio of IgG1/IgG2a was calculated as 112.09, which was also unexpectedly high.
  • epitope mapping according to Example 14 showed recognition of the specific HERV- K (HML-2) Env sequence DRKPFY (SEQ ID NO: 35) with a sum of fluorescence intensities: 522391 A.U. Fluorescence intensity is the readout of the peptide array assay used for epitope mapping in Example 14 and it is indicative of the level of antibodies binding to a particular region of the protein.
  • FIG.14 is a Clustal Omega amino acid sequence comparison showing the alignment of the HERV-K (HML-2) Env analog (SEQ ID NO: 24) and its corresponding recognized epitope (SEQ ID NO: 35).
  • SEQ ID NO: 46 met the design goal of a HERV-K (HML-2) Env analog-Fc fusion protein with a manufacturing yield greater than 50 mg/L. In addition, IgG titer and ratio were high.
  • the HERV-K (HML-2) Env analog selection combined with the long linker and IgG1 in SEQ ID NO: 46 met the required target criteria for a HERV-K (HML-2) Env analog-Fc fusion protein.
  • HERV-K (HML-2) Env analog-Fc fusion protein As an eighth attempt in creating a novel HERV-K (HML-2) Env analog-Fc fusion protein, a different portion of the extracellular domain (SEQ ID NO: 5) of the full-length HERV- K (HML-2) Env consensus sequence of SEQ ID NO: 8 was selected to create the HERV-K (HML-2) Env analog of SEQ ID NO: 25. It was expected that a longer HERV-K (HML-2) Env analog (65 amino acids in length) may potentially incorporate more epitopes which may improve binding and therefore increase immunogenicity.
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 25 is shown below.
  • FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F are a Clustal Omega amino acid sequence comparison that includes the alignment of the full-length HERV-K (HML- 2) Env consensus sequence (SEQ ID NO: 8) with the HERV-K (HML-2) Env analog of SEQ ID NO: 25.
  • HERV-K (HML-2) Env analog comprising amino acids 384-448 of the HERV-K (HML-2) Env consensus sequence of SEQ ID NO: 8 belongs to the extracellular domain of the Env protein it was anticipated that endogenous HERV-K (HML-2) Env protein might be accessible to the circulating antibodies generated by the immunization with the compound, facilitating the binding.
  • the HERV-K (HML-2) Env analog of SEQ ID NO: 25 contains 2 different peptides predicted to bind to B-cell receptors, as analyzed by BepiPred Linear Epitope Prediction 2.0, which is necessary to induce humoral immunity: 1) NW (NW EPITOPE); and 2) DRPWEASP (SEQ ID NO: 37).
  • FIG. 15 is a Clustal Omega amino acid sequence comparison showing the alignment of the HERV-K (HML-2) Env analog (SEQ ID NO: 25) and its corresponding predicted epitopes for binding to B-cell receptors (NW EPITOPE and SEQ ID NO: 37).
  • HERV-K (HML-2) Env analog of SEQ ID NO: 25 was linked via the long linker GGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 3) to the IgG1 Fc fragment of SEQ ID NO: 1.
  • the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 47 was manufactured in CHO cells according to Example 4 and was purified according to Example 6.
  • the Fc fusion protein structure of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 47 was confirmed according to Example 7, and sequence identification is performed according to Example 8.
  • the resultant yield of the HERV-K (HML-2) Env analog-Fc fusion protein produced was 57 mg/L.
  • Immunization studies to evaluate the effectiveness of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 47 were performed in 6- to 8-week-old BALB/c mice according to Example 11.
  • mice were administered three doses of 30 ⁇ g of the HERV-K (HML- 2) Env analog-Fc fusion protein of SEQ ID NO: 47 with and without adjuvant 4 weeks apart (on Day 0, Day 28 and Day 56) via subcutaneous (s.c.) injection.
  • Blood was obtained from each mouse for antibody titer analysis via ELISA via submandibular venipuncture 21 to 28 days prior to the first injection and then 21 to 28 days after each injection.
  • the binding capacity of the mice antisera to the HERV-K (HML-2) Env protein and the total levels of IgG anti-HERV-K (HML-2) Env Ab titers in the mice antisera were measured according to Example 12.
  • the resultant total IgG anti-HERV-K (HML-2) Env Ab titers yield in the antisera from mice immunized with the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 47 was 373.3 ⁇ g/mL, which was very high.
  • the IgG1 and IgG2a anti-HERV-K (HML-2) Env Ab titers in the mice antisera were measured according to Example 13, and the ratio of IgG1/IgG2a was calculated as 86.99, which was also relatively high.
  • epitope mapping according to Example 14 showed recognition of the specific HERV-K (HML-2) Env sequences DRPW (SEQ ID NO: 38) and RAREGVW (SEQ ID NO: 39) with a sum of fluorescence intensities: 673320 A.U. Fluorescence intensity is the readout of the peptide array assay used for epitope mapping in Example 14 and it is indicative of the level of antibodies binding to a particular region of the protein.
  • FIG. 16 is a Clustal Omega amino acid sequence comparison showing the alignment of the HERV-K (HML-2) Env analog (SEQ ID NO: 25) and its corresponding recognized epitopes (SEQ ID NO: 38 and SEQ ID NO: 39).
  • SEQ ID NO: 47 met the design goal of a HERV-K (HML-2) Env analog-Fc fusion protein with a manufacturing yield greater than 50 mg/L. In addition, IgG titer and ratio were acceptable and the binding affinity was also significant.
  • the evaluations of SEQ ID NO: 46 and SEQ ID NO: 47 demonstrated that each of these two sequences had different relative advantages although both sequences met the design goals.
  • SEQ ID NO: 46 had a higher manufacturing yield (127 mg/L) than did SEQ ID NO: 47 (57 mg/L), and the ratio of IgG1 to IgG2a anti-HERV-K (HML-2) Env Ab titers in the mice antisera was calculated as 112.09 for SEQ ID NO: 46 and 86.99 for SEQ ID NO: 47.
  • total IgG anti-HERV-K (HML-2) Env Ab titers yield in the antisera from mice immunized with the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 47 was 373.3 ⁇ g/mL and the equivalent value for SEQ ID NO: 46 was 99.5 ⁇ g/mL
  • epitope mapping resulted in a higher fluorescence intensity for SEQ ID NO: 47 (673320 A.U.) than for SEQ ID NO: 46 (522391 A.U.)
  • FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F are a Clustal Omega amino acid sequence comparison showing the alignment of the full-length HERV-K (HML-2) Env consensus sequence (SEQ ID NO: 8) with the eight HERV-K (HML-2) Env analogs (SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 25).
  • FIG.17A and FIG.17B show a Clustal Omega amino acid sequence comparison of the three HERV-K (HML-2) Env analog-Fc fusion proteins (SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42) that use a short linker (SEQ ID NO: 2).
  • FIG. 18A and FIG.18B show a Clustal Omega amino acid sequence comparison of the five HERV- K (HML-2) Env analog-Fc fusion proteins (SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47) that use a long linker (SEQ ID NO: 3).
  • the protein yields of the HERV-K (HML-2) Env analog-Fc fusion proteins of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47 are shown below in Table 1.
  • FIG. 20 is a graph showing the measured total IgG anti-HERV-K (HML-2) Env Ab titers (individual and median) for the HERV-K (HML-2) Env analog-Fc fusion proteins of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46.
  • SEQ ID NO: 40 SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46.
  • 21 is a graph showing the measured total IgG anti-HERV-K (HML-2) Env Ab titers (individual and median) for the HERV-K (HML-2) Env analog-Fc fusion proteins of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46 that includes additional data points for SEQ ID NO: 46 from a second TWI-004PCT/60767-PCT3 immunogenicity study.
  • FIG.23 is a graph showing the measured total IgG anti-HERV-K (HML- 2) Env Ab titers (individual and median) for the HERV-K (HML-2) Env analog-Fc fusion proteins of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 47.
  • SEQ ID NO: 40 SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 47.
  • FIG. 24 is a graph showing the measured total IgG anti-HERV-K (HML-2) Env Ab titers (individual and median) for the HERV-K (HML-2) Env analog-Fc fusion proteins of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 47 that includes additional data points for SEQ ID NO: 47 from a second immunogenicity study.
  • SEQ ID NO: 46 had the maximum median anti-HERV-K (HML-2) Env IgG titer across all of the sequences in FIG.20 and FIG.21
  • SEQ ID NO: 47 had the maximum median anti-HERV- K (HML-2) Env IgG titer across all of the sequences in FIG.23 and FIG.24.
  • FIG.22 is a graph showing the measured IgG1/IgG2a ratios (individual and median) for the HERV-K (HML-2) Env analog-Fc fusion proteins of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46.
  • FIG. 40 shows the measured IgG1/IgG2a ratios (individual and median) for the HERV-K (HML-2) Env analog-Fc fusion proteins of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46.
  • HERV-K (HML-2) Env Analog-Fc Fusion Proteins for Use as a Therapeutic Vaccine A HERV-K (HML-2) Env analog-Fc fusion protein may be used as a vaccine.
  • the HERV-K (HML-2) Env analog-Fc fusion protein is provided in a pharmaceutical composition. Injection of any protein into a patient (e.g., a human) may induce an immune response, the magnitude and type of which is highly dependent on the “status” of the respective immune system. For example, injection of a foreign antigen (Ag) relative to a self Ag may induce a greater immune response in an immune system that maintains central and peripheral tolerance mechanisms.
  • Ag foreign antigen
  • an Ag-na ⁇ ve immune system has na ⁇ ve B and T lymphocytes that have a much higher threshold of activation than do the Ag-primed “memory” cells of a Ag-primed immune system, such that the antigen-presenting cells (APCs) that present Ag require much less Ag to activate primed memory T cells, and 2) due to expansion of memory T cells during the Ag priming exposure, there are inherently greater numbers of such cells upon re-exposure to an injected Ag, such as an injected self Ag or self Ag analog.
  • APCs antigen-presenting cells
  • FIG.19 is a schematic diagram depicting example modes in which an antigen may interact with an antigen presenting cell, e.g., a dendritic cell.
  • injection of a self Ag is designed to induce an immune response against a self Ag (e.g., a patient’s endogenously-produced antigen or endogenously-produced protein, e.g., endogenously-produced HERV-K (HML-2)) but needs to overcome immune system central and peripheral tolerance mechanisms.
  • the antigen is exposed to the immune system to cause an immune response (e.g., generate antibodies against the self Ag) in a manner that overcomes these tolerance mechanisms.
  • an immune response e.g., generate antibodies against the self Ag
  • this is done through the use of a carrier (e.g., an adjuvant).
  • this can be done through linking of the self Ag or a self Ag analog to an Fc fragment in order to increase the antigen presentation of the self Ag via Fc fragment-Fc(gamma)R binding as depicted in FIG. 19.
  • this is done through linking of the self Ag or self Ag analog to an Fc fragment and using a carrier (e.g., an adjuvant).
  • Generation of an immune response against a self Ag may be done through a priming approach.
  • Boosting can be done through one or more follow-on injections after a priming injection.
  • Boosting e.g., booster injections
  • Boosting may increase the level of antibodies against a self Ag, create higher affinity antibodies against a self Ag, or both increase the level and the affinity of antibodies against a self Ag.
  • Antigen presenting cells can influence both the “magnitude” and “type” of response to an Ag.
  • B cells participate in the immune response directly by humoral immunity (antibody production) and also participate in the T cell immune response as specific APCs that TWI-004PCT/60767-PCT3 selectively capture and present antigens to T cells. Both of these B cell functions are achieved through activation of the surface B cell receptor (BCR), which is essentially a membrane bound antibody that binds specifically to a particular antigen. Multivalent soluble antigens such as the Fc-fusion homodimer containing the specific antigen can be recognized by BCRs and activate them.
  • BCR surface B cell receptor
  • the HERV-K (HML-2) Env analog-Fc fusion protein homodimers can 1) activate B cells through antigen-specific BCR activation leading to an increase in antibody production, and 2) through B cell mediated APC activity, increase T cell recognition and reactivity directed specifically against the HERV-K (HML-2) Env epitopes.
  • these fusion proteins can activate either humoral immunity, cellular immunity or a combination of humoral and cellular immunity after administration.
  • Adjuvants [0228]
  • APC activation is the conceptual basis of many immune enhancing substances called adjuvants.
  • Dominant APCs are dendritic cells (DCs) and macrophages that present Ag in complex with Major Histocompatibility Complex (MHC) molecules on their surface to T cell Ag receptors.
  • Some adjuvants are designed to trick the immune system into reacting to the injected vaccine Ag as if it were part of an ongoing infection (i.e., infectious agents provide such natural viral or bacterial adjuvant substances). Therefore, adjuvants activate APCs for greater Ag-presentation capabilities necessary to overcome the high activation threshold of na ⁇ ve T cells, in addition to shaping their development into the Th1 immune system response, Th2 immune system response, or a mixture of Th1 and Th2 immune system responses.
  • DCs dendritic cells
  • MHC Major Histocompatibility Complex
  • the novel HERV-K (HML-2) Env analog-Fc fusion protein used as a vaccine may be co-administered with an adjuvant to enhance or otherwise alter the immune response in the target patient.
  • known adjuvants may be used in a pharmaceutical composition of the HERV-K (HML-2) Env analog-Fc fusion protein to enhance the induction of anti-HERV-K (HML-2) Env antibodies.
  • adjuvants examples include but are not limited to oil-in-water, amorphous aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate (Alum), Freund’s adjuvant (complete and/or incomplete), squalene, AS01B, AS02, AS03, AS04, MF59, MF59 (Novartis, Basel, Switzerland), QS-21 (SaponiQx, Massachusetts, USA), CpG 1018 (Dynavax, California, USA), Immune Stimulating Complexes TWI-004PCT/60767-PCT3 (ISCOMS), MontanideTM ISA-51, MontanideTM ISA-720, MontanideTM GEL 01 PR, MontanideTM GEL 02 PR, Sepivac SWE (Seppic, Castres, France), polylactide co-glycolide (PLG), monophosphoryl lipid A
  • AAHS amorphous aluminum hydroxyphosphate s
  • the selected adjuvant may be MF59 (Novartis, Basel, Switzerland) and AS-03 (GlaxoSmithKline).
  • a custom formulation of MF59 (Novartis) or an equivalent such as AddaVax (Invivogen) or other Advax-based vaccine adjuvants from Vaxine Pvt Ltd. (Australia) may be used in a pharmaceutical composition of the HERV-K (HML-2) Env analog-Fc fusion protein.
  • the HERV-K (HML-2) Env analog-Fc fusion protein is co-administered with the Sepivac SWE (Seppic, Castres, France) adjuvant to enhance or otherwise alter the immune response in the target patient.
  • the HERV-K (HML-2) Env analog-Fc fusion protein formulation is prepared onsite for administration.
  • the HERV-K (HML-2) Env analog-Fc fusion protein is mixed with an adjuvant onsite under sterile mixing conditions.
  • the HERV-K (HML-2) Env analog-Fc fusion protein and adjuvant are thoroughly mixed and/or emulsified to prepare a homogenous emulsion for administration to the patient.
  • the adjuvanted formulation of the HERV-K (HML-2) Env analog-Fc fusion protein or a pharmaceutical composition thereof is administered to a patient by subcutaneous (s.c.) injection or intramuscular (i.m.) injection, as the s.c. or i.m. injection sites are more likely to induce a strong antibody response due to there being more dendritic cells (DCs) in the subcutaneous and intramuscular spaces.
  • DCs dendritic cells
  • an adjuvant may be especially advantageous in older patients who experience altered immune competence with increasing age, so-called immunosenescence, which is the result of changes at multiple levels of the immune system over time.
  • immunosenescence Once a patient has measurable antibodies, upon re-challenge with HERV-K (HML-2) or a HERV-K (HML-2) Env analog-Fc fusion protein, the patient will exhibit rapid development of anti-HERV-K (HML-2) Env antibodies.
  • HERV-K (HML-2) Env Analog-Fc Fusion Protein Vaccines Evaluated in Mice The efficacy of exemplary HERV-K (HML-2) Env analog-Fc fusion proteins of this disclosure or pharmaceutical compositions thereof may be initially evaluated in mice immunization studies for their capacity to induce anti-HERV-K (HML-2) Env protein IgG titers when administered according to the procedure in Example 11.
  • BALB/c mice are a relevant animal model that has been extensively used for preclinical immunogenicity assessment of vaccines. This strain generates robust Ab responses when immunized with adjuvanted and non- adjuvanted vaccine candidates.
  • mouse-specific reagents are widely available for evaluating the kinetics and characteristics of a variety of immune responses to vaccination, including relevant Ab isotypes and T helper cell responses (e.g., Th1 vs. Th2 responses). Therefore, the BALB/c mouse model was selected to evaluate the immunogenicity of HERV-K (HML-2) Env analog-Fc fusion protein vaccines with respect to dose, potentiation by adjuvants, routes of administration, and dosing frequency required to achieve optimal Ab responses.
  • HML-2 HML-2
  • target mice e.g., BALB/c mice
  • HML-2 exemplary HERV-K
  • HML-2 Env analog-Fc fusion protein
  • Serum was collected before the first injection and then at regular intervals following the first injection (e.g., every 7-14 days beginning at Day 14).
  • anti-HERV-K (HML-2) Env IgG antibody titers were measured according to Example 12.
  • the kinetic response that is the duration of response, to dose levels varying from 1 ⁇ g to 100 ⁇ g after 1, 2, and 3 doses is expected to demonstrate increasing anti-HERV-K (HML- 2) Env protein antibody titers at all dose levels up to at least 56 days post vaccination.
  • HERV-K (HML-2) Env Analog-Fc Fusion Proteins for Use as a Booster Vaccine [0239]
  • an exemplary HERV-K (HML-2) Env analog-Fc fusion protein of this TWI-004PCT/60767-PCT3 disclosure for example the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 46 or the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 47, may be used as a booster vaccine.
  • HERV-K (HML-2) Env analog-Fc fusion protein may amplify their antibody titers and increase their neutralization against endogenously produced HERV-K (HML-2).
  • HERV-K (HML-2) Env analogs are synthesized to maximize antigenicity and overall manufacturability, while the Fc region prolongs antigen residence time and/or binding to Fc(gamma)R receptors present on APCs.
  • the naturally glycosylated Fc fragment will help bind Fc(gamma) receptors on antigen-presenting cells (APCs), which will in turn cause greater presentation of the HERV-K (HML-2) Env analog antigen to T-cells and/or B-cells, which is expected to produce a strong immune response (e.g., antibody titers) to the HERV-K (HML-2) Env antigen present on the HERV-K (HML-2) Env analog-Fc fusion protein, and that these antibody titers will be able to bind and neutralize the endogenously produced target protein (e.g., the HERV-K (HML-2) Env protein).
  • APCs antigen-presenting cells
  • the APCs can internalize the HERV-K (HML-2) Env antigen via Fc(gamma) receptors (the Fc portion of the vaccine compound will bind to the Fc receptor on the membrane of APCs) or through B-cell receptors (BCRs) (the HERV-K (HML-2) Env analog can be recognized by BCRs), and then process and present HERV-K (HML-2) Env fragments to CD4+ Th cells that in turn promote (“help”) B cell activation and anti-HERV-K (HML-2) Env IgG (i.e., Ab) production.
  • BCRs B-cell receptors
  • Antigen-presenting cells may be, for example, dendritic cells (DCs), monocytes or macrophages that can internalize the molecules of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 46 or the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 47 via Fc-receptor mediated phagocytosis or endocytosis (e.g., through the Fc region of the HERV-K (HML-2) Env analog-Fc fusion protein binding to the Fc(gamma) receptors in immune cells).
  • DCs dendritic cells
  • monocytes or macrophages that can internalize the molecules of the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 46 or the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 47 via Fc-receptor mediated phagocytos
  • the antigen After Fc-mediated uptake of the HERV-K (HML-2) Env analog-Fc fusion protein by APCs, the antigen will be processed and presented on the MHC II complex where it can be recognized by T-cell receptors (TCRs) specific for the same antigen of na ⁇ ve CD4 T- helper cells, which can get differentiated into Th2 or Th1.
  • TCRs T-cell receptors
  • B-cells that have recognized the HERV-K (HML-2) Env will also process and present the antigen on the MHC II complex, where it can be recognized by Th2 cells specific to the same antigen.
  • the TCR of the helper T cell recognizes the antigen, and the T TWI-004PCT/60767-PCT3 cell’s CD4 molecule interacts with MHC II on the B cell.
  • the coordination between B cells and helper T cells that are specific to the same antigen is referred to as linked recognition.
  • Th2 cells Once activated by linked recognition, Th2 cells produce and secrete cytokines, such as IL-4, IL-5 and IL-6, that activate the B cells and cause proliferation into clonal cells.
  • Th2 cells stimulate the differentiation of activated B cell clones into memory B cells, which will quickly respond to subsequent exposures to the same protein epitope, and plasma cells, which will secrete anti- HERV-K (HML-2) Env antibodies.
  • the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 46 or the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 47 may increase exposure of the HERV-K (HML-2) Env analog to APCs over a protracted period of time due to the presence of the Fc fragment and the Fc-FcRn receptor interactions that enable the HERV-K (HML-2) Env analog-Fc fusion protein to have a prolonged in vivo pharmacokinetic half-life.
  • the glycosylated Fc fragment in the HERV-K (HML- 2) Env analog-Fc fusion protein of SEQ ID NO: 46 or the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 47 is expected to help induce a strong immune response directed to the therapeutic or antigen portion of the fusion protein through binding of the Fc to Fc(gamma)R receptors on immune cells, thereby increasing HERV-K (HML-2) Env analog uptake and processing in a manner described in FIG. 19.
  • These properties in combination are expected to significantly increase the amount of anti-HERV-K (HML-2) Env antibodies while also decreasing the amount of antigen necessary to produce the required immune response.
  • a therapy comprising treatment of a patient with the HERV-K (HML- 2) Env analog-Fc fusion protein of SEQ ID NO: 46, the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 47, or a pharmaceutical composition thereof, may consist of a booster vaccine administered to patients that are already antibody-positive to HERV-K (e.g., patients that may already have received an initial dose of HERV-K (HML-2) Env analog-Fc fusion protein vaccine), as a means to amplify their antibody titers and affinity.
  • a therapy comprising a treatment of a patient with the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 46, the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 47, or a pharmaceutical composition of one of these two fusion proteins thereof, may be administered as a booster vaccine to patients that have been previously immunized with a vaccine against HERV-K (HML-2) as a means to amplify their antibody titers and affinity specifically against HERV-K (HML-2).
  • Such a therapy is important in cases where priming vaccines are not 100% effective and/or where the induced antibody titers wane over time.
  • the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 46, the HERV- K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 47, or a pharmaceutical composition of one of these two fusion proteins thereof may be administered to a patient by subcutaneous injection (s.c.) or intramuscularly (i.m.), as the s.c. or i.m. injection sites are more likely to induce a strong antibody response with minimal side effects.
  • Fc Fusion Protein Production [0245]
  • a fusion protein can be expressed by a cell as described in more detail in the Examples section.
  • Cells may be transfected with a nucleic acid molecule (e.g., vector) encoding the HERV-K (HML-2) Env analog-Fc fusion protein (e.g., where the entire HERV-K (HML-2) Env analog-Fc fusion protein is encoded by a single nucleic acid molecule).
  • a nucleic acid molecule e.g., vector
  • HML-2 HERV-K
  • Env analog-Fc fusion protein e.g., where the entire HERV-K (HML-2) Env analog-Fc fusion protein is encoded by a single nucleic acid molecule.
  • CHO cells may be transfected with a vector that encodes for the HERV-K (HML-2) Env analog-Fc fusion protein, but this process only results in temporary expression of the HERV-K (HML-2) Env analog-Fc fusion protein for a period of time (e.g., 3 days, 4 days, 5, days, 7 days, 10 days, 12 days, 14 days, or more) before the host cell stops expressing appreciable levels of the HERV-K (HML-2) Env analog-Fc fusion protein (i.e., transient transfection).
  • a period of time e.g., 3 days, 4 days, 5, days, 7 days, 10 days, 12 days, 14 days, or more
  • HERV-K (HML-2) Env analog-Fc fusion protein in transiently transfected CHO cells is described in Example 4, and synthesis and methods of making a HERV-K (HML-2) Env analog-Fc fusion protein in stably transfected CHO cells is described in Example 5.
  • the HERV-K (HML-2) Env analog-Fc fusion protein may be purified or isolated from the cells (e.g., by lysis of the cells).
  • the HERV-K (HML-2) Env analog-Fc fusion protein is secreted by the cells and may be purified or isolated from the cell culture media in which the cells were grown.
  • Purification of the HERV-K (HML-2) Env analog-Fc fusion protein can include using column chromatography (e.g., affinity chromatography) or using other separation methods based on differences in size, charge, and/or affinity for certain molecules.
  • Purification of the HERV-K (HML-2) Env analog-Fc fusion protein involves selecting or enriching for proteins containing an Fc fragment, e.g., by using Protein A beads or a Protein A column that cause proteins containing an Fc fragment to become bound with affinity at neutral solution pH to the Protein A covalently conjugated to the Protein A beads.
  • the purified HERV-K (HML-2) Env analog-Fc fusion protein can be characterized, e.g., for purity, protein yield, structure, and/or activity, using a variety of methods, e.g., absorbance at 280 nm (e.g., to determine protein yield), size exclusion or capillary electrophoresis (e.g., to determine the molecular weight, percent aggregation, and/or purity), mass spectrometry (MS) and/or liquid chromatography (LC-MS) (e.g., to determine purity and/or glycosylation), and/or ELISA (e.g., to determine extent of binding, e.g., affinity, to a HERV-K (HML-2) Env antibody or a cell receptor such as a CD98 cell receptor).
  • absorbance at 280 nm e.g., to determine protein yield
  • size exclusion or capillary electrophoresis e.g., to determine the molecular
  • the protein yield of a HERV-K (HML-2) Env analog-Fc fusion protein after production in transiently transfected CHO cells and protein A purification may be greater than 50 mg/L.
  • compositions and Routes of Administration The amount and concentration of the HERV-K (HML-2) Env analog-Fc fusion protein in the pharmaceutical compositions, as well as the quantity of the pharmaceutical composition administered to a patient, can be selected based on clinically relevant factors, such as medically relevant characteristics of the patient (e.g., age, weight, gender, other medical conditions, and the like), the solubility of compounds in the pharmaceutical compositions, the potency and activity of the compounds, and the manner of administration of the pharmaceutical compositions.
  • Formulations of the present disclosure include those suitable for parenteral administration.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by intravenous, intramuscular, or subcutaneous injection or by intranasal administration.
  • suitable aqueous and non-aqueous carriers include water, saline, ethanol, salts, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate, buffering agents, such as potassium and/or sodium phosphates, pH buffers, such as hydrochloric acid and/or sodium hydroxide, and the like.
  • the pharmaceutical composition (e.g., as described herein) comprises a Tween-like surfactant, e.g., Tween-80, at a concentration between about 0.001% and about 2%, or between about 0.005% and about 0.1%, or between about 0.01% and about 0.5%.
  • Tween-like surfactant e.g., Tween-80
  • the HERV-K (HML-2) Env analog-Fc fusion protein may be administered as a bolus, infusion, or an intravenous push, or administered through syringe injection, pump, pen, needle, or indwelling catheter.
  • the HERV-K (HML-2) Env analog-Fc fusion protein may be administered by a subcutaneous bolus injection.
  • the HERV-K (HML-2) Env analog-Fc fusion protein or a pharmaceutical composition thereof is administered to a patient by subcutaneous injection (s.c.) or intramuscularly (i.m.), as the s.c. or i.m. injection sites are more likely to induce a strong antibody response with minimal side effects.
  • Methods of introduction may also be provided by rechargeable or biodegradable devices.
  • Various slow- release polymeric devices have been developed and tested in vivo in recent years for the TWI-004PCT/60767-PCT3 controlled delivery of drugs, including proteinaceous biopharmaceuticals.
  • biocompatible polymers including hydrogels
  • biocompatible polymers including hydrogels
  • Additional pharmaceutically acceptable ingredients for use in the compositions include buffering agents, salts, stabilizing agents, diluents, preservatives, antibiotics, isotonic agents, and the like.
  • HERV-K (HML-2) Env analog-Fc fusion protein elicits an immune response in the patient, and more specifically an immune response against HERV-K (HML-2), relieving symptoms and conditions associated with ALS (e.g., motor neuron degeneration and cell death, muscular weakness, paralysis, and respiratory failure).
  • ALS e.g., motor neuron degeneration and cell death, muscular weakness, paralysis, and respiratory failure.
  • the immune response is demonstrated by a lack of observable clinical symptoms, or reduction of clinical symptoms or disease biomarkers normally displayed by an afflicted patient.
  • a method of activating immune cells at a site of infection or disease comprising administering a therapeutically effective amount of the HERV-K (HML-2) Env analog-Fc fusion protein to a patient.
  • a method of increasing antibody production in a patient comprising administering a therapeutically effective amount of the HERV-K (HML-2) Env analog-Fc fusion protein to a patient.
  • HML-2 HERV-K
  • Env analog-Fc fusion protein HERV-K (HML-2) Env analog-Fc fusion protein
  • Actual dosage levels of the HERV-K (HML-2) Env analog-Fc fusion protein can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular fusion protein employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular fusion protein employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a suitable dose of a HERV- K (HML-2) Env analog-Fc fusion protein will be the amount that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the immunogenic formulation is provided, in various aspects, in unit dosage form TWI-004PCT/60767-PCT3 for ease of administration and uniformity of dosage.
  • Unit dosage form as used herein generally means physically discrete units suited as unitary dosages for the patient to be treated, each unit containing a predetermined quantity of the HERV-K (HML-2) Env analog-Fc fusion protein calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • HML-2 HERV-K
  • Env analog-Fc fusion protein calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms is dictated by and are directly dependent on the unique characteristics of the excipient(s) and therapeutic agent(s) and the particular biological effect to be achieved.
  • the formulation is provided in a kit of components for administration of the HERV-K (HML-2) Env analog-Fc fusion protein to the patient.
  • a pharmaceutical composition comprising the HERV-K (HML-2) Env analog-Fc fusion protein dispersed in a suitable carrier is provided in a unit dosage form (e.g., vial).
  • the kit further comprises a discrete unit dosage form (e.g., vial) containing an adjuvant and/or other carrier system for onsite mixing of the HERV-K (HML-2) Env analog-Fc fusion protein for administration.
  • the kit comprises one or more emulsifying needles and syringes for onsite mixing of the immunogenic formulation for administration. In one or more embodiments, the kit comprises one or more dosing syringes for administering the prepared immunological composition to the patient. In one or more embodiments, the kit further comprises instructions for preparing the immunogenic composition and/or administering the immunogenic composition.
  • the present disclosure contemplates formulation of a HERV-K (HML-2) Env analog-Fc fusion protein in any of the aforementioned pharmaceutical compositions and preparations. Furthermore, the present disclosure contemplates administration via any of the foregoing routes of administration.
  • the microarray was pre-stained with the secondary antibody and the background signal was subtracted from the final measurement.
  • the peptide microarrays were incubated in washing buffer (phosphate-buffered saline (PBS) with 0.05% Tween20, pH 7.4) for 15 min at room temperature (RT) and in blocking buffer (Rockland Blocking Buffer MB-070) for 30 min at RT, followed by incubation with secondary antibody diluted 1:5000 in staining buffer (PBS with 0.05% Tween20 and 10% blocking buffer, pH 7.4) for 45 min.
  • washing buffer phosphate-buffered saline (PBS) with 0.05% Tween20, pH 7.4
  • blocking buffer Rockland Blocking Buffer MB-070
  • microarrays were washed three times in washing buffer for 1 min each and in dipping buffer (1 mM Tris buffer, pH 7.4) three times until all visible contamination was removed.
  • the microarrays were dried in a pressurized air stream from top to bottom and scanned (GenePix 4300A, Molecular Devices LLC, CA, USA).
  • Serum samples were diluted 1:200 in staining buffer and 200 ⁇ L were added to each array, which were incubated overnight at 4°C on an orbital shaker (85 rpm).
  • Peptide microarrays were washed three times for 1 min in washing buffer and stained with the secondary antibody (1:5000 in staining buffer) for 45 min.
  • the means of these percentages in ALS and control samples were calculated, generating a value for each peptide which was called epitope recognition score (ERS).
  • the ERS for all peptides was compared between ALS and controls by Mann-Whitney test.
  • the median ERS of the controls was defined as a positivity threshold: peptides above this value were considered positive in eliciting a humoral response.
  • the percentage of positive peptides was compared between ALS and controls by Fisher’s exact test. To search for the actual epitopes eliciting a differential response in ALS sera compared to controls, adjacent peptides were analyzed to find a common sequence between 4 and 12 amino acids.
  • Example 2 PEPperMAP IgG Epitope Mapping of Mouse Sera Samples against the Envelope Protein of HERV-K (HML-2)
  • HML-2 The sequence of the envelope protein of HERV-K (HML-2) (SEQ ID NO: 10, UniProtKB: Q69384) was elongated with neutral GSGSGSG linkers at the C- and N-terminus to avoid truncated peptides.
  • the elongated antigen sequence was converted into linear 15 amino acid peptides with a peptide-peptide overlap of 14 amino acids.
  • HERV-K (HML- 2) Env peptide microarrays contained 699 different peptides printed in duplicate (1,398 spots) and were framed by additional HA (YPYDVPDYAG, 90 spots) control peptides.
  • One hundred and twenty (120) mouse sera samples comprised pre- and post- treatment pairs of animals treated with a HERV-K (HML-2) Env analog-Fc fusion protein, with or without adjuvant and at doses of 1 ⁇ g, 10 ⁇ g or 30 ⁇ g.
  • the washing buffer used was PBS, pH 7.4 with 0.005% Tween 20; washing for 2 x 10 sec after each incubation step.
  • the blocking buffer used was Rockland blocking buffer MB- 070; for 30 min before first assay.
  • the incubation buffer used was washing buffer with 10% blocking buffer.
  • Assay conditions include serum dilution of 1:200 in incubation buffer; incubation for 16 h at 4°C and orbital shaking at 140 rpm.
  • a HERV-K (HML-2) Env peptide microarray was pre-stained with the secondary antibody (goat anti-mouse IgG (Fc) DyLight680 (0.2 ⁇ g/mL)) in incubation buffer for 45 min at RT to investigate background interactions with the antigen-derived peptides that could interfere with the main assays.
  • a software algorithm broke down fluorescence intensities of each spot into raw, foreground and background signals, and calculated averaged median foreground intensities and spot-to-spot deviations of spot duplicates. Based on averaged median foreground intensities, intensity maps were generated and interactions in the peptide maps highlighted by an intensity color code with red for high and white for low spot intensities. A maximum spot-to-spot deviation of 40% was tolerated, otherwise the corresponding intensity value was zeroed. This could be bypassed by manual flagging of peptides as “Artifact” or “Valid”.
  • iPSC derived (control line WC-30, male) spinal motor neurons and cortical GABAergic neurons were thawed and cultured for seven days.
  • Cells were seeded at 30K cells/well onto 96-well PDL coated plates for use in determining the toxicity of HERV-K (HML- 2) Env protein.
  • Cells were treated with different concentrations of recombinant HERV-K (HML-2) Env (MyBiosource (MBS1391552) diluted in cell culture media: 0, 20, 40, 80, 160, 320, 640 and 1280 nM.
  • HERV-K (HML-2) Env analog-Fc fusion proteins were synthesized as follows.
  • a gene sequence of interest was constructed using proprietary software (Curia, Belmont, CA) and was cloned into a high expression mammalian vector.
  • CHO cells were seeded in a shake flask 24 hours before transfection and were grown using serum-free chemically defined media.
  • a DNA expression construct that encodes the HERV-K (HML-2) Env analog-Fc fusion protein of interest was transiently transfected into a suspension of CHO cells using the (Curia, Belmont, CA) standard operating procedure for transient transfection. After 20 hours, the cells were counted to determine the viability and viable cell count, and the titer was measured by FortéBio® Octet® (Pall FortéBio LLC, Fremont, CA).
  • Example 5 Synthesis and Methods of Making a HERV-K (HML-2) Env Analog-Fc Fusion Protein by Integration of a Stable Expression Vector in CHO Cells.
  • a CHO cell line is originally derived from CHO-K1 (Curia, Belmont, CA), and the endogenous glutamine synthetase (GS) genes are knocked out by recombinant technology using methods known in the art.
  • Stable expression DNA vectors are designed and optimized for CHO expression and GS selection and are incorporated into a high expression mammalian vector (Curia, Belmont, CA).
  • the sequence of each completed construct is confirmed prior to initiating TWI-004PCT/60767-PCT3 scale up experiments.
  • the suspension-adapted CHO cells are cultured in a humidified 5% CO2 incubator at 37°C in a chemically defined media (CD OptiCHO; Invitrogen, Carlsbad, CA). No serum or other animal-derived products are used in culturing the CHO cells.
  • the transfected cells are seeded into CD OptiCHO selection media containing between 0-100 ⁇ M methionine sulfoximine (MSX) at a cell density of 0.5 ⁇ 10 6 cells/mL in a shaker flask and are incubated at 37oC with 5% CO2. During the selection process, the cells are spun down and resuspended in fresh selection media every 2-3 days until the CHO stable pool recovers its growth rate and viability. The cell culture is monitored for growth and titer. [0276] The cells are grown to 2.5 ⁇ 10 6 cells per mL. At the time of harvest for cell banking, the viability is expected to be above 95%.
  • MSX methionine sulfoximine
  • the cells are then centrifuged, and the cell pellet is resuspended in the CD OptiCHO media with 7.5% dimethyl sulfoxide (DMSO) to a cell count of 15 ⁇ 10 6 cells per mL per vial. Vials are cryopreserved for storage in liquid nitrogen.
  • DMSO dimethyl sulfoxide
  • a small-scale-up production is performed using the CHO cells as follows. The cells are scaled up for production in CD OptiCHO growth medium containing 100 ⁇ M MSX at 37°C and are fed every 2-4 days as needed, with CD OptiCHO growth medium supplemented with glucose and additional amino acids as necessary for approximately 14-21 days. The conditioned media supernatant harvested from the stable pool production run is clarified by centrifuge spinning.
  • the protein is run over a Protein A (MabSelect, GE Healthcare, Little Chalfont, United Kingdom) column pre-equilibrated with binding buffer. Washing buffer is then passed through the column until the OD280 value (NanoDrop, Thermo Scientific) is measured to be at or near background levels.
  • the HERV-K (HML-2) Env analog-Fc fusion protein is eluted using a low pH buffer, elution fractions are collected, and the OD280 value of each fraction is recorded. Fractions containing the target HERV-K (HML-2) Env analog-Fc fusion protein are pooled and optionally further filtered using a 0.2 ⁇ m membrane filter.
  • the cell line is optionally further subcloned to monoclonality and is optionally further selected for high titer HERV-K (HML-2) Env analog-Fc-fusion protein-expressing clones using the method of limiting dilution, a method known to those skilled in the art.
  • HML-2 high titer HERV-K
  • HERV-K (HML-2) Env analog-Fc fusion protein-expressing cell line production of the HERV-K (HML-2) Env analog-Fc fusion protein is accomplished as described above in growth medium without MSX, or optionally in growth medium containing MSX, to obtain a cell culture supernatant containing the recombinant, CHO-made, HERV-K (HML-2) Env analog-Fc fusion protein.
  • the MSX concentration is optionally increased over time to exert additional selectivity for clones capable of yielding higher product titers.
  • Example 6 Purification of a HERV-K (HML-2) Env Analog-Fc Fusion Protein Manufactured in CHO Cells.
  • HML-2 HERV-K
  • HML-2 Env analog-Fc fusion protein
  • Conditioned media supernatants containing the secreted HERV-K (HML-2) Env analog-Fc fusion protein were harvested from the CHO production runs and were clarified by centrifugation. The supernatant containing the desired HERV-K (HML-2) Env analog-Fc fusion protein was run over a Protein A column, washed and eluted using a low pH gradient.
  • the eluted fractions containing the desired protein were pooled and buffer exchanged into 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer.
  • a final filtration step was performed using a 0.2 ⁇ m membrane filter.
  • the final protein concentration was calculated from the solution optical density at 280 nm.
  • Capillary Electrophoresis using Sodium Dodecyl Sulfate (CE-SDS) Analysis of the size and purity of the target protein was performed using a LabChip GXII (Perkin Elmer).
  • a HERV-K (HML-2) Env analog-Fc fusion protein sample for analysis was prepared in loading buffer ( ⁇ reductant; e.g., beta-mercaptoethanol) and denatured at 70°C for 10 min before being loaded into the NuPAGETM Gel system (ThermoFisher Scientific).
  • the gel was stained with SimplyBlueTM SafeStain. Under non-reducing conditions, the sample was run against known molecular weight (MW) protein standards, and the eluting peak represented the ‘apparent’ MW of the fusion protein homodimer.
  • MW molecular weight
  • the sample is first treated to remove naturally occurring glycan that might interfere with the MS analysis.
  • 100 ⁇ L of a 2.5 mg/mL HERV-K (HML-2) Env analog-Fc fusion protein dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer solution is first buffer exchanged into 0.1 M Tris, pH 8.0 buffer containing 5 mM EDTA using a Zeba desalting column (Pierce, ThermoFisher Scientific, Waltham, MA).
  • PNGase F enzyme Prozyme N-glycanase
  • the sample is then analyzed via LC-MS TWI-004PCT/60767-PCT3 (NovaBioassays, Woburn, MA) resulting in a molecular mass of the molecule which corresponds to the desired homodimer without the glycan.
  • This mass is then further corrected since the enzymatic process used to cleave the glycan from the N glycosylation site also deaminates the asparagine side chain to form an aspartic acid, and in doing so the enzymatically treated homodimer gains 2 Da overall, corresponding to a mass of 1 Da for each chain present in the homodimer.
  • the actual molecular mass is the measured mass minus 2 Da to correct for each of the enzymatic modifications of the HERV-K (HML-2) Env analog-Fc fusion protein structure in the analytical sample.
  • Example 9 %Homodimer by Size-Exclusion Chromatography for a HERV-K (HML-2) Env Analog-Fc Fusion Protein.
  • Size-exclusion chromatography (SEC-HPLC) of HERV-K (HML-2) Env analog-Fc fusion proteins is carried out using a Waters 2795HT HPLC (Waters Corporation, Milford, MA) connected to a 2998 Photodiode array at a wavelength of 280 nm.
  • a sample containing a HERV-K (HML-2) Env analog-Fc fusion protein of interest is injected into a MAbPac SEC-1, 5 ⁇ m, 4 ⁇ 300 mm column (ThermoFisher Scientific, Waltham, MA) operating at a flow rate of 0.2 mL/min and with a mobile phase comprising 50 mM sodium phosphate, 300 mM NaCl, and 0.05% w/v sodium azide, pH 6.2.
  • the MAbPac SEC-1 column operates on the principle of molecular size separation.
  • HML-2 larger soluble HERV-K (HML-2) Env analog-Fc aggregates (e.g., multimers of HERV-K (HML-2) Env analog-Fc fusion protein homodimers) elute at earlier retention times, and the non-aggregated homodimers elute at later retention times.
  • HML-2 Large soluble HERV-K
  • HML-2 Env analog-Fc fusion protein homodimers
  • Example 10 In Vitro Fc (Gamma) and FcRn Receptor Binding Affinity for a HERV-K (HML- 2) Env Analog-Fc Fusion Protein.
  • HML-2 The binding of a HERV-K (HML-2) Env analog-Fc fusion protein to Fc(gamma) receptors at pH 7.4 is conducted using an ELISA assay as follows. Human Fc(gamma) receptors I, IIa, IIb, III and the FcRn receptor are used as mammalian receptors.
  • a HERV-K (HML-2) Env analog-Fc fusion protein is diluted to 10 ⁇ g/mL in sodium bicarbonate buffer at pH 9.6 and coated on Maxisorp (Nunc) microtiter plates overnight at 4°C, after which the microplate strips are washed 5 times with PBST (PBS/0.05% Tween-20) buffer and blocked with Superblock TWI-004PCT/60767-PCT3 blocking reagent (ThermoFisher).
  • microtiter plate is incubated for 1 hour at room temperature after which the microplate strips are washed 5 times with PBST and then are loaded with 100 ⁇ L/well of streptavidin-HRP diluted 1:10000 in PBST/10% Superblock buffer. After incubating for 45 min, the microplate strips are washed again 5 times with PBST. Trimethylbenzidine (TMB) is added to reveal the bound Fc(gamma) or FcRn receptor proteins and is stopped with ELISA stop reagent (Boston Bioproducts).
  • TMB Trimethylbenzidine
  • the plate is read in an ELISA plate reader at 450 nm, and the OD values (proportional to the binding of each rhFc(gamma) or FcRn receptor to the HERV-K (HML-2) Env analog-Fc fusion protein) are plotted against log concentrations of each rhFc(gamma) receptor or FcRn receptor added to each well to generate binding curves using GraphPad Prism software.
  • mice were randomly assigned to 10 animals per study group, were housed 5 per cage in ventilated housing racks, and were given standard irradiated feed and filtered water ad libitum. Mice were ear tagged individually for identification, the study cages were labeled with animal IDs, study name, and study group identification, and the study records including individual and group immunization sheets, blood/serum collection sheets, weight measurement, and health observation records were maintained per standard operating procedures (SOPs) and specific study protocols.
  • SOPs standard operating procedures
  • HERV-K (HML-2) Env analog-Fc fusion proteins were administered at doses ranging from 0.1 ⁇ g to 100 ⁇ g with and without an adjuvant (Sepivac SWE adjuvant).
  • mice were restrained at the neck scruff with the thumb and forefinger such that a tent of skin was formed over the scruff.
  • the needle was inserted parallel to the skin and was directed toward the posterior of the animal.
  • Injections were administered with a 23-gauge needle and injection volumes did not exceed 2 mL. Mice were observed for 1-3 hrs after administration for any immediate reactions and then daily for general health. Blood was obtained from each mouse via submandibular venipuncture 21-28 days prior to the first test article injection and then 21 to 28 days after each injection. Fresh blood was collected in micro-vacutainer tubes and allowed to clot, and then serum was separated by centrifugation and was stored frozen in aliquots for antibody titer analysis via ELISA.
  • mice that showed substantial antibody responses and the unimmunized control group may be kept for an additional 60-90 days until their antibody levels wane down to baseline/minimum levels, and then given an additional booster injection to evaluate the recall (memory) immune response. This would require up to three additional serum collections at 60 and 90 days after the last serum collection (i.e., Days 144 and 174, respectively), and then at 28 days after the final booster injection.
  • HERV-K (HML-2) Env analog-Fc fusion protein and adjuvant 50 to 100 ⁇ L would be mixed immediately prior to immunization of 50 to 100 ⁇ L injections.
  • Example 12 Evaluation of Binding Capability of Mice Antisera to the HERV-K (HML-2) Env Protein and the Total levels of IgG Anti-HERV-K (HML-2) Env Ab Titers in Mice Antisera.
  • a standard curve was created by serial dilutions of anti-HERV-K (HML-2) antibody added to recombinant HERV-K (HML-2) coated wells.
  • Antibody titers in the samples were analyzed by interpolating on the 4-PL standard curve fitting using SoftMax software.
  • Example 13 ELISA for Measurement of IgG1 and IgG2 Anti-HERV-K (HML-2) Env Ab Titers in Mice Anti- Sera.
  • ELISA anti-IgG1, IgG2a, IgG2b, or IgG3 antibodies present in mouse serum samples were captured by recombinant HERV-K (HML-2) Env (Catalog# CBS- EP724336HUaO; Cusabio) coated on microtiter plate strip wells (coating buffer: 0.05 M Carb/Bicarb Buffer pH9.6 (Catalog# C3041; Sigma)).
  • Mouse IgG subclass antibodies (Monoclonal Mouse IgG1 Isotype Control (Catalog# NBP1-97005; Novus Biologicals), Mouse IgG2a isotype control antibody (Catalog# NBP1-96778; Novus Biologicals), Mouse G2b isotype control antibody (Catalog# NBP1-96969; Novus Biologicals), Recomb.
  • Mouse IgG3 isotype control antibody (Catalog# MAB007; R&D Systems)) were serially diluted in sample dilution buffer (SBD) (PBST + 10%SB, 10%HS, 2mM EDTA, Na Azide) and were immobilized on 96-well plates for final quantitation of mouse antibody titers.
  • SBD sample dilution buffer
  • HERV-K (HML-2) Env peptide microarrays contained 699 different peptides printed in duplicate (1,398 spots) and were framed by additional HA (YPYDVPDYAG, 90 spots) control peptides.
  • a software algorithm broke down fluorescence intensities of each spot into raw, foreground and background signals and calculated averaged median foreground intensities and spot-to-spot deviations of spot duplicates. Based on averaged median foreground intensities, intensity maps were generated and interactions in the peptide maps were highlighted by an intensity color code with red for high and white for low spot intensities. A maximum spot-to-spot deviation of 40% was tolerated, otherwise the corresponding intensity value was zeroed. [0295] Averaged spot intensities of the assays with the mouse serum samples against the microarray content from the N- to the C-terminus of the envelope protein of HERV-K (HML- 2) were further plotted to visualize overall spot intensities and signal-to-noise ratios.
  • Example 15 Evaluation of Binding Affinity of Mice Antisera to the HERV-K (HML-2) Env Protein by Surface Plasmon Resonance.
  • Antisera of mice immunized with vaccine compounds were analyzed with a Surface Plasmon Resonance assay (Carterra) to measure their binding affinity to recombinant HERV-K Env protein.
  • a HC30M (Polycarboxylate hydrogel, medium charge density, 30nm coating thickness) chip was used.
  • Goat Anti-Mouse IgG Fc UNLB was covalently immobilized at 0.070 mg/ml to capture the antibodies from the mouse sera.
  • Up to four dilutions of mice sera (1:100, 1:1000, 1:10000, 1:100000) in HBSTE + 0.5 g/L BSA were applied to the chip in duplicate.
  • Anti-human Endogenous Retrovirus type K (HERV-K) envelope protein antibody from Austral Biologicals was used as a positive control.
  • Six concentrations (1 ⁇ M - 0.00412 ⁇ M) and (0.65 ⁇ M - 0.0025 ⁇ M) of HERV-K Env recombinant protein from Cusabio were injected into the chips.
  • n represents the number of replicates used for the mean calculations.
  • ka is the on-rate and kd is the off-rate, as TWI-004PCT/60767-PCT3 determined from a 1:1 Langmuir binding model to which direct observations of binding association and dissociation have been fit.
  • Kinetic evaluations were performed by injecting a concentration series of the analyte (i.e., the HERV- K (HML-2) Env analog-Fc fusion protein) and Rmax is the assessed saturation point of the association rate.
  • RU is an acronym corresponding to Response Unit or Resonance Unit.
  • Table 3 Binding Affinity Results for Cusabio Sequence Dilution n Mean ka Mean kd Mean KD Mean Rmax .
  • HERV-K (HML-2) Env recombinant protein (MyBiosource; MBS1391552) (80 nM and 320 nM), as determined according to Example 3, was preincubated for 1 hour at RT with: Only culture media, four different dilutions of mice antisera in culture media: 1:1, 1:10, 1:100, 1:1000, a commercial HERV-K (HML-2) Env antibody (Austral Biologicals, HERM-1811-5; 3 ⁇ g/mL) as positive control and a mouse IgG isotype control (Invitrogen, #31903, 3 ⁇ g/mL) as a negative control.
  • Example 17 Generation of Acute Lesions by Injecting HERV-K (HML-2) Env Protein in the TWI-004PCT/60767-PCT3 Motor Cortex Through Stereotaxia. or older) previously immunized with a HERV-K (HML-2) non-immunized mice were stereotaxically injected in the motor cortex with recombinant HERV-K (HML-2) Env (32 ng or 64 ng), or vehicle and brains were collected for further immunohistochemistry analysis, according to the following protocol. Body weights were collected from all mice and animals’ health status was recorded.
  • HERV-K (HML-2) Env protein (Catalog# CBS-EP724336HUaO; Cusabio) was diluted to a final injection dosage of 32 ng or 64 ng per mouse (2 ⁇ L were injected per animal) in vehicle (TE Buffer (10mM tris-HCl pH 8.0; 1mM EDTA (Qiagen #1018499)) with 10% glycerol). Mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg).
  • the left motor cortex was injected with 2 ⁇ L of HERV-K (HML-2) Env in vehicle or only vehicle at a rate of 0.1 ⁇ L/min using a microinjector.
  • Mice were euthanized 1 week after the injection. Before euthanasia, mice were weighed and had terminal bleeding. Blood was processed to serum or plasma and aliquots were sent for antibody analysis.
  • Example 18 Assessment of In Vivo Protection Against Acute Lesions Caused by Stereotaxic Injection of HERV-K (HML-2) Env Protein in the Motor Cortex of Mice Vaccinated with a HERV-K (HML-2) Env Analog-Fc Fusion Protein.
  • the specimens are embedded, with up to 40 brain hemispheres per block, arranged for coronal TWI-004PCT/60767-PCT3 sectioning in a gelatin matrix using MultiBrain ® Technology (NeuroScience Associates, Knoxville, TN).
  • MultiBrain ® Technology NeuroScience Associates, Knoxville, TN.
  • the blocks After curing with a formaldehyde solution, the blocks are rapidly frozen by immersion in 2-methylbutane chilled with crushed dry ice and are mounted on a freezing stage of an AO 860 microtome.
  • the MultiBrain ® blocks are sectioned in the coronal plane with a setting on the microtome of 35 ⁇ m. All sections are cut through the entire hemisphere of the brain and collected sequentially into a series of 24 cups.
  • Immunohistochemistry For immunohistochemistry (IHC), a set of every twelfth section (an interval of 420 microns) for Iba1, NeuN and Caspase-3 is stained free-floating. All incubation solutions from the primary antibody onward use Tris buffered saline (TBS) with Triton X100 as the vehicle; all rinses are with TBS. [0304] After a hydrogen peroxide treatment and rinses, each set of sections is immunostained with the primary antibodies as shown below, overnight at room temperature. Vehicle solutions contain Triton X100 for permeabilization. Following rinses, a biotinylated secondary antibody (anti IgG of host animal in which the primary antibody is produced) is applied.
  • TBS Tris buffered saline
  • Vector Lab’s ABC solution Catalog # PK-6100 (avidin-biotin-HRP complex; details in instruction for VECTASTAIN ® Elite ABC, Vector, Burlingame, CA) at a dilution specified in the tables below is applied.
  • the sections are again rinsed, then treated with a chromagen: diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide, nickel (II) sulfate to create a visible reaction product.
  • DAB diaminobenzidine tetrahydrochloride
  • II nickel
  • the sections are mounted on gelatin coated glass slides, then air dried.
  • the mounted Caspase-3 slides are counterstained with Neutral Red counterstain.
  • the Iba1 and NeuN stained slides are dehydrated in alcohols, cleared in xylene and coverslipped.
  • Iba1 IHC Stain Primary Antibody: Iba1 IHC Secondary Antibody: Anti-Rabbit Biotinylated TWI-004PCT/60767-PCT3 Catalog #: ab178846 Catalog #: BA-1000 Host: Rabbit Host: Goat Primary Antibody: NeuN IHC Secondary Antibody: Anti-Rabbit Biotinylated Source: Abcam Source: Vector aspase- a Primary Antibody: Caspase-3 Secondary Antibody: Anti-Rabbit Biotinylated [0305] Neutral Red Counterstain (N.R.) – The Caspase-3 IHC stained mounted slides are carried through the following sequence: deionized water (dH2O), neutral red solution made in an acetate buffer; pH4.5, dH 2 O rinses, differentiated in 70% ethanol, 95% ethanol, 100% ethanol, 1:1 100% ethanol/xylene, xylene, then coverslipped with Permount as a bonding medium.
  • dH2O dei
  • mice show weight loss, motor-neuronal symptoms, grouping of muscle fibers characteristic of ALS and short life span. Moreover, these animals show signs of TDP-43 proteinopathy in the brain, the hallmark of ALS. The appearance of symptoms starts very early, at around 1.5 months of age thus, vaccination against HERV-K Env to prevent the disease should be done at neonatal age. However, the neonatal immune system in mice and humans is still immature and, compared to the adult one, it is switched towards immune tolerance to prevent autoimmunity. Thus, it is first necessary to test whether the neonatal transgenic mice can mount an appropriate humoral response after vaccination with the compounds.
  • the suitability of the model is determined by analyzing the evolution of HERV-K Env antibody titers following immunization as measured by ELISA. If an appropriate immune response is confirmed, the animals are followed up to evaluate the protective effect of the vaccine against the appearance of clinical symptoms, neuropathology, and neurodegeneration biomarkers. [0310] Twenty (20) mixed sex hemizygous C57BL/6-Tg (Thy1-env) E337Nath/J (JAX Stock #28326) and twenty (20) mixed sex non-transgenic littermates are generated by IVF. [0311] Mice are enrolled in the study at 1 month-old as described in Table 4 below.
  • Table 4 Study Design e n TWI-004PCT/60767-PCT3 Starting at 1- month-old: e at Day 0, at Day 21, at Day 42 and at Day 105 of the study. Groups 1 and 3 receive injections of only adjuvant on the same days. Before each injection and two weeks after the final injection (D119) non-terminal bleeds are collected and processed to serum. Terminal bleeds are conducted at Day 180. Blood is processed to serum for analysis of HERV-K Env antibody titers by ELISA and for Neuron-derived exosome isolation and biomarker analysis. If an appropriate humoral response against HERV-K Env in the vaccinated mice is observed, the animals continue to be followed up to assess neuroprotection of the vaccines.
  • HERV-K nucleic acids and proteins and levels of neurodegenerative proteins are measured in the NDEs by qPCR and sensitive immunoassays (ELISA, Luminex and MSD).
  • ELISA qPCR and sensitive immunoassays
  • a statistical comparison of the levels of HERV-K and neurodegenerative proteins is performed between WT and transgenic mice and between vaccinated and unvaccinated mice. The levels are also correlated with the clinical symptoms and other established markers of neurodegeneration such as Nfl and GFAP levels in serum, as measured by Luminex.
  • Brain and muscle tissue collected from the study mice are analyzed for neuropathological changes by IHC.
  • mice brains and spinal cord are stained by IHC for: TWI-004PCT/60767-PCT3 ⁇ HERV-K Env protein, to assess whether the vaccination affects the expression of the antigen.
  • ⁇ Neuronal and motor neuronal markers to assess whether the vaccination rescues from neuronal and motor neuronal death caused by the target protein.
  • ⁇ Inflammatory markers, such as Iba1 to assess whether the vaccination rescues from brain inflammation caused by the target protein.
  • TDP-43 to assess if the vaccination protects against the formation of TDP-43 cytoplasmic aggregates.
  • Proteins of the TDP-43 pathway such as UNC13A and stathmin-2, to assess whether the vaccination protects against the loss of TDP-43 functionality.
  • Sections of the tibialis anterior muscle from the mice are stained for myosin heavy chain, to assess whether the vaccines protect against the fiber grouping in skeletal muscle characteristic of motor neuronal disease.
  • HML-2 General Preclinical Evaluation of the Effectiveness of HERV-K (HML-2) Env Analog-Fc Fusion Proteins in Inducing an Anti-HERV-K (HML-2) Env Titer in Non- Human Primates
  • HML-2 General Vaccination Protocol of HERV-K (HML-2) Env Analog-Fc Fusion Proteins in Cynomolgus Monkeys (Macaca fascicularis).
  • An automatic lighting system is set to provide a 12-hour light/dark cycle, except during designated procedures.
  • PMI LabDiet ® Fiber-Plus ® Monkey Diet 5049 biscuits are provided at an appropriate daily ration.
  • the diet is routinely analyzed by the manufacturer for contaminants to be within Testing Facility’s specifications.
  • Drinking water is provided ad libitum.
  • the water is routinely analyzed TWI-004PCT/60767-PCT3 for contaminants.
  • Fruits, vegetables, treats, as well as enrichment devices (such as perches, music, movies, floor toys, and/or foraging/hanging devices) are provided throughout the course of the study.
  • Veterinary care is available throughout the course of the study and animals are examined by veterinary staff as warranted by clinical signs or other changes.
  • Formulation is vortexed for 10-20 sec prior to injection.
  • Experimental observations and measurements listed in the subsequent sections are conducted for all animals Procedure Details Mortality/moribundity At least twice daily (AM and PM) check To assess general animal health and wellness (except on the first and last day of the study where it is conducted at least once). Food Evaluation Visual inspection for overall appetite is conducted concomitantly with the mortality/moribundity checks. Cage side Observations Once daily, beginning of the second day of acclimation, and 1 hour ( ⁇ 0.5 hour) after dosing on dosing days. Additional cage side observations are conducted as necessary to properly monitor the animal’s health condition.
  • Example 21 Evaluation of the Total Levels of IgG Anti-HERV-K (HML-2) Env Ab Titers in Cynomolgus Monkeys’ Antisera.
  • HML-2 anti-HERV-K
  • HML-2 Env IgG antibodies present in serum samples (antisera) taken from monkeys immunized with a HERV-K (HML-2) Env analog-Fc fusion protein are captured by recombinant HERV-K (HML-2) (Catalog# CBS EP724336HUaO; Cusabio) coated on microtiter plate strip wells (coating buffer: 0.05 M Carb/Bicarb Buffer pH9.6 (# C3041; Sigma)), overnight at 2 ⁇ 8°C.
  • Soluble antibody standards are included in the assay for quantitating an IgG titer.
  • Soluble standards prepared from diluting anti-HERV-K (HML-2) rabbit anti-human HERV-K (HML-2) (Catalog#CSB PA724336LA01HU; Cusabio) in SDB), assay controls, and study serum samples, diluted at 1:100 or higher in Sample Dilution Buffer (SDB: PBST + 10% SB, 10% HS, 2mM EDT, 0.05% Na Azide), are added to the protein coated strip wells for analysis. Plates are incubated for 14-21 hours at 4°C.
  • PBST PBS/tween
  • SB108a Mouse Anti- Monkey IgG-HRP
  • TMB reagent is added and incubated for 25 minutes.
  • the enzyme substrate reaction is then stopped by the addition of Stop Reagent (1% H2SO4) and the color intensity (optical density, OD) is measured in a microplate reader at 450 nm wavelength.
  • a standard curve is created by serial dilutions of anti-HERV-K (HML-2) antibody added to recombinant HERV-K (HML-2) coated wells.
  • Antibody titers in the samples are analyzed by interpolating on the 4-PL standard curve fitting using SoftMax software.
  • Example 22 Epitope Mapping to Analyze Specific HERV-K (HML-2) Env Epitope Recognition Using Non-Human Primate Antisera.
  • a HERV-K (HML-2) Env peptide microarray is pre-stained with the secondary antibody in incubation buffer to investigate background interactions with the antigen-derived TWI-004PCT/60767-PCT3 peptides that could interfere with the main assays.
  • the microarray content is the sequence of the envelope protein of HERV-K (HML-2) (UniProtKB: Q69384) elongated with neutral GSGSGSG linkers at the C- and N-terminus to avoid truncated peptides.
  • the elongated antigen sequence is converted into linear 15 amino acid peptides with a peptide-peptide overlap of 14 amino acids.
  • the resulting HERV-K (HML-2) Env peptide microarrays contain 699 different peptides printed in duplicate (1,398 spots) and are framed by additional HA (YPYDVPDYAG, 90 spots) control peptides.
  • HA peptides framing the peptide microarrays are subsequently stained with the control antibody (mouse monoclonal anti-HA (12CA5) DyLight800 (0.2 ⁇ g/mL)) for 45 min in incubation buffer at RT, as internal quality control to confirm the assay performance and the peptide microarray integrity.
  • control antibody mouse monoclonal anti-HA (12CA5)
  • DyLight800 0.2 ⁇ g/mL
  • Quantification of spot intensities and peptide annotation are based on the 16-bit gray scale TIFF files that exhibit a higher dynamic range than the 24-bit colorized TIFF files.
  • Microarray image analysis is done with PepSlide ® Analyzer (SICASYS Software GmbH, Heidelberg, Germany) and is summarized in Excel files.
  • a software algorithm breaks down fluorescence intensities of each spot into raw, foreground and background signals and calculates averaged median foreground intensities and spot-to-spot deviations of spot duplicates. Based on averaged median foreground intensities, intensity maps are generated and interactions in the peptide maps are highlighted by an intensity color code with red for high and white for low spot intensities. A maximum spot-to-spot deviation of 40% is tolerated, otherwise the corresponding intensity value is zeroed. [0330] Averaged spot intensities of the assays with the monkey serum samples against the microarray content from the N- to the C-terminus of the envelope protein of HERV-K (HML- 2) are further plotted to visualize overall spot intensities and signal-to-noise ratios.
  • the intensity plots are correlated with peptide and intensity maps as well as with visual inspection of the microarray scans to identify epitopes of the monkey serum samples.
  • TWI-004PCT/60767-PCT3 It is expected that epitope mapping of serum from animals immunized with the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 46 or the HERV-K (HML-2) Env analog-Fc fusion protein of SEQ ID NO: 47 will show recognition of one or more specific HERV-K (HML-2) Env sequences contained in SEQ ID NO: 46 or SEQ ID NO: 47, respectively.
  • ELISpot to Analyze Type of Th Immune Response in Immunized Non-Human Primates.
  • ELISpot is a technique related to ELISA that was developed for the detection of secreted proteins, such as cytokines and growth factors. It is also called enzyme-linked immunospot.
  • ELISpot is performed using a PVDF or nitrocellulose membrane 96-well plate pre- coated with an antibody specific to the secreted protein. Cells are added to the plate, where they attach to the coated membrane. Cells are then stimulated, and the secreted protein binds to the antibody. Next, a detection antibody is added that binds specifically to the bound protein.
  • the resulting antibody complex is detected either through enzymatic action to produce a colored substrate or with fluorescent tags.
  • An advantage to using fluorescence is the ability to identify more than one secreted protein at a time.
  • the membrane is analyzed by manually counting the spots or with an automated reader designed for this purpose. Each secreting cell appears as a spot of color or fluorescence; thus this is a quantitative method for evaluating protein secretion.
  • PVDF membranes are prepared in 96-well plates by incubating in 35% ethanol for 30 seconds and then washing thoroughly with PBS. Any remaining ethanol can affect cell viability, as well as antibody binding.
  • Each 96-well plate is coated with capture antibody (anti- IL4, anti-IL-5 and anti-IFN gamma) diluted in phosphate buffered saline (PBS). Approximately 0.5–1 ⁇ g per well of antibody is used for well-defined spots. Kits are optimized with capture concentrations for best performance (100 ⁇ L per well). Plates are incubated overnight at 4°C and then are washed manually with PBS. A plate washer is not used at this stage. One hundred (100) ⁇ L of 2% dry skim milk is added per well to block non-specific binding to the membrane. Plates are incubated for 2 h at room temperature and then are washed 3 times in PBS.
  • capture antibody anti- IL4, anti-IL-5 and anti-IFN gamma
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • Both freshly prepared and cryopreserved cells may be used in the assay.
  • frozen cells are left to rest at least 1 hour after thawing to allow the removal of cell debris before addition to the plate.
  • Cells are counted using a viability dye like trypan blue and TWI-004PCT/60767-PCT3 should be over 95% viable.
  • Cells are diluted to the required concentration and the cell suspension is added to wells. Typically, cell numbers should usually range from between 2 ⁇ 10 5 to 4 ⁇ 10 5 PBMC cells per well. Serum-free media is used since serum contains many proteins which may affect the results.
  • Cells are cultured overnight at 37°C in CO2 incubator without shaking the plates. During the overnight incubation, the cells secrete cytokine, which binds to the primary antibody. Cells are stimulated with peptide pools of the target HERV-K Env protein and with PMA/Ionomycin as positive controls to stimulate IL-4 and IL-5 and PHA to stimulate IFN gamma. Cells are washed and the unbound cytokine is washed away by incubating with PBS 0.1% Tween 20 for 10 min. For the detection of antibodies, the secondary conjugated-antibody is diluted in PBS 1% BSA, added to the wells and incubated for 1–2 h at room temperature.
  • Plates are washed 3 times with PBS 0.1% Tween 20 to remove non-specific detection antibody binding.
  • the base is taken off the bottom of the plate to enable thorough washing of the membrane before substrate/chromogen is added.
  • the enzyme substrate (TMB (3,3’,5,5’-tetramethylbenzidine) is added to each well and is incubated with gentle agitation on a plate shaker, as directed by the manufacturer. After the base is replaced and the substrate is added, spot formation is carefully monitored. Reaction is stopped by gently washing the plate with PBS 0.1% Tween 20 once development appears to slow. Both sides of the membrane are washed with distilled water to stop the spot formation and membranes are dried at room temperature.
  • the number of spots corresponds to the number of cells that secreted the protein, allowing for quantification of the immune response.
  • Each plate is read 3 times and results are averaged in order to minimize errors in the measurements.
  • It is expected that the number of cells secreting IL-4 and IL-5 will be higher than the number of cells secreting IFN gamma, which would be indicative of a Th2 response induced by the vaccine.
  • Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the disclosure includes TWI-004PCT/60767-PCT3 embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
  • the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim.
  • any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
  • elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the disclosure, or aspects of the disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and/or features.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Genetics & Genomics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Psychiatry (AREA)
  • Hospice & Palliative Care (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente divulgation concerne des protéines de fusion fabriquées par recombinaison comprenant un fragment de protéine Env de HERV-K (HML-2) ou un analogue de celui-ci lié à un fragment Fc humain. Des modes de réalisation comprennent l'administration des protéines de fusion à des patients ayant une maladie ou un trouble ayant l'intention d'atténuer et/ou de réduire la durée de symptômes associés à l'état ou à la maladie (par exemple, mais sans s'y limiter, la faiblesse musculaire, la paralysie et l'insuffisance respiratoire), et/ou la prévention des symptômes associés à l'état ou à la maladie, par exemple, en empêchant la dégénérescence des motoneurones et la mort cellulaire chez des patients atteints de SLA associées à l'état ou à la maladie. Par conséquent, le "traitement" signifie généralement à la fois un traitement thérapeutique et des mesures prophylactiques ou préventives. L'amélioration après traitement peut se manifester sous la forme d'une diminution ou d'une élimination de tels symptômes, par exemple, par une diminution ou une élimination de symptômes associés à la SLA, et/ou par une diminution de la durée de tels symptômes.
PCT/US2025/040127 2024-08-01 2025-07-31 Protéines de fusion analogiques env de herv-k (hml-2) pour immunothérapie spécifique à l'antigène et procédés d'utilisation Pending WO2026030596A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202463678342P 2024-08-01 2024-08-01
US202463678369P 2024-08-01 2024-08-01
US63/678,369 2024-08-01
US63/678,342 2024-08-01

Publications (1)

Publication Number Publication Date
WO2026030596A1 true WO2026030596A1 (fr) 2026-02-05

Family

ID=97027883

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/040127 Pending WO2026030596A1 (fr) 2024-08-01 2025-07-31 Protéines de fusion analogiques env de herv-k (hml-2) pour immunothérapie spécifique à l'antigène et procédés d'utilisation

Country Status (1)

Country Link
WO (1) WO2026030596A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003106634A2 (fr) * 2002-06-13 2003-12-24 Chiron Corporation Vecteurs pour l'expression de polypeptides hml-2
US20090297530A1 (en) 2006-05-22 2009-12-03 Feng Wang-Johanning Herv-k antigens, antibodies, and methods
WO2018107117A1 (fr) 2016-12-09 2018-06-14 Akston Biosciences Corporation Fusions d'insuline-fc et méthodes d'utilisation
EP3351265A1 (fr) 2017-01-20 2018-07-25 Geneuro SA Anticorp du herv-k enveloppe et ses utilisations
WO2019043127A1 (fr) * 2017-09-01 2019-03-07 Inprother Aps Vaccin à utiliser dans la prophylaxie et/ou le traitement d'une maladie
WO2020006529A1 (fr) 2018-06-29 2020-01-02 Akston Biosciences Corporation Protéines de fusion insuline-fc à action ultra-longue et procédés d'utilisation
WO2021207599A1 (fr) * 2020-04-10 2021-10-14 Akston Biosciences Corporation Immunothérapie spécifique d'un antigène pour des protéines de fusion de covid-19 et procédés d'utilisation
WO2023118508A1 (fr) * 2021-12-23 2023-06-29 Bavarian Nordic A/S Virus mva recombinants pour administration intrapéritonéale pour le traitement du cancer

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003106634A2 (fr) * 2002-06-13 2003-12-24 Chiron Corporation Vecteurs pour l'expression de polypeptides hml-2
US20090297530A1 (en) 2006-05-22 2009-12-03 Feng Wang-Johanning Herv-k antigens, antibodies, and methods
WO2018107117A1 (fr) 2016-12-09 2018-06-14 Akston Biosciences Corporation Fusions d'insuline-fc et méthodes d'utilisation
EP3351265A1 (fr) 2017-01-20 2018-07-25 Geneuro SA Anticorp du herv-k enveloppe et ses utilisations
US10981977B2 (en) 2017-01-20 2021-04-20 Geneuro Sa Anti-HERV-K envelope protein antibodies and methods for detecting HERV-K envelope protein in patients with sporadic amyotrophic lateral sclerosis
WO2019043127A1 (fr) * 2017-09-01 2019-03-07 Inprother Aps Vaccin à utiliser dans la prophylaxie et/ou le traitement d'une maladie
WO2020006529A1 (fr) 2018-06-29 2020-01-02 Akston Biosciences Corporation Protéines de fusion insuline-fc à action ultra-longue et procédés d'utilisation
WO2021207599A1 (fr) * 2020-04-10 2021-10-14 Akston Biosciences Corporation Immunothérapie spécifique d'un antigène pour des protéines de fusion de covid-19 et procédés d'utilisation
WO2023118508A1 (fr) * 2021-12-23 2023-06-29 Bavarian Nordic A/S Virus mva recombinants pour administration intrapéritonéale pour le traitement du cancer

Non-Patent Citations (45)

* Cited by examiner, † Cited by third party
Title
"Uniprot", Database accession no. Q69384
ARRU G ET AL.: "HERV-K Modulates the Immune Response in ALS Patients", MICROORGANISMS., vol. 9, no. 8, 23 August 2021 (2021-08-23), pages 1784
AZADEH BEYGMORADIAHMAD HOMAEIROOHULLAH HEMMATIPEDRO FERNANDES: "Recombinant protein expression: Challenges in production and folding related matters", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, vol. 233, 2023, pages 123407, XP087285089, ISSN: 0141-8130, DOI: 10.1016/j.ijbiomac.2023.123407
BHARDWAJ N.MONTESION MROY FCOFFIN JM.: "Differential expression of HERV-K (HML-2) proviruses in cells and virions of the teratocarcinoma cell line Tera-1", VIRUSES, vol. 7, no. 3, 2015, pages 939,968
BUSCHER K.TREFZER U.HOFMANN MSTERRY W.KURTH R.DENNER J.: "Expression of human endogenous retrovirus K in melanomas and melanoma cell lines.", CANCER RES, vol. 65, no. 10, 2005, pages 4172 - 4180, XP007914067
BUSCHER KHAHN SHOFMANN MTREFZER UOZEL MSTERRY W.LOWER J.LOWER R,KURTH RDENNER J.: "Expression of the human endogenous retrovirus-K transmembrane envelope, Rec and Np9 proteins in melanomas and melanoma cell lines", MELANOMA RES, vol. 16, no. 3, 2006, pages 223 - 234
COBB BA: "The history ofIgG glycosylation and where we are now", GLYCOBIOLOGY, vol. 30, no. 4, 20 March 2020 (2020-03-20), pages 202 - 213
CZAJKOWSKY DANIEL M. ET AL: "Fc-fusion proteins: new developments and future perspectives", EMBO MOLECULAR MEDICINE, vol. 4, no. 10, 1 October 2012 (2012-10-01), US, pages 1015 - 1028, XP055803855, ISSN: 1757-4676, DOI: 10.1002/emmm.201201379 *
DEWANNIEUX MHARPER FRICHAUD A,LETZELTER CRIBET DPIERRON GHEIDMANN T.: "Identification of an infectious progenitor for the multiple-copy HERV-K human endogenous retroelements", GENOME RES., vol. 16, no. 12, December 2006 (2006-12-01), pages 1548 - 56
DICK, LW., BIOTECHNOL BIOENG., vol. 100, no. 6, 2008, pages 132 - 43
DOUVILLE RLIU JROTHSTEIN J.NATH A. ET AL.: "Identification of active loci of a human endogenous retrovirus in neurons of patients with amyotrophic lateral sclerosis", ANN NEUROL, vol. 69, no. 1, 2011, pages 141 - 151, XP055242264, DOI: 10.1002/ana.22149
GARCIA-MONTOJO ET AL.: "Antibody Response to HML-2 May Be Protective in Amyotrophic Lateral Sclerosis", ANN NEUROL., vol. 92, no. 5, November 2022 (2022-11-01), pages 782 - 792
GARCIA-MONTOJO MARTA ET AL: "Human endogenous retrovirus-K (HML-2): a comprehensive review", CRITICAL REVIEWS IN MICROBIOLOGY, vol. 44, no. 6, 14 October 2018 (2018-10-14), GB, pages 715 - 738, XP093171025, ISSN: 1040-841X, DOI: 10.1080/1040841X.2018.1501345 *
GARCIA-MONTOJO MARTA: "Development of an Fc-fusion HERV-K vaccine to treat amyotrophic lateral sclerosis and frontotemporal dementia", NEUROTHERAPEUTICS : THE JOURNAL OF THE AMERICAN SOCIETY FOR EXPERIMENTAL NEUROTHERAPEUTICS, vol. 22, no. 4, 1 July 2025 (2025-07-01), pages e00657, XP093327403, ISSN: 1878-7479, DOI: 10.1016/j.neurot.2025.e00657 *
GARCIA-MONTOJO MDOUCET-O'HARE THENDERSON LNATH A.: "Human endogenous retrovirus-K (HML-2): a comprehensive revie¡,v", CRIT REVMICROBIOL, vol. 4-1, no. 6, November 2018 (2018-11-01), pages 715 - 738, XP093171025, DOI: 10.1080/1040841X.2018.1501345
GARCIA-MONTOJO MSIMULA ERFATHI SMCMAHAN CGHOSALABERRY JDCUDKOWICZ MELKAHLOUN AJOHNSON KNORCZTO G: "Antibody Response to HML-2 May Be Protective in Amyotrophic Lateral Sclerosis.", ANN NEUROL., vol. 92, no. 5, November 2022 (2022-11-01), pages 782 - 792
GARCIA-MONTOJO, M.SIMULA, E.R.FATHI, S.MCMAHAN, C.GHOSAL, A.BERRY, J.D.CUDKOWICZ, M.ELKAHLOUN, A.JOHNSON, K.NORA TO, G.: "Antibody Response to HNIL-2 May Be Protective in Amyotrophic Lateral Sclerosis", ANN NEUROL., vol. 92, 2022, pages 782 - 792
GARCIA-MONTOJO, M.SIMULA, E.R.FATHI. SMCMAHAN, C.GHOSAL, A.BERRY, J.D.CUDKOWICZ, MELKAHLOUN, A.JOHNSON, K.NORATO, G.: "Antibody Response to HML-2 May Be Protective in Amyotrophic Lateral Sclerosis.", ANN NEUROL, vol. 92, 2022, pages 782 - 792
HERBST HSAUTER MMUELLER-LANTZSCH N.: "Expression of human endogenous retrovirus K elements in germ cell and trophoblastic tumors", AM J PATHOL, vol. 1-19, no. 5, 1996, pages 1727 - 1735, XP009009504
HOHN OHANKE KBANNERT N.: "HERV-K(HML-2), the Best Preserved Family of HERVs: Endogenization, Expression, and Implications in Health and Disease", FRONT ONCOL., vol. 3, 20 September 2013 (2013-09-20), pages 246
HOSSEINIPORGHAM SEPIDEH ET AL: "Anti-HERV-K Drugs and Vaccines, Possible Therapies against Tumors", VACCINES, vol. 11, no. 4, 28 March 2023 (2023-03-28), CH, pages 751, XP093225390, ISSN: 2076-393X, DOI: 10.3390/vaccines11040751 *
JOHANNING GLMALOUF GGZHENG XESTEVA FJWEINSTEIN JNWANG-JOHANNING F. SU X.: "Expression of human endogenous retrovirus-K is strongly associated with the basal-like breast cancer phenotype", SCI REP., vol. 7, 6 February 2017 (2017-02-06), pages 41960, XP055940398, DOI: 10.1038/srep41960
JUSTIZ VAILLANT AAJAMAL ZPATEL P ET AL., IMMUNOGLOBULIN., 28 August 2023 (2023-08-28), Retrieved from the Internet <URL:ncbi.nlm.nih.gov/books/NBK513-160/>
KRAUS BENJAMIN ET AL: "Vaccination Directed against the Human Endogenous Retrovirus-K Envelope Protein Inhibits Tumor Growth in a Murine Model System", PLOS ONE, vol. 8, no. 8, 30 August 2013 (2013-08-30), US, pages e72756, XP093327724, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0072756 *
KURTH RBANNERT N.: "Beneficial and detrimental effects of human endogenous retroviruses", INT J CANCER, vol. 126, no. 2, 2010, pages 306 - 314, XP071285694, DOI: 10.1002/ijc.24902
LI WLEE MHHENDERSON LTYAGI RBACHANI MSTEINER JCAMPANAC EHOFFMAN DAVON GELDERN GJOHNSON K: "Human endogenous retrovirus-K contributes to motor neuron disease", SCI TRANSL MED., vol. 7, no. 307, 30 September 2015 (2015-09-30), pages 307, XP055353030, DOI: 10.1126/scitranslmed.aac8201
LOWER RLOWER JFRANK II.HARZMANN R.KURTH R.: "Human teratocarcinomas cultured in vitro produce unique retrovirus-like viruses", J GEN VIROL, vol. 65, 1984, pages 887,898
LU DAI ET AL: "Development of human endogenous retrovirus type K- related treatments for human diseases", JOURNAL OF MEDICAL VIROLOGY, JOHN WILEY & SONS, INC, US, vol. 96, no. 3, 18 March 2024 (2024-03-18), pages n/a, XP072610247, ISSN: 0146-6615, DOI: 10.1002/JMV.29532 *
MASSIMO STEFANI: "Protein misfolding and aggregation: new examples in medicine and biology of the dark side of the protein world", BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - MOLECULAR BASIS OF DISEASE, vol. 1739, 2004, pages 5 - 25, XP004679591, ISSN: 0925-4439, DOI: 10.1016/j.bbadis.2004.08.004
MCCORMICK ALBROWN RH JR.CUDKOWICZ MEAL-CHALABI AGARSON JA.: "Quantification of reverse transcriptase in ALS and elimination of a novel retroviral candidate", NEUROLOGY, vol. 70, no. 4, 2008, pages 278 - 283
MUSTER TWALTENBERGER A.GRASSAUER AHIRSCHL SCAUCIG PROMIRER 1FODINGER DSEPPELE HSCHANAB 0MAGIN-LACHMANN C ET AL.: "An endogenous retrovirus derived. from human melanoma cells.", CANCER RES, vol. 63, no. 24, 2003, pages 8735 - 874, XP002495984
NAPODANO, CECILIAMARINO, MARIAPAOLASTEFANILE, ANNUNZIATAPOCINO, KRIZIASCATENA, ROBERTOGULLI, FRANCESCARAPACCINI, GIANNOCI, STEFANO: "Immunological Role of IgG Subclasses", IMMUNOLOGICAL INVESTIGATIONS, vol. 50, 2020, pages 1 - 18
ORICCHIO ESCIAMANNA 1BERALDI RTOLSTONOG GVSCHUMANN GGSPADAFORA C.: "Distinct roles for LINE-1 and HERV-K retroelements in cell proliferation, differentiation and tumor progression.", ONCOGENE, vol. 26, no. 29, 2007, pages 4226 - 4233, XP037743209, DOI: 10.1038/sj.onc.1210214
REIS ΒSJUNGBLUTH AAFROSINA DHOLZ MRITTER ENAKAYAMA EISHIDA TOBATA YCARVER BSCHER H ET AL.: "Prostate cancer progression correlates with increased humoral immune response to a human endogenous retrovirus GAG protein", CLIN CANCER RES, vol. 19, no. 22, 2013, pages 6112 - 6125
RICHEL ELIE ET AL: "Antigen-dependent modulation of immune responses to antigen-Fc fusion proteins by Fc-effector functions", FRONTIERS IN IMMUNOLOGY, vol. 14, 5 October 2023 (2023-10-05), Lausanne, CH, XP093278917, ISSN: 1664-3224, DOI: 10.3389/fimmu.2023.1275193 *
RUGGIERI AMALDENER ESAUTER MMUELLER-LANTZSCH NMEESE EFACKLER OTMAYER J.: "Human endogenous retrovirus HERV-K(HML-2) encodes a stable signal peptide with biological properties distinct from Rec.", RETROVIROLOGY., vol. 6, 16 February 2009 (2009-02-16), pages 17, XP021051453, DOI: 10.1186/1742-4690-6-17
SCHMITT KREICHRATH JROESCH A.MEESE E.MAYER J.: "Transcriptional Profiling of Human Endogenous Retrovirus Group HERV-K(HML-2) Loci in Melanoma", GENOME BIOL EVOL, vol. 3, no. 2, 2013, pages 307 - 328
SHAH AHRIVAS SRDOUCET-O'HARE TTGOVINDARAJAN VDEMARINO CWANG T. AMPIE LZHANG 3BANASAVADI-SIDDEGOWDA YKWALBRIDGE SMARIC D: "Human endogenous retrovirus K contributes to a stem cell niche in glioblastoma", J CLIN INVEST., vol. 133, no. 13, 3 July 2023 (2023-07-03), pages 167929
SIGCTL LH.: "IgG subclasses", J CLIN RHEURNATOL., vol. 18, no. 6, 2012, pages 31618
STEELE AJAL-CHALABI AFERRANTE KCUDKOWICZ MEBROWN RH JR.GARSON JA.: "Detection of serum reverse transcriptase activity in patients with ALS and unaffected blood relatives", NEUROLOGY, vol. 6-1, no. 3, 2005, pages 454 - 458
STEINER JPBACHANI MMALIK NDEMARINO CLI WSAMPSON KLEE MHKOWALAK J.BHASKAR MDOUCET-O 'HARE T.: "Human Endogenous Retrovirus K Envelope in Spinal Fluid of Amyotrophic Lateral Sclerosis Is Toxic", ANN NEUROL., vol. 92, no. 4, October 2022 (2022-10-01), pages 545 - 561, XP093277111, DOI: 10.1002/ana.26452
VIOLA MVFRAZIER MWHITE LBRODY JSPIEGELMAN S.: "RNA-instructed DNA polymerase activity in a cytoplasmic particulate fraction in brains from Guamanian patients", J EXP MED, vol. 142, no. 2, 1975, pages 483 - 494
WANG-JOHANNING FLIU JRYCAJ KHUANG MTSAI KROSEN DGCHEN DTLU DWBARNHART KFJOHANNING GL.: "Expression of multiple human endogenous retrovirus surface envelope proteins in ovarian cancer", INT J CANCER, vol. 120, no. 1, 2007, pages 81 - 90, XP055034826, DOI: 10.1002/ijc.22256
WILDSCHUTTE JHRAM DSUBRAMANIAN RSTEVENS VLCOFFIN JM.: "The distribution of insertionally polymorphic endogenous retroviruses in breast cancer patients and cancer-free controls", RETROVIROLOGY, vol. 11, 2014, pages 62, XP021193812, DOI: 10.1186/s12977-014-0062-3
XUE BEI ET AL: "Human Endogenous Retrovirus K (HML-2) in Health and Disease", FRONTIERS IN MICROBIOLOGY, vol. 11, 17 July 2020 (2020-07-17), XP093087796, DOI: 10.3389/fmicb.2020.01690 *

Similar Documents

Publication Publication Date Title
CN105524160B (zh) 药物组合物
CN111542534B (zh) 新型支架式hiv-1疫苗免疫原
RU2664197C2 (ru) Иммуногенная композиция, включающая ее вакцина, набор для приготовления вышеуказанной композиции и способ лечения заболеваний, связанных с патологией секреции гастрина
US20250345413A1 (en) Sars-cov-2 subunit vaccine
US20210079054A1 (en) Peptide immunogens of il-31 and formulations thereof for the treatment and/or prevention of atopic dermatitis
CN113631573A (zh) 通过靶向新Tau物种治疗Tau蛋白病的方法
US20260035433A1 (en) Pd-l1 analog fusion proteins for antigen specific immunotherapy and methods of use
US20090169549A1 (en) Conformational isomers of alpha-synuclein, antibodies thereto and methods of their manufacture and use
US20260035415A1 (en) Herv-k (hml-2) env analog fusion proteins for antigen specific immunotherapy and methods of use
US20260035414A1 (en) Herv-k (hml-2) env analog fusion proteins for antigen specific immunotherapy and methods of use
US20240400634A1 (en) Peptide immunogens targeting islet amyloid polypeptide (iapp) and formulations thereof for prevention and treatment of disorders related to aggregated iapp
WO2026030596A1 (fr) Protéines de fusion analogiques env de herv-k (hml-2) pour immunothérapie spécifique à l&#39;antigène et procédés d&#39;utilisation
WO2026030592A1 (fr) Protéines de fusion analogues de herv-k (hml-2) env pour immunothérapie spécifique à l&#39;antigène et procédés d&#39;utilisation
WO2026030590A1 (fr) Protéines de fusion analogiques env de herv-k (hml-2) pour immunothérapie spécifique à l&#39;antigène et procédés d&#39;utilisation
JP2024517927A (ja) ウイルス感染に対するペプチドワクチン
KR102914577B1 (ko) Ige 매개 알레르기 질환의 치료를 위한 막 결합 ige를 표적하는 펩티드 면역원 및 이의 제제
US20240115674A1 (en) Vaccine and methods for preventing filariasis and dirofilariasis
WO2024163327A1 (fr) Immunogènes de la glycoprotéine 42 du virus epstein-barr pour la vaccination et la découverte d&#39;anticorps
US20100322956A1 (en) Methods and substances for the treatment of alzheimer&#39;s
WO2026085175A1 (fr) Protéines de fusion analogues du ngf pour l&#39;immunothérapie spécifique à l&#39;antigène et procédés d&#39;utilisation
WO2026085180A2 (fr) Protéines de fusion analogues du ngf pour l&#39;immunothérapie spécifique à l&#39;antigène et procédés d&#39;utilisation
WO2026085182A1 (fr) Protéines de fusion analogues de ngf pour immunothérapie spécifique d&#39;un antigène et procédés d&#39;utilisation
JP2026507778A (ja) 水痘帯状疱疹ウイルスのｇＥ及びｇＩの融合タンパク質及び使用
CN120897758A (zh) Covid-19融合蛋白的核衣壳抗原免疫疗法和使用方法
WO2026085178A1 (fr) Protéines de fusion analogues de ngf pour immunothérapie spécifique d&#39;un antigène et procédés d&#39;utilisation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25765358

Country of ref document: EP

Kind code of ref document: A1